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Investigation of the phosphatidylinositol 3-kinase pathway in B cells Ma, Kewei 2009

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Investigation of the Phosphatidylinositol 3-Kinase Pathway in B cells by KEWEI MA M.Sc. (Genetics), Peking Union Medical College & China Academy of Medical Sciences, 2001 A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver) January 2009 © 2009 Kewei Ma  ii Abstract There is hardly a cellular process that is not regulated in some way by phosphoinositides, which makes biochemical and physiological studies of these lipids extremely important. PI 3-kinases are key regulators of phosphoinositide metabolism and have been shown to affect a large variety of cellular responses. The key products of PI 3-kinases that have functional activity in higher eukaryotic cells are PI(3,4,5)P3 and PI(3,4)P2. PI(3,4,5)P3 is universally accepted as one of the most important second messengers in signal transduction. However, our knowledge of the functions of PI(3,4)P2 as a lipid second messenger is much less precise. In this dissertation, work was undertaken to elucidate the regulation of PI(3,4,5)P3 and PI(3,4)P2 production and downstream signaling in B cells. Cells with membrane targeted exogenous SHIP were utilized to manipulate phosphoinositide levels. The relationship of PI(3,4,5)P3 and PI(3,4)P2 levels to downstream PKB phosphorylation and activation was studied. PI(3,4,5)P3  and PI(3,4)P2 levels were found to closely correlate with PKB phosphorylation levels at Thr308 and Ser473, respectively. In addition, PI(3,4)P2  levels determine the PKB activity in the cytosol; while PI(3,4,5)P3  levels determine PKB activity at the plasma membrane. Different doses and different forms of B cell receptor (BCR) agonists were used for stimulation. PI 3-kinase activation was studied carefully following stimulation with low doses of anti-BCR antibody and F(ab')2 fragments. Low concentrations of F(ab')2 fragments produced higher levels of PI(3,4,5)P3 than did a high concentration of F(ab')2 fragments. Downstream PKB signaling was studied in these models. Similar conclusions were drawn from both SHIP over-expressing BJAB cells and dose-dependent BCR  iii stimulations. We speculated that phosphoinositides’ regulation of the kinetics of PKB phosphorylation at Ser473 and Thr308 might be mediated by additional proteins. Investigation of plasma membrane-associated PKB showed that it formed a protein complex of around 400KD, which we attempted to characterize further with respect to PKB phosphorylation and association with lipids. In conclusion, phosphoinositide production is intricately regulated in vivo to control downstream signaling. The levels of PI(3,4)P2  and PI(3,4,5)P3  have precise and profound effects on PKB and other molecules such as TAPP and Bam32. This study has contributed new insight into the PI 3-kinase signaling pathway from the aspect of phosphoinositide lipid function.  iv Table of Contents Abstract ..........................................................................................................................ii Table of Contents ........................................................................................................ iv List of Tables ................................................................................................................vi List of Figures..............................................................................................................vii Abbreviations................................................................................................................ ix Acknowledgement ......................................................................................................xii Chapter 1. Overview. ................................................................................. 1 1.1 Introduction to phosphoinositides and the PI 3-kinase pathway. ................... 1 1.1.1 The phosphatidylinositol lipids...................................................................... 1 1.1.2 PI metabolism. ................................................................................................ 3 1.1.3 Three classes of PI 3-kinase. ....................................................................... 5 1.1.4 Different tissue and organelle distribution of the PI 3-kinase family....... 6 1.1.5 The functions of PIP lipids in sub-cellular compartments ........................ 8 1.1.6 Phosphoinositides regulate downstream effectors through phosphoinositide binding domains....................................................................... 11 1.1.7 PI 3-kinase activation mechanisms downstream of different receptors. ................................................................................................................................... 14 1.1.8 Down-regulation of the PI 3-kinase/PKB pathway. ................................. 15 1.1.9 PIP lipids co-coordinate the PDK1-PKB pathway. .................................. 17 1.1.10 Protein-protein interactions downstream of PI(3,4,5)P3 result in intricate regulation of PIP downstream pathways. ............................................ 19 1.1.11 PKB Ser473 kinases and significance of the mTOR pathway. ........... 22 1.1.12 PKB membrane detachment: an important step in PKB function....... 23 1.1.13 Diseases related to abnormal phosphoinositide metabolism and therapeutic intervention of PI 3-kinase inhibitors in the treatment of common diseases. .................................................................................................................. 24 1.1.14  Approaches to study phosphoinositides ................................................ 29 1.2 Hypothesis and strategies .................................................................................. 33 Chapter 2. Materials and Methods....................................................... 35 Reagents and Antibodies ...................................................................................... 35 Cell lines, Constructs and transfection................................................................ 35 B cell stimulation, preparation of cell lysates and immunoblotting ................. 36 Crude plasma membrane and cytosol preparation ........................................... 37 Lipid labeling, extraction and HPLC separation................................................. 38 Flow cytometry to measure antibody binding to B cell receptors ................... 39 PKB kinase assay................................................................................................... 40 Immunoprecipitatation of the Flag-tagged protein............................................. 41 Gel staining with silver stain or colloidal Coomassie ........................................ 41 Gel filtration. ............................................................................................................ 42 Chapter 3.  In vivo regulation of membrane recruitment of TAPP, Bam32 and Btk by levels of PI(3,4)P2 and PI(3,4,5)P3..................... 43 3.1 Introduction ........................................................................................................... 43  v 3.2 Results ................................................................................................................... 45 3.3 Discussion ............................................................................................................. 56 Chapter 4. Low concentrations of B cell receptor agonists stimulate distinct waves of PI(3,4)P2 and PI(3,4,5)P3 production and downstream signaling in B cells. ................................................ 59 4.1 Introduction ........................................................................................................... 59 4.2 Results ................................................................................................................... 61 4.3 Discussion ............................................................................................................. 77 Chapter 5. PI(3,4)P2-correlated PKB Ser473 phosphorylation involves kinetics independent of PI(3,4,5)P3 and Thr308 phosphorylation ....................................................................................... 82 5.1 Introduction ........................................................................................................... 82 5.2 Results ................................................................................................................... 84 5.3 Discussion ........................................................................................................... 104 Chapter 6. Summary .............................................................................. 112 6.1 General conclusions .......................................................................................... 112 6.2 Future directions ................................................................................................ 117 References ............................................................................................... 120 Appendix. Proteomics and lipidomics: Flag-PKB overexpression. ...................................................................................... 138 Introduction ................................................................................................................ 138 Results and Discussion ........................................................................................... 139  vi List of Tables Table 1. 1 Location of PI kinases and phosphatases ................................................. 4 Table 1. 2 Three classes of PI 3-kinase ....................................................................... 5 Table 1. 3 Phosphoinositide binding domains ........................................................... 12 Table 1. 4 Diseases related to abnormal phosphoinositide metabolism enzymes ................................................................................................................................... 26 Table 3. 1 Optimization of phosphoinositide analysis............................................... 47 Table Appendix-1A Mass-spectrometry data of Flag immunoprecipitation bands………………………………………………………………………….….143 Table Appendix-1B Mass-spectrometry data of Flag immunoprecipitation bands……………………………………………………………………………..144  vii List of Figures Figure 1. 1 Structure of phosphatidylinositol. ............................................................... 2 Figure 1. 2 The PIP metabolism network is composed of phosphoinositide kinases and phosphatases. .................................................................................... 3 Figure1.3 Hypothesis ..................................................................................................... 33 Figure 3.1 HPLC profile of orthophosphate labeled deacylated phosphoinositide (Without normalization)..........................................................................................50 Figure 3. 2 HPLC profile of deacylated phosphoinositides corresponding to PI(3,4,5)P3, PI(3,4)P2 levels in BJAB cells. ........................................................ 51 Figure 3. 3 TAPP and Bam32 membrane recruitment by PI(3,4,5)P3 and PI(3,4)P2. .................................................................................................................. 52 Figure 3. 4 Peak PI(3,4,5)P3/PI(3,4)P2  ratio. ............................................................ 53 Figure 3. 5 Comparison of Ser473 phosphorylation in BJAB cells and BJAB cells expressing TAPP2.................................................................................................. 55 Figure 4.1 PI(3,4)P2 and PI(3,4,5)P3  production by F(ab')2 and intact titration. .. 62 Figure 4.2 Membrane recruitment of EGFP-PH domain proteins in response to low versus high doses of BCR ligand.................................................................. 64 Figure 4.3 PKB phosphorylation at Thr308 and Ser473 sites in response to dose stimulation of F(ab')2 and intact antibody stimulation at 2 minutes in BJAB cells........................................................................................................................... 67 Figure 4.4 PKB activity in response to dose stimulation of F(ab')2 and intact antibody stimulation at 2 minutes in BJAB cells. ............................................... 68 Figure 4.5 Lipid analysis showing that low concentration of intact antibody caused higher levels of PI(3,4,5)P3 and PI(3,4)P2 than did F(ab')2. ............... 70  viii Figure 4.6 Low concentration of intact antibody caused higher Ser473 and Thr308 phosphorylation than did F(ab')2. ........................................................... 71 Figure 4.7 Blocking of Fc RIIB receptor with 2.4G2 shows that the positive signal of intact antibody is independent of Fc RIIB binding. ....................................... 74 Figure 4.8 Intact antibody binds more B cell receptor than F(ab')2 does. ............. 76 Figure 5. 1 The kinetics of PI(3,4,5)P3 and PI(3,4)P2 production in A20 cells...... 86 Figure 5. 2 PKB phosphorylation in BJAB and MS19 cells showing that Thr308 phosphorylation and Ser473 phosphorylation correlate with PI(3,4,5)P3 and PI(3,4)P2 levels, respectively. ............................................................................... 90 Figure 5. 3 PKB  assay  following  B  cell  stimulation.............................................. 92 Figure 5. 4 GSK-3ß Ser9 phosphorylation in BJAB and MS19 cells. .................... 92 Figure 5. 5 Membrane preparation to show the membrane and cytosol portion of phosphorylated PKB and total PKB..................................................................... 96 Figure 5. 6 PKB kinase activity in membrane and cytosol fractions. ..................... 97 Figure 5. 7 Live cell imaging of GFP-flag-PKB. ....................................................... 100 Figure 5. 8 PKB forms a large protein complex at the plasma membrane, but not cytosol. ................................................................................................................... 103 Figure 5. 9 Functions of PTEN and SHIP in regulating lipid levels and downstream signaling in B cells. ........................................................................ 109 Figure 5. 10 Model of roles of PI(3,4,5)P3 and PI(3,4)P2 in regulating PKB. ...... 111 Figure appendix 1  Flag-PKB over-expression in BJAB cells. .............................. 141 Figure appendix 2 Silver staining and Coomassie staining of lysates after anti- Flag immunoprecipitation. ................................................................................... 142  ix Abbreviations AGC Protein A, Protein G and protein C ARNO ADP-ribosylation factor nucleotide-binding site opener Bam32 B cell adaptor protein of 32 kDa (DAPP) BCR B cell receptor Btk Bruton’s tyrosine kinase CCV Clathrin Coated Vesicles CD Clusters of differentiation CISK cytokine-independent survival kinase CMT4B Charcot-Marie-Tooth disease type 4B CTMP Carboxyl-Terminal Modulator Protein DAG Diacylglycerol DAPP Dual adaptor of phosphotyrosine 3-phosphoinositides (Bam32) DNA-PK DNA-dependent protein kinase EEA1 Early endosome autoantigen 1 EGFP Enhanced green fluorescent protein ENTH/ANTH Epsin N-terminal homology ER Endoplasmic reticulum FPLC Fast performance liquid chromatography FRET Fluorescence Resonance Energy Transfer FYVE Fab1p, YOTB, Vac1p and EEA1 GAP GTPase activating protein GEF Guanine gucleotide exchange factor GFP Green fluorescent protein GPCR G protein coupled receptor GRP General receptor for phosphoinositides-1 HPLC High pressure liquid chromatography HRS Hepatocyte growth factor-regulated tyrosine kinase substrate IC Immune complex  x IL Interleukin ILK  Integrin linked kinase ING2 Inhibitor of growth 2 IRS Insulin receptor substrate ITAM Immunoreceptor tyrosine-based activation motif ITIM Immunoreceptor tyrosine-based inhibition motif JIP JNK-interacting protein kDa Kilodalton LY LY 294002 Mi Mitochondria MS Membrane targeted SHIP MSK Mitogen- and stress- activated protein kinase MTM1 Myotubularin MTMR Myotubular myopathy-related protein mTOR Mammalian target of rapamycin mTORC Mammalian target of rapamycin complex MW Molecular weight OCRL Oculocerebrorenal syndrome of Lowe phosphatase PDGF Platelet derived growth factor PDK1 Phosphoinositide dependent kinase-1 PH Pleckstrin homology PI             Phosphoinositide, phosphatidylinositol PIKE Phosphatidylinositol 3(PI3)-kinase enhancer PIP           Phosphoinositide phosphate PIPP proline-rich inositol polyphosphates-phosphatase PKB         Protein kinase B (Akt) PLC phosphoinositide phospholipase C PM Plasma membrane PP2A Type 2 protein serine/threonine phosphatase  xi PRK Protein kinase C-related kinase PTB Phosphorylated tyrosine binding PTEN Phosphatase and tension homolog deleted on chromosome 10 PX Phox RSK p90 ribosomal S6 kinase S6K p70 ribosomal S6 kinase SDS Sodium dodecyl sulphate SGK Serum and glucocorticoid inducible kinase SH Src homolog SHIP SH2 domain containing inositol phosphatase SKIP skeletal muscle and kidney enriched inositol phosphatase SNX Sorting nexin TAPP Tandom PH domain containing protein TH Tec homolog TPIP TPTE and PTEN homologous inositol lipid phosphatase TPTE transmembrane phosphatase with tensin homology XLMTM X-linked myotubular myopathy  xii Acknowledgement I would like to give my first appreciation to my supervisor, Dr. Vincent Duronio, who led me into this active phosphoinositide signaling research, which I believe is one of the most promising and fastest developing research area in life science. Most of this thesis work was done in the close collaboration with Dr. Aaron Marshall, who provided supportive data, as well as precise interpretation of my data. My committee, Drs. Michael Gold, Gerald Krystal, Alice Mui gave invaluable discussions. Dr. Marc Germain also provided intellectual input. All of the scholarly attitude toward science I witnessed in these excellent scientists during my Ph.D training will be a tremendous asset in my future career. My gratitude also goes to Dr. Juergen Kast and Ms. Shujun Lin for their work with mass spectrometry.  1 Chapter 1. Overview. 1.1 Introduction to phosphoinositides and the PI 3-kinase pathway. 1.1.1 The phosphatidylinositol lipids. Phosphatidylinositol (PI) lipids, a minor proportion (less than 10% in eukaryotic cells (Payrastre, 2001)) of total phospholipids within the plasma membrane, have been shown to be among the most important second messengers in cellular signal transduction, with involvement in almost all cellular processes. Phosphatidylinositol lipids, or the inositol-containing glycerophospholipids, are also collectively known as phosphoinositides. More than 90% of the phosphatidylinositol pool consists of PI, which is the unphosphorylated phosphatidylinositol (Figure 1.1). Unphosphorylated PI has not been shown to have signaling roles unless it is phosphorylated at the D-3, 4, or 5 position(s), forming phosphorylated phosphatidylinositols (or PIPs). All of the seven PIPs (PI3P, PI4P, PI5P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2, PI(3,4,5)P3) have been identified, with PI3P, PI4P and PI(4,5)P2 being the most abundant. Metabolism of PIPs is mediated by a network of inositol kinases and phosphatases (Vanhaesebroeck, 2001). The percentage of individual PIPs in different cell types can vary greatly owing to the different expression levels of inositol kinases and/or phosphatases. PI5P, PI(3,5)P2, PI(3,4)P2 and PI(3,4,5)P3 comprise smaller portions of total PIPs. PI 3-kinase’s biochemical function is to add a phosphate at the D-3 position to produce PI3P, PI(3,4)P2 or PI(3,4,5)P3. PI(3,5)P2 is not discussed in this thesis, since its production and function are unclear. PI 3-kinase’s physiological functions include regulating cell survival, motility, immunity, glucose metabolism, transcription, translation, cell cycle control and cardiovascular functions, just to name a few (reviewed by Marone, 2005; Vanhaesebroeck, 2001; Vanhaesebroeck  2 and Alessi, 2000). Our knowledge of D-3 phosphoinositides functions was mostly acquired through in vitro binding assays or in vitro kinase activity assays, as well as blockade of the PI 3-kinase pathway. However, the complexity of PI 3-kinase’s roles results from the spatial and temporal control of lipid production and from a variety of signaling functions performed by each individual lipid species in regulating downstream proteins. Figure 1. 1 Structure of phosphatidylinositol. OH OH OH OH OH P O O O O 1' 2' 3' 4' 5' 6'  3 1.1.2 PI metabolism. Consideration of the entire PI metabolism network is inevitable when one discusses PI 3-kinase, because PI 3-kinase substrates and products themselves are substrates and products of other inositol kinases and phosphatases. D-3 PIP levels are regulated by all the potential enzymes in the network as shown in Figure 1.2. For example, PIP-4 kinase can regulate PI(3,4,5)P3 and PI(3,4)P2 levels  (Carricaburu et al., 2003).  Besides their specific biochemical properties, these enzymes’ individual spatial distributions also contribute to the regulation of PIP production as shown in Table 1. Figure 1. 2 The PIP metabolism network is composed of phosphoinositide kinases and phosphatases. PI 3-kinase is centrally located in this network and is the best studied phosphoinositide kinase. Only general names of  PI kinases and phosphatases are shown. PI3K, PI4K and PI5K add phosphate at the corresponding position on the inositol ring; whereas 3- phosphatase, 4-phosphatase and 5-phosphatase remove the phosphate from the inositol ring at the corresponding position. PI(3,4,5)P3 PI(3,4)P2PI3P PI4P PI PI(4,5)P2 PI3K Class I, II, III PI3K Class I, II PI3K Class I 3-phosphatase 3-phosphatase 3-phosphatase 4-phosphatase 4-phosphatase 5-phosphatase SHIP PI5K PI5K PI4K PI4K DAG +IP3 PLC  4 Table 1. 1 Location of PI kinases and phosphatases  kinase  phosphatase Enzyme location Enzyme location PI 3-kinase  3-phosphatases P110 a-d  PM, nucleus PTEN PM, nucleus, GC PI3K-C2a GC, endosomes, nucleus, CCV MTM1 PM PI3K-C2ß  PM, nucleus MTMR1-8 PM PI3K-C2  GC TPIPa, ß,   ER, GC Vps34 GC, endosome 4-phosphatases PI 4-kinase  Synaptojanin 1,2 CCV, Mi PI4KIIa  GC, endosomes HSac1,2,3 GC, ER PI4KIIß  PM, GC Type I, II Not known PI4KIIIa  GC, ER 5-phosphatases PI4KIIIß  GC, nucleus SHIP 1, 2 PM, nucleus PIP 4-kinase  SKIP ER, PM PIP4Ka, ß  PM PIPP PM PIP4K  ER Ocrl GC, endosomes, lysosomes PI 5-kinase  72-kDa-5-phosphatase GC PIK-FYVE Late endosomes 5-phosphatase IV Not known PIP 5-kinase  Synaptojanin 1,2 Mi, synaptic vesicles PIP5Ka, ß,   PM 5-phosphatase II Not known Distribution of PI kinases and phosphatases in cellular organelles. These proteins are mostly located in intracellular membrane and plasma membrane structures, with some proteins such as PTEN also located in the nucleus. Besides their functions in cell signaling, these proteins play important role in the transportation of vesicles. Summarized from several reviews: Antonietta De Matteis et al., 2005; Matteis and Godi, 2004; Balla and Varnai, 2002; Balla et al., 2000. (GC: Golgi Complex; Mi: Mitochondria; ER: Endoplasmic reticulum; CCV: Clathrin Coated Vesicles)  5 1.1.3 Three classes of PI 3-kinase. Table 1. 