Costimulation through CD28 Suppresses T Cell Receptor-dependent Activation of the Ras-like Small GTPase Rap1 in Human T Lymphocytes

Members of the Ras superfamily of small molecular weight GTPases play diverse and critical roles in mediating cellular responses to extracellular stimuli, including mitogenesis, cytoskeletal maintenance and rearrangement, and integrin activation. In T lymphocytes, biochemical and genetic evidence demonstrate that Ras plays an essential role in coupling T cell receptor ligation to signaling cascades required for T cell proliferation and development. Recent observations that C3G, a guanine nucleotide exchange factor specific for the Ras-related GTPase Rap1, is recruited into tyrosine-phosphorylated protein signaling complexes in activated T cells have suggested that Rap1 may also play a role in T cell activation. Utilizing a recently developed technique for detection of endogenous, GTP-bound Rap1, we have found that Rap1, but not Rap2, is transiently activated following T cell receptor stimulation of normal human T lymphocytes. Increases in intracellular calcium is both necessary and sufficient to induce Rap1 activation. Remarkably, costimulation of T cells with mitogenic anti-CD28 antibody completely abolished T cell receptor-dependent activation of Rap1. This report demonstrates a potential role for Rap1 in T cell receptor signaling and suggests inactivation of Rap1 as a candidate target of CD28-dependent costimulatory signals required for T cell antigen responsiveness. Members of the Ras superfamily of small molecular weight GTPases play diverse and critical roles in mediating cellular responses to extracellular stimuli, including mitogenesis, cytoskeletal maintenance and rearrangement, and integrin activation. In T lymphocytes, biochemical and genetic evidence demonstrate that Ras plays an essential role in coupling T cell receptor ligation to signaling cascades required for T cell proliferation and development. Recent observations that C3G, a guanine nucleotide exchange factor specific for the Ras-related GTPase Rap1, is recruited into tyrosine-phosphorylated protein signaling complexes in activated T cells have suggested that Rap1 may also play a role in T cell activation. Utilizing a recently developed technique for detection of endogenous, GTP-bound Rap1, we have found that Rap1, but not Rap2, is transiently activated following T cell receptor stimulation of normal human T lymphocytes. Increases in intracellular calcium is both necessary and sufficient to induce Rap1 activation. Remarkably, costimulation of T cells with mitogenic anti-CD28 antibody completely abolished T cell receptor-dependent activation of Rap1. This report demonstrates a potential role for Rap1 in T cell receptor signaling and suggests inactivation of Rap1 as a candidate target of CD28-dependent costimulatory signals required for T cell antigen responsiveness. Biochemical, mutational, and genetic studies have demonstrated that physical and functional coupling of the T cell receptor (TCR) 1The abbreviations used are: TCR, T cell receptor; PTK, protein-tyrosine kinases; IL, interleukin; MAP, mitogen-activated protein; TPA, 12-O-tetradecanoylphorbol-13-acetate; ERK, extracellular-regulated kinase; GST, glutathioneS-transferase; RBD, Rap binding domain; Ral-GDS, Ral guanine nucleotide dissociation stimulator; PAGE, polyacrylamide gel electrophoresis. to associated Src (Fyn and Lck) and Syk (Syk, ZAP-70) family protein-tyrosine kinases (PTKs) plays a critical role in both T cell activation and development (reviewed in Refs. 1Weiss A. Littman D. Cell. 1994; 76: 263-274Google Scholar and 2Qian D. Weiss A. Curr. Opin. Cell. Biol. 1997; 9: 205-212Google Scholar). The generation of lipid second messengers, mobilization of calcium stores, and gene transcription critical for T cell proliferation and cytokine secretion is entirely dependent on TCR-associated PTK activity (1Weiss A. Littman D. Cell. 1994; 76: 263-274Google Scholar, 3Cantrell D. Annu. Rev. Immunol. 