UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The role of docking proteins and adapter proteins in signalling by the B cell receptor Ingham, Robert J. 1999

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2000-486591.pdf [ 8.53MB ]
Metadata
JSON: 831-1.0089627.json
JSON-LD: 831-1.0089627-ld.json
RDF/XML (Pretty): 831-1.0089627-rdf.xml
RDF/JSON: 831-1.0089627-rdf.json
Turtle: 831-1.0089627-turtle.txt
N-Triples: 831-1.0089627-rdf-ntriples.txt
Original Record: 831-1.0089627-source.json
Full Text
831-1.0089627-fulltext.txt
Citation
831-1.0089627.ris

Full Text

THE ROLE OF DOCKING PROTEINS A N D ADAPTER PROTEINS IN SIGNALLING BY THE B CELL RECEPTOR by ROBERT J. I N G H A M B.Sc, Simon Fraser University, 1994 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Microbiology and Immunology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 1999 © Robert J. Ingham, 1999 in presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of M\CA t> bo (D O{ y 1 jTnv*-u/r-«(„« u Tt V The University of British Columbia Vancouver, Canada Date Dec 2 / , /If*} DE-6 (2/88) Abstract The production of antibodies (Abs) by B cells is an important part of the immune response. In order for B cells to differentiate into Ab-producing plasma cells, they require signals that are generated by the B cell receptor (BCR) when it binds antigen (Ag). The B C R activates multiple signalling pathways and a common theme of these pathways utilized by the B C R is that they consist of components that are physical ly separated in resting cells. Therefore, i n order for efficient signal transmission, these components must be brought together dur ing B C R signalling. M y hypothesis was that two groups of proteins, docking proteins and adapter proteins, play an important role i n this process. Docking and adapter proteins contain protein/protein and pro te in / l ip id interaction domains that allow them to mediate the formation of signalling complexes and recruit signall ing proteins to different areas of the cell. In this thesis, I w i l l show that the B C R utilizes a number of docking and adapter proteins to perform these functions. I w i l l present data showing that the BCR uses the Cas, c-Cbl, and G a b l docking proteins to recruit SH2 domain-containing signalling proteins including Crk, Ptdlns 3-kinase, SHP-2, She, and Grb2 to cellular membranes. This may be important for br inging these s igna l l ing proteins i n close p rox imi ty to membrane-associated substrates. Furthermore, I w i l l show that the B C R uses the Crk adapter proteins to recruit the C 3 G guanine nucleotide exchange factor to membrane-associated docking proteins. This may be important for bringing C3G close to its targets, R a p l and R-Ras, both of which associate wi th cellular membranes. Finally, I w i l l show that i n order for the She adapter protein to become tyrosine phosphorylated and function as a docking site for Grb2 complexes, both the She SH2 and PTB domains are required. Interestingly, the She-binding protein, SHIP, is also required for phosphorylation of She by the BCR. Thus, it appears that the B C R uses a number of docking and adapter proteins to mediate the formation of signalling complexes and to recruit signalling i i molecules to different areas of the cell. This l ikely improves the efficiency of signalling by the BCR. iii Table of Contents Abstract i i Table of Contents i v List of Figures v i i i List of Abbreviations x i Acknowledgments x i i i Chapter 1 Introduction 1 1.0 Role of B cells during the immune response 1 1.1 Regulation of Ab production 2 1.2 Structure of the BCR 2 1.3 Proximal B C R signalling events 5 1.4 Signalling pathways regulated by the BCR 6 1.4.1 Ptdlns 3-kinase pathway 6 1.4.2 PLC-y pathway •. . . . 10 1.4.3 Ras pathway 13 1.4.4 Other signalling pathways regulated by the BCR . . . 13 1.5 Activation of signalling pathways by the B C R requires the coupling of components which are physically separated i n resting cells . . . 16 1.6 Protein-protein and protein-lipid interaction domains are important mediators of signal transduction . . . 16 1.6.1 SH2 domains 16 1.6.2 PTB domains 19 1.6.3 SH3 domains 20 1.6.4 P H domains 20 1.7 Docking proteins and adapter proteins 21 1.8 Docking proteins and adapter proteins used by the BCR . . . 22 1.9 Thesis Goals 23 1.10 Thesis summary 24 Chapter 2 Materials and Methods 26 2.0 Reagents 26 2.0.1 Antibodies 26 2.0.2 Glutathione S-transferase (GST) fusion proteins . . . 27 2.1 Cel l culture and stimulations 28 2.1.1 Cel l lines 28 2.1.2 Cel l culture 29 2.1.3 Cel l stimulation 29 2.1.4 Preparation of particulate and soluble fractions. . . 30 2.1.5 Preparation of lysates for Rap activation assay . . . 30 2.2 Precipitation experiments 31 2.2.1 Immunoprecipitation 31 2.2.2 Precipitation of GST fusion proteins (except GST-RalGDS(RBD)) 31 2.2.3 Precipitation wi th GST-RalGDS(RBD) 31 2.2.4 Precipitation wi th biotinylated She peptides . . . . 32 2.3 SDS-PAGE and immunoblotting 32 2.4 F A R Western blotting experiments 33 2.5 Molecular biology methods 33 2.5.1 Restriction endonuclease, blunt ending, and phosphatase reactions 33 2.5.2 Agarose gel electrophoresis 34 2.5.3 Gel purification of D N A 34 2.5.4 Ligation of purified D N A fragments 34 2.5.5 Transformation of competent bacteria 35 2.5.6 Small scale isolation of plasmid D N A 35 2.5.7 Large scale isolation of plasmid D N A 35 2.6 Plasmids 36 2.7 Retroviral infection of B cell lines 37 2.7.1 Production of retroviruses using the BOSC 23 packaging cell line 37 2.7.2 Infection of B cell lines 38 2.7.3 Placing cells i n drug selection 38 2.8 Enzyme Assays 39 2.8.1 Ptdlns 3-kinase enzyme assays 39 Chapter 3 The BCR uses the Crk adapter proteins to form signalling complexes 40 3.0 Introduction 40 3.1 Results 45 3.1.1 Crk proteins associate wi th tyrosine-phosphorylated proteins after BCR ligation 45 3.1.2 Cas proteins inducibly associate wi th Crk proteins in BCR-stimulated cells 48 3.1.3 c-Cbl is tyrosine phosphorylated and binds directly to the Crk SH2 domain in BCR-stimulated cells 49 3.1.4 Crk and Grb2 associate wi th different exchange factors in R A M O S B cells. . 57 3.1.5 Subcellular localization of Crk, Cas, and c-Cbl i n BCR-stimulated cells 61 3.2 Discussion 62 v Chapter 4 Activation of the R a p l GTPase by the B C R involves a diacylglycerol-dependent pathway that is independent of Crk signalling complexes 74 4.0 Introduction 74 4.1 Results 75 4.1.1 R a p l is activated by the B C R 75 4.1.2 PdBu, but not ionomycin, is sufficient to activate R a p l i n B cells 76 4.1.3 PLC-y-mediated D A G production is required for the activation of R a p l by the BCR 79 4.1.4 Association of Crk wi th pl30Cas and c-Cbl is neither necessary nor sufficient for activation of R a p l i n B cells 84 4.2 C a l D A G - 1 , a DAG-regulated activator of R a p l 85 4.3 Results 88 4.3.1 Ca lDAG-1 is expressed in a number of B cell lines . . 88 4.3.2 Overexpression of C a l D A G - 1 , but not C 3 G , i n DT40 cells enhances BCR-mediated R a p l activation . 88 4.4 Discussion 93 Chapter 5 G a b l is a membrane docking site for a number of signalling proteins involved in signalling by the B C R 99 5.0 Introduction 99 5.1 Results 100 5.1.1 G a b l is tyrosine phosphorylated and associates wi th tyrosine-phosphorylated proteins i n response to B C R ligation 100 5.1.2 The She and Grb2 adapter proteins associate wi th G a b l after BCR engagement 101 5.1.3 Ptdlns 3-kinase associates wi th G a b l after B C R engagement 108 5.1.4 SHP-2 associates wi th G a b l after B C R engagement. . . I l l 5.1.5 The SH2 domains of She, Ptdlns 3-kinase, and SHP-2 bind directly to G a b l whereas the Grb2 SH2 domain binds to Gabl-associated SHP-2 and She. . . I l l 5.1.6. G a b l is a membrane-associated docking protein i n B cells 117 5.2 Discussion 124 Chapter 6 Overexpression of G a b l i n WEHI-231 cells augments the signalling function of Gabl-associated proteins. . . . 134 6.0 Introduction 134 vi 6.1 Results 135 6.1.1 Gabl expressed in WEHI-231 cells associates with similar signalling proteins as Gab l expressed in R A M O S cells 135 6.1.2 Gabl expressed in WEHI-231 cells associates with Ptdlns 3-kinase in response to BCR stimulation. . . 138 6.1.3 Overexpression of Gabl in WEHI-231 cells enhances BCR-mediated Akt activation 138 6.1.4 Gabl expressed in WEHI-231 cells associates with SHP-2 in response to BCR stimulation 141 6.1.5 Overexpression of Gabl in WEHI-231 cells enhances BCR-mediated SHP-2 tyrosine phosphorylation and Grb2 binding to SHP-2 146 6.1.6 The Gabl P H domain is required for efficient tyrosine phosphorylation of Gabl and for association of Gabl with signalling proteins in BCR-stimulated cells 149 6.2 Discussion 154 Chapter 7 The inositol phosphatase SHIP is required for efficient tyrosine phosphorylation of She in response to BCR engagement 157 7.0 Introduction 157 7.1 Results 159 7.1.1 Efficient tyrosine phosphorylation of She in response to BCR crosslinking requires both the SH2 and PTB domain of She 159 7.1.2 Binding of SHIP to She depends on both the SH2 and PTB domain of She 160 7.1.3 BCR-induced binding of Grb2 to She depends on both the SH2 and PTB domain of She 163 7.1.4 Tyrosine phosphorylation of She in response to BCR engagement requires SHIP. 163 7.2 Discussion 168 Chapter 8 Discussion f . . . . 173 8.0 Summary of Thesis 173 8.1 The BCR may use other signalling modules 174 8.2 Recruitment of proteins to microdomains in BCR-stimulated cells 176 8.3 Co-localization and membrane localization of signalling proteins as a strategy in signal transduction 178 References 180 v i i List of Figures Figure 1.1 Schematic representation of the B C R showing the different subunits 4 Figure 1.2 Downstream targets of Ptdlns 3-kinase 8 Figure 1.3 Downstream targets of PLC-y. 12 Figure 1.4 Downstream targets of Ras 15 Figure 1.5 Signalling components must be brought together for efficient signal transmission 18 Figure 3.1. Crk proteins and anti-Crk Abs 42 Figure 3.2. Crk proteins are tyrosine phosphorylated and associate wi th tyrosine-phosphorylated proteins after BCR crosslinking 47 Figure 3.3. Crk proteins associate wi th Cas proteins i n BCR-stimulated cells . . 51 Figure 3.4. c-Cbl is tyrosine phosphorylated and associates wi th Crk proteins after BCR crosslinking 54 Figure 3.5. c-Cbl binds directly to the SH2 domain of c-Crk 56 Figure 3.6 Association of SOS and C 3 G with Crk and Grb2 60 Figure 3.7. Subcellular localization of Crk, Cas, and c-Cbl 64 Figure 3.8. Subcellular localization of C r k / C a s and C r k / c - C b l complexes. . . . 66 Figure 3.9. Proposed role of Crk proteins i n BCR signalling. 73 Figure 4.1. Activation of R a p l following BCR ligation 78 Figure 4.2. R a p l is activated in B cells by PdBu but not by ionomycin . . . . . 81 Figure 4.3. Activation of R a p l by the BCR requires PLC-y but does not require IP3 receptor-mediated increases i n intracellular C a 2 + 83 Figure 4.4. Association of C r k L wi th pl30Cas and c-Cbl is neither necessary nor sufficient for activation of R a p l in B cells 87 viii Figure 4.5. Ca lDAG-1 is expressed i n a number of B cell lines 90 Figure 4.6. Overexpression of Ca lDAG-1 i n DT40 B cells enhances BCR-mediated R a p l activation 92 Figure 5.1. G a b l is tyrosine phosphorylated after B C R ligation 103 Figure 5.2. pl30Cas and c-Cbl do not co-precipitate wi th G a b l 105 Figure 5.3. G a b l associates wi th tyrosine-phosphorylated proteins i n BCR-stimulated cells 107 Figure 5.4. She and Grb2 associate wi th G a b l after BCR ligation 110 Figure 5.5. Ptdlns 3-kinase associates wi th G a b l i n BCR-stimulated cells. . . . 113 Figure 5.6. SHP-2 is tyrosine phosphorylated and associates wi th G a b l after B C R ligation 115 Figure 5.7. The SH2 domains of She, SHP-2, and Ptdlns 3-kinase can b ind directly to G a b l precipitated from anti-IgM-stimulated R A M O S cells 119 Figure 5.8. The Grb2 SH2 domain binds to Gabl-associated SHP-2 and She. . . 121 Figure 5.9. G a b l is present i n the particulate fraction of R A M O S cells and associates wi th signalling proteins in this fraction 123 Figure 5.10. G a b l associates wi th She, Grb2, Ptdlns 3-kinase, and SHP-2 in the particulate fraction of R A M O S cells after B C R ligation . . . 126 Figure 5.11. G a b l associates wi th signalling proteins i n BCR-stimulated cells . 128 Figure 6.1. G a b l expressed i n WEHI-231 cells is tyrosine phosphorylated and associates wi th tyrosine-phosphorylated proteins i n BCR-stimulated cells 137 Figure 6.2. G a b l expressed i n WEHI-231 cells associates wi th Ptdlns 3-kinase in BCR-stimulated cells 140 ix Figure 6.3. Expression of G a b l i n WEHI-231 cells enhances A k t phosphorylation in response to BCR crosslinking 143 Figure 6.4. G a b l expressed i n WEHI-231 cells associates wi th SHP-2 i n BCR-stimulated cells 145 Figure 6.5. Expression of G a b l i n WEHI-231 cells enhances SHP-2 phosphorylation i n response to B C R crosslinking 148 Figure 6.6. Expression of G a b l i n WEHI-231 cells enhances Grb2 binding to SHP-2 i n response to BCR crosslinking . .151 Figure 6.7. The G a b l P H domain is required for efficient tyrosine phosphorylation of G a b l and for association of G a b l wi th signalling proteins in BCR-stimulated cells 153 Figure 7.1. BCR-induced tyrosine phosphorylation of She depends on both the SH2 and PTB domains of She . . 162 Figure 7.2. Association of SHIP wi th She i n BCR-stimulated cells requires both the SH2 and PTB domains of She 165 Figure 7.3. BCR-induced binding of Grb2 to She depends on both the SH2 and PTB domains of She 167 Figure 7.4. BCR-induced tyrosine phosphorylation of She depends on the binding of SHIP to the PTB domain of She 170 x List of Abbreviations A b antibody A g antigen B C R B cell receptor B S A bovine serum albumin D A G diacylglycerol diCs dioctanoylglycerol E B V Epstein-Barr virus ECL enhanced chemiluminescence EGF epidermal growth factor E R K extracellular signal-regulated kinase H A hemagglut in in G A P GTPase-activating protein G D S GDP-dissociation stimulator GPI glycosylphosphatidylinositol G N E F guanine nucleotide exchange factor GSK -3 glycogen synthase kinase-3 G S T glutathione S-transferase I L K integrin-linked kinase IP3 inositol trisphosphate IPTG isopropylthio-fJ-galactoside I T A M immunoreceptor tyrosine-based activation motif L B Luria-Bertani L P A lysophosphatidic acid M A P K mitogen-activated protein kinase m l g membrane immunog lobu l in xi M K K M A P K k i n a s e M K K K M A P K k i n a s e k i n a s e N F - A T n u c l e a r f ac to r o f a c t i v a t e d T ce l l s P - T y r p h o s p h o t y r o s i n e P d B u p h o r b o l d i b u t y r a t e P D K p h o s p h o i n o s i t i d e - d e p e n d e n t k i n a s e P H p l e c k s t r i n h o m o l o g y P K C p r o t e i n k i n a s e C P L C p h o s p h o l i p a s e C P M S F p h e n y l m e t h y l s u l f o n y l f l u o r i d e P T B p h o s p h o t y r o s i n e - b i n d i n g P t d l n s p h o s p h a t i d y l i n o s i t o l P R K 2 p r o t e i n k i n a s e C - r e l a t e d k i n a s e 2 R B D R a p l - b i n d i n g d o m a i n S H S r c h o m o l o g y T C R T c e l l r e c e p t o r x i i Acknowledgments I w o u l d l ike to thank all those who either directly or indirectly helped me wi th this thesis. First, I wou ld like to thank my supervisor Dr. M i k e G o l d and all past and present members of the G o l d lab. I w o u l d like to thank my supervisory committee and Dr. Linda Matsuuchi for helpful comments. I w o u l d like to thank Dr. M i k i Matsuda and his lab for providing me wi th many reagents and al lowing me to vis i t their lab. Final ly , I w o u l d l ike to thank Julinor, for helping me throughout my degree. x i i i Chapter 1 Introduction 1.0 Role of B cells during the immune response The immune system is comprised of a number of different cell types and soluble factors that work i n concert to r i d the body of pathogenic agents. A n important element of the immune system is the B cell which circulates between the blood and the lymphatic system looking for its specific antigen (Ag) which may be a bacterium, virus, or other foreign substance. After encountering its specific A g and receiving additional T cell-derived signals, a B cell proliferates and differentiates into a plasma cell which secretes antibodies (Abs). It is these Abs that participate in the immune response. Abs contribute in several ways to the immune response (1). Firstly, they can b ind and sequester toxins thereby preventing the toxins from entering and damaging the body's cells. Secondly, Abs mark bacteria and viruses for phagocytosis by cells like macrophages which have receptors that recognize A g / A b complexes. Finally, Abs can contribute to the lysis of bacteria by recruiting complement proteins to Ab-coated bacteria. Thus, many aspects of the immune response are regulated by B cells and the importance of B cells i n the immune response is highlighted by the fact that patients lacking mature B cells are immunocompromised and have trouble clearing bacterial infections (2). Whi le Abs can clear the body of infection, they can also contribute to pathological conditions. For example, the inappropriate production of Abs against self proteins can lead to autoimmune diseases such as systemic lupus erythematosus and myasthenia gravis (1). Furthermore, the production of Abs against innocuous foreign proteins such as pet dander and various pollens can cause allergies i n some people (1). Therefore, understanding how a resting B cell is dr iven to differentiate into an antibody-producing plasma cell is not only important for understanding 1 how the immune response responds to infection, but should also provide some insight into the cause of pathological conditions mediated by Abs. 1.1 Regulation of A b production B cell activation and A b production are regulated by a number of positive and negative signals received by the B cell. Of paramount importance is a signal through the B cell receptor (BCR). The B C R binds A g and when it does so, this causes the B cell to enter the cell cycle (3). However, i n many cases this signal alone is not sufficient to cause a B cell to proliferate and begin producing Abs. This requires other signals that act i n concert wi th the B C R such as signals through CD40 (4), CD19 (5), and cytokine receptors such as those for IL 4, 5, and 6 (6). In addition to receptors that promote B cell activation, there are also receptors that negatively regulate B cell activation. For example, co-engagement of the FcyRII-Bl receptor or CD22 wi th the BCR interferes wi th B C R signalling and limits B cell proliferation (7). Both FcyRII-Bl and CD22 associate w i t h the tyrosine phosphatase SHP-1 (8,9) wh ich is known to negatively regulate B C R signalling (10,11). Thus, i n addit ion to receptors that act wi th the B C R to promote B cell activation, there are also receptors that either block B C R signall ing or l imi t its duration. These receptors can prevent inappropriate or excessive B cell activation. 1.2 Structure of the B C R The B C R is a modular receptor consisting of 4 different polypeptide chains (Fig. 1.1) (12). The membrane immunoglobulin (mlg) is composed of two identical heavy chains and two identical light chains that are covalently l inked. Each m l g therefore has two identical antigen binding sites formed by one heavy chain and one light chain. The signalling function of the B C R is mediated by the Iga and IgP 2 Figure 1.1 Schematic representation of the B C R . The heavy chains (dark blue), light chains (light blue) and Iga and Igfi subunits (green) of the B C R are shown. The I T A M motifs of Iga and IgP are coloured red. A g binding sites are noted by arrows. Intermolecular and intramolecular disulphide bonds are not shown. 3 4 subunits of the BCR. Igoc and Ig(3 are encoded by the mb-1 and B-29 genes respectively and form a covalently-linked heterodimer (12). These Igoc and IgP heterodimers non-covalently associate w i t h the m l g and it is thought that 2 Igoc/(3 heterodimers associate wi th each mlg molecule (12). Crit ical to the function of Igoc and Igp are motifs w i t h i n their cytoplasmic tai l termed immunoreceptor tyrosine-based activation motifs (ITAMs) (12b,13). These motifs are common to many immunoreceptors including the T cell receptor (TCR) C D 3 chain components as wel l as the y chain of the FceRI receptor which binds IgE (13). A l l I T A M motifs consist of tyrosine residues w i t h i n the core sequence Y X X L / I - ( X ) 6 - 8 - Y X X L / I (single-letter code, where X = any amino acid). A s w i l l be outl ined i n the next section, these tyrosine residues wi th in the I T A M motifs play a key role i n initiating signalling by the BCR. 1.3 Proximal BCR signalling events Clustering of the B C R by multivalent Ags or by anti-mlg Abs initiates a biochemical cascade that ultimately leads to gene transcription. The most proximal signalling event initiated by the B C R is the activation of tyrosine kinases (14), specifically tyrosine kinases of the Src family (15) as wel l as the Syk (16) and Btk (17) tyrosine kinases. The Src kinases are thought to be activated first and their main substrate appears to be the tyrosine residues wi th in the I T A M motifs of the Igoc and Ig(3 components of the B C R (17,18). Once phosphorylated these I T A M motifs can serve as binding sites for the Src family kinases (19) via motifs termed Src homology (SH) 2 domains wh ich w i l l be discussed i n more detail i n Section 1.6. This recruitment of Src kinases to the phosphorylated I T A M motifs increases the intrinsic kinase activity of the Src kinases (20) and brings these kinases i n close proximi ty to I T A M motifs on nearby B C R complexes. This facilitates the phosphorylat ion of addit ional I T A M motifs and allows for the recruitment of additional Src family kinases to the BCR. 5 Whi le the Src kinases phosphorylate the I T A M motifs, Syk appears to phosphorylate most other tyrosine-phosphorylated proteins i n BCR-stimulated cells (18). Syk, like the Src kinases, also associates wi th the I T A M motifs of the B C R and does so using its two SH2 domains (16,19,21). Recruitment of Syk to the I T A M motifs enhances its catalytic activity (22) and is critical for B C R signall ing (8). Furthermore, phosphorylation of Syk by Src kinases may also contribute to Syk activation (22a). Once Syk is activated, it goes on to activate a number of signalling pathways inc lud ing those regulated by phosphatidylinosi tol (Ptdlns) 3-kinase, phospholipase (PLC)-y, and Ras (23-25). 1.4 Signalling pathways regulated by the BCR B C R ligation results i n the activation of a number of key signalling enzymes inc luding Ptdlns 3-kinase, PLC-y , and Ras. Whi le my work has focused on the upstream events mediating the activation of these signalling pathways by the BCR, I wou ld first like to briefly summarize what is known of the downstream signalling events regulated by these signalling molecules. In particular, I w i l l highlight the downstream signalling events mediated by these proteins i n B cells. 1.4.1 Ptdlns 3-kinase pathway Ptdlns 3-kinases are l i p id kinases that phosphorylate the inositol phospholipids Ptdlns, PtdIns(4)P, and PtdIns(4,5)P2 at the 3' position of the inositol ring, converting these lipids into PtdIns(3)P, PtdIns(3,4)P2, and PtdIns(3,4,5)P3, respectively (26,27) (Fig. 1.2). While there are several classes of Ptdlns 3-kinases, the most important in BCR signal transduction are the Class IA Ptdlns 3-kinases. Class IA Ptdlns 3-kinases can phosphorylate both PtdIns(4)P and PtdIns(4,5)P2 and the resulting products, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 / are increased after BCR crosslinking (28). Class IA Ptdlns 3-kinases are heterodimers, consisting of a catalytic 6 Figure 1.2 Downstream targets of Ptdlns 3-kinase. Signalling events mediated by the Class IA Ptdlns 3-kinase products PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are indicated. Briefly, production of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 by Ptdlns 3-kinase results i n the activation of P D K 1 , P D K 2 , Ak t , and various P K C isoforms. P D K 1 and P D K 2 phosphorylate and activate A k t which in turn phosphorylates a number of proteins. Phosphory la t ion of Forkhead transcript ion factors by A k t results i n their accumulation i n the cytoplasm thereby inhibi t ing their transcriptional activity. Phosphorylat ion of Bad and caspase 9 by A k t inhibits their ability to promote apoptosis while phosphorylation of GSK-3 inhibits GSK-3 activity, allowing N F - A T C and (3-catenin to accumulate in the nucleus where they can initiate transcription. It has also recently been reported that A k t phosphorylates and activates the I K B kinases which phosphorylate the inhibitor of N F K B , I K B . Phosphorylation of IKB targets it for degradation and allows N F K B to translocate to the nucleus and activate transcription (not shown i n diagram). ^PtdIns(M)P, PtdIns(3,4,5)P3 • P D K 1 , 2 V * PKC 8, e, r|, £ ^ #Akt ^ Forkhead J - , ^ see Fig. 1.3 ^transcription V ^ GSK-3 factors ^ Bad, Caspase 9 ^ + NF-ATC and p-catenin • transcription • anontosis n u c l e a r localization •transcription 8 subunit and an adapter subunit (26,27). Three mammalian catalytic subunits (a,(3, and y) and five adapter subunits have been identified (27). The adapter subunits all possess two SH2 domains which, as w i l l be talked about later (see Section 1.6.1), are important for recruiting Ptdlns 3-kinase to cellular membranes i n response to receptor signalling. Several targets have been reported for the inositol phospholipid products of Ptdlns 3-kinase. Certain protein kinase C (PKC) isoforms including 8, e, n , £ can be activated in vitro by Ptdlns 3-kinase products (29,30) and all these P K C isoforms are expressed i n B cells (31,32). Phorbol esters, which activate P K C enzymes, are potent activators of extracellular signal-regulated kinases (ERKs) 1 and 2 i n B cells (33,77). As outlined i n Section 1.4.3, ERKs are involved i n the regulation of a number of transcription factors. However, the major target of Pdlns 3-kinase products may be the serine/threonine kinase A k t (also referred to as PKB) which has been shown to be activated by the B C R (34-37). The Ptdlns 3-kinase product PtdIns(3,4)P2 can activate A k t (38-40) in vitro and both PtdIns(3,4)P2 and PtdIns(3,4,5)P3 can directly b ind to the pleckstrin homology (PH) domain (see Section 1.6.4) of A k t (38,40). Likewise, the Ptdlns 3-kinase products PtdIns(3,4)P2 and PtdIns(3,4,5)P3 can b ind to the P H domain of the A k t activator, phosphoinositide-dependent protein kinase 1 (PDK1) (41). This leads to the activation P D K 1 wh ich phosphorylates A k t on threonine 308, thereby faci l i tat ing the act ivat ion of A k t (41). The P H domain-mediated binding of Ptdlns 3-kinase products, facilitates the recruitment of P D K 1 and A k t to the plasma membrane wh ich may be important for co-localizing P D K 1 and Akt . Maximal activation of A k t also requires that it be phosphorylated on serine 473 by a kinase that has been called P D K 2 but which has not been identified definit ively (41a). Recent work by Balendran et al. showed that P D K 1 can phosphorylate serine 473 when associated wi th protein kinase C-related kinase-2 (PRK2) (42). Integrin-linked kinase (ILK) can also phosphorylate A k t on serine 473 (43) w h i l e H e m m i n g s and colleagues bel ieve that P D K 2 is a nove l 9 rapamycin-sensitive kinase (44,45). Once activated, A k t phosphorylates a number of proteins including Bad (46,47), caspase 9 (48), Forkhead family transcription factors (49,50), glycogen synthase kinase-3 (GSK-3) (51),IKB kinase (52-54), and m T O R (54a). Through phosphorylating Bad and caspase 9, A k t can inhibit the pro-apoptotic functions of these proteins (48,55). Phosphorylation of Forkhead proteins by A k t causes these proteins to be retained i n the cytoplasm where they cannot initiate transcription (49,50,56-58). Phosphorylation of GSK-3 by A k t inhibits GSK-3 (59). Since G S K - 3 is a negative regulator of N F - A T C (60,61) and ^-catenin (62) transcription factors, Akt-mediated inhibit ion of GSK-3 may increase the ability of these factors to promote transcription. Stimulation of the B C R is known to enhance GSK-3 phosphorylation and inactivation i n a Ptdlns 3-kinase sensitive manner (36). Likewise, it has also recently been reported that A k t phosphorylates and activates the I KB kinases which phosphorylate the inhibitor of N F K B , I K B (52-54). This targets I K B for degradation and allows N F K B to translocate to the nucleus and activate transcription. Furthermore, A k t mediated activation of mTor may allow A k t to activate p70S6Kinase (54a). Thus, A k t l ikely regulates several aspects of B C R signalling including apoptosis and gene transcription. 1.4.2 PLC-Y pathway Both P L C - y l and PLC-y2 are activated by the B C R (63-66). P L C - y cleaves PtdIns(4,5)P2 into the second messengers diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (IP3) (Fig. 1.3) (67). D A G is important for the activation of several P K C isoforms whi le IP3 causes the release of C a 2 + from intracellular stores thereby increasing the concentration of intracellular C a 2 + . This increase i n intracellular C a 2 + helps activate some P K C enzymes and also activates the serine/threonine phosphatase ca lc ineur in (68). C a l c i n e u r i n dephosphorylates the N F - A T C transcription factor al lowing N F - A T C to translocate to the nucleus and regulate gene 10 Figure 1.3 Downstream targets of PLC-y. Signalling events mediated by the P L C - y products IP3 and D A G are indicated. Briefly, activation of P L C - y results i n the b reakdown of PtdIns(4,5)P2 into IP3 and D A G . IP3 mediates the increase i n intracel lular C a 2 + leading to the activation of the phosphatase calcineurin. Calcineurin dephosphorylates N F - A T C thereby al lowing N F - A T c to translocate to the nucleus and initiate transcription. D A G activates P K C s and some P K C isoforms require increases i n intracellular C a 2 + as wel l . P K C s have many functions but one appears to be activation of the E R K kinase cascade. Once activated, E R K translocates to the nucleus where it phosphorylates and activates transcription factors (see Fig. 1.4), resulting i n increased gene transcription. 11 PtdIns(4,5)P2 £2 <^ IPs D A G 0 ^ # intracellular ^ ^ P K C s J I other targets T calcineurin X • N F - A T C # E R K •transcription 12 transcription (68). B C R signalling is known to cause N F - A T C to translocate to the nucleus (69). 1.4.3 Ras pathway Ras is GTPase that acts as a "molecular switch", shuttling between an inactive, GDP-bound state and an active, GTP-bound state (70). Several groups have shown that Ras is activated i n response to B C R engagement (33,71,72). Active Ras has several effector proteins inc lud ing Raf-1 (73), Ptdlns 3-kinase (74), and Ral GDP-dissociation stimulator (GDS) (75) (Fig. 1.4). The best studied Ras effector is Raf-1 wh ich regulates transcription (76). Raf-1 activates the serine/threonine kinases M E K 1 and 2 which i n turn phosphorylates and activates the E R K 1 and 2. The B C R has been shown to activate all these kinases (33,77,78). Once activated, E R K s migrate to the nucleus where they phosphorylate and activate Ets domain-containing transcription factors inc luding Ets-1, Ets-2, E lk-1 , and Sapla (79-81). These transcription factors induce the expression of number of early response genes inc luding c-fos and egr-1. B C R ligation is k n o w n to induce the expression of Egr-1 and its expression can be blocked by the expression of a dominant negative form of Ras (82). 1.4.4 Other signalling pathways regulated by the BCR In addit ion to activating the Ptdlns 3-kinase, PLC-y , and Ras pathways, the B C R is also known to activate other signalling pathways including those regulated by HS-1 (83) and Vav (84). HS-1 is a hematopoietic-specific protein that appears to regulate BCR-media ted proliferation and apoptosis (85,86). V a v is tyrosine phosphorylated i n response to B C R cross- l inking (84) and is required for BCR-mediated proliferation of B cells (87-89). Vav is also k n o w n to activate the Rac l GTPase (90). Rac l -GTP may mediate activation of the Jnk kinase by the BCR. Rac l also activates Ptdlns 4-phosphate 5-kinases (91) which produce PtdIns(4,5)P2, 13 Figure 1.4 D o w n s t r e a m targets of Ras. Signall ing events mediated by active, GTP-bound Ras are indicated. Briefly, i n its active, GTP-bound state Ras associates wi th a number of effectors including Ptdlns 3-kinase, Raf-1, and RalGDS. Activation of Raf-1 by Ras initiates a serine/threonine kinase cascade that ultimately leads to the activation and translocation of E R K s 1 and 2 to the nucleus. Once i n the nucleus, ERKs phosphorylates Ets family transcription factors thereby increasing the transcriptional activity of these proteins. The G N E F for Ral , RalGDS is also a target of Ras. Ras-GTP binds RalGDS and facilitates its recruitment to the membrane where it can activate Ral. The function of Ral is currently unknown. 14 • Ptdlns 3-kinase V # RalGDS see Fig. 1.2 -^MEK1/2 * R a l - G T P • ERK 1/2 ? -f-Elk-1, Ets-domain-containing txn factors 0 •transcription 15 the substrate of PLC-y and Ptdlns 3-kinase. Thus, activation of Rac l may be required for sustained P L C - y and Ptdlns 3-kinase signalling. 1.5 Activation of signalling pathways by the BCR requires the coupling of components which are physically separated in resting cells A common theme i n B C R signal l ing pathways is that they consist of components that are physically separated i n resting cells (Fig 1.5). For example, the Ras activator SOS is a cytosolic enzyme that must translocate to the membrane i n order to activate Ras which is tethered to the inner face of the plasma membrane. Similarly, PLC-y and Ptdlns 3-kinase are cytosolic enzymes whose l ip id substrates are located i n the inner leaflet of the plasma membrane. Thus, controll ing the intracellular loca l iza t ion of s ignal l ing proteins is key to regulat ing signal transduction and mechanisms to recruit signalling proteins to different cellular compartments must exist. 1.6 Protein-protein and protein-lipid interaction domains are important mediators of signal transduction Bringing together the components of signalling pathways is important for signal transmission. A s mentioned previously, recruitment of signall ing proteins to different regions i n the cell is important for al lowing enzymes to gain access to substrates. Furthermore, the co-localizing of signal proteins allows for multiple components of a signalling pathway to more efficiently interact. In order to facilitate this, signalling proteins possess a variety of modular domains that allow for the formation of signalling complexes and the recruitment of signalling molecules to different areas of the cell. Of particular importance are domains that mediate protein-protein interactions such as SH2, SH3, and phosphotyrosine-binding (PTB) domains and domains that bind to lipids such as P H domains. 16 Figure 1.5 Signalling components must be brought together for efficient signal transmission. A, In resting cells, the cytosolic signalling enzymes PLC-y, Ptdlns 3-kinase, and Ras are physically separated from their targets which are associated with the plasma membrane. B, BCR stimulation results in the recruitment of PLC-y, Ptdlns 3-kinase, and Ras to the plasma membrane through association with membrane-associated proteins (here represented as a question mark, "?", to denote that the identity of many of these proteins is unknown). This brings these enzymes in close proximity to their targets al lowing for signal transmission. 17 A R e s t i n g c e l l s M e m b r a n e - a s s o c i a t e d s u b s t r a t e s C y t o s o l i c e n z y m e s B B C R - s t i m u l a t e d c e l l s IP3 + DAG PIP3 18 1.6.1 SH2 domains A s mentioned i n Section 1.4, SH2 domains b ind to phosphorylated tyrosine residues. These domains are about 100 amino acids i n size and crystallography data has shown that SH2 domains possess a cleft that contains a positively-charged arginine residue which interacts wi th the negatively charged phosphate group of the phosphotyrosine molecule (92,93). This arginine residue is critical for SH2 domain function as mutation of this residue is sufficient to abrogate phosphotyrosine binding to the SH2 domain. Whi le SH2 domains from different proteins all have the common feature of binding phosphotyrosine residues, not all SH2 domains bind the same phosphotyrosine residues. Two studies have used peptide libraries to look at the b inding of different SH2 domains to different phosphotyrosine-containing peptides (94,95). These studies found that the specificity of a given SH2 domain is generally governed by amino acids immedia te ly carboxy-terminal to the phosphotyrosine residue. The reason for this specificity appears to be that a second b ind ing cleft is present immediately carboxy-terminal to the phosphotyrosine b ind ing pocket. For example, the Src SH2 domain prefers b i n d i n g to a phosphotyrosine residue within a YEEI motif (96). The isoleucine i n the +3 position associates w i t h a smal l , hydrophobic pocket conferring specificity to small , hydrophobic residues at the +3 position (92). In contrast, the carboxy-terminal SH2 domain of P L C - y l favours 3 hydrophobic residues immediately carboxy-terminal to the phosphotyrosine (96). The reason being that this SH2 domain possesses a large hydrophobic cleft immediately carboxy-terminal to the phosphotyrosine binding pocket (93). • 1.6.2 PTB domains PTB domains are another phosphotyrosine-binding module. The first PTB domain was first identified i n the She adapter protein (97,98) but PTB domains have subsequently been identified i n the IRS-1/2 docking proteins (99) as wel l as in the 19 Numb protein (100). In contrast to SH2 domains, PTB domains recognize phosphotyrosines in the context of the amino acids immediately amino-terminal to the phosphotyrosine residue. The consensus recognition sequence for PTB domain of She and IRS-1 is the NPXY motif (97-99) while Numb prefers a GPY motif (100). Other residues more amino-terminal appear to contribute to the specificity of binding of specific PTB domains (101,102). Interestingly, it has been found that some PTB domains, such as that in the Numb protein, bind with high affinity to unphosphorylated motifs (100). This suggests that the PTB domain is not exclusively a phosphotyrosine-binding motif. 