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Molecular mechanisms of cellular activation Grill, Brock 2003

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Molecular Mechanisms of Cellular Activation By: Brock Grill B.Sc. (Honours), University of Alberta, 1998 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE R E Q U I R E M E N T S FOR THE D E G R E E O F DOCTOR OF PHILOSOPHY in THE FACULTY OF G R A D U A T E STUDIES Experimental Medicine Program, Department of Medicine We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 2003 © Brock Grill, 2003 Wednesday, January 8, 2003 UBC Rare Books and Special Collections - Thesis Authorisation Form Page. 1 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e h e a d o f my d e p a r t m e n t o r b y h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r , C a n a d a D a t e http.//www. library, ubc.ca/spcoll/th esauth.html ABSTRACT There is no biochemical evidence for the activation of Rac and Cdc42 in hemopoietic cells, nor is the mechanism of activation of these small GTPases well characterized in hemopoietic cells. We demonstrate here that lnterleukin-3 (IL-3) induced activation of endogenous Rac-1, Rac-2 and Cdc42. Rac-1 was also activated by colony-stimulating factor-1 (CSF-1), Steel locus factor (SLF), granulocyte-macrophage colony-stimulating factor (GM-CSF) , lnterleukin-5 (IL-5), or by cross-linking the B-lymphocyte receptor for antigen (BCR). The molecules found to be upstream activators of Rac-1 in hemopoietic cells were P l -3K, p21 Ras, and M-Ras. The activation of Rac-1, Rac-2, and Cdc42 by IL-3 and other hemopoietic growth factors is likely to be an important component of their actions in promoting growth, survival and function. While studying the Rac G E F , smgGDS, we serendipitously observed a protein which was up-regulated in activated lymphocytes. We purified, from actively dividing T-lymphocytes this novel, highly conserved cytoplasmic phospho-protein, which we term Caprin-1. We found that expression of endogenous Caprin-1 correlates with the proliferative status of cells, being up-regulated in actively dividing cells and down-regulated in quiescent cells. We identified Caprin-1 and a homologous protein, Caprin-2, as members of a novel protein family characterized by two novel protein domains, termed homology regions-1 and - 2 (HR-1, HR-2). We also observed that over-expression of a fusion protein of G F P and Caprin-1 induced a specific, dose-dependent ii suppression of the proliferation of NIH 3T3 cells, further supporting a role for Caprin-1 in cellular proliferation. TABLE OF CONTENTS Abstract ii Table of Contents iv List of Figures vii Abbreviations ix Preface xi Acknowledgements xiii Fo reward xv Chapter 1: Introduction 1.1 An introduction to signal transduction 1 1.2 Small GTPases 2 1.3 Ras GTPases 3 1.4 Rho GTPases 6 1.5 Activation of Rac and Cdc42 8 1.6 MAP kinases 12 1.7 Proteomics and Genomics 13 Chapter 2: Experimental Procedures 2.1 For Chapter 3 2.1.1 cDNA constructs, antibodies and reagents 16 2.1.2 Cell culture 17 2.1.3 Cell stimulation 18 2.1.4 Electroporation 18 iv 2.1.5 Assay for activation of Rac-1, Rac-2 and Cdc42 19 2.1.6. Assay for activation of p38 MAPK, Akt or Erk. 20 2.2 For Chapter 4 2.2.1 cDNA constructs and antibodies 21 2.2.2 Generation of cDNA consensus sequences and 22 intron-exon boundaries 2.2.3 Cells 23 2.2.4 Immunoblotting and immunoprecipitation 24 2.2.5 In-gel digestion and mass spectrometric analysis 24 2.2.6 Flow cytometric analysis of Caprin-1 and cell division 25 2.2.7 Immunofluorescence 25 2.2.8 3 2P-labelling of Caprin-1 25 Chapter 3: Activation of Rac-1, Rac-2 and Cdc42 by hemopoietic growth factors or cross-linking of the B lymphocyte receptor for antigen 3.1 Introduction 26 3.2 Results 3.2.1 Activation of endogenous Rac-1, Rac-2 and Cdc42 by 27 multiple hemopoietic growth factors 3.2.2. Activation of endogenous Rac-1 by cross-linking of 35 the B C R 3.2.3 Different requirements for PI-3K activity for activation 36 of Rac-1 induced by IL-3 or cross-linking of the B C R 3.2.4 Ras-mediated activation of Rac-1 is PI-3K 39 independent V 3.2.5. Activation of Ras or Rac-1, but not PI-3K, is sufficient 45 for activation of p38 MAPK in hemopoietic cells 3.3 Discussion 48 Chapter 4: Identification and characterization of Caprin-1 and the Caprin family of proteins 4.1 Introduction 56 4.2 Results 4.2.1. A 116 kDa cytoplasmic protein is up-regulated in 57 proliferating T or B lymphoblasts 4.2.2. Expression of p116 in tissues and other cell-lines 61 4.2.3. Caprin-1 is expressed at high levels in dividing 61 thymocytes 4.2.4 Structure of Caprin-1 63 4.2.5 HR-1, a novel protein domain highly conserved in 72 Vertebrates and insects 4.2.6 Caprin-2 73 4.2.7 Genomic organization 75 4.2.8 Caprin-1 is a phospho-protein that exists in a multi- 85 protein complex 4.2.9 Levels of Caprin-1 decrease when factor-dependent 79 Baf/3 cells were deprived of IL-3 or the M-1 leukemia was induced to differentiate with IL-6 4.2.10 Over-expression of GFP-Caprin-1 results in specific, 80 dose-dependent inhibition of cell division vi 4.3 Discussion 85 Chapter 5: Conclusions and future experiments 87 References 90 LIST OF FIGURES Chapter 1 Figure 1.1. A dendrogram comparing the level of amino acid homology between representative members of the Ras superfamily of small GTPases Figure 1.2 Rac related signal transduction 11 Chapter 3 Figure 3.1 Specificity of Rac antibodies and activation of Rac-1 by (3-common family cytokines Figure 3.2 IL-3 activates multiple Rho family GTPases in bone marrow-derived mast cells Figure 3.3 Activation of Rac-1 stimulated by hemopoietic growth factors acting through receptor tyrosine kinases Figure 3.4 Activation of Rac-1 induced by cross-linking of the B C R Figure 3.5 Effects of the PI-3K inhibitor, Ly294002, on activation of Rac-1 and p38 MAPK induced by IL-3 or cross-linking of the B C R 31 33 34 35 38 vn Figure 3.6 Figure 3.7 Figure 3.8 Figure 3.9 Chapter 4 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Figure 4.9 Over-expression of activated mutants of H-Ras, N-Ras or 42 M-Ras results in activation of Rac-1 via a mechanism, which is not inhibited by Ly294002 Over-expression of a dominant negative mutant of H-Ras 44 blocks IL-3 induced activation of endogenous Rac-1 Over-expression of activated H-Ras or Rac-1, but not 46 p110 PI-3K, are sufficient for activation of p38 MAPK in hemopoietic cells Enhanced activation of p38 MAPK in the presence of the 47 PI-3K inhibitor Ly 294002 Expression pattern and localization of p116 59 Caprin-1 expression is elevated in large thymocytes 63 Structure of Caprin-1 and its identity with p116 67 The Caprin-1 and insect HR-1 domains, and the human 69 Caprin-1 and Caprin-2 proteins and genes Phosphorylation of Caprin-1 76 Caprin-1 is capable of forming mulitmeric complexes 78 Expression of Caprin-1 decreases when cells cease to divide 79 Loss of Caprin-1 G F P fusion protein expression over time 82 Over-expression of GFP-Caprin-1 blocks division of 3T3 cells 83 vi i i ABBREVIATIONS ADP adenosine diphosphate Akt v-Akt murine thymoma viral oncogene homolog AP-1 activator protein-1 A P anti-peptide Arf ADP-ribosylation factor ATF-1, 2 activating transcription factor-1, 2 ATP adenosine triphosphate B C R B cell receptor for antigen BHP Bombyx HR-1 containing protein B M M C bone marrow-derived mast cell cDNA complementary DNA Caprin cytoplasmic activation/proliferation related protein Cdc42 cell division cycle 42 ConA Concanavalin A CRIB domain Cdc42/Rac interactive binding domain CSF-1 colony-stimulating factor-1 DNA deoxyribonucleic acid DH Dbl homology domain DHP Drosophila HR-1 containing protein Erk extracellular-regulated kinase EST expressed sequence tag fMLP formyl-methionine-leucine-phenylalanine F C S fetal calf serum G - C S F granulocyte-colony stimulating factor G A P GTPase activating protein GDI G D P dissociation inhibitor G D P guanosine diphosphate G D S GDP-dissociation stimulator G E F guanine nucleotide exchange factor G F P green fluorescent protein G M - C S F granulocyte/macrophage-colony stimulating factor Grb2 growth factor receptor-bound protein 2 G S T glutathione S-transferase G T P guanosine triphosphate GTPase guanosine triphosphatase HA hemaglutinin HR-1 or 2 homology region-1 or 2 IL-2, 3, 4, 5, 6 interleukin-2, 3, 4, 5, 6 ix IgG, IgM immunoglobulin-G, M IP immunoprecipitation JNK c-Jun N-terminal kinase kb kilo base pairs kDa kilo dalton L P S lipopolysaccharide MAPK mitogen activated protein kinase M A P K A P mitogen activated protein kinase activated protein MEK MAPK/Erk kinase MKK MAPK kinase P A G E polyacrylamide gel electrophoresis PAK p21-activated kinase P C R polymerase chain reaction P D G F platelet-derived growth factor PH domain pleckstrin homology domain PKB, P K C protein kinase B, C PLC phospholipase C PI-3K phosphatidyl-inositol-3-kinase PIP2 phosphatidyl inositol-(4,5)-bisphosphate PIP3 phosphatidyl inositol-(3,4,5)-triphosphate PMA phorbol-12-myristate-13-acetate Rab Ras-associated protein Rac Ras-related C3 botulinum toxin substrate Ral v-ral simian leukemia viral oncogene homolog Ran Ras-related nuclear protein Ras Rat sarcoma RasGRF-1 , 2 Ras protein-specific guanine nucleotide-releasing factor-1, 2 RGK Rad/Rem/Gem/Kir Rho Ras homolog RNA ribonucleic acid RSK ribosomal S6 kinase R T - P C R reverse transcriptase-PCR S D S sodium dodecylsulfate S F M serum-free media SH2 src homology domain-2 She SH2-containing protein S L F Steel locus factor Sos son of sevenless TCR T cell receptor TNF-a tumor necrosis factor alpha W C L whole cell lysate PREFACE This thesis includes 5 chapters. The first chapter is an introduction to the signal transduction pathways necessary to understand the studies performed regarding the Rac small GTPases . This chapter also includes an introduction to the complexities associated with characterizing and determining the function of a novel protein family. The second chapter is a detailed description of the experimental procedures used for all the experiments presented in this thesis. Chapter 3 and 4 focus on results contained in the published and submitted manuscripts listed below. Gary Wilson is a technician who was instrumental in the study of Caprin-1 described in Chapter 4 . He was involved in the experiment shown in Figure 4.7B, and generated all cDNA constructs that were employed for the study of Caprin-1. Kai-Zin Zhang is a post-doctoral fellow who performed the immunofluorescence shown in Figure 4.3H. Regis Doyonnas is a post-doctoral fellow who provided the training necessary to perform the bioinformatic studies shown in Figures 4.3 and 4.4, and was instrumental in deciphering the consensus sequence of Caprin-2 shown in Figure 4.4. Manfredo Quadroni is a principal investigator from the University of Lausanne, Switzerland, who performed ms/ms mass spectrometric analysis to identify two peptide sequences from Caprin-1. Chapter 5 is an overall discussion of future studies that may follow up the work contained in this thesis. xi Publications obtained during the course of this thesis: Grill B, and Schrader JW. Activation of Rac-1 , Rac-2 , and Cdc42 by hemopoietic growth factors or cross-linking of the B-lymphocyte receptor for antigen. Blood. 100(9):3183-92, 2002. Schrader J , Schallhorn A, Grill B, Guo X. Activation of small GTPases of the Ras and Rho family by growth factors active on mast cells. Mol Immunol. 38(16-18):1181, 2002. Grill B, Wilson G M , Zhang KX, Bin Wang, Regis Doyonnas, Quadroni M, and Schrader JW. Cytoplasmic activation/proliferation-related protein (Caprin)-1: prototype of a new family of proteins. In submission. 2003. Publications not discussed in this thesis Grill B*, Schubert P*. Gary M. Wilson, and John W. Schrader. Splicing of smgGDS provides support for the theory that individual Armadillo domains are encoded by single exons. manuscript in preparation. 2003. * Both authors contributed equally to this work. xi i ACKNOWLEDGEMENTS I would like to thank Dr. John Schrader for his patience and supervision which have made this thesis possible, as well as his endless hours of teaching. I appreciate the wealth of expertise and experience that Gary Wilson and Regis Doyonnas have shared with me on so many occasions. Each has been an invaluable mentor and friend. I would also like to thank Drs. Kelly McNagny and Fabio Rossi for their discussions, insight, and guidance. I would also like to acknowledge several collaborators who have provided reagents for my studies. These generous individuals include Drs. A. Ambrosini, Rob Kay, Frank 'McCormick, Lewis T. Williams, Alan Hall, Paul Luzio, and the late Dr. Ian Clark-Lewis. On a more personal level, I feel a great sense of gratitude to the people who provided the support and encouragement throughout my life: Lois, Francis, Regan and Lindsay. It has been my desire since I was a small child to be a scientist. "Your love, support and belief in me over the years have brought me to the achievement of this thesis and the realization of my dream of becoming a scientist". Finally, I would like to thank the love of my life, Stephanie, for bringing light to my life in so many ways, making my time in Vancouver something I will never forget, and teaching me how to "live life" xi i i For the greatest finder I know. Blue eyes FOREWORD Biology is the study of life, and life is diversity. As such, it is my belief that a biologist must seek to embody diversity by gaining an understanding of as many subjects, topics and technologies as possible. This thesis is a product of my best efforts at diversity. XV CHAPTER 1: INTRODUCTION 1.1. An introduction to signal transduction All cells, whether a single, simple prokaryotic cell, or a eukaryotic cell that is part of a complex multicellular organism, must be capable of sensing and responding to their environment. Signal transduction is the process by which a cell converts external stimuli or environmental information into cellular changes, such as alterations in gene expression, or enzymatic or cellular activities. These intracellular changes allow organisms to adjust to ever-changing environmental conditions and the demands of survival on earth. With regard to multicellular eukaryotes, such as humans, an array of extracellular proteins, such as hormones and cytokines, have evolved which allow for intercellular communication critical for the maintenance of homeostasis. The immune system, which has evolved only in Vertebrate organisms, is particularly dependent upon intercellular communication being composed of a diverse range of cells spatially distributed throughout the body in several different hemopoietic organs and non-hemopoietic tissues. A diverse array of receptors has evolved which, once bound by ligand, activate a variety of membrane proximal signaling molecules such as tyrosine kinases. The activity of these kinases then leads to the recruitment and/or activation of other protein and lipid kinases, phospholipases, and small GTPases. Activation of these molecules triggers cascades of intracellular signals, which result in the activation of transcription factors and alterations in gene expression, ultimately leading to cellular changes. 1 The specific focus of this thesis will be the cells of the immune system, the ligand-receptor systems associated with these cells (specifically receptors for hemopoietic growth factors, and the B cell receptor for antigen (BCR)), and the signal transduction pathways activated by these receptors. 1.2. Small GTPases All GTPases cycle between an inactive, GDP-bound form and an active, GTP-bound form which has affinity for a series of effector proteins that control signal transduction cascades. Small GTPases of the Ras superfamily do not contain multiple subunits as is the case for heterotrimeric G-proteins. Rather, they are monomeric proteins whose activity is regulated by a series of positive and negative regulatory molecules. When bound to G D P , and inactive, small GTPases are targets for the binding of a class of activating molecules, the guanine nucleotide exchange factors (GEF). G E F are recruited by many different types of receptors including those of the cytokine receptor superfamily (e.g. receptor for IL-3) which lack intrinsic tyrosine kinase activity, receptor tyrosine kinases (e.g. CSF-1) , and antigen receptors expressed on lymphocytes (e.g. BCR) 1" 3. Once recruited to stimulated receptors, G E F are positioned to bind small GTPases and cause the release of G D P 4 ' 6 . This allows the GTPase to load with G T P due to the excess of G T P in the cytosol. Thus activated, these small GTPases bind to and activate/recruit effectors which mediate cellular c h a n g e s 4 7 , 8 . Although small GTPases have intrinsic enzymatic activity which causes the hydrolysis of G T P leading to the generation of G D P and inactivation of the GTPase, this 2 occurs at a slow rate for most small GTPases . As such, two groups of negative regulatory molecules allow for tighter, more rapid control of small GTPase inactivation. These negative regulators are the GTPase activating proteins (GAP) and the G D P dissociation inhibitors (GDI). G A P bind to GTP-bound, active small GTPases and enhance the intrinsic G T P a s e activity leading to more rapid inactivation 6 . GDI molecules are more complex and their physiological roles in signaling remain obscure. They are uniquely capable of binding to both G D P and GTP-bound small GTPases 6 9 . As a result, they perform a negative regulatory role by binding to GDP-bound small G T P a s e s , inhibiting G D P dissociation and, hence, G T P binding and activation. However, by binding to GTP-bound GTPases they also inhibit G T P hydrolysis and hence prolong activation of small GTPases. 