1996; 14: 259-274Google Scholar). In normal human T lymphocytes, ligation of the TCR alone is insufficient to induce proliferation and cytokine secretion, and instead induces anergy, a long term unresponsiveness to antigen stimulation. Costimulation of T cells by soluble interleukins (ILs) (e.g.. IL-2, -4, -7, or -15) or by ligation of the T cell accessory protein CD28 by antibodies or B7–1 and B7–2 ligands on antigen presenting cells avoids induction of anergy and leads to a full proliferative T cell response (4Lenschow D. Walunas T. Bluestone J. Annu. Rev. Immunol. 1996; 14: 233-258Google Scholar, 5Boussiotis V. Barber D. Nakarai T. Freeman G. Gribben J. Bernstein G. D'Andrea A. Ritz J. Nadler L. Science. 1994; 266: 1039-1042Google Scholar). The mechanism(s) by which CD28 stimulation prevents anergy and cooperates with TCR-derived signals to induce T cell proliferation are poorly understood. CD28 can activate both Src and Tek family PTKs (reviewed in Ref. 6Ward S. Biochem. J. 1996; 318: 361-377Google Scholar). CD28-dependent PTK activity phosphorylates a number of T cell signaling proteins previously shown to be tyrosine-phosphorylated in response to TCR stimulation, including phospholipase C-γ1, SLP-76, p36/38, and the proto-oncogene products Vav and Cbl (1Weiss A. Littman D. Cell. 1994; 76: 263-274Google Scholar, 2Qian D. Weiss A. Curr. Opin. Cell. Biol. 1997; 9: 205-212Google Scholar, 6Ward S. Biochem. J. 1996; 318: 361-377Google Scholar, 7Nunès J. Collette Y. Truneh A. Olive D. Cantrell D. J. Exp. Med. 1994; 180: 1067-1076Google Scholar, 8Nunès J. Truneh A. Olive D. Cantrell D. J. Biol. Chem. 1996; 271: 1591-1598Google Scholar). Similarly, CD28 stimulation also leads to activation of phosphatidylinositol 3-kinase and calcium mobilization in T lymphocytes (reviewed in Ref. 6Ward S. Biochem. J. 1996; 318: 361-377Google Scholar). However, CD28 stimulation also generates second messengers distinct from TCR signaling, including ceramide (9Chan G. Ochi A. Eur. J. Immunol. 1995; 25: 1999-2004Google Scholar) and reactive oxygen intermediates (10Los M. Schenk H. Hexel K. Baeuerle P. Dronge W. Schulz-Osthoff K. EMBO J. 1995; 14: 3731-3740Google Scholar), which may play a role in the ability of CD28 stimulation to synergistically activate Jun kinases and enhance IL-2 mRNA transcription and stability in CD3-stimulated T cells (4Lenschow D. Walunas T. Bluestone J. Annu. Rev. Immunol. 1996; 14: 233-258Google Scholar, 6Ward S. Biochem. J. 1996; 318: 361-377Google Scholar). In T cells, stimulation by either anti-CD3 or anti-CD28 antibodies results in recruitment of the Ras nucleotide exchange factor Sos to tyrosine-phosphorylated Shc and p36/38 proteins via the adaptor protein Grb2 (7Nunès J. Collette Y. Truneh A. Olive D. Cantrell D. J. Exp. Med. 1994; 180: 1067-1076Google Scholar, 8Nunès J. Truneh A. Olive D. Cantrell D. J. Biol. Chem. 1996; 271: 1591-1598Google Scholar, 11Buday L. Egan S. Rodriguez Viciana P. Cantrell D. Downward J. J. Biol. Chem. 1994; 269: 9019-9023Google Scholar, 12Ravichandran K. Lee K. Songyang Z. Cantley L. Burn P. Burakoff S. Science. 