Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

B Cell Receptor-induced Phosphorylation of Pyk2 and Focal Adhesion Kinase Involves Integrins and the Rap GTPases and Is Required for B Cell Spreading

2009; Elsevier BV; Volume: 284; Issue: 34 Linguagem: Inglês

10.1074/jbc.m109.013169

ISSN

1083-351X

Autores

Kathy W.K. Tse, May Dang-Lawson, Rosaline L. Lee, Doris Vong, Anica Bulic, Leonard Buckbinder, Michael R. Gold,

Tópico(s)

Immunotherapy and Immune Responses

Resumo

Signaling by the B cell receptor (BCR) promotes integrin-mediated adhesion and cytoskeletal reorganization. This results in B cell spreading, which enhances the ability of B cells to bind antigens and become activated. Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related cytoplasmic tyrosine kinases that regulate cell adhesion, cell morphology, and cell migration. In this report we show that BCR signaling and integrin signaling collaborate to induce the phosphorylation of Pyk2 and FAK on key tyrosine residues, a modification that increases the kinase activity of Pyk2 and FAK. Activation of the Rap GTPases is critical for BCR-induced integrin activation as well as for BCR- and integrin-induced reorganization of the actin cytoskeleton. We now show that Rap activation is essential for BCR-induced phosphorylation of Pyk2 and for integrin-induced phosphorylation of Pyk2 and FAK. Moreover Rap-dependent phosphorylation of Pyk2 and FAK required an intact actin cytoskeleton as well as actin dynamics, suggesting that Rap regulates Pyk2 and FAK via its effects on the actin cytoskeleton. Importantly B cell spreading induced by BCR/integrin co-stimulation or by integrin engagement was inhibited by short hairpin RNA-mediated knockdown of either Pyk2 or FAK expression and by treatment with PF-431396, a chemical inhibitor that blocks the kinase activities of both Pyk2 and FAK. Thus Pyk2 and FAK are downstream targets of the Rap GTPases that play a key role in regulating B cell morphology. Signaling by the B cell receptor (BCR) promotes integrin-mediated adhesion and cytoskeletal reorganization. This results in B cell spreading, which enhances the ability of B cells to bind antigens and become activated. Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related cytoplasmic tyrosine kinases that regulate cell adhesion, cell morphology, and cell migration. In this report we show that BCR signaling and integrin signaling collaborate to induce the phosphorylation of Pyk2 and FAK on key tyrosine residues, a modification that increases the kinase activity of Pyk2 and FAK. Activation of the Rap GTPases is critical for BCR-induced integrin activation as well as for BCR- and integrin-induced reorganization of the actin cytoskeleton. We now show that Rap activation is essential for BCR-induced phosphorylation of Pyk2 and for integrin-induced phosphorylation of Pyk2 and FAK. Moreover Rap-dependent phosphorylation of Pyk2 and FAK required an intact actin cytoskeleton as well as actin dynamics, suggesting that Rap regulates Pyk2 and FAK via its effects on the actin cytoskeleton. Importantly B cell spreading induced by BCR/integrin co-stimulation or by integrin engagement was inhibited by short hairpin RNA-mediated knockdown of either Pyk2 or FAK expression and by treatment with PF-431396, a chemical inhibitor that blocks the kinase activities of both Pyk2 and FAK. Thus Pyk2 and FAK are downstream targets of the Rap GTPases that play a key role in regulating B cell morphology. Antibodies (Abs) 2The abbreviations used are: AbantibodyBCRB cell receptorIgimmunoglobulinECMextracellular matrixLFA-1lymphocyte function-associated antigen-1Pyk2proline-rich tyrosine kinaseFAKfocal adhesion kinaseVLA-4very late antigen-4Tyr(P)phosphotyrosineLPSlipopolysaccharideshRNAshort hairpin RNAGCgerminal centerSH2Src homology 2Erkextracellular signal-regulated kinaseRTreverse transcriptionGFPgreen fluorescent proteinIL-4interleukin-4GAPGTPase-activating protein. 