1. 3. Ras GTPases The Ras superfamily of small GTPases is a broad group of molecules involved in a variety of functions. To date, there are six subfamilies of the Ras superfamily (Ras, Rho, Ran, Arf, Rab, and R G K (Rad/Rem/Gem/Kir)) which are involved in a variety of processes including growth and proliferation, cytoskeletal morphology, nuclear import/export, and vesicle trafficking 4 , 1 ° . The Ras subfamily is composed of several subgroups (Fig. 1.1). First are the classical Ras molecules, H-Ras, N-Ras, K-Ras (K-Ras 4A and B splice forms), which have an apparent molecular weight of approximately 21 kDa when separated by reducing SDS-polyacrylamide gel electrophoresis (PAGE) . The non-classical Ras 3 molecules have a slower Mr of approximately 28 kD and include M-Ras, R-Ras, and TC21. Finally, there are the Rap and Ral GTPases. The Ras molecules are principally associated with activation of a variety of effector molecules which affect gene expression. All members of the Ras subfamily are characterized by two regions of high homology, known as "switch I and II". Switch I is the principal effector-binding region. Three principle mutations within the switch I region characterize the interaction of p21 Ras molecules with the effectors Raf, PI-3K and RalGDS 1 1 . The Raf effector molecules of which there are three known isoforms (Raf- i , B-Ras and A-Raf) act to trigger a cascade of kinase activation (Fig. 1.2). As a result of Raf activation by Ras, Raf phosphorylates the dual specificity kinases (MEK1 and 2), which subsequently phosphorylate the extracellular-regulated kinases (Erk) on a threonine and a tyrosine residue. Activated Erk then leads to the phosphorylation and activation of transcription factors such as Elk-1, leading to alterations in gene expression 1 0 . The activation of PI-3K and Ra lGDS by Ras also causes the activation of signaling cascades leading to the activation of transcription factors and induction of gene expression 1 2 , 1 3 . The non-classical Ras molecule M-Ras, like p21 Ras, has been shown to activate PI-3K, Ra lGDS and Raf. However, the activation of Raf and RalGDS is much weaker than with classical Ras molecules 1 4 ' 1 6 . Ultimately, alterations in gene expression induced by both classical and non-classical Ras molecules lead to the progression of the cell cycle. Further, when these 4 Ras proteins are constitutively activated by mutations, and allowed to signal unchecked, they are associated with transformation and oncogenesis 1 0 , 1 7 . R a p l A R h o E Figure 1.1. A dendrogram comparing the level of amino acid homology between representative members of the Ras superfamily of small GTPases. 5 1.4. Rho GTPases Pioneering studies within the laboratory of Alan Hall utilized micro-injection of constitutively active mutants of Rho subfamily GTPases to provide the first functional classification of these molecules 1 8" 2 0. Cdc42, Rac and Rho were segregated based on the cytoskeletal effects induced which were filopodia, lamellipodia (membrane ruffles), and stress fibres, respectively. In addition to sharing a role in controling cytoskeletal architecture, Rho subfamily GTPases also share a high degree of sequence homology with one another (Fig. 1.1). Three isoforms of Rac, Rac-1, Rac-2, and Rac-3, form a closely related subgroup of the Rho GTPases . Cdc42 is more distantly related and, like Rac-1, is expressed ubiquitously 2 1 , 2 2 (Fig. 1.1). In contrast, the expression of Rac-2 is restricted to cells of the lymphohemopoietic system 2 1 . Rac-3 is the most recently described isoform and seems to have no discernable pattern of expression 2 3 . The specific functional role of Rac-3 awaits further study. Most clues to the function of the Rac family have come from experiments involving either over-expression of constitutively active or dominant-negative mutants of Rac-1, or micro-injection of constitutively active Rac-1 7 ' 2 4 ' 2 5 . Such technologies have implicated Rac-1 in far more than formation of lamellipodia. The roles for Rac-1 have included progression of cells through the GA phase of cell cycle, and activation of the p21 activated kinase (PAK) family of serine/threonine kinases 7 ' 2 4" 2 6 . Over-expression of dominant-active or -inhibitory mutants of Rac have also implicated it in the activation of 6 both families of "stress activated kinases" (p38 M A P K and JNK) in fibroblasts 2 7" 3 0 , and of JNK in hemopoietic cells 3 1 , 3 2 . Similar approaches have implicated Rac and Cdc42 in a variety of functions critical to the operation of the immune system including production of oxidants by neutrophils, phagocytosis, chemotaxis, cell-mediated cytotoxicity, and the differentiation of lymphocytes of the T helper-1 subclass 3 3" 4 0. Certainly there is strong evidence that the Rac and Cdc42 GTPases are of vital importance to eukaryotic cell function and are involved in a variety of immunologically relevant processes that are indispensable to the survival of Vertebrate organisms. An obvious caveat to experiments that involve supra-physiological levels of constitutively active or dominant-negative mutant proteins is that they may not accurately identify physiological functions. The interpretation of experiments using dominant-negative mutants of Rac is particularly problematic since there are exchange factors with two catalytic domains {e.g. mSos-1, Ras GRF1 and 2) that are able to bind to and activate members of the Ras and Rac families 7 ' 4 1 4 4 . Thus, dominant-negative Rac mutants will bind to and sequestrate activators of Rac that also activate members of the Ras pathway, and vice versa. Gene-targeting experiments have yielded a variety of insights into the immunological importance of Rac-2. Analysis of hemopoietic cells from gene-targeted mice lacking Rac-2 has provided clear evidence for specific functions of Rac-2 that are not complemented by other Rac family members. The absence of Rac-2 resulted in defects in chemotaxis, adhesion and super-oxide production in neutrophils 4 5 , and 7 growth, survival, chemotaxis, adhesion and degranulation in mast cells 4 6 . There were also surprises not predicted by previous experimentation with the methodologies discussed above, including indications that Rac-2 was upstream of Erk MAP kinase 4 5 and A k t 4 6 . There were also observations revealing an inter-regulation of Rac-2 and Rac-1 and Cdc42. Mast cells from mice lacking Rac-2 expressed much higher levels of Rac-1 (which were not diminished when Rac-2 was reintroduced) 4 6, while hemopoietic stem cells lacking Rac-2 exhibited increased activation of Cdc42 4 7 . These observations emphasize the limitations to our current picture of signaling events involving Rac. 1.5. Activation of Rac and Cdc42 There is only one report of a direct biochemical examination of the ability of hemopoietic growth factors to activate endogenous Rac. This study concluded that, whereas treatment of neutrophils with fMetLeuPhe (fMLP) induced strong activation of Rac, treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF) , or TNF-a (tumor necrosis factor- a) did not 4 8 . Biochemical evidence of the activation of Rac by platelet-derived growth factor (PDGF), fMLP, leukotrienes, and cross-linking of the T cell receptor has been previously reported 4 9 " 5 2 . The general mechanisms of activation are thought to involve the recruitment to the plasma membrane of guanine-nucleotide exchange factors (GEF) which trigger the exchange of Rac-bound G D P for G T P . With one notable exception, smgGDS 5 3 , 5 4 , the G E F that are active on the Rho family contain pleckstrin homology domains (PH) capable of binding membrane lipids, such as PIP3, generated by 8 phosphatidyl-inositol-3-kinase (PI-3K) 5 5 , 5 6 . Moreover, studies with pharmacological inhibitors of PI-3K (Ly294002 and wortmannin) have demonstrated that PI-3K is necessary for activation of Rac by many st imul i 4 9 - 5 1 - 5 7 . However, activation of Rac-2 by treatment of neutrophils with phorbol myristate acetate (PMA) was not blocked by an inhibitor of PI-3K activity, pointing to the existence of Rac G E F that do not require PI-3K activity for their action on R a c 5 1 . Although PI-3K can be directly recruited by a receptor, both classical and non-classical Ras molecules are also potentially capable of recruiting PI-3K to the plasma membrane and inducing the generation of PIP3 which results in activation of Rac. The role of Ras molecules as activators of Rac has been supported by evidence that constitutively active and dominant negative mutants of Ras induce and inhibit membrane ruffling, respectively 1 1 , 1 8 . However, biochemical studies on Ras-mediated activation of Rac have not been performed to date. Figure 1.2 summarizes the signal transduction events leading to the activation of Rac and Cdc42 in response to extracellular stimulation of cells. First, interaction of antigens or soluble ligands {e.g. hemopoietic growth factors) with the B cell antigen receptor (BCR) or cytokine receptors, respectively, leads to a series of intracellular tyrosine phosphorylation events. This allows the activation of Rac or Cdc42 via one of several pathways. Firstly, phospho-tyrosine residues allow the recruitment of adaptor molecules, such as She and Grb2, which subsequently recruit Ras G E F , such as Sos, to the plasma membrane 1 0 . This recruitment is thought to localize specific G E F near Ras GTPases which are anchored in the plasma membrane via prenylation and either 9 palmitoylation or a stretch of poly-basic amino acids 4 , 6 , 1 ° . The subsequent activation of Ras leads to the recruitment of PI-3K and generation of PIP3 5 8 . This in turn allows recruitment and activation of G E F specific for Rac 4 2 , 5 6 . The second mechanism of Rac activation can occur via direct recruitment of PI-3K by a receptor. In this case, the p85 subunit of PI-3K binds via its SH2 domain to phosphorylated tyrosine residues on the ligand-stimulated receptor 5 8 . This allows localization of the catalytic subunit of PI-3K (pi 10), which is bound to the p85 subunit, next to the plasma membrane. Once at the plasma membrane, the p110 subunit leads to the activation of Rac as described above 4 2 , 5 6 . It has also been shown that Rac can be activated downstream of Cdc42 1 9 . 10 extracellular space cell surface receptor (e.g. IL-3 receptor) PIP2 PIP3 Rho GEF ^ (e.g. Vav) [ GDP nucleus transcriptional activation (Elk-j) (alterations in gene expression) Fig. 1.2. Basic overview of Rac and Ras related signal transduction 11 1.6. MAP kinases Hemopoietic growth factors, whether acting through receptor tyrosine kinases (e.g. Steel locus factor (SLF) or colony-stimulating factor-1 (CSF-1)), or receptors of the cytokine receptor superfamily (e.g. interleukin-3 (IL-3) or GM-CSF) activate Erk, c-jun N-terminal kinase (JNK), and p38 mitogen activated protein kinases (MAPK) 3 1- 5 9- 6 1 . The Erk kinases, Erk1 and 2, are activated by phosphorylation by the upstream kinases MEK1 and 2 6 2 " 6 4 . An Erk kinase, Erk5, which is activated by M E K 5 has also been recently discovered 6 5 . The p38 MAPK include four isoforms, a , (3, Sand y, which are activated by the upstream kinases MKK3 and 6 6 6 . Finally, the J N K kinases include three isoforms, JNK1 , 2, and 3, that are activated by MKK4 and 7 3 2 ' 6 7 ' 6 8 . These three broad groups of M A P K are activated by dual phosphorylation of both a threonine and tyrosine residue in the activation loop of the kinase by the specific M E K or MKK described above 6 9 - 7 1 . The phosphorylated tripeptide motif, TXY , is specific for each family of M A P K . The generation of antibodies which specifically recognize the phosphorylated forms of these three families of M A P K have been highly useful in assessing their activation by a range of stimuli in different cellular settings. Activation of any of these three families of M A P K leads to the phosphorylation and activation of kinases (e.g. R S K 7 2 and other M A P K activated protein (MAPKAP) kinases 7 3 ' 7 5) and transcription factors (e.g. ATF-1 and 2, AP-1 and Elk-1) leading to alterations in gene expression 6 9 ' 7 2 7 3 . 12 1.7. Proteomics and Genomics Science has entered the post-genomic era with the sequencing of the human genome 7 6 , 7 7 , and other eukaryotic and metazoan genomes 7 8 ' 8 3 . All areas of biological research, including signal transduction, have progressed as the number genes within known protein families has rapidly expanded 8 4 , 8 5 . However, approximately 40% of genes identified in the human genome have no homology with known proteins 7 6 or the approximately 1800 known protein domains 7 7 . Without homology as a key, alternative strategies must be employed to determine the function of these novel proteins and protein families. Comparative analysis of multiple eukaryotic genomes will allow prioritization of novel genes/gene families for scientific study whose evolutionary conservation suggests their function is critical to basic cellular processes 76'77>86-88. Similarly, genes present only in higher Vertebrate genomes (such as mammalian genomes) with no homologous sequence in the genomes of organisms such as Drosophila melanogaster or yeast may also be considered high priority. Such genes may play critical roles in the immune and central nervous systems which are expanded or highly developed in higher Vertebrates 76,77,86 s ince signal transduction is a critical process in eukaryotic organisms and is central to the functioning of higher Vertebrate systems, it is highly likely that these prioritization schemes will identify novel proteins involved in signal transduction. The fact that these proteins were not identified in genetic or phenotypic screens used to date should be attributed to the saturation or bias of these strategies, rather than to the lack of importance of the proteins. 13 Having prioritized these novel proteins for study, proteomics strategies and systems biology will be particularly useful for investigating their function. Mass spectrometry, which has greatly advanced in recent years, will be invaluable in identifying proteins from expansive systems biology experiments 8 7 , 8 9 . In combination with sequenced genomes, mass spectrometry should allow the identification of all novel proteins contained in multi-protein complexes, if the complex can be purified or isolated 9 0 , 9 1 . Microarray technology will certainly complement mass spectrometry in providing clues as to the functions of novel genes by linking their up- or down-regulation to particular stimuli {e.g. hemopoietic growth factors), stages of cell cycle and development, and differing environmental conditions {e.g. osmolarity and variation in media composit ion) 9 2. However, even with the use of mass spectrometry, proteome analysis will remain particularly challenging given the large amount of splicing that occurs in eukaryotic organisms 9 3 , 9 4 . Nonetheless, studying splice forms will be absolutely critical given that alternative splicing may allow a single gene present in a given eukaryotic genome to direct the production of multiple proteins during different stages of development and in different tissues. Further, some of the proteins generated by alternative splicing will certainly have varied functions in vivo. In Chapter 4 of this thesis, we have employed mass spectrometry in the identification of a novel protein that was purified from activated lymphocytes. Further, by analysing genomic and E S T databases for sequence homology, we have identified a second molecule called Caprin-2. Our studies on the Caprins represent another 14 example of how mass spectrometry and genomics allows one to move from a purified unknown protein to the identification and study of an entire gene family. Future proteomics-based studies will be instrumental in determining the exact function, and mechanism of action of the Caprins. 