2 Three classes of PI 3-kinase Catalytic subunit Regulatory subunit Substrate Class IA p110a, p110ß , p110d  p85a, p85ß, p55  PI=PI4P=PI(4,5)P2 Class IB p110  p101 PI=PI4P=PI(4,5)P2 Class II pI3K-C2a ,PI3K-C2ß, PI3K-C2  None PI>PI4P>PI(4,5)P2 Class III Vps34p p150 PI Different classes of PI 3-kinase have specificity for different substrates and therefore produce different profiles of PIP lipids. The structures of different classes of PI 3-kinase are explained in more detail in the main text. Reviewed in Fruman and Cantley, 2002; Vanhaesebroeck, 2001; Koyasu, 2003. PI 3-kinase proteins comprise a family of enzymes with several subclasses (Table 1.2): Class IA and IB, class II, and class III. Class I PI 3-kinases are able to produce all the D-3 phosphoinositides: PI(3,4,5)P3, PI(3,4)P2 and PI3P. Class I PI 3-kinases are composed of a regulatory p85 subunit and a catalytic p110 subunit. The p85 subunits of class IA include p85 , p55 , p50 , p85 , p85 , while the p110 subunit of class IA includes p110 , p110 , p110 . p85 , p55 and p50 are alternate splicing products of the same gene, and the others are products of different genes. Different types of subunits in class IA can combine in multiple ways to form a kinase holoenzyme. The p85 subunit has two SH2 domains that bind phosphorylated tyrosines, and an inter-SH2 domain that binds to p110 subunits.  By virtue of the SH2 domain in the regulatory subunits, the enzymes are activated through tyrosine phosphorylated receptors/adaptor proteins which will be discussed in section 1.1.7. Class IB PI 3-kinase is composed of a regulatory p101 subunit and a catalytic p110  subunit. The p101 subunit does not contain an SH2 domain.  6 Its activation is through G-protein coupled receptors (GPCR) (Fruman and Cantley, 2002; Vanhaesebroeck, 2001; Koyasu, 2003). Class II PI 3-kinase mainly produces PI(3,4)P2 and PI3P. It is a monomeric molecule and contains a Phox (PX) domain with affinity for PI3P and PI(3,4)P2. Thus regulation of class II involves negative feedback from its products. Class II PI 3-kinases contain three subclasses, which are PI 3-kinase C2 , C2 and C2 . Class III PI 3-kinase produces only PI3P. Most of its functions were identified in Saccharomyces cerevisiae. Its mammalian homologue regulates vesicle trafficking through Fab1p, YOTB, Vac1p and EEA1 (FYVE) domain-containing proteins binding to PI3P (Vanhaesebroeck, 2001; Koyasu, 2003).  1.1.4 Different tissue and organelle distribution of the PI 3-kinase family Class IA and IB are the best-studied PI 3-kinases in mammalian cells. Knockout studies have shown that the different subclasses contribute to PI 3-kinase functions in different ways, depending upon cell types, tissues or organs. PI 3-kinase class IA is universally expressed in tissues and cell types (Fruman, 2004). PI 3-kinase IB is expressed mainly in leukocytes (Krugmann et al., 1999). Genetic ablation studies have shown the relative importance of the PI 3-kinase subclasses in different cells. For example, in B cells, p85a and p110d are important in maintaining PI 3-kinase functions, while in T cells, p85ß and p110 are also important. p110a and p110ß have the utmost importance as shown by the embryonic lethality that results from their genetic ablation (Vanhaesebroeck 2005; Fruman 2004) and by a number of cancers being caused by  7 p110a gene mutations  (Samuels et al., 2004; reviewed in Bader et al., 2005). Of the three classes of class II PI 3-kinase, C2a and C2ß are widely expressed in many tissues, while C2 is confined to the liver (Harada et al., 2005). Members of PI 3-kinase subclasses not only function in a tissue-specific manner, as discussed above, but also function differentially in different cellular compartments/organelles. One regulator of PI 3-kinase IA in the nucleus is PIKE, a nuclear GTPase that enhances PI 3-kinase activity in a GTP-dependent manner. PIKE is able to mediate its stimulatory effect on the PI 3-kinase/PKB signaling pathway (Ahn et al., 2004). PI 3-kinase IB is activated in a G-protein-dependent manner in both the nucleus and cytosol (Bacqueville et al., 2001). C2 has been identified in the trans-Golgi network, clathrin coated vesicles and nuclear speckles (Didichenko and Thelen, 2001; Domin et al., 2000), as shown in Table 1.1. C2  has been found to function in the nucleus (Sindic et al., 2001), with activity being correlated with the G2/M transition (Visnjic D, 2003). Although class II PI 3-kinases were speculated to be constitutively located in subcellular locations where they normally function (Domin et al., 2000), class II a and ß are also found to be recruited to the EGF receptor (Arcaro et al., 2000). Biochemical separation and studies examining in vivo PIP lipids in cellular organelles have been done primarily with the nucleus, mostly with PI(4,5)P2 and inositol phosphates  (York et al., 1994; Divecha, 1993; Irvine, 2003), but data related to D-3 PIPs in the nucleus have rarely been reported. Although people have achieved some understanding of PI 3-kinase signaling in the nucleus (Ahn et al., 2004; Deleris et al., 2003; Ye and Snyder, 2004), the biochemical roles of nuclear phosphoinositides are poorly understood. It is still difficult to understand how phosphoinositides locate to membrane-depleted nuclear structures. A  8 study showing that nuclear PIP is embedded in nuclear speckles through its fatty acid tail rather than its inositol head has made this situation even more complicated. PI 3-kinase activity and the Plecstrin homology (PH) domain of PDK1 are indispensable for PDK1 phosphorylation in the nucleus and nuclear shuttling (Scheid et al., 2005).  Therefore, it is clear that nuclear PI 3-kinase/PDK1/PKB pathway regulation will be an important research focus in the future. 1.1.5 The functions of PIP lipids in sub-cellular compartments It is also interesting to know how each individual PIP lipid functions differentially within different cellular compartments, including the plasma membrane, the endo- membrane system (endosome, trans-Golgi network) and nucleus. PI4P is involved in endomembrane transport and exocytosis (Matteis et al., 2006). PI(4,5)P2’s function in the nucleus has been well characterized (Irvine, 2003). Cytoplasmic ERK/MAPK activates PLCß1, which translocates into the nucleus and degrades PI(4,5)P2  into DAG and IP3. The nuclear DAG attracts and activates PKCa/PKBßII. The nuclear IP3 transforms into other forms of inositol phosphate, as well as mobilizes calcium. However the source of calcium is controversial. No direct study of nuclear PI(3,4,5)P3 and PI(3,4)P2 lipids has shown the specific functions of each D-3 PIP lipid in the nucleus, although the existence in that location has been proven (Watt et al., 2004). Meanwhile, in the cytoskeletal and endomembrane systems there has been some evidence for D-3 PIP lipids function, as discussed below.  PI(3,4,5)P3, PI(4,5)P2 and PI(3,4)P2 in cytoskeleton function.  9 Both PI(3,4,5)P3 and PI(4,5)P2 function in actin polymerization. However, they have different roles during this process. After neutrophils are stimulated with a chemoattractant, both PI(3,4,5)P3 and actin are observed at one pole of the cells with similar distribution and kinetics (Insall et al., 2001; Leslie et al., 2005). Meanwhile, PI(4,5)P2 remains evenly distributed throughout the membrane, without any significant quantitative change. The role of PI(3,4,5)P3 in actin polymerization is instructive because PI(3,4,5)P3 is not only sufficient to induce actin polymerization, it is also produced at the right time to instruct where and when actin polymerization should occur (Insall RH, 2001). Both PI 3-kinase (Puri et al., 2005) and PI 3-kinase (Sadhu et al., 2003) have been shown to be essential in neutrophil trafficking and to direct lymphocyte movement. The mechanism of PI(3,4,5)P3’s function in actin polymerization is likely to be mediated by one or more Rho family GTPases. Cdc42 and Rac are candidates, and both can be activated by the GEF Vav that has a PH domain that binds to PI(3,4,5)P3. GEFs for Rac, such as SOS and Pix, also have a similar ability to bind PI(3,4,5)P3. Phosphoinositide delivery experiments showed that both PI(3,4,5)P3 and PI(4,5)P2 can cause actin polymerization. Overexpression of metabolic enzymes that increase PI(4,5)P2 levels also showed similar effects. Sequestration of PI(4,5)P2 can impair actin polymerization. However, the mechanism of PI(4,5)P2 action during this process is definitely different from that of PI(3,4,5)P3. First, PI(4,5)P2 does not have a corresponding re-distribution after stimulation. Thus, PI(4,5)P2’s function is not instructive, although it is indispensable during actin polymerization.  PI(4,5)P2 is more likely to be a marker that is always on the plasma membrane (Insall RH, 2001). Second, being one of the abundant types of phosphoinositides within the plasma membrane, the  10 overall PI(4,5)P2 level after stimulation is relatively stable, which is distinctly different from the kinetics of PI(3,4,5)P3 (Gold and Aebersold, 1994; Marshall et al., 2002). PI(3,4)P2 has not attracted as much attention in cytoskeletal function as the two lipids listed above, but there is some evidence of its involvement. The PI(3,4)P2 specific binding protein TAPP has been shown to be localized in the actin-rich membrane ruffles in B cells (Marshall et al., 2002). A more detailed study by Hogan et al. (2004) showed that TAPP1 is involved in actin cytoskeletal rearrangement in NIH-3T3 cells. Since TAPP1/2 membrane recruitment is regulated by PI(3,4)P2, this suggests that PI(3,4)P2 plays some role in actin cytoskeleton rearrangement as well. In conclusion, all three lipids, PI(3,4,5)P3, PI(4,5)P2 and PI(3,4)P2, are involved in cytoskeleton rearrangement. More information will be required to know how each process is regulated by each lipid, how they function separately and how these functions are coordinated.   D-3 phosphoinositides produced in the endomembrane system. The endomembrane system is made up of endoplasmic reticulum (ER) and the Trans-Golgi Network. After stimulation with PDGF, and endocytosis of the PDGF receptor in CHO cells, PI(3,4,5)P3 was observed by FRET analysis to be produced in situ in the endomembrane system (Sato et al., 2003). This suggested that the signaling pathways downstream of PI(3,4,5)P3 are activated at intracellular compartments far away from the plasma membrane (Sato et al., 2003). Due to different PI 3-kinase subclasses’ distribution in sub-cellular compartments, it will be interesting to show, with similar approaches, the in situ cellular distribution of corresponding enzymes that are responsible  11 for this PI(3,4,5)P3 production. Due to differentially subcompartmentalized phosphoinositide metabolism, a “PI-fingerprint” of each cell membrane compartment has been proposed (De Matteis et al., 2005). Each cell membrane compartment is special in terms of phosphoinositide makeup and metabolism, which is closely linked to respective functions. However, the technical means to measure the precise levels of the PIPs in the different compartments is not yet available. 1.1.6 Phosphoinositides regulate downstream effectors through phosphoinositide binding domains A variety of protein domains bind phosphoinositides (Lemmon, 2003), including PH, PX (phox), FYVE, ANTH/ENTH and PHD. All of these, except PHD domains, can bind to one or more D-3 phosphoinositides (Table 1.3). PI(3,4,5)P3  has the largest number of identified binding partners, including PKB, Btk, Bam32, Vav, Sos, Gab, Grp1, PLC 1, ARNO, PDK1 and cytohesin1. The common concept that PI(3,4,5)P3 is the most important phosphoinositide second messenger is mostly due to the large number of identified effectors. While regulation is usually mediated by membrane recruitment, PDK1 is unique since it has not been observed to be recruited to the membrane upon stimulation. PDK1 is found to be a constitutively active kinase, both at the plasma membrane and in cytosol. However, PI 3-kinase activity and increased levels of PI(3,4,5)P3 may  still be  indispensable  to  anchor  PDK1 at  the  plasma  membrane (Anderson et al., 1998). Besides PH domains, another protein domain binding to PI(3,4,5)P3 and to PI(3,4)P2 is the PX domain (reviewed in Ellson et al., 2002). Both p47phox and PI 3-kinase  12 C2 also bind PI(3,4)P2 through their PX domains. Proteins with high affinity for PI(3,4)P2 include PKB, TAPP1/2 and Bam32, with PKB and Bam32 having dual affinity for both PI(3,4,5)P3 and PI(3,4)P2. Table 1. 3 Phosphoinositide binding domains Protein domain PIP specificity Representative Protein PH PI(3,4,5)P3 PKB, Btk, Bam32, Vav, Sos, Gab, Grp1, PLC 1, ARNO, PDK1, cytohesin1, centaurin (Hayashi et al., 2006) PI(3,4)P2  PKB, TAPP1/2, Bam32, PDK1 PI(4,5)P2  PLCd1, Dynamin 1, 2 PI(3,5)P2  Centaurin PI3P PLCß1 (Razzini et al., 2000) PI4P OSBP (Levine and Munro, 2002) FAPP1 (Dowler, 2000) PX PI(3,4,5)P3 CISK PI(3,4)P2  p47phox, PI 3-kinase C2 PI(3,5)P2  Sorting nexin-1, CISK PI3P p40phox, SNX 2, 5, 7, 13, Vam7p PI5P SNX13 FYVE PI3P EEA1, Hrs, Sara, Fens-1, Endofin PHD PI5P ING2 PI4P EpsinR PI(4,5)P2  Epsin1,2, AP180, CALM, HIP ENTH/ANTH PI(3,5)P2  Ent3p and Ent5p This is a list of phosphoinositide-binding protein domains and proteins. It is worth noting that most of the lipid-binding properties are proven by in vitro binding assays, which are most of the proposals of their in vivo regulations by lipid production are based on. Reviewed in Gozani et al., 2003; Razzini et al., 2000; Balla, 2005; Antonietta De Matteis et al., 2005; Cho and Stahelin, 2005; Overduin et al., 2001.  13 Different cells have different kinetics of PI(3,4,5)P3 and PI(3,4)P2 production in response to different agonists (Gold and Aebersold, 1994; Gray and Downes, 1999). In the stimulation of B cells, PI(3,4,5)P3  and PI(3,4)P2 actually initiate two waves of cell signaling by recruiting PH domain containing proteins such as Btk, Bam32 and TAPP (Krahn et al., 2004). Our knowledge of the signaling significance of PI(3,4)P2 was mostly acquired through studies of Bam32 and TAPP. Bam32 works as an adaptor protein upstream of the MAPK pathway (Han et al., 2003; Niiro et al., 2002). Another adaptor protein linking PI(3,4,5)P3 to MAPK is centaurin (Hayashi et al., 2006). Centaurin-a1 is known to be a PI(3,4,5)P3-binding protein that has two PH domains and a putative ADP ribosylation factor GTPase-activating protein domain. Centaurin-a1 contributes to ERK activation in growth factor signaling, linking the PI 3-kinase pathway to the ERK mitogen-activated protein kinase pathway through its ability to interact with PI(3,4,5)P3. TAPP2 expression enhances BCR-induced calcium flux and NF-AT activation (Krahn et al., 2004). In another study, TAPP showed negative effects by activating a protein phosphatase (Kimber et al., 2003). Thus it seems that TAPP has several distinct roles in mediating PI(3,4)P2 induced downstream signaling. In platelets, long lasting PI(3,4)P2 production was correlated with platelet aggregation (Trumel et al., 1999; Sultan et al., 1991). It is possible that this function may be related to TAPP due to the finding that TAPP is involved in actin cytoskeletal rearrangement in NIH-3T3 cells (Hogan et al., 2004) and colocalized in actin-rich membrane ruffles in B cells (Marshall et al., 2002). Proteins containing PX domains include p40phox and p47phox, both related to oxidative burst and phagosome maturation (Kanai et al., 2001). Another phosphoinositide binding  14 domain is the FYVE domain, which binds to PI3P (Burd and Emr, 1998). FYVE domain containing proteins are involved in vesicle trafficking (Balla, 2005). The identification of phosphoinositide binding domains has elucidated some of the key functions of phosphoinositides. However, the exact relationship between phosphoinositides and downstream proteins is not straightforward (Balla, 2005). For example, not all “phosphoinositide binding” domains have affinity for PIP lipids. Most of the PH and PX domains do not have affinity for PIP lipids at all, or just have low affinity for PIP lipids (Yu et al., 2004). This certainly elicits great interest and arguments about the functions of PH and PX domains, which will be discussed in detail in section 1.1.10. 1.1.7 PI 3-kinase activation mechanisms downstream of different receptors. PI 3-kinase can be activated downstream of a variety of agonists and receptors. Different mechanisms are responsible for signal transduction between receptors and PI 3- kinase. Receptor tyrosine kinase family members, including the PDGF receptor, c-Kit receptor and CSF-1 receptor, interact directly with the p85 regulatory subunit of PI 3- kinase.  These receptors can auto-phosphorylate intermolecularly on several tyrosines including the YXXM motif in the cytoplasmic tail. The phosphorylated YXXM motif then attracts the SH2 domain of the p85 subunit (Reedijk et al, 1992; Serve et al., 1994). Cytokine receptors lacking intrinsic enzyme activity, such as the IL-3, IL-5 and GM-CSF receptors, initiate signaling by activating Jak family tyrosine kinases, leading to the phosphorylation of the ß-common chain and recruitment of adaptor proteins, such as Shc and IRS-2. These proteins are subsequently phosphorylated at several tyrosines, including their YXXM consensus sequence, allowing p85 recruitment (Gu et al., 2000; Zamorano  15 et al., 1996). The common subunit of the IL-7 receptor contains a YXXM sequence in the cytoplasmic tail, which can be phosphorylated by Jak and then recruit p85 directly (Venkitaraman et al., 1994). Apart from Jak dependent activation, IL-7R induced PI 3- kinase activation can also be mediated by the YXXM containing protein IRS-2, as in the case of the IL-4R. Adaptor proteins Gab1, Gab2 and Gab3 also contain YXXM motifs and are involved in growth factor-induced PI 3-kinase recruitment. In insulin and IGF-I signaling, the docking proteins IRS-1/2 contain YXXM motifs and recruit PI 3-kinase class IA. In the case of B cells, the mechanism of upstream tyrosine phosphorylation is much more complex, involving the tyrosine phosphorylation of B cell receptor subunits, CD19 co-stimulatory receptor, tyrosine kinases Syk, Lyn, Fyn and Btk (Kurosaki, 2002). In mast cells the mechanism of Fc RI induced PI 3-kinase activation is relatively simple: p85 is recruited to the phosphorylated tyrosine in the receptor cytoplasmic tail after Fc RI dimerization (Wilson et al., 2002). 1.1.8 Down-regulation of the PI 3-kinase/PKB pathway. Inositol phosphatases: PTEN and SHIP The most well-known down-regulators of the PI 3-kinase pathway are SHIP and PTEN, both of which are inositol phosphatases using PI(3,4,5)P3 as substrate. PTEN mutations have been found in a broad range of cancers. PTEN is a D-3 phosphoinositide phosphatase, dephosphorylating PI(3,4,5)P3 and PI(3,4)P2 at the D-3 position. PTEN also has enzyme activity as a protein phosphatase (Leslie et al., 2002). Loss of the suppressive effect on the PI 3-kinase pathway by the PTEN tumor suppressor plays a key role in the tumorigenesis process, probably due to the up-regulation of  PI(3,4,5)P3 and PI(3,4)P2  16 levels and thus PKB activity, as well as cell cycle control through Cyclin D (Radu et al., 2003). High basal PKB phosphorylation and activity have been observed in a variety of PTEN deficient cells. However, in normal cells, the mechanism by which PTEN is recruited to its substrate and activated is still not fully understood. It is believed that PTEN may be anchored to the membranes by electrostatic forces (Das et al., 2003). However, how the regulation of this anchorage occurs is unclear. In migratory lymphocytes, the PTEN protein is restricted to the rear part of the cell, thus leaving accumulated PI(3,4,5)P3 on the front part of the cell (Comer and Parent, 2002; Wang et al., 2002). Meanwhile, controversial evidence suggests that PTEN influences cell migration regardless of PI(3,4,5)P3 (Leslie et al., 2005), indicating more complex roles for PTEN in regulating cell migration. Whether or not PTEN-regulated PKB activity simultaneously regulates migration is not clear. The in vitro substrates of PTEN include all D-3 phosphoinositides: PI(3,4,5)P3, PI(3,4)P2, PI(3,5)P2 and PI3P, but over-expression of PTEN had little effect on the level of the latter two (Leslie and Downes, 2002). SHIP dephosphorylates PI(3,4,5)P3 to yield PI(3,4)P2, as well as acting upon IP(1,3,4,5)P4 to generate IP(1,3,4)P3 (Rauh et al., 2003). Initially identified and named as an inositol phosphatase, SHIP has been found to also have important roles as an adaptor through its SH2 domain, proline rich domain and NPXY motif (Rohrschneider et al., 2000). The relative roles of PTEN and SHIP in regulating lipid levels will be discussed in more detail in Chapter 5. Other down-regulators: PP2A, staurosporin, ILK inhibitor and CTMP There are several ways to down-regulate PKB: eg., PP2A, staurosporin, ILK inhibitor and CTMP (Carboxyl-Terminal Modulator Protein). Previous results in our  17 laboratory showed that ceramide-induced PP2A activity is responsible for the dephosphorylation of the Ser473 site and inhibition of PKB (Schubert et al., 2000). Staurosporine works by inhibiting PDK1 (Hill et al., 2001). Special interest is placed on the ILK inhibitor because this inhibitor can specifically inhibit the phosphorylation on Ser473. Biochemical data showed that this inhibitor has higher specificity for ILK than other kinases (Yoganathan et al., 2000). However, due to the uncertainty of the Ser473 phosphorylation mechanism, the precise action of this inhibitor is still unclear. Whether this ILK inhibitor works on some other ILK associated protein or even influences upsteam phosphoinositide levels will be interesting to elucidate. Other negative regulators of PKB include PKB associated proteins, such as CTMP (Maira et al., 2001). CTMP binds specifically to the carboxyl-terminal regulatory domain of PKBa at the plasma membrane. Binding of CTMP reduces the activity of PKB by inhibiting phosphorylation on Serine 473 and Threonine 308. It is possible that this level of regulation might be coordinated with phosphoinositides. 1.1.9 PIP lipids co-coordinate the PDK1-PKB pathway. Both PDK1 and PKB belong to the same kinase family, the AGC family, which has some common structural characteristics: a phosphorylation site in the activation loop and another phosphorylation site in the hydrophobic motif (HM) on the carboxyl terminus. The AGC kinase family includes PKB, PDK1, S6K, SGK, MSK and PRK (Frodin et al., 2002). Some members of the AGC family kinases, such as the PKC related kinase-1 (PRK1/PKN), possess a negatively charged acidic residue (Asp or Glu), instead of a serine/threonine phosphorylation site in the hydrophobic motif at the C-teriminus.  18 PDK1 is the kinase responsible for the phosphorylation of the activation loop site of AGC family kinases. Although PKB Ser473 phosphorylation is indispensable for PI 3-kinase activity, which is indicated by LY294002 (LY) inhibition, not all the phosphorylation of AGC family kinases is inhibited by LY. Some other AGC kinase family members such as RSK and MSK are effectors of ERK and p38mitogen-activated protein (MAP) kinases, respectively. Thus, HM site phosphorylation mechanisms in the various AGC family kinases are different.  The activation mechanisms of AGC kinases are divided into common and divergent mechanisms (Frodin et al., 2002), which have explained the differential phosphorylation mechanisms of AGC kinase HM sites. PDK1 is activated after the binding of the phosphate pocket in PDK1’s own kinase domain to its substrate’s HM phosphorylation sites. In the case of  AGC family members such as RSK, S6K or SGK, their HM kinases are located in the cytosol. Then PDK1 can perform its kinase function without being recruited to the plasma membrane (Frodin et al., 2002). In the case of PKB, it seems that the PKB HM site kinase is located at the plasma membrane, where PKB is phosphorylated at Ser473. Thus, PDK1 needs to perform its activation loop site (Thr308) kinase function at the plasma membrane by binding phosphorylated Ser473 within its phosphorylation pocket. PIP lipids have the function of locating PDK1 and PKB together on the membrane. Another study by Hill, et al (2002) supported this idea that Ser473 kinase is located at the plasma membrane. Data in this thesis also strongly support the conclusion that the Ser473 kinase is located at the plasma membrane. However, the exact function of PI(3,4)P2 or PI(3,4,5)P3 in the regulatory events has not been conclusively demonstrated. Results in Chapter 5 of this thesis will address these questions.  