1.6.3 SH3 domains SH3 domains bind proline-rich sequences in target proteins with the consensus sequence PXXP (103). SH3 domain ligands can be divided into two categories, class I and class II, based on whether they bind the SH3 domain in a amino-terminal to carboxy-terminal orientation (class I) or a carboxy-terminal to amino-terminal orientation (class II) (104). The polyproline ligand of the SH3 domain has been shown to form a left-handed polyproline type II helix (105,106). Unlike SH2 and PTB domains, post-translational modification of the motifs recognized by SH3 domains is not required for SH3 domain-binding. This means that SH3 domain-mediated interactions are present in resting cells as well as in activated cells. However, it should be noted that phosphorylation of serine/threonine residues adjacent to proline-rich sequences can abolish SH3 domain binding (107). Furthermore, conformational changes in proteins may also reveal proline-rich sequences able to bind SH3 domains. For example, a proline-rich sequence within the SH2 domain of the Crk adapter protein is much better able to bind SH3 domains when the Crk SH2 domain is engaged by a phosphotyrosine molecule (108). 20 1.6.4 PH domains U n l i k e S H 2 , S H 3 , a n d P T B d o m a i n s , P H d o m a i n s d o n o t m e d i a t e p r o t e i n - p r o t e i n i n t e r a c t i o n s b u t r a t h e r a l l o w p r o t e i n s t h a t p o s s e s s t he se m o t i f s t o b i n d i n o s i t o l p h o s p h o l i p i d s (109) . D i f f e r e n t P H d o m a i n s b i n d d i f f e r e n t i n o s i t o l l i p i d s a n d t he se h a v e b e e n d i v i d e d i n t o 4 g r o u p s b a s e d o n t h e i r P t d l n s l i p i d b i n d i n g p r e f e r e n c e (110,111) . O f p a r t i c u l a r i n t e r e s t to t he s t u d y o f s i g n a l t r a n s d u c t i o n are the g r o u p I a n d III P H d o m a i n - c o n t a i n i n g p r o t e i n s w h i c h b i n d P t d l n s 3 - k i n a s e p r o d u c t s . G r o u p I P H d o m a i n s s u c h as t ha t i n f o u n d B t k , b i n d P t d I n s ( 3 , 4 , 5 ) P 3 w h i l e the G r o u p III P H d o m a i n s o f A k t a n d P D K 1 b i n d b o t h P t d I n s ( 3 , 4 ) P 2 a n d P t d l n s ( 3 , 4 , 5 ) P 3 (110,111). S i n c e P t d l n s 3 - k i n a s e p r o d u c t s a r e c o m p o n e n t s o f t h e i n n e r - l e a f l e t o f t he p l a s m a m e m b r a n e , p r o t e i n s p o s s e s s i n g P H d o m a i n s tha t c a n b i n d t o these p r o d u c t s a re a b l e to t r a n s l o c a t e t o t h e p l a s m a m e m b r a n e w h e n P t d l n s 3 - k i n a s e i s a c t i v a t e d . T h i s p r o v i d e s a m e a n s o f r e g u l a t i n g the i n t r a c e l l u l a r l o c a t i o n o f P H d o m a i n - c o n t a i n i n g s i g n a l l i n g p r o t e i n s i n r e s t i n g v e r s u s a c t i v a t e d c e l l s . 1.7 Docking proteins and adapter proteins A s m e n t i o n e d i n the p r e v i o u s p a r a g r a p h s , p r o t e i n - p r o t e i n a n d p r o t e i n - l i p i d i n t e r a c t i o n m o d u l e s p l a y a n i m p o r t a n t r o l e i n m e d i a t i n g t h e f o r m a t i o n o f s i g n a l l i n g c o m p l e x e s a n d i n r e c r u i t i n g s i g n a l l i n g p r o t e i n s to d i f f e r e n t a reas o f t he c e l l . T h e r e f o r e , i n o r d e r to u n d e r s t a n d s i g n a l t r a n s d u c t i o n , i n c l u d i n g t h a t b y the B C R , i t i s i m p o r t a n t t o u n d e r s t a n d t h e f u n c t i o n o f p r o t e i n s t h a t p o s s e s s t h e s e m o t i f s . T h e s e i n c l u d e t h e S H 2 d o m a i n - b i n d i n g d o c k i n g p r o t e i n s a n d the S H 2 / S H 3 d o m a i n - c o n t a i n i n g a d a p t e r p r o t e i n s . T h e S H 2 d o m a i n - b i n d i n g d o c k i n g p r o t e i n f a m i l y i n c l u d e s I R S - 1 , I R S - 2 , t h e C a s p r o t e i n s , c - C b l , S L P - 7 6 , L A T , B l n k , a n d t h e D o k p r o t e i n s (112-115) . T h e s e p r o t e i n s h a v e m u l t i p l e p r o t e i n - p r o t e i n i n t e r a c t i o n m o t i f s i n c l u d i n g p r o l i n e - r i c h s e q u e n c e s , S H 2 a n d S H 3 d o m a i n s , a n d P T B d o m a i n s . S e v e r a l d o c k i n g p r o t e i n s a l s o p o s s e s s P H d o m a i n s w h i c h m a y a l l o w these p r o t e i n s to a s s o c i a t e w i t h m e m b r a n e s . 21 However, the most striking feature of these proteins is that they are phosphorylated on multiple tyrosine residues in response to receptor signalling which allows them to bind SH2 domain-containing signalling proteins. For example, tyrosine phosphorylation of IRS-1 in response to insulin stimulation, allows Fyn, Ptdlns 3-kinase, SHP-2, Grb2, and Nek to associate with IRS-1 (112). By binding SH2 domain-containing proteins, docking proteins can co-localize signalling components and recruit signalling components to areas of the cell where they perform their functions. The family of ubiquitously-expressed SH2/SH3 domain-containing adapter proteins includes She, Grb2, Crk, Nek, and the p85 subunit of Ptdlns 3-kinase. Each of these proteins can bind a number of different signalling proteins via their SH2 and SH3 domains and may therefore participate in the formation of multiple signalling complexes. While most adapter proteins possess no intrinsic catalytic activity, they do associate with enzymes. Perhaps, the best studied adapter protein is the SH2/SH3 adapter Grb2. Grb2 uses its SH3 domain(s) to bind to SOS, a guanine nucleotide exchange factor (GNEF) for Ras, while using its SH2 domain to either directly or indirectly (through the She and SHP-2 adapter proteins) associate with activated growth factor receptors (104). In this way, SOS is recruited to the membrane allowing it to more efficiently activate Ras. Thus, SH2 /SH3 adapter proteins can couple cytosolic signalling proteins to the membrane. 1.8 Docking proteins and adapter proteins used by the BCR. Since Ptdlns 3-kinase, PLC-y, and SOS are cytosolic enzymes that act on membrane-bound substrates (PtdIns(4,5)P2 and Ras), activation of these enzymes by the BCR likely involves the use of docking proteins and adapter proteins. When work on this thesis started little was known about docking proteins and adapter proteins used by the BCR to activate these signalling pathways. 22 It was known that the SH2 domains Ptdlns 3-kinase can bind to the transmembrane protein CD19 in BCR-stimulated cells (116) and that Ptdlns 3-kinase may also be recruited to the membrane in B cells through its association with the Src kinase Lyn (117). In this thesis I will show that Gabl is an additional docking protein used by the BCR to recruit Ptdln 3-kinase, as well as other signalling proteins, to the membrane in response to BCR stimulation. Furthermore, when work on this thesis started it was known that the She and Grb2 adapter proteins were used by the BCR to recruit SOS to the membrane in order to bring it in close proximity to Ras (118,119). However, it was not known how Shc»Grb2»SOS complexes associated with the membrane. In this thesis I will address the questions of (1) how She may be recruited to cellular membranes after BCR ligation and (2) how She is phosphorylated after BCR ligation. Finally, since the B cells express several other docking proteins and adapter proteins I investigated the role of some of these proteins in BCR signalling. In this thesis, I will show that the BCR uses the Cas and c-Cbl docking proteins to recruit signalling complexes containing the Crk adapter proteins to cellular membranes. 1.9 Thesis goals Hypothesis: The BCR uses multiple docking and adapter proteins to mediate the formation of signalling complexes and recruit signalling proteins to cellular membranes. 1. To identify membrane docking proteins used by the BCR 2. To identify signalling proteins/adapter proteins recruited to these docking proteins 3. To determine where signalling protein/docking protein complexes are localized in B cells 23 4. To show that recruitment of signalling proteins to membrane docking proteins is important for the signalling function of these signalling proteins 1.10 Thesis summary This thesis can be divided into three parts. In the first section, I w i l l discuss work done i n the lab by Danielle Krebs and I on the role of Crk adapter proteins in B C R signalling. We found that Crk is used by the B C R to recruit C 3 G , a guanine nucleotide exchange factor for R a p l and R-Ras, to cellular membranes (120). This may provide a means for C 3 G to more efficiently activate R a p l and/or R-Ras. Sarah McLeod in the lab and I went on to show that R a p l is indeed activated by the BCR, but surprisingly, activation of R a p l by the B C R appears to be dependent on PLC-y-media ted D A G production and independent of the b ind ing of Crk»C3G complexes to tyrosine-phosphorylated docking proteins (121). This section also includes some preliminary investigations into how D A G may be activating R a p l i n B cells. In the second section, I w i l l discuss my work on the role of the G a b l docking protein i n B C R signalling. I found that G a b l is a membrane-associated docking site for three SH2 domain-containing signalling proteins involved i n B C R signalling, She, SHP-2, and Ptdlns 3-kinase (122). Thus, G a b l may l ink the B C R to multiple signalling pathways. To test this hypothesis, I overexpressed G a b l i n WEHI-231 cells wh ich normal ly express little if any G a b l . U s i n g this system, I found that BCR-induced Ptdlns 3-kinase signalling and SHP-2 phosphorylation were enhanced. Finally, I w i l l discuss a study i n which I investigated the functions of the SH2 and PTB domains of the She adapter protein. I found that BCR-induced tyrosine phosphorylat ion of She requires both the SH2 and PTB domains of She (123). Moreover, work done i n collaboration w i t h Hidetaka Okada (Kansai Med ica l 24 Univers i ty , M o r i g u c h i , Japan) showed that the b ind ing of the SHIP inosi tol phosphatase to the She PTB domain may play a key role i n this process. Thus, the work presented i n this thesis shows that adapter proteins and docking proteins are invo lved i n many B C R signal l ing pathways where they function as mediators of signalling complex formation. The formation of signalling complexes allows for the co-localization of signalling proteins and the recruitment of signalling proteins to cellular membranes where their substrates are located. A l l of these processes likely play a role i n the initiation and regulation of B C R signalling pathways. Publications arising from work i n this thesis Ingham, R.J., D.L. Krebs, S.M. Barbazuk, C .W. Turck, H . Hi ra i , M . Matsuda, and M . R . Go ld . 1996. B cell antigen receptor signaling induces the formation of signaling complexes containing the Crk adapter proteins. /. Biol. Chem. 271: 32306-32314. McLeod , S.J., R.J. Ingham, J.L. Bos, T. Kurosaki, and M.R. Gold . 1998. Activation of the R a p l GTPase by the B cell antigen receptor. /. Biol Chem. 273: 29218-29223. Ingham, R.J., M . Holgado-Madruga, C. Siu, A.J . Wong, and M.R. Gold . 1998. The G a b l protein is a docking site for multiple proteins involved in signaling by the B cell antigen receptor. /. Biol. Chem. 273: 30630-30637. Ingham, R.J., H . Okada, M . Dang-Lawson, J. Dinglasan, P. van der Geer, T. Kurosaki, and M.R. Gold . 1999. Tyrosine phosphorylation of She in response to B cell antigen receptor engagement depends on the SHIP inositol phosphatase. /. Immunol. 163: 5891-5895. 25 Chapter 2 Materials and Methods 2.0 Reagents 2.0.1 Antibodies The goat anti-mouse I g M , goat anti-human I g M , and goat-anti-mouse IgG Abs used for stimulation of B cells were purchased from Bio-Can (Mississauga, ON) . DT40 chicken B cells were stimulated wi th the 2-9B10 mouse anti-chicken I g M monoclonal antibody (mAb) (a gift from Dr. Michael Ratcliffe; M c G i l l University, Montreal, QC). The rabbit polyclonal Abs raised against C r k L , c-Crk II, c-Cbl, C 3 G , c-Abl (affinity-purified rabbit Ig control), R a p l , Grb2, and SHP-2 were from Santa Cruz Biotechnology, (Santa Cruz, C A ) . The anti-glutathione S-transferase (GST) m A b was also from Santa Cruz Biotechnology. The mAbs against c-Crk (anti-c-Crk 102-304 -which was raised against amino acids 102-304 of human c-Crk II but recognizes c-Crk I, II and CrkL) , Grb2, SOS1/SOS2, and pl30Cas were from Transduction Laboratories (Lexington, K Y ) . The 4G10 anti-phosphotyrosine (P-Tyr) m A b as w e l l as the anti-Gabl, She, SOS1, and SHIP polyclonal Abs were from Upstate Biotechnology, Inc. (Lake Placid, N Y ) . The M 2 an t i -FLAG m A b was from B A B C O (San Francisco, C A ) whi le the anti-phospho-Akt (Ser 473) and ant i -Akt Abs were from N e w England Biolabs (Beverly, M A ) . The 3A8 m A b which recognizes the SH2 domain of human c-Crk was a gift from Dr. M i c h i y u k i Matsuda (International Medical Center of Japan, Tokyo, Japan). The rabbit anti-Cas2 A b was from Dr. Hisamaru Hi ra i (University of Tokyo, Tokyo, Japan). The 2D9 ant i -CalDAG-1 m A b was from Drs. H i roak i Kawasaki and Anne Graybiel (Massachusetts Institute of Technology, Cambridge, M A ) . The polyclonal A b against the p85 subunit of Ptdlns 3-kinase was from Dr. Lewis Wi l l iams (University of California, San Francisco, San Francisco, C A ) , and the rabbit anti-GST 26 polyclonal A b was from Dr. Steve Robbins (University of Calgary, Calgary, AB) . Rabbit IgG, used as a control, was purified from normal rabbit serum (Sigma, St. Louis, M O ) using protein A-Sepharose 4B (Sigma). Horseradish-peroxidase-conjugated sheep-anti-mouse IgG, goat-anti-rabbit IgG, Protein A , and Protein G used for immunoblot t ing were purchased from Bio-Rad (Mississauga, ON) . 2.0.2 G S T fusion proteins c D N A s encoding the following GST fusion proteins were obtained from the following people: GST fused to the amino-terminal SH3 domain of c-Crk was from Dr. Mich iyuk i Matsuda (International Medical Center of Japan, Tokyo, Japan); GST fused to the SH2 domain of c-Crk or the SH2 domains of p85 was from Dr. Tony Pawson (Samuel Lunenfeld Research Institute, Moun t Sinai Hospi ta l , Toronto, O N ) ; GST fused to full length Grb2 or the Grb2 SH2 domain was from Dr. Gary Koretzky, Universi ty of Iowa, Iowa Ci ty , I A ) ; GST fused to the 97 amino acid R a l G D S - R a p l b ind ing domain (RBD) was from Dr. Johannes Bos (University of Utrecht, Utrecht, Netherlands); GST fused to the either the SH2 or PTB domains of She was from Dr. Melanie Welham, (University of Bath, Bath, U K ) ; GST fused to the tandem SH2 domains of SHP-2 was from Dr. Frank Jirik, (University of British Columbia, Vancouver, BC); and GST alone was from Dr. Steve Robbins (University of Calgary, Calgary, AB). For preparation of fusion proteins, a colony from a freshly streaked bacterial plate was used to inoculate a 20 m l Luria-Bertani (LB) broth culture. After incubating overnight, the 20 m l culture was used to seed a 1 L L B culture. The 1L culture was grown to an OD600 of between 0.8-1.0 and isopropylthio-P-galactoside (IPTG) (GIBCO-BRL, Burlington, O N ) was added to a final concentration of 100 u M . The culture was then incubated overnight at 26°C. The next morning, cells were pelleted and lysed i n lysis buffer (50 m M Tr i s -HCl p H 7.5, 150 m M N a C l , 1% Triton 27 X-100, 1 m g / m l lysozyme, 0.1 m g / m l D N A s e I, 10 | i g / m l leupeptin, 10 u g / m l soybean trypsin inhibitor, 1 u g / m l aprotinin, and 1 m M phenylmethylsulfonyl fluoride (PMSF)) for 30 min on ice. To ensure lysis, the cells were sonicated for 2 min on ice using a needle probe and a Misonix X L sonicator (Farmingdale, N Y ) set at setting 4. Insoluble material was removed by ultracentrifugation at 30,000 rpm for 45 min at 4°C using a Beckman L8-70 ultracentrifuge wi th a 70Ti rotor (Beckman, Palo Al to , C A ) . Fusion protein was collected on glutathione-Sepharose 4B beads (Pharmacia, Baie d'Urfe, QC) for 1 h at 4°C. The beads were then washed three times w i t h wash buffer (25 m M T r i s - H C l p H 7.5, 150 m M N a C l , 0.1% Tri ton X-100, 10 | i g / m l leupeptin, 10 ( i g / m l soybean trypsin inhibitor, 1 u g / m l aprotinin, and 1 m M PMSF). Bound fusion protein was eluted wi th elution buffer (50 m M Tris-base, 20 m M glutathione (final p H -8)) and eluates were dialyzed against 10 m M Tr i s -HCl p H 7.5 to remove free glutathione. The purity and integrity of the purified fusion proteins were analyzed by S D S - P A G E followed by Coomassie blue staining. Fusion protein concentrations were estimated by comparison to known amounts of bovine serum albumin (BSA) electrophoresed on the same gel. 2.1 Cell culture and stimulations 2.1.1 Cell lines The WEHI-231 (murine B lymphoma), A20 (murine B lymphoma) and R A M O S (human B lymphoma) cell lines were from the Amer ican Type Culture Collection (Manassas, V A ) . The B A L I 7 (murine B lymphoma) cell line was from Dr. Tony DeFranco (University of California, San Francisco, San Francisco, C A ) . DT40 cells were a gift from Dr. Tomohiro Kurosaki (Kansai Medica l Universi ty, Moriguchi , Japan). BOSC 23 cells were a gift from Dr. Warren Pear (Massachusetts 28 Institute of Technology, Cambridge, M A ) . DT40 cells expressing the murine ecotropic retrovirus receptor were produced in our lab by M a y Dang-Lawson. 2.1.2 C e l l culture The R A M O S , A20, and B A L I 7 cell lines as wel l as the WEHI-231 cell line and its variants were grown i n RPMI-1640 supplemented wi th 10% heat-inactivated fetal calf serum, 2 m M L-g lu tamine , 1 m M s o d i u m pyruvate , and 50 | i M 2-mercaptoethanol. DT40 cells and variant cell lines were grown i n the same R P M I media supplemented wi th 1% chicken serum and 4 m M L-glutamine instead of 2 m M . Variants of both WEHI-231 and DT40 cells expressing genes of interest were grown in the presence of 0.25 ^ g / m l puromycin (Calbiochem, La Jolla, C A ) . The BOSC 23 cells were grown in D M E M supplemented wi th 10% heat-inactivated fetal calf serum, 2 m M L-glutamine and 1 m M sodium pyruvate. The selection and maintenance of BOSC 23 cells have been described previously (124). 2.1.3 C e l l st imulation The cells were pelleted and then washed in a modified HEPES-buffered saline (25 m M H E P E S , 125 m M N a C l , 5 m M KC1, 1 m M C a C l 2 , 1 m M N a H 2 P 0 4 , 0.5 m M M g S 0 4 , 5.56 m M glucose, 2 m M L-glutamine, 1 m M sodium pyruvate, 50 | i M 2-mercaptoethanol). The cells were then resuspended to 2.5 x 1 0 7 / m l in the same modif ied Hepes-buffered saline and stimulated w i t h goat-anti-human I g M or goat-anti-mouse I g M Abs at a final concentration of 100 | i g / m l . DT40 chicken B cells were stimulated w i t h the 2-9B10 m A b at a final concentration of 50 (_ig/ml. Reactions were stopped by adding cold phosphate-buffered saline containing 1 m M Na3V04 and cells were pelleted by centrifugation at 1,500 rpm for 5 m in at 4°C. Cells were then solubilized at 5 x 1 0 7 / m l i n Triton X-100 lysis buffer (1% Triton X-100, 20 m M Tr i s -HCl , p H 8,137 m M N a C l , 10% glycerol, 2 m M E D T A , 1 m M N a 3 V 0 4 , 1 m M P M S F , 10 | i g / m l leupept in , 1 | i g / m l aprot inin) . After 10 m i n on ice, 29 detergent-insoluble material was removed by centrifugation. Protein concentrations were determined using the bicinchoninic acid assay (Pierce, Rockford IL). 2.1.4 Preparation of particulate and soluble fractions Cells were stimulated and washed as above, resuspended i n sonication buffer (20 m M Tr i s -HCl , p H 8, 137 m M N a C l , 10% glycerol, 5 m M E D T A , 1 m M N a 3 V 0 4 , 1 m M Na3MoC>4, 5 u M (3-methylaspartic acid, 1 m M P M S F , 10 u g / m l leupeptin, 10 Ltg/ml soybean trypsin inhibitor, 1 u g / m l aprotinin), and broken wi th five 5 s bursts using a needle probe and a Misonix X L sonicator set at setting 4. The efficiency of cell disruption was monitored by trypan blue staining. Unbroken cells and nuclei were removed by centrifuging at 14,000 rpm for 2 m in at 4°C using an Eppendorf 5415C microcentrifuge. The post-nuclear supernatant was centrifuged at 60,000 rpm for 20 min i n a Beckman TL-100 ultracentrifuge wi th a T L A 100.3 rotor. The soluble fraction was removed and Triton X-100 was added to a final concentration of 1%. The pellet containing the particulate fraction was rinsed wi th sonication buffer and then resuspended i n sonication buffer containing 1% Triton X-100. The pellet was dispersed by brief sonication and detergent-insoluble material was removed by centrifuging at 14,000 rpm for 3 minutes i n the cold using an Eppendorf 5415C microcentrifuge. Protein concentrations were determined using the bicinchoninic acid assay. 2.1.5 Preparation of cell lysates for Rap activation assay Cells were stimulated as described i n Section 2.1.2 but reactions were stopped by adding an equal volume of Nonidet P-40 lysis buffer (1% Nonidet P-40, 50 m M Tr i s -HCl , p H 7.5, 200 m M N a C l , 2 m M M g C l 2 , 10% glycerol, 1 m M N a 3 V 0 4 , 1 m M P M S F , 10 u g / m l leupeptin, 1 u g / m l aprotinin) directly to the cell suspension. The cells were lysed for 10 minutes on ice wi th occasional agitation and then centrifuged at 14,000 rpm for 15 min in the cold. The supernatant was then added to washed 30 glutathione-Sepharose 4B beads to which GST-RalGDS(RBD) fusion protein had been pre-bound (see Section 2.2.3 for Rap assay and ref. 125). 2.2 Precipitation experiments 2.2.1 Immunoprecipitation Cel l lysates (0.5-1 mg protein; 1-2 x 10 7 cell equivalents) were pre-cleared for 30 min at 4°C wi th 10 ul protein A or G-Sepharose and then mixed wi th Abs (1-4 (ig) for 1-3 h at 4°C. For cell fractionation experiments, the cytosolic fraction (-900 (ig protein) or membrane fraction (~600 | ig protein) from 3 x 10 7 cell equivalents was used. Immune complexes were collected w i t h 10 | i l prote in A - or protein G-Sepharose 4B for l h . The beads were then washed three times wi th Triton X-100 lysis buffer (see Section 2.1.2) before bound proteins were eluted wi th S D S - P A G E sample buffer containing 100 m M dithiothreitol. 2.2.2 Precipitation with GST fusion proteins (except GST-RalGDS(RBD)) Cel l lysates (0.5-1 mg of protein; 1-2 x 10 7 cell equivalents) were pre-cleared for 1 h w i t h 15 | i l glutathione-Sepharose 4B and then mixed w i t h 10 jig of purified fusion protein for 3 h at 4°C. The fusion protein complexes were collected wi th 15 | i l glutathione-Sepharose 4B for 1 h. The beads were then washed three times wi th Triton X-100 lysis buffer before bound proteins were eluted wi th SDS-PAGE sample buffer containing 100 m M dithiothreitol. 2.2.3 Precipitation with GST-RalGDS(RBD) Bacterial cell lysate (30 | i l) containing GST-RalGDS(RBD) was incubated wi th 15 | i l glutathione-Sepharose 4B beads for 30-60 min at 4°C. After washing once with Nonidet P-40 lysis buffer (see Section 2.1.4), the immobil ized GST-RalGDS(RBD) was 31 incubated wi th cell lysates (1.25 x 10 7 cell equivalents) for 1 h at 4°C. The beads were then washed three times wi th Nonidet P-40 lysis buffer before bound proteins were eluted wi th S D S - P A G E sample buffer containing 100 m M dithiothreitol. 2.2.4 Precipitation wi th biotinylated She peptides The biotinylated She peptide ( b i o t i n - E L F D D P S Y V N V Q N L D K ) , biotinylated phospho-Shc peptide ( b i o t i n - E L F D D P S p Y V N V Q N L D K ) , and biotinylated control phosphopeptide ( b i o t i n - L Q S D p Y M N M T P ) were gifts from Dr . Chr is Turck (University of California, San Francisco, San Francisco, C A ) . Cel l lysates were pre-cleared for 1 h wi th 25 ul avidin-agarose (Pierce) before being mixed for 2 h wi th 25 ul avidin-agarose beads to which 5 ug of biotinylated peptide had been adsorbed. The beads were then washed three times wi th Triton X-100 lysis buffer before bound proteins were eluted w i t h S D S - P A G E sample buffer conta in ing 100 m M dithiothreitol . 2.3 S D S - P A G E and immunoblott ing Proteins were separated on 1.5 mm-thick S D S - P A G E mini-gels and transferred to nitrocellulose filters for 75 min at 70 V . Molecular mass standards were visualized by staining wi th Ponceau S (Sigma, St. Louis , M O ) . For anti-P-Tyr blots, filters were blocked wi th 5% B S A i n Tris-buffered saline (TBS) (10 m M T r i s - H C l , p H 8, 150 m M N a C l ) whi le all other blots were blocked w i t h TBS containing 5% milk powder. Primary antibodies were diluted i n T B S / 1 % BSA and incubated wi th the filter for 3 h at room temperature or overnight i n the cold. After washing wi th TBS/0.05% Tween-20, the filters were incubated 1 h wi th horseradish peroxidase-conjugated goat-anti-rabbit IgG (1:20,000 i n TBS), sheep-anti-mouse IgG (1:10,000 i n TBS), protein A (1:2500 i n TBS), or protein G (1:2500 i n TBS). The filters were washed extensively w i t h T B S / 0 . 1 % Tween-20 and immunoreactive bands 32 were v isua l ized by enhanced chemiluminescence detection (ECL) (Amersham, Oakvil le , Ontario). To reprobe filters, bound Abs were removed by washing the filter wi th several changes of TBS, p H 2 for 1 h. The stripped filters were then blocked and probed as above. Western blotting experiments shown i n this thesis are representative of at least 2 experiments i n which similar results were obtained. 2.4 FAR Western blotting experiments Filters were blocked for 1 h w i t h T B S / 5 % B S A and then incubated overnight in the cold w i t h 1-2 H g / m l of GST fusion protein i n 20 m M T r i s - H C l , p H 7.5, 150 m M N a C l , 5 m M DTT, 0.02% azide. After washing wi th multiple changes for 30 m i n w i t h TBS/0 .05% Tween-20, the filters were incubated for 1 h at room temperature wi th 1 u g / m l of the anti-GST m A b or the rabbit anti-GST A b (1:5000) i n TBS. The filters were washed for 30 min w i t h TBS/0.05% Tween-20 and then incubated 1 h at room temperature w i t h horseradish peroxidase-conjugated sheep anti-mouse IgG (1:10,000 i n TBS). After washing the filters for 30 min w i t h TBS/0 .1% Tween-20, immunoreactive bands were visualized by E C L . F A R Western blot t ing experiments shown i n this thesis are representative of at least 2 experiments i n which similar results were obtained. 2.5 Molecular biology methods 2.5.1 Restriction endonuclease, blunt-ending, and phosphatase reactions Restriction endonucleases were purchased from N e w England Biolabs or G i b c o / B R L . Restriction enzyme reactions were carried out as recommended by the manufacturers w i th the amount of endonuclease added always being 5% or less of the total volume. To screen mini-preps, D N A was digested for 1 h whereas D N A used for ligation reactions was digested for a min imum of 3 h. To create blunt ends, dNTPs (0.5 m M of each nucleotide) and Klenow fragment (2 units) were added to 33 restriction enzyme digests and incubated at 25°C for an additional 20 min. The blunting reaction was terminated by incubating at 75°C for 10 min. In cases where blunt-end ligations were to be performed, the vector D N A was dephosphorylated wi th 5 units of calf intestinal alkaline phosphatase (New England Biolabs) for 30 min at 37°C. The dephosphorylation reaction was terminated by incubating at 75°C for 10 min. 2.5.2 Agarose gel electrophoresis Agarose gels (0.5-2% agarose i n TBE buffer (90 m M Tris-borate, 2 m M EDTA)) containing 25 | i g / m l ethidium bromide (Sigma) were electrophoresed at 100-200V to resolve D N A fragments. D N A i n the agarose gel was v i sua l i zed by U V i l l u m i n a t i o n and the image was captured us ing an A l p h a Innotech gel documentation system (Alpha Innotech Corporation, San Leandro, C A ) 2.5.3 Gel purification of DNA D N A was extracted from agarose gels using a clean scalpel and purified using the Qiaex II or QIAquick Gel Extraction Kits (Qiagen, Mississauga, O N ) according to the manufacturer's protocols. 2.5.4 Ligation of purified DNA fragments Ligations were performed using the Rapid D N A Ligat ion K i t (Boehringer Mannheim Canada, Laval , QC) essentially according to the manufacturer's protocol. Briefly, 0.5 u l of vector D N A was added to 1.5-3.5 | i l of insert D N A to achieve an approximate molar ratio of insert to vector of 3. Distilled, deionized water was then added to a final volume of 4 ul. To this, 1 ul of 5X D N A dilution buffer, 5 ul of 2X T4 D N A Ligase Buffer, and 0.5 ul (2.5 U) of T4 D N A ligase was added. The ligation reaction was carried out at room temperature for 10 min. 34 2.5.5 Transformation of competent bacteria Competent E. coli strains, DH5oc or HB101, were prepared by the technician in the lab, M a y Dang-Lawson. Competent bacteria (100 ul) were added to the D N A (50-100 ng of intact plasmid or entire ligation mixtures) and incubated on ice for 15-20 min. The bacteria were heat-shocked for 2 min at 42°C and then incubated on ice for 5 min. The entire transformation mixture was then plated onto L B plates containing 100 u g / m l ampicil l in and incubated overnight at 37°C. 2.5.6 Sma l l scale isolat ion of p l a smid D N A Small scale isolation of plasmid D N A was carried out using the QIAprep miniprep kit (Qiagen). Briefly, a single bacterial colony was transferred into 2 m l of L B medium containing 100 | i g / m l ampicil l in and incubated overnight at 37°C wi th shaking. The fo l lowing day, 1.5 m l of the culture was transferred to a microcentrifuge tube and centrifuged at 10,000 rpm for 1 min. The supernatant was removed by aspiration and plasmid D N A was isolated from the bacterial pellet according to the manufacturer's protocol using a modified alkaline-lysis mini-prep procedure. Puri f ied p lasmid D N A was quantified using a spectrophotometer (Pharmacia Biotech, Cambridge, U .K. ) and stored at -20°C. Us ing this procedure, approximately 20 | ig of plasmid D N A were routinely obtained. 2.5.7 Large scale isolation of plasmid D N A Large scale preparation of plasmid D N A was carried out using Nucleobond A X cartridges (Clonetech, Palo Al to , C A ) according to the manufacturer's protocol. Purif ied p lasmid D N A was quantified using a spectrophotometer (Pharmacia Biotech) and stored at -20°C. Using this procedure, approximately 500 | ig of plasmid D N A were routinely obtained from a 150 ml culture. 35 2.6 Plasmids p M S C V F L A G - C 3 G The c D N A encoding human C 3 G was cut out of the p C A G G S expression vector (a gift from Dr. M i c h i y u k i Matsuda, International Medica l Center of Japan, Tokyo, Japan) wi th Xhol and NotI and ligated in-frame into the Sail and NotI sites of the p M S C V F L A G retroviral expression vector (a gift from Dr . M i c h i y u k i Matsuda, International Medical Center of Japan, Tokyo, Japan). pMX-pie-C3G The c D N A encoding human C 3 G was cut out of the p M S C V F L A G vector by digesting w i t h Hindlll and NotI. The fragment was then ligated into the Hindlll and NotI sites of pBluescript (Stratagene, La Jolla, C A ) to generate pBluescript-C3G. The C3G c D N A was then cut out of pBluescript wi th Xhol and Not I and ligated into the Xhol and NotI sites of the pMX-pie retroviral expression vector (a gift from Dr. Alice M u i , University of British Columbia, Vancouver, BC). pMX-p ie -Ca lDAG-1 Mur ine C a l D A G - 1 c D N A i n the eukaryotic expression vector, p C M V S p o r t ( G i b c o / B R L ) , was a gift from Drs. H i r o a k i K a w a s a k i and A n n Greyb ie l (Massachusetts Institute of Technology, Cambridge, M A ) . The C a l D A G - 1 c D N A was cut out of the p C M V S p o r t vector w i th Sail and NotI and cloned into the Xhol and NotI sites of pMX-p ie . p M S C V F L A G - w i l d type She, R175M She, and R401M She . The FLAG-tagged human She c D N A s were a gift from Dr. Peter van der Geer (University of California, San Diego, La Jolla, C A ) . c D N A s encoding She proteins wi th either a point mutation that inactivates the SH2 domain (R401M) or the PTB 36 domain (R175M) were generated by P C R and amino-terminally tagged w i t h the F L A G epitope. The c D N A s were then subcloned into the p M S C V p a c murine retrovirus expression vector (126). p M X - G a b l Hemagglut in in (HA)-tagged murine G a b l c D N A s i n the L T R 2 expression vector were obtained from M a r i n a Holgado-Madruga and Dr . Alber t W o n g (Kimmel Cancer Institute, Philadelphia, P A ) . The APtdlns 3-kinase and A S H P - 2 mutants were generated by P C R wi th the APtdlns 3-kinase G a b l c D N A being generated by mutat ing the codons encoding tyrosines 448, 473, and 590 to phenylalanine while the ASHP-2 G a b l c D N A was generated by mutating the codons encoding tyrosine 628 to phenylalanine. The A P H mutant is a truncation of the G a b l c D N A that removes the codons for amino acids 1-115. The G a b l constructs were excised from L T R 2 by digestion wi th BamHI and EcoRI and ligated into the BamHI and EcoRI sites of the p M X retroviral expression vector (127) (a gift from Dr. Al ice M u i , Univers i ty of Brit ish Columbia , Vancouver, BC) . This resulted i n removal of the H A tag. 2.7 Retroviral infection of B cell lines 2.7.1 Production of retroviruses using the B O S C 23 packaging cell l ine The production of retroviruses using the BOSC 23 cell line was performed essentially as described by Pear et al. (128) but optimized for B cells i n our lab by Danielle Krebs and Yvonne Yang (124). Briefly, on the day prior to transfection, 1 confluent 10 cm dish of BOSC 23 cells was trypsinized and the cells were plated into each wel l of two 6 well-dishes . The next day, the medium was aspirated and 1 ml of fresh medium containing 25 ( iM chloroquine was added. To prepare the D N A for transfection, 2 | ig plasmid D N A followed by 200 | i l of 250 m M C a C l 2 was added to a 37 12 x 75 m m polystyrene tube. To this mixture, 200 ul of 2X HEPES-buffered saline (50 m M sodium H E P E S , p H 7.05, 10 m M KC1, 12 m M dextrose, 280 m M N a C l , 1.5 m M Na2HPC>4) was added dropwise over a period of 5 s while vortexing at low speed. The mixture was vortexed for an additional 10 s and added to the BOSC 23 cells. BOSC 23 cells were then returned to the incubator for 8-10 h, after which the medium was aspirated from the cells and replaced w i t h 2 m l of fresh medium. Thirty-six hours after starting the transfection, the media was replaced again. A t approximately 48 h post-transfection, the supernatant was collected and sterile-filtered using a 0.22 u M low protein-binding filter (Mil l ipore, Bedford, M A ) . The retrovirus-containing cell supernatants were then used immediately to infect WEHI-231 cells or DT40 cells expressing the murine ecotropic retrovirus receptor. 2.7.2 Infect i on of B ce l l l i nes O n the day of infection, 5 x 105-1 x 10 6 WEHI-231 cells or ecotropic retrovirus receptor-expressing DT40 cells were resuspended i n 0.5 m l of medium and were added to each wel l of a 6-well dish. The filtered, virus-containing medium from one we l l of a 6-well dish of BOSC 23 cells (~2 ml) was added to a 12 x 75 m m polystyrene tube containing 2.5 ul of 10 m g / m l polybrene (hexadimethrine bromide, Sigma). The tube was gently mixed and added to the B cells for approximately 24 h. Cells were then collected, pelleted by centrifugation and resuspended i n fresh m e d i u m . 2.7.3 P l a c i ng cel ls i n d r u g se lect ion A t 48 hr post-infection, the cells were centrifuged and placed i n medium containing puromycin at 0.25 u g / m l . After approximately 1 week of puromycin selection, the stable, bulk populations were used for experiments. Bulk populations were kept for no more than one month. This retroviral-mediated gene transfer procedure routinely results i n >95% of the surviving puromycin-resistant cells expressing the gene of interest (124). 38 2.8 Enzyme Assays 2.8.1 Ptdlns 3-kinase enzyme assays T r i t o n X - 1 0 0 ex t r ac t s (see S e c t i o n 2.1.3) f r o m 3-5 x 1 0 6 c e l l s (7 x 1 0 6 c e l l s fo r c e l l f r a c t i o n a t i o n e x p e r i m e n t s ) w e r e i m m u n o p r e c i p i t a t e d as d e s c r i b e d i n S e c t i o n 2 .2 .1 . T h e i m m u n o p r e c i p i t a t e s w e r e w a s h e d t w i c e w i t h T r i t o n X - 1 0 0 l y s i s b u f f e r (Na3VC>4 r e d u c e d to 50 u M ) a n d th ree t i m e s w i t h 10 m M T r i s - H C l p H 7.5. T e n m i c r o g r a m s o f P t d l n s ( A v a n t i P o l a r L i p i d s , A l a b a s t e r , A L ) w h i c h h a d b e e n d r i e d u n d e r n i t r o g e n gas , r e s u s p e n d e d to 1 m g / m l i n 30 m M H e p e s , p H 7.4, a n d s o n i c a t e d w e r e a d d e d to e a c h t u b e . A f t e r 10 m i n o n i c e , r e a c t i o n s w e r e i n i t i a t e d b y a d d i n g 4 0 u l o f k i n a s e a s s a y b u f f e r (30 m M H e p e s , p H 7.5, 30 m M M g C l 2 , 200 u M a d e n o s i n e , 50 u M A T P , 10 u C i 3 2 P - A T P ) . R e a c t i o n s w e r e c a r r i e d o u t f o r 15 m i n at r o o m t e m p e r a t u r e a n d s t o p p e d w i t h 0.1 m l 1 M H C 1 . T h e l i p i d s w e r e e x t r a c t e d w i t h 0.2 m l c h l o r o f o r m / m e t h a n o l (1:1) a n d 2 0 u l o f t h e l o w e r p h a s e w a s s p o t t e d o n t o o x a l a t e - t r e a t e d s i l i c a T L C p l a t e s ( V W R S c i e n t i f i c , T o r o n t o , O N ) . T h e c h r o m a t o g r a m s w e r e d e v e l o p e d i n c h l o r o f o r m / m e t h a n o l / w a t e r / a m m o n i a (18:14:3:1). A f t e r d r y i n g , the p l a t e s w e r e e x p o s e d t o f i l m at - 8 0 ° C . P t d l n s 3 - k i n a s e e n z y m e a s s a y e x p e r i m e n t s s h o w n i n t h i s t h e s i s a r e r e p r e s e n t a t i v e o f a t l e a s t 2 e x p e r i m e n t s i n w h i c h s i m i l a r r e s u l t s w e r e o b t a i n e d . 39 Chapter 3 The B C R uses Crk adapter proteins to form signall ing complexes 3.0 Introduction Recruitment of signalling proteins to cellular membranes is important for bringing cytosolic signalling proteins i n close proximity to membrane-associated substrates. For example, the BCR uses the She, SHP-2, and Grb2 adapter proteins to recruit the Ras activator SOS and the Ptdlns 5'-phosphatase SHIP to the plasma m e m b r a n e w h e r e Ras a n d i n o s i t o l p h o s p h o l i p i d s are l o c a t e d (118,119,122,123,129,130). This recruitment is facilitated by the SH2 domains of the adapter proteins b inding to tyrosine-phosphorylated membrane docking proteins. Another group of SH2 domain-containing adapter proteins are the Crk proteins (115). The Crk adapter proteins are structurally very similar to Grb2, consisting of a single SH2 domain wi th either one or two SH3 domains (see Fig. 3.1) and possessing no apparent catalytic activity. Given this fact, the Crk proteins are thought to mediate the formation of signalling complexes and recruit signall ing proteins to different areas wi th in the cell. Therefore, I was interested i n determining if the BCR used Crk proteins for these purposes. In particular, I was interested i n determining which proteins were complexed wi th Crk i n BCR-stimulated cells and whether Crk proteins recruited these proteins to specific areas within the cell. v -Crk was first identified as the gene product responsible for the transforming activity of both the CT10 (131) and ASV-1 (132) avian retroviruses. v-Crk consists of a fusion between the c-Crk I protein and the viral Gag protein and possesses both an SH2 domain and an SH3 domain (see Fig. 3.1) which allow v-Crk to interact wi th other signalling proteins. 