15 CHAPTER 2: EXPERIMENTAL PROCEDURES 2.1. Procedures for Chapter 3 2.1.1. - cDNA constructs, antibodies and reagents. The vectors encoding G12V H-Ras, Q61K N-Ras, V12L Rac-1, and constitutively active PI-3K (p110*) were gifts from Dr. A. Ambrosini (DiBiT, H. San Raffaele, Milan, Italy), Dr. Rob Kay (The Terry Fox Laboratories, Vancouver, Canada), Dr. Frank McCormick (University of California, San Francisco Cancer Research Institute, CA) , and Dr. Lewis T. Will iams (Chiron Corporation, Emeryville, CA), respectively. M-Ras was cloned as described previously 1 7 . The bacterial expression vector, pGEX, encoding a Glutathione-S-transferase (GST) - p21 activated kinase (PAK) fusion protein (amino acids 59 to 145 of human PAK-1(3) (GST-PAK) and the mammalian expression vector, pRK5, encoding myc tagged Rac-1 were kind gifts from Dr. Alan Hall (MRC Laboratory for Molecular Cell Biology, London, UK). The p E G F P - C 1 vector was purchased from Clontech. The Rac-1 mouse monoclonal antibody was purchased from Up State Biotechnology Inc. Rabbit polyclonal antibodies specific for Rac^2 5 1 and Cdc42 (Fig 1A and B and data not shown) were from Santa Cruz Biotechnology. Antibodies specific for phosphorylated molecules, including phospho-Erk (mouse monoclonal), phospho-serine 473 of Akt (rabbit polyclonal), and phospho-p38 MAPK (rabbit polyclonal) were from New England Biolabs (Beverly, MA). Polyclonal rabbit antibodies recognizing unphosphorylated Erk and p38 M A P K were from Santa Cruz Biotechnology (Santa Cruz, CA) and Akt was from New England Biolabs (Beverly, MA). The F(ab') 2 fragments of goat anti-mouse IgM were purchased from Jackson ImmunoResearch Laboratories. Recombinant 16 murine cytokines: colony stimulating factor-1 (CSF-1), Steel locus factor (SLF), and interleukin-5 (IL-5) were obtained from R & D Systems (Windsor, Ontario). The late Dr. Ian Clark-Lewis (The Biomedical Research Centre, Vancouver, Canada) generously provided synthetic murine IL-3 and G M - C S F . Ly294002 was obtained from Calbiochem-Novabiochem Corp. and stock solutions were made at 50 mM in 100 % ethanol unless indicated otherwise. 2.1.2. - Cell culture. Cells were grown at 37°C in humidified incubators gassed with 5% C 0 2 using RPMI 1640 medium (Stem Cell Technologies, Vancouver, Canada), supplemented with 10% (v/v) fetal calf serum (FCS) (Cansera), 50 uM (3-mercaptoethanol, 0.2 mM L-glutamine, and 1 mM sodium pyruvate. The IL-3 dependent cell lines, Baf/3, WEHI 274.3, R6/X and MC/9, were cultured in 4% WEHI 3B conditioned medium as a source of IL-3. Primary bone marrow-derived mast cells (BMMC) were generated from 4-8 week old (C57BL/6 x DBA/2) F1 hybrid mice (BDF1) or Balb/c mice by culturing bone marrow cells in 4% WEHI-3B conditioned medium for 3-4 weeks to generate mature B M M C as described previously 9 5 . Primary B cell blasts were generated from the spleens of 4-8 week old BDF1 hybrid mice. A single cell suspension of splenocytes was obtained and red cells lysed using red cell removal buffer (0.017 M NH 4 CI, 0.14 M Tris, pH 7.2). After depletion of adherent cells by incubation for 2 hours on tissue-culture treated plastic dishes in 10 % F C S and RPMI, splenocytes were resuspended at 2 X 10 6 cells/mL with 15 ng/mL lipopolysaccharide (LPS) and cultured for 72 hours to generate B cell blasts. 17 2.1.3. - Cell stimulation. Prior to stimulation, factor-dependent cell-lines or B M M C were cultured overnight in one-tenth the concentration of growth factor in which they were normally grown. Cells were then washed two times in serum free media containing 20 mM Hepes (pH 7.2) (SFM). MC/9 or WEHI 274.3 were resuspended at 10 7 cells/mL and B M M C at 1.5 x 10 7 cells/mL in S F M for 1.5 hours at 37°C. Cells were stimulated in 1 mL of S F M by addition of saturating concentrations of recombinant or synthetic cytokines: IL-5 (50 ng/mL), S L F (50 ng/mL), CSF-1 (200 ng/mL), IL-3 (5 ug/mL), and G M - C S F (10 u,g/mL). After incubation with L P S for 72 hours, cultures of splenocytes were washed two times in S F M , resuspended in S F M at 1.5 x 10 7 cells/mL and incubated for 3 hours at 37°C in S F M . B- lymphocytes (1.5 x 10 7 cells in 1 mL) were stimulated by addition of 40 jxg of F(ab)' 2 fragments of goat anti-mouse IgM. Ly294002 or solvent was added to the cells during the final 15 minutes of serum starvation (prior to stimulation) where indicated. 2.1.4. - Electroporation. 2.0 x 10 7Baf/3, MC/9 or R6/X cells were incubated in 500 uL of S F M with 10 mg/mL DEAE for 20 minutes at 37°C. The suspension was transferred to a 0.4 cm electroporation cuvette and electroporated at 300 Volts and 975 uF using a Gene Pulser II (Bio-rad) yielding a time constant of 20 - 25 mseconds. After electroporation, cells were incubated at 37°C in RPMI, 10 % F C S , and 4 % WEHI-3B conditioned medium for 10 to 16 hours at 10 6 cells/mL. Cells were then washed twice in S F M and incubated at 1.0 x 10 7 cells/mL in S F M for 2 hours. Where indicated, cells were treated with Ly294002 for 30 minutes during the last half-hour of serum starvation. 18 2.1.5. - Assay for activation of Rac-1, Rac-2 and Cdc42. Recombinant G S T - P A K was prepared by growing Escherichia coli (DH5a) to an optical density (600 nm) of 0.6 at 37°C and inducing fusion protein production for 2 hours at 25°C with 0.1 mM isopropyl P-D-thiogalactoside (IPTG). Cells were pelleted and sonicated in P B S containing 1% Triton X-100 and protease inhibitors (protease inhibitor cocktail tablets (Boehringer Mannheim)). The soluble fraction of bacterial sonicate was applied to Glutathione (GT) Sepharose 4B beads (Amersham Pharmacia Biotech) for 30 minutes at 4°C which were washed twice in P B S containing 0.1% Tween-20 (PBST) and 1 mM dithiothreitol (DTT) and twice in P B S T without DTT. To assess Rac/Cdc42 activation, equal numbers of cells were lysed in a buffer containing 50 mM Tris pH 7.5, 10% glycerol, 1% Nonidet P-40, 30 mM MgCI 2 , 150 mM NaC l , 1mM sodium molybdate, 200 mM sodium orthovanadate, 1 mM sodium fluoride, 50 mM p-glycerol phosphate, 1 ng/mL microcystin, 10 u,g/ml_ aprotinin, 10 ng/ml_ soy bean trypsin inhibitor, 2 u,g/mL leupeptin, 0.7 |ig/mL pepstatin, and 40 ug/mL P M S F . Total protein in whole cell lysates (WCL) was quantified using a B C A protein assay kit (Pierce). Aliquots of W C L were added to 20 \iL of GT beads with bound G S T - P A K and were rotated for 30 minutes at 4°C. The supernatant was removed from the beads and retained for immunobloting. Beads were then washed three times with 1 mL of the same buffer used to lyse the cells. Sodium dodecyl sulfate (SDS) sample buffer was applied to aliquots of whole cell lysates or bead pellets and heated to 80°C for 10 minutes. Known aliquots of S D S eluates of beads or whole cell lysates were subject to S D S - P A G E (12% acrylamide) and immunobloting. Blots were probed with a Rac-1 mouse monoclonal antibody or rabbit 19 polyclonal antibodies specific for Cdc42 or Rac-2 to quantify levels of the respective activated G T P a s e s . The ratio of GTP-bound-GTPase in stimulated cells versus unstimulated controls was estimated from measurements of arbitrary units of optical density of bands corresponding to the G T P a s e in immunoblots of the respective samples. The ratio of the optical density of the band corresponding to the GTPase in immunoblots of eluates from the GST-PAK-coated beads, and in an immunoblot of a known aliquot of the WCL, provided an estimate of the absolute amount of GTP-bound GTPase as a fraction of the total cellular GTPase . The amount of total Rac/Cdc42 in the sample of W C L which was subjected to S D S - P A G E and immunobloting corresponded to 1 to 4% of that applied to the G S T - P A K coated beads as indicated. The degree of activation of GTPases was also expressed as the ratio of the optical density of the immunoblotted band of activated GTPase precipitated from stimulated cells, to the optical density of the immunoblotted band of activated GTPase precipitated from unstimulated cells. Goat antibodies specific for rabbit or mouse immunoglobulins and conjugated to horseradish peroxidase (Dako) were used as secondary antibodies. Immunoblots were developed using enhanced chemiluminescence (Amersham Pharmacia Biotech). All experiments are a representative of three or more experiments with the exception of Fig. 3.7 which was performed twice. 2.1.6. - Assay for activation of p38 MAPK, Akt or Erk. Proteins from WCL (50-100 u.g of total protein) were separated on S D S - P A G E , transferred to nitrocellulose membranes and probed with phospho-specific antibodies to assess levels of activated p38 MAPK, 20 Akt or Erk (as described in section 2.1.5.). To confirm equivalency of loading, membranes were stripped of bound antibodies by exposure to a solution containing 62.5 mM Tris, 0.2 % S D S , and 100 mM |3-mercaptoethanol for 30 minutes at 55°C, followed by washing with P B S T and re-probing with antibodies specific for the unphosphorylated forms of p38 MAPK, Erk, or Akt. 2.2. Procedures for Chapter 4 2.2.1. - cDNA constructs and antibodies. A cDNA encoding Caprin-1 (BC001731) was obtained from Research Genetics (Huntsville, AL). Full-length Caprin-1, or the indicated fragments were amplified by P C R and cloned into the mammalian expression vectors, p E G F P - C 1 (Clontech), p E B G (a gift from Dr. Leonard Zon, Dana-Farber Cancer Institute, Boston, Massachusetts), or the retroviral vector pMX-PIE (a gift from Dr. Alice Mui, Jack Bell Research Center, Vancouver General Hospital, Vancouver, Canada) to generate constructs encoding N-terminal G F P - or GST-fusion proteins, or a carboxy-terminal HA-epitope tagged version of Caprin-1, respectively. A cDNA encoding p137 9 6 , as well as an anti-serum against p137 were the kind gifts of Dr. Paul Luzio (University of Cambridge, UK). Aff in i ty pur i f ied rabbit an t ibod ies spec i f i c for the pept ide , E K L M D L L D R H V E D G N V T V Q H A , termed A P antibodies, were prepared from hyper-immune serum. 21 2.2.2. - Generation of cDNA consensus sequences and intron-exon boundaries. cDNA consensus sequences for human, mouse and Xenopus laevis Caprin-1, and human Caprin-2 were generated using a series of overlapping EST clones from the National Center for Biotechnology Information (NCBI) databases (www.ncbi.nlm.nih.gov). All sequence was covered by a minimum of 3 overlapping E S T clones. If only two overlapping EST clones were present, the sequence was confirmed using human or mouse genomic sequence. Consensus sequences were assembled using Sequencher (Gene Codes Corporation) and Macvector (Accelrys) software. Clones used to generate the Caprin-1 sequences included: for the human BC001731, BG720819, BG772489, BI253821, AU126290, BE902810, AL040074, AU131547, BG25298, AU1322904, BG742970, AU141221, BG681409, BI259287, AU119855, BG291262, BG563335; for the mouse BE288344, AM 1506, BF137301, X89571, BI152150, BI437836, BI1965135, BF467450, BF461675, AA066083, BE951949, AA140302, BF099994, AW913584, BI156548, AA183811, AI007156, BI695199, BF101269, BG072052, BG084803; and for X. laevis AW872143, BG408625, BE679984, BG408527, BE491274, BF048081, BE679686, BG406719, AW200655, BG346761, BG360366, BG021521, BF614499, AW200485, BE491444, BG486636, BE491455, BG345580. Clones used to generate the consensus sequence for human Caprin-2 included: AY074490, AW892778, BG944982, AU117060, BE734486, AL536436, AA301060, AI092672, Z444678, AU143985, BG250521, AA187575, BG202526, BE155964, 22 R55924, BF727469, BE378048, BG198633, BG201727, AL566335, AW022120, AI689091, BF589574, BG941654. Signal peptide and protein domain searches were performed using the SignalP sof tware (www.cbs .d tu .dk / se rv i ces /S igna lP ) and the Pros i te software (www.expasy.org), respectively. Dendrograms were generated using the Treetop software available at the Genebee website (www.genebee.msu.su). Exon boundaries were derived by analysis of acceptor-donor sites in contiguous, human genomic sequence (NCBI) (AC090469, AC068306, and AL049652 for caprin-1, and AC010198 for caprin-2), and confirmed using the BLAT t o o l 9 7 (University of Santa Cruz: www.genome.cse.ucsc.edu). Databases of Fugu rubripes and Ciona intestinalis genomes were provided by the DOE Joint Genome Institute (JGI: www.jgi.doe.gov). 2.2.3. - Cells. Cultured as described previously 1 7 or as described earlier in section 2.1.2, and transfected with 7 jig of DNA using lipofectamine reagent (Invitrogen) according to the manufacturer's instructions. Primary T- and B-lymphoblasts were generated from mouse splenocytes by stimulation with Concavalin A and IL-2 for 48 hours, or an anti-CD3 monoclonal antibody (2C11, Pharmigen) and IL-2 (2% of a 10 fold concentrate of medium conditioned by X063 cells secreting IL-2) for 3 days (or for the length of time indicated), or lipopolysaccharide for 3 days (as described earlier in section 2.1.2.), respectively. Bone-marrow cells from (CBA x C57BL6) F1 hybrid mice were cultured in RPMI supplemented with 10% F C S and 4% WEHI-3B-conditioned medium as a source of IL-3, or 20% L-cell-conditioned media as a source of C S F - 1 . 23 Differentiation of M1 leukemia cells was stimulated using recombinant, murine IL-6 (Intergen). 2.2.4. - Immunobloting and immune-precipitation.. Cells or tissues were lysed with buffer (described in section 2.1.5.) containing 1% Nonidet P-40, or for multimerization/co-precipitation experiments, buffer containing 0.2% Triton X-100. The lysates were normalized for total protein content. For immunobloting, samples were run on S D S -P A G E , and the separated proteins transferred to nitrocellulose and immunoblotted (as described in sections 2.1.5 and 6) with the indicated rabbit polyclonal antibodies (anti-G F P from Clontech or ant i-GST from Molecular Probes), or a mouse monoclonal antibody against the HA epitope (Covanse). Immune complexes were purified using protein-A Sepharose beads, and GST-fusion proteins using glutathione (GT) beads (Amersham/Pharmicia). All experiments were performed three or more times and a representative experiment was shown. 2.2.5. - In-gel digestion and mass spectrometric analysis. Gel bands stained with Coomassie blue were excised, digested with trypsin and analyzed as described 9 8 . Peptide mixtures were desalted on P O R O S R2 beads (Applied Biosystems) and analyzed by nanospray ionization on a quadrupole time-of-flight mass spectrometer (SCIEX O S T A R Pulsar). Database searches with the peptide fragmentation spectra were performed using the Mascot software (www.matrixscience.com), and analyzed manually to deduce the peptide sequences. 24 2.2.6. - Flow cytometric analysis of Caprin-1 and cell division. A FacsCalibur instrument, and "Cellquest" analysis software (BD Biosciences) were utilized for flow cytometry. For staining of intracellular antigens, cells were fixed in paraformaldehyde (1%) and permeabilized with saponin (0.3%) and B S A (1%). Fluorescein-conjugated goat-anti rabbit antibodies were obtained from Jackson Immunoresearch Laboratories (West Grove, PA). Analysis of cell division was performed by loading 3T3 cells with the red fluorescent dye, PKH26, according to the manufacturer's instructions (Sigma). All experiments are a representative of three or more experiments. 2.2.7. - Immunofluorescence. Cel ls were fixed in paraformaldehyde (4%), and permeabilized with saponin (0.3%) or methanol. After blocking with P B S containing Tween-20 (0.1%) (PBST) and goat serum (5%), cells were stained with A P antibodies or an anti-HA mouse monoclonal antibody followed by goat anti-rabbit or anti-mouse antibodies conjugated to AlexaFluor (594) from Molecular Probes. 2.2.8. - 32P-labelling of Caprin-1. Baf/3 cells were incubated at 2.5 x 10 6 cells/mL in phosphate-free DMEM (Invitrogen) with 10% dialyzed F C S , saturating concentrations of murine IL-3 (as described in section 2.1.2.), and Hepes (10 mM) for 2 hours. 2 mCi of 3 2 P-label led phosphoric acid (ICN) was added, and 1- 6 hours later Caprin-1 was immunoprecipitated from cell-lysates, and the precipitates subjected to S D S - P A G E , autoradiography, and immunobloting. 25 CHAPTER 3: ACTIVATION OF RAC-1, RAC-2 AND CDC42 BY HEMOPOIETIC GROWTH FACTORS OR CROSS-LINKING OF THE B LYMPHOCYTE RECEPTOR FOR ANTIGEN 3.1 Introduction Since there is only one report of a direct biochemical examination of the ability of a hemopoietic growth factor, G M - C S F , to activate endogenous Rac and this growth factor was unable to activate Rac 4 8 further study was warranted. Here a direct investigation of activation of endogenous Rac-1, Rac-2 and Cdc42 was performed. We tested the ability of a variety of hemopoietic growth factors active on two classes of receptors and ligation of the B C R to activate endogenous Rac-1 . To determine the molecular mechanism of activation of Rac-1, a variety of candidate activating molecules including PI-3K, p21 Ras GTPases , and the non-classical Ras, M-Ras, were tested. We also investigated whether activation of Rac-1 correlates with the activation of a known downstream effector of Rac, p38 MAPK. 26 3.2. Results 3.2.1. Activation of endogenous Rac-1, Rac-2 and Cdc42 by multiple hemopoietic growth factors. The activation of Rac-1, Rac-2 and Cdc42 was measured by specifically co-precipitating GTP-bound forms of these GTPases using a recombinant fusion protein (GST-PAK) of Glutathione-S-transferase (GST) and amino acid residues 59-145 of p21 activated kinase-1 p (PAK-1P), including the Cdc42/Rac-interactive binding domain 9 9 ' 1 0 0 . The G S T - P A K allows assessment of activation of Rac or Cdc42 as it specifically interacts with GTP-bound , activated Rac or Cdc42, but not the GDP-bound , inactive conformations of these GTPases as demonstrated previously 5 1 . Further, we have shown that a mutant of Rac-1 which is incapable of hydrolyzing G T P and hence is consitutively activated interacts with G S T - P A K , while a dominant negative mutant of Rac-1 which is constitutively bound with G D P and inactive does not significantly interact with G S T - P A K (data not shown). Since the G S T - P A K co-precipitates a variety of activated Rho family GTPases, it was critical to use antibodies specific for the desired GTPase . To assess the specificity of the antibodies we have used to identify Rac-1 and Rac-2, we expressed myc-tagged constitutively active, mutants of these proteins in Baf/3 cells. We co-precipitated the exogenous GTPases with G S T - P A K and split the precipitates into aliquots which were subjected to S D S - P A G E and immunobloting with either a monoclonal antibody specific 27 for Rac-1 or polyclonal antibodies specific for Rac-2. As shown in Figure 3.1 A, the Rac-1 specific monoclonal antibody specifically recognized constitutively active Rac-1 while detecting absolutely none of the constitutively active Rac-2 present. The converse was observed with immunobloting with polyclonal antibodies specific for Rac-2 (Fig. 3.1 A), in agreement with previous assessments of the specificity of these antibodies 5 1 . Moreover, as shown in Figure 3.1 B, the Rac-1 specific monoclonal antibody was able to specifically detect over-expressed myc-tagged Rac-1, but not myc-tagged Rac-2 while recognizing endogenous Rac-1. The converse was observed when fractions of the same co-precipitation were immunoblotted with the Rac-2 specific polyclonal antibodies. These results indicated that the antibodies used to detect Rac-1 and Rac-2 were indeed operationally specific for their respective targets and had the necessary specificity and sensitivity for use in assessing activation of specific endogenous isoforms which occur naturally as a mixture in murine hemopoietic cells. The polyclonal antibodies specific for Cdc42 have been used previously 4 9 5 1 and we confirmed that they recognized recombinant Cdc42, but did not cross-react with Rac-1 or p21 Ras (Fig. 3.1C). The mouse mast cell-like cell-line, MC/9, was chosen as a convenient model for study of the activation of endogenous Rac-1 , because it responds to multiple h e m o p o i e t i c growth fac to rs . T h e s e i nc lude in te r leuk in -3 ( IL-3), granulocyte/macrophage-colony stimulating factor (GM-CSF) , and IL-5, all of which act through receptors of the hemopoietin family, as well as Steel locus factor (SLF), which acts through a receptor-tyrosine kinase. 