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Regulation of lipin phosphorylation and lipid homeostasis by glycogen synthase kinase 3 Chan, Leslie Jing 2018

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Regulation of lipin phosphorylation and lipid homeostasis by glycogen synthase kinase 3  by  Leslie Jing Chan  B.Sc., The University of British Columbia, 2012  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (CELL AND DEVELOPMENTAL BIOLOGY)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2018  © Leslie Jing Chan, 2018 ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Regulation of lipin phosphorylation and lipid homeostasis by glycogen synthase kinase 3  submitted by Leslie Jing Chan in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Cell and Developmental Biology  Examining Committee: Dr. Christopher Loewen Supervisor  Dr. Calvin Roskelley Supervisory Committee Member   Supervisory Committee Member Dr. Susanne Clee University Examiner Dr. Elizabeth Conibear University Examiner  Additional Supervisory Committee Members: Dr. Robert Nabi Supervisory Committee Member Dr. Thibault Mayor Supervisory Committee Member iii  Abstract It is imperative for cell survival and function to maintain proper steady-state lipid levels, or lipid homeostasis. This has significant physiological consequences, as lipid homeostasis is disrupted in metabolic diseases including obesity and diabetes, which necessitates a greater understanding of this cellular phenomenon. The lipin family of phosphatidic acid phosphatases are conserved enzymes that control the cellular balance of phospholipid and triglyceride synthesis, and mammalian lipins can also regulate lipid synthesis through interacting with transcription factors in the nucleus. Unsurprisingly, lipins are tightly regulated enzymes and a conserved mechanism of lipin regulation is phosphorylation by kinases, which can control the subcellular localization of lipins from the cytoplasm to other cellular compartments. To date, various kinases have been identified that phosphorylate lipins including the mechanistic target of rapamycin complex 1 (mTORC1), which controls lipin 1 localization from the cytoplasm to the nucleus and the ability of lipin 1 to repress sterol regulatory element binding protein (SREBP) target-gene transcription and thus cholesterol and fatty acid biosynthesis and uptake.     A high-throughput screen seeking novel kinase regulators of lipins has never been performed. In this work, we designed an overexpression screen in yeast and identified Mck1, a glycogen synthase kinase 3 (GSK3) kinase, as a novel regulator of lipins and lipid homeostasis. We further discovered that this relationship was conserved from yeast to mammals by characterizing that mammalian GSK3 phosphorylates lipin 1 directly. GSK3 activity, downstream of the PI3K/Akt pathway, towards lipin 1 was found to control its localization, and in the absence of GSK3 activity, lipin 1 translocated to the nucleus and repressed SREBP target-gene expression. We observed that regulation of SREBP-target gene expression in this pathway iv  was dependent on lipin 1 and additionally that both GSK3 paralogs, GSK3α and GSK3β, appeared to be involved. Finally, we characterized the role of GSK3 in lipid metabolism using mouse models and found that mice lacking GSK3α or GSK3β in the liver demonstrated resistance to some effects of diet-induced obesity including weight gain and the expression of certain SREBP target genes, suggesting that GSK3 in the liver plays a role in the development of these phenotypes.   v  Lay Summary It is vital for cells to have the appropriate balance of lipids, which are biomolecules best known for creating cell membranes and for energy storage as fat. The balance of lipids is unbalanced in diseases including obesity, so it is important to study enzymes that act directly on lipids. Here, we were interested lipins, enzymes that convert a membrane lipid, phosphatidic acid, to a fat storage lipid, diacylglycerol. We sought to identify other enzymes that controlled lipin function, and using yeast and mouse/human cells, we discovered that GSK3 was an enzyme that directly mediated the ability of lipin to affect lipid synthesis. This was the first time GSK3 was found to play such a direct role in lipid metabolism, and thus we further tested its role in mouse models of diet-induced obesity. We found that mice missing GSK3 only in their livers were moderately resistant to effects of diet-induced obesity.  vi  Preface The studies described in this dissertation were designed, performed, and analyzed by the author LJ Chan except where noted below, and with assistance as highlighted below. All components of this thesis were written by LJ Chan with editing and input from CJ Loewen, CD Roskelley, IR Nabi, T Mayor, and MR Hughes.   Data from Chapter 2 will be submitted for publication. LJ Chan and CJ Loewen designed the experiments, except as indicated hereafter. The SDL screen was designed by CJ Loewen and performed and analyzed by M Briggs and BP Young. In vitro kinase assay involving Mck1 and Pah1 (Figure 2.8) were designed by JM McQueen and performed by JM McQueen and LJ Chan. All other experiments were performed and analyzed by LJ Chan.  Data from Chapter 3 will be submitted for publication. LJ Chan and CJ Loewen designed the experiments, except as indicated hereafter. Mass spectrometry (Figure 3.2), western blots (Figure 3.3), in vitro kinase assay involving GSK3β and lipin 1 (Figure 3.7), and reverse transcriptase quantitative PCR of SREBP target genes from mouse embryonic fibroblasts (Figure 3.12) were designed, performed, and analyzed by TR Peterson while in the laboratory of DM Sabatini at the Whitehead Institute at the Massachusetts Institute of Technology. Phosphoproteomic analysis of FLAG-lipin 1 (Figure 3.6) was performed by the UBC Proteomics Core. Generation of mice used in animal studies was designed and performed by MR Hughes from the laboratory of KM McNagny at the University of British Columbia. Histology (Figure 3.30) was performed by Wax-it Histology Services Inc. (Vancouver, BC, Caanda). All other experiments were performed and analyzed by LJ Chan.  vii   Animal studies described in Chapter 3 of this thesis were approved by the University of British Columbia Animal Care Committee (Certificate #A17-0138) and followed the ethical guidelines of the Canadian Committee on Animal Care.  viii  Table of Contents Abstract .......................................................................................................................................... iii Lay Summary ...................................................................................................................................v Preface............................................................................................................................................ vi Table of Contents ......................................................................................................................... viii List of Tables ...................................................................................................................................x List of Figures ................................................................................................................................ xi List of Abbreviations .....................................................................................................................xv Acknowledgements .................................................................................................................... xviii Dedication ......................................................................................................................................xx Chapter 1: Introduction ....................................................................................................................1 1.1 Obesity and Lipid Homeostasis ...................................................................................... 1 1.2 Sterol regulatory element binding proteins ..................................................................... 2 1.3 Phosphatidic acid is a central player in lipid homeostasis .............................................. 5 1.3.1 The glycerol-3-phosphate pathway ............................................................................. 5 1.3.2 Phosphatidic acid signaling and the Opi1 pathway .................................................... 7 1.4 Lipins are conserved phosphatidic acid phosphatase enzymes ....................................... 8 1.4.1 Discovery and characterization ................................................................................... 8 1.4.2 Function in lipid metabolism and lipid homeostasis................................................. 12 1.4.3 Lipins and disease ..................................................................................................... 16 1.4.4 Regulation of lipins by phosphorylation ................................................................... 17 1.4.4.1 The yeast lipin Pah1 is regulated by phosphorylation ...................................... 18 1.4.4.2 Lipin 1 regulation by phosphorylation .............................................................. 20 ix  1.4.4.3 mTORC1 phosphorylates lipin 1 to control its subcellular localization and SREBP target gene expression.......................................................................................... 22 1.5 Glycogen synthase kinase 3 .......................................................................................... 26 1.5.1 Regulation ................................................................................................................. 29 1.5.2 GSK3 substrates: mechanism and guidelines ........................................................... 30 1.6 Thesis Investigation ...................................................................................................... 33 Chapter 2: Synthetic dosage lethality screening identifies novel kinase regulators of the yeast lipin Pah1 .......................................................................................................................................36 2.1 Introduction ................................................................................................................... 36 2.2 Materials and Methods .................................................................................................. 39 2.3 Results ........................................................................................................................... 42 2.4 Discussion ..................................................................................................................... 60 Chapter 3: Chapter 3: GSK3 controls lipin 1 subcellular localization and SREBP target gene expression ......................................................................................................................................67 3.1 Introduction ................................................................................................................... 67 3.2 Materials and Methods .................................................................................................. 73 3.3 Results ........................................................................................................................... 85 3.4 Discussion ................................................................................................................... 149 Chapter 4: Conclusions and Future Directions ............................................................................161 References ....................................................................................................................................172 Chapter 5: Appendices .................................................................................................................185 Appendix A: RNA Sequencing Preliminary Results .............................................................. 185 Appendix B: SDL Screen Hits and Mass Spectrometry Peptide Results ............................... 187 x  List of Tables Appendix B: SDL Screen Hits and Mass Spectrometry Peptide Results Table 1. Pah1 synthetic dosage lethality screen hits ................................................................... 187 Table 2. Peptides identified at lipin 1 S468 and S472 in mass spectrometry analysis of FLAG-lipin 1 WT immunoprecipitated from HEK 293T cells treated with DMSO ............................. 204 Table 3. Peptides identified at lipin 1 S468 and S472 in mass spectrometry analysis of FLAG-lipin 1 S468A immunoprecipitated from HEK 293T cells treated with DMSO......................... 209 Table 4. Peptides identified at lipin 1 S468 and S472 in mass spectrometry analysis of FLAG-lipin 1 S472A immunoprecipitated from HEK 293T cells treated with DMSO......................... 215 Table 5. Peptides identified at lipin 1 S468 and S472 in mass spectrometry analysis of FLAG-lipin 1 WT immunoprecipitated from HEK 293T cells treated with PI-103 .............................. 221 Table 6. Peptides identified at lipin 1 S468 and S472 in mass spectrometry analysis of FLAG-lipin 1 S468A immunoprecipitated from HEK 293T cells treated with PI-103 ......................... 226 Table 7. Peptides identified at lipin 1 S468 and S472 in mass spectrometry analysis of FLAG-lipin 1 S472A immunoprecipitated from HEK 293T cells treated with PI-103 ......................... 231 xi  List of Figures Figure 1.1: The glycerol-3-phosphate pathway of triglyceride synthesis ........................................6 Figure 1.2: Domain architecture of the yeast lipin Pah1 and mammalian lipin 1 .........................11 Figure 1.3: mTORC1 regulation of lipin 1 subcellular localization controls SREBP-target gene expression ......................................................................................................................................25 Figure 2.1: The role of lipin family phosphatidic acid phosphatases in lipid metabolism ............44 Figure 2.2: Synthetic dosage lethality overexpression screening to identify regulators of Pah1 ..45 Figure 2.3: Increased PAP activity negatively affects cell growth and causes inositol auxotrophy  ........................................................................................................................................................47 Figure 2.4: GAL-PAH1 overexpression in wild type yeast cells causes increased lipid droplet staining by nile red .........................................................................................................................48 Figure 2.5: Pah1 synthetic dosage lethality screen identified eleven potential kinase regulators of Pah1................................................................................................................................................50 Figure 2.6: Four kinases identified in the Pah1 SDL screen cause yeast cells to exhibit inositol auxotrophy when knocked out .......................................................................................................51 Figure 2.7: Pah1 purified from Δmck1 cells demonstrates a mobility shift ...................................54 Figure 2.8: In vitro kinase assay demonstrates that Mck1 phosphorylates Pah1...........................55 Figure 2.9: Deletion of the transcriptional repressor Opi1 rescues the inositol auxotrophy of Δmck1 cells ....................................................................................................................................57 Figure 2.10: Expression of catalytically active Mck1 rescues the inositol auxotrophy of Δmck1 cells ................................................................................................................................................58 Figure 2.11: Δmck1 cells have increased neutral lipid accumulation as assayed by nile red staining ...........................................................................................................................................59 xii  Figure 2.12: Lipin 1 possesses multiple GSK3-consensus phosphorylation sites .........................64 Figure 3.1: Lipin 1 possesses multiple GSK3-consensus phosphorylation sites ...........................86 Figure 3.2: Mass spectrometry reveals lipin 1 phosphorylation at S468/S472 is dependent on the PI3K/Akt pathway and GSK3 activity ...........................................................................................88 Figure 3.3: Phosphorylation-specific antibody to lipin 1 S468/S472 reveals lipin 1 phosphorylation at S468/S472 is dependent on the PI3K/Akt pathway and GSK3 activity .........91 Figure 3.4: Expression of FLAG-lipin 1 S468A, S472A, and S468AS472A mutants in HEK 293T cells .......................................................................................................................................92 Figure 3.5: Validation of phosphorylation-specific antibodies using FLAG-lipin 1 S468A and S472A mutants expressed in HEK 293T cells ...............................................................................93 Figure 3.6: Phosphoproteomic analysis of FLAG-lipin 1 WT, S468A, and S472A reveals that S468 phosphorylation is potentially dependent on S472 phosphorylation ....................................96 Figure 3.7: In vitro kinase assay using phosphorylation-specific antibodies demonstrates that GSK3ß phosphorylates lipin 1 .....................................................................................................100 Figure 3.8: HAP1 Lpin1 KO cells lack lipin 1 protein expression ..............................................103 Figure 3.9: HAP1 Lpin1 KO cells lack lipin 1 staining in immunofluorescence assay ...............104 Figure 3.10: Lipin 1 translocates to the nucleus from the cytoplasm under conditions of dual PI3K/Akt pathway and GSK3 inhibition .....................................................................................105 Figure 3.11: Lipin 1 protein abundance remains unchanged upon inhibitor treatments .............107 Figure 3.12: Dual inhibition of the PI3K/Akt pathway and GSK3 causes a decrease in SREBP-target gene expression in a lipin 1-dependent manner in mouse embryonic fibroblasts .............108 Figure 3.13: NIH 3T3 cells treated with the inhibitors LY294002 and GSK3 IX demonstrate decreased FASN expression ........................................................................................................112 xiii  Figure 3.14: HEK 293T cells treated with Torin 1 demonstrate significantly decreased SREBP-target gene expression ..................................................................................................................113 Figure 3.15: FLAG-Lipin 1 S468A and S472A mutants localize more to the nucleus and to the nuclear periphery compared to wild type FLAG-lipin 1 .............................................................114 Figure 3.16: Transient expression of phosphorylation-deficient lipin 1 mutants at S468 and S472 causes a decrease in FASN expression ........................................................................................116 Figure 3.17: HAP1 GSK3α and GSK3β knockout cells lack GSK3α and GSK3β protein expression ....................................................................................................................................120 Figure 3.18: HAP1 GSK3α and GSK3β knockout cells show sensitivity to PI-103 treatment in relation to FASN expression ........................................................................................................121 Figure 3.19: C57Bl/6J mice fed two different HFDs gain significantly more weight than mice fed a chow diet ...................................................................................................................................124 Figure 3.20: C57Bl/6J mice fed either of two high fat diets demonstrate qualitative signs of diet-induced obesity ............................................................................................................................125 Figure 3.21: C57Bl/6J mice fed one of the high fat diets tested possess increased FASN expression in the liver ..................................................................................................................126 Figure 3.22: Control, li-GSK3α, and li-GSK3β mice consume the same weekly amount of chow or HFD per animal per cage over 10 weeks .................................................................................128 Figure 3.23: Control, li-GSK3α, and li-GSK3β mice percent weight gain from starting weight over 10 weeks on a chow diet ......................................................................................................129 Figure 3.24: Control, li-GSK3α, and li-GSK3β mice percent weight gain over 10 weeks on a HFD compared to a chow diet .....................................................................................................132 xiv  Figure 3.25: Control, li-GSK3α, and li-GSK3β mice final percent weight gain on a HFD after 10 weeks............................................................................................................................................134 Figure 3.26: Representative images of control, li-GSK3α, and li-GSK3β mice fed a chow diet versus a high fat diet demonstrate qualititative signs of diet-induced obesity ............................135 Figure 3.27: Livers from control, li-GSK3α, and li-GSK3β mice fed a chow or high fat diet demonstrate hepatic steatosis .......................................................................................................136 Figure 3.28: Liver weights from control, li-GSK3α, and li-GSK3β mice fed a chow or high fat diet................................................................................................................................................137 Figure 3.29: Epidydimal fat pad weights from control, li-GSK3α, and li-GSK3β mice fed a chow or high fat diet ..............................................................................................................................139 Figure 3.30: Histological analysis of liver sections of control, li-GSK3α, and li-GSK3β mice fed a chow or high fat diet .................................................................................................................143 Figure 3.31: SREBP-target gene expression measured from livers of control, li-GSK3α, and li-GSK3β mice fed a chow or high fat diet .....................................................................................147 Figure 3.32: GSK3 directly phosphorylates lipin 1 to control its subcellular localization and SREBP-target gene expression ....................................................................................................155   xv  List of Abbreviations AGPAT  Acylglycerol-3-phosphate Acyltransferase BSA   Bovine Serum Albumin CIP   Calf Intestinal Phosphatase CKII   Casein Kinase II CLIP   Carboxy Terminal Lipin Domain CTD-NEP1  C-terminal Domain Nuclear Envelope Phosphatase 1 CWI   Cell Wall Integrity  DAG   Diacylglycerol  DGAT   Diacylglycerol Acyltransferase DMEM  Dulbecco’s Modified Eagle Media ER   Endoplasmic Reticulum  FASN   Fatty Acid Synthase FBS   Fetal Bovine Serum fld   Fatty Liver Dystrophy  GPAT   Glycerol Phosphate Acyltransferase  GSK3   Glycogen Synthase Kinase 3 HAD   Haloacid Dehalogenase HFD   High Fat Diet HMGCR  HMG-CoA Reductase IF   Immunofluorescence  IMDM   Iscove’s Modified Dulbecco’s Media INSIG   Insulin Induced Gene xvi  IP   Immunoprecipitation LPA   Lyso-Phosphatidic Acid MEF   Mouse Embryonic Fibroblast mTORC1  Mechanistic Target of Rapamycin Complex 1 MAPK   Mitogen-activated Protein Kinase MAPKK  Mitogen-activated Protein Kinase Kinase MAPKKK  Mitogen-activated Protein Kinase Kinase Kinase  NEP1-R1  Nuclear Envelope Phosphatase 1-Regulatory Subunit 1  NGS   Normal Goat Serum NLIP   Amino Terminal Lipin Domain NLS   Nuclear Localization Signal PA   Phosphatidic Acid PAP   Phosphatidic Acid Phosphatase PBD   Polybasic Domain PBS   Phosphate Buffered Saline PCR   Polymerase Chain Reaction PGC-1α  Peroxisome Proliferator-activated Receptor γ Coactivator 1α PKA   Protein Kinase A PKC   Protein Kinase C PI3K   Phosphatidylinositol-3-kinase  PKD   Protein Kinase D PPAR   Peroxisome Proliferator-activated Receptor  RIP   Regulated Intramembrane Proteolysis xvii  (RT-)qPCR  (Reverse Transcriptase) Quantitative Polymerase Chain Reaction SCAP   SREBP Cleavage Activating Protein SCD1   Stearoyl-CoA Desaturase SDL   Synthetic Dosage Lethality  S1P   Site 1 Protease S2P   Site 2 Protease SRD   Serine Rich Domain SRE   Sterol Regulatory Element  SREBP  Sterol Regulatory Element Binding Protein TAG   Triacylglycerol  TBS   Tris Buffered Saline TBS-T   Tris Buffered Saline Tween TOR   Target of Rapamycin   xviii  Acknowledgements I would first like to acknowledge my supervisor, Dr. Christopher Loewen, for giving me the opportunity to pursue my PhD in the Loewen Lab. He has guided my research project with a steady hand and provided moments of insight, encouragement, and levity. I would also like to thank Dr. Calvin Roskelley, Dr. Robert Nabi, and Dr. Thibault Mayor for being a helpful and resourceful supervisory committee. The Loewen Lab has been a lively and supportive environment, and this is because of the people that have been part of the lab. I extend my thanks to the lab members that I have had the pleasure to learn from and work with including Dr. John Shin, Dr. Jenny McQueen, Dr. Barry Young, Dr. Jesse Chao, Dr. Shabnam Tavassoli, Jigyasa Verma, Seeva Swaminathan, Kathryn Post, and Nicole Ng. Finally, I offer my most sincere gratitude to Andrew Wong, Analise Hofmann, and Peter Liu, the students whose tenures in the lab have most overlapped with mine, for being fantastic labmates and even better friends. Our top-notch banter brightened my days and your words of encouragement always steeled my resolve in the face of whatever graduate school threw at me.    As the aforementioned Andrew succinctly stated, “the best part of grad school is the grad students”, and I have been lucky to make amazing friends over the course of my degree. To Tak Poon, Erin Bell, Arif Arif, Connie Leung, Emily Lostchuck, and Stephanie Campbell, I thank you all for your friendship.   I have received technical help throughout my PhD and would like to extend thanks to Dr. Michael Hughes, Jigyasa Verma, Tak Poon, Duke Sheen, and Nicole Ng for their assistance.  xix   To Martin Cheung, Ricky Tsui, and Kenneth Cho, it’s hard to tell in the moment which of your childhood friends will be the ones that stick around, but I’m glad it was you guys.   I did not have pets growing up, but I have been lucky enough to have had two canine companions, Fido and Cooper, all through graduate school to remind me of what means most in life: good food, long walks, and the company of those you love.   It has been invaluable to belong to a supportive and present family throughout my life and especially through graduate school. I have been blessed to always know that I have so many people from my grandparents, aunts and uncles, cousins, family friends, and godfather, who are invested in my success and care about my wellbeing. A most special shout out goes to my sister, Cody, who I am lucky is not just my sibling, but also one of my best friends.     There are no people who are more responsible for my success than my parents, Louisa and Alex. It is because of them that I have had the luxury to never worry about anything except achieving my goals and dreams, no matter how lofty or ambitious. They have taught me to be humble but confident, to always make time for family and friends, and most of all, the value of honest hard work.     Finally, to Evelyn Lee – my co-dog walker, sous chef, racecar driver, and everything else in between – thanks for being with me every step of the way. Whether it’s running a marathon, finishing my PhD, or getting into law school, everything seems within reach with you cheering me on.  xx  Dedication  I dedicate my thesis to my Aunt Rose, who passed away earlier this year, and to all four of my grandparents who created worthy family legacies to both live up to and to build upon.  1  Chapter 1: Introduction 1.1 Obesity and Lipid Homeostasis The obesity epidemic is a serious and growing global health concern. As of 2015, the proportion of the world population afflicted with obesity was estimated to be 12% and 5% for adults and children, respectively, and the rate of overall obesity incidence had at least doubled since 1980 in over 70 countries [1]. Obesity increases the risk of other medical conditions and complications including – but not limited to – insulin resistance, type 2 diabetes, cardiovascular disease, and a variety of cancers [1]. Obesity is characterized by excessive or abnormal fat accumulation in a manner that is potentially detrimental to health, and this excess fat can disrupt the proper maintenance of steady-state intracellular lipid levels, or lipid homeostasis, and thereby cause adverse downstream effects. It has been found, for instance, that in obesity excess fatty acids can be released by adipocytes and aberrantly stored by cells that do not normally do so such as those that comprise the liver, pancreas, and skeletal muscle. This fatty acid excess and triglyceride storage in non-adipose tissue is strongly associated with insulin resistance [2, 3]. Furthermore, core lipid biosynthetic pathways are disrupted in obesity and insulin resistance. For example, it has been observed that lipogenesis is increased under these conditions at least in part due to dysregulation of the sterol regulatory element binding protein (SREBP) pathway, a pivotal cellular pathway that regulates the biosynthesis of fatty acid and cholesterol biosynthesis and uptake at the transcriptional level [4-8]. Therefore, studying the cellular pathways that govern lipid homeostasis is imperative to better understanding how obesity and its associated diseases alter these pathways and potential avenues to ultimately treat the effects or onset of these conditions. 2  Lipids are biomolecules that are essential in fundamental cellular processes including biological membrane creation, energy storage, cell signaling, and the formation of membrane-protein interactions. In animals, lipids are consumed through dietary intake and are processed by the digestive system and subsequently distributed to cells throughout the body. Other tissues, most prominently the liver, can also synthesize lipids de novo using glucose in a process called lipogenesis [9]. In the context of cells, fatty acids are positioned as a central hub of lipid metabolism because they can be used by cellular pathways to create phospholipids for membrane production, synthesize triglycerides or cholesterol esters for energy storage in lipid droplets, and provide energy for cellular processes via fatty acid oxidation. Unsurprisingly, maintaining and regulating lipid homeostasis is crucial to proper cellular function and survival, and cells possess a multitude of specialized enzymes that can act directly on lipids and in concert with a variety of molecular pathways. These enzymes and pathways are important areas of study in the path to understanding the mechanisms that govern cellular lipid metabolism and homeostasis in both healthy and disease states.  1.2 Sterol regulatory element binding proteins SREBPs are transcription factors that regulate the de novo synthesis and uptake of fatty acids and cholesterol in cells, and thereby exert significant influence on lipid homeostasis and metabolism. In humans, the Srebf1 and Srebf2 genes encode SREBP-1 and SREBP-2, respectively. SREBP-1 has two splice variants, SREBP-1a and SREBP-1c. SREBP-1c is responsible for upregulation of fatty acid synthesis genes, while SREBP-2 is responsible for upregulation of cholesterol synthesis genes, and SREBP-1a is known to regulate transcription of both fatty acid and cholesterol synthesis genes [10, 11]. Genes upregulated by SREBPs contain 3  the sterol regulatory element (SRE) in their promoters, which consists of the sequence TCACNCCAC [12]. Together, SREBP proteins regulate the expression of genes encoding key lipid and sterol synthesis and uptake proteins including the low-density lipoprotein receptor, fatty acid synthase, HMG-CoA reductase, and stearoyl-CoA desaturase [10, 13]. The SREBP pathway has been found to be downstream of critical metabolic signaling regulators including the nutrient and growth factor sensing kinase complex mTORC1, the liver X receptor, insulin, AMP-activated protein kinase, and the PI3K/Akt pathway thus allowing signal integration from these pathways to an effector that controls cellular lipid homeostasis [11, 14-16].  Unsurprisingly, SREBPs have been shown to be crucial in regulating physiological lipid metabolism and homeostasis. In seminal studies by the laboratories of Brown and Goldstein that first characterized key elements of the SREBP pathway, a dominant negative version of SREBP-1a was expressed in mice and this resulted in massive overproduction of triglycerides and cholesterol in the livers of these animals along with severe hepatic steatosis (fatty liver) [17]. It is now appreciated that the liver is the major site of SREBP activity. Hepatic SREBP-1c protein levels have been observed to be significantly elevated in humans suffering from hepatic steatosis [15], which is most commonly caused by obesity, and insulin signaling via the insulin receptor IRS1 also results in increased hepatic SREBP-1c protein abundance and fatty acid synthesis [16]. Furthermore, the SREBP pathway has been shown to be directly responsible in the development of hepatic steatosis and carbohydrate-induced triglyceride excess in insulin resistant contexts in mice [18]. It has thus been suggested that SREBPs or other members of the SREBP regulatory pathway could be attractive therapeutic targets in alleviating or preventing co-morbidities of obesity such as hepatic steatosis.  4   SREBPs are precisely regulated in a negative feedback loop that involves sterol sensing, vesicular transport, and specialized protein cleavage. Research into the mechanisms by which SREBPs are regulated led to the first full characterization of a regulatory mechanism termed regulated intramembrane proteolysis (RIP), in which transmembrane proteins are cleaved to liberate cytosolic fragments that translocate to the nucleus to affect transcription [19]. Despite their role as nuclear transcription factors, full length SREBPs are transmembrane proteins that span the endoplasmic reticulum (ER) membrane [10]. In the presence of sterols, SREBPs remain localized to ER because they are bound to SREBP Cleavage Activating Protein (SCAP), which in turn interacts with another integral ER membrane protein, Insulin Induced Gene (INSIG). SCAP is a cholesterol binding protein and binding with cholesterol decreases the affinity for SCAP to associate with COPII proteins necessary for vesicular transport, and INSIG further promotes this sterol-mediated inhibition. In the absence of cholesterol, the INSIG and SCAP interaction is weakened and SCAP interacts with COPII proteins to transport itself and SREBP to the Golgi Apparatus via COPII vesicles. At the Golgi, SREBP is cleaved by two proteases, the site 1 protease (S1P) and site 2 protease (S2P), respectively, and this liberates the N terminus of the protein from the membrane-spanning regions of the protein. This cleaved fragment contains the transcription factor regions of SREBP and it then translocates from the cytoplasm to the nucleus to upregulate SRE-containing lipid and cholesterol synthesis and uptake genes [10]. Ultimately, the sterol binding/sensing by SCAP provides this pathway with an elegant negative feedback loop. When sterol levels increase due to SREBP activity in the nucleus, for instance, cholesterol and INSIG inhibit the ability of SCAP to interact with COPII proteins as mentioned earlier, and SREBPs are once again retained at the ER membrane as a result [10].  5   1.3 Phosphatidic acid is a central player in lipid homeostasis 1.3.1 The glycerol-3-phosphate pathway Fatty acids are utilized in the glycerol-3-phosphate pathway to generate phospholipids and triglycerides (Figure 1.1). This highly conserved pathway consists of a series of enzymes that are mostly integral membrane proteins which reside on the surface of the ER. In the first steps of this pathway acyl-CoA, synthesized from a fatty acid, and a glycerol-3-phosphate backbone are converted into lyso-phosphatidic acid (LPA), which is then converted to phosphatidic acid (PA). The enzymes responsible for these reactions are glycerol phosphate acyltransferases (GPATs) and acylglycerol-3-phosphate acyltransferases (AGPATs), respectively. PA can then be used as a precursor to synthesize a number of phospholipid species including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, or be dephosphorylated by phosphatidic acid phosphatase (PAP) enzymes to generate the neutral lipid diacylglycerol (DAG). In the ultimate step of triglyceride synthesis, DAG is further esterified into triacylglycerol (TAG) by diacylglycerol acyltransferases (DGATs). DAG and TAG are neutral lipids that can then be stored in lipid droplets. Of all the enzymes in the glycerol-3-phosphate pathway, only PAP enzymes are not integral ER membrane proteins, and are instead cytosolic proteins that are recruited to the ER membrane to convert PA into DAG. This offers unique regulatory potential to PAP enzymes in the context of this pathway, which will be discussed in further detail in section 1.4.   6    Figure 1.1: The glycerol-3-phosphate pathway of triglyceride synthesis Schematic depicting key enzymes in the glycerol-3-phosphate pathway of triglyceride synthesis. Enzymes highlighted are glycerol phosphate acyltransferase (GPAT), acylglycerol 3-phosphate acyltransferase (AGPAT), phosphatidic acid phosphatase (PAP), and diacylglycerol acyltransferases (DGAT). The intersection between PA and DAG mediated by the PAP reaction is highlighted to emphasize the key role PAP enzymes play in regulating phospholipid synthesis and lipid storage.    7  1.3.2 Phosphatidic acid signaling and the Opi1 pathway  The glycerol-3-phosphate pathway positions PA at a significant intersection point between phospholipid synthesis and membrane biogenesis, and neutral lipid synthesis and fat storage (Figure 1.1). However, PA has emerged as more than just a precursor lipid for phospholipids and neutral lipids, but also a key signaling lipid. Through interactions with effector proteins, PA has been implicated in roles including cytoskeletal rearrangement, cell differentiation, secretion, vesicular trafficking, and cell survival and proliferation [20]. From studies in the budding yeast Saccharomyces cerevisiae, it is now also appreciated that PA coordinates cellular signals to directly regulate lipid metabolism. In yeast, Opi1 is a transcription factor that binds to and represses the Ino2-Ino4 transcriptional activator complex at UASINO-containing promoters, which are present in the promoters of over 30 phospholipid metabolic genes [21, 22]. It has been found that Opi1 is repressed by directly binding PA found specifically at the ER, which sequesters Opi1 at the ER membrane and away from the nucleus [22]. Thus, PA at the ER promotes phospholipid gene transcription, phospholipid biosynthesis, and membrane biogenesis by alleviating the repressive effects of Opi1.   In 2010, our lab discovered that PA binding to Opi1 was regulated by intracellular pH [23]. This finding functionally connected the stark decrease in intracellular pH that occurs when yeast cells are starved of glucose and lipid metabolism at the transcriptional level through Opi1. Intriguingly, the glucose availability signal worked directly through PA, which acted as a pH biosensor in this pathway. In response to glucose starvation, the pH of yeast cells drops rapidly from ~7 to ~6 and we found that this change in internal pH causes Opi1 to unbind PA and translocate from the ER membrane to the nucleus where it represses the transcription of UASINO-8  containing phospholipid synthesis genes [23]. Moreover, the binding of Opi1 to PA was determined to be pH-dependent. The head group of PA has a pKa in the physiological range and in a relatively acidic environment, such as that in a yeast cell starved of glucose, it becomes protonated [24]. This is consistent with the electrostatic hydrogen bond switch mechanism proposed by Kooijman et al. for charge-dependent association between proteins and PA [24]. We further found that Opi1 preferentially binds non-protonated PA and has lower affinity for protonated PA, and ultimately that this charge-dependent PA binding is what linked glucose availability, PA, Opi1, and lipid metabolism [23].   1.4 Lipins are conserved phosphatidic acid phosphatase enzymes 1.4.1 Discovery and characterization  PAP activity was first observed in various rat tissues and chicken livers by Smith et al. in 1957 [25]. However, the family of enzymes responsible for carrying out this fundamental enzymatic activity was not fully elucidated for almost 50 years. In 2006, O’Hara et al. discovered that SMP2, a gene of unknown molecular function, was the phosphatidic acid phosphatase gene in the budding yeast Saccharomyces cerevisiae that converted PA to DAG in a Mg2+-dependent manner in the glycerol-3-phosphate pathway [26]. The gene was renamed PAH1 (for phosphatidic acid phosphohydrolase 1) and the PAH1 protein product was found to share homology with previously discovered mammalian proteins called lipins encoded by the Lpin1, Lpin2, and Lpin3 genes [26]. Although known to play significant roles in lipid metabolism [27, 28], the molecular function of the mammalian lipins were unknown until the discovery of the Pah1 PAP enzyme in yeast. This discovery helped established the lipin family of PAP enzymes as highly conserved players in triglyceride synthesis and lipid metabolism. Lipin-family PAP 9  enzymes have been additionally identified in organisms including flies, worms, and plants, highlighting the high degree of conservation of this enzyme family [29-31].  It is important to note that overall cellular PAP activity is divided between that which is Mg2+-dependent and that which is Mg2+-independent. However, the enzymes that contribute to these PAP activities have been found to have both different cellular roles and substrate specificities. In budding yeast, for instance, Dpp1 and Lpp1 are Mg2+-independent PAP enzymes, while App1 and Pah1 are Mg2+-dependent PAP enzymes. Dpp1 and Lpp1 are integral membrane proteins localized to the vacuole and ER, respectively, and have relatively broad substrate specificity [32, 33]. They are both able to catalyze the dephosphorylation of lipids including – but not limited to – PA and lyso-PA, and are thought to be involved in lipid signaling as opposed to lipid synthesis [32-34]. Of the Mg2+-dependent enzymes, App1 is a peripheral membrane protein conserved only in fungi and has relatively broad substrate specificity [35]. In contrast, Pah1 is a peripheral membrane protein with PAP activity that is highly specific for PA, is conserved from yeast to mammals, and has been found to play crucial roles in lipid synthesis and metabolism [21, 26, 36, 37]. Therefore, it appears that Pah1 is the sole PAP enzyme in yeast that is specific to PA contributes to DAG production in triglyceride synthesis via the glycerol-3-phosphate pathway.   