2The abbreviations used are: AbantibodyBCRB cell receptorIgimmunoglobulinECMextracellular matrixLFA-1lymphocyte function-associated antigen-1Pyk2proline-rich tyrosine kinaseFAKfocal adhesion kinaseVLA-4very late antigen-4Tyr(P)phosphotyrosineLPSlipopolysaccharideshRNAshort hairpin RNAGCgerminal centerSH2Src homology 2Erkextracellular signal-regulated kinaseRTreverse transcriptionGFPgreen fluorescent proteinIL-4interleukin-4GAPGTPase-activating protein. made by B lymphocytes play a critical role in host defense against infection. Antigen-induced signaling by the B cell receptor (BCR) initiates an activation program that leads to B cell proliferation and subsequent differentiation into Ab-producing cells. BCR clustering by antigens or by anti-immunoglobulin (anti-Ig) Abs used as surrogate antigens initiates multiple signaling pathways that control gene expression, cell survival, and proliferation pathways (1Dal Porto J.M. Gauld S.B. Merrell K.T. Mills D. Pugh-Bernard A.E. Cambier J. Mol. Immunol. 2004; 41: 599-613Crossref PubMed Scopus (414) Google Scholar, 2Kurosaki T. Nat. Rev. Immunol. 2002; 2: 354-363Crossref PubMed Scopus (173) Google Scholar, 3Niiro H. Clark E.A. Nat. Rev. Immunol. 2002; 2: 945-956Crossref PubMed Scopus (503) Google Scholar).BCR signaling also promotes integrin activation (4Spaargaren M. Beuling E.A. Rurup M.L. Meijer H.P. Klok M.D. Middendorp S. Hendriks R.W. Pals S.T. J. Exp. Med. 2003; 198: 1539-1550Crossref PubMed Scopus (166) Google Scholar, 5Harris E.S. McIntyre T.M. Prescott S.M. Zimmerman G.A. J. Biol. Chem. 2000; 275: 23409-23412Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), localized actin polymerization, reorganization of the actin cytoskeleton, and changes in B cell morphology (6Lin K.B. Freeman S.A. Zabetian S. Brugger H. Weber M. Lei V. Dang-Lawson M. Tse K.W. Santamaria R. Batista F.D. Gold M.R. Immunity. 2008; 28: 75-87Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 7Westerberg L. Greicius G. Snapper S.B. Aspenström P. Severinson E. Blood. 2001; 98: 1086-1094Crossref PubMed Scopus (77) Google Scholar), all of which may facilitate B cell activation. Integrin activation and cell spreading is critical for the activation of B cells by membrane-bound antigens. Macrophages, dendritic cells, and follicular dendritic cells can present arrays of captured antigens to B cells (8Bergtold A. Desai D.D. Gavhane A. Clynes R. Immunity. 2005; 23: 503-514Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 9Qi H. Egen J.G. Huang A.Y. Germain R.N. Science. 2006; 312: 1672-1676Crossref PubMed Scopus (404) Google Scholar), and this may be one of the main ways in which B cells encounter antigens (10Bajénoff M. Egen J.G. Koo L.Y. Laugier J.P. Brau F. Glaichenhaus N. Germain R.N. Immunity. 2006; 25: 989-1001Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar). BCR-induced integrin activation prolongs the interaction between the B cell and the antigen-presenting cell and also allows the B cell to spread on the surface of the antigen-presenting cell such that more BCRs can encounter and bind membrane-bound antigens (11Fleire S.J. Goldman J.P. Carrasco Y.R. Weber M. Bray D. Batista F.D. Science. 2006; 312: 738-741Crossref PubMed Scopus (328) Google Scholar). Subsequent contraction of the B cell membrane allows the B cells to gather the BCR-bound antigen into an immune synapse in which clustered antigen-engaged BCRs are surrounded by a ring of ligand-bound integrins. Formation of this immune synapse reduces the amount of antigen that is required for B cell activation (12Carrasco Y.R. Fleire S.J. Cameron T. Dustin M.L. Batista F.D. Immunity. 