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Regulation of Rap2 in B lymphocytes Li, Anson H.Y. 2002

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REGULATION OF RAP2 IN B L Y M P H O C Y T E S By A N S O N H . Y . LI B.Sc., University of British Columbia, 2000 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF M A S T E R OF SCIENCE In THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Microbiology and Immunology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A August 2002 ©Anson H . Y . L i , 2002 U B C Rare Books and Special Collections - Thesis Authorisation Form Page 1 of 1 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may b e g r a n t e d b y t h e h e a d o f my d e p a r t m e n t o r b y h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f M \ ' c r o \ > 0 \ Q Q a & Xrn fT t O O p l T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r , C a n a d a http://www.library.ubc.ca/spcoll/thesauth.html 8/15/2002 A B S T R A C T Ras and members of the Ras-related family of small GTP-binding proteins are involved in many biological functions. These include regulation of cell growth, differentiation, and activation; actin cytoskeleton rearrangement, membrane trafficking, vesicle trafficking and nuclear transport. These GTP-binding proteins cycle between an inactive GDP-bound form or an active GTP-bound form which can bind downstream effectors. Rapl A , RaplB, Rap2A, and Rap2B proteins share approximately 50% sequence identity with Ras. In addition, Rap proteins share a similar effector region as Ras, but have different flanking amino acids. Despite the similarities, the functions of Rapl and Rap2 have not been completely elucidated although recent studies suggest that both Rap proteins may be involved in cell migration and adhesion. Previous work has shown that Rapl is activated by many receptors. In our laboratory, we showed that Rapl was activated by B C R cross-linking and CXCR4 stimulation. Hence, the first aim of this thesis was to determine i f these receptors also activated Rap2. This is because Rap2 may or may not have the same function as Rapl in B lymphocytes. The second objective was to uncover how B C R cross-linking and CXCR4 activate Rap proteins. A PLC inhibitor was used to elucidate the mechanism of this Rap activation signalling pathway. Finally, the third goal was to develop loss of function approaches for blocking Rapl and Rap2 activation. Expression of the Rap-specific GAP proteins, RapGapH and Spa-1, was used to inhibit activation of Rapl and Rap2 in B cell lines. These experiments have resulted in three major findings. First I showed that anti-Ig antibodies, stromal cell-derived factor-1 (SDF-1), and phorbol 12,13-dibutyrate (PdBu) all activate Rap2 in B cells with activation kinetics similar to that for Rapl. Second, I showed that the B C R activates Rap2 via PLC-y, and Rap2 activation can be inhibited by l-[6-[[ 170-3-ii methoxyestra-l,3,5(10)-trien-17-yl]amino]hexyl]-lH-pyr^ (U73122). Finally, I showed that RapGapU and Spa-1 differentially inhibit the activation of Rap 1 and Rap2. iii T A B L E OF C O N T E N T S Abstract . . . . . . . . . . i i Table of contents . . . . . . . . . iv List of figures . . . . . . . . . . vi List of abbreviations . . . . . . . • v i i Acknowledgments . . . . . . . . . ix Dedication . . . . . . . . . . x CHAPTER I Introduction 1.0 B Cells and their f u n c t i o n . . . . . 1 1.0.1 Signal transduction by key receptors in B lymphocytes . . . . . 1 1.0.2 The role oftheBCR in B cell activation . 2 1.0.3 Signal transduction by the B C R . . . 3 1.0.4 The role o fCXCR4 in B cell function . 6 1.0.5 Signal transduction via CXCR4 . . . 8 1.1 Ras and Ras-like proteins . . . . . 9 1.1.1 The importance of Ras and Ras-family members . 10 1.1.2 GTPases 11 1.1.3 GEFs and GAPs 11 1.2 Rap 1 andRap2 . . . . . . 12 1.2.1 General characteristics of Rap 1 and Rap2 . . 12 1.2.2 Known functions of Rap proteins . . . 14 1.2.3 Rap proteins in B lymphocytes . . . 16 1.3 WEHI-231, A20, and 2PK3 B cell lines as model systems . . . . . . 17 1.3.1 WEHI-231 cells 17 1.3.2 A20 cells 18 1.3.3 2PK-3 cells 18 1.4 Thesis objectives . . . . . . 18 CHAPTER LT. Materials and Methods 2.0 Antibodies and other reagents . . . . 20 2.1 B C e l l Lines " 2.1.1 WEHI-231, A20, 2PK-3 cells . . . 20 2.1.2 pMSCV RapGapU-expressing B cell lines . . 21 2.1.3 p L X S N Spa-1-expressing B cell lines . . 21 2.1.4 pMX-PIE Spa-1-expressing B cell lines . . 21 2.2 Cell culture . . . . . . 22 2.3 Rap activation assay . . . . . 22 2.4 Protein analysis 2.4.1 SDS-PAGE and western blotting . . . 23 iv 2.4.2 Quantitation and statistical analysis. . . 24 CHAPTER UI Activation of the Rap2 proteins in B lymphocytes 3.0 Rationale and summary . . . . . 25 3.1 Activation of Rap2 by anti-Ig antibodies . . . 26 3.2 Activation of Rap2 by SDF-1 . . . . 29 3.3 Mechanism of BCR-induced Rap2 activation . 32 3.3.1 Activation of Rap2 by phorbol esters . . 32 3.3.2 Inhibition of Rap2 activation by the PLC inhibitor U73122 34 3.4 Conclusion . . . . . . 38 CHAPTER TV Developing loss of function approaches to study the roles of the Rapl and Rap2 proteins in B lymphocytes 4.1 Rationale and summary . . . . . 39 4.2 The effect of RapGapU expression on Rapl and Rap2 activation . . . . . . 39 4.3 The effect of Spa-1 expression on Rapl and Rap2 activation . . . . . . 46 4.4 Conclusion . . . . . . 52 CHAPTER V Discussion 5.1 Summary . . . . . . . 53 5.2 Coordinate activation of Rapl and Rap2 in B cells . 53 5.3 Activation of Rap2 via the PLC pathway. . 54 5.4 Differential regulation of Rapl and Rap2 by RapGapII and Spa-1 . . . . . . 55 5.5 The role of Rap proteins in cell migration and cell adhesion . . . . . . 56 5.6 Further work . . . . . . 59 References . . . . . . . . . . 61 Appendix I Overview of B C R signalling pathway . . . 73 Appendix U Overview of CXCR4 signalling pathway . . . 74 Appendix HI Phylogenic tree of Rap and Ras family GTPases. . . 75 v LIST OF FIGURES Fig. 3.1 Anti-IgM activates Rapl and Rap2 in WEHI-231 cells 27 Fig. 3.2 Anti-IgG activates Rapl and Rap2 in A20 cells . 28 Fig. 3.3 SDF-1 activates Rapl and Rap2 in WEHI-231 cells . 30 Fig. 3.4 SDF-1 activates Rapl and Rap2 in 2PK-3 cells . 31 Fig. 3.5 Rapl and Rap2 are activated by the D A G mimic PdBu in WEHI-231 cells 33 Fig. 3.6 The PLC inhibitor U73122 partially inhibits BCR-induced Rapl and Rap2 activation in WEHI-231 cells . . . . . . . 36 Fig. 3.7 Relative levels of Rap2 and Rapl activation in U73122 and U73343 pre-treated WEHI-231 cells . . 37 Fig. 4.1 Effects of RapGapJJ expression on anti-IgM-induced Rapl and Rap2 activation in WEHI-231 cells . 41 Fig. 4.2 Effects of RapGapU expression on anti-IgG-induced Rapl and Rap2 activation in A20 cells . . 42 Fig. 4.3 Effects of RapGapE expression on SDF-1 -induced Rapl and Rap2 activation in WEHI-231 cells. . . 43 Fig. 4.4 Effects of RapGapH expression on SDF-1-induced Rapl and Rap2 activation in 2PK-3 cells . . . 44 Fig. 4.5 Effects of RapGapU expression on PdBu-induced Rapl and Rap2 activation in WEHI-231 cells . . 45 Fig. 4.6 Effects of Spa-1 expression on anti-IgM-induced Rapl and Rap2 activation in WEHI-231 cells. . 48 Fig. 4.7 Effects of Spa-1 expression on anti-IgG-induced Rapl and Rap2 activation in A20 cells . . . 49 Fig. 4.8 Effects of Spa-1 expression on SDF-1 induced Rapl and Rap2 activation in 2PK-3 cells . . . 50 Fig. 4.9 Effects of Spa-1 expression on PdBu-induced Rapl and Rap2 activation in WEHI-231 cells . 51 vi LIST OF A B B R E V I A T I O N S B C R B cell antigen receptor B L N K B cell linker protein D A G Diacylglycerol E C L Enhanced chemiluminescence EGF Epithelial growth factor E R K Extracellular signal-regulated kinase FDC Follicular dendritic cell GNEF Guanine-nucleotide exchange factor HRP Horseradish peroxidase I C A M Intercellular adhesion molecule n>3 Inositol trisphosphate L P A Lysophosphatidic acid LESTR Leukocyte-derived seven-transmembrane domain receptor mlg Membrane immunoglobulin NFAT Nuclear factor of activated T cells PDGF Platelet-derived growth factor PdBu Phorbol 12,13-dibutyrate PI3K Phosphatidylinositol 3-kinase PI(4,5)P2 Phosphatidylinositol 4,5-biphosphate PI(3,4,5)P3 Phosphatidylinositol 3,4,5-trisphosphate P K A Protein kinase A P K C Protein kinase C PLC Phospholipase C vii PTK Protein tyrosine kinase RasGRP Ras guanyl nucleotide-releasing protein Rl f RalGDS-like factor RPIP8 Rap2-interacting protein 8 SDF-1 Stromal cell-derived factor-1 SOS Son of sevenless TP A 12-O-tetradecanoylphorbol 13-acetate U73122 l-[6-[[17(3-3-methoxyestra-l,3,5(10)-trien-17-yl]amino]hexyl]-lH pyrrole-2,5-dione U73343 l-[6-[[17p-3-methoxyestra-l,3,5(10)-trien-17-yl]amino]hexyl]-lH pyrrole-2,5-dione vm A C K N O W L E D G M E N T S I would like to take the opportunity now to express my appreciation for my supervisor, Dr. Michael Gold, for allowing me to do my Masters degree in the laboratory. His guidance and supervision of the laboratory environment has enhanced my knowledge of immunology immensely and made my two years at U B C a memorable and pleasurable experience. I would also like to give thanks to my committee members, Dr. Gerry Weeks and Dr. Jim Kronstad, for all their friendly advice and suggestions. A special thank you is extended to all Gold laboratory members: Anita Burgess, Sherri Christian, May Dang-Lawson, Rosaline Lee, Sarah McLeod, Blazej Szczygielski, and Mindy Zhu. You have not only contributed to my learning, but you have been there as my friends as well. Finally, I would like to express my gratitude to the rest of my friends and family. Your continual support either financially or emotionally will forever be cherished and remembered. ix To my mother, Ticky Li, who suffers from myasthenia gravis. x C H A P T E R O N E I N T R O D U C T I O N 1.0 B Cells and their function B lymphocytes play a vital role in the host immune system by protecting the body from foreign pathogens. The two primary roles of B lymphocytes are to present foreign antigens to T lymphocytes and to produce specific antibodies directed against foreign antigens (Peakman and Vergani, 1997). In short, B lymphocytes sample the microenvironment with their receptors. If a threat is present, a specific population of B lymphocytes is activated by the combination of antigen stimulation and T lymphocyte-derived signals. These activated B cells will proliferate and differentiate into antibody-producing plasma cells. Antibodies eliminate pathogens by three main mechanisms: neutralization, opsonization, and complement activation. Although each B cell recognizes only one or a few related antigens via their B cell antigen receptor (BCR), the diversity of antigen binding sites on the population of B cells is so abundant that the repertoire for antibody specificity within an individual exceeds 10 and presumably can recognize any pathogen one might encounter (Anderson, 1999). How B lymphocytes develop, mature, become activated, and differentiate is therefore of much interest. As the following thesis is primarily focused on the effects of certain external cues being transmitted to the interior of the B cell, a brief discussion on signal transduction in B lymphocytes is explained below. 1.0.1 Sifinal transduction by key receptors in B lymphocytes Receptors are important components of any cell, and many are located at the plasma membrane. When a surface receptor binds to a specific extracellular molecule, a signalling cascade is initiated. First the receptor is activated and then the signal is relayed to other 1 intracellular signalling molecules. The net result is that changes in cell functions occur (Campbell, 1996). These changes vary from gene expression to cytoskeletal rearrangements. Many receptors regulate B cell development, activation, and tolerance. The following thesis will look at two important receptors that reside on the surface membrane of B lymphocytes: B C R and CXCR4. Briefly, B C R signalling leads to activation or tolerance. The combination of antigen-induced B C R signalling and co-stimulatory signals provided by helper T lymphocytes causes B cells to proliferate and then differentiate into antibody-secreting plasma cells (Sharon, 1998). In contrast, for immature B cells, self-antigen-induced B C R signalling in the bone marrow often leads to B cell apoptosis or B cell anergy. Thus, tolerance to self-antigens in the body is maintained. CXCR4 signalling is also important because it directs B cells and B cell progenitors to the proper location. The ligand for CXCR4, SDF-1, helps recruit naive B cells into lymphoid organs where they can encounter foreign antigens (Okada et al., 2002). SDF-1, made by stromal cells in the bone marrow, retains B progenitors in the bone marrow where B cell maturation can take place (Janeway et al., 2001). 