19 1.1.10 Protein-protein interactions downstream of PI(3,4,5)P3 result in intricate regulation of PIP downstream pathways. The PH domain is the 11th most abundant protein domain in the human genome and most proteins with PH domains do not appear to have affinity for phosphoinositide lipids at all, or just have very low affinity, and bind to other proteins instead (Yu et al., 2004). Except in a few circumstances, most of the phosphoinositide-protein binding is not highly specific (Kavran et al., 1998). Upon increased phosphoinositide production, only specific downstream signaling pathways are activated. It is reasonable to speculate that activation of protein effectors is regulated by some mechanism that enhances or abolishes membrane recruitment/detachment, besides precisely controlled phosphoinositide production. It is also possible that associated proteins will modify the effector proteins’ activity. Given the fact that phosphoinositides regulate many cellular processes at the same time, it can be speculated that different downstream signaling pathways are precisely modified by protein partners, along with spatially regulated phosphoinositide production. Protein binding characteristics of PH domains are becoming more important with accumulating evidence in the case of PI(3,4,5)P3-directed signaling (Varnai et al., 2005). In the study of Varnai et al., overexpression of different PH domains was found to differentially influence downstream signaling pathways, suggesting that PI(3,4,5)P3/PI(3,4)P2 binding properties are not the only factors that determine PH domain-containing proteins’ membrane activation and downstream signaling. The reasons could be that PH domain binding to PI lipids are affected by PH domain binding to other proteins, or conversely, the binding partners of the PH-containing protein can  20 affect the interaction of the PH domain with lipids. More evidence exists in the case of GTPase proteins (Kim et al., 2002a; Jaffe et al., 2004) and PIP kinase recruitment (Godi et al., 2004), showing that these PH domain-containing proteins have important protein- protein interactions at the membrane. However, it is not yet clear how PI lipid production, via effects on the PH domains, may be affecting these protein-protein interactions. The PKB PH domain was found to be the structural basis for PKB association with the cytoskeleton (van den Heuvel et al., 2002; Cenni V, 2003) as well as PKB dimer formation (Datta et al., 1995; Gold, 2003) and other protein associations (Du and Tsichlis, 2005). With more evidence suggesting that PH domain-containing proteins interact with other membrane proteins in addition to PIP lipids, identification of these protein associations at the plasma membrane in future studies will be important to obtain a complete understanding of phosphoinositide-directed signaling. The field of signal transduction research aims to elucidate not only how specific events can be stimulated by specific agonists, but also the cross-talk among signal cascades and how signaling specificity is achieved. Since PH domains not only interact with phosphoinositides, but also with proteins, this causes a competition between PIs and proteins at the membrane. In addition, PI(3,4,5)P3 interacting proteins Btk, PLC , Gab1, Gab2 and Vav also contain other protein interacting domains to form a larger “signalosome” to fulfill the cross-talk function. Btk contains an N-terminal PH domain, SH2, SH3 domains, kinase domain and a Tec homology (TH) domain, which allow Btk to interact with over 20 proteins identified so far (Qiu et al., 2000). Vav and PLC also contain a PH domain, SH2 domain, SH3 domain and tyrosine phosphorylation sites. Gab contains a PH domain, proline rich domain and tyrosine phosphorylation sites. The exact  21 functions of most of these interactions are still unknown, especially how regulation is achieved by PH domains’ interaction with PIs. For example, some Btk-mediated downstream pathways have been attributed to PI 3-kinase activity since they are inhibited by the specific PI 3-kinase inhibitors LY294002 or wortmannin. However, a controversial study has also shown that Btk signaling might not be PI 3-kinase related (Suzuki et al., 2003). Together these studies suggest that protein interactions with the PH domain of Btk PH can also play a key role in Btk signaling. SHIP does not contain a PH domain, but it can also be involved in signalling crosstalk due to its regulation of PI levels as well as its interaction with other proteins that regulate different pathways. SHIP contains an SH2 domain, a proline-rich domain and an NPXY motif (Y is the tyrosine phosphorylation site). Thus, SHIP’s role as an inositol phosphatase and its protein-binding adaptor functions might be functioning in the midst of a crosstalk network. SHIP was cloned on the basis of its association with Shc, an adaptor protein that also functions in a complex with Grb2 and Sos to activate Ras. Upon recruitment to ITIM/ITAM motifs (detailed discussion in Chapter 4) to dephosphorylate PI(3,4,5)P3 to PI(3,4)P2, the SH2 domain of SHIP is no longer bound to Shc, allowing Grb2 to bind to Shc and resulting in activation of the Ras pathway. Also, with SHIP activation, its NPXY site is tyrosine phosphorylated and associates with the PTB domain on Dok. Dok is an adaptor protein associated with Ras-GAP, which is a GTPase activating protein and negatively regulates Ras. Therefore SHIP membrane recruitment causes negative effects on both the PI 3-kinase and Ras pathways by association with Shc and Dok through protein interacting domains (Robson et al., 2004; Abramson and Pecht, 2002). In Chapter 4 of this dissertation, SHIP is described as having activity downstream  22 of B cell receptor ITAM and ITIM motifs to regulate PI production. We can also speculate that SHIP protein associations may change correspondingly under those ITAM/ITIM activation conditions. Some conflicting results must be mentioned regarding the roles of SHIP in the regulation of PKB activity and cell survival. SHIP and another 5- phosphatase have been shown to promote apoptosis (Kisseleva et al., 2002; Valderrama- Carvajal et al., 2002). Conversely, SHIP has also been shown to attenuate Fc RIIB induced apoptosis (Pearse et al., 1999). SHIP’s dual functions as an adaptor protein and a PI 5-phosphatase may have opposing effects that can modify its downstream effects, depending upon the context of the studies. 1.1.11 PKB Ser473 kinases and significance of the mTOR pathway. The discussion and investigation of the potential kinase that phosphorylates PKB at the Ser473 site have been of interest for more than ten years. Initially, PDK1 and MAPKK were the candidates (Vanhaesebroeck, 2000). Then, ILK was proposed (Delcommenne et al., 1998). However, none of these has received unanimous recognition as the Ser473 kinase. Recently, different Ser473 kinases were identified in different cell types, i.e. DNA PK in HEK293 cells (Feng et al., 2004), PKC beta II in mast cells (Kawakami et al., 2004), and PKC alpha in endothelial cells (Simons, 2004). The most recent data suggested that mTOR (mammalian target of rapamycin), may serve as the Ser473 kinase (Sarbassov et al., 2005; Jacinto et al., 2006; Polak et al., 2006). TOR is a conserved Ser/Thr kinase that regulates cell growth and metabolism. TOR is part of two distinct multiprotein complexes, TOR complex 1 (TORC1), which is sensitive to rapamycin, and TORC2, which is not. MTORC1 specifically contains raptor,  23 while mTORC2 specifically contains rictor and mSIN1. Ser473 phosphorylation is not sensitive to acute rapamycin treatment, and thus mTOR has not previously been considered as the Ser473 kinase. TORC2, which is the rictor-mTOR complex, has been shown to phosphorylate Ser473 both in vivo and in vitro. The mechanism of mTORC2 activating PKB is still elusive. PKB activates mTORC1, thus mTORC2 could indirectly activate mTORC1. However, knockdown of mTORC2 does not affect the mTORC1 effector S6K1, suggesting that mTORC2 activates a pool of PKB that is not upstream of mTORC1 (Sarbassov et al., 2004).  This speculation is supported by a more recent study showing that the rictor-mTOR complex regulates PKB activity and specificity by means of affecting a subset of PKB targets in vivo, including FOXO1/3a, while other PKB targets, TSC2 and GSK3, and the TORC1 effectors, S6K and 4E-BP1, are unaffected (Jacinto et al., 2006). 1.1.12 PKB membrane detachment: an important step in PKB function One of the most recent advances in the study of PKB is the finding that PKB membrane detachment is an important step in regulating PKB activity (Ananthanarayanan et al., 2007; Kunkel et al., 2005; Dong et al., 2007).  This conclusion is mainly drawn from the usage of FRET and observation of the kinetics of the active form of PKB. The mechanism of this detachment is suggested by Dong et al. (2001), which showed that the PH domain of PKB is associated with calmodulin near the plasma membrane, thus interrupting the association of PKB with PIs. The release of PKB from the plasma membrane is thus an active, rather than a passive process that occurs with the diminished PI levels on plasma membranes. As a matter of fact, FRET has been utilized to observe an active form of PKB that is released from plasma membranes and present in  24 the nucleus, even though the membrane phosphoinositide level is still high (Kunkel et al, 2005). A recent study by Zhang’s group suggested that PKB Thr308 phosphorylation could be a factor affecting PKB membrane association (Ananthanarayanan et al., 2007). 1.1.13 Diseases related to abnormal phosphoinositide metabolism and therapeutic intervention of PI 3-kinase inhibitors in the treatment of common diseases. Enzymes involved in PI metabolism are involved in a number of diseases (Table 1.4). Underlying mechanisms for the malfunction of such enzymes are divergent, including point mutations, gene amplification, overexpression or increased phosphorylation. It is worth noting that most of the abnormalities have been identified genetically, and detailed mechanisms of those mutation leading to oncogenesis from a signal transduction perspective, are still largely unknown. Disruptions of different parts of the PI 3-kinase pathway induce divergent syndromes.  Recent identification of a large number of cancers that have mutations in the p110 gene, PIK3CA, suggested that drugs targeted to this protein could be potential treatments for those cancers (Bader et al., 2005). Specific inhibitors targeted to PI3K  and d have been developed (Stephens et al., 2005; Ward et al., 2003), which have been shown to be effective in blocking the temporal activation of these two inositol kinases (Condliffe et al., 2005) and to have encouraging therapeutic effects in mouse models of rheumatoid arthritis (Camps et al., 2005). A SHIP activator has also been examined and has anti-inflammatory properties (Ong et al., 2007) (Dr. Alice Mui, personal communication). Studies of specific cellular and molecular pathophysiological mechanisms regulating individual cancers will be important for determining the exact role of PI 3-kinase in cancer development. For example, over 50  25 point mutations in the PIK3CA gene are related to cancer, and it is possible that all these mutations may not lead to the same abnormal signaling events. An ultimate objective is to develop corresponding drugs fitted to individual oncogenic mechanisms of such cancers: i.e., to target the production of specific lipids and/or the specific cellular compartment under specific circumstances or in specific cell types. Abnormalities in the p110 subunit of PI 3-kinase has also affected functions in myocardiac cells, vascular smooth muscle cells and macrophages, resulting in cardiac hypertrophy, heart failure and atherosclerosis (McMullen et al., 2007; Chang et al., 2007). A more detailed list of human diseases linked to abnormal PI3K pathway signaling is shown in Table 1.4.  26 Table 1. 4 Diseases related to abnormal phosphoinositide metabolism enzymes Gene Abnormality  References p110 Cancers Cardiohypertrophy Atherosclerosis (Samuels et al., 2004) Review in (Bader et al., 2005) (McMullen et al., 2007) (Shioi et al., 2000) (Chang et al., 2007) p85 Cancers (Jimenez et al., 1998) Review in (Bader et al., 2005) PTEN Cancers Cancer predisposition syndrome: (Cowden disease Bannayan-Zonana syndrome, etc) Neuron hypertrophy and behavior changes Skeletal abnormality (Mehenni et al., 2005) Review in (Sulis and Parsons, 2003) (Kwon et al., 2006) (Greer et al., 2006) (Liu et al., 2007) SHIP1 Inhibitory mutation associated with a case of AML and chemotherapy resistance Potential involvement in Paget’s disease? Review in (Rohrschneider et al., 2000) SHIP2 Type 2 diabetes (Clement et al., 2001) (Marion et al., 2002) MTM1 X-linked myotubular myopathy (Kim et al., 2002b) MTMR2 Charcot-Marie-Tooth disease 4B1 (Kim et al., 2002b) OCRL1 Lowe syndrome (Sharon F.  Suchy, 2002) An increasing numbers of diseases are linked to PI 3-kinase pathway. PTEN, SHIP, MTM, MTMR and OCRL are phosphoinositide phosphatases. Besides cancer, PTEN mutations also cause abnormalities in the functions of central nervous system and skeletal  27 muscular development and metabolism. The other phosphoinositide phosphatases are linked to different syndromes manifested mainly in skeletomuscular system in Paget’s disease, X-linked myotubular myopathy and Charcot-Marie-Tooth disease. Sporadic cases of leukemia and type 2 diabtes were reported to be linked to SHIP. Specific PI 3-kinase inhibitors have also shown encouraging effects in ischemic heart disease (Doukas et al., 2006), which is the number 1 killer in most developed countries. In Doukas et al.’s study, a PI 3-kinase /  inhibitor has been shown to limit myocardial infarct development in an infarction/reperfusion model. Surprisingly, they showed that this beneficial effect happens several hours after reperfusion. This is therapeutically important, because one major limitation in anti-ischemic therapies is the requirement of early delivery.  Current guidelines for thrombolytic therapy is that it be administered within 12 hours after a clot forms in the coronary artery, however many patients do not have access to medical care during this period. The therapeutic effects of this / inhibitor is surprising because PI 3-kinase pathways are considered beneficial events that should not be disrupted. This is the reason that general PI 3-kinase inhibitors do not help in reversing the lesion from ischemia/reperfusion. The important result in this study may be due to two reasons. One is that the inhibitor is unique: rather than broadly inhibiting both  and , it is a relatively small molecule more specifically targeted to PI 3- kinase . The second is that the drug is delivered after reperfusion, thus it will not disrupt PI 3-kinase’s prosurvival effect during infarction. Meanwhile, it should also be noted that PI 3-kinase and PI 3-kinase are characterized as proinflammatory (or anti-tissue survival) during the inflammatory process, which is also the mechanism of action in the rheumatoid arthritis model (Camps et al., 2005).  This anti-inflammatory/anti-edema  28 effect is also beneficial in ameliorating the ischemic tissue lesion. Besides, endothelial cell mitogenesis, a repair process important to tissue survival after ischemic damage, was not disrupted. All these results have shown that if given by an appropriate route (i.e. intravenous) at an appropriate period (post-reperfusion), PI 3-kinase inhibition can be a very promising therapy for myocardial infarction treatment. Asthma and COPD (chronic obstructive pulmonary disease) are also major debilitating and lethal diseases that PI 3-kinase inhibitors can target. The current long- term control of the chronic forms of these diseases include: steroids for controlling inflammation; mast cell stabilizer for prophylaxis; and leukotriene modifiers for reduction of leukotrine production. Steroids are the only first line drug in this group, but many of these patients become insensitive to steroids after long-term usage. Given the anti-inflammatory effect of PI 3-kinase and inhibitors, the outcomes of drug development in these diseases are eagerly awaited.  Besides interrupting the expression and activation of inflammatory pathways, PI 3-kinase and inhibitors also modify inflammatory cell recruitment and immune cell functions, which is one of the major mechanisms in the pathophysiology of chronic respiratory inflammation. Thus, it was not surprising when a study by Lee et al. (2006) showed that a PI 3-kinase inhibitor attenuated allergic airway inflammation and hyperresponsiveness in an animal model of asthma. PI 3-kinase has also been shown to be important in the development of asthma (Wymann et al., 2003). New PI 3-kinase inhibitors were recently reported based on the structural design and X-ray crystallography of complexes formed by inhibitors bound to PI 3-kinase  (Pomel et al., 2006). To date, there are no publications showing the effects of these PI 3-kinase  inhibitors on inflammatory respiratory diseases.  29 1.1.14  Approaches to study phosphoinositides  Delivery of phosphoinositides Delivery of PIPs into cells is the most straightforward way to study the functions of one particular phosphoinositide. Although some labs including ours have had a hard time in reproducing these experiments, many successful experiments have also been reported (Ishiki et al., 2005). Factors such as the cells’ health due to the delivery vehicle and the inability to incorporate those PIs are the main difficulties. Better delivery systems still need to be developed to make such experiments more reliable.  Phosphoinositide antibodies Numerous autoimmune diseases including anti-phospholipids syndrome have been well characterized clinically (Font et al., 1991). It is therefore reasonable to develop antibodies targeted to specific phospholipids in the lab. Antibodies to almost all the PIP lipids have been developed, and cytochemistry, immunohistochemistry and flow cytometry approaches have been carried out based on such antibodies (Chen et al., 2002; Niswender et al., 2003). With FITC-conjugated PI(3,4,5)P3 antibody, the PI(3,4,5)P3 peak can be detected by flow-cytometry after stimulation. The sensitivity is claimed to be three orders of magnitude higher for binding PI(3,4,5)P3 than PI(4,5)P2 (Chen et al., 2002). However, the ability to differentiate PI(3,4,5)P3 from PI(3,4)P2 is not that dramatic, which has been the biggest concern when those antibodies are utilized.  30  In vitro biochemical study So far the quantification and functional study of PI5P still largely relies on the in vitro kinase assay using PIP4-kinase to evaluate the relative PI5P level (Gozani et al., 2003). Although the analysis of PI5P was done using HPLC by Cantley’s group  (Rameh et al., 1997-1), this study is the only one that has shown the in vivo PI5P levels, to the best of our knowledge. From our own experience in B cells, the PI5P peak is very low and is easily masked by the more abundant PI4P, but it is feasible to detect. Due to the uncertainty of PI5P’s functions, it is still unknown what stimulation condition in B cells can induce the PI5P peak.  In vivo cell labeling This is the traditional way to study PIs and still the most reliable approach (Gold et al., 1994) Pioneering work to show the rapid fluctuation in overall levels of PIs have yielded a good understanding of how PIs regulate cell signaling. The shortcoming is the lack of sensitivity, since tens of millions of cells are required. A detailed method is described in Chapter 2 Materials and Methods: Lipid labeling, extraction and HPLC separation Phosphoinositides protein overlay assay This is a convenient way to identify the PI-binding properties of a new protein, as shown for ING2 (Gozani et al., 2003). This method enables the identification of the lipid ligands with which lipid binding proteins interact. This assay also provides qualitative  31 information on the relative affinity with which a protein binds to a lipid. In the assay, serial dilutions of different lipids are spotted onto a nitrocellulose membrane to which they attach. These membranes are then incubated with a lipid binding protein possessing an epitope tag. The membranes are washed and the protein, still bound to the membrane by virtue of its interaction with lipid(s), is detected by immunoblotting with an antibody recognizing the epitope tag. On the basis of this protein-lipid binding assay, more in vitro enzyme assays containing the corresponding PI can be performed to study the biochemical properties of the new protein. Based on the PI binding proteins found so far, this in vitro approach has been shown to correlate well with the in vivo binding character. GFP fusion proteins GFP-PH fusion proteins are indirect markers for the production and re- distribution of the PIs and remain the most accurate means of detecting changes in membrane PI’s in live cells (Gray et al., 1999; Marshall et al., 2002). However, the kinetics of the fusion PH domain movement cannot precisely mimic that of the whole protein, for the simple reason that the PH domain may not form protein complexes that the intact protein may form, and which will have an important influence on the recruitment and detachment of proteins. Fluorescence Resonance Energy Transfer (FRET) This is a unique means of studying the interaction of the PI with protein in situ. In a study by Sato et al., a membrane-targeted construct was designed, composed of CFP  32 and YFP, with a PH domain in the middle of the two fluorescent proteins. PH domain binding to specific PIs serve as an on-off switch for observing specific colors in a single cell, which provide a unique way to observe the location and kinetics of in vivo PI production (Sato et al., 2003). This technique has also been adapted to monitor the kinetics of active forms of PKB by observing its dynamic migration in different cellular subsets (Kunkel et al., 2005; Ananthanarayanan et al., 2007). Genetic dissection of PI pathways To understand D-3 PI signaling pathways (rather than the PI molecules themselves) during hematopoietic cells’ development and differentiation, a series of knockout animal models have been made. P110 and p110 knockout are lethal at the embryonic stage (Fruman, 2004; Vanhaesebroeck, 2005). p110 and p110 are expressed mainly in hematopoietic cells and show profound developmental defects (Fruman, 2004; Vanhaesebroeck, 2005). Combined with data from human diseases in which alterations in the PI 3-kinase pathway are implicated (Table 1.4), these genetic dissection data reveal a more detailed scenario. There is also the potential that the genetic data from animal models will reveal much more about the underlying human diseases. For example, mice deficient in Pten in osteoblasts were of normal size but demonstrated a dramatic and progressively increasing bone mineral density throughout life (Liu et al., 2007), which suggests a critical role for this tumor-suppressor gene in regulating osteoblast lifespan and thus can likely explain the skeletal abnormalities in patients carrying germ-line mutations of PTEN.  33 1.2 Hypothesis and strategies Figure1.3 Hypothesis The basic hypothesis driving the work presented in this thesis is that as one of the major products of PI 3-kinase, PI(3,4)P2 has functions in cell signaling in its own right. With the cloning of TAPP1/2 by our colleagues, which were shown to selectively bind PI(3,4)P2 (Marshall et al., 2001), we were curious about how this new PH domain- containing protein could function in signal transduction. With previous work in our laboratory showing that the level of PI(3,4)P2 is related to PKB Ser473 phosphorylation in bone marrow mast cells, our primary question was the potential function of TAPP1/2 in linking PI(3,4)P2 to PKB Ser473 phosphorylation in B cells. Although not providing any obvious link between TAPP1/2 recruitment and PKB Ser473 phosphorylation, these SHIP PI(3,4)P2 PTEN PI(3,4,5)P3 PTEN PI3K PI3K  PI 3-kinase products Btk Bam32 PKB TAPP 308P PI(4)P PI(4,5)P2 473P  34 initial studies provided biochemical proof of TAPP1/2 PH domain binding to PI(3,4)P2 specifically. Following the initial study described in Chapter 3, I was interested in the regulation of other PH domain-containing proteins by PIs, as well as regulation of PKB phosphorylation and membrane recruitment. Two major strategies were adopted. One set of studies investigated the dose response of B cell receptor (BCR) to different forms of ligands, intact anti-IgM and F(ab')2 portions of  the same antibodies. This was the focus of the studies described in Chapter 4. Second, in Chapter 5, I described how plasma membrane-targeted SHIP over-expression was used to modulate levels of PIPs as another means of investigation to study downstream signaling, including effects upon PKB, TAPP1/2, Bam32 and Btk.  