4 0 Figure 3.1. C r k proteins and ant i -Crk Abs . The structures of the four C r k proteins are shown. The ability of the four different anti-Crk Abs to precipitate C r k L and C r k II were determined experimental ly and are indicated to the right. The location of the epitope recognized by the 3A8 m A b is indicated by the asterisk. This epitope is not present i n C rkL . The epitopes recognized by the rabbit Abs to c-Crk II and C r k L are contained w i th in the carboxy-terminal 20 amino acids of the respective C r k protein. Since c-Crk I has the same electrophoretic mobi l i ty as the endogenous Ig l ight cha in of R A M O S cells as w e l l as the Ig l ight cha in of A b s u sed for immunoprecip i tat ion, we were unable to study c-Crk I. Based on its sequence, we w o u l d expect that c-Crk I w o u l d be immunoprec ip i tated by the anti -Crk (102-304) m A b and the 3A8 m A b . 41 c-Crk II 40/42 kDa c-Crk I 28 kDa CrkL 38 kDa M] anti-c-Crk 3A8 anti-CrkL anti-c-Crk n (102-304) anti-c-Crk (rabbit Ab) (rabbit Ab) (mAb) (mAb) — +++ +++ +++ +/- ++ v-Crk SH2 f~J SH3| GagE 42 Two cellular homologues of the v-crk gene have been identified. The c-crk gene encodes two proteins, c-Crk I which is similar to v-Crk, consisting of a single S H 2 and a single S H 3 domain , and c-Crk II, w h i c h has an addi t iona l carboxy-terminal SH3 domain (ref. 133 and Fig 3.1). The crkL gene encodes for a protein that is structurally similar to c-Crk II in having one SH2 domain and two SH3 domains (ref. 134 and Fig . 3.1). Whi le C r k L has an overal l amino acid similarity of only 60% wi th c-Crk II, the amino acid sequences of the SH2 domain and amino-terminal SH3 domain are 97% identical (134). This suggests that c-Crk II and C r k L interact w i th similar proteins and may therefore have overlapping or redundant functions. Expression of the different Crk proteins varies. c-Crk II appears to be ubiquitously expressed while c-Crk I is expressed in a smaller number of tissues (133). C r k L is expressed predominantly i n cells of the hematopoietic lineage (134). G iven that Crk proteins, l ike other adapter proteins, contain no apparent catalytic activity, their role i n signal transduction is to use their SH2 and SH3 domains to form signalling complexes. Thus, the signalling pathways they regulate are governed by proteins that associate wi th their SH2 and SH3 domains. The SH2 domain of Crk binds growth factor receptors (e.g. platelet-derived growth factor (PDGF) f5-receptor), membrane-associated docking proteins (e.g. c-Cbl, IRS-1), and cytoskeletal proteins (e.g. paxil l in, the Cas proteins), and thus appears to help recruit Crk proteins to the membrane and cytoskeleton (115). The SH3 domains of Crk, i n particular the amino-terminal SH3 domain, also associate w i t h a number of proteins (115). In addition to the Ras activator SOS (135), the amino-terminal SH3 domain can associate wi th the A b l and A r g tyrosine kinases (136), DOCK180 (137), H P K 1 and KHS-1 (138), Eps l5 and Epsl5R (139), and C 3 G (135,140). D O C K 180 binds to and promotes the activation of the Rac GTPase (141). H P K 1 and K H S are related to germinal center kinase and may be involved i n the activation of Jnk (142). Eps l5 and Eps l5R are involved i n receptor-mediated endocytosis (143) and the G N E F C3G 43 is an exchange factor for the R a p l A and R a p l B proteins (hereafter collectively termed R a p l ) (144). The diversi ty of proteins that can b i n d to the C r k amino-terminal SH3 domain suggests that Crk proteins may regulate mult iple signalling pathways. Since Ras is l ikely a key mediator of B C R signalling, I was interested in whether Crk proteins were involved i n the regulation of Ras by the BCR. Given that Crk can b ind SOS, one possibility is that Crk plays a similar role to Grb2 in recruiting SOS to cellular membranes to activate Ras. Alternatively, Crk proteins may negatively regulate Ras signalling i n B cells by binding C 3 G , an activator of R a p l . The R a p l GTPase is thought to antagonize Ras-mediated signalling (145-147) and the B C R may use C r k to recruit C 3 G to cellular membranes where R a p l is located. Thus, C r k proteins may have either a positive or negative influence on BCR-induced signalling events mediated by Ras. To determine if Crk proteins participated i n B C R signalling, I, wi th the help of Danielle Krebs i n the lab, asked whether B C R engagement caused the formation of signalling complexes involving either C r k L or c-Crk II. In the R A M O S B cell line, we found that B C R ligation caused the SH2 domains of C r k L and c-Crk II to bind to the Cas proteins and to c-Cbl , membrane-associated docking proteins that are tyrosine phosphorylated i n response to B C R engagement. The Cas proteins and c-Cbl contain mul t ip le protein-protein interaction motifs and may l ink C r k complexes to other signall ing proteins i n addit ion to local izing C r k proteins to cellular membranes. We also found that the SH3 domains of both C r k L and c-Crk II bound primarily to C3G in R A M O S cells as opposed to SOS. Thus, Crk proteins may be involved i n the negative regulation of the Ras pathway i n B cells. Finally, we found that B C R ligation increased the amount of Crk i n the particulate fraction of R A M O S cells, suggesting that Crk proteins may move C 3 G from the cytosol to cellular membranes where R a p l is located 4 4 3.1 Results 3.1.1 C r k proteins associate w i t h tyrosine-phosphorylated proteins after B C R ligation S H 2 / S H 3 adapter proteins assemble signalling complexes by using their SH2 domains to b ind proteins that are tyrosine phosphorylated. Thus, if Crk proteins are involved in B C R signaling, BCR ligation should induce tyrosine phosphorylation of proteins that can b ind to the Crk SH2 domain. To test this, we incubated lysates from the R A M O S B cell line wi th a GST fusion protein containing the SH2 domain of c-Crk. We found that crosslinking the B C R on this cell line w i t h anti-IgM Abs stimulated the tyrosine phosphorylation of several proteins that could b ind in vitro to the GST-c-Crk SH2 domain fusion protein (Fig. 3.2A). It is l ikely that these phosphoproteins w o u l d also b ind to the SH2 domain of C r k L given that the SH2 domains of c-Crk and C r k L are highly conserved. Consistent w i th the in vitro results, we found that B C R crosslinking caused tyrosine-phosphorylated proteins to b ind to C r k L and c-Crk II i n R A M O S cells (Fig. 3.2B, upper panel). The 120-kDa and the 60-kDa Crk-associated phosphoproteins were always the most prominent. The c-Crk II/phosphoprotein complexes were less abundant than the CrkL/phosphopro te in complexes (Fig. 3.2B, upper panel), but could be readily observed wi th longer exposures (Fig. 3.2C, upper panel). This difference may be due to C r k L being expressed at higher levels than c-Crk II i n R A M O S cells. The association of these tyrosine-phosphorylated proteins wi th c-Crk II was evident w i th in 2 m i n of adding anti-IgM Abs to the R A M O S cells and persisted for at least 1 h (Fig. 3.3C, upper panel). Further analysis showed that two 120-kDa phosphoproteins that associated wi th C r k L and c-Crk II after B C R ligation d id so via the Crk SH2 domain (see below). 45 Figure 3.2. Crk proteins are tyrosine phosphorylated and associate with tyrosine-phosphorylated proteins after BCR crosslinking. A , R A M O S cells were incubated for 2 m i n w i t h (+) or without (-) ant i -IgM Abs . C e l l lysates were precipitated w i t h either GST or the GST-c-Crk II SH2 domain fusion protein. Precipitated proteins were immunoblot ted w i t h the 4G10 anti-P-Tyr m A b . Molecular mass standards (in kDa) are indicated to the left. B, R A M O S cells were incubated for 2 m i n wi th (+) or without (-) ant i-IgM Abs . C e l l lysates were precipitated wi th the anti-CrkL A b , the anti-c-Crk II A b , or wi th rabbit Ig (control). In the lane marked L B , immunoprecipitations were carried out w i t h lysis buffer instead of cell lysate. Precipitated proteins were analyzed by anti-P-Tyr immunoblotting. The filter was then stripped and reprobed wi th the anti-CrkL A b . The ant i -CrkL A b was highly selective for C r k L while the anti-c-Crk II A b was specific for c-Crk II and d id not precipitate C r k L (Fig 3.1). C , R A M O S cells were st imulated w i t h an t i - IgM A b s for the indicated times. C e l l lysates were immunoprecipi ta ted w i t h the anti-c-Crk II A b and analyzed by anti-P-Tyr immunoblotting. The filter was then stripped and re-probed wi th the anti-c-Crk II A b . 46 B ppt : st im: 200. 116. 97' + + 66 -A 45 —Ij Anti-P-Tyr Blot ippt : anti-c-Crk II stim (min): 0 2 5 15 60 pp!20 c-Crk II Anti-P-Tyr Blot 45H c-Crk II Anti-c-Crk (102-304) Blot ippt : c° pp!20 C r k L Anti-P-Tyr Blot 45 -^ C r k L Anti-CrkL Blot 47 In add i t ion to causing C r k L and c-Crk II to associate w i t h several ty ros ine-phosphory la ted proteins, B C R l i g a t i o n s t imula ted the tyrosine phosphorylation of C r k L and c-Crk II. When R A M O S cells were activated through the B C R , a 38-kDa tyrosine-phosphorylated protein was observed i n ant i -CrkL immunoprecipitates (Fig. 3.2B, upper panel) while a 40-kDa tyrosine-phosphorylated protein was seen i n anti-c-Crk II immunoprecipitates (Fig. 3.2C, upper panel). The molecular masses of these proteins suggested that they could be C r k L and c-Crk II, respectively. Reprobing these blots showed that the 38-kDa tyrosine-phosphorylated protein i n ant i -CrkL immunoprecipitates had the same electrophoretic mobility as C r k L . Figure 3.2C (lower panel) shows that B C R crosslinking caused some of the c-Crk II to migrate wi th a higher apparent molecular mass. Such bandshifts are often indicative of phosphorylation and this higher molecular mass form of c-Crk II had the same electrophoretic mobi l i ty as the tyrosine-phosphorylated 40-kDa protein seen i n anti-c-Crk II immunoprecipitates from activated R A M O S cells. Thus, it appears that both C r k L and c-Crk II are tyrosine phosphorylated i n response to B C R ligation. This may allow other SH2 or PTB-containing proteins to b ind to CrkL or c-Crk Et. 3.1.2 Cas proteins associate wi th C r k proteins i n BCR-stimulated cells. To elucidate the role of Crk proteins i n B C R signalling, we tried to identify the tyrosine-phosphorylated proteins that associate wi th C r k L and c-Crk II after BCR crosslinking. In fibroblasts, v-src transformation causes substantial tyrosine phosphorylation of the 120- to 130-kDa Cas protein at multiple Y X X P sequences to which the Crk SH2 domain can bind (148). These phosphorylated Y X X P motifs are the preferred binding motif of the Crk proteins (95). Therefore, we asked whether pl30Cas was the 120-kDa tyrosine-phosphorylated protein that associated wi th Crk proteins i n activated R A M O S cells (see F i g 3.2B and C , upper panels) Immunoblott ing w i t h a m A b raised against p l30Cas revealed two proteins of 48 approximately 120 k D a and 105 k D a that bound to C r k L and c-Crk II i n anti-IgM-stimulated R A M O S cells (Fig. 3.3A). These proteins associated wi th the Crk proteins to some extent i n resting cells, but B C R ligation significantly increased their binding to C r k L and to c-Crk II. The higher molecular mass protein is likely to be the Cas protein that has been described by Hi ra i and colleagues (148) while the 105-kDa protein is likely the lymphocyte-specific Cas protein, C a s L / H e f l (149,150). To determine how the Cas proteins interact wi th Crk proteins, we incubated R A M O S cell lysates wi th GST fusion proteins containing either the c-Crk II SH2 domain or the c-Crk II amino-terminal SH3 domain (Fig. 3.3B). Both forms of Cas bound specifically to the c-Crk II SH2 domain but not to the amino-terminal c-Crk II SH3 domain or to a GST-Grb2 SH2 domain fusion protein. The c-Crk II SH2 fusion protein precipitated a small amount of Cas from unstimulated R A M O S cells but much more from anti-IgM-stimulated cells. This suggested that tyrosine residues in Cas that mediate b inding to Crk proteins were phosphorylated at low levels i n unstimulated R A M O S cells and that phosphoryla t ion of these residues was increased by B C R ligation. Consistent w i th this idea, anti-P-Tyr immunoblotting showed that B C R ligation stimulated tyrosine phosphorylation of the 130-kDa Cas protein and to a lesser extent, the pl05Cas protein i n R A M O S cells (Fig. 3.3C, upper panel). Thus, these data clearly show that BCR ligation induces Crk proteins to bind to pl30Cas and a 105-kDa protein that is likely CasL. 3.1.3 c -Cb l is tyrosine phosphory lated and b inds directly to the C r k SH2 doma in in BCR-st imulated cells Another candidate tyrosine-phosphorylated protein that could bind to the Crk SH2 domain i n B cells is the 120-kDa c-Cbl protein. c-Cbl is tyrosine phosphorylated i n response to B C R cross-linking (151,152) and has been shown to b ind to C r k proteins i n activated T cells (153,154). We found that B C R ligation induced tyrosine phosphorylation of c-Cbl i n R A M O S cells and that tyrosine-phosphorylated c-Cbl had the same electrophoretic mobility as the Crk-associated 120-kDa phosphoprotein 49 Figure 3.3. C r k proteins associate w i t h Cas proteins i n BCR-s t imula ted cells. R A M O S cells were incubated for 2 min wi th (+) or without (-) anti-IgM Abs. A , Cel l lysates were precipitated wi th the anti-CrkL A b , the anti-Crk II A b , or wi th rabbit IgG (control). Precipitated proteins were analyzed by blotting wi th an anti-pl30Cas m A b which recognizes both the pl30 and pi05 Cas proteins. Molecular mass standards (in kDa) are indicated to the left. B , C e l l lysates were precipitated w i t h the indicated fusion proteins or wi th the anti-pl30Cas mAb. Precipitated proteins were analyzed by blotting wi th the anti-pl30Cas mAb. C , Ce l l lysates were precipitated wi th the rabbit anti-Cas2 A b or wi th a control A b . Precipitated proteins were analyzed by anti-P-Tyr immunoblotting. The filter was then stripped and reprobed wi th the anti-pl30Cas m A b . 50 T f i 2 558 I ^ ] Cas proteins Anti-Cas Blot B <f <f / #/ ~|Cas proteins Anti-Cas Blot ippt : stim : 116H + + •p!30 Cas Anti-P-Tyr Blot 116 J«wg»«««» I ~ | Cas proteins Anti-Cas Blot 5 1 (Fig. 3.4A). The anti-Crk (102-304) m A b , which recognizes both C r k L and c-Crk II (data not shown), precipitated a small amount of c-Cbl from lysates of unstimulated R A M O S cells, but much greater amounts of c-Cbl from lysates of anti-IgM-treated cells (Fig. 3.4B). Moreover , C r k proteins could be detected i n ant i -c-Cbl immunoprecipitates from anti-IgM-stimulated cells but not from unstimulated cells (Fig. 3.4C). Immunoprecipitating w i t h Abs specific for either C r k L or c-Crk II showed that B C R ligation induced the formation of both C r k L * c - C b l complexes and c-Crk II•c-Cbl complexes, wi th C r k L * c - C b l complexes being more prevalent (data not shown). While C r k L and c-Crk II associated wi th c-Cbl i n a BCR-dependent manner, Grb2 associated constitutively wi th c-Cbl i n R A M O S cells (Fig. 3.4D), pointing out a functional difference between the Crk and Grb2 adapter proteins. The inducible association of c-Cbl wi th C r k L and c-Crk II suggested that c-Cbl, l ike the Cas proteins, binds to the SH2 domain of Crk proteins. Indeed, the GST-c-Crk II SH2 fusion protein precipitated significant amounts of c-Cbl from lysates of anti-IgM-stimulated R A M O S cells but very little c-Cbl from lysates of unstimulated cells (Fig. 3.5A). While a small amount of c-Cbl from both stimulated and unstimulated R A M O S cells bound to the GST-c-Crk II amino-terminal SH3 domain fusion protein, c-Cbl bound primari ly to the c-Crk II SH2 domain and its ability to do so correlated wi th its phosphorylation on tyrosine residues. To determine whether c-Cbl bound directly to the SH2 domain of C r k proteins, we used the GST-Crk II SH2 domain fusion protein to probe blots of anti-Crk and anti-c-Cbl immunoprecipitates (Fig. 3.5B, upper panel). The c-Crk II SH2 domain bound directly to the C r k II-associated 120-kDa protein and to immunoprecipitated c-Cbl. BCR ligation increased the ability of the GST-c-Crk II SH2 domain fusion protein to b i n d to immunoprec ip i ta ted c-Cbl . Thus, BCR-induced tyrosine phosphorylation of c-Cbl creates b inding sites for the SH2 domain of Crk proteins. 52 Figure 3.4. c-Cbl is tyrosine phosphorylated and associates w i t h C r k proteins after B C R c ross l ink ing . R A M O S cells were incubated for 2 m i n wi th (+) or without (-) anti-IgM Abs . A , Cel l lysates were precipitated wi th the anti-c-Crk (102-304) m A b which recognizes both C r k L and c-Crk II, wi th the anti-c-Cbl A b , or wi th rabbit IgG (control). Precipitated proteins were analyzed by anti-P-Tyr immunoblot t ing. Molecular mass standards (in kDa) are indicated to the left. B , Ce l l lysates were precipitated wi th the anti-c-Crk (102-304) m A b or wi th an isotype-matched m A b (control) and analyzed by immunoblotting wi th an anti-c-Cbl A b . C , Ce l l lysates were precipitated wi th the anti-c-Cbl A b or wi th rabbit IgG (control) and analyzed by immunoblotting wi th the anti-c-Crk (102-304) mAb. D, Cel l lysates were precipitated wi th the anti-c-Cbl A b or wi th rabbit IgG (control) and analyzed by immunoblotting wi th an anti-Grb2 m A b . Ce l l lysate from 5 x 10 5 cells was included as a positive control. 53 'C-Cbl Anti-P-Tyr Blot • IgH •Crk Anti-c-Crk (102-304) Blot B — h ^ - c - C b l D i p p t : s t i m : 31H tf G r b 2 Anti-c-Cbl Blot Anti-Grb2 Blot 54 F i g u r e 3.5. c - C b l b i n d s d i r e c t l y t o t h e S H 2 d o m a i n o f c - C r k . R A M O S c e l l s w e r e i n c u b a t e d f o r 2 m i n w i t h (+) o r w i t h o u t (-) a n t i - I g M A b s . A , C e l l l y s a t e s w e r e p r e c i p i t a t e d w i t h t h e i n d i c a t e d f u s i o n p r o t e i n s o r w i t h t h e a n t i - c - C b l A b a n d t h e n a n a l y z e d b y i m m u n o b l o t t i n g w i t h the a n t i - c - C b l A b . M o l e c u l a r m a s s s t a n d a r d s ( i n k D a ) a re i n d i c a t e d t o t h e left . B , C e l l l y s a t e s w e r e p r e c i p i t a t e d w i t h t h e a n t i - c - C r k I I A b , t h e a n t i - c - C b l A b , o r w i t h r a b b i t I g G ( c o n t r o l ) . P r e c i p i t a t e d p r o t e i n s w e r e s e p a r a t e d b y S D S - P A G E a n d t r a n s f e r r e d to n i t r o c e l l u l o s e . T h e f i l t e r w a s p r o b e d f i r s t w i t h t he G S T - c - C r k SH2 d o m a i n f u s i o n p r o t e i n ( u p p e r p a n e l ) a n d t h e n r e p r o b e d w i t h the a n t i - c - C b l A b ( l o w e r p a n e l ) . 55 Anti-c-Cbl Blot c-Cbl GST-c-Crk SH2 Blot 116 -97 -c-Cbl Anti-c-Cbl Blot 56 A l t h o u g h c - C r k II b i n d s to S h e i n P C 1 2 c e l l s (135) , w e f o u n d tha t C r k L a n d c - C r k II d i d n o t b i n d to S h e i n a c t i v a t e d R A M O S c e l l s ( d a t a n o t s h o w n ) . T h i s i s i n c o n t r a s t t o G r b 2 , w h o s e S H 2 d o m a i n b i n d s p h o s p h o r y l a t e d S h e (118,119,130) a n d S H P - 2 (122) i n B C R - s t i m u l a t e d R A M O S c e l l s . T h u s , t h e S H 2 d o m a i n s o f C r k a n d G r b 2 h a v e d i f f e r e n t t a rge t s i n a c t i v a t e d B ce l l s . T h i s s u g g e s t s t h a t p r o t e i n s t h a t b i n d to the S H 3 d o m a i n s o f C r k a n d G r b 2 c o u l d b e d i r e c t e d t o d i f f e r e n t c e l l u l a r l o c a t i o n s . 3.1.4 C r k a n d G r b 2 a s s o c i a t e w i t h d i f f e r e n t e x c h a n g e f ac to r s i n R A M O S B c e l l s H a v i n g s h o w n tha t B C R l i g a t i o n c a u s e s C a s p r o t e i n s a n d c - C b l t o b i n d to the S H 2 d o m a i n s o f C r k p r o t e i n s , i t w a s i m p o r t a n t t o c h a r a c t e r i z e t h e p r o t e i n s tha t b i n d to the S H 3 d o m a i n s o f C r k L a n d c - C r k II i n B ce l l s . C r k p r o t e i n s h a v e b e e n s h o w n to a s soc i a t e w i t h t w o g u a n i n e n u c l e o t i d e e x c h a n g e f ac to r s , S O S a n d C 3 G (135). S O S i s a n a c t i v a t o r o f R a s (155,156) w h i l e C 3 G ac t i va t e s R a p l (144), a G - p r o t e i n t ha t m a y act as a n e g a t i v e r e g u l a t o r o f t h e R a s p a t h w a y (145-147) . T h u s , R a s - m e d i a t e d s i g n a l l i n g m a y re f lec t a b a l a n c e b e t w e e n the a c t i o n s o f S O S a n d C 3 G . I n a d d i t i o n to b i n d i n g C r k p r o t e i n s , S O S a n d C 3 G c a n a l s o a s soc i a t e w i t h G r b 2 (135). W h i l e t h i s s u g g e s t s t h a t C r k a n d G r b 2 c o u l d h a v e s i m i l a r r o l e s i n r e g u l a t i n g t h e R a s p a t h w a y , i t i s n o t k n o w n w h e t h e r C r k » S O S , C r k » C 3 G , G r b 2 « S O S , a n d G r b 2 » C 3 G c o m p l e x e s are a l l p r e s e n t i n s i g n i f i c a n t a m o u n t s i n B c e l l s . T o d e t e r m i n e w h i c h a d a p t e r p r o t e i n / e x c h a n g e f ac to r c o m p l e x e s w e r e m o s t p r e v a l e n t i n B c e l l s , w e f i r s t a s s e s s e d the a b i l i t y o f S O S a n d C 3 G to b i n d G S T f u s i o n p r o t e i n s c o n t a i n i n g e i t h e r t h e c - C r k II a m i n o - t e r m i n a l S H 3 d o m a i n o r t h e e n t i r e G r b 2 p r o t e i n . W e f o u n d t h a t S O S b o u n d e q u a l l y w e l l t o t he c - C r k II a m i n o - t e r m i n a l S H 3 a n d G r b 2 G S T f u s i o n p r o t e i n s in vitro ( F i g . 3 . 6 A , u p p e r p a n e l ) . T h e a n t i - S O S m A b w e u s e d f o r i m m u n o b l o t t i n g i n t h i s e x p e r i m e n t r e c o g n i z e s b o t h S O S 1 a n d S O S 2 . I n c o n t r a s t t o S O S , C 3 G b o u n d m u c h be t t e r to the c - C r k II a m i n o - t e r m i n a l S H 3 d o m a i n t h a n t o G r b 2 ( F i g . 3 . 6 A , l o w e r p a n e l ) . 57 We next investigated whether the relative abilities of SOS and C 3 G to b ind Crk and Grb2 in vitro reflected which complexes were present i n the R A M O S B cell line. C r k L , c-Crk II, and Grb2 were precipitated from cell lysates and the precipitates were probed wi th Abs to SOS or C 3 G (Figs. 3.6B, C , and D). In these experiments, c-Crk II was specifically precipitated wi th the 3A8 m A b which recognizes an epitope i n the SH2 domain of c-Crk II that is not present i n C r k L (157). Unl ike other anti-Crk II Abs that b ind epitopes near the SH3 domains, the 3A8 m A b can precipitate Crk II w i th proteins bound to its SH3 domains. Since the binding of proteins to the SH3 domains of Grb2 can also block A b binding, we precipitated Grb2 wi th a phosphotyrosine-containing peptide ( E L F D D P S p Y V N V Q N L D K ; single-letter code, p Y = phosphotyrosine) based on one of the sequences i n She that binds to the Grb2 SH2 domain. This peptide precipitated a substantial portion of the Grb2 i n R A M O S cells (data not shown). Consistent w i t h the fusion protein experiments, C 3 G preferentially associated wi th Crk as opposed to Grb2 i n R A M O S cells. C3G was precipitated by the anti-CrkL A b and by the 3A8 anti-c-Crk II m A b but not by the She phosphopeptide that precipitates Grb2 (Figs. 3.6B, C, and D). The interaction of C 3 G wi th C r k L and c-Crk II was evident i n unstimulated R A M O S cells and d id not change upon ligation of the B C R w i t h anti-IgM Abs (Fig. 3.6D). Al though SOS bound the SH3 domains of C r k and Grb2 equally we l l in vitro, SOS preferentially bound to Grb2 i n R A M O S cells (Fig. 3.6B, upper panel). M u c h less SOS bound to C r k L than to Grb2 and SOS could not be detected i n anti-c-Crk II immunoprecipitates (data not shown). The weak binding of SOS to the Crk proteins in vivo may reflect the ability of C 3 G to compete more effectively for binding to Crk. C 3 G may have higher affinity for the Crk SH3 domain than SOS (158) or it may be expressed at higher levels than SOS1 and SOS2. Thus, C 3 G binds exclusively to the C r k proteins i n R A M O S cells whi le SOS associates primarily wi th Grb2 and to a lesser extent wi th C r k L . 58 Figure 3.6 Association of SOS and C 3 G wi th C r k and Grb2. A , R A M O S cell lysates were precipitated w i t h the indicated fusion proteins. Precipitated proteins were analyzed by immunoblotting wi th the anti-C3G A b . The filter was then stripped and reprobed wi th a m A b that recognizes both SOS1 and SOS2. Cel l lysate from 5 x 10 5 cells was included as a positive control. Molecular mass standards (in kDa) are indicated to the left. B, Ce l l lysates were precipitated wi th the indicated peptides immobil ized on beads, wi th the 3A8 anti-c-Crk II m A b , or w i th an isotype-matched control m A b . The She P-Tyr peptide ( E L F D D P S - p Y V N V Q N L D K ) is based on the sequence surrounding Y317 of human She which is known to b ind the Grb2 SH2 domain. The non-phosphorylated version of this peptide, as wel l as an irrelevant P-Tyr-containing peptide ( L Q S D p Y M N M T P ) , neither of wh ich precipitated Grb2 (data not shown), were used as controls. Sequential immunoblot t ing w i t h the anti-C3G A b and then wi th the anti-SOS m A b was performed as i n A . Ce l l lysate from 5 x 10 5 cells was included as a positive control. Note that the anti-SOS m A b reacted w i t h a band i n the 3A8 immunoprecipitates from R A M O S cells (lane marked R). However, this band had a different electrophoretic mobility than SOS and is l ikely to be a contaminant i n the anti-c-Crk II A b preparation since it was present when the cell lysate was omitted from the reaction and replaced wi th Triton X-100 lysis buffer (lane marked LB). C , Cel l lysates were precipitated wi th the She P-Tyr peptide, w i th a CrkL-specific A b or w i th rabbit IgG (control). Sequential immunoblot t ing w i t h the ant i-C3G A b and then w i t h the anti-SOS m A b was performed as i n A . Ce l l lysate from 5 x 10 5 cells was included as a positive control. D , R A M O S cells were incubated for 2 min wi th (+) or without (-) anti-IgM Abs. Cel l lysates were precipitated wi th the indicated Abs or wi th purified rabbit IgG (control) and immunoblot ted w i t h the anti-C3G A b . Ce l l lysate from 5 x 10 5 cells was included as a positive control. 59 ppt: 200 116 CP CP CP SOS Anti-SOS Blot 200 H 116 H SOS Anti-SOS Blot 200 116 -L C 3 G Anti-C3G Blot C 3 G Anti-C3G Blot B D ppt: / ippt: L B R s t i m : 200 H 116H 200-116-1 Anti-SOS Blot SOS 200 - | 116 J C 3 G Anti-C3G Blot •C3G Anti-C3G Blot 60 3.1.5 Subcellular localization of Crk, Cas, and c-Cbl in BCR-stimulated cells Both Crk and C 3 G are cytosolic proteins whereas R a p l is targeted to the cytosolic face of cellular membranes by a l i p i d modification (70). The ability of C r k * C 3 G complexes to regulate R a p l may therefore require translocation of these complexes from the cytoplasm to cellular membranes. To see i f this occurred i n R A M O S cells, we analyzed the subcellular localization of the C r k proteins before and after B C R ligation using a sonication/ultracentrifugation procedure (see Section 2.1.4). Immunoblotting the soluble and particulate fractions of R A M O S cells wi th the anti-Crk (102-304) m A b showed that the majority of Crk protein was i n the soluble fraction (Fig. 3.7A). Whi le a small amount of Crk was present i n the particulate fraction of unstimulated cells, B C R ligation increased the amount of Crk proteins in the particulate fraction. This suggested that B C R engagement caused Crk proteins to translocate from the cytosol to cellular membranes. Since the Crk SH2 domain binds to Cas proteins and c-Cbl, we asked whether Cas proteins and c-Cbl were i n the particulate fraction of R A M O S cells. Immunoblotting wi th the anti-pl30Cas m A b (Fig. 3.7B) or w i th an anti-c-Cbl A b (Fig. 3.7C) showed that the majority of these proteins were i n the soluble fraction of R A M O S cells. However, significant amounts of Cas protein and c-Cbl were present i n the particulate fractions of both unstimulated and anti-IgM-stimulated R A M O S cells. B C R ligation d id not significantly alter the subcellular distribution of Cas or c-Cbl. Thus, small but significant amounts of Cas and C b l were i n the particulate fraction of R A M O S cells, even before B C R ligation. Tyrosine phosphorylation of membrane-associated Cas proteins and c-Cbl could provide binding sites for the Crk SH2 domain. This wou ld allow Crk proteins to bring C 3 G and other proteins that bind to their SH3 domains to the membrane. To test this model , we asked whether Crk*Cas or C r k * c - C b l complexes could be found i n the particulate fraction of BCR-stimulated R A M O S cells. We found that BCR ligation increased the amount of CrkL*Cas complexes i n both the particulate 61 and soluble fractions of R A M O S cells (Fig. 3.8A). Similarly, B C R crosslinking caused a large increase in the amount of C r k L * c - C b l complexes i n the particulate fraction of R A M O S cells (Fig. 3.8B). C r k L * c - C b l complexes were also found i n the soluble fraction of R A M O S cells. We were not able to detect membrane-associated c-Crk II* Cas complexes or c-Crk II* c-Cbl complexes, perhaps because c-Crk II is expressed at lower levels than CrkL . Nevertheless, our data show that B C R crosslinking induced the formation of membrane-associated C r k L * C a s complexes and C r k L * c - C b l complexes i n R A M O S cells. 3.2 Discussion We have made several novel observations concerning the role of Crk adapter proteins i n B C R signalling. We found that both C r k L and c-Crk II are tyrosine p h o s p h o r y l a t e d i n response to B C R l i g a t i o n a n d that s eve ra l tyrosine-phosphorylated proteins associate wi th Crk proteins after B C R crosslinking. We identified two of these phosphoproteins as pl30Cas and c-Cbl and showed that both bound to the SH2 domains of Crk proteins after B C R engagement. We also show that, i n the R A M O S B cell line, the amino-terminal SH3 domain of c-Crk II preferentially binds the C 3 G nucleotide exchange factor over SOS. Since C 3 G can activate R a p l , it suggests that C r k proteins may be invo lved i n the negative regulation of Ras-mediated signalling i n B cells. Our cell fractionation studies showed that Cas proteins and c-Cbl are present to some extent i n the particulate fraction of BCR-stimulated R A M O S cells and may therefore provide docking sites that can recruit Crk complexes to cellular membranes. Consistent wi th this idea, we found that B C R ligation increased the amount of Crk i n the particulate fraction of R A M O S cells and induced the formation of CrkL*Cas and C r k L * c - C b l complexes i n the particulate fraction. Crk-mediated translocation of C 3 G to cellular membranes may be important for C 3 G to activate R a p l and for other Crk-associated proteins to perform their functions. 62 Figure 3.7. Subce l l u l a r loca l i za t ion of C r k , Cas, and c -Cb l . R A M O S cells were incubated for 3 m in wi th (+) or without (-) anti-IgM Abs before preparing particulate and soluble fractions. Particulate and soluble fractions from "5 x 10 5 cell equivalents were separated by S D S - P A G E and analyzed by immunoblot t ing w i t h (A) the anti-c-Crk (102-304) m A b or w i t h (B) the anti-pl30Cas m A b . Molecular mass standards (in kDa) are indicated to the left. C, Particulate and soluble fractions from 1 x 10 6 cells were immunoprec ip i ta ted w i t h the an t i -c -Cbl A b and then immunoblotted wi th the same A b . 63 fraction : stim : 45-C r k Anti-c-Crk (102-304) Blot B fract ion: ^ e>° stim : 116. 97' ] Cas proteins Anti-Cas Blot ippt: fraction : stim : Anti-c-Cbl 116H c-Cbl Anti-c-Cbl Blot 64 Figure 3.8. Subcellular localization of Crk/Cas and Crk / c -Cb l complexes. R A M O S cells were incubated for 3 min wi th (+) or without (-) anti-IgM Abs. Particulate and soluble fractions from 2.5 x 10 7 cells were immunoprecipitated w i t h the anti-CrkL A b and immunoblotted wi th (A) the anti-pl30Cas m A b or (B) the anti-c-Cbl A b . Molecular mass standards (in kDa) are indicated to the left. 65 ippt: anti-CrkL fraction : stim : 116- ""1 Cas proteins Anti-Cas Blot ippt: anti-CrkL fraction : stim: + - + Anti-c-Cbl Blot 66 We have identified the Cas proteins and c-Cbl as two major targets of the Crk SH2 domain i n activated B cells. Smit et al. (159) have also shown that c-Cbl binds to Crk proteins after B C R crosslinking. Cas proteins and c-Cbl can be considered part of a family of proteins termed docking proteins, that includes IRS-1, IRS-2, G a b l , and the Drosophila D O S protein. Whi l e these proteins contain various protein interaction motifs, their common feature is that they can be phosphorylated on mu l t i p l e tyrosine residues and thus serve as d o c k i n g sites for SH2 domain-containing proteins. pl30Cas and p l05CasL both have an SH3 domain as wel l as 15 and 13 Y X X P motifs, respectively, that could b ind the SH2 domain of Crk proteins (115). Similarly, c-Cbl has 17 proline-rich motifs that could potentially bind SH3 domains (160) and is also strongly phosphorylated on tyrosine residues i n response to B C R l igat ion (151,152). Thus, mult iple s ignal l ing proteins could simultaneously b ind a single molecule of Cas or c-Cbl. In B cells, c-Cbl binds Grb2 and Ptdlns 3-kinase i n addition to Crk (152,161). This may allow crosstalk between Crk-associated proteins and signalling pathways involving Grb2 and Ptdlns 3-kinase. There is also some evidence that c-Cbl acts as a negative regulator of immunoreceptor signalling. c-Cbl can bind to Syk i n BCR-stimulated cells (161) and co-transfection of c D N A encoding for a C D 8 / C D 3 £ chain fusion (extracellular and transmembrane domains of C D 8 fused to the intracellular domain of the T C R £ chain), Syk, and c-Cbl in COS-7 cells showed that transfecting increasing amounts of c -Cbl -encodingcDNA decreased the amount of detectable Syk protein i n cell lysates (162). However, co-transfection of a c-Cbl protein that could not interact wi th Syk d id not downregulate Syk protein levels even when large amounts of mutant c-Cbl c D N A where introduced. This downregulation of Syk may be due to the fact that Syk is being ubiquitinated and degraded as c-Cbl as been shown to enhance the ubiquitination, internalization, and degradation of the epidermal growth factor (EGF) receptor (163) and P D G F receptor stimulation (164). 67 pl30Cas and c-Cbl may also link tyrosine kinases to Crk and Crk-associated proteins. In B cells, c-Cbl associates wi th the F y n and Btk tyrosine kinases (151,161). pl30Cas has also been reported to b ind Src kinases (148). These tyrosine kinases could phosphorylate the Crk proteins as wel l as proteins bound to the Crk SH3 domains. Tyros ine phosphory la t ion of C r k L and c-Crk II cou ld a l low SH2-containing proteins to b ind to them and may provide another means by which Crk proteins could mediate the formation of signalling complexes. In R A M O S cells, we found that C r k proteins b o u n d C 3 G v i a their amino-terminal SH3 domain. Smit et al. (159) have also reported that C r k L binds C3G in R A M O S cells but that c-Crk II does not. The A b they used to precipitate c-Crk II recognizes an epitope i n the amino-terminal SH3 domain of c-Crk II. We also found that this A b d i d not precipitate c-Crk II«C3G complexes (data not shown), presumably because the epitope on c-Crk II is masked by the b ind ing of C3G. However, when we used the 3A8 anti-c-Crk II m A b which recognizes an epitope in the SH2 domain of c-Crk II (but not CrkL) , we were able to clearly show that c-Crk II does bind C 3 G i n R A M O S cells. Thus, C r k L and c-Crk II are l ikely to have similar functions i n B cells. One target of C 3 G is R a p l . R a p l may be a key signalling molecule since loss-of-function mutations i n Drosophila rapl are lethal (145). Several studies have shown that R a p l is a negative regulator of the Ras s igna l l ing pathway. Overexpression of R a p l inhibits fibroblast transformation by oncogenic versions of Ras (146) and blocks Ras-dependent germinal vesicle b reakdown i n Xenopus oocytes (147). Similarly, a gain-of-function mutation i n the Drosophila rapl gene blocks Ras-dependent development of the R7 photoreceptor cell (145). Activated R a p l does not prevent Ras activation (165) but instead blocks the ability of Ras to interact w i t h and activate downstream effectors. R a p l has an almost identical effector-interaction sequence as Ras (166) and this allows R a p l - G T P to compete with Ras-GTP for binding to Ras effectors. Rap l -GTP is thought to sequester Ras effectors 68 and prevent them from being activated by Ras. Potential downstream effectors of Ras include Raf-1 (73), Ptdlns 3-kinase (74), RasGAP (167), and RalGDS, a nucleotide exchange factor that activates another member of the Ras family called Ral (168,169). R a p l - G T P can b ind all of these proteins (169-171) and thus could potentially inhibit many, if not all , Ras-mediated signalling events. Competition between Ras-GTP and R a p l - G T P for binding to Raf-1 may be of particular significance. The binding of Ras-GTP to Raf-1 initiates a protein kinase cascade that culminates i n activation of the E R K family of mitogen-activated protein kinases. The E R K s are important regulators of cell growth and differentiation that phosphorylate and activate transcription factors such as Elk-1 (172). Activat ion of the R a s / R a f / E R K pathway by the BCR and other receptors is usually transient. For example, BCR-induced activation of Raf-1 is maximal after 1 m i n and declines to near basal levels by 15 min (33). Prolonged Ras signalling may be deleterious to cells and this may be prevented by activation of R a p l which can bind and sequester Ras effectors such as Raf-1. Overexpression of constitutively-active R a p l A i n fibroblasts has been shown to block Ras-dependent activation of ERKs by E G F (165). Whether R a p l normally limits the magnitude or duration of E R K activation i n B cells or other cells remains to be determined. The regulation of downstream effectors of Ras (e.g. Raf-1) may involve a balance between SOS-mediated activation of Ras and C3G-mediated activation of R a p l . Our data suggest that i n B cells the Crk and Grb2 adapter proteins may have opposing functions in this process since they preferentially b ind different G N E F s that activate G-proteins that may oppose each other. In R A M O S cells, Grb2 bound SOS but d id not b ind detectable amounts of C3G. Thus, She and Grb2 may promote BCR- induced Ras activation. C r k L * S O S complexes may also make a minor contribution to BCR-induced Ras activation. However , we found that the C r k proteins associate pr imari ly wi th C 3 G i n R A M O S cells (see Fig. 3.6C) and w o u l d 69 therefore more l ike ly be invo lved i n Rapl -media ted down-regulat ion of Ras signalling pathways. While several studies have suggested a role for R a p l i n the regulation of Ras signalling (145-147), these studies have relied on the overexpression of R a p l . It may be that R a p l does not normal ly regulate Ras s ignal l ing, but does so when overexpressed. In support of this, Zwartkruis et al. have reported that endogenous R a p l does not interfere wi th Ras-mediated E R K activation (173). Thus, R a p l may really function to regulate other s ignal l ing pathways. There is evidence i n Drosophila that R a p l regulates the Dacapo gene product, wh ich shows sequence similarity to the cyclin-dependent kinase inhibitor p 2 7 K i p ! (174). Furthermore, R a p l can also b ind B-Raf, a Raf isoform, that unlike Raf-1, can be activated by R a p l (175) . Thus, i n some situations R a p l can activate E R K . However, it is unlikely that R a p l activates E R K i n B cells as B-Raf does not appear to be expressed i n B cells (Phil Stork; personal communication). In addition to acting as a G N E F for R a p l , C 3 G can also act as a G N E F for R-Ras (176) . It is possible that the real role of the recruitment of C 3 G to membranes by Crk proteins is to activate R-Ras. Activation of R-Ras by Crk»C3G complexes has been shown to regulate integrin affinity (177). C3G has also been reported to be involved i n Jnk activation (178). Jnk is known to be activated by the B C R (77), but it is unknown if R-Ras or C r k » C 3 G complexes are involved i n Jnk activation by the BCR. In addi t ion to C 3 G and perhaps SOS, C r k proteins may control the interactions and subcellular localization of other proteins that b ind to their SH3 domains such as D O C K 180, H P K 1 and K H S , A b l and A r g , and Eps l5 and Epsl5R. Whi le D O C K 1 8 0 does not appear to be expressed i n B cells (R.J. Ingham; unpublished data), it is not known whether these other proteins associate wi th Crk proteins i n B cells. 