2004; 20: 589-599Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 13Carrasco Y.R. Batista F.D. EMBO J. 2006; 25: 889-899Crossref PubMed Scopus (117) Google Scholar).Recent work has shown that B cells in lymphoid organs may contact soluble antigens by extending membrane processes into a highly organized network of lymph-filled conduits (14Roozendaal R. Mempel T.R. Pitcher L.A. Gonzalez S.F. Verschoor A. Mebius R.E. von Andrian U.H. Carroll M.C. Immunity. 2009; 30: 264-276Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). These conduits are created by fibroblastic reticular cells that partially ensheathe collagen fibrils. In addition to being rich in collagen, fibronectin, and other extracellular matrix (ECM) components, the fibroblastic reticular cells that form these conduits express high levels of intercellular adhesion molecule-1, the ligand for the αLβ2 integrin (lymphocyte function-associated antigen-1 (LFA-1)) on B cells (10Bajénoff M. Egen J.G. Koo L.Y. Laugier J.P. Brau F. Glaichenhaus N. Germain R.N. Immunity. 2006; 25: 989-1001Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar). Thus B cells interacting with these conduits are likely to be in contact with integrin ligands, and integrin-dependent spreading may enhance the ability of B cells to extend membrane processes into the fibroblastic reticular cell conduit.In addition to promoting cell spreading, integrins can act as co-stimulatory receptors that enhance signaling by many receptors including the T cell receptor and the BCR (15Bachmann M.F. McKall-Faienza K. Schmits R. Bouchard D. Beach J. Speiser D.E. Mak T.W. Ohashi P.S. Immunity. 1997; 7: 549-557Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 16Schwartz M.A. Ginsberg M.H. Nat. Cell Biol. 2002; 4: E65-68Crossref PubMed Scopus (675) Google Scholar, 17McLeod S.J. Shum A.J. Lee R.L. Takei F. Gold M.R. J. Biol. Chem. 2004; 279: 12009-12019Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Thus signaling proteins that regulate B cell spreading and that are also targets of BCR/integrin co-stimulation may play a key role in the activation of B cells by membrane-bound antigens as well as soluble antigens that are delivered to lymphoid organs by fibroblastic reticular cell conduits.Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related non-receptor protein-tyrosine kinases that integrate signals from multiple receptors and play an important role in regulating cell adhesion, cell morphology, and cell migration in many cell types (18Rovida E. Lugli B. Barbetti V. Giuntoli S. Olivotto M. Dello Sbarba P. Biol. Chem. 2005; 386: 919-929Crossref PubMed Scopus (16) Google Scholar, 19van Buul J.D. Anthony E.C. Fernandez-Borja M. Burridge K. Hordijk P.L. J. Biol. Chem. 2005; 280: 21129-21136Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 20Okigaki M. Davis C. Falasca M. Harroch S. Felsenfeld D.P. Sheetz M.P. Schlessinger J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 10740-10745Crossref PubMed Scopus (221) Google Scholar). Integrins, receptor tyrosine kinases, antigen receptors, and G protein-coupled chemokine receptors all stimulate tyrosine phosphorylation of Pyk2 and FAK, a modification that increases the enzymatic activity of these kinases and allows them to bind SH2 domain-containing signaling proteins (21Avraham H. Park S.Y. Schinkmann K. Avraham S. Cell. Signal. 2000; 12: 123-133Crossref PubMed Scopus (407) Google Scholar). FAK, which is expressed in almost all tissues (21Avraham H. Park S.Y. Schinkmann K. Avraham S. Cell. Signal. 2000; 12: 123-133Crossref PubMed Scopus (407) Google Scholar), is a focal adhesion component that mediates integrin-dependent cell migration (22van Seventer G.A. Salmen H.J. Law S.F. O'Neill G.M. Mullen M.M. Franz A.M. Kanner S.B. Golemis E.A. van Seventer J.M. Eur. J. Immunol. 