Lipin family PAP enzymes possess significant sequence and domain homology (Figure 1.2). From Pah1 to mammalian lipins, a high degree of sequence homology exists in the amino terminal lipin domain (NLIP) and carboxy terminal lipin domain (CLIP) regions of the protein [27]. Significantly, the haloacid dehalogenase-like (HAD-like) domain resides within the CLIP 10  domain of lipins and contains the DxDxT catalytic motif that confers PAP activity to all members of the lipin family [36]. The HAD-like domain additionally contains the LxxIL transcription factor interaction motif, which has been found to be required for mammalian lipin 1 to act as a transcriptional activator in the nucleus [38]. In the yeast lipin Pah1 it has been found that an amphipathic helix at the N-terminus of the protein before the NLIP domain is required for membrane insertion and association [39]. In mammalian lipins, the polybasic domain (PBD) is a region near the NLIP domain consisting of a stretch of lysine and arginine residues that has been characterized as the domain responsible for binding the lipin substrate PA in both lipin 1 and lipin 2 [40-42]. The binding of PA by lipins appears to be consistent with the electrostatic hydrogen bond switch model proposed by Kooijman et al. for charge-dependent protein interaction with PA [24]. Intriguingly, the PBD was initially characterized as a nuclear localization signal (NLS), and since lipins are known to translocate to the nucleus to perform many cellular functions [14, 38, 42, 43], it is possible that the PBD region of the protein is responsible for both PA binding and nuclear translocation.    11    Figure 1.2: Domain architecture of the yeast lipin Pah1 and mammalian lipin 1 Schematic highlighting domains of murine lipin 1 and yeast Pah1. NLIP and CLIP regions represent regions of high homology between all lipins. HAD is the haloacid dehalogenase domain, which confers PAP activity to lipins. PBD is the polybasic domain shown to bind phosphatidic acid in lipin 1, while the SRD is the serine-rich domain. The amphipathic helix in Pah1 is required for membrane association.    12  1.4.2 Function in lipid metabolism and lipid homeostasis Even before the molecular function of the lipin family of PAP enzymes was discerned, mammalian lipins had already emerged as potentially crucial regulators of lipid metabolism and homeostasis. In 2001, Peterfy et al. characterized mice harboring a spontaneous null mutation in the Lpin1 gene that caused severe lipid-related phenotypes. These mice were termed fld (fatty liver dystrophy) mice due to the fatty liver and elevated triglyceride levels they exhibited in the neonatal stage [27]. Additionally, fld mice exhibited lipodystrophy manifesting as a severe lack of adipose tissue and impaired adipocyte differentiation, along with peripheral neuropathy, and insulin resistance [27]. The same study determined that mice and humans possess three lipin genes due to sequence similarity: Lpin1, Lpin2, and Lpin3. Finally, a subsequent study by the same group further demonstrated that overexpression of lipin 1 in mice resulted in the development of obesity even when fed a chow diet compared to wild type controls [28]. The extreme metabolic phenotypes resulting from loss or overexpression of lipin 1 thus positioned lipins as central players in the physiological regulation of lipid homeostasis even before their molecular function was known.   Since the initial characterization of fld mice and the eventual discovery that lipin family proteins were conserved PAP enzymes, the field of lipin biology has expanded to better delineate the specific functions of lipin enzymes in yeast and mammals. Yeast has been an ideal model organism to study lipin function and regulation because it possesses a single lipin ortholog, Pah1, and is relatively easy to manipulate genetically compared to other eukaryotic systems. Research on Pah1 has demonstrated its importance in regulating lipid homeostasis in the yeast cell and highlights the control it exerts in mediating phospholipid synthesis and lipid storage. Cells 13  lacking Pah1 possess a number of severe phenotypes included slow growth, temperature sensitivity, and a significantly increased accumulation of PA and a corresponding increase in phospholipids [36, 37]. One of the consequences of this aberrant accumulation of phospholipids in these cells is the striking expansion of the nuclear/ER membrane [37, 44]. Δpah1 cells also expectedly possess a significant reduction in cellular TAG levels, but surprisingly do not demonstrate an overall decrease in neutral lipids [36, 45]. However, the number of lipid droplets is still starkly reduced in cells lacking Pah1, which has been attributed to the direct role Pah1 plays in lipid droplet formation/biogenesis [45]. Given the importance of lipins in maintaining cellular lipid homeostasis, it is not surprising they are tightly regulated and that regulation of lipins is tied closely to their function. Studies on Pah1 have been invaluable in aiding our understanding of the pathways and enzymes that regulate cellular lipin function in yeast and mammals, and this will be discussed in greater detail in section 1.4.4.   The mammalian lipin orthologs lipin 1, lipin 2, and lipin 3 have been studied to varying degrees since their discovery and initial characterization. All three of these enzymes have been confirmed as being capable of dephosphorylating PA to form DAG in vitro, with lipin 1 possessing the highest specific activity for PA of the three, followed by lipin 2 and then lipin 3 [46]. The tissue-specific expression levels of three lipin orthologs has been found to differ and sometimes to overlap, but the role that this variation in expression relates to specific functions remains an area of ongoing research [46]. Lipin 1 has been found to be most highly expressed in adipose tissue and skeletal muscle but is also expressed moderately in the liver, whereas lipin 2 is highly expressed in the liver and brain, and lipin 3 is expressed at low levels in the intestine, kidney, and liver [46]. The overlap of lipin expression in tissues and cell types undoubtedly adds 14  to the complexity of studying the function of specific lipin orthologs and is a factor that must always be accounted for. Recent work, however, has begun to better characterize both tissue-specific roles of lipins and the cooperative role of the lipin orthologs in certain contexts. For instance, lipin 1 in the liver has been specifically implicated in the regulation of SREBP and its associated target genes in response to mechanistic target of rapamycin complex 1 (mTORC1) signaling [14]. There is also evidence for lipin 1 and lipin 3 working cooperatively to regulate PAP activity in adipocytes [47]. Further evidence of a tissue-specific or compensatory/cooperative model of lipin function in mammals can be derived from the fact that unlike fld mice, individual Lpin2 and Lpin3 knockout mice demonstrate only subtle abnormal phenotypes, if any [47, 48].  Of the three mammalian lipin orthologs, lipin 1 has received the most intensive study to date. Characterization of lipin 1 has elucidated its role not just as a PAP enzyme recruited from the cytoplasm to the ER to convert PA to DAG in triglyceride synthesis, but also as a protein capable of translocating to the nucleus to interact with transcription factors and influence gene transcription. The role of lipin 1 in influencing the activity of transcription factors has been borne out in an increasing number of studies. Hepatic lipin 1 was first found in 2006 to physically interact with the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and its target PPARα, acting as an amplifier of lipid metabolism in hepatocytes via this interaction with the PGC-1α pathway [38]. A subsequent study defined the role of lipin 1 in directly interacting with the transcription factor PPARγ in pre-adipocytes to amplify PPARγ and C/EBPα signaling in these cells, which is necessary to promote adipogenesis and maintain adipocyte function [49]. Other transcription factors now known to interact with lipin 1 include the nuclear factor of 15  activated T-cells c4 (NFATc4) in adipocytes and myocyte enhancer factor 2 (MEF2) in neuronal cells [50, 51]. More recently, mTORC1 was discovered to regulate lipin 1 translocation to the nucleus via phosphorylation. Lipin 1 translocation to the nucleus in this context repressed SREBP nuclear protein abundance to repress SREBP target gene expression, and therefore fatty acid and cholesterol biosynthesis at the transcriptional level [14]. It is evident, therefore, that lipin 1 plays an important role in a diverse array of molecular pathways through its interactions with transcription factors, many of which are central to the adipogenic program or lipid metabolism and synthesis.   Another interesting finding from the studies investigating the role of lipin 1 interaction with transcription factors is the variable requirement of lipin 1 PAP activity in these contexts. It has been suggested that lipin 1 may act as a molecular scaffold for transcription factors [52], but the fact that PAP activity is sometimes required for the effects of lipin 1 on transcription may indicate that there are additional or separate mechanisms at play. It has been recently observed that lipin 1 PAP activity is absolutely required for its role in regulating SREBP in the nucleus. Specifically, even a phosphorylation deficient form of lipin 1 that translocates fully to the nucleus but does not possess catalytic PAP activity cannot induce the repression of SREBP target gene expression [14]. In other contexts, including the role of lipin 1 in the PGC-1α pathway and NFATc4 pathway, lipin 1 PAP activity has been found not to be required [38, 50]. Complicating this area of lipin 1 biology, however, is the fact that lipin proteins have been found to form homo- and hetero-oligomers [53] and it is possible then that catalytically dead versions of lipin 1 can form oligomers with endogenous lipin 1 or potentially even with lipin 2 or lipin 3. Further 16  study will therefore be needed to better delineate the requirement of PAP activity in the ability of lipin 1 to regulate transcription, and the role of oligomerization in lipin function overall.   1.4.3 Lipins and disease Loss of function mutations in Lpin1 and Lpin2 have been attributed to rare human diseases, while no diseases have currently been ascribed to Lpin3 mutations. However, it is interesting to note that neither of these diseases associated with Lpin1 and Lpin2 phenocopy the phenotypes observed in mice lacking Lpin1 or Lpin2. Several deleterious mutations in Lpin1 have been found to cause acute myoglobinuria due to recurrent rhabdomyolysis that persists through childhood [54-56]. Rhabdomyolysis is characterized by the periodic breakdown of skeletal muscle resulting in the release of muscle proteins such as myoglobin into the bloodstream leading to adverse effects. Importantly, Lpin1 mutations associated with this condition have been demonstrated in vitro to have lost PAP activity due to the inability to bind PA, mapping the cause of this disease at least partly to a lack of lipin 1 PAP activity [55]. Mutations in Lpin2 have only been identified in three families in the world, and are known to cause the rare autoinflammatory disease Majeed’s syndrome [57-59]. This condition manifests as symptoms including recurrent fevers, inflammation of the bone and skin, and dyserythropoietic anemia [57-59]. Work by Donkor et al. in 2009 determined that loss of PAP activity in lipin 2 is the likely culprit of the symptoms observed in Majeed’s syndrome [60]. It remains an open question as to why loss of lipin 1 or lipin 2 function in humans does not result in similar phenotypes as in murine models, though it can be surmised that perhaps in humans lipin orthologs compensate in a different manner than in mice.   17  Although mutations in Lpin1 have not been directly linked to diseases associated with lipid metabolism and homeostasis, as perhaps expected given the phenotypes of fld mice, Lpin1 has been strongly linked to prevalent metabolic diseases and traits. Multiple studies have found that Lpin1 polymorphisms and expression levels are associated with glucose homeostasis, insulin sensitivity, metabolic syndrome traits, and obesity-related phenotypes [61-66]. This suggests that lipin 1 plays a significant role in human metabolism. However, it remains unknown how and which cellular functions of lipin 1 contribute to these disease traits and sensitivities, and this remains an active area of study. Interestingly, it has recently been discovered that lipin 1 PAP activity in skeletal muscle plays a role in autolysosome maturation/formation through activation of protein kinase D (PKD) signaling and that this may have implications in human health [67]. Statins, commonly prescribed cholesterol lowering drugs, are known to decrease PKD activation due to decreasing cellular DAG levels, and in some cases can be an underlying cause in statin-induced myopathy. In conjunction with lipin 1 deficiency such as in Lpin1 heterozygous missense mutations, which would also result in decreased cellular DAG, statin treatments could therefore increase the risk of myopathy. This finding may also provide the basis for the rhabdomyolysis observed in individuals with homozygous Lpin1 loss of function mutations [54].   1.4.4 Regulation of lipins by phosphorylation  As mentioned previously, lipins are the only enzyme in the glycerol-3-phosphate triglyceride synthesis pathway that are not integral membrane proteins residing in the ER membrane. This unique feature which is conserved from yeast to mammals positions lipins as attractive targets of regulation in this pathway because they need to be recruited from the cytoplasm to the ER or other subcellular compartments to perform their cellular functions. Also, 18  the conversion of PA to DAG represents an important branchpoint between phospholipid synthesis and neutral lipid synthesis, further highlighting the importance of the PA/DAG flux mediated by lipins. It is unsurprising, then, that lipins have been found to be tightly regulated enzymes. The overarching theme of lipin regulation is post-translational modification. Recent work has, for instance, demonstrated the functional roles of sumoylation [51] and acetylation [68] in controlling lipin 1 localization and function. However, the major mechanism of lipin regulation appears to be through phosphorylation by kinases, which will be discussed in this section. Studies on both the yeast lipin Pah1 and mammalian lipin 1 have been insightful in shedding light on the crucial role phosphorylation plays in mediating lipin function and in identifying the molecular pathways that influence lipid homeostasis via regulating lipins.   1.4.4.1 The yeast lipin Pah1 is regulated by phosphorylation  As a model organism, yeast is in many ways ideal for studying lipin function and regulation because it contains a single lipin enzyme (as opposed to the three lipin enzymes found in mammals), is easily genetically manipulated, and possesses better characterized lipid metabolic pathways and phenotypes compared to more complex eukaryotes. Indeed, research focused on Pah1 in budding yeast has been instrumental in uncovering the kinases and pathways that phosphorylate lipins and the specific roles of phosphorylation in regulating lipin function. It should be noted that there exist key differences between the regulation of Pah1 and mammalian lipins by phosphorylation, and that known phosphorylation sites of the yeast and mammalian proteins have not been found to be conserved, but nevertheless many of the main concepts of lipin regulation by phosphorylation appear to be consistent through evolution.   19  Initial studies on Pah1 established that it was one of the most phosphorylated proteins in yeast [36]. Phosphorylation has now emerged as the major mechanism in regulating Pah1 and is known to affect its enzymatic PAP activity, subcellular localization, and abundance/degradation [26, 39, 69-73]. These regulatory mechanisms are closely tied to the fact that Pah1, like all lipins, is a peripheral membrane protein that requires recruitment to subcellular compartments to carry out its functions. It has been observed that Pah1 is localized to the cytoplasm in its phosphorylated form and recruitment to the nuclear/ER membrane is dependent on its dephosphorylation by the Nem1/Spo7 phosphatase complex [39, 44, 69]. Moreover, phosphorylation has been found to inhibit Pah1 PAP activity. This is illustrated succinctly when looking at Pho85-Pho80 cyclin-dependent kinase, which was discovered to directly phosphorylate Pah1 at seven sites [26, 70]. Mutation of the seven phosphorylated serine and threonine residues to alanine residues, thus creating a phosphorylation deficient version of Pah1, resulted in an almost two-fold increase in PAP activity when assayed in an in vitro PAP activity assay [26]. Conversely, a separate study demonstrated phosphorylation of the same seven sites in purified Pah1 protein caused a marked decrease in PAP activity [70]. It is also telling that cells lacking either Nem1 or Spo7, the enzymatic and regulatory subunits of the phosphatase complex, respectively, exhibit similar phenotypes as cells completely lacking Pah1 including a greatly expanded nuclear/ER membrane [44]. This strongly suggests that Nem1/Spo7 complex-mediated dephosphorylation of Pah1 is necessary for Pah1 to contribute cellular PAP activity in vivo. Lastly, phosphorylation has been found to regulate Pah1 protein abundance through ubiquitin-independent degradation by the 20S proteasome [73-75]. It has been found that phosphorylation has a protective effect towards Pah1 half-life, whereas dephosphorylation promotes Pah1 degradation [74]. Therefore, phosphorylation and dephosphorylation appear to govern multiple 20  aspects critical to Pah1 function and highlights the intricate and layered nature of cellular lipin regulation.   In addition to Pho85-Pho80, several other kinases have been found to directly phosphorylate Pah1 with variable effects on Pah1 localization, activity, and stability. These include Cdc28-cyclin B, which phosphorylates three of the same residues also phosphorylated by Pho85-Pho80; protein kinase A (PKA) which phosphorylates five serine residues; protein kinase C (PKC) which phosphorylates four serine residues, three of which it shares with PKA; and casein kinase II (CKII) which phosphorylates six residues [69-72, 75]. Interestingly, the three sites phosphorylated by Cdc28-cyclin B have been found to not alter Pah1 PAP activity. However, PKA phosphorylation of Pah1 has been found to occur in conjunction with Cdc28-cyclin B and Pho85-Pho80 phosphorylation, and inhibits Pah1 PAP activity and membrane association [71]. Phosphorylation of Pah1 by PKC appears to have little to no effect on Pah1 activity or subcellular localization [72], while casein kinase II phosphorylation has been identified as playing a key role on mediating the role of Pah1 in lipid homeostasis and its proteasomal degradation [74, 75].   1.4.4.2 Lipin 1 regulation by phosphorylation  Much of the research conducted on the regulation of mammalian lipins by phosphorylation has been focused on lipin 1. Like Pah1 in yeast, mammalian lipin 1 has been found to be a highly phosphorylated protein [14, 76]. Intriguingly, the mechanisms by which phosphorylation regulates lipin 1 appear to be appreciably different than what has been observed in Pah1, though it must also be noted that these mechanisms have been better characterized in 21  yeast to date. In contrast to Pah1, for instance, phosphorylation does not seem to inhibit the PAP activity of lipin 1 [14, 76]. Specifically, phosphorylated lipin 1 isolated from adipocytes was observed to have the same PAP activity as dephosphorylated lipin 1 in in vitro PAP activity assays [76]. Lipin 1 subcellular localization does, however, appear to be significantly mediated by its phosphorylation state, though the detailed mechanisms by which this regulation of localization occurs have not yet been uncovered. Insulin-mediated multi-site phosphorylation of lipin 1 has long been known to control lipin 1 localization and causes lipin 1 to localize primarily to the cytoplasm as opposed to associating with membranes [76].  Additionally, it has been observed that phosphorylation at sites within the lipin 1 serine rich domain (SRD) mediate its interaction with 14-3-3 proteins, which function as molecular scaffolds that typically sequester phosphorylated binding partners to the cytoplasm [43]. The function of the 14-3-3 interaction with lipin 1 appears to be to constrain the localization of phosphorylated lipin 1 to the cytoplasm in adipocytes, further highlighting the importance of lipin 1 subcellular localization to its function [43]. However, many of the experimentally-determined phosphorylation sites of lipin 1 exist outside this SRD region, suggesting additional mechanisms of lipin 1 regulation by phosphorylation [14, 43, 76]. To our knowledge, at present the only kinase or kinase complex known to directly impart phosphorylation to lipin 1 to regulate its function is the mTORC1 complex, and the regulatory interaction between mTORC1 and lipin 1 will be discussed in detail in section 1.4.4.3. Dephosphorylation of lipin 1 in mammalian systems is less well-characterized than with Pah1 in yeast, but recent work has demonstrated that Nem1 and Spo7, which together form a phosphatase complex in yeast, appears to have mammalian orthologues in the C-terminal domain nuclear envelope phosphatase 1 (CTD-NEP1) and the nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1), respectively [77]. These proteins were found to complement 22  yeast cells lacking Nem1 and Spo7, and CTD-NEP1 was found to dephosphorylate lipin 1 in a manner dependent on NEP1-R1 [77]. This suggests that at least some aspects of lipin 1 activation through dephosphorylation are conserved through evolution.  1.4.4.3 mTORC1 phosphorylates lipin 1 to control its subcellular localization and SREBP target gene expression Initial studies discovered that phosphorylation of Ser106 of lipin 1 was responsive to insulin and sensitive to treatment by rapamycin, the well-characterized mTORC1 inhibitor [78]. This positioned lipin 1 as a potential regulatory target of mTORC1, a key nutrient and growth factor sensing kinase complex, and/or insulin signaling and the phosphatidylinositol-3-kinase (PI3K)/Akt pathway. Insulin signaling has since become a well-known stimulus of lipin 1 phosphorylation and regulation as mentioned previously [76]. mTORC1 has been observed to promote SREBP activity [79], but the mechanism was not known until more recently. In 2011, Peterson et al. discovered that lipin 1 functionally links mTORC1 signaling and SREBP and its downstream transcriptional targets [14] (Figure 1.3). Specifically, mTORC1 was found to directly phosphorylate lipin 1 at multiple serine and threonine residues, including the insulin-responsive Ser106, and that this mTORC1-dependent phosphorylation promotes lipin 1 cytoplasmic localization. Inhibition of mTORC1 or mutation of 17 of 19 mTORC1-dependent phosphorylation sites to alanine residues caused lipin 1 to translocate from the cytoplasm to the nucleus and a corresponding repression of SREBP target gene expression, thus downregulating cellular lipid and sterol synthesis (Figure 1.3). The stark repression of SREBP target gene expression in these conditions was demonstrated to be lipin 1-dependent as the repressive effect on these genes was alleviated in lipin 1-deficient fld mouse embryonic fibroblasts (MEFs) when 23  compared to wild type MEFs. Furthermore, nuclear lipin 1 appears to repress SREBP target gene expression by decreasing SREBP protein abundance in the nucleus and causing SREBP redistribution to the nuclear periphery by a currently uncharacterized mechanism. Lipin 1 catalytic PAP activity was also found to be necessary to mediate SREBP target gene expression in this pathway, with catalytically inactive lipin 1 unable to repress SREBP target gene expression even in a phosphorylation-deficient form that translocates completely to the nucleus. Together, these findings demonstrate the role of catalytically active and dephosphorylated lipin 1 in the nucleus in regulating lipid homeostasis at the transcriptional level.   The mTORC1-mediated lipin 1 regulation of SREBP has physiological consequences in diet-induced hepatic steatosis and weight gain when evaluated in mouse models [14]. When challenging mice possessing a postnatal liver-specific knockout of raptor, a crucial mTORC1 regulatory subunit, with a high fat and high cholesterol “Western” diet, it was found that these mice gain significantly less weight when compared with wild type control animals. Fatty liver phenotypes, and elevated liver triglyceride and plasma cholesterol levels exhibited by the control mice fed the Western diet were each greatly diminished or alleviated in the liver-specific raptor knockout mice as well. Furthermore, SREBP target gene expression in the liver of the liver-specific raptor knockout mice was repressed compared to control mice both fed chow and Western diets. Significantly, additional full animal shRNA knockdown of lipin 1 caused SREBP target gene expression to no longer undergo repression, even in the liver-specific raptor knockout background, strongly suggesting that the repression of SREBP is largely dependent on lipin 1 [14]. Lipin 1, therefore, appears to work antagonistically to mTORC1 in terms of SREBP target gene expression. When mTORC1 is active, lipin 1 is phosphorylated and localizes to the cytosol, 24  and SREBP downstream targets are transcribed; conversely, when mTORC1 signaling is inactive, it no longer phosphorylates lipin 1 which then translocates to the nucleus and represses SREBP target gene expression. Based on these findings, mTORC1 and lipin 1 are likely important targets to study in relation to diet-induced hepatic steatosis, adiposity, and lipogenesis.    25    Figure 1.3: mTORC1 regulation of lipin 1 subcellular localization controls SREBP-target gene expression mTORC1 has been found to directly phosphorylate lipin 1 to control its subcellular localization from the cytoplasm to the nucleus. When mTORC1 signaling is active (left), it phosphorylates multiple sites on lipin 1, which promotes lipin 1 to localize to the cytoplasm. In this scenario, SREBP-target gene expression is undisturbed. When mTORC1 signaling is inactive (right), such as through pharmacological inhibition, lipin 1 becomes dephosphorylated and translocates from the cytoplasm to the nucleus where it represses SREBP-target gene expression. This figure is based on the work of Peterson et al., 2011 [14].   26   1.5 Glycogen synthase kinase 3 Glycogen synthase kinase 3 (GSK3) family kinases are widely expressed serine/threonine dual-specificity kinases that are highly conserved from yeast to mammals and are involved in a variety of cellular functions including cell growth and differentiation, development, and metabolism. Mammals including mice and humans possess two separate genes that encode the paralogous GSK3 kinases GSK3α and GSK3β. To date, GSK3β has been the better characterized of the two GSK3 paralogs. These two kinases share 85% sequence homology overall and 98% sequence homology in their respective kinase domains, while the main sequence variability occurs at the N and C termini of the respective proteins [80, 81]. Despite the high degree of conservation there have been reports of GSK3α- and GSK3β-specific pathways and functions in the cell, although there are contexts where the kinases have been shown to be interchangeable as well [82-85]. Intriguingly, studies in mice have determined that full animal knockout of GSK3β is lethal in the late developmental stages due to hepatic apoptosis [84], whereas GSK3α knockout mice are viable [86]. This may suggest that there exist functions that are tissue-specific or specific for the individual paralogs, or that gene dosage may be important in overall GSK3 function.   GSK3 was initially discovered as a kinase that functioned in glucose homeostasis by phosphorylating glycogen synthase, the rate limiting enzyme in glycogen production [87-89]. In this role, GSK3 is active in the absence of insulin and phosphorylates glycogen synthase to inhibit its enzymatic activity. The presence of insulin activates the PI3K/Akt pathway through the insulin receptor IRS1 and Akt directly phosphorylates GSK3 to inhibit its kinase activity, 27  resulting in inactivation of glycogen synthase and thus glycogen production. It is now well established that GSK3 plays significant roles in numerous other critical cellular pathways. Perhaps the most prominently known role of GSK3 kinases is the regulation of β-catenin stability and abundance in the canonical Wnt signaling pathway, which is crucial in cell fate determination, differentiation, migration, and growth in mammals [90, 91]. In this pathway, GSK3α and GSK3β are known to be functionally redundant, and constitute part of the “destruction complex” along with Axin, APC, casein kinase I, and β-catenin [83]. As part of the destruction complex, in the absence of Wnt ligands GSK3 phosphorylates β-catenin to target it for ubiquitination by an E3 ubiquitin ligase and subsequent proteasome-mediated degradation. In the presence of Wnt ligands, the destruction complex disassociates, and β-catenin is no longer targeted for proteasomal degradation by GSK3 and can translocate to the nucleus to activate the transcription of its target genes. GSK3 is also known to play key roles in Hedgehog signaling, circadian rhythms, and apoptosis [81, 92].   GSK3 kinases have been implicated in various physiological roles and human diseases. From early findings that GSK3 phosphorylated glycogen synthase [87, 88] and tau [93], a major protein associated with Alzheimer’s disease, GSK3 has been a major focus of research in the context of insulin resistance and type 2 diabetes, and neurological/neurodegenerative diseases. Indeed, further investigation has increased our understanding in how aberrant GSK3 activity can arise from high glucose conditions and impair insulin signaling via GSK3 phosphorylation of the insulin receptor, IRS1, which ultimately targets the receptor for proteasomal degradation [94, 95]. Additionally, GSK3 activity is greatly increased under conditions of insulin resistance, and GSK3 inhibition has been found to improve glucose metabolism and the action of insulin in 28  skeletal muscle [96, 97]. Therefore, it is possible that dysregulation of GSK3 activity resulting from impaired insulin signaling can then further amplify insulin resistance via a feed-forward loop. In the realm of neurobiology and neurodegenerative disorders, GSK3 has been implicated in mood disorders including schizophrenia and bipolar disorder, and in the development of Alzheimer’s disease [98]. Lithium has been used for a relatively long period of time as an effective treatment of bipolar disorder in humans, at least partly through its ability to inhibit GSK3 activity [99, 100]. The formation of amyloid plaques and neurofibrillary tangles are hallmarks of Alzheimer’s disease, and GSK3 is thought to play significant contributing roles in both these pathophysiological phenomena [98]. Specifically, GSK3 has been found to promote the production of amyloid beta protein [85], which ultimately form amyloid plaques, and is known to directly phosphorylate tau, which in its phosphorylated form is a major constituent of neurofibrillary tangles [101]. GSK3 has also emerged as potentially playing important roles in inflammation and cancer [98].   GSK3 has unsurprisingly been regarded as an attractive target for pharmacological treatment of human diseases [102]. Lithium, as mentioned previously, has been used for some time as an effective treatment for bipolar disorder and is a well-characterized inhibitor of GSK3 activity, although the precise mechanism of how lithium alleviates symptoms of bipolar disorder through GSK3 remains unknown [98]. Due to the ubiquitous nature of GSK3 expression in essentially all cell types and tissues, and its involvement in a multitude of cellular processes and biological functions, pharmacological modulation of GSK3 activity is a complex proposition that necessitates further study of GSK3 and specific inhibitors. Two prominent issues arising from using GSK3 inhibition as an approach to treat diseases are potential side effects and specificity. 29  The ubiquity of GSK3 in cells and tissues, and its central role in diverse cellular pathways and functions likely increases the likelihood of undesired consequences or potential toxicity when GSK3 is inhibited. Additionally, it has been noted that the development of specific inhibitors that discriminate between GSK3α and GSK3β may not be possible because of the high degree of sequence homology, particularly in the kinase domain [81]. This would lead to roadblocks in attempting to modulate GSK3α- or GSK3β-specific functions in cells. However, as an overall therapeutic strategy, it has been suggested that the fact that GSK3 activity has been found to be abnormally elevated in many disease states that treatment with inhibitors with the aim of restoring the basal level of GSK3 activity would be a promising avenue [102].   1.5.1 Regulation GSK3-family kinases are unique in that they are constitutively active under resting or unstimulated conditions, and therefore require multiple layers of regulation to mediate their activity in the cell. Regulation of GSK3 kinases occurs at the most direct level by phosphorylation of the kinase at key regulatory residues that inhibit or activate its ability to bind substrates. Reversible phosphorylation at an N-terminal serine residue, Ser21 of GSK3α and Ser9 of GSK3β, for instance, is known to rapidly inhibit GSK3 kinase activity, while dephosphorylation by protein phosphatase 1 has been demonstrated to activate GSK3 activity [103, 104]. Phosphorylation of these N-terminal serine residues has been observed to result from external stimuli including insulin, growth factors, and serum [103, 104], and causes the formation of an N-terminal “pseudosubstrate” that blocks the positively-charged active site of GSK3 from being accessed by substrates [105]. Additionally, phosphorylation at a specific 30  tyrosine residue, Tyr279 for GSK3α and Tyr216 for GSK3β, positively regulates GSK3 activity by causing a conformational change that allows for substrate binding [106].   GSK3 activity in cells can also be regulated by intracellular sequestration, as demonstrated in canonical Wnt signaling discussed previously. The basis of this regulation is separate from GSK3 regulation by phosphorylation and mainly involves the formation of a destruction complex consisting of GSK3, axin, APC, casein kinase I, and β-catenin, which allows for GSK3 to phosphorylate β-catenin to target it for ubiquitination and proteasomal degradation [81]. This in turn regulates the cellular abundance of β-catenin in the absence of Wnt ligands. In this way the Wnt pathway regulates GSK3 function via protein-protein interactions and complex formation instead of phosphorylation. In the presence of Wnt ligands, the functional dissolution of the destruction complex effectively blunts the ability of GSK3 to phosphorylate β-catenin. Under certain conditions, GSK3 has also been observed to translocate to different subcellular compartments including the nucleus and to mitochondria [107], but the mechanisms that control GSK3 subcellular localization have not yet been fully delineated.   1.5.2 GSK3 substrates: mechanism and guidelines Another defining characteristic of GSK3 kinases is the almost absolute requirement for GSK3 substrates to be pre-phosphorylated by another kinase. This “priming phosphorylation” adds yet another layer of potential regulation and fine-tuning to cellular GSK3 activity/function. The consensus GSK3 phosphorylation motif can be summarized as S/T-X-X-X-S/T where the N-terminal serine or threonine is the GSK3 target phosphorylation residue, X is any amino acid, and the C-terminal serine or threonine is the priming residue that is phosphorylated by another 31  kinase [81]. It is thought that the primed substrate interacts with high affinity to the positively-charged binding pocket of GSK3 and additionally allows for correct substrate orientation within the active site [92]. The requirement of a priming phosphorylation contributed by another kinase allows for signal integration from different cellular pathways and the priming kinases can additionally be regarded as GSK3 regulators in this context. Many priming kinases for GSK3 substrates have been identified including casein kinase I as part of the canonical Wnt signaling pathway where it primes β-catenin [108], casein kinase II priming of glycogen synthase in glucose metabolism [109], and protein kinase A priming Cubitus interruptus in the Hedgehog signaling pathway [110]. A relatively small number of GSK3 substrates have seemingly bypassed the need of priming phosphorylation by possessing a negatively charged amino acid at the priming site, including, but not limited to, c-Jun, histone H1.5, and c-Myc [111-113]. Interestingly, SREBP-1 has also been found to be a GSK3 phosphorylation target that may not require a priming phosphorylation, and it has been further proposed that GSK3 may provide the priming phosphorylation itself [114], which would be unusual, and remains unconfirmed. These studies have positioned GSK3 activity as having a negative regulatory role on the SREBP pathway and thereby lipid synthesis via phosphorylating SREBP-1, which promotes the Fbw7 ubiquitin ligase to target SREBP-1 for proteasomal degradation [114-116].   Over a hundred substrates for GSK3 have been identified at present and to varying degrees of confidence, further highlighting the central and multifaceted role for GSK3 in a variety of cellular processes across numerous cell types and biological contexts. While some of these GSK3 substrate interactions have been well studied and characterized, many currently do not meet the criteria for bona fide GSK3 substrates initially proposed by Frame and Cohen and 32  reviewed recently in Sutherland, 2011, and thus potentially could represent false positives [92]. The GSK3 consensus phosphorylation motif is not particularly complex and commonly occurs in protein sequences, but clearly not every protein that contains these motifs and not every single instance of this motif in a given protein sequence is phosphorylated by GSK3. Additionally, in vitro phosphorylation by GSK3 does not necessarily predict in vivo phosphorylation, nor does it ascribe physiological function to the phosphorylation. As such, it is important to highlight and summarize the proposed guidelines for investigating whether a proposed substrate is a true physiological phosphorylation target of GSK3 [92]:   1) Purified GSK3 should phosphorylate the substrate in vitro, and additionally the phosphorylated residues should be phosphorylated in in vivo contexts. 2) The phosphorylation state at the putative phosphorylation site(s) identified in vitro should change in response to altering GSK3 activity in vivo such as by pharmacological inhibition by well-characterized GSK3 inhibitors.  3) A physiological function of the substrate should be altered in response to the change in the putative phosphorylation site(s) and/or GSK3 activity in the cell. Furthermore, creation of a serine/threonine to alanine phosphorylation-deficient form of the proposed substrate at the identified sites should result in the substrate no longer responding to alterations in GSK3 activity.  4) Mutation of the priming residue in many cases should also result in the substrate becoming insensitive to manipulations of GSK3 activity. This should only be used as additional validation because it has been observed that a small number of characterized substrates do not require a priming phosphorylation [81].  33   It is especially important in the case of GSK3 to have stringent criteria when establishing which potential substrates are likely true physiological targets because of the multitude of roles that GSK3 has been ascribed in cellular regulation and function. In separating what are true physiological targets of GSK3, it will help in the path to understanding how GSK3 specifically plays roles in these cellular pathways and processes. It will also undoubtedly be of great utility in evaluating the potential of pharmacologically altering GSK3 activity in the treatment human diseases, both regarding overall feasibility and potential auxiliary effects to expect so they can be considered with appropriate foresight.   1.6 Thesis Investigation Phosphorylation has been well-established to be a major mechanism in regulating the function of the conserved lipin family of phosphatidic acid phosphatase enzymes, which are key players in cellular lipid synthesis and metabolism. However, a high throughput genome-wide screen has never been performed to comprehensively identify novel kinase regulators of lipins in an unbiased manner. The overarching goal of this dissertation was to use high throughput synthetic dosage lethality (SDL) screening in the budding yeast Saccharomyces cerevisiae to first identify and then characterize in detail novel kinase regulators of the yeast lipin Pah1. Additionally, when appropriate, we would subsequently aim to further investigate whether the regulatory relationships we discovered in yeast were conserved in mammalian systems using tissue culture cells and mouse models as necessary.   34  In Chapter 2 of this thesis, we describe the results of our high throughput SDL screen in yeast where we overexpressed Pah1 in each individual deletion mutant strain in the yeast deletion collection array, comprising over 4800 deletion mutants. We sought synthetic lethal combinations with Pah1 overexpression and specifically narrowed our focus to strains missing kinases because we were interested in identifying potential kinases that phosphorylated Pah1. From our screen, we identified eleven candidate kinases, each of which when deleted caused synthetic lethality upon Pah1 overexpression. We subsequently performed low throughput secondary screens to further narrow our candidate genes by investigating which of the kinase null strains exhibited phenotypes consistent with decreased PA levels and analyzing Pah1 protein isolated from some strains to analyze for mobility shifts. Ultimately, we identified the yeast GSK3-family kinase Mck1 as our primary kinase of interest. We then performed in vitro and in vivo studies to understand the relationship between Mck1 and Pah1, discovering that Pah1 is a phosphorylation target of Mck1, and that cells lacking Mck1 show phenotypes consistent with dysregulated Pah1 PAP activity including decreased PA and increased neutral lipid accumulation.   Chapter 3 uses the kinase-substrate relationship between Mck1 and Pah1 we discovered and characterized in yeast as a foundation, and studies whether this relationship is conserved in between mammalian GSK3 and lipin 1. Lipin 1 contains numerous confirmed phosphorylation sites, some of which fit the GSK3 consensus phosphorylation motif. Using proteomic and biochemical approaches, we confirmed a GSK3 phosphorylation site within lipin 1 consisting of serine 468 (S468) and serine 472 (S472) in the canonical S/T-x-x-x-S/T motif that shares an experimentally confirmed phosphorylation site with mTORC1 at the S472 residue. We 35  additionally found evidence that the mTORC1 phosphorylation at the S472 residue was required as a priming phosphorylation that allowed GSK3 phosphorylation at S468. Next, we found that GSK3 phosphorylation of lipin 1 promoted the cytosolic localization of lipin 1, whereas dephosphorylation resulted in lipin 1 translocation to the nucleus. Mutation of lipin 1 S468 or S472 to phosphorylation-deficient residues additionally resulted in lipin 1 favoring a more nuclear distribution and a significant accumulation at the nuclear periphery. Lipin 1 translocation to the nucleus has been characterized by previous studies to repress SREBP target gene expression. Indeed, our subsequent findings when investigating this avenue suggested that GSK3-mediated lipin 1 phosphorylation also regulated the function of lipin 1 in repressing SREBP. Having linked GSK3 to SREBP activity via lipin 1, in the last part of this chapter we moved to beginning to study this pathway in in vivo mouse models of diet-induced obesity. In a preliminary study, we found that liver-specific knockout of either GSK3 paralog, GSK3α or GSK3β, appeared to confer moderate resistance to the effects of a high fat diet including weight gain and the expression of certain SREBP target genes, suggesting that GSK3 in the liver plays a role in the development of phenotypes associated with diet-induced obesity.     