1993; 262: 902-905Google Scholar). Subsequent conversion of Ras to its active, GTP-bound form (13Downward J. Graves J. Warne P. Rayter S. Cantrell D. Nature. 1990; 346: 719-723Google Scholar) allows membrane localization and activation of Raf, which initiates a cascade of serine/threonine kinase activation, culminating in activation of transcription factors, gene transcription, and mitogenesis (14Izquierdo M. Reif K. Cantrell D. Immunol. Today. 1995; 16: 159-164Google Scholar). Studies in transgenic mice and fetal thymic organ cultures have confirmed that an intact Ras signaling pathway is required for proper T cell development and function (15Swan K. Alberola-Ila J. Gross J. Appleby M. Forbush K. Thomas J. Perlmutter R. EMBO J. 1995; 14: 276-285Google Scholar, 16Alberola-Ila J. Forbush K. Seger R. Krebs E. Perlmutter R. Nature. 1995; 373: 620-623Google Scholar, 17Whitehurst C. Geppert T. J. Immunol. 1996; 156: 1020-1029Google Scholar, 18Crompton T. Gilmour K. Owen M. Cell. 1996; 86: 243-251Google Scholar, 19Rayter S. Woodrow M. Lucas S. Cantrell D. Downward J. EMBO J. 1992; 11: 4549-4556Google Scholar), and blocks in Ras signaling pathways are found in anergized T cells (20Li W. Whaley C. Mondino A. Mueller D. Science. 1996; 271: 1272-1275Google Scholar,21Fields P. Gajewski T. Fitch F. Science. 1996; 271: 1276-1278Google Scholar). Recent studies in B and T lymphocytes have suggested a novel signaling pathway in lymphocytes involving the Ras-related GTPase Rap1. Antigen receptor-dependent tyrosine phosphorylation of the proto-oncogene product Cbl induces Cbl association with SH2 domain-containing adaptor proteins, including Crk family proteins (22Reedquist K. Fukazawa T. Panchamoorthy G. Langdon W. Shoelson S. Druker B. Band H. J. Biol. Chem. 1996; 271: 8435-8442Google Scholar, 23Buday L. Khwaja A. Sipeki S. Farago A. Downward J. J. Biol. Chem. 1996; 271: 6159-6163Google Scholar, 24Smit L. van der Horst G. Borst J. J. Biol. Chem. 1996; 271: 8564-8569Google Scholar) and the p85 subunit of phosphatidylinositol 3-kinase (25Fukazawa T. Reedquist K. Trub T. Soltoff S. Panchamoorthy G. Druker B. Cantley L. Shoelson S. Band H. J. Biol. Chem. 1995; 270: 19141-19150Google Scholar, 26Meisner H. Conway B. Hartley D. Czech M. Mol. Cell. Biol. 1995; 15: 3571-3578Google Scholar, 27Panchamoorthy G. Fukazawa T. Miyake S. Solftoff S. Reedquist K. Druker B. Shoelson S. Cantley L. Band H. J. Biol. Chem. 1996; 271: 3187-3194Google Scholar, 28Kim T. Kim Y.-T. Pillai S. J. Biol. Chem. 1995; 270: 27504-27509Google Scholar). In these studies, it was observed that CrkL was constitutively associated via its SH3 domains to the guanine nucleotide exchange factor C3G, which has guanine nucleotide exchange activity specific for Rap1 (29Tanaka S. Morishita T. Hashimoto Y. Hattori S. Nakamura S. Shibuya M. Matuoka K. Takenawa T. Kurata T. Nagashima K. Matsuda M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3443-3447Google Scholar, 30Gotoh T. Hattori S. Nakamura S. Kitayama H. Noda M. Takai Y. Kaibuchi K. Matsui H. Hatase O. Takahashi H. Kurata T. Matsuda M. Mol. Cell. Biol. 1995; 15: 6746-6753Google Scholar). As C3G could be detected in complex with tyrosine-phosphorylated Cbl (22Reedquist K. Fukazawa T. Panchamoorthy G. Langdon W. Shoelson S. Druker B. Band H. J. Biol. Chem. 1996; 271: 8435-8442Google Scholar), it was suggested that Rap1 may be activated as a consequence of TCR stimulation. Rap1 was originally identified by its ability to revert viral Kras oncogenic transformation in fibroblasts, yet potential roles and functions for Rap1 in physiological signaling pathways remain poorly understood (31Kitayama H. Sugimoto Y. Matsuzaki T. Ikawa Y. Noda M. Cell. 1990; 56: 77-84Google Scholar,32Pizon V. Chardin P. Lerosey I. Olofsson B. Tavitian A. Oncogene. 