2001; 31: 1417-1427Crossref PubMed Scopus (76) Google Scholar), cell spreading, and cell adhesion (18Rovida E. Lugli B. Barbetti V. Giuntoli S. Olivotto M. Dello Sbarba P. Biol. Chem. 2005; 386: 919-929Crossref PubMed Scopus (16) Google Scholar) in adherent cells as well as co-clustering of LFA-1 with the T cell receptor in lymphocytes (23Giannoni E. Chiarugi P. Cozzi G. Magnelli L. Taddei M.L. Fiaschi T. Buricchi F. Raugei G. Ramponi G. J. Biol. Chem. 2003; 278: 36763-36776Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Pyk2 is expressed mainly in hematopoietic cells, osteoclasts, and the central nervous system (24Andreev J. Simon J.P. Sabatini D.D. Kam J. Plowman G. Randazzo P.A. Schlessinger J. Mol. Cell. Biol. 1999; 19: 2338-2350Crossref PubMed Scopus (146) Google Scholar) and is critical for chemokine-induced migration of B cells, macrophages, and natural killer cells (20Okigaki M. Davis C. Falasca M. Harroch S. Felsenfeld D.P. Sheetz M.P. Schlessinger J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 10740-10745Crossref PubMed Scopus (221) Google Scholar, 25Guinamard R. Okigaki M. Schlessinger J. Ravetch J.V. Nat. Immunol. 2000; 1: 31-36Crossref PubMed Scopus (434) Google Scholar, 26Gismondi A. Jacobelli J. Strippoli R. Mainiero F. Soriani A. Cifaldi L. Piccoli M. Frati L. Santoni A. J. Immunol. 2003; 170: 3065-3073Crossref PubMed Scopus (51) Google Scholar) as well as the spreading of osteoclasts on vitronectin (27Lakkakorpi P.T. Bett A.J. Lipfert L. Rodan G.A. Duong le T. J. Biol. Chem. 2003; 278: 11502-11512Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). FAK and Pyk2 are thought to mediate overlapping but distinct functions because Pyk2 expression only partially reverses the cell adhesion and migration defects in FAK-deficient fibroblasts (28Sieg D.J. Iliæ D. Jones K.C. Damsky C.H. Hunter T. Schlaepfer D.D. EMBO J. 1998; 17: 5933-5947Crossref PubMed Scopus (286) Google Scholar).In B cells, clustering of the BCR, β1 integrins, or β7 integrins induces tyrosine phosphorylation of both Pyk2 and FAK (29Astier A. Avraham H. Manie S.N. Groopman J. Canty T. Avraham S. Freedman A.S. J. Biol. Chem. 1997; 272: 228-232Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 30Freedman A.S. Rhynhart K. Nojima Y. Svahn J. Eliseo L. Benjamin C.D. Morimoto C. Vivier E. J. Immunol. 1993; 150: 1645-1652PubMed Google Scholar, 31Manie S.N. Astier A. Wang D. Phifer J.S. Chen J. Lazarovits A.I. Morimoto C. Freedman A.S. Blood. 1996; 87: 1855-1861Crossref PubMed Google Scholar, 32Glodek A.M. Honczarenko M. Le Y. Campbell J.J. Silberstein L.E. J. Exp. Med. 2003; 197: 461-473Crossref PubMed Scopus (76) Google Scholar, 33Mlinaric-Rascan I. Yamamoto T. FEBS Lett. 2001; 498: 26-31Crossref PubMed Scopus (17) Google Scholar). FAK is involved in the chemokine-induced adhesion of B cell progenitors (34Glodek A.M. Le Y. Dykxhoorn D.M. Park S.Y. Mostoslavsky G. Mulligan R. Lieberman J. Beggs H.E. Honczarenko M. Silberstein L.E. Leukemia. 2007; 21: 1723-1732Crossref PubMed Scopus (51) Google Scholar), and Pyk2 is required for chemokine-induced migration of mature B cells (25Guinamard R. Okigaki M. Schlessinger J. Ravetch J.V. Nat. Immunol. 2000; 1: 31-36Crossref PubMed Scopus (434) Google Scholar). However, the role of these kinases in BCR- and integrin-induced B cell spreading has not been investigated, and the signaling pathways that link the BCR and integrins to tyrosine phosphorylation of Pyk2 and FAK have not been elucidated.We have shown previously that the ability of the BCR to induce integrin activation, B cell spreading, and immune synapse formation requires activation of the Rap GTPases (6Lin K.B. Freeman S.A. Zabetian S. Brugger H. Weber M. Lei V. Dang-Lawson M. Tse K.W. Santamaria R. Batista F.D. Gold M.R. Immunity. 