36  Chapter 2: Synthetic dosage lethality screening identifies novel kinase regulators of the yeast lipin Pah1 2.1 Introduction Maintaining lipid homeostasis is critical to the growth and fitness of all organisms, and the increasing prevalence of lipid-associated disorders including obesity and type 2 diabetes necessitates a more comprehensive understanding of the fundamental pathways governing lipid metabolism. The lipin family of phosphatidic acid phosphatase (PAP) enzymes have emerged as critical regulators of lipid homeostasis and are associated with metabolic diseases. The molecular function of lipins is to convert the lipid phosphatidic acid (PA) into diacylglycerol (DAG) at the ER membrane in the penultimate step of triacylglycerol (TAG) synthesis and fat storage [26]. Lipins thereby exert direct influence on cellular lipid homeostasis. Accordingly, it has been found that loss of lipin 1 function in mice causes severe lipodystrophy, hepatic steatosis, and insulin resistance [27], whereas overexpression of lipin 1 results in increased lipogenesis and obesity in mice even on a chow diet [28]. Furthermore, lipin 1 and lipin 2 mutations in humans result in the metabolic diseases myoglobinuria and Majeed syndrome, respectively, and lipin 1 and lipin 2 polymorphisms have been found to be associated with obesity and diabetes traits [61, 64, 65]. It is also intriguing that mammalian lipins have been found to translocate to the nucleus and play key roles in regulating the transcription of genes related to both the adipogenic program and lipid synthesis [14, 38, 49, 50].   Given their importance in maintaining lipid homeostasis, it is unsurprising that lipins are tightly regulated enzymes. It is now known that lipins are highly phosphorylated enzymes and 37  that phosphorylation is the major mechanism of lipin regulation. Work studying the yeast lipin Pah1 has been invaluable in uncovering the roles of phosphorylation in regulating lipin function. Phosphorylation by kinases has been discovered to control the subcellular localization, abundance, enzymatic activity, and degradation of Pah1 [70-75]. Specifically, phosphorylated Pah1 is localized to the cytoplasm and is enzymatically inactive [36]. Dephosphorylation by the Nem1-Spo7 phosphatase complex, conversely, is required for the recruitment of Pah1 to the ER, activation of enzymatic PAP activity, and additionally results in an increased susceptibility of Pah1 to degradation by the 20S proteasome [73, 74]. The control of Pah1 function by phosphorylation/dephosphorylation positions the kinases that phosphorylate Pah1 as key regulators of lipid homeostasis. Several kinases in yeast including Pho85-Pho80 cyclin-dependent kinase, Cdc28-cyclin B, protein kinase A, protein kinase C, and casein kinase II have been found to directly phosphorylate the yeast lipin Pah1 to variable effect [70-72, 75].   In mammalian systems, lipin 1 is the best characterized of three mammalian lipin orthologs (the others being lipin 2 and lipin 3), and phosphorylation and other post-translational modifications have been found to significantly control its function. Like Pah1 in yeast, lipin 1 is a highly phosphorylated protein [76] and phosphorylation similarly appears to be the major form of lipin regulation in mammals, but does not appear to inhibit lipin 1 PAP activity [14, 38]. Instead, phosphorylation largely appears to regulate lipin 1 subcellular localization to control cellular lipin activity. For instance, insulin signaling results in the multi-site phosphorylation of lipin 1 and promotes its cytoplasmic localization as opposed to membrane association [76]. Furthermore, the mechanistic target of rapamycin complex 1 (mTORC1) is currently the only kinase or kinase complex known to directly phosphorylate lipin 1, and controls lipin 1 38  translocation from the cytoplasm to the nucleus [14]. In this instance, mTORC1 phosphorylation causes lipin 1 to localize to the cytoplasm, whereas loss of mTORC1 phosphorylation results in lipin 1 translocation to the nucleus where it represses the sterol regulatory element binding protein (SREBP) and its downstream transcriptional targets, which are critical in cellular fatty acid and cholesterol synthesis and uptake [14]. The activity of mTORC1 towards lipin 1 therefore mediates the effects of mTORC1 signaling on the SREBP pathway. Recently, acetylation of lipin 1 by Tip60, the catalytic subunit of the conserved NuA4 acetyltransferase complex, has been discovered to control lipin 1 recruitment from the cytoplasm to the ER to facilitate cellular triacylglycerol (TAG) synthesis in mice [68]. The same study additionally characterized that the Tip60 homologue in yeast, ESA1, is required for TAG accumulation in a pathway likely involving the acetylation of the yeast lipin Pah1. This highlights that some regulatory pathways of lipins are potentially conserved from yeast to mammals, and the potential utility of using yeast to study lipin regulation and function.   We aimed to identify additional kinases that regulate lipins by conducting a systematic genome-wide overexpression screen in yeast using its sole lipin homologue, Pah1. Importantly, a screen seeking to identify novel kinase regulators of lipins has never been performed. This screen, which was a variant on synthetic dosage lethality (SDL) screening, yielded 322 SDL interactions including 11 kinases that when deleted demonstrated significantly decreased cell growth upon Pah1 overexpression. This suggested that these kinases may play a role in regulating Pah1. Upon further investigation, we determined that the yeast GSK3 homologue, Mck1, directly phosphorylates Pah1 in vitro and regulates lipid metabolism in yeast. Specifically, cells lacking Mck1 demonstrate phenotypes consistent with decreased phosphatidic 39  acid levels in a regulatory pathway involving Opi1, the master transcriptional regulator of phospholipid biosynthesis in yeast [22, 117]. Additionally, these cells have increased neutral lipid content as evidenced by lipid droplet staining.   2.2 Materials and Methods Yeast Media and Growth Conditions Yeast media was prepared using standard methods with yeast extract and yeast nitrogenous bases from Difco and amino acids supplied by Bioshop except for media and agar plates for inositol assays which were made according to an established SGA protocol either including or excluding 1mM inositol. Yeast cultures were grown at 30°C unless otherwise stated and transformed with standard methods unless stated otherwise.  Plasmids pGAL1/10-GST-6XHis-PAH1 and pGH312 HA-Pah1 plasmids were kind gifts from the laboratory of Dr. George Carman at Rutgers University (New Jersey, USA). Mck1 plasmids containing wild type Mck1 and catalytically inactive Mck1 D164A were the same as used in McQueen et al., 2012 [118].  SDL Screen The synthetic dosage lethality (SDL) screen was performed by mating the SGA starting strain transformed with the pGAL1/10-GST-6XHis-PAH1 plasmid to the yeast haploid deletion collection (Open Biosystems) in a 384-array format using a ROTOR high throughput screening machine (Singer Instruments, Somerset, United Kingdom). Colony size was determined under 40  inducing conditions (2% galactose) and ratios determined by comparing colony size on control (2% dextrose) versus experimental (2% galactose) plates using the analysis software Balony [119]. SDL hits were defined as those that had a ratio of colony size of 0.800 or less, and a p<0.05 in all three biological replicates.   Pah1 Mobility Shift Assays For HA-Pah1 mobility-shift experiments, log phase yeast transformed with the pGH312 plasmid were lysed by methods mentioned previously. 160μg of protein lysate was then separated on a 7% poly-acrylamide gel and probed with anti-HA primary antibody (Roche, Switzerland) and goat anti-mouse IRdye 800 CE fluorescent secondary antibody (Li-Cor Biosciences, Nebraska, USA), and imaged with Odyssey Infrared imaging system (Li-Cor Biosciences, Nebraska, USA).   In vitro Kinase Assays Kinase assays for Mck1 and Pah1 were performed using Mck1-myc, Mck1-D164A-myc, and Pah1-HA purified from whole cell lysates incubated with anti-myc or anti-HA affinity matrix beads (Covance, New Jersey, USA), respectively. 0.02 mCi γ-32P-ATP was added to each reaction containing purified Mck1-myc or Mck1-D164A-myc and Pah1-HA and incubated for 40 min at 30˚C to allow kinase reactions to occur. Subsequently, the reactions were stopped by adding 2X SDS loading buffer and were run on polyacrylamide gels. Finally, the gels were dried using a gel dryer and imaged using film.     41  Semi-Quantitative Reverse Transcriptase PCR RNA was harvested from log phase yeast cultures using Qiagen RNeasy RNA extraction kit after isolating ~20 OD units of yeast and digesting using zymolase. RNA extraction was performed using manufacturer-recommended protocol and RNA concentration was determined using a NanoDrop™ spectrophotometer (ThermoFisher, Massachusetts, USA). cDNA was then generated via reverse transcription using reverse transcriptase (New England Biolabs, Massachusetts, USA). PCR reactions were performed using gene-specific primers for ACT1 and INO1, and PCR products were analyzed on 1% agarose gels and then using ImageJ software to determine relative expression of INO1 normalized to ACT1.   Confocal Microscopy Microscopy was performed on a Zeiss Pascal Laser Scanning Microscope (Zeiss, Germany).   Nile Red Lipid Droplet Staining  Mid-log phase yeast cells were collected and washed in media 2 times before staining. Cells were then stained in the dark for 5 minutes with 0.01 mg/mL nile red added to media. Care was taken to store stained cells in the dark before microscopy was performed. Confocal microscopy was performed on a Pascal Laser Scanning Microscope (Zeiss) and lipid droplet fluorescence intensity was analyzed using CellProfiler software [120]. Lipid droplet number was counted manually.     42  Data and Statistical Analysis Data is presented as mean ± SD or mean ± SEM where appropriate and as indicated in figure legends. Data was analyzed using unpaired student’s t-test for data comparing two groups, which comprised all statistical comparisons in this chapter. Statistical analysis was performed using Microsoft Excel software with statistical significance set at p<0.05.   2.3 Results PAH1 Synthetic Dosage Lethality Screen  Since phosphorylation is known to be a major regulatory mechanism of lipin localization and/or activity, we aimed to identify novel kinases that phosphorylate and regulate lipins. To do this, we used the budding yeast Saccharomyces cerevesiae as a model because it possesses a single lipin homologue, Pah1, which has been demonstrated to play a critical role in triglyceride synthesis and neutral lipid storage (Figure 2.1). Additionally, yeast has 129 protein kinases, with more than half having close metazoan homologues [121]. 93 of these kinases are represented in the yeast deletion collection which is comprised of the ~4800 nonessential yeast gene deletion mutants.   Even though phosphorylation by kinases is known to regulate lipins, a genome-wide screen for lipin regulators has never been performed. Therefore, we designed an unbiased high-throughput overexpression screen to systematically analyze the nonessential yeast deletion collection for novel kinase regulators of the lipin Pah1 (Figure 2.2). The screen was a variation of synthetic dosage lethality (SDL) screening in which PAH1 was overexpressed in each of the individual deletion strains in the nonessential yeast deletion collection, and each strain was 43  subsequently assayed for colony size as an indicator of cellular fitness. Previous studies have shown that PAH1 overexpression is moderately detrimental to cell growth [26] and thus we rationalized that overexpression of PAH1 in deletion mutants lacking regulators of Pah1 would demonstrate an even more pronounced decrease in cellular fitness. Additionally, SDL screening has been previously used as a robust method to identify substrate-kinase relationships [122, 123].    44    Figure 2.1: The role of lipin family phosphatidic acid phosphatases in lipid metabolism Schematic depicting the role of lipins in lipid metabolism. In yeast and mammals, lipins convert the phospholipid precursor phosphatidic acid to the neutral lipid diacylglycerol. In yeast, PA represses the transcriptional repressor Opi1, which represses the Ino2-Ino4 transcriptional activator. Black boxes, phospholipids; gray boxes, neutral lipids; PC, phosphatidylcholine; PI, phosphatidylinositol; PA, phosphatidic acid; DAG, diacylglycerol; TAG, triacylglycerol.   45    Figure 2.2: Synthetic dosage lethality overexpression screening to identify regulators of Pah1 Schematic overview of synthetic dosage lethality (SDL) screening in yeast. A query strain transformed with the GAL-PAH1 overexpression plasmid is mated to the viable single deletion mutant array. Diploids are sporulated, haploids are selected, and the resulting arrays are plated on non-inducing or inducing conditions. Synthetic dosage lethal phenotypes are determined by comparing colony size in the control (non-inducing) condition versus the experimental (inducing) condition using Balony software (Young and Loewen, BMC Bioinformatics, 2013).   46  To validate that our GAL-PAH1 construct increases cellular PAP activity, we showed that overexpression using the construct in our query strain causes the expected changes in PA and neutral lipid homeostasis. PA in the yeast ER directly binds and sequesters Opi1, the master transcriptional regulator of many phospholipid synthesis genes including the inositol synthesis gene INO1 [22, 23]. A decrease in cellular PA allows Opi1 to translocate to the nucleus and suppress INO1 expression [22] (Figure 2.1). Without supplemented inositol, cells require INO1 transcription to synthesize inositol and to grow; cells that cannot grow without supplemented inositol are inositol auxotrophs. PAH1 overexpression and the resulting decrease in cellular PA have been demonstrated to cause inositol auxotrophy [26]. Similarly, our query strain exhibited slow growth on media lacking inositol upon PAH1 overexpression with our construct, demonstrating a decrease in cellular PA levels (Figure 2.3). An increase in cellular PAP activity should also lead to an increase in neutral lipid accumulation. Using nile red, a fluorescent lipophilic dye that stains lipid droplets and has been used to estimate neutral lipid content, we found that overexpression of PAH1 causes a 50% increase in the average intensity per lipid droplet stained when compared to a vector control (Figure 2.4). This demonstrated an increase in neutral lipid accumulation introduced by our construct.  Finally, we noticed that our query strain showed only a modest decrease in the rate of log phase growth upon PAH1 overexpression (Figure 2.3), indicating that a cell possessing all regulatory kinases of Pah1 could buffer PAP activity effectively even when overexpressing Pah1, thus providing us a large dynamic range in identifying hits from our screen.      47   Figure 2.3: Increased PAP activity negatively affects cell growth and causes inositol auxotrophy A. Yeast inositol auxotrophy growth assays of wild type cells transformed with a control vector or GAL-PAH1 overexpression vector under non-inducing (Dextrose) or inducing (Galactose) conditions. B. Growth curve analysis of indicated strains in minimal media with 2% galactose. Doubling time of GAL-PAH1 is 3.15 hours and vector is 2.68 hours (p=1.6 X 10-9) in galactose media.   48   Figure 2.4: GAL-PAH1 overexpression in wild type yeast cells causes increased lipid droplet staining by nile red A. Scatter plot of lipid droplets stained in wild type cells by nile red (vector, n=347 lipid droplets; GAL-PAH1, n=355 lipid droplets; linear regression, vector, r2 = 0.8948; GAL-PAH1, r2 =0.8623). B. Nile red staining of lipid droplets in wild type cells transformed with either control vector or GAL-PAH1 overexpression vector grown under inducing conditions and imaged by confocal microscopy. Quantification of intensity of nile red staining of lipid droplets by CellProfiler software is plotted (n > 300 lipid droplets for each; * p < 1.05 x 10-5; scale bar: 2 μm; error bars, SEM).   49   PAH1 SDL screen identifies eleven kinases including the yeast GSK3β homologue Mck1  Our screen identified 322 SDL hits including 11 kinases that when deleted caused synthetic dosage lethality upon Pah1 overexpression (Appendix B Table 1, and Figure 2.5). Four of these kinase deletion mutants: Δdbf2, Δbck1, Δbub1, and Δmck1, possessed slow growth on media lacking supplemented inositol, indicating possible decreases in cellular PA due to an increase in Pah1 PAP activity (Figure 2.6). Δdbf2 and Δbck1 were previously identified as inositol auxotrophs from two independent genome-wide screens for inositol auxotrophy [23, 124], and Δmck1 was identified by one of these screens [124]. The kinases identified by our screen therefore likely represent novel kinases regulators of Pah1 and lipid homeostasis.   We next aimed to assay Pah1 phosphorylation state in the kinases null mutants using mobility shift in SDS-polyacrylamide gels as this method has demonstrated to be effective in previous studies [26, 69]. We first expressed and purified HA-Pah1 from yeast cultures with wild type, Δdbf2, Δbck1, Δbub1, and Δmck1 backgrounds. Subsequent analysis demonstrated that HA-Pah1 from wild type, Δdbf2, Δbck1, and Δbub1 cells migrated at similar rates in the assay, while HA-Pah1 from the Δmck1 cells showed a significantly faster migrating species of Pah1, suggesting a potential decrease in Pah1 phosphorylation in this strain background (Figure 2.7). This established Mck1 as a prime candidate to study as a novel regulatory kinase of Pah1.    50   Figure 2.5: Pah1 synthetic dosage lethality screen identified eleven potential kinase regulators of Pah1 A. SDL kinase interactions identified in the genome-wide screen. Plotted are the normalized colony size measurements for the indicated kinase mutants alone (no plasmid) or overexpressing GAL-PAH1 (n=3; p < 0.05; error bars, SD). B. Growth assay with wild type and kinase null strains transformed with empty vector or GAL-PAH1 in non-inducing (Dextrose) or inducing conditions (Galactose). 51    Figure 2.6: Four kinases identified in the Pah1 SDL screen cause yeast cells to exhibit inositol auxotrophy when knocked out  Growth assays for the indicated mutants lacking each of the eleven kinases identified in our Pah1 SDL screen. Strains are serially diluted from a starting OD600 of 0.1, plated on synthetically defined media with or without supplemented inositol, and grown at 37°C for 3 days.   52  Mck1 phosphorylates Pah1 and regulates lipid homeostasis Mck1 is one of four glycogen synthase kinase 3 (GSK3) homologues in yeast and is a dual-specificity serine/threonine kinase known to be involved in cell signaling and cell cycle regulation[125]. Notable roles for Mck1 include calcium stress signaling [126], inhibiting protein kinase A in response to poor nutrient availability [127], and inhibiting the main cyclin-dependent kinase Clb2-Cdk1 [118]. Recently, Mck1 has also been identified as a regulator of quiescent entry and exit, and chronological lifespan [128]. In mammals, GSK3 kinases are ubiquitously expressed in essentially every cell and tissue, and are known as central hub of cellular regulation, mediating a number of processes including glucose homeostasis, cell differentiation, apoptosis, and development [98]. GSK3 has also been linked to many diseases including insulin resistance, type 2 diabetes, various cancers, and neurological disorders including Alzheimer’s disease [81, 98]. As such, it is unsurprising that GSK3 remains an active focus of ongoing research and seen as a potential therapeutic target [102]. It is also important to note that Mck1 was the only yeast GSK3 kinase of four total to be identified by our screen, suggesting that this may be a role specific to this paralog [129].    We further investigated the mobility shift of HA-Pah1 purified from Δmck1 cells. To support that the faster mobility was due to a decrease in phosphorylation, we treated Pah1 from wild type and Δmck1 cells with calf intestinal phosphatase (CIP) and observed that CIP treatment caused Pah1 from both strains to shift as faster migrating species to the same degree (Figure 2.7).  This suggested that faster migrating Pah1 species are less phosphorylated than slower migrating species. Additionally, it appeared that Mck1 was likely not the only kinase that directly phosphorylates Pah1 because the CIP treated Pah1 from Δmck1 was further dephosphorylated 53  compared to the non-CIP treated control. Next, to test whether Mck1 could directly phosphorylate Pah1, we performed an in vitro kinase assay with Mck1-myc and HA-Pah1 expressed and immunoprecipitated from yeast cells. We assayed for phosphorylation of Pah1 by Mck1 in this manner by adding the immunoprecipitated proteins in a reaction together with radiolabelled γ-32P-ATP. We found that Pah1 was phosphorylated by wild type Mck1 and that this phosphorylation did not occur when a catalytically inactive D164A Mck1 mutant was used (Figure 2.8). As an internal control for Mck1 catalytic activity we also observed Mck1 auto-phosphorylation that only occurred with wild type Mck1 (Figure 2.8). This data supports that our purified Mck1-myc was catalytically active and directly phosphorylates our purified HA-Pah1, and that generally Mck1 is capable of phosphorylating Pah1.       54    Figure 2.7: Pah1 purified from Δmck1 cells demonstrates a mobility shift A. Immunoprecipitated Pah1-HA purified from wild type, Δmck1, Δbub1, Δdbf2, and Δbck1 yeast strains separated on a SDS polyacrylamide gel and visualized with anti-HA antibody by western blot. B. Immunoprecipitated Pah1-HA from wild type (MCK1) and the Δmck1 mutant with (+) and without (-) treatment with calf intestinal alkaline phosphatase (CIP) visualized by western blot with an anti-HA antibody.   55    Figure 2.8: In vitro kinase assay demonstrates that Mck1 phosphorylates Pah1 Mck1-myc and Mck1-D164A-myc purified from yeast lysates and incubated with purified Pah1-HA (+) or a control purification from yeast lacking Pah1-HA (-), and γ-ATP32 for 40 minutes at 30°C. An additional control was performed using a strain without Mck1-myc (untagged). Phosphorylated Pah1 (Pah1-P) and phosphorylated Mck1 (Mck1-P) as indicated on the figure itself. Mck1-P was detected because catalytically active Mck1 auto-phosphorylates itself.    56  To investigate Mck1 regulation of Pah1 PAP activity physiologically, we then studied the role of Mck1 in cellular PA and neutral lipid homeostasis. Since we observed that Δmck1 cells were inositol auxotrophs, likely due to decreased cellular PA levels and increased repression of INO1 expression by Opi1, we additionally deleted Opi1 in Δmck1 cells. The Δmck1Δopi1 double mutant demonstrated complete growth rescue on media lacking inositol, strongly supporting the fact that Δmck1 cells possessed decreased cellular PA levels (Figure 2.9a). Furthermore, when assayed for INO1 expression by semi-quantitative reverse transcription PCR, the Δmck1 strain showed a ~50% decrease in INO1 expression compared to wild type and was restored in the Δmck1Δopi1 strain (Figure 2.9b). We also further demonstrated that Mck1 kinase activity is necessary for its role in PA homeostasis by rescuing the inositol auxotrophy phenotype of Δmck1 cells by expressing wild type Mck1 on a plasmid in these cells (Figure 2.10). Conversely, Δmck1 cells transformed with a plasmid expressing the catalytically inactive D164A Mck1 still exhibited inositol auxotrophy in media lacking inositol (Figure 2.10). Therefore, Mck1 regulates INO1 expression via Opi1, functionally linking Mck1 to cellular PA levels and Pah1 PAP activity.  Finally, Δmck1 cells should also exhibit increased neutral lipid storage due to increased Pah1 PAP activity. We assayed Δmck1 cells with nile red to estimate neutral lipid content in lipid droplets and found an increase in the intensity of nile red staining per lipid droplet and number of lipid droplets in Δmck1 cells compared to wild type (Figure 2.11).    57   Figure 2.9: Deletion of the transcriptional repressor Opi1 rescues the inositol auxotrophy of Δmck1 cells A. Inositol auxotrophy growth assays comparing the growth of wild type, Δino1, Δmck1, and Δmck1Δopi1 yeast strains on synthetically-defined media containing or excluding supplemented inositol. Growth assays were incubated at 37°C for 3 days before imaging.  B. INO1 expression in wild type (WT) and the indicated mutants determined by semi-quantitative RT-PCR (n=3; * WT vs Δmck1, p < 0.01; * WT vs Δmck1Δopi1, p < 5 X 10-4).   58    Figure 2.10: Expression of catalytically active Mck1 rescues the inositol auxotrophy of Δmck1 cells Inositol auxotrophy growth assays with the Δmck1 mutant transformed with the plasmids: vector alone (vector); wild type Mck1(MCK1); and kinase-dead Mck1 (mck1-D164A). Yeast strains were grown on synthetically-defined media containing or excluding supplemented inositol and incubated at 37°C for 3 days before imaging.   59    Figure 2.11: Δmck1 cells have increased neutral lipid accumulation as assayed by nile red staining  A. Lipid droplet number per cell in wild type (WT) and Δmck1 cells as determined by nile red staining (n > 75 cells for each; * indicates p < 7.286E X 10-9; scale bar: 2 μm; error bars, SEM). B. Quantification of nile red staining of lipid droplets in wild type (WT) and Δmck1 cells imaged by confocal microscopy using CellProfiler software (n > 350 lipid droplets foreach; * indicates p < 0.002; scale bar: 2 μm; error bars, SEM).   60  2.4 Discussion Through our SDL screen for novel kinases regulators of the yeast lipin Pah1 and subsequent work, we have characterized a role for the yeast GSK3 kinase homologue Mck1 in phosphorylating Pah1 and regulating cellular lipid homeostasis. The screen we performed reinforces the utility of high-throughput SDL screening using the yeast deletion collection for identifying kinase-substrate relationships, and our unbiased screening approach yielded additional candidate kinases and many other genes that could be studied in the future (Appendix B Table 1). Mck1 phosphorylation of Pah1 establishes a novel role for GSK3-family kinases in regulating lipid homeostasis through the lipin family of phosphatidic acid phosphatases. This is especially true in the context of yeast, as the characterized functions of Mck1 primarily involve its role in cell signaling and the cell cycle [118, 129]. Here we have demonstrated that Mck1 directly phosphorylates Pah1 in vitro, and that cells lacking Mck1 possess phenotypes consistent with dysregulated cellular PAP activity including inositol auxotrophy, suggesting decreased cellular PA levels, and increased neutral lipid content and lipid droplet number. Furthermore, we have mapped the inositol auxotrophy of Δmck1 cells to the well-characterized relationship between PA and the master transcriptional regulator Opi1, strongly supporting our link between Mck1 and cellular PA levels.   Taken together, our data strongly suggests that Mck1 phosphorylation of Pah1 plays an overall inhibitory role in regulating Pah1 function in the context of PA and DAG homeostasis. Further work will be required to investigate the specific phosphorylation sites at which Mck1 phosphorylates Pah1, as Pah1 contains multiple Mck1/GSK3 consensus phosphorylation motifs throughout the length of the protein (Figure 2.12). Additionally, the specific effects of Mck1 61  phosphorylating Pah1 have not yet been elucidated and could be related to previously characterized roles of phosphorylation by kinases in controlling the localization, enzymatic activity, and degradation of Pah1. Mck1 phosphorylation of Pah1 could, for instance, promote Pah1 localization to the cytoplasm as opposed to ER association, which is similar to what has been ascribed to other kinases that phosphorylate Pah1. Mck1 phosphorylation could alternatively or additionally inhibit or modulate the PAP activity of Pah1 directly to maintain cellular PA and DAG levels, and an in vitro PAP activity assay of Pah1 purified from wild type and Δmck1 would be informative in this regard. Finally, dephosphorylation of Pah1 is known to result in its ubiquitin-independent degradation by the 20S proteasome [73-75]. Mck1 phosphorylation could therefore possibly have a protective effect on Pah1 degradation. However, it is important to note that we did not observe evidence of a difference in Pah1 protein levels or stability in our analysis of purified Pah1 from wild type and Δmck1 cells.    Mck1 shares a conserved consensus phosphorylation motif with other GSK3-family kinases including mammalian GSK3α and GSK3β [81, 129]. This motif, which allows for optimal affinity of GSK3-family kinases to their substrates, consists of S/T-X-X-X-S/T where the first S/T represents the GSK3 phosphorylation residue, X represents any amino acid and the second S/T residue is the site of a priming phosphorylation by another kinase [98]. The requirement of a priming phosphorylation by another kinase, in addition to sequence specificity, exists as a cellular mechanism to regulate GSK3 kinase activity and function [81, 98]. As such, the kinase or kinases that provide the priming phosphorylation are also of interest in the regulation of substrates of GSK3-family kinases such as Pah1. In the current work, we have not identified the kinase or kinases responsible for priming Pah1 for phosphorylation by Mck1. Since 62  our purified Mck1 was able to phosphorylate purified Pah1 in our in vitro kinase assays, presumably this Pah1 already possessed the priming phosphorylation necessary for Mck1 phosphorylation. Alternatively, it is possible that the priming kinase was pulled down in addition to Mck1 or Pah1. This is perhaps less likely because when analyzing our purified preparations of Mck1 and Pah1 by western blot both migrated at their typical molecular weights.   The identity of the priming kinase may be one or more of the other ten kinase hits from our screen, as each of these kinases when deleted also resulted in synthetic lethality upon Pah1 overexpression. Of these kinases, however, seven did not demonstrate significant inositol auxotrophy when deleted in yeast. Further, Pah1 purified from the remaining kinase null strains that did demonstrate inositol auxotrophy did not show a mobility shift when assayed except for Δmck1. This suggests that either it is not these kinases individually that impart the priming phosphorylation, or that it is a combination of these kinases (or potentially others not identified by our screen) that are contributing the priming phosphorylation necessary for Mck1 to ultimately phosphorylate Pah1.  It is imperative to validate GSK3 substrates given the ubiquitous nature of GSK3 expression in cells and their multi-faceted roles in cellular regulation and function. As currently constituted, our evidence that Pah1 is a validated GSK3 substrate falls short of proposed guidelines for defining bona fide GSK3 substrates as first proposed by Frame and Cohen and summarized recently in a review by Sutherland [92]. To summarize, to be considered a true GSK3 substrate, a phosphorylation target must have specific phosphorylation sites confirmed by a method such as mass spectrometry, and modulation of GSK3 activity must alter the 63  phosphorylation state of the putative site. Additionally, the phosphorylation site should alter a physiological function that can be disrupted by altering GSK3 activity and by mutating the phosphorylation site to a phosphorylation-deficient form. We have provided compelling evidence in our current work evidence that Pah1 can be phosphorylated specifically by Mck1 in vitro, and that cells lacking Mck1 demonstrate phenotypes consistent with overactive Pah1 including decreased PA levels and increased neutral lipids. Further work should therefore focus on delineating the specific Mck1 phosphorylation sites of Pah1 and linking them directly to physiological effects such as inositol auxotrophy and/or neutral lipid storage in lipid droplets.   Potential future studies in mammalian systems  Interestingly, mammalian lipin 1 contains a number of GSK3 consensus phosphorylation sites among some of its experimentally-defined phosphorylation sites (Figure 2.12), which may provide future avenues of study. One of these phosphorylation pairs is composed of serine 468 and serine 472 positioned in the canonical S/T-X-X-X-S/T GSK3 motif. Serine 472 has been found previously to be a residue directly phosphorylated by the mTORC1 complex [14]. This serine 472 residue is also in the proper location relative to serine 468 to potentially provide the priming phosphorylation for GSK3 to phosphorylate serine 468. Though none of this constitutes direct evidence that mammalian GSK3 phosphorylates lipin 1, the knowledge that there is an experimentally validated priming phosphorylation site is promising nonetheless. If GSK3 were to phosphorylate lipin 1 at S468, it could represent crosstalk between the mTORC1 and GSK3 to regulate lipid homeostasis through lipin 1. As there have been so few kinases characterized to directly phosphorylate lipin 1 and because GSK3 is amongst the most central of cellular regulatory kinases discovered to date, this avenue may be a promising avenue to pursue.  64    Figure 2.12: Lipin 1 possesses multiple GSK3-consensus phosphorylation sites Schematic depicting some experimentally determined lipin 1 phosphorylation sites labelled in black. GSK3 consensus phosphorylation motifs are indicated. Sites in grey (S349 and T718) represent residues in GSK3 consensus phosphorylation sites not yet confirmed by mass spectrometry.   65  Other kinases identified by our SDL Screen  The other hits identified by our screen also represent potential regulators of Pah1. In particular, the three other kinase null strains that demonstrated inositol auxotrophy – Δbck1, Δbub1, and Δdbf2 – are strong candidates because they likely have decreased cellular PA levels like Δmck1 cells. Unlike Δmck1, however, Pah1 purified from these three strains did not possess a noticeable mobility shift when analyzed on a polyacrylamide gel. It is possible that these kinases do not phosphorylate Pah1 at enough sites to cause an appreciable shift. As mentioned previously, these kinases may also less directly affect Pah1 phosphorylation and function by providing the priming phosphorylation for Mck1 phosphorylation.   Looking more specifically at two of these kinases, it is intriguing that Dbf2 and Bub1 have mammalian orthologues with clear links to human disease. The Dbf2 orthologue is LATS1, a ubiquitously expressed AGC family kinase, which similarly to Dbf2, functions as a nuclear mitotic exit network kinase. This is a signaling pathway that coordinates cyclin-dependent kinase inactivation, sister chromatid decondensation, and cytokinesis at the end of mitosis [130, 131]. This kinase also functions in the Hippo pathway and mutations in LATS1 have been associated with human cancers [132, 133]. Although a direct link has yet to be established, its involvement in the Hippo pathway is likely key in this role [133, 134]. Bub1 is also conserved from yeast to mammals and is a spindle assembly checkpoint kinase that functions with a network of at least thirteen other proteins that monitors proper bipolar attachment of microtubules to the kinetochore [135]. It localizes to the kinetochore in G2 phase and is thought to function as a platform for recruitment of other checkpoint proteins [136]. Consistent with this role, loss of Bub1 results in aneuploidy and increased tumor susceptibility in mice [137], and Bub1 expression and mutations 66  are risk factors for human cancers [138, 139]. Further study of Dbf2/LATS1 and Bub1 in their relationship to lipins could therefore add insight into the role of lipins in the cell cycle and development, and as part of pathways that are implicated in human cancers.      Lastly, Bck1 is a mitogen-activated protein kinase kinase kinase (MAPKKK) in yeast that is part of the protein kinase C signaling pathway. Upon activation by the yeast protein kinase C homologue Pkc1, Bck1 continues a phosphorylation cascade consisting of two redundant MAP kinase kinases (MAPKK), Mkk1 and Mkk2, and a MAP kinase, Mpk1 [140]. This signaling cascade plays a central role in the yeast cell wall integrity (CWI) pathway, which regulates cell wall remodeling during cell growth and division and in response to environmental stresses [140]. Intriguingly, it has been found that the cell wall integrity pathway is required for proper phospholipid homeostasis, with cells lacking Mpk1 exhibiting phenotypes including inositol auxotrophy and DAG and TAG accumulation [141]. Our finding that Bck1 has an SDL relationship with Pah1 further reinforces the role of the CWI pathway in lipid metabolism and may offer hints regarding the effectors that the pathway signals through to regulate lipid homeostasis.  67  Chapter 3: GSK3 controls lipin 1 subcellular localization and SREBP-target gene expression  3.1 Introduction In Chapter 2, we utilized a high throughput overexpression screen in budding yeast to uncover potentially novel kinase regulators of lipins, conserved phosphatidic acid phosphatase enzymes that convert phosphatidic acid (PA) to diacylglycerol (DAG) in the penultimate step of triglyceride synthesis and lipid storage. From the results of this screen, we discovered and characterized a role for Mck1, a yeast glycogen synthase 3 (GSK3) kinase, in phosphorylating the yeast lipin Pah1 to regulate cellular lipid homeostasis. This represents a novel role for GSK3-family kinases in phosphorylating a target that directly acts on lipids as an enzyme in the glycerol-3-phosphate pathway of triglyceride synthesis and is an established mediator of cellular lipid metabolism. In this current work, we describe studies to establish whether the relationship between GSK3 kinases and lipins is functionally conserved from yeast to mammals by using in vitro tissue culture systems and in vivo using mouse models.   Obesity is an increasingly prevalent global health concern and increases the risk of various co-morbidities including insulin resistance, type 2 diabetes, cardiovascular disease, and numerous cancers [1]. A hallmark of obesity is the dysregulation of steady-state intracellular lipid levels, which results in adverse downstream effects. The disruption of proper lipid homeostasis in obesity and other metabolic conditions thus necessitates an increased understanding of the molecular players and cellular pathways that govern cellular lipid 68  metabolism. Maintaining lipid homeostasis is critical to cellular function and survival, and cells possess numerous specialized enzymes capable of acting directly on lipids to direct appropriate lipid synthesis and balance. Working in conjunction with signaling inputs from other cellular pathways, these enzymes are key regulators in establishing and maintaining lipid homeostasis.   The lipin family of phosphatidic acid phosphatase (PAP) enzymes is conserved from yeast to mammals, and members of this family have emerged as key regulators of lipid homeostasis and are linked to metabolic diseases [26-28, 36, 61, 64]. Work in budding yeast first identified lipins as the elusive Mg2+-dependent PAP enzymes that convert phosphatidic acid (PA) to diacylglycerol (DAG) in the glycerol-3-phosphate triglyceride synthesis pathway [36] (Figure 1.1). Unlike other enzymes in this pathway, which are exclusively integral membrane proteins embedded in the endoplasmic reticulum (ER) membrane, lipins are cytosolic proteins that must be recruited to the ER to access their substrate. Through influencing the flux between the phospholipid precursor PA and the neutral lipid DAG, lipins are positioned at a crucial branchpoint between membrane biogenesis and lipid storage. Subsequent work in yeast, which possesses a single lipin ortholog, Pah1, has shown the critical role of lipins in mediating the balance between phospholipid synthesis and lipid storage. Cells lacking Pah1, for instance, demonstrate slow growth and temperature sensitivity, and have been found to have decreased TAG levels, fewer lipid droplets, and elevated PA and phospholipid levels resulting in the severe expansion of the nuclear/ER membrane [36, 37].    Mammals express three lipin orthologs: lipin 1, lipin 2, and lipin 3. All three of these enzymes share homology with yeast Pah1 and possess PAP activity when assayed in vitro [36, 69  46]. Of the three mammalian lipins, lipin 1 has received the most intensive characterization to date. Lipin 1 was initially discovered as the gene product that was deleteriously mutated in mice termed fatty liver dystrophy (fld) mice due to the fatty liver and elevated triglyceride levels they possessed in the neonatal stage [27]. These lipin 1-deficient fld mice also exhibited other severe lipid-related phenotypes including lipodystrophy manifesting as a stark lack of adipose tissue, impaired adipocyte differentiation, and insulin resistance [27]. Therefore, lipin 1 was immediately positioned as an important physiological regulator of lipid homeostasis. Further characterization of mammalian lipin 1 has shed light on its role not only as a PAP enzyme involved in triglyceride synthesis at the ER, but additionally as an enzyme that can translocate to the nucleus and influence the transcription of genes related to the adipogenic program and lipid synthesis by interacting directly with transcription factors including peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and its target PPARα in hepatocytes, and PPARγ in pre-adipocytes [14, 38, 49, 50].    Lipin function is tightly regulated in cells and this has been a major area of study in lipin biology. An overarching theme in lipin regulation is post-translational modification. For instance, sumoylation [51] and acetylation [68] of lipin 1 have been found to control lipin 1 localization and function. However, the major mechanism of lipin 1 regulation appears to be phosphorylation by kinases, and studies in both yeast and mammalian systems have contributed to uncovering the crucial role of phosphorylation in controlling lipin function and identifying cellular pathways that regulate lipid homeostasis via lipins. Both lipin 1 and Pah1 are highly phosphorylated proteins [36, 76] and regulation of lipins is intimately related to the fact that they are cytosolic proteins that need to be recruited to subcellular compartments to perform their 70  cellular functions. Studies in yeast have established that phosphorylation and dephosphorylation events can modulate the enzymatic activity, subcellular localization, and protein abundance of Pah1 [36, 69-75]. It is now appreciated that phosphorylation of Pah1 can inhibit its catalytic PAP activity and promote its cytosolic localization, while dephosphorylation by the conserved Nem1/Spo7 phosphatase complex (CTD-NEP1/NEP1-R1 in mammals) is required for Pah1 membrane association and PAP activity [69, 70]. Dephosphorylation of Pah1 is additionally known to promote its proteasome-mediated degradation, whereas phosphorylation can have a stabilizing effect on Pah1 abundance [73-75]. Kinases including the Pho80-Pho85 cyclin-dependent kinase, Cdc28-cyclin B, protein kinase A (PKA), protein kinase C (PKC), and casein kinase II (CKII) have been characterized to directly phosphorylate Pah1 to variable effect [69-72, 75].    Mammalian lipin 1, like yeast Pah1, has been found to be regulated by phosphorylation by kinases. However, the mechanisms by which phosphorylation modulates lipin 1 function appear to be different from Pah1 in appreciable ways. Most notably, in contrast to Pah1, phosphorylation has not been observed to alter the catalytic PAP activity of lipin 1 [14, 38]. Lipin 1 subcellular localization does, however, change based on its phosphorylation state, and this appears to be the major effect of phosphorylation on lipin 1. Insulin signaling, for instance, is known to cause multi-site phosphorylation of lipin 1 and promotes its localization to the cytoplasm instead of interacting with membranes [43, 76]. To date, the mechanistic target of rapamycin complex 1 (mTORC1) is the only kinase or kinase complex known to directly phosphorylate lipin 1 to regulate its intracellular localization [14]. A key nutrient- and growth factor-sensing complex, mTORC1 is a critical regulator of cellular metabolism and is known to 71  positively regulate lipid metabolism through the sterol regulatory element binding protein (SREBP) pathway [14]. SREBPs are transcription factors that are central to maintaining lipid homeostasis by controlling fatty acid and cholesterol biosynthesis and uptake [7, 10]. It is now known that mTORC1 mediates SREBP-target gene expression via regulating the subcellular localization of lipin 1. Specifically, mTORC1 phosphorylates lipin 1 to promote its cytosolic localization, whereas absence of mTORC1 phosphorylation results in lipin 1 translocation to the nucleus where it represses SREBP-target gene expression by a presently uncharacterized mechanism that requires lipin 1 PAP activity [14]. Thus, lipin 1 has been found to act antagonistically to mTORC1 function in regulating SREBP-mediated gene transcription.   