70 Our cell fractionation studies showed that B C R l iga t ion induced the appearance of C r k L * C a s and C r k L * c - C b l complexes i n the membrane-enriched particulate fraction of R A M O S cells. This correlated wi th an increase in the amount of Crk proteins i n the particulate fraction. In contrast, a similar amount of Cas proteins and c-Cbl were present i n the particulate fraction before and after B C R ligation. This suggests a model in which BCR-induced tyrosine phosphorylation of membrane-associated Cas and c-Cbl creates binding sites for the Crk SH2 domain and thereby recruits Crk proteins to cellular membranes (Fig. 3.9). While it is not clear how the Cas proteins and c-Cbl associate wi th membranes, it may be due to their ability to b ind Src kinases such as F y n wh ich are anchored to cellular membranes by l i p id modifications. The constitutive membrane association of the Cas proteins and c-Cbl may be due to the fact that growth factors i n the serum the cells are grown i n are recruiting Cas and c-Cbl to cellular membranes. It wou ld be interesting to look at the localizat ion of C r k proteins before and after B C R stimulation i n normal B cells or serum-starved B cell lines. It is possible that i n the absence of other receptor signalling, B C R signalling recruits c-Cbl and the Cas proteins to cell membranes. In support of this, c-Cbl is known to be recruited to the epidermal growth factor EGF receptor in response to E G F stimulation (179). The significance of BCR-induced translocation of Crk protein complexes to cellular membranes remains to be determined, but it may be critical for C 3 G to activate either R a p l and/or R-Ras. Microscopy studies w i l l be required to determine wh ich cellular membranes R a p l and R-Ras are associated w i t h i n B cells and whether C r k * C 3 G complexes translocate to those membranes after B C R ligation. In summary, we have shown that the C r k L and c-Crk II adapter proteins are used by the B C R to promote the formation of signalling protein complexes. The assembly of these complexes may initiate signall ing reactions by co-localizing components of s igna l l ing pathways, thereby, a l l owing for efficient s ignal transmission. 71 Figure 3.9. Proposed role of C r k proteins i n B C R s i gna l l ing . Recruitment of C r k * C 3 G complexes to cellular membranes by binding to Cas proteins and c-Cbl. Cas proteins and c-Cbl b ind to the SH2 domain of C r k whi le C 3 G binds to the amino-terminal SH3 domain of Crk. See discussion for details. Note that the membrane-associated protein wi th the question mark, "?", is meant to represent an unknown protein that the Cas and/or c-Cbl proteins might associate wi th at cellular membranes. 72 73 Chapter 4 Activation of the R a p l GTPase by the B C R involves a diacylglycerol-dependent pathway that is independent of C r k signall ing complexes 4.0 Introduction The results i n the previous chapter suggested that R a p l might be a target of BCR signalling. C 3 G , an exchange factor that activates both R a p l A and R a p l B (144,180), is constitutively associated wi th the Crk adapter proteins i n B cells (120). In response to B C R engagement, the membrane-associated Cas and c-Cbl docking proteins are tyrosine phosphorylated and serve as binding sites for the SH2 domains of Crk proteins (120,159). Thus, B C R signalling recruits C r k * C 3 G complexes to cellular membranes where R a p l is located. This may allow C 3 G to activate R a p l . The two mammalian R a p l proteins, R a p l A and R a p l B , are 97% identical at the amino acid level are closely related to Ras (70). R a p l A (also k n o w n as Krev-1) was first identified by its ability to reverse the transformation of N I H 3T3 cells by an activated form of Ki-Ras (146). Expression of activated R a p l also blocks the actions of Ras i n Drosophila eye development (145) and i n the maturation of Xenopus oocytes (147). These results suggest that activation of endogenous R a p l may be a way i n which the magnitude and/or duration of Ras-mediated signalling is limited. R a p l inhibits Ras signalling by binding and sequestering Ras effectors (170). The effector b inding domain of R a p l is almost identical to that of Ras, suggesting that R a p l - G T P competes w i t h Ras-GTP for downstream effectors. In vitro experiments have shown that R a p l - G T P binds to Raf-1 and other targets of Ras but does not activate them (170,171). In vivo, R a p l may also keep Ras effectors physically separated from Ras since R a p l is located primarily on the cytoplasmic face of the Golg i apparatus (181) while Ras is at the inner face of the plasma membrane. Consistent w i t h the idea that R a p l - G T P sequesters Ras effectors i n inactive 74 complexes, expressing const i tut ively-act ive R a p l A i n fibroblasts inhibi ts Ras-dependent activation of the E R K kinases (165). Whi l e there is considerable data showing that R a p l can regulate Ras signalling, little is known about how endogenous R a p l is regulated and whether receptors regulate R a p l activation. The data presented i n the previous chapter showing that C r k # C 3 G complexes translocate to cellular membranes i n response to BCR stimulation suggested that the R a p l might be a target of the BCR. Therefore, we investigated whether R a p l was activated by the BCR. Work done by Sarah M c L e o d and I, showed that B C R ligation does lead to activation of R a p l . However , activation of R a p l by the B C R appears to be independent of the binding of C r k # C 3 G complexes to membrane-associated docking proteins. Instead, BCR-induced activation of R a p l involves a PLC-y-med ia ted pathway that is dependent upon D A G production. 4.1 Results 4.1.1 R a p l is activated by the B C R R a p l opposes the actions of Ras and may therefore be a key regulator of cell growth. To determine if the B C R activates R a p l , we used a novel assay for R a p l activation developed by Franke et al. (125). This assay is based on the observation that the RalGDS protein has high affinity for active R a p l - G T P but does not b ind the inactive GDP-bound form of R a p l (182). Therefore, we used a GST fusion protein containing the Rap l -b ind ing domain (RBD) of Ra lGDS to selectively precipitate activated R a p l . The recovery of activated R a p l was monitored by immunoblotting wi th an anti-Rapl A b which recognizes both R a p l A and Rap lB . Us ing this assay, we found that initiating B C R signalling wi th anti-Ig Abs increased the amount of activated R a p l i n the IgM+ WEHI-231 immature B lymphoma cell line, the mature IgM+ BAL17 murine B cell line, and the I g G + A20 75 murine cell l ine wh i ch is thought to resemble a memory B cell (Fig. 4.1). R a p l was also found to be activated in mature resting B cells isolated f rom mouse spleen, the I g M + R A M O S h u m a n B cell line as wel l as the I g M + DT40 chicken B cell l ine (ref. 121 and data not shown). Thus , BCR - i nduced activation of R a p l is a c o m m o n feature of B C R engagement in many different B cells. 4.1.2 PdBu , but not ionomyc in , is suff icient to activate R a p l i n B cells H a v i n g shown that R a p l is activated by the BCR, we were interested in determining the mechanism by w h i c h the B C R activates R a p l . O u r previous work suggested that C r k » C 3 G complexes might be invo lved (ref. 120 and see Chapter 3). H o w e v e r , F r a n k e et al. s h o w e d that there is another m e c h a n i s m for receptor- induced activation of R a p l (125). They found that increases in cytoplasmic C a 2 + concentrations were sufficient to activate R a p l i n platelets and that R a p l activation i n platelets i n response to thrombin treatment requ i red increases in cytoplasmic C a 2 + concentrations. Since B C R engagement also leads to increases in cytoplasmic C a 2 + concentrations through the activation of P L C - y (183), the B C R could activate R a p l either v ia the C a 2 + - d e p e n d e n t pathway or v ia the b ind ing of C r k » C 3 G complexes to tyrosine-phosphorylated docking proteins. T o d ist inguish between these two possibilities, we first asked whether the C a 2 + - d e p e n d e n t pathway for R a p l activation is present in B cells. W e used the C a 2 + - s e l e c t i v e i onophore , i o n o m y c i n , to increase intrace l lu lar C a 2 + levels i n WEHI-231 mur ine B cells. W e found that an ionomyc in concentration of 500 n M , wh ich causes similar increases in intracellular C a 2 + levels as does B C R stimulation (184), d i d not activate R a p l (Fig. 4.2). H igher concentrations of i onomyc in (1 uM), w h i c h cause larger increases i n intracellular C a 2 + concentrations, also fa i led to activate R a p l i n B cells (S. J. M c L e o d ; persona l communicat ion ) . W h i l e the C a 2 + - d e p e n d e n t pathway for R a p l activation appears to be absent in B cells, we found that the other PLC-y-der ived second messenger, D A G , cou ld activate R a p l i n 76 Figure 4.1. Act ivat ion of R a p l fo l lowing BCR l igation. WEHI-231, B A L 17, and A20 cells were incubated for 2 m i n wi th (+) or without (-) the appropriate anti-Ig Abs. C e l l lysates were incubated w i t h the GST-Ra lGDS(RBD) fusion protein and precipitated proteins were analyzed by anti-Rap 1 immunoblotting. Molecular mass standards (in kDa) are indicated to the left. These experiments were performed by Sarah J. McLeod i n our lab. 77 cell line : stim. : 31.5 21.5 Anti-Rap1 Blot Rapl 78 B cells. Phorbol dibutyrate (PdBu), a phorbol ester that mimics the action of D A G , caused strong activation of Rapl in the WEHI-231 murine B cell line (Fig. 4.2) and in other B cell lines (121). Furthermore, simultaneous treatment of cells with both PdBu and ionomycin did not enhance or inhibit the level of Rapl activation seen in cells treated with PdBu alone. Thus, a DAG-dependent pathway for Rapl activation is present in B cells. 4.1.3 PLC-y-mediated D A G production is required for the activation of R a p l by the BCR To test whether the BCR activates Rapl via this DAG-dependent pathway, we made use of several variants of the DT40 chicken B cell line. To determine whether activation of Rapl by the BCR is dependent on the generation of P L C - y - d e r i v e d second messengers, we asked whether the BCR could activate Rapl in a variant of the DT40 chicken B cell line in which the genes encoding PLC-y2 have been disrupted (183). Since these cells do not express P L C - y l , they are unable to produce IP3 or D A G in response to BCR ligation (183). We found that BCR-induced activation of Rap l was dramatically reduced in the PLC-y-deficient DT40 cells as compared to wild type DT40 cells (Fig. 4.3A). In contrast, PdBu caused strong activation of Rap l in both the wild type and PLC-y-deficient DT40 cells, indicating that Rapl could still be activated in the PLC-y-deficient DT40 cells. Thus, P L C - y expression, and presumably activation, is required for the BCR to activate Rapl . Since P L C - y is necessary for the majority of BCR-induced Rapl activation (Fig. 4.3A) and PdBu can activate Rapl (Fig. 4.2), it is likely that the BCR activates Rapl via a DAG-dependent pathway. To confirm that activation of Rapl by the BCR does not require increases in intracellular C a 2 + concentrations in addition to D A G production, we asked whether the BCR could activate Rapl in a variant of the DT40 cell line in w h i c h the genes encoding all three IP3 receptors 79 Figure 4.2. Rapl is activated in B cells by PdBu but not by ionomycin. WEHI-231 cells were left untreated (-) or incubated for 5 min with 100 Hg/ml anti-IgM (ocIgM), 10 or 100 n M PdBu, 500 n M ionomycin, or a combination of 100 n M PdBu and 500 n M ionomycin. Cel l lysates were incubated with the GST-RalGDS(RBD) fusion protein and precipitated proteins were analyzed by anti-Rapl immunoblotting. Molecular mass standards (in kDa) are indicated to the left. These experiments were performed by Sarah J. McLeod in our lab. 80 stim. : 31.5 H 5$» ti — * 10 100 *P A 21.5 H Anti-Rapl Blot WEHI-231 cells 81 Figure 4.3. Activation of R a p l by the B C R requires PLC-y but does not require IP3 receptor-mediated increases in intracellular C a 2 + . A , W i l d type (wt) and PLC-y-deficient (PLC-y-/-) DT40 B cells were left untreated (-) or incubated for 5 min with 50 p g / m l anti-IgM or 100 n M PdBu. Cell lysates were incubated with the GST-RalGDS(RBD) fusion protein and precipitated proteins were analyzed by anti-Rapl immunoblotting. Molecular mass standards (in kDa) are indicated to the left. B, Wild type (wt) and IP3 receptor-deficient (IP3R -/-) DT40 B cells were left untreated (-) or incubated for 5 min with 50 p g / m l anti-IgM or 100 n M PdBu. Cell lysates were precipitated with the GST-RalGDS(RBD) fusion protein and precipitated proteins were analyzed by anti-Rapl immunoblotting. Molecular mass standards (in kDa) are indicated to the left. These experiments were performed by Sarah J. McLeod in our lab. 82 A wt P L C - Y - / -cell line: : DT40 DT40 stim. : 31.5 — 21.5 — ^ — — — 14— Anti-Rapl Blot B I P 3 R - / -cell line :: DT40 DT40 stim. : 31.5 — mmmm — — —• 21.5 — Anti-Rapl Blot R a p l 83 have been disrupted (185). The IP3 receptors are responsible for the release of C a 2 + from intracellular stores in response to IP3 b inding and in these IP3 receptor-deficient DT40 cells there is no increase in intracellular C a 2 + following BCR engagement (185). We found that BCR ligation caused strong activation of Rapl in the IP3 receptor-deficient DT40 cells (Fig. 4.3B) and the extent of Rapl activation in these cells was similar to that caused by engaging the BCR on wild type DT40 cells. This suggests that D A G production is sufficient to induce maximal activation of Rapl . Consistent with this idea, we found that increasing intracellular C a 2 + concentrations with ionomycin did not potentiate the ability of PdBu to activate Rapl (Fig. 4.2). In sum, these data argue that increases in intracellular C a 2 + are neither necessary nor sufficient for activation of Rapl by the BCR. 4.1.4 Association of Crk with pl30Cas and c-Cbl is neither necessary nor sufficient for activation of R a p l in B cells Our data indicate that a novel DAG-dependent pathway for Rapl activation exists in B cells. Our previous work suggested that the BCR might activate Rapl by recruiting C r k * C 3 G complexes to cellular membranes where Rapl is located (ref. 120 and see Chapter 3). In this model, translocation of C r k » C 3 G complexes to cellular membranes was mediated by the binding of the C r k SH2 domain to membrane-associated docking proteins (e.g. Cas proteins, c-Cbl) that are tyrosine phosphorylated in response to BCR ligation. To determine whether the formation of these Crk complexes is involved in the DAG-dependent activation of Rap l , we investigated whether PdBu induced CrkL to associate with c-Cbl or pl30Cas. Figure 4.4A shows that B C R ligation caused C r k L to associate with an approximately 120-kDa tyrosine-phosphorylated protein in R A M O S cells. However, treatment of R A M O S cells wi th P d B u d i d not cause C r k L to associate with tyrosine-phosphorylated proteins. Immunoblotting with specific Abs confirmed that PdBu did not cause CrkL to associate with pl30Cas (Fig. 4.4B) or c-Cbl (Fig. 4.4C). 84 Thus, the b ind ing of C r k « C 3 G complexes to the Cas proteins, c-Cbl , or other tyrosine-phosphorylated proteins is not necessary for PdBu-induced R a p l activation. Since the B C R appears to activate R a p l via a PLC-y- and DAG-dependent pathway, it is l ikely that the binding of Crk to tyrosine-phosphorylated docking proteins is also not necessary for BCR-induced R a p l activation. Further experiments indicated that the binding of C r k * C 3 G complexes to the Cas proteins and c-Cbl does not appear to be sufficient to induce R a p l activation (Fig. 4.4D). The binding of C r k to tyrosine-phosphorylated docking proteins was normal i n PLC-y-deficient DT40 cells i n which BCR-induced R a p l activation was greatly reduced. This suggests that even i f the b i n d i n g of C r k proteins to tyrosine-phosphorylated docking proteins induces R a p l activation, that this plays only a minor role in BCR-induced R a p l activation. 4.2 C a l D A G - 1 , a D A G - r e g u l a t e d activator of R a p l Although the B C R activates R a p l via a DAG-dependent mechanism, it is not known how this occurs. One possibility is that the B C R activates or recruits C3G by a mechanism that does not depend on the binding of Crk to tyrosine-phosphorylated docking proteins. The recent cloning of Ca lDAG-1 by Kawasaki et al. (186) suggested an alternative mechanism. C a l D A G - 1 is a G N E F for R a p l that is similar to classical P K C enzymes i n possessing both C a 2 + and DAG-b ind ing motifs. Both the C a 2 + and D A G - b i n d i n g domains of C a l D A G - 1 are functional and can mediate the activation of C a l D A G - 1 . In 293T cells expressing C a l D A G - 1 , both the calcium ionophore, A23187, and the phorbol ester, P M A , can enhance R a p l activation (186). Given our data suggesting R a p l activation by the B C R was dependent upon PLC-y-mediated D A G production, we were interested i n determining if C a l D A G - 1 was the G N E F responsible for the activation of R a p l by the BCR. We found that C a l D A G - 1 is expressed in a number of B cell lines and that overexpression of C a l D A G - 1 i n DT40 chicken B cells enhanced BCR-mediated R a p l 85 Figure 4.4. Association of CrkL with pl30Cas and c-Cbl is neither necessary nor sufficient for activation of Rapl in B cells. A-C, RAMOS cells were left untreated (-) or incubated for 2 min with 100 ^ig/ml anti-IgM or 100 nM PdBu. Cell lysates were incubated with an anti-CrkL Ab and precipitated proteins were analyzed by immunoblotting with (A) the 4G10 anti-P-Tyr mAb, (B) anti-pl30Cas mAb, or (C) anti-c-Cbl Ab. D , Wild type (wt) and PLC-y-deficient (PLC-y -/-) DT40 cells were left untreated (-) or incubated for 2 min with 50 pg/ml anti-IgM or 100 nM PdBu. Cell lysates were incubated with an anti-CrkL Ab and precipitated proteins were analyzed by anti-P-Tyr immunoblotting. Molecular mass standards (in kDa) are indicated to the left of each panel. 86 (/CrkL ippts. Anti P-Tyr Blot pp120 pp39 B ippt. stim. 200 Anti-Cas Blot p130Cas Anti-Cbl Blot c-Cbl cell line: stim. 200—I aCrkL ippts. wt PLC-y-/-DT40 DT40 pp120 Anti P-Tyr Blot 87 activation. In contrast, overexpression of C 3 G d id not augment BCR-mediated R a p l activation. This suggests that C a l D A G - 1 could be the R a p l activator used by BCR. 4.3 Results 4.3.1 C a l D A G - 1 is expressed i n a number of B cell lines If C a l D A G - 1 is the R a p l activator used by the BCR, then C a l D A G - 1 must be expressed i n B cells. Kawasaki et al. reported that C a l D A G - 1 m R N A was present at high levels i n tissues r ich i n B cells such as spleen, l ymph node, bone marrow, and peripheral blood leukocytes (186). This strongly suggested that C a l D A G - 1 might be expressed i n B cells. To determine if this was true, we looked at whether Ca lDAG-1 was expressed i n various B cell lines using a m A b antibody directed against murine C a l D A G - 1 . A s shown i n Figure 5.1, C a l D A G - 1 is highly expressed i n the BAL17 murine B cell line but is present at very low levels in the WEHI-231 and R A M O S B cell lines. Since C a l D A G - 1 is expressed i n B cells, albeit sometimes at very low levels, it could mediate R a p l activation by the BCR. 4.3.2 Overexpress ion of C a l D A G - 1 , but not C 3 G , i n D T 4 0 cells enhances BCR-mediated R a p l activation To address whether the B C R could use C a l D A G - 1 to activate R a p l , we expressed C a l D A G - 1 i n the DT40 chicken B cell line. This was accomplished by using retrovirus particles containing the C a l D A G - 1 c D N A to infect DT40 cells that had been transfected wi th the gene encoding for the murine ecotropic retroviral receptor. A s a control, we also overexpressed C 3 G i n these cells. We then asked whether overexpressing C a l D A G - 1 in DT40 cells wou ld enhance R a p l activation by the BCR. Figure 5.2A shows that DT40 cells overexpressing C a l D A G - 1 showed increased R a p l activation i n response to B C R crosslinking compared to w i l d type 88 F i g u r e 4.5. C a l D A G - 1 i s e x p r e s s e d i n a n u m b e r o f B c e l l l i n e s . A n t i - C a l D A G - 1 i m m u n o p r e c i p i t a t e s o f t h e i n d i c a t e d c e l l l i n e s w e r e i m m u n o b l o t t e d w i t h t h e a n t i - C a l D A G - 1 m A b . T o t a l c e l l l y s a t e (10 [ ig) f r o m W E H I - 2 3 1 c e l l s i n f e c t e d w i t h r e t r o v i r u s p a r t i c l e s c o n t a i n i n g m u r i n e C a l D A G - 1 ( W E H I - 2 3 1 + C a l D A G - l ) w a s i n c l u d e d as a p o s i t i v e c o n t r o l . M o l e c u l a r m a s s s t a n d a r d s ( i n k D a ) a re i n d i c a t e d to the lef t . 89 cell l i n e : 66 o ' 1 o 3 £ A ^ & & £ £ 45—I C a l D A G - 1 ant i -Ca lDAG-1 Blot 90 Figure 4.6. Overexpression of C a l D A G - 1 i n DT40 B cells enhances BCR-mediated R a p l activation. A , BOSC 23 cells were transfected wi th the pMX-p ie alone (vector) or wi th pMX-pie containing the c D N A s encoding for C a l D A G - 1 (CalDAG-1) or C 3 G (C3G). The BOSC 23 culture supernatant containing the resulting virus particles were used to infect DT40 cells expressing the murine ecotropic retrovirus receptor. The DT40 cells were either left unstimulated (-) or stimulated for 2 m i n wi th anti-IgM Abs (+). Ce l l lysates were incubated w i t h the GST-RalGDS(RBD) fusion protein and precipitates were immunoblotted wi th the anti-Rapl A b . B , Total cell lysate (20 pg) was blotted wi th the anti-C3G A b (upper panel) or the anti-CalDAG-1 m A b (lower panel) to show expression of the introduced c D N A s . Note that these are relatively short exposures to highlight the overexpression of the introduced c D N A s . W i t h longer exposures of the anti-C3G blot, we were able to detect the endogenous C3G. However, even after very long exposures of the ant i -CalDAG-1 blot we were unable to detect endogenous Ca lDAG-1 i n the total cell lysates of DT40 cells. 91 GST-RalGDS(RBD) ppt. DT40 c e l l s s t i m . A* 0 <3 f - + - + - + • R a p l - G T P 6 anti-Rapl blot DT40 c e l l s A* C? stim. — + — _|_ — _|_ anti-C3G blot anti-CalDAG-1 blot •C3G •CalDAG-1 • I g H 92 DT40 cells. In contrast, DT40 cells expressing human C 3 G d i d not show enhanced BCR-mediated R a p l activation (Fig. 5.2A) despite the fact that C 3 G was highly overexpressed i n these cells (Fig. 5.2B, upper panel). In fact, overexpressing C3G in the DT40 cells may even have slightly inhibited BCR-mediated R a p l activation. Regardless, these results show that the B C R can use C a l D A G - 1 but not C 3 G to activate R a p l . 4.4 Discussion By acting as an antagonist of Ras-mediated signalling, the R a p l GTPase may play a key role i n regulating cell growth and survival. Us ing an assay that allows us to selectively precipitate the activated, GTP-bound form of R a p l , we found that engaging the B C R activates R a p l in several B cell lines as we l l as i n normal murine B cells. Moreover, our data suggest that BCR-induced R a p l activation proceeds via a novel DAG-dependent pathway that may involve the D A G - b i n d i n g R a p l G N E F Ca lDAG-1 . Several other examples of receptor-induced activation of R a p l have been published (187). In addit ion to the activation of R a p l i n platelets, where various agonists including thrombin can activate R a p l (125), R a p l has been shown to be activated by the TCR, various growth factor receptors, as wel l as receptors coupled to heterotrimeric G-proteins (187). Thus, it appears that R a p l activation is common theme i n receptor signalling. However, not all receptors appear able to activate R a p l , as it has been reported that the insul in receptor does not activate R a p l i n response to insul in treatment (173). While many receptors appear to activate R a p l , the mechanism by which they do so varies. In platelets, increases i n intracellular free C a 2 + levels are both necessary and sufficient for activation of R a p l . While BCR-induced R a p l activation required P L C - y , experiments using IP3 receptor-deficient DT40 cells showed that increases i n intracellular free C a 2 + were not required for the B C R to activate R a p l 93 (Fig 4.3B). Moreover , it appears that the Ca 2 + -dependen t pathway for R a p l activation is not present i n B cells since C a 2 + ionophores could not stimulate R a p l activation (Fig 4.2). Al though C a 2 + increases are not involved i n activation of R a p l in B cells, BCR-induced R a p l activation was dramatically reduced i n PLC-y-deficient DT40 B cells. This suggested that the other PLC-y-derived second messenger, D A G , may be involved i n activation of R a p l by the BCR. Indeed, PdBu , which mimics the action of D A G , as wel l as dioctanoylglycerol (diCs), a synthetic diacylglycerol, caused strong activation of R a p l i n variety of B cells (ref. 121 and see Fig. 4.2). These results suggest that the B C R activates R a p l via a DAG-dependent pathway. In addit ion to C a 2 + and D A G , c A M P is also k n o w n to activate R a p l through the activation of protein kinase A and other cAMP-responsive proteins (187).. Thus, while R a p l activation is a common event i n receptor signalling, different cell types and different receptors activate R a p l via distinct mechanisms. The components that l ink D A G to R a p l activation i n B cells are not known and we do not know if the ultimate target of D A G is a G N E F for R a p l or a R a p l G A P . We found that the R a p l G N E F , C a l D A G - 1 , is expressed to some extent i n all the B cell lines tested (Fig 4.5). Furthermore, i n all of these cells R a p l is activated by the BCR and R a p l activation can be induced by treating the cells wi th PdBu (ref. 121 and S.J. M c L e o d ; personal communicat ion) , suggesting that B C R - i n d u c e d R a p l activation i n all these cell lines is mediated by the same DAG-dependent pathway seen i n DT40 B cells. The fact that C a l D A G - 1 can be used by DT40 cells to enhance BCR-mediated R a p l activation (Fig 4.6), further supports that idea that Ca lDAG-1 is the R a p l activator used by the BCR. If C a l D A G - 1 is the R a p l activator used by the B C R i n all these cell lines, then it means that the low levels of Ca lDAG-1 expression seen i n some of the cell lines is sufficient to activate R a p l . It should be noted that i n B A L I 7 cells, which express the highest levels of C a l D A G - 1 of the B cell lines tested (see F ig . 4.5), R a p l is not activated to a significantly greater extent than i n other cell lines (see Fig. 4.1). In fact, 94 R a p l activation i n BAL17 cells is weaker than i n many of the other B cell lines (S.J. McLeod ; personal communication). This observation at first seems inconsistent wi th our data showing that overexpression of C a l D A G - 1 i n DT40 B cells enhances R a p l activation (see F ig . 5.2). However , it may be that B A L 1 7 cells have compensated i n some way to tolerate the high levels of C a l D A G - 1 expression in order to prevent excessive R a p l activation. Excessive R a p l activation may be deleterious to B cells as attempts to express both w i l d type and constitutively-active R a p l A i n various B cell lines have been unsuccessful (R.J. Ingham and S.J. McLeod; unpublished observation). Presumably, this is due to the fact that excessive R a p l activity is toxic to the cells. Whi le our data show that the B C R can use C a l D A G - 1 to activate R a p l , we cannot conclude that C a l D A G - 1 is the G N E F used by the B C R to activate R a p l . To address this question, we plan to knock-out chicken C a l D A G - 1 i n DT40 cells to see if BCR-mediated R a p l activation is inhibited. Al though we were unable to detect C a l D A G - 1 protein i n DT40 chicken B cells using the m A b used to detect the human and murine protein (R.J. Ingham, S.J. M c L e o d , and S. Chris t ian; unpublished observations), it may be that this m A b does not recognize chicken C a l D A G - 1 . While we found that overexpression of C a l D A G - 1 was able to enhance R a p l activation by the BCR, we found that overexpression of another R a p l G N E F , C3G, d id not enhance R a p l activation and may have even inhibited R a p l activation by the B C R (see Fig. 3.6). It is not clear why overexpression of C 3 G i n the DT40 cells inhibited BCR-mediated R a p l activation but may be due to some non-specific effect related to the high level of expression of the introduced C3G. Therefore, it is l ikely that C 3 G (and presumably Crk proteins) are not involved i n BCR-mediated R a p l activation and play some other role i n B C R signalling. Our data showing that Crk complex formation is neither necessary nor sufficient for R a p l activation (see Fig. 4.4) supports this notion. However when we overexpressed C 3 G i n the DT40 cells, it is possible that the exogenous C 3 G was unable to participate i n B C R signalling 95 because endogenous Crk proteins were l imit ing. In our model, association of Crk wi th C 3 G is important for shuttling C3G to different areas i n the cell. If there was not enough Crk i n the DT40 cells to b ind the overexpressed C3G, the B C R might not be able to recruit this additional C3G to sites where R a p l is located. Therefore, we plan to simultaneously overexpress both Crk and C 3 G i n DT40 cells and look at whether BCR-mediated R a p l activation is enhanced. Furthermore, we plan to knock-out C 3 G i n DT40 cells to unequivocally determine if C 3 G has any role in BCR-mediated R a p l activation. A n alternative target for the DAG-dependent R a p l activation pathway used by the B C R are P K C enzymes. In prel iminary experiments, we found that BCR-induced R a p l activation was significantly inhibited by chelerythrine chloride, a P K C inhibitor that targets the catalytic domain of P K C enzymes (188) (S.J. McLeod; personal communication). Further work is needed to determine whether P K C enzymes are used by the B C R to activate R a p l or whether this result reflects non-specific effects of chelerythrine chloride. It w i l l be interesting to see if overexpression of various P K C isoforms i n B cells as any effect on BCR-mediated R a p l activation. A s mentioned earlier, we do not know whether the ultimate target of this DAG-dependent pathway is an activator of R a p l or whether this DAG-dependent pathway inhibits a R a p l G A P . Four R a p l G A P s have been identified, S P A - 1 , R a p G A P s 1 and 2, and tuberin (refs. 189-191 and Drs. N a o k i M o c h i z u k i and i M i c h i y u k i Matsuda; personal communication). SPA-1 is expressed pr imari ly i n lymphoid cells (189) and it w i l l be interesting to determine if D A G regulates its activity or subcellular localization. Our ini t ial model for R a p l activation by the B C R involved the binding of Crk»C3G complexes to tyrosine-phosphorylated docking proteins (120). Since c-Cbl and the Cas proteins can associate wi th cellular membranes, this interaction could recruit cytosolic C r k » C 3 G complexes to membranes where R a p l is located. 96 However, we found that the association of Crk complexes w i t h c-Cbl, pl30Cas, or other tyrosine-phosphorylated proteins was neither necessary nor sufficient for the DAG-dependent activation of R a p l . PdBu caused strong activation of R a p l but d id not cause C r k to associate wi th c-Cbl, pl30Cas, or other tyrosine-phosphorylated proteins (Figs 4.4 A - C ) . Moreover, BCR-induced R a p l activation was dramatically reduced i n PLC-y-def ic ien t DT40 cells even though C r k associated w i t h tyrosine-phosphorylated proteins i n a normal manner (Fig 4.4D). The small amount of residual R a p l activation seen i n the PLC-y-deficient DT40 cells following BCR engagement may reflect the contribution of the C a s / c - C b l * Crk »C3G pathway wi th the majority of BCR-induced R a p l activation proceeding v ia the independent DAG-regulated pathway. Our findings do not however rule out a role for Crk and C 3 G i n the activation of R a p l by the BCR. It is possible that the DAG-dependent pathway regulates C 3 G or Crk»C3G complexes in some way that does not involve the binding of Crk to tyrosine-phosphorylated docking proteins. The function of R a p l i n B C R signalling remains to be elucidated. As an antagonist of Ras, R a p l may play a key role i n regulating B cell activation. Since activated Ras can transform B cells (192), the activation of Ras by the B C R most l ikely promotes B cell proliferation. The concomitant activation of R a p l may oppose this by l imi t ing Ras-mediated signalling. Thus, the balance between the activation of Ras and R a p l may be a key parameter that determines the biological outcome of B C R ligation. Whi le there is considerable evidence that R a p l can function as a negative regulator of Ras-mediated signall ing, the majority of these studies have used systems where R a p l is overexpressed. There is now evidence that R a p l may activate signall ing pathways that are distinct from Ras. Studies i n Drosophila suggest that endogenous R a p l does not regulate Ras signalling but rather regulates distinct pathways involved i n morphogenesis (193) and the regulation of cell cycle inhibitors such as Dacapo, an orthologue of the p 2 7 K i p ! cyclin-dependent kinase 97 inhibitor (174). Furthermore, activating R a p l w i th T P A i n Rat-1 cells does not interfere wi th the Ras-dependent activation of ERK2 i n response to P D G F and E G F st imulation (165). Thus, endogenous R a p l may not function to regulate Ras signalling, but rather, may mediate signalling pathways that are distinct from Ras. R a p l may also have other functions i n B cells. M a l y et al. have shown that the ability of the B C R to stimulate the oxidative burst is dependent on R a p l A (194). In neutrophils, R a p l A associates wi th cytochrome b558 and this may be important for assembly of the N A D P H oxidase complex which produces reactive oxygen species (195). The production of these reactive oxygen species may allow B cells to k i l l bacteria that they b ind via their BCR. Thus, there are several potential targets for R a p l - G T P i n B cells and our lab is interested i n identifying signalling pathways that are regulated by R a p l during BCR signalling. 98 Chapter 5 G a b l is a docking site for a number of signall ing proteins involved i n signall ing by the B C R 5.0 Introduction The B C R uses many different SH2 domain-containing enzymes and SH2 domain-containing adapter proteins that need to be recruited to membranes where their substrates are located. To recruit these SH2 domain-containing proteins to the membrane and form signalling complexes, the B C R uses a number of membrane docking proteins. As shown in Chapter 3, the Cas proteins and c-Cbl are examples of docking proteins that recruit C r k » C 3 G complexes to cellular membranes i n BCR-stimulated cells (120). c-Cbl also recruits Ptdlns 3-kinase i n BCR-stimulated cells (152,161). The B C R also uses the Blnk adapter protein to provide a membrane binding site for P L C - y , Grb2, and Vav (195a,195b). The requirement for Blnk to recruit these proteins appears to be critical in B cells as mice and humans deficient i n Blnk fail to develop mature B cells (195c,195d). Since the B C R uses many other SH2 domain-containing proteins, it is l ikely that the B C R uses multiple docking proteins to recruit SH2 domain-conta ining s igna l l ing proteins to cellular membranes. In 1996, Wong and colleagues reported the cloning of G a b l , a docking protein that bound mult iple SH2 domain-containing proteins in fibroblasts (196). G a b l , which stands for Grb2-associated binder-1, is tyrosine phosphorylated i n response to stimulation of a number of receptors including those for insul in and E G F (196), nerve growth factor (197) and cytokines such as IL-3, IL-6, and IFNy (198). In add i t ion , receptors that activate heterotr imeric G-prote ins such as the lysophosphatidic acid (LPA) receptor have been reported to use G a b l (199). Receptor-induced phosphorylation of G a b l allows it to bind the SH2 domains of the She and Grb2 adapter proteins, Ptdlns 3-kinase, PLC-y, and the tyrosine phosphatase 99 SHP-2 (196). G iven that many of the SH2 domain-containing proteins known to bind to G a b l are involved i n B C R signalling, I tested the hypothesis that the B C R uses G a b l as a membrane docking protein for SH2 domain-containing signalling proteins. In the R A M O S human B cell line, I found that B C R ligation induced strong tyrosine phosphorylation of G a b l and resulted i n the binding of She, Grb2, Ptdlns 3-kinase and SHP-2 to G a b l . F A R Western analysis showed that the SH2 domains of She, Ptdlns 3-kinase, and SHP-2 bound directly to G a b l while Grb2 bound indirectly to G a b l via its SH2 domain b inding to tyrosine-phosphorylated She and SHP-2 bound to G a b l . Furthermore, these Gabl / s igna l l ing protein complexes were present in the membrane-enriched particulate fraction of activated R A M O S cells. Thus, the B C R appears to use G a b l to recruit mult iple s ignal l ing proteins to cellular membranes where they can act on membrane-associated targets. 