2008; 28: 75-87Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 17McLeod S.J. Shum A.J. Lee R.L. Takei F. Gold M.R. J. Biol. Chem. 2004; 279: 12009-12019Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). In addition to binding effector proteins such as RapL and Rap1-interacting adaptor molecule (RIAM) that promote integrin activation (35Katagiri K. Maeda A. Shimonaka M. Kinashi T. Nat. Immunol. 2003; 4: 741-748Crossref PubMed Scopus (397) Google Scholar, 36Lafuente E.M. van Puijenbroek A.A. Krause M. Carman C.V. Freeman G.J. Berezovskaya A. Constantine E. Springer T.A. Gertler F.B. Boussiotis V.A. Dev. Cell. 2004; 7: 585-595Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 37Han J. Lim C.J. Watanabe N. Soriani A. Ratnikov B. Calderwood D.A. Puzon-McLaughlin W. Lafuente E.M. Boussiotis V.A. Shattil S.J. Ginsberg M.H. Curr. Biol. 2006; 16: 1796-1806Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar), the active GTP-bound forms of Rap1 and Rap2 bind multiple proteins that control actin dynamics and cell morphology (38Bos J.L. Curr. Opin. Cell Biol. 2005; 17: 123-128Crossref PubMed Scopus (392) Google Scholar). Moreover we showed that BCR/integrin-induced phosphorylation of Pyk2 in B cells is dependent on Rap activation (17McLeod S.J. Shum A.J. Lee R.L. Takei F. Gold M.R. J. Biol. Chem. 2004; 279: 12009-12019Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). However, this previous study did not address how Rap-GTP links the BCR and integrins to Pyk2 phosphorylation, whether Rap activation is important for FAK phosphorylation in B cells, or whether B cell spreading is regulated by Pyk2 or FAK. We now show that Pyk2 and FAK are differentially expressed and localized in B cells, that Pyk2 and FAK are important for B cell spreading, and that integrin engagement enhances BCR-induced phosphorylation of Pyk2 and FAK, a process that depends on both Rap activation and actin dynamics.DISCUSSIONThe binding of antigens by B cells often occurs in the context of integrin engagement. Integrin-dependent cell spreading enhances the ability of B cells to contact antigens, and integrin signaling may synergize with BCR signaling to promote both B cell spreading and activation. The Pyk2 and FAK kinases are key regulators of cell morphology, and in this report we showed that the kinase activities of Pyk2 and FAK are important for BCR/integrin-induced B cell spreading. Moreover we showed that integrins enhance the ability of the BCR to phosphorylate Pyk2 and FAK on their auto/transphosphorylation sites, the initial step in the activation of these kinases. Finally we showed that both Rap activation and actin dynamics were critical for BCR/integrin-induced phosphorylation of Pyk2 and FAK.We had shown previously that integrin engagement enhances BCR-induced Pyk2 phosphorylation (17McLeod S.J. Shum A.J. Lee R.L. Takei F. Gold M.R. J. Biol. Chem. 2004; 279: 12009-12019Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), and we have now shown that the same is true for FAK phosphorylation. Moreover by clustering integrins with Abs, we showed that integrin signaling was sufficient to induce tyrosine phosphorylation of Pyk2 and FAK in B cells. Thus signaling by antigen-clustered BCR complexes and ligand-bound integrins can have additive effects on the phosphorylation of Pyk2 and FAK. This highlights the ability of integrins to act as co-stimulatory receptors that collaborate with lymphocyte antigen receptors. Pyk2 and FAK appeared to be selective targets of the BCR/integrin collaboration as integrin engagement did not enhance BCR-induced phosphorylation of