Title: Fyn and ZAP-70 Are Required for Vav Phosphorylation in T Cells Stimulated by Antigen-presenting Cells
Abstract: In T cells, triggering of the T cell antigen receptor or of the co-stimulatory receptor CD28 can direct tyrosine phosphorylation of the signaling protein Vav. We investigated the role played by the protein tyrosine kinases Fyn, Lck, and ZAP-70 in these processes in a T cell hybridoma after physiological stimulation of the T cell receptor (TCR) and CD28. A dominant-negative mutant approach based on overexpression of catalytically inactive alleles of these kinases showed that CD28-induced Vav phosphorylation preferentially requires Fyn, whereas ZAP-70 had no role. Consistently, Vav was strongly phosphorylated in Lck-deficient JCAM-1 cells after CD28 ligation. In contrast, ZAP-70 appeared to control TCR-directed Vav phosphorylation. However, overexpression of ZAP-70 carrying a mutated Tyr315, contained within a motif previously suggested to be a Vav Src homology 2 domain binding site, had little or no effect. Immunoprecipitation assays showed that phosphorylated Vav associated with Fyn after CD28 triggering and that this interaction, likely to involve binding of Fyn Src homology 2 domain to Vav, was more strongly detectable after concomitant CD28 and TCR stimulation. These data suggest that Fyn plays a major role in controlling Vav phosphorylation upon T cell activation and that the mechanism implicating ZAP-70 in this process may be more complex than previously anticipated. In T cells, triggering of the T cell antigen receptor or of the co-stimulatory receptor CD28 can direct tyrosine phosphorylation of the signaling protein Vav. We investigated the role played by the protein tyrosine kinases Fyn, Lck, and ZAP-70 in these processes in a T cell hybridoma after physiological stimulation of the T cell receptor (TCR) and CD28. A dominant-negative mutant approach based on overexpression of catalytically inactive alleles of these kinases showed that CD28-induced Vav phosphorylation preferentially requires Fyn, whereas ZAP-70 had no role. Consistently, Vav was strongly phosphorylated in Lck-deficient JCAM-1 cells after CD28 ligation. In contrast, ZAP-70 appeared to control TCR-directed Vav phosphorylation. However, overexpression of ZAP-70 carrying a mutated Tyr315, contained within a motif previously suggested to be a Vav Src homology 2 domain binding site, had little or no effect. Immunoprecipitation assays showed that phosphorylated Vav associated with Fyn after CD28 triggering and that this interaction, likely to involve binding of Fyn Src homology 2 domain to Vav, was more strongly detectable after concomitant CD28 and TCR stimulation. These data suggest that Fyn plays a major role in controlling Vav phosphorylation upon T cell activation and that the mechanism implicating ZAP-70 in this process may be more complex than previously anticipated. Src homology antibodies antigen antigen-presenting cell kinase-defective interleukin 2 monoclonal antibody protein tyrosine kinase Dulbecco's modified Eagle's medium T cell antigen receptor wild-type. Vav is a signaling protein expressed almost exclusively in cells of hematopoietic origin and is phosphorylated on tyrosine residues in response to a wide variety of stimuli (1Collins T.L. Deckert M. Altman A. Immunol. Today. 1997; 18: 221-225Abstract Full Text PDF PubMed Scopus (96) Google Scholar). Vav contains two SH31 domains spaced by an SH2 domain, a cysteine-rich zinc-binding domain, a pleckstrin homology domain, and a Dbl homology region characteristic of guanine nucleotide exchange factors for the small GTPases of the Rho family (1Collins T.L. Deckert M. Altman A. Immunol. Today. 1997; 18: 221-225Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 2Romero F. Fisher S. Cell. Signalling. 1996; 8: 545-553Crossref PubMed Scopus (47) Google Scholar). Recent studies have provided some clues as to the function of Vav. Thus, its role in positive regulation of lymphocyte activation is inferred from the severe defect in TCR and B cells antigen receptor-mediated activation in Vav-null mice (3Turner M.T. Mee J.P. Walters A.E. Quinn M.E. Mellor A.L. Zamoyska R. Tybulewicz V.L.J. Immunity. 1997; 7: 451-460Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 4Fischer K.