In Chapter 2 of this thesis, we characterized the yeast GSK3 kinase Mck1 as a novel regulator of Pah1 that was capable of phosphorylating purified Pah1 in vitro. Cells lacking Mck1 additionally exhibited phenotypes consistent with decreased PA levels and increased neutral lipid levels, suggesting that Pah1 PAP activity was elevated in this background. This represented the first evidence of GSK3 kinases potentially regulating an enzyme directly involved in lipid synthesis and metabolism pathways. GSK3 kinases are serine/threonine dual-specificity kinases that are ubiquitously expressed in essentially all cells and tissues and are known to be central in critical cellular pathways and processes including cell growth and differentiation, development, and glucose metabolism [80, 81]. GSK3 has also been implicated in many diseases including insulin resistance and type 2 diabetes, mood disorders such as bipolar disorder, neurodegenerative diseases, inflammation, and cancer [80, 81, 98], and as such is seen as a potential pharmacological target [81, 98, 102].    72  To further establish a role for GSK3 kinases in regulating lipid homeostasis through the phosphorylation of lipins, we next looked to investigate the relationship between mammalian GSK3 and lipin 1, which will be discussed in this chapter. We found that lipin 1 contains multiple GSK3-consensus phosphorylation motifs, including one at S468 and S472 that includes a residue phosphorylated by mTORC1, and that pharmacological modulation of GSK3 and its upstream regulatory pathways affects the phosphorylation state of this lipin 1 site in a manner dependent on GSK3 activity. Our findings also suggest that mTORC1 provides the priming phosphorylation required at S472 of lipin 1 for GSK3 to phosphorylate S468. We then confirmed that purified GSK3β is capable of phosphorylating purified lipin 1 in vitro at S468. Furthermore, lipin 1 localization changes from cytosolic to nuclear upon conditions of GSK3 inhibition, suggesting that GSK3 phosphorylation controls lipin 1 subcellular localization and promotes its cytoplasmic localization. GSK3-mediated regulation of lipin 1 localization controls SREBP-target gene expression; in conditions of GSK3 inhibition where lipin 1 translocates to the nucleus, we observed a corresponding decrease in SREBP-target gene expression that was dependent on lipin 1. We characterized phosphorylation-deficient serine to alanine mutants at S468 and S472 of lipin 1 and found that each of these mutant forms of lipin 1 localized more to the nucleus and nuclear periphery compared to wild type lipin 1, and caused moderate decreases in SREBP-target gene expression when expressed in cells. Using proteomics approaches, we further found that S468 phosphorylation was likely dependent on S472 phosphorylation, consistent with S472 being a priming phosphorylation site for GSK3 to phosphorylate lipin 1.  Finally, having defined a link between GSK3 and lipid metabolism via lipin 1 and the SREBP pathway, we sought to perform a preliminary pilot study to test the role of GSK3 in 73  animal models of diet-induced obesity. As previous studies have demonstrated that pharmacological inhibition of GSK3 in mice alleviated effects of diet-induced obesity [142, 143] and that actions of mTORC1 regulation of lipin 1 in the liver had similar effects [14], we rationalized to challenge liver-specific GSK3α and GSK3β knockout mice on a high fat diet. We found that these mice were moderately resistant to the effects of diet-induced obesity including weight gain and the expression of the SREBP-target gene SCD1 compared to their control counterparts fed the same high fat diet. Further studies will be required to increase statistical power and to define whether our findings are indeed due to GSK3 regulation of SREBP via lipin 1, but our initial pilot study appears promising as a foundation in this route of inquiry.    It is important to note that some of the work described in this chapter are the work of Dr. Timothy Peterson. His specific contributions are indicated in figure legends and in the preface section of this thesis.   3.2 Materials and Methods Plasmids pRK5 FLAG wildtype, catalytic active lipin 1 was a gift from David Sabatini (Addgene plasmid #32005). pLKO-puro FLAG wildtype, catalytic active lipin 1 was a gift from David Sabatini (Addgene plasmid #32010). Both of the aforementioned lipin 1 expression plasmids were used to create Serine to Alanine phosphorylation-deficient forms of lipin 1 at Serine 468 and Serine 472, respectively.    74  Site Directed Mutagenesis Site directed mutagenesis of plasmid constructs was performed using the Q5 site-directed mutagenesis kit (E0554, New England Biolabs, Massachusetts, USA). Primers were designed using the NEBaseChanger software (www.nebasechanger.neb.com). Mutagenesis PCR reactions and subsequent kinase, ligase, and Dpn1 treatment steps were performed using manufacturer-recommended instructions. Final mutagenized plasmids were transformed into NEB Stable chemically-competent cells (C3040, New England Biolabs, Massachusetts, USA) and plated on selective plates as appropriate.   Tissue Culture Media All cell lines were cultured using standard protocols in DMEM media (#11965, ThermoFisher, Massachusetts, USA) containing 10% FBS except HAP1 cell lines, which were cultured in IMDM media (#12440, ThermoFisher, Massachusetts, USA) containing 10% FBS.   HAP1 Cell lines HAP1 parental and knockout cell lines for GSK3α, GSK3β, and lipin 1 were obtained from Horizon Genomics (United Kingdom). We confirmed these cell lines by western blot (Figure 3.8 and Figure 3.17)  Inhibitors Torin 1 was purchased from Tocris Biosciences (Bristol, United Kingdom). LY294002 was purchased from Sigma-Aldrich (Missouri, USA). PI-103 was purchased from Sigma-Aldrich 75  (Missouri, USA). GSK3 IX was purchased from Sigma-Aldrich (Missouri, USA). All inhibitors were dissolved in DMSO, aliquoted into single use aliquots, and stored at -20°C.   Transfection Transfections were performed on tissue culture cells using Lipofectamine 2000 Transfection Reagent (#11668019) (ThermoFisher, Massachusetts, USA) using manufacturer-recommended protocols for reagent and plasmid construct concentration per reaction. Cells were incubated with lipofectamine-DNA complex for 16 hours before fresh media free of transfection reagents was added.   Mouse lines  B6 congenic Gsk3a floxed (Gsk3afl/fl), Gsk3b floxed (Gsk3bfl/fl) and albumin-Cre (AlbCre) (from B6.Cg-Tg (Alb-Cre)21Mgn/J) mice were a kind gift from Dr. Jim Woodgett (University of Toronto, Canada) [144]. These mice were first backcrossed onto C57Bl/6J once and then intercrossed to obtain liver specific deletion of Gsk3a and/or Gsk3b alleles.  Tissue from the ear pinna was used for PCR genotyping (BRC Genotyping Facility (Vancouver, BC, Canada)).  In the results section, the abbreviations li-GSK3 and li-GSK3denote mice with genotype Gsk3afl/fl.AlbCre+ or Gsk3bfl/fl.AlbCre+,  respectively.  PCR Genotyping Primers: Gsk3a WT (650/525 bp) or floxed (650/1000 bp) amplicons FORWARD: 5’-CTT GAA CCT TTT GTC CTG AAG AAC C-3’ REVERSE: 5’-CCC CCA CCA AGT GAT TTC ACT GCT A-3’  76  Gsk3b WT (886 bp) or Floxed (1095 bp) amplicons FORWARD: 5’-GGG GCA ACC TTA ATT TCA TT-3’ REVERSE: 5’-GTG TCT GTA TAA CTG ACT TCC TGT GGC-3’  AlbCre (generic Cre) (700 bp) FORWARD: GTG CAA GTT GAA TAA CCG GAA ATG G REVERSE: AGA GTC ATC CTT AGC GCC GTA AAT CAAT  Mouse Diets Irradiated high fat diets for mice were purchased from BIO-SERV (distributed through VWR Cat#89067-471) (Flemington, NJ, USA) and (Catalog# D12492) Research Diets Inc. (New Brunswick NJ, USA). The full ingredients of the formulations are available from the suppliers. Both the BIO-SERV and Research Diets HFD formulations contained 60% of kilocalories from fat. Regular mouse diet (chow) consisted of a 50:50 mixture of irradiated Picolab Rodent Diet 20 5058 and Picolab Mouse Diet 20 5053 (Lab Diet, St. Louis, MO, USA).  Mouse HFD experiments and sample collection Animal experiments were conducted with approval of the UBC Animal Care Committee (A17-0138) and followed ethical guidelines of the Canadian Committee on Animal Care. Mice were weaned on to a regular chow diet until about 8 weeks old before transitioning to a high fat diet (HFD) for 10 weeks (or remaining on regular chow as a control). Mouse weight was monitored and recorded weekly for 10 weeks, and food consumption per cage per animal was also recorded for both chow and high fat diets. After 10 weeks, mice were sacrificed and processed as follows: Animals were individually photographed, and epidydimal fat pads collected and weighed. Livers were collected, imaged, and weighed. Livers and fat pads were frozen on dry ice and 77  subsequently stored at -80°C, stored in RNAlater RNA stabilization reagent (#76104, Qiagen, Germany), or fixed in 10% formalin for future analysis.  Cell/tissue lysis and protein sample preparation  Tissue culture cells  Cells were grown to the appropriate confluence, washed 2x with PBS, and scraped down into 50uL for 6-well dish or 500uL for a 10cm dish, of 1x RIPA lysis buffer (150mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 50mM Tris pH 7.5 in distilled water) containing cOmplete mini protease inhibitor cocktail (#4693159001, Sigma-Aldrich, Missouri, USA) and PhosStop phosphatase inhibitor cocktail (#4906845001, Sigma-Aldrich, Missouri, USA) added at manufacturer-recommended amounts. Collected cells were rotated for 30 mins at 4°C and centrifuged for 20 minutes at 13,200rpm at 4°C. Lysate was transferred to a new tube and protein concentration was determined using the Pierce™ BCA Protein Assay Kit (#23227, ThermoFisher, Massachusetts, USA). Protein samples were diluted to 1ug/uL working samples using 1xRIPA lysis buffer and 4x SDS sample buffer (16% glycerol, 3.2% SDS, 0.016% Bromophenol Blue, 8% 2-mercaptoethanol, 80mM Tris pH 6.8 in distilled water) and boiled at 95°C for 5 minutes.   Mouse livers Liver samples, previously stored at -80°C, were thawed on ice and subsequently submerged in a 1.5mL microcentrifuge tube containing 200-300uL 1X RIPA lysis buffer containing protease and phosphatase inhibitors as mentioned previously. Livers associated into lysis buffer by gently homogenizing using a VWR Pellet Mixer (#47747-370, VWR, Pennsylvania, USA) on ice. 78  Homogenized liver samples were then rotated for 30 mins at 4°C and centrifuged for 20 minutes at 13,200rpm at 4°C. Lysate was transferred to a new tube and protein concentration was determined using the Pierce™ BCA Protein Assay Kit (#23227, ThermoFisher, Massachusetts, USA). Protein samples were diluted to 1ug/uL working samples using 1xRIPA lysis buffer and 4x SDS sample buffer and boiled at 95°C for 5 minutes.   Western Blotting Samples were analyzed on polyacrylamide gels (6-8%) alongside PrecisionPlus protein standards (BioRad, California, USA). Transfer was performed on to PVDF membranes and subsequently blocked using 5% bovine serum albumin (BSA) in TBS-T (150mM NaCl, 50mM Tris pH 7.5, and 0.1% Tween 20 in distilled water). Primary antibodies were prepared in 5% BSA in TBS-T and membranes were incubated overnight at 4°C with rocking. Membranes were then washed 3 times with TBS-T and incubated with HRP-conjugated secondary antibodies prepared at 1:10000 dilutions in 5% BSA in TBS-T for 1 hour at room temperature. Membranes were then washed 3 times with TBS-T and 3 times with TBS (150mM NaCl and 50mM Tris pH 7.5). Blots were exposed on CL-Xposure X-ray exposure film (#34090) from ThermoFisher (Massachusetts, USA).   Antibodies Primary Antibodies Primary antibodies to lipin 1 (#5195), FLAG (#8146), GSK3α (#4337), GSK3β (#9832), phospho-S9-GSK3β, phospho-T308 Akt, phospho-S65-4EBP1, and 4EBP1 were obtained from Cell Signaling Technology (Massachusetts, USA). Antibodies to Actin (ab8226) and Beta 79  Tubulin (ab6046) were obtained from Abcam (Cambridge, United Kingdom). All primary antibodies were used at supplier-recommended dilutions and incubation times.   Secondary Antibodies Goat anti-rabbit IgG (H+L)-HRP Conjugate (#1721019) and Goat anti-mouse IgG (H+L)-HRP Conjugate (#1721011) secondary antibodies were obtained from Biorad (California, USA). All secondary antibodies were prepared at 1:10000 dilutions in 5% BSA in TBS-T.  Phosphorylation-specific Lipin 1 Antibodies  Phosphorylation-specific antibodies for lipin 1 S472-P and S468-P/S472-P were previously used in [14] and were provided by Cell Signaling Technology (Massachusetts, USA). No serial numbers were provided.    Immunoprecipitation Immunoprecipitations were performed for FLAG-lipin 1 expressed in HEK293T cells using the EZview ™ Red ANTI-FLAG ® M2 Affinity Gel (F2426) from Sigma-Aldrich (Missouri, USA). Beads were washed 2 times in 0.1M glycine, pH 3.5, and then 3 times with 0.25M Tris pH 7.4. Beads were then washed 1 time in IP lysis buffer 1 (150mM NaCl, 20mM Tris pH 8, 5mM EDTA pH 8, 0.1% SDS) and then blocked using IP lysis buffer 1 containing 0.1% BSA for 1 hour at 4°C. Lysate prepared as described previously was added to beads and 300ug of protein were used per immunoprecipitation. Lysate was incubated with beads overnight at 4°C. Beads were then washed 3 times with IP buffer 2 (150mM NaCl, 20mM Tris pH 8, 5mM EDTA pH 8, 0.1% SDS, 0.5% Triton X-100 in distilled water), 3 times with IP buffer 3 (500mM NaCl, 20mM 80  Tris pH 8, 0.5% Triton X-100, and 0.1% SDS in distilled water), and 1 time with IP buffer 4 (50mM Tris pH 8 in distilled water). Samples were then diluted in 1x sample buffer and boiled at 95°C for 5 minutes to elute the immunoprecipitated protein before analysis by western blot or mass spectrometry.   RNA extraction and qPCR RNA was extracted from tissue culture cells and mouse livers using the Qiagen RNeasy RNA extraction kit using manufacturer-recommended protocols (#74104, Qiagen, Germany). Extracted RNA concentration was measured and then working stocks were made for each sample to 20ng/uL using RNase- and DNase-free Ultrapure Distilled Water (#10977-015, ThermoFisher, Massachusetts, USA). qPCR was performed in 96-well format in technical triplet per sample using the Power SYBR Green RNA-to-CT 1-step kit from ThermoFisher (Massachusetts, USA) with all reagents and reactions made up using Ultrapure Distilled Water where appropriate. Quantification of relative abundance of transcripts was performed using the Applied Biosystems 7900HT and associated StepOne software (Applied Biosystems, California, USA). Target genes were measured to a GAPDH control on the same plate for each sample. Relative expression was determined by normalizing target genes to GAPDH expression and to a control treatment when necessary.   Primers for qPCR are as follows:  GAPDH (Mus musculus):  Forward: 5’-TCA CCA TCT TCC AGG AGG GA-3’ Reverse: 5’-GCA TTG CTG ACA ATC TTG AGT GAG-3’ 81   FASN (Mus musculus) Forward: 5’-CAG CAG AGT CTA CAG CTA CCT-3’ Reverse: 5’-ACC ACC AGA GAC CGT TAT GC-3’ HMGCR (Mus musculus) Forward: 5’-CTT GTG GAA TGC CTT GTG ATT G-3’ Reverse: 5’-AGC CGA AGC AGC ACA TGA T-3’ SCD1 (Mus musculus) Forward: 5’-ACG CCG ACC CTC ACA ATT C-3’ Reverse: 5’-CAG TTT TCC GCC CTT CTC TTT-3’  Immunofluorescence Assays  Immunofluorescence (IF) assays were performed on tissue culture cells plated on cover slips in 12-well dishes. Cells were seeded on to cover slips in appropriate media and allowed to grow to the appropriate confluence. Inhibitors and transfection reagents and plasmid constructs were applied as necessary. To fix cells on the cover slips, cells were washed 1 time with 1X PBS and then ~1mL of 4% paraformaldehyde was added to each well and cells were incubated in this solution for 20 minutes at room temperature. Subsequently, each cover slip was washed 2 times with 1X PBS and kept in 1X PBS until detergent extraction. Detergent extraction was performed using 0.1% Triton X-100 made up in 1X PBS, and cover slips were incubated in this solution for 10 minutes before washing 3 times with 1X PBS. Cover slips were then blocked in 10% normal goat serum (NGS) in 1% BSA in 1X PBS for 30 minutes at room temperature. Primary antibody diluted appropriately in 1% BSA in 1X PBS was then added to cover slips and incubated 82  overnight at 4°C while covered and in the dark. Cover slips were then washed 3 times with 1X PBS. Secondary antibodies diluted appropriately in 1% BSA in 1X PBS were applied to cover slips for 1 hour at room temperature. Cover slips were then washed 3 times with 1X PBS. For DAPI staining, when necessary, DAPI was added to a 1:10000 dilution in 1% BSA in 1X PBS and cover slips were incubated for 2 minutes at room temperature and subsequently washed 4 times in 1X PBS. Cover slips were then mounted on to microscopy slides using DABCO antifade (D2522) reagent (Sigma-Aldrich, Missouri, USA) and allowed to set overnight at 4°C. Cover slips were imaged using an Olympus FV1000 Confocal Microscope or a Leica SP5 confocal microscope, as described in the next section.   Microscopy Microscopy was performed on an Olympus FV1000 confocal microscope (Olympus, Pennsylvania, USA), except for FLAG-lipin 1 localization studies (Figure 3.15), which were performed on a Leica SP5 confocal microscope (Leica, Germany).   Lipin 1 Localization Assays Endogenous lipin 1 localization assays (Figure 3.10) were performed on an Olympus FV1000 confocal microscope (Olympus, Pennsylvania, USA). NIH 3T3 cells were treated with inhibitors as described in Figure 3.10 and immunofluorescence assays were performed as described previously in this section with DAPI staining for nuclei and lipin 1 labelling using lipin 1 antibody #5195 from Cell Signaling Technology (Massachusetts, USA). After acquiring images, CellProfiler software [120] was used to identify nuclei in an automated manner from DAPI staining. This DAPI-positive area was considered the nucleus for the purposes of this experiment 83  and used as a region of interest (ROI) to measure corresponding nuclear lipin 1 labelling for each image, thus allowing an unbiased quantification of nuclear lipin 1 labelling.  Mass Spectrometry In Figure 3.2, lipin 1 phosphorylation sites were identified by mass spectrometry of trypsin-digested FLAG-lipin 1 purified from HEK 293T cells overexpression FLAG-lipin 1. Label-free quantification of lipin 1 phosphorylation sites was done using BioWorks Rev3.3 software utilizing the methodology used previously in [145] to determine the area under the curve and relative abundance for the identified phosphopeptides.  In Figure 3.6, FLAG-lipin 1 wild type, S468A and S472A mutants were expressed in HEK 293T cells and immunoprecipitated as described previously in this section. Samples were then provided to the UBC proteomics core facility for phosphoproteomic mass spectrometric analysis. Immunoprecipitated samples were separated and analyzed by SDS-PAGE by the facility and routine in-gel digestion of protein bands by trypsin was also performed by the facility. Due to the high purity and quantity of our samples, no enrichment procedures for phosphopeptides were necessary or utilized in our analysis. Phosphoproteomic analysis was performed using a Bruker (Massachusetts, USA) impact II QTOF instrument. Peptides were analyzed using Byonic software (California, USA) against both mouse and human protein databases with phosphorylated S/T modifications specifically included in the search parameters. When analyzing phosphopeptides, we considered DeltaMod scores above 10.0 as a high likelihood of the correct placement of modifications for a given peptide, as defined and suggested by the 84  Byonic support materials, which can be found at https://www.proteinmetrics.com/support-information/.   Cell Sorting by FACS Cells to be sorted by FACS were lifted from plates by incubating with 0.05% trypsin at 37°C until no longer adhered, centrifuged for 5 minutes at 1200rpm, and resuspended in 0.5-10 mL pre-warmed PBS + 2% FBS and transferred to an appropriate FACS tube. Cells were sorted for GFP fluorescence using the BD FACSAria IIu (Becton-Dickinson, New Jersey, USA) into tubes containing pre-warmed DMEM media containing 10% FBS. The sorted GFP-positive cells were processed immediately after collection.  Histology Fixed liver tissue was placed in 20% sucrose prior to embedding in OCT and snap-frozen on dry ice. Sectioning and staining for triglyceride content with Oil Red O was performed by Wax-it Histology Services Inc (Vancouver, BC, Canada).  Data and Statistical Analysis Data is presented as mean ± SD or mean ± SEM where appropriate and as indicated in figure legends. Data was analyzed using unpaired student’s t-test for data comparing two groups, which comprised all statistical comparisons in this chapter. Statistical analysis was performed using Microsoft Excel or GraphPad Prism 6 software with statistical significance set at p<0.05.   85  3.3 Results The PI3K/Akt pathway and GSK3 regulate lipin 1 S468 an S472 phosphorylation  Lipin 1 contains a number of experimentally-determined phosphorylation sites, including multiple putative GSK3 consensus phosphorylation sites, which can be summarized as S/T-X-X-X-S/T where the first serine or threonine is the GSK3 target residue, X is any amino acid, and the last serine or threonine is a required priming residue that must be pre-phosphorylated by another kinase [81, 98] (Figure 3.1). One of these consensus sites is comprised of Serine 468 (S468) and Serine 472 (S472), and S472 is a residue phosphorylated directly by mTORC1 [14]. Phosphorylation of S472 by mTORC1 in conjunction with other experimentally-determined phosphorylation sites controls lipin 1 subcellular localization and cellular lipid homeostasis via regulation of SREBP-target gene expression [14]. The location of S472 relative to S468 in lipin 1 suggested that mTORC1 phosphorylation of S472 could provide the priming phosphorylation for GSK3 to phosphorylate lipin 1 at S468. In Chapter 2, we determined that in yeast the GSK3 homologue, Mck1, phosphorylates the yeast lipin 1 homologue, Pah1, directly in an in vitro kinase assay. As GSK3-family kinases share a conserved consensus phosphorylation motif, this further strengthened the rationale that GSK3 could phosphorylate lipin 1 in a mammalian context.    86    Figure 3.1: Lipin 1 possesses multiple GSK3-consensus phosphorylation sites Schematic depicting some experimentally determined lipin 1 phosphorylation sites labelled in black. GSK3 consensus phosphorylation motifs are indicated. Sites in grey (S349 and T718) represent residues in GSK3 consensus phosphorylation sites not yet confirmed by mass spectrometry.   87  To investigate this possibility, we examined the phosphorylation states of S468 and S472 in lipin 1 in response to pharmacological inhibition of mTORC1 and GSK3 in HEK 293T cells. Using mass spectrometry, we found that in vehicle treated cells S472 phosphorylation was present but S468/S472 dual phosphorylation was not detected (Figure 3.2). Addition of the mTORC1 inhibitor, Torin 1, abolished S472 phosphorylation as observed in previous studies [14]. However, treatment with the GSK3 inhibitor, GSK3 IX, resulted in no changes in S472 or S468/S472 phosphorylation state compared to the vehicle treatment. However, it is well-established that the PI3K/Akt pathway is upstream of and represses GSK3 activity in response to serum and insulin [146]. Therefore, we next tested whether inhibition of the PI3K/Akt pathway, and therefore activation of GSK3, could alter the phosphorylation of lipin 1 S468 and S472. Indeed, inhibition of PI3K/Akt pathway by the inhibitor PI-103 resulted in the dual phosphorylation of S468 and S472. This suggested that activated GSK3 could be providing the phosphorylation at S468, and to provide evidence for this we additionally treated cells concurrently with PI-103 and GSK3 IX (Figure 3.2). In this dual inhibitor condition, where both the PI3K/Akt pathway and GSK3 were inhibited, S472 phosphorylation remained but dual phosphorylation at S468/S472 was lost. This strongly suggested that GSK3 was responsible for phosphorylation of S468 in lipin 1.    88    Figure 3.2: Mass spectrometry reveals lipin 1 phosphorylation at S468/S472 is dependent on the PI3K/Akt pathway and GSK3 activity  Quantitative mass spectrometry to determine phosphorylation state of FLAG-lipin 1 immunoprecipitated from HEK 293T cells treated with 250 nM Torin, 1.4 μM IX, and/or 250 nM PI-103 for 1 hour. “-” indicates phosphorylated form is absent, “+” indicates phosphorylated form is present, while “++” indicates the phosphorylated form is more abundant than “+” as determined by measuring the ratio of the area under the curves for the phosphorylated peptide vs total peptide. This experiment was performed by Dr. Timothy Peterson during his time in the laboratory of David Sabatini (Massachusetts Institute of Technology).    89  To examine this role further, we raised phosphorylation-specific antibodies to the single phosphorylated S472 and the dual phosphorylated S468/S472 cassettes of lipin 1 and performed similar inhibitor treatments as before again using HEK 293T cells. Using the S468/S472 antibody, we observed similar dual phosphorylation of lipin 1 in the presence of PI-103, which was eliminated by inclusion of GSK3 IX (Figure 3.3). We confirmed that addition of PI-103 reduced the inhibitory S9 phosphorylation of GSK3ß, consistent with the increased activity of GSK3ß observed towards lipin 1. PI-103 addition eliminated the stimulatory T308 phosphorylation of Akt and reduced both the insulin-stimulated S106 phosphorylation of lipin 1, and S65 phosphorylation of 4EBP1 by mTORC1, consistent with inhibition of the PI3K/Akt pathway (Figure 3.3). We next generated phosphorylation-deficient serine to alanine mutants of FLAG-lipin 1 at S468 and S472, individually and in combination, to further validate our phosphorylation-specific antibodies and to assay the roles of these phosphorylated serine residues in the regulation of lipin 1 by GSK3 in future studies. Mutants were created using site directed mutagenesis of a lipin 1 expression construct used in previous studies [14]. First, we confirmed that these FLAG-lipin 1 mutants were properly expressed by western blot in transfected HEK 293T cells. We found that the FLAG-lipin 1 S468A, FLAG-lipin 1 S472A, and FLAG-lipin 1 S468AS472A mutants were all expressed at the same level as FLAG-lipin 1 wild type (Figure 3.4). However, the FLAG-lipin 1 S468AS472A migrated at a considerably lower molecular weight compared to wild type FLAG-lipin 1 and the S468A and S472A mutants, which migrated at the expected molecular weight of ~130 kDa, suggesting that this version of the protein exhibited some form of instability or degradation (Figure 3.4). We thus proceeded with using the individual FLAG-lipin 1 S468A and S472A mutants in subsequent studies. Using the S468A and S472A mutants, we validated that our phosphorylation-specific antibodies were 90  specific for S472 phosphorylation and S468/S472 dual phosphorylation, respectively (Figure 3.5a). Finally, we also verified that these antibodies could detected changes in S472 and S468/S472 phosphorylation upon treatment with Torin1 (Figure 3.5b and 3.5c).    91    Figure 3.3: Phosphorylation-specific antibody to lipin 1 S468/S472 reveals lipin 1 phosphorylation at S468/S472 is dependent on the PI3K/Akt pathway and GSK3 activity  HEK 293T cell lysates, from cells treated as in Figure 3.2, western blotted for levels and phosphorylation states of the specified proteins using the specified antibodies for lipin 1 phosphorylated at S468/S472, GSK3β phosphorylated at S9, Akt phosphorylated at T308, lipin 1 phosphorylated at S106, 4EBP1 phosphorylated at S65, and total 4EBP1. This experiment was performed by Dr. Timothy Peterson during his time in the laboratory of David Sabatini (Massachusetts Institute of Technology).   92    Figure 3.4: Expression of FLAG-lipin 1 S468A, S472A, and S468AS472A mutants in HEK 293T cells FLAG-lipin 1 wild type, S468A, S472A, and S468AS472A mutants expressed in HEK 293T cells. Cell lysates were probed with an anti-FLAG antibody in a western blot and reveals that wild type, S468A and S472A mutants migrate at the same molecular weight, while the S468AS472A mutant does not.    93   Figure 3.5: Validation of phosphorylation-specific antibodies using FLAG-lipin 1 S468A and S472A mutants expressed in HEK 293T cells A. FLAG-lipin 1 WT, S468A, and S472A expressed and isolated from HEK 293T cells. Cell lysates probed with lipin 1, lipin 1 S472-P and S468/S472-P phosphorylation-specific antibodies. The lipin 1 S472-P antibody fails to label the lipin 1 S472A mutant, while the lipin 1 S468/S472-P shows moderate labelling in the S468A mutant but fails to label the S472A mutant.  B. Endogenous lipin 1 and lipin 1 S472-P phosphorylation-specific antibody assayed from HEK 293T lysates from cells treated with DMSO or increasing concentrations of Torin 1. Increasing Torin 1 treatment causes a decrease in lipin 1 S472 phosphorylation, while total lipin 1 remains stable.  C. Immunoprecipitated FLAG-Lipin 1 wild type from HEK 293T cells treated with DMSO or Torin 1 probed for FLAG and lipin 1 S468/S472-P phosphorylation-specific antibody. Torin 1 treatment results in decreased dual phosphorylation at lipin 1 S468/S472.    94  Using phosphoproteomic approaches, we further investigated the potential dependency of lipin 1 S468 phosphorylation on S472 phosphorylation, and vice versa (Figure 3.6 and Appendix B Table 2 to Table 7). Here, we immunoprecipitated FLAG-lipin 1 wild type, S468A, and S472A expressed in HEK 293T cells treated with DMSO or PI-103 and analyzed the phosphorylation state of the lipin 1 peptide containing the S468/S472 cassette using mass spectrometry (Figure 3.6a). Peptides identified containing the S468/S472 cassette can be found in Appendix B Table 2 to Table 7. PI-103 treatment was used as a stimulating condition as we previously found that PI3K inhibition by this inhibitor resulted in dual lipin 1 S468/S472 phosphorylation (Figure 3.2). In FLAG-lipin 1 wild type from cells treated with DMSO, we found peptides unphosphorylated at both S468 and S472 and singly phosphorylated at S472, and no peptides phosphorylated at S468 alone or dually phosphorylated at S468/S472 (Figure 3.6b). FLAG-lipin 1 WT from cells treated with PI-103 corroborated our data from Figure 3.2, as we found dual phosphorylation at S468/S472 in this condition (Figure 3.6b), although only in a single instance. It is important to note that this single instance had relatively high confidence according to Byonic software (California, USA), with which we performed our analysis, and that our analysis did not specifically enrich for phosphopeptides which a future experiment could aim to do. FLAG-lipin 1 S468A from cells treated with DMSO or PI-103 still possessed multiple peptides where S472 was phosphorylated (Figure 3.6c), suggesting that S468 does not affect the ability for S472 to be phosphorylated even under conditions of PI3K pathway inhibition. Conversely, FLAG-lipin 1 S472A from cells treated with either DMSO or PI-103 demonstrated no instances of S468 phosphorylation, indicating that even when the PI3K pathway was inhibited, S468 was not phosphorylated when S472 was unable to be phosphorylated (Figure 3.6c). Overall, this data 95  further supports that S468 phosphorylation is dependent on PI3K pathway inhibition (Figure 3.6b) and S472 phosphorylation (Figure 3.6c).   96    Figure 3.6: Phosphoproteomic analysis of FLAG-lipin 1 WT, S468A, and S472A reveals that S468 phosphorylation is potentially dependent on S472 phosphorylation A. Peptide from trypsin digest of lipin 1 containing the S468/S472 cassette with residue numbers labelled accordingly.  B. Phosphorylation state of the lipin 1 S468/S472 cassette as indicated from FLAG-lipin 1 wild type (WT) expressed and immunoprecipitated from HEK 293T cells treated with either DMSO or 250nM PI-103 for 16 hours. Phosphorylation is indicated by (P) beside the indicated residue. “-” indicates that the phosphorylation state indicated was not found in any peptides in the 97  analysis, while a “+” indicates that the phosphorylation state indicated was found in multiple peptides above a DeltaMod threshold of 10.0 as suggested by Byonic software (California, USA). “+*” indicates the condition for which we observed only a single instance of the dually phosphorylated S468/S472 cassette, but that this instance was identified as a relatively high confidence hit as defined by Byonic software (DeltaMod score of above 10.0).  C. Phosphorylation state of the lipin 1 S468/S472 cassette as indicated from FLAG-lipin 1 S468A and S472 phosphorylation-deficient mutants expressed and immunoprecipitated from HEK 293T cells treated with either DMSO or 250nM PI-103 for 16 hours. Phosphorylation is indicated by (P) beside the indicated residue. “-” indicates that the phosphorylation state indicated was not found in any peptides in the analysis, while a “+” indicates that the phosphorylation state indicated was found in multiple peptides above a DeltaMod threshold of 10.0 as suggested by Byonic software (California, USA).   98  GSK3β directly phosphorylates lipin 1 in vitro Since our mass spectrometry analysis and phosphorylation-specific antibodies suggested that activated GSK3 phosphorylates lipin 1 at S468, we next tested whether GSK3 could phosphorylate lipin 1 in an in vitro kinase assay (Figure 3.7). To determine if GSK3 directly regulated lipin 1 by phosphorylation, we purified GSK3ß and performed in vitro kinase assays on purified murine lipin 1. Since both GSK3α and GSK3β share significant sequence homology, particularly in the kinase domain, we elected to test only GSK3β in our assay [81]. We detected robust GSK3ß-catalyzed S468/S472 lipin 1 phosphorylation only when GSK3ß was obtained from cells pretreated with Torin 1, and not vehicle; which was abolished by treatment with GSK3 IX in vitro (Figure 3.7a, lane 5). Torin 1 pre-treatment reduced S9 GSK3ß phosphorylation (Figure 3.7b) consistent with the increased GSK3ß activity observed toward lipin 1. GSK3ß kinase activity is known to be indirectly regulated by mTORC1 through S6K1-dependent regulation of GSK3ß S9 phosphorylation [147]. As a reflection of the specificity of our S468/S472 phosphorylation-specific antibody, mutating the S468/S472 sites to alanine (as part of a mutant where fifteen of the nineteen other lipin 1 phosphorylation sites, including S106, were mutated to alanine, hereafter referred to as "17xA-lipin 1" [14], abolished GSK3ß-dependent phosphorylation (Figure 3.7a lane 7). Additionally, GSK3ß-mediated phosphorylation of lipin 1 at S468 and S472 was significantly decreased by in-cell pretreatment of lipin 1 with Torin 1 (Figure 3.7 lane 6), which reduced both S106 and S472 phosphorylation (Figure 3.7a lane 6, and Figure 3.7c). This suggested that mTORC1-dependent lipin 1 phosphorylation, likely on S472, served as a priming event for its phosphorylation by GSK3ß on S468. In aggregate, these data supported that GSK3ß acted as a kinase for the S468/S472 phosphorylation cassette and were consistent with mTORC1 regulating GSK3ß activity toward lipin 1 by two 99  mechanisms: indirectly, by controlling inhibitory S9 phosphorylation of GSK3ß; and directly, by controlling the priming phosphorylation of lipin 1 on S472 for its subsequent phosphorylation by GSK3ß on S468.   100    Figure 3.7: In vitro kinase assay using phosphorylation-specific antibodies demonstrates that GSK3ß phosphorylates lipin 1 A. In vitro kinase assays for GSK3ß. myc-GSK3ß, myc-rap2a, FLAG-lipin 1, and FLAG-17xA-lipin 1 were purified from HEK-293T cells treated with vehicle, Torin1, and/or GSK3 IX as indicated for 1 hour. Anti-myc immunoprecipitates under the given conditions (kinase) were combined with anti-FLAG immunoprecipitates under the given conditions (substrate) and assayed for S468/S472 phosphorylation of lipin 1 by Western blotting (IP: myc in vitro kinase assay). This experiment was performed by Dr. Timothy Peterson during his time in the laboratory of David Sabatini (Massachusetts Institute of Technology). B. myc-GSK3β immunoprecipitates from A assayed for S9 phosphorylation with and without Torin1 treatment for 1 hour. This experiment was performed by Dr. Timothy Peterson during his time in the laboratory of David Sabatini (Massachusetts Institute of Technology). C. FLAG-lipin 1and FLAG-17xA-lipin 1 immunoprecipitates from A assayed for S106 phosphorylation with or without 250nM Torin 1 treatment for 1 hour. This experiment was performed by Dr. Timothy Peterson during his time in the laboratory of David Sabatini (Massachusetts Institute of Technology).  101  GSK3 controls lipin 1 subcellular localization and SREBP-dependent gene expression  Having established strong evidence that S468 of lipin 1 can be directly phosphorylated by GSK3β, we next looked to uncover what cellular function GSK3 phosphorylation of lipin 1 served. Lipin 1 is known to have its subcellular localization regulated by phosphorylation [14, 76], and thus we first investigated potential changes in lipin 1 localization under conditions of pharmacological inhibition of the PI3K/Akt pathway and GSK3 with the same inhibitors used to test lipin 1 phosphorylation in our previous experiments. Additionally, lipin 1 localization is a useful characteristic to study because dephosphorylation and recruitment of lipin 1 to subcellular compartments is required for its cellular functions in triglyceride synthesis and transcriptional regulation [14, 38, 49, 68, 76]. To validate our lipin 1 antibody, we first obtained commercially available HAP1 Lpin1 knockout cells generated by Cas9/CRISPR. We confirmed that these cells were lacking lipin 1 protein expression (Figure 3.8) and did not exhibit lipin 1 labelling in immunofluorescence assays (Figure 3.9) using our lipin 1 antibody. Subsequently, we moved on to testing NIH 3T3 cells with our slate of inhibitors to characterize lipin 1 localization and found that cells treated with our vehicle control demonstrated primarily cytoplasmic lipin 1 localization that was excluded from the nucleus (Figure 3.10a and 3.10b). Treatment with the mTORC1 inhibitor Torin 1 was used as a positive control and expectedly resulted in substantial translocation of lipin 1 to the nucleus (Figure 3.10a and 3.10b). Individual addition of the PI3K/Akt pathway inhibitor, PI-103, or GSK3 inhibitor, GSK3 IX, respectively, caused a modest nuclear relocalization of lipin 1. Dual inhibition of PI3K/Akt pathway and GSK3, however, resulted in an increased translocation of lipin 1 from the cytoplasm to the nucleus similar to Torin 1 treatment, indicating that PI3K and GSK3 activity is necessary to promote lipin 1 retention in the cytoplasm, perhaps in an additive manner (Figure 3.10a and 3.10b). In a separate 102  experiment, we assayed lipin 1 protein levels in NIH 3T3 cells treated by Torin 1, PI-103, GSK3 IX, and both PI-103 and GSK3 IX together by western blot and found that lipin 1 levels remain unchanged in all these conditions (Figure 3.11). This demonstrated that these inhibitors were not causing an increase in lipin 1 protein expression, but instead causing existing cytoplasmic lipin 1 pools to translocate to the nucleus, thus shifting the nuclear to cytoplasmic ratio of the enzyme.    Since dual pharmacological inhibition of the PI3K/Akt pathway and GSK3 causes similar lipin 1 translocation from the cytoplasm to the nucleus as inhibition of mTORC1 by Torin 1, we next investigated whether GSK3 was also regulating lipin 1 subcellular localization to ultimately affect lipid homeostasis though SREBP-target gene expression. Using mouse embryonic fibroblasts (MEFs) generated from wild type and fld mice missing the LPIN1 gene, we analyzed the expression of three SREBP-target genes – FASN, HMGCR, and SCD1 – in response to PI3K/Akt pathway and/or GSK3 inhibition with PI-103 and GSK3 IX, respectively, using RT-qPCR. We additionally confirmed the fld MEFs did not express Lpin1 mRNA (Figure 3.12). In wild type MEFs, we found that PI3K/Akt pathway inhibition alone by PI-103 generally did not result in a significant decrease in SREBP-target gene expression. However, additional inhibition of GSK3 by GSK3 IX in combination with PI-103 resulted in a sharp decrease in all three genes analyzed (Figure 3.12). Strikingly, when assaying the fld MEFs under the same conditions, the effects of dual inhibition by PI-103 and GSK IX on SREBP-target gene expression seen in wild type MEFs were ameliorated (Figure 3.12). This strongly suggested that lipin 1 was directly and solely responsible for the decrease in gene expression seen in the wild type fibroblasts, and in combination with our localization data, suggested that it was specifically nuclear localized lipin 1 that was repressing SREBP-target gene expression.  103   Figure 3.8: HAP1 Lpin1 KO cells lack lipin 1 protein expression HAP1 Lpin1 knockout cell lysates assayed for lipin 1 protein expression by western blot with an anti-lipin 1 antibody.   104    Figure 3.9: HAP1 Lpin1 KO cells lack lipin 1 staining in immunofluorescence assay HAP1 control and Lpin1 knockout cells labelled with anti-lipin 1 antibody (green) and DAPI (blue) in an immunofluorescence assay. HAP1 Lpin1 knockout cells lack lipin 1 staining present in the control cells (Scale bar = 20µm).     105   Figure 3.10: Lipin 1 translocates to the nucleus from the cytoplasm under conditions of dual PI3K/Akt pathway and GSK3 inhibition A. NIH 3T3 cells labelled in an immunofluorescence assay to determine the subcellular localization of lipin 1. Endogenous lipin 1 was labelled using an anti-lipin 1 antibody (green channel) and the nucleus was visualized with DAPI (blue channel). Cells were treated with vehicle, 250nM torin 1, 250nM PI-103, 1.4uM GSK IX, or a combination of 250nM PI-103 and 1.4uM GSK3 IX as indicated for 8 hours before fixation before staining (scale bar = 20µm).  106  B. Summary of nuclear lipin 1 localization. Relative lipin 1 nuclear localization was determined from confocal microscopy images using CellProfiler image analysis. CellProfiler was used to first identify nuclei in each image using DAPI-stained channel and this was deemed to represent the area of the nucleus. This area was subsequently used as the region of interest (ROI) for measuring lipin 1 integrated total fluorescence intensity in corresponding lipin 1-labelled channels, and this was measured for each nucleus identified and for each condition (n>43 for each condition; error bars, SEM; * indicates p<0.05)   107    Figure 3.11: Lipin 1 protein abundance remains unchanged upon inhibitor treatments Lipin 1 protein levels remain consistent in HEK 293FT cells upon treatment with DMSO, 250nM Torin 1, 250nM PI-103, 1.4µM GSK3 IX, and 250nM PI-103 + 1.4µM GSK3IX for 16 hours. Cell lysates were probed with an anti-lipin 1 antibody in a western blot.    108    Figure 3.12: Dual inhibition of the PI3K/Akt pathway and GSK3 causes a decrease in SREBP-target gene expression in a lipin 1-dependent manner  Relative mRNA expression of LPIN1 and the SREBP target genes FASN, SCD1, and HMGCR, by qPCR in wild type and fld MEFs. Addition of vehicle, 250nM PI-103 and 1.4μM GSK3 IX as indicated. (n=4; error bars, SEM; * indicates p < 0.02). This experiment was performed by Dr. Timothy Peterson during his time in the laboratory of David Sabatini (Massachusetts Institute of Technology).   