I utilized B cells as a model system due to the fact that the PI metabolizing enzymes, such as PI 3-kinase and SHIP, have been well described in B cells.  However, there are many unanswered questions regarding the precise regulation of this signaling network. I felt that it was necessary to clarify some of the key questions, such as the mostly ignored functions of PI(3,4)P2 in the process of SHIP recruitment, and PI(3,4,5)P3/PI(3,4)P2’s functions in determining PKB Thr308 and Ser473 phosphorylation. I pursued my studies using PI analysis in the B cell model system. I hope that these studies will also lead to more investigations of  PI signaling in other cell types.  35 Chapter 2. Materials and Methods. Reagents and Antibodies Goat anti-human IgM F(ab')2 and intact goat anti-human IgM,  rabbit anti-mouse IgG F(ab')2 and intact rabbit anti-mouse IgG, and Cy5 conjugated mouse anti-rabbit light chain specific antibodies were purchased from Jackson ImmunoResearch. PKB antibody for immunoprecipitation, 4G10 antibody against phosphorylated tyrosine, and histone H2B for PKB kinase assay were purchased from Upstate. PKB phospho-Ser473 antibody and PKB phospho-Thr308 antibody were from Cell Signaling Technologies. PKB antibody for immunoblotting was purchased from Stressgen (Victoria, British Columbia, Canada). GSK-3ß phospho-Ser9 was from BD Pharmingen. 2.4G2 antibody for in vivo block of Fc RIIB (without azide) was from BD Pharmingen. Anti-Flag M2 affinity gel was from Sigma. Cell lines, Constructs and transfection BJAB cell line is derived from human B lymphoma; whereas A20 and IIA1.6 cell lines are mouse B cell lines. BJAB cells and A20 cells were cultured in RPMI 1640 medium supplemented with 10% FCS, antibiotics, 50 µM 2-mercaptoethanol (A20 cells only). IIA1.6 cells (kindly provided by Dr. Mark Coggeshall, Oklahoma Health Foundation, Oklahoma City) were cultured under the same conditions as A20 cells. Membrane SHIP-expressing BJAB cells were provided by Dr. Aaron Marshall (University of Manitoba, Winnipeg, Canada). Human PKB wildtype and Ser473 Asp mutant in FLAG-CMV2 transient expression vectors were kind gifts from Dr. Takashi Tsuruo (Institute of Cellular and Molecular Bioscience, University of Tokyo, Japan). The  36 FLAG-PKB expression vector was cut by EcoRI and the released Flag-PKB was re- cloned into the stable expression vector 3×flag-CMV10 vector (Sigma). Plasmids were linearized with BamH1 and purified with QiaEx (Qiagen). Cells were fed with fresh medium the day before transfection. Ten million cells were resuspended in 0.4 ml pre- chilled complete medium and mixed with 20 µg DNA in an electroporation cuvette, which was then incubated on ice for 10-20 minutes. Electroporation was done with a BioRad Gene Pulser, under the condition of 260 V and 950 µF. The cuvette was kept on ice for 5-10 minutes, and cells were transferred to a 10 cm diameter petri dish. After 24 hours, G418 was added to the medium at a concentration of 2 mg/ml. After selection for 7 to 10 days, cells were plated in 96-well plates at a concentration of 0.3 cells/well with medium containing G418 (1.5 mg/ml). Two weeks later, clones were checked for FLAG and PKB expression levels by immunoblotting. The EGFP-PH domain transient transfection and confocal microscopic study of EGFP-PH domain membrane recruitment were done in Dr. Aaron Marshall’s laboratory, as described in Krahn et al. (2004). B cell stimulation, preparation of cell lysates and immunoblotting To reduce the basal signaling caused by the components in serum, cells were starved in RPMI 1640 with 1% serum for 16 hours. After washing three times with RPMI 1640 containing 25 mM Hepes, pH 7.4, cells were re-suspended at 107 cells/ml and warmed at 37oC for 20 minutes. BJAB cells were stimulated with goat anti-human IgM F(ab')2 or intact goat anti-human IgM at the specified time points, and for A20 cells, rabbit anti-mouse F(ab')2 and intact antibodies were used. For 2.4G2 block experiments,  37 10 µg/ml 2.4G2 antibody was pre-incubated with B cells for 15 minutes before BCR stimulation. Reactions were terminated by pelleting the cells for 10 seconds and immediately lysing in solubilization buffer (2% Nonidet P-40, 100 mM NaCl, 10 mM NaF, 0.2 mM sodium orthovanadate, 10% glycerol, 40 µg/ml phenylmethylsulfonyl fluoride, 1 mM sodium molybdate, 1:1000 protease inhibitors cocktail (Sigma), 1 µg/ml microcystin-LR). After cell lysates were centrifuged at 15,000 rpm for 1 minute to pellet the nuclei, the supernatants were used for immunoblotting. Cell extracts were separated on SDS-PAGE and then transferred onto nitrocellulose membranes. The membrane was blocked with 5% skim milk in TBST and then incubated in primary antibody at 4oC overnight. Primary antibodies were dissolved in TBST/1% BSA/0.02% NaN3. The membrane was then washed with TBS and TBST for 30 minutes and incubated with appropriate HRP-conjugated secondary antibody for 1 hour at room temperature. Immuno-reactive bands were visualized with ECL reagent (Amersham Pharmacia Biotech, Baie d’Urfe, Quebec, Canada) or SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology, Rockford, IL) and images captured digitally using a FluorChem™ Imaging System (Alpha Innotech, San Leandro, CA) To re-probe the membranes to verify equal loading of cell fractions, membranes were incubated in stripping buffer (62.5 mM Tris-HCL, pH 6.8, 2% SDS, 100 mM ß-mercaptoethanol) for 30 minutes at 50oC, and re-blocked and re-probed with appropriate antibody as above. Crude plasma membrane and cytosol preparation Preparation of crude plasma membrane was performed as described (Scheid et al., 2002a) with minor modification. Briefly, cells were pelleted and the medium was  38 removed after stimulation with 10 µg/ml F(ab')2 or intact antibody at specific time points. The cells were lysed in ice-cold hypotonic lysis buffer (10 mM Hepes (pH 7.4), 1 mM EDTA, 40 µg/ml phenylmethylsulfonyl fluoride, 1 mM sodium molybdate, 1:1000 protease inhibitor cocktail, 0.2 mM Na orthovanadate) for 30 minutes. Cell nuclei and unbroken cells were spun down at 2000 × g for 15 minutes at 4oC. The supernatant was centrifuged at 100,000 × g in a TL-100 ultracentrifuge (Beckman) for 15 minutes to pellet the membrane fraction. The supernatant was collected as the cytosol preparation (1 107 cell equivalent/ml). The pellet was resuspended at 1 108 cell equivalents/ml of hypotonic lysis buffer containing 1% Nonidet P-40 by pipetting and then sonicating for 30 seconds. The samples of cytosol or membrane were subject to immunoblotting as described above. Lipid labeling, extraction and HPLC separation For the studies described in Chapter 4, cells were labeled with [3H] inositol.  Cells were starved in inositol free DMEM medium (ICN) with 10% dialysed FBS for 48 hours (Gray et al., 1999). Cells were then labeled with myo-inositol at 10-40 µCi/ml for 48 to 72 hours during which time the cells remained healthy by appearance. After being washed and transferred to fresh medium, cells were stimulated at the indicated time points and reaction was stopped by addition of 2.7M HCl. Lipids were extracted in methanol/chloroform and deacylated by methylamine reagents (Scheid et al., 2002a). Samples were analyzed by separation over a SAX 10 HPLC column (Whatman, Clifton, NJ). Inositol(1,3,4,5)P4, inositol(1,4,5)P3 and de-acylated phosphatidylinositol (PI) (Perkin Elmer, Boston, MA) were used as standards.  39 For the studies in Chapter 3 and 5, [32P]-orthophosphate labeling was used. Cells were starved of phosphate in 99% phosphate free RPMI 1640 medium for one hour at 37oC, re-suspended at 107/ml in phosphate free RPMI1640 medium and labeled by addition of 0.5 mCi carrier-free [32P]-orthophosphate (ICN, Costa Mesa, CA) for 1.5 hours at 37oC.  After being washed and transferred to fresh medium, cells were stimulated at the indicated time points and reaction was stopped by addition of 2.7 M HCl. Lipid extraction and HPLC (Waters) separation procedures were the same as for inositol labeling. Flow cytometry to measure antibody binding to B cell receptors Flow cytometry was utilized to measure the binding of antibody and F(ab’)2 fragment to the B cell receptor. A20 cells were incubated with rabbit F(ab’)2 or intact antibodies at 4oC for the various time points. Unbound antibodies were washed off with FACS buffer (PBS and 2% FCS) at indicated time courses and cells were kept on ice for 1 hour to minimize receptor internalization. 2% mouse serum was used for blocking for 15 minutes on ice. The detection antibody, Cy5 conjugated mouse anti-rabbit light chain specific (Jackson ImmunoResearch), was then added for 1 hour at 4oC. Samples were washed with FACS buffer again before being re-suspended in 400 µl FACS buffer and analyzed by flow cytometry. B cells only and B cells with cross-linking antibody only were used as controls.  40 PKB kinase assay Cell lysates were incubated with PKB antibody for 1 hour at 4oC. The antibody was captured by mixing with 20 µl Protein G Sepharose beads at 4oC for 1 hour. Beads were washed three times with fresh solubilization buffer containing 500 mM NaCl and one time with kinase assay buffer (20 mM MOPS, 5 mM EGTA, 2 mM EDTA, 20 mM MgCl2, 250 µM DTT, 5 µM -methyl aspartic acid, 25 mM -glycerophosphate, 1:1000 protease inhibitor cocktail, 40 µg/ml phenylmethylsulfonyl fluoride, 1 mM sodium molybdate, 0.2 mM Na orthovanadate, 1 µg/ml microcystin-LR). Beads were re- suspended in 30ul kinase assay buffer with 3 µg Histone H2B as substrate. 10 µl ATP solution containing 200 µM ATP, 5 µCi [32P]-ATP, 75 mM MgCl2 was added, followed by incubation at 30oC for 15 minutes with constant shaking. Reactions were stopped by adding 2  SDS-PAGE loading buffer containing 2% -mercaptoethanol and boiling for 5 minutes. Histone H2B was separated by SDS-PAGE. The gel was dried and analyzed by phosphorimager (BioRad, Hercules, CA). The histone H2B bands were cut out and counted in a liquid scintillation counter (Beckman Coulter, Fullerton, CA).  When Crosstide was used as substrate, the beads were then resuspended in 25 µl of kinase buffer containing 60 µM Crosstide (Upstate Biotechnology). ATP solution (5 µl) (200 µM ATP, 10 µCi of [32P]ATP in kinase buffer) was added, followed by incubation at 30 °C for 15 min. Reactions were stopped by spotting 20 µl onto 2-cm2 pieces of P81 filter paper (Whatman), followed by extensive washing in 1% (v/v) phosphoric acid. Measurement of associated radioactivity was by liquid scintillation counting.             A kit from Cell Signaling Technologies was used for nonradioactive kinase assays, which has a similar mechanism to that described above, but with a different  41 substrate. Briefly, after cell stimulation and nucleus disposal, cell lysate was incubated with anti-PKB antibody-bound beads overnight. The beads were washed three times with the lysis buffer and incubated with a GSK-3 protein fragment and ATP at 30oC for 30 minutes. Reactions were stopped with SDS-PAGE loading buffer. The phosphorylated GSK-3 protein fragment was detected by a specific antibody provided in the kit. The result of this assay was found to be highly comparable to that of the radioactive PKB kinase assay in comparing PKB activity in BJAB and MS19 cells. Immunoprecipitatation of the Flag-tagged protein Flag immunoprecipitation was done with the anti-Flag M2 beads. Briefly, beads were washed with TBS three times, then 15 µl beads was put into the cell lysate with rotation for 2 hours. Beads were spun down at 15,000 rpm for 5 seconds and washed with TBS three times. 20 µl sample buffer was put in the beads and the mixture was boiled for 3 minutes to elute the proteins. Gel staining with silver stain or colloidal Coomassie For silver staining, SDS-PAGE gels were fixed in 50% methanol and 10% acetic acid for 30 minutes, then incubated in 5% methanol and 1% acetic acid for 15 minutes. Gels were then washed in water for 5 minutes three times before incubation with freshly made 0.2 g/L sodium thiosulfate for 90 seconds, then 3 × 30 seconds water washes before incubation with 0.2 g/100ml silver nitrate. After 3 × 60 seconds water washes, gels were developed until bands appeared. Six percent acetic acid was used to stop the reaction. Water was again used for washing before gels were dried.  42 Colloidal Coomassie blue staining was utilized to visualize bands sent for mass spectrometry. Briefly, gels were fixed in 50% ethanol and 3% phosphoric acid for two hours before washing in water 3 × 20 minutes. Gels were stained in modified Neuhoff Solution (16 g/100ml ammonium sulfate, 25% methanol, 5% phosphoric acid, 0.1 g/100ml Coomassie brilliant blue G250, add Milli-Q water to 100 ml) up to 3 days, then washed in water and stored in 20% ammonium sulfate at 4%. Selected bands were cut out and sent for trypsin digestion and MS/MS analysis in the laboratory of Dr. Juergen Kast (UBC Biomedical Research Center). Gel filtration. FPLC (Amersham) separation on a Superdex200 10/100 GL column (Amersham) was used to study the PKB protein complex. Protein standards were used to calibrate the column. Membrane and cytosol preparations were run with the buffer composed of 20 mM Tris-HCl pH7.5, 20 mM NaCl, 2 mM EDTA and 0.1% NP-40. Collected fractions were precipitated with four volumes of acetone overnight at -20 oC, and then spun down at 15,000 rpm for 15 minutes. Pellets were dissolved in sample buffer and processed for immunoblotting.  43 Chapter 3.  In vivo regulation of membrane recruitment of TAPP, Bam32 and Btk by levels of PI(3,4)P2 and PI(3,4,5)P3 3.1 Introduction The B cell receptor (BCR) consists of an antigen-binding subunit and a signaling subunit. The BCR recognizes the antigenic determinants and initiates downstream signaling to regulate B cell development, survival, apoptosis and antibody production (Gold, 2002). The strength and quality of BCR signaling plays a key role in determining B cell fate to maintain normal B cell functions and immune responses (Gold, 2002). The BCR can initiate several signaling pathways, including the PI 3-kinase, Ras, Rac, Rap GTPase, and PLC-DAG pathways (Gold et al., 2000; Niiro and Clark, 2002). Fc RIIB is a low affinity Fc receptor located on a variety of cell types, including B cells, follicular dendritic cells and epithelial cells (Ravetch and Lanier, 2000; Coggeshall et al., 2002). The most important function of Fc RIIB is to initiate negative signals (Coggeshall et al., 2002). Fc RIIB has an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic portion that can recruit the SH2 domain- containing inositol 5-phosphatase (SHIP), which dephosphorylates PI(3,4,5)P3 at the D-5 position, simultaneously reducing the level of PI(3,4,5)P3 and increasing the level of PI(3,4)P2. The action of SHIP can therefore result in inhibitory effects by its effects on PI(3,4,5)P3 levels, but it can also have positive effects, since the  importance of PI(3,4)P2 has been reported by our lab and others (Scheid et al., 2002a; Kimber et al., 2002).  44           TAPP (tandem PH-domain-containing protein) was initially cloned by Alessi’s group  and identified to have specific binding to PI(3,4)P2 (Dowler et al., 2000). Meanwhile, our collaborator Dr. Aaron Marshall cloned the same protein using distinct methodology (Marshall et al., 2002). TAPP1 and TAPP2 are widely expressed. Bam32, which was cloned by Dr. Aaron Marshall, is expressed by B lymphocytes, but not T lymphocytes or nonhematopoietic cells (Marshall et al., 2002). TAPP1, TAPP2 and Bam32 are all single-copy genes residing on distinct chromosomes: TAPP1 on chromosome 10, TAPP2 on chromosome 8, and Bam32 on chromosome 4q25–q27. These three molecules share several characteristics, including small size (32 to 47 kDa), lack of enzymatic domains, high conservation between humans and mice, and the presence of PH domains near the C-terminus. The structural difference between TAPP and Bam32 is that the N-terminal regions of TAPP1 and TAPP2 contain a second PH domain. In vitro binding assays showed that TAPP1/2 have high affinity for PI(3,4)P2, while Bam32 has high affinity for both PI(3,4)P2 and PI(3,4,5)P3. However, how these molecules function in vivo in response to phosphoinositide production is still unclear.             Given the similarities and differences in structure and in vitro binding specificity of TAPP, Bam32 and Btk (Bruton tyrosine kinase), I was interested in how the production of phosphoinositides in vivo might regulate TAPP, Btk and Bam32 recruitment to the plasma membrane. Using BJAB cells and BJAB cells stably over- expressing a membrane-targeted SHIP protein, I addressed these questions in depth.  45 3.2 Results             Optimization of phosphoinositide analysis. We felt it was a priority to make the measurement of phosphoinositide levels in different sets of experiments reproducible, especially due to the fact that it is not feasible to handle duplicate samples, and as there were over 20 samples to deal with at the same time. Normalization based upon the total labeling of each sample was felt to be the most reasonable way to perform the normalization, based on the assumption that during an acute stimulation the total phosphoinositide pool should remain constant. Similar normalization methods were used previously by others (Gold et al., 1994; Gupta et al., 1999; Scharenberg et al., 1998). This way of normalization indeed has its advantage when investigating the relative changes in lipid levels, thus allowing one to determine whether the level of one kind of lipid is relatively constant or changes after stimulation (Gold et al., 1994; Marshall, 2002). However, after cells are lysed and lipids are extracted and deacylated, the phosphoinositide pool still only constitutes a small part of all the phospholipids; for example, in endothelial cells phosphatidylinositol is just 3.1% of all the phospholipids (Murphy, 1992). The percentage of bi- or tri-phosphorylated PIs, which are present at a much lower level than than mono-phosphorylated PIs, is even lower in the phospholipid pool (Vanhaesebroeck, 2001). Practically, we found that total lipid labeling was difficult to quantify due to large errors in sampling of the CHCl3/MeOH-extracted lipids. For example, pipeting a small 2 µl aliquot of phospholipid yielding total cpm in the range of 106 can lead to large errors in calculating the total lipid in 140 µl of sample that was  46 loaded onto the HPLC. Also, dilution would not solve this problem because only a limited volume of sample could be loaded on the HPLC. In our studies, we decided to normalize the variations among samples using the level of PI(4,5)P2, which is relatively constant after stimulation. Our group has previously used this way of normalization (Scheid et al., 2002a). Considering the small proportion of poly-phosphoinositide in the much larger PI(4,5)P2 phospholipid pool, the minor undulation of PI(4,5)P2 levels is more reliable for normalization when the objective is to measure the amount of one of the other  phosphoinositides. To confirm this idea, preliminary experiments were performed with duplicate cell populations, which were separately labeled with orthophosphate, and stimulated under the same conditions. After cell lysis, lipid extraction and deacylation, HPLC results from the duplicated cell preparations were compared. The degree of similarity of duplicate samples would show how well the two different normalization methods worked.  From the results shown in Table 3.1, it is apparent that normalization with PI(4,5)P2 was indeed more accurate.  47 Table 3. 1 Optimization of phosphoinositide analysis Before normalization PI(3,4,5)P3 PI(3,4)P2 PI(4,5)P2 Whole count (1/70 aliquot) BJAB control 1147 /937 11077 /9175 163372 /190030 93728 /99007 Sample /Duplicate BJAB F(ab)2 2minutes 3555 /4392 33799 /39536 210759 /161452 99650 /96733 Normalization with the average values of whole count PI(3,4,5)P3 PI(3,4)P2 PI(4,5)P2 PI(3,4,5)P3 SD/Mean PI(3,4)P2 SD/Mean BJAB control 1190 /921 6595 /4697 169561 /186713 12.7% 16.8% Sample /Duplicate BJAB F(ab)2 2minutes 3471 /4416 15600 /23821 205744 /162363 12.0% 20.0% Normalization with PI(4,5)P2 PI(3,4,5)P3 PI(3,4)P2 PI(3,4,5)P3 SD/Mean PI(3,4)P2 SD/Mean BJAB control 1033/982 9976/9612 2.5% 1.8% Sample / Duplicate BJAB F(ab)2 2minutes 4131/3909 39269/35188 2.7% 5.4% BJAB cells were stimulated with F(ab')2 for 2 minutes, and lipid extracts were prepared. Duplicate samples were analyzed by HPLC as described in Materials and Methods. The top panel shows the original cpm values from HPLC and sample aliquots.  Due to variations in the cell lipid extraction, the values for the PIP lipids were normalized to the original lipid extract (middle panel) or to PI(4,5)P2 (bottom panel). Normalization shown in the middle panel was calculated as follows: Normalized cpm of sample 1= (average cpm of sample 1 to 4 / cpm of sample 1)  sample 1 specific peak For example, PI(3,4,5)P3  normalized with whole counts = (93728+99007+99650+96733)/4/93728  1147= 1190 Similar formula was used for the normalization with PI(4,5)P2. After normalization, the mean “SD” was calculated for each PIP, and the percentage of deviation of each duplicate sample is shown under the column SD/Mean (middle and bottom panel).  48 Membrane recruitment of TAPP, Bam32 and Btk by PI(3,4)P2  and PI(3,4,5)P3. BJAB is a human B cell lymphoma cell line. BJAB cells have been shown to be stimulated by either goat-anti-human IgM F(ab')2 or intact antibodies (Krahn et al., 2004). Intact antibody is commonly regarded as an “inhibitory” antibody because it co- ligates the Fc RII receptor, which recruits and activates the inositol phosphatase SHIP. We also over-expressed membrane-targetted SHIP (provided by Dr. Aaron Marshall) in BJAB cells and chose a stable MS19 (Membrane-SHIP) cell line in the study.  Figure 3.1 shows the typical HPLC profiles of the deacylated lipids and Figure 3.2 presents a summary of data that were normalized as discussed above.  