-D. Zmuidzinas A. Gardner S. Barbacid M. Bernstein A. Guidos C. Nature. 1995; 374: 474-476Crossref PubMed Scopus (286) Google Scholar, 5Tarakhosky A. Turner M. Schaal S. Mee P.J. Duddy L.P. Rajewsky K. Tybulewicz V.L.J. Nature. 1995; 374: 467-469Crossref PubMed Scopus (389) Google Scholar, 6Zhang R. Alt F.W. Davidson L. Orkin S.H. Swat W. Nature. 1995; 374: 470-473Crossref PubMed Scopus (374) Google Scholar) and from studies showing that Vav synergizes with TCR stimulation for IL-2 gene transcription (7Wu J. Katzav S. Weiss A. Mol. Cell. Biol. 1995; 15: 4337-4346Crossref PubMed Scopus (166) Google Scholar). Moreover, Vav has been reported to associate with PTKs, adapter proteins such as Grb2, SLP-76, Nck, Crk, as well as cytoskeletal proteins (1Collins T.L. Deckert M. Altman A. Immunol. Today. 1997; 18: 221-225Abstract Full Text PDF PubMed Scopus (96) Google Scholar) suggesting its potential implication in different signaling pathways. Tyrosine phosphorylation of Vav results in an augmentation of its GDP/GTP exchange activity for the Rho family GTPases, Rac, CDC42, and RhoA (8Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (678) Google Scholar, 9Han J. Das B. Wei W. Van Aelst L. Mosteller R.D. Khosravi-Far R. Westwick J.K. Der C.J. Broek D. Mol. Cell. Biol. 1997; 17: 1346-1353Crossref PubMed Scopus (276) Google Scholar). These proteins regulate signaling leading to actin cytoskeleton changes (10Machesky L.M. Hall A. Trends Cell Biol. 1996; 6: 304-310Abstract Full Text PDF PubMed Scopus (253) Google Scholar), and interestingly, T cells lacking Vav are defective in actin cap formation induced by TCR triggering (11Holsinger L.J. Graef I.A. Swat W. Chi T. Bautista D.M. Davidson L. Lewis R.S. Alt F.W. Crabtree G.R. Curr. Biol. 1998; 8: 563-572Abstract Full Text Full Text PDF PubMed Google Scholar, 12Fischer K.-D. Kong Y.-Y. Nishina H. Tedford K. Marengère L.E.M. Kozieradski I. Sasaki T. Starr M. Chan G. Gardener S. Nghiem M.P. Bouchard D. Barbacid M. Bernstein A. Penninger J.M. Curr. Biol. 1998; 8: 554-562Abstract Full Text Full Text PDF PubMed Google Scholar). In addition, Vav may be implicated, via the Rho family of GTPases, in the activation of c-Jun N-terminal kinase (8Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (678) Google Scholar,13Olson M. Pasteris N.G. Gorski J.L. Hall A. Curr. Biol. 1996; 6: 1628-1633Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar).In T cells, Vav becomes tyrosine-phosphorylated upon stimulation of the TCR by antibodies (14Margolis B. Hu P. Katzav S. Oliver J.M. Ullrich A. Weiss A. Schlessinger J. Nature. 1992; 356: 71-74Crossref PubMed Scopus (302) Google Scholar) or of the co-stimulatory receptor CD28 by its natural ligands B7-1 (CD80) or B7-2 (CD86) and by specific antibodies (15Nunès J.A. Truneh A. Olive D. Cantrell D.A. J. Biol. Chem. 1996; 271: 1591-1598Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Why Vav is one of the few PTK substrates whose phosphorylation is directed by the TCR and CD28 (15Nunès J.A. Truneh A. Olive D. Cantrell D.A. J. Biol. Chem. 1996; 271: 1591-1598Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) is unclear. However, this dual control on Vav may reflect an important regulatory mechanism in lymphocyte activation. Indeed, TCR ligation alone by antigen-major histocompatibility complexes is usually insufficient to trigger full T cell activation and requires co-engagement of CD28 (16Lenschow D. Walunas T.L. Bluestone J.A. Annu. Rev. Immunol. 1996; 14: 233-258Crossref PubMed Scopus (2338) Google Scholar). Moreover, also in B cells, Vav phosphorylation is controlled by both the antigen receptor and co-stimulatory molecules (1Collins T.L. Deckert M. Altman A. Immunol. Today. 1997; 18: 221-225Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 17Tedder T.F. Inaoki M. Sato S. Immunity. 1997; 6: 107-118Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Thus, establishing how these receptors control Vav activation is important for understanding its specific function in the context of lymphocyte activation.The identity of the PTKs that phosphorylate Vav during T cell activation, that is the PTKs connecting the TCR and CD28 to Vav, remains unclear as little effort has been made to address this question in a physiological setting. Co-expression experiments in heterologous cell systems and in vitro kinase assays with recombinant proteins indicated that either Lck, Fyn, Syk, or ZAP-70 could phosphorylate Vav (8Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (678) Google Scholar, 18Gulbins E. Coggeshall K.M. Baier G. Katzav S. Burn P. Altman A. Science. 