109   We proceeded to investigate the robustness of this pathway by testing PI3K/Akt pathway inhibition and GSK3 inhibition using a different PI3K/Akt inhibitor and in cell types that we would use going forward. As such, we treated NIH 3T3 cells with an alternate PI3K/Akt pathway, LY294002, and dual inhibition in combination with GSK3 IX resulted in a substantial decrease in FASN expression compared to the vehicle control (Figure 3.13). Interestingly, treatment of LY294002 alone did not significantly alter FASN expression in these cells, but treatment with GSK3 IX alone resulted in a moderate decrease. This suggests that at least in some cell lines or conditions, GSK3 is phosphorylating and regulating lipin 1 independent of PI3K/Akt signaling. It is also possible that another upstream pathway we have not presently identified is contributing to this basal activation of GSK3. Next, because we were going to be using HEK 293T cells extensively in some of our future experiments, we ensured that the treatment with Torin 1 resulted in the expected robust repression of the SREBP pathway. Indeed, treatment with Torin 1 resulted in significant decreases in the expression of the SREBP target genes FASN, SCD1, and HMGCR (Figure 3.14).    To further investigate the direct function of S468 and S472 in the role of lipin 1 in regulating SREBP-target gene expression, we proceeded to express our FLAG-lipin 1 S468A and FLAG-lipin 1 S472A mutants in HEK 293T cells and investigate the localization of these lipin 1 mutants by immunofluorescence assay using an anti-FLAG antibody (Figure 3.15). We found that each of our wild type and mutant constructs localized to distinct localizations that we categorized as cytoplasmic (only in the cytoplasm, without distinguishable nuclear localization), cytoplasmic and nuclear (both distinguishable cytoplasmic and nuclear localization), nuclear (nuclear localization without other localization), and nuclear periphery (clearly localized to the 110  periphery of the nucleus) (Figure 3.15a). We observed that in comparison to wild type FLAG-lipin 1 expressed in these cells, both the lipin 1 S468A and S472A mutants demonstrated a clear enrichment of localization to the nuclear periphery (Figure 3.15b). Further, by comparing the relative total cytoplasmic localization (all categories that included any cytoplasmic localization) to nuclear localization (all categories that included nuclear or nuclear periphery localization), it appeared that both mutants generally localized to the nucleus more than FLAG-lipin 1 wild type (Figure 3.15c). This suggested that lipin 1 S468 and S472 are both important for regulating lipin 1 subcellular localization from the cytoplasm to the nucleus. Having established that the S468A and S472A mutants localized more to the nucleus than wild type lipin 1, we next measured the effect of expressing these mutants on SREBP-target gene expression by reverse transcriptase quantitative PCR (RT-qPCR). In these experiments, cells were transiently co-transfected with an n1-eGFP construct along with either FLAG-lipin 1 wild type, S468A, or S472A constructs. We also transfected cells with n1-eGFP alone as a control. Cells were subsequently sorted by FACS to select for GFP-positive cells with the aim of enriching for cells containing the FLAG-lipin 1 constructs and expressing wild type, S468A, or S472A lipin 1. Cell lysates and RNA was extracted from these FACS sorted cells for western blot and RT-qPCR analysis. Our western blot analysis confirmed that in our experiment each of the lipin 1 constructs expressed at the same level and migrated at the expected molecular weight (Figure 3.16a). From our RT-qPCR analysis of FASN expression, we observed that cells expressing n1-eGFP alone and wild type lipin 1 had the same level of FASN expression, while cells expressing either the lipin 1 S468A or S472A mutants exhibited a moderate decrease in FASN expression (Figure 3.16b). In this analysis, we defined the lowest possible FASN expression as that of HEK 293T cells treated with Torin 1 (Figure 3.14) and considered the decrease in FASN expression caused by overexpression of the 111  lipin 1 S468A and S472A mutants relative to this scale (Figure 3.16b). Taken together, our findings suggest that both S468 and S472 of lipin 1 play roles in controlling lipin 1 subcellular localization from the cytoplasm to the nucleus and SREBP-target gene expression.    112   Figure 3.13: NIH 3T3 cells treated with the inhibitors LY294002 and GSK3 IX demonstrate decreased FASN expression Relative mRNA expression of the SREBP target gene FASN by qPCR in NIH 3T3 cells treated with DMSO, 20µM LY294002 and 1.4μM GSK3 IX as indicated. FASN expression is normalized to GADPH expression and FASN expression for the indicated inhibitor treatments are relative to DMSO treatment (n=3; error bars, SEM; * indicates p < 0.05)   113   Figure 3.14: HEK 293T cells treated with Torin 1 demonstrate decreased SREBP-target gene expression by qPCR A. Relative mRNA expression of the SREBP target gene FASN from HEK 293T cells treated with DMSO or 250nM Torin 1 for 16 hours. FASN expression is normalized to GADPH expression and FASN expression for the Torin 1 treatment is relative to DMSO treatment (n=3; error bars, SEM; * indicates p<0.05). B. Relative mRNA expression of the SREBP target gene SCD1 from HEK 293T cells treated with 250nM Torin 1 for 16 hours. SCD1 expression is normalized to GADPH expression and SCD1 expression for the Torin 1 treatment is relative to DMSO treatment (n=3; error bars, SEM; * indicates p<0.05). C. Relative mRNA expression of the SREBP target gene HMGCR from HEK 293T cells treated with 250nM Torin 1 for 16 hours. HMGCR expression is normalized to GADPH expression and HMGCR expression for the Torin 1 treatment is relative to DMSO treatment (n=3; error bars, SEM; * indicates p<0.05).   114   115  Figure 3.15: FLAG-lipin 1 S468A and S472A mutants localize more to the nucleus and to the nuclear periphery compared to wild type FLAG-lipin 1  A. Representative images of FLAG-lipin 1 localization categories in HEK 293T cells with DAPI staining in blue representing nuclei and green representing anti-FLAG labelling of FLAG-lipin 1 wild type, S468A, or S472A (scale bar = 10µm). B. Proportion of cells in each localization category for cells expressing FLAG-lipin 1 WT, S468A, or S472A. Cytoplasm only (C) refers to localization observed only in the cytoplasm and without distinguishable nuclear or other localization, Cytoplasm and Nucleus (CN) describes localization that is distinguishable both in the nucleus as identified by corresponding DAPI staining and in the surrounding cytoplasm, Nucleus only refers to localization only in the nucleus as identified by corresponding DAPI staining, and Nuclear Periphery describes localization concentrated at the nuclear periphery as identified by corresponding DAPI staining.  C. Nuclear to cytoplasmic ratio of FLAG-lipin1 wild type, S468A, and S72A. Nuclear to cytoplasmic ratio determined by combining instances of localization from Figure 3.15B that contained cytoplasmic (C + CN) or nuclear (CN + N + NP) localization of FLAG-lipin 1 and normalizing total instances of nuclear localization to total instances of cytoplasmic localization.     116   117  Figure 3.16: Transient expression of phosphorylation-deficient lipin 1 mutants at S468 and S472 causes a decrease in FASN expression A. Western blot of FLAG-lipin 1 WT, S468A and S472A expressed in HEK 293T cells either transfected with n1-eGFP alone (lanes 1-3) or co-transfected with n1-eGFP and FLAG-lipin 1 wild type (WT), S468A, or S472A constructs, as indicated (lanes 4-12). GFP-positive cells were identified and sorted by FACS and some were used to prepare cell lysates that were subsequently analyzed for lipin 1 expression using an anti-lipin 1 antibody. The remainder of the sorted cells were used in Figure 3.16B for expression analysis.  B. Relative mRNA expression of the SREBP target gene FASN by qPCR from sorted HEK 293T cells transiently transfected with n1-eGFP, FLAG-lipin 1 wild type, FLAG-lipin 1 S468A, and FLAG-lipin 1 S472A from the same experiment as Figure 3.16A. Cells were either transfected with n1-eGFP alone or co-transfected with n1-eGFP and a lipin 1 construct, and GFP-positive cells were isolated by FACS. RNA was isolated from some of these GFP-positive cells and RT-qPCR was performed. Relative FASN expression was set relative to Torin 1 treated HEK 293T cells from Figure 3.14 because this was likely the lowest level of FASN expression possible in this pathway in HEK 293T cells. FASN expression is normalized to GADPH expression and FASN expression in cells expressing FLAG-lipin 1 WT, S468A, and S472A are relative to cells expressing n1-eGFP alone (n=3; error bars, SEM; * indicates p < 0.05).   118  Chemical-genetic interactions in GSK3α and GSK3β knockout HAP1 cells  We next sought to establish chemical-genetic interactions between the PI3K/Akt pathway inhibition by PI-103 and GSK3 by using commercially available GSK3α- and GSK3β-knockout cells that were generated by the Cas9/CRISPR system in a HAP1 background. By using cell lines individually knocked out for GSK3α or GSK3β, we rationalized that we could also specifically narrow down the contributions of GSK3α and GSK3β in regulating SREBP-target gene expression via lipin 1. It has been observed that some GSK3 pathways and functions are GSK3α- or GSK3β-dependent, while in others such as the Wnt signaling pathway, the paralogs are interchangeable [82-85]. In the case of GSK3 regulation of lipin 1, our data suggests that a full cellular complement of both GSK3α and GSK3β is required to mediate the effects of GSK3 on lipin 1 and SREBP-target gene expression. We first checked that our HAP1 GSK3α- and GSK3β-knockout cell lines indeed lacked expression of GSK3α and GSK3β, respectively (Figure 3.17a and 3.17b). Subsequently, HAP1 parental, HAP1 GSK3α knockout, and GSK3β knockout cells were treated with DMSO, Torin 1, or PI-103, and FASN expression was measured by RT-qPCR (Figure 3.18). Torin 1 treatment expectedly caused a sharp decrease in FASN expression in all the HAP1 cell lines, highlighting that HAP1 cells likely possessed similar signaling pathways as the other cell lines and MEFs we had tested previously. Significantly, the HAP1 GSK3α and GSK3β knockout cells exhibited a decrease in FASN expression when treated with PI-103 alone, but the HAP1 control cells did not (Figure 3.18), indicating that GSK3 is required to regulate the repressive effects of lipin 1 on SREBP-target gene expression. Additionally, this suggests that both GSK3α and GSK3β are involved in this pathway and are required together to regulate the effects of lipin 1 nuclear translocation and the SREBP pathway.  119  120   Figure 3.17: HAP1 GSK3α and GSK3β knockout cells lack GSK3α and GSK3β protein expression A. Western blot of HAP1 GSK3α knockout cell lysate using an anti-GSK3α antibody demonstrates that HAP1 GSK3α knockout cells lack protein expression of GSK3α, whereas HAP1 control cells do possess GSK3α protein expression. B. Western blot of HAP1 GSK3β knockout cell lysate using an anti-GSK3β antibody demonstrates that HAP1 GSK3β knockout cells lack protein expression of GSK3β, whereas HAP1 control cells do possess GSK3β protein expression.   121   Figure 3.18: HAP1 GSK3α and GSK3β knockout cells show sensitivity to PI-103 treatment in relation to FASN expression A. Relative mRNA expression of the SREBP target gene FASN by RT-qPCR in HAP1 control and GSK3ß knockout cells. Addition of vehicle, 250nM Torin 1, or 250nM PI-103 as indicated. FASN expression is normalized to GAPDH expression and FASN expression for inhibitor treatments are relative to DMSO treatment (n=3; error bars, SEM; * indicates p < 0.05). B. Relative mRNA expression of the SREBP target gene FASN by RT-qPCR in HAP1 control and GSK3α knockout cells. Addition of vehicle, 250nM Torin 1, or 250nM PI-103 as indicated. FASN expression is normalized to GAPDH expression and FASN expression for inhibitor treatments are relative to DMSO treatment (n=3; error bars, SEM; * indicates p < 0.05). 122  Liver-specific loss of GSK3 alleviates some effects of diet-induced obesity in mice  Both GSK3 and lipin 1 have been separately implicated in playing key roles in diet-induced obesity and/or insulin resistance by previous studies. GSK3 activity has long been known to be increased in insulin-responsive tissues in mice susceptible to obesity and diabetes [148], and in patients suffering from type 2 diabetes [96]. Our results suggest that increased GSK3 activity due to insulin resistance could result in increased phosphorylation and repression of lipin 1, an increase in SREBP-target gene expression, and elevated lipid synthesis. Furthermore, recent studies have found that whole-animal treatment with GSK3 inhibitors alleviates weight gain phenotypes in mice fed a high fat diet, and in one case treatment with an inhibitor ameliorated diet-induced hepatic steatosis [142, 143]. Increased lipin 1 activity in the liver resulting from genetic mTORC1 inhibition has also been found to be protective in regard to weight gain and hepatic steatosis in mice fed a high fat diet [14]. Importantly, the alleviation of these phenotypes appears to be largely dependent on lipin 1 and its function in repressing SREBP-target gene expression, as additional knockdown of lipin 1 reverses the protective effects and SREBP gene expression patterns [14]. We therefore looked next to investigate the physiological role of GSK3 regulation of lipin 1 specifically in the liver using mouse models.   First, we aimed to ensure that mice would gain weight predictably in our facilities when fed a high fat diet (HFD) in comparison to similar studies. We decided to test two different HFDs, one used in a previous study [142] from Research Diets (New Jersey, USA), and another from VWR (Pennsylvania, USA). C57Bl/6J mice were fed a chow diet until 8 weeks old and were subsequently switched to Research Diets HFD or VWR HFD for 10 weeks. A cohort of control mice were fed a chow diet throughout the course of the experiment. The weight of each 123  animal was recorded weekly to chart weight gain (Figure 3.19). We found that while both HFDs produced significant weight gain in the animals compared to their chow counterparts (Figure 3.19a and 3.19b), the Research Diets HFD produced a greater and more consistent overall weight gain and thus decided to use this HFD in our subsequent experiments. Mice on both HFDs developed consistent hepatic steatosis (Figure 3.20a and 3.20b), as expected with diet-induced obesity models. Finally, in investigating the expression of FASN in the livers of the mice fed the Research Diets HFD, we found that FASN expression was greatly increased in livers from these mice compared to livers of mice fed the chow diet (Figure 3.21), suggesting a dysregulated SREBP pathway caused by diet-induced obesity.    124   Figure 3.19: C57Bl/6J mice fed two different HFDs gain significantly more weight than mice fed a chow diet A. Wild type C57Bl/6J mice fed a VWR (HFD – VWR, left) or Research Diets HFD (HFD – RD, right) both gain significantly more weight than mice fed a chow diet (Regular Chow) over 11 weeks. Weight gain is charted as percent weight gain from starting weight (n as indicated on the figure itself; error bars, SD; * indicates p<0.05). B. Weight gain charted as percent weight gain from starting weight for wild type C57Bl/6J mice fed a chow (Regular Chow), VWR (HFD – VWR) or Research Diets HFD (HFD – RD) to compare between the three diets over 11 weeks (n as indicated on the figure itself; error bars, SD). 125     Figure 3.20: C57Bl/6J mice fed either of two high fat diets demonstrate qualitative signs of diet-induced obesity A. Representative images of wild type C57Bl/6J mice fed a chow diet (Chow) or a VWR (HFD – VWR) or Research Diets (HFD – RD) high fat diet. B. Representative images of the livers of wild type C57Bl/6J mice fed a chow diet (Chow) or a VWR (HFD – VWR) or Research Diets (HFD – RD) high fat diet. Livers from mice fed either HFD demonstrate hepatic steatosis.    126   Figure 3.21: C57Bl/6J mice fed one of the high fat diets tested possess increased FASN expression in the liver FASN expression measured by qPCR from RNA extracted from livers of wild type C57Bl/6J mice fed a chow diet (Chow) or Research Diets HFD (HFD – RD) indicates that FASN expression is increased in the liver in mice fed the Research Diets HFD. FASN expression was normalized to GAPDH expression and FASN expression for HFD - RD is relative to FASN expression on Chow (n=4 (Chow) and n=5 (HFD - RD); error bars, SEM; * indicates p<0.05).   127  We next obtained B6 congenic Gsk3a floxed (Gsk3afl/fl), Gsk3b floxed (Gsk3bfl/fl) and albumin-Cre (AlbCre) mice [144], to generate liver-specific GSK3α and GSK3β knockout mice for our next set of experiments. These mice were backcrossed to C57Bl/6J mice and intercrossed to obtain liver-specific deletion of Gsk3a or Gsk3b alleles. We set up an experiment consisting of our Gsk3afl/flAlbCre+ or Gsk3bfl/flAlbCre+ mice, referred to as li-GSK3α and li-GSK3β from here onward, which were homozygous knockouts for GSK3α or GSK3β in the liver, respectively. We had two cohorts each of control (AlbCre- mice floxed at either Gsk3a or Gsk3b), li-GSK3α, and li-GSK3β mice, and one cohort of each genotype was fed a chow diet and the other the Research Diets HFD (hereafter referred to as HFD). It is important to note that we treated this as a pilot study and additionally due to unforeseen circumstances, our cohort numbers were limiting for the appropriate statistical power. Again, we fed both cohorts of mice chow diets until 8 weeks old, and subsequently switched one cohort of mice to the HFD. We then monitored weight gain and food consumption per cage over the course of 10 weeks and subsequently collected blood, epididymal fat pad, and liver samples for further analysis. We found no difference in weekly food consumption per animal per cage (Figure 3.22) and that mice on the chow diet, regardless of genotype, gained the same amount of weight over the course of the experiment (Figure 3.23a and 3.23b), indicating that the li-GSK3α and li-GSK3β genotypes did not appear to alter the basal metabolism of these mice.    128   Figure 3.22: Control, li-GSK3α, and li-GSK3β mice consume the same weekly amount of chow or HFD per animal per cage over 10 weeks Weekly chow (Chow) or Research Diets HFD (HFD) consumption over 10 weeks measured for individual cages normalized to number of mice in each cage shows that weekly food consumption of either diet per animal per cage is not significantly different (n= 7 cages (chow) and n= 9 cages (HFD); error bars, SEM). Cages contained mixed genotypes of mice including control, li-GSK3α, and li-GSK3β. li-GSK3α, and li-GSK3β mice are Gsk3afl/fl.AlbCre+ or Gsk3bfl/fl.AlbCre+,  respectively. Control mice are AlbCre- and possess floxed Gsk3a or Gsk3b.  129   Figure 3.23: Control, li-GSK3α, and li-GSK3β mice percent weight gain from starting weight over 10 weeks on a chow diet A. Weight gain charted as percent from starting weight weekly over 10 weeks in control, li-GSK3α, and li-GSK3β mice fed a chow diet (n as indicated in figure legend). li-GSK3α, and li-GSK3β mice are Gsk3afl/fl.AlbCre+ or Gsk3bfl/fl.AlbCre+,  respectively. Control mice are AlbCre- and possess floxed Gsk3a or Gsk3b.  130  B. Weight gain represented as percent from starting weight at experimental end point at week 10. Final percent weight gain from starting weight is charted for control, li-GSK3α, and li-GSK3β mice fed a chow diet. Statistical comparisons are between control and li-GSK3α or li-GSK3β, respectively, as indicated.   131  Mice on the HFD, when compared to their corresponding counterparts fed the chow diet, each demonstrated substantial weight gain as expected (Figure 3.24a and 3.24b) and developed hepatic steatosis (Figure 3.26 and 3.27), except for a single li-GSK3β animal which appeared to have a normal liver (Figure 3.27, bottom right panel). This animal that did not exhibit signs of hepatic steatosis was also the li-GSK3β mouse that gained the least weight on the HFD over the course of the experiment (Figure 3.24b). When comparing between the genotypes fed the HFD, we found that the li-GSK3α and li-GSK3β mice ultimately gained roughly 20% less weight than the control mice on average, suggesting a potential resistance to weight gain on the HFD (Figure 3.24a and Figure 3.25). Looking at the final weights of these mice fed the HFD, we found statistical significance between control and li-GSK3α mice, but not between control and li-GSK3β mice, likely due to our limited number of control mice fed the HFD (Figure 3.25). Additionally, we noticed that the final weights of li-GSK3β mice possessed greater variability than in control and li-GSK3α (Figure 3.25). Liver and epidydimal fat pad weights matched the trend seen in total weight, and the relationships between control, li-GSK3α, and li-GSK3β mice fed the HFD were significantly different when compared to their chow diet counterparts, but not when compared to each other (Figure 3.28a, 3.28b, and 3.28c, and Figure 3.29a, 3.29b, and 3.29c.    132   Figure 3.24: Control, li-GSK3α, and li-GSK3β mice percent weight gain over 10 weeks on a HFD compared to a chow diet A. Weight gain charted as percent from starting weight weekly over 10 weeks in control, li-GSK3α, and li-GSK3β mice fed a HFD (n as indicated in the figure itself). li-GSK3α, and li-GSK3β mice are Gsk3afl/fl.AlbCre+ or Gsk3bfl/fl.AlbCre+,  respectively. Control mice are AlbCre- and possess floxed Gsk3a or Gsk3b. 133  B. Final percent weight gain at Week 10 charted for control, li-GSK3α, and li-GSK3β mice fed a chow versus high fat diet. Statistical comparisons are between Chow and HFD for each genotype indicated (n as indicated on the figure itself; error bars, SEM; * indicates p<0.05).    134   Figure 3.25: Control, li-GSK3α, and li-GSK3β mice final percent weight gain on a HFD after 10 weeks Weight gain at Week 10 represented as percent from starting weight at experimental end point (Week 10). Final percent weight gains charted for control, li-GSK3α, and li-GSK3β mice fed a HFD for 10 weeks (n as indicated; error bars, SEM, * indicates p<0.05 for li-GSK3α and li-GSK3β compared to control, respectively). li-GSK3α, and li-GSK3β mice are Gsk3afl/fl.AlbCre+ or Gsk3bfl/fl.AlbCre+,  respectively. Control mice are AlbCre- and possess floxed Gsk3a or Gsk3b.  135    Figure 3.26: Representative images of control, li-GSK3α, and li-GSK3β mice fed a chow diet versus a high fat diet demonstrate qualitative signs of diet-induced obesity Representative images of control, li-GSK3α, and li-GSK3β mice fed a chow or HFD. li-GSK3α, and li-GSK3β mice are Gsk3afl/fl.AlbCre+ or Gsk3bfl/fl.AlbCre+,  respectively. Control mice are AlbCre- and possess floxed Gsk3a or Gsk3b.  136    Figure 3.27: Livers from control, li-GSK3α, and li-GSK3β mice fed a chow or high fat diet demonstrate hepatic steatosis Representative images of livers from control, li-GSK3α, and li-GSK3β mice fed a chow or HFD. Livers collected from mice fed the HFD demonstrate clear signs of hepatic steatosis in most cases except for one liver found in the li-GSK3β background (bottom right panel). li-GSK3α, and li-GSK3β mice are Gsk3afl/fl.AlbCre+ or Gsk3bfl/fl.AlbCre+,  respectively. Control mice are AlbCre- and possess floxed Gsk3a or Gsk3b. 137   Figure 3.28: Liver weights from control, li-GSK3α, and li-GSK3β mice fed a chow or high fat diet A. Liver mass charted for livers collected from control, li-GSK3α, and li-GSK3β mice fed a chow diet or HFD. li-GSK3α, and li-GSK3β mice are Gsk3afl/fl.AlbCre+ or Gsk3bfl/fl.AlbCre+,  respectively. Control mice are AlbCre- and possess floxed Gsk3a or Gsk3b. When comparing within genotypes, liver weights are significantly different between chow and HFD (n as indicated; error bars, SEM; * indicates p<0.05).  138  B. Liver mass charted for livers collected from control, li-GSK3α, and li-GSK3β mice fed a chow diet (n as indicated; error bars, SEM; * indicates p<0.05 comparing to Control HFD).  C. Liver mass charted for livers collected from control, li-GSK3α, and li-GSK3β mice fed a HFD (n as indicated; error bars, SEM; * indicates p<0.05 comparing to Control HFD). 139   Figure 3.29: Epididymal fat pad weights from control, li-GSK3α, and li-GSK3β mice fed a chow or high fat diet A. Epididymal fat pad mass charted for fat pads collected from control, li-GSK3α, and li-GSK3β mice fed a chow or HFD. li-GSK3α, and li-GSK3β mice are Gsk3afl/fl.AlbCre+ or Gsk3bfl/fl.AlbCre+,  respectively. Control mice are AlbCre- and possess floxed Gsk3a or Gsk3b. When comparing within genotypes, fat pad weights are significantly different between chow and HFD for control and li-GSK3β (n as indicated; error bars, SEM; * indicates p<0.05).  140  B. Epididymal fat pad mass charted for fat pads collected from control, li-GSK3α, and li-GSK3β mice fed a chow diet (n as indicated; error bars, SEM; * indicates p<0.05 comparing to WT HFD).  C. Epididymal fat pad mass charted for fat pads collected from control, li-GSK3α, and li-GSK3β mice fed a HFD (n as indicated; error bars, SEM; * indicates p<0.05 comparing to WT HFD).   141  We then performed histological analysis of a subset of liver sections collected from the control, li-GSK3α, and li-GSK3β mice on both diets, and confirmed the accumulation triglycerides in the liver sections of the mice fed the HFD by staining with Oil Red O, a fat-soluble dye that stains triglycerides (Figure 3.30a, 3.30b, and 3.30c). This analysis corresponded to the hepatic steatosis observed in the livers of mice fed the HFD, as livers sections from control, li-GSK3α, and li-GSK3β mice fed a chow diet each demonstrated minimal staining by Oil Red O, while the HFD counterparts largely exhibited intense staining by the dye (Figure 3.30, 3.30b, and 3.30c). From the sections we analyzed, the li-GSK3α and li-GSK3β liver sections from mice fed the HFD demonstrated more variability in triglyceride staining than control liver sections on the same diet, with some sections showing regions with less intense staining. Unsurprisingly the liver from the li-GSK3β animal that did not exhibit hepatic steatosis in Figure 3.27 (bottom right panel) also showed much less intense staining by Oil Red O (Figure 3.30c, bottom right panel). We also observed regions with weaker Oil Red O staining in some li-GSK3α liver sections, even though all the livers from these mice showed hepatic steatosis at a macroscopic level (Figure 3.30b, right column). Analyzing more of the livers collected from this experiment and increasing the number of control mice on a HFD in future experiments would be helpful in assessing whether these potential differences in Oil Red O staining, and thus triglyceride content, are likely a consequence of liver-specific loss of GSK3α or GSK3β.   Next, we isolated RNA from livers of a subset of the mice from each genotype to investigate potential changes in SREBP-target gene expression (Figure 3.31a, 3.31b, and 3.31c). Here, we found variable effects of liver-specific loss of either GSK3α or GSK3β, depending on target gene. Expression of FASN, for instance, was consistent with what we observed in our pilot 142  study when comparing control mice on chow versus the HFD (Figure 3.21), but in both li-GSK3α and li-GSK3β backgrounds FASN expression was unexpectedly elevated on chow and further increased on the HFD (Figure 3.31a). Conversely, expression of SCD1 was substantially increased in control mice fed the HFD but this effect was largely ameliorated in both li-GSK3α and li-GSK3β backgrounds fed the HFD (Figure 3.31b). Finally, expression of HMGCR remained relatively unchanged in all conditions (Figure 3.31c). Importantly, in both li-GSK3α and li-GSK3β backgrounds, the change in SREBP-target gene transcription of the three genes tested was consistent in effect and magnitude.    143   144    145   146  Figure 3.30: Histological analysis of liver sections of control, li-GSK3α, and li-GSK3β mice fed a chow or high fat diet  A. Liver sections of livers collected from control mice (AlbCre- and possess floxed Gsk3a or Gsk3b) fed either a chow diet (Chow) or high fat diet (HFD) stained with Oil Red O, a fat-soluble dye that stains neutral triglycerides (images were obtained at a magnification of 20X, scale bar = 100µM). B. Liver sections of livers collected from li-GSK3α mice (Gsk3afl/fl.AlbCre+) fed either a chow diet (Chow) or high fat diet (HFD) stained with Oil Red O, a fat-soluble dye that stains neutral triglycerides (images were obtained at a magnification of 20X, scale bar = 100µM). C. Liver sections of livers collected from li-GSK3β mice (Gsk3bfl/fl.AlbCre+) fed either a chow diet (Chow) or high fat diet (HFD) stained with Oil Red O, a fat-soluble dye that stains neutral triglycerides (images were obtained at a magnification of 20X, scale bar = 100µM).  147    Figure 3.31: SREBP-target gene expression measured from livers of control, li-GSK3α, and li-GSK3β mice fed a chow or high fat diet A. FASN expression measured by qPCR from RNA extracted from livers of control, li-GSK3α, and li-GSK3β mice fed a chow (Chow, black bars) or high fat diet (HFD, grey bars). li-GSK3α, and li-GSK3β mice are Gsk3afl/fl.AlbCre+ or Gsk3bfl/fl.AlbCre+,  respectively. Control mice are AlbCre- and possess floxed Gsk3a or Gsk3b. FASN expression was normalized to GAPDH expression and expression of FASN on HFD is relative to expression on Chow (n=2 for Control HFD, n=3 for all other conditions; error bars, SEM; * indicates p<0.05). 148  B. SCD1 expression measured by qPCR from RNA extracted from livers of control, li-GSK3α, and li-GSK3β mice fed a chow (Chow, black bars) or high fat diet (HFD, grey bars). li-GSK3α, and li-GSK3β mice are Gsk3afl/fl.AlbCre+ or Gsk3bfl/fl.AlbCre+,  respectively. Control mice are AlbCre- and possess floxed Gsk3a or Gsk3b. SCD1 expression was normalized to GAPDH expression and expression of SCD1 on HFD is relative to expression on Chow (n=2 for Control HFD, n=3 for all other conditions; error bars, SEM; * indicates p<0.05). C. HMGCR expression measured by qPCR from RNA extracted from livers of control, li-GSK3α, and li-GSK3β mice fed a chow (Chow, black bars) or high fat diet (HFD, grey bars). li-GSK3α, and li-GSK3β mice are Gsk3afl/fl.AlbCre+ or Gsk3bfl/fl.AlbCre+,  respectively. Control mice are AlbCre- and possess floxed Gsk3a or Gsk3b. HMGCR expression was normalized to GAPDH expression and expression of HMGCR on HFD is relative to expression on Chow (n=2 for Control HFD, n=3 for all other conditions; error bars, SEM; * indicates p<0.05).   149  Although some of our findings presented from our work in in vivo mouse models are not statistically significant, likely due to our limited sample size, these results represent compelling evidence to design future experiments to further investigate the role of GSK3 in the liver in the development of diet-induced obesity. In particular, the fact that SCD1 expression decreases in the livers of both li-GSK3α and li-GSK3β mice on the HFD compared to control mice on the same diet is intriguing because SCD1 is a SREBP-target gene known to play important roles in lipid storage and the onset of diet-induced obesity [149, 150], which will be discussed in the next section. Presently, we are analyzing the plasma and livers we collected from this experiment to measure both circulating and hepatic levels of cholesterol and triglyceride content, which we believe will further refine our results in regard to the role of GSK3 in the liver in the physiological consequences of diet-induced obesity.   3.4 Discussion Following our work in Chapter 2, we have now defined a role for GSK3 kinases in regulating lipins through direct phosphorylation that appears to be conserved from yeast to mammals. Our work in characterizing the role of GSK3 phosphorylation in mediating lipin 1 subcellular localization and SREBP-target gene expression adds to the already expansive repertoire of cellular pathways and processes that GSK3 kinases are known to be involved in. This represents the first time GSK3 has been implicated in directly controlling lipid synthesis and metabolism via a lipid metabolic enzyme such as lipin 1. Our work also identifies the PI3K/Akt pathway and mTORC1 pathways as playing key upstream roles in mediating GSK3 phosphorylation of lipin 1. Crosstalk between these pathways, GSK3, and lipin 1 may therefore represent an important mechanism to integrate insulin signaling and nutrient and growth factor 150  availability to lipid synthesis via the SREBP pathway. Finally, our work further highlights the critical nature of regulating lipin 1 subcellular localization through phosphorylation to control cellular lipin 1 function.    Given the ubiquitous nature of GSK3 in cells and tissues, and its known involvement in a multitude of cellular processes and functions, it is imperative to thoroughly separate true targets of GSK3 phosphorylation from potential false positives [92]. This, in turn, will facilitate a more refined understanding of the specific role GSK3 plays in the cell and allow for better characterization of GSK3 as a potential therapeutic target. Criteria for defining a validated GSK3 phosphorylation substrate were initially proposed by Frame and Cohen and were reviewed recently by Sutherland in 2011 [92]. These criteria include the need for a validated substrate to contain a GSK3 consensus phosphorylation site that is experimentally-determined to be phosphorylated, the ability for purified GSK3 to phosphorylate the target serine or threonine residue(s) on the putative substrate, identification of the priming residue(s), and that modulation of GSK3 activity causes both a change in phosphorylation at the GSK3 target residue(s) and a physiological effect [92]. In accordance with these criteria, we believe we have reasonably established that lipin 1 is a bona fide substrate of GSK3 phosphorylation. We identified that lipin 1 S468, which is part of the S468/S472 GSK3-consensus phosphorylation motif, is capable of being phosphorylated by mass spectrometry, using phosphorylation-specific antibodies specific for dual S468/S472 lipin 1 phosphorylation, and by in vitro kinase assays involving purified GSK3 and purified lipin 1. We additionally modulated GSK3 activity pharmacologically using the inhibitor PI3K inhibitor PI-103 and the GSK3 inhibitor GSK3 IX, individually and in combination, to activate and inhibit GSK3, respectively. As a result, we found that these 151  treatments corresponded well to GSK3-mediated phosphorylation of S468; that is, PI-103 inhibited the PI3K/Akt pathway to activate GSK3 and this caused dual phosphorylation at S468/S472, while inhibition of both PI3K/Akt and GSK3 abolished the phosphorylation at S468 but not S472, the putative priming site. In seeking a physiological role for GSK3 phosphorylation of lipin 1, we discovered – once again using pharmacological inhibition with PI-103 and GSK3 IX – that GSK3 mediates the subcellular localization of lipin 1, which in turn regulates SREBP-target gene expression. GSK3 phosphorylation promotes lipin 1 cytoplasmic localization, while conditions where GSK3 activity is inhibited results in lipin 1 translocation to the nucleus and a repression in the expression of SREBP downstream targets. Finally, when mutating either the target site or priming site from serine residues to alanine residues, we noticed both a change in localization from the cytoplasm to the nucleus and a decrease in SREBP-target gene expression when these phosphorylation-deficient forms of lipin 1 were expressed in cells.    Additional studies could be pursued into further defining GSK3 phosphorylation sites in lipin 1 that may exist aside from the site characterized at S468/S472 in this study. Although our findings suggest that that S468 is a GSK3-mediated lipin 1 phosphorylation site that contributes to regulating the ability of lipin 1 to translocate from the cytoplasm to the nucleus, we cannot exclude the possibility that other GSK3 sites exist. Firstly, several other GSK3 consensus sites exist that span the length of the lipin 1 protein sequence, many of which include experimentally determined phosphorylation sites (Figure 3.1). Furthermore, some of our studies were performed utilizing pharmacological inhibition of GSK3 in cells that would likely abolish all GSK3-mediated phosphorylation of lipin 1. This would be especially important to consider regarding our lipin 1 localization studies because the inhibitors could be affecting the phosphorylation of 152  multiple GSK3 phosphorylation sites including S468/S472 that result in the changes in lipin 1 localization we observed. Potentially, additional GSK3 lipin 1 phosphorylation sites could explain the moderate repression in SREBP we observed when expressing phosphorylation-deficient S468A and S472A forms of lipin 1 in cells. It has been found in other studies, notably, that multiple phosphorylations are required for full lipin 1 repression, with one such study requiring mutations of 17 phosphorylated residues, including S472, to observe full lipin 1 translocation to the nucleus from the cytoplasm [14]. Thus, it appears that lipin 1 phosphorylation may be multifactorial and cooperative in nature. Another possible explanation for our phosphorylation-deficient mutants causing only moderate repression of SREBP is compensation by lipin 2 or lipin 3. Lipin 1 and lipin 2 have been observed to be able to compensate each other in certain contexts [48], but it is unknown at present whether lipin 2 can function redundantly to lipin 1 in regulating transcription in the nucleus and the whether the mechanisms that lipin 2 are conserved from lipin 1. Additionally, it is known that mice lacking both lipin 1 and lipin 2 are not viable, suggesting lipin 3 cannot compensate for total lipin function [48]. Further research characterizing the regulation and function of lipin 2 and lipin 3 will therefore be required in this line of investigation.   It remains unknown how lipin 1 represses SREBP-target gene expression in the nucleus, and further studies will be required to characterize this mechanism. Previous reports have noted that lipin 1 PAP activity is a variable requirement for its effects on transcription in the nucleus, suggesting different mechanisms exist. Catalytic activity is not required, for instance, for lipin 1 to mediate its effects on the PGC-1α pathway in hepatocytes or PPARγ in adipocytes [38, 49], and it has been proposed that lipin 1 may act as a molecular scaffold for transcription factors in 153  these pathways [52]. Significantly, PAP activity has been observed to be necessary for the effect of lipin 1 in repressing SREBP-target gene expression [14]. Even a phosphorylation-deficient form of lipin 1 that translocates fully to the nucleus but did not possess catalytic activity was found to be unable to repress SREBP downstream targets [14]. Why the PAP activity of lipin 1 is required in its repressive functions on SREBP will therefore require more study. It has been noted in one previous study that lipin 1 nuclear localization caused nuclear remodeling resulting in elongated nuclei. Additionally, nuclear lipin 1 causes a decrease in SREBP protein levels, but also results in the remaining SREBP to localize to the nuclear periphery and to colocalize to nuclear lamins [14]. Therefore, it is possible that lipin 1 affects SREBP target-gene transcription through its effects on lamins and nuclear morphology. Whether lipin 1 directly interacts with SREBP proteins has not yet been characterized and would be of great utility in delineating this aspect of lipin 1 repression of SREBP-target gene expression.    This is the second report of kinases or kinase complexes, preceded by mTORC1 [14], directly phosphorylating lipin 1 to regulate its subcellular localization to control lipid metabolism via the expression of SREBP target genes. In identifying GSK3 as a kinase that regulates lipin 1 in a manner that requires priming by mTORC1 and is downstream of the well-characterized PI3K/Akt pathway, it is intriguing to consider the implications of signal integration between both pathways to mediate lipid homeostasis through lipin 1. Since mTORC1 individually imparts phosphorylation at multiple lipin 1 sites including S472 [14], it is possible that the function of mTORC1 phosphorylation of S472 is to prime GSK3 as a means to provide a graded response to signals from mTORC1 or PI3K/Akt signaling. The PI3K/Akt pathway is known to be downstream of insulin signaling via the insulin receptor [151], while mTORC1 is a key nutrient 154  and growth factor sensing kinase [152]. There is already known crosstalk between these pathways, and in our experiments, we found that inhibition of the PI3K/Akt pathway resulted in loss of lipin 1 phosphorylation at the mTORC1 phosphorylation site S106, but not S472. This suggests that a portion of mTORC1 activity towards lipin 1 is mediated by the PI3K/Akt pathway and lends support to the idea that in a condition where insulin signaling is not active, but growth factors and/or nutrients are present, GSK3 could then phosphorylate lipin 1 due to the remaining S472 priming by mTORC1. This would then result in lipin 1 cytoplasmic localization, promote SREBP-target gene expression, and provide a graded response to extracellular signals by the PI3K/Akt and mTORC1 towards lipid synthesis (Figure 3.32).   155    Figure 3.32: GSK3 directly phosphorylates lipin 1 to control its subcellular localization and SREBP-target gene expression GSK3 directly phosphorylates lipin 1 at S468 when S472 is primed by mTORC1, to regulate lipin 1 subcellular localization and to promote SREBP-target gene expression. Under conditions when the PI3K/Akt pathway is active (left), GSK3 is inhibited and mTORC1 phosphorylates lipin 1 at multiple sites including serine 106 (S106) and serine 472 (S472), which results in lipin 1 localizing to the cytoplasm and maintains SREBP activity in the nucleus. Upon PI3K/Akt pathway inhibition (center), GSK3 is active and mTORC1 still provides phosphorylation at S472, which primes lipin 1 to be phosphorylated by GSK3 at serine 468 (S468). Dual phosphorylation of lipin 1 at S468 and S472 promotes lipin 1 cytoplasmic localization and thus also promotes SREBP-target gene expression. Inhibition of the PI3K/Akt pathway and GSK3 (right) results in loss of S468 phosphorylation and results in lipin 1 translocation to the nucleus and repression of SREBP-target gene expression.   156   Our findings potentially complement previously characterized effects of insulin resistance, GSK3 activity, and hepatic steatosis on SREBP activity in the liver. In insulin resistant contexts in mice, for instance, the SREBP pathway has been found to play a major and direct role in the development of hepatic steatosis and carbohydrate-induced triglyceride excess [18]. Furthermore, SREBP-1c protein levels and SREBP pathway activity are elevated in humans exhibiting hepatic steatosis and have additionally been linked to increased insulin signaling via the insulin receptor [15, 16]. GSK3, as a target directly downstream of the PI3K/Akt pathway, is known to be overly active in insulin resistant contexts due to impaired insulin signaling [148, 153] and GSK3 has been found to potentially exacerbate insulin resistance by phosphorylating the insulin receptor IRS1, which inhibits its activity, and could thus further dampen insulin signaling [95]. Under conditions of insulin resistance where GSK3 is aberrantly active, it is possible that it phosphorylates lipin 1 at S468 and causes lipin1 to localize to the cytoplasm instead of translocating to the nucleus, thus promoting excessive SREBP activity and fatty acid and cholesterol biosynthesis and uptake. This would correlate with the role of SREBP in causing hepatic steatosis, and insulin resistance and type 2 diabetes resulting in hypertriglyceridemia [18]. Looking forward, inhibition of GSK3 using GSK3 inhibitors could perhaps represent an effective treatment for these conditions.   