5.1 Results 5.1.1 G a b l is tyrosine phosphorylated and associates w i th tyrosine-phosphorylated proteins i n response to B C R ligation If the B C R uses G a b l as a docking site for SH2 domain-containing proteins, then G a b l should be phosphorylated on tyrosine residues i n response to B C R engagement. I initiated B C R signalling i n the R A M O S human B lymphoma cells w i t h ant i - IgM Abs and then examined the phosphorylat ion state of G a b l by anti-P-Tyr immunoblotting. I found that a 110- to 120-kDa protein that had the same electrophoretic mobility as G a b l was strongly tyrosine phosphorylated when anti-IgM antibodies were added to the cells (Fig. 5.1A, upper panel). This response persisted for at least 30 m i n fol lowing B C R engagement (Fig. 5.IB, upper panel). Reprobing these blots w i t h ant i -Gabl Abs showed that B C R l igat ion caused a decrease i n the electrophoretic mobili ty of G a b l (Fig. 5.1A and B, lower panels) 100 which correlated wi th the tyrosine phosphorylation of G a b l . However, since B C R ligation also induces tyrosine phosphorylation of pl30Cas and c-Cbl (see Chapter 3), I wanted to rule out the possibility that the tyrosine-phosphorylated 110- to 120-kDa band seen i n the ant i-Gabl immunoprecipitates was either Cas or c-Cbl that had co-precipitated wi th G a b l . I found that neither Cas nor c-Cbl could be detected in anti-Gabl immunoprecipitates (Fig. 5.2), even after very long exposures (data not shown). Al though I cannot definitively rule out the possibility that an unidentified tyrosine-phosphorylated 110- to 120-kDa protein co-precipitated w i t h G a b l , the simplest interpretation of the results is that B C R l iga t ion induces tyrosine phosphorylation of G a b l . The finding that G a b l is tyrosine phosphorylated i n response to B C R ligation suggested that it could act as a docking site for SH2 domain-containing signalling proteins. Since many of these proteins are themselves tyrosine phosphorylated i n response to receptor signalling, I asked whether tyrosine-phosphorylated proteins associated w i t h G a b l i n ant i -IgM-st imulated R A M O S cells. I found that tyrosine-phosphorylated proteins of approximately 150 kDa, 90-97 kDa , 70 kDa, 55 kDa , and 50 kDa bound to G a b l after B C R ligation (Fig. 5.3). Our group had previously shown that BCR engagement causes She (118) and Ptdlns 3-kinase (data not shown) to associate w i t h tyrosine-phosphorylated proteins of 110-120 kDa. Therefore, I asked whether these signall ing proteins associated w i t h G a b l i n activated R A M O S B cells. 5.1.2 The She and Grb2 adapter proteins associate wi th G a b l after B C R engagement The p52 and p46 forms of the She adapter pro te in are s trongly tyrosine-phosphorylated in response to B C R engagement and phosphorylated She is a major target of the SH2 domain of the Grb2 adapter protein i n activated B cells (118). Since Grb2 associates wi th the Ras activator SOS, She may play a key role i n activating Ras by directing Grb2«SOS complexes to the plasma membrane where Ras 101 Figure 5.1. G a b l is tyrosine phosphorylated after B C R l igat ion. A , R A M O S cells were incubated for 2 min wi th (+) or without (-) anti-IgM Abs. B , R A M O S cells were incubated w i t h an t i - IgM A b s for the indicated times. C e l l lysates were immunoprec ip i t a ted w i t h either an an t i -Gab l A b or w i t h an irrelevant affinity-purified rabbit A b (control). Precipitated proteins were analyzed by immunoblotting wi th the 4G10 anti-P-Tyr m A b (upper panels). The filters was then stripped and reprobed wi th the anti-Gabl A b (lower panels). 102 A B •PPt-stimulation + - + 116-J 97" 116-1 97" Anti-P-Tyr Blot Anti-Gabl Blot ippt.: anti-Gabl stimulation (min): 0 2 5 15 30 116-97-116— 97- - - Anti-P-Tyr Blot Anti-Gabl Blot 103 Figure 5.2. pl30Cas and c-Cbl do not co-precipitate w i th G a b l . R A M O S cells were incubated for 2 m i n w i t h (+) or without (-) ant i -IgM Abs . C e l l lysates were immunoprecipitated wi th Abs to G a b l , pl30Cas, c-Cbl or w i th rabbit IgG (control). Precipitated proteins were analyzed by blotting wi th an anti-pl30Cas m A b (upper panel) or wi th an anti-c-Cbl A b . Molecular mass standards (in kDa) are indicated to the left. Note that even after very long exposures of these blots to f i lm, no pl30Cas or c-Cbl was detected i n anti-Gabl immunoprecipitates. 104 A ippt. : stimulation : 200 Anti-Cas Blot I Cas proteins B ippt.: stimulation : 200 . / V .f Anti-Cbl Blot c-Cbl 105 F i g u r e 5.3. G a b l associates w i t h t y r o s i n e - p h o s p h o r y l a t e d p r o t e i n s i n BCR-s t imu la ted cells. R A M O S cells were incubated for 2 m i n w i th (+) or without (-) ant-IgM Abs. Ce l l lysates were immunoprecipitated wi th either the ant i -Gabl A b or w i th an irrelevant a f f in i ty -pur i f ied rabbit A b (control) and then ana lyzed by immunob lo t t i ng w i t h the 4G10 ant i -P-Tyr m A b . The filter was s t r ipped and reprobed w i th the ant i -Gab l A b (not shown) The phosphory lated 110- to 120-kDa protein i n this f igure had the same electrophoretic mobi l i ty as G a b l . Molecular mass standards (in kDa) are indicated to the left. Note that the molecular masses of the Gab l -assoc iated proteins are rough estimates based on their electrophoretic mobilities relative to the molecular mass standards. 106 ippt.: stimulation : 200 pp150 G a b l pp90-97 pp70 pp55 pp50 Anti-P-Tyr Blot 107 is located. She also recruits Grb2»SHIP complexes i n BCR-stimulated B cells (129). SHIP is an inosi to l phosphatase that converts the Ptd lns 3-kinase product PtdIns(3,4,5)P3 to PtdIns(3,4)P 2 (200). Given the potentially important role of She in B C R s igna l l ing , I asked whether the Gabl-associa ted phosphoproteins of approximately 55-kDa and 50-kDa (see Fig. 5.3) were the two isoforms of She. Immunoblott ing ant i -Gabl immunoprecipitates w i t h anti-She Abs showed that both the p52 and p46 forms of She bound to G a b l i n anti-IgM-stimulated R A M O S cells but not i n unstimulated cells (Fig. 5.4A). Thus, both isoforms of She bind to G a b l after BCR ligation. The binding of She to G a b l after BCR engagement suggests that G a b l could be a docking site for Grb2«SOS or Grb2«SHIP complexes i n activated B cells. Therefore, I determined whether Grb2 bound to G a b l i n B cells. Figure 5.4B shows that there was a small amount of Grb2 associated wi th G a b l i n unstimulated R A M O S cells but that B C R stimulation significantly increased the amount of Grb2 bound to G a b l . Thus, G a b l may participate i n Grb2-mediated signalling by the B C R by providing a docking site for Grb2»SOS complexes and/or Grb2»SHIP complexes. 5.1.3 Ptdlns 3-kinase associates wi th G a b l after B C R engagement Ptdlns 3-kinase is another cytosolic signalling enzyme that must be recruited to the plasma membrane i n order to gain access to its substrates. Ptdlns 3-kinase phosphorylates inositol phospholipids and B C R ligation is k n o w n to increase the levels of Ptdlns 3-kinase products in B cells (28). To determine whether G a b l might also be a docking site for Ptdlns 3-kinase, I immunoprecipitated G a b l from R A M O S cells and assayed for the presence of the p85 subunit of Ptdlns 3-kinase and for Ptdlns 3-kinase enzyme activity. Immunoblotting wi th an anti-p85 A b showed that B C R ligation caused Ptdlns 3-kinase to associate wi th G a b l (Fig. 5.5A). Similarly, Ptdlns 3-kinase enzyme activity was present i n an t i -Gabl immunoprecipitates from anti-IgM-stimulated cells but not from unstimulated cells (Fig. 5.5B). Thus i n B 108 Figure 5.4. She and Grb2 associate w i th G a b l after B C R l igation. R A M O S cells were incubated for 2 m i n w i th (+) or without (-) anti- IgM Abs. A , cell lysates were immunoprec ip i ta ted w i th either the ant i -Gab l A b , an anti-She A b or rabbit IgG (control). The filter was probed wi th the anti-She Ab. Note that the band present in the control lane and in the ant i -Gab l immunoprecip i tate lane f r om unst imulated R A M O S cells is the Ig heavy chain of the A b used for immunoprecip i tat ion and that its electrophoretic mobi l i ty is slightly different than that of the p46 form of She. B , cell lysates were immunoprec ip i ta ted w i th either the an t i -Gab l A b or rabbit IgG (control). " C e l l lysate" represents 40 u g of total cellular prote in (6 x 10 5 cell equivalents). The filter was p robed w i th an anti -Grb2 m A b . Mo lecu la r mass standards (in kDa) are indicated to the left. 109 A ippt. : stimulation : 66—\ 4? * + -45-p52 She p46 She Anti-She Blot B ippt. : stimulation : 31 + - + Grb2 Anti-Grb2 Blot n o cells, G a b l appears to be a docking site for Ptdlns 3-kinase and may recruit Ptdlns 3-kinase to the plasma membrane where its substrates are located. 5.1.4 SHP-2 associates wi th G a b l after B C R engagement In A431 cells, E G F stimulation causes the 70-kDa SHP-2 (PTP-1D, Syp) tyrosine phosphatase to associate w i t h G a b l (196). In R A M O S cells, I found that a tyrosine-phosphorylated protein of similar molecular mass bound to G a b l after B C R ligation (Fig. 5.3). Therefore, I asked whether this Gabl-associated protein was SHP-2. I found that SHP-2 bound to G a b l i n anti-IgM-stimulated cells but not i n unst imulated cells (Fig. 5.6A). Because the electrophoretic mobi l i ty of the Gabl-associated SHP-2 was similar to the 70-kDa tyrosine-phosphorylated protein seen i n anti-Gabl immunoprecipitates (Fig. 5.3), this suggested that SHP-2 might be tyrosine phosphorylated in response to B C R ligation. Figure 5.6B shows that SHP-2 was strongly tyrosine phosphorylated i n response to B C R crosslinking. Moreover, tyrosine-phosphorylated proteins of approximately 150 k D a and 110-120 kDa co-precipitated w i t h SHP-2 from anti-IgM-stimulated cells. The 110- to 120-kDa protein is l ikely G a b l . The identity of the 150-kDa protein is not known but a tyrosine-phosphorylated protein of similar molecular mass associates wi th G a b l in anti-IgM-stimulated cells (Fig. 5.3). Thus, SHP-2 is a target of BCR-activated tyrosine kinases and uses G a b l as a docking site. 5.1.5 The SH2 domains of She, Ptdlns 3-kinase, and SHP-2 b i n d directly to G a b l whereas the Grb2 SH2 domain binds to Gabl-associated SHP-2 and She H u m a n G a b l contains tyrosine residues, which if phosphorylated, w o u l d be consensus b inding sites for the SH2 domains of She, Grb2, Ptdlns 3-kinase, and SHP-2. There are several tyrosine residues wi th hydrophobic residues i n the +1 and +3 positions that could be binding sites for the She SH2 domain (96), tyrosine 48 is 111 Figure 5.5. Ptd lns 3-kinase associates w i th G a b l i n BCR-s t imu la ted cells. R A M O S cells were incubated for 2 m i n with (+) or without (-) anti-IgM Abs. Ce l l lysates were immunoprec ip i tated w i th either the ant i -Gab l A b , an anti-p85 A b , or an irrelevant aff inity-purif ied rabbit A b (control). The precipitated proteins were analyzed by A , immunoblot t ing w i th the anti-p85 A b or B, in vitro Ptdlns 3-kinase enzyme assays us ing Ptd lns as a substrate. Reaction products f rom the l i p id kinase assays were separated by T L C and radiolabeled Ptdlns-phosphate (Ptdlns-P04) was visual ized by au to rad i og raphy . N o t e that the anti-p85 i m m u n o p r e c i p i t a t i o n s i n these experiments were not quantitative and are intended only to indicate the migration of p85 and Ptdlns-P04. 112 A ippt. stimulation 3r + — 9 7 - f 66-p85 Anti-p85 Blot B ippt. stimulation + f9 Ptdlns-P04 in vitro kinase assay 113 F i g u r e 5.6. S H P - 2 i s t y r o s i n e p h o s p h o r y l a t e d a n d a s s o c i a t e s w i t h G a b l a f t e r B C R l i g a t i o n . R A M O S c e l l s w e r e i n c u b a t e d f o r 2 m i n w i t h (+) o r w i t h o u t (-) a n t i - I g M A b s . A , c e l l l y s a t e s w e r e i m m u n o p r e c i p i t a t e d w i t h t h e a n t i - G a b l A b . " C e l l l y s a t e " r e p r e s e n t s 4 0 u g o f t o t a l c e l l u l a r p r o t e i n (6 x 1 0 5 c e l l e q u i v a l e n t s ) . T h e f i l t e r w a s p r o b e d w i t h a n a n t i - S H P - 2 A b . B , c e l l l y s a t e s w e r e i m m u n o p r e c i p i t a t e d w i t h t he a n t i - S H P - 2 A b o r w i t h a n i r r e l e v a n t a f f i n i t y - p u r i f i e d r a b b i t A b ( c o n t r o l ) . I m m u n o p r e c i p i t a t e s w e r e p r o b e d w i t h t he 4 G 1 0 a n t i - P - T y r m A b ( u p p e r p a n e l ) . T h e f i l t e r w a s t h e n s t r i p p e d a n d r e p r o b e d w i t h t h e a n t i - S H P - 2 A b ( l o w e r p a n e l ) . M o l e c u l a r m a s s s t a n d a r d s ( i n k D a ) are i n d i c a t e d t o the left . N o t e t ha t t he m o l e c u l a r m a s s e s o f t h e S H P - 2 - a s s o c i a t e d p r o t e i n s i n B a re r o u g h e s t i m a t e s b a s e d o n t h e i r e l e c t r o p h o r e t i c m o b i l i t i e s r e l a t i v e to t h e m o l e c u l a r m a s s s t a n d a r d s . 114 A ippt. stimulation 66-H< O O V • + -SHP-2 Anti-SHP-2 Blot B ippt.: stimulation : 200-Anti-P-Tyr Blot 116-97-66 H 45-I 97-1 Anti-SHP-2 Blot 66-^ pp150 pp110-120 SHP-2 SHP-2 115 part of a Y K N D motif wh ich is optimal for b inding the Grb2 SH2 domain (96), tyrosines 447, 472, and 589 are all part of Y V P M sequences wh ich are optimal for binding the SH2 domains of Ptdlns 3-kinase (96), and tyrosine 627 is part of a Y L D L sequence that is optimal for binding the SH2 domains of SHP-2 (96). This suggests that BCR-induced tyrosine phosphorylation of G a b l could create binding sites for the SH2 domains of She, Grb2, Ptdlns 3-kinase, and SHP-2 and that this could be the basis for the association of these proteins wi th G a b l i n activated R A M O S cells. To directly test this model, I performed F A R Western assays i n which I probed blots of an t i -Gabl immunoprecipitates w i t h GST fusion proteins containing the SH2 domains of She, Grb2, Ptdlns 3-kinase, and SHP-2. Figure 5.7A (upper panel) shows that a GST fusion protein containing the SH2 domain of She could b ind directly to a 110- to 120-kDa protein that was immunoprecipitated wi th ant i -Gabl Abs from anti-IgM-stimulated R A M O S cells but not from unstimulated R A M O S cells. Reprobing this blot wi th the anti-Gabl A b showed that this 110- to 120-kDa band had the same electrophoretic mobility as G a b l (Fig. 5.7A, lower panel). The She SH2 domain fusion protein d id not b ind to G a b l precipitated from unstimulated R A M O S cells, consistent w i th the idea that it binds only to tyrosine-phosphorylated G a b l . I found that GST fusion proteins containing the tandem SH2 domains of SHP-2, the amino-terminal SH2 domain of the Ptdlns 3-kinase p85 subunit or the carboxy-terminal SH2 domain of p85 could also bind directly to G a b l from anti-IgM-stimulated R A M O S cells (Figs. 5.7B,C, and D , respectively). A s a specificity control, I showed that a GST fusion protein containing the She PTB domain d id not b ind to G a b l or any other protein i n ant i -Gabl immunoprecipitates (Fig. 5.7E). Thus it appears that BCR- induced tyrosine phosphorylation of G a b l creates binding sites for the SH2 domains of She, Ptdlns 3-kinase and SHP-2. Al though Grb2 binds directly to tyrosine-phosphorylated G a b l precipitated from EGF-stimulated A431 cells (196), I found that a GST fusion protein containing 116 t h e S H 2 d o m a i n o f G r b 2 d i d n o t b i n d d i r e c t l y t o G a b l p r e c i p i t a t e d f r o m a n t i - I g M - s t i m u l a t e d R A M O S c e l l s ( F i g u r e 5 . 8 A ) . I n s t e a d , t h e G r b 2 S H 2 d o m a i n b o u n d d i r e c t l y to a 7 0 - k D a p r o t e i n a n d a 5 2 - k D a p r o t e i n t h a t w e r e p r e s e n t i n a n t i - G a b l i m m u n o p r e c i p i t a t e s f r o m a n t i - I g M - s t i m u l a t e d R A M O S c e l l s ( F i g u r e 5 . 8 A ) . T h e m o l e c u l a r m a s s e s o f these p r o t e i n s s u g g e s t e d t h a t t h e y c o u l d b e S H P - 2 a n d S h e . W e h a d p r e v i o u s l y s h o w n t h a t t y r o s i n e - p h o s p h o r y l a t e d S h e w a s a m a j o r t a rge t o f t he G r b 2 S H 2 d o m a i n i n B c e l l s (118) w h i l e i n a m y e l o i d c e l l l i n e G r b 2 h a s b e e n r e p o r t e d t o b i n d t o t y r o s i n e - p h o s p h o r y l a t e d S H P - 2 (201) . R e p r o b i n g the b l o t s w i t h A b s t o S H P - 2 a n d t o S h e s h o w e d t h a t t he 7 0 - k D a b a n d t h a t t he G r b 2 S H 2 d o m a i n b o u n d t o i n t h e a n t i - G a b l i m m u n o p r e c i p i t a t e s h a d t h e s a m e e l e c t r o p h o r e t i c m o b i l i t y as S H P - 2 ( F i g u r e 5 .8B) w h i l e t he 5 2 - k D a b a n d c o - m i g r a t e d w i t h t h e p 5 2 f o r m o f S h e ( F i g u r e 5 . 8 C ) . T h u s , i n B C R - s t i m u l a t e d R A M O S B c e l l s , G r b 2 u se s i t s S H 2 d o m a i n to b i n d d i r e c t l y to S H P - 2 a n d S h e t h a t i s a s s o c i a t e d w i t h G a b l . 5.1.6 G a b l i s a m e m b r a n e - a s s o c i a t e d d o c k i n g p r o t e i n i n B c e l l s T h e d a t a s u g g e s t t h a t G a b l i s a m u l t i f u n c t i o n a l d o c k i n g p r o t e i n t h a t b i n d s She , G r b 2 , P t d l n s 3 - k i n a s e , a n d S H P - 2 i n r e s p o n s e to B C R l i g a t i o n . S i n c e a l l o f these p r o t e i n s m u s t t r a n s l o c a t e f r o m t h e c y t o s o l t o c e l l u l a r m e m b r a n e s i n o r d e r to a c t i v a t e s i g n a l l i n g p a t h w a y s , I a s k e d w h e t h e r G a b l i s a s s o c i a t e d w i t h c e l l u l a r m e m b r a n e s a n d w h e t h e r i t ac t s as a m e m b r a n e d o c k i n g s i t e f o r t h e s e s i g n a l l i n g p r o t e i n s i n B c e l l s . T o tes t t h i s , I s e p a r a t e d R A M O S c e l l s i n t o a s o l u b l e c y t o s o l i c f r a c t i o n a n d a m e m b r a n e - e n r i c h e d p a r t i c u l a t e f r a c t i o n u s i n g a s o n i c a t i o n / u l t r a c e n t r i f u g a t i o n p r o c e d u r e (see S e c t i o n 2.1.4). F i g u r e 5 . 9 A s h o w s tha t G a b l w a s p r e s e n t i n b o t h the s o l u b l e a n d p a r t i c u l a t e f r a c t i o n s o f R A M O S c e l l s . I n r e s t i n g c e l l s , t h e m a j o r i t y o f G a b l w a s i n the s o l u b l e f r a c t i o n b u t t he re w a s a s i g n i f i c a n t p o r t i o n i n the p a r t i c u l a t e f r a c t i o n w h i c h m a y b e a b l e to act as a d o c k i n g s i te f o r s i g n a l l i n g p r o t e i n s . I n d e e d , 117 Figure 5.7. The SH2 domains of She, SHP-2, and Ptdlns 3-kinase can b ind directly to G a b l precipitated from anti-IgM-st imulated R A M O S cells. R A M O S cells were incubated for 2 m i n w i t h (+) or without (-) ant i -IgM Abs . C e l l lysates were immunoprecipitated w i t h the ant i -Gabl A b or w i t h rabbit IgG (control). F A R Western assays were performed i n which the blots were probed w i t h GST fusion proteins containing A , the She SH2 domain, B, the tandem SH2 domains of SHP-2, C , the amino- (N) terminal SH2 domain of the Ptdlns 3-kinase p85 subunit, D, the carboxy- (C) terminal SH2 domain of the Ptdlns 3-kinase p85 subunit, or E , the She PTB domain. Reprobing these blots wi th the anti-Gabl A b showed that the 110- to 120-kDa band recognized by the fusion proteins in panels A through D had the same electrophoretic mobi l i ty as G a b l . The "*" indicates the heavy chain of the immunoprecipitating A b . Molecular mass standards (in kDa) are indicated to the left. 118 ippt.: stimulation : 200 B ippt.: stimulation : Gabl 2 0 0 • c> GST-She SH2 Blot 200-116—1 97-Anti-Gabl Blot ippt.: stimulation : 200 G a b l GST-SHP-2 (N+C) SH2 G a b l ippt.: stimulation : 200 G a b l G a b l GST-p85 (N) SH2 Blot GST-p85 (C) SH2 Blot ippt.: stimulation : 200 GST-She PTB Blot 119 Figure 5.8. The Grb2 SH2 domain binds to Gabl-associated SHP-2 and She. R A M O S cells were incubated for 2 m in wi th (+) or without (-) anti-IgM Abs. Cel l lysates were immunoprecipitated wi th the anti-Gabl A b or w i th rabbit IgG (control). A , a F A R Western assay was performed i n wh ich the blot was probed w i t h a GST fusion protein containing the Grb2 SH2 domain. Reprobing this blot showed that the bands detected by the Grb2 SH2 domain fusion protein had the same electrophoretic mobility as SHP-2 (B) and the p52 isoform of She (C). Note that i n panel A the p46 She band is obscured by the heavy chain of the A b (marked as "*") used for immunoprecipitating G a b l . Molecular mass standards (in kDa) are indicated to the left. 120 B ippt . : s t imu l a t i on : 200 + - + O <V S H P - 2 p52 S h e p46 S h e GST-Grb2 SH2 Blot Anti- SHP-2 Blot Anti- She Blot 121 Figure 5.9. G a b l is present i n the particulate fraction of R A M O S cells and associates w i th s i gna l l i ng proteins i n this fraction. R A M O S cells were incubated for 2 m i n wi th (+) or without (-) anti- IgM Abs. Particulate and soluble fractions f rom equal numbers of cells were immunoprecip i tated wi th the ant i -Gab l A b and analyzed by immunob lo t t ing w i th either A , the ant i -Gab l A b or B, the 4G10 anti-P-Tyr m A b . Molecular mass standards (in kDa) are indicated to the left. Note that the molecular masses of the Gabl -assoc iated proteins i n B are rough estimates based on their electrophoretic mobilities relative to the moleular mass standards. 122 A Anti-Gab 1 Ippts. fraction stimulation - + - + 1 1 6 H 97 , — J G a b l Anti-Gabl Blot B Anti-Gabl Ippts. fraction stimulation 200 pp150 G a b l pp90-97 p p 7 0 pp55 pp50 Anti-P-Tyr Blot 123 reprobing the blot i n Figure 5.9A wi th the anti-P-Tyr m A b showed that substantial amounts of the Gabl / ty ros ine phosphoprotein complexes were present i n both the particulate and soluble fractions of anti-IgM-stimulated cells (Fig. 5.9B). In Figures 5.3 through 5.6 I showed that BCR ligation caused She, Grb2, Ptdlns 3-kinase, and SHP-2 to b ind to G a b l . The anti-P-Tyr blot i n Figure 5.9B suggested that complexes of G a b l w i t h these s ignal l ing proteins were present i n the membrane fraction of activated R A M O S cells. To assess this direct ly, I immunoprecipitated G a b l from the particulate and soluble fractions of R A M O S cells and looked for its association w i t h these signal l ing proteins (Fig. 5.10). Complexes of She, Grb2, Ptdlns 3-kinase, and SHP-2 wi th G a b l were clearly present i n both the particulate and soluble fraction of anti-IgM-stimulated R A M O S cells. Thus, tyrosine-phosphorylated G a b l is associated w i t h cellular membranes and provides b inding sites that can recruit She, Grb2, Ptdlns 3-kinase, and SHP-2 to cellular membranes i n response to B C R engagement. 5.2 Discussion We and others have previously shown that the B C R uses the Cas and c-Cbl docking proteins to co-localize components of signalling pathways (see Chapter 3 and refs, 120,151,157,159, 161). Here I show that i n the R A M O S human B cell line, G a b l is a major docking site for a number of proteins involved i n B C R signalling. In response to B C R ligation, G a b l is tyrosine phosphorylated and binds She, Grb2, Ptdlns 3-kinase and SHP-2. I estimated that between 1% and 5% of the total Grb2, Ptdlns 3-kinase, and SHP-2 in R A M O S cells bound to G a b l after B C R ligation while up to 10% of the She was bound to G a b l i n activated R A M O S cells. This is similar to the level of Grb2 and Ptdlns 3-kinase reported to associate w i t h the C b l docking protein i n activated R A M O S cells (120,161). Further analysis showed that a significant por t ion of these G a b l / s i g n a l l i n g protein complexes were i n the membrane-enriched particulate fraction of anti-IgM-stimulated R A M O S cells. 124 Figure 5.10. G a b l associates w i t h She, Grb2 , Ptdlns 3-kinase, and SHP-2 i n the particulate fraction of R A M O S cells after B C R ligation. R A M O S cells were incubated for 2 m i n wi th (+) or without (-) anti-IgM Abs. Particulate and soluble fractions from 3 x 10 7 cells were immunoprecipitated wi th the anti-Gabl A b and analyzed by immunoblott ing w i t h the A , anti-She A b , B , anti-Grb2 m A b , C, anti-p85 A b , or D , anti-SHP-2 A b . "Ce l l lysate" represents 40 ug of total cellular protein (6 x 10 5 cell equivalents). Molecular mass standards (in kDa) are indicated to the left. E , In vitro Ptdlns 3-kinase enzyme assays using Ptdlns as a substrate. Reaction products from the kinase assays were separated by T L C and radiolabeled Ptdlns-phosphate (Ptdlns-POj) was visualized by autoradiography. 125 B Anti-Gabl Ippts. fraction : stimulation : 66-45-+ — + — + fraction stimulation Anti-Gabl Ippts. p52 She p46 She 2 1 . 5 H Anti-She Blot Anti-Grb2 Blot Anti-Gabl Ippts. fraction : stimulation : ^ ^ f £ & ~* - J p85 Anti-Gabl Ippts fraction : stimulation : 97-- + - + 66 H Anti-SHP-2 Blot SHP-Anti-Gab1 Ippts. fraction : / *f stimulation : — + — + • • Ptdlns-P04 in vitro kinase assay 126 Figure 5.11. G a b l associates w i t h s igna l l ing proteins i n BCR-s t imula ted cells. Signalling proteins associated wi th G a b l i n BCR-stimulated cells and the signalling pathways they may regulate during B C R signalling are indicated (see text for details). The proposed mechanism of association of the different proteins wi th G a b l is based on the F A R Western data (see Figs 5.7 and 5.8). The P H domain-mediated association of G a b l wi th cellular membranes is based on the work of others studying G a b l in other systems ( refs. 202,203 and see text). 127 128 Thus, the B C R may use G a b l to recruit cytosolic signalling enzymes to cellular membranes where their substrates are located. This could be an important step i n the activation of signalling pathways by the BCR. D o c k i n g proteins such as IRS-1, the Cas proteins, and c -Cb l are phosphorylated on mult iple tyrosine residues after receptor signall ing, a l lowing them to b ind the SH2 domains of a variety of signalling proteins. In this way, receptors use docking proteins to form signalling complexes and to recruit signalling proteins to specific cellular locations. I found that the B C R uses G a b l i n this manner i n R A M O S cells. Performing F A R Western experiments, I found that BCR-induced tyrosine phosphorylation of G a b l created b inding sites for the SH2 domains of She, Ptdlns 3-kinase, and SHP-2 (Fig. 5.7). Interestingly, the SH2 domain of Grb2 d id not b ind directly to G a b l , but rather associated indirectly wi th G a b l through b ind ing Gabl-associated SHP-2 and She (Fig. 5.8). Thus, this SH2 domain-mediated recruitment of signall ing proteins to G a b l w o u l d concentrate these cytosolic signalling proteins at specific sites in the cell. A significant por t ion of G a b l was present i n the membrane-enriched particulate fraction of both unstimulated and anti-IgM-stimulated R A M O S cells (see Fig. 5.9). This particulate fraction contains cellular membranes and is interpreted as being a membrane-enriched fraction. In performing these experiments I found that docking proteins like G a b l , the Cas proteins, and c-Cbl are present at relatively high levels i n the particulate fraction (see Figs. 6.9 and 3.7) while cytosolic signalling proteins like Crk are present at very low levels i n the particulate fraction (see Fig. 3.7). This supports the idea that docking proteins specifically associate wi th cellular membranes and that the presence of docking proteins i n this fraction is l ikely not an artifact of the preparation. One limitation of using this biochemical technique to look at the localization of G a b l i n BCR-stimulated cells is it does not differentiate between different cellular membranes. In part to address this problem, we have 129 started looking at the localization of a GFP-tagged G a b l i n WEHI-231 cells as wel l as i n AtT20 endocrine cells i n wh ich B C R signalling has been reconstituted. Several groups have now shown that association of G a b l w i t h cellular membranes is mediated by the P H domain of G a b l (202,203). P H domains can bind the inositol phospholipids PtdIns(4,5)P2, PtdIns(3,4)P 2 / or Ptdlns(3,4,5)P 3 wi th the P H domains of different proteins prefering different lipids (109). PtdIns(4,5)P2 is present in the membranes of both unstimulated and anti-IgM-stimulated B cells (28) and may be responsible for the constitutive membrane association of G a b l i n B cells. However, two groups have shown that the likely target of the G a b l P H domain are Ptdlns 3-kinase products (202,203). Isakoff et al . showed that expressing a constitutively-active Ptdlns 3-kinase could target GFP-tagged G a b l to the membrane i n Cos cells (203) while Maroun et al. showed that the Ptdlns 3-kinase inhibitor, LY294002, could interfere wi th the membrane localization of G a b l i n M D C K cells grown i n serum (202). Given that engagement of the BCR increases the levels of the Ptdlns 3-kinase products, PtdIns(3,4)P 2 and PtdIns(3,4,5)P3 (28), B C R signalling might recruit G a b l to the membrane i n B cells. However , I observed constitutive membrane localization of G a b l in R A M O S cells. This may be due to the fact that the cells were grown i n 10% serum and signalling by receptors for serum growth factors may result i n the continual product ion of PtdIns(3,4,5)P 3. Maroun et al. have shown that i n M D C K cells grown i n serum, G a b l is constitutively at the membrane and specifically at sites of cell-cell contact (202). Our lab plans to investigate whether we can observe BCR- induced recruitment of G a b l to cellular membranes i n serum-starved R A M O S cells. M y results suggest that G a b l could play a role i n several key B C R signalling pathways inc luding the Ptdlns 3-kinase and Ras pathways. Act iva t ion of Ras involves the recruitment of Grb2*SOS complexes to the plasma membrane where Ras is located. Our lab has previously shown that a major target for the Grb2 SH2 domain i n anti-IgM-stimulated R A M O S B cells is tyrosine-phosphorylated She and 130 that B C R l igat ion induces the formation of S h c # G r b 2 « S O S complexes (118). However, it was not clear how She was recruited to the plasma membrane after B C R engagement. In vitro, the She SH2 domain can b ind to the phosphorylated I T A M motifs of the B C R Ig -a /p subunits (204). However , I am unable to detect this interaction in vivo, even in cells overexpressing She (R.J. Ingham; unpublished observation). In contrast, I could readily detect G a b l ' S h c complexes i n both detergent extracts and particulate fractions from activated R A M O S cells. Moreover, I could show that the She SH2 domain could b ind directly to G a b l precipitated from anti-IgM-stimulated R A M O S cells. G a b l has several tyrosine residues w i t h hydrophobic residues i n the +1 and +3 positions that the She SH2 domain could bind to (96) and this may account for the large amount of She that binds to G a b l in anti-IgM-stimulated R A M O S B cells. In addition to showing that She binds to G a b l i n the particulate fraction of anti-IgM-stimulated R A M O S cells, I found that the Grb2 SH2 domain could b ind directly to the Gabl-associated She (Fig. 5.8). This wou ld leave the SH3 domains of Grb2 free to b ind SOS and/or SHIP. Thus, G a b l could recruit S h c » G r b 2 * S O S and She • G r b 2 » SHIP complexes to the plasma membrane i n B cells. This is contrast to other Grb2 /dock ing protein interactions. For example, Grb2 binds to C b l via its SH3 domains (117,156) suggesting that C b l , unlike G a b l , may sequester Grb2 and prevent it from binding SOS or SHIP. I also found that G a b l may recruit Grb2»SOS and Grb2»SHIP complexes to the plasma membrane i n B cells by using SHP-2 as an adapter protein. I found that B C R ligation caused SHP-2 to b ind to G a b l (Fig. 5.6A) and that this interaction was mediated by the SHP-2 SH2 domains (Fig. 5.7B). SHP-2 was strongly phosphorylated on tyrosine residues after B C R ligation (Fig. 5.6B) and the Grb2 SH2 domain could bind directly to the Gabl-associated SHP-2 (Fig. 5.8). Thus, G a b l may use both She and SHP-2 to recruit Grb2 complexes. In addition to recruiting Grb2 complexes to G a b l i n B cells, SHP-2 has the potential to dephosphorylate G a b l and negatively regulate the formation of Gab l / s i gna l l i ng protein complexes. In support of this, 131 Nishida et al. showed that G a b l is a substrate of SHP-2 (205). Also , the Drosophila G a b l orthologue, D O S , is a substrate of the SHP-2 orthologue i n Drosophila, Corkscrew (206). It remains to be determined whether the primary role of SHP-2 in B C R signalling is to function as an adapter protein or as a tyrosine phosphatase. I also showed that G a b l may contribute to activation of the Ptdlns 3-kinase pathway by the B C R (Fig. 5.5). B C R ligation leads to increased levels of Ptdlns 3-kinase products i n B cells (28). These l i p i d second messengers have been implicated i n a number of important cellular processes including the prevention of apoptosis and regulation of transcription (59). Since the inositol l i p id substrates of Ptdlns 3-kinase are part of the plasma membrane, Ptdlns 3-kinase must be recruited from the cytoplasm to the plasma membrane after B C R engagement. Previous work has identified the transmembrane protein CD19 as we l l as the c-Cbl docking protein as major b inding sites for Ptdlns 3-kinase i n activated B cells (116,151,161). I have shown that G a b l is another target for Ptdlns 3-kinase in B cells and that G a b l •Ptdlns 3-kinase complexes are present i n the particulate fraction of activated R A M O S cells. The use of multiple membrane docking sites may be a common strategy by which receptors recruit important s ignall ing proteins such as Ptdlns 3-kinase to cell membranes. Alternatively, each docking protein may recruit signalling proteins to a different intracellular locale. G a b l may also be involved i n addit ional B C R signall ing pathways. For example, I found that small amounts of the C r k L adapter protein associated wi th G a b l after B C R ligation i n R A M O S cells (R.J. Ingham; unpublished observation). Since C r k L constitutively binds the C 3 G guanine nucleotide exchange factor i n B cells (ref. 120 and see Chapter 3) and C 3 G has been implicated i n activation of the R a p l (144) and R-Ras (176) GTPases, G a b l may regulate the activation of these GTPases by the BCR. Furthermore, the unidentified 150-kDa and 90- to 97-kDa tyrosine-phosphorylated proteins that associate w i t h G a b l i n anti-IgM-stimulated R A M O S cells (see Fig. 5.3) may also be signalling proteins. It has been reported that 132 SHIP can b ind G a b l (207) and given that She and Grb2 are important for SHIP binding i n B cells (refs. 123,129 and see also Chapter 7), this might be the way SHIP is recruited to G a b l . Recruitment of SHIP to G a b l w o u l d provide another means by which G a b l could regulate the Ptdlns 3-kinase pathway. A closely related protein to G a b l , Gab2, has recently been described (205,208). While human Gab2 is only 37% identical at the amino acid level to human G a b l (200,203), they are structurally very similar. Gab2, like G a b l , possesses a P H domain and most of the tyrosine phosphorylation sites and the relative order i n which they are found are conserved between the two proteins (205,208). Not surprisingly, Gab2 appears to associate wi th many of the same proteins as G a b l (205,208). Some B cells lines (e.g. R A M O S ) appear to express G a b l but not Gab2 while the WEHI-231 B cell line expresses Gab2 but not G a b l (refs. 205,208 and R.J. Ingham, unpublished observation). It is not known if some B cell lines express both G a b l and Gab2 nor is it known which are expressed by normal B cells. Since G a b l and Gab2 associate wi th similar SH2 domain-containing signall ing proteins, they may have redundant functions. However , Zhoa et al. showed that overexpression of G a b l i n 293 cells could enhance Ras- and EGF-mediated Elk-1 activation whi le overexpression of Gab2 in these cells actually inhibited Elk-1 activation under these same conditions (209). Thus, further work is needed to determine if G a b l and Gab2 have similar or distinct signalling functions. Signalling by the B C R involves the recruitment of a number of cytosolic SH2 domain-containing signalling proteins to cellular membranes. To recruit a diverse array of SH2-containing signall ing proteins, the BCR-activated tyrosine kinases must create an equally diverse array of b inding sites. I have shown here that tyrosine phosphorylation of G a b l by the B C R creates binding sites for a number of key proteins involved i n B C R signalling and that G a b l recruits these proteins to cellular membranes where their targets are located. 