1993; 260: 822-825Crossref PubMed Scopus (229) Google Scholar, 19Deckert M. Tartare-Deckert S. Couture C. Altman A. Immunity. 1996; 5: 591-604Abstract Full Text PDF PubMed Scopus (244) Google Scholar, 20Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 155-164Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Moreover, Vav was reported to form a complex with Lck (21Gupta S. Weiss A. Kumar G. Wang S. Nel A. J. Biol. Chem. 1994; 269: 17349-17351Abstract Full Text PDF PubMed Google Scholar) or with ZAP-70 in anti-TCR-stimulated Jurkat T cells (19Deckert M. Tartare-Deckert S. Couture C. Altman A. Immunity. 1996; 5: 591-604Abstract Full Text PDF PubMed Scopus (244) Google Scholar, 22Huby R.D.J. Carlile G.W. Ley S.C. J. Biol. Chem. 1995; 270: 30241-30244Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 23Katzav S. Sutherland M. Packham G. Yi T. Weiss A. J. Biol. Chem. 1994; 269: 32579-32585Abstract Full Text PDF PubMed Google Scholar). However in other works (24Wu J. Motto D.G. Koretzky G.A. Weiss A. Immunity. 1996; 4: 593-602Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 25Tuosto L. Michel F. Acuto O. J. Exp. Med. 1996; 184: 1161-1166Crossref PubMed Scopus (173) Google Scholar), the association of Vav with ZAP-70 was not clearly detected.In this work, we investigated which PTKs control Vav phosphorylation in T cells upon physiological engagement of CD28 and TCR by their respective ligands. Toward this end, we expressed catalytically inactive mutants of Lck, Fyn, and ZAP-70 in a T cell hybridoma and monitored the effect on Vav phosphorylation. This approach provided evidence for a preferential role of Fyn in the phosphorylation of Vav after CD28 ligation. Moreover, following CD28 stimulation, Vav physically associated with Fyn, and this interaction was more strongly detected upon concomitant engagement of CD28 and TCR. We also show that ZAP-70 influences Vav phosphorylation after TCR engagement by antigen-major histocompatibility complex, but mutation of Tyr315 of ZAP-70, a site previously suggested to be a Vav-binding site, has little or no influence on phosphorylation of the latter.DISCUSSIONIn this work we investigated which of the PTKs, Lck, Fyn, and ZAP-70 that play a major role in T cell activation, control Vav phosphorylation upon TCR and CD28 engagement with Ag/major histocompatibility complex and B7-1, respectively. By employing a dominant-negative mutant approach, we show that Vav phosphorylation due to CD28 ligation is dependent on the Src PTKs, in particular on Fyn. In contrast, ZAP-70 did not appear to play any role in CD28-induced Vav phosphorylation. We show also that co-engagement of CD28 and TCR leads to additional accumulation of phosphorylated Vav. We have recently demonstrated that under conditions that mimic physiological stimulation (e.g. Ag presented by APC), the absence of CD28 engagement results in a dramatic reduction of TCR-proximal signaling capacity (e.g. tyrosine phosphorylation of ζ and ZAP-70) (45Tuosto L. Acuto O. Eur. J. Immunol. 1998; 28: 2131-2142Crossref PubMed Scopus (91) Google Scholar), suggesting that some CD28 signals may directly feed into the TCR. Thus, the sole contribution of the TCR to Vav phosphorylation could not be formally analyzed under the stimulatory conditions used here (e.g. antigen presentation). However, our data strongly suggest that ZAP-70 is implicated in this pathway, as the increment of Vav phosphorylation due to TCR engagement was abolished by overexpression of the ZAP-70-KD mutant. The observation that a sizable fraction of phosphorylated Vav was co-immunoprecipitated with Fyn after Ag stimulation (Fig. 4) supports the idea that Fyn is also implicated in controlling Vav activation via the TCR. Although Fyn-KD inhibited also TCR-mediated Vav phosphorylation (data not shown), this latter result cannot be unambiguously interpreted as this mutant may interfere with immunoreceptor tyrosine-based activation motif phosphorylation and therefore with ZAP-70 activation. The substantial levels of phosphorylated Vav already attained with CD28 alone and the additive accumulation after TCR triggering suggest that the co-stimulatory signal plays a major role in Vav activation. Thus, it is possible that when a T cell encounters a professional APC (e.g. a dendridic cell expressing high levels of B7 family proteins) Vav phosphorylation is primarily directed by CD28 due to the higher density of its ligands compared with the density of TCR ligands (e.g. orders of magnitude lower) and have a critical role in helping to sustain TCR signaling (45Tuosto L. Acuto O. Eur. J. Immunol. 