1989; 3: 201-204Google Scholar). Rap1 has an effector domain nearly identical to Ras and binds to many of the same effector proteins as Ras, including Raf-1, the catalytic subunit p110 of phosphatidylinositol 3-kinase and the Ral guanine nucleotide exchange factors in vitro, suggesting that Rap1 might antagonize Ras signaling by sequestering potential Ras effectors in inactive complexes (33Hata Y. Kikuchi A. Sasaki T. Schaber M. Gibbs J. Takai Y. J. Biol. Chem. 1990; 265: 7104-7107Google Scholar, 34French M. John J. Pizon V. Chardin P. Tavitian A. Clark R. McCormick F. Wittinghofer A. Science. 1990; 249: 169-171Google Scholar, 35Nassar N. Horn G. Hermann C. Block C. Jankecht R. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 723-729Google Scholar, 36Hermann C. Horn G. Spaargaren M. Wittinghofer A. J. Biol. Chem. 1996; 271: 6794-6800Google Scholar). Recent observations that Rap1 can mediate cAMP-dependent B-Raf and MAP kinase activation in PC12 cells (37Vossler M. Yao H. York R. Pan M.-G. Rim C. Stork P. Cell. 1997; 89: 73-82Google Scholar) and can enhance mitogenic signaling pathways in Swiss 3T3 responses to insulin (38Yoshica Y. Kawata M. Miura Y. Musha T. Sasaki T. Kikuchi A. Takai Y. Mol. Cell. Biol. 1992; 12: 3407-3414Google Scholar) suggest that in certain cells, Rap1 could contribute positive signals to mitogenic responses. In this study, we utilize a recently developed technique for detecting GTP-bound Rap proteins (39Franke B. Akkerman J.-W. Bos J. EMBO J. 1997; 16: 252-259Google Scholar) and demonstrate that Rap1 is transiently activated in TCR-stimulated normal human T cell PHA blasts and the cytotoxic T cell clone D11 but not in the leukemic T cell line Jurkat. Although TCR-mediated Rap1 activation is calcium-dependent, either phorbol ester or calcium ionophore alone are sufficient to induce Rap1 activation. Remarkably, activation of Rap1 by could be completely by costimulation suggesting that Rap1 may play a role in the of costimulation activation. The human T cell cell line was as previously (25Fukazawa T. Reedquist K. Trub T. Soltoff S. Panchamoorthy G. Druker B. Cantley L. Shoelson S. Band H. J. Biol. Chem. 1995; 270: 19141-19150Google Scholar). T cell PHA blasts from human from and the lymphocytes by The and of by cells for with PHA and IL-2 The in fetal and from and for an in IL-2 PHA blasts from IL-2 to and stimulation of the D11 cytotoxic human T cell clone was as previously R. M. van B. R. J. Immunol. Scholar). cells in and for on in the or of antibodies a of CD28 a mitogenic antibody by van R. M. R. M. E. L. W. Scholar). and in cells activated by in antibodies and and and was in and cells for In cells by in or cells with or from the in and with antibodies as antibodies used and antibodies in by of with for to kinase antibodies have previously A. B. S. C. Bos J. Nature. 1992; Scholar). to of the Rap binding domain of and of protein have previously C. Horn G. Spaargaren M. Wittinghofer A. J. Biol. Chem. 1996; 271: 6794-6800Google Scholar). for by binding to cell for with of to of proteins by and detection by and or antibodies as previously (39Franke B. Akkerman J.