other signaling proteins such as Erk, Akt, and paxillin.We also showed that activation of the Rap GTPases was critical for BCR/integrin signaling to induce the phosphorylation of Pyk2 and FAK on their auto/transphosphorylation sites as well as tyrosine residues in their activation loops. Although Rap-GTP likely contributes to Pyk2 and FAK phosphorylation by activating integrins on B cells (17McLeod S.J. Shum A.J. Lee R.L. Takei F. Gold M.R. J. Biol. Chem. 2004; 279: 12009-12019Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), we found that activated Rap also acts downstream of the BCR to promote Pyk2 phosphorylation and downstream of integrins to promote the phosphorylation of Pyk2 and FAK. The active GTP-bound form of Rap binds multiple effector proteins that promote actin polymerization and the stabilization of F-actin polymers (38Bos J.L. Curr. Opin. Cell Biol. 2005; 17: 123-128Crossref PubMed Scopus (392) Google Scholar). Many of the downstream consequences of Rap activation may therefore reflect its role in reorganization of the actin cytoskeleton. Indeed we found that the Rap-dependent steps in Pyk2 and FAK phosphorylation were also blocked by actin-disrupting drugs. This suggests that Rap-GTP promotes Pyk2 and FAK phosphorylation via its ability to remodel the actin cytoskeleton. Rap1 activation was not dependent on actin dynamics, suggesting that the requirement for actin remodeling lies downstream of Rap activation.Although Pyk2 phosphorylation has been shown to require an intact actin cytoskeleton in a number of cell types (21Avraham H. Park S.Y. Schinkmann K. Avraham S. Cell. Signal. 2000; 12: 123-133Crossref PubMed Scopus (407) Google Scholar), how this contributes to Pyk2 phosphorylation is not clear. Phosphorylation of Pyk2 on Tyr402 may involve Pyk2 dimerization and subsequent transphosphorylation (51Park S.Y. Avraham H.K. Avraham S. J. Biol. Chem. 2004; 279: 33315-33322Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Rap-dependent actin polymerization could create a cytoskeletal platform that promotes Pyk2 dimerization. However, we found that treating B cells with the actin-stabilizing agent jasplakinolide also prevented tyrosine phosphorylation of Pyk2, indicating that polymerized F-actin is not sufficient to support receptor-induced Pyk2 phosphorylation. Dynamic remodeling of the actin cytoskeleton may be required for efficient Pyk2 dimerization. Alternatively Pyk2-dependent phosphorylation in vivo may require cycles of actin polymerization and depolymerization that regulate either the kinase activity of Pyk2 or the accessibility of its catalytic site.In contrast to Pyk2, Rap activation and actin dynamics were required for integrin-induced FAK phosphorylation but not for BCR-induced FAK phosphorylation in A20 B lymphoma cells. For integrin-induced FAK phosphorylation, Rap activation was required for the initial step in FAK activation, phosphorylation of Tyr397, an event that is initiated by transphosphorylation and that can be amplified by Src family kinase (56Zeng L. Si X. Yu W.P. Le H.T. Ng K.P. Teng R.M. Ryan K. Wang D.Z. Ponniah S. Pallen C.J. J. Cell Biol. 2003; 160: 137-146Crossref PubMed Scopus (118) Google Scholar). How Rap activation and F-actin contribute to integrin-induced FAK Tyr397 phosphorylation is not clear. Our microscopy data suggest that FAK constitutively co-localizes with integrins in B cells. Rap activation and actin polymerization could therefore contribute to the recruitment and/or stabilization of other proteins that regulate FAK Tyr397 phosphorylation. In adherent cells that form focal adhesions, integrin activation results in the recruitment of talin to the integrin α and β chain cytoplasmic domains (57Schlaepfer D.D. Mitra S.K. Ilic