1998; 28: 2131-2142Crossref PubMed Scopus (91) Google Scholar). Interestingly, Klasen et al. (47Klasen D. Pages F. Peyron J.-F. Cantrell D.A. Olive D. Int. Immunol. 1998; 10: 481-489Crossref PubMed Scopus (49) Google Scholar) reported that CD28-induced Vav phosphorylation is more persistent than the one stimulated via the TCR.Three lines of evidence suggest that Fyn may play a major role in CD28-driven phosphorylation of Vav. First, when expressed at comparably low mutant/wild-type ratios in T8.1 cells, Fyn-KD was more effective than Lck-KD as a dominant-negative mutant to inhibit Vav phosphorylation. Second, upon CD28 ligation Fyn was found associated with Vav, an interaction that may promote efficient Vav phosphorylation (discussed below). We found no gross difference between Fyn and Lck expression in T8.1 cells with the antisera used in this study, whereas in Jurkat cells the same reagents evidenced higher levels of Lck over Fyn (data not shown). It is therefore unlikely that our results were biased by an abnormally higher expression of Fyn over Lck. Third, Vav phosphorylation occurred efficiently in CD28-stimulated JCAM-1 cells which lack Lck but express Fyn. This result is of relevance since in this cell line TCR-driven tyrosine phosphorylation of cellular substrates is dramatically inhibited (37Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar). Fyn (and Lck) can phosphorylate Tyr173 (48Raab M. Cai Y.-C. Bunnel S.C. Heyeck S.D. Berg L.J. Rudd C.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8891-8895Crossref PubMed Scopus (157) Google Scholar) in the intracellular portion of CD28. However, the inhibition of Vav phosphorylation produced by Fyn-KD (and to a minor extent by Lck-KD) cannot be due to an indirect effect on Tyr173 as it was recently shown that a Y173F mutation does not modify CD28-induced Vav phosphorylation (47Klasen D. Pages F. Peyron J.-F. Cantrell D.A. Olive D. Int. Immunol. 1998; 10: 481-489Crossref PubMed Scopus (49) Google Scholar). Nevertheless, the neighboring sequence C-terminal to Tyr173 was found to be critical for Vav phosphorylation (47Klasen D. Pages F. Peyron J.-F. Cantrell D.A. Olive D. Int. Immunol. 1998; 10: 481-489Crossref PubMed Scopus (49) Google Scholar, 49Kim H.-H. Tharayil M. Rudd C.E. J. Immunol. 1998; 273: 296-301Google Scholar), but the underlying mechanism remains to be understood. Alternatively, Itk, a PTK reported to interact with, and be activated by, CD28 could be responsible for Vav phosphorylation. Since Itk is activated by Src PTKs, including Lck (50August A. Sadra A. Dupont B. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11227-11232Crossref PubMed Scopus (150) Google Scholar, 51Heyeck S.D. Wilcox H.M. Bunnell S.C. Berg L.J. J. Biol. Chem. 1997; 272: 25401-25408Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 52Gibson S. August A. Branch D. Dupont B. Mills G.B. J. Biol. Chem. 1996; 271: 7079-7083Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), the effects of Fyn-KD on Vav could be explained by an inhibition of Itk activation. However, Gibson et al. (52Gibson S. August A. Branch D. Dupont B. Mills G.B. J. Biol. Chem. 1996; 271: 7079-7083Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) have reported that Itk is not activated via CD28 in JCAM-1 and requires expression of Lck, whereas Vav phosphorylation occurs in this mutant cell line, as shown here. Moreover, mutation of Tyr173considerably reduced CD28-induced Itk activation (53King P. Sadra A. Teng J.M.C. Liu X. Han A. Selvakumar A. August A. Dupont B. J. Immunol. 1997; 158: 580-590PubMed Google Scholar) but not Vav phosphorylation (47Klasen D. Pages F. Peyron J.-F. Cantrell D.A. Olive D. Int. Immunol. 