-W. Bos J. EMBO J. 1997; 16: 252-259Google Scholar). Rap binding of cell used for binding on and for Rap1 to that of Rap1 for binding not Recent have suggested that Rap1 may be in B and T cell antigen receptor signaling in lymphocytes, as these studies recruitment of the guanine nucleotide exchange factor C3G to tyrosine-phosphorylated an and of antigen via the CrkL adaptor protein (22Reedquist K. Fukazawa T. Panchamoorthy G. Langdon W. Shoelson S. Druker B. Band H. J. Biol. Chem. 1996; 271: 8435-8442Google Scholar). TCR stimulation activated Rap1 in T lymphocytes, we a recently developed novel technique the of to GTP-bound Rap1 from cell proteins with to GTP-bound Rap1 and binding to Rap proteins C. Horn G. Spaargaren M. Wittinghofer A. J. Biol. Chem. 1996; 271: 6794-6800Google Scholar) and can be as for detecting activated GTP-bound Rap1 proteins in cell has previously used this technique to report the activation of Rap1 in human with to activation of Rap1 (39Franke B. Akkerman J.-W. Bos J. EMBO J. 1997; 16: 252-259Google Scholar). of GTP-bound Rap1 in normal human T cell PHA blasts or stimulation. T cells in the or of anti-CD3 by and activation by antibody for GTP-bound Rap1 was with protein and detected by of activated Rap1 detected in T cells, and TCR stimulation, a distinct in GTP-bound Rap1 was of cell confirmed that of Rap1 for binding in not The of GTP-bound Rap1 but in of an in Rap1 was the activation of Rap1, we the of Rap1 activation in T cells TCR of binding that Rap1 activation was of TCR a of activation stimulation. of Rap1 and stimulation. In Rap1 activation was found to to activation by not Rap1 is and transiently to its GTP-bound form TCR stimulation. Rap1 activation was also observed in TCR-stimulated D11 cells, Rap1 activation was and In we have to activation of Rap1 in cell tyrosine phosphorylation of Cbl was observed and not it is that either C3G catalytic activity or Rap1 in recruitment of C3G into signaling complexes may not be sufficient for Rap1 activation, and an as yet novel exchange factor or in normal T cells may be in Jurkat. studies in human demonstrated that of with phorbol ester or calcium ionophore was sufficient to induce Rap1 activation, and activation of Rap1 by was completely dependent on calcium (39Franke B. Akkerman J.-W. Bos J. EMBO J. 1997; 16: 252-259Google Scholar). activation of Rap1 in T cells was we the ability of phorbol ester and calcium alone or in to activate Rap1 in T cell PHA T cells with demonstrated activation of Rap1 was of to TPA, by activation was to be that observed by stimulation of PHA blasts with the calcium ionophore also in activation of Rap1 However, Rap1 activation was and with a of in activation of Rap1 that to a of the observed and In this Rap1 activation was stimulation but for on of Rap1 with cell of protein used in binding with of cellular Rap1 to its GTP-bound In stimulation of with or alone or in to activate Rap1 also TCR stimulation of PHA blasts could also induce activation of the However, of binding with antibody detected small of GTP-bound in cells, and in was observed TCR stimulation of PHA blasts or T cells not Although TCR stimulation to activate in which Rap1 activation was we and could induce activation. of shown in and with antibodies a activation of by alone also activation of not of and shown demonstrated that with a Rap1 and that activation not signals from GTP-bound by of cellular cell of cellular protein used in binding is this activation of a exchange factor by calcium and or the exchange factor Rap1 has activity C3G not exchange activity for N. R. Horn G. Wittinghofer A. Oncogene. 