D. Biochim. Biophys. Acta. 2004; 1692: 77-102Crossref PubMed Scopus (367) Google Scholar). This allows FAK to interact with paxillin and undergo autophosphorylation. At the same time, activation of Src family kinases by protein-tyrosine phosphatase α increases the phosphorylation of FAK at Tyr397. Further work is required to determine whether Rap and F-actin promote integrin-dependent FAK phosphorylation by regulating these steps in B cells. Interestingly Rap activation and F-actin were not required for BCR-induced phosphorylation of FAK when the cells were in suspension, a situation in which there is minimal integrin engagement. FAK has been reported to associate constitutively with the Src family kinase Lyn and with the BCR in WEHI-231 B lymphoma cells (33Mlinaric-Rascan I. Yamamoto T. FEBS Lett. 2001; 498: 26-31Crossref PubMed Scopus (17) Google Scholar). FAK phosphorylation could therefore be a proximal Rap-independent BCR signaling event that is initiated by BCR.A key finding was that Pyk2 and FAK are important for B cell spreading that is initiated by BCR/integrin co-stimulation or by integrin clustering. This is consistent with Pyk2 and FAK being downstream targets of Rap because blocking Rap activation also prevents B cell spreading (6Lin K.B. Freeman S.A. Zabetian S. Brugger H. Weber M. Lei V. Dang-Lawson M. Tse K.W. Santamaria R. Batista F.D. Gold M.R. Immunity. 2008; 28: 75-87Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 17McLeod S.J. Shum A.J. Lee R.L. Takei F. Gold M.R. J. Biol. Chem. 2004; 279: 12009-12019Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Knocking down the expression of either Pyk2 or FAK in A20 B lymphoma cells reduced the ability of these cells to undergo cell spreading, whereas PF-431396, a dual specificity inhibitor of the kinase activities of both Pyk2 and FAK, substantially inhibited A20 cell spreading. This suggests that both Pyk2 and FAK contribute to the ability of A20 B lymphoma cells to undergo cell spreading. Moreover the use of PF-431396 showed that the kinase activities of Pyk2 and FAK were critical for B cell spreading.Although it is not known how Pyk2 and FAK promote B cell spreading, these kinases may coordinate the activation of Rac, Cdc42, and RhoA, GTPases that control cytoskeletal organization. In T lymphocytes, Pyk2 binds Vav (50Ostergaard H.L. Lysechko T.L. Immunol. Res. 2005; 31: 267-282Crossref PubMed Scopus (49) Google Scholar), an exchange factor that activates Rac. Both Pyk2 and FAK can interact with the RhoA activator p190RhoGEF (58Lim Y. Lim S.T. Tomar A. Gardel M. Bernard-Trifilo J.A. Chen X.L. Uryu S.A. Canete-Soler R. Zhai J. Lin H. Schlaepfer W.W. Nalbant P. Bokoch G. Ilic D. Waterman-Storer C. Schlaepfer D.D. J. Cell Biol. 2008; 180: 187-203Crossref PubMed Scopus (177) Google Scholar), and in fibroblasts Pyk2 associates with Wrch1, a Cdc42-like GTPase that promotes the formation of filopodia (59Ruusala A. Aspenström P. Mol. Cell. Biol. 2008; 28: 1802-1814Crossref PubMed Scopus (29) Google Scholar). Pyk2 and FAK can also bind the p85 subunit of phosphoinositide 3-kinase following integrin ligation (60Sarkar S. Svoboda M. de Beaumont R. Freedman A.S. Leuk. Lymphoma. 2002; 43: 1663-1671Crossref PubMed Scopus (21) Google Scholar, 61Mitra S.K. Hanson D.A. Schlaepfer D.D. Nat. Rev. Mol. Cell Biol. 2005; 6: 56-68Crossref PubMed Scopus (1926) Google Scholar). Phosphatidylinositol 3,4,5-trisphosphate produced by phosphoinositide 3-kinase activates Vav and promotes Rac-dependent actin polymerization and cytoskeletal rearrangement. Pyk2 and FAK can also bind and phosphorylate the scaffolding proteins p130Cas and paxillin, which can then recruit the Rac activators DOCK180 and PAK-interacting exchange factor (PIX), leading to Rac-dependent