28 In contrast to published data indicating that G M - C S F failed to activate Rac in human neutrophils 4 8 , we observed rapid activation of Rac-1 by G M - C S F (Fig. 3.1 D). This activation was maximal at 1 minute, and was maintained for at least ten minutes. Stimulation of MC/9 cells with IL-3 also induced activation of Rac-1, although the kinetics were slower with maximal activation occurring after 5 minutes and being maintained for at least ten minutes (Fig 3.1 D). Similar results were observed with IL-5 (Fig. 3.1 D). The levels of Rac-1 G T P reached after stimulation by these three growth factors was estimated to range from 0.8 to 1.4% (0.8% for G M - C S F , 1.2 % for IL-3, and 1.4 % for IL-5) as a percentage of the total Rac-1 present in whole cell lysate (WCL). The ratio of the optical density of the band corresponding to the activated GTPase in immunoblots of eluates from the GST-PAK-coated beads, and in an immunoblot of a known aliquot of the WCL, provided an estimate of the absolute amount of GTP-bound GTPase as a fraction of the total cellular GTPase. We also used cultures of bone marrow-derived mast cells (BMMC) to confirm that IL-3 activated endogenous Rac-1 in primary cells. IL-3 treatment of BMMC resulted in greater increases in the levels of activated Rac-1 over those seen in resting cells (4.3 fold), than those seen in MC/9 (2.4 fold) (Fig. 3.2A compared to Fig. 3.1 C). The estimated percentage of total cellular Rac-1 activated in IL-3 treated B M M C cells was also greater than that seen in IL-3 treated MC/9 cells (3.6 % in B M M C compared with 1.2% in MC/9). Because Rac-2 is specifically expressed in hemopoietic cells, it was interesting to examine activation of this Rac isoform 2 1 , 1 0 1 . Endogenous Rac-2, as detected by 29 antibodies specific for Rac-2 (Fig. 3 .1 ) 5 1 , was clearly activated by IL-3 in B M M C (Fig. 3.2B). However, limitations of the Rac-2 antibodies (could only be used on precipitated Rac-2, not whole cell lysates) prevented accurate quantitation of the percentage of the total cellular Rac-2 that was activated. IL-3 stimulation of B M M C also induced increased levels of GTP-bound Cdc42 (activation of approximately 1% of total cellular Cdc42) (Fig. 3.2C). We observed that colony stimulating factor-1 (CSF-1) and S L F , two other hemopoietic growth factors, acting through a different class of receptor (tyrosine kinase receptors), also activated Rac-1. S L F stimulation of MC/9 cells induced maximal increases in levels of Rac-1 G T P by one minute, but levels had dropped considerably by five minutes (Fig. 3.3A). S L F also induced activation of endogenous Rac-1 in primary B M M C (Fig. 3.3B). Stimulation of the myelomonocytic cell line, WEHI 274.3, with C S F - 1 , led to rapid activation of Rac-1, with levels reaching a maximum at one minute, and dropping significantly by five minutes (Fig. 3.3C). The percentages of Rac-1 G T P (as a fraction of the total Rac-1 pool present) in cells activated by S L F (4% in B M M C and 2.4 % in MC/9) or by CSF-1 (5%) were again relatively small. 30 Figure 3.1. Specificity of Rac antibodies and activation of Rac-1 by p-common family cytokines. Baf/3 cel ls were electroporated with 15|a,g of constitutively act ive Rac-1 (Rac-1 myc) or R a c - 2 (Rac-2 myc). 16 hours post-electroporat ion, cel ls were lysed and both over -expressed and endogenous R a c G T P a s e s were precipi tated us ing recombinant G S T - P A K bound to G T beads . Ant ibodies speci f ic for Rac-1 or R a c - 2 were used for immunoblot ing A) over -expressed , myc- tagged Rac-1 or Rac -2 as indicated or B) endogenous and over -expressed Rac-1 or Rac -2 i so fo rms. C) Baf /3 ce l ls were e lect roporated with 30 u.g of p lasmid encod ing consti tut ively act ive, myc- tagged H - R a s (H-Ras) , or Rac-1 (Rac-1) , or constitutively act ive HA- tagged C d c 4 2 (Cdc42) . Immunoblots of ce l l lysates were made and probed with an t i -Cdc42 speci f ic antibody, or ant i-myc antibody. D) M C / 9 cel ls were incubated for 5 minutes with P B S (con), G M - C S F (10 utj/mL), IL-3 (5 ug/mL) , or IL-5 (50 ng/mL) and lysed. Immunoblots of Rac-1 G T P bound by beads and in W C L were performed using a Rac-1 speci f ic monoc lona l ant ibody (Rac-1 G T P ) . One-ha l f of the total G T P - b o u n d Rac-1 precip i tated w a s run in immunob lo ts . T h e W C L cor responded to 1.3 %, 1.2 %, and 1.2 % of the total R a c avai lab le for precipitation for G M - C S F , IL-3, and IL-5, respect ive ly . N u m b e r s represent the rat ios of the opt ica l dens i ty of the immunoblotted band of G S T - P A K precipitated Rac-1 from st imulated ce l ls to the optical density of the immunoblotted band of G S T - P A K precipitated Rac-1 from control, unstimulated cel ls. 31 Rac-1 myc Rac-2 myc 1 1/2 1/4 1 1/2 1/4 anti-Rac-1 Ab anti-Rac-2 Ab Rac-1 myc Rac-2 myc 1 1/2 1/4 Rac-1 myc — endo Rac Rac-2 myc endo Rac 1 1/2 1/4 anti-Rac-1 Ab anti-Rac-2 Ab HA Cdc42 endo Cdc42 4? o N anti-Cdc42 blot anti-myc blot GM-CSF IL-3 V 5' 10' Rac-1 GTP — — — 1.0 2.8 2.7 3.0 1' 5" 10" 1.0 1.2 2.4 2.5 IL-5 5' 1.0 3.2 B Rac-1 GTP WCL 1.0 4.3 Rac-2 GTP WCL 1.0 2.8 . 4 cdc42 GTP WCL 1.0 1.9 Figure 3.2. IL-3 activates multiple Rho family GTPases in bone marrow-derived mast cells. Bone marrow-derived mast cells (BMMC) generated from BDF1 mice were incubated for 5 minutes with PBS (con) or IL-3 (5 ug/mL). Cells were lysed and GST-PAK was used to precipitate endogenous, GTP-bound Rac-1, Rac-2 or Cdc42. Immunoblots were performed with A) a Rac-1 specific monoclonal antibody, B) polyclonal antibodies specific for Rac-2, or C) polyclonal antibodies specific for Cdc42. Immunoblots of Rac/Cdc42 present in WCL were performed with the same antibodies as used to visualize Rac/Cdc42-GTP precipitated with GST-PAK (except for the case of Rac-2 where, for technical reasons, the Rac-1 antibody was used to probe the immunoblot of the WCL). One-half of total GTP-bound Rac-1 precipitated was run in the Rac-1 immunoblot; the WCL corresponded to 3 % of total Rac-1 available for precipitation. The total amount of GTP-bound Cdc42 precipitated was run in the Cdc42 specific immunoblot; the WCL corresponded to 4 % of the total Cdc42 available for precipitation. 33 Rac-1 GTP WCL Rac-1 GTP WCL B u 30" 1' 5' 10' 1.0 3.6 7.1 4.1 4.0 1.0 3.5 CSF-1 1' 5' 10" 1.0 12.0 8.5 4.5 Figure 3.3. Activation of Rac-1 stimulated by hemopoietic growth factors acting through receptor tyrosine kinases. A) MC/9 cells were stimulated with SLF (50 ng/mL), B) BMMC from BDF1 mice were stimulated with SLF (50 ng/mL), or C) WEHI 274.3 cells were stimulated with CSF-1 (200 ng/mL) for the indicated number of minutes or with PBS for 5 minutes (con). Following stimulation, lysates were assayed for GTP-bound/activated endogenous Rac-1 (Rac-1 GTP) as specified earlier. Immunoblots were performed using a Rac-1 specific monoclonal antibody. One-half of the total GTP-bound Rac-1 precipitated was run in immunoblots for A, B, and C. The WCL corresponded to 1.9 %, 2.6 %, and 2.1 % of the total Rac-1 available for precipitation for A, B, and C, respectively. 34 3.2.2. Activation of endogenous Rac-1 by cross-linking of the BCR. To investigate whether Rac-1 was activated by ligation of the endogenous receptor for antigen on B-lymphocytes (BCR), we used primary cultures of B lymphoblasts that had been generated by treatment of resting splenocytes with L P S . We cross-linked the B C R without engaging the Fc-receptors by using the F(ab)' 2 fragment of antibodies to IgM. This resulted in rapid activation of Rac-1, with maximal stimulation occurring at one minute (5.3 fold over background) (Fig. 3.4). This level of activated Rac-1 was maintained for ten minutes. Once again, the absolute amount of Rac-1 activated by BCR-mediated stimulation represented only a small fraction of the total cellular Rac-1 (about 1%). a-IgM / V 5' 10' • ^ ____ _mmm Rac-1 GTP — ~ — 1.0 5.3 5.3 4.2 WCL Figure 3.4. Activation of Rac-1 induced by cross-linking of the BCR. Splenocytes from BDF1 mice were stimulated with lipopolysaccharide (LPS) (15 u,g/mL) for 72 hours to generate B-lymphoblasts. B-lymphoblasts were stimulated with F(ab)'2 fragments of anti-mouse IgM (40 ng/mL) for the indicated length of time or treated with PBS for 5 minutes (con). Following stimulation, lysates were assayed for GTP-bound endogenous Rac-1 (Rac-1 GTP) as specified earlier. Immunoblots were performed using a Rac-1 monoclonal antibody. One-half of the total GTP-bound Rac-1 precipitated was run and the WCL corresponded to 1.4 % of the total Rac-1 available for precipitation. 35 3.2.3. Different requirements for PI-3K activity for activation of Rac-1 induced by IL-3 or cross-linking of the BCR. All known Rho family guanine nucleotide exchange factors (GEF) which contain the Dbl homology (DH) catalytic domain, also contain a plecksfrin homology (PH) domain that is likely to bind phosphoinositides generated by phosphatidyl-inositol-3-kinase (PI-3K) 5 5 , 1 0 2 . Thus if activation of Rac-1 by hemopoietic growth factors or ligation of the B C R involves these G E F s , it is likely to depend on PI-3K. To test this hypothesis, we investigated the effect of a pharmacological inhibitor of PI-3K, Ly294002. The activation of Rac-1 induced by cross-linking the B C R was reduced by approximately 75% with concentrations of Ly294002 (3.1 u,M) that reduced Akt phosphorylation to background levels (Fig. 3.5B). This suggested that the remaining 25 % of Rac-1 activation did not require increases in levels of PI-3K activity. With higher concentrations of Ly294002, levels of Akt phosphorylation were reduced to well below the background level in unstimulated cells. We observed that when background PI-3K activity was so reduced, Rac-1 activation was also reduced to near background levels (93% inhibition at 25 uM Ly294002) (Fig. 3.5B). This suggests that low background levels of overall PI-3K activity may be permissive for Rac-1 activation. Similar results were obtained with IL-3 stimulation of B M M C , where activation of Rac-1 was only partially inhibited (by 70%) by Ly294002 even at a high concentration (50 u,M) (Fig. 3.5A). Immunobloting of cell lysates with an antibody specific for phosphorylated Akt confirmed that the IL-3 induced increase in PI-3K activity had been completely inhibited. These results suggested the possible existence of a pool of Rac-1 36 which was activated by IL-3 or ligation of the B C R through mechanisms that did not require stimulus-mediated increases in PI-3K activity. However, because levels of Akt phosphorylation were never reduced below background to undetectable levels, we cannot exclude the possibility that a low level of PI-3K activity was permissive for IL-3 induced Rac-1 activation. There were also differences in the sensitivity to inhibition by Ly294002 of activation of the p38 M A P K and Erk induced by IL-3 or by cross-linking of the B C R . Thus, Ly294002 had no inhibitory effect on the IL-3 induced activation of p38 M A P K or Erk (Fig. 3.5A). The absence of any effect of Ly294002 on activation of these MAP family kinases was surprising given evidence implicating the Rac pathway in their activation 2 8 ' 3 0 , and our finding that Ly294002 inhibited IL-3 induced activation of Rac by 70%. These results suggested that, in the context of signaling through endogenous IL-3 receptors, part of the activation of Rac and all of the activation of p38 M A P K and Erk, was independent of IL-3 induced increases in PI-3K activity. However, in the case of ligation of the B C R , the presence of Ly294002 resulted in partial inhibition of the activation of both p38 M A P K and Erk 1/2 (Fig. 3.5B). Thus, activation of Erk and p38 M A P K downstream of the B C R involved both PI-3K dependent and PI-3K independent pathways. 37 Figure 3.5. Effects of the PI-3K inhibitor, Ly294002, on activation of Rac-1 and p38 MAPK induced by IL-3 or cross-linking of the BCR. A) B M M C from Ba lb /c mice or B) L P S st imulated B lymphob las ts from B D F 1 mice were pretreated with L y 2 9 4 0 0 2 or with ethanol (solvent), and subsequent ly st imulated for 5 minutes with P B S (con), IL-3 (1 ng/mL), or F(ab) ' 2 f ragments of ant i -mouse IgM (40 ng/mL), after which cel ls were l ysed . Act iva ted, endogenous Rac-1 (Rac-1 G T P ) w a s precipi tated by G S T - P A K bound to G T b e a d s . Immunoblots were per formed using a Rac-1 speci f ic monoc lona l antibody. Act ivat ion leve ls of endogenous Akt, Erk, and p38 M A P K were m e a s u r e d f rom W C L us ing an t ibod ies c a p a b l e of spec i f ica l ly recogn iz ing the phospho ry la ted form of these m o l e c u l e s ( p - A K T , p-Erk , p-p38 M A P K ) . Equ iva lency of loading w a s conf i rmed by re-probing phospho-b lo ts with ant ibodies capab le of recognizing both phosphory lated and non-phosphorylated proteins (Akt, Erk, p38 M A P K ) . 