From Figure 3.1 and 3.2 we can see there were decreased levels of PI(3,4,5)P3  with SHIP recruitment and over- expression of membrane-targeted SHIP. Intact antibody stimulation/SHIP recruitment caused decreased levels of PI(3,4)P2 compared to that observed in F(ab')2 antibody stimulation; whereas SHIP over-expression in MS19 cells caused higher levels of PI(3,4)P2  compared to that observed in parental BJAB cells. Additional details regarding phosphoinositide metabolism in this model system are discussed in Chapter 5. Dr. Aaron Marshall’s laboratory provided the data for TAPP, Bam32 and Btk membrane recruitment in this model system (Figure 3.3). Perhaps due to technological limitations in recording of fluorescence in vivo, the membrane recruitment data in Figure 3.3 did not show absolute distinctions between different conditions in this model system, especially at later time points with TAPP and Bam32 recruitment. However, it was consistently observed that MS19 cells and intact antibody stimulation showed a faster membrane reruitment of Bam32 and TAPP at early time points such as 0.5 minute. Although the lower levels of PI(3,4,5)P3  may at least partially account for inhibition of Btk  49 recruitment under inhibitory signaling conditions, it did not appear that differences in the absolute levels of PI(3,4)P2  can solely account for the more rapid recruitment of Bam32 and TAPP in MS19 cells at early time points.  In considering the relationship of the levels of PI(3,4,5)P3  and PI(3,4)P2  to the recruitment of Bam32 and TAPP, we calculated the ratios of PI(3,4,5)P3/PI(3,4)P2, and these appeared to be more closely related to the changes in membrane recruitment that were observed. Levels of PI(3,4,5)P3  peaked at around 0.5 to 1 minute and dropped afterwards, in accord with the post-one minute time that PI(3,4)P2  levels started to increase. The peak ratio of PI(3,4,5)P3/PI(3,4)P2  dropped dramatically after 1 minute, as conditions shown in Figure 3.4. We attribute the cause of this more rapid recruitment of Bam32 and TAPP at early time points to the ratio of PI(3,4,5)P3/PI(3,4)P2, which dropped dramatically with inhibitory signaling (Krahn et al, 2004).  50 Figure 3.1 HPLC profile of orthophosphate labeled deacylated phosphoinositide (Without normalization). BJAB cells were labeled with orthophosphate and stimulated with F(ab')2 or intact antibody at a concentration of 10 µg/ml. Lipids were extracted, deacylated and run through a Partisil SAX 10 HPLC column. The cpm per fraction is shown for a representative set of experiments using BJAB and MS19 cells stimulated with F(ab')2 or intact antibodies for 1 minute to show the relative PI(3,4,5)P3 levels and for 2  minute to show the relative PI(3,4)P2 levels at these four different stimulation conditions. (A) The PI(3,4,5)P3  peak is eluted around 94 minutes. (B) The PI(3,4)P2 peak is eluted around 70 minutes. The peaks were determined by inositol standards as described previously (Scheid et al., 2002a). Total cpm value in each peak is calculated as level of respective phosphoinositide lipid.  51  Figure 3. 2 HPLC profile of deacylated phosphoinositides corresponding to PI(3,4,5)P3, PI(3,4)P2 levels in BJAB cells. BJAB or MS19 cells were labeled with 32P-orthophosphate and stimulated with F(ab')2 or intact antibody at the concentration of 10 µg/ml at the time points shown on the graphs. For A and B, black bars: BJAB cells stimulated with F(ab')2; white bars: BJAB cells stimulated with intact antibody; bars with cross hatching: MS19 cells stimulated with F(ab')2; bars with horizontal lines: MS19 cells stimulated with intact antibody. Lipids were extracted, deacylated and run through Partisil SAX 10 HPLC column to determine relative levels of PI(3,4,5)P3 (A) and PI(3,4)P2 (B), based on cpm of deacylated glycerophosphoinositides. Panel C shows results of similar experiments done using A20 cells stimulated with F(ab')2 or intact antibody. Results shown are means ± standard deviation of results from 3 independent experiments. Levels of incorporation were normalized in each experiment based on total 32P incorporation.  52 Figure 3. 3 TAPP and Bam32 membrane recruitment by PI(3,4,5)P3 and PI(3,4)P2. (data obtained by Dr. Aaron Marshall) WT and MS19 cells were transiently transfected and stimulated with antibodies as shown in the figure. Cells were scored at each frame for visible accumulation of fluorescence at the membrane (50–100 cells/stimulation condition). The y-axis indicates the percentage of cells scored positive for membrane recruitment at each time point. Similar results were found for TAPP1 (not shown). A, Kinetics of Bam32 PH domain-directed membrane recruitment under different conditions. B, TAPP2. C, Btk Note the more rapid recruitment of Bam32 and TAPP2 under inhibitory signaling through Fc RII and/or SHIP.  53    Figure 3. 4  PI(3,4,5)P3/PI(3,4)P2  ratio. The ratio of  PI(3,4,5)P3  to PI(3,4)P2 were calculated. This ratio peaks around 0.5 to 1 minute. This ratio reflects the relative production of these two lipids, and therefore is used to reflect the regulation of downstream signaling. The faster TAPP membrane recruitment under negative signaling conditions does not correlate to absolute levels of PI(3,4,5)P3  or PI(3,4)P2, but does inversely correlate to this ratio reversely. The results shown are the means ± standard deviation of results from 3 independent experiments.  54 TAPP2 over-expressing cells do not show altered levels of PKB Ser473 phosphorylation. Our group was the first to propose the that PI(3,4)P2  may contribute to PKB phosphorylation at Ser473 (Scheid et al., 2002a). Thus, the identification of TAPP2 as a PI(3,4)P2  binding protein brought up the question of whether there may be a potential link between TAPP1/2 and PKB Ser473 phosphorylation. We used two clones of BJAB cells that stably over-expressed TAPP, obtained from Dr. Aaron Marshall, to determine whether there were any changes in the levels of PKB phosphorylation, especially at the Ser473 site. These TAPP over-expressing cell lines have been shown previously to have upregulated of downstream signalling (Krahn et al, 2004). Stimulation of both cell types with F(ab')2 and intact antibody induced similar degree of PKB phosphorylation at the Ser473 site. Therefore, our results did not show any positive correlation between TAPP and PKB Ser473 phosphorylation in these cell lines (Figure 3.5).  55  Figure 3. 5 Comparison of Ser473 phosphorylation in BJAB cells and BJAB cells expressing TAPP2. BP5 is a cell line with stable TAPP2 overexpression provide by Dr. Aaron Marshall. Both parental BJAB cells and BP5 cells were starved and stimulated under the same conditions for 2 minutes. Western blotting was used to show PKB phosphorylation at the Ser473 site. No difference is seen in BP5 cells compared to BJAB parental cells. The membrane was re-probed using anti-PKB antibody to ensure equal loading. This is representative of more than 5 experiments using two different cell lines stably expressing the TAPP2 protein.  The dose response of BCR agonists is discussed in more detail in Chapter 4.  56 3.3 Discussion Our priors study has established firm correlation between the in vivo production of PI(3,4)P2  and TAPP membrane recruitment by showing that they have similar kinetics (Marshall et al., 2002), soon after the cloning of TAPP and the identification of its in vitro phosphoinositide binding properties (Kimber et al.,2002; Marshall et al., 2002). In the current study, we are using a more complicated model system to study in more detail the in vivo production of PI(3,4)P2  and its regulation on the membrane recruitment of TAPP. Meanwhile, in this model system, we were also intereted in knowing how other PH domains such as Bam32 and Btk are regulated by PI(3,4)P2  and PI(3,4,5)P3, respectively. Surprisingly, under inhibitory signaling condtions with intact antibody, which produces lower levels of PI(3,4)P2 than F(ab')2 antibody does, there is a faster Bam32 recruitment. And in cells expressing active SHIP, which also produces lower levels of PI(3,4)P2  than parental cells do, there is also faster TAPP and Bam32 recruitment, especially at early time point such as 0.5 minute.  These results were surprising in the year 2002, when the traditional view was still prevalent that intact antibody stimulation can only induce negative effects compared to stimulation with F(ab')2 , due to the recruitment of SHIP as a result of Fc RII co-ligation. One report found that overexpression of full-length Btk lead to increased cellular levels of PI(3,4,5)P3, suggesting that in some cases PH domain proteins may be able to regulate levels of their lipid ligands, perhaps as a result of protecting them from degradation by lipid phosphatases (Scharenberg et al., 1998). Although its not clear whether this is a generalized phenomenon for PH domain proteins, we cannot rule out the possibility that PI(3,4)P2 levels may differ between cells transiently expressing Bam32 or TAPP2 PH  57 domains and the untransfected cells used for lipid measurements. It is also possible that inhibitory signaling results in higher localized concentrations of PI(3,4)P2 in plasma membrane niches supporting rapid recruitment of Bam32 and TAPP2, or that the observed rapid recruitment reflects the alteration in the PI(3,4,5)P3 to PI(3,4)P2 balance, as a reduction in the PI(3,4,5)P3/PI(3,4)P2 ratio consistently correlates with rapid recruitment in our study. Our working hypothesis is that the altered PI(3,4,5)P3/PI(3,4)P2 balance during "inhibitory" signaling leads to fundamental alterations in the assembly of membrane-proximal signaling complexes, perhaps favoring the stable membrane docking of PI(3,4)P2 effector molecules such as Bam32 and TAPP2. The question of PI(3,4,5)P3/PI(3,4)P2 ratio or absolute PI(3,4)P2 level determining downstream protein signaling will be addressed in more detail in the discussion of PKB signaling in Chapter 5.             Both Bam32 and TAPP1/2 were reported to bind specifically to the SHIP product PI(3,4)P2 in vitro (Dowler et al., 1999; 2000); thus, their differential responses to SHIP inhibitory signaling in the context of B cells fit with their in vitro lipid binding specificities. However, it does not appear to be universally possible to predict in vivo responses from in vitro binding results. For example, Bam32 was also found to bind PI(3,4,5)P3 with similar affinity to PI(3,4)P2, whereas the TAPPs showed no PI(3,4,5)P3- binding ability; however, in our model, both proteins behave similarly, and we have not observed recruitment of Bam32 at early time points corresponding to peak PI(3,4,5)P3 accumulation and Btk recruitment. This suggests that PI(3,4,5)P3 is not an important determinant of Bam32 recruitment in this cellular environment. However, other functions  58 for PI(3,4,5)P3 binding cannot be ruled out. Therefore, it is essential that the functional significance of in vitro binding activities be verified in a relevant cellular context.            A study from Alessi’s group showed a potential linkage between TAPP and PKB phosphorylation at Ser473, which could be mediated by a phosphatase called protein tyrosine phosphatase PTPL1. They showed a direct association between TAPP1 and PTPL1 (Kimber et al., 2003). Knock-down of TAPP1 induced decreased activity of PTPL1 and thus enhanced PKB phosphorylation at Ser473.  According to their study, the regulatory mechanism of the phosphatase may be PI(3,4)P2-directed, and PI(3,4)P2  will down-regulate PKB Ser473 phosphorylation. This is especially interesting if we look at previous data from our laboratory (Scheid et al., 2002a) showing the positive correlation between PI(3,4)P2 and Ser473 phosphorylation in mast cells. Therefore, in the next chapters, we investigated the correlation between PI(3,4)P2  and PKB Ser473 phosphorylation in B cell model systems. In conclusion, although previously the in vivo correlation between PI(3,4)P2 and TAPP was firmly established (Marshall et al.,2002), our current study investigated the more intricate PI(3,4)P2’s regulation of  TAPP recruitment. Our data suggest that TAPP recruitment correlates inversely to the PI(3,4,5)P3/PI(3,4)P2  ratio, especially at early time points, when this ratio achieve its peak around 0.5 to 1 minute.  59 Chapter 4. Low concentrations of B cell receptor agonists stimulate distinct waves of PI(3,4)P2 and PI(3,4,5)P3 production and downstream signaling in B cells. 4.1 Introduction Protein kinase B/Akt (PKB) is a well-established effector of PI 3-kinase signaling pathways in B cells (Craxton et al., 1999; Gold et al., 2000; Gold et al., 1999; Li et al., 1999; Marshall et al., 2000). PI(3,4)P2 has a similar function to that of PI(3,4,5)P3 in in vitro kinase assays of PKB activity (Alessi et al., 1997). Prior work in our laboratory demonstrated that PI(3,4)P2 has a critical role in PKB Ser473 phosphorylation (Scheid et al., 2002a). PH domains with high affinity to PI(3,4)P2, but not PI(3,4,5)P3 have also been identified (Marshall et al., 2002). We have previously shown that SHIP not only decreases PI(3,4,5)P3 levels, but it also elicits another wave of signaling protein recruitment by producing PI(3,4)P2 (Krahn et al., 2004).   There are numerous knockout studies that have shown the importance of PI 3- kinase activity in maintaining normal immune function (Okkenhaug et al., 2003). Since the major function of PI 3-kinase is to produce second messenger phosphoinositide lipids, we have used biochemical analysis to quantify these lipid products as a direct way to study the function of PI 3-kinase activity in immune cells. In this part of the study, we carried out a dose titration of F(ab')2 and intact antibodies directed against the B cell receptor to determine the profile of lipid production at different doses of  antibody stimulation. Meanwhile, our collaborator Dr. Aaron Marshall monitored membrane recruitment of two different GFP-PH domains with affinities for either PI(3,4,5)P3 or  60 PI(3,4)P2, and we monitored phosphorylation of PKB at Thr308 and Ser473 sites as well as PKB activity. Strikingly, we found that stimulation with low concentrations of BCR ligands produced higher levels of PI(3,4,5)P3 and PI(3,4,5)P3-associated responses and lower levels of PI(3,4)P2 and PI(3,4)P2-associated responses, compared to those observed at higher concentrations. We also found that under conditions where intact antibody does not bind to Fc RIIB, it caused stronger activation of PI 3-kinase signaling than the corresponding molar concentration of F(ab’)2 antibody. This part of the study investigated for the first time the phosphoinositide production following stimulation with various doses of BCR ligands, showing that the dose and form of the ligand can profoundly affect the subsequent production of PI(3,4)P2 and PI(3,4,5)P3.  61 4.2 Results Biochemical analysis of PI(3,4)P2 and PI(3,4,5)P3 following stimulation with various concentrations of BCR ligand. Following 3H-inositol labeling of BJAB human B lymphoma cells, we quantified the levels of PI(3,4)P2 and PI(3,4,5)P3 produced in response to B cell receptor stimulation. A dose-response analysis demonstrated that stimulation with 2 µg/ml F(ab')2 anti-IgM for 2 minutes caused a higher PI(3,4,5)P3 peak compared to stimulation with higher concentrations such as 5 to 15 µg/ml (Fig. 4.1A). When PI(3,4)P2 levels were measured, more of this lipid was generated following stimulation with the higher doses of F(ab')2 although the difference is not statistically significant. Stimulation of B cells with the same concentrations of intact anti-IgM antibody for the same time period caused a similar effect (Fig. 4.1B), but the intact antibody’s effect was stronger than F(ab')2 at low doses, which will be discussed below. At high doses, such as 10 µg/ml or 15 µg/ml, intact antibody had a less potent effect than F(ab')2. This was likely the result of the well described negative effect of intact antibody, since the Fc RIIB receptor is engaged by intact antibody at these higher concentrations (Chacko et al., 1996). To our knowledge, the increased production of PI(3,4,5)P3 and PI(3,4)P2 production in response to low versus high dose BCR stimulation has not been previously reported and thus we investigated it further with respect to PH domain containing protein recruitment and PKB phosphorylation.  62  Figure 4.1 PI(3,4)P2 and PI(3,4,5)P3  production by F(ab')2 and intact titration. BJAB cells were labeled with 3H inositol and stimulated by F(ab')2 (A) or intact antibody (B) at 2 minutes using the indicated concentrations. Lipids were extracted and analyzed as described in Materials and Methods.  Data presented as Mean SD are calculated from three indepedent experiments.  63 Stimulation with low concentrations of BCR ligand enhances membrane recruitment of the Btk-PH domain.  Dr. Aaron Marshall’s group tested whether low concentrations of BCR ligands also differentially affected membrane recruitment of PH domains binding to these lipids.  BJAB cells expressing EGFP-Btk-PH domain, which is PI(3,4,5)P3-specific (Bolland, 1998; Salim, 1996; Scharenberg et al., 1998) or TAPP2 PH domain, which is PI(3,4)P2-specific (Dowler et al., 2000; Kimber et al., 2002; Marshall et al., 2002), were stimulated with various concentrations of BCR ligand and membrane recruitment was determined by live cell confocal imaging.  Time courses showed a markedly increased and sustained recruitment of Btk PH domain at 1 µg/ml F(ab')2 antibody compared to 10 µg/ml (Figure 4.2). Similarly, increased recruitment of Btk PH domain was observed following stimulation with low dose intact antibody. TAPP2 PH- mediated recruitment was slightly reduced at 0.1 µg/ml, and similar levels were observed at 1 or 10 µg/ml, although slightly lower recruitment of TAPP2 was observed at 10 µg/ml compared to 1 µg/ml intact antibody. These results show that the PI(3,4,5)P3-dependent recruitment of Btk is much more pronounced at low concentration of B cell receptor ligands, correlating with the hgher amount of PI(3,4,5)P3. The differences in PI(3,4)P2- dependent recruitment of TAPP2 were much less pronounced between the low and high concentrations of stimulating ligands, but the trend in each case correlated with the levels of PI(3,4)P2.  64  Figure 4.2 Membrane recruitment of EGFP-PH domain proteins in response to low versus high doses of BCR ligand.  (Experiment performed in  Dr. Aaron Marshall’s laboratory) Quantitative analysis was done as described in Figure 3.3, to show the membrane recruitment of EGFP-Btk-PH or EGFP-TAPP2-PH following stimulation with various concentrations of F(ab')2 or intact antibody. Cells were analyzed at 2 minutes post- stimulation.  65 Low doses of F(ab')2 cause more PKB Thr308 phosphorylation and less Ser473 phosphorylation than higher doses. Data from our laboratory and others (Carricaburu et al., 2003; Scheid et al., 2002a) suggest that the levels of PI(3,4)P2 can somehow contribute to PKB phosphorylation at the Ser473 site. Thus, we examined phosphorylation at both Thr308 and Ser473 sites of PKB following stimulation with various concentrations of antibody. The results in Figure 4.3A and B show representative immunoblots and the tabulated data from Ser473 phosphorylation and Thr308 phosphorylation. The level of phospho-Thr308 and phospho-Ser473 corresponded to the levels of PI(3,4,5)P3  and PI(3,4)P2, respectively, as we might have predicted. At low dose of F(ab')2, there was higher Thr308 phosphorylation compared to the higher doses, corresponding to the higher levels of PI(3,4,5)P3. On the other hand, the relative level of Ser473 phosphorylation tended to follow the changes in PI(3,4)P2 levels. By contrast, cells treated with a low dose of intact antibody had elevated levels of both Thr308 phosphorylation and Ser473 phosphorylation (Figure 4.3A, B), consistent with the elevated levels of both lipids under these conditions. The responses in terms of phosphorylation at these regulatory sites on PKB also gave us the opportunity to examine the relative PKB activity under the various stimulation conditions. The results in Figure 4.4 show that F(ab')2  caused a higher PKB activity at higher concentration (10 µg/ml), which corresponds to conditions in which there was a higher level of Ser473 phosphorylation; on the other hand, at lower concentration of intact antibody, there is a comparable high PKB activity, which corresponds to elevated levels of both Thr308 and Ser473 phosphorylation. Higher concentrations of intact antibody caused slightly lower PKB activity at all the time points,  66 and both Ser473 phosphorylation and Thr308 phosphorylation were also lower under these conditions. However, at low doses of intact antibody, this inhibitory effect was lost, fitting with the increased phosphorylation seen under these conditions.  67  Figure 4.3 PKB phosphorylation at Thr308 and Ser473 sites in response to dose stimulation of F(ab')2 and intact antibody stimulation at 2 minutes in BJAB cells. A. Cells were stimulated with various concentrations of F(ab')2 or intact anti-Ig. Representative blots using anti-phosphoSer473 and anti-phosphoThr308 are shown, with relative values obtained by densitometry. B. Relative measures of Ser473 and Thr308 phosphorylation were determined and represented as the mean standard deviation. Values are normalized to PKB quantity. The results are representative of three independent experiments.  68  Figure 4.4 PKB activity in response to dose stimulation of F(ab')2 and intact antibody stimulation at 2 minutes in BJAB cells. PKB assays following B cell stimulation. BJAB cells were stimulated with 1 or 10 g/ml of F(ab')2 or intact antibody as indicated. Assay results are averaged from triplicate samples. This is representative of at least 4 experiments using Crosstide as substrate.  69 Low dose of intact antibody causes a greater increase in levels of PI(3,4,5)P3, PI(3,4)P2 and PKB phosphorylation than a corresponding dose of F(ab')2. At higher doses, we could see an “inhibitory” effect of intact versus F(ab')2 anti-IgM antibody, but at low doses we did not see any inhibitory effect of intact antibody on the production of either lipid (Figure 4.1) or on PKB phosphorylation and activity (Figure 4.3, 4.4).  Thus, we did a time course of lipid production at low doses of both F(ab')2 and intact antibody. Figure 4.5 shows that at all the time points, the equivalent concentrations of intact antibody produced higher levels of both PI(3,4,5)P3 and PI(3,4)P2. This increased potency of low doses of intact antibody was also evident when we examined Ser473 phosphorylation and Thr308 phosphorylation at all the time points in both BJAB cells and A20 cells (Figure 4.6). While Fc RIIB receptor is a low affinity receptor and thus the intact antibody is not likely to have an inhibitory effect at these lower concentrations, we predicted that the same molar dose of intact antibody should have the same potency as F(ab')2, not an increased effect. To exclude the possibility that this was due to variable quality of different antibody batches, we examined four different batches of antibodies and found consistently increased PKB phosphorylation with low dose intact antibody stimulation. Similar results were observed using human BJAB cells and murine A20 cells, stimulated with goat anti-human IgM or rabbit anti-mouse IgG, respectively (Figure 4.6).  70  Figure 4.5 Lipid analysis showing that low concentration of intact antibody caused higher levels of PI(3,4,5)P3 and PI(3,4)P2 than did F(ab')2. PI(3,4)P2 and PI(3,4,5)P3 levels were measured in a time course (0 to 10 minutes) following stimulation of BJAB cells with 2 µg/ml F(ab')2, 2 µg/ml intact antibody or 3 µg/ml intact antibody This result is representative of two independent experiments that gave similar results.  71  Figure 4.6 Low concentration of intact antibody caused higher Ser473 and Thr308 phosphorylation than did F(ab')2.  