membrane ruffling (61Mitra S.K. Hanson D.A. Schlaepfer D.D. Nat. Rev. Mol. Cell Biol. 2005; 6: 56-68Crossref PubMed Scopus (1926) Google Scholar).An interesting observation was that when B cells were activated with LPS plus IL-4 Pyk2 levels decreased, but FAK levels increased significantly. B cells activated in this manner resemble antigen-activated germinal center (GC) B cells, which proliferate within lymphoid organ follicles and undergo somatic hypermutation of their Ig genes. These GC B cells then compete for limiting amounts of antigen that are displayed on the surface of follicular dendritic cells, which provide the B cells with survival signals. GC B cells interacting with follicular dendritic cells in vivo exhibit a spread morphology with multiple membrane processes (62Allen C.D. Okada T. Tang H.L. Cyster J.G. Science. 2007; 315: 528-531Crossref PubMed Scopus (577) Google Scholar, 63Hauser A.E. Junt T. Mempel T.R. Sneddon M.W. Kleinstein S.H. Henrickson S.E. von Andrian U.H. Shlomchik M.J. Haberman A.M. Immunity. 2007; 26: 655-667Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). This presumably increases their ability to detect antigens on the surface of the follicular dendritic cell. The activation-induced increase in FAK expression may reflect a switch from the motile phenotype of a circulating B cell to the more adhesive phenotype of an activated GC B cell. FAK expression and activation are associated with sustained adhesion at least in B cell progenitors (34Glodek A.M. Le Y. Dykxhoorn D.M. Park S.Y. Mostoslavsky G. Mulligan R. Lieberman J. Beggs H.E. Honczarenko M. Silberstein L.E. Leukemia. 2007; 21: 1723-1732Crossref PubMed Scopus (51) Google Scholar). A number of adhesion molecules including α6 integrin are up-regulated in activated GC B cells (64Ambrose H.E. Wagner S.D. Immunology. 2004; 111: 400-406Crossref PubMed Scopus (8) Google Scholar, 65Klein U. Tu Y. Stolovitzky G.A. Keller J.L. Haddad Jr., J. Miljkovic V. Cattoretti G. Califano A. Dalla-Favera R. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 2639-2644Crossref PubMed Scopus (337) Google Scholar), and gene expression profiling has shown that FAK mRNA levels are elevated in GC B cells (66Alizadeh A.A. Eisen M.B. Davis R.E. Ma C. Lossos I.S. Rosenwald A. Boldrick J.C. Sabet H. Tran T. Yu X. Powell J.I. Yang L. Marti G.E. Moore T. Hudson Jr., J. Lu L. Lewis D.B. Tibshirani R. Sherlock G. Chan W.C. Greiner T.C. Weisenburger D.D. Armitage J.O. Warnke R. Levy R. Wilson W. Grever M.R. Byrd J.C. Botstein D. Brown P.O. Staudt L.M. Nature. 2000; 403: 503-511Crossref PubMed Scopus (7919) Google Scholar). Thus, the increased expression of FAK after B cell activation may be part of a proadhesion gene expression program in which FAK promotes integrin-dependent adhesion and cell spreading, which facilitate BCR-antigen interactions that provide survival signals for GC B cells. Similarly the change in Pyk2 mRNA splicing in activated B cells may allow Pyk2 to interact with additional proteins that control cell adhesion or cytoskeletal reorganization. In summary, we have shown that Pyk2 and FAK are downstream targets of the Rap GTPases that play an important role in B cell spreading, a process that contributes to B cell activation. Antibodies (Abs) 2The abbreviations used are: AbantibodyBCRB cell receptorIgimmunoglobulinECMextracellular matrixLFA-1lymphocyte function-associated antigen-1Pyk2proline-rich tyrosine kinaseFAKfocal adhesion kinaseVLA-4very late antigen-4Tyr(P)phosphotyrosineLPSlipopolysaccharideshRNAshort hairpin RNAGCgerminal centerSH2Src homology 2Erkextracellular signal-regulated kinaseRTreverse transcriptionGFPgreen fluorescent proteinIL-4interleukin-4GAPGTPase-activating protein. 2The abbreviations used

Referência(s)