However, our findings potentially contradict previous work that has established a role for GSK3 in directly phosphorylating SREBP-1 to downregulate its abundance and SREBP activity [114-116]. In this role, GSK3 has been proposed to phosphorylate SREBP-1, which promotes its ubiquitination by the ubiquitin ligase Fbw7 and subsequent proteasomal degradation [114]. Additionally, the PI3K/Akt pathway, which is directly upstream of GSK3 and inhibits GSK3 157  activity, is considered to positively regulate SREBP activity overall [11]. Our discovery that GSK3 phosphorylation of lipin 1 promotes lipin 1 cytoplasmic localization and therefore also SREBP-target gene expression positions GSK3 activity in this context as upregulating SREBP activity. The contrasting roles of GSK3 described in previous reports and our current work may be due to subtle differences in cell lines or experimental conditions and strategy, or that GSK3 plays both these roles within different cell types of cellular contexts. It is important to note, however, that pharmacological inhibition or downregulation of GSK3 activity were not tested in relation to SREBP-target gene expression in the three studies highlighted previously [114-116]. In our work, we modulated GSK3 activity using both pharmacological inhibition and cell lines lacking GSK3α or GSK3β and did not observed the increase in SREBP-target gene expression that would be expected if GSK3 activity negatively regulated SREBP activity. Indeed, in one instance we even found that NIH 3T3 cells treated only with our GSK3 inhibitor, GSK3 IX, showed a decrease in FASN expression (Figure 3.13). Another key difference is that our work defines lipin 1 as a canonical GSK3 substrate that requires a priming phosphorylation at S472, likely at least partly imparted by mTORC1, to facilitate S468 phosphorylation, whereas in previous studies SREBP-1 has been proposed to be a non-canonical GSK3 substrate that somehow does not require a priming kinase and additionally does not possess a negatively-charged amino acid where the priming site should be positioned relative to the GSK3 phosphorylation site [114]. It has been further suggested that GSK3 may act as its own priming kinase to phosphorylate SREBP-1, but this remains unconfirmed and would be unusual in the realm of GSK3 substrates [114].  158  Potentially, GSK3 may play a multifaceted role in SREBP regulation. That GSK3 may play both positive and negative regulatory roles for SREBP activity in a given cell type or tissue would be unsurprising given the ubiquitous nature of GSK3 signaling. It could also potentially have contributed to the more moderate or variable phenotypes we have observed in our work, particularly in animal models where we found that liver-specific knockout of GSK3 caused an unexpected increase in FASN expression, but also a stark decrease in SCD1 expression, in the livers of these mice fed a high fat diet compared to control mice fed the same diet in parallel (Figure 3.31). An additional study that could be conducted to better characterize the role for GSK3 in regulating SREBP in the livers of these mice would be to assay SREBP phosphorylation at the sites identified in [114] between the control and liver-specific GSK3 knockout mice on both a chow or high fat diet. We could also seek to assay the expression of more SREBP-target genes from the livers of these mice to potentially delineate which subset of SREBP-target genes are regulated by GSK3, as it is known that different isoforms of SREBP regulate the transcription of different sets of fatty acid and cholesterol biosynthesis genes [10, 11].   Our preliminary results from our liver-specific GSK3α and GSK3β knockout mice adds evidence to GSK3 playing a key role in lipid metabolism with physiological consequences related to diet-induced obesity. Previous studies have noted that mice treated with GSK3 inhibitors ameliorates weight gain in animals fed a high fat diet [142, 143]. Our work builds on these prior findings and identifies that the activities of GSK3α and GSK3β specifically in the liver are potentially responsible for these phenotypes, as we found that mice lacking either GSK3 paralog in the liver did not appear to exhibit the degree of weight gain that control mice did on 159  the same HFD. This decrease in diet-induced weight gain was accompanied by corresponding decreases in the expression of the SREBP target gene SCD1, and trends in liver and epidydimal fat pad mass. Significantly, in all of our genetic backgrounds fed a chow diet, we saw no differences in weight gain, indicating that loss of GSK3α and GSK3β in the liver did not alter the basal metabolism of these mice and it was only when fed an HFD that differences in the genetic backgrounds manifested. Due to our limitations on sample size in our study, statistical significance was not attained for some of the parameters measured; however, we did find a significant difference in weight gain of li-GSK3α mice on the HFD compared to control (Figure 3.25), and in the stark decrease of SCD1 expression exhibited by both li-GSK3α and li-GSK3β mice fed the HFD (Figure 3.31b). SCD1 encodes the enzyme stearoyl-CoA desaturase, which converts the saturated fatty acids palmitate and stearate into the monounsaturated fatty acids palmitoleate and oleate, respectively, and thereby plays a pivotal role in lipid storage [149]. Furthermore, SCD1-deficient mice were previously found to be resistant to diet-induced obesity [150] and SCD1 polymorphisms in humans are associated with decreased body weight and improved insulin sensitivity [154], highlighting the importance of SCD1 in lipid metabolism. Since we found SCD1 to be highly upregulated in the livers of control mice fed the HFD and that liver-specific knockout of GSK3 greatly alleviated this upregulation, it is possible that the decrease in weight gain on the HFD we observed in li-GSK3α and li-GSK3β mice could be due to decreased SCD1 expression in the liver. Our findings thus serve as a promising foundation to perform future studies towards elucidating the role of GSK3 in the liver in diet-induced obesity phenotypes with particular focus on SREBP regulation via lipin 1 and SREBP-target genes such as SCD1.   160  It is exciting to speculate that the moderate HFD-resistant phenotypes exhibited by our li-GSK3α and li- GSK3β knockout mice may be due to the role of GSK3 in regulating lipin 1 and SREBP-target gene expression, especially that of SCD1. SREBP is known to be the major pathway of lipogenesis in the liver and to be significantly upregulated under conditions of insulin resistance and hepatic steatosis, two closely-related co-morbidities of obesity. Additionally, as highlighted previously, SCD1 plays crucial roles in lipid metabolism and the onset of diet-induced obesity. However, further study needs to be conducted to confirm that the phenotypes exhibited by the li-GSK3α and li-GSK3β knockout mice are reproducible and if they are indeed due to the role of GSK3 in phosphorylating and regulating lipin 1 outlined in this work. We are presently analyzing the circulating and liver levels of cholesterol and triglyceride content of the mice from our study, which will identify more physiological consequences of liver-specific GSK3 activity in diet-induced obesity. Further work within our current li-GSK3α and li-GSK3β HFD trial would also include assaying the livers of these mice for the level of SREBP, SCD1, total lipin 1, and lipin 1 phosphorylated at S468 and S472 using the appropriate antibodies. Similar to a previous study characterizing the role of mTORC1 phosphorylation of lipin 1 to control SREBP-target gene expression, a key future study could involve knocking down lipin 1 expression in li-GSK3α and li-GSK3β knockout mice fed an HFD using previously validated lipin 1 shRNA constructs [14]. This would demonstrate whether the effects seen in our initial studies are in fact dependent on lipin 1.  Lipin 1-deficient fld mice have been well characterized and could also be used in these HFD studies, either by creating fld mice that additionally lack liver-specific GSK3α or GSK3β expression, or by treating these mice with GSK3 inhibitors in conjunction with an HFD [14, 27].  161  Chapter 4: Conclusions and Future Directions Conclusions We have established that GSK3-family kinases play a conserved role in directly phosphorylating members of the lipin family of phosphatidic acid phosphatase (PAP) enzymes. We used high-throughput screening in Saccharomyces cerevisiae to identify novel kinase regulators of the yeast lipin Pah1. This screen identified the yeast GSK3 kinase, Mck1, as a novel regulator of Pah1. We validated this hit in part by demonstrating that Mck1 is capable of directly phosphorylating Pah1 in vitro. Since lipins and GSK3 kinases are both highly conserved through evolution, we were subsequently interested in whether the kinase-substrate interaction we discovered in yeast was also conserved in mammalian contexts. We next uncovered that mammalian GSK3 directly phosphorylated mammalian lipin 1, in response to signals through the PI3K/Akt and mTORC1 pathways, to ultimately control lipin 1 subcellular localization and its function in repressing SREBP in the nucleus. This represented a novel role for GSK3 in regulating a lipid synthesis enzyme to regulate the SREBP pathway. Finally, we demonstrated the role of GSK3 in lipid metabolism physiologically using animal models with mice lacking either GSK3α and GSK3β in the liver. These liver-specific knockout mice exhibited resistance to effects of diet-induced obesity including weight gain and the expression of the SREBP-target gene SCD1, indicating that GSK3 activity in the liver is potentially linked to the development of diet-induced obesity.    Our investigations into the role of Mck1 in phosphorylating and regulating Pah1 provided evidence that Pah1 was directly regulated by Mck1, and that Mck1 phosphorylation likely inhibited Pah1 enzymatic activity or membrane association in some manner. Our subsequent 162  studies focused on uncovering characteristics of Mck1 that would be consistent with a role in regulating Pah1 through phosphorylation and therefore cellular PA/DAG balance. Indeed, yeast cells lacking Mck1 demonstrated well-characterized phenotypes associated with decreased cellular PA levels including, most notably, inositol auxotrophy. By additionally demonstrating that only catalytically active Mck1 could rescue the inositol auxotrophy of Δmck1 strains, we further strengthened the premise that Mck1 phosphorylation of Pah1 contributed to PA/DAG homeostasis (Figure 2.10). Δmck1 cells also exhibited increased neutral lipid accumulation as assayed by staining of lipid droplets, which was consistent with phenotypes we observed in wild type strains overexpressing Pah1 to increase cellular PAP activity (Figures 2.4 and 2.11). Taken together, it is evident that Mck1 plays a key role in maintaining lipid homeostasis in the yeast cell, potentially through its phosphorylation and regulation of Pah1.   We identified serine 468 (S468) and serine 472 (S472) of lipin 1 as a GSK3-consensus phosphorylation motif that is directly phosphorylated by GSK3 with physiological relevance in cells. Intriguingly, GSK3 phosphorylation of lipin 1 controls the subcellular localization of lipin 1 to ultimately mediate SREBP-target gene expression and is downstream of the PI3K/Akt pathway. It also intersects closely with mTORC1 phosphorylation of lipin 1, which is already known to mediate the effects of mTORC1 on the SREBP pathway [14], because we found that mTORC1 provides the priming phosphorylation for GSK3 phosphorylation at S472 of lipin 1. Thus, we believe we have uncovered a signaling nexus between the PI3K/Akt pathway and GSK3, and mTORC1 to control fatty acid and cholesterol synthesis and uptake through SREBP in response to upstream signals through these pathways. Finally, since the liver is a major site of SREBP activity and based on previous reports that inhibition of GSK3 provided protective 163  effects on weight gain in animal models of diet-induced obesity, we investigated whether liver-specific knockout of either GSK3α or GSK3β resulted in similar effects. Indeed, both liver-specific GSK3α or GSK3β knockout mice appeared to exhibit resistance to some diet-induced obesity phenotypes including weight gain and the expression of the SREBP-target gene SCD1.    We believe that we have identified lipin 1 as a bona fide phosphorylation substrate of GSK3 at its S468/S472 consensus motif based on conventions reviewed by Sutherland in [92]. This is significant because despite the over 100 kinases described to be GSK3 phosphorylation targets, relatively few have met all these criteria [81, 92]. In thoroughly validating the S468/S472 phosphorylation site, it is important to note that we have not uncovered any evidence that disqualifies other sites that fit the GSK3-consensus phosphorylation motif on lipin 1 as potential GSK3 phosphorylation targets. It is possible that GSK3 phosphorylates lipin 1 at more sites than just S468/S472, and more work will need to be done to confirm or refute this claim. Lipin 1 contains multiple GSK3-consensus phosphorylation motifs in addition to S468/S472 (Figure 3.1), and the presence of additional sites that GSK3 could act on may represent some limitations of the results presented in Chapter 3. For instance, one of our major findings involved the stark nuclear re-localization of lipin1 in NIH 3T3 cells treated with both PI3K/Akt and GSK3 inhibitors (Figure 3.10), but this is likely the result of loss of all GSK3 phosphorylation on lipin 1 and does not necessarily link this phenotype specifically to S468/S472. Furthermore, when testing specific phosphorylation-deficient mutants of lipin 1 at S468 and S472, we observed that expression of these mutants caused only a moderate repression in SREBP-target gene expression (Figure 3.16b) compared to pharmacological inhibition (Figure 3.12, Figure 3.13, and Figure 3.14), perhaps signaling that other phosphorylation sites may be in play. Thus, a major future 164  study would be to characterize whether there are other sites that GSK3 phosphorylates on lipin 1 apart from S468/S472, and to characterize the roles of these sites, individually and together with all GSK3 sites on lipin 1, in regulating lipin 1 function.   We have proposed that GSK3 positively regulates SREBP activity via its phosphorylation of lipin 1. This potentially contrasts with previous reports that GSK3 directly phosphorylates SREBP-1 to target it for proteasomal degradation and in this way negatively regulates SREBP activity and gene expression [114-116], and with roles attributed to the PI3K/Akt pathway in positively regulating SREBP activity overall [11]. However, our work has characterized that lipin 1 is a bona fide GSK3 substrate and that both pharmacological inhibition and knockout of GSK3 in vitro result in changes in lipin 1 subcellular localization and decreases in SREBP-target gene expression. We also identify lipin 1 as a canonical GSK3 substrate. This differs from SREBP-1 which has been suggested, but not yet confirmed, to be a GSK3 substrate that is somehow either primed by GSK3 itself or does not require a priming phosphorylation [114], which would be an unusual finding. Furthermore, the previous studies that have characterized GSK3 phosphorylation of SREBP-1 were mainly focused on the Fbw7 ubiquitin ligase that targets phosphorylated SREBP-1 for degradation [114]. As such, in these studies it was not demonstrated that GSK3 inhibition alters SREBP-target gene expression [114-116]. In our work, under conditions of GSK3 inhibition or in cell lines lacking GSK3α or GSK3β expression, we did not observe increases in SREBP-target gene expression that would be excepted if GSK3 played a negative regulatory role on the SREBP pathway. Potentially, both the PI3K/Akt pathway and GSK3 play both positive and negative regulatory roles on SREBP-target gene expression depending on cell or tissue type, and more study will be required to better understand 165  the conditions upon which GSK3 promotes or inhibits SREBP activity. We acknowledge that our studies in this work were primarily focused on lipin 1 and not SREBP. Therefore, investigation of SREBP abundance, stability, and phosphorylation state using the same cell lines and experimental conditions utilized in this study would be illuminating.  Future Directions  There remain avenues of study that could be pursued in the future to better validate the role of Mck1 in phosphorylating Pah1 to regulate cellular PAP activity and lipid homeostasis. We did not pursue these lines of investigation primarily because we transitioned to focusing on elucidating the role of mammalian GSK3 in regulating mammalian lipin 1, as detailed in Chapter 3 of this thesis. We believe the work in Chapter 3 complements and strengthens our initial results in yeast and provides more support for Mck1 regulating Pah1, but also acknowledge there are limits to extrapolating findings in mammalian systems to yeast without more work. As such, we acknowledge that our investigation of Mck1 regulation of Pah1 falls short of the criteria set out by Frame and Cohen (recently summarized by Sutherland in [92]) for defining Pah1 as a bona fide GSK3 substrate. We have demonstrated that purified Mck1 specifically phosphorylates Pah1 in vitro, but future work should define the specific phosphorylation site(s) that fit the canonical GSK3-consensus motif (S/T-X-X-X-S/T) [98] that are phosphorylated by Mck1, likely using proteomics approaches. After defining the specific site or sites that Mck1 imparts phosphorylation on Pah1, these sites can then be functionally linked to the phenotypes of decreased PA and increased neutral lipid accumulation seen in Δmck1 cells. Since it has been observed in yeast that phosphorylation can inhibit Pah1 PAP activity [36] and membrane association [44], these are aspects of Pah1 function that can be assayed for in Δmck1 166  backgrounds using in vitro PAP activity assays and localization studies, for instance. Additionally, modulation of Mck1 activity using catalytically inactive forms or through pharmacological inhibition, and mutation of defined phosphorylation sites to phosphorylation-deficient alanine residues should be confirmed as being able to impart similar phenotypes as seen in Δmck1 cells. Together, this proposed future work would concretely define Pah1 as a validated substrate of Mck1 in yeast.    We further demonstrate the utility of high-throughput screening in yeast to identify previously uncharacterized kinase-substrate interactions that are conserved from yeast to mammals. In the context of lipin regulation, the finding that Mck1/GSK3 both phosphorylate Pah1/lipin 1 is significant because although many kinases have been observed to phosphorylate and regulate lipins in yeast and mammals [14, 36, 69-72, 75], none have been shown to perform this function in both contexts. This is despite the fact that Pah1/lipin 1 and some of these kinases are known to be conserved from yeast to mammals. However, this could be attributed to the lack of conservation in phosphorylation sites between Pah1 and lipin 1 that exists. Indeed, even the GSK3-consensus phosphorylation motifs that span Pah1 do not align with those in lipin 1, despite Mck1 and GSK3 phosphorylating Pah1 and lipin 1, respectively, in vitro. Furthermore, having established that GSK3 phosphorylates Pah1 and lipin 1, it would be interesting to investigate whether mTORC1 phosphorylation of lipin 1 [14] is conserved in yeast, as this could potentially offer a useful background to study general TOR regulation of lipid metabolism. SDL screening, then, appears to be an effective method for identifying kinases that directly regulate a substrate, and in the context of our screen may identify our other Pah1 SDL interactions as promising future avenues of research. Two of the other kinases identified by our SDL screen that 167  additionally exhibited inositol auxotrophy when deleted in yeast were Dbf2 and Bub1 (Figure 2.6), both of which have highly conserved mammalian homologs in LATS1 and BUB1, respectively. Therefore, Dbf2 and Bub1, along with their mammalian counterparts, could potentially represent novel regulators of lipin function and could be studied in a similar manner as our approach to investigating the relationship between Mck1/GSK3 and Pah1/lipin 1.    Our work further highlights the importance of regulating lipin 1 subcellular localization and the role of lipin 1 in regulating gene expression in the nucleus. It is intriguing that lipin 1 regulation of SREBP target-gene expression has been observed to require its catalytic PAP activity [14], because this suggests that PA or DAG levels could play a direct role in regulating SREBP. Lipin 1 activity in the nucleus or nuclear envelope, for instance, could decrease PA levels or increase DAG levels in those compartments to transmit a repressive signal to SREBP. Both PA and DAG are known to be signaling lipids, acting as second messengers in various cellular pathways [20, 156], and the role of lipin 1 in adipogenesis appears to at least partially involve PA and its activation of the ERK pathway, which then inhibits induction of PPAR𝛾 and the adipogenic program [157]. The nucleus and nuclear envelope are likely the sites to investigate in this context because lipin 1 nuclear localization and activity are both required to repress SREBP. It is also possible that modulation of PA or DAG levels could contribute to the nuclear morphology changes seen in [14], which accompanies lipin 1 nuclear translocation and repression of SREBP. Future studies in studying the role of PA and DAG in lipin 1 regulation of SREBP would thus be well warranted.   168  GSK3 regulation of lipin 1 was found to be downstream of PI3K/Akt signaling, which has long been known to inhibit GSK3 function [146], and mTORC1, which provides the necessary priming phosphorylation for GSK3 to phosphorylate lipin 1. Along with the fact that mTORC1 directly phosphorylates lipin 1 at multiple sites to control its subcellular localization and SREBP-target gene expression [14], this signaling network of PI3K/Akt, mTORC1, and GSK3 may exist to provide a graded response to integrate signals to mediate the SREBP pathway via lipin 1. As we found in our investigations, inhibition of the PI3K/Akt pathway by inhibitors caused loss of some mTORC1-directed phosphorylation of lipin 1, but notably that phosphorylation at S472 persisted, and this was the only scenario when S468 was also phosphorylated in a manner dependent on GSK3 activity (Figure 3.2). This suggests that when PI3K/Akt signaling is inactive, but mTORC1 signaling is active, that GSK3 phosphorylates lipin 1 at S468 to promote its localization to the cytoplasm. This may represent a mechanism of fine-tuned signal integration as the PI3K/Akt pathway is known to be downstream of insulin signaling [151] and mTORC1 is well known as a growth factor and nutrient sensing complex [152]. This may also have implications in insulin resistance, which is known to cause dampening of insulin signaling and the PI3K/Akt pathway. In insulin-resistant contexts, GSK3 is known to be overactive due to this attenuated PI3K/Akt signaling [148] and thought to additionally exacerbate the insulin-resistant condition by phosphorylating the insulin receptor IRS1 and further inhibiting its function [94]. Future work should therefore focus on the role of GSK3 in insulin-resistant contexts, as excessive GSK3 activity, through its effects on lipin 1 localization, may promote SREBP-target gene expression and thus fatty acid and cholesterol biosynthesis. This may, for instance, contribute the hepatic steatosis, hypercholestrolemia and hypertriglyceridemia observed commonly as symptoms of obesity and type 2 diabetes.  169    Our work in mouse models have offered initial insights into the role of GSK3 in the liver and the development of diet-induced obesity. In finding that mice lacking either paralog of GSK3, GSK3α or GSK3β, in the liver appear to possess moderate resistance to weight gain on a high fat diet, we have built upon previous studies that found similar results via pharmacological inhibition with a variety of GSK3 inhibitors [142, 143]. We also found trends suggesting these mice exhibit decreased weight gain specifically in liver and epididymal fat pads, and in the expression of at least one SREBP target gene, SCD1, in the liver. Significantly, SCD1, which encodes the enzyme stearoyl-CoA desaturase, is known to be a key enzyme in lipid metabolism and the development of diet-induced obesity [149]; mice lacking SCD1, for instance, have been characterized previously to be resistant to weight gain on a high fat diet [150]. Paradoxically, we found that expression of another SREBP-target gene, FASN, increased in the livers of our liver-specific GSK3 knockout mice fed the HFD, and that HMGCR expression remained unchanged under all conditions (Figure 3.31). These findings may be attributed to the previously proposed role for GSK3 in negatively regulating the SREBP pathway by targeting SREBP-1 for ubiquitination and proteasomal degradation [114-116], or that GSK3 or lipin 1 may regulate specific SREBP isoforms because it is known, for instance, that SREBP-2 regulates cholesterol synthesis genes whereas SREBP-1c regulates fatty acid synthesis genes [10, 11]. We are presently analyzing samples collected from this experiment to analyze circulating and liver cholesterol and triglyceride levels, which will be useful in better assessing the effects of GSK3 activity in the liver on lipid metabolism on a high fat diet in our study. Overall, this highlights the need for further study of the roles of GSK3 and lipin 1 in SREBP regulation. Indeed, we have now performed preliminary RNA-sequencing analysis of livers from control and li-GSK3β mice 170  fed a high fat diet and confirmed our qPCR data that SCD1 expression is elevated in the control livers and is greatly reduced in the li-GSK3β livers (Appendix A Figure 1). Furthermore, we found that li-GSK3β mice have significantly decreased cholesterol and neutral lipid synthesis genes compared to the control livers as well (Appendix A Figure 2).  Our study was treated as a pilot study, and with our limited cohort size the differences between control mice and the liver-specific GSK3α and GSK3β knockout mice were not necessarily statistically significant but provide a good foundation for future studies, particularly with the preliminary RNA-sequencing data (Appendix A). A pivotal future study will be to repeat this experiment with larger cohorts of animals to increase statistical power and demonstrate the reproducibility of our initial findings. In conjunction with our work in defining a role for GSK3 in regulating lipin 1 and the SREBP pathway, it is possible the effects we observed in the li-GSK3α and li-GSK3β animals could be due to this relationship. Specifically, in the absence of either GSK3α or GSK3β in the liver, our other findings would suggest that lipin 1 would be less constrained to the cytoplasm and could translocate more readily to the nucleus to repress SREBP-target gene expression. The result of this decrease in fatty acid and/or cholesterol biosynthesis and uptake in the liver could thus result in the decrease in weight gain exhibited by these mice on an HFD compared to their control counterparts.   In the long term, our results may provide some groundwork for future studies on the potential of pharmacological intervention using GSK3 inhibitors in treating or delaying the onset of obesity or related metabolic disorders. GSK3 is an attractive drug target, given its involvement in a multitude of cellular pathways and processes, and a number of GSK3 inhibitors are at 171  various stages of clinical trials. One of the main concerns of using GSK3 inhibition as a therapeutic strategy is the potential for side effects because of the central nature of its functions [81, 98]. However, it has been proposed that GSK3 inhibition would be particularly useful in contexts where GSK3 is already overactive and thus the therapeutic approach would be to bring GSK3 activity back into balance [102]. Furthermore, in some instances knocking out a single allele of GSK3 has been found to alter the downstream effects of GSK3 activity [158]. Based on the hypothesis that GSK3 activity is overactive in insulin resistance, then, it is possible that inhibition of GSK3 would alleviate the negative effects of overactive GSK3 in these contexts. Additionally, if the weight gain resistance shown by our li-GSK3α and li-GSK3β mice are indeed confirmed to be through GSK3 regulation of lipin 1 and SREBP in the liver, our in vitro work supports that it is a function of GSK3 that requires a full complement of both GSK3α and GSK3β. Taken together, we believe there may be potential in pharmacologically modulating GSK3 activity specifically in the liver in metabolic disorders such as obesity.  172  References 1. Afshin, A., M.H. Forouzanfar, M.B. Reitsma, P. Sur, K. Estep, A. Lee, L. Marczak, A.H. Mokdad, M. Moradi-Lakeh, M. Naghavi, J.S. Salama, T. Vos, K.H. Abate, C. Abbafati, M.B. Ahmed, Z. Al-Aly, A. Alkerwi, R. Al-Raddadi, A.T. Amare, A. Amberbir, et al., Health Effects of Overweight and Obesity in 195 Countries over 25 Years. N Engl J Med, 2017. 377(1): p. 13-27. 2. Nagle, C.A., E.L. Klett, and R.A. Coleman, Hepatic triacylglycerol accumulation and insulin resistance. J Lipid Res, 2009. 50 Suppl: p. S74-9. 3. 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Synapse, 2011. 65(3): p. 234-48.  185  Chapter 5: Appendices Appendix A: RNA Sequencing Results Figure 1: RNA-sequencing analysis reveals changes in SCD1 expression in li-GSK3β livers  186   Figure 2: RNA-sequencing analysis reveals global transcriptional changes with respect to cholesterol synthesis and neutral lipid synthesis in li-GSK3β livers  187  Appendix B: SDL Screen Results and Mass Spectrometry Peptide Results ORF Name Gene Ctrl Ctrl SD Exp Exp SD Ratio  Ratio SD Diff p-value YJL200C ACO2 0.379 0.013 0.050 1.1E-04 0.13238 0.00431 -0.32862 7.37E-04 YNL220W ADE12 0.968 0.117 0.050 6.9E-18 0.05236 0.00582 -0.91785 0.007986 YNL220W ADE12 0.844 0.147 0.050 6.9E-18 0.06127 0.01185 -0.79446 0.016729 YNL220W ADE12 0.773 0.074 0.050 6.9E-18 0.06526 0.00601 -0.72302 0.005211 YNL220W ADE12 0.663 0.117 0.050 6.9E-18 0.07754 0.01214 -0.61309 0.017621 YDR226W ADK1 0.442 0.083 0.211 1.3E-02 0.49086 0.07209 -0.23107 0.04281 YER093C-A AIM11 1.121 0.053 0.105 6.9E-02 0.09399 0.06127 -1.01584 0.003774 YJL046W AIM22 1.103 0.031 0.195 6.1E-02 0.17854 0.05941 -0.90751 0.004903 YJR080C AIM24 0.793 0.026 0.613 2.9E-02 0.77286 0.01493 -0.17982 0.001566 YJR005W APL1 0.460 0.017 0.071 7.8E-03 0.15486 0.01219 -0.3887 3.78E-04 YJR005W APL1 0.459 0.010 0.074 2.0E-02 0.16105 0.04447 -0.38534 0.002078 YNL094W APP1 0.826 0.100 0.286 9.6E-02 0.34632 0.09915 -0.54016 0.020097 YGL105W ARC1 0.695 0.031 0.236 4.1E-02 0.33851 0.05388 -0.4592 0.003147 YLR370C ARC18 1.074 0.096 0.486 5.8E-02 0.4557 0.06802 -0.58774 0.016212 YDL192W ARF1 1.021 0.078 0.309 1.1E-02 0.30377 0.01275 -0.71195 0.004441 YBR164C ARL1 0.979 0.065 0.721 6.4E-02 0.73747 0.05921 -0.25832 0.032254 YHR129C ARP1 0.987 0.021 0.673 8.4E-02 0.68299 0.09181 -0.31395 0.043293 YMR116C ASC1 0.906 0.052 0.050 6.9E-18 0.05535 0.00307 -0.85619 0.00185 YJL115W ASF1 0.730 0.027 0.385 5.7E-02 0.52548 0.05749 -0.34467 0.003682 YCL038C ATG22 0.975 0.066 0.756 6.4E-02 0.77431 0.01405 -0.21905 7.72E-05 YNL315C ATP11 0.705 0.055 0.050 6.9E-18 0.07136 0.00548 -0.65489 0.003475 YNL315C ATP11 0.690 0.067 0.050 6.9E-18 0.07315 0.00701 -0.63994 0.005437 YNL315C ATP11 0.672 0.071 0.050 6.9E-18 0.07524 0.00829 -0.62236 0.006395 YNL315C ATP11 0.638 0.086 0.050 6.9E-18 0.07988 0.01117 -0.58796 0.010551 YNL315C ATP11 0.371 0.090 0.050 6.9E-18 0.14245 0.03145 -0.32058 0.036881 188  YML081C-A ATP18 1.116 0.060 0.383 6.9E-02 0.3461 0.07244 -0.73291 0.011596 YPR020W ATP20 0.755 0.096 0.306 6.9E-02 0.40779 0.09097 -0.44894 0.020533 YNR020C ATP23 1.105 0.051 0.050 6.9E-18 0.04535 0.00218 -1.05508 0.001186 YLR114C AVL9 0.460 0.154 0.147 7.5E-02 0.30061 0.09711 -0.31329 0.047727 YBL089W AVT5 1.203 0.008 0.809 8.4E-02 0.67157 0.06576 -0.39477 0.018462 YOR113W AZF1 0.961 0.015 0.717 6.1E-02 0.7469 0.06519 -0.24347 0.032935 YJL095W BCK1 1.038 0.076 0.505 7.4E-02 0.49153 0.09548 -0.5328 0.026277 YER167W BCK2 1.017 0.067 0.692 5.3E-02 0.67965 0.01443 -0.32561 0.002025 YBR200W BEM1 0.737 0.069 0.180 1.5E-01 0.23879 0.18941 -0.55667 0.030629 YER155C BEM2 0.958 0.007 0.355 8.1E-02 0.37027 0.0817 -0.60247 0.00752 YER155C BEM2 0.892 0.068 0.350 2.1E-02 0.39523 0.0418 -0.54154 0.009812 YER016W BIM1 1.111 0.044 0.629 1.7E-02 0.56683 0.02014 -0.482 0.003176 YER016W BIM1 1.121 0.039 0.699 6.1E-02 0.62317 0.04344 -0.42182 0.006064 YER016W BIM1 1.098 0.017 0.693 1.0E-01 0.63 0.08719 -0.40517 0.02457 YER016W BIM1 1.110 0.059 0.707 8.9E-02 0.6408 0.09694 -0.40266 0.044784 YDL074C BRE1 0.218 0.010 0.050 6.9E-18 0.23012 0.01092 -0.16777 0.001901 YNR051C BRE5 0.713 0.010 0.115 5.1E-02 0.16086 0.06907 -0.59781 0.002422 YPR057W BRR1 1.239 0.007 0.659 7.9E-02 0.53161 0.06532 -0.5808 0.009999 YGR188C BUB1 1.046 0.057 0.294 1.3E-01 0.2773 0.11741 -0.75265 0.009329 YOR026W BUB3 0.726 0.049 0.437 2.3E-02 0.60307 0.02098 -0.28892 0.005943 YCR047C BUD23 0.768 0.019 0.233 1.1E-01 0.30653 0.14197 -0.53465 0.025119 YDL151C BUD30 0.847 0.050 0.087 3.9E-02 0.10042 0.03865 -0.75987 1.21E-04 YCR063W BUD31 0.916 0.047 0.117 4.2E-02 0.12529 0.03806 -0.79968 4.62E-05 YCR063W BUD31 0.974 0.012 0.154 6.1E-02 0.15712 0.06098 -0.81998 0.001967 YCR063W BUD31 0.855 0.029 0.190 9.9E-02 0.22063 0.11507 -0.66545 0.009141 YCR063W BUD31 0.855 0.048 0.254 1.1E-02 0.29814 0.01482 -0.60042 0.002591 YGR262C BUD32 0.309 0.057 0.050 6.9E-18 0.16763 0.03205 -0.25906 0.023229 YMR275C BUL1 0.889 0.027 0.612 5.4E-02 0.68956 0.06071 - 0.020647 189  0.27616 YML102W CAC2 0.800 0.041 0.341 9.7E-02 0.42943 0.13239 -0.45819 0.029173 YGR217W CCH1 1.169 0.071 0.881 6.6E-02 0.75268 0.01723 -0.28854 0.001902 YLR110C CCW12 0.818 0.094 0.424 2.8E-02 0.52453 0.0569 -0.3935 0.024314 YBR131W CCZ1 0.401 0.079 0.050 6.9E-18 0.12916 0.02265 -0.35094 0.02439 YER061C CEM1 0.989 0.062 0.595 7.0E-02 0.60356 0.07033 -0.39332 0.020919 YGR157W CHO2 0.843 0.022 0.402 6.7E-02 0.47942 0.09331 -0.44097 0.019506 YMR198W CIK1 1.332 0.116 0.528 1.2E-01 0.40407 0.10747 -0.80381 0.032581 YMR198W CIK1 1.124 0.081 0.669 1.4E-01 0.58888 0.08816 -0.45491 0.008883 YNL298W CLA4 1.199 0.023 0.512 8.6E-02 0.42631 0.06575 -0.68683 0.005609 YNL298W CLA4 1.135 0.093 0.702 5.0E-02 0.62289 0.06371 -0.43244 0.028222 YPR119W CLB2 1.289 0.010 0.853 1.1E-01 0.66145 0.08287 -0.43591 0.027992 YDL155W CLB3 0.923 0.085 0.534 6.3E-03 0.58443 0.06072 -0.38848 0.024966 YNL225C CNM67 0.366 0.099 0.054 5.9E-03 0.15381 0.02185 -0.31206 0.042175 YNL225C CNM67 0.335 0.069 0.050 6.9E-18 0.15491 0.02804 -0.28521 0.027906 YNL225C CNM67 0.297 0.069 0.050 6.9E-18 0.17944 0.04888 -0.24748 0.037144 YNL051W COG5 0.882 0.011 0.420 1.1E-01 0.47541 0.11596 -0.46136 0.021503 YNL041C COG6 1.061 0.060 0.612 5.5E-02 0.57613 0.03068 -0.44895 0.002935 YML071C COG8 1.004 0.025 0.779 3.4E-02 0.77534 0.01734 -0.22518 0.001904 YNL052W COX5A 1.133 0.049 0.327 1.3E-01 0.28763 0.1044 -0.8064 0.010997 YDL142C CRD1 1.116 0.098 0.050 6.9E-18 0.04512 0.00379 -1.06628 0.004204 YLR087C CSF1 0.732 0.048 0.411 8.3E-03 0.56345 0.03311 -0.32116 0.009067 YMR048W CSM3 1.043 0.044 0.547 4.0E-02 0.52372 0.02587 -0.4964 0.001606 YLR381W CTF3 0.993 0.058 0.575 4.6E-02 0.57798 0.01624 -0.41849 8.03E-04 YJL006C CTK2 0.503 0.039 0.086 3.0E-02 0.16884 0.04886 -0.41703 0.001445 YJL006C CTK2 0.475 0.024 0.088 2.7E-02 0.18628 0.06088 -0.38763 0.006331 YJL006C CTK2 0.488 0.011 0.108 4.2E-02 0.22254 0.08739 -0.37981 0.007562 YJL006C CTK2 0.504 0.024 0.120 3.3E-02 0.23913 0.06941 -0.3841 0.006976 190  YJL006C CTK2 0.442 0.040 0.121 4.6E-02 0.27025 0.0934 -0.32188 0.008149 YJL006C CTK2 0.456 0.007 0.148 5.3E-02 0.32478 0.11749 -0.30851 0.016617 YML112W CTK3 0.629 0.011 0.050 6.9E-18 0.07957 0.00145 -0.57856 1.92E-04 YML112W CTK3 0.615 0.006 0.085 1.4E-02 0.1387 0.02208 -0.52948 4.22E-04 YML112W CTK3 0.640 0.008 0.095 5.3E-02 0.14805 0.08314 -0.54533 0.005152 YML112W CTK3 0.644 0.060 0.128 7.4E-02 0.21168 0.14261 -0.51646 0.031972 YJR048W CYC1 1.038 0.067 0.338 6.2E-02 0.32569 0.05416 -0.69987 0.005464 YAL012W CYS3 0.257 0.011 0.050 6.9E-18 0.19505 0.0086 -0.20683 0.001428 YKL087C CYT2 0.806 0.152 0.504 1.3E-01 0.6185 0.07697 -0.30197 0.019829 YIR023W DAL81 0.735 0.098 0.234 2.4E-02 0.31913 0.0113 -0.50156 0.010955 YGR092W DBF2 0.998 0.056 0.549 1.1E-01 0.54644 0.08877 -0.44956 0.014053 YCL016C DCC1 0.840 0.064 0.567 8.7E-02 0.67159 0.05535 -0.27324 0.007768 YFR012W DCV1 0.860 0.024 0.622 5.5E-02 0.72482 0.07416 -0.23784 0.038746 YFL001W DEG1 1.005 0.075 0.439 7.0E-02 0.43509 0.0409 -0.56534 0.001373 YFL001W DEG1 1.045 0.082 0.462 1.6E-01 0.44265 0.15457 -0.58324 0.036365 YFL001W DEG1 1.010 0.088 0.464 6.6E-02 0.458 0.04565 -0.5469 0.005288 YFL001W DEG1 0.968 0.077 0.478 7.4E-02 0.4942 0.06964 -0.4897 0.011806 YOR030W DFG16 0.498 0.084 0.122 3.3E-02 0.24212 0.02431 -0.3755 0.009642 YKR035W-A DID2 1.003 0.093 0.447 1.1E-01 0.43925 0.06873 -0.55626 2.63E-04 YKL213C DOA1 0.769 0.105 0.579 6.2E-02 0.7553 0.02308 -0.1906 0.024156 YGL240W DOC1 0.650 0.011 0.377 6.9E-02 0.57835 0.09682 -0.27302 0.023112 YGL240W DOC1 0.598 0.032 0.387 5.4E-02 0.64555 0.06499 -0.21055 0.010751 YNL136W EAF7 0.959 0.162 0.150 4.9E-02 0.15173 0.02876 -0.8091 0.009641 YKL204W EAP1 0.573 0.032 0.050 6.9E-18 0.08755 0.00472 -0.52284 0.001873 YLR443W ECM7 1.154 0.039 0.867 1.6E-02 0.75186 0.01232 -0.28684 0.003455 YOR133W EFT1 1.240 0.105 0.258 2.5E-01 0.19596 0.17798 -0.98149 0.014995 YKL048C ELM1 0.521 0.048 0.353 5.4E-02 0.67493 0.06349 -0.16789 0.015548 YMR312W ELP6 0.648 0.072 0.050 6.9E-18 0.07804 0.00811 -0.59821 0.007236 191  YDR512C EMI1 1.227 0.128 0.050 6.9E-18 0.04125 0.00464 -1.17658 0.005876 YDR512C EMI1 1.200 0.077 0.050 6.9E-18 0.04185 0.00279 -1.14975 0.002231 YDR512C EMI1 1.178 0.088 0.050 6.9E-18 0.04269 0.00334 -1.12801 0.003034 YDR512C EMI1 1.127 0.125 0.050 6.9E-18 0.04498 0.00542 -1.0768 0.006718 YDR512C EMI1 0.997 0.083 0.050 6.9E-18 0.05049 0.00413 -0.94702 0.003828 YBR026C ETR1 0.996 0.071 0.194 5.7E-02 0.19379 0.05231 -0.80208 0.002914 YFR019W FAB1 1.011 0.059 0.512 5.3E-02 0.50724 0.05592 -0.49925 0.009565 YJL157C FAR1 0.886 0.252 0.388 3.9E-01 0.35723 0.29079 -0.49773 0.039435 YFR008W FAR7 1.211 0.035 0.902 6.0E-02 0.74444 0.03567 -0.30896 0.008674 YMR058W FET3 0.589 0.034 0.051 1.8E-03 0.08734 0.00598 -0.53788 0.001981 YMR058W FET3 0.571 0.005 0.050 6.9E-18 0.08752 7.07E-04 -0.52131 3.95E-05 YMR058W FET3 0.526 0.045 0.050 6.9E-18 0.09569 0.00824 -0.47638 0.004411 YMR058W FET3 0.536 0.043 0.053 3.7E-03 0.09861 0.00793 -0.48304 0.003646 YLR404W FLD1 1.066 0.071 0.741 6.4E-03 0.69807 0.04518 -0.32506 0.021858 YIL134W FLX1 1.197 0.030 0.086 4.7E-02 0.07122 0.03686 -1.11113 1.99E-04 YIL098C FMC1 1.084 0.012 0.534 1.9E-02 0.49244 0.02018 -0.55037 0.001108 YIL098C FMC1 1.057 0.029 0.561 8.8E-02 0.53076 0.08365 -0.496 0.015843 YIL098C FMC1 0.961 0.051 0.588 5.7E-02 0.61615 0.09268 -0.37334 0.037038 YER145C FTR1 0.600 0.017 0.266 1.1E-01 0.4395 0.17512 -0.33413 0.042627 YHR059W FYV4 0.820 0.067 0.216 5.0E-02 0.2643 0.05952 -0.60366 0.006862 YGR252W GCN5 0.323 0.017 0.050 6.9E-18 0.15528 0.00816 -0.27288 0.001908 YGR252W GCN5 0.310 0.011 0.050 6.9E-18 0.16171 0.00577 -0.25959 9.21E-04 YGR252W GCN5 0.322 0.020 0.057 9.2E-03 0.17766 0.04014 -0.26586 0.005671 YGR252W GCN5 0.289 0.014 0.063 5.1E-03 0.2174 0.00977 -0.22573 9.92E-04 YJR040W GEF1 1.039 0.060 0.631 4.7E-02 0.60833 0.04414 -0.40762 0.009558 YJR040W GEF1 1.020 0.015 0.655 2.4E-02 0.64262 0.02105 -0.36446 0.001695 YJR040W GEF1 0.970 0.082 0.633 1.8E-02 0.65658 0.05239 -0.337 0.02573 YDR506C GMC1 1.121 0.041 0.823 2.3E-02 0.73468 0.02325 - 0.006754 192  0.29809 YDR508C GNP1 1.113 0.024 0.491 2.2E-02 0.44199 0.02882 -0.62181 0.002712 YJR090C GRR1 0.665 0.044 0.069 1.6E-02 0.10419 0.0264 -0.59675 0.003926 YJR090C GRR1 0.612 0.072 0.114 2.9E-02 0.19089 0.05802 -0.49843 0.016122 YJR090C GRR1 0.615 0.048 0.137 5.1E-02 0.23028 0.10032 -0.47764 0.019834 YJR090C GRR1 0.685 0.035 0.218 2.9E-02 0.32106 0.05597 -0.4666 0.007827 YML121W GTR1 0.947 0.053 0.536 1.4E-01 0.5611 0.12761 -0.41148 0.029874 YCR065W HCM1 1.486 0.048 1.013 4.8E-02 0.68177 0.02825 -0.47318 0.00519 YLR192C HCR1 0.906 0.063 0.657 6.3E-02 0.72562 0.05801 -0.24956 0.026098 YNL021W HDA1 0.372 0.035 0.072 1.6E-02 0.19477 0.04604 -0.29973 0.006549 YDR295C HDA2 0.310 0.034 0.146 4.2E-02 0.46437 0.0894 -0.16428 0.009913 YOR202W HIS3 0.310 0.064 0.051 1.2E-03 0.17217 0.03955 -0.25933 0.028887 YOR202W HIS3 0.279 0.021 0.054 4.3E-03 0.19587 0.01803 -0.22456 0.004103 YJR055W HIT1 0.664 0.016 0.100 2.8E-02 0.15025 0.0425 -0.56411 0.00161 YJR055W HIT1 0.313 0.025 0.051 1.2E-03 0.16344 0.01277 -0.26204 0.004329 YJR055W HIT1 0.679 0.049 0.118 4.1E-02 0.17012 0.04686 -0.56079 1.38E-04 YJR055W HIT1 0.669 0.020 0.125 4.5E-02 0.18448 0.06115 -0.54421 0.001123 YJR055W HIT1 0.624 0.014 0.116 2.7E-02 0.18739 0.04699 -0.50757 0.003249 YJR055W HIT1 0.483 0.032 0.147 4.0E-02 0.30962 0.09353 -0.33574 0.01796 YDR174W HMO1 0.809 0.032 0.355 6.8E-02 0.44202 0.09579 -0.45419 0.021229 YJR139C HOM6 0.733 0.032 0.082 2.9E-02 0.11304 0.04315 -0.651 0.00395 YMR186W HSC82 1.112 0.056 0.799 6.9E-02 0.71874 0.05494 -0.31306 0.019149 YHL002W HSE1 1.130 0.111 0.649 1.9E-02 0.57879 0.0437 -0.48063 0.02048 YHR067W HTD2 0.757 0.052 0.050 6.9E-18 0.06639 0.00471 -0.70682 0.002654 YOL012C HTZ1 0.564 0.113 0.420 6.7E-02 0.75102 0.03519 -0.14448 0.047909 YNL037C IDH1 0.922 0.027 0.245 2.1E-02 0.26518 0.0155 -0.6768 7.54E-05 YFL013C IES1 0.324 0.034 0.052 2.5E-03 0.16345 0.01554 -0.27143 0.007249 YFL013C IES1 0.345 0.041 0.067 1.5E-02 0.19979 0.05373 -0.27735 0.015435 193  YFL013C IES1 0.332 0.063 0.070 2.9E-02 0.22071 0.0995 -0.26189 0.038443 YFL013C IES1 0.325 0.053 0.081 2.5E-02 0.26416 0.09903 -0.244 0.041125 YJR118C ILM1 0.506 0.019 0.050 6.9E-18 0.09903 0.00375 -0.45559 8.37E-04 YJL082W IML2 0.863 0.058 0.542 2.8E-02 0.62938 0.03378 -0.32124 0.010613 YBR107C IML3 0.833 0.026 0.541 7.6E-02 0.64794 0.0781 -0.2918 0.018945 YOL108C INO4 0.356 0.010 0.094 3.2E-02 0.26399 0.08856 -0.26231 0.00701 YPL017C IRC15 1.139 0.045 0.641 4.8E-02 0.56551 0.06235 -0.49759 0.016591 YKR019C IRS4 0.902 0.060 0.590 9.5E-02 0.65024 0.0631 -0.31183 0.007152 YDL115C IWR1 1.130 0.029 0.068 2.4E-02 0.05982 0.01939 -1.06154 1.18E-04 YPR061C JID1 1.090 0.104 0.050 6.9E-18 0.04627 0.00435 -1.0404 0.004975 YDR532C KRE28 0.363 0.013 0.050 6.9E-18 0.13776 0.00499 -0.3134 8.39E-04 YNL071W LAT1 1.176 0.084 0.050 6.9E-18 0.04274 0.003 -1.12571 0.002773 YJR070C LIA1 0.681 0.054 0.079 2.4E-02 0.1198 0.04253 -0.60182 0.00803 YFL018C LPD1 1.042 0.034 0.050 6.9E-18 0.04805 0.00161 -0.99168 5.88E-04 YFL018C LPD1 1.027 0.045 0.050 6.9E-18 0.04876 0.00213 -0.97736 0.001078 YFL018C LPD1 0.995 0.046 0.050 6.9E-18 0.05038 0.00241 -0.94466 0.001207 YFL018C LPD1 0.972 0.035 0.050 6.9E-18 0.05148 0.00187 -0.92244 7.13E-04 YDL240W LRG1 0.793 0.026 0.551 2.3E-02 0.69547 0.01165 -0.24136 9.51E-04 YJL124C LSM1 1.449 0.115 0.357 3.7E-02 0.24992 0.04404 -1.09151 0.008975 YNL147W LSM7 1.587 0.020 0.227 8.0E-02 0.14308 0.05181 -1.36026 0.002211 YNL147W LSM7 1.615 0.014 0.263 7.5E-02 0.16261 0.04644 -1.35231 0.001425 YNL147W LSM7 1.600 0.050 0.326 9.8E-02 0.20298 0.05885 -1.27459 0.002473 YNL147W LSM7 1.590 0.039 0.333 2.4E-02 0.20994 0.01782 -1.25654 9.47E-04 YNL268W LYP1 0.969 0.042 0.539 9.5E-02 0.56063 0.12283 -0.43022 0.04393 YDR034C LYS14 0.712 0.051 0.059 1.2E-02 0.0829 0.01917 -0.65381 0.003587 YJL013C MAD3 1.077 0.015 0.698 9.0E-03 0.6479 0.01763 -0.3796 0.002014 YJL013C MAD3 1.149 0.073 0.747 1.1E-02 0.65364 0.05107 -0.40163 0.020646 YPR051W MAK3 1.266 0.063 0.569 1.6E-02 0.45054 0.02799 -0.6974 0.00484 YNL307C MCK1 0.608 0.073 0.206 1.3E-02 0.34476 0.05843 - 0.018596 194  0.40257 YNL307C MCK1 0.526 0.089 0.224 1.4E-02 0.4333 0.04512 -0.30184 0.030426 YNL307C MCK1 0.573 0.067 0.267 6.4E-02 0.46208 0.08069 -0.3059 0.012072 YPR046W MCM16 1.210 0.031 0.698 1.1E-01 0.57756 0.09802 -0.512 0.02744 YDR318W MCM21 1.255 0.091 0.618 7.4E-02 0.49683 0.08013 -0.63618 0.021922 YOL009C MDM12 0.655 0.119 0.278 2.0E-02 0.43306 0.0562 -0.37773 0.034626 YOR147W MDM32 0.979 0.089 0.190 8.1E-02 0.2013 0.09877 -0.78975 0.019484 YOR147W MDM32 0.990 0.039 0.214 5.7E-02 0.21418 0.05021 -0.77673 7.61E-04 YGL219C MDM34 1.101 0.082 0.279 4.9E-02 0.25868 0.06582 -0.82122 0.012379 YKL053C-A MDM35 0.852 0.023 0.093 3.0E-02 0.10912 0.03669 -0.75986 0.001885 YPR070W MED1 0.443 0.048 0.050 6.9E-18 0.11408 0.01156 -0.39317 0.007435 YIR033W MGA2 0.775 0.115 0.447 5.3E-02 0.58269 0.06205 -0.32796 0.036518 