1997; 15: Scholar), and exchange factors for have yet to be these that Rap1 is activated following TCR stimulation. As these results demonstrated that both phorbol and intracellular calcium sufficient to induce Rap1 activation in T cells, we either protein kinase or calcium was required for Rap1 activation with results in Rap1 activation was in a by of PHA blasts with the intracellular calcium of Rap1 activation was observed with with completely Rap1 activation and In of T cells with the protein kinase on Rap1 activation protein kinases with J. R. J. Biochem. Scholar). that could Rap1 activation we in a the of with the tyrosine kinase A. with completely abolished Rap1 activation TCR and with the for tyrosine kinases in signals from the TCR and the of Rap1 activation by recruitment of C3G to tyrosine-phosphorylated Cbl (22Reedquist K. Fukazawa T. Panchamoorthy G. Langdon W. Shoelson S. Druker B. Band H. J. Biol. Chem. 1996; 271: 8435-8442Google Scholar). However, activation of Rap1 by both and TCR stimulation in and T cells, calcium of the of C3G that C3G calcium binding as as found in the binding domains of the Ras exchange factors nucleotide factor and C. N. L. A. M. L. Nature. 1995; N. W. Z. H. M. Mol. Cell. Biol. 1997; Scholar). the of Rap1 is activated in protein of C3G in human are nearly A. a exchange factor or may be mediating Rap1 activation in has recently that ligation of the T cell accessory protein TCR stimulation, also leads to tyrosine phosphorylation of the Cbl proto-oncogene J. Truneh A. Olive D. Cantrell D. J. Biol. Chem. 1996; 271: 1591-1598Google Scholar), and we that CD28 ligation might also induce Rap1 activation in T lymphocytes. T cells with alone or or a of anti-CD3 and anti-CD28 by the of GTP-bound Rap1 as with T cells In stimulation of CD28 to activate Rap1. costimulatory anti-CD28 with anti-CD3 completely Rap1 activation. of GTP-bound Rap1 observed in T results observed in either alone or in with anti-CD3 to activation of Rap1 stimulation by anti-CD28 or anti-CD28 anti-CD3 antibodies that the of CD28 on Rap1 activation a in Rap1 activation a in activation of T cells with anti-CD28 antibodies alone in a in GTP-bound Rap1, as with in cells a of to nearly Costimulation TCR and CD28 to a in of GTP-bound Rap1 these GTP-bound Rap1 could be detected stimulation. CD28 or TCR CD28 stimulation in activation of not stimulation CD28 initiates signals that in a in GTP-bound Rap1 and completely blocks conversion of Rap1 to its that costimulation CD28 was not signaling we the ability of anti-CD3 and anti-CD28 antibodies to MAP kinase activation in PHA As in stimulation with either anti-CD3 or anti-CD28 antibodies both gel with of the MAP kinase costimulation CD28 not antagonize MAP kinase activation and activation. results demonstrate that of Rap1 activation is a specific consequence of CD28 The by which CD28 costimulation blocks activation is and could of or of potential the Tek family kinase S. Biochem. J. 1996; 318: 361-377Google Scholar), which could in phosphorylation of Cbl association with or phosphorylation of CrkL binding to both the SH2 and SH3 domains of S. B. H. EMBO J. 1994; T. C. J. K. J. Druker B. J. Biol. Chem. 1994; 269: Scholar). second messengers to CD28 signaling, including ceramide and reactive oxygen might Rap1 or its exchange or activate proteins, as or protein P. B. T. McCormick F. Proc. Natl. Acad. Sci. U. S. A. Scholar, R. A. J. J. Biol. Chem. 1995; 270: Scholar, M. N. B. K. H. H. N. Mol. Cell. Biol. 1995; 15: Scholar). and CD28 stimulation both induce calcium in T cells (1Weiss A. Littman D. Cell. 1994; 76: 263-274Google Scholar, 6Ward S. Biochem. J. 1996; 318: 361-377Google Scholar), it is that of Rap1 of the Rap1 was originally identified in for of viral into potential roles for Rap1 in have from that the of and Rap1 on receptor signaling with the ability of Rap1 to transformation by viral of Rap1 MAP kinase activation and factor stimulation of V. Freeman G. A. Barber D. Nadler L. Science. 