133 Chapter 6 Overexpression of G a b l in WEHI-231 cells augments the signall ing function of Gabl-associated proteins 6.0 Introduction A critical step i n the initiation of signalling pathways is bringing cytosolic signalling proteins to the plasma membrane where their substrates are located. This requires the creation of membrane docking sites for S H 2 domain-containing signalling proteins or adapter proteins associated wi th signalling proteins. In B cells this docking protein function is carried out by proteins such as Iga /p , CD19, c-Cbl, the Cas proteins, and as shown i n the previous chapter, G a b l . G a b l is a membrane-associated docking protein i n B cells and the B C R uses G a b l to recruit a number of SH2 domain-containing signalling proteins to cellular membranes. The binding of these cytosolic signalling proteins to membrane-associated G a b l may be important for these signalling proteins to carry out their functions. For example, the recruitment of Ptdlns 3-kinase to the membrane by G a b l w o u l d bring Ptdlns 3-kinase close to the plasma membrane lipids which are its substrates. Furthermore, recruitment of Grb2»SOS and Grb2»SHIP complexes to G a b l w o u l d bring SOS i n close proximity Ras and SHIP i n close proximity to PtdIns(3,4,5)P3 w h i c h are associated w i t h the plasma membrane. Thus, G a b l may play a key role i n the activation of several signalling pathways by facilitating the recruitment of signalling proteins to membranes where their substrates are located. Based on my work i n the previous chapter, my hypothesis was that G a b l potentiates BCR-induced signalling by recruiting signalling proteins to the plasma membrane. To test this hypothesis, my approach was to overexpress G a b l in WEHI-231 cells, wh ich express very little if any G a b l (R.J. Ingham; unpublished observation). M y prediction was that providing additional membrane binding sites for G a b l - b i n d i n g proteins w o u l d enhance their function. I found that 134 overexpression of G a b l i n WEHI-231 cells enhanced the BCR-mediated activation of the serine/threonine kinase A k t whose activation is dependent upon Ptdlns 3-kinase. In add i t ion , overexpression of G a b l enhanced BCR-med ia t ed phosphorylat ion of the SHP-2 tyrosine phosphatase w h i c h correlated w i t h an increased binding of Grb2 to SHP-2. Finally, I found that the G a b l P H domain was required for phosphorylation of G a b l by the BCR. Since the P H domain l ikely mediates the membrane localization of G a b l in B cells, this suggests that G a b l must be at the membrane to potentiate signalling events such as activation of Ptdlns 3-kinase and phosphorylation of SHP-2. Thus, my data shows that G a b l plays an important role i n regulating the signalling proteins it binds and provides further evidence that membrane localization of signalling proteins is important for their function. 6.1 Results 6.1.1 Gabl expressed in WEHI-231 cells associates with similar signalling proteins as Gabl expressed in RAMOS cells In order to look at the effect of G a b l overexpression on B C R signalling, I chose to express G a b l i n WEHI-231 cells which express very little if any endogenous G a b l (ref. 205 and data not shown). Given that G a b l is not normally expressed i n WEHI-231 cells, I first wanted to determine if G a b l could be used by the BCR i n these cells. Figure 6.1 shows that G a b l expressed i n WEHI-231 cells is tyrosine phosphorylated i n response to B C R crossl inking and associates w i t h similar tyrosine-phosphorylated proteins as G a b l i n anti-IgM stimulated R A M O S cells (see Chapter 5; Figure 5.3). This indicates that G a b l functions similarly i n WEHI-231 and R A M O S cells. I then investigated whether expression of G a b l i n WEHI-231 cells potentiated the function of signalling proteins that bind to G a b l . 135 Fig. 6.1. G a b l expressed i n WEHI-231 cells is tyrosine phosphorylated and associates w i t h tyrosine-phosphorylated proteins i n BCR-s t imula ted cells. The BOSC 23 packaging cell line was transfected wi th the p M X vector (vector) or p M X containing c D N A encoding for w i l d type G a b l (wt Gabl ) . The resulting retrovirus particles were used to infect WEHI-231 cells. The WEHI-231 cells were stimulated wi th ant i - IgM Abs for 2 m i n (+) or left unst imulated (-) and cell lysates were immunoprecipi tated w i t h the ant i -Gabl A b . Precipitated proteins were then analyzed by immunoblotting wi th the 4G10 anti-P-Tyr m A b (upper panel). The blot was then stripped and reprobed wi th the anti-Gabl A b (lower panel). Molecular mass standards (in kDa) are indicated to the left. Note that the molecular masses of the Gabl-associated proteins are rough estimates based on their electrophoretic mobilities relative to the molecular mass standards. 136 construct anti-IgM anti-P-Tyr blot anti-Gabl reprobe anti-Gabl ippts wt Gabl vector ppl50 Gabl SHP-2 ++m p52Shc ^ p46Shc | ^ Gabl 137 6.1.2 Gabl expressed in WEHI-231 cells associates with Ptdlns 3-kinase in response to B C R stimulation I previously showed that G a b l serves as membrane b inding site for Ptdlns 3-kinase i n BCR-stimulated R A M O S cells (ref. 122 and see Chapter 6). I postulated that recruitment of Ptdlns 3-kinase to G a b l w o u l d provide a means for Ptdlns 3-kinase to gain access to its phosphol ipid substrates wh ich are present i n the inner-leaflet of the plasma membrane. Thus, overexpression of G a b l i n WEHI-231 cells should provide additional membrane b inding sites for Ptdlns 3-kinase and allow more Ptdlns 3-kinase to be recruited to the membrane. Presumably this w o u l d al low Ptdlns 3-kinase to produce more PtdIns(3,4,5)P3 and PtdIns(3,4,)P2 which w o u l d enhance signalling events mediated by these phospholipids. I first asked whether Ptdlns 3-kinase could bind G a b l after B C R ligation i n WEHI-231 cells, as it does i n R A M O S cells. Figure 6.2 shows that the p85 subunit of Ptdlns 3-kinase associates w i t h G a b l i n BCR-stimulated cells. However , a mutant G a b l protein lacking 3 tyrosine residues that, based on their surrounding sequences, w o u l d provide binding sites for the SH2 domains of the p85 subunit of Ptdlns 3-kinase (96), d id not b ind Ptdlns 3-kinase after B C R ligation (Fig. 6.2). Thus, G a b l can associate wi th Ptdlns 3-kinase in WEHI-231 cells. 6.1.3 Overexpression of Gabl in WEHI-231 cells enhances BCR-mediated Akt activation One of the consequences of Ptdlns 3-kinase activation is activation of the serine/threonine kinase A k t (59). A k t plays a key role i n Ptdlns 3-kinase-mediated signalling by phosphorylating several proteins including GSK-3 , members of the Forkhead family of transcription factors, caspase 9, as we l l as the pro-apoptotic protein Bad (59). Ac t iva t ion of A k t correlates w i t h its phosphorylat ion on threonine 308 and serine 473 residues. Therefore, monitoring the phosphorylation of these specific residues, using phospho-specific Abs, provides a convenient means 138 Fig. 6.2. G a b l expressed i n WEHI-231 cells associates w i t h P td lns 3-kinase i n BCR-s t imula ted cells. The BOSC 23 packaging cell line was transfected wi th the p M X vector (vector) or p M X containing c D N A encoding for w i l d type G a b l (wt Gabl) or APtdlns 3-kinase G a b l (API3K Gabl ) . The resulting retrovirus particles were used to infect WEHI-231 cells. Cells were stimulated wi th anti-IgM Abs for 2 min (+) or left unstimulated (-) and cell lysates were immunoprecipitated wi th the anti-Gabl A b . The precipitated proteins were analyzed by immunoblotting wi th the anti-p85 A b (upper panel). The blot was then stripped and reprobed wi th the anti-G a b l A b (lower panel). Molecular mass standards (in kDa) are indicated to the left. 139 anti-Gabl ippts construct: vector wt A PI3K G a b l G a b l an t i - IgM: — anti-p85 blot ant i -Gabl reprobe p85 G a b l 140 of measuring A k t activation. It has previously been shown that the B C R activates A k t i n a P td lns 3-kinase-dependent manner (34-37). Therefore, us ing phosphorylation of serine 473 as a measure of A k t activation, I looked at whether overexpression of G a b l i n WEHI-231 cells enhanced A k t activation i n response to B C R stimulation. Figure 6.3 shows that BCR-induced phosphorylation of A k t on serine 473 was significantly enhanced i n Gabl-expressing WEHI-231 cells compared to the parenta l W E H I - 2 3 1 cells . Fur thermore , augmenta t ion of A k t phosphorylation required functional Ptdlns 3-kinase binding sites as cells expressing the mutant form of G a b l lacking the Ptdlns 3-kinase-binding sites showed little if any enhancement of A k t phosphorylation over the parental cells (Fig. 6.3). Thus, overexpression of G a b l i n WEHI-231 cells augments signalling events mediated by Ptdlns 3-kinase and suggests that providing additional membrane binding sites for P td lns 3-kinase d u r i n g B C R s igna l l ing is sufficient to enhance P td lns 3-kinase-mediated signalling events. 6.1.4 G a b l expressed i n WEHI-231 cells associates wi th SHP-2 i n response to B C R s t imulat ion Given that Ptdlns 3-kinase-dependent signalling events were augmented i n Gabl -express ing cells, this suggested that the s ignal l ing function of other Gabl-associated proteins w o u l d also be enhanced i n these cells. In Chapter 5 I showed that the SHP-2 tyrosine phosphatase associated w i t h G a b l i n BCR-stimulated cells. Therefore, I was interested i n determining if G a b l expressed i n WEHI-231 cells associated w i t h SHP-2 and whether this enhanced SHP-2's signalling function. Figure 6.4 shows that SHP-2 bound to G a b l after B C R ligation i n Gabl-expressing WEHI-231 cells. In contrast, a G a b l protein i n which the major SHP-2 binding site on murine SHP-2 , tyrosine 628 (210), had been mutated showed greatly decreased SHP-2 binding compared to w i l d type G a b l . Thus, G a b l expressed i n WEHI-231 cells associates wi th SHP-2 and tyrosine 628 appears to be the major 141 Fig. 6.3. Expression of G a b l in WEHI-231 cells enhances A k t phosphorylat ion in response to BCR crosslinking. The BOSC 23 packaging cell line was transfected wi th the p M X vector (vector) or p M X containing c D N A encoding for w i l d type G a b l (wt Gabl) or APtdlns 3-kinase G a b l (API3K Gabl ) . The resulting retrovirus particles were used to infect WEHI-231 cells. Cells were stimulated wi th anti-IgM Abs for 2 min (+) or left unstimulated (-). Total cell lysates were analyzed by immunoblotting wi th the anti-phospho-Akt A b (upper panel). The blot was then stripped and reprobed wi th the anti-Akt A b (middle panel) and the anti-Gabl A b (lower panel). Molecular mass standards (in kDa) are indicated to the left. 142 construct: vector Akt reprobe anti-Gabl reprobe anti-IgM: — anti-P-Akt blot wt Gabl A P I 3 K Gab l + - + - + P-Akt Akt Gab l 143 Fig. 6.4. G a b l expressed i n WEHI-231 cells associates wi th SHP-2 i n B C R - stimulated cells. The BOSC 23 packaging cell line was transfected wi th the p M X vector (vector) or p M X containing c D N A encoding for w i l d type G a b l (wt Gab l ) or ASHP-2 G a b l (ASHP-2 Gabl ) . The resulting retrovirus particles were used to infect WEHI-231 cells. Cells were stimulated wi th anti-IgM Abs for 2 m i n (+) or left unstimulated (-) and cell lysates were immunoprecipitated wi th the anti-Gabl A b . The precipitated proteins were analyzed by immunoblotting wi th the anti-SHP-2 A b (upper panel). The blot was then stripped and reprobed wi th the ant i -Gabl A b (lower panel). Molecular mass standards (in kDa) are indicated to the left. 144 anti-Gabl ippts construct: anti-IgM : anti-SHP-2 blot anti-Gabl reprobe vector wt A SHP-2 Gabl Gabl - + - + - + SHP-2 Gabl 145 SHP-2 binding site. 6.1.5 Overexpression of G a b l i n WEHI-231 cells enhances BCR-mediated SHP-2 tyrosine phosphorylation and Grb2 b ind ing to SHP-2 In Chapter 5 I showed that B C R l iga t ion i n d u c e d the tyrosine phosphorylation of SHP-2 and the binding of SHP-2 to G a b l . Moreover, I found that Grb2 could b ind to tyrosine-phosphorylated SHP-2 associated w i t h G a b l (see Fig. 5.11). The b i n d i n g of G r b 2 » S O S complexes to SHP-2 that is bound to membrane-associated G a b l wou ld provide a means for bringing SOS close to Ras. Alternatively, since Grb2 can also bind SHIP i n B cells (refs. 123,129 and see Chapter 7) recrui tment of G r b 2 » S H I P complexes to S H P - 2 that is b o u n d to membrane-associated G a b l wou ld bring SHIP i n close proximity to its phospholipid substrates. In order for SHP-2 to act as an adapter protein i n this way, it needs to be both bound to G a b l and tyrosine phosphorylated. I hypothesized that the binding of SHP-2 to G a b l w o u l d bring it close to membrane-associated tyrosine kinases and promote its tyrosine phosphorylation. To test this hypothesis, I looked at whether tyrosine phosphorylation of SHP-2 was enhanced i n Gabl-expressing WEHI-231 cells. Figure 6.5 shows that expression of G a b l i n WEHI-231 cells greatly increased BCR- induced tyrosine phosphoryla t ion of SHP-2 compared to the parental WEHI-231 cell line. Expressing the G a b l mutant lacking the major SHP-2 binding site also enhanced SHP-2 phosphorylation but was less efficient at doing so (Fig 6.5). Thus, G a b l overexpression in WEHI-231 cells enhances SHP-2 phosphorylation. Given that association of Grb2 wi th SHP-2 is mediated by the SH2 domain of Grb2 (see Chapter 5), this suggested to me that the increased tyrosine phosphorylation of SHP-2 seen in Gabl-expressing cells (Fig. 6.5) may allow SHP-2 to b ind more Grb2. Therefore, I looked at whether there was more Grb2 associated wi th SHP-2 i n Gabl-expressing cells. Figure 6.6 shows i n Gabl-expressing cells, more Grb2 co-precipitated wi th SHP-2. Expression of the mutant G a b l protein which was 146 Fig. 6.5. Expression of G a b l in WEHI-231 cells enhances SHP-2 phosphorylation in response to BCR crosslinking. The BOSC 23 packaging cell line was transfected with the p M X vector (vector) or p M X containing c D N A encoding for wild type Gabl (wt Gabl) or ASHP-2 Gabl (ASHP-2 Gabl). The resulting retrovirus particles were used to infect WEHI-231 cells. Cells were stimulated with anti-IgM Abs for 2 min (+) or left unstimulated (-) and cell lysates were immunoprecipitated with the anti-SHP-2 Ab. The precipitated proteins were analyzed by immunoblotting with the 4G10 anti-P-Tyr mAb (upper panel). The blot was then stripped and reprobed with the anti-SHP-2 Ab. Molecular mass standards (in kDa) are indicated to the left. 147 anti-SHP-2 ippts construct: anti-IgM : anti-P-Tyr blot anti-SHP-2 reprobe wt A SHP-2 vector Gabl Gabl — + — + — + P-SHP-2 SHP-2 148 severely reduced i n SHP-2 binding (Fig. 6.4) d id not enhance association of Grb2 wi th SHP-2 as much as expression of w i l d type G a b l protein but still significantly enhanced Grb2 binding to SHP-2 over that seen i n the parental WEHI-231 cells (Fig. 6.6). Therefore, it appears that expression of G a b l i n WEHI-231 cells provides a means to more efficiently couple Grb2 to SHP-2 during B C R signalling. This in turn may enhance the signalling function of proteins bound to the SH3 domains of Grb2. 6.1.6 The G a b l PH domain is required for efficient tyrosine phosphorylation of G a b l and for association of G a b l wi th signall ing proteins i n BCR-stimulated cells Membrane localization of G a b l is key to my model of how G a b l functions during B C R signalling. Several groups have shown that recruitment of G a b l to the plasma membrane i n other systems is dependent upon the P H domain of G a b l binding to Ptdlns 3-kinase products (202,203). One potential consequence of G a b l being recruited to the plasma membrane is that it may make G a b l a better substrate for membrane-associated tyrosine kinases. Phosphorylation of G a b l is critical for G a b l to provide binding sites for SH2 domain-containing proteins. To see if the P H domain of G a b l was required for BCR-induced phosphorylation of G a b l and the subsequent association of signalling proteins wi th G a b l , I expressed i n WEHI-231 cells a mutant G a b l protein lacking the P H domain. Figure 6.7 shows that the P H domain was required for efficient BCR-induced tyrosine phosphorylation of G a b l . Furthermore, the association of tyrosine-phosphorylated proteins w i t h G a b l was greatly reduced by removing the P H domain (Fig. 6.7). Thus, i n order for G a b l to function as a docking site for signalling proteins during B C R signalling, a functional P H domain is required. 149 Fig. 6.6. Expression of G a b l i n WEHI-231 cells enhances Grb2 b ind ing to SHP-2 i n response to BCR crossl inking. The BOSC 23 packaging cell line was transfected wi th the p M X vector (vector) or p M X containing c D N A encoding for w i l d type G a b l (wt Gabl) or ASHP-2 G a b l (ASHP-2 Gabl ) . The resulting retrovirus particles were used to infect WEHI-231 cells. Cells were stimulated wi th anti-IgM Abs for 2 m in (+) or left unstimulated (-) and cell lysates were immunoprecipitated w i t h the anti-SHP-2 A b . The precipitated proteins were analyzed by immunoblotting wi th an anti-Grb2 A b (upper panel). The blot was then stripped and reprobed wi th the anti-SHP-2 A b . Molecular mass standards (in kDa) are indicated to the left. 150 anti-SHP-2 ippts construct: anti-IgM: anti-Grb2 blot anti-SHP-2 reprobe wt A SHP-2 vector Gabl Gabl - + - + - + Grb2 SHP-2 151 Fig. 6.7. The G a b l P H domain is required for efficient tyrosine phosphorylation of G a b l and for association of G a b l w i th s ignal l ing proteins i n BCR-st imulated cells. The BOSC 23 packaging cell line was transfected wi th the p M X vector (vector) or p M X containing c D N A encoding for w i l d type G a b l (wt Gab l ) or A P H G a b l ( A P H Gabl) . The resulting retrovirus particles were used to infect WEHI-231 cells. Cells were stimulated w i t h anti-IgM Abs for 2 m i n (+) or left unstimulated (-) and cell lysates were immunoprecipitated wi th the anti-Gabl A b . Precipitated proteins were analyzed by immunoblott ing wi th the 4G10 anti-P-Tyr m A b (upper panel). The blots were then stripped and reprobed w i t h the an t i -Gabl A b (lower panel). Molecular mass standards (in kDa) are indicated to the left. Note that this blot was not reprobed w i t h an anti-She A b and the bands labelled "p52 She" and "p46Shc" were identif ied based solely on their molecular masses and comparing the electrophoretic mobil i ty of other ant i -Gabl immunoprecipitates w i t h an anti-She A b . 152 anti-Gabl ippts construct: anti-IgM: anti-P-Tyr blot anti-Gabl reprobe wt APH vector Gabl Gabl - + - + - + Gabl A PH Gabl p52Shc p46Shc Gabl A PH Gabl 153 6.2 Discussion I have shown that G a b l plays an important role i n the activation of several signalling pathways used by the BCR. M y studies show that expression of G a b l i n WEHI-231 cells, wh ich do not normally express G a b l , enhances the signall ing function of Gab l -b ind ing proteins during B C R signalling. Furthermore, I found that the P H domain of G a b l was required for BCR-induced tyrosine phosphorylation of G a b l and for the association of signalling proteins wi th G a b l . Thus, my data show that membrane-associated docking proteins such as G a b l play an important role in regulating B C R signalling pathways. I found that overexpression of G a b l i n WEHI-231 cells resulted i n enhanced activation of the serine/threonine kinase A k t i n response to B C R engagement (Fig. 6.3) and that the ability of G a b l to do so correlated wi th Gabl ' s ability to associate wi th Ptdlns 3-kinase (Fig. 6.2). Since the activation of A k t i n B cells is dependent upon Ptdlns 3-kinase activity (34-37), it is not surprising that uncoupling Ptdlns 3-kinase from G a b l (Fig. 6.2) ablated the abili ty of G a b l to enhance Ptdlns 3-kinase-mediated signalling events (Fig. 6.3). M y data, support the model that G a b l functions as a membrane docking site for Ptdlns 3-kinase dur ing B C R signalling wh ich allows Ptdlns 3-kinase access to its membrane-associated phosphol ip id substrates. M y data also show that tyrosine phosphorylation of SHP-2 was enhanced i n Gabl-expressing cells and this correlated wi th increased binding of Grb2 to SHP-2 (Fig. 6.5). Our lab previously noted that there was little phosphorylation SHP-2 i n response to B C R engagement i n WEHI-231 cells (T. Saxton and M . R . G o l d ; unpublished observation). In contrast, i n R A M O S cells I found that SHP-2 is heavily tyrosine-phosphorylated i n response to B C R engagement (ref. 122 and see Chapter 5). It is tempting to speculate that this difference i n SHP-2 phosphorylation is due to the fact that R A M O S cells express G a b l while WEHI-231 cells express little 154 or no G a b l . If this is the case, then G a b l is an important regulator of SHP-2 function. Whi le it is clear that SHP-2's ability to recruit Grb2 is clearly enhanced i n BCR-stimulated cells expressing G a b l , it is l ikely that G a b l regulates other SHP-2 functions as wel l . It has been reported that tyrosine phosphorylation of SHP-2 correlates wi th an increase in its phosphatase activity (211) and that engagement of its SH2 domains by tyrosine-phosphorylated proteins also enhances its activity by al lowing SHP-2 to assume an active conformation (212). Thus, given that SHP-2 phosphorylation is enhanced i n G a b l expressing cells (Fig. 6.5) and that recruitment of SHP-2 to G a b l is mediated by the SH2 domains of SHP-2 (see Chapter 5), it is likely that recruitment of SHP-2 to G a b l enhances SHP-2's phosphatase activity. While G a b l appears to regulate SHP-2 function i n B cells, it is also likely that SHP-2 regulates G a b l function as wel l during B C R signalling. Two groups have shown that G a b l , and the related protein Gab2, are substrates of SHP-2 (205,208). I have some prel iminary data showing that more Ptdlns 3-kinase bound to the ASHP-2-mutant G a b l than w i l d type G a b l and that A k t phosphorylat ion was enhanced to a greater degree i n the ASHP-2 mutant-expressing cells than i n the w i l d type Gabl-expressing cells (R.J. Ingham; unpublished observation). This suggests that SHP-2 dephosphorylates the Ptdlns 3-kinase b inding sites on G a b l and is therefore a negative regulator of Ptdlns 3-kinase signalling. Indeed, BCR-induced A k t phosphorylat ion is enhanced i n SHP-2-deficient DT40 B cells (M.R. G o l d ; unpublished observation) and SHP-2 has been shown to inhibit Ptdlns 3-kinase binding to the IRS-1 docking protein (213). I also found that the P H domain of G a b l is important for G a b l function. BCR-induced tyrosine phosphorylation of G a b l is clearly decreased when the P H domain is removed, presumably because the mutant G a b l is not targeted to the membrane where the BCR-activated tyrosine kinases that can phosphorylate G a b l are located (Fig 6.7). Furthermore, the decreased phosphorylat ion of the A P H 155 mutant protein correlated w i t h decreased b ind ing of tyrosine-phosphorylated proteins to G a b l (Fig. 6.7). I have preliminary data showing that proteins known to b ind to G a b l (e.g. Ptdlns 3-kinase) do not b ind the A P H domain mutant (R.J. Ingham; unpublished observation). Surprisingly, I still saw considerable binding of two phosphoproteins of approximately 45 and 50 kDa to the A P H domain-mutant G a b l w h i c h are l ike ly the p46 and p52 k D a She proteins. If these two phosphoproteins are the She proteins then it suggests that She may associate to some extent w i t h G a b l i n a phosphotyrosine-independent manner. However , additional experiments are needed to confirm this observation. While the A P H domain mutant results clearly show that the P H domain is important for G a b l function during B C R signalling, I have not shown that the P H domain is involved i n recruiting G a b l to cellular membranes. To address this, I have expressed GFP-tagged forms of the w i l d type and A P H domain-mutant G a b l proteins i n WEHI-231 cells and i n AtT20 endocrine cells expressing all components of the B C R as wel l as the Syk tyrosine kinase. Our lab plans to examine the cellular localization of these two proteins in unstimulated versus BCR-stimulated cells. In summary, my data show that G a b l plays an important role i n regulating the s igna l l ing funct ion of its associated proteins d u r i n g B C R signal l ing . Furthermore, my data suggest that membrane localization of G a b l is critical for it to function as a docking protein. These results support a model i n which the binding of SH2 domain-containing s ignal l ing proteins to phosphotyrosine-containing sequences on membrane-associated docking proteins is a cri t ical step i n the initiation of B C R signalling pathways. 156 Chapter 7 The inositol phosphatase SHIP is required for efficient tyrosine phosphorylation of She i n response to B C R engagement 7.0 Introduction In previous chapters I discussed how the adapter protein She was important for recruit ing Grb2 complexes to the plasma membrane i n response to B C R engagement. She, like other adapter proteins, consists exclusively of protein-protein interaction domains and therefore may play an important role i n B C R signalling i n facilitating the formation of signalling complexes. The SH2 and PTB domains of She can b ind to phosphotyrosine-containing sequences i n other proteins whi le tyrosine phosphorylation of She i n response to B C R crosslinking creates binding sites for the SH2 domain of the Grb2. In addi t ion to inducing the formation of Shc»Grb2»SOS complexes, B C R ligation causes the SHIP inositol phosphatase to b ind to She (118,119). SHIP dephosphorylates the 5' position of the Ptdlns 3-kinase product PtdIns(3,4,5)P3 (200). Dephosphory la t ion of PtdIns(3,4,5)P3 by SHIP may l imi t the magnitude and duration of B C R signalling events that are dependent upon PtdIns(3,4,5)P3 including activation of the Btk tyrosine kinase (214) and A k t serine/threonine kinase (35), as well as increases i n intracellular C a 2 + (215). The mechanism by which SHIP associates wi th She is complex. BCR-induced tyrosine phosphorylation of SHIP creates binding sites for the PTB domain of She. The binding of the She PTB domain to these sites on SHIP is required for She to bind SHIP (216). However, other interactions also contribute to the binding of SHIP to She. Whi le the SH2 domain of SHIP may be able to b ind to phosphotyrosine residues on She (217), Harmer at al. have recently shown that another interaction, one mediated by Grb2, is required to stabilize S h c ' S H I P complexes (129). In their model, two Grb2 molecules that are bound v ia their SH3 domains to proline-rich 157 regions i n SHIP use their SH2 domains to b ind to phosphotyrosine residues on She. The importance of this Grb2-mediated interaction between She and SHIP is highlighted by the fact that SHIP is unable to associate wi th She i n Grb2-deficient B cells (129). Thus, the interaction of Grb2 wi th She is essential for She to b ind both SOS and SHIP. Both SOS and SHIP are cytosolic enzymes whose substrates are localized to the inner face of the plasma membrane. SOS activates Ras (155) which is tethered to the inner face of the plasma membrane by a l i p i d anchor w h i l e SHIP dephosphorylates PtdIns(3,4,5)P3, a plasma membrane phospholipid (200). Thus, both SOS and SHIP must be recruited to the plasma membrane to perform their functions. This may be accomplished, at least i n part, by their Grb2-mediated binding to tyrosine-phosphorylated She. In B cells stimulated through the BCR, b o t h G r b 2 » S O S c o m p l e x e s a n d G r b 2 * S H I P c o m p l e x e s b i n d to tyrosine-phosphorylated She (118,129,218) and Shc«Grb2«SOS complexes are found i n the membrane-rich particulate fraction of the cells (118). Whi le She is found i n the cytoplasm of resting B cells, it is recruited to the membrane after B C R ligation (118,119). This is l ikely to be mediated by the SH2 d o m a i n of She b i n d i n g to phospho ty ros ine -con t a in ing sequences on membrane-associated proteins. A s shown i n Chapter 5, i n the R A M O S human B cell line She binds via its SH2 domain to G a b l , a membrane-associated docking protein that is tyrosine phosphorylated in response to B C R ligation. A s wel l , the SH2 domain of She has been shown in vitro to b ind the phosphorylated I T A M s of the B C R Iga and Igp chains (204,219). Thus, recruitment of She to the plasma membrane and the subsequent tyrosine phosphorylation of She may allow the BCR to recruit Grb2-containing signalling complexes to the cell membrane. Since She must be tyrosine phosphorylated i n order to b ind Grb2 and to recruit Grb2-associated signalling proteins, it is important to understand how the B C R regulates She phosphorylation. The simplest model is that after B C R ligation, 158 She uses its SH2 domain to b ind to the phosphorylated B C R I T A M s or to tyrosine-phosphorylated G a b l and this brings She i n close proximi ty to the BCR-associated tyrosine kinases. To test the hypothesis that the She SH2 domain is required for phosphorylation of She by the BCR, I expressed i n WEHI-231 B cells a mutant She protein i n wh ich the SH2 domain had been inactivated by a point mutation. A s expected, I found that BCR-induced tyrosine phosphorylation of this SH2 domain mutant was significantly lower than tyrosine phosphorylation of w i l d type She. Surprisingly, a She protein i n which the PTB domain was inactivated by a point mutat ion exhibited an even greater reduction i n BCR- induced tyrosine phosphorylation. This suggested that the PTB domain of She was also required for tyrosine phosphorylation of She. In support of this idea, in a collaboration wi th Dr. Hidetaka Okuda (Kansai Medical University, Moriguchi , Japan) we found that SHIP, a protein that binds to the PTB domain of She is required for efficient tyrosine phosphorylation of She by the BCR. Moreover, we showed that the ability of SHIP to b ind to the PTB domain of She correlated wi th its ability to promote tyrosine phosphorylation of She. Thus, these data suggest a novel role for SHIP i n B C R signalling i n regulating the tyrosine phosphorylation of She. 7.1 Results 7.1.1 Efficient tyrosine phosphorylat ion of She i n response to B C R crossl inking requires both the SH2 and PTB domains of She B C R l igation causes She to b ind via its SH2 domain to I g a / | 3 a n d other membrane-associated docking proteins. These interactions may bring She i n close p rox imi ty to BCR-ac t iva ted tyrosine kinases and a l low these kinases to phosphorylate She. This model implies that She must have a functional SH2 domain i n order to become tyrosine phosphorylated after B C R engagement. To test this hypothesis, I expressed i n WEHI-231 cells a mutant form of She i n which the 159 SH2 domain was rendered non-functional by a point mutation (She R401M). I then asked whether She R401M could be tyrosine phosphorylated i n response to B C R crosslinking. Figure 7.1 shows that the She R401M SH2 domain mutant exhibited decreased BCR-induced tyrosine phosphorylation compared to the w i l d type She protein. However, when I reprobed this blot wi th the anti-She A b (Fig. 7.1A, lower panel) I found that the R401M She protein was not expressed at the same level as the w i l d type protein. But even when this decreased level of expression was taken into account, densitometry showed that the relative level of phosphorylat ion of the R401M mutant was less than the w i l d type protein (data not shown). Thus, this shows that the SH2 domain of She is important for She to be tyrosine phosphorylated after BCR ligation. In addit ion to its SH2 domain, She also has a PTB domain that can b ind phosphotyrosine-containing sequences. To determine whether the She PTB domain contributed to the ability of She to be phosphorylated by the BCR, I expressed in WEHI-231 cells a She protein i n which the PTB domain was inactivated by a point mutat ion (She R175M). Surpr is ingly , I found that this muta t ion reduced BCR-induced tyrosine phosphorylation of She to an even greater extent than the SH2 domain mutation (Figure 7.1). Thus, the PTB domain of She also appears to be important for phosphorylation of She by the BCR. 7.1.2 B ind ing of SHIP to She depends on both the SH2 and P T B domains of She Figure 7.1 shows that mutating the SH2 and PTB domains of She reduced the ability of She to b ind other tyrosine-phosphorylated proteins. Association of She wi th an unidentified 70-kDa phosphoprotein (that d id not react w i th anti-Syk Abs (data not shown)) required a functional SH2 domain but was unaffected by inact ivat ion of the PTB domain . In contrast, the b i n d i n g of a 140-kDa phosphoprotein to She was completely ablated by mutating the She PTB domain and 160 Fig. 7.1. BCR-induced tyrosine phosphorylation of She depends on both the SH2 and P T B domains of She. The BOSC 23 packaging cell line was transfected wi th the p M S C V p a c vector (vector) or p M S C V p a c containing the c D N A encoding FLAG-tagged w i l d type She (FLAG-wt She), a She protein i n which the SH2 domain was inactivated (FLAG-Shc R401M), or a She protein i n which the PTB domain was inactivated (FLAG-Shc R175M). The resulting retrovirus particles were used to infect WEHI-231 cells. The WEHI-231 cells were then stimulated wi th anti-IgM Abs for the indicated times. Ce l l lysates were immunoprecipi tated w i t h the M 2 a n t i - F L A G m A b and the precipitated proteins were analyzed by immunoblott ing w i t h the 4G10 ant i -P-Tyr m A b (upper panel). The posi t ions of the tyrosine-phosphorylated FLAG-tagged She proteins (P-FLAG-Shc) are indicated by an arrow. The blot was then stripped and reprobed wi th the anti-She A b (lower panel). Note that the bands i n the 'vector' lanes of the anti-She reprobe (lower panel) are the heavy chain of the A b used for immunoprecipi ta t ion. Molecular mass standards (in kDa) are indicated to the left. Note that the molecular masses of the She-associated proteins ("ppl40" and "pp70") are rough estimates based on their electrophoretic mobilities relative to the molecular mass standards. 161 anti-FLAG ippts She protein anti-IgM (min) 200 anti-P-Tyr blot ex anti-She reprobe pp140 pp70 P-FLAG-Shc IgH FLAG-Shc 162 greatly reduced by mutating the She SH2 domain. Since SHIP binds to the She PTB domain and has a molecular mass of 135-140 kDa , I asked whether the binding of SHIP to She showed the same dependency on both the She PTB domain and the She SH2 domain. Immunoblotting wi th anti-SHIP Abs showed that after B C R ligation SHIP bound strongly to w i l d type She (Fig. 7.2). As expected, SHIP d id not bind to the She R175M mutation which has a non-functional PTB domain. Surprisingly, very little SHIP bound to She R401M which has a non-functional SH2 domain. Thus, the SH2 domain of She is required for one or more of the steps involved i n the association of SHIP w i t h She. Moreover, the extent of SHIP b inding to She correlated w i t h the degree to wh ich She was tyrosine phosphorylated after B C R ligation. 7.1.3 BCR- induced b i n d i n g of Grb2 to She depends on both the SH2 and P T B domains of She To determine if the decreased BCR-induced tyrosine phosphorylation of the mutant She proteins had any functional consequence, I looked at the ability of these mutant She proteins to bind Grb2. Harmer et al. showed that the binding of Grb2 to She i n BCR-st imulated B cells was dependent pr imar i ly on phosphorylation of tyrosine 239 of She, and to a lesser extent on phosphorylation of tyrosine 313 (220). I found that the BCR-induced association of Grb2 wi th She was greatly decreased by a mutation i n either the SH2 domain or the PTB domain of She (Fig. 7.3). Thus, She requires both a functional SH2 domain and a functional PTB domain i n order for the BCR to phosphorylate it on sites that are important for Grb2 binding. 7.1.4 Tyrosine phosphorylat ion of She i n response to B C R engagement requires SHIP M y data indicate that tyrosine phosphorylat ion of She at sites that are important for Grb2 binding depends on the PTB domain of She. A major target of 163 Fig. 7.2. Association of SHIP wi th She i n BCR-stimulated cells requires both the SH2 and PTB domains of She. The BOSC 23 packaging cell line was transfected wi th the p M S C V p a c vector (vector) or p M S C V p a c containing the c D N A encoding FLAG-tagged w i l d type She (FLAG-wt She), a She protein i n wh ich the SH2 domain was inactivated (FLAG-Shc R401M), or a She protein i n which the PTB domain was inactivated (FLAG-Shc R175M). The resulting retrovirus particles were used to infect WEHI-231 cells. The WEHI-231 cells were then stimulated wi th anti-IgM Abs for the indicated times. Ce l l lysates were immunoprecipi tated w i t h the M 2 a n t i - F L A G m A b and the precipitated proteins were analyzed by immunoblott ing wi th an anti-SHIP A b (upper panel). The blot was then stripped and reprobed wi th the M 2 a n t i - F L A G m A b (lower panel). Molecular mass standards (in kDa) are indicated to the left. 164 anti-FLAG ippts She protein anti-IgM (min) anti-SHIP 2 0 0 " blot 116-9 9 0 2 0 2 0 2 0 2 anti-FLAG reprobe 66H SHIP — I ^ - F L A G - S h e 165 F i g . 7.3. B C R - i n d u c e d b i n d i n g o f G r b 2 to S h e d e p e n d s o n b o t h t h e S H 2 a n d P T B d o m a i n s o f S h e . T h e B O S C 23 p a c k a g i n g c e l l l i n e w a s t r a n s f e c t e d w i t h t h e p M S C V p a c v e c t o r ( v e c t o r ) o r p M S C V p a c c o n t a i n i n g t h e c D N A e n c o d i n g F L A G - t a g g e d w i l d t y p e S h e ( F L A G - w t She ) , a Sh e p r o t e i n i n w h i c h t h e S H 2 d o m a i n w a s i n a c t i v a t e d ( F L A G - S h c R 4 0 1 M ) , o r a S h e p r o t e i n i n w h i c h t h e P T B d o m a i n w a s i n a c t i v a t e d ( F L A G - S h c R 1 7 5 M ) . T h e r e s u l t i n g r e t r o v i r u s p a r t i c l e s w e r e u s e d to i n f e c t W E H I - 2 3 1 c e l l s . T h e W E H I - 2 3 1 c e l l s w e r e t h e n s t i m u l a t e d w i t h a n t i - I g M A b s f o r t h e i n d i c a t e d t i m e s . C e l l l y s a t e s w e r e i m m u n o p r e c i p i t a t e d w i t h t h e M 2 a n t i - F L A G m A b a n d t h e p r e c i p i t a t e d p r o t e i n s w e r e a n a l y z e d b y i m m u n o b l o t t i n g w i t h a n a n t i - G r b 2 A b ( u p p e r p a n e l ) . T h e b l o t w a s t h e n s t r i p p e d a n d r e p r o b e d w i t h t h e M 2 a n t i - F L A G m A b ( l o w e r p a n e l ) . M o l e c u l a r m a s s s t a n d a r d s ( i n k D a ) a re i n d i c a t e d t o the left . 