YMR115W MGR3 0.969 0.035 0.050 6.9E-18 0.05169 0.00188 -0.91852 7.35E-04 YJR077C MIR1 0.978 0.027 0.433 1.6E-01 0.44586 0.17425 -0.54469 0.049409 YBR084W MIS1 1.158 0.044 0.059 1.2E-02 0.0512 0.0129 -1.09907 0.00132 YEL007W MIT1 1.275 0.042 0.939 6.0E-02 0.73581 0.02304 -0.33577 0.001506 YNL076W MKS1 1.228 0.026 0.050 6.9E-18 0.04074 8.63E-04 -1.17772 2.45E-04 YMR167W MLH1 1.212 0.038 0.775 3.2E-02 0.64095 0.04626 -0.43705 0.012737 YLL006W MMM1 0.904 0.059 0.489 5.7E-02 0.54592 0.09613 -0.41547 0.031819 YLR320W MMS22 0.761 0.043 0.435 1.3E-02 0.57353 0.02516 -0.32532 0.006041 YDR245W MNN10 1.131 0.057 0.374 8.0E-02 0.3332 0.07996 -0.75657 0.011212 YDR245W MNN10 1.021 0.094 0.342 5.6E-02 0.33717 0.05333 -0.67846 0.01001 YDR245W MNN10 1.109 0.068 0.476 3.5E-02 0.43122 0.04497 -0.63279 0.008445 YDR245W MNN10 1.172 0.054 0.537 3.2E-02 0.45863 0.03295 -0.63578 0.00473 YJR074W MOG1 1.009 0.051 0.087 2.9E-02 0.08696 0.02909 -0.92225 0.002565 YBR122C MRPL36 0.918 0.008 0.529 7.7E-02 0.5769 0.08665 -0.38897 0.021713 YBR122C MRPL36 0.753 0.032 0.521 1.2E-02 0.69291 0.03523 -0.23218 0.011154 YBR122C MRPL36 0.896 0.029 0.621 2.7E-02 0.69444 0.04273 - 0.013173 195  0.27457 YBR122C MRPL36 0.692 0.030 0.504 2.2E-02 0.72844 0.01012 -0.18801 0.001787 YML009C MRPL39 0.970 0.084 0.092 3.9E-02 0.09389 0.03362 -0.87752 0.002784 YML128C MSC1 1.193 0.098 0.380 1.6E-02 0.32063 0.02352 -0.81229 0.006483 YMR037C MSN2 0.579 0.061 0.189 6.2E-02 0.32204 0.09487 -0.39059 0.010398 YHR151C MTC6 0.908 0.068 0.636 3.6E-02 0.70094 0.0196 -0.2727 0.008214 YMR100W MUB1 0.867 0.030 0.507 6.9E-02 0.5844 0.07298 -0.35988 0.014296 YMR004W MVP1 0.968 0.053 0.702 5.8E-03 0.72772 0.04741 -0.26599 0.023372 YDR493W MZM1 0.914 0.060 0.480 2.5E-02 0.52851 0.05225 -0.43389 0.014122 YDR493W MZM1 0.887 0.093 0.518 1.8E-02 0.58917 0.04557 -0.36869 0.020387 YPR131C NAT3 0.578 0.055 0.050 6.9E-18 0.08733 0.00859 -0.52799 0.005462 YPR131C NAT3 0.496 0.033 0.050 6.9E-18 0.10127 0.00685 -0.44595 0.002745 YNL119W NCS2 0.727 0.151 0.131 5.0E-02 0.19113 0.0911 -0.5958 0.039369 YML120C NDI1 1.140 0.051 0.762 4.4E-02 0.66843 0.0185 -0.37773 0.001939 YDR456W NHX1 0.854 0.003 0.051 7.5E-04 0.0592 0.00103 -0.80304 9.37E-06 YGR089W NNF2 0.992 0.043 0.644 1.8E-02 0.65133 0.04504 -0.34772 0.015101 YBR170C NPL4 0.901 0.111 0.050 6.9E-18 0.05643 0.00747 -0.85067 0.008343 YBR170C NPL4 0.891 0.106 0.050 6.9E-18 0.05692 0.00674 -0.84096 0.007875 YDL167C NRP1 0.966 0.017 0.544 3.2E-02 0.56239 0.02787 -0.4226 0.001641 YDL167C NRP1 0.948 0.027 0.552 4.0E-02 0.58276 0.03613 -0.39515 0.00348 YDL167C NRP1 0.898 0.035 0.537 7.2E-02 0.60111 0.09686 -0.36083 0.036428 YDL167C NRP1 0.915 0.014 0.601 4.0E-02 0.65653 0.0401 -0.31427 0.006335 YKR082W NUP133 0.844 0.051 0.416 4.7E-02 0.49125 0.02785 -0.4279 1.83E-04 YML103C NUP188 1.161 0.090 0.866 6.7E-02 0.746 0.01156 -0.29492 0.004296 YDL116W NUP84 0.965 0.042 0.326 3.9E-02 0.33808 0.04324 -0.63921 0.00417 YBR025C OLA1 0.928 0.043 0.425 4.2E-02 0.45787 0.03054 -0.50218 0.001367 YHL020C OPI1 0.876 0.048 0.641 1.8E-02 0.7349 0.0515 -0.23456 0.03023 YPR044C OPI11 0.693 0.031 0.050 6.9E-18 0.07232 0.00313 -0.64274 0.001137 196  YDL096C OPI6 1.170 0.053 0.799 7.2E-02 0.68341 0.05245 -0.37035 0.01404 YPR075C OPY2 1.008 0.057 0.606 8.9E-02 0.60167 0.07967 -0.40145 0.022576 YGR038W ORM1 1.282 0.044 0.929 1.8E-02 0.72572 0.02575 -0.35262 0.007671 YDR071C PAA1 0.972 0.021 0.727 1.8E-02 0.74788 0.01215 -0.24508 0.001629 YJL128C PBS2 1.091 0.037 0.566 1.0E-02 0.51943 0.02306 -0.52515 0.003327 YBR035C PDX3 1.220 0.048 0.050 6.9E-18 0.04105 0.00165 -1.16984 8.54E-04 YBR035C PDX3 1.101 0.078 0.050 6.9E-18 0.04564 0.00307 -1.05081 0.002719 YBR035C PDX3 0.927 0.089 0.063 1.8E-02 0.06679 0.01331 -0.86382 0.003576 YBR035C PDX3 0.946 0.089 0.085 5.0E-02 0.08741 0.04481 -0.86095 0.002785 YBL017C PEP1 1.194 0.060 0.843 1.3E-02 0.70739 0.02876 -0.35095 0.010928 YJL053W PEP8 0.524 0.036 0.359 5.2E-02 0.68149 0.05271 -0.16499 0.004627 YNL097C PHO23 0.643 0.087 0.137 6.8E-02 0.23078 0.13724 -0.50672 0.043752 YMR123W PKR1 0.951 0.057 0.359 5.1E-02 0.37579 0.03781 -0.59268 0.001823 YPL268W PLC1 1.171 0.058 0.860 2.5E-02 0.73501 0.01552 -0.31116 0.005842 YAL023C PMT2 1.097 0.018 0.741 2.6E-02 0.67545 0.02873 -0.35651 0.004877 YOR266W PNT1 0.740 0.055 0.050 6.9E-18 0.06796 0.00528 -0.69002 0.003213 YNL055C POR1 0.837 0.065 0.460 7.6E-02 0.55112 0.09232 -0.37674 0.023362 YNL055C POR1 0.946 0.093 0.669 6.5E-02 0.70788 0.03108 -0.27704 0.011816 YNL279W PRM1 1.172 0.031 0.698 1.2E-01 0.59341 0.09093 -0.47446 0.021043 YHL011C PRS3 0.665 0.021 0.050 6.9E-18 0.07526 0.00232 -0.61497 5.80E-04 YCR079W PTC6 1.444 0.087 0.567 5.1E-02 0.39377 0.03949 -0.87697 0.005282 YJR059W PTK2 0.788 0.109 0.050 6.9E-18 0.06456 0.00817 -0.73826 0.010773 YDR496C PUF6 0.852 0.096 0.441 7.6E-02 0.51568 0.04718 -0.41104 0.007749 YDL104C QRI7 1.098 0.171 0.130 5.8E-02 0.12283 0.05841 -0.9687 0.02044 YNL250W RAD50 0.846 0.048 0.055 4.5E-03 0.06582 0.00862 -0.79031 0.002137 YER095W RAD51 0.787 0.014 0.275 5.5E-02 0.35016 0.07433 -0.51204 0.008345 YJR033C RAV1 1.026 0.030 0.532 1.1E-01 0.52161 0.12365 -0.49446 0.037783 197  YNR018W RCF2 1.331 0.126 0.330 2.5E-02 0.24995 0.02384 -1.00042 0.007189 YDR028C REG1 0.347 0.025 0.050 6.9E-18 0.14497 0.01082 -0.29681 0.003665 YBR050C REG2 1.146 0.036 0.774 7.4E-02 0.67574 0.06872 -0.37279 0.025238 YBR267W REI1 0.952 0.017 0.451 4.7E-02 0.47478 0.0558 -0.50088 0.007135 YPL066W RGL1 1.239 0.020 0.823 1.1E-02 0.66458 0.0193 -0.41583 0.002739 YLR039C RIC1 0.322 0.034 0.082 2.8E-02 0.2613 0.10482 -0.23976 0.021102 YMR154C RIM13 0.344 0.082 0.050 6.9E-18 0.15464 0.03952 -0.29398 0.036624 YOR275C RIM20 0.426 0.038 0.050 6.9E-18 0.1185 0.01115 -0.37553 0.005116 YER070W RNR1 0.554 0.006 0.050 6.9E-18 0.09028 9.95E-04 -0.5039 7.24E-05 YER070W RNR1 0.525 0.037 0.050 6.9E-18 0.09576 0.00671 -0.47469 0.002978 YER070W RNR1 0.454 0.057 0.050 6.9E-18 0.11204 0.01535 -0.40411 0.0099 YER070W RNR1 0.433 0.048 0.050 6.9E-18 0.1169 0.01306 -0.38305 0.007757 YJR063W RPA12 0.409 0.009 0.143 4.3E-02 0.35203 0.1112 -0.26625 0.017974 YBR191W RPL21A 0.736 0.012 0.065 2.2E-02 0.0891 0.031 -0.67082 0.001228 YHR010W RPL27A 0.412 0.061 0.050 6.9E-18 0.12444 0.02033 -0.36181 0.014061 YHR010W RPL27A 0.273 0.029 0.050 6.9E-18 0.1853 0.02141 -0.22323 0.008551 YHR010W RPL27A 0.402 0.072 0.094 6.2E-02 0.21871 0.11005 -0.30794 0.008524 YDL075W RPL31A 0.440 0.036 0.050 6.9E-18 0.11429 0.0089 -0.39028 0.004251 YDL075W RPL31A 0.424 0.030 0.050 6.9E-18 0.11866 0.00897 -0.37367 0.003298 YDL075W RPL31A 0.391 0.044 0.050 6.9E-18 0.12946 0.0149 -0.34132 0.008371 YDL075W RPL31A 0.372 0.083 0.050 6.9E-18 0.14048 0.02749 -0.32218 0.031759 YDL075W RPL31A 0.288 0.050 0.050 6.9E-18 0.1784 0.02758 -0.23799 0.021346 YIL148W RPL40A 0.995 0.036 0.740 1.0E-02 0.74423 0.03672 -0.25592 0.015599 YPR043W RPL43A 0.703 0.056 0.050 6.9E-18 0.07159 0.00597 -0.65304 0.003594 YML073C RPL6A 0.965 0.077 0.173 3.2E-02 0.18275 0.04669 -0.79207 0.008934 YML073C RPL6A 1.004 0.122 0.188 8.2E-02 0.19961 0.11075 -0.81591 0.028485 YML073C RPL6A 0.951 0.131 0.188 4.7E-02 0.20812 0.07735 -0.7631 0.025904 198  YML073C RPL6A 0.961 0.050 0.210 2.5E-02 0.2204 0.03714 -0.75083 0.004735 YLR448W RPL6B 0.907 0.025 0.596 5.9E-02 0.65908 0.08488 -0.31131 0.034471 YHR200W RPN10 0.835 0.031 0.516 1.7E-02 0.61951 0.03874 -0.3187 0.009151 YDL081C RPP1A 0.448 0.099 0.050 6.9E-18 0.11683 0.02348 -0.3977 0.029473 YDR025W RPS11A 0.845 0.073 0.455 6.1E-02 0.53652 0.03231 -0.3901 0.00229 YBR048W RPS11B 0.955 0.082 0.444 5.9E-02 0.47201 0.09612 -0.51143 0.030138 YDR447C RPS17B 1.208 0.014 0.828 3.3E-02 0.68585 0.03447 -0.37985 0.007294 YDR450W RPS18A 1.006 0.054 0.454 8.5E-02 0.45555 0.09628 -0.55108 0.023852 YLR441C RPS1A 0.960 0.113 0.586 2.6E-02 0.61657 0.05794 -0.37424 0.034895 YJL136C RPS21B 0.991 0.004 0.491 2.3E-02 0.49535 0.024 -0.50003 0.00118 YHR021C RPS27B 0.587 0.028 0.181 7.0E-02 0.31058 0.12657 -0.40686 0.021229 YHR021C RPS27B 0.557 0.007 0.197 1.0E-01 0.35221 0.18105 -0.36007 0.035607 YHR021C RPS27B 0.570 0.046 0.221 5.2E-02 0.3905 0.09701 -0.34986 0.022094 YHR021C RPS27B 0.615 0.018 0.243 6.2E-02 0.39474 0.09696 -0.37207 0.012868 YDL061C RPS29B 0.647 0.023 0.194 1.2E-01 0.30063 0.18661 -0.45352 0.03421 YJR145C RPS4A 0.846 0.084 0.308 1.1E-01 0.36144 0.12359 -0.53769 0.016809 YOR096W RPS7A 0.602 0.023 0.101 4.9E-02 0.16984 0.08334 -0.50114 0.007976 YBR189W RPS9B 0.942 0.040 0.355 9.4E-02 0.37605 0.09568 -0.58654 0.010099 YBL103C RTG3 1.384 0.109 0.050 6.9E-18 0.03634 0.00272 -1.33412 0.003295 YBL103C RTG3 1.081 0.035 0.135 1.1E-01 0.12507 0.10031 -0.94561 0.007275 YBL103C RTG3 1.105 0.028 0.261 1.8E-01 0.23249 0.15481 -0.84467 0.016568 YOR014W RTS1 0.905 0.041 0.483 4.8E-02 0.5351 0.05646 -0.42166 0.010759 YLL002W RTT109 0.870 0.041 0.365 9.4E-02 0.41606 0.09343 -0.50432 0.00658 YBR095C RXT2 0.884 0.016 0.127 1.1E-01 0.14541 0.12532 -0.75749 0.012612 YBR095C RXT2 1.038 0.063 0.151 3.4E-02 0.14659 0.03786 -0.88716 0.004682 YBR095C RXT2 0.959 0.041 0.215 2.0E-02 0.22512 0.02604 -0.74384 0.002498 YDL076C RXT3 0.618 0.106 0.142 3.4E-02 0.24182 0.09048 -0.47617 0.034623 YDR159W SAC3 1.003 0.041 0.714 4.5E-02 0.71098 0.01582 - 1.06E-04 199  0.28927 YMR060C SAM37 0.635 0.104 0.143 7.6E-02 0.21287 0.08773 -0.49162 0.002966 YFR040W SAP155 0.924 0.037 0.592 8.8E-02 0.6409 0.09672 -0.33232 0.034761 YMR263W SAP30 1.059 0.081 0.498 1.3E-01 0.47426 0.13568 -0.56042 0.040343 YBR037C SCO1 1.100 0.082 0.286 1.1E-01 0.25927 0.10223 -0.81459 0.010497 YBR024W SCO2 1.301 0.043 0.241 2.4E-02 0.18571 0.02197 -1.06043 0.001585 YDR469W SDC1 0.601 0.048 0.130 3.5E-02 0.21984 0.06705 -0.4708 0.01131 YDL168W SFA1 1.164 0.098 0.853 5.4E-02 0.73432 0.01481 -0.31065 0.009936 YLR403W SFP1 0.258 0.018 0.057 1.0E-02 0.22074 0.02752 -0.20112 0.001491 YLR403W SFP1 0.270 0.010 0.062 1.7E-02 0.22983 0.06227 -0.20764 0.004197 YLR403W SFP1 0.230 0.018 0.057 1.0E-02 0.24855 0.03193 -0.17242 0.002563 YOR035C SHE4 0.410 0.068 0.154 2.9E-02 0.38703 0.10404 -0.25637 0.0384 YLR079W SIC1 0.529 0.010 0.356 1.4E-02 0.67184 0.0266 -0.1738 0.003867 YHR206W SKN7 0.744 0.029 0.482 4.3E-02 0.64677 0.04471 -0.26238 0.006822 YGR271W SLH1 1.160 0.050 0.825 1.1E-02 0.71328 0.03983 -0.33455 0.015215 YBR077C SLM4 0.746 0.045 0.511 3.4E-02 0.68636 0.04626 -0.23497 0.015252 YGR229C SMI1 0.815 0.028 0.146 2.7E-02 0.17919 0.03119 -0.66915 0.001318 YGR229C SMI1 0.827 0.014 0.183 3.3E-02 0.22148 0.03831 -0.64372 0.001059 YGR229C SMI1 0.860 0.012 0.195 2.7E-02 0.22649 0.0323 -0.66539 0.001166 YDR477W SNF1 0.873 0.098 0.116 4.4E-02 0.13174 0.04759 -0.75723 0.006558 YDR477W SNF1 0.911 0.071 0.132 4.1E-02 0.14784 0.05664 -0.77977 0.008849 YDR477W SNF1 0.860 0.008 0.149 7.3E-02 0.17249 0.08367 -0.71113 0.004235 YDR477W SNF1 0.899 0.041 0.273 1.9E-01 0.2997 0.21562 -0.62686 0.039822 YDR477W SNF1 0.908 0.018 0.330 1.2E-01 0.36164 0.12913 -0.57767 0.016853 YDR477W SNF1 0.942 0.033 0.368 1.2E-01 0.38872 0.11829 -0.57424 0.017015 YGL115W SNF4 0.656 0.099 0.070 2.7E-02 0.11555 0.06309 -0.5856 0.021668 YPR101W SNT309 0.965 0.058 0.633 4.9E-02 0.65548 0.02296 -0.33219 0.00308 YDR006C SOK1 0.932 0.149 0.201 9.2E-03 0.2206 0.03031 -0.73103 0.019409 200  YPR069C SPE3 0.531 0.038 0.124 4.2E-02 0.22995 0.0611 -0.40637 5.34E-04 YJL127C SPT10 0.780 0.063 0.050 6.9E-18 0.06452 0.00548 -0.73023 0.003651 YER161C SPT2 0.690 0.038 0.457 1.1E-02 0.66508 0.05024 -0.23284 0.018934 YKR092C SRP40 0.677 0.070 0.393 7.5E-02 0.5753 0.05429 -0.28378 1.58E-04 YDL229W SSB1 0.973 0.080 0.679 4.1E-02 0.69944 0.01615 -0.29368 0.008947 YPL106C SSE1 0.580 0.081 0.322 4.2E-02 0.56124 0.06596 -0.25755 0.031818 YLR006C SSK1 0.902 0.034 0.633 2.7E-02 0.70311 0.03899 -0.2687 0.012814 YLR452C SST2 1.073 0.084 0.490 7.4E-02 0.46183 0.09127 -0.58381 0.026622 YAL011W SWC3 1.009 0.109 0.691 3.2E-02 0.68886 0.04341 -0.3188 0.028101 YBR231C SWC5 1.027 0.046 0.571 6.2E-02 0.55461 0.03666 -0.45564 8.92E-04 YDR334W SWR1 1.083 0.012 0.609 1.6E-01 0.56162 0.14503 -0.47408 0.048981 YKL081W TEF4 1.176 0.118 0.773 2.9E-02 0.66158 0.04722 -0.40319 0.028382 YOL072W THP1 0.898 0.039 0.397 4.1E-02 0.4419 0.0421 -0.50101 0.003708 YPR045C THP3 0.680 0.027 0.057 9.9E-03 0.08434 0.01728 -0.62305 0.001508 YCR053W THR4 0.568 0.120 0.050 6.9E-18 0.09218 0.01999 -0.51807 0.025625 YGR162W TIF4631 0.756 0.080 0.291 5.1E-02 0.39593 0.10892 -0.46514 0.03445 YGR162W TIF4631 0.833 0.048 0.345 2.1E-02 0.41755 0.04807 -0.4873 0.009691 YPR040W TIP41 0.910 0.020 0.429 6.8E-02 0.47084 0.06698 -0.4806 0.006276 YPR133W-A TOM5 1.132 0.026 0.050 6.9E-18 0.0442 0.00102 -1.08184 2.94E-04 YPR133W-A TOM5 1.126 0.049 0.050 6.9E-18 0.04448 0.00194 -1.07626 0.001038 YPR133W-A TOM5 1.072 0.047 0.050 6.9E-18 0.04673 0.00209 -1.02201 0.00106 YPR133W-A TOM5 1.067 0.037 0.050 6.9E-18 0.0469 0.00162 -1.01734 6.73E-04 YNL070W TOM7 1.060 0.109 0.252 8.2E-02 0.2459 0.09524 -0.80739 0.023674 YNL121C TOM70 1.266 0.272 0.538 3.8E-02 0.43733 0.06018 -0.72817 0.048843 YLR435W TSR2 0.874 0.028 0.402 5.8E-02 0.45831 0.05061 -0.47184 0.002055 YBR058C UBP14 0.693 0.028 0.379 2.1E-02 0.54812 0.03923 -0.31386 0.006857 YER151C UBP3 0.515 0.083 0.050 6.9E-18 0.0998 0.01698 -0.46503 0.015547 YER151C UBP3 0.502 0.085 0.050 6.9E-18 0.10235 0.01594 -0.45179 0.017402 201  YMR223W UBP8 0.813 0.030 0.545 6.1E-02 0.67207 0.08909 -0.26807 0.038565 YBR173C UMP1 0.443 0.019 0.050 6.9E-18 0.11307 0.00481 -0.39303 0.001219 YBR173C UMP1 0.415 0.038 0.050 6.9E-18 0.12165 0.01153 -0.36462 0.005419 YBR173C UMP1 0.404 0.042 0.050 6.9E-18 0.12515 0.0123 -0.35365 0.00702 YBR173C UMP1 0.428 0.032 0.060 1.4E-02 0.13834 0.02401 -0.36865 0.002207 YOR106W VAM3 0.921 0.039 0.511 6.8E-02 0.55718 0.08599 -0.4094 0.021833 YGL212W VAM7 0.519 0.030 0.242 6.5E-02 0.46026 0.1039 -0.27701 0.009098 YOR054C VHS3 1.129 0.011 0.848 1.5E-02 0.75109 0.01576 -0.28108 0.00235 YOR089C VPS21 0.840 0.058 0.636 4.4E-02 0.75807 0.04435 -0.2044 0.024902 YLR360W VPS38 0.988 0.016 0.688 4.1E-02 0.69652 0.03943 -0.29986 0.008455 YKR020W VPS51 0.676 0.062 0.441 4.3E-02 0.65834 0.09128 -0.23503 0.047968 YJL029C VPS53 0.328 0.097 0.114 3.2E-02 0.34993 0.0558 -0.21397 0.048677 YOL129W VPS68 1.098 0.044 0.633 5.2E-02 0.57681 0.05228 -0.46558 0.010069 YML041C VPS71 0.911 0.076 0.291 8.9E-02 0.31357 0.06848 -0.62056 7.46E-04 YDR485C VPS72 0.955 0.027 0.251 1.2E-02 0.26325 0.0203 -0.70416 0.001499 YGL104C VPS73 0.908 0.099 0.644 3.1E-02 0.71522 0.05417 -0.26339 0.037906 YML097C VPS9 0.330 0.039 0.052 2.5E-03 0.15918 0.01909 -0.2779 0.009618 YJL012C VTC4 1.196 0.037 0.671 6.6E-02 0.56237 0.0671 -0.52564 0.016238 YJL012C VTC4 1.205 0.056 0.762 8.3E-02 0.63559 0.0881 -0.44284 0.036313 YGL173C XRN1 0.566 0.026 0.149 5.6E-02 0.26825 0.10778 -0.41689 0.018477 YGL173C XRN1 0.575 0.019 0.168 4.9E-02 0.29025 0.07704 -0.4071 0.004258 YBR174C YBR174C 0.676 0.029 0.352 5.2E-02 0.51951 0.05939 -0.32365 0.005566 YBR277C YBR277C 0.967 0.069 0.695 5.0E-02 0.72217 0.06954 -0.27223 0.044643 YNL064C YDJ1 0.578 0.067 0.127 3.0E-02 0.22126 0.05473 -0.45117 0.010154 YDL032W YDL032W 0.742 0.082 0.050 6.9E-18 0.06813 0.00705 -0.69233 0.006952 YDL118W YDL118W 1.219 0.024 0.364 1.6E-01 0.29696 0.13084 -0.85517 0.014594 YDL119C YDL119C 1.199 0.075 0.313 5.0E-02 0.26224 0.04375 -0.88564 0.004965 YDL157C YDL157C 1.148 0.108 0.266 5.2E-02 0.2384 0.07018 -0.88161 0.016134 202  YDL172C YDL172C 0.928 0.083 0.477 2.7E-02 0.51562 0.02103 -0.45106 0.008239 YDL180W YDL180W 0.899 0.114 0.538 2.0E-02 0.60556 0.06218 -0.36138 0.039253 YDR455C YDR455C 0.726 0.063 0.050 6.9E-18 0.06943 0.00618 -0.67577 0.004348 YER137C YER137C 1.117 0.083 0.866 5.5E-02 0.77594 0.01123 -0.25111 0.006716 YDL072C YET3 0.671 0.059 0.141 6.5E-02 0.21631 0.10863 -0.52957 0.018389 YDL072C YET3 0.719 0.057 0.164 1.3E-01 0.21903 0.16661 -0.55568 0.017002 YDL072C YET3 0.519 0.071 0.210 3.8E-02 0.41281 0.09644 -0.30897 0.030019 YFL013W-A YFL013W-A 0.324 0.077 0.050 6.9E-18 0.16206 0.0332 -0.27428 0.037184 YGL072C YGL072C 0.821 0.157 0.234 1.8E-01 0.25911 0.1552 -0.58758 0.009135 YGL088W YGL088W 0.985 0.049 0.252 5.9E-02 0.25945 0.07214 -0.73327 0.010685 YGL088W YGL088W 0.978 0.043 0.296 5.6E-02 0.30512 0.06497 -0.68134 0.008236 YGL088W YGL088W 0.978 0.059 0.328 7.9E-03 0.33731 0.02857 -0.64959 0.005176 YGL088W YGL088W 0.895 0.027 0.324 1.3E-01 0.35815 0.12865 -0.57079 0.014844 YGL149W YGL149W 0.617 0.035 0.166 7.2E-02 0.26592 0.11237 -0.4513 0.008397 YGL149W YGL149W 0.629 0.015 0.226 8.5E-02 0.36009 0.13353 -0.40287 0.022067 YGL218W YGL218W 0.997 0.029 0.050 6.9E-18 0.05019 0.00152 -0.94707 4.84E-04 YGL218W YGL218W 0.998 0.007 0.060 1.3E-02 0.05967 0.01365 -0.93851 1.65E-04 YGL218W YGL218W 1.036 0.050 0.231 5.9E-02 0.2254 0.0641 -0.80469 0.007181 YGL218W YGL218W 1.092 0.026 0.248 7.1E-02 0.22733 0.06595 -0.84421 0.004478 YGL235W YGL235W 1.085 0.093 0.061 9.7E-03 0.05548 0.00478 -1.0249 0.003389 YIL092W YIL092W 1.142 0.020 0.625 2.5E-02 0.54789 0.02821 -0.51673 0.002989 YJL169W YJL169W 0.797 0.022 0.520 7.9E-02 0.6505 0.08019 -0.27687 0.020842 YJR018W YJR018W 0.346 0.040 0.050 6.9E-18 0.14638 0.01553 -0.29579 0.008868 YJR018W YJR018W 0.328 0.026 0.050 6.9E-18 0.15362 0.01298 -0.27769 0.004398 YJR120W YJR120W 1.093 0.049 0.105 5.0E-02 0.09448 0.04034 -0.98816 2.21E-05 YLR200W YKE2 0.534 0.035 0.334 2.8E-02 0.62573 0.01589 -0.19952 0.001546 YML090W YML090W 0.573 0.033 0.050 6.9E-18 0.08757 0.00505 -0.52287 0.001982 203  YMR057C YMR057C 0.782 0.039 0.152 2.3E-02 0.19356 0.01984 -0.62979 3.25E-04 YMR057C YMR057C 0.777 0.040 0.169 4.0E-02 0.21626 0.04131 -0.60769 4.29E-04 YMR057C YMR057C 0.774 0.034 0.182 5.6E-03 0.23582 0.00564 -0.5919 0.001238 YMR057C YMR057C 0.767 0.041 0.193 3.4E-02 0.25032 0.03629 -0.57444 0.001612 YNL120C YNL120C 0.798 0.135 0.091 4.2E-02 0.11792 0.06209 -0.70706 0.021319 YNL122C YNL122C 1.102 0.021 0.783 4.3E-02 0.71147 0.05278 -0.31903 0.019075 YNL140C YNL140C 0.878 0.162 0.473 6.1E-02 0.54441 0.03895 -0.40516 0.033373 YNL170W YNL170W 0.588 0.032 0.350 9.0E-02 0.59012 0.13426 -0.23816 0.040153 YNL171C YNL171C 0.769 0.040 0.050 6.9E-18 0.06516 0.00332 -0.71938 0.001529 YNL235C YNL235C 0.980 0.120 0.668 6.9E-02 0.68241 0.01425 -0.31287 0.012898 YNR005C YNR005C 1.343 0.055 0.344 2.7E-02 0.2562 0.01256 -0.99852 5.68E-04 YNR025C YNR025C 0.694 0.033 0.477 7.5E-02 0.68378 0.07336 -0.21701 0.018092 YOR309C YOR309C 1.035 0.038 0.487 1.2E-02 0.47128 0.00758 -0.54729 0.001213 YOR309C YOR309C 1.089 0.040 0.512 7.3E-02 0.47283 0.08354 -0.57724 0.017444 YOR309C YOR309C 0.673 0.019 0.327 5.6E-02 0.48453 0.07471 -0.34619 0.008663 YOR343C YOR343C 1.170 0.028 0.873 8.4E-02 0.74609 0.07012 -0.29727 0.036629 YPL150W YPL150W 1.206 0.021 0.872 8.0E-02 0.72271 0.05894 -0.3338 0.02033 YML001W YPT7 0.618 0.073 0.231 1.1E-01 0.36689 0.15648 -0.38704 0.030286 YJL056C ZAP1 0.653 0.121 0.129 1.9E-02 0.21216 0.07723 -0.52389 0.033906 YNL241C ZWF1 1.095 0.036 0.700 1.1E-01 0.64047 0.11095 -0.39501 0.045675  Table 1. Synthetic dosage lethal (SDL) interactions identified by Pah1 SDL screen Table detailing SDL interactions identified in the Pah1 SDL screen sorted alphabetically by gene name. Hits were identified using Balony software [119] and are listed with their control (non-inducing, no Pah1 overexpression) and experimental (inducing, Pah1 overexpression) colony size measurements, and the ratio between experimental and control plates. SDL hits were defined as those that had a ratio of colony size of 0.800 or less between experimental and control conditions, and a p<0.05 in all three biological replicates. 204  Protein Rank Peptide < ProteinMetrics Confidential > Modification Type(s) Observed m/z z Observed (M+H) Calc. mass (M+H) Off-by-x error Mass error (ppm) Score Delta Delta Mod |Log Prob| 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.022 4 4313.067 4314.054 -1 3.8 444.3 444.3 444.3 3.49 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.027 4 4313.085 4314.054 -1 8.0 363.0 363.0 363.0 0.93 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.024 4 4313.073 4314.054 -1 5.2 322.4 322.4 322.4 1.48 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.696 3 4314.074 4314.054 0 4.6 878.8 878.8 244.7 11.25 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.696 3 4314.075 4314.054 0 4.8 752.9 752.9 222.1 9.25 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.696 3 4314.072 4314.054 0 4.2 732.9 732.9 219.6 8.55 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.874 5 4785.341 4785.323 0 3.8 991.3 991.3 215.4 13.28 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.273 4 4314.072 4314.054 0 4.1 1266.5 1266.5 201.4 30.54 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.874 5 4785.343 4785.323 0 4.2 665.7 665.7 192.2 7.28 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.695 3 4314.071 4314.054 0 3.9 731.2 731.2 185.9 8.55 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.875 5 4785.348 4785.323 0 5.2 1237.8 1237.8 179.5 30.54 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.273 4 4314.071 4314.054 0 4.1 1125.6 1125.6 173.5 16.50 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.876 5 4785.349 4785.323 0 5.4 1068.1 1068.1 172.9 14.98 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.273 4 4314.070 4314.054 0 3.8 1263.6 1263.6 165.7 30.54 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.697 3 4314.077 4314.054 0 5.4 498.8 498.8 161.2 4.13 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.696 3 4314.075 4314.054 0 4.8 645.5 645.5 145.5 6.25 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1197.091 4 4785.340 4785.323 0 3.6 753.2 753.2 144.9 9.18 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.273 4 4314.069 4314.054 0 3.5 1138.1 1138.1 140.8 16.50 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.875 5 4785.344 4785.323 0 4.3 1004.1 1004.1 138.0 13.98 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.273 4 4314.071 4314.054 0 3.9 766.2 766.2 135.4 9.65 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.273 4 4314.070 4314.054 0 3.7 979.6 979.6 126.7 13.65 205  1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.697 3 4314.077 4314.054 0 5.3 630.0 630.0 125.8 6.25 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.697 3 4314.077 4314.054 0 5.4 483.0 483.0 123.9 3.15 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1197.092 4 4785.345 4785.323 0 4.5 427.2 427.2 123.4 3.67 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.273 4 4314.071 4314.054 0 3.9 761.6 761.6 115.8 9.65 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.874 5 4785.340 4785.323 0 3.5 446.4 446.4 114.9 3.12 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.695 3 4314.071 4314.054 0 4.0 314.0 314.0 102.0 1.57 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1262.082 2 2523.157 2523.144 0 5.2 462.7 462.7 99.7 4.46 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1262.080 2 2523.152 2523.144 0 3.1 755.9 755.9 97.7 10.92 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1262.081 2 2523.155 2523.144 0 4.4 560.2 560.2 82.9 6.62 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.273 4 4314.068 4314.054 0 3.4 937.5 937.5 81.2 12.65 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.874 5 4785.341 4785.323 0 3.8 341.7 341.7 75.4 1.68 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.876 5 4785.350 4785.323 0 5.6 1038.9 1038.9 74.6 13.98 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.272 4 4314.065 4314.054 0 2.7 786.5 786.5 72.9 9.24 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.271 4 4314.064 4314.054 0 2.3 640.7 640.7 71.3 6.24 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.270 4 4314.058 4314.054 0 1.0 647.3 647.3 70.2 5.90 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1262.090 2 2523.173 2523.144 0 11.2 488.3 488.3 68.8 3.04 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1197.088 4 4785.332 4785.323 0 1.8 203.1 203.1 63.5 0.11 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.696 3 4314.072 4314.054 0 4.2 502.4 502.4 63.0 4.44 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1262.078 2 2523.148 2523.144 0 1.6 400.0 400.0 61.2 3.96 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1439.037 3 4315.097 4314.054 1 9.3 263.6 263.6 58.5 0.20 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.876 5 4785.348 4785.323 0 5.3 433.1 433.1 56.1 3.77 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1262.081 2 2523.154 2523.144 0 4.0 370.3 370.3 55.0 4.28 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1262.078 2 2523.149 2523.144 0 2.0 820.7 820.7 52.1 11.52 206  1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1217.083 4 4865.311 4865.289 0 4.4 695.6 695.6 51.3 6.88 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.271 4 4314.063 4314.054 0 2.2 661.4 652.8 49.6 7.24 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], Q[+1] 1439.363 3 4316.075 4315.038 1 7.9 495.0 495.0 49.0 3.24 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1262.077 2 2523.146 2523.144 0 0.6 595.0 595.0 45.2 5.05 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1301.568 2 2602.129 2602.126 0 0.8 676.1 676.1 44.2 8.18 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], Q[+1] 1079.773 4 4316.069 4315.038 1 6.5 281.8 281.8 43.2 0.84 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.273 4 4314.071 4314.054 0 3.9 587.0 587.0 41.8 5.65 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], Q[+1] 1079.273 4 4314.068 4315.038 -1 7.8 546.7 546.7 39.6 3.48 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1262.082 2 2523.157 2523.144 0 5.2 618.0 618.0 38.0 7.62 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.270 4 4314.058 4314.054 0 1.0 649.6 649.6 37.3 5.90 1 R.SANQSPQS[+79.966]VGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1216.830 4 4864.297 4865.289 -1 2.3 218.0 218.0 36.3 0.11 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1301.570 2 2602.132 2602.126 0 2.2 701.8 701.8 33.6 9.52 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1217.082 4 4865.307 4865.289 0 3.5 784.0 784.0 32.0 9.18 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1301.569 2 2602.130 2602.126 0 1.3 818.0 818.0 31.7 11.18 1 R.SAN[+0.984]Q[+0.984]SPQSVGGSGIDSGVESTSDSLR.D N[+1], Q[+1] 1262.578 2 2524.148 2524.128 0 7.7 233.5 233.5 30.5 0.38 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1217.083 4 4865.308 4865.289 0 3.9 635.0 635.0 30.5 6.18 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1301.571 2 2602.134 2602.126 0 2.9 838.2 838.2 27.0 11.92 1 R.SANQSPQS[+79.966]VGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1465.351 3 4394.038 4394.020 0 4.1 299.4 299.4 25.8 1.49 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1301.567 2 2602.126 2602.126 0 -0.1 455.8 455.8 22.3 4.02 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1301.568 2 2602.128 2602.126 0 0.5 671.1 671.1 21.8 7.19 1 R.SANQ[+0.984]SPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1]*2 1262.579 2 2524.150 2524.128 0 8.8 364.9 364.9 20.8 1.74 1 R.SANQ[+0.984]SPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1]*2 1262.579 2 2524.150 2524.128 0 8.6 211.9 211.9 20.6 0.04 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], Q[+1] 957.875 5 4785.343 4786.307 -1 8.3 276.4 276.4 20.4 0.30 207  1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1217.078 4 4865.291 4865.289 0 0.2 219.3 219.3 19.5 0.02 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1465.352 3 4394.041 4394.020 0 4.8 318.3 318.3 19.4 1.28 1 R.SANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1301.566 2 2602.125 2602.126 0 -0.7 279.6 279.6 18.7 1.76 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1301.565 2 2602.122 2602.126 0 -1.7 473.5 473.5 17.4 3.88 1 R.SANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1301.566 2 2602.125 2602.126 0 -0.6 302.2 302.2 17.2 1.96 1 R.SANQ[+0.984]SPQSVGGSGIDSGVESTSDSLR.D Q[+1] 1262.079 2 2523.151 2523.144 0 2.6 804.4 804.4 16.6 11.52 1 R.SANQSPQ[+0.984]S[+79.966]VGGSGIDSGVESTSDSLR.D Q[+1], S[+80] 1302.066 2 2603.124 2603.110 0 5.4 276.1 71.7 15.0 0.71 1 R.SANQSPQS[+79.966]VGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1465.353 3 4394.044 4394.020 0 5.4 379.4 379.4 13.7 2.83 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1217.082 4 4865.307 4865.289 0 3.5 443.1 443.1 13.2 4.01 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], Q[+1] 1079.271 4 4314.064 4315.038 -1 6.8 429.6 425.9 12.1 2.66 1 R.SANQSPQS[+79.966]VGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1465.353 3 4394.045 4394.020 0 5.6 364.9 364.9 10.9 1.82 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1217.083 4 4865.309 4865.289 0 4.0 393.0 393.0 10.3 2.76 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1301.568 2 2602.128 2602.126 0 0.5 327.5 327.5 10.0 1.97 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], Q[+1] 1197.090 4 4785.337 4786.307 -1 7.0 317.2 317.2 8.2 0.71 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1262.079 2 2523.151 2523.144 0 2.6 452.1 452.1 8.0 4.22 1 R.SANQS[+79.966]PQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1], S[+80] 1302.066 2 2603.124 2603.110 0 5.3 335.9 117.4 7.4 1.04 1 R.SANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1301.570 2 2602.133 2602.126 0 2.4 548.7 548.7 5.8 5.55 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1301.567 2 2602.127 2602.126 0 0.0 342.1 342.1 5.6 2.45 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1301.568 2 2602.129 2602.126 0 0.8 724.6 724.6 5.3 9.18 1 R.SANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1301.569 2 2602.131 2602.126 0 1.6 552.5 552.5 5.1 5.55 1 R.SANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1301.571 2 2602.134 2602.126 0 2.8 574.4 574.4 3.2 6.92 1 R.SANQ[+0.984]SPQSVGGSGIDSGVESTSDSLR.D Q[+1] 1262.079 2 2523.151 2523.144 0 2.8 786.3 786.3 1.9 10.92 1 R.SAN[+0.984]QSPQSVGGSGIDSGVESTSDSLR.D N[+1] 1262.079 2 2523.150 2523.144 0 2.4 513.4 513.4 1.5 4.28 208  Table 2. Peptides identified at lipin 1 S468 and S472 in mass spectrometry analysis of FLAG-lipin 1 wild type immunoprecipitated from HEK 293T cells treated with DMSO Peptides identified by mass spectrometry analysis of FLAG-lipin 1 wild type immunoprecipitated from HEK 293T cells treated with DMSO for 16 hours. Analysis was performed using Byonic Software (California, USA) and peptides are ranked by DeltaMod score. When analyzing phosphopeptides, we considered DeltaMod scores above 10.0 as a high likelihood of the correct placement of modifications for a given peptide, as defined and suggested by the Byonic support materials (https://www.proteinmetrics.com/support-information/).   209  Protein Rank Peptide < ProteinMetrics Confidential > Modification Type(s) Observed m/z z Observed (M+H) Calc. mass (M+H) Off-by-x error Mass error (ppm) Score Delta Delta Mod |Log Prob| 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.594 2 2506.180 2506.165 0 6.0 954.9 73.5 73.5 13.27 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.575 2 2586.143 2586.132 0 4.4 931.9 95.9 57.1 11.56 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.366 3 4298.084 4298.059 0 5.8 361.7 53.3 53.3 1.04 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.366 3 4298.084 4298.059 0 5.7 939.9 50.0 50.0 10.49 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.574 2 2586.142 2586.132 0 3.9 788.6 48.9 48.9 9.27 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1213.086 4 4849.324 4849.295 0 6.0 755.9 52.3 45.2 6.22 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.577 2 2586.146 2586.132 0 5.5 830.5 63.5 45.1 10.27 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.366 3 4298.082 4298.059 0 5.4 777.8 45.1 45.1 7.49 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1460.024 3 4378.056 4378.025 0 7.1 588.4 44.7 44.7 3.02 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.570 2 2586.133 2586.132 0 0.6 721.7 71.7 44.2 7.26 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.095 4 4769.358 4769.328 0 6.3 1019.9 43.7 43.7 11.22 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.094 4 4769.355 4769.328 0 5.7 630.8 43.4 43.4 3.69 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1213.087 4 4849.327 4849.295 0 6.7 758.9 42.9 42.9 6.22 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.575 2 2586.142 2586.132 0 4.2 641.0 59.5 41.3 6.27 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.367 3 4298.088 4298.059 0 6.7 668.8 40.9 40.9 5.02 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.576 2 2586.145 2586.132 0 5.4 939.2 112.3 39.1 11.56 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.574 2 2586.141 2586.132 0 3.5 467.1 50.0 38.9 3.14 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.094 4 4769.355 4769.328 0 5.7 836.3 38.0 38.0 7.69 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.574 2 2586.140 2586.132 0 3.2 681.6 37.1 37.1 7.27 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.592 2 2506.177 2506.165 0 4.9 781.5 36.9 36.9 9.27 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1460.024 3 4378.058 4378.025 0 7.4 747.3 36.7 36.7 6.02 210  1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.677 5 4769.357 4769.328 0 6.1 1102.5 36.1 36.1 13.31 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.575 2 2586.142 2586.132 0 4.0 805.4 75.1 36.1 10.27 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1460.024 3 4378.057 4378.025 0 7.2 665.8 35.6 35.6 5.02 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1213.087 4 4849.324 4849.295 0 6.1 1019.0 35.4 35.4 11.22 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.023 4 4297.069 4298.059 -1 3.0 296.3 35.0 35.0 0.32 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.021 4 4297.063 4298.059 -1 1.8 353.1 34.9 34.9 0.39 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.594 2 2506.180 2506.165 0 5.9 792.7 34.5 34.5 9.27 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.573 2 2586.138 2586.132 0 2.5 664.0 33.8 33.8 6.26 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1213.087 4 4849.324 4849.295 0 6.1 1015.3 33.3 33.3 11.22 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.276 4 4298.081 4298.059 0 5.0 1066.6 32.7 32.7 13.58 1 R.AANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1254.085 2 2507.162 2507.149 0 5.1 703.3 32.2 32.2 8.27 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.590 2 2506.173 2506.165 0 3.1 732.2 30.7 30.7 8.27 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.365 3 4298.081 4298.059 0 5.2 414.0 30.3 30.3 1.16 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.578 2 2586.148 2586.132 0 6.5 762.3 30.2 30.2 8.80 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1459.687 3 4377.045 4378.025 -1 5.2 306.5 34.7 29.6 0.73 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1460.024 3 4378.057 4378.025 0 7.3 612.8 29.3 29.3 4.02 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.577 2 2586.146 2586.132 0 5.6 359.0 60.2 29.3 2.79 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.362 3 4298.073 4298.059 0 3.2 500.4 39.3 28.1 2.40 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.593 2 2506.178 2506.165 0 5.1 734.5 27.5 27.5 8.27 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.588 2 2506.169 2506.165 0 1.3 441.9 26.9 26.9 1.99 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.585 2 2506.163 2506.165 0 -1.0 670.2 26.7 26.7 6.14 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.574 2 2586.141 2586.132 0 3.7 656.9 72.9 26.6 7.27 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.367 3 4298.085 4298.059 0 6.0 224.0 26.5 26.5 0.07 211  1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.592 2 2506.176 2506.165 0 4.4 822.0 26.2 26.2 10.27 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.094 4 4769.355 4769.328 0 5.7 664.5 26.1 26.1 4.69 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1213.086 4 4849.320 4849.295 0 5.3 671.7 25.8 25.8 4.69 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.575 2 2586.143 2586.132 0 4.6 460.2 25.4 25.4 2.99 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1460.024 3 4378.057 4378.025 0 7.2 716.8 25.0 25.0 6.02 1 R.AANQSPQSVGGS[+79.966]GIDSGVESTSDSLR.D S[+80] 1293.576 2 2586.145 2586.132 0 5.1 326.9 26.9 25.0 2.48 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.592 2 2506.177 2506.165 0 4.5 823.6 24.9 24.9 10.27 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.476 5 4768.350 4769.328 -1 5.4 516.2 24.5 24.5 1.69 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.094 4 4769.353 4769.328 0 5.1 575.8 24.2 24.2 2.69 1 R.AANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1254.089 2 2507.171 2507.149 0 8.6 588.2 24.0 24.0 4.80 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1293.575 2 2586.143 2586.132 0 4.3 271.2 23.8 23.2 2.32 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1460.023 3 4378.055 4378.025 0 6.7 740.6 22.5 22.5 6.02 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.677 5 4769.354 4769.328 0 5.3 453.8 22.4 22.4 1.64 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.592 2 2506.177 2506.165 0 4.8 386.5 21.9 21.9 3.01 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.574 2 2586.142 2586.132 0 3.9 637.6 69.6 21.6 6.27 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.276 4 4298.082 4298.059 0 5.3 238.5 21.4 21.4 0.13 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.592 2 2506.176 2506.165 0 4.4 441.1 21.2 21.2 3.14 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.024 4 4297.073 4298.059 -1 4.0 383.4 21.2 21.2 1.20 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.589 2 2506.171 2506.165 0 2.5 620.5 21.0 21.0 5.26 1 R.AANQ[+0.984]SPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], Q[+1] 1433.363 3 4298.076 4299.043 -1 8.4 701.1 54.4 20.7 6.02 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.594 2 2506.181 2506.165 0 6.1 680.2 18.3 18.3 6.80 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.575 2 2586.142 2586.132 0 4.0 562.5 59.2 18.2 5.27 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.094 4 4769.355 4769.328 0 5.7 367.9 44.7 18.1 0.46 212  1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.587 2 2506.167 2506.165 0 0.5 569.4 17.3 17.3 4.26 1 R.AANQ[+0.984]SPQSVGGSGIDSGVESTSDSLR.D Q[+1] 1254.084 2 2507.160 2507.149 0 4.4 761.2 26.8 16.9 9.27 1 R.AANQSPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], Q[+1] 1193.595 4 4771.358 4770.312 1 9.0 491.8 16.6 16.6 0.52 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.591 2 2506.174 2506.165 0 3.6 649.1 16.5 16.5 6.27 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1460.023 3 4378.054 4378.025 0 6.5 407.0 15.0 15.0 0.74 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.276 4 4298.080 4298.059 0 5.0 1097.7 14.8 14.8 13.58 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1293.573 2 2586.138 2586.132 0 2.4 269.9 14.6 14.6 1.10 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.367 3 4298.087 4298.059 0 6.5 710.1 14.4 14.4 6.02 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1460.022 3 4378.052 4378.025 0 6.1 411.6 28.2 14.1 0.74 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.024 4 4297.075 4298.059 -1 4.6 215.4 13.7 13.7 0.21 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.095 4 4769.356 4769.328 0 5.8 676.9 12.8 12.8 4.69 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.575 2 2586.142 2586.132 0 4.1 670.7 42.9 12.7 7.27 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.574 2 2586.141 2586.132 0 3.8 362.6 48.0 11.8 2.87 1 R.AANQSPQSVGGS[+79.966]GIDSGVESTSDSLR.D S[+80] 1293.573 2 2586.138 2586.132 0 2.4 431.6 29.6 11.6 1.91 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.678 5 4769.359 4769.328 0 6.4 1135.8 11.6 11.6 13.31 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.024 4 4297.073 4298.059 -1 4.2 322.1 11.3 11.3 0.85 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.677 5 4769.355 4769.328 0 5.6 403.4 10.9 10.9 1.56 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1293.576 2 2586.145 2586.132 0 5.0 223.3 11.7 10.4 0.82 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.274 4 4298.073 4298.059 0 3.3 535.8 10.2 10.2 2.64 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.587 2 2506.166 2506.165 0 0.2 506.2 10.0 10.0 2.19 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.574 2 2586.141 2586.132 0 3.6 527.9 30.1 9.0 4.34 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.592 2 2506.176 2506.165 0 4.5 691.2 8.4 8.4 7.27 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1293.576 2 2586.145 2586.132 0 5.0 580.0 23.2 8.0 5.27 213  1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.591 2 2506.174 2506.165 0 3.6 345.7 7.5 7.5 2.48 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.275 4 4298.080 4298.059 0 4.8 1236.5 7.0 7.0 16.48 1 R.AANQSPQSVGGS[+79.966]GIDSGVESTSDSLR.D S[+80] 1293.575 2 2586.142 2586.132 0 4.1 334.6 42.9 6.8 2.86 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1293.576 2 2586.144 2586.132 0 5.0 393.8 27.4 6.8 2.79 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.276 4 4298.083 4298.059 0 5.5 623.4 6.1 6.1 4.58 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.577 2 2586.146 2586.132 0 5.6 265.7 45.4 5.4 2.08 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.276 4 4298.083 4298.059 0 5.5 1215.7 5.3 5.3 16.48 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.586 2 2506.165 2506.165 0 0.0 582.5 5.0 5.0 4.14 1 R.AAN[+0.984]QSPQSVGGSGIDSGVESTSDSLR.D N[+1] 1254.085 2 2507.163 2507.149 0 5.7 791.7 25.8 4.9 9.27 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.574 2 2586.140 2586.132 0 3.3 538.3 12.4 4.5 4.34 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1293.573 2 2586.138 2586.132 0 2.6 705.5 28.3 4.5 7.26 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.276 4 4298.083 4298.059 0 5.5 292.0 17.4 4.4 0.81 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.676 5 4769.352 4769.328 0 5.0 688.8 3.9 3.9 4.78 1 R.AANQ[+0.984]SPQSVGGSGIDSGVESTSDSLR.D Q[+1] 1254.083 2 2507.159 2507.149 0 3.9 519.0 18.7 3.8 4.18 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.676 5 4769.350 4769.328 0 4.6 1040.3 3.7 3.7 11.78 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1293.575 2 2586.144 2586.132 0 4.7 228.9 6.9 3.5 1.27 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1460.024 3 4378.057 4378.025 0 7.2 300.7 3.2 3.2 0.40 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.367 3 4298.088 4298.059 0 6.7 373.5 2.4 2.4 0.71 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.590 2 2506.173 2506.165 0 3.0 438.3 2.2 2.2 2.05 1 R.AANQSPQSVGGSGIDS[+79.966]GVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1617.112 3 4849.321 4849.295 0 5.4 266.3 9.0 1.2 0.04 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.574 2 2586.142 2586.132 0 3.9 506.3 22.5 0.8 3.20 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.274 4 4298.074 4298.059 0 3.5 722.8 0.0 0.0 6.58 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1617.113 3 4849.323 4849.295 0 5.9 288.1 0.0 0.0 0.09 214  Table 3. Peptides identified at lipin 1 S468 and S472 in mass spectrometry analysis of FLAG-lipin 1 S468A immunoprecipitated from HEK 293T cells treated with DMSO Peptides identified by mass spectrometry analysis of FLAG-lipin 1 S468A immunoprecipitated from HEK 293T cells treated with DMSO for 16 hours. Analysis was performed using Byonic Software (California, USA) and peptides are ranked by DeltaMod score. When analyzing phosphopeptides, we considered DeltaMod scores above 10.0 as a high likelihood of the correct placement of modifications for a given peptide, as defined and suggested by the Byonic support materials (https://www.proteinmetrics.com/support-information/).   