38 3.2.4. Ras-mediated activation of Rac-1 is PI-3K independent. Our observation that IL-3 induced activation of Rac-1 was not completely inhibited by concentrations of Ly294002 which completely blocked increases in PI-3K mediated activation of Akt, suggested the existence of alternative mechanisms of activating Rac-1 that did not require increased PI-3K activity. There is evidence that Ras proteins are upstream activators of Rac ™ , and hemopoietic growth factors are known to activate Ras family members 1 0 3 ' 1 0 4 . To test the hypothesis that members of the Ras family could activate Rac-1 in hemopoietic cells, and to investigate whether this occurred through a mechanism involving increased PI-3K activity, we co-expressed wild-type Rac-1 and a constitutively active mutant of one of two p21 Ras isoforms, H-Ras and N-Ras, in the IL-3 dependent cell-line, Baf/3. A plasmid encoding G F P , p E G F P - C 1 , was co-expressed in all samples to monitor cell viability and electroporation efficiency. Over-expression of either a constitutively active mutant of p21 H-Ras (G12V H-Ras) or N-Ras (Q61K N-Ras) induced activation of co-expressed Rac-1 (Fig. 3.6A). To investigate whether Ras-induced activation of Rac-1 was dependent on Ras-mediated increases in PI-3K activity, we examined the effects of treating cells with Ly294002. To determine the concentration of Ly294002 needed to give maximal inhibition of Akt phosphorylation in Baf/3 cells exhibiting high levels of PI-3K activity, we treated Baf/3 cells transfected with either constitutively active H-Ras (G12 H-Ras) or PI-3K (p110*) with titrated doses of Ly294002 (Fig. 3.6C). We observed that 25 uM Ly294002 resulted in the reduction of Akt phosphorylation to background levels in cells over-expressing either the G12 H-Ras or the constitutively active PI-3K. Moreover, there was no further decrease in Akt 39 phosphorylation in cells treated with 50 u.M Ly294002. These experiments showed that, despite the fact that Ly294002 (25 u.M) reduced the phosphorylation of Akt induced by expression of G12 H-Ras or Q61 N-Ras to background levels, it had no effect on the activation of exogenous Rac-1 (Fig. 3.6A). Thus, expression of constitutively active p21 H or N-Ras activated co-expressed Rac-1 through a mechanism which did not depend on overall increases in PI-3K activity. Because we have shown that the method used to assay activation of "p21 Ras" by hemopoietic growth factors in published studies does not discriminate between isoforms of p21 Ras or the "non-classical" member of the Ras family, M-Ras 1 7 , it was important to determine whether M-Ras was upstream of Rac-1 as well. This was particularly relevant, as we have shown (Schallhorn and Schrader, unpublished observations) that hemopoietic growth factors such as IL-3 were strong activators of, M-Ras, but not of p21 H-Ras. Expression of a constitutively active mutant of M-Ras (Q71L M-Ras) in Baf/3 cells resulted in activation of co-expressed Rac-1 (Fig. 3.6B). Moreover, treatment of cells expressing constitutively active M-Ras with Ly294002 (25 (xM) had no effect on activation of co-expressed Rac-1, although the phosphorylation of Akt induced by expression of Q71L M-Ras was reduced to background levels (Fig. 3.6B). Therefore, we have concluded that both p21 Ras isoforms (H-Ras and N-Ras) and M-Ras are capable of activating exogenous Rac-1 through mechanisms that do not depend on overall increases in PI-3K activity. To determine whether increased PI-3K activity alone was sufficient for activation of Rac in cells of hemopoietic origin, we performed parallel experiments in which groups of cells were co-transfected with Rac-1 and a constitutively active mutant of the p110 40 catalytic subunit of PI-3K. These experiments demonstrated that over-expression of activated PI-3K in hemopoietic cells, was sufficient for activation of co-expressed Rac-1 (Fig. 3.6A). In order to examine whether Ras molecules were upstream of Rac-1 in a physiological setting, we examined the activation of endogenous or over-expressed Rac-1 by IL-3 in the presence or absence of a dominant negative mutant of p21 H-Ras (S17N H-Ras). Although use of dominant negative mutants of Ras molecules is problematic for reasons discussed in section 3.3, we nonetheless observed that dominant negative H-Ras noticeably inhibited activation of endogenous Rac-1 induced by IL-3 stimulation (Fig. 3.7). Dominant negative H-Ras also reduced levels of activated, over-expressed Rac-1 (Fig. 3.7). However, this was a reduction below background levels, since detectable activation of over-expressed Rac-1 was not observed with IL-3 stimulation. Since IL-3 stimulation had activated endogenous Rac-1 in these Baf/3 cells, this was a surprising result we propose might be explained in two ways. First, this observation may be a result of mislocalization of over-expressed Rac-1. One might envision a situation in which there is a limited amount of Rac-1 that can exist at any given time in the appropriate plasma membrane compartment (such as a lipid raft) for activation by IL-3. Because the endogenous Rac-1 was present prior to the over-expressed Rac-1 , it is possible that endogenous Rac-1 may have saturated this particular signaling compartment. As a result, over-expressed Rac-1 would be mislocalized, and fail to be activated by IL-3 stimulation, while endogenous Rac-1 would 41 be activated normally. Alternatively, it remains a formal possibility that Rac-1 is not activated by IL-3 and we have been misled by the anti-Rac-1 antibody which is in fact not specific for Rac-1. If this were true, we would likely be observing the activation of the Rac-3 isoform, since the data in Figure 3.1 rules out that our anti-Rac-1 antibody can detect Rac-2. A definitive explanation of this unexpected result will require more sophisticated reagents capable of specifically detecting Rac-3 and an answer as to whether or not Rac-3 is expressed in Baf/3 cells, or the B M M C used to study IL-3 induced activation of Rac-1 in section 3.2.1. Figure 3.6. Over-expression of activated mutants of H-Ras, N-Ras or M-Ras results in activation of Rac-1 via a mechanism, which is not inhibited by Ly294002. Baf/3 cel ls were co-e lect roporated with 3 up; of p E G F P - C 1 empty vector, 3 uo of wi ld- type, myc tagged R a c - 1 , and 15 pg of A and C) control p lasmid (vector), a constitutively act ive mutant of PI-3K (p110 *), or constitutively act ive mutants of one of two p21 R a s isoforms (G12 H - R a s and Q61 N-Ras) (B) control p lasmid (vector), wild type M - R a s (WT M-Ras ) , or a consti tut ively act ive mutant of M-R a s (Q71 M - R a s ) . 16 hours post-electroporat ion, ce l ls were incubated with Ly294002 (25 u M d isso lved in D M S O ) for 2 hours for A) N - R a s or incubated with Ly294002 (25 u M d isso lved in ethanol) for 30 minutes for A) H -Ras , B), and C) and lysed. Act ivated Rac-1 myc (Rac-1 myc G T P ) w a s precip i tated from lysates us ing G S T - P A K bound to G T b e a d s and detected by immunoblot ing with a Rac-1 speci f ic monoclonal ant ibody. T h e activation status of Akt and Erk w a s a lso determined from the s a m e lysates us ing phospho-spec i f i c ant ibodies(p-Akt , p-Erk). Equiva lency of loading was conf irmed as descr ibed earlier. 42 Rac-1 myc GTP Rac-1 myc (WCL) p-Akt Akt p-Erk Erk B Rac-1 myc GTP Rac-1 myc (WCL) o$? oS? el? p-Akt Akt p-Erk Erk X p-Akt <5 <0 & ° \ V V myc Rac-1 GTP M * * * * " Gndo x Rac-1 (WCL) J J . - ^ p-ERK ERK Figure 3.7. Over-expression of a dominant negative mutant of H-Ras blocks IL-3 induced activation of endogenous Rac-1. Baf/3 cells were electroporated with 3 |xg of plasmid encoding myc-tagged, wild-type Rac-1 and where indicated with 15 uxj of plasmid encoding a fusion protein of GFP and dominant negative H-Ras (S17N H-Ras). 16 hours post-electroporation, the cells were cultured for 2 hours in the absense of serum and IL-3. Following this factor starvation, cells were stimulated for 5 minutes with PBS (con), or IL-3 (1 ug/mL). Cells were then lysed and both activated endogenous (Rac-1 GTP, endo) and over-expressed (Rac-1 GTP, myc) Rac-1 were precipitated by GST-PAK bound to GT beads. Immunoblots were performed using a Rac-1 specific monoclonal antibody. Activation levels of endogenous Erk were measured from WCL using a monoclonal antibody capable of specifically recognizing the phosphorylated form of Erk (p-Erk). Equivalency of loading was confirmed by re-probing phospho-blots with antibodies capable of recognizing both phosphorylated and non-phosphorylated proteins (Erk). 44 3.2.5. Activation of Ras or Rac-1, but not PI-3K, is sufficient for activation of p38 MAPK in hemopoietic cells. To assess whether activation of Ras, Rac-1, or PI-3K was sufficient for activation of p38 M A P K in hemopoietic cells, constitutively active mutants were transiently over-expressed in two IL-3 dependent cell lines, Baf/3 and R6/X. In both cell lines, over-expression of G12V H-Ras, Q61K N-Ras or V12L Rac-1 was sufficient for the induction of increased p38 M A P K phosphorylation (Fig. 3.8A, B). However, over-expression of constitutively active PI-3K did not result in increased phosphorylation of p38 M A P K in either cell type (Fig. 3.8A, B). Microscopic observation of levels of G F P expression in transfected cells ruled out the possibility that over-expression of the p110* construct was toxic (data not shown). Moreover, immunobloting for phospho-Akt demonstrated not only that over-expression of p110* resulted in increased PI-3K activity, but that the levels induced were considerably higher than those observed in cells which were over-expressing G12V H-Ras, and in which activation of p38 M A P K was induced. To investigate the role of PI-3K in the induction of p38 M A P K activation downstream of Ras, cells expressing Q61K N-Ras were treated with Ly294002. Treatment with Ly294002 did not inhibit activation of p38 M A P K induced by over-expression of activated Q61K N-Ras, instead inducing a reproducible increase in phosphorylation of p38 MAPK (Fig. 3.9). 45 •4* p-p38 MAPK p38 MAPK myc Figure 3.8. Over-expression of activated H-Ras or Rac-1, but not p110 PI-3K, are sufficient for activation of p38 MAPK in hemopoietic cells. A) Baf/3 cells or B) R6/X cells were electroporated with 5 ug of pEGFP-C1 empty vector, and 30 u,g (unless otherwise specified) of control plasmid (vector), constitutively active p21 Ras (G12 H-Ras or Q61 N-Ras), constitutively active Rac-1 (V12 Rac-1), or constitutively active PI-3K (p110*). 16 hours post-electroporation, activation of p38 MAPK or PI-3K was measured using phospho-p38 MAPK (p-p38 MAPK) or phospho-Akt (p-Akt) specific antibodies, respectively. Equivalency of loading was confirmed using antibodies capable of recognizing both the phosphorylated and non-phosphorylated form of p38 MAPK (p38 MAPK), or Akt (Akt). The relative expression levels of constitutively active mutants of Ras and Rac-1 were estimated using a monoclonal antibody specific for an epitope tag from human c-myc (myc) common to both. 46 X X 0< ^ Qp OP QP phospo-p38 MAPK p38 MAPK Figure 3.9. Enhanced activation of p38 MAPK in the presence of the PI-3K inhibitor Ly 294002. M C / 9 cel ls were electroporated with constitutively act ive mutants of N - R a s . 16 hours post-electroporat ion, the cel ls were starved of serum and IL-3 for 2 hours and then treated with 25 u M Ly 2 9 4 0 0 2 for 30 minutes prior to genera t ion of who le ce l l l ysa tes wh ich were immunoblot ted with ant ibod ies spec i f i c for the phosphory la ted (phospo -p38 M A P K ) or non-phosphorylated (p38 M A P K ) form of p38 M A P K . 47 3.3 Discussion We show here using both primary cells and cell-lines that endogenous Rac-1 is activated by stimulation of receptors of the hemopoietin family (the receptors for IL-3, G M - C S F , or IL-5), of the tyrosine kinase family (receptors for S L F and CSF-1) , or of the receptor for antigen on B-lymphocytes (BCR). IL-3 induced activation of both Rac-2 and the related G T P a s e , Cdc42 (Fig. 3.2). Although stimulation of endogenous receptors induced significant increases in levels of GTP-bound Rac-1 or Cdc42 relative to those in unstimulated cells (2.5 to 12-fold factors of induction) (Fig. 3.1-4), the absolute amount of activated Rac-1 or Cdc42 remained small (ranging from approximately 1 to 5 percent depending on the stimuli). These results are in broad agreement with reported levels of GTP-bound Rac-2 in neutrophils stimulated with fMLP (2.5% of total cellular Rac-2), or in porcine aortic endothelial cells stimulated with P D G F (4% of total cellular Rac -1 ) 4 9 ' 5 0 . These results should be taken as minimal estimates of Rac activation, as in vivo levels of Rac-1 or 2 G T P will rapidly decrease due to the high intrinsic GTPase activity of Rac GTPases . Moreover, some activated Rac may be associated with a detergent insoluble fraction, for example the cytoskeleton, meaning it will be unavailable for detection by this assay. Our conclusion that IL-3 induced activation of Rac-1 and -2, was based on the use of antibodies, which we demonstrated were operationally specific for each Rac isoform (Fig. 3.1 A, B). The notion that IL-3 induces activation of Rac-1 is consistent with observations that bone-marrow cells from mice that are homozygous null for Rac-2 as a result of gene-targeting, still retain the capacity to proliferate, survive and 48 differentiate to B M M C in response to IL-3 4 6 . The defects that are observed in these Rac-2 deficient cells reflect the differences in the specific functions of Rac-1 and Rac-2 4 6 . Antibodies specific for Rac-3 were not available, so there is also the caveat that some of the Rac-GTP we detected may be Rac-3. A detailed analysis of this question will require the development of specific antibodies capable of differentiating between Rac-1, Rac-2 and Rac-3. One likely component of the PI-3K dependent mechanism mediating the activation of Rac-1 downstream of the B C R is the Rac G E F , Vav. Vav contains a PH domain, has enhanced G E F activity in the presence of lipid products of PI-3K 5 6 , and is critical for activation of B cells via the B C R 1 0 5 . Moreover, Vav becomes tyrosine phosphorylated (an event associated with enhanced G E F activity) in response to IL-3, S L F or G M - C S F 1 0 6 , and thus may be responsible for the PI-3K dependent activation of Rac-1 induced by IL-3. Since the Tec family kinase, Btk, is required for a portion of the activation of Erk induced by ligation of the B C R 1 0 7 and is activated by products of PI-3K activity 1 0 8 , it is likely to be involved in the portion of Erk activation that we observed was dependent upon PI-3K (Fig. 3.5B). Our observations that over-expression of constitutively active PI-3K resulted in activation of Rac-1, but not of p38 M A P K (Fig. 3.6A and 7), are consistent with the report that over-expression of constitutively active PI-3K was sufficient to induce membrane ruffling, but not activation of transcription factors known to require phosphorylation by p38 M A P K 1 0 9 . Nevertheless, these observations with over-expressed proteins do not establish that physiological increases in PI-3K activity are sufficient for activation of Rac-1 . Indeed, although we made 49 extensive efforts with more than 5 independent experiments, we were unable to observe activation of Rac-1 by lnterleukin-4 (data not shown), even though we have previously shown that IL-4 induces increases in PI-3K activity As IL-4 also fails to activate the Ras /MAPK pathways in lymphohemopoietic cells s 9- 6 0. 1 0 4. 1 1 1, this is compatible with the notion that physiologically relevant activation Rac-1 requires both signals downstream of PI-3K and others downstream of Ras. It was somewhat unexpected to find that a component of the activation of Rac-1 downstream of the IL-3 receptor and the B C R was not dependent on receptor induced increases in PI-3K activity (Fig. 3.5A). In this connection, however, it was of interest that expression of constitutively active mutants of H-Ras, N-Ras or M-Ras resulted in activation of co-expressed Rac-1 through mechanisms which did not depend on increases in PI-3K activity (Fig. 3.6). There is indirect evidence consistent with our observation. Thus dominant active mutants of H-Ras induced membrane ruffling that probably reflected activation of Rac and was not inhibited by Ly294002 or wortmannin 1 1 ' 2 0 . In Rat-1 fibroblasts, both PI-3K dependent and independent pathways led from Ras to the Rac effector PAK 1 1 2 . Certainly, our results indicate that in hemopoietic cells, activation of Rac-1 downstream of Ras occurs without simultaneous increases in overall PI-3K activity. It should be noted that our results do not exclude that small levels of P l -3K activity corresponding to that in resting, unstimulated cells might still have a permissive role in Rac-1 activation. Indeed this is consistent with our data in B-lymphocytes, where high doses of Ly294002 that in these cells (but not mast cells) reduced levels of PI-3K activity to below background levels, also blocked Rac-1 50 activation beyond the maximal levels of inhibition seen with mast cells. The molecular mechanism for PI-3K independent activation of Rac-1 is unclear. Only, one candidate Rac G E F lacks a PH domain. This protein smgGDS 5 3 , 5 4 is structurally unrelated to the other Ras and Rho family G E F s and lacks a DH or cdc25 catalytic domain. Nevertheless, smgGDS has G E F activity on a broad range of small GTPases, including Rac, Rap, Ral and some members of the Ras family such as Ki-Ras 4B 1 1 3 1 1 5 , and M-Ras (Korherr, Quadroni and Schrader, unpublished observations). However, there is no evidence that smgGDS is downstream of Ras or is involved in any responses to extra-cellular signals. Our results raise the possibility that the activation of Rac-1 induced by IL-3 which occurs in the absence of increased PI-3K activity may be mediated by activation of p21 Ras isoforms, M-Ras, or both. We and others reported that IL-3 and S L F activate Ras isoforms that bound to the monoclonal antibody Y13-259 and were hitherto thought to be p21-Ras alone 1 0 3 ' 1 0 4 , but are now known to include M-Ras 1 7 . However, we have recently shown that IL-3 activates M-Ras and to a lesser extent p21 H-Ras (Schallhorn and Schrader, unpublished observation), making both candidate components of IL-3 mediated mechanisms for activation of Rac-1. The question of whether some or all of the Rac activation induced by hemopoietic growth factors is downstream of Ras is difficult to address experimentally. Transient over-expression of a dominant-negative mutant of p21-Ras, S17N H-Ras, resulted in decreased activation of Rac-1 by IL-3 (Fig. 3.7). However, dominant-negative Ras mutants such as S17N H-Ras will sequestrate Ras G E F , such as mSos-1 and Ras GRF-1 and -2, that are also direct activators of Rac 4 1 ' 4 3 . Therefore we cannot 51 exclude that S17N H-Ras might be exerting its inhibitory effect on activation of Rac-1, not through inhibiting activation of Ras, but by directly sequestrating a Rac G E F or targeting it for destruction. Our observations on the lack of correlation between activation of Rac-1 and of p38 M A P K in hemopoietic cells, contrast with evidence from experiments with other types of cells which suggest that activation of Rac was necessary and sufficient for activation of JNK and p38 M A P K 2 7 3 ° . Our observations that the activation of Rac-1 resulting from stimulation of the receptors for hemopoietic growth factors or the B C R , could be inhibited without corresponding proportional decreases in activation of p38 M A P K (Fig. 3.5), suggested that activation of p38 M A P K was not entirely dependent on activation of Rac-1. Thus, whereas inhibition of PI-3K activity below background levels almost completely abolished the activation of Rac-1 induced by ligation of the BCR, the effect on p38 M A P K activation was much less (Fig. 3.5B). Even greater differences in the effect of Ly294002 on activation of Rac-1 and p38 M A P K occurred in response to IL-3, where Ly294002 reduced activation of Rac-1 by approximately 70%, while having no effect on activation of p38 M A P K (Fig. 3.5A). Similar results were reported in mast cells stimulated with SLF, where PI-3K inhibitors failed to block p38 M A P K activation 1 1 6 , although not the activation of J N K 1 1 6 1 1 7 . The latter suggests differences in the mechanisms of activation of JNK and p38 MAPK by SLF . This notion is also supported by our observation that over-expression of activated p21 Ras was sufficient to activate p38 M A P K in Baf/3 cells (Fig. 3.8), whereas our published data and that of others shows that over-expression of activated Ras was not sufficient to activate J N K 3 1 , 3 2 . 