1997; Scholar), and a in the of Rap1, with development S. T. H. Proc. Natl. Acad. Sci. U. S. A. 1997; Scholar). In of Rap1 can also mitogenesis in Swiss 3T3 fibroblasts, and of cAMP-dependent kinase signaling by Rap1 in PC12 cells (37Vossler M. Yao H. York R. Pan M.-G. Rim C. Stork P. Cell. 1997; 89: 73-82Google Scholar), that in cell Rap1 can mitogenic signaling pathways as Rap1 is to Ras, and the GTPase effector domain is completely identical Rap1 and This has to the that the of Rap1 on signaling from by Rap1 for binding to the same effector proteins by Ras N. Horn G. Hermann C. Block C. Jankecht R. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 723-729Google Scholar). Rap1 binds with in to and family of activated Rap1 with a Ras effector protein could either pathways to Ras, or the effector in an inactive signaling TCR stimulation in the of costimulation results in antigen of normal T lymphocytes, costimulatory signals of IL-2 and Recent studies demonstrate that an intact Ras signaling pathway is critical for proliferative T cell and that blocks in this pathway induce an in T cells (15Swan K. Alberola-Ila J. Gross J. Appleby M. Forbush K. Thomas J. Perlmutter R. EMBO J. 1995; 14: 276-285Google Scholar, 16Alberola-Ila J. Forbush K. Seger R. Krebs E. Perlmutter R. Nature. 1995; 373: 620-623Google Scholar, 17Whitehurst C. Geppert T. J. Immunol. 1996; 156: 1020-1029Google Scholar, 18Crompton T. Gilmour K. Owen M. Cell. 1996; 86: 243-251Google Scholar, 19Rayter S. Woodrow M. Lucas S. Cantrell D. Downward J. EMBO J. 1992; 11: 4549-4556Google Scholar). in anergized T cells, is a in MAP kinase activation (20Li W. Whaley C. Mondino A. Mueller D. Science. 1996; 271: 1272-1275Google Scholar, P. Gajewski T. Fitch F. Science. 1996; 271: 1276-1278Google Scholar). this was in that of GTP-bound Rap1 constitutively in an anergized T cell with cells V. Freeman G. A. Barber D. Nadler L. Science. 1997; Scholar). found to with a in activation and association with in to that Rap1 could play a role in and stimulation, Rap1 may also play a role in T cell anergy, As we have observed that Rap1 is activated in normal T cells following stimulation by but not costimulatory signaling CD28 is it be of to Rap1 plays a role in T cells to and proliferative the of Rap1 to antagonize Ras signaling it is to that Rap1 activation, in the of might to Ras signaling by sequestering or potential Ras effector proteins phosphatidylinositol This in might signaling cascades a certain required for mitogenesis and contribute to T cell Although stimulation with anti-CD3 and anti-CD28 but not CD28 ligands B7–1 and B7–2 J. Truneh A. Olive D. Cantrell D. J. Biol. Chem. 1996; 271: 1591-1598Google Scholar), can both induce Ras and MAP kinase activation, of the and of Ras effector activation in the and of CD28 costimulation have not Rap might induce activation distinct from In this it is that of C3G can enhance kinase activation, and C3G can and kinase activation S. T. H. Proc. Natl. Acad. Sci. U. S. A. 1997; Scholar). Rap1 may also signaling pathways to Rap1 has demonstrated to with in cells F. L. O. C. G. J. Biol. Chem. 1994; 269: Scholar, T. C. P. R. 1995; Scholar), in the Rap1 protein in E. J. I. Proc. Natl. Acad. Sci. U. S. A. 1997; Scholar). Rap1 might also play a role in the secretion of cytotoxic in T lymphocytes. studies in are the by which and CD28 Rap1 binding to and the role of Rap1 in T cell activation. van for and for are to and van for critical we and and in for critical of this