BJAB or A20 cells were stimulated with 2 µg/ml F(ab')2 compared to 2 µg/ml or 3 µg/ml intact antibody, showing that at this lower concentration, equal quantities of the intact antibodies produced increased PKB phosphorylation. A representative blot (representative of three experiments with similar results) is shown; results of densitometry (arbitrary unit given by FluorChem imaging system, as described in Materials and Methods) are shown below each lane.  72 The underlying mechanism of the increased effects of low dose intact antibody on the PI 3-kinase pathway is inherent to intact antibody and independent of Fc RIIB binding. The difference between F(ab')2 and intact antibody is that intact antibody has the Fc portion, through which the intact antibody may be having additional effects on BCR signaling. To compare intact- and F(ab')2-induced signaling, independent of the influence of Fc RIIB binding to the Fc portion of the antibody, we used 2.4G2 antibody to block Fc RIIB on A20 cells. 2.4G2 does not have stimulatory effects on A20 cells (Figure 4.7 A), which is consistent with the original paper describing ITIM motifs in the Fc RIIB receptor (Muta et al., 1994). Our results show that, under condition of Fc RIIB blockade, intact antibody more strongly activated PKB phosphorylation at all doses (Figure 4.7 B and C). At high doses of intact antibody, PKB phosphorylation was markedly increased by Fc RIIB blockade, whereas Fc RIIB blockade had little effect at lower doses, presumably reflecting a lack of binding of intact antibody to Fc RIIB at low doses. Thus, the enhanced effect that was observed with lower doses of intact antibody, compared to that of F(ab')2 fragments, was independent of Fc RIIB. A dose-response analysis is shown in Figure 4.7D, which highlights the effect of the blocking antibody only at higher concentration of intact anti-IgG antibody. Increasing concentrations of intact antibody caused a reduced effect on PKB phosphorylation, and blocking of the Fc RIIB receptor not only eliminated the inhibitory effect, but it caused an even greater increase in strength of the signal. Using the Fc RIIB deficient A20 cell line, IIA1.6, we also consistently saw results similar to the conditions in Figures 4.7A and B using 2.4G2 blocking (Figure 4.7 E and F).  73  74 Figure 4.7 Blocking of Fc RIIB receptor with 2.4G2 shows that the positive signal of intact antibody is independent of Fc RIIB binding. 2.4G2 antibody was incubated with A20 cells at 37oC prior to stimulation with intact antibody to block binding to Fc RIIB. All samples are blotted with Ser473 antibody. A. 2.4G2 antibody did not show stimulation effects on PKB phosphorylation at either control condition or high and low concentrations of 2 µg/ml F(ab')2 and 10 µg/ml F(ab')2, respectively. B. Stimulation at high concentration of 10 µg/ml F(ab')2 and 15 µg/ml intact antibody with or without pre-incubation with 2.4G2 blocking antibody.. C. Stimulation at low concentration of 2 µg/ml F(ab')2 and 3 µg/ml intact antibody with or without pre- incubation with 2.4G2 blocking antibody. D. Dose response of intact antibody stimulation with or without 2.4G2 block. With the increasing concentration of intact antibody, Ser473 phosphorylation decreased, while blocking with 2.4G2 antibody caused increasing Ser473 phosphorylation. Densitometry values are shown below the corresponding bands. E, F. stimulation with IIA1.6, Fc RIIB receptor deficient B cells to show intact antibody has stronger effect than F(ab')2 at both low and high dose stimulations.  75 Intact antibody can result in more BCR binding than F(ab’)2 does. Finally, we compared the BCR binding ability of F(ab')2 and intact antibodies. After BCR crosslinking by the respective antibodies, Cy5 conjugated light chain specific secondary antibody was used to analyze the respective quantity of F(ab')2 or intact antibody bound to the BCR. Figure 4.8.A. shows that intact antibody actually has more BCR binding potential than F(ab')2. This explains why a low dose of intact antibody causes a stronger effect than F(ab')2 on downstream signaling rather than a equal one. This explains the results in Figure 4.5 and 4.6 showing that the positive effect of low dose intact antibody is inherent to intact antibody. Also, we tested the binding of F(ab')2 compared to intact antibody in the Fc RIIB-negative IIA1.6 cell line, and found the same relative difference in binding (Fig.4.8.B), which suggests that additional binding to the Fc receptor cannot explain higher binding of intact antibody.  76  Figure 4.8 Intact antibody binds more B cell receptor than F(ab')2 does. A20 cells (A) or  Fc RIIB receptor deficient IIA1.6 cells (B) are stimulated with rabbit anti-mouse antibodies at the indicated time course. Cy5 conjugated mouse anti-rabbit secondary antibody light chain specific was used to detect the F(ab')2 and intact antibodies bound to B cell receptors. Binding was calculated from fluorescence measurements and is presented in arbitrary units showing the mean of two experiments.  77 4.3 Discussion This part of our studies has revealed two interesting findings that give new insight into the PI 3-kinase signaling pathway: first, when low concentrations of antibody are used to stimulate the B cell receptor, the increase in PI(3,4,5)P3 and PI(3,4)P2 production is quite distinct from that seen with standard high dose stimulation; the second observation was that low doses of intact antibody had a more potent effect than equivalent concentrations of F(ab')2. Although the latter observation was easily attributed to the lower extent of F(ab')2 binding on a molar basis, the former observation may have important implications regarding the effect of dose and form of BCR ligands on signal quantity and quality, which are relevant for widely-used experimental models of BCR signaling and perhaps for normal B cell biology, as discussed below. Our finding that lower concentrations of F(ab')2 can produce higher levels of PI(3,4,5)P3, is likely due to decreased SHIP activity induced under these conditions. There are several previous studies showing that both F(ab')2 and intact antibody cause SHIP tyrosine phosphorylation, suggesting that SHIP may play a critical role in establishing the thresholds for BCR signaling under both activating and inhibitory conditions (Chacko et al., 1996; Crowley et al., 1996; D'Ambrosio et al., 1996; Saxton et al., 1994). Cambier’s group observed that with F(ab')2 stimulation, SHIP-/- cells have increased PI(3,4,5)P3 level and decreased PI(3,4)P2 level compared to wild type cells (Brauweiler et al., 2000), which was the first report suggesting that there is BCR induced SHIP activation. These conclusions were made primarily based on the results of lipid analysis. They also showed that BCR-induced SHIP activity is a key regulator of BCR-  78 mediated signaling, B cell activation, and B cell development, which is consistent with the BCR’s crucial function during these processes. Our data indicate that BCR-induced co-ordination of SHIP and PI 3-kinase activities may be intricately regulated by the concentration of BCR ligands. Specifically, our results suggest that SHIP activation by the BCR has a higher dose threshold than PI 3-kinases, such that PI(3,4,5)P3 production is optimal within an intermediate signaling “window” at which PI 3-kinases are efficiently activated, while SHIP is not. This may also be generally true for ITAM- dependent immunoreceptor signaling, since a recent study suggested that the effect of SHIP in attenuating Fc RI activation of mast cells occurs mainly at high IgE/antigen doses (Gimborn et al., 2005). Our data may be able to explain some observations by Helgason et al. (2000) using SHIP -/- B cells. In that study, it was shown that SHIP-/- B cells had a higher proliferation rate than wild-type cells, but only when stimulated with higher concentrations of antibody, which they attributed to the potential higher amount of PI(3,4,5)P3 in SHIP-/- B cells. However, at lower concentrations of antibody stimulation, SHIP -/- cells have a similar or lower proliferation rate than wild-type cells.  In light of our results, this finding could reflect the differential activation of SHIP at high, but not lower doses of BCR ligand. The B cell receptor (BCR) plays important roles in recognition of foreign antigens and self-components to allow the immune system to make appropriate antibody responses. For this reason, there is considerable interest in understanding how the BCR transmits signals to influence B cell development and differentiation into antibody secreting plasma cells versus inducing anergy or apoptosis after encountering foreign or  79 self-antigens, respectively (Healy et al., 1998). Identifying the effect of antigen dose on the quantity and quality of downstream signaling lipids and proteins not only increases our understanding of these important physiological processes but may also allow us to determine the molecular basis for autoimmune diseases or immunodeficiency diseases that are due to defects in BCR signaling. High doses of antigen stimulation in the absence of co-stimulatory signals are thought to favor anergy/tolerance induction over activation under some circumstances (Healy et al., 1998).  PI(3,4,5)P3 responses have been invariably associated with B cell activation, and we now show that they are preferentially induced by lower doses of BCR ligand. Since PI(3,4)P2 responses are preferentially generated  by high antigen doses, it is tempting to speculate that they may be involved in induction of the anergic state. BCR clustering causes a rapid increase of PI(3,4,5)P3 and PI(3,4)P2 (Gold and Aebersold, 1994), recruitment of the PH domain containing adaptor proteins Btk, Bam32, TAPP2 (Krahn et al., 2004), and activation of PKB/Akt (Craxton et al., 1999; Gold et al., 1999; Li et al., 1999). Knockout studies by several groups show that in B cell development and mature B cell proliferation, the response to anti-IgM antibody is seriously impaired when PI 3-kinase signaling is disrupted (Fruman et al., 1999; Okkenhaug et al., 2002). Considering these knockout studies and our current results, we may ask whether those knockout studies can completely reflect the complexity of physiological PI 3-kinase regulation under which B cells respond to different doses of foreign or self antigens. Immature B cells undergo apoptosis after BCR engagement, whereas activation of mature B cells after BCR engagement must be due to the involvement of different downstream pathways (King et al., 2000). Other studies have  80 shown that different developmental stages of B cells respond differently to the same kind of BCR ligand, likely due to the diverse influences on gene expression profiles (Glynne et al., 2000). Combined with our findings with in vitro stimulation using low doses of BCR ligands, it is evident that the subtle differences in BCR signaling response must take into account the potency of antigen receptor signaling. It will be important in future studies to determine whether different developmental stages of B cells differ in their sensitivity to activation of PI(3,4,5)P3 versus PI(3,4)P2 responses over a range of antigen doses. Another issue addressed in this chapter is the relative importance of Thr308 and Ser473 sites in their contribution to PKB activity. According to the studies done to date, the phosphorylation mechanism for these two sites are distinct (Williams et al., 2000). A study by Alessi et al. (1997) reported that Thr308 phosphorylation is essential for PKB activity, whereas Ser473 phosphorylation can contribute another 10-fold increase in activity of the enzyme. Scheid et al. (2002b) showed that when Thr308 is mutated to alanine, PKB activity is dramatically decreased, but when Ser473 is mutated to alanine, PKB activity is only mildly decreased, which suggests that Thr308 is a more important phosphorylation site for PKB activity. However, a more recent study by Newton’s group (Gao et al., 2005) in which a Ser473-specific phosphatase, PHLPP (PH domain leucine- rich repeat protein phosphatase), was cloned, suggests that the Ser473P phosphatase can inactivate PKB by directly dephosphorylating Ser473. This study strongly supports the importance of the Ser473 site in maintaining PKB activity. A study by O’toole et al. (2001) showed that tumor necrosis factor-alpha activation of PKB in WEHI-164 cells is accompanied by increased phosphorylation of Ser473, but not Thr308. All these data  81 suggested that the phosphorylation at these two sites can be independent. In the titration with F(ab')2, we observed that at low dose there was higher Thr308 phosphorylation and lower Ser473 phosphorylation. Although there is a transient high PKB activity at 2 minutes at lower dose, which might be due to the sustained Thr308 phosphorylation, PKB activity at higher dose is sustained and elevated at all time points. Thus, Ser473 phosphorylation status must also contribute to PKB activity. In fact, the results suggest that Ser473 can compensate for reduced Thr308 phosphorylation and this may play an important role in sustained activity of PKB. This regulatory role of Ser473 may not be easily detected in studies using PKB with a mutation to alanine at Thr308, which results in a much more drastic change in the protein. Regarding the relative importance of Thr308 and Ser473 in contributing to PKB activity, further investigation using another strategy was carried out in B cells and will be discussed in Chapter 5. In conclusion, by titration of F(ab')2 and intact antibody used to stimulate B cells, we detected some interesting new features of PI 3-kinase pathway regulation in B cells. These findings provide a striking example of altered signal quality regulated at the level of concentrations and forms of ligands and point out the importance of considering ligand dose in the regulation of immunoreceptor signaling.  82 Chapter 5. PI(3,4)P2-correlated PKB Ser473 phosphorylation involves kinetics independent of PI(3,4,5)P3 and Thr308 phosphorylation 5.1 Introduction SHIP, which serves as the major PI 5-phosphatase in hematopoietic cells, can have an inhibitory effect on the activation of a number of PH domain containing proteins, such as PKB, Btk and Bam32 (Marshall et al., 2000). One of the major explanations for this inhibition is that activation of these proteins can be inhibited due to decreased PI(3,4,5)P3 levels. Previous studies showed that SHIP can down-regulate PKB activity, because SHIP can decrease PI(3,4,5)P3 levels (Aman et al., 1998; Jacob et al., 1999). However, we noticed that measurement of corresponding PI(3,4)P2  levels were not taken into account in these studies. It has been reported that PI(3,4)P2 has an indispensable effect on PKB activation  (Franke et al., 1997; Scheid et al., 2002a). Therefore, it is important to know how changes in PI(3,4)P2 levels by SHIP may be having an inhibitory effect on PKB in B cell negative signaling. Two previous studies have provided data on PI(3,4)P2 levels after SHIP membrane recruitment in the B-lymphoma cell line A20, which is interesting because they showed that after SHIP recruitment, PI(3,4)P2 levels decreased along with the dramatic decrease of PI(3,4,5)P3 (Gupta et al., 1999 ; Scharenberg et al., 1998). These observations provided evidence that the decreased PKB phosphorylation may not be attributed solely to decreased PI(3,4,5)P3 levels. There are two possibilities for this decreased level of PI(3,4)P2. One is that there might be some D- 3 phosphatase or D-4 phosphatase activated at the same time. The other possibility  83 suggested by the data of Gupta et al. (1999), is that there is an association between the SH2 domain of the p85 subunit of PI 3-kinase and the C-terminal tyrosine phosphorylation site of SHIP. This association would compete the binding of p85 to intracellular portion of BCR, effectively decrease PI 3-kinase activity and thus decrease both PI(3,4)P2 and PI(3,4,5)P3 levels. In this study, we investigated in greater detail the kinetics of PI(3,4)P2 and PI(3,4,5)P3 generation during positive and negative B cell stimulation, and  how metabolism of these lipids influences PKB phosphorylation. We used the human cell line, BJAB, as well as BJAB cells expressing a membrane targeted mouse SHIP construct, Fc RIIB-SHIP- SH2, which had been shown in our previous studies to have constitutive membrane SHIP activity (Krahn et al., 2004) (shown in Chapter 3). This membrane SHIP would coordinate with endogenous SHIP to regulate PI(3,4,5)P3 and PI(3,4)P2 levels under different stimulation conditions. We found that phosphorylation of PKB at Thr308 and Ser473 correspond to observed changes in the levels of PI(3,4,5)P3 and PI(3,4)P2, respectively. Ser473 phosphorylation was found to correlate with PI(3,4)P2 levels and to be independent of PI(3,4,5)P3 levels and Thr308 phosphorylation. On the other hand, membrane/cytosol separation showed that Ser473 phosphorylation, which was correlated with PI(3,4)P2 levels, was found to be present only in cytosol. Furthermore, overall PKB activity, primarily due to the cytosolic enzyme, was dependent upon levels of PI(3,4)P2, while only membrane-associated PKB activity was dependent upon PI(3,4,5)P3  levels. This part of the study showed for the first time in vivo the distinct roles of PI(3,4,5)P3 and PI(3,4)P2 in determining PKB phosphorylation and activity.  84 5.2 Results SHIP membrane recruitment induced lower level of PI(3,4)P2. As we reported previously, BJAB cells express human Fc RIIB, which can cause inhibitory effects after co-ligation with B cell antigen receptor by the stimulation of goat anti-human IgG (Krahn et al., 2004).  There were decreased PI(3,4,5)P3 levels with SHIP recruitment, resulting from stimulation with intact antibody, in both BJAB and MS19 cells (Figure 3.2). As previously reported by other groups using the A20 cell line (Gupta et al., 1999; Scharenberg et al., 1998), in the BJAB cell line we a saw a decreased PI(3,4)P2 level after intact antibody stimulation compared to the same dose of F(ab')2 (Figure 3.2). Similar results were also verified in A20 cells (Figure 5.1). Intact antibody stimulation is known to cause SHIP recruitment to the cytoplasmic portion of Fc RIIB, and the recruited SHIP can de-phosphorylate PI(3,4,5)P3 to PI(3,4)P2. Theoretically, the level of PI(3,4)P2 should increase with the dephosphorylation of PI(3,4,5)P3 by SHIP, and thus would be predicted to be higher after stimulation with intact antibody than with F(ab')2 antibody. However, this was not observed. The reason that SHIP membrane recruitment did not produce elevated PI(3,4)P2 in B cells may be attributed to decreased PI 3-kinase activity as a result of SHIP/p85 association (Gupta et al., 1999). Therefore, lower levels of PI(3,4,5)P3 with intact antibody than with F(ab')2 antibody may be due to SHIP recruitment and decreased PI 3-kinase activity; whereas lower levels of PI(3,4)P2  with intact antibody than with F(ab')2 antibody may be due to decreased PI 3-kinase activity.  85 Membrane-targeted SHIP induced increased PI(3,4)P2, as well as reduced PI(3,4,5)P3. In MS19 cells, which were derived from BJAB cells and express a membrane-targeted SHIP lacking the SH2 domain (mem-SHIP construct), we consistently observed higher PI(3,4)P2 levels, particularly at longer times of stimulation. Although there was increased PI(3,4)P2 in MS19 cells, the PI(3,4,5)P3  level was dramatically decreased compared to BJAB cells (Figure 3.2). The sharply altered levels of PI(3,4)P2 and PI(3,4,5)P3 in BJAB and MS19 cells following different means of stimulation (Figure 3.2) provided us with a unique opportunity to study the roles of these two PI 3-kinase products in regulating PKB phosphorylation. In particular, the relative lack of knowledge regarding the function of PI(3,4)P2 in PKB regulation made us want to pursue this question using this model system.  86  Figure 5. 1 The kinetics of PI(3,4,5)P3 and PI(3,4)P2 production in A20 cells. A20 cells were stimulated with rabbit anti-mouse antibodies. Lipid analysis is done as describled in Materials and Methods and Figure 3.1 and Figure 3.2.  Error bars are standard error from three independent experiments.  87 In general, the levels of PI(3,4,5)P3 and PI(3,4)P2 determine the levels of PKB Thr308 phosphorylation and Ser473 phosphorylation, respectively. We investigated how PKB phosphorylation at Thr308 and Ser473 changed in MS19 cells. Thr308 phosphorylation was decreased dramatically in MS19 cells compared to BJAB (Figure 5.2A), which  can be explained by the lower PI(3,4,5)P3 level in MS19 (Figure 3.2). Interestingly, although MS19 cells have dramatically decreased PI(3,4,5)P3, Ser473 phosphorylation was still comparable to that in BJAB cells (Figure 5.2A). Because we and others showed before that PI(3,4)P2 is important in maintaining Ser473 phosphorylation (Scheid et al., 2002a; Carricaburu et al., 2003), we attribute the sustained Ser473 phosphorylation and activity to the sustained PI(3,4)P2 level. These initial experiments were done with 10 µg/ml of either intact antibody or F(ab')2 fragments. Although this dose of intact antibody caused a dramatic decrease in PI(3,4,5)P3 level in MS19 cells than that of BJAB cells, it did not show obvious inhibition of Ser473 phosphorylation. In order to clarify the inhibitory effect of the intact antibody which would be co-ligating BCR and Fc RIIB, we continued with a time course analysis using the same molar concentration of intact antibody (10 µg/ml F(ab')2 antibody and 15 µg/ml intact antibody) (Figure 5.2C), which caused less stimulation of Ser473 phosphorylation compared to F(ab')2. Changes in Thr308 and Ser473 phosphorylation in these two cell lines did not follow the same trend with the former increased and the latter well maintained in MS19 cells (Figure 5.2A and 5.2B); this different trend of changes on these two sites phosphorylation can make one conclude that there must be some PKB molecules that are phosphorylated only on Ser473. If this is the case, we cannot exclude  88 the possibility of the existence of PKB phosphorylated only on Thr308, which will be discussed below. This is an interesting phenomenon due to the uncertain mechanism of Ser473 phosphorylation and the uncertain roles that PI(3,4)P2 and PI(3,4,5)P3 play during the process of PKB membrane recruitment and phosphorylation. It is widely accepted that phosphorylation of PKB at both Thr308 and Ser473 sites occurs at the plasma membrane (Andjelkovic et al., 1997; Thomas et al., 2002; Bellacosa et al., 1998; Watton et al., 1999). Thus, we next separated the plasma membrane and cytosol fractions to further clarify the relationship between plasma membrane PKB protein and phosphoinositide lipids.  89   90  Figure 5. 2 PKB phosphorylation in BJAB and MS19 cells showing that Thr308 phosphorylation and Ser473 phosphorylation correlate with PI(3,4,5)P3 and PI(3,4)P2 levels, respectively. A. Under the same stimulation conditions as used for the lipid analysis shown in Figure 5.1, PKB phosphorylation at Ser473 and Thr308 and PKB activity were analysed. Total cell lysates were analyzed by immunoblotting using antibodies toward the specified phosphorylation sites and corresponding re-blots for loading controls. The panel of another experiment is to show different kinetics of Thr308 phosphorylation in different sets of experiments.  B. Densitometry of PKB phosphorylation at Thr308 and Ser473. Data were normalized with PKB reblotting.  Values shown represent the average values obtained from triplicate analysis of one experiment. This is representative of two separate experiments in which the same trend was observed. C. Stimulation of BJAB or A20 cells was done with intact antibody used at the same molar concentration as the F(ab')2, highlighting the lower Ser473 phosphorylation in response to intact antibody. This figure is representative of five experiments giving similar results.  