52 Despite the lack of correlation between increases in activity of Rac-1 and p38 M A P K seen when inhibitors of PI-3K were used to reduce levels of activation of endogenous Rac-1, over-expression of constitutively active V12L Rac-1 was sufficient for activation of p38 MAPK (Fig. 3.8). However, in that we and others have shown that the levels of activated Rac-1 induced by physiological stimuli are relatively low (less than 5% of cellular Rac), results of experiments involving over-expression of constitutively active Rac-1 (where cellular levels of activated Rac-1 exceed those observed with physiological activation of endogenous Rac-1) may not be biologically relevant. Consistent with this notion, over-expression of the Rac G E F , Vav, which probably resulted in levels of activated endogenous Rac-1 that are closer to physiological levels, failed to induce activation of p38 M A P K in RBL cells 1 1 8 . The notion that physiologically relevant levels of activated Rac-1 are alone, insufficient to activate p38 MAPK, is supported by our observation that over-expression of constitutively active PI-3K in two IL-3 dependent cell lines was insufficient for activation of p38 MAPK (Fig. 3.8), although it activated co-expressed, exogenous Rac-1 (Fig. 3.6A). These results are consistent with the report that over-expression of constitutively active PI-3K was sufficient to induce membrane ruffling, but not activation of transcription factors known to require phosphorylation by p38 MAPK, including AP-1 , and Elk-1 1 0 9 . The differential effect of inhibition of PI-3K activity on activation of Rac-1 and p38 MAPK by IL-3 might be explained in several ways. First, p38 M A P K activation might not depend on activation of Rac-1 at all. In that we have shown that IL-3 activates Cdc42 (Fig. 3.2C) and the activation of Cdc42 induced by P D G F occurs independent of PI-3K 53 catalytic activity 1 1 9 , it is possible that Cdc42 is responsible for the PI-3K independent activation of p38 MAPK. However, we failed to observe consistent activation of p38 M A P K in cells of hemopoietic origin (Baf/3 or R6X) in which constitutively activated Cdc42 had been over-expressed (data not shown). Alternatively, IL-3 might activate two pools of Rac-1 in hemopoietic cells, the minor one of which is activated via a PI-3K independent mechanism and is responsible for activation of p38 MAPK. Finally, it is possible that the 30% of IL-3-induced Rac-1 activation that was independent of increases in PI-3K activity may synergize with another signal to activate p38 MAPK. Our observations that over-expression of activated Ras (H-Ras, N-Ras or M-Ras) induced activation of Rac-1 and p38 M A P K (Fig. 3.6, 3.8), the latter through mechanisms which could only involve physiological levels of endogenous Rac-1, raise the possibility that this second pathway may involve Ras. Activation of p38 M A P K induced by granulocyte-colony stimulating factor (G-CSF) was dependent on activation of Ras 6 1 . Certainly our data show that increased PI-3K activity alone, even at levels greater than those associated with p38 M A P K activation in cells over-expressing constitutively active p21 Ras (Fig. 3.8), was insufficient for activation of p38 MAPK. Finally, activation of p38 M A P K by IL-3 may involve Rac-independent mechanisms, which as discussed above, appear to be involved in activation of p38 M A P K downstream of cross-linking of the BCR, as well. The activation of Rac by hemopoietic growth factors is likely to be involved in many of their actions in the regulation of growth, survival, differentiation and function, all 54 of which are influenced by the absence of Rac-2 4 5 , 4 6 . One critical function of the Rac pathway in growth may involve stathmin, first recognized as a protein over-expressed in leukemic blast cells and now known to be a key regulator of microtubule assembly 1 2 ° . The Rac and Cdc42 pathways activate p65 P A K (PAK-1) which phosphorylates stathmin on serine 16, inactivating it so that it releases tubulin 1 2 1 . We identified stathmin as one of the proteins undergoing serine/threonine phosphorylation after stimulation with IL-3, and showed that one of the sites phosphorylated was serine 16 (Quadroni and Schrader, unpublished observation). Our demonstration that IL-3 induces activation of Rac-1, Rac-2 and Cdc42, accounts for the IL-3 induced activation of PAK-1 1 2 2 and phosphorylation of stathmin on serine 16, an essential prelude to cell division. Over-expression of activated Rac promotes survival of hemopoietic cells 1 2 3 and hemopoietic cells from Rac-2 deficient mice exhibited decreased expression of the anti-apoptotic protein, Bcl-X L , and increased expression of the pro-apoptotic molecule, BAD 4 6 . Hemopoietic growth factors such as G M - C S F , S L F and CSF-1 are capable of inducing cellular migration and chemotaxis, which in the case of CSF-1 has been shown to be Rac dependent 3 7 , 1 2 4 . Finally, our demonstration that IL-3 and CSF-1 induce activation of Rac provides a molecular basis for observations that these cytokines induce membrane ruffling 1 2 4 1 2 5 . Our findings that hemopoietic growth factors activate Rac-1, Rac-2 and Cdc42 confirms the central role of these GTPases in growth factor-mediated signaling. Further, it raises new questions about the existence of novel mechanisms of activating these GTPases, which do not depend on increases in PI-3K activity and potentially involve novel effectors of the Ras family. 55 CHAPTER 4: IDENTIFICATION AND CHARACTERIZATION OF CAPRIN-1 AND THE CAPRIN FAMILY OF PROTEINS 4.1 Introduction The cellular elements of the blood and immune system are generated continuously throughout adult life through tightly controlled cellular proliferation. The large-scale generation of erythrocytes and leukocytes is dependent on the proliferation of committed progenitor cells in the bone-marrow 1 2 6 . Likewise the continuous generation of naive B- or T-lymphocytes depends on the extensive proliferation in the bone-marrow or thymus of the precursors from which mature lymphocytes are selected. The ability of the immune system to respond to pathogens also depends upon the rapid proliferation of both antigen-specific lymphocytes and the progenitors that generate granulocytes, macrophages and mast cells. Since, approximately 40% of genes identified in the human genome have no homology with known proteins 7 6 or the approximately 1800 known protein domains 7 7 , it is probable many proteins and protein families relevant to the immune system remain to be identified. Here we describe the observation of a protein that was up-regulated in proliferating lymphocytes. We have identified this protein, performed molecular characterization of its localization, post-translational modification, and sequence similarity to a related protein, and attempted to determine its function. 56 4.2 Results 4.2.1. A 116kDa cytoplasmic protein is up-regulated in proliferating T or B lymphoblasts We performed immunoblots of proliferating T- lymphoblasts using affinity-purified (AP) antibodies specific for a peptide sequence from the guanine-nucleotide exchange factor, smgGDS 1 2 7 . We observed, not the expected 55 or 50 kDa smgGDS splice-forms (data not shown), but instead a 116 kDa protein (p116) that occurred at much higher levels in activated T-lymphoblasts, than in splenocytes (Fig. 4.1 A, B). Similar results were seen when T-lymphocytes were activated by ligation of CD3 with a monoclonal antibody in the presence of lnterleukin-2 (Fig. 4.1C). These A P antibodies also detected increased levels of p116 in blast cells derived from B-lymphocytes (Fig. 1B). We also observed high levels of expression of p116 in immunoblots of a variety of lymphohemopoietic cell-lines including R6X (IL-3 dependent mast cell/megakaryocytic cells), MC/9 (IL-3 dependent mast cells), WEHI-231 B lymphoma, the IL-3-dependent cell-line Baf/3 (Fig. 4.1 D), and M-1 monocytic leukemia cells (Fig. 4.6). Immunoblots of lymphoblasts (Fig. 4.1 A), or Baf/3 cells (Fig. 4.1 D), or of immunoprecipitates made with A P antibodies from lysates of Baf/3 cells (Fig. 4.1 E), demonstrated that A P antibodies detected only p116 in these cells. Therefore, with these cells, we were able to use flow cytometry and A P antibodies to compare levels of expression of p116 in individual cells, and to investigate its subcellular localization. T-lymphoblasts (Fig. 4.1 F) or B-lymphoblasts (data not shown) exhibited approximately 15-fold greater staining with A P antibodies than resting lymphocytes, consistent with the 57 results of immunobloting (Fig. 4.1 A). Likewise, "blast" cells generated from normal murine bone-marrow by culture with conditioned medium containing either IL-3 or C S F -1, stained strongly with A P antibodies (Fig. 4.1G). Unfixed, live Baf/3 cells did not react with A P antibodies, while cells that were fixed and permeabilized did (Fig. 4.1 H), suggesting that p116 was not expressed on the cell surface. The specificity of this staining was confirmed by the demonstration that it was inhibited by competition with the specific peptide targeted by A P antibodies but not with an irrelevant peptide (Fig. 4.1 H). We used A P antibodies and immunofluorescence to examine the intracellular localization of p116 in Baf/3 cells. We observed that the fluorescence (shown to be specific by inhibition by competing peptide) was evenly distributed throughout the cytoplasm and excluded from the nucleus (Fig. 4.11). 58 Figure 4.1. Expression pattern and localization of p116. W h o l e cy top lasm ic l ysa tes (normal ized for total protein content) of sp lenocy tes , T - l ymphob las ts (sp lenocytes s imula ted with Concanava l i n A and IL-2), or B- lymphoblasts (sp lenocytes st imulated with 15 ug/mL L P S ) from A) ( C 5 7 B L 6 x D B A ) F1 hybrid (BDF1) mice, or B) Ba lb /c mice were run on S D S - P A G E , and immunoblot ted with A P ant ibodies. Equ iva lency of loading w a s conf i rmed by re-probing blots with ant ibod ies aga ins t Erk (anti-Erk). C) sp lenocy tes f rom C 5 7 B L / 6 m ice were t reated with the an t i -CD3 antibody (2 u.g/mL 2C11) for the indicated length of t ime. W h o l e cytoplasmic lysates were normal ized for total protein content and ana l yzed a s for A and B. D) W h o l e cy top lasmic lysates (normal ized for total protein content) f rom a variety of different cel l l ines were ana l yzed with A P ant ibodies as for A and B E) A P ant ibodies were used to precipitate p116 (*) from Baf/3 cel ls. F) Intracellular staining of sp lenocytes ( — A P ; secondary alone) or T- lymphoblasts ( A P ; secondary alone) from BDF1 mice a s s e s s e d by flow cytometry. G) Mur ine bone-marrow cel ls were cultured for 6 days with condi t ioned med ia containing either IL-3 or C S F - 1 as indicated. Shown are the profiles of the large "blast" cel ls ( — A P ; secondary alone). H) Stain ing with A P ant ibodies of live Baf /3 cel ls , or Baf/3 cel ls f ixed with P F A and permeabi l ized with saponin ( secondary a lone; ~ ~ A P ; A P + speci f ic peptide; A P + non-speci f ic peptide). I) Cy tospun Baf /3 cel ls were f ixed in P F A , methanol permeabi l i zed, and s ta ined with A P ant ibodies with or without speci f ic peptide. A P binding is shown in red and DAPI staining of the nuclei , in blue. J) Immunoblot with A P antibodies of indicated t issues from an adult Ba lb /c mouse . 59 60 4.2.2. Expression of p116 in tissues and other cell-lines We observed high levels of p116 in A P immunoblots of every dividing cell examined, including cell-lines of epithelial origin (scp-2 murine mammary cells, HEK 293 human embryonic kidney cells) and mesenchymal origin (3T3 fibroblasts, L929 endothelial cells, C2 myoblasts) (Fig. 4.1 D and data not shown). Levels of p116 were highest in the thymus and spleen, whereas kidney, muscle or liver showed very little immunoreactivity (Fig. 4.1J), consistent with the notion that levels of p116 correlated with the frequency of dividing cells. One exception to this generalization was the brain, which exhibited high levels of p116. However there are other precedents for proteins (such as members of the Ras and M A P kinase pathways) that play important roles in cellular proliferation, but are also involved in activation and signaling in certain non-dividing cells such as those of the brain 1 2 8 . Based on the general correlation of levels of p116 protein expression with cellular activation and proliferation, and its cytoplasmic localization, we gave the protein the operational name of "cytoplasmic activation/proliferation-related protein-1" (Caprin-1). 4.2.3. Caprin-1 is expressed at high levels in dividing thymocytes All thymocytes stained with the A P antibodies, including both CD4 and CD8 single positive cells and the CD4 and CD8 double-positive or double-negative cells (Fig. 4.2A and data not shown). However there was clear heterogeneity in the levels of staining, with the staining profile of thymocytes as a whole showing a shoulder of brightly staining 61 cells (Fig. 4.2A). By gating on either the large dividing "blast" cells or the small non-dividing thymocytes, we observed that it was the dividing "blast" cell population that was responsible for the shoulder of brightly staining cells (Fig. 4.2B). Comparison of the mean fluorescence intensities of the large and small thymocytes indicated that the dividing thymocytes exhibited levels of staining with A P antibodies that were 3-fold higher than those of the population of small, non-dividing thymocytes. 62 A o o 1 B A Figure 4.2. Caprin-1 expression is elevated in large thymocytes. Intracellular staining of thymocytes with goat anti-rabbit secondary antibody ( ), or AP antibodies and goat anti-rabbit secondary antibody ( — ), gated on (A) the whole thymocyte population (R.1), or on (B) the "small" thymocytes (R2) or the "large" thymocytes (R3) as determined by forward light scatter. 63 4.2.4. Structure of Caprin-1 We used A P antibodies to immunoprecipitate Caprin-1 from lysates of T-lymphoblasts. Coomassie staining of the immunoprecipitate after S D S - P A G E revealed the expected 116 kDa band, which was not present in a control preparation made in parallel using immunoglobulins from unimmunized rabbits (data not shown). The 116 kDa band was excised and analyzed by mass spectrometry. The two peptide sequences we obtained (LNQDQLDAVSK and YEVTNNLEFAK) were both present in the sequence of a human protein termed "p137" 9 6 or "GPI-anchored membrane protein" 1 2 9 . However, analysis of ESTs and human draft genomic sequence (NCBI) indicated that the published data contained a series of important errors. First, a single base deletion in the "p137" cDNA had resulted in a frame-shift. As a result, the carboxy-terminus of both the deduced protein and the recombinant protein lacked 80 amino acids and included, instead, an artifactual region. This included the presumptive site for GPI-linkage, which thus does not exist in the authentic protein. Another error at the 5' end of the published cDNA sequence introduced an artifactual stop codon. This resulted in misidentification of the initiating methionine and the truncation of the first 53 amino acids from the authentic N-terminus. There was no evidence of a signal peptide. EST sequences were assembled and translated to generate consensus sequences for Caprin-1 in the human, mouse, and Xenopus laevis (Fig. 4.3A). There was a striking degree of amino-acid conservation, with the human and mouse sequences being 97% conserved (96% identical), and the human and X. laevis 80% conserved (68% identical). 