1.0.2 The role of the B C R in B cell activation The B C R consists of an antigen-binding subunit (mlg) and a signalling subunit (CD79a/b). The membrane immunoglobulin (mlg) consists of four polypeptides - two heavy chains of either 50 kDa (IgG) to 72 kDa (IgM) in molecular weight and two 25 kDa light chains - that are held together by disulfide bonds (Gold and Matsuuchi, 1995). At the amino termini of both the heavy and light chains are the variable regions. These regions give each B C R its unique antigen binding specificity. Associated with the mlg in a non-covalent manner are two disulfide-bonded transmembrane polypeptides, Iga (CD79a) and Igp (CD79b). These two polypeptides 2 are the main signalling components of the B C R complex because their carboxyl termini extend across the membrane and into the cytoplasm and provide the only significant cytoplasmic domains present in the B C R complex (Campbell, 1999; Kurosaki, 1997). The B C R is important for two main reasons: It controls the development and maturation of B cell progenitors, and it controls the survival and activation of mature B lymphocytes (Gold and Matsuuchi, 1995; Janeway et al., 2001). During B cell development in the bone marrow, gene rearrangements are occurring on the heavy chain genes in an attempt to generate a pre-BCR. Signalling by the pre-BCR serves as an important checkpoint for B cell development and allows for further gene rearrangements of the light chain genes. Successful light chain gene arrangement leads to cell-surface expression of the B C R and then each B cell undergoes negative selection in order to eliminate those with BCR's that react strongly to self-antigens. It is unknown at the present time how B cells are able to sense a functional B C R and thereby receive additional signals for maturation. After development, B cells are released from the bone marrow and they circulate within the body's lymphatic system and blood stream. It has been shown that continuous expression of the B C R and signalling via the B C R in B cells are required for continued survival in the periphery (Lam et al., 1997). Most importantly, upon recognizing pathogen-associated antigens, the B cell is activated. Again, B C R signalling occurs. Hence, B C R signalling appears to control survival, activation, development, and death. Mutations in the B C R that lead to constitutive signalling or absence of signalling could cause immunodeficiency, autoimmunity, and even malignancy. This is why understanding how signal transduction works within B lymphocytes is important. 1.0.3 Signal transduction by the B C R 3 The initial B C R signalling events begin at the receptor when the B C R binds tightly with an antigen, which causes multiple antigen receptors to cluster (See Appendix I). Membrane Ig cross-linking or clustering is required for signal generation and is the initial step to B cell activation (Gold et al., 1992). This is because clustering allows receptor associated protein tyrosine kinases (PTK) of the Src family - Lyn, Fyn, Blk, and Lck - to come in close proximity to activate one another and phosphorylate the immunoreceptor tyrosine-based activation motifs (ITAM; [D/E]xxYxx[L/I]xxxxxxxYxx[L/I]) of other CD79a/b chains (Campbell, 1999; DeFranco, 1997; Kurosaki, 1997). ITAMS that are approximately 26 amino acid in length (Kurosaki, 1997) and found on the cytoplasmic domains of the B C R receptor complex are phosphorylated by these PTKs. Phosphorylated ITAMs provide binding sites for proteins that possess Src homology 2 (SH2) domains, such as Syk - another PTK. Once Syk is recruited and binds to the phosphorylated ITAMs on the Iga and Ig(3 chains of the B C R complex, Syk, itself, gets phosphorylated and activated by the Src-family tyrosine kinases, and its activity is greatly enhanced (DeFranco, 1997). Another tyrosine kinase that gets activated after BCR engagement is the Tec-family kinase Bruton's tyrosine kinase (Btk). Syk recruitment to the B C R is a crucial event because Syk activates a multitude of downstream signalling proteins which lead to the activation of three major signalling pathways: the phospholipase C-y (PLC-y) pathway, the phosphatidylinositol 3-kinase (PI3K) pathway, and the Ras pathway (Kurosaki, 1997). Adaptor proteins, such as B cell linker protein (BLNK), Cbl, Crk, Grb2, and She, are vital to the activation of these signal transduction pathways even though these proteins possess no intrinsic enzymatic function (Peterson et al., 1998). Their role is to mediate protein-protein interactions and to localize certain proteins to membranes. Activation of the PLC-y pathway begins with PLC-y recruitment to the membrane via the binding of PLC-y2's SH2 domain to B L N K , an adaptor protein that is phosphorylated by Syk 4 (Campbell, 1999). Once PLC-y is brought to the plasma membrane, it is phosphorylated and activated by Btk. PLC-y then cleaves the plasma membrane phospholipid phosphatidylinositol 4,5-biphosphate (PI(4,5)P2) into inositol trisphosphate (IP3) and diacylglycerol (DAG) (Scharenberg and Kinet, 1998). IP3 causes the release of C a 2 + ions from the endoplasmic reticulum while D A G activates multiple isoforms of the protein kinase C (PKC) family of serine/threonine kinases (Gold, 2000). The liberation of calcium activates a phosphatase known as calcineurin, and calcineurin ultimately dephosphorylates the transcription factor nuclear factor of activated T cells (NFAT). N F A T then translocates to the nucleus and stimulates transcription. PKC, on the other hand, activates the transcription factor, N F - K B , by initiating a pathway that leads to the degradation of the inhibitory subunit IKB (Gold, 2000). This permits the migration of N F - K B into the nucleus. BCR engagement also activates PI3K (Gold et al., 1992), which consists of two subunits, the regulatory subunit p85 and the catalytic subunit p i 10 (Okkenhaug and Vanhaesebroeck, 2001). PI3K activation can occur by several means. First the CD19 co-receptor on B lymphocytes contain two Y x x M motifs that are tyrosine phosphorylated after B C R engagement and provide the correct binding sites for the two SH2 domains on p85. The binding of p85 induces a conformational change within PI3K that increases its specific activity (Gold, 2000). The secondary method of PI3K activation is via adaptor proteins. For example, it has been shown that PI3K can bind via its SH2 domains to Cbl and Gabl (Gold et al., 2000; Ingham et al., 1998). These adaptor proteins localize PI3K to its substrates, which are located in the plasma membrane. This is possible because adaptor proteins either contain PH domains which are capable of binding to certain membrane phospholipids, thus allowing for membrane recruitment, or they contain other protein interaction domains, for example PTB/SH2-like domains, that allow for interactions with membrane proteins. 5 The main function of P B K is to phosphorylate PI(4,5)P2 on the 3-position of the inositol ring, creating phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) (Rameh and Cantley, 1999). PI(4,5)P2 is the substrate of PLC-y whereas PI(3,4,5)P3 is involved in recruiting PH domain-containing signalling proteins to the membrane (Rameh and Cantley, 1999). Some of these PH domain-containing signalling proteins include PDK1, Akt kinases, and Btk. The third signalling pathway to get activated after B C R cross-linking is the Ras pathway (Harwood and Cambier, 1993; Saxton et al., 1994; Su and Karin, 1996). Ras activation in B lymphocytes is most likely activated by the D A G -"> Ras guanyl nucleotide-releasing protein (RasGRP) pathway (Tognon et al., 1998) with minor contributions by the Shc/Grb/Son of Sevenless (SOS) pathway. It is believed that PLC-y2-generated D A G recruits the RasGRP protein to the plasma membrane where Ras is located. RasGRP is a guanine-nucleotide exchange factor (GNEF) that activates Ras by triggering the displacement of GDP from Ras in order to generate an active GTP bound form of Ras. The other pathway begins with the membrane recruitment of the adaptor proteins She (Saxton et al., 1994) and Grb2. She and Grb2 mediate the recruitment of the GNEF, SOS, to the membrane where it activates membrane bound Ras. It has been suggested that the Shc/Grb2/SOS complex is recruited to the plasma membrane by Gabl or B L N K (Fu et al., 1998; Ingham et al., 1998) after B C R ligation. Active Ras binds to and activates the serine/threonine kinase, Rafl . This subsequently sets off a cascade of kinases (Kolch, 2000) in which Mek phosphorylates and activates the extracellular signal-regulated kinases (ERK). This leads to the phosphorylation of the transcription factor, Elk, and the induction of Fos expression. Without E R K activation, B cell proliferation does not occur (Richards et al., 2001). 1.0.4 The role of CXCR4 in B cell function 6 Chemokines are small chemoattractant proteins that stimulate the migration and activation of cells. In B cells, the chemokine SDF-1 retains B cell progenitors in the bone marrow microenvironment and allows for B cell maturation. SDF-1, SLC, and ELC recruit B cells into lymphoid organs while B L C recruits activated B cells into lymphoid follicles where they proliferate (Okada et al., 2002). Finally SDF-1 recruits plasma cells back to the bone marrow where they can survive for long periods of time (Hargreaves et al., 2001). In this thesis, I will focus on the signalling by the receptor for SDF-1, CXCR4. Originally called leukocyte-derived seven-transmembrane domain receptor (LESTR), CXCR4 was isolated in 1994 by Loetscher et al. (Loetscher et al., 1994). At the time, much research was focused on chemotactic agonists, such as interleukin-8 (IL-8) and related cytokines, and how they could attract leukocytes by binding to seven-transmembrane domain, G protein-coupled receptors. CXCR4 was such a receptor. Today, CXCR4 is most often recognized as the entry cofactor for T cell line-trophic (T-trophic) Human Immunodeficiency Virus-1 (HIV-1) (Hori et al., 1998) and the receptor that can bind to the chemokine, stromal cell-derived factor-1 (SDF-1) Briefly, CXCR4 is a seven-transmembrane domain receptor coupled to heterotrimeric Gj proteins. It was named after the type of chemokine to which it binds. Chemokines, such as SDF-1, are potent chemoattractants and are named according to the position of their first two conserved and closely paired cysteines (CXC, C X 3 C , CC, or C) (Murdoch, 2000). As SDF-1 binds LESTR exclusively (Baggiolini, 1998) and is grouped under the superfamily of C X C chemokines, LESTR was redesignated as CXCR4. The interaction between CXCR4 and SDF-1 is quite important for many reasons. First, CXCR4 is expressed by all B lymphocytes and monocytes, expressed by most T lymphocytes, and weakly expressed in natural killer cells (NK cells) (Hori et al., 1998). SDF-1, formerly known as pre-B-cell growth-stimulating factor (PBSF), is secreted by bone marrow stromal cells 7 (D'Apuzzo et al., 1997). CXCR4 knockout mice are deficient in B cell progenitors in the fetal liver and bone marrow (Ma et al., 1999). SDF-1 knockout mice also contain severely reduced B-cell progenitors in the fetal liver and bone marrow (Nagasawa et al., 1996) which suggests that both the ligand and the receptor have similar functions. Together, it has been shown that SDF-1 and CXCR4 are responsible for confining B cell precursors within the bone marrow environment and promoting their survival, which allows them to mature and differentiate (Baggiolini, 1998; Bleul et al., 1996; DApuzzo et al., 1997). SDF-1 and CXCR4 have also been implicated in recruiting mature B cells into lymphoid organs and plasma cells to the bone marrow (Hargreaves et al., 2001; Okada et al., 2002). Because the CXCR4 receptor is responsible for a multitude of B cell functions (Murdoch, 2000) , it is important to understand how these signals are conveyed upon activation of the receptor. 