91               PI(3,4)P2-directed Ser473 phosphorylation is a key determinant of total PKB activity. In both BJAB and MS19 cells, stimulation with either F(ab')2 or intact IgG results in similar increases in PI(3,4)P2, while the levels of PI(3,4,5)P3 are much lower in MS19 due to the expression of active SHIP (Fig. 3.2). Also, as shown in Fig. 5.2, the levels of  Ser473  and  Thr308  phosphorylation  corresponds  to  the  levels  of  PI(3,4)P2 and PI(3,4,5)P3, respectively. As can be seen in Figure 5.3, stimulation with either agonist resulted in parallel increases in PKB activity in both BJAB and MS19 cells. These observations suggest that elevated levels of PI(3,4)P2 , and corresponding increases in PKB Ser473 phosphorylation, are directly correlated with PKB activity measurements, whereas the level of Thr308 phosphorylation does not appear to determine the PKB activity. To further confirm that BJAB and MS19 cells have similar PKB activities, PKB downstream protein GSK-3 beta Ser9 phosphorylation was used to monitor PKB activity. Figure 5.4 shows that BJAB and MS19 cells have similar levels of GSK-3 beta Ser9 phosphorylation. The importance of Ser473 phosphorylation in determining PKB activity was described by Gao et al. (2005) when studying the regulatory function of a phospho- Ser473 phosphatase PH domain leucine-rich repeat protein phosphatase (PHLPP). Thus, our studies in B cells provide supporting evidence that Ser473 phosphorylation is a key determinant of PKB activity.  92 Figure 5. 3 PKB  assay  following  B  cell  stimulation. BJAB or MS19  cells  were stimulated  with  10 µg/ml  F(ab')2   or  intact  antibody  as indicated.  Assay results are representative of at least 4 experiments using both radioactive and non-radioactive assays with either crosstide, Histone H2B or GSK3 protein fragment as substrate. Results shown are results of experiments using crosstide as substrate. There was no difference in the levels of activity in BJAB compared to MS19 cells in all the assays. Figure 5. 4 GSK-3ß Ser9 phosphorylation in BJAB and MS19 cells. BJAB  or  MS19  cells  were stimulated  with  10 µg/ml  F(ab')2   or  intact  antibody  as indicated in Figure 5.3. GSK-3ß Ser-9 phosphorylation in those cells was analysed. Total cell lysates were analyzed by immuno-blotting using antibodies toward phospho-GSK-3ß Ser-9 and p85 re-blots for loading controls.  93 PI(3,4,5)P3 determines PKB activity on membranes. It is widely accepted that PKB is phosphorylated at Thr308 and Ser473 sites at the plasma membrane (Andjelkovic et al., 1997; Thomas et al., 2002; Bellacosa et al., 1998; Watton et al., 1999). Thus, we wished to determine the effects of altered PI(3,4)P2 and PI(3,4,5)P3 levels on phosphorylation  and  activity  of  the  membrane-associated  and  cytosolic  PKB.  We separated the plasma membrane and cytosol fractions from stimulated BJAB and MS19 cells and blotted for PKB Thr308 and Ser473 phosphorylation in each. We found that the quantity of membrane-associated PKB that is phosphorylated at Thr308 correlated with the level of PI(3,4,5)P3 (Figure 5.5 A and B). As expected, there was very little phospho- Thr308 associated with the membrane in MS19 cells, which have very low levels of PI(3,4,5)P3. However, the Ser473 phosphorylation of cytosolic PKB was much higher than that of membrane-associated PKB in MS19 cells and was equivalent to that of cytosolic PKB in BJAB cells (Figure 5.5 A and B), accounting for the equivalent overall levels of PKB Ser473 phosphorylation in the two cells types (Figure 5.2).  It is important to note that as shown from the normalized intensity of the bands, and as shown in the kinase assay data below, there is approximately ten-fold  higher  amounts  of  PKB  in cytosol  compared  to  membrane preparations. Also, preparation of membranes and cytosol yielded much lower basal levels of phosphorylated PKB than what we might have expected based on whole cell lysates of BJAB cells, but this was consistently observed in many experiments. Next we determined the PKB kinase activity that was present in the membrane and cytosol fractions prepared as described above. As can be seen in Figure 5.6, the level of cytosolic PKB activity, which accounts for the majority of total activity, was similar in  94 both BJAB and MS19 cells. In membranes from BJAB cells, there was a substantial increase in the PKB kinase activity following stimulation. However, there was almost no detectable activity in MS19 cell membranes. Comparison of F(ab')2 and intact antibody stimulation consistently showed that there was higher activity following stimulation with F(ab’)2. Together, these results suggest that while cytosolic PKB activity correlated more closely with the level of PI(3,4)P2 , it was clear that the membrane-associated PKB kinase activity correlated with the level of PI(3,4,5)P3. These results show that the production of the two major lipid products that are elevated following activation of PI 3-kinase, PI(3,4)P2 and PI(3,4,5)P3, can have distinct effects in regulating PKB kinase activity. The role of PI(3,4,5)P3 in membrane-localized activity of PKB is evident from the striking reduction in membrane-associated PKB activity in MS19 cells. However, we have also shown an essential role for elevated PI(3,4)P2 in  determining  the  activity  of  cytosolic  PKB,  which  represents  the  major component of total PKB activity in cells. Our data suggest that elevated PI(3,4)P2, together with elevated Ser473 phosphorylation, can compensate for a reduced level of PI(3,4,5)P3 and correspondingly lower levels of Thr308 phosphorylation. However we cannot rule out the possibility that the total PKB activity may still be mediated by a small fraction of the total PKB enzyme pool which is doubly phosphorylated at both these sites. Even if the latter were true, an important conclusion that we can make from our data is that experimental measurement of the level of Ser473 phosphorylation must be considered a more accurate reflection of cellular PKB activity than the measurement of phosphorylation at Thr308.  95   96  Figure 5. 5 Membrane preparation to show the membrane and cytosol portion of phosphorylated PKB and total PKB. Cell stimulation with either F(ab')2 antibody or intact antibody and sample preparation are described in Materials and Methods. Membrane preparation was made at the indicated time points following stimulation of BJAB and MS19 cells. A. Membrane and cytosol preparations were blotted with anti-phospho-Thr308 PKB and anti-phospho- Ser473 PKB. All blots were exposed for the same time in order to measure the relative quantity of membrane/cytosol distribution in B. Densitometry values are shown below the corresponding bands. All results are representative of at least three independent experiments. B. Quantification of different forms of phosphorylated PKB in the membrane versus cytosol. Data were normalized by cell equivalents, which are 15 µl loading of membrane (107 cell equivalents/100 µl) and 15 µl loading of cytosol (107 cell equivalents/800 µl). Densitometry was done with FluorChem Imaging system and arbitrary values were given by the system. Error bars are standard errors from the three independent experiments. Differences between the average values of MS19 and BJAB cells were significant (p 0.01) except for the level of cytosolic phospho-Ser473. student’s t-test was used for statistic analysis.  97  Figure 5. 6 PKB kinase activity in membrane and cytosol fractions. Membrane and cytosol preparations, as prepared in Fig. 5.5, using 107 cell equivalents per sample, were solubilized and PKB immunoprecipitated prior to kinase assay. The radioactive kinase assay was performed using crosstide as substrate. Results are averaged from 2 duplicate samples. This is representative of more than three independent experiments.  98 Recruitment of EGFP-PKB to plasma membrane correlates with levels of PI(3,4)P2. Investigations of the in vivo binding of the PKB PH domain to phosphoinositides have yielded divergent results  and  conclusions  regarding  the  roles of  PI(3,4,5)P3 and  PI(3,4)P2 in  PKB membrane recruitment (Gray et al., 1999; Astoul et al., 1999; Costello et al., 2002). Two papers from Cantrell’s group utilized the PKB PH domain as a means of monitoring PI(3,4,5)P3  kinetics (Astoul et al., 1999; Costello et al., 2002). On the other  hand, Downes’ group  showed  that  the  kinetics  of  PI(3,4,5)P3 correlated  with  membrane association of the GRP1 PH domain, and the PI(3,4)P2 correlated with  recruitment of the PKB PH domain (Gray et al., 1999). To further investigate the kinetics of PKB membrane recruitment relative to PI levels in the BJAB model, our collaborator, Dr. Aaron Marshall used an EGFP-PKB construct to monitor its membrane vs. cytosolic distribution using confocal microscopy and digital image analysis. Figure 5.7 shows a comparison of membrane EGFP-PKB recruitment in BJAB cells treated with F(ab')2 or intact anti-IgM. In both cases, the level of EGFP-PKB at the membrane is increased within 1 min, and is sustained for up to 30 min, which correlates more closely with the levels of PI(3,4)P2 in these cells. Dr. Marshall’s lab also investigated the membrane/cytosol distribution of EGFP-PKB in MS19 cells. Surprisingly, despite the dramatically lower level of PI(3,4,5)P3 in MS19 cells, recruitment of EGFP-PKB was similar to that seen in BJAB cells. Comparison of F(ab')2 and intact antibody stimulation showed that the level of recruitment was lower in cells treated with intact antibody. Again, these membrane recruitment data suggest that the association of PKB with the plasma membrane is dependent upon the level of PI(3,4)P2. An alternative explanation may be that PKB recruitment is dependent upon elevated  99 levels of either PI(3,4,5)P3 or PI(3,4)P2, but importantly, our results suggest that an elevated level of PI(3,4)P2 can substitute for PI(3,4,5)P3 in these events.  100 Figure 5. 7 Live cell imaging of GFP-flag-PKB. (Study performed by Dr. Aaron Marshall as a collaborative project.) A. E-GFP-flag-PKB in BJAB cells, following F(ab')2 or intact antibody stimulation, was monitored by live cell confocal imaging to determine the relative level of PKB membrane recruitment from 1 minute till 30 minutes. B. EGFP-flag-PKB in MS19 cells was monitored as in A. Cells were stimulated with F(ab')2 or intact antibody as indicated for 1 to 5 min. Detailed method are describled in Ma et al. (2008).  101 PKB complex of 400kDa exists only on plasma membrane, and does not change with stimulation. We hypothesized that the complex kinetics of PKB membrane recruitment and detachment might involve binding of PKB to some other proteins on the plasma membrane. Separation of cell extracts by gel filtration showed that PKB was found within a protein complex of around 400kDa at the plasma membrane, but not in cytosol (Figure 5.8.A.). Similar results were observed in both BJAB parental cells and stable cell lines over-expressing flag-PKB mutants. In cytosol, only PKB monomers can be identified, suggesting that any association between PKB and cytosolic proteins must be transient and cannot be detected by this gel filtration approach. Unlike the cytosolic PKB monomer, membrane PKB exists both within the complex and as a monomer, suggesting that the function of the membrane PKB association might be different from the kinase-substrate function in cytosol. Separation of PKB on a longer gel showed that both the PKB monomer and the PKB complex appeared to be phosphorylated based on the presence of a slower migrating form of PKB (Figure 5.8.B). This upper band shows up only after stimulation and can be detected by PKB antibody, thus it is likely to be the active phospho-PKB. Similar results were observed in Flag-473D mutant. It is interesting to notice that Flag-PKB-473D mutant showed a similar migration on SDS polyacrylamide gel as the potential phosphorylated Flag-PKB.  102   103 Figure 5. 8 PKB forms a large protein complex at the plasma membrane, but not cytosol. A. Superdex100 gel filtration shows that PKB is present in a protein complex of around 400kDa only at the membrane, but not in cytosol. The Flag-tagged PKB protein, or the Flag-PKB-473D mutant was detected with anti-Flag antibody. The cytosol samples were over-exposed compared to the membrane to show that none of the high molecular weight complex containing PKB could be detected. Numbers are FPLC column collections. B. Plasma membrane portion of BJAB cells and stable cell lines over-expressing Flag- PKB. Western blot with anti-PKB antibody. BJAB parental cells unstimulated and stimulated and Flag-PKB over-expressing cells of wildtype and 473D showed similar pattern in protein complex and PKB monomer. Numbers are FPLC column collections. BJAB cells were stimulated with F(ab')2 for 10 minutes to show the stimulation condition.  104 5.3 Discussion The PI 3-kinase/PKB pathway is a well-established survival pathway, in which PKB phosphorylation is entirely dependent upon PI 3-kinase activity. Although it is known that PI 3-kinase has two main products, PI(3,4,5)P3 and PI(3,4)P2, which are responsible for PKB activation, the exact functions of these two lipids in PKB activation are still not completely characterized. In vitro studies showed that either PI(3,4,5)P3 or PI(3,4)P2 can activate PKB to its full extent (Vanhaesebroeck and Alessi, 2000). Due to the incompletely characterized mechanism of PKB Ser473 phosphorylation and the importance of this phosphorylation site (Gao et al., 2005), it is important to understand how PI(3,4)P2 and PI(3,4,5)P3 regulate PKB phosphorylation in vivo. A prior study from our laboratory using mast cells from SHIP knockout mice showed that PI(3,4,5)P3 is essential, but not sufficient for PKB activation. It was shown that PI(3,4)P2  is necessary for full activation of PKB (Scheid et al., 2002a). In this study, we tried to continue to pursue this question by probing into the mechanism by which PI(3,4)P2 regulates Ser473 phosphorylation. We used a B cell system in which we had studied the PI(3,4)P2 binding proteins TAPP2 and Bam32 in Chapter 3 (Krahn et al., 2004). There have been several studies showing SHIP’s negative effect on PKB in B cells (Aman et al., 1998; Jacob et al., 1999). Those authors attributed the decreased Thr308 and Ser473 phosphorylation levels to SHIP’s function of lowering PI(3,4,5)P3 level. However, due to the uncertainty of the mechanism by which Ser473 is phosphorylated, it has never been shown that the level of PI(3,4,5)P3 is directly correlated with Ser473 phosphorylation. We thought it was important to clarify the exact relationship between Ser473 phosphorylation and PI(3,4,5)P3 and/or PI(3,4)P2 in B cells  105 based on the numerous prior publicatons in this cell system. Interestingly, in another study using several other human malignant B cell lines (Choi et al., 2002), the authors did not see any inhibitory effects of SHIP or SHIP2 on PKB. Instead, they saw PTEN’s inhibitory effects on PKB activity in the same cell lines. Both SHIP and PTEN can reduce PI(3,4,5)P3 levels, but SHIP does not dephosphorylate PI(3,4)P2, so reduction in PI(3,4)P2  rather than PI(3,4,5)P3 was causing this difference. The difference between the functions of SHIP and PTEN as inositol phosphatases is that PTEN dephosphorylates phosphoinositide at the D-3 position, which would emphasize the importance of D-3 phosphoinositides other than PI(3,4,5)P3. PTEN can also use PI(3,4)P2 as substrate but SHIP cannot (Walker SM, 2001; Leslie and Downes, 2002), which can be an important factor. PTEN decreases the amount of both of these lipids, while SHIP just modifies the lipid ratio of PI(3,4,5)P3/PI(3,4)P2. We can regard PI(3,4,5)P3 and PI(3,4)P2 as the products of PI 3-kinase that serve as “core lipids”. PTEN and SHIP have different effects in regulating the levels of these “core lipids”, which is shown in Figure 5.9. Unlike SHIP, PTEN directly regulates the quantity of the “core lipids”. In this study, we showed more clearly that PI(3,4,5)P3 and PI(3,4)P2 have distinct functions in determining PKB phosphorylation. PI(3,4)P2, but not PI(3,4,5)P3, is important in determining the quantity of Ser473 phosphorylation, which is a novel regulatory mechanism of PKB phosphorylation by PI 3-kinase products. As introduced in Chapter 1.1.10, although different kinases has been identified to be the Ser473 kinase in different cells, our data and those of others have shown that in B cells, mast cells and HEK293 cells (Scheid et al., 2002a; Carricaburu et al., 2003; Ma et al, 2008), PI(3,4)P2  levels correlate with Ser473 phosphorylation level, suggesting that this is a common phenomenon and there  106 might be a common mechanism by which PI(3,4)P2  controls the phosphorylation of PKB Ser473. In vitro studies showed that PI(3,4,5)P3 and PI(3,4)P2 have a similar effect on PKB phosphorylation at Thr308 and Ser473 sites (Alessi et al., 1997). The reason may be that PI(3,4,5)P3 and PI(3,4)P2 have a similar effect in changing the conformation of PKB and thus releasing the inhibitory effect of the PH domain on PKB activation. Structural studies have provided further details about the mechanism, showing that the D3 and D4 position phosphates fit into the PH domain, while the D5 phosphate does not form significant interaction with any residue on the PH domain, which explains why PKB interacts with similar affinity with both PI(3,4)P2 and PI(3,4,5)P3 (Thomas et al., 2002). Our data demonstrated that in vivo the mechanism for phosphoinositide regulation on PKB phosphorylation is more complicated. In each of our analysis, we have clearly shown that PI(3,4)P2 levels can influence Ser473 phosphorylation independently of the levels of  PI(3,4,5)P3 and phosphorylation at Thr308, the latter two also being co- regulated. In terms of PKB kinase activity, we have shown that overall activity is determined by the level of PI(3,4)P2, rather than PI(3,4,5)P3. However, when assaying the kinase activity present in membrane preparations, there was a clear correlation with the level of PI(3,4,5)P3. This discrepancy may be due to regulation of the kinetics of PKB dissociation from the plasma membrane, which is not yet well understood, but is a key event since the majority of PKB targets are in the cytosol and nucleus. Recently, an interesting potential role for Ca2+/calmodulin in this process was reported (Dong et al., 2007). Calmodulin was found to bind to the PH domain of PKB and potentially disrupt its binding to lipids, thereby implicating elevated Ca2+ levels in the regulation of PKB  107 dissociation. Another interesting question is the involvement of the Ser473 kinase, identified by various means in many studies, and its potential direct or indirect regulation by PI(3,4)P2. Figure 5.10.A shows a summary of functions of PI(3,4,5)P3  and PI(3,4)P2  in determining PKB phosphorylation and activity. Ananthanarayanan et al. (2007) proposed that PKB membrane detachment is an active process which is determined by PKB phosphorylation and could influence PKB activity. We proposed a model that is supplemental to this idea of active detachment (Figure 5.10.B). Due to methodological limitations, although we cannot know at the current stage that this active detachment process is mediated by PI(3,4,5)P3  or PI(3,4)P2,  it is possible that the PI(3,4)P2- determined cytosolic PKB Ser473 phosphorylation may be regulated by PI(3,4)P2 itself, with or without the help of a potential PKB protein complex. However, there is also some other possibilities, for example, PI(3,4)P2  may down-regulate some cytosolic phosphatase which could also increase the cytosolic PKB phosphorylation. No matter what the more detailed mechanism is, this part of the study suggest that we need to consider the roles that both lipids may play when we investigate PI 3-kinase’s regulation on PKB function, which includes membrane recruitment, phosphorylation and membrane detachment. Besides, we think that PIP’s regulation of PKB may not be performed by lipids themselves, but with the help from other proteins on membrane. In conclusion, we have investigated PI 3-kinase-generated lipid products following B cell stimulation, and determined their effects on PKB regulation. We have shown that PI(3,4)P2 levels determine the level of PKB phosphorylation at Ser473, while PI(3,4,5)P3 levels determine the level of PKB phosphorylation at Thr308. Furthermore,  108 while membrane-associated PKB activity is determined by levels of PI(3,4,5)P3, the association of PKB with membranes, as measured by monitoring a EGFP-tagged PKB, appears to be determined primarily by levels of PI(3,4)P2. Furthermore, PI(3,4)P2 levels are a direct indicator of cytosolic PKB kinase activity, which represents the majority of the kinase activity. Thus, the balance of PI(3,4,5)P3/PI(3,4)P2, which is regulated by enzymes such as PTEN and SHIP, regulates a complex set of events that together determine the downstream effects of PKB.  109 Figure 5. 9 Functions of PTEN and SHIP in regulating lipid levels and downstream signaling in B cells. Being produced by PI 3-kinase, PI(3,4,5)P3 and PI(3,4)P2 are located in the center of PI 3-kinase signaling. PTEN decreases the levels of both PI(3,4,5)P3  and PI(3,4)P2, while SHIP modifies the ratio of these two lipids by turning PI(3,4,5)P3  into PI(3,4)P2. PKB PI(3,4,5)P3 PI(3,4)P2 Bam32 Btk TAPP 308P 473P SHIP PTENPTEN PI 3-kinase products PI3K PI3K Downstream signaling  110 A   111 B  Figure 5. 10 Model of roles of PI(3,4,5)P3 and PI(3,4)P2 in regulating PKB. As shown in Figure A, PI(3,4,5)P3 and PI(3,4)P2 have specific roles in determining the distribution of Thr308 phosphorylation levels, Ser473 phosphorylation levels and PKB activity in the membrane and cytosol. Figure B combines the discussions in 1.1.9 and Ananthanarayanan et al., as well as our model of PIP lipids regulating PKB membrane recruitment and association. Both lipids are involved in PKB membrane recruitment. Phosphorylated PKB is actively detached from membrane (Ananthanarayanan et al., 2007).  We propose that PIP lipid(s) may also be involved in this active detachment process, which determines the cytosolic PKB phosphorylation level.  112 Chapter 6. Summary 6.1 General conclusions The initial study not shown in this thesis was that the in vivo PI(3,4)P2 production following BCR stimulation corresponds to the kinetics of membrane recruitment of TAPP (Marshall et al. 2002). Comparison of phosphoinositide production in BJAB cells (Figure 3.2) and A20 cells (Figure 5.1) shows that BJAB cells have much higher levels of PI(3,4,5)P3 and PI(3,4)P2. The abnormal phosphoinositide metabolism in BJAB cells might contribute to the oncogenesis process. A better understanding of how this process is regulated might help to design treatment plans in some cases of lymphoma. Multiple myeloma, which is another lymphoid malignancy, is a disorder of plasma cells. Evidence showed that PTEN is down-regulated in multiple myeloma (Choi et al., 2002), and PI 3- kinase inhibitors selectively kill PTEN-null myeloma cells (Zhang et al., 2003). Although our initial work showed the same kinetics of PI(3,4)P2  production and TAPP membrane recruitment (Marshall et al., 2002), based on which the firm correlation between PI(3,4)P2  and TAPP recruitment was established, further investigation in Chapter 3 using a more complicated model system suggested that the relationship between PI(3,4)P2  and TAPP recruitment is not just this simple (Krahn et al., 2004). Following stimulation with intact antibody, we observed decreased levels of both PI(3,4)P2 and PI(3,4,5)P3 compared to stimulation with F(ab’)2. However, the changes of TAPP PH domain recruitment and Ser473 phosphorylation are different: TAPP PH domain recruitment increased with intact antibody stimulation compared to F(ab’)2  113 stimulation, while Ser473 phosphorylation decreased with intact antibody stimulation compared to F(ab')2 stimulation. The conclusion based on these data is that TAPP PH domain recruitment inversely correlates with PI(3,4,5)P3/ PI(3,4)P2 ratio (Chapter 3), while Ser473 phosphorylation correlates with PI(3,4)P2 levels (Chapter 5). Although both TAPP recruitment and PKB Ser473 phosphorylation correlate with PI(3,4)P2, our data from different model system suggest that these correlations are not exactly the same. Similar to bone marrow mast cells, in B cells we saw again the correlation between levels of PI(3,4)P2 and PKB Ser473 phosphorylation, which can be independent of PI(3,4,5)P3 levels and PKB Thr308 phosphorylation. PI(3,4)P2 might contribute to PKB kinetics in two different ways: one is membrane recruitment, which is demonstrated by the sustained increase of both PI(3,4)P2 production and PKB plasma membrane recruitment up to 20 minutes; the other is membrane association, which is suggested by the accumulation of Ser473-phosphorylated PKB only in cytosol. In BJAB cells, the production of PI(3,4)P2 can only be detected after 1 minute, while the peak production of PI(3,4,5)P3 can be detected at 30 seconds or even earlier. In support of the dogma that PI(3,4,5)P3 is responsible for the plasma membrane recruitment of PKB, our studies suggested that PI(3,4,5)P3 correlates with earlier PKB membrane recruitment, but both PI(3,4,5)P3  and PI(3,4)P2  may be involved in this recruitment process. Another major part of these studies showed that different doses and forms of BCR ligands induce distinct production of PI(3,4)P2 and PI(3,4,5)P3 compared to higher dose. Lower dose of F(ab')2 produced higher levels of PI(3,4,5)P3 and lower levels of PI(3,4)P2 than higher dose. The immunological significance of this finding needs further clarification since this kind of titration study on PI 3-kinase has never been shown.  