166 anti-FLAG ippts She protein anti-IgM (min) anti-Grb2 3 1 ~ blot anti-FLAG 66-reprobe 0 2 0 2 0 2 0 2 Grb2 FLAG-Shc 167 the She PTB domain is the SHIP inositol phosphatase (216) which has been shown to associate wi th the Syk tyrosine kinase (218). Thus, it is possible that the binding of SHIP to the PTB domain of She brings Syk i n close proximity to She and that this facilitates She phosphoryla t ion. To address whether SHIP is required for phosphorylation of She by the BCR, we made use of DT40 chicken B cells i n which the genes encoding SHIP have been disrupted (208). Figure 7.4A shows that tyrosine phosphorylation of the three isoforms of chicken She was reduced i n SHIP-deficient DT40 cells compared to w i l d type DT40 cells. Expressing an exogenous w i l d type SHIP i n the SHIP-deficient DT40 B cells was able to restore tyrosine phosphorylation of She by the B C R (Fig. 7.4B). In contrast, expressing a mutant form of SHIP (SHIP Y917F/Y1020F), that does not efficiently associate wi th She i n BCR-stimulated cells (Fig. 7.3C), d id not restore She phosphorylation. Thus, tyrosine phosphorylation of She by the B C R depends not only on the expression of SHIP but also on the binding of SHIP to the PTB domain of She. 7.2 Discussion We have shown for the first time that BCR-induced tyrosine phosphorylation of She strongly depends on the binding of SHIP to the She PTB domain. Thus, i n addi t ion to dephosphorylat ing PtdIns(3,4,5)P3, a second role for SHIP i n B C R signalling is to promote the tyrosine phosphorylation of She. One model that explains the requirement for the S H I P / S h c interaction in promoting the tyrosine phosphorylation of She is that SHIP brings a tyrosine kinase close to She. This kinase is likely to be Syk since BCR-induced phosphorylation of She does not occur i n cells lacking Syk (18,221). Al though SHIP has been shown to b ind to both She and Syk i n anti-Ig-stimulated B cells (129,218), Shc»SHIP»Syk complexes have not been detected (218). It is possible that such ternary complexes may be of low abundance, exist only transiently, or dissociate dur ing the immunoprecipitat ion procedure. 168 Fig. 7.4. BCR- induced tyrosine phosphorylation of She depends on the b ind ing of SHIP to the P T B domain of She. (A) W i l d type (wt) or SHIP-deficient (SHIP-/-) DT40 cells were stimulated wi th the M 4 anti-IgM m A b for the indicated times. Cel l lysates were incubated w i t h the anti-She A b and immunoprecipitates were d iv ided into two equal fractions. One fraction was immunoblotted w i t h the anti-P-Tyr m A b (upper panel) while the other fraction was blotted w i t h the anti-She A b . (B ,C) SHIP-deficient (SHIP-/-) DT40 cells expressing either a transfected w i l d type SHIP protein (wt SHIP) or a transfected SHIP protein i n wh ich the tyrosine residues critical for b ind ing the She PTB had been mutated (Y917F/Y1020F SHIP) were stimulated w i t h the M 4 anti-IgM A b for the indicated times. Ce l l lysates were immunoprecipi ta ted w i t h the anti-She A b and the immunoprecipitates were divided into two equal fractions. The phosphorylation of She was analyzed i n panel B. One fraction was analyzed by immunoblotting wi th the anti-P-Tyr m A b (upper panel) while the other fraction was blotted wi th the anti-She A b (lower panel). The b inding of SHIP to She was analyzed i n C. One fraction was analyzed by immunoblotting wi th the anti-SHIP A b (upper panel) while the other fraction was blotted wi th the anti-She A b (lower panel). Molecular mass standards (in kDa) are indicated to the left. This experiment was performed by Dr. Hidetaka Okada (Department of Obstetrics and Gynecology, Kansai Medical University, Moriguchi , Japan) 169 A anti-She ippts cell line wt DT40 SHIP-/-DT40 anti-IgM (min) o 3 1 0 0 3 1 0 66—r anti-P-Tyr blot 46" anti-She reprobe B cell line SHIP protein anti-She ippts SHIP-/-DT40 anti-P-Tyr blot 46—1 anti-She reprobe wt Y917F/Y1020F SHIP SHIP 0 1 3 10 0 1 3 10 — — — cell line SHIP protein anti-IgM (min) anti-She ippts SHIP-/-DT40 wt Y917F/Y1020F SHIP SHIP anti-SHIP blot anti-She reprobe 220—1 97.4-SHIP 170 The b inding of Grb2»SHIP complexes to She could also protect She from dephosphorylation by tyrosine phosphatases and i n this way contribute to the maintenance of She phosphorylation. Stable binding of SHIP to She requires two distinct interactions, (i) the b inding of phosphotyrosine-containing sequences on SHIP to the She PTB domain and (ii) the bridging of SHIP to She by Grb2. Grb2's SH3 domains b ind to proline-rich regions on SHIP while Grb2's SH2 domain binds to phosphotyrosine-containing sequences on She. In the SHIP-deficient DT40 cells, even if She were tyrosine phosphorylated, there wou ld be no Grb2«SHIP complexes to b ind to the phosphotyrosines on She and protect them from phosphatases. Similarly, the She R175M protein i n which the PTB domain has been inactivated would be unable to recruit and stably bind Grb2«SHIP complexes. Even if She is tyrosine phosphorylated i n the absence of SHIP, Grb2»SOS complexes appear not to be sufficient to protect She from dephosphorylation. One possibility is that Grb2«SOS complexes are much less abundant than Grb2»SHIP complexes i n B cells. Alternatively, Grb2»SOS complexes may not be able to bind stably to phosphorylated She if SHIP is not also present i n the complex. The interactions between SHIP, Syk, She, Grb2, and SOS are very complex and make it difficult to design unequivocal experiments to determine whether the b i n d i n g of S H I P to She promotes the phosphory la t ion of She, protects phosphorylated She from tyrosine phosphatases, or facilitates both of these processes. Nevertheless, we have clearly shown for the first time that BCR-induced tyrosine phosphorylation depends on the binding of SHIP to the PTB domain of She. In addition to the PTB domain, the SH2 domain of She is also important for BCR-induced tyrosine phosphorylation of She. By b inding membrane-associated docking proteins such as Igoc, Ig(3 (204,219), and G a b l (122), the She SH2 domain may bring She close to Syk and SHIP. In BCR-st imulated cells, Syk binds to the phosphorylated B C R I T A M s and SHIP is found i n the membrane fraction (218,222). 171 In summary, we have shown that BCR-induced tyrosine phosphorylation of She depends on both its SH2 domain and its PTB domain. Furthermore, interaction between She and SHIP is also required for phosphorylation of She by the BCR 172 Chapter 8 Discussion 8.0 Summary of thesis The objective of this thesis was to study the role of docking proteins and adapter proteins i n signalling by the BCR. I found that the B C R utilizes several docking and adapter proteins to mediate the formation of signalling complexes and localize signalling proteins to different areas of the cell. In Chapter 3, data was presented that showed that the Crk adapter proteins are used by the B C R to recruit C 3 G to cellular membranes. Crk uses its amino-terminal SH3 domain to b ind proline-rich motifs i n C 3 G and uses its SH2 domain to b ind membrane-associated docking proteins such as c-Cbl and the Cas proteins that are tyrosine phosphorylated after B C R ligation. Since C 3 G is a G N E F for the R a p l and R-Ras GTPases, the recruitment of C 3 G to cellular membranes may be important for allowing C 3 G to activate R a p l and/or R-Ras. In Chapter 4 we showed B C R crosslinking does indeed lead to activation of R a p l . However , rather than being mediated by the b ind ing of Crk proteins to tyrosine-phosphorylated docking proteins, BCR-induced R a p l activation occurs via a pathway that is dependent upon the production of D A G by PLC-y. I also presented some preliminary data showing that C a l D A G - 1 , a C a 2 + / D A G - b i n d i n g Rapl-specific G N E F , may be used by the BCR to activate R a p l . In Chapter 5, I showed that G a b l acts as a membrane b inding site for a number of SH2 domain-containing signall ing proteins invo lved B C R signalling including She, Grb2, SHP-2, and Ptdlns 3-kinase. Recruiting these proteins to the plasma membrane w o u l d provide these signall ing proteins w i t h access to their membrane-associated substrates. Indeed, i n Chapter 6 I showed that expression of G a b l in WEHI-231 cells, which express little if any endogenous G a b l , enhanced the signalling function of Gabl-associated proteins. For example, i n Gabl-expressing 173 WEHI-231 cells, BCR-induced activation of A k t , a downstream target of Ptdlns 3-kinase, was enhanced. In addi t ion, the abil i ty of SHP-2 to be tyrosine phosphorylated and to bind Grb2 i n response to B C R ligation was enhanced in these cells. Furthermore, it appears that association of G a b l wi th cellular membranes is critical for G a b l to enhance the signalling function of its associated proteins. Final ly , i n Chapter 7 I showed that tyrosine phosphorylat ion of the She adapter protein by the B C R requires both the SH2 and PTB domains of She. A s wel l , the She PTB domain-binding protein, SHIP, is also important for phosphorylation of She by the BCR. The overall conclusion from my work is that the B C R uses multiple adapter proteins and docking proteins to assemble s ignal l ing complexes and recruit signall ing molecules to cellular membranes. This facilitates the activation of multiple signalling pathways by the BCR. 8.1 The B C R may use other s ignal l ing modules M y work has focused on the role of a l imited number of protein-protein and protein-lipid interaction modules during B C R signalling. Several other modular domains are present in signalling proteins but it is unclear if signalling proteins w i t h these domains are i n v o l v e d i n B C R s ignal l ing . P a x i l l i n , a 70-kDa phosphoprotein involved i n focal adhesion assembly (223), associates wi th the SH2 domain of C r k proteins when tyrosine phosphorylated (224-226). Pax i l l i n also possesses 4 L I M domains which it uses to associate wi th focal adhesions (226). We were unable to detect the association of Crk wi th paxi l l in i n our studies (data not shown) , but i t is not clear that our lys is p ro toco l w o u l d l iberate cytoskeleton-associated paxill in. If Crk proteins do b ind paxi l l in i n BCR-stimulated cells, then it is possible that Crk may be involved i n the recruitment of signalling molecules to the cytoskeleton and in particular to focal adhesions. 174 W W and P D Z domains are two other protein-protein interaction motifs that may be important i n B C R signalling. W W domains are small domains (38 amino acids) that are characterized by two tryptophan residues separated by 20-22 amino acids (227). Like SH3 domains, W W domains b ind proline-rich sequences wi th in a consensus X P P X Y motif (227). W W domains have also been reported to b ind phosphorylated serine and threonine residues (228). Several signalling proteins possess W W domains including the Yes-associated protein, Y A P (229), as we l l as N E D D 4 which binds to and suppresses the activity of the (3-subunit of the epithelial sodium channel ((3ENaC) (230). P D Z domains bind proteins possessing the consensus sequence S / T X V - C O O " (with other hydrophobic amino acids able to replace the valine) at the extreme carboxy-terminus of the target protein (231-233). The best s tudied P D Z domain-containing protein is the Drosophila photoreceptor-specific protein, InaD (234). InaD possesses 5 P D Z domains which it uses to simultaneously bind several proteins involved i n converting the signal of photon b ind ing into an electrical signal i n Drosophila photoreceptor neurons (234). In addit ion to b inding the C a 2 + channel Trp, InaD binds to PLC-(3 and a Drosophila eye-specific P K C called eye-PKC. By simultaneously binding all these proteins, InaD forms signalling complexes at the cytoplasmic tail of Trp (234) wh ich l ikely allows for more efficient signal transmission. P D Z domain-containing proteins are l ikely to be important i n other signalling systems since P D Z domain b inding sites have been found i n several receptors inc luding the metabotropic glutamate receptor (235) as we l l as several cytoskeletal proteins (236,237). Whi le there is no published evidence that W W and P D Z domain-containing proteins play any role i n B C R signalling, studies looking at the Epstein-Barr virus (EBV) protein, L M P 2 A , suggest that they might. E B V is the causative agent of infectious mononucleosis. Once infected w i t h E B V , individuals remain so for life as a latent infection is maintained wi th in the body's B cells (238). The maintenance 175 of viral latency is dependent upon the L M P 2 A protein. L M P 2 A is a transmembrane protein that possesses 12 membrane-spanning regions and a large amino-terminal intracellular domain. W i t h i n this amino-terminal domain are a number of tyrosine residues including two of which conform to a consensus I T A M motif (239). In EBV-infected cells, L M P 2 A is tyrosine phosphorylated and associates w i t h Src family tyrosine kinases as wel l as Syk. By binding these kinases L M P 2 A is thought to sequester them away from the B C R and thus inhibit B C R signalling. Inhibiting B C R signal l ing is crit ical to prevent the EBV-infected B cells from becoming activated and a l lowing the virus to remain latent. Whi le E B V can b ind and sequester the Src kinases as wel l as Syk, it l ikely sequesters other signalling proteins as wel l . Since 8 tyrosine residues i n L M P 2 A are known to be phosphorylated in EBV-transformed cells (239,240), L M P 2 A may b i n d and sequester other SH2 domain-containing proteins. L M P 2 A also possesses consensus SH3 and W W domain b ind ing sites as we l l as a potential P D Z domain b ind ing site at its carboxy-terminus. Whi le it has not been shown that these motifs i n L M P 2 A bind signall ing proteins, it is tempting to speculate that these S H 3 , W W , and P D Z domain-binding motifs may be used to sequester proteins possessing these motifs away from the B C R thereby preventing these proteins from participating i n BCR signalling. 8.2 Recruitment of proteins to microdomains i n BCR-st imulated cells. Recruitment of s ignal l ing proteins to cellular membranes is a common theme i n signal transduction. However, signalling proteins need to be directed to specific cellular membranes and perhaps even to specific subdomains wi th in a particular membrane. One such subdomain important for signal transduction are l i p i d rafts (241-243). L i p i d rafts are areas of the plasma membrane enriched i n g lycosphingol ip ids , cholesterol, and g lycosylphospht idy l inos i to l (GPI)- l inked proteins. Glycosphingol ip ids generally have long, relatively unsaturated acyl 176 chains, w h i c h a l low them to form a compact, ordered structure w i t h i n the membrane (242,243). In T cells it is known that the T C R moves into l ip id rafts i n response to T C R st imulat ion (244) and that a number of s ignal l ing proteins including the Src kinases Lek and Fyn, Syk, Ras, PLC-y , c-Cbl , She, and Ptdlns 3-kinase are found i n these rafts i n TCR-stimulated cells (244,245). B cells have l ip id rafts (246) and there is some unpublished data showing that the B C R as wel l as PLC-y move into l i p i d rafts i n response to B C R signalling (Bennett Weintraub; personal communication). It is also interesting to note that both G a b l and Gab2 possess sites at their amino-termini that are similar to the myristoylation and palmitoylation sites found i n some of the Src kinases (247). These modifications are important for directing these kinases to l ip id rafts and it w i l l be interesting to determine if the Gab proteins localize to l i p id rafts. Another microdomain that might be important for the co-localization of signalling proteins i n BCR-stimulated cells are focal adhesions. Focal adhesions are clusters of cytoskeletal and signalling proteins that generally formed during integrin stimulation (248). The Crk-associated proteins, paxi l l in and pl30Cas, are known to associate wi th focal adhesions and in B cells, pl30Cas is known to associate wi th Pyk2, a relative of focal adhesion kinase ( p l 2 5 F A K ) (249). Furthermore, B C R ligation leads to phosphorylation of Pyk2 and this requires an intact cytoskeleton as pre-treatment of cells w i th cytochalasin B inhibits BCR-mediated phosphorylation of Pyk2 (249). This data suggests that focal adhesion or focal adhesion-like structures are involved i n B C R signalling and that Crk proteins may be involved in recruiting signalling proteins to these structures. Whi le my work has shown that B C R ligation leads to the formation of signalling complexes i n the membrane-enriched particulate fraction, further work is required to characterize the precise location of these s ignal l ing complexes i n BCR-stimulated cells. This w i l l l ikely involve additional biochemical fractionation 177 techniques as w e l l as immunofluorescence microscopy and perhaps even immunoelectron microscopy 8.3 Co-localization and membrane localization of signall ing proteins as a strategy i n signal transduction Co-localizing signalling proteins and recruiting signalling proteins to specific compartments wi th in the cell are important for efficient signal transmission. For example, the translocation of signalling molecules such as SOS, PLC-y , and Ptdlns 3-kinase to the membrane is cr i t ical for these proteins to interact w i t h membrane-associated targets. While it is obvious that signalling proteins like PLC-y and Ptdlns 3-kinase must translocate to membranes because their substrates are l ipids, Weng et al. have suggested that membrane localization of signalling proteins may have other benefits (250). They argued that recruiting proteins to membranes creates a more favourable two-dimensional reaction environment over the three-dimensional environment found i n the cytoplasm. This w o u l d increase the effective concentration of signall ing components and may favourably alter the or ientat ion of s igna l l ing components a l l o w i n g for more efficient s ignal transmission (250). The efficiency of signal transmission is l ikely even further enhanced by recruiting signal proteins to specific subdomains such as l ip id rafts and focal adhesions. Docking proteins can also act as scaffolding proteins to co-localize the various components of a signall ing pathway. This is illustrated most elegantly i n the docking proteins used by the M A P K kinase cascades. The proteins at the different levels of the M A P K cascades ( M A P K kinase kinase ( M K K K ) , M A P K kinase ( M K K ) and M A P K ) are co-localized to a single docking protein w h i c h al lows the components to more efficiently interact (76). For example, the yeast protein Ste5 acts a scaffolding protein for the Ste20, S t e l l ( M K K K ) , Ste7 ( M K K ) , and Fus3 ( M A P K ) components of the M A P K pathway regulating the yeast mating pathway (251-253). 178 L i k e w i s e , i n m a m m a l i a n c e l l s , J IP1 c o - l o c a l i z e s t h e c o m p o n e n t s o f t he J n k p a t h w a y , H P K , M L K 3 ( M K K K ) , M K K 7 ( M K K ) , a n d J n k ( M A P K ) (254). A n o t h e r a d v a n t a g e o f c o - l o c a l i z i n g s i g n a l l i n g e n z y m e s to the s a m e d o c k i n g p r o t e i n i s t h a t i t a l l o w s f o r c r o s s t a l k b e t w e e n s i g n a l l i n g p a t h w a y s . G a b l a p p e a r s to d o t h i s b y c o - l o c a l i z i n g P t d l n s 3 - k i n a s e , S H P - 2 , a n d S H I P to r e g u l a t e t he p r o d u c t i o n o f i n o s i t o l p h o s p h o l i p i d s e c o n d m e s s e n g e r s . R e c r u i t i n g P t d l n s 3 - k i n a s e t o t h e m e m b r a n e - a s s o c i a t e d G a b l w o u l d a l l o w i t t o p h o s p h o r y l a t e i t s i n o s i t o l l i p i d s u b s t r a t e s . T h e s i m u l t a n e o u s r e c r u i t m e n t o f S H P - 2 to G a b l m a y a l l o w S H P - 2 t o d e p h o s p h o r y l a t e G a b l a n d d i s r u p t t he b i n d i n g o f P t d l n s 3 - k i n a s e to G a b l . T h i s m a y b e a w a y i n w h i c h the c e l l l i m i t s t h e m a g n i t u d e a n d / o r d u r a t i o n o f P t d l n s 3 - k i n a s e s i g n a l l i n g . F u r t h e r m o r e , r e c r u i t m e n t o f S H I P to G a b l v i a S h c # G r b 2 a n d S H P - 2 » G r b 2 w o u l d p r o v i d e a n o t h e r m e a n s o f d o w n r e g u l a t i n g the P t d l n s 3 - k i n a s e p a t h w a y b y c o n v e r t i n g P t d I n s ( 3 , 4 , 5 ) P 3 t o P t d I n s ( 3 , 4 ) P 2 . T h u s , G a b l m a y b e a f o c a l p o i n t f o r s i g n a l l i n g m o l e c u l e s t ha t r e g u l a t e i n o s i t o l p h o s p h o l i p i d s . T h i s c o u l d b e t e s t e d u s i n g t h e m u t a n t G a b l p r o t e i n s w h i c h l a c k the b i n d i n g s i tes f o r S H P - 2 a n d P t d l n s 3 - k i n a s e a n d the D T 4 0 c e l l m u t a n t s l a c k i n g S H P - 2 o r S H I P . I n c o n c l u s i o n , d o c k i n g a n d a d a p t e r p r o t e i n s p l a y a n i m p o r t a n t r o l e i n B C R s i g n a l l i n g . T h e u s e o f d o c k i n g p r o t e i n s b y the B C R a l l o w s the B C R to u t i l i z e S H 2 d o m a i n - c o n t a i n i n g s i g n a l l i n g p r o t e i n s t h a t i t w o u l d n o t n o r m a l l y b e a b l e to r e c r u i t g i v e n t h a t t h e I T A M m o t i f s o f t h e B C R a r e l i m i t e d i n t h e t y p e s o f S H 2 d o m a i n - c o n t a i n i n g p r o t e i n s t h e y c a n b i n d . A d a p t e r p r o t e i n s a l l o w the B C R to u s e s i g n a l l i n g p r o t e i n s t h a t d o n o t h a v e t h e i r o w n S H 2 d o m a i n s a n d a l l o w s t h e s e s i g n a l l i n g p r o t e i n s t o b e c o u p l e d t o m e m b r a n e d o c k i n g p r o t e i n s . F u r t h e r m o r e , s p e c i f i c d o c k i n g p r o t e i n s m a y r e c r u i t s i g n a l l i n g p r o t e i n s t o d i f f e r e n t s u b c e l l u l a r l o c a t i o n s s u c h as l i p i d raf ts o r f o c a l a d h e s i o n - l i k e s t r u c t u r e s . T h u s , d o c k i n g p r o t e i n s a n d a d a p t e r p r o t e i n s a l l o w t h e B C R t o n o t o n l y u t i l i z e a g r e a t e r n u m b e r o f s i g n a l l i n g m o l e c u l e s , b u t a l s o i m p r o v e s the e f f i c i e n c y o f B C R s i g n a l l i n g . 179 References 1. Janeway, C. A . and P Travers. 1997. Immunobiology: The Immune System i n Health and Disease. Garland Publishing Inc., N e w York. 2. Ochs, H . D. and C. I. Smith. 1996. X-l inked agammaglobulinemia. A clinical and molecular analysis. Medicine 75:287-299. 3. DeFranco, A . L . , E. S. Raveche, and W. E. Paul . 1985. Separate control of B lymphocyte early activation and proliferation i n response to anti-IgM antibodies. /. Immunol. 135:87-94. 4. Klaus, G . G. , M . S. Choi , E. W. Lam, C. Johnson-Leger, and J. Cliff. 1997. CD40: a pivotal receptor i n the determination of l ife/death decisions i n B lymphocytes. Int. Rev. Immun. 15:5-31. 5. Fujimoto, M . , J. C. Poe, M . Inaoki, and T. F. Tedder. 1998. CD19 regulates B lymphocyte responses to transmembrane signals. Sem. Immunol. 10:267-277. 6. Takatsu, K . 1997. Cytokines involved i n B-cell differentiation and their sites of action. Proc. Soc. Exp. Biol. Med. 215:121-133. 7. O'Rourke, L . , R. Tooze, and D. T. Fearon. 1997. Co-receptors of B lymphocytes. Curr. Opin. Immunol. 9:324-329. 8. D'Ambrosio , D . , K . L . Hippen, S. A . Minskoff, I. Mel lman, G . S. Pani, and J. C. Cambier. 1995. Recruitment and activation of PTP1C i n negative regulation of antigen receptor signaling by Fc gamma RIIB1. Science 268:293-297. 180 9. Doody, G . M . , L . B. Justement, C. C. Delibrias, R. J. Matthews, J. L i n , M . L . Thomas, and D . T. Fearon. 1995. A role in B cell activation for CD22 and the protein tyrosine phosphatase SHP. Science 269:242-244. 10. Cyster, J. G . and C. C. Goodnow. 1995. Protein tyrosine phosphatase 1C negatively regulates antigen receptor signaling i n B lymphocytes and determines the thresholds for negative selection. Immunity 2:1-20. 11. Pani, G . , M . Koz lowsk i , J. C. Cambier, G . B. M i l l s , and K . A . Siminovitch. 1995. Identif icat ion of the tyrosine phosphatase P T P 1 C as a B cel l antigen receptor-associated protein involved i n the regulation of B cell signaling. / . Exp. Med. 181:2077-2084. 12. Gold , M . R. and L . Matsuuchi. 1995. Signal transduction by the antigen receptors of B and T lymphocytes. Int. Rev. Cytol. 157:181-276. 12b. Flaswinkel, H . and M . Reth. 1994. Duyal role of the tyrosine activation motif of the Ig-a protein during signal transduction via the B cell antigen receptor.EMBO /. 13:13-19. 13. Isakov, N . 1998. I T A M s : immunoregulatory scaffolds that l ink immunoreceptors to their intracellular signaling pathways. Receptors & Channels 5:243-253. 14. Gold , M . R., D. A . Law, and A . L. DeFranco. 1990. Stimulation of protein tyrosine phosphorylation by the B-lymphocyte antigen receptor. Nature 345:810-813. 181 15. Burkhard t , A . L . , M . Brunswick , J. B. Bolen, and J. J. M o n d . 1991. A n t i - i m m u n o g l o b u l i n s t imulat ion of B lymphocytes activates src-related protein-tyrosine kinases. Proc. Natl. Acad. Sci. USA 88:7410-7414. 16. Hutchcroft, J. E. , M . L . Harrison, and R. L . Geahlen. 1992. Association of the 72 kDa protein-tyrosine kinase PTK72 w i t h the B cell antigen receptor. / . Biol. Chem. 267:8613-8619. 17. de Weers, M . , G . S. Brouns, S. Hinshelwood, C. Kinnon , R. K . B. Schuurman, R. W. Hendriks, and J. Borst. 1994. B-cell antigen receptor stimulation activates the h u m a n Bruton ' s tyros ine k inase , w h i c h is def ic ient i n X - l i n k e d agammaglobulinemia. /. Biol. Chem. 269:23857-23860. 18. Richards, J. D. , M . R. Go ld , S. L . Hourihane, A . L . DeFranco, and L . Matsuuchi. 1996. Reconstitution of B cell antigen receptor-induced signaling events i n a nonlymphoid cell line by expressing the Syk protein-tyrosine kinase. / . Biol. Chem. 271:6458-6466. 19. Law, D. A . , M . R. Gold , and A . L . DeFranco. 1992. Examination of B lymphoid cell lines for membrane immunoglobulin-stimulated tyrosine phosphorylation and src-family tyrosine kinase m R N A expression. Moi. Immunol. 29:917-926. 20. Clark, M . R., S. A . Johnson, and J. C. Cambier. 1994. Analysis of Ig-a-tyrosine kinase interaction reveals two levels of b ind ing specificity and tyrosine phosphorylated Ig-oc stimulation of Fyn activity. EMBO J. 13:1911-1919. 182 21. Shiue, L . , M . J. Zol le r , and J. S. Brugge. 1995. Syk is activated by phosphotyrosine-containing peptides representing the tyrosine-based activation motifs of the h igh affinity receptor for IgE. /. Biol. Chem. 270:10498-10502. 22. Kurosaki, T., S. A . Johnson, L . Pao, K . Sada, and H . Yamamura. 1995. Role of the Syk autophosphorylation site and SH2 domains i n B cell antigen receptor signaling. /. Exp. Med. 182:1815-1823. 22a Chan A . C , M . Dalton, R. Johnson, G . H . Kong , T. Wang, R. Thoma, and T. Kurosaki . 1995. Act iva t ion of ZAP-70 kinase activity by phosphorylat ion of tyrosine 493 is required for lymphocyte antigen receptor function EMBO }. 14:2499-2508. 23. DeFranco, A . L . 1997. The complexity of signaling pathways activated by the BCR. Curr. Opin. Immunol. 9:296-308. 24. Kurosaki , T. 1999. Genetic Analysis of B Cel l Ant igen Receptor Signaling. Ann. Rev. Immunol. 17:555-592. 25. G o l d , M . R. 1999. Intermediary Signaling Effectors Coup l ing the B C R to the Nucleus. Curr. Top. Microbiol. Immunol. 245:77-134. 26. Toker, A . and L . C. Cantley. 1997. Signall ing through the l i p i d products of phosphoinositide-3-OH kinase. Nature 387:673-676. 27. Vanhaesebroeck, B. , S. J. Leevers, G . Panayotou, and M . D . Waterfield. 1997. Phosphoinositide 3-kinases: a conserved family of signal transducers. Trends Biochem. Sci. 22:267-272. 183 28. Go ld , M . R. and R. A . Aebersold. 1994. Both phosphatidylinositol 3-kinase and phosphat idyl inosi to l 4-kinase products are increased by antigen receptor signaling i n B lymphocytes. /. Immunol. 152:42-50. 29. Toker, A . , M . Meyer, K. K. Reddy, J. R. Falck, R. Aneja,, S. Aneja, A . Parra, D. J. Burns, L . M . Ballas, and L . C. Cantley. 1994. Activation of protein kinase C family members by the novel polyphosphoinositides PtdIns-3,4-P2 and PtdIns-3,4,5-P3. /. Biol. Chem. 269:32358-32367. 30. Nakanishi, H . , K . A . Brewer, and J. H . Exton. 1993. Activation of the £ isozyme of protein kinase C by phosphat idyl inosi tol 3,4,5-trisphosphate. / . Biol. Chem. 268:13-16. 31. Mischak, H . , W. Kolch, J. Goodnight, W. F. Davidson, TJ. Rapp, S. Rose-John, and J. F. Mushinski . 1991. Expression of protein kinase C genes i n hemopoietic cells is cell-type- and B cell-differentiation stage specific. /. Immunol. 147:3981-3987. 32. Sidorenko, S. P., C. -L . Law, S. J. Klaus, K . A . Chandran, M . Takata, T. Kurosaki, and E. A . Clark. 1996. Protein kinase C | i (PKCp) associates wi th the B cell antigen receptor complex and regulates lymphocyte signaling. Immunity 5:353-363. 33. Tordai, A . , R. F. Frankl in, H . Patel, A . M . Gardner, G . L . Johnson, and E. W. G e l f a n d . 1994. C r o s s - l i n k i n g of surface I g M s t imu la t e s the R a s / R a f - l / M E K / M A P K cascade i n human B lymphocytes . / . Biol. Chem. 269:7538-7543. 184 34. L i , H . L . , W. W . Davis, E. L . Whiteman, M . J. Birnbaum, and E. Pure. 1999. The tyrosine kinases Syk and L y n exert opposing effects on the activation of protein kinase A k t / P K B i n B lymphocytes. Proc. Natl. Acad. Sci. 96:6890-6895. 35. Aman , M . J., T. D. Lamkin, H . Okada, T. Kurosaki, and K . S. Ravichandran. 1998. The inositol phosphatase SHIP inhibits A k t / P K B activation i n B cells. /. Biol. Chem. 273:33922-33928. 36. Gold , M . R., M . P. Scheid, L . Santos, M . Dang-Lawson, R. A . Roth, L . Matsuuchi, V . Duronio, and D. L . Krebs. 1999. The B cell antigen receptor activates the A k t (Protein Kinase B ) / g l y c o g e n synthase kinase-3 s igna l l ing pathway v i a phosphatidylinositol 3-kinase. /. Immunol. 166:1894-1905. 37. Astoul , A . , S. Watton, and D. Cantrell. 1999. The dynamics of Protein Kinase B regulation during B cell antigen receptor engagement. /. Cell. Biol. 145:1511-1520. 38. Franke, T. F., D. R. Kaplan, L . C. Cantley, and A . Toker. 1997. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science 275:665-668. 39. K l i p p e l , A . , W . M . Kavanaugh, D . Pot, and L . T. Wil l iams. 1997. A specific product of phosphatidylinositol 3-kinase directly activates the protein kinase A k t through its pleckstrin homology domain. Moi. Cell. Biol. 17:338-344. 40. Freeh, M . , M . Andjelkovic , E. Ingley, K . K . Reddy, J. R. Falck, and B. A . H e m m i n g s . 1997. H i g h affinity b i n d i n g of i n o s i t o l phosphates and phosphoinositides to the pleckstrin homology domain of R A C / P r o t e i n kinase B and their influence on the kinase activity. /. Biol. Chem. 272:8474-8481. 185 41. Alessi, D. R., S. R. James, C. P. Dowries, A . B. Holmes, P. R. J. Gaffney, C. B. Reese, and P. Cohen. 1997. Characterization of a 3-phosphoinositide-dependent protein kinase w h i c h phosphorylates and activates protein kinase B a . Curr. Biol. 7:261-269. 41a. Alessi , D. R., M . Andjelkovic, B. Caudwell , P. Cron, N . Morrice, P. Cohen and B. A . Hemmings. 1996. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 15:6541-6551. 42. Balendran, A . , A . Casamayor, M . Deak, A . Paterson, P. Gaffney, R. Currie, and D. R. Alessi. 1999. P D K 1 acquires P D K 2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2. Curr. Biol. 9:393-404. 43. Delcommenne, M . , C. Tan, V . Gray, L . Rue, J. Woodgett, and S. Dedhar. 1998. Phosphoinos i t ide-3-OH kinase-dependent regulation of glycogen synthase kinase-3 and protein kinase B / A k t by the integrin-linked kinase. Proc. Natl. Acad. Sci. USA 95:11211-11216. 44. Andjelkovic, M . , D. R. Alessi , R. Meier, A . Fernandez, N . J. C. Lamb, M . Freeh, P. Cron , J. M . Lucocq, and B. A . Hemmings. 1997. Role of translocation i n the activation and function of protein kinase B. /. Biol. Chem. 272:31515-31524. 45. Ziegler, W . H . , D . B. Parekh, J. A . Le Good, R. D. Whelan, J. J. Ke l ly , B. A . Hemmings, and P. J. Parker. 1999. Rapamycin-sensitive phosphorylation of P K C on a carboxy-terminal site by an atypical P K C complex. Curr. Biol. 9:522-529. 186 46. del Peso, L . , M . Gonzales-Garcia, C. Page, R. Herrera, and G . Nunez. 1997. Interleukin-3-induced phosphorylation of B A D through the protein kinase Akt . Science 278:687-689. 47. Datta, S. R., H . Dudek, X . Tao, S. Masters, H . Fu, Y. Gotoh, and M . E. Greenberg. 1997. A k t phosphorylation of B A D couples survival signals to the cell-intrinsic death machinery. Cell 91:231-241. 48. Cardone, M . H . , N . Roy, H . R. Stennicke, G . S. Salvesen, T. F. S. Franke, S. Frisch, and J. C . Reed. 1998. Regula t ion of cell death protease caspase-9 by phosphorylation. Science 282:1318-1321. 49. Brunet, A . , A . Bonni, M . J. Zigmond, M . Z . L i n , P. Juo, L . S. H u , M . J. Anderson, J. Blenis, and M . E. Greenberg. 1999. A k t promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857-868. 50. Kops, G. J., N . D. de Ruiter, A . M . de Vries-Smits, D . R. Powel l , J. L . Bos, and BurgeringBM.. 1999. Direct control of the Forkhead transcription factor A F X by protein kinase B. Nature 398:630-634. 51. Cross, D . A . E. , D. R. Alessi , P. Cohen, M . Andjelkovic, and B. A . Hemmings. 1995. Inhibition of glycogen synthase kinase-3 by insulin mediated protein kinase B. Nature 378:785-789. 52. Kane, L . P., V . Smith Shapiro, D. Stokoe, and A . Weiss. 1999. Induction of N F - K B by the A k t / P K B kinase. Curr. Biol. 9:601-604. 187 53. Ozes O. N. , L . D. Mayo, J. A . Gustin, S. R. Pfeffer, L . M . Pfeffer and D. B. Dormer. 1999. N F - K B activation by tumour necrosis factor requires the A k t serine-threonine kinase. Nature 401:82-85. 54. Romashkova J. A . and S. S. Makarov . 1999. N F - K B is a target of A K T in anti-apoptotic P D G F signalling. Nature 401:86-89. 54a. Scott, P. H . , G . J. Brunn, A . D . Kohn , R. A . Roth, J. C. Lawrence Jr. 1998. Evidence of insu l in-s t imula ted phosphory la t ion and act ivat ion of the mammal ian target of rapamycin mediated by a protein kinase B signaling pathway. Proc. Natl. Acad. Sci. 95(13):7772-7777 55. Zha , J., H . Harada, E. Yang, J. Jockel, and S. J. Korsmeyer. 1996. Serine phosphorylation of death agonist B A D in response to survival factor results i n binding to 14-3-3 not B C L - X . Cell 87:619-628. 56. Guo , S., G . Rena, S. C ichy , X . H e , P. Cohen, and T. Unterman. 1999. Phosphorylation of serine 256 by protein kinase B disrupts transactivation by F K H R and mediates effects of insu l in on insulin-l ike growth factor-binding protein-1 promoter activity through a conserved insul in response sequence. / . Biol. Chem. 274:17184-17192. 57. Tang, E. D. , G . Nunez, F. G . Barr, and K. L . Guan. 1999. Negative regulation of the forkhead transcription factor F K H R by Akt . /. Biol. Chem. 274:16741-16746. 58. Biggs, W . H . , J. Meisenhelder, T. Hunter, W . K . Cavenee, and K . C. Arden. 1999. Protein kinase B/Akt-media ted phosphorylation promotes nuclear exclusion of the winged helix transcription factor F K H R 1 . Proc. Natl. Acad. Sci. 96:7421-7426. 188 59. Downward , J. 1998. Mechanisms and consequences of activation of protein kinase B / A k t . Curr. Opin. Cell Biol. 10:262-267. 60. Beals, C. R., C. M . Sheridan, C. W. Turck, P. Gardner, and G . R. Crabtree. 1997. Nuclear export of N F - A T c enhanced by glycogen synthase kinase-3. Science 275:1930-1933. 61. K lemm, J. D. , C . R. Beals, and G . R. Crabtree. 1997. Rapid targeting of nuclear proteins to the cytoplasm. Curr. Biol. 7:638-644. 62. Rubinfeld, B., I. Albert, E. Porfiri , C. F io l , S. Munemitsu, and P. Polakis. 1996. Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. Science 272:1023-1026. 63. Bijsterbosch, M . K . , C. J. Meade, G . A . Turner, and G . G . B. Klaus. 1985. B lymphocyte receptors and polyphosphoinositide degradation. Cell 41:999-1006. 64. Fahey, K . A . and A . L . DeFranco. 1987. Cross-l inking membrane I g M induces production of inositol trisphosphate and inositol tetrakisphosphate i n WEHI-231 B lymphoma cells. /. Immunol. 138:3935-3942. 65. Carter, R. H . , D . J. Park, S. G . Rhee, and D . T. Fearon. 1991. Tyrosine phosphorylat ion of phospholipase C induced by membrane immunoglobul in crosslinking i n B lymphocytes. Proc. Natl. Acad. Sci. USA 88:2745-2749. 66. Hempe l , W . M . , R. C . Schatzman, and A . L . DeFranco. 1992. Tyrosine phosphorylation of phospholipase C-y2 upon crosslinking of membrane Ig on murine B lymphocytes. /. Immunol. 148:3021-3027. 189 67. Berridge, M . J. 1993. Inositol trisphosphate and calcium signaling. Nature 361:315-325. 68. Rao, A . , C. Luo, and P. G. Hogan. 1997. Transcription factors of the N F A T family: regulation and function. Ann. Rev. Immunol. 15:707-747. 69. Healy, J. I., R. E. Dolmetsch, L . A . Timmerman, J. G . Cyster, M . L . Thomas, G . R. Crabtree, R. S. Lewis , and C. C. Goodnow. 1997. Different nuclear signals are activated by the B cell receptor dur ing positive versus negative signaling. Immunity 6:419-428. 70. Boguski, M . S. and F. McCormick. 1993. Proteins regulating Ras and its relatives. Nature 366:643-654. 71. Harwood , A . E. and J. C . Cambier. 1993. B cell antigen receptor cross-linking triggers rapid P K C independent activation of p 2 1 r a s . /. Immunol. 151:4513-4522. 72. Lazarus, A . H . , K . Kawauch i , M . J. Rapoport, and T. J. Delovi tch. 1993. Ant igen- induced B lymphocyte activation involves the p 2 1 r a s and r a sGAP signaling pathway. /. Exp. Med. 178:1765-1769. 73. Cook, S. J. and F. McCormick . 1993. Inhibition by c A M P of Ras-dependent activation of Raf. Science 262:1069-1072. 74. Rodriguez-Viciana, P., P. H . Warne, R. Dhand, B. Vanhaesebroeck, I. Gout, M . J. Fry, M . D . Waterfield, and J. Downward. 1994. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370:527-532. 190 75. Wolthius, R. M . F, B. Bauer, L . J. van't Veer, A . M . M . de Vries-Smits, R. H . Cool , M . Spaargaren, A . Wittinghofer, B. M . T. Burgering, and J. L . Bos. 1996. RalGDS-l ike factor (Rlf) is a novel Ras and RaplA-associating protein. Oncogene 13:353-362. 76. Schaeffer, H . J. and M . J. Weber. 1999. Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Moi. Cell. Biol. 19:2435-2444. 77. Sutherland, C. L . , A . W . Heath, S. L . Pelech, P. R. Young, and M . R. Gold . 1996. Differential activation of the E R K , J N K , and p38 mitogen-activated protein kinases by CD40 and the B cell antigen receptor. /. Immunol. 157:3381-3390. 78. Gold , M . R., J. S. Sanghera, J. Stewart, and S. L . Pelech. 1992. Selective activation of p42 M A P kinase i n mur ine B l y m p h o m a cel l l ines by membrane immunoglobul in crosslinking. Evidence for protein kinase C-independent and -dependent mechanisms of activation. Biochem. J. 287:269-276. 79. Su, B. and M . Kar in . 1996. Mitogen-activated protein kinase cascades and regulation of gene expression. Curr. Opin. Immunol. 8:402-411. 80. Bijsterbosch, M . K . and G . G . K laus . 1985. C r o s s l i n k i n g of surface immunoglobu l in and Fc receptors on B lymphocytes inhbits st imulation of inosi tol phospho l ip id b reakdown v ia the antigen receptors. / . Exp. Med. 162:1825-1836. 81. Janknecht, R. and T. Hunter. 1997. Convergence of M A P kinase pathways on the ternary complex factor Sap-la. EMBO J. 16:1620-1627. 