1.0.5 Signal transduction via CXCR4 Most chemokine receptors, like CXCR4, signal through heterotrimeric G proteins (Baird et al., 1999; Mohle et al., 2001; Ward and Westwick, 1998), which consist of three subunits: Ga , G(3, and Gy (See Appendix II). CXCR4 likely signals via Ga, since SDF-1-induced migration is blocked by pertussis toxin, a toxin which prevents the activation of Ga; (Thelen, 2001). When chemokine receptors are in their inactive state, the G a subunit is GDP-bound and the complex is not associated with the receptor. Upon chemokine signalling, the trimeric G protein complex associates with the receptor, and the GDP bound to the G a subunit is displaced by GTP. The heterotrimeric protein then dissociates into two components: GTP bound G a and G[3y (Thelen, 2001) . The two subunits then go and activate other effector molecules such as PLC and PI3K. In T cells, SDF-1 stimulation leads to the activation of PLC and P K C (Yonezawa et al., 2000). As a result of PLC activation, intracellular C a 2 + is increased, D A G is produced, and 8 ERK2 is activated. In other cell lines - human megakaryoblasts, human platelets, and hematopoietic progenitors - SDF-1 signalling activates Akt, focal adhesion components (RAFTK, Cas, Paxillin, F A K , Crk, CrkL), PI3K, ERK, SHP2, SHIP, Cbl, Fyn, and N F - K B (Chernock et al., 2001; Majka et al., 2000; Tilton et a l , 2000; Wang et al., 2000). Akt activation promotes cell survival by inactivating several apoptosis-mediating proteins such as B A D , and caspase-9. Activation of focal adhesion components is important in cell migration because it is responsible for the formation of focal adhesions and the reorganization of actin cytoskeleton. Other proteins such as SHP2, Cbl, and Fyn are also involved in CXCR4 signalling and may act as key mediators of SDF-1-induced responses. Whether all of these proteins are also activated in B lymphocytes has not been confirmed. In this thesis, I wil l show that CXCR4 activates the Rapl and Rap2 GTPases, which are members of the Ras family of GTPases. 1.1 Ras and Ras-like proteins The Ras superfamily of proteins are small, 20-25kDa GTPases that regulate a wide variety of cellular functions. The superfamily is subdivided into several subfamilies based on the similarities that exist in their effector domain: Ras, Rho/Rac, Rab, Ran, Rad, and Arf (Geyer and Wittinghofer, 1997; Zwartkruis and Bos, 1999). Each subfamily seems to have distinct biological functions. For example, the Rab, Arf, and Arf-like GTPases are involved in the assembly, loading, targeting, budding and fusion of vesicles to their appropriate cellular compartments. The Rac/Rho subfamily controls cytoskeletal organization, mitogenesis, transformation, protein kinase cascades, phospholipid metabolism and transcriptional regulation. Lastly, Ran GTPases are necessary for nuclear transport (Macara et al., 1996; Rebhun et al., 2000). Hence Ras and Ras-like proteins control a multitude of functions in cells. 9 Rap proteins belong to this subfamily of Ras proteins, and Rapl was first identified as a putative antagonist of Ras signalling (Kitayama et al., 1989). Therefore I will first discuss Ras and Ras signalling. 1.1.1 The importance of Ras and Ras-family members Originally, Ras GTPases were intensely studied because 10-20% of human tumors contain mutated versions of these genes (Bos, 1998; Macara et al., 1996), and it was speculated that Ras proteins somehow contributed to the aberrant regulation of growth stimulatory signalling pathways in these cells (Campbell et al., 1998). Now it is known that Ras proteins are part of an important signalling pathway of protein kinases, as described before, that often link surface receptor signalling to gene expression. By being situated at the inner surface of the plasma membrane (Campbell et al., 1998) where most receptors are located, they serve as signal transducing switches that control cell growth, transformation, differentiation, and apoptosis (Bos, 2001; Kolch, 2000). Presently, more and more signalling pathways are being discovered that emanate from Ras. Activated forms of Ras interact with many downstream molecules. Some of these include Rafl , A-Raf, RalGDS, PI3K, RalGDS-like factor (Rlf), Rgl, and Rin l (Bos, 1998; Macara et al., 1996; Zwartkruis and Bos, 1999). Rafl and A-Raf regulate the E R K signalling pathway. Rlf, RalGDS and Rgl are members of the RalGEF family and serve to connect Ras to the small GTPase Ral. PI3K, as mentioned before, is involved in generating PI(3,4,5)P3, which regulates the activity of proteins involved in cell motility, cell cycle control, and cell differentiation. The function of Rin l is unknown. Other Ras subfamily members include R-Ras, TC21/R-Ras2, M-Ras/R-Ras3, Rapl A , Rap IB, Rap2A, Rap2B, RalA, RalB, Rit, Rin, Dex-Ras, Rheb, Rhes, NOEY2, KB -Rasl , K B -Ras2, Ha-Ras, K-Ras, and N-Ras (Rebhun et al., 2000). Not a lot is known about these Ras-like 10 proteins relative to what we know about Ras. However, their interactions with downstream effectors often hint at their possible role in cell function. These downstream effectors are however often shared, and cross-talk may occur. 1.1.2 GTPases Proteins that belong to the GTPase superfamily are viewed as molecular switches that can be turned on by binding to GTP and turned off by hydrolysing GTP back to GDP. In this cycle, GTPases have three conformational states. The first is a GDP-bound inactive state. When the GDP is released, the GTPase is in its empty state. Under physiological conditions, the GTPase is now more likely to take up GTP in its empty guanine nucleotide binding site than GDP because of the higher concentration of GTP within the cytosol (Bourne et al., 1991). This overall reaction can be driven forward by the excess of GTP over GDP in the cytoplasm, by a greater affinity of the GTPase for GTP than GDP, or with the help of other regulatory molecules, which wil l be described below. Upon GTP binding, the GTPase is now in its active state until it reverts back to its inactive state when GTP is hydrolysed. There are many known classes of GTPases. A l l of them share a conserved fold and a general mechanism of action (Corbett and Alber, 2001). 1.1.3 GNEFs and GAPs As described from above, there are regulatory proteins that can facilitate and catalyze the release of bound GDP from GTPases and promote its replacement by GTP. These proteins are known as GNEFs. Conversely, GAPs are regulatory proteins that accelerate GTP hydrolysis by GTPases. GNEFs and GAPs for Ras may not exclusively regulate Ras; they may also regulate other Ras-like proteins as well. GNEFs that have been reported to catalyze GDP-GTP exchange on Ras are SOS, RasGRF, and RasGRP (Bos, 1998; Zwartkruis and Bos, 1999). Finally, GAPs that 11 have been reported to stimulate the intrinsic GTPase ability of Ras are RasGAP and neurofibromin (NF1) (Campbell et al., 1998; Zwartkruis and Bos, 1999). 1.2 Rap l and Rap2 Presently, four Ras-related Rap proteins have been identified: RaplA, RaplB, Rap2A, and Rap2B (Reuther and Der, 2000). RaplA, a 21 kDa protein, was discovered by Kitayama et al. in 1989 via its ability to apparently reverse the transformed phenotype of human fibroblasts expressing activated forms of Ras (Kitayama et al., 1989). Since Rap proteins are very similar in structure to Ras, it was suggested at the time that Rap might antagonize Ras-induced responses by competing with its effectors. Additionally, there was also the presumption that Rap proteins may be involved in growth control. However, over-expressed Rap2 does not share the same characteristic in Ras-transformed murine fibroblasts as RaplA, nor does it interfere with Ras-induced transformation (Jimenez et al., 1991). Recently, experiments looking at the over-expression of Rapl in other cell lines did not observe interference or antagonism within the Ras signalling pathway (Zwartkruis et al., 1998). The ability of over-expressed activated Rapl to block the Ras -> Erk pathway may just be an artefact generated by the high concentration of constitutively active Rapl. It is believed that under physiological conditions, even maximal levels of endogenous activated Rapl could never sequester all the Ras effectors away from Ras. Since many GTPases have important signalling functions, our laboratory has investigated the role of Rap in B cell signalling. A short description of Rap proteins is included in order for the reader to understand the molecular experiments explained later on in the thesis. 1.2.1 General characteristics of Rapl and Rap2 RaplA, RaplB, Rap2A and Rap2B proteins share approximately 50% amino acid sequence identity with Ras (Janoueix-Lerosey et al., 1992; Jimenez et al., 1991) (See Appendix IU). More specifically, RaplA and RaplB are 95% identical (Janoueix-Lerosey et al., 1998), 12 Rap2A and Rap2B are 90% identical (Nancy et al., 1999), and Rapl and Rap2 proteins are 60% identical in terms of sequence identity (Beranger et al., 1991; Jimenez et al., 1991; van den Berghe et al., 1999). In terms of structural organization, Rapl proteins have the exact same effector region as Ras, but have different flanking amino acids. Because the effector region confers the ability of Ras to interact with its unique set of effector molecules, it was believed that Rapl could also interact with this same set of molecules. The effector domain in Rap2 differs from Rapl by one amino acid substitution at position 39 - phenylalanine for serine. However, this amino acid substitution has no effect on Rap proteins since it has been shown experimentally that Rap lA altered in this way still displays the same set of properties (van den Berghe et al., 1999). As to cellular localization, it is known that Ras proteins are located at the plasma membrane whereas Rap proteins are concentrated near nuclear membranes (Bos, 2001). Due to the structural similarities and a common effector region, one might expect that Rap and Ras proteins would have a shared set of regulatory proteins and effector molecules. For instance, a GNEF specific for both Rapl and Ras is RasGRP2 (Clyde-Smith et a l , 2000), and effector molecules that interact with both Ras and Rap are Raf, PI3K, and Rl f (Janoueix-Lerosey et al., 1998; Nancy et al., 1999; Traver et al., 2000). However, the similarities end here. More and more regulatory proteins and effector molecules are being discovered that are only specific for Rap proteins and not Ras, whereas some are specific for Rap and other Ras-like proteins. For example, PDZ-GEF1 is a GNEF specific for Rapl and Rap2 but not Ras (de Rooij et al., 1999). Even amongst the Rap-associated proteins, some of these regulatory proteins and effector molecules have been characterized to interact with Rapl but not Rap2 and vice versa. An example would be Rap2-interacting protein 8 (RPIP8), which interacts exclusively with Rap2 but not Rapl or Ras (Janoueix-Lerosey et al., 1998). The other variable to consider is that some of these regulatory molecules and effector molecule interactions with Rap proteins only occur in certain cell types because of cell-type specific expression. 13 At present time, GNEFs that are known to activate both Rapl and Rap2 are Epacl, Epac2 (de Rooij et al., 2000; de Rooij et al., 1998), and PDZ-GEF1 (de Rooij et al., 1999). Other GNEFs that have been shown to activate Rapl are CalDAG-GEFI (Clyde-Smith et al., 2000), CalDAG-GEFIII (Yamashita et al., 2000), C3G (van den Berghe et al., 1999), smgGDS (Ichiba et al., 1997), and guanine nucleotide exchange factor for Rapl (GFR) (Ichiba et al., 1999). It is not known whether these GNEFs also activate Rap2. GAPs that can inactivate Rapl and Rap2 are RapGapI (Janoueix-Lerosey et al., 1992), RapGapH (Mochizuki et a l , 1999; McLeod, L i et al., JI, 2002) and Spa-1 (Hattori et al., 1995; Nur-E-Kamal, 1996; Tsukamoto et al., 1999; Wada et al., 1997). Another GAP that has been reported to regulate Rapl is tuberin (Wienecke et al., 1995). While the Rap proteins share a structural similarity with Ras, their effector regions are almost exactly the same. The ability of Rap proteins to compete with Ras effectors was inferred from this observation. For example, activated forms of Rapl can bind to the Ras effector molecules: Raf, Rlf, and PI3K (Nancy et al., 1999; Traver et al., 2000; van den Berghe et al., 1999). However, Rapl cannot bind to all of the subset of Ras effectors. In addition, Rapl has its own subset of effectors that includes Ral-GEF (van den Berghe et al., 1999), RalGDS, and B-Raf. Rap2 can also bind RalGDS, but it has its own subset of effectors as well which includes RPIP8 (Janoueix-Lerosey et a l , 1998) and RGS14 (Traver et al., 2000). 