114 However, the results of this study also emphasized the importance of PI(3,4)P2 in cell signaling. We showed that intact antibodies actually have better BCR binding than F(ab')2, which is the reason that lower dose of intact antibodies produce higher PI 3- kinase activation than F(ab')2. As discussed in Chapter 4, this might be an artificial effect of F(ab')2 antibody production or it could possibly have in vivo immunogical significance. However, this question cannot be answered by in vitro stimulation at this stage. The regulation of PH domain containing proteins might not be solely via phosphoinositides. The regulation of Ser473-phosphorylated PKB by PI(3,4)P2 has suggested this possibility. The specific kinetics of the membrane recruitment and detachment of Ser473 phosphorylated PKB suggested that this process might also involve other proteins.  By over-expression of Flag-PKB, we identified a variety of potential PKB-associated proteins, but they were not readily detected following immunoprecipitating of endogenous PKB (see Appendix). Even though they are not detectable, the possibility still exists that these represented transient associations that were below detection limits when immunoprecipatating endogenous PKB. I did obtain some evidence of possible PKB association with other proteins by showing that PKB formed a large protein complex only on the plasma membrane, but not in cytosol. Thus the PKB-associated proteins we tried to identify are possibly related to PKB’s function on the plasma membrane. More studies will have to be pursued to further characterize the potential PKB-associated proteins, but we cannot rule out the possibility that the high molecular weight complex formed by PKB was due to aggregation of PKB molecules at the plasma membrane.  115 PIP’s regulation of PKB function were summarized in Figure 5.10A. We realized that there is no perfect match of Thr308 phosphorylation and PI(3,4,5)P3  kinetics in BJAB cells (Figure 5.2A). The relationship between PI(3,4,5)P3  and PKB Thr308 is not obviouse,  which is the reason that we need to develop MS19 cells to uncover the potential mechanisms. There are several factors that need to be considered in the process of PKB Thr308 phosphorylation: PKB membrane recruitment, activity of  the PKB Thr308 kinase PDK1 and PDK1 membrane kinetics. Because PKB is downstream of PI 3-kinase, lipids production is an indispensable factor in PKB Thr308 phosphorylation, however, it is not the only factor. Therefore, we do not simply see the correlation between PI(3,4,5)P3  and PKB Thr308 phosphorylation in most of the circumstances. This is also why we need to develop different model systems and strategies like MS19 cells and different antibody stimulations to elucidate the underlying mechanisms. Basing on the study in Chapter 5, we can also draw some conclusions regarding the properties of the potential PKB Ser473 kinase. The levels of PI(3,4,5)P3  determine the levels of PKB Ser473 phosphorylation on membrane. PKB is phosphorylated on membrane, therefore we think that the process PKB Ser473 phosphorylation may also be under the influence of PI(3,4,5)P3, and the kinase may be just on the membrane. This supports the data by Hill et al. (2002) showing that this potential kinase is located on plasma membrane. Our prior study showed that PI(3,4)P2  may contribute to Ser473 phosphorylation in some uncertain way (Scheid, et al., 2002a). The current study showed more evidence and investigated the potential mechanisms of PI(3,4)P2’s contribution to Ser473 phosphorylation in more depth. Interestingly, data from Alessi’s group showed that there is a PKB Ser473 phosphatase called protein tyrosine phosphatase (PTPL1)  116 working downstream of TAPP (Kimber et al., 2003).  TAPP is positively correlated to the levels of PI(3,4)P2. According to that study, PI(3,4)P2  should down-regulate PKB Ser473 phosphorylation. Unfortunately, this has never been observed in any our model system in B cells, mast cells and others in HEK293 cells (Ma et al., 2008; Scheid et al., 2002a; Carricaburu et al., 2003). Different methods unavoidably have their limitations. Traditional biochemical method is to lyse the cells completely; whereas cell biology techniques keep the cells intact but do not own the advantage of biochemical separations. The final model that we proposed regarding the PIP’s regulation of PKB function involves the active membrane detachment of PKB (Figure 5.10B), which is largely a cell biology process and supported later by FRET. The GFP and FRET techniques just won the 2008 Nobel Prize, which laureates the importance of cell biology methods in modern life science research. This study merits the combination of completely different expertises by drawing a series of conclusions in PIP’s regulation of PH domain and PH domain-containing protein membrane recruitment, PKB membrane recruitment/detachment and PKB phosphorylation and activity.  117 6.2 Future directions A number of the studies undertaken in this work have lead to further questions that should be addressed in future studies, as outlined below. 1. Functional studies of phosphoinositides. There are many different inositol phosphatases, kinases and phosphoinositides present in different cellular compartments whose actions must be coordinated to create a highly organized network. How the phosphoinositides interact with the various proteins to influence downstream cell functions requires a systemic study to reveal the whole scenario in cells. With the realization of the importance of lipid research, study of the complex functions of lipids has been termed “lipidomics”. The conjugation of lipidomics and proteomics might help in the studies of signal transduction downstream of lipid and lipid metabolizing enzymes. For example, the directions could be to look at the whole scenario of dynamic changes of both phosphoinositides and proteins following cell stimulation. 2. The relationship between phosphoinositides and PH domain containing proteins such as PKB. PH domains performs their functions not only by binding phosphoinositides, but also by interacting with other proteins. A series of PKB PH domain mutants may be able to reveal in more detail how PKB protein associations influence PKB downstream signaling. The A20 and BJAB cell lines are still good models to develop this story further. Similarly, other important PH domains  in B cells such as Btk can be also tested to look at the specificity and universality of the characteristics of these PH domains. Proteomics and  118 bioinformatics approach can be utilized with these PH domain mutants to get a whole profile of protein associations and phosphoinositide binding, each of which may influence the other. 3. PI 3-kinase pathway in B cells. Understanding of the of PI 3-kinase activity under some other stimulation conditions such as via CD40, or Toll-like receptors is still at an early stage. In vivo stimulation of B cells is far more complicated than simply studying BCR stimulation. How the PI 3-kinase/PKB pathway is activated under the co-ordinated stimulation of different receptors in vivo also needs further investigation. 4. PKB Ser473 kinase. Different kinases have been identified in different cell types. These candidate kinases can be knocked down by RNAi to find which is the major Ser473 kinase in B cells. After identification of this kinase, kinase activity can be assayed in BJAB cells to look at the correlation between PI(3,4)P2 production and this kinase activity to further demonstrate the relationship between phosphoinositide lipids and the activity of the identified Ser473 kinase in B cell lines. 5. The kinetics of Ser473-phosphorylated PKB. Recently the FRET technique was adapted to show the kinetics of membrane detachment of Thr308 phosphorylated PKB (Ananthanarayanan et al., 2007). A similar study could also be used in the study of Ser473 phosphorylated PKB. 6. Role of the cytoskeletion in PIP lipid function and PKB phosphorylation. Monitoring the level of PI(3,4)P2 by means of TAPP’s membrane/cytosol distribution strongly suggested that this lipid can be co-localized with  119 cytoskeleton (Hogan et al., 2004; Marshall et al., 2002). There is evidence showing that PKB is associated with cytoskeleton, which acts as a scaffold (Cenni et al., 2003). No matter what the Ser473 kinase is in different cell systems, Ser473 phosphorylation level is correlated to PI(3,4)P2 levels. In this study we have shown that membrane Ser473 phosphorylation is correlated to the level of PI(3,4,5)P3 at the membrane. Cytoskeleton disruption and/or lipid delivery conditions can be developed to elucidate the relationships among cytoskeleton, lipids, PKB Ser473 kinase and PKB kinetics. 7. PI 3-kinase inhibitors and lipid metabolism modifying drugs. Drug that inhibit PI 3-kinase have shown some exciting progress in rheumatoid arthritis, myocardial infarction, and respiratory inflammatory diseases. 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Yu JW, Audhya A, Singh S, Keleti D, DeWald DB, Murray D, Emr SD, Lemmon MA. (2004) Genome-wide analysis of membrane targeting by S. cerevisiae pleckstrin homology domains. Mol. Cell. 13, 677-688.  137 Zamorano J, Wang H, Wang L, Pierce J, and Keegan A. (1996) IL-4 protects cells from apoptosis via the insulin receptor substrate pathway and a second independent signaling pathway. J. Immunol.157, 4926-4934. Zhang J, Choi Y, Mavromatis B, Lichtenstein A, Li W. (2003) Preferential killing of PTEN-null myelomas by PI3K inhibitors through Akt pathway. Oncogene. 18;22(40):6289-95.  138 Appendix. Proteomics and lipidomics: Flag-PKB overexpression. Introduction Over 20 proteins have been found to associate with PKB (Brazil et al., 2002; Du and Tsichlis, 2005). Many of these protein associations influence PKB activity in some way. For example, one prior study showed that heat shock protein Hsp90 increases PKB activation, and disruption of the association between heat shock protein and PKB decreased PKB activation and lead to apoptosis (Sato et al., 2000).  A recent report also suggested that calmodulin interacts directly with PH domain of PKB (Dong et al., 2007). Although there are considerable data regarding PKB associated proteins, we realized that there is no published proteomic data of PKB associated proteins to date. After the finding of a PKB complex on plasma membrane (Figure 5.10.), we proposed to get a profile of PKB associated proteins by means of Flag-PKB overexpression and Flag immunoprecipitation.  139 Results and Discussion             Figure appendix-1 shows the expression of of Flag-PKB-wildtype and Flag-PKB- 473D in BJAB cells. Like the original transient expression plasmid (Sato et al., 2000), Flag-PKB protein runs at the same level as endogenous PKB on SDS-PAGE gel. After Flag-IP with whole cell lysate from the two cell lines, silver staining showed that several specific bands were immunoprecipitated by anti-Flag  (Figure appendix-2). Each specific band was cut and sent for mass spectrometry for identification. Results were shown in table appendix-1A. Surprisingly, all of the 9 bands turned out to be PKBa, except one is PABP (poly-A binding protein) (Table appendix 1.A). There are two possibilities that could explain why many bands from MW 150 to 50 kDa were identified as PKB. One is that PKB might form dimers or trimers. Another is that in PKB might form strong associations with other proteins that are hard to separate on SDS-PAGE. Evidence of the first possibility comes from TCL (T cell lymphoma protein) studies. TCL protein, as a PKB coactivator, can cause PKB oligomirization (Kunstle et al., 2002). Earlier work also showed that PKB forms dimers through PH domain (Datta et al., 1995). A model was proposed in which PKB dimer formation is important for its activation (Gold, 2003), based on which we conclude that PKB may also forms trimer under some circumstances. The band below Flag-PKB is likely due to normal degradation of over-expressed PKB protein. This degraded Flag-PKB protein might oligomirize as well, forming even more bands at high molecular weight on SDS- PAGE gel.  140 The gel that was left over after the 9 bands were cut out in table appendix-1.A. was divided again into four equal segments and sent for mass spectrometry. The results are shown in table appendix 1.B. The four segments are from high molecular weight to low molecular weight. BJAB-4 and W14-4 are the gel cut below the PKB band. Compared to control immunoprecipitation in parental cells, flag tagged PKB over- expression cells produced more protein binding after Flag immunoprecipitation (Table Appendix 1.B.). Next we tested the immunoprecipitated proteins with Western blotting. We chose two candidate proteins in this profile, HSP90 and EF-2. However, Western blotting failed to show that these two proteins indeed associated with PKB in either parental cells or Flag-PKB over-expressed cells. PKB was found to associate with the cytoskeleton, which means PKB subcellular movement is facilitated by interaction with non-substrate ligands (Cenni et al., 2003). Theoretically, this type of association should be detected biochemically, and if so, should result in PKB migrating at a high molecular weight in size exclusion chromatography. The PKB N-terminal PH domain may mediate the interaction between PKB and cytoskeleton. This interaction is modulated by growth factor stimulation (Cenni et al., 2003). Also, this interaction seems to be related to Thr308 and Ser473 phosphorylation. Mutants to alanine abrogated the interaction between PKB and actin. As discussed previously in Chapter 5, PKB may also associate with other proteins through its PH domain. However, our knowledge of protein ligands of the PKB PH domain is still minimal, especially how these associations may influence PKB membrane kinetics and activity. The dual binding property of PKB PH domain for both phosphoinositide and  141 protein needs further clarifications with more sensitive proteomic approaches in the future.  Figure appendix 1  Flag-PKB over-expression in BJAB cells. Two BJAB cell lines stably expressing Flag-tagged wild type (W28) or 473D (D14) PKB were chosen for analysis. The D14 and W28 cells had more than a 10-fold higher expression of PKB than the parental BJAB cells (left panel). As a loading control, the equal expression of p85 was detected in all cell types (right panel).  142 Figure appendix 2 Silver staining and Coomassie staining of lysates after anti-Flag immunoprecipitation. Lysates of BJAB, BJAB-D14, and BJAB-W28 cells (50×106 cells) were used for immunoprecipitation of Flag-PKB using anti-Flag antibody conjugated to beads. Beads were washed Five times with TBS (A) or with TBA containing 500mM NaCl (high salt wash) (B and C), and the bound protein were resolved by SDS-PAGE and detected by silver staining ( A and B) or by staining with Coomassie Blue (C).  143 Table Appendix-1.A. Mass-spectrometry data of Flag immunoprecipitation bands The bands on the SDS-PAGE gel shown in Figure Appendix 2.C were cut and sent to UBC biomedical research center proteomic facility for the analysis of the eluted protein after immunoprecipitating with anti-Flag antbody-conjugated beads from the lysate of the stably transfected cells shown in Figure Appendix 2.C. Only Nine obvious bands were cut. Their samples from #1 to #9 are from high to low MW. Sample ID Accession # Mass Peptide matched Protein name #1 P31749 56050 15 (AKT1_HUMAN) RAC-alpha serine/threonine-protein kinase (EC  (RAC-PK-alpha) (Protein kinase B) (PKB) (C-AKT) #2 P31749 56050 2 (AKT1_HUMAN) RAC-alpha serine/threonine-protein kinase (EC  (RAC-PK-alpha) (Protein kinase B) (PKB) (C-AKT) #3 P31749 56050 3 (AKT1_HUMAN) RAC-alpha serine/threonine-protein kinase (EC  (RAC-PK-alpha) (Protein kinase B) (PKB) (C-AKT) #4 P31749 56050 7 (AKT1_HUMAN) RAC-alpha serine/threonine-protein kinase (EC  (RAC-PK-alpha) (Protein kinase B) (PKB) (C-AKT) #5 P31749 56050 5 (AKT1_HUMAN) RAC-alpha serine/threonine-protein kinase (EC  (RAC-PK-alpha) (Protein kinase B) (PKB) (C-AKT) #6 P31749 56050 13 (AKT1_HUMAN) RAC-alpha serine/threonine-protein kinase (EC  (RAC-PK-alpha) (Protein kinase B) (PKB) (C-AKT) #7 P31749 56050 8 (AKT1_HUMAN) RAC-alpha serine/threonine-protein kinase (EC  (RAC-PK-alpha) (Protein kinase B) (PKB) (C-AKT) #8 P11940 52032 5 (PABP1_HUMAN) Splice isoform 1; Variant Displayed; from P11940 Polyadenylate-binding protein 1(Poly(A)-binding protein 1) #9 P31749 56050 7 (AKT1_HUMAN) RAC-alpha serine/threonine-protein kinase (EC  (RAC-PK-alpha) (Protein kinase B) (PKB) (C-AKT)  144 Table appendix-1.B. Mass-spectrometry data of Flag immunoprecipitation bands Sample ID Accession # Mass Total Ion Scores Protein Name Peptide Matched BJAB-1 PAB1_HUMAN 70854 242 (P11940) Polyadenylate-binding protein 1  8 IF4B_HUMAN 69240 195 (P23588) Eukaryotic translation initiation factor 4B  9 KRAC_HUMAN 56080 162 (P31749) RAC-alpha serine/threonine-protein kinase  7 HS9B_HUMAN 83423 92 (P08238) Heat shock protein HSP 90-beta 3 W14-1 KRAC_HUMAN 56080 1659 (P31749) RAC-alpha serine/threonine-protein kinase 39 HS9B_HUMAN 83423 935 (P08238) Heat shock protein HSP 90-beta  19 HS9A_HUMAN 84889 946 (P07900) Heat shock protein HSP 90-alpha  18 SKB1_HUMAN 73322 375 (O14744) Protein arginine N-methyltransferase 5 9 EF2_HUMAN 96115 200 (P13639) Elongation factor 2 6 TRAL_HUMAN 80246 95 (Q12931) Heat shock protein 75 kDa, mitochondrial precursor 2 IF4B_HUMAN 69240 83 (P23588) Eukaryotic translation initiation factor 4B  3 THIO_HUMAN 11884 74 (P10599) Thioredoxin (ATL-derived factor) 2 BJAB-2 TCPZ_HUMAN 58313 270 (P40227) T-complex protein 1, zeta subunit  8 KRAC_HUMAN 56080 293 (P31749) RAC-alpha serine/threonine-protein kinase 10 TCPQ_HUMAN 60022 306 (P50990) T-complex protein 1, theta subunit 9 EF11_HUMAN 50451 254 (P68104) Elongation factor 1-alpha 1 6 TCPA_HUMAN 60819 278 (P17987) T-complex protein 1, alpha subunit 8 TCPG_HUMAN 60934 250 (P49368) T-complex protein 1, gamma subunit 6 TCPH_HUMAN 59842 245 (Q99832) T-complex protein 1, eta subunit 7 TCPE_HUMAN 60089 149 (P48643) T-complex protein 1, epsilon subunit 5 TCPB_HUMAN 57663 142 (P78371) T-complex protein 1, beta subunit 6 TCPD_HUMAN 58401 107 (P50991) T-complex protein 1, delta subunit 2 EF1G_HUMAN 50298 104 (P26641) Elongation factor 1-gamma 3  145 W14-2 KRAC_HUMAN 56080 1786 (P31749) RAC-alpha serine/threonine-protein kinase 36 TCPQ_HUMAN 60022 574 (P50990) T-complex protein 1, theta subunit 11 TCPH_HUMAN 59842 554 (Q99832) T-complex protein 1, eta subunit  11 SKB1_HUMAN 73322 414 (O14744) Protein arginine N-methyltransferase 5 10 TBAK_HUMAN 50804 255 (P68363) Tubulin alpha-ubiquitous chain 7 TCPD_HUMAN 58401 244 (P50991) T-complex protein 1, delta subunit 6 TCPG_HUMAN 60934 242 (P49368) T-complex protein 1, gamma subunit 5 HSB1_HUMAN 22826 223 (P04792) Heat-shock protein beta-1 5 TCPZ_HUMAN 58313 201 (P40227) T-complex protein 1, zeta subunit 5 TBB1_HUMAN 50095 214 (P07437) Tubulin beta-1 chain 6 TCPB_HUMAN 57663 160 (P78371) T-complex protein 1, beta subunit 4 EF11_HUMAN 50451 148 (P68104) Elongation factor 1-alpha 1  4 TBA1_HUMAN 50634 147 (P68366) Tubulin alpha-1 chain 5 TCPA_HUMAN 60819 127 (P17987) T-complex protein 1, alpha subunit 3 RO52_HUMAN 55162 121 (P19474) 52 kDa Ro protein 3 DJC7_HUMAN 57203 87 (Q99615) DnaJ homolog subfamily C member 7 2 ST38_HUMAN 54498 76 (Q15208) Serine/threonine-protein kinase 38 2 TCPE_HUMAN 60089 74 (P48643) T-complex protein 1, epsilon subunit 3 BJAB-3 EF11_HUMAN 50451 533 (P68104) Elongation factor 1-alpha 1 11 EF12_HUMAN 50780 335 (Q05639) Elongation factor 1-alpha 2 7 EF1G_HUMAN 50298 254 (P26641) Elongation factor 1-gamma 7 RSSA_HUMAN 32816 254 (P08865) 40S ribosomal protein SA  5 ACTB_HUMAN 42052 228 (P60709) Actin, cytoplasmic 1 5 IF35_HUMAN 37654 216 (O00303) Eukaryotic translation initiation factor 3 subunit 5 6 IF41_HUMAN 46353 201 (P60842) Eukaryotic initiation factor 4A-I 5 RUV2_HUMAN 51296 157 (Q9Y230) RuvB-like 2  4 EF1D_HUMAN 31086 131 (P29692) Elongation factor 1-delta 4 YB1_HUMAN 35903 108 (P67809) Nuclease sensitive element binding protein 1 2  146 RO52_HUMAN 55162 92 (P19474) 52 kDa Ro protein 3 ROC_HUMAN 33725 84 (P07910) Heterogeneous nuclear ribonucleoproteins C1/C2  3 IF36_HUMAN 52587 50 (P60228) Eukaryotic translation initiation factor 3 subunit 6 2 IF34_HUMAN 35874 51 (O75821) Eukaryotic translation initiation factor 3 subunit 4 2 W14-3 RO52_HUMAN 55162 754 (P19474) 52 kDa Ro protein  18 KRAC_HUMAN 56080 527 (P31749) RAC-alpha serine/threonine-protein kinase 13 EF11_HUMAN 50451 405 (P68104) Elongation factor 1-alpha 1 7 TBAK_HUMAN 50804 431 (P68363) Tubulin alpha-ubiquitous chain 9 TBB1_HUMAN 50095 382 (P07437) Tubulin beta-1 chain  9 ME50_HUMAN 37442 372 (Q9BQA1) Methylosome protein 50 7 TBA1_HUMAN 50634 265 (P68366) Tubulin alpha-1 chain 6 ARG1_HUMAN 44982 64 (Q8N6T3) ADP-ribosylation factor GTPase activating protein 1 2 BJAB-4 RS3_HUMAN 26842 280 (P23396) 40S ribosomal protein S3 9 RS4X_HUMAN 29676 92 (P62701) 40S ribosomal protein S4, X isoform  4 RS2_HUMAN 31590 81 (P15880) 40S ribosomal protein S2  3 RL8_HUMAN 28104 68 (P62917) 60S ribosomal protein L8 2 RS6_HUMAN 28834 74 (P62753) 40S ribosomal protein S6  3 RS3A_HUMAN 30023 62 (P61247) 40S ribosomal protein S3a 3 RL7A_HUMAN 30017 50 (P62424) 60S ribosomal protein L7a  2 RLA0_HUMAN 34423 50 (P05388) 60S acidic ribosomal protein P0  2 W14-4 ME50_HUMAN 37442 386 (Q9BQA1) Methylosome protein 50 8 GFAP_HUMAN 49907 96 (P14136) Glial fibrillary acidic protein, astrocyte  2 The SDS-PAGE gel shown in Figure Appendix 2.C was equally cut into four pieces from top to bottom, no matter the bands are visible or not with Coomassie Blue staining. The gels were sent to UBC biomedical research center proteomic facility to analyze the eluted proteins after immunoprecipitating with anti-Flag antbody-conjugated beads from the cell lysate of the stably transfected cells  147 shown in Figure Appendix 2.C. BJAB is parental cell line as negative control. W14 is Flag-PKB expressing BJAB cell line. The numbers –1 to –4 after BJAB and W14 designate the gel pieces cut from top to bottom of the SDA-PAGE gel


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