64 During the course of our study, a human cDNA (BC 001731), and a model human mRNA (XM_011991) and protein (XP_011991) corresponding to the protein we deduced, were added to the public database (NCBI). The 709 amino acids predicted a protein of 78.4 kDa, far smaller than the apparent size (116 kDa) of the native protein we had purified and observed in immunoblots. However, a fusion protein of G S T and the protein encoded by the cDNA BC 001731, that had a predicted Mr of approximately 106K, migrated anomalously slow on S D S - P A G E (Mr 140 K) (Fig. 4.3B). This aberrant migration was not due to post-translational modification specific for mammalian cells because it occurred when fusion proteins were expressed in either mammalian or bacterial cells. The ability of A P antibodies to recognize nanogram quantities of this fusion protein (Fig. 4.3C) confirmed that the protein encoded by the cDNA B C 001731 was identical to p116 (Fig. 4.3C). In reciprocal experiments, a rabbit anti-serum raised against a recombinant GST-fusion protein that included amino-acids 189-629 of the protein encoded by the BC001731 cDNA, recognized the 116kDa band that was immunoprecipitated from Baf/3 cells by A P antibodies (Fig. 4.3D). This anti-serum also detected a 116 kDa band that was up-regulated in T or B-lymphoblasts (Fig. 4.3E), and was expressed in various tissues (Fig. 4.3F) at the same relative levels as the 116 kDa band detected by the A P antibodies. Thus, the 116 kDa protein we termed Caprin-1 was identical to the protein encoded by the cDNA B C 001731 and represented the authentic version of the protein that was previously described as "GPI-anchored membrane protein p137". 65 It was conceivable that some Caprin-1 was present on the cell-surface in a form that was not recognized by the A P antibodies. Therefore, we expressed full-length Caprin-1 (cDNA BC001731) fused at the N-terminus with G F P (GFP-Caprin-1) or at the C-terminus with a HA-epitope tag (Caprin-1-HA) and examined Bosc 293 cells or 3T3 cells by fluorescence microscopy or immunofluorescence, respectively. In neither case could we detect localization at the membrane, and the tagged-recombinant Caprin-1 exhibited the same cytosolic distribution and exclusion from the nucleus as was seen with staining of endogenous Caprin-1 using A P antibodies (Fig. 4.3G). Caprin-1 is encoded in humans by the gene termed "membrane component, chromosome 11, surface marker 1" (M11S1) at 11 p13 on Chromosome 11, and in mice by the Gpiapl ("GPI-anchored membrane protein 1") gene on murine chromosome 2. Both names appear misleading. Caprin-1 transcripts, perhaps as a result of usage of two promoters, exhibit two alternative 5' untranslated regions (UTR), one of which contains exon 1 and has an in-frame stop codon 5' to the initiating methionine (Fig. 4.4C). Examination of ESTs and RT-PCR (data not shown) indicate that both UTR are conserved in mouse and human. There was also EST evidence for alternative splicing that would result in an alternative carboxy-terminus encoded by the alternative Exon 18'. This predicts replacement of the 20 carboxy-terminal amino acids with five new residues, "NILWW" (conserved in mouse (BQ829672) and human (BG708967)). Exon 18' also includes a long 3' UTR that exhibits a high level of conservation between mouse and human. The Caprin-1 cDNA that includes exons 18 and 19 has a long 1.1Kb 3' UTR (Fig. 4.4C) that is also highly 66 conserved in mouse and human (overall identity 87%), as well as chicken, with a smaller 204 bp region being 88% identical in human and X. laevis. The remarkable conservation of these 3' UTR suggests they are involved in important functions, which may include post-transcriptional regulation of levels of Caprin-1 or sub-cellular localization of its mRNA 1 3 0 . Figure. 4.3. Structure of Caprin-1 and its identity with p116. A) Capr in-1 amino ac id s e q u e n c e s f rom X. laevis, M. musculus, a n d H. sapiens. B ) Immunoblot with a n t i - G S T ant ibod ies of recombinant G S T - C a p r i n - 1 precipi tated from B o s c 2 9 3 ce l ls (mam) or E. coli (bac) precipitated using G T - b e a d s . C ) Immunoblot with A P ant ibodies of the indicated amounts of GST-Cap r i n -1 purified from B o s c 293 cel ls . Immunoblots with anti-Caprin-1 ant ibodies: D) an immunoprecipi tate of Baf /3 cel ls made with A P ant ibodies; E) lysates of the indicated cel ls of Ba lb /c origin (equ iva lency of loading w a s conf i rmed by re-probing with an ant i -Erk) , or F) homogena tes of the indicated Ba lb /c t i ssues . G) Loca l izat ion of G F P - C a p r i n - 1 exp ressed in B o s c 2 9 3 ce l ls . H) Immunof luorescent staining with ant i -HA ant ibody of 3 T 3 cel ls retrovirally infected with HA- tagged Caprin-1 (Capr in-1-HA) or parental 3T3 cel ls. 67 A X. laevis M . rrujsctjlus ! H sapiens 1 X. laevis 75 M. musculus 99 H sapiens 10* X. laevis 175 M. musculus 186 H sapiens 201 X. laevis 276 M musculus 196 H. sapiens 193 X. laevis 372 M. musculus 394 H. sapiens 396 X laevis 469 M musculus -194 H. sapiens 496 X. laevis M. musculus S94 H. sapiens 596 X. laevis 663 M. musculus ',93 H. sapiens 695 M P 3 A T 3 | S - - - - - - K|AV P G 5 | S I) A|V|| f ^ l [5^1 O S E A M K Q l L G I I D K K L R N L D K K K G K I D D Y O P fl|JU K G F R I N O P t ^ j R a 1/ T U V C M P S A T S H S G S G S K S S G P P P P S G S S G S l I | A A A G A A A P A S Q M P A T GT G A V Q T £ A M K QI L G V t D K K L R H L E K K K S K L 0 D V Q E R M N K G £ P L N Q D Q L D A V S K Y C M P S A T S H S G S G 3 K S S S P P P P S G S S G S E A M A G A S A A A P A S Q H P A T G T S A V O T E A M K O i L G V I D K K L R N L E K K K G K L D D Y Q E R M N K G E R L N Q D Q L O A V S K Y C I T ] v - J ^ E F G R E L q p y F ] J A T | G J Q D I Q K SI K K|A|A R R E Q L | L J R E E A E O K R L K T V L E j j c F V L D K L G D E E V PJNIO L K Q ' E V T N N L E F A K E L Q R S F M A L S Q D I Q K T K K T A R R E Q L M R E E A E Q K R L K T V L E L C Y V l D K E G D D D V R T D L K Q G T ] E » T W M L E F A K E L Q R S F M A L S O D I O K T K K T A R R E Q L M R E E A E O K P L K T V L E L Q Y V L D K L G D D E V F I T t>L K OSI .TTG V p I L S E E E l S L L D E F Y K L V D P E R O M s l | N G V P L V S E E E L | I J L L D E F V K L V|N|P E R D I A J S V P ! L S E E E L S L L D E F Y K L V D P E R O M S >' R I J S | D Q V E | Q 1 A S T T | H L W D V/L C | S | K 0 K | S | V C S T T Y K | - I | L K D I I D R I l l IdslciY F 0 S|A QlN H O N G L C E E E E E L R L N E Q Y E HASF H L W D L L E G K E K P V C G T T Y SAL K E I V E R V F Q S N Y F D S T H N H O N G L C E E E EP L R L N E Q V E H A S I H L W Q L L E G K E K P V C G T T Y K V L K E I V E R V F Q S N Y F D S T H N H C N G L C E E E E V E E Q A P E L EP EP VE E Y T E|TJSE V E S T E F V N f l V E DQV (A]E A E P E P A E E Y T E Q S E V E S T E Y V N R V F D Q V P E A E P F . P A E E Y T E Q S E V E S T E Y V N R | Q F '4 Iii I • >I I K E O V D E W T V E T V E WHS I O C | A A 7 P [ | j l P E PJL_AJ[|NAJI V Q V Q P D P I V R R Q R V Q D u M A Q M O G P Y N F l N J Q O S M L E F E I J Q V I J D P A l V S A G P I * | Q F M A E T Q F S S G E K E C t V D E WT V E T V E V V N S L Q Q Q P Q A A S P S V P E P H S L T P V A G|S - - I D P L V R R Q R V 0 D L M A O M Q G P Y N F F O D S l l l O F E » Q T L D P A I V S A G P 1 . I G F M A E T G F T S G E K E Q V D E WT V E T V E V V N S L Q Q Q P Q A A S P S V P E P H S L T P V A Q A] | D P L V FI R Q R V Q D I M A Q M Q G P Y N F I 0 0 S M L C F E M O T L D P A l V S A S P k _ C P P V H S E | p [ R l , - s K P I I Q V P I |DTlfFQv|A|l V S S l l f e E A Y T i . s l F ll Y O P SHIP I lE |A |RMgNrD|A |Mr 01 6 A S L S L NIFEIPFIQT I IssTI: I A A S I I IF Q V p 'a l i N P T Q N M D M P Q L V C P F J V H S E S R L A a £ | N a V P V Q P E • A T Q V P L V S S T S E G Y T A S Q » L Y Q P S H A T E O R P Q K E P M D Q I G A T I S L K J D a T l i s S S L P A A S Q P Q V F O J N P T O N M O M P Q l V C P P V H S E S R L A O P H O V F V Q P E A T Q V P L V S S T S E G V T A S O P L Y Q P S H A T E O R P Q K E P O T P Q S A T I S L U T P O T T A S S S L F A A S O F Q V F Q A G l ] j K P L H S S G I N V N A A P F Q S M O T V F N M N A P V P P V N E P E T L K Q r . -G T S K P L H S S G I N V N A A P F Q S M O T V F N M N A P V P P A N E P E T L K O C S G T S K P L H S S G I N V N A A P F Q S M Q T V F N M N A P V P P V N E P E T L K OQINIQY Q A S Y N Q S F S S Q P H Q V E Q T E l Q Q E Q L G T V V G T Y H G to? O A S Y N OTFII I - ^ Q P H Q V E O T E L Q j r f E Q L Q T V V|Np Q Y O A I Y N Q S F S S O P H Q V E Q T E L Q O C Q L G T V V G T Y H G S l 3 fG]A |H 0 A p|s ^fTc C D C P H Q V P G N H C P D df IH Q V F I G N H C F p R | N | S g p F Y N | N | R ^ M G d o l ^ ^ r j R ^ N / | M N G Y n G I G S I N G F R G G Y D G Y P J A A | F 1 P | N T P N S G Y | P H A ( Q F 1 N I A P R P Y S I N I N Y Q R D G Y Q G N ' F K R ' Q ] A ^ ^ 3 ' J G P R V A P R ' G I F P R S S O P Y Y M S R G V S R G G S R G A R G L M N G Y R G P A N G f R G G Y 0 G Y R p S F S N T P T 1 S G Y S O S Q F r A P R D Y S G T Y O R D G Y Q G N F K R G S G Q S G P H G A P R G R G G P P I G F P R S T N I Q P Y Y N S R G V S R G G S H G A R G l M N G Y R G P A N G F R G G Y D G Y p P S F S N T P N S G Y r Q S Q F S A P R D V s d l Y Q R D G Y Q G N F K R G S G O S G P R G A P R G R G G P P 3 P N R G M P Q M N T Q Q V N | H P N R G M P O M N T Q Q V N I B GST-Caprin-1 200 — GST Caprin-1 25 12 ng 1 1 6 _ IB: anti-Caprin-1 I B : anti-Capri n-1 »y <^ s<r & ^ ^> I B : Erk I B : anti-Caprin-1 68 Figure. 4.4. The Caprin-1 and insect HR-1 domains, and the human Caprin-1 and Caprin-2 proteins and genes. A) Al ignment (Clustal-W) of the HR-1 doma ins f rom B H P , D H P , and human Cap r i n -1 , and a dendrogram of these plus the HR-1 doma in of human Cap r i n -2 . B) Al ignment of human Capr in-1 and Capr in -2 and their HR-1 ( ) and H R - 2 ( ) domains , and the C R D ( ) of Capr in -2 . The first three N-terminal methionine res idues of Capr in -2 are highl ighted in red. C) Capr in-1 and Capr in -2 g e n e s with the exons encod ing H R - 1 , H R - 2 and the C R D indicated. A l so shown are the alternative 5' and 3' U T R of Capr in -1 . 69 A hCapr in-1 BHP DHP 1 fo Q Q L I 1- G V ' I i F T i i D K K L R N L E K K K | G | K L D | D [ 7 | Q f E l R M N K E H K I R N L E K R K S K L f f f s Y R D LTSIK A E H K I R N L E K R K T K L E S Y R | A T | Q ] S S hCapr in -1 51 L E" F " A K E L BHP 61 L E F A R D L DHP 51 L E F A R E L R L K [T]V |J K I K T v L c K 1 R E V L 1 1 Q] |M S L G Q [ S ] D V S i ie 150 150 hCapr in-1 147 R L N E j Q l Y E BHP 151 G[F1H|L Q I|T DHP 151 | P l I IAITIA [Q hCaprin-2 hCaprin-1 • BHP DHP B Capiin-1 i Caprin-2 1 Caprin-1 7 Caprin-2 76 Caprin-1 '57 Caprin-2 225 Caprin-1 232 Caprin-2 300 Caprin-1 300 I Caprin-2 375 I Caprin-1 331 Caprin-2 450 M E V Q V S Q A S L Q F E L T S V E K S L R B W S R L S R E V I A W L C P I I P N t I L N F P P M P S I A 3 S V S M V Q L F S S P F G Y Q S P S G AITIS a His!!-: 75 i.) i. I I l ! A r l Ik ~ . t | II M | _ l I i i h i Ik I I 1.1 0 v v i.l ••• O V V L I « r m nn n n i . i 3 ; n [3 • S ET TTjK-|T|s | r5 lvraH A I S - T I H . k v n L I. t GIK.IE K I P I V I | ~ T T T T C 1 s L i s |V|P|L> U J M | E | O ; | S L | , |W U L L E a i s l e K |A |VJV |G T T V KI IL K r, 11^  S I N I V * L> S I T 11 N II I . | N S | I |V $ 1 | 1- V P K | £ A J K F. K E Y P L » 1 LI p i A P A V E D K T 5 T I P t A I B P L P A [ H I . v T | B . Fl p - ! s r|« > vHMglFI O j j s K |r.| V K P | i | i . M | E | P |K j P | l P I. M k | I I III O l F T l S i G E l K !|V D I V J N K [ C F J I (j I' \> I • Y A R K P N L P K R W D M L T E P D G Q E K K Q i R K Q D T S K L 449 . T i [pjn r c K KIQIT-FTTIKI^ IKIPI^  | P | P Q w|j]v DJTPIK S K @ O Y V Q E E Q K K Q E T P K L W P V Q L Q K F . Q D P K K Q T P K S W T P S M 0 S E Q N 5 M Caprin-1 352 Caprin-2 525 Caprin-1 352 Caprin-2 600 Caprin-1 414 Caprin-2 665 T T K S W T T P M C E E Q D S K 1 1 K S W E N N V Q S Q I S P K S W G V A T A S l . I P N D Q 1 H Q P V G S S S T L P K I P I V R R ? t I. R K R V Q D L M l ' iMlMpTT; o m- Mfi loj i k> ti 0 i> siMii. n v K I N o r i r> p-flTTIv ODE] • P R K l . N T E P K D V P K P V 599 N M D M P Q I. V C P p r n H R ] R H A Q B B A T Q V P Lipis[r|lplfilv T[A-SIO P I. V N | L ] S S g s p. i. i i i H|gi o A I S s p|v i | |s S | N | A | I I.|V T|T U H A 1 D O H O A T E A A g j P P 0 Caprin-1 488 Caprin-2 737 KP^HF T| ( iriNF^l^lA p F V f NJMIN'A'P v P * l v N f q p h r f T S l y o N u|71n A >R A | S | S S|S|Y|T|I N|I |A P r Q | \ | . M Q T V r N | \ |N A P I. \> P\K k [ i j ... | K|I S P (YJS .>.;[* Y H I S f - -j... Caprin-1 558 Caprin-2 807 Caprin-1 632 Caprin-2 881 c L p | £ ] i l £ j - t e ] < ; NJY <j|i |Q F | I A I |K I Y s r , R P | N | F Q Q l ^ h * R vl • i -• - ; | o p R A IN --.|g A|<J W S D S S v s ^ J u Caprin-1 697 I Caprin-2 953 I 1 E T F N S O D S G Q G D J i' v i) v i' v ; INII- A .• I L P V H V Y P L P Q Q M R V A F S A A R T S N L A P G T L D Q P I V F D L L L N N L G E 1027 Caprin-2 1028 T F D L Q L G R F N C P V N G T Y V P 1 P H W L K L A Y N Y P L Y V N L M K N B E V L V S A Y A N 0 G A P D H B ' Caprin-2 H03 L H R G A I Y G S S W K Y S T F S O Y L L Y Q D 1126 A 3 N H A 1 L Q L F Q G D Q I W L R 1102 70 c 1 Exon2(0.2kb) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 5' UTR ATG Caprin-1 Q ^ ^ 0.5kb 19.0 0.09 4.2 0.08 3*0 3.0 0 09 3*0 0.09 3.0 0.7 0.2 1.2 4*4 0.5 0.4 1.5 X G A 1 Alt 3' UTR 18' 1 Exon2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 (0.06kb) 5' UTR ATG Caprin-21 ^ f j j | B i I I I I I I I • I • I I 2.2 kb 10.0 5.8 1.3 2.1 1.1 0.9 2.9 1.5 1.1 2^3 1.6 2.4 1.2 1.1 2.2 3' UTR 71 4.2.5. HR-1, a novel protein domain highly conserved in Vertebrates and insects We found no homologs of Caprin-1 in yeast or Caenorhabditis elegans, but the Drosophila melanogaster genome encoded a novel hypothetical protein of 961 amino acids (AAG22572), which included a region with 32% identity and 52% similarity with a region near the N-terminus of human Caprin-1. We termed this "homologous region-1" (HR-1) and the predicted D. melanogaster protein, "Drosophila HR-1-containing Protein" (DHP). We also identified Bombyx mori ESTs (AV398342, AV398325, AV398082, and AU003960) which encoded part of a third novel protein that exhibits a well-conserved HR-1 domain (38% identity and 61% conservation with the HR-1 domain of human Caprin-1) (Fig. 4.4A), and which we termed "Bombyx HR-1-containing Protein" (BHP). DHP, BHP and human Caprin-1 exhibited no homology in any region other than HR-1. Interestingly, the two insect HR-1 domains were no more similar to each other than to the HR-1 domain of human Caprin-1 (Fig 4.4A). The level of sequence conservation of the HR-1 in these three distinct proteins is remarkable, and suggests strongly that HR-1 represents a novel, independently-folding protein domain that is likely to have a conserved function. In Vertebrates, we found HR-1 only in Caprin-1 and a paralog of Caprin-1 that we identified in the EST and cDNA databases (NCBI) and termed Caprin-2. 72 4.2.6. Caprin-2 Well-conserved orthologs of Caprin-2 (Fig. 4) were identified in mammals, X. laevis and other amphibians, Danio rerio and Fugu rubripes. The Caprin-2 gene was located on human chromosome 12 and murine chromosome 6. The predicted Caprin-2 protein exhibited homology with Caprin-1 in two regions (Fig. 4.4B). One region was an HR-1 domain which was 73% conserved (51% identical), and the second region, which we termed "homologous region-2" (HR-2), exhibited 54% conservation and 36% amino acid identity with 334 amino acids near the C-terminus of Caprin-1 (Fig. 4.4B). HR-2 exhibited no similarities with known domains, or structural motifs suggestive of function, and occurs only in Caprin-1 and -2. Caprin-2 differed from Caprin-1 in the presence of a third domain at the carboxy-terminus (Fig. 4.4B, C) that was highly conserved in human and mouse Caprin-2 (98% identity) and was homologous to the globular domain of C1q. We termed this the "C1q-related domain" (CRD) (Fig. 4.4). The C1q domain and the related region in tumor necrosis factor-alpha are involved in multimerization 1 3 1 , and it is possible the C R D of Caprin-2 may have a similar function. We used R T - P C R with primers spanning HR-2 and the C R D to demonstrate that mature, spliced Caprin-2 transcripts that included the C R D were expressed in murine spleen (data not shown). Analyses of the EST, cDNA and S A G E databases (NCBI) confirmed that, as with Caprin-1, Caprin-2 mRNA is present in hemopoietic cells, including erythroid progenitor cells (AY074491, AY074490) and in the brain. Caprin-2 is annotated in the databases as a distinct hypothetical protein "FLJ22569", with a corresponding provisional gene FLJ22569. Analysis of the EST and 73 cDNA databases predict that in humans, alternative splicing generates two proteins with the predicted sizes of 126 and 117 kDa and the N-termini "MEQV" and "MKSAK" (Fig 4.4B, highlighted in red), respectively. Neither of these proteins includes a signal peptide. There is E S T evidence for an X. laevis protein that starts at the intervening methionine in the conserved "MQVLF" motif also indicated in Figure 4.4B. The NCBI databases provide evidence for multiple splice-variants of Caprin-2 involving differential use of internal exons (BG944982, AL536436, BI769244, AA187575, BE155973, and AY074490). Human Caprin-2 has a polymorphism (K/R) at residue 237, and differential splicing can result in the addition of an alanine at residue 823. 4.2.7 Genomic organization The human Caprin-1 and Caprin-2 genes exhibit a similar organization of exons and introns (Fig. 4.4C). The number and size of exons encoding HR-1 (6 exons) and HR-2 (9 exons) are the same in both genes (Fig. 4.4C), consistent with their origin from gene duplication. The C R D of Caprin-2 is encoded by a single exon. There is conservation of synteny for both caprin-1 and Caprin-2 loci. In the murine and F. rubripes genomes, orthologs of the human genes for LMO-2, (Lim domain only-2 protein), Caprin-1 and FLJ10774 (annotated as an acetyltransferase) are linked in the same order and orientation as in the human. Both the human Caprin-2 gene and its F. rubripes ortholog are adjacent to orthologous genes encoding the RAN-binding protein 8. The most primitive organism in which we have evidence for a Caprin is the Urochordate, Ciona 74 intestinalis. Its unassembled genome contains sequences that encode a well-conserved Caprin HR-1 and a part of HR-2. 