1.2.2 Known functions of Rap proteins Despite the numerous studies looking at Rap protein effectors, regulatory proteins, and activators, the functions of Rapl and Rap2 have not been fully elucidated. Much of what is known has come from the study of Rapl. Rapl has been implicated in the control of platelet activation, T cell anergy, neuronal differentiation, cell migration, and cell adhesion. 14 Rapl has been found to be expressed at high levels in human neutrophils and human platelets. Rapl is activated by a large variety of stimulating agents in neutrophils: fMLP, PAF, GM-CSF, phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), and IgG-coated particles (M'Rabet et al., 1998). Stimulation of human platelets by von Willebrand factor (vWF) causes the activation of RaplB (Franke et al., 2000; Torti et al., 1999). Zwartkruis et al. have also tested for the ability of Rapl proteins to be activated in a variety of cell lines. In these studies, Zwartkruis et al. showed that Rapl is activated by a variety of growth factor receptors including receptor tyrosine kinases such as the platelet-derived growth factor (PDGF) receptor and epithelial growth factor (EGF) receptor, and G protein-coupled receptors including the receptors for lysophosphatidic acid (LPA), thrombin, and endothelin (Zwartkruis et al., 1998). York et al. showed that Rapl was involved in nerve growth factor signalling, which is important for neuronal differentiation (York et al., 1998). More specifically, protein kinase A (PKA) activation is required for Rapl activation and this leads to B-Raf activation, sustained activation of ERK, and neuronal differentiation via an ERK-dependent pathway (Vossler et al., 1997). In T lymphocytes, activated Rapl blocks IL-2 gene transcription (Boussiotis et al., 1997). This is consistent with the later observation that Rapl is activated in anergic T cells (Carey et al., 2000) that have been stimulated by TCR engagement alone in the absence of co-stimulatory signals. If the co-stimulatory molecule, CD28, is engaged, Rapl activation was blocked. Increased Rapl expression has been shown to alter cell morphology in Dictyostelium discoideum by altering cytoskeletal function (Rebstein et al., 1997). In contrast, Rapl loss-of-function mutations in Drosophila disrupt cell migrations and cause abnormalities in cell shape during morphogenesis of cells in the eye disk (Asha et al., 1999). 15 Lately, multiple studies have shown that Rap proteins regulate cell adhesion. For example, activated Rapl promotes cell adhesion in Hela cells, and inhibiting Rap activation via the expression of Spa-1 Rap-GAP opposes adhesion (Tsukamoto et al., 1999). Suga et al. have demonstrated that activated Rapl leads to the activation of the P2-integrin LFA-1 , which binds intercellular adhesion molecule-1 (ICAM-1) (Suga et al., 2001). Activated Rapl also increases T cell adhesion to the LFA-1-ligand I C A M . In addition, Rap activation regulates the avidity of the V L A - 4 (oc4Pi) integrin which binds vascular cell adhesion molecule-1 (VCAM-1) (Reedquist et al., 2000). In related studies, Rapl was found to positively regulate integrin activation in T cells by promoting the clustering of LFA-1 and V L A - 4 (Katagiri et al., 2000; Sebzda et al., 2002). The ability of Rap-GTP to promote integrin activation in T cells suggests that it could be important for T cell extravasation. Despite the many studies on Rapl, not much is known about Rap2. It is clear that certain cellular properties of Rap2 are markedly different from Ras and Rapl. As mentioned before, overexpression of Rap2 does not oppose Ras-transformation of murine fibroblasts whereas Rapl does. While activated Rapl and Rap2 share common effectors, they also activate their own subset of effectors. A l l these facts suggest that there may be biological differences between Rapl and Rap2. 1.2.3 Rap proteins in B lymphocytes In previous experiments conducted in our laboratory, it was shown that BCR engagement activates Rapl , but that Rapl does not modulate Ras signalling (McLeod and Gold, 2001; McLeod et al., 1998). The BCR activates Rapl via PLC-y2-dependent production of D A G . Rapl activation is greatly reduced in PLC-y2 deficient variant of the DT40 chicken B cell line. Recent experiments in our laboratory have also shown that Rapl and /or Rap2 promote B cell adhesion and migration towards chemokines (Gold et al., 2000; McLeod, L i et al., JI, 2002). 16 1.3 WEHI-231, A20, and 2PK3 B cell lines as model systems Three mouse monoclonal cell lines were employed to study Rap activation in B cells. B cell lines offer excellent models for the study of signal transduction and the biochemistry of signalling proteins. This is because B cell lines are easy to grow to large numbers and have been extensively characterized and widely used for signalling studies. The use of a mouse model is applicable since Rap proteins are conserved across species. For example, human and mouse Rapl proteins are 88% identical in terms of amino acid sequence. Rap2 proteins between the two species are also 88% identical. Thus, results in these mouse cell lines can be used to infer similar cellular processes that take place in humans and other mammalian systems. A brief description of three B cell lines used in the following study is described below. 1.3.1 WEHI-231 cells WEHI-231 is a mouse tumor cell line that expresses surface IgM, but does not secrete immunoglobulin (Ig) (Lanier and Warner, 1981) unless stimulated with lipopolysaccharide (LPS). The special feature about these immature B cells is that when their BCRs are cross-linked with anti-Ig antibodies used as a surrogate for antigen, they will undergo growth arrest and apoptosis (Boyd et al., 1981). This characteristic of WEHI-231 makes them the favourite model system to study negative selection (King and Monroe, 2000) of self-reactive B cells as immature B lymphocytes going through development in the bone marrow undergo a similar process of growth arrest and cell death when their prototypic receptors bind strongly to self antigens. Rap proteins are known to regulate B cell migration towards the chemokines SDF-1 (McLeod, L i , et al., JI, 2002), and this process is thought to be important for confining immature B cells with stromal cells within the bone marrow microenvironment. Therefore, WEHI-231 cells are also a good model for SDF-1 signalling studies. 17 1.3.2 A20 cells The A20 B cell lymphoma line was first characterized by Kim et al. (Kim et al., 1979). These mature B cells express IgG and were derived from a BALB/c inbred mouse. As A20 B cells have been used previously for adhesion studies, they will be employed to study Rap proteins as Rap proteins are implicated in the activation of integrins. 1.3.3 2PK-3 cells The 2PK-3 B cell lymphoma line was also derived from a BALB/c inbred mouse. 2PK-3 s express IgG2 at the surface and are representative of mature B lymphocytes prior to the plasma cell stage of differentiation (Warner et al., 1979). 2PK-3 have been utilized in migration studies (Reif and Cyster, 2000) as they respond to SDF-1 and other chemokines. Hence, this cell line will also be used for the study of Rap proteins. 1.4 Thesis objectives The role of Rap proteins in regulating cell function is not completely understood. Not much is known about their role in B cells. Importantly, a lot of work has been done on Rapl but little is known about Rap2. The first objective of this thesis was to characterize the activation of Rap2 proteins by B C R cross-linking and CXCR4 stimulation in B cells. Previous experiments have shown that anti-Ig antibodies and SDF-1 can activate Rapl in B cells. The goal was to determine i f there is coordinate regulation of Rapl and Rap2 proteins. If there is, this suggests that maybe Rapl and Rap2 possess the same upstream activators. The second objective was to elucidate how B C R cross-linking activates Rap proteins. Phorbol 12,13-dibutyrate (PdBu) and a PLC inhibitor were used to test the hypothesis that Rap2 is activated by PLC-dependent production of D A G , as is the case for Rapl in B cells. 18 The third objective was to develop loss of function approaches to study the roles of Rapl and Rap2 in B cells. As stated before, many GAPs regulate Rap proteins. Two Rap-GAPs were expressed in B cell lines in order to inhibit Rap activation and to determine i f both suppress Rapl and Rap2 or if they had differential effects on Rap proteins. This would allow us to explore the roles of Rapl and Rap2 in B cells. Note: Some of the work in this thesis (SDF-1-induced activation of Rap2) has been published in Sarah J. McLeod, Anson H . Y . L i , Rosaline L. Lee, Anita E. Burgess, and Michael R. Gold, The Rap GTPases Regulate B Cell Migration Toward the Chemokine Stromal Cell-Derived Factor-1 (CXC Chemokine Ligand 12): Potential Role for Rap2 in Promoting B Cell Migration, Journal of Immunology, 2002, In Press. 19 C H A P T E R T W O M A T E R I A L S A N D M E T H O D S 2.0 Antibodies and other reagents Goat anti-mouse IgM (u. chain specific) and rabbit anti-mouse IgG (Fey fragment specific) antibodies used for B C R ligation were purchased from Jackson ImmunoResearch Laboratories (Mississauga, Ontario, Canada). Mouse SDF-1 a used to stimulate the CXCR4 receptor was obtained from R & D Systems (Minneapolis, MN), and the phorbol ester, PdBu, was procured from Sigma (Oakville, Ontario, Canada). The PLC inhibitor, l-[6-[[ 17(3-3-Methoxyestra-l,3,5(10)-trien-17-yl]amino]hexyl]-lH-pyrrole-2,5-dione (U73122), and its negative control and structural analogue, l-[6-[[17P-3-methoxyestra-l,3,5(10)-trien-17-yl]amino]hexyl]-lH-pyrrole-2,5-dione (U73343), were obtained from Biomol (Plymouth • Meeting, PA). Glutathione Sepharaose 4B beads used for immunoprecipitation studies were acquired from Amersham Biosciences (Baie d'Urfe, Quebec, Canada). The anti-Rap 1 rabbit polyclonal IgG was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and the anti-Rap2 mouse monoclonal IgG2a was obtained from BD Transduction Laboratories (Mississauga, Ontario, Canada). The secondary reagents used for chemiluminescence, protein A-horseradish peroxidase (HRP) and goat anti-mouse-HRP, were acquired from Bio-Rad (Hercules, CA). The western blotting detection reagent, ECL, was purchased from Amersham Biosciences. 2.1 B cell lines 2.1.1 WEHI-231. A20. 2PK-3 cells The WEHI-231, A20, and 2PK-3 B cell lymphomas have been described previously (Kim et al., 1979; Lanier and Warner, 1981; Warner et a l , 1979). A l l B cell lines can be obtained from American Type Culture Collection (Rockville, MD). 20 2.1.2 pMSCV RapGapTJ-expressing B cell lines The cDNA for FLAG-tagged RapGapH (Mochizuki et al., 1999) in the pMSCV retroviral vector was a gift from Dr. Michiyuki Matsuda (Osaka University, Japan). The empty pMSCV vector or the pMSCV RapGapU vector was transfected into BOSC23 packaging cell lines and the resulting virus particles were used to infect WEHI-231 and 2PK-3 cells as described (Krebs et al., 1999). After selection in puromycin, stable bulk populations of cells were maintained in the presence of puromycin. A20 cells were transfected by electroporation. Individual puromycin-resistant clones were selected, expanded, and maintained in medium containing puromycin. Transfection of the WEHI-231 and 2PK-3 B cells was done by Sarah McLeod from our laboratory whereas the A20 transformants were prepared by May Dang-Lawson from our laboratory. FLAG-tagged RapGapII expression was assessed by immunoblotting with anti-F L A G M2 monoclonal antibodies (Sigma) and with anti-RapGapU antibodies (Mochizuki et al., 1999). 2.1.3 p L X S N Spa-1-expressing B cell lines The cDNA for FLAG-tagged Spa-1 (Hattori et al., 1995) in the p L X S N retroviral vector was a gift from Dr N . Minato (Kyoto University, Japan). This vector or the empty p L X S N vector was transfected into BOSC23 cells and used to generate retroviruses for infection of WEHI-231 cells. Again electroporation was used for A20 cells. Selection was achieved in medium containing G418. FLAG-tagged Spa-1 expression was assessed by immunoblotting with anti-FLAG M2 monoclonal antibodies and with anti-Spa-1 antibodies, which was prepared by Blazej Szczygielski from our laboratory. 2.1.4 pMX-PIE Spa-1-expressing B cell lines 21 The pMX-PIE vector was a gift from Dr. A . Mui (University of British Columbia, British Columbia, Canada). The cDNA for FLAG-tagged Spa-1 was subcloned into the vector by Sarah McLeod and used for retroviral-mediated gene transfer into 2PK-3 B cells. 