215   Protein Rank Peptide < ProteinMetrics Confidential > Modification Type(s) Observed m/z z Observed (M+H) Calc. mass (M+H) Off-by-x error Mass error (ppm) Score Delta Delta Mod |Log Prob| # of unique peptides 1 R.SANQAPQSVGGSGIDS[+79.966]GVESTSDSLR.D S[+80] 1293.575 2 2586.143 2586.132 0 4.4 634.9 22.2 0.6 5.36 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.696 3 4299.073 4298.059 1 2.4 574.0 1.0 1.0 2.56 299 1 R.SANQAPQSVGGSGIDS[+79.966]GVESTSDSLR.D S[+80] 1293.574 2 2586.141 2586.132 0 3.8 427.5 51.2 2.3 2.14 299 1 R.SANQAPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1254.085 2 2507.163 2507.149 0 5.3 640.1 37.5 3.9 5.49 299 1 R.SANQAPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], Q[+1] 1075.273 4 4298.070 4299.043 -1 7.2 393.8 29.4 4.9 0.60 299 1 R.SANQAPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], Q[+1] 1075.776 4 4300.082 4299.043 1 8.2 201.4 32.7 6.8 0.02 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.092 4 4769.346 4769.328 0 3.7 353.9 38.2 7.2 0.11 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.364 3 4298.076 4298.059 0 3.9 556.4 39.4 7.3 1.99 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.592 2 2506.176 2506.165 0 4.3 303.0 7.5 7.5 1.66 299 1 R.SANQAPQSVGGS[+79.966]GIDSGVESTSDSLR.D S[+80] 1293.577 2 2586.147 2586.132 0 6.0 217.2 33.0 8.1 0.62 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   836.059 3 2506.162 2506.165 0 -1.5 236.4 9.5 9.5 0.32 299 1 R.SANQAPQSVGGSGIDS[+79.966]GVESTSDSLR.D S[+80] 1293.576 2 2586.145 2586.132 0 5.2 591.8 65.3 10.3 4.49 299 1 R.SANQ[+0.984]APQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], Q[+1] 1075.522 4 4299.067 4299.043 0 5.5 714.8 39.2 11.9 6.19 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.676 5 4769.352 4769.328 0 5.0 218.7 22.1 12.1 0.01 299 1 R.SANQAPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], Q[+1] 1075.273 4 4298.068 4299.043 -1 6.7 335.6 50.9 13.2 0.56 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.278 4 4298.090 4298.059 0 7.1 308.1 13.2 13.2 0.52 299 1 R.SANQAPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], Q[+1] 1075.275 4 4298.076 4299.043 -1 8.5 359.7 32.8 13.6 0.34 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.364 3 4298.077 4298.059 0 4.1 441.1 38.2 15.2 0.90 299 1 R.SANQ[+0.984]APQSVGGSGIDSGVESTSDSLR.D Q[+1] 1254.086 2 2507.164 2507.149 0 6.0 739.7 82.1 18.2 7.07 299 1 R.SANQAPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1254.086 2 2507.164 2507.149 0 5.9 620.1 60.9 19.2 5.49 299 216  1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.276 4 4298.083 4298.059 0 5.6 229.2 20.9 20.9 0.11 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.676 5 4769.349 4769.328 0 4.3 491.6 21.4 21.4 0.79 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.579 2 2506.152 2506.165 0 -5.4 214.2 22.3 22.3 0.12 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.034 3 4297.086 4298.059 -1 7.1 202.2 24.9 24.9 0.05 299 1 R.SANQAPQSVGGSGIDS[+79.966]GVESTSDSLR.D S[+80] 1293.575 2 2586.143 2586.132 0 4.4 585.5 43.1 25.1 4.36 299 1 R.SANQAPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], Q[+1] 1075.280 4 4298.099 4299.043 -1 13.8 228.6 38.0 26.7 0.00 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.679 5 4769.367 4769.328 0 8.1 261.6 27.6 27.6 0.02 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.525 4 4299.079 4298.059 1 3.9 434.4 28.7 28.7 1.83 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.587 2 2506.167 2506.165 0 0.8 294.7 29.3 29.3 1.13 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.031 4 4297.102 4298.059 -1 10.7 214.7 29.8 29.8 0.01 299 1 R.SANQAPQSVGGSGIDS[+79.966]GVESTSDSLR.D S[+80] 1293.575 2 2586.144 2586.132 0 4.6 298.5 37.1 30.2 1.66 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.593 2 2506.178 2506.165 0 5.1 424.5 32.6 32.6 2.09 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.588 2 2506.168 2506.165 0 1.0 286.8 33.4 33.4 1.28 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.366 3 4298.085 4298.059 0 6.0 284.0 34.2 34.2 0.42 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1590.457 3 4769.357 4769.328 0 6.0 248.1 35.6 35.6 0.01 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.368 3 4298.088 4298.059 0 6.9 274.8 35.7 35.7 0.27 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.024 4 4297.075 4298.059 -1 4.5 281.7 36.0 36.0 0.51 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.593 2 2506.179 2506.165 0 5.6 289.3 36.6 36.6 1.79 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.677 5 4769.358 4769.328 0 6.3 810.5 36.9 36.9 6.70 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.276 4 4298.081 4298.059 0 5.1 473.5 38.0 38.0 2.06 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.677 5 4769.357 4769.328 0 6.1 872.0 38.2 38.2 7.70 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.024 4 4297.073 4298.059 -1 4.1 416.4 38.7 38.7 1.83 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.029 4 4297.094 4298.059 -1 8.8 292.8 38.9 38.9 0.15 299 217  1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.093 4 4769.349 4769.328 0 4.4 356.3 39.9 39.9 0.07 299 1 R.SANQAPQSVGGSGIDS[+79.966]GVESTSDSLR.D S[+80] 1293.577 2 2586.147 2586.132 0 6.1 526.7 54.2 39.9 3.24 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.676 5 4769.352 4769.328 0 5.0 628.4 56.9 40.0 2.98 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.880 5 4770.369 4769.328 1 7.9 263.4 49.2 40.4 0.02 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.029 4 4297.093 4298.059 -1 8.7 268.6 40.7 40.7 0.05 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.677 5 4769.354 4769.328 0 5.3 999.0 40.8 40.8 9.98 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.694 3 4299.067 4298.059 1 1.0 380.2 41.6 41.6 0.37 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.367 3 4298.085 4298.059 0 6.2 848.3 42.1 42.1 7.80 299 1 R.SANQAPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], Q[+1] 1075.275 4 4298.079 4299.043 -1 9.2 722.8 52.6 42.6 5.15 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.678 5 4769.361 4769.328 0 6.9 848.8 42.9 42.9 6.70 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.677 5 4769.356 4769.328 0 5.8 815.2 43.0 43.0 6.98 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.024 4 4297.074 4298.059 -1 4.3 525.4 43.1 43.1 2.10 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.591 2 2506.174 2506.165 0 3.5 494.9 43.6 43.6 3.22 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.678 5 4769.360 4769.328 0 6.7 1146.1 44.0 44.0 12.70 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.678 5 4769.360 4769.328 0 6.7 1104.1 44.8 44.8 12.70 299 1 R.SANQAPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1254.085 2 2507.163 2507.149 0 5.6 615.5 45.0 45.0 5.49 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.591 2 2506.175 2506.165 0 3.8 612.4 47.1 47.1 5.36 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.877 5 4770.355 4769.328 1 4.8 617.6 47.1 47.1 2.98 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.677 5 4769.358 4769.328 0 6.3 792.8 47.5 47.5 5.70 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.275 4 4298.078 4298.059 0 4.5 1066.9 48.9 48.9 13.06 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.025 4 4297.077 4298.059 -1 5.1 381.4 49.1 49.1 0.83 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.588 2 2506.169 2506.165 0 1.5 574.7 49.2 49.2 3.96 299 1 R.SANQAPQSVGGSGIDS[+79.966]GVESTSDSLR.D S[+80] 1293.574 2 2586.142 2586.132 0 3.9 536.6 70.4 49.5 3.39 299 218  1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.096 4 4769.362 4769.328 0 7.1 262.6 49.6 49.6 0.02 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.590 2 2506.172 2506.165 0 2.8 441.9 49.6 49.6 1.86 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.677 5 4769.357 4769.328 0 6.0 944.5 49.8 49.8 8.98 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.369 3 4298.091 4298.059 0 7.5 737.6 51.0 51.0 5.40 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.588 2 2506.169 2506.165 0 1.6 443.4 51.4 51.4 1.69 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.589 2 2506.170 2506.165 0 2.1 414.5 52.5 52.5 1.75 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.591 2 2506.175 2506.165 0 3.9 388.5 52.6 52.6 1.93 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.677 5 4769.358 4769.328 0 6.2 715.4 52.7 52.7 4.70 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.025 4 4297.080 4298.059 -1 5.6 629.0 52.9 52.9 4.19 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.677 5 4769.355 4769.328 0 5.5 793.9 52.9 52.9 5.98 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.677 5 4769.356 4769.328 0 5.8 746.3 53.1 53.1 4.98 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.025 4 4297.077 4298.059 -1 5.1 317.5 53.3 53.3 0.73 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.591 2 2506.175 2506.165 0 4.0 688.8 53.4 53.4 6.36 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.589 2 2506.170 2506.165 0 1.9 652.7 54.6 54.6 5.96 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.587 2 2506.167 2506.165 0 0.9 570.1 55.6 55.6 3.96 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.591 2 2506.175 2506.165 0 3.8 652.0 56.0 56.0 6.36 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.095 4 4769.359 4769.328 0 6.5 495.4 57.7 57.7 0.59 299 1 R.SANQAPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], Q[+1] 1193.594 4 4771.355 4770.312 1 8.3 418.2 58.2 58.2 0.04 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.366 3 4298.084 4298.059 0 5.8 813.7 60.0 60.0 8.08 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.591 2 2506.175 2506.165 0 4.0 590.1 61.0 61.0 4.36 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.590 2 2506.173 2506.165 0 3.2 684.4 61.7 61.7 5.96 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.368 3 4298.090 4298.059 0 7.1 799.3 62.2 62.2 6.80 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.593 2 2506.179 2506.165 0 5.4 702.6 62.3 62.3 7.49 299 219  1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.275 4 4298.079 4298.059 0 4.7 813.6 62.3 62.3 8.19 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.588 2 2506.168 2506.165 0 1.0 531.8 63.5 63.5 3.00 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.368 3 4298.090 4298.059 0 7.3 677.8 63.6 63.6 4.40 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.094 4 4769.355 4769.328 0 5.7 780.0 64.9 64.9 5.87 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.590 2 2506.174 2506.165 0 3.3 727.3 68.5 68.5 6.96 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.588 2 2506.169 2506.165 0 1.7 649.1 68.7 68.7 4.96 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.589 2 2506.171 2506.165 0 2.2 728.4 69.3 69.3 6.96 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   836.064 3 2506.177 2506.165 0 4.8 222.1 70.4 70.4 0.67 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.368 3 4298.089 4298.059 0 6.9 700.9 70.5 70.5 5.80 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.277 4 4298.086 4298.059 0 6.2 857.6 70.9 70.9 8.91 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.274 4 4298.073 4298.059 0 3.3 904.4 71.3 71.3 9.67 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.368 3 4298.091 4298.059 0 7.4 802.2 71.5 71.5 7.40 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.592 2 2506.176 2506.165 0 4.5 665.4 72.9 72.9 6.36 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.277 4 4298.087 4298.059 0 6.6 1263.0 73.8 73.8 30.38 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.595 2 2506.182 2506.165 0 6.7 737.1 74.6 74.6 6.79 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.592 2 2506.176 2506.165 0 4.5 626.0 76.1 76.1 4.94 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.096 4 4769.360 4769.328 0 6.7 429.9 77.5 77.5 0.04 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.591 2 2506.175 2506.165 0 4.1 737.5 79.4 79.4 6.94 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.277 4 4298.086 4298.059 0 6.3 1178.3 80.4 80.4 14.49 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.592 2 2506.177 2506.165 0 4.7 715.6 80.5 80.5 6.94 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.596 2 2506.184 2506.165 0 7.4 732.0 81.2 81.2 6.39 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.277 4 4298.087 4298.059 0 6.5 1317.8 81.8 81.8 30.38 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.094 4 4769.356 4769.328 0 5.8 645.4 88.9 82.0 2.45 299 220  1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.277 4 4298.087 4298.059 0 6.6 1169.9 83.3 83.3 14.49 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.094 4 4769.354 4769.328 0 5.4 832.3 84.6 84.6 6.45 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.277 4 4298.086 4298.059 0 6.4 1255.2 85.7 85.7 16.33 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.591 2 2506.176 2506.165 0 4.1 789.2 85.9 85.9 7.94 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.094 4 4769.353 4769.328 0 5.1 732.6 88.4 88.4 4.45 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.094 4 4769.354 4769.328 0 5.4 829.7 90.4 90.4 6.45 299 1 R.SANQAPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1254.087 2 2507.166 2507.149 0 6.6 725.2 90.6 90.6 6.79 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.094 4 4769.354 4769.328 0 5.4 1125.0 92.2 92.2 12.45 299 1 R.SANQAPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1254.086 2 2507.165 2507.149 0 6.1 741.9 95.9 95.9 6.79 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.095 4 4769.357 4769.328 0 6.1 1021.1 96.1 96.1 10.17 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.095 4 4769.359 4769.328 0 6.6 862.6 96.5 96.5 7.17 299 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.596 2 2506.184 2506.165 0 7.4 928.1 117.3 117.3 10.49 299 Table 4. Peptides identified at lipin 1 S468 and S472 in mass spectrometry analysis of FLAG-lipin 1 S472A immunoprecipitated from HEK 293T cells treated with DMSO Peptides identified by mass spectrometry analysis of FLAG-lipin 1 S472A immunoprecipitated from HEK 293T cells treated with DMSO for 16 hours. Analysis was performed using Byonic Software (California, USA) and peptides are ranked by DeltaMod score. When analyzing phosphopeptides, we considered DeltaMod scores above 10.0 as a high likelihood of the correct placement of modifications for a given peptide, as defined and suggested by the Byonic support materials (https://www.proteinmetrics.com/support-information/).    221  Protein Rank Peptide < ProteinMetrics Confidential > Modification Type(s) Observed m/z z Observed (M+H) Calc. mass (M+H) Off-by-x error Mass error (ppm) Score Delta Delta Mod |Log Prob| 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.592 2 2522.176 2522.160 0 6.4 916.8 916.8 916.8 16.00 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.590 2 2522.172 2522.160 0 4.8 869.2 869.2 869.2 15.30 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.591 2 2522.174 2522.160 0 5.7 829.4 829.4 829.4 14.30 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.592 2 2522.176 2522.160 0 6.4 794.2 794.2 794.2 13.00 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.590 2 2522.173 2522.160 0 4.9 791.8 791.8 791.8 13.30 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.590 2 2522.173 2522.160 0 5.1 752.3 752.3 752.3 13.30 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.589 2 2522.170 2522.160 0 4.1 738.5 738.5 738.5 12.30 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.590 2 2522.172 2522.160 0 4.9 722.1 722.1 722.1 12.30 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.590 2 2522.172 2522.160 0 4.7 639.4 639.4 639.4 10.30 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.587 2 2522.167 2522.160 0 2.7 620.3 620.3 620.3 9.37 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.582 2 2522.158 2522.160 0 -1.0 609.5 609.5 609.5 9.40 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.589 2 2522.171 2522.160 0 4.3 583.1 583.1 583.1 9.30 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.584 2 2522.160 2522.160 0 -0.1 571.2 571.2 571.2 8.40 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.589 2 2522.171 2522.160 0 4.2 560.4 560.4 560.4 9.30 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.590 2 2522.174 2522.160 0 5.4 540.2 540.2 540.2 8.44 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.590 2 2522.173 2522.160 0 5.2 527.1 527.1 527.1 7.44 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.584 2 2522.160 2522.160 0 0.1 508.8 508.8 508.8 7.31 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.590 2 2522.173 2522.160 0 5.0 496.6 496.6 496.6 7.21 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.588 2 2522.169 2522.160 0 3.4 494.7 494.7 494.7 7.09 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.590 2 2522.173 2522.160 0 5.0 483.5 483.5 483.5 7.09 1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.590 2 2522.172 2522.160 0 4.8 448.1 448.1 448.1 8.18 222  1 R.SANQSPQSVGGSGIDSGVESTSDSLR.D   1261.592 2 2522.177 2522.160 0 6.5 362.0 362.0 362.0 5.27 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.024 4 4313.072 4314.054 -1 5.1 308.7 308.7 308.7 4.43 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.026 4 4313.083 4314.054 -1 7.5 233.3 233.3 233.3 3.06 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.699 3 4314.081 4314.054 0 6.4 577.1 577.1 223.6 7.33 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.877 5 4785.357 4785.323 0 7.0 820.9 820.9 199.4 12.62 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.700 3 4314.085 4314.054 0 7.3 614.5 614.5 189.0 8.33 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.276 4 4314.083 4314.054 0 6.8 825.3 825.3 187.0 12.71 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.699 3 4314.081 4314.054 0 6.4 467.5 467.5 174.5 6.22 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1197.094 4 4785.354 4785.323 0 6.3 570.2 570.2 168.3 7.25 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.697 3 4314.078 4314.054 0 5.5 662.6 662.6 163.8 9.63 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.877 5 4785.357 4785.323 0 7.0 966.8 966.8 163.7 15.62 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.276 4 4314.083 4314.054 0 6.7 1054.7 1054.7 156.7 17.71 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.878 5 4785.359 4785.323 0 7.4 839.1 839.1 154.9 12.62 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.877 5 4785.355 4785.323 0 6.7 942.9 942.9 151.2 14.62 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.700 3 4314.084 4314.054 0 7.1 489.9 489.9 138.4 6.24 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1197.094 4 4785.354 4785.323 0 6.4 557.3 557.3 128.4 6.39 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.275 4 4314.077 4314.054 0 5.2 1051.8 1051.8 128.0 18.01 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1197.093 4 4785.350 4785.323 0 5.6 373.7 373.7 123.3 5.35 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.274 4 4314.074 4314.054 0 4.7 1080.3 1080.3 117.2 18.01 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.876 5 4785.349 4785.323 0 5.4 667.8 667.8 113.2 9.92 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.876 5 4785.352 4785.323 0 6.0 536.8 536.8 103.5 7.06 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.276 4 4314.083 4314.054 0 6.7 1064.1 1064.1 99.8 17.71 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.876 5 4785.353 4785.323 0 6.3 548.3 548.3 89.5 7.06 223  1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1262.082 2 2523.157 2523.144 0 4.9 730.0 730.0 88.4 12.30 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.696 3 4314.073 4314.054 0 4.5 241.6 241.6 78.7 3.28 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.273 4 4314.071 4314.054 0 3.9 760.8 760.8 74.7 12.01 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1262.084 2 2523.161 2523.144 0 6.5 591.3 591.3 66.6 9.00 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.877 5 4785.356 4785.323 0 6.9 312.8 312.8 66.5 4.06 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1262.087 2 2523.166 2523.144 0 8.5 419.5 419.5 57.5 6.70 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.272 4 4314.065 4314.054 0 2.6 456.1 456.1 56.8 5.96 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.273 4 4314.068 4314.054 0 3.3 345.3 345.3 51.6 3.74 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1301.569 2 2602.132 2602.126 0 2.0 734.6 734.6 50.0 11.37 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1217.086 4 4865.321 4865.289 0 6.4 620.9 620.9 49.0 8.25 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 957.876 5 4785.349 4785.323 0 5.4 446.0 446.0 47.0 6.81 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1301.575 2 2602.143 2602.126 0 6.3 698.8 698.8 45.4 11.30 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1301.573 2 2602.139 2602.126 0 4.7 714.9 714.9 43.7 12.30 1 R.S[+79.966]ANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80]*2 1341.553 2 2682.099 2682.093 0 2.3 301.7 246.2 41.5 4.18 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1197.092 4 4785.344 4785.323 0 4.4 307.9 307.9 40.2 3.68 1 R.SANQSPQ[+0.984]S[+79.966]VGGSGIDSGVESTSDSLR.D Q[+1], S[+80] 1302.070 2 2603.132 2603.110 0 8.4 296.9 103.8 40.0 3.37 1 R.SANQS[+79.966]PQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1], S[+80] 1302.067 2 2603.128 2603.110 0 6.6 471.9 471.9 37.1 7.88 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1439.036 3 4315.093 4314.054 1 8.4 211.0 211.0 36.4 2.93 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.274 4 4314.073 4314.054 0 4.3 357.1 357.1 35.5 4.61 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.273 4 4314.068 4314.054 0 3.4 560.0 560.0 32.7 8.01 1 R.SANQSPQS[+79.966]VGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1217.084 4 4865.313 4865.289 0 4.7 298.0 298.0 32.0 3.68 1 R.SANQSPQSVGGS[+79.966]GIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1217.087 4 4865.326 4865.289 0 7.4 363.8 363.8 30.9 5.05 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.274 4 4314.073 4314.054 0 4.4 588.1 588.1 24.2 8.01 224  1 R.SANQ[+0.984]SPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1]*2 1262.580 2 2524.152 2524.128 0 9.5 260.7 260.7 23.4 3.92 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1301.571 2 2602.135 2602.126 0 3.3 717.6 717.6 22.9 11.37 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1262.089 2 2523.170 2523.144 0 10.3 394.0 394.0 21.2 6.05 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.698 3 4314.078 4314.054 0 5.7 299.5 299.5 21.0 3.75 1 R.SANQS[+79.966]PQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1], S[+80] 1302.067 2 2603.127 2603.110 0 6.2 531.6 463.6 20.6 8.14 1 R.SANQSPQSVGGSGIDS[+79.966]GVESTSDSLR.D S[+80] 1301.573 2 2602.139 2602.126 0 4.8 517.2 517.2 20.1 8.21 1 R.SANQ[+0.984]SPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1]*2 1262.583 2 2524.159 2524.128 0 12.2 349.4 349.4 19.7 3.86 1 R.SANQ[+0.984]SPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], Q[+1]*2 1080.016 4 4317.043 4316.022 1 4.2 534.6 534.6 18.9 7.15 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1465.355 3 4394.050 4394.020 0 6.8 308.6 308.6 17.3 3.71 1 R.SANQ[+0.984]SPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1]*2 1262.583 2 2524.159 2524.128 0 12.2 311.7 311.7 15.7 3.70 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1301.575 2 2602.142 2602.126 0 5.9 592.1 592.1 15.5 9.30 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1301.574 2 2602.140 2602.126 0 5.3 666.1 666.1 15.0 11.30 1 R.SANQ[+0.984]SPQSVGGSGIDSGVESTSDSLR.D Q[+1] 1262.082 2 2523.156 2523.144 0 4.6 747.1 747.1 14.7 12.30 1 R.SANQ[+0.984]SPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], Q[+1]*2 1080.018 4 4317.049 4316.022 1 5.6 408.3 408.3 14.5 5.83 1 R.SANQSPQS[+79.966]VGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1217.078 4 4865.289 4865.289 0 -0.1 214.8 214.8 13.3 2.93 1 R.SANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1262.080 2 2523.153 2523.144 0 3.5 428.7 428.7 13.1 7.00 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1438.700 3 4314.084 4314.054 0 7.1 339.4 339.4 12.7 3.65 1 R.SANQS[+79.966]PQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1], S[+80] 1302.068 2 2603.129 2603.110 0 7.0 405.2 309.2 12.6 5.61 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1465.354 3 4394.048 4394.020 0 6.3 504.5 504.5 12.4 6.24 1 R.SANQ[+0.984]SPQSVGGSGIDSGVESTSDSLR.D Q[+1] 1262.089 2 2523.171 2523.144 0 10.7 468.6 468.6 12.2 6.37 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1465.353 3 4394.045 4394.020 0 5.6 237.7 237.7 9.4 3.00 1 R.SANQ[+0.984]SPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], Q[+1] 1438.696 3 4314.073 4315.038 -1 8.9 315.7 315.7 9.1 3.51 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.274 4 4314.075 4314.054 0 4.9 293.4 293.4 7.0 4.09 225  1 R.SANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1301.574 2 2602.141 2602.126 0 5.5 217.0 217.0 5.9 3.76 1 R.SANQ[+0.984]SPQSVGGSGIDSGVESTSDSLR.D Q[+1] 1262.079 2 2523.151 2523.144 0 2.6 294.3 294.3 5.5 4.06 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1217.085 4 4865.320 4865.289 0 6.2 457.1 457.1 3.8 5.43 1 R.SANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1301.568 2 2602.128 2602.126 0 0.5 536.9 536.9 3.3 6.52 1 R.SANQ[+0.984]SPQSVGGSGIDSGVESTSDSLR.D Q[+1] 1262.083 2 2523.158 2523.144 0 5.6 696.0 696.0 1.4 11.30 1 R.SANQ[+0.984]S[+79.966]PQSVGGSGIDSGVESTSDSLR.D Q[+1], S[+80] 1302.067 2 2603.127 2603.110 0 6.3 462.8 437.6 1.1 6.89 1 R.S[+79.966]ANQSPQSVGGS[+79.966]GIDSGVESTSDSLR.D S[+80]*2 1341.551 2 2682.095 2682.093 0 1.0 260.8 260.8 0.6 4.18 1 R.SANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1079.275 4 4314.076 4314.054 0 5.2 206.6 206.6 0.2 2.96 Table 5. Peptides identified at lipin 1 S468 and S472 in mass spectrometry analysis of FLAG-lipin 1 wild type immunoprecipitated from HEK 293T cells treated with PI-103 Peptides identified by mass spectrometry analysis of FLAG-lipin 1 wild type immunoprecipitated from HEK 293T cells treated with 250nM PI-103 for 16 hours. Analysis was performed using Byonic Software (California, USA) and peptides are ranked by DeltaMod score. When analyzing phosphopeptides, we considered DeltaMod scores above 10.0 as a high likelihood of the correct placement of modifications for a given peptide, as defined and suggested by the Byonic support materials (https://www.proteinmetrics.com/support-information/).    226  Protein Rank Peptide < ProteinMetrics Confidential > Modification Type(s) Observed m/z z Observed (M+H) Calc. mass (M+H) Off-by-x error Mass error (ppm) Score Delta Delta Mod |Log Prob| 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.596 2 2506.184 2506.165 0 7.7 924.8 68.6 68.6 11.48 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.575 2 2586.142 2586.132 0 4.1 761.1 63.3 54.1 8.86 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.094 4 4769.353 4769.328 0 5.3 573.5 53.4 53.4 2.34 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.095 4 4769.359 4769.328 0 6.4 602.6 52.8 52.8 2.96 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.574 2 2586.141 2586.132 0 3.6 851.9 82.2 51.3 10.86 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.095 4 4769.358 4769.328 0 6.3 629.7 46.1 46.1 2.96 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.577 2 2586.147 2586.132 0 6.0 759.5 74.5 44.5 8.86 1 R.AANQS[+79.966]PQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1], S[+80] 1294.070 2 2587.133 2587.116 0 6.8 633.7 54.8 43.2 5.48 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.367 3 4298.085 4298.059 0 6.1 281.7 40.9 40.9 0.93 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.094 4 4769.356 4769.328 0 5.7 775.0 39.5 39.5 6.34 1 R.AANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1254.084 2 2507.161 2507.149 0 4.8 489.6 37.7 37.7 3.81 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.577 2 2586.147 2586.132 0 6.1 926.3 118.1 37.3 11.86 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.592 2 2506.176 2506.165 0 4.3 761.8 36.3 36.3 8.86 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.592 2 2506.176 2506.165 0 4.4 399.8 35.9 35.9 2.72 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.365 3 4298.081 4298.059 0 5.1 212.6 37.1 35.3 0.24 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.570 2 2586.133 2586.132 0 0.5 617.9 43.6 34.3 4.61 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.595 2 2506.183 2506.165 0 7.3 719.8 34.1 34.1 7.48 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.572 2 2586.136 2586.132 0 1.8 619.3 51.5 33.8 4.61 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.368 3 4298.090 4298.059 0 7.3 489.7 33.4 33.4 2.09 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.026 4 4297.083 4298.059 -1 6.4 238.9 32.7 32.7 0.15 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.580 2 2586.153 2586.132 0 8.4 427.0 32.2 32.2 2.11 227  1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1213.087 4 4849.325 4849.295 0 6.3 722.2 32.1 32.1 4.96 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.678 5 4769.363 4769.328 0 7.3 224.7 33.9 31.3 0.02 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1460.025 3 4378.060 4378.025 0 7.9 348.7 30.3 30.3 0.79 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.577 2 2586.147 2586.132 0 5.9 733.6 35.6 29.6 7.86 1 R.AANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1254.091 2 2507.175 2507.149 0 10.4 476.1 29.5 29.5 1.60 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1460.024 3 4378.058 4378.025 0 7.4 335.9 29.2 29.2 0.64 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.593 2 2506.179 2506.165 0 5.4 722.3 29.2 29.2 7.86 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.593 2 2506.179 2506.165 0 5.7 765.7 28.0 28.0 8.86 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.570 2 2586.132 2586.132 0 0.1 607.2 39.2 27.9 4.43 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.593 2 2506.178 2506.165 0 5.0 718.5 27.6 27.6 7.86 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.591 2 2506.174 2506.165 0 3.7 478.7 27.2 27.2 2.81 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.577 2 2586.146 2586.132 0 5.5 660.7 58.4 27.1 6.86 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.095 4 4769.358 4769.328 0 6.2 579.5 27.0 27.0 2.34 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.572 2 2586.137 2586.132 0 2.2 481.3 49.5 26.4 2.56 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1293.576 2 2586.145 2586.132 0 5.4 327.6 45.4 26.4 1.81 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.369 3 4298.093 4298.059 0 7.9 521.2 26.1 26.1 2.25 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.589 2 2506.171 2506.165 0 2.1 543.6 25.9 25.9 2.72 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.576 2 2586.145 2586.132 0 5.2 688.9 71.6 25.5 6.86 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1293.575 2 2586.143 2586.132 0 4.5 489.1 25.3 25.3 2.89 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1293.574 2 2586.142 2586.132 0 3.9 458.2 51.0 24.9 2.81 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.593 2 2506.179 2506.165 0 5.7 682.1 23.9 23.9 6.86 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.593 2 2506.179 2506.165 0 5.7 493.1 23.4 23.4 3.81 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.593 2 2506.179 2506.165 0 5.4 563.5 22.9 22.9 4.86 228  1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.592 2 2506.176 2506.165 0 4.4 537.8 20.4 20.4 3.97 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.677 5 4769.358 4769.328 0 6.2 368.8 20.2 20.2 0.93 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1213.088 4 4849.329 4849.295 0 7.0 257.7 31.4 20.1 0.10 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.677 5 4769.358 4769.328 0 6.2 945.0 19.8 19.8 9.09 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1213.090 4 4849.338 4849.295 0 8.9 354.6 19.6 19.6 0.13 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.277 4 4298.085 4298.059 0 6.0 876.1 19.6 19.6 9.63 1 R.AANQ[+0.984]S[+79.966]PQSVGGSGIDSGVESTSDSLR.D Q[+1], S[+80] 1294.069 2 2587.130 2587.116 0 5.7 828.7 103.2 19.2 9.86 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.574 2 2586.142 2586.132 0 3.9 510.4 59.7 18.5 3.81 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.677 5 4769.356 4769.328 0 5.9 952.4 18.4 18.4 10.46 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.275 4 4298.078 4298.059 0 4.3 258.8 18.1 18.1 0.95 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1293.573 2 2586.138 2586.132 0 2.7 252.8 33.9 17.7 0.76 1 R.AANQ[+0.984]S[+79.966]PQSVGGSGIDSGVESTSDSLR.D Q[+1], S[+80] 1294.070 2 2587.134 2587.116 0 6.9 689.6 45.7 17.6 6.48 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.595 2 2506.183 2506.165 0 7.0 722.0 17.5 17.5 7.48 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.367 3 4298.087 4298.059 0 6.6 423.1 17.5 17.5 1.97 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.575 2 2586.142 2586.132 0 4.0 357.5 34.5 17.4 2.44 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1460.024 3 4378.058 4378.025 0 7.5 550.0 17.2 17.2 2.25 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.275 4 4298.079 4298.059 0 4.7 249.4 16.9 16.9 0.95 1 R.AANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1254.087 2 2507.167 2507.149 0 7.0 681.5 16.5 16.5 6.48 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.094 4 4769.353 4769.328 0 5.2 403.6 16.3 16.3 1.19 1 R.AANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1254.089 2 2507.171 2507.149 0 8.9 465.4 16.3 16.3 2.15 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.095 4 4769.358 4769.328 0 6.3 509.1 16.1 16.1 0.97 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1293.577 2 2586.147 2586.132 0 6.2 440.1 37.8 15.8 2.81 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1293.575 2 2586.143 2586.132 0 4.3 302.7 19.0 15.8 2.03 229  1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.677 5 4769.358 4769.328 0 6.3 811.0 15.6 15.6 7.09 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.095 4 4769.357 4769.328 0 6.0 573.1 15.3 15.3 2.34 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.095 4 4769.360 4769.328 0 6.6 350.6 15.2 15.2 0.16 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.277 4 4298.087 4298.059 0 6.5 927.3 15.0 15.0 10.26 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.576 2 2586.144 2586.132 0 4.8 301.5 14.7 14.7 1.79 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.679 5 4769.366 4769.328 0 8.0 780.7 13.2 13.2 6.09 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.577 2 2586.147 2586.132 0 6.0 722.0 51.9 12.9 7.86 1 R.AANQSPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1254.086 2 2507.165 2507.149 0 6.4 435.5 12.8 12.8 2.39 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], S[+80] 1460.023 3 4378.056 4378.025 0 7.0 278.7 12.4 12.4 0.54 1 R.AANQ[+0.984]SPQSVGGSGIDSGVESTSDSLR.D Q[+1] 1254.087 2 2507.167 2507.149 0 7.0 719.2 27.3 11.7 7.48 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1213.087 4 4849.325 4849.295 0 6.3 220.8 37.8 11.1 0.01 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.574 2 2586.140 2586.132 0 3.2 581.2 59.6 10.4 3.61 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.578 2 2586.149 2586.132 0 6.7 306.5 11.4 10.3 1.86 1 R.AANQ[+0.984]S[+79.966]PQSVGGSGIDSGVESTSDSLR.D Q[+1], S[+80] 1294.074 2 2587.141 2587.116 0 9.8 406.3 33.5 10.1 1.56 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.592 2 2506.176 2506.165 0 4.3 580.2 10.1 10.1 4.86 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.574 2 2586.141 2586.132 0 3.8 279.4 9.7 9.7 2.32 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.575 2 2586.142 2586.132 0 4.2 395.1 20.5 9.3 2.45 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.593 2 2506.178 2506.165 0 5.3 321.2 9.2 9.2 2.25 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57], S[+80] 1213.087 4 4849.326 4849.295 0 6.4 279.2 9.1 9.1 0.10 1 R.AANQSPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], Q[+1] 1075.276 4 4298.083 4299.043 -1 10.1 384.6 8.9 8.9 0.47 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.591 2 2506.175 2506.165 0 3.7 529.4 8.6 8.6 3.97 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.575 2 2586.142 2586.132 0 4.2 376.6 20.5 8.3 2.53 1 R.AAN[+0.984]QSPQSVGGSGIDSGVESTSDSLR.D N[+1] 1254.087 2 2507.166 2507.149 0 6.7 786.5 19.6 7.9 8.48 230  1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1293.574 2 2586.141 2586.132 0 3.7 512.1 6.0 6.0 2.89 1 R.AANQS[+79.966]PQSVGGSGIDSGVESTSDSLR.D S[+80] 1293.576 2 2586.144 2586.132 0 5.0 513.3 44.7 5.9 3.81 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1293.572 2 2586.136 2586.132 0 1.6 454.6 10.0 5.7 2.48 1 R.AANQSPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.276 4 4298.081 4298.059 0 5.2 704.1 4.9 4.9 6.63 1 R.AANQSPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1293.577 2 2586.148 2586.132 0 6.2 386.9 23.2 4.5 2.53 1 R.AANQSPQSVGGSGIDS[+79.966]GVESTSDSLR.D S[+80] 1293.578 2 2586.149 2586.132 0 6.7 549.2 29.1 3.0 3.60 1 R.AANQSPQSVGGS[+79.966]GIDSGVESTSDSLR.D S[+80] 1293.574 2 2586.140 2586.132 0 3.2 348.9 29.9 1.8 1.22 1 R.AANQSPQ[+0.984]S[+79.966]VGGSGIDSGVESTSDSLR.D Q[+1], S[+80] 1294.078 2 2587.149 2587.116 0 12.7 361.7 1.8 1.8 0.71 1 R.AANQSPQSVGGSGIDSGVESTSDSLR.D   1253.592 2 2506.177 2506.165 0 4.7 652.5 1.7 1.7 6.86 1 R.AANQSPQSVGGS[+79.966]GIDSGVESTSDSLR.D S[+80] 1293.577 2 2586.147 2586.132 0 6.1 440.4 37.4 0.2 2.53 Table 6. Peptides identified at lipin 1 S468 and S472 in mass spectrometry analysis of FLAG-lipin 1 S468A immunoprecipitated from HEK 293T cells treated with PI-103 Peptides identified by mass spectrometry analysis of FLAG-lipin 1 S468A immunoprecipitated from HEK 293T cells treated with 250nM PI-103 for 16 hours. Analysis was performed using Byonic Software (California, USA) and peptides are ranked by DeltaMod score. When analyzing phosphopeptides, we considered DeltaMod scores above 10.0 as a high likelihood of the correct placement of modifications for a given peptide, as defined and suggested by the Byonic support materials (https://www.proteinmetrics.com/support-information/).    231  Protein Rank Peptide < ProteinMetrics Confidential > Modification Type(s) Observed m/z z Observed (M+H) Calc. mass (M+H) Off-by-x error Mass error (ppm) Score Delta Delta Mod |Log Prob| 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.276 4 4298.082 4298.059 0 5.4 928.7 86.3 86.3 10.33 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.094 4 4769.356 4769.328 0 5.8 659.6 85.8 85.8 4.86 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.596 2 2506.185 2506.165 0 7.8 683.3 84.5 84.5 5.78 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.594 2 2506.180 2506.165 0 5.8 717.2 81.9 81.9 7.24 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.095 4 4769.359 4769.328 0 6.5 544.8 81.2 81.2 2.13 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.597 2 2506.186 2506.165 0 8.4 753.6 80.9 80.9 7.78 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.594 2 2506.181 2506.165 0 6.5 654.1 80.1 80.1 6.43 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.595 2 2506.182 2506.165 0 6.9 699.9 78.4 78.4 6.43 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.594 2 2506.181 2506.165 0 6.2 477.4 78.3 78.3 2.35 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.096 4 4769.361 4769.328 0 6.9 822.6 75.6 75.6 8.04 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.276 4 4298.083 4298.059 0 5.7 708.9 71.9 71.9 6.33 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.277 4 4298.087 4298.059 0 6.5 937.6 70.3 70.3 10.51 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.593 2 2506.178 2506.165 0 5.2 651.5 69.0 69.0 6.24 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.595 2 2506.182 2506.165 0 6.9 704.6 68.9 68.9 7.43 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.596 2 2506.184 2506.165 0 7.6 702.4 67.8 67.8 7.43 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.095 4 4769.357 4769.328 0 6.0 441.8 66.8 66.8 1.94 1 R.SANQAPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1254.087 2 2507.167 2507.149 0 7.3 701.3 66.1 66.1 7.43 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.594 2 2506.180 2506.165 0 5.8 579.5 65.6 65.6 4.24 1 R.SANQAPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1254.086 2 2507.165 2507.149 0 6.3 690.6 65.0 65.0 6.43 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.594 2 2506.181 2506.165 0 6.3 580.2 60.0 60.0 4.43 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.367 3 4298.086 4298.059 0 6.4 651.5 59.8 59.8 5.90 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.59 2 2506.18 2506.16 0 6.2 700.0 57.0 57.0 6.43 232  4 1 5 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.277 4 4298.085 4298.059 0 6.1 1161.9 56.6 56.6 15.51 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.593 2 2506.178 2506.165 0 5.2 604.1 53.5 53.5 5.24 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.590 2 2506.174 2506.165 0 3.4 610.5 53.0 53.0 5.05 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.679 5 4769.364 4769.328 0 7.4 241.5 51.9 51.9 0.28 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.678 5 4769.360 4769.328 0 6.7 482.1 50.4 50.4 1.60 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.368 3 4298.090 4298.059 0 7.3 770.3 49.7 49.7 7.90 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.593 2 2506.180 2506.165 0 5.8 364.6 47.7 47.7 1.93 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.367 3 4298.086 4298.059 0 6.3 480.2 46.5 46.5 2.83 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.592 2 2506.177 2506.165 0 4.8 477.9 45.2 45.2 3.13 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 1193.096 4 4769.362 4769.328 0 7.0 496.2 59.5 44.0 1.98 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.367 3 4298.087 4298.059 0 6.5 560.6 43.7 43.7 3.90 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.369 3 4298.091 4298.059 0 7.4 264.1 43.6 43.6 1.36 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.877 5 4770.358 4769.328 1 5.4 278.1 43.3 43.3 0.26 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.275 4 4298.080 4298.059 0 4.9 860.0 42.7 42.7 9.33 1 R.SANQAPQ[+0.984]SVGGSGIDS[+79.966]GVESTSDSLR.D Q[+1], S[+80] 1294.079 2 2587.150 2587.116 0 13.5 423.8 41.9 41.9 0.68 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.366 3 4298.082 4298.059 0 5.4 269.1 40.7 40.7 1.19 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.593 2 2506.179 2506.165 0 5.7 535.7 39.8 39.8 3.32 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.678 5 4769.359 4769.328 0 6.5 621.8 38.4 38.4 3.66 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.592 2 2506.177 2506.165 0 4.8 700.6 37.6 37.6 7.24 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.677 5 4769.356 4769.328 0 5.9 738.7 55.1 36.8 5.47 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.678 5 4769.360 4769.328 0 6.6 883.3 36.5 36.5 8.66 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.591 2 2506.174 2506.165 0 3.6 273.2 36.0 36.0 1.51 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDH C[+57] 1075.27 4 4298.08 4298.05 0 6.2 606.7 35.2 35.2 4.51 233  R.E 7 5 9 1 R.SANQAPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1254.092 2 2507.177 2507.149 0 11.1 360.1 33.5 33.5 0.97 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.678 5 4769.359 4769.328 0 6.5 1047.8 33.4 33.4 11.66 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.024 4 4297.075 4298.059 -1 4.5 259.8 32.4 32.4 0.72 1 R.SANQAPQS[+79.966]VGGSGIDSGVESTSDSLR.D S[+80] 1293.575 2 2586.142 2586.132 0 4.1 277.3 30.3 30.3 1.37 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.594 2 2506.180 2506.165 0 5.8 239.0 29.0 29.0 0.59 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.594 2 2506.181 2506.165 0 6.2 293.8 27.6 27.6 1.83 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1433.366 3 4298.083 4298.059 0 5.6 565.2 27.3 27.3 3.72 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.591 2 2506.174 2506.165 0 3.6 343.2 25.2 25.2 1.64 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.591 2 2506.175 2506.165 0 3.8 212.3 24.2 24.2 0.46 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.588 2 2506.169 2506.165 0 1.7 205.0 23.9 23.9 0.08 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.594 2 2506.180 2506.165 0 5.8 209.3 18.1 18.1 0.81 1 R.SANQ[+0.984]APQSVGGSGIDSGVESTSDSLR.D Q[+1] 1254.087 2 2507.166 2507.149 0 6.8 700.9 80.3 18.0 7.43 1 R.SANQAPQ[+0.984]SVGGSGIDSGVESTSDSLR.D Q[+1] 1254.088 2 2507.170 2507.149 0 8.1 540.7 51.9 18.0 2.86 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHREITK.D C[+57] 954.678 5 4769.359 4769.328 0 6.5 580.4 16.0 16.0 2.66 1 R.SANQAPQSVGGSGIDS[+79.966]GVESTSDSLR.D S[+80] 1293.579 2 2586.151 2586.132 0 7.4 704.7 57.4 14.7 7.43 1 R.SANQAPQSVGGSGIDSGVESTSDSLR.D   1253.593 2 2506.178 2506.165 0 5.0 255.5 10.3 10.3 1.11 1 R.SANQAPQSVGGSGIDS[+79.966]GVESTSDSLR.D S[+80] 1293.581 2 2586.155 2586.132 0 9.1 243.6 25.7 9.0 0.94 1 R.SANQAPQ[+0.984]SVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57], Q[+1] 1075.277 4 4298.084 4299.043 -1 10.4 708.6 52.8 7.3 5.46 1 R.SANQAPQSVGGSGIDSGVESTSDSLRDLPSIAISLC[+57.021]GGLSDHR.E C[+57] 1075.276 4 4298.083 4298.059 0 5.5 496.4 30.9 3.4 2.26 1 R.SANQAPQSVGGSGIDS[+79.966]GVESTSDSLR.D S[+80] 1293.577 2 2586.147 2586.132 0 5.9 541.8 57.1 1.9 3.32 Table 7. Peptides identified at lipin 1 S468 and S472 in mass spectrometry analysis of FLAG-lipin 1 S472A immunoprecipitated from HEK 293T cells treated with PI-103 234  Peptides identified by mass spectrometry analysis of FLAG-lipin 1 S472A immunoprecipitated from HEK 293T cells treated with 250nM PI-103 for 16 hours. Analysis was performed using Byonic Software (California, USA) and peptides are ranked by DeltaMod score. When analyzing phosphopeptides, we considered DeltaMod scores above 10.0 as a high likelihood of the correct placement of modifications for a given peptide, as defined and suggested by the Byonic support materials (https://www.proteinmetrics.com/support-information/).     

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