4.2.8. Caprin-1 is a phospho-protein that exists in a multi-protein complex X. laevis Caprin-1 was previously identified in a screen for cDNA encoding proteins that were phosphorylated by mitotically-activated kinases present in an extract of fertilized oocytes 1 3 2 . However, because of its previous misidentification as a GPI-anchored membrane protein, it was concluded that its phosphorylation by these kinases was an artifact of the screening strategy and was not physiologically relevant. We used A P antibodies to immunoprecipitate Caprin-1 from Baf/3 cells labeled for 1-6 hours with 3 2 P - l a b e l l e d ortho-phosphate. In 3 independent experiments, autoradiographs demonstrated a major phosphorylated species of Mr 116K, together with two doublets of Mr ~66K and ~33K (Fig. 4.5A). The upper band (116K) was recognized by anti-recombinant Caprin-1 antibodies confirming its identity as phosphorylated Caprin-1. The 33K and 66K phospho-proteins also co-precipitated with transiently expressed GST-Caprin-1, but not with G S T alone from 3 2 P orthophosphate-labeled cells (Fig. 4.5B). Surprisingly, unlike endogenous Caprin-1, GST-Caprin-1 did not incorporate any 3 2 phosphate. The most likely explanation for this was that the N-terminal G S T tag was preventing phosphorylation of GST-Caprin-1. This result could be explained by protein folding. It is possible that the G S T is causing the GST-Caprin-1 to fold in such a way that the potentially phosphorylated residue(s) is sequestered away from the phosphorylating kinase. Along a similar line of thought, altered protein folding may 75 destabalize the GST-Caprin-1 tertiary structure such that the fusion protein is no longer recognized as a target for phosphorylation. In other experiments we explored the possibility that Caprin-1 existed in multimeric complexes. We observed that immunoprecipitation of a GFP-Caprin-1 fusion protein using antibodies to G F P , resulted in co-precipitation of co-expressed HA-tagged Caprin-1 (Fig. 4.6 B, C). Precipitation of a GFP-fusion of a deletion mutant (HR-1) that corresponded essentially to HR-1 also resulted in co-precipitation of HA-tagged Caprin-1 (Fig. 4.6). In contrast, only small amounts of HA-tagged Caprin-1 were co-precipitated with a fusion protein of G F P and essentially HR-2. These results suggest that the HR-1 region is important for formation of a complex containing two or more Caprin-1 molecules. Figure. 4.5. Phosphorylation of Caprin-1. A) Autoradiograph (left) and immunoblot with anti-Caprin-1 (right) of 3 2P-labelled Caprin-1 (*) immunoprecipitated with AP antibodies from Baf/3 cells cultured in 32P-labelled phosphoric acid for the indicated time. B) Baf/3 cells were electroporated with a 30 u.g plasmid encoding either GST, or a fusion protein of GST and Caprin-1. 16 hours post-electroporation, cells were cultured in 32P-labelled phosphoric acid for the indicated time. Shown are autoradiographs, or the same nitrocellulose membranes probed with an anti-GST antibody, of the same samples run on 7.5 or 10% polyacrylamide gels. For A and B Co-precipitating phospho-proteins are also indicated ( • ) 76 autoradiograph IB: anti-Caprin-1 *v SJ^ ^ N <b <b N <b to 200 — 200 — 97 — 97 — 66 — • 66 45 — 45 W i t • B Autoradiograph IB: anti-GST GST GST-Caprin-1 GST GST-Caprin-1 200 — 116 — 97— 45— e e — • 7 . 5 % gel 116 — 97 — 66— 45— 31— 10% gel • 77 B lysates IPanti-GFP ( ? & N <y o x <-? & & Caprin-1-HA — IB: anti-HA blot GFP-Caprin-1 — GFP-HR-2 — IB: anti-GFP blot GFP-HR-1 — GFP Figure. 4.6. Caprin-1 is capable of forming multimeric complexes. Caprin-1 tagged at the C-terminus with a HA tag (Caprin-1-HA) was transiently co-expressed in Bosc 293 cells with GFP, or the indicated GFP- fusion proteins of Caprin-1 or a series of the indicated A) deletion mutants of Caprin-1. Shown in B) is an immunoblot with an anti-HA monoclonal antibody (IB: anti-HA) of cell lysates (lysates) or of anti-GFP immunoprecipitates (IP anti-GFP), detecting Caprin-1-HA that co-precipitates with the GFP-fusion proteins and in C) the same blot re-probed with anti-GFP antibodies (IB: anti-GFP) to demonstrate precipitation of the GFP-fusion proteins. 78 4.2.9. Levels of Caprin-1 decrease when factor-dependent Baf/3 cells were deprived of IL-3 or the M-1 leukemia was induced to differentiate with IL-6 To investigate changes in levels of Caprin-1 when cells ceased to divide, we deprived IL-3 dependent Baf/3 cells of IL-3. We observed a marked decrease in the levels of Caprin-1 relative to total proteins (Fig. 4.7A). We also treated the mouse leukemic cell-line M-1 with IL-6, which induces it to differentiate into adherent, non-proliferating macrophages, and observed that cessation of growth and differentiation was accompanied by decreased levels of Caprin-1 (Fig. 4.7B). A IB: ant i-Caprin-1 IB: Erk Figure. 4.7. Expression of Caprin-1 decreases when cells cease to divide. Immunoblots with ant i-Caprin-1 ant ibodies of lysates normal ized for total protein of A ) IL-3 dependent , Baf/3 ce l ls cul tured with or without IL-3 for 16 hours, or B) M1 l eukem ia ce l l s t reated with IL-6 (50ng/mL) for the indicated number of days to induce differentiation to non-div id ing adherent cel ls. Blots were re-probed with anti- Erk antibodies to confirm equiva lency of loading. B 6^ V V ^ 6 ^ 4.2.10. Over-expression of GFP-Caprin-1 results in specific, dose-dependent inhibition of cell division We used flow cytometry to determine whether over-expression of Caprin-1 affected proliferation of 3T3 cells. We observed that over-expression of GFP-Caprin-1 resulted in dose-dependent decreases in cell numbers relative to those of cells expressing equimolar amounts of G F P alone (Fig. 4.8). To determine whether this was due to interference with cell division, we transfected 3T3 cells with either GFP-Caprin-1 or G F P alone, and labeled them with a fluorescent dye, PKH26, that associates permanently with the plasma membrane and is diluted two-fold each time a cell divides. Using two-colour flow cytometry we compared the number of divisions undergone by cells expressing various levels of GFP-Capr in-1, or equimolar levels of the control protein G F P . As shown in Figure 4.9, cells expressing mean levels of GFP-Caprin-1 that were 3-fold in excess of endogenous levels of Caprin-1 (mean level, L) exhibited significantly longer doubling times than control, untransfected cells in the same culture, or cells expressing G F P alone. This effect was dose-dependent and cells expressing G F P -Caprin-1 at mean levels that were at 12-fold (mean level, M), or 48-fold (mean level, H) excesses over endogenous levels of Caprin-1, divided infrequently or failed to divide at all (Fig. 4.9). We saw no evidence that over-expression of Caprin-1 led to increased cell death from apoptosis (data not shown). When GFP-Caprin-1 was over-expressed, although cellular proliferation was severely impeded, we observed no changes in the fraction of cells at each stage of the cell cycle (G1, S, and G2/M-phase) compared to cells that were either untransfected or 80 expressing G F P alone. Thus, over-expression of GFP-Caprin-1 did not block NIH 3T3 cells at any particular point in the cell cycle. This was not unexpected given that levels of endogenous Caprin-1 did not fluctuate with different phases of the cell cycle in these cells (data not shown). 81 B 100% 80 . _ relative 6 0 _ i — fold change in cell number 40 . _ . 20 r~~] o , T B 1 ' ' 1 1 ' I 1 1 1 1 GFP GFP- L M H alone Caprin-1 Figure. 4.8. Loss of Caprin-1 GFP fusion protein expression over time. 3T3 cells were transiently transfected with either pEGFP-C1 empty vector or pEGFP-C1 encoding a GFP-Caprin-1 fusion protein. A) GFP or GFP-Caprin-1 expression was analyzed by flow cytometry 24 and 72 hours post transfection. The expression of both GFP and GFP-Caprin-1 was analyzed based on 4 equivalent, half-log gates representing low (L), medium (M), and high (H) equimolar expression of GFP or GFP fusion protein. B) Absolute numbers of GFP-Caprin-1 expressing cells were normalized to absolute numbers of GFP expressing cells in the same gate at 72 hours compared to 24 hours post-transfection. 82 Figure. 4.9. Over-expression of GFP-Caprin-1 blocks division of 3T3 cells. 3 T 3 cel ls were transiently t ransfected with p E G F P - C 1 encod ing either G F P a lone or G F P - C a p r i n - 1 , and at 24 hours were labe led with the l ipophi l ic dye, P K H 2 6 . A) S h o w s on a logar i thmic sca le , the exp ress ion of G F P - C a p r i n - 1 in three gates represent ing low (L), med ium (M), and high (H) cor respond ing , respect ively to mean levels of G F P - C a p r i n - 1 (24 hours after transfection) that are 3, 12 and 48 fold above that of endogenous Cap r i n -1 . B) S h o w s levels of P K H 2 6 dye f luorescence, immediately after labeling ( ),or 3 days later ( ) of cel ls with levels of G F P - C a p r i n - 1 cor respond ing to those in each of the gates spec i f ied in A . C) S h o w s the number of cel l d iv is ions over 3 days of cel ls express ing the s a m e three mean levels of G F P -Caprin-1 or of cel ls express ing equimolar levels of G F P , or of untransfected 3T3 cel ls growing in the s a m e culture. 83 A B GFP-Caprin-1 GFP GFP-Caprin-1 L M 84 4.3 Discussion Levels of Caprin-1 in cells of the lymphohemopoietic system correlated tightly with their proliferative status. Activation of T or B-lymphocytes to proliferating blast cells was associated with 15-fold increases in the amount of Caprin-1 per cell (Fig. 4.1). Hemopoietic progenitors proliferating in response to IL-3 or CSF-1 also expressed high levels of Caprin-1 (Fig. 1G). In contrast, levels of Caprin-1 decreased when proliferating, factor-dependent cells were deprived of IL-3 (Fig. 4.7A), or M-1 leukemia cells were induced to differentiate to non-dividing, adherent macrophages (Fig. 4.7B). Likewise the tissue with the highest level of expression of Caprin-1 was the thymus, a site of continuous cellular proliferation. Significantly, while all thymocytes contained Caprin-1, the cells with the highest levels were the large, dividing "blast" cells (Fig. 4.2). In contrast, Caprin-1 levels were low in tissues such as the kidney or muscle that have a low proportion of dividing cells. The one exception was the adult brain, which expressed significant levels of Caprin-1 despite having a low percentage of dividing cells. It is possible that Caprin-1 resembles components of the Ras and MAP kinase pathways, which are critical for cell growth but also have key functions in non-dividing cells in the brain 1 2 8 . A role for Caprin-1 in cell growth would also be consistent with the identification of X. laevis Caprin-1 in a screen for novel proteins that were phosphorylated by a mitotically activated kinase 1 3 2 . The nature of the role of Caprin-1 in proliferation and the mechanism of the dose-dependent suppression of cell-division we observed when we over-expressed GFP-Caprin-1 require further investigation. Given our evidence that 85 Caprin-1 exists in multimeric complexes that include unidentified phospho-proteins (Fig. 4.5), one possibility is that it acts as a scaffold for formation of a critical complex of proteins. Excess Caprin-1 may inhibit assembly of this complex by titrating out binding partners. It is also conceivable that, like other proteins involved in growth (e.g. p21 Ras or c-myc), Caprin-1 exerts both positive and negative effects, and that when in excess, its inhibitory effects may predominate. Finally, the presence of G F P at the N-terminus may interfere with its function by creating a dominant inhibitory protein. Given that both Caprin-1 and -2 are expressed in lymphohemopoietic cells, it seems likely that they will have important roles in the generation and function of the blood and immune system. 86 CHAPTER 5: CONCLUSIONS AND FUTURE DIRECTIONS We describe here the activation of Rac-1, -2 and Cdc42 by IL-3. The mechanism of activation of Rac-1 by IL-3 was studied in detail and we have demonstrated biochemically, using endogenous systems, that Rac-1 is activated by both a PI-3K-dependent and -independent mechanism. We provide evidence that p21 Ras isoforms, and the non-classical Ras molecule M-Ras, may be responsible for PI-3K independent activation of Rac-1. Further, we showed that blocking activation of Rac-1 did not result in inhibition of p38 M A P K activity. This was an interesting and unexpected result given that constitutively active Rac-1 was sufficient for activation of p38 M A P K in hemopoietic cells. While investigating the promiscuous Ras family G E F , smgGDS, we discovered and provided the initial characterization of Caprin-1, the prototype of a novel family of proteins. We have shown that Caprin-1 is a cytoplasmic phospho-protein associated with cellular proliferation. We also describe the two novel protein domains which are distinct to Caprin-1 and -2. Several questions remain to be addressed regarding Rac in hemopoietic cells. As this thesis has not touched on the role of Rac-3, it would be interesting to study the expression and activation of this newly described protein in hemopoietic cells. However, this will require the generation of Rac-3 specific antibodies. The antibodies shown to be functionally specific for Rac-1 and -2 in this thesis should also be evaluated with regard to Rac-3. Since the presence of G E F capable of activating both Rho and Ras subfamily GTPases make the use of dominant negative mutants problematic for studying these molecules (discussed earlier), it would be important to revisit questions addressed previously with dominant negative molecules. This could be done by conjugating a GTPase binding domain (e.g. CRIB domain) to antibodies specific for a single Ras GTPase . Such "inhibitory antibody conjugates", once micro-injected into cells, would bind specifically to a single target GTPase via the antibody. Antibody binding would 87 localize the GTPase binding domain in close proximity to the GTPase allowing efficient and specific binding to the GTPase. Once bound, the GTPase would not be capable of interacting with endogenous effectors and signaling would be blocked. A similar strategy that might be effective would be over-expression of the G T P a s e binding domain of an effector that is specific for a single Ras family small GTPase , allowing inhibition of that molecule but no other small GTPases. Using either of these strategies, one would target a specific GTPase , titrate out binding of endogenous effectors, and inhibit GTPase signaling. In the case of a novel protein family such as the Caprins there are any number of directions the work might be taken in the future. This thesis has provided some initial indications that Caprin-1 may be important for proliferation, but the exact function of Caprin-1 remains to be discovered. One strategy to investigate this might be the use of immunoprecipitation and mass spectrometry to co-precipitate and identify, respectively, Caprin-1 binding proteins. If such binding proteins linked Caprin-1 to a particular signal transduction pathway or cellular function, this would be the first step in understanding Caprin-1 function. The identification of the domain (HR-1 or -2 or a specific motif) required for interaction with binding proteins would be a further advance in understanding Caprin-1. Similarly, the crystal structure of Caprin-1, HR-1, or HR-2 would be invaluable. Mice which are homozygous null for Caprin-1 may have a phenotype that reveals the function of Caprin-1. However, since Caprin-1 seems to be expressed in a variety of tissues and related to proliferation one might expect disruption of both alleles of Caprin-1 to be lethal. Therefore, the use of a tissue specific knock-out using the Cre recombinase might prove useful in averting such lethality, and examining the role of Caprin-1 in a specific tissue, such as the brain, or thymus. We observed that over-expression of GFP-Caprin-1 causes the rounding and loss of adherence of a significant proportion (approximately 50%) of NIH 3T3 cells (data not shown), a phenotype associated with loss of Rac or Rho function. It may be possible to rescue this by over-expressing constitutively active mutants of these 88 GTPases. If such an experiment were successful, it would functionally link Caprin-1 to Rho family GTPases, and the actin cytoskeleton. With regard to Caprin-2, the future is wide open. The expression pattern of both RNA and protein needs to be determined for this protein, as does the predominant splice form that exists in different tissues. Although the function of Caprin-1 might provide some starting points, the same strategies as discussed for Caprin-1 will likely have to be employed to address the function of Caprin-2. 89 REFERENCES 1. Ihle J N , Witthuhn B A , Quelle F W , Yamamoto K , Silvennoinen O. Signaling through the hematopoietic cytokine receptors. 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