2.2 Cell culture The WEHI-231, A20, and 2PK-3 murine B cell lines were grown in RPMI-1640 (Gibco, Grand Island, N Y ) supplemented with 10% heat-inactivated fetal calf serum (Hyclone, Logan, UT), 2 m M L-glutamine, 100 m M sodium pyruvate, 50 u M 2-mercaptoethanol, 50 units per ml of penicillin-G, and 50 \ig per ml of streptomycin (Pen/Strep, Gibco). To reduce the basal signalling background in WEHI-231 cells, these cells were grown overnight in medium containing 1% heat-inactivated fetal calf serum before performing the Rap activation assays. 2.3 Rap activation assay Cells were resuspended to 2.5 x 10 per ml in modified Hepes-buffered saline (Saxton et al., 1994), and stimulated with 100 u.g per ml anti-mouse Ig antibodies, 100 ng per ml (12.5 nM) recombinant murine SDF-1, or 100 nM PdBu. In studies using the PLC inhibitor, U73122, and its structural analogue, U73343 (Bleasdale et al., 1990; Smith et al., 1990), cells were pre-treated with these compounds at a final concentration of 20 uM for 20 minutes prior to stimulation. The cell suspension was solubilized in an equal volume of Rap lysis buffer (50 m M Tris-H C l pH 7.4, 10% glycerol, 1% IGEPAL, 200 m M NaCl, 2 m M M g C l 2 , 1 m M vanadate, 1 m M phenylmethylsulfonyl fluoride, 10 ug per ml leupeptin, 1 p.g per ml aprotinin) to terminate the reaction and placed on ice for 10 minutes. Detergent-insoluble material was removed by centrifugation at 14,400 R P M for 15 minutes at 4°C, and cell lysates were used for the Rap activation assay as described below. 22 The assay uses a GST-RalGDS fusion protein to selectively precipitate the active GTP-bound forms of Rapl and Rap2. The preparation of GST-RalGDS has been described before (McLeod et al., 1998). Fifteen microlitres of the fusion protein was used per sample and mixed with 20 ul of glutathione Sepharose 4B beads in the cold for one hour. The beads were subsequently washed twice with Rap lysis buffer before being mixed with the cell lysates in the cold for another hour. The beads were then washed three times with Rap lysis buffer to wash away unbound proteins. Bound proteins were then eluted with SDS-PAGE sample buffer containing 100 m M dithiothreitol. 2.4 Protein analysis 2.4.1 SDS-PAGE and western blotting Eluted proteins were separated using 1.5 mm-thick SDS-PAGE mini-gels and transferred to a nitrocellulose filter (Schleicher & Schuell, Keene, NH). Molecular mass standards were visualized by staining with Ponceau S (Sigma). The filters were blocked with 5% milk powder in TBST (10 m M Tris-HCl, pH 8, 150 m M NaCl)/0.1% Tween 20 at room temperature for one hour. Mouse anti-Rap2 antibodies were diluted to 0.1 (ig per ml in 5% milk powder in TBST, added to the filters and incubated overnight at 4°C. The filters were washed with TBST for 30 minutes at room temperature and then incubated with goat anti-mouse-HRP antibodies diluted at 1:2000 in 5% milk powder in TBST for 30 minutes at room temperature. The filters were washed once again with TBST for 30 minutes at room temperature and visualized by enhanced chemiluminescence detection. Filters were subsequently reprobed by first removing the bound antibodies on the filter by washing the filter with several changes of stripping solution (10 m M Tris-HCl, pH 8, 150 m M NaCl, pH adjusted to 2.0) for one hour. The filters were again blocked with 5% milk powder in TBST at room temperature for one hour. Rabbit anti-Rapl antibodies were diluted to 0.5 ug per ml in 5% milk powder in TBST and added to the filters and incubated 23 overnight at 4°C. The filters were washed with TBST for 15 minutes at room temperature and then incubated with Protein A-HRP diluted at 1:2500 in 5% milk powder in TBST for 45 minutes at room temperature. The filters were washed with TBST for 15 minutes at room temperature and finally visualized by enhanced chemiluminescence (ECL) detection. 2.4.2 Quantitation and statistical analysis Immunoreactive bands corresponding to activated Rap proteins were visualized by ECL. The X-ray films were scanned and saved as TIFF files. Densitometry was performed using ImageQuant (Molecular Dynamics, Sunnyvale, CA) software to analyze the intensity of the bands. Each experiment was repeated at least twice. 24 CHAPTER THREE ACTIVATION OF THE RAP2 PROTEINS IN B LYMPHOCYTES 3.0 Rationale and summary Previous work in our laboratory showed that Rapl is activated when B lymphocytes are stimulated via the B C R or CXCR4. The aim of this chapter was to determine whether these receptors also activated Rap2 and to determine i f they did so via a similar signalling pathway. McLeod et al. have shown that Rapl is activated in B lymphocytes after B C R ligation (McLeod and Gold, 2001) via PLC-y2-dependent production of the second messenger D A G (McLeod et al., 1998). This was demonstrated using a PLC-y2-deficient variant of the DT40 chicken B cell line. BCR-induced activation of Rapl in PLC-y2-deficient DT40 cells was significantly reduced when compared to the wild-type control. Addition of PdBu, a phorbol ester that mimics the action of D A G , into these cells caused Rapl activation in these B cell lines (McLeod et al., 1998), suggesting that D A G mediates Rap activation. The other PI(4,5)P2-derived second messenger does not activate Rap in B cells (McLeod et a l , 1998). Many chemokine receptors are coupled to heterotrimeric Gj proteins and signal via PLC-P activation, and this ultimately leads to an increase in intracellular D A G (Thelen, 2001). Our laboratory showed that SDF-1-induced signalling via CXCR4, a chemokine receptor found on B lymphocytes, also activates Rapl (McLeod, L i , et al., JI, 2002). This work indicated that activation of the Rap GTPases is important for B cell migration in response to SDF-1 binding to CXCR4 (McLeod, L i , et al., JI, 2002). Since Rapl and Rap2 are closely related and regulated by similar GNEFs and GAPs, I hypothesized that B C R cross-linking and CXCR4 stimulation would also activate Rap2 via a PLC-y2 -> D A G pathway. In this chapter, I show that anti-Ig, SDF-1, and PdBu all activated 25 Rap2 in B cells with kinetics similar to that for Rapl activation. I also show that the B C R activated Rap2 via PLC-y2 and that Rap2 activation could be inhibited by PLC inhibitor, U73122, but not its structural analogue, U73343. 3.1 Activation of Rap2 by anti-Ig antibodies In order to characterize Rap2 activation in B lymphocytes, I stimulated WEHI-231 and A20 cells with anti-Ig antibodies in order to cross-link the BCR. A 60 minute Rap activation time course was done to look at the kinetics of Rap2 activation, Rap activation was assessed using GST-RalGDS to pull down the activated GTP-bound forms of Rap proteins. After SDS-P A G E and transfer to nitrocellulose, filters were immunoblotted for Rap2 and then reprobed for Rapl in order to detect the activated Rapl and Rap2. Figure 3.1 and 3.2 show that Rap2 was activated in WEHI-231 cells and A20 cells after B C R engagement. Rap2 activation was evident after two minutes of stimulation, was maximal at 10 to 30 minutes, and was still elevated even after one hour. Rapl was also activated with similar kinetics. 26 A WEHI-231 cells GST-RalGDS pull down Anti-IgM (min) 0 2 5 10 15 30 60 21.5 H •Rap2-GTP Anti-Rap2 Blot B WEHI-231 cells GST-RalGDS pull down Anti-IgM (min) 0 2 5 10 15 30 60 21.5 n Anti-Rap1 Blot •Rap1-GTP Figure 3.1. Ant i - IgM activates Rap l and Rap2 in WEHI-231 cells. WEHI-231 cells were stimulated with 100 [ig/ml anti-IgM antibodies for the indicated times. A G S T - R a l G D S fusion protein was used to selectively precipitate the activated GTP-bound forms of the Rap proteins. Western blotting was used to detect the activated forms of (A) Rap2 and (B) R a p l . Molecular mass markers (in kDa) are shown to the left. Similar results were obtained in two independent experiments. 27 A A20 cells GST-RalGDS pull down Anti-IgG (min) 0 2 5 10 15 30 60 21.5 H •Rap2-GTP B Anti-Rap2 Blot A20 cells GST-RalGDS pull down Anti-IgG (min) 0 2 5 10 15 30 60 21.5 H •Rap1-GTP Anti-Rap1 Blot Figure 3.2. Anti-IgG activates Rapl and Rap2 in A20 cells. A20 cells were stimulated with 100 p.g/ml anti-IgG antibodies for the indicated times. A GST-RalGDS fusion protein was used to selectively precipitate the activated GTP-bound forms of the Rap proteins. Western blotting was used to detect the activated forms of (A) Rap2 and (B) Rapl. Molecular mass markers (in kDa) are shown to the left. Similar results were obtained in two independent experiments. 28 3.2 Activation of Rap2 by SDF-1 Because B lymphocytes migrate towards SDF-1 and Rap proteins are involved in adhesion in T cells and migration in Drosophila, we speculated that Rap proteins would also play a role in B cell migration. Our laboratory had previously shown that SDF-1 activates Rapl in WEHI-231 and 2PK-3 cells (McLeod, L i , et a l , JI, 2002). To determine whether SDF-1 also activates Rap2 GTPases, we stimulated WEHI-231 and 2PK-3 cells with SDF-1 and assayed Rap activation as in previous experiments. These experiments showed that Rapl and Rap2 were activated in WEHI-231 and 2PK-3 cells after SDF-1 stimulation. Both Rapl and Rap2 were activated within two minutes and remained activated for 30 minutes. The kinetics of Rapl and Rap2 activation were similar. Maximal levels of Rap activation occurred between 10 to 30 minutes although there was some variability as to when this was achieved. It is possible that Rap proteins are continuously cycling between the activated and inactivated states during this time where a majority of the Rap proteins are activated. This would account for the lack of consistency in identifying maximal levels of Rap activation at a specific moment in time. Interestingly in 2PK-3 cells, the activated Rap2 appeared as a doublet. It is unknown what this doublet represents. It could be that these two proteins are Rap2A and Rap2B. In summary, Figures 3.3 and 3.4 show that Rap2 is activated by SDF-1. The kinetics of Rap2 activation were similar to BCR-induced activation of WEHI-231 and A20 cells. This suggested that Rapl and Rap2 were activated co-ordinately. 29 WEHI-231 cells GST-RalGDS pull down SDF-1 (min) 0 2 5 10 15 30 60 21.5 •Rap2-GTP Anti-Rap2 Blot B WEHI-231 cells GST-RalGDS pull down SDF-1 (min) 0 2 5 10 15 30 60 21.5 H •Rap1-GTP A n t i - R a p ! B l o t were Figure 3.3. SDF-1 activates Rapl and Rap2 in WEHI-231 cells. WEHI-231 cells stimulated with 100 ng/ml (12.5 nM) SDF-1 for the indicated times. A GST-RalGDS fusion protein was used to selectively precipitate the activated GTP-bound forms of the Rap proteins. Western blotting was used to detect for the activated forms of (A) Rap2 and (B) Rapl. Molecular mass markers (in kDa) are shown to the left. Similar results were obtained in two independent experiments. 30 A 2PK-3 cells GST-RalGDS pull down SDF-1 (min) 0 2 5 10 15 30 60 21.5 H B Anti-Rap2 Blot 2PK-3 cells GST-RalGDS pull down • R a p 2 - G T P SDF-1 (min) 0 2 5 10 15 30 60 21.5 H •Rap1 -GTP Anti-Rap! Blot Figure 3.4. SDF-1 activates Rapl and Rap2 in 2PK-3 cells. 2PK-3 cells were stimulated with 100 ng/ml (12.5 nM) SDF-1 for the indicated times. A GST-RalGDS fusion protein was used to selectively precipitate the activated GTP-bound forms of the Rap proteins. Western blotting was used to detect for the activated forms of (A) Rap2 and (B) Rapl . Molecular mass markers (in kDa) are shown to the left. Similar results were obtained in two independent experiments. 31 3.3 Mechanism of BCR-induced Rap2 activation We showed previously that BCR-induced Rapl activation was via a B C R PLC-y2 -> D A G -> Rapl pathway. Since Rapl and Rap2 are regulated by similar GEFs and GAPs (de Rooij et al., 2000; Hattori et al., 1995; Ichiba et al., 1997; Mochizuki et al., 1999; Tsukamoto et al., 1999), we hypothesized that Rap2 would be activated by the same pathway. We performed two tests to examine this. First, Rap2 should be activated by phorbol esters which mimic D A G . Second, Rap2 activation should be blocked by PLC inhibitors. 