1998; 10: 481-489Crossref PubMed Scopus (49) Google Scholar). These data argue against Itk being implicated in Vav phosphorylation. Thus, in light of our results, Fyn is the most likely candidate to phosphorylate Vav directly.We found that ZAP-70 catalytic activity was critical for directing phosphorylation of Vav after physiological engagement of the TCR. However, Tyr315 present in the linker region of ZAP-70, thought to be important for Vav phosphorylation (23Katzav S. Sutherland M. Packham G. Yi T. Weiss A. J. Biol. Chem. 1994; 269: 32579-32585Abstract Full Text PDF PubMed Google Scholar, 46Wu J. Zhao Q.H. Kurosaki T. Weiss A. J. Exp. Med. 1997; 185: 1877-1882Crossref PubMed Scopus (77) Google Scholar), appears to be essentially dispensable for this function (Fig. 5) and also for IL-2 production.2 Our results contrast with those of Wu et al. (46Wu J. Zhao Q.H. Kurosaki T. Weiss A. J. Exp. Med. 1997; 185: 1877-1882Crossref PubMed Scopus (77) Google Scholar) who found that the same mutation could not complement a Syk PTK-deficient chicken B cell lymphoma for a defective phosphorylation of several signaling proteins including Vav and for nuclear factor of activated T cell activation. This discrepancy may be due to the different cellular systems utilized. However, we also found that, compared with ZAP-70-KD, ZAP-70Y315F exerted a very weak effect on TCR-induced nuclear factor of activated T cell activation when overexpressed in Jurkat cells.2 Thus, additional experiments are needed to definitively establish whether Tyr315 of ZAP-70 has a role in Vav phosphorylation, leaving open the question of how ZAP-70 exerts this function during TCR ligation. In this context, it is intriguing that, despite the inhibiting effect of ZAP-70-KD on TCR-directed Vav phosphorylation, we found that Fyn, but not ZAP-70 (this work and Refs. 24Wu J. Motto D.G. Koretzky G.A. Weiss A. Immunity. 1996; 4: 593-602Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar and 25Tuosto L. Michel F. Acuto O. J. Exp. Med. 1996; 184: 1161-1166Crossref PubMed Scopus (173) Google Scholar), was detected associated with Vav. In line with this observation, others (21Gupta S. Weiss A. Kumar G. Wang S. Nel A. J. Biol. Chem. 1994; 269: 17349-17351Abstract Full Text PDF PubMed Google Scholar) have previously reported that in Jurkat cells Vav forms a complex with Lck after TCR stimulation. The difference with our results in the T8.1 cells can be explained by the non-physiological high levels of Lck expressed in Jurkat cells compared with T cell hybridomas (Ref. 54Olszowy M.W. Leuchtmann P.L. Veillette A. Shaw A.S. J. Immunol. 1995; 155: 4236-4240PubMed Google Scholar and data not shown) and by the largely, although not completely, redundant roles of Lck and Fyn in T cell activation (55Lowell C.A. Soriano P. Genes Dev. 1996; 10: 1845-1857Crossref PubMed Scopus (252) Google Scholar, 56Groves T. Smiley P. Cooke M.P. Forbush K. Perlmutter R.M. Guidos C.J. Immunity. 1996; 5: 417-428Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 57Oers N.S.C. Lowin-Kropf B. Finlay D. Connolly K. Weiss A. Immunity. 1996; 5: 429-436Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Our result indicating that Vav-Fyn interaction requires tyrosine phosphorylation is consistent with the observation that recombinant SH2 domain of Fyn binds to phosphorylated Vav (58Bonnefoy-Berard N. Munshi A. Yron I. Wu S. Collins T.L. Deckert M. Shalom-Barack T. Giampa L. Herbert E. Hernandez J. Meller N. Couture C. Altman A. Stem Cells. 1996; 14: 250-268Crossref PubMed Scopus (24) Google Scholar). A direct SH2-mediated association is likely to promote an effective catalytic reaction as it has been suggested for Fyn and Src with some of their known substrates (38Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1079) Google Scholar, 59Songyang Z. Carraway K.L. Eckm M.J. Harrison S.C. Feldman R.A. Mohammmadi M. Schlessinger J. Hubbard S.R. Smith D.P. Eng C. Lorenzo M.J. Ponder B.A.J. Mayer B.J. Cantley L.C. Nature. 1995; 373: 536-539Crossref PubMed Scopus (841) Google Scholar,60DeMali K.A. Kazlauskas A. Mol. Cell. Biol. 1998; 18: 2014-2022Crossref PubMed Google Scholar). According to a processive mechanism proposed for Src PTKs (59Songyang Z. Carraway K.L. Eckm M.J. Harrison S.C. Feldman R.A. Mohammmadi M. Schlessinger J. Hubbard S.R. Smith D.P. Eng C. Lorenzo M.J. Ponder B.A.J. Mayer B.J. Cantley L