191 82. McMahon , S. B. and J. G . Monroe. 1995. Activation of the p 2 1 r a s pathway couples antigen receptor stimulation to induction of the primary response gene egr-1 i n B lymphocytes. /. Exp. Med. 181:417-422. 83. Yamanashi, Y . , M . Okada, T. Semba, T. Yamori , H . Umemori , S. Tsunasawa, K . Toyoshima, D . Kitamura, T. Watanabe, and T. Yamamoto. 1993. Identification of HS1 protein as a major substrate of protein-tyrosine kinase(s) upon B-cell antigen receptor-mediated signaling. Proc. Natl. Acad. Sci. USA 90:3631-3635. 84. Gulbins, E., C. Langlet, G . Baier, N . Bonnefoy-Berard, E. Herbert, A . Airman, and K . M . Coggeshall . 1994. Tyrosine phosphorylat ion and activation of Vav G T P / G D P exchange activity i n antigen receptor-triggered B cells. /. Immunol. 152:2123-2129. 85. Taniuchi, I., D . Kitamura, Y. Maekawa, T. Fukuda, H . K i sh i , and T. Watanabe. 1995. Antigen-receptor induced clonal expansion and deletion of lymphocytes are impaired i n mice lacking HS1 protein, a substrate of antigen-receptor-coupled tyrosine kinases EMBO }. 14:3664-3678. 86. Yamanashi, Y . , T. Fukuda, H . Nish izumi , T. Inazu, K . -I. Higashi , D. Kitamura, T. Ishida, H . Yamamura, T. Watanabe, and T. Yamamoto. 1997. Role of tyrosine phosphorylation of HS1 i n B cell antigen receptor-mediated apoptosis. /. Exp. Med. 185:1387-1392. 87. Bachmann, M . F., L . Nitschke, C. Krawczyk, K . Tedford, P. S. Ohashi, and K . D. Fischer. 1999. The guanine-nucleotide exchange factor Vav is a crucial regulator of B cell receptor activation and B cell responses to nonrepetitive antigens. / . Immunol. 163:137-142. 192 88. Tarakhovsky, A . , M . Turner, S. Schaal, P. J. Mee, L . P. Duddy, K . Rajewsky, and V. L. J. Tybulewicz. 1995. Defective antigen receptor-mediated proliferation of B and T cells i n the absence of Vav. Nature 374:467-470. 89. Zhang, R., F. W. A l t , L . Davidson, S. H . Ork in , and W. Swat. 1995. Defective signalling through the T- and B-cell antigen receptors i n lympho id cells lacking the vav proto-oncogene product. Nature 374:470-473. 90. Crespo, P., K . E. Schuebel, A . A . Ostrom, J. S. Gutkind , and X. R. Bustelo. 1997. Phosphorylation-dependent activation of rac-1 G D P / G T P exchange by the vav proto-oncogene product. Nature 385:169-172. 91. Tolias, K. F., L . C. Cantley, and C. L . Carpenter. 1995. Rho family GTPases bind to phosphoinositide kinases. / . Biol. Chem. 27'0:17'656-17659. 92. Waksman, G. , S. E. Shoelson, N . Pant, D. Cowburn, and J. Kur iyan. 1993. Binding of a h igh affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms. Cell 72:779-790. 93. Pascal, S. M . , A . TJ. Singer, G . Gish, T. Yamazaki, S. E. Shoelson, L . E. Kay, and J. D. Forman-Kay. 1994. Nuclear magnetic resonance structure of an SH2 domain of phospholipase C-gamma 1 complexed wi th a h igh affinity b inding peptide. Cell 77:461-472. 94. Songyang, Z . , S. E. Shoelson, M . Chaudhuri , G . Gish, T. Pawson, W . G . Haser, F. K i n g , T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G . Neel , R. B. Birge, J. E. Fajardo, M . M . Chou, H . Hanafusa, B. Schaffhausen, and L . C. Cantley. 1993. SH2 domains recognize specific phosphopeptide sequences. Cell 72:767-778. 193 95. Songyang, Z . , S. E. Shoelson, J. McGlade, P. Olivier, T. Pawson, X . R. Bustelo, M . Barbacid, H . Sabe, H . Hanafusa, T. Y i , R. Ren, D. Baltimore, S. Ratnofsky, R. A . Feldman, and L . C. Cantley. 1994. Specific motifs recognized by the SH2 domains of Csk , 3BP2, fps/fes, G R B - 2 , H C P , S H C , Syk, and Vav . Moi. Cell. Biol. 14:2777-2785. 96. Songyang, Z . and L . C. Cantley. 1995. Recognition and specificity i n protein tyrosine kinase-mediated signalling. Trends Biochem. Sci. 20:470-475. 97. Kavanaugh, W. M . and L . T. Wil l iams. 1994. A n alternative to SH2 domains for binding tyrosine-phosphorylated proteins. Science 266:1862-1865. 98. Blaikie, P., D. Immanuel, J. W u , N . L i , V . Yajnik, and B. Margolis. 1994. A region i n She distinct from the SH2 domain can b ind tyrosine-phosphorylated growth factor receptors. /. Biol. Chem. 269:32031-32034. 99. Gustafson, T. A . , W. He , A . Craparo, C. D. Schaub, and T. J. O ' N e i l l . 1995. Phosphotyrosine-dependent interaction of S H C and insul in receptor substrate 1 wi th the N P E Y motif of the insul in receptor via a novel non-SH2 domain. Moi. Cell. Biol. 15:2500-2508. 100. L i , S. C , Z . Songyang, S. J. Vincent, C. Zwahlen, S. Wiley, L . Cantley, L . E. Kay, and T. Pawson . 1997. High-af f in i ty b i n d i n g of the Drosoph i l a N u m b phosphotyrosine-binding domain to peptides containing a Gly-Pro-(p)Tyr motif. Proc. Natl. Acad. Sci. 94:7204-7209. 194 101. Trub, T., W . E. Cho i , G . Wolf , E. Ottinger, Y . Chen, and M . Weiss. 1995. Specificity of the PTB domain of She for beta turn-forming pentapeptide motifs amino-terminal to phosphotyrosine. / . Biol. Chem. 270:18205-18208. 102. van der Geer, P., S. Wiley, V . K . La i , R. Stephens, M . F. White, D. Kaplan, and T. Pawson. 1996. Identification of residues that control specific b inding of the She phosphotyrosine-binding domain to phosphotyrosine sites. Proc. Natl. Acad. Sci.USA 93:963-968. 103. Alexandropoulos, K . , G . Cheng, and D. Baltimore. 1995. Proline-rich sequences that b ind to Src homology 3 domains wi th ind iv idua l specificities. Proc. Natl. Acad. Sci.USA 92:3110-3114. 104. Pawson, T. 1995. Protein modules and signalling networks. Nature 373:573-580. 105. L i m , W . A . , F. M . Richards, and R. O. Fox. 1994. Structural determinants of peptide-binding orientation and of sequence specificity i n SH3 domains Nature 372:375-379. 106. Feng, S., J. K . Chen, H . Y u , }. A . Simon, and S. L . Schreiber. 1994. Two binding orientations for peptides to the Src SH3 domain: development of a general model for SH3-ligand interactions. Science 266:1241-1247. 107. Chen D. , S. B. Waters, K . H . Holt , and J. E. Pessin. 1996. SOS phosphorylation and disassociation of the Grb2-SOS complex by the E R K and J N K signaling pathways. /. Biol. Chem. 271:6328-6332. 195 108. Anafi , M . , M . K. Rosen, G . D. Gish, L . E. Kay, and T. Pawson. 1996. A potential SH3 domain-binding site in the Crk SH2 domain. /. Biol. Chem. 271:21365-21374. 109. Lemmon, M . A . , M . Falasca, K . M . Ferguson, and J. Schlessinger. 1997. Regulatory recruitment of s ignal l ing molecules to the cell membrane by pleckstrin-homology domains. Trends Cell Biol. 7:237-242. 110. Fruman, D. A . , L . E. Rameh, and L . C. Cantley. 1999. Phosphoinositide binding domains: embracing 3-phosphate. Cell 97:817-820. 111. Rameh, L . E. , Arvidsson A k . , K . L . Carraway, A . D. Couvi l lon , A . Crompton, B. VanRenterghem, M . P. Czech, S. J. Burakoff, D. S. Wang, C. S. Chen, and L . C. Cantley. 1997. A comparative analysis of the phosphoinositide binding specificity of pleckstrin homology domains. /. Biol. Chem. 272:22059-22066. 112. White, M . 1997. The IRS-signalling system during insul in and cytokine action. Bioessays 19:491-500. 113. Rudd , C . E. 1999. Adaptors and molecular scaffolds in immune cell signaling. Cell 96:5-8. 114. Lupher Jr, M . L , N . Rao, M . J. Eck, and H . Band. 1999. The C b l protooncoprotein: a negative regulator of immune receptor signal transduction. Immunol. Today 20:375-382. 115. Feller, S. M . , G. Posern, J. Voss, C. Kardinal , D. Sakkab, and B. S. Knudsen. 1998. Physiological signals and oncogenesis mediated through C r k family adapter proteins. /. Cell. Phys. 177:535-552. 196 116. Tuveson, D. A v R. H . Carter, S. P. Soltoff, and D. T. Fearon. 1993. CD19 of B cells as a surrogate kinase insert region to b ind phosphatidylinositol 3-kinase. Science 260:986-989. 117. Yamanish i , Y . , Y . F u k u i , B. Wongsasant, Y . Kinosh i ta , Y . Ichimori , K . Toyoshima, and T. Yamamoto. 1992. Act iva t ion of Src-like protein-tyrosine kinase L y n and its association wi th phosphatidylinositol 3-kinase upon B-cell antigen receptor-mediated signaling. Proc. Natl. Acad. Sci. USA 89:1118-1122. 118. Saxton, T. M . , I. van Oostveen, D. Bowtell, R. Aebersold, and M . R. Gold . 1994. B cell antigen receptor cross-linking induces phosphorylation of the Ras activators S H C and m S O S l as we l l as assembly of complexes containing S H C , GRB-2, m S O S l , and a 145-kDa tyrosine-phosphorylated protein. /. Immunol. 153:623-636. o 119. Lankester, A . C , G . M . van Schijndel, P. M . Rood, A . J. Verhoven, and R. A . van Lier. 1994. B cell antigen receptor cross-linking induces tyrosine phosphorylation and membrane translocation of a multimeric She complex that is augmented by CD19 co-ligation. Eur. f. Immunol. 24:2818-2825. 120. Ingham, R. J., D. L . Krebs, S. M . Barbazuk, C. W. Turck, H . Hi ra i , M . Matsuda, and M . R. Gold . 1996. B cell antigen receptor signaling induces the formation of complexes containing the Crk adapter proteins. /. Biol. Chem. 271:32306-32314. 121. M c L e o d , S. J., R. J. Ingham, J. L . Bos, T. Kurosaki , and M . R. Go ld . 1998. Act iva t ion of the R a p l GTPase by the B cell antigen receptor. /. Biol. Chem. 273:29218-29233. 197 122. Ingham, R. J., M . Holgado-Madruga, C. Siu, A . J. Wong, and M . R. Gold . 1998. The G a b l protein is a docking site for multiple proteins involved i n signaling by the B cell antigen receptor. /. Biol. Chem. 273:30630-30637. 123. Ingham, R. J., H . Okada, M . Dang-Lawson, J. Dinglasan, P. van der Geer, and M . R. G o l d . 1999. Tyrosine phosphorylation of She i n response to B cell antigen receptor engagement depends on the SHIP inositol phosphatase. / . Immunol. In press: 124. Krebs, D. L . , Y . Yang, M . Dang, J. Haussmann, and M . R. Gold . 1999. Rapid and efficient retrovirus-mediated gene transfer into B cell lines. Meth. Cell. Sci. 21:57-68. 125. Franke, B., J-W. Akkerman,N. , and J. Bos. 1997. Rapid C a 2 + - m e d i a t e d activation of R a p l i n human platelets. EMBO J. 16:252-259. 126. Hawley, R. G. , F. H . Lieu, A . Z . Fong, and T. S. Hawley. 1994. Versatile retroviral vectors for potential use in gene therapy. Gene Therapy 1:136-138. 127. Onishi , M . , S. Kinoshita, Y . Morikawa, A . Shibuya, J. Phil l ips, L . L . Lanier, D . M . Gorman, G . P. No lan , A . Miyaj ima, and T. Ki tamura. 1996. Applicat ions of retrovirus-mediated expression cloning. Exp. Hemat. 24:324-329. 128. Pear, W . S., G . P. Nolan , M . L . Scott, and D. Baltimore. 1993. Production of high-titer helper-free retroviruses by transient transfection. Proc. Natl. Acad. Sci. USA 90:8393-8398. 198 129. Harmer, S. L . and A . L . DeFranco. 1999. The Src homology domain 2-containing inositol phosphatase SHIP forms a ternary complex wi th She and Grb2 i n antigen receptor-stimulated B cells. /. Biol. Chem. 274:12183-12191. 130. Smit, L . , M . M . de Vries-Smits, J. L . Bos, and J. Borst. 1994. B cell antigen receptor s t imulat ion induces formation of a Shc-Grb2 complex containing mult iple tyrosine-phosphorylated proteins. /. Biol. Chem. 269:20209-20212. 131. Mayer, B. J., M . Hamaguchi , and H . Hanafusa. 1988. A novel v i ra l oncogene wi th structural similarity to phospholipase C. Nature 332:272-275. 132. Tsuchie, H . , C. H . Chang, M . Yoshida, and P. K . Vogt. 1989. A newly isolated avian sarcoma virus, A S V - 1 , carries the crk oncogene. Oncogene 4:1281-1284. 133. Matsuda, M . , S. Tanaka, S. Nagata, A . Kojima, T. Kurata, and M . Shibuya. 1992. Two species of human C R K c D N A encode proteins w i t h distinct biological activities. Moi. Cell. Biol. 12:3482-3489. 134. ten Hoeve, ]., C. Morr is , N . Heisterkamp, and J. Groffen. 1993. Isolation and chromosomal loca l iza t ion of C R K L , a human crk- l ike gene. Oncogene 8:2469-2474. 135. Matsuda, M . , Y. Hashimoto, K . Muroya , H . Hasegawa, T. Kurata, S. Tanaka, S. N a k a m u r a , and S. Ha t to r i . 1994. C R K prote in b inds to two guanine nucleotide-releasing proteins for the Ras family and modulates nerve growth factor-induced activation of Ras i n PC12 cells. Moi. Cell. Biol. 14:5495-5500. 199 136. Feller, S. M . , B. Knudsen, and H . Hanafusa. 1994. c -Abl kinase regulates the protein binding activity of c-Crk. EMBO J. 13:2341-2351. 137. Hasegawa, H . , E. Kiyokawa, S. Tanaka, K . Nagashima, N . Gotoh, M . Shibuya, T. Kurata, and M . Matsuda. 1996. DOCK180, a major Crk-binding protein, alters cell morpho logy upon translocation to the cel l membrane. Moi. Cell. Biol. 16:1770-1776. 138. Oehrl , W. , C . Kardinal , S. Ruf, K . Adermann, J. Groffen, G . S. Feng, J. Blenis, and S. M . Feller. 1998. The germinal center kinase (GCK)-related protein kinases H P K 1 and K H S are candidates for highly selective signal transducers of Crk family adapter proteins. Oncogene 17:1893-1901. 139. Schumacher, C , B. S. Knudsen, T. Ohuchi , P. P. D i Fiore, R. H . Glassman, and H . Hanafusa. 1995. The SH3 domain of Crk binds specifically to a conserved proline-rich motif i n Eps l5 and Epsl5R. /. Biol. Chem. 270:15341-15347. 140. Tanaka, S., T. Morishita, Y. Hashimoto, S. Hattori, S. Nakamura, M . Shibuya, K . Matuoka, T. Takenawa, T. Kurata, K . Nagashima, and M . Matsuda. 1994. C3G, a guanine nucleotide-releasing protein expressed ubiquitously, binds to the Src homology 3 domains of Crk and G R B 2 / A S H proteins. Proc. Natl. Acad. Sci. USA 91:3443-3447. 141. K iyokawa , E. , Y . Hashimoto, S. Kobayashi, H . Sugimura, T. Kurata, and M . Matsuda. 1998. Activation of Rac l by a Crk SH3-binding protein, DOCK180. Genes Dev. 12:3331-3336. 200 142. Ling, P., Z . Yao, C. F. Meyer, X . S. Wang, W. Oehrl, S. M . Feller, and T. H . Tan. 1999. Interaction of hematopoietic progenitor kinase 1 wi th adapter proteins Crk and C r k L leads to synergistic activation of c-Jun N-terminal kinase. Moi. Cell. Biol. 19:1359-1368. 143. Marsh, M . and H . T. McMahon . 1999. The structural era of endocytosis. Science 285:215-220. 144. Gotoh, T., S. Hat tor i , S. Nakamura , H . Ki tayama, M . N o d a , Y . Takai , K . Kaibuchi , H . Matsui , O. Hatase, H . Takahashi, T. Kurata, and M . Matsuda. 1995. Identification of R a p l as a target of the C r k SH3 domain-binding guanine nucleotide-releasing factor C3G. Moi. Cell. Biol. 15:6746-6753. 145. Hariharan, I. K . , R. W. Carthew, and G. M . Rubin . 1991. The Drosophila roughened mutation: activation of a rap homolog disrupts eye development and interferes wi th cell determination. Cell 67:717-722. 146. Kitayama, H . , Y . Sugimoto, T. Matsuzaki , Y . Ikawa, and M . Noda . 1989. A ras-related gene wi th transformation suppressor activity. Cell 56:77-84. 147. Campa, M . J., K . J. Chang, V . Mol ina , B. R. Rep, and E. G . Lapetina. 1991. Inhibition of ras-induced germinal vesicle breakdown i n Xenopus oocytes by rap-IB. Biochem. Biophys. Res. Commun. 174:1-5. 148. Sakai, R., A . Iwamatsu, N . Hirano, S. Ogawa, T. Tanaka, H . Mano, Y. Yazaki, and H . Hi ra i . 1994. A novel signaling molecule, p l30 , forms stable complexes in vivo 201 with v-Crk and v-Src i n a tyrosine phosphorylation-dependent manner. EMBO J. 13:3748-3756. 149. Minegishi , M . , K . Tachibana, T. Sato, S. Iwata, and Y . Nojima. 1996. Structure and function of Cas-L, a 105-kD Crk-associated substrate-related protein that is involved i n beta 1 integrin-mediated signaling i n lymphocytes. /. Exp. Med. 184:1365-1375. 150. Astier, A . , S. N . Manie, S. F. Law, T. Canty, N . Haghayghi, B. J. Druker, R. Salgia, and A . S. Freedman. 1997. Association of the Cas-like molecule HEF1 wi th C r k L fol lowing integrin and antigen receptor signaling i n human B-cells: potential relevance to neoplastic lymphohematopoietic cells. Leuk. and Lymph. 28:65-72. 151. Cory, G . O. C , R. C . Lovering, S. Hinshelwood, L . McCar thy-Morrogh , R. J. Levinsky, and C. Kinnon. 1995. The protein product of the c-cbl protooncogene is phosphorylated after B cell receptor stimulation and binds the SH3 domain of Bruton's tyrosine kinase. /. Exp. Med. 182:611-615. 152. K i m , T. J., Y . - T . K i m , and S. P i l l a i . 1995. Assoc ia t ion of activated phosphatidylinositol 3-kinase wi th p l 2 0 c ^ i n antigen receptor-ligated B cells. / . Biol. Chem. 270:27504-27509. 153. Buday, L . , A . Khwaja, S. Sipeki, A . Farago, and J. Downward. 1996. Interactions of C b l w i th two adaptor proteins, Grb2 and Crk, upon T cell activation. /. Biol. Chem. 271:6159-6163. 154. Reedquist, K . A . , T. Fukazawa, G . Panchamoortyhy, W . Y . Langdon, S. E. Shoelson, B. J. Druker , and H . Band. 1996. Stimulat ion through the T cell 202 receptor induces C b l association w i t h the C r k proteins and the guanine nucleotide exchange protein C3G. /. Biol. Chem. 271:8435-8442. 155. Egan, S. E. , B. W. Giddings, M . W. Brookds, L . Buday, A . M . Sizeland, and R. A . Weinberg. 1993. Association of Sos Ras exchange protein wi th Grb2 is implicated i n tyrosine kinase signal transduction and transformation. Nature 363:45-51. 156. Buday, L . and J. Downward . 1993. Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor. Cell 73:611-620. 157. Matsuda, M . , S. Nagata, S. Tanaka, K . Nagashima, and T. Kurata. 1993. Strutural requirement of C R K SH2 region for b ind ing to phosphotyrosine-containing proteins. /. Biol. Chem. 268:4441-4446. 158. Knudsen, B.S., J. Zheng, S .M. Feller, J.P. Mayer, S.K. Burrell, D. Cowburn, and H . Hanafusa. 1995. Affinity and specificity requirements for the first Src homology 3 domain of the Crk proteins. EMBO J.14:2191-2198. 159. Smit, L . , G . van der Horst, and J. Borst. 1996. Sos, Vav, and C 3 G participate in B cell receptor-induced signaling pathways and differentially associate w i t h Shc-Grb2, Crk, and Crk -L adaptors. /. Biol. Chem. 271:8654-8569. 160. Blake, T., J. Shaprio, H . Morse, and W . Y . Langdon. 1991. The sequences of the human and mouse c-cbl proto-oncogenes show v-cbl was generated by a large truncation encompassing a proline-rich domain and a leucine zipper-like motif. Oncogene 6:653-657. 203 161. Panchamoorthy, G v T. Fukazawa, S. Miyake, S. Soltoff, K . Reedquist, B. Druker, S. Shoelson, L . Cantley, and H . Band. 1996. p l 2 0 c ^ is a major substrate of tyrosine phosphorylation upon B cell antigen receptor stimulation and interacts in vivo with Fyn and Syk tyrosine kinases, Grb2 and She adaptors, and the p85 subunit of phosphatidylinositol 3-kinase. /. Biol. Chem. 271:3187-3194. 162. Lupher, Jr., M . L . , N . Rao, N . L . L i l l , C. E. Andoniou , S. Miyake, E. A . Clark, B. Druker and H . Band. 1998. Cbl-mediated negative regulation of the Syk tyrosine kinase. /. Biol. Chem. 273:35273-35281. 163. Miyake, S., M . L . Lupher, Jr., B. Druker, and H . Band. 1998. The tyrosine kinase regulator C b l enhances the u b i q u i t i n a t i o n and degrada t ion of the platelet-derived growth factor alpha.Proc. Natl Acad. Sci. USA .95:7927-7932. 164. Levkowi tz , G . , H . Waterman, E. Zamir , Z . K a m , S. Oved, W . Y . Langdon, L . Beguinot, B. Geiger, Y. Yarden. 1998. c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor. Genes Dev. 12:3663-3674 165. Cook, S. J., B. Rubinfeld, I. Albert, and F. McCormick. 1993. RapV12 antagonizes Ras-dependent activation of ERK1 and ERK2 by L P A and E G F i n Rat-1 fibroblasts. EMBO J. 12:3475-3485. 166. Wittinghoffer, A . and C. Herrmann. 1995. Ras-effector interactions, the problem of specificity. FEBS Lett. 369:52-56. 167. Mar t in , G . A . , A . Yatani, R. Clark, L . Conroy, P. Polakis, A . M . Brown, and F. McCormick. 1992. G A P domains responsible for ras p21-dependent inhibition of muscarinic atrial K+ channel currents. Science 255:192-194. 204 168. Hofer, F v S. Fields, C. Schneider, and G . S. Mart in. 1994. Activated Ras interacts w i t h the Ra l guanine nucleotide dissociation stimulator. Proc. Natl. Acad. Sci. USA 91:11089-11093. 169. Spaargaren, M . and J. R. Bischoff. 1994. Identification of the guanine nucleotide dissociation stimulator for Ral as a putative effector molecule of R-Ras, H-Ras, K-Ras and Rap. Proc. Natl. Acad. Sci. USA 91:12609-12613. 170. Nassar, N . , G . H o r n , C . Hermann, A . Scherer, F. M c C o r m i c k , and A . Wittinghoffer. 1995. The 2.2A crystal structure of the Ras-binding domain of the serine/threonine kinase c-Raf-1 i n complex wi th R a p l A and a G T P analogue. Nature 375:554-560. 171. Freeh, M . , J. John, V . Pizon, P. Chardin, A . Tavitian, R. Clark, F. McCormick , and A . Wittinghofer. 1990. Inhibition of GTPase activating protein stimulation of Ras-p21 GTPase by the Krev-1 gene product. Science 249:169-171. 172. Davis , R. J. 1993. The mitogen-activaed protein kinase signal transduction pathway. /. Biol. Chem. 268:14553-14556. 173. Zwartkruis, F. J., R. M . Wolthuis, N . M . Nabben, B. Franke, and J. L . Bos. 1998. Extracellular signal-regulated activation of R a p l fails to interfere i n Ras effector signalling. EMBO }. 17:5905-5912. 174. de Nooij , J. C , M . A . Letendre, and I. K . Hariharan. 1996. A cyclin-dependent kinase inhibitor, Dacapo, is necessary for timely exit from the cell cycle during Drosophila embyogenesis. Cell 87:1237-1247. 205 175. Ohtsuka, T., K . Sh imizu , B. Yamamur i , S. Kuroda , and Y . Takai . 1996. Act ivat ion of B-raf protein kinase by R a p l B small GTP-binding . /. Biol. Chem. 271:1258-1261. 176. Gotoh, T., Y. Ni ino , M . Tokuda, O. Hatase, S. Nakamura, and S. Hattori. 1997. Act iva t ion of R-Ras by Ras-guanine nucleotide-releasing factor. /. Biol. Chem. 272:18602-18607. 177. A r a i , A . , Y . Nosaka, H . Kohsaka, N . Miyasaka , and O. M i u r a . 1999. C r k L activates integrin-mediated hematopoietic cell adhesion through the guanine nucleotide exchange factor C3G. Blood 93:3713-3722. 178. Tanaka, S., T. Ouchi , and H . Hanafusa. 1997. Downstream of C r k adaptor signaling pathway: Act iva t ion of Jun kinase by v -Crk through the guanine nucleotide exchange protein C3G. Proc. Natl. Acad. Sci. USA 94:2356-2361. 179. Fukazawa, T., S. Miyake, V . Band, and H . Band. 1996. Tyrosine phosphorylation of Cb l upon epidermal growth factor (EGF) stimulation and its association wi th E G F receptor and downstream signaling proteins. /. Biol. Chem. 271:14554-14559. 180. van den Berghe, N . , R. H . Cool , G . H r n , and A . Wittinghofer. 1997. Biochemical characterization of C3G: an exchange factor that discriminates between R a p l and Rap2 and is not inhibited by RaplA(S17N). Oncogene 15:845-850. 181. Beranger, F., B. Goud , A . Tavitian, and J. de Gunzburg. 1991. Association of the Ras-antagonistic Rap/Krev-1 proteins wi th the Golg i complex. Proc. Natl. Acad. Sci. USA 88:1606-1610. 206 182. Herrmann, C , G . Horn , M . Spaargaren, and A . Wittinghofer. 1996. Differential interaction of the Ras family GTP-binding proteins H-Ras, R a p l A , and R-Ras w i t h the putative effector molecules Raf kinase and Ral-guanine nucleotide exchange factor. /. Biol. Chem. 271:6794-6800. 183. Takata, M . , Y . Homma, and T. Kurosaki . 1995. Requirement of phospholipase C-gamma 2 activation i n surface immunoglobulin M-induced B cell apoptosis. /. Exp. Med. 182:907-914. 184. Page, D. M . and A . L . DeFranco. 1988. Role of phosphoinositide-derived second messengers i n mediat ing ant i - IgM-induced growth arrest of WEHI-231 B lymphoma cells. / . Immunol. 140:3717-3726. 185. Sugawara, H . , M . Kurosaki, M . Takata, and T. Kurosaki. 1997. Genetic evidence for involvement of type 1, type 2, and type 3 inositol 1,4,5-trisphosphate receptors i n signal transduction through the B cell antigen receptor. EMBO }. 16:3078-3088. 186. Kawasaki, H . , G . M . Springett, S. Toki, J. J. Canales, P. Harlan, J. P. Blumenstiel, I. A . Bany, N . Mochizuk i , A . Ashbacher, M . Matsuda, D. E. Housman, and A . M . Greybiel. 1998. A Rap guanine nucleotide exchange factor enriched highly i n the basal ganglia. Proc. Natl. Acad. Sci. USA. 95:13278-13283. 187. Bos, J. L . 1998. A l l i n the family? N e w insights and questions regarding interconnectivity of Ras, R a p l and Ral. EMBO J. 17:6776-6782. 188. Herbert, J. M . , J. M . Augereau, J. Gleye, and J. P. Maffrand. 1990. Chelerythrine is a potent and specific inhibitor of protein kinase C . Biochem. Biophys. Res. Comm. 172:993-999. 207 189. Kurachi , H . , Y. Wada, N . Tsukamoto, M . Maeda, H . Kubota, M . Hattori, K . Iwai, and N . Minato . 1997. H u m a n SPA-1 gene product selectively expressed i n lymphoid tissues is a specfic GTPase-activating protein for R a p l and Rap2. /. Biol. Chem. 272:28081-28088. 190. Rubinfeld, B v S. Munemitsu, R. Clark, L . Conroy, W. J. Crosier, F. McCormick, and P. Polakis. 1991. Molecular cloning of a GTPase activating protein for the Krev-1 protein p21 r ap l . Cell 65:1033-1042. 191. Wienecke, R., A . Konig , and J. E . DeClue. 1995. Identification of tuberin, the tuberous sclerosis-2 product. Tuberin possesses specific R a p l G A P activity. /. Biol. Chem. 270:16409-16414. 192. Siranian, M . I., A . Marchetti, G . D i Rocco, G . Starace, R. Jucker, and S. Nasi . 1993. Ras oncogene transformation of human B lymphoblasts is associated w i t h lymphocyte activation and wi th a block of differentiation. Oncogene 8:157-163. 193. Asha, H . , N . D . de Ruiter, M . G . Wang, and I. K . Hariharan. 1999. The R a p l GTPase functions as a regulator of morphogenesis i n vivo. EMBO }. 18:605-615. 194. M a l y , F. E. , L . A . Qui l l i am, O. Dorseuil , C. J. Der, and G . M . Bokoch. 1994. Activated or dominant inhibitory mutants of R a p l A decrease the oxidative burst of Epstein-Barr virus- transformed human B lymphocytes . / . Biol. Chem. 269:18743-18746. 195. Gabig, T. G. , C D. Crean, P. L . Mantel, and R. Rosli. 1995. Function of wild-type or mutant Rac2 and Rap l a GTPases i n differentiated HL60 cell N A D P H oxidase activation. Blood 85:804-811. 208 195a. Ishiai M . , M . Kurosaki , R. Pappu, K . Okawa, I. Ronko, C. Fu , M . Shibata, A . Iwamatsu, A . C. Chan and T. Kurosaki. 1999. B L N K is required for coupling Syk to P L C gamma 2 and Rac l - JNK i n B cells. Immunity. 10:117-125. 195b. Fu C , C. Turck, T. Kurosaki , and A . C . Chan. 1998. B L N K : a central linker protein i n B cell activation. Immunity. 9:93-103. 195c. Pappu R., A . M . Cheng, B. L i , Q. Gong, C. C h i u , N . Griff in, M . White, B. P. Sleckman, and A . C. Chan. 1999. Requirement for B Cel l Linker Protein (BLNK) i n B Ce l l Development. Science. 286:1949-1954. 195d. Minegish i Y . , J. Rohrer, E. Coustan-Smith, H . M . Lederman, R. Pappu, D . Campana, A . C. Chan, and M . E. Conley. 1999. A n Essential Role for B L N K i n H u m a n B Cel l Development. Science. 286:1954-1957. 196. Holgado-Madruga, M . , D. R. Emlet, D. K . Moscatello, A . K . G o d w i n , and A . J. Wong. 1996. A Grb2-associated docking protein i n E G F - and insulin-receptor signalling. Nature 379:560-564. 197. Holgado-Madruga, M . , D. K . Moscatello, D . R. Emlet, R. Dieterich, and A . J. Wong. 1997. Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor. Proc. Natl. Acad. Sci. USA. 94:12419-12424. 198. Takahashi-Tezuka, M . , Y. Yoshida, T. Fukada, T. Ohtani , Y . Yamanaka, K . Nish ida , K . Nakajima, M . H i b i , and T. Hirano. 1998. G a b l acts as an adapter molecule l ink ing the cytokine receptor gpl30 to E R K mitogen-activated protein kinase. Moi. Cell. Biol. 18:4109-4117. 209 199. Daub, H v C . Wallasch, A . Lankenau, A . Herr l ich, and A . Ul l r i ch . 1997. Signal characteristics of G protein-transactivated E G F receptor. EMBO J. 16:7032-7044. 200. Damen, J. E. , L . L i u , P. Rosten, R. K. Humphries, A . B. Jefferson, P. W . Majerus, and G . Krysta l . 1996. The 145-kDa protein induced to associate w i t h She by mulitple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5 trisphosphate 5-phosphatase. Proc. Natl. Acad. Sci. USA 93:1689-1693. 201. Welham, M . J., U . Duchert, K . B. Leslie, F. Jirik, and J. W . Schrader. 1994. Interleukin (IL) -3 and granulocyte/macrophage colony-stimulating factor, but not IL-4, induce tyrosine phosphorylation, activation, and association of SHPTP2 wi th Grb2 and phosphatidylinositol 3'-kinase. /. Biol. Chem. 269:23764-23768. 202. Maroun, C. R., M . Holgado-Madruga, I. Royal , M . A . Naujokas, T. M . Fournier, WongAJ. , and M . Park. 1999. The G a b l P H domain is required for localization of G a b l at sites of cell-cell contact and epithelial morphogenesis downstream from the met receptor tyrosine kinase. Moi Cell Biol 19:1784-1799. 203. Isakoff, S. J., T. Cardozo, J. Andreev, Z . L i , K . M . Ferguson, R. Abagyan, M . A . Lemmon, A . Aronheim, and E. Y . Skolnik. 1998. Identification and analysis of P H domain-containing targets of phosphatidylinositol 3-kinase using a novel i n vivo assay i n yeast. EMBO f. 17:5374-5387. 204. D 'Ambrosio , D. , K . L . Hippen , and J. C. Cambier. 1996. Distinct mechanisms mediate S H C association wi th the activated and resting B cell antigen receptor. Eur. J. Immunol. 26:1960-1965. 210 205. Nishida , K . , Y. Yoshida, M . Itoh, T. Fukada, T. Ohtani, T. Shirogane, T. Atsumi , M . Takahashi-Tezuka, K . Ishihara, M . H i b i , and T. Hirano. 1999. Gab-family adapter proteins act downstream of cytokine and growth factor receptors and T-and B-cell antigen receptors. Blood 93:1809-1816. 206. Herbst, R., P. M . Carroll , J. D. Al la rd , J. Schilling, and T. Raabe. 1996. Daughter of sevenless is a substrate of the phosphotyrosine phosphatase Corkscrew and functions during sevenless signaling. Cell 85:899-909. 207. Lecoq-Lafon, C , F. Verdier, S. Fichelson, S. Chretien, S. Gisselbrecht, and P. Mayeux. 1999. Erythropoietin induces the tyrosine phosphorylation of G A B 1 and its association wi th S H C , SHP2, SHIP, and phosphatidylinositol 3-kinase. Blood 93:2578-2585. 208. G u , H . , J. C. Pratt, S. J. Burakoff, and B. G. Neel. 1998. Cloning of p97/Gab2, the major SHP2-binding protein i n hematopoietic cells, reveals a novel pathway for cytokine-induced gene activation. Molec. Cell. 2:729-740. 209. Chunmei , Z . , Y . De-Hua, R. Shen, G . -S. Feng. 1999. Gab2, a new pleckstrin homology domain-containing adapter protein, acts to uncouple signaling from E R K kinase to Elk-1. /. Biol. Chem. 274:19649-19654. 210. Rocchi, S., S. Tartare-Deckert, J. Murdaca, M . Holgado-Madruga, A . J. Wong, and E. V a n Obberghen. 1998. Determination of G a b l (Grb2-Associated Binder-1) interaction w i t h insul in receptor-signalling molecules. Moi. Endo. 12:914-923. 211 211. Voge l , W . , R. Lammers, J. Huang , and A . U l l r i c h . 1993. Act iva t ion of a phospho ty ros ine phosphatase by tyrosine p h o s p h o r y l a t i o n . Science 259:1611-1614. 212. Barford, D . and B. G . Neel . 1998. Revealing mechanisms for SH2 domain mediated regulation of the protein tyrosine phosphatase SHP-2 . Structure 6:249-254. 213. Myers , M . G.,Jr., R. Mendez, P. Shi, J. H . Pierce, R. Rhoads, and M . F. White. 1998. The COOH-te rmina l tyrosine phosphorylation sites on IRS-1 b ind SHP-2 and negatively regulate insul in signaling. /. Biol. Chem. 273:26908-26914. 214. Bolland, S., R. N . Pearse, T. Kurosaki, and J. V . Ravetch. 1998. SHIP modulates immune receptor responses by regulat ing membrane association of Btk. Immunity 8:508-516. 215. Okada, H . , S. Bolland, A . Hashimoto, M . Kurosaki, Y. Kabuyama, M . lino, and T. Kurosaki . 1998. Role of the inositol phosphatase SHIP i n B cell receptor-induced Ca2+ oscillatory response. /. Immunol. 161:5129-5132. 216. L a m k i n , T. D . , S. F. Walk , L . L i u , J. E . Damen, G . Krys ta l , and K . S. Ravichandran. 1997. She interaction w i t h Src homology 2 domain containing inositol phosphatase (SHIP) in v ivo requires the Shc-phosphotyrosine binding d o m a i n and two specific phosphotyros ines on S H I P . / . Biol. Chem. 272:10396-10401. 217. L i u , L . , J. E. Damen, M . R. Hughes, I. Babic, F. R. Jirik, and G . Krystal. 1997. The Src homology 2 (SH2) domain of SH2-containing inositol phosphatase (SHIP) is 212 essential for tyrosine phosphorylation of SHIP, its association wi th She, and its induction of apoptosis. /. Biol. Chem. 272:8983-8988. 218. Crowley, M . T., S. L . Harmer, and A . L . DeFranco. 1996. Activation-induced association of a 145-kDa tyrosine-phosphorylated protein wi th She and Syk i n B lymphocytes and macrophages. /. Biol. Chem. 271:1145-1152. 219. Baumann, G . , D. Maier, F. Freuler, C. Tschopp, K . Baudisch, and J. Wienands. 1994. In vitro characterization of major ligands for Src homology 2 domains derived from protein tyrosine kinases, from the adapter protein She and from GTPase-activating protein i n Ramos B cells. Eur. J. Immunol. 24:1799-1807'. 220. Harmer, S. L . and A . L . DeFranco. 1997. She contains two Grb2 binding sites needed for efficient formation of complexes wi th SOS i n B lymphocytes. Moi. Cell. Biol. 17:4087-4095. 221. Naga i , K . , M . Takata, H . Yamamura , and T. Kurosak i . 1995. Tyrosine phosphorylation of She is mediated through L y n and Syk i n B cell receptor signaling. /. Biol. Chem. 270:6824-6829. 222. Kurosaki , T. 1997. Molecular mechanisms i n B cell antigen receptor signaling. Curr. Opin. Immunol. 9:309-318. 223. Turner, C. E. 1998. Paxill in. Int. }. Biochem. 30:955-959. 224. Birge, R. B., J. E. Fajardo, C. Reichman, S. E. Shoelson, Z . Songyang, L . C. Cantley, and H . Hanafusa. 1993. Identification and characterization of a 213 high-affinity interaction between v-Crk and tyrosine-phosphorylated paxi l l in i n CTlO-transformed fibroblasts. Moi. Cell Biol 13:4648-4656. 225. Beitner-Johnson, D. , V . A . Blakesley, Z . Shen-Orr, M . Jimenez, B. Stannard, L . M . Wang, J. Pierce, and D . LeRoith. 1996. The proto-oncogene product c-Crk associates w i t h insul in receptor substrate-1 and 4PS. Modu la t ion by insul in growth factor-I (IGF) and enhanced IGF-I signaling. /. Biol Chem. 271:9287-9290. 226. Turner, C. E. and J. T. Mi l l e r . 1994. Primary sequence of pax i l l in contains putative SH2 and SH3 domain b ind ing motifs and mult iple L I M domains: ident i f ica t ion of a v i n c u l i n and p p l 2 5 F a k - b i n d i n g region. / . Cell. Sci. 107:1583-1591. 227. Sudol , M . 1996. The W W module competes w i t h the SH3 domain? Trends Biochem. Sci. 21:161-163. 228. L u , P. J., X . Z . Zhou, M . Shen, and K . P. L u . 1999. Function of W W domains as phosphoserine- or phosphothreonine-binding modules. Science 283:1325-1328. 229. Sudol, M . , P. Bork, A . Einbond, K . Kastury, T. Druck, N e g r i n i M . , K . Huebner, and D . Lehman. 1995. Characterization of the mammalian Y A P (Yes-associated protein) gene and its role i n defining a novel protein module, the W W domain. /. Biol. Chem. 270:14733-14741. 230. Staub, O., S. Dho, P. Henry, J. Correa, T. Ishikawa, J. McGlade, and D. Rotin. 1996. W W domains of Nedd4 b ind to the proline-rich PY motifs i n the epithelial Na+ channel deleted i n Liddle's syndrome. EMBO J. 15:2371-2380. 214 231. K i m , E. , M . Niethammer, A . Rothschild, Y . N . Jan, and M . Sheng. 1995. Clus te r ing of Shaker-type K+ channels by interaction w i t h a family of membrane-associated guanylate kinases. Nature 378:85-88. 232. Kornau, H . C , L . T. Schenker, M . B. Kennedy, and P. H . Seeburg. 1995. Domain interaction between N M D A receptor subunits and the postsynaptic density protein PSD-95. Science 269:1737-1740. 233. Songyang, Z . , A . S. Fanning, C. Fu , J. X u , S. M . Marfatia, A . Crompton, A . C. Chan , J. M . Anderson , and L . C . Cantley. 1997. Recogni t ion of unique carboxyl-terminal motifs by distinct P D Z domains. Science 275:73-77. 234. Ranganathan, R. and E. M . Ross. 1997. P D Z . d o m a i n proteins: scaffolds for signaling complexes. Curr. Biol. 7:R770-R773. 235. Brakeman, P. R., A . A . Lanahan, R. O'Brien, K . Roche, C. A . Barnes, and R. L . Huganir . 1997. Homer: a protein that selectively binds metabotropic glutamate receptors. Nature 386:284-288. 236. Gomperts, S. N . 1996. Clustering membrane proteins: It's a l l coming together wi th the PSD-95/SAP90 protein family. Cell 84:659-662. 237. Ponting, C. P., C. Phi l l ips , K . E. Davies, and D . J. Blake. 1997. P D Z domains: targeting signalling molecules to sub-membranous sites. Bioessays 19:469-479. 238. Longnecker, R. and C. L . Mil ler . 1996. Regulation of Epstein-Barr virus latency by latent membrane protein 2. Trends MicrobiolA:38-42. 215 239. Longnecker, R., B. Druker, T. M . Roberts, and E. Kieff. 1991. A n Epstein-Barr virus protein associated wi th cell growth transformation interacts wi th a tyrosine kinase. J.Virol .65:3681-3692. 240. M i l l e r , C. L . , R. Longnecker, and E. Kieff. 1993. Epstein-Barr virus latent membrane protein 2 A blocks calcium mobil izat ion i n B lymphocytes. /. Virol. 67:3087-3094. 241. Jacobson, K . and C. Dietrich. 1999. Looking at l i p i d rafts? Trends Cell Biol. 9:87-91. 242. Horejsi, V . , K . Drbal, M . Cebecauer, J. Cerny, T. Brdicka, P. Angelisova, and H . Stockinger. 1999. GPI-microdomains: a role i n signalling via immunoreceptors. Immunol. Today 20:356-361. 243. Simons, K . and E. Ikonen. 1997. Functional rafts i n cell membranes. Nature 387:569-572. 244. M o n t i x i , C , C. Langlet, A . M . Bernard, J. Thimonier , C. W . Dubois, J. P. Chauvin, M . Pierres, and H . T. He. 1998. Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains. EMBO J. 17:5334-5348. 245. Xavier, R., T. Brennan, Q. L i , C. McCormack, and B. Seed. 1998. Membrane compartmentation is required for efficient T cell activation. Immunity 8:723-732. 216 246. Deans, J. P., S. M . Robbins, M . J. Polyak, and J. A . Savage. 1998. Rap id redis t r ibut ion of CD20 to a low density detergent-insoluble membrane compartment. /. Biol. Chem. 273:344-348. 247. M u m b y , S. M . 1997. Reversible palmitoylation of signaling proteins. Curr. Opin. Cell. Biol. 9:148-154. 248. Dogic, D. , B. Eckes, and M . Aumail ley. 1999. Extracellular matrix, integrins and focal adhesions. Curr. Top. Pathol. 93:75-85. 249. Astier , A . , H . Avraham, S. N . Manie , J. Groopman, T. Canty, and A . S. F r e e d m a n . 1997. The re la ted adhes ion focal t y ros ine kinase is tyrosine-phosphorylated after betal-integrin stimulation i n B cells and binds to pl30cas. /. Biol. Chem. 272:228-232. 250. Weng, G . , U . S. Bhalla, and R. Iyengar. 1999. Complexity i n biological signaling systems. Science 284:92-96. 251. Cho i , K . Y . , B. Satterberg, D. M . Lyons, and E. A . E l ion , 1994. Ste5 tethers multiple protein kinases i n the M A P kinase cascade required for mating in S. cerevisiae. Cell. 78:499-512. 252. Marcus, S., A . Polverino, M . Barr, and M . Wigler. 1994. Complexes between STE5 and components of the pheromone-responsive mitogen-activated protein kinase module. Proc. Natl. Acad. Sci. USA 91:7762-7766. 253. Printen, J. A . and G. F. Sprague Jr. 1994. Protein-protein interactions i n the yeast pheromone response pathway: Ste5p interacts w i t h al l members of the M A P kinase cascade. Genetics 138:609-619. 217 254. Whitmarsh, A . J., J. Cavanagh, C. Tournier, J. Yasuda, and R. J. Davis. 1998. A mammalian scaffold complex that selectively mediates M A P kinase activation. Science 281:1671-1674. 218 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0089627/manifest

Comment

Related Items