3.3.1 Activation of Rap2 by phorbol esters First, I tested whether the D A G mimic PdBu could activate Rap2 in WEHI-231 cells. A 60 minute Rap activation assay was done as in previous experiments. Figure 3.5 shows that Rapl and Rap2 were activated in WEHI-231 cells after PdBu stimulation. The kinetics were similar to that for anti-Ig- and SDF-1-induced Rap2 activation, with Rap2 being activated from two to 60 minutes. Reprobing of the filter showed that PdBu activated Rapl with similar activation kinetics. Figure 3.5 (A) shows that Rap2 activation at 60 minutes dropped back down to basal levels whereas in Figure 3.5 (B), Rapl activation at 60 minutes is still quite strong. Again, not much is known about Rap activation beyond the initial 15 minutes. There are insufficient time points to suggest that PdBu-induced Rap2 activation has stopped at 60 minutes. Preliminary experiments in our lab suggest that Rap proteins cycle between their active and inactive states in waves after one hour of stimulation. What is clear is that Rap2 can be activated by PdBu and the initial activation kinetics are very similar to Rapl . This suggested that Rap2 might also be activated via a PLC -"> D A G pathway. 32 WEHI-231 cells GST-RalGDS pull down PdBu (min) 0 2 10 15 30 60 Anti-Rap2 Blot •Rap2-GTP B 21.5 H WEHI-231 cells GST-RalGDS pull down > 0 2 5 10 15 30 60 - — •Rap1-GTP Anti-Rapl Blot Figure 3.5. Rapl and Rap2 are activated by the D A G mimic PdBu in WEHI-231 cells. WEHI-231 cells were stimulated with 100 nM PdBu, a phorbol ester that mimics the effect of D A G , for the indicated times. A GST-RalGDS fusion protein was used to selectively precipitate the activated GTP-bound forms of the Rap proteins. Western blotting was used to detect the activated forms of (A) Rap2 and (B) Rapl. Molecular mass markers (in kDa) are shown to the left. Similar results were obtained in two independent experiments. 3 3 3.3.2 Inhibition of Rap2 activation by the PLC inhibitor U73122 Based on the above experiment, a loss of function approach was used to test i f indeed the PLC -> D A G pathway leads to the activation of Rap2. The PLC inhibitor, U73122, was used to block D A G production. A structural analogue of U73122, U73343, was used as a negative control. WEHI-231 cells were either pre-treated with U73122 or U73343 or left untreated and then stimulated with anti-Ig antibodies for 10 minutes. Filters were immunoblotted for Rap2 and then reprobed for Rapl . In order to quantitate the relative levels of Rapl and Rap2 activation or inhibition in these cells, densitometry studies were performed using ImageQuant software. The intensity of each band that appeared on the filter is indicative of the amount of Rap activation at a given time. Obtaining a pixel volume for each U73122 band from the U73122 treatment and dividing this value with the pixel volume of its U73343 treatment control on the blot would give us the relative levels of Rap activation at each time point. Figure 3.6 shows that Rap2 activation was partially inhibited by U73122. In contrast, Rapl activation was almost completely inhibited when WEHI-231 cells were pre-treated with U73122. In both cases, Rapl and Rap2 activation was still observed in U73343 pre-treated cells and was comparable to untreated cells. In addition, dimethyl sulfoxide (DMSO), the solvent used to dissolve U73122 and U73343, did not activate Rapl or Rap2 and did not block Rap activation. Figure 3.7 shows that Rapl activation was inhibited 60-70% whereas Rap2 activation was inhibited 40-50% in U73122 pre-treated cells. In conclusion, PLC inhibitor, U73122, partially inhibited Rapl and Rap2 activation in WEHI-231 cells. The partial inhibition seen could be a result of insufficient amounts of inhibitor used to block D A G production or that the inhibitor is not very efficient at blocking PLC in WEHI-231 cells. To test whether PLC activation was completely inhibited, one could measure IP3 production, or one could look at other DAG-dependent events like the activation of the 34 Ras -> E R K pathway and immunoblot for phosphorylated forms of ERK. An alternative way to assess the role of PLC-y2 in Rap2 activation would be to use PLC-y2 deficient DT40 cells, as it had been done for Rap 1. 35 WEHI-231 cel ls GST-RalGDS pull down U73122 U73343 Untreated Ant i - IgM (min) 0 2 5 10 0 2 5 10 0 5 21.5 H Rap2-GTP Anti-Rap2 Blot B WEHI-231 cel ls GST-RalGDS pull down U73122 U73343 Untreated Ant i - IgM (min) 0 2 5 10 0 2 5 10 0 5 21.5 H -Rap1-GTP Ant i -Rap! Blot Figure 3.6. The PLC inhibitor U73122 partially inhibits BCR-induced Rapl and Rap2 activation in WEHI-231 cells. WEHI-231 cells were pre-treated with 20 u M U73122 or 20 uM of U73343 for 20 minutes or left untreated for 20 minutes. The cells were then stimulated with 100 ug/ml anti-IgM antibodies for the indicated times. A GST-RalGDS fusion protein was used to selectively precipitate the activated GTP-bound forms of the Rap proteins. Western blotting was used to detect the activated forms of (A) Rap2 and (B) Rapl . Molecular mass markers (in kDa) are shown to the left. Similar results were obtained in two independent experiments. 36 100% C 7 5 % A 2 5 10 Time (min) • Rap l • R a p 2 Figure 3.7. Relative levels of Rapl and Rap2 activation in U73122 pre-treated WEHI-231 cells. Densitometry was performed on Figure 3.6 using ImageQuant. Relative levels of Rapl and Rap2 activation in U73122 pre-treated WEHI-231 cells were calculated by taking the pixel volume for each time point, a representative of the amount of Rap activation, and dividing it by the pixel volume of its corresponding U73343 value. This ratio was expressed as a percentage. 37 3.4 Conclusion This chapter has shown that anti-Ig and SDF-1 can stimulate the activation of Rap2. This is the first report of receptor-induced activation of Rap2. The activation kinetics of Rap2 activation were similar to Rapl. These studies suggest that Rapl and Rap2 activation are co-ordinately regulated in B lymphocytes. This is not surprising since Rapl and Rap2 share a common subset of GNEFs and GAPs. The mechanism of Rap2 activation was also examined in this chapter. The ability of Rap2 to be activated by a PLC -> D A G pathway was tested. PdBu addition stimulated Rap2 activation in WEHI-231 cells. The use of PLC inhibitor, U73122, in BCR-induced WEHI-231 showed that Rap2 activation is at least partly dependent on PLC activity. Parallel experiments conducted in the laboratory using U73122 in SDF-1-induced 2PK-3 cells showed that SDF-1 -induced Rap2 activation was also PLC dependent. These two experiments therefore show that BCR-induced and SDF-1-induced activation of Rap2 is at least partly dependent on PLC. Since the efficacy of U73122 was not assessed, it is possible that these results underestimate the role of PLC in Rap2 activation. A more direct test of the role of PLC-y2 in BCR-induced Rap2 activation would be to use the PLC-y2-deficient DT40 cells. 38 C H A P T E R F O U R D E V E L O P I N G LOSS OF F U N C T I O N A P P R O A C H T O STUDY T H E ROLES OF T H E RAP1 A N D RAP2 PROTEINS I N B L Y M P H O C Y T E S 4.1 Rationale and summary To study the roles of Rapl and Rap2 in B cells, we wanted to use a loss of function approach to inhibit the activation of the endogenous Rapl and Rap2 in B cells. To do this, we expressed two different Rap-GAPs in B cells. RapGapU and Spa-1 are two GAPs that accelerate the rate of GTP hydrolysis in Rapl and Rap2 converting them to an inactive GDP-bound form (Ohba et al., 2000). Thus, RapGapn or Spa-1 was expressed individually in B cell lines and a Rap activation assay was performed to look for Rapl and Rap2 inhibition when using the aforementioned stimuli from the previous chapter. I found that RapGapU and Spa-1 differentially inhibited Rapl versus Rap2 activation. Both RapGapU and Spa-1 completely inhibited Rapl activation. RapGapU inhibited Rap2 substantially but not completely whereas Spa-1 only caused modest inhibition of Rap2. 4.2 The effect of RapGapI I expression on R a p l and Rap2 activation Preliminary experiments in our laboratory had shown that Rapl activation was completely inhibited in RapGapU expressing and Spa-1 over-expressing cell lines (S. McLeod et al., unpublished results). I examined whether Rap2 activation was inhibited by these GTPases. First, WEHI-231 and A20 cells transfected with FLAG-tagged RapGapU or the empty vector, pMSCV were stimulated with anti-Ig antibodies and Rap activation was assayed over a 15 minute time course. Filters were immunoblotted for Rap2 and then reprobed for Rapl . The effects of Rap activation in cells expressing RapGapU were compared with vector control cells. I found that Rapl activation was completely inhibited in RapGapU-expressing WEHI-231 and A20 cells. This was a good control to test that the cells were indeed expressing 39 RapGapH because it had been shown before that Rapl activation was inhibited in these cell lines. Figure 4.1 and 4.2 also show that RapGapH partially inhibited Rap2 activation in WEHI-231 and A20 cells. This inhibitory effect of Rap2 was more pronounced at later time intervals than at the beginning of the time course. Therefore, this difference in Rap inhibition showed that RapGapII differentially inhibited Rapl and Rap2 activation in BCR-induced B cells, being more effective at inhibiting Rapl than Rap2. After looking at BCR-induced Rap2 activation in RapGapU-expressing WEHI-231 and A20 cells, we decided to look at SDF-1-induced Rap2 activation in RapGapU-expressing WEHI and 2PK-3 cells. WEHI-231 and 2PK-3 cells transfected with FLAG-tagged RapGapH or the empty vector, pMSCV, were stimulated with SDF-1. Figure 4.3 and 4.4 show that RapGapn expression completely inhibited Rapl activation but only partially inhibited Rap2 activation, similar to what was seen for B C R stimulation. To conclude the studies using RapGapH, PdBu was used to stimulate WEHI-231 cells transfected with FLAG-tagged RapGapH or the empty vector, pMSCV, to see i f the same partial inhibitory effect on Rap2 activation was consistent. Figure 4.5 shows that RapGapn blocked Rapl activation but failed to block Rap2 activation completely. In all experiments, the inhibitory effect of Rap2 activation by RapGapH is much stronger later in the time course. In summary, RapGapH completely inhibited BCR-induced, SDF-1-induced, and PdBu-induced Rapl activation but only partially inhibited Rap2 activation. This indicates that RapGapH is a more effective inhibitor of Rapl than Rap2. Nevertheless, RapGapn did cause substantial inhibition of Rap2 activation. 40 WEHI-231 cel ls Vector RapGapll Ant i - IgM (min) 0 2 5 10 15 0 2 5 10 15 21.5 H •Rap2-GTP Anti-Rap2 Blot B WEHI-231 cel ls Vector RapGapll Ant i - IgM (min) 0 2 5 10 15 0 2 5 10 15 21.5H •Rap1-GTP Anti-Rap1 Blot Figure 4.1. Effects of RapGapll expression on anti-IgM-induced Rapl and Rap2 activation i WEHI-231 cells. Stable populations of WEHI-231 cells expressing the empty vector pMSCV pMSCV-containing cDNA encoding FLAG-tagged RapGapll were stimulated with 100 ug/ml anti-IgM antibodies for the indicated times. A GST-RalGDS fusion protein was used to selectively precipitate the activated GTP-bound forms of the Rap proteins. Western blotting used to detect the activated forms of (A) Rap2 and (B) Rapl. Molecular mass markers (in kDa) are shown to the left. Similar results were obtained in two independent experiments. was 4 1 A20 cells Vector RapGapII Ant i- IgG (min) 0 2 5 10 15 0 2 5 10 15 21.5 H •Rap2-GTP B Anti-Rap2 Blot A20 cells Vector RapGapII Ant i- IgG (min) 0 2 5 10 15 0 2 5 10 15 21.5 H •Rap1-GTP Anti-Rap1 Blot Figure 4.2. Effects of RapGapII expression on anti-IgG-induced Rapl and Rap2 activation in A20 cells. Stable populations of A20 cells expressing the empty vector p M S C V or pMSCV-containing cDNA encoding FLAG-tagged RapGapII were stimulated with 100 (j.g/ml anti-IgG antibodies for the indicated times. A GST-RalGDS fusion protein was used to selectively precipitate the activated GTP-bound forms of the Rap proteins. Western blotting was used to detect the activated forms of (A) Rap2 and (B) Rapl. Molecular mass markers (in kDa) shown to the left. Similar results were obtained in two independent experiments. are 42 WEHI-231 cel ls Vector RapGapll SDF-1 (min) 0 2 5 10 15 0 2 5 10 15 21.5 H •Rap2-GTP Anti-Rap2 Blot B WEHI-231 cel ls Vector RapGapll SDF-1 (min) 0 2 5 10 15 0 2 5 10 15 21.5 •Rap1-GTP Anti-Rap1 Blot Figure 4.3. Effects of RapGapll expression on SDF-1-induced Rapl and Rap2 activation in WEHI-231 cells. Stable populations of WEHI-231 cells expressing the empty vector pMSCV or pMSCV-containing cDNA encoding FLAG-tagged RapGapll were stimulated with 100 ng/ml (12.5 nM) SDF-1 for the indicated times. A GST-RalGDS fusion protein was used to selectively precipitate the activated GTP-bound forms of the Rap proteins. Western blotting was used to detect the activated forms of (A) Rap2 and (B) Rapl. Molecular mass markers (in kDa) shown to the left. Similar results were obtained in two independent experiments. 43 are 2PK-3 cel ls Vector RapGapII SDF-1 (min) 0 5 15 30 0 5 15 30 21.5 H •Rap2-GTP B Anti-Rap2 Blot 2PK^3 cel ls Vector RapGapII SDF-1 (min) 0 5 15 30 0 5 15 30 21.5 H •Rap1-GTP Anti-Rap1 Blot Figure 4 .4. Effects of RapGapII expression on SDF-1-induced Rapl and Rap2 activation in 2PK-3 cells. Stable populations of 2PK-3 cells expressing the empty vector pMSCV or pMSCV-containing cDNA encoding FLAG-tagged RapGapII were stimulated with 100 ng/ml (12.5 nM) SDF-1 for the indicated times. A GST-RalGDS fusion protein was used to selectively precipitate the activated GTP-bound forms of the Rap proteins. Western blotting was used to detect the activated forms of (A) Rap2 and (B) Rapl. Molecular mass markers (in kDa) are shown to the left. Similar results were obtained in four independent experiments. 44 WEHI-231 cel ls Vector RapGapII PdBu (min) 0 2 5 10 15 0 2 5 10 15 21.5 H •Rap2-GTP Anti-Rap2 Blot B WEHI-231 cel ls Vector RapGapII PdBu (min) 0 2 5 10 15 0 2 5 10 15 21.5 H •Rap1-GTP Ant i -Rap! Blot Figure 4.5. Effects of RapGapII expression on PdBu-induced Rapl and Rap2 activation in WEHI-231 cells. Stable populations of WEHI-231 cells expressing the empty vector pMSCV oi pMSCV-containing cDNA encoding FLAG-tagged RapGapII were stimulated with 100 n M PdBu for the indicated times. A GST-RalGDS fusion protein was used to selectively precipitate the activated GTP-bound forms of the Rap proteins. Western blotting was used to detect the activated forms of (A) Rap2 and (B) Rapl. Molecular mass markers (in kDa) are shown to the left. Similar results were obtained in two independent experiments. 45 4.3 The effect of Spa-1 expression on R a p l and Rap2 activation The second GAP to be utilized for the loss of function study was Spa-1. Similar to RapGapU, previous work had shown that Rapl activation was completely inhibited in Spa-1 over-expressing cell lines (S. McLeod et al., unpublished results). The experiments in section 4.2 have already shown that Rapl activation was more efficiently inhibited by RapGapU than was Rap2 activation. WEHI-231 and A20 cells transfected with FLAG-tagged Spa-1 or the empty p L X S N vector were stimulated with anti-Ig antibodies. Figures 4.6 and 4.7 show that Spa-1 inhibited Rapl activation completely, as was shown previously. In contrast, Spa-1, like RapGapU, only partially inhibited anti-Ig-induced Rap2 activation in WEHI-231 cells. There was very little inhibition in anti-Ig-induced Rap2 activation in A20 cells when compared to WEHI-231 cells. This could be a result of different amounts of Rap2 being activated in different cell lines and Spa-1 's inability to inhibit Rap2 activation efficiently. The ability of Spa-1 to inhibit Rap2 activation following CXCR4 stimulation with SDF-1 was investigated next. 2PK-3 cells transfected with FLAG-tagged Spa-1 or the empty pMX-PIE vector were stimulated with SDF-1. The use of the pMX-PIE vector, which encodes EGFP after an internal ribosomal empty site, provided an advantage for migration studies being conducted in the laboratory. Figure 4.8 showed that Spa-1 only partially inhibited SDF-l-induced Rap2 activation whereas Spa-1 completely blocked SDF-l-induced Rapl activation. Finally, PdBu was used to stimulate WEHI-231 cells transfected with FLAG-tagged Spa-1 or the empty p L X S N vector to see i f Spa-1 had any inhibitory effect on PdBu-induced Rap2 activation. Figure 4.9 again shows that Spa-1 partially inhibited Rap2 activation while completely inhibiting Rapl activation in WEHI-231 cells. Thus, these experiments have shown that activation of Rapl is completely blocked by RapGapU and Spa-1. In contrast, Rap2 is less effectively inhibited by RapGapU and Spa-1. 46 RapGapH caused significant but not complete inhibition of Rap2 activation. In contrast, Spa-1 caused only modest inhibition of Rap2. 47 WEHI-231 cel ls Vector Spa-1 Ant i - IgM (min) 0 2 5 10 15 0 2 5 10 15 21.5 Rap2-GTP Anti-Rap2 Blot B WEHI-231 cel ls Vector Spa-1 Ant i - IgM (min) 0 2 5 10 15 0 2 5 10 15 21.5 •Rap1-GTP Anti-Rap1 Blot Figure 4.6. Effects of Spa-1 expression on anti-IgM-induced Rapl and Rap2 activation in WEHI-231 cells. Stable populations of WEHI-231 cells expressing the empty vector p L X S N c pLXSN-containing cDNA encoding FLAG-tagged Spal were stimulated with 100 ug/ml anti-IgM antibodies for the indicated times. A GST-RalGDS fusion protein was used to selectively precipitate the activated GTP-bound forms of the Rap proteins. Western blotting was used to detect the activated forms of (A) Rap2 and (B) Rapl. Molecular mass markers (in kDa) are shown to the left. Similar results were obtained in two independent experiments. 48 A20 cells Vector Spa-1 Anti-IgG (min) 0 2 5 10 15 0 2 5 10 15 21.5 H .„ — k - R a P 2 - G T P Anti-Rap2 Blot B A20 cells Vector Spa-1 Anti-IgG (min) 0 2 5 10 15 0 2 5 10 15 21.5 i •Rap1-GTP Anti-Rap! Blot Figure 4.7. Effects of Spa-1 expression on anti-IgG-induced Rapl and Rap2 activation in A20 cells. Stable populations of A20 cells expressing the empty vector p L X S N or pLXSN-containing cDNA encoding FLAG-tagged Spal were stimulated with 100 ng/ml anti-IgG antibodies for the indicated times. A GST-RalGDS fusion protein was used to selectively precipitate the activated GTP-bound forms of the Rap proteins. Western blotting was used to detect the activated forms of (A) Rap2 and (B) Rapl. Molecular mass markers (in kDa) are shown to the left. Similar results were obtained in two independent experiments. 49 2PK-3 cel ls Vector Spa-1 SDF-1 (min) 0 2 5 1015 0 2 5 10 15 21.5 Rap2-GTP Anti-Rap2 Blot B 2PK-3 cel ls Vector Spa-1 SDF-1 (min) 0 2 5 10 15 0 2 5 10 15 21.5 H Rap1-GTP Anti-Rap1 Blot Figure 4.8. Effects of Spa-1 expression on SDF-l-induced Rapl and Rap2 activation in 2PK-3 cells. Stable populations of 2PK-3 cells expressing the empty vector pMX-PIE or pMX-PIE-containing cDNA encoding FLAG-tagged Spal were stimulated with 100 ng/ml (12.5 nM) SDF-1 for the indicated times. A GST-RalGDS fusion protein was used to selectively precipitate the activated GTP-bound forms of the Rap proteins. Western blotting was used to detect the activated forms of (A) Rap2 and (B) Rapl. Molecular mass markers (in kDa) are shown to the left. Similar results were obtained in five independent experiments. 50 WEHI-231 cel ls Vector Spa-1 PdBu (min) 0 2 5 10 15 0 2 5 10 15 21.5 H •Rap2-GTP Anti-Rap2 Blot B WEHI-231 cel ls Vector Spa-1 PdBu (min) 0 2 5 10 15 0 2 5 10 15 21.5 H •Rap1-GTP Ant i -Rap! Blot Figure. 4.9. Effects of Spa-1 expression on PdBu-induced Rapl and Rap2 activation in WEHI-231 cells. Stable populations of WEHI-231 cells expressing the empty vector p L X S N or pLXSN-containing cDNA encoding FLAG-tagged Spal were stimulated with 100 nM PdBu for the indicated times. A GST-RalGDS fusion protein was used to selectively precipitate the activated GTP-bound forms of the Rap proteins. Western blotting was used to detect the activated forms of (A) Rap2 and (B) Rapl. Molecular mass markers (in kDa) are shown to the left. Similar results were obtained in two independent experiments. 51 4.4 Conclusion This chapter has shown that both RapGapH and Spa-1 completely inhibit Rapl activation in B cells when stimulated with anti-Ig antibodies, SDF-1, and PdBu. RapGapH inhibits Rap2 substantially but not completely whereas Spa-1 only causes modest inhibition of Rap2. One possible explanation for this partial inhibitory effect is that perhaps at lower dosages of stimuli, better inhibition would be seen because less Rap2 proteins would become activated. A dose response would need to be conducted to test this assumption. Figures 4.1 to 4.9 also showed that it was easier to inhibit Rapl than Rap2. There are several explanations for this. First, there could be more activated forms of Rap 2 than Rapl in B cells. Second, Ohba et al. have alluded to the fact that Rap2 has a low sensitivity to Rap-GAPs (Ohba et al., 2000), and this may account for the high levels of activated Rap2 observed in anti-Ig-induced, SDF-1-induced, and PdBu-induced RapGapH- or Spa-1-expressing B cell lines. Parallel studies conducted in the laboratory have shown that RapGapH significantly inhibits B cell adhesion and migration. The inability of RapGapH to completely inhibit Rap2 activation could account for the small population of adherent and migratory B cells. Spa-1 also does not inhibit migration or adhesion very much. This could be because there is still sufficient Rap2 activation in these cells. Therefore, more studies are required to quantitate the difference in inhibition of Rap2 by RapGapH and Spa-1. 52 C H A P T E R F I V E DISCUSSION 5.1 Summary One of the primary aims of my thesis was to see i f Rap2 was activated by the same stimuli as Rapl. This was shown in Chapter 3 when we observed Rap2 activation in B cells that were stimulated with anti-Ig antibodies or SDF-1. The other goal of this thesis was to investigate how B C R engagement and CXCR4 signalling activated Rap2. We saw that addition of a D A G mimic, PdBu, induced Rap2 activation whereas the use of a PLC inhibitor, U73122, significantly inhibited Rap2 activation. This implied that Rap2 was activated at least in part, through a PLC -> D A G pathway. The last aim of my thesis was to develop loss of function approaches for blocking Rapl and Rap2 activation. We observed that RapGapU and Spa-1 both inhibited Rapl activation. RapGapU caused significant inhibition of Rap2 activation while Spa-1 was less effective at inhibiting Rap2 activation. The significance of these findings will be discussed below. 5.2 Coordinate activation of Rap l and Rap2 in B cells While Rapl has been shown to be activated by many receptors, little was known about Rap2 activation. M y work is the first report of receptor-induced activation of Rap2. More specifically, I showed for the first time that Rap2 is activated by both the B C R and CXCR4 in B cells. Previously, Rap2 was only shown to be activated by P M A and by cell adhesion. Initially, our laboratory looked at Rap2 because we wanted to know i f it was redundant in function with Rapl in B lymphocytes. This is because the amino acid sequence identity between Rap2 and Rapl is quite high, 60% (Janoueix-Lerosey et al., 1998; Nancy et al., 1999; Traver et al., 2000), and both Rap proteins possess a virtually identical effector region. On top of this, 53 many of the GNEFs and GAPs that regulate Rapl can interact with Rap2 (Ohba et a l , 2000). However, there exists the possibility that Rap2 may have overlapping functions with Rapl or alternatively, Rap2 may have a completely different function from Rapl. This is because while Rap2 shares most of the effector proteins with Rapl (Ohba et al., 2000), both Rap proteins possess their own distinct subset of effector molecules that they can bind to. I showed that Rap2 was activated by both the B C R and CXCR4 and that the activation kinetics between Rap2 and Rapl were quite similar. Hence, this observation raised the possibility that Rap2 could be co-ordinately regulated by the same regulatory proteins as Rapl. Many receptors, when stimulated, are capable of activating Rapl (Bos, 1998). This is because Rap-GNEFs are capable of coupling almost any receptor signalling pathway to Rapl . Consistent with this idea, I showed that Rap2, like Rapl , is activated at least in part, through a PLC -> D A G pathway in B cells. Although many of these studies have not looked at Rap2 activation, I speculate that many other receptors that activate Rapl will activate Rap2 as well. 5.3 Activation of Rap2 via the P L C pathway Previous experiments had shown that B C R activated Rapl via a B C R -> PLC-y2 -> D A G -> Rapl pathway. We hypothesized that Rap2 would also be activated by the same pathway. M y results indicate that B C R activates Rap2 via PLC-dependent production of D A G . This is because the phorbol ester, PdBu, activated Rap2 whereas the PLC inhibitor, U73122, blocked Rap2 activation. Since ionomycin, a calcium ionophore, did not activate Rap2 (S. McLeod, unpublished results), it supports the idea that D A G is the PLC-derived second messenger that activates Rap2. If Rapl and Rap2 activation are downstream of D A G , the interesting question raised is which Rap-GNEFs and Rap-GAPs are regulated by D A G in B cells. Known Rap-GNEFs that 54 are regulated by D A G are CalDAG-GEFI (Kawasaki et al., 1998), RasGRP2 (Clyde-Smith et al., 2000), and CalDAG-GEFIH (Yamashita et al., 2000). However, most of these GNEFs are also activated by intracellular C a 2 + increases whereas addition of ionomycin in B cells did not stimulate Rapl (McLeod et al., 1998) or Rap2 activation (S. McLeod, unpublished results). It is also not known whether these GNEFs are expressed in B cells. Thus, there may be another DAG-regulated GNEF in B cells that does not respond to Ca 2 + . As for Rap-GAPs, Spa-1, RapGapI, RapGapU, Tuberin G A P I P 4 B P , and E6fPl are known to stimulate GTPase Rapl activity (McLeod and Gold, 2001). However, only Spa-1, RapGapI, and RapGapU are known to regulate Rap2 and only Spa-1 is known to be expressed in B cells (McLeod and Gold, 2001). Clearly more studies need to be done. In order to investigate which GNEFs and GAPs couple the B C R and CXCR4 to Rapl/2 activation, loss of function studies can be utilized. Generating specific GNEF/GAP-deficient B cell lines and then looking for Rapl and Rap2 activation will be quite useful in seeing which GNEFs and GAPs regulate Rapl and Rap2. A complementation study can be used to follow-up on this experiment in which the GNEF and GAP that was missing is replaced. Another approach would be to over-express certain GNEFs and GAPs in these B cell lines and to see i f they have any effect on Rapl and Rap2 activation. GNEFs and GAPs that do not affect Rapl and Rap2 in B cells will have no visible effects. 5.4 Dif ferential regulation of R a p l and Rap2 by R a p G a p l l and Spa-1 We knew previously that RapGapU inhibited Rapl activation. Since Rap2 could have redundant functions as Rapl, we needed to know whether RapGapll could also inhibit Rap2. Because using one approach could give us artefacts, it was important to use a second loss-of-function approach to study Rapl and Rap2 inhibition. Hence, we over-expressed Spa-1, another Rap-specific GAP (McLeod and Gold, 2001). 55 Our results showed that Spa-1 effectively inhibited Rapl but not Rap2. In contrast, RapGapII caused significant inhibition in Rapl and Rap2 activation. This is supported by experiments conducted by Ohba et al. in NIH 3T3 cells. They showed that Rap2 functions as a slowly responding molecular switch compared to Rapl because Rap2 appears to have a low sensitivity to GAP proteins resulting in a high GTP/GDP ratio on Rap2 (Ohba et al., 2000). Since RapGapII and Spa-1 differentially inhibit Rapl versus Rap2 activation, this can be used to identify functions that can be carried out by Rap2 in the absence of Rapl activation. This is discussed in the following section. 5.5 The role of Rap proteins in cell migrat ion and cell adhesion When Rapl (then called Krev-1) was first cloned, it was believed to have the ability to revert Ras transformed cells (Kitayama et al., 1989). Because the 21 kDa protein, Rapl, shared a 50% amino acid sequence identity with Ras, it was believed that the function of Rapl was to suppress Ras signalling by interacting with Ras effectors via competitive inhibition. This theory was further strengthened when it was revealed that Rapl and Ras had the same effector region (Kitayama et al., 1989). However, this theory began to lose support when it was demonstrated that Rap2, which also shared a virtually identical effector region as Ras, neither exhibited growth-promoting nor growth-inhibitory effects in several independent cell lines (Jimenez et al., 1991). Later, Zwartkruis et al. showed that Rapl activation did not inhibit Ras effector signalling when Rapl signalling was activated by stimulating several growth factor receptors such as PDGF, EGF, and the G protein-coupled receptors for LP A , thrombin and endothelin. It is believed that Ras inhibition by Rapl was an artefact due to over-expressing activated Rapl. Subcellular localization studies, while interesting, have not fully elucidated the functions of Rap proteins either. We know that Rap2 proteins undergo post-translational modifications that allow them to interact with membranes (Beranger et al., 1991). However, Rap proteins seem 56 to be associated with different membranes than Ras, which is located at the plasma membrane (Pizon et al., 1994). This brings into question whether Rap proteins could interact with Ras effectors at all since Ras effectors are localized at the plasma membrane and Rap proteins are confined to distinct areas of the cell such as the peri-nuclear membrane region (Bos, 2001) and the endoplasmic reticulum (Beranger et al., 1991). Recent studies using fluorescent resonance energy transfer have shown that activation of Rapl begins outside the nucleus and spreads outwards towards the plasma membrane over time (Bos, 2001). The ability of Rap proteins to translocate to discrete areas of the cell was also confirmed in human platelets. Franke et al. showed that platelet aggregation correlated with the translocation of Rapl proteins to detergent insoluble cytoskeletal fractions (Franke et al., 2000). This led us to believe that perhaps Rap proteins interacted with cytoskeletal components in B lymphocytes and somehow influenced adhesion and migration. Studies of Rap translocation to the cytoskeleton in B cell lines were inconclusive however (unpublished results). More and more investigations are showing that Rapl is involved in cell adhesion and cell migration. For example, Tsukamoto et al. showed that transiently expressing a Rapl GAP, Spa-1, in HeLa cells induced rounding up of cells whereas transiently expressing the Rap-GNEF, C3G, induced extensive cell spreading (Tsukamoto et al., 1999). In another study in T cells, Reedquist et al. showed that activated Rapl promotes V L A - 4 and LFA-1-dependent adhesion (Reedquist et al., 2000). They speculate that Rapl may coordinate adhesion-dependent signals during T cell migration or extravasation through epithelial cells. Finally Arai et al. demonstrated that CrkL-mediated cell adhesion in hematopoietic cells can be inhibited by a dominant negative mutant of Rapl , RaplA-17N (Arai et al., 2001). These reports made us curious as to whether Rapl and Rap2 could also mediate cell adhesion and migration in B lymphocytes. Recently, our laboratory has shown that expressing RapGapU blocked B cell adhesion via LFA-1 and V L A - 4 (S. McLeod, unpublished results). Also, treating B cells with SDF-1 induces 57 Rap activation, and we have shown that SDF-1-induced migration requires activation of the Rap GTPases (McLeod, L i , et al., JI, 2002). Expressing RapGapII in these cells blocked Rap activation and reduced SDF-1-induced migration significantly whereas expressing a constitutively active form for Rap2 - Rap2V12 - increased SDF-1-induced B cell migration. In other studies in the laboratory, expressing Rap2V12 in A20 cells promotes morphological changes (S. McLeod, unpublished results). The cells seemed to adhere to surfaces better than control cells. Taken together, these experiments indicate that Rap proteins clearly play a role in B cell adhesion and migration. If Rap proteins are important for B cell adhesion and migration, the next question to ask is exactly how Rap proteins regulate these processes. Increase in cell adhesion depends on the activation of the LFA-1 and V L A - 4 integrins in B cells. Sarah McLeod has shown that Rap activation is important for activation of both LFA-1 and V L A - 4 by anti-Ig antibodies and by phorbol esters (S. McLeod, unpublished results). While the mechanism for integrin activation is not known, we do know that integrin activation requires integrin clustering which is dependent on their release from actin cytoskeleton. Maybe, activated Rap proteins regulate this process. There are several situations during B cell development and activation in which adhesion and migration processes are required. First, B cell progenitor binding to bone marrow stromal cells is necessary for the production of mature B cells (Melchers and Rolink, 1999). Second, the ability for B cells to migrate across the endothelial cells in blood vessels and enter the lymphoid organs requires integrins (Melchers and Rolink, 1999). Third, antigen presentation between B and T cells requires cell-cell adhesion, which is mediated by integrins (Janeway et al., 2001). Finally, B cell survival in the periphery is dependent on cell-cell interactions with follicular dendritic cells (FDC). This process requires the B cells to adhere to the FDCs in order to receive survival signals. Therefore, it would be interesting to see i f Rap proteins play a role in these processes. 58 5.6 Further work Many questions still remain unanswered regarding the regulation and function of Rap proteins in B lymphocytes. I have shown that Rapl and Rap2 can be activated by a variety of stimulants: anti-Ig antibodies, SDF-1, and PdBu. We also know that the activation kinetics of Rapl are very similar to Rap2 suggesting coordinate regulation by the same upstream activators. The other finding of this thesis is that Rap2, like Rapl , seems to be activated via a PLC -> D A G pathway. However, we do not know which GNEF or GAP regulates Rap activation/inhibition. Loss of function experiments creating B cell lines that are deficient in one specific GNEF or GAP will have to be generated in order to observe which GNEFs and GAPs couple the B C R and CXCR4 to Rap activation. While over-expressing GAPs to inhibit Rap function is an alternative method to replace the loss of function effect of Rap proteins, it could nevertheless introduce artefacts in our studies as these GAPs might not completely inhibit Rap in certain specific situations, and they could interfere with other signalling pathways in B lymphocytes. Experiments conducted in our laboratory have shown that anti-Ig, SDF-1, and PdBu stimulation in several B cell lines expressing RapGapU does not interfere with ERK, p38, INK, and Rac activation (R. Lee, S. McLeod, S. Christian et al., unpublished results). A better approach would be to create a conditional Rap 1/2 knockout mutant. However, this would require multiple knockouts of RaplA, RaplB, Rap2A, and Rap2B. A n alternative is to couple loss of function experiments with gain of function experiments, like over-expressing Rap2V12, a constitutively active form of Rap2. We know that Spa-1 is found in B cells (S. McLeod, M . Gold, unpublished results). Results from Chapter 4 of the thesis also showed that Spa-1 was more active on Rapl than Rap2. Since Spa-1 is ineffective at inhibiting Rap2, this suggests the existence of another GAP that 59 could regulate Rap2 activation in B lymphocytes. A yeast 2-hybrid analysis of proteins which interact with Rap2 could uncover other Rap-GAPs in B lymphocytes. The best way to address whether Rapl and Rap2 have the same or different functions in B lymphocytes would be to over-express RaplV12 and Rap2V12 in B cell lines. 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Uchiyama. 2000. SDF-1 has Costimulatory Effects on Human T Cells: Possible Involvement of MapK (ERK2) Activation. Microbiology and Immunology. 44:135-141. York, R.D., H . Yao, T. Dillon, C L . Ellig, S.P. Eckert, E.W. McCleskey, and P. J.S. Stork. 1998. Rapl Mediates Sustained M A P Kinase Activation Induced by Nerve Growth Factor. Nature. 392:622-626. Zwartkruis, F.J.T., and J.L. Bos. 1999. Ras and Rapl: Two Highly Related Small GTPases with Distinct Function. Experimental Cell Research. 253:157-165. Zwartkruis, F.J.T., R.M.F. Wolthuis, N .M.J .M. Nabben, B. Franke, and J.L. Bos. 1998. Extracellular Signal-Regulated Activation of Rapl Fails to Interfere in Ras Effector Signalling. EMBO. 17:5905-5912. 72 A P P E N D I X I Overview of B C R signalling pathway Syk-BCR clustering Protein tyrosine kinases M. PLC-y Ca 2 + PKC Calcineurin NF-KB V N F A T i V PI3K DAG PI(3,4,5)P, Akt RasGRP Ras Raf-1 Mek1,2 V ERK1.2 Elk — + Fos 73 APPENDIX II Overview of CXCR4 signalling pathway CXCR4-S D F - 1 Gai G £ r PI3K PI3K r PLC-13 Akt t K I \ t ^ I V IP3 DAG Ca 2 + PKC Calcineurin NF-KB N F A T 74 A P P E N D I X III Phvlogenic tree of Rap and Ras family GTPases 0.1 Note: The following phylogenic tree was generated by aligning the amino acid sequences using ClustalW and drawn using TreeViewPPC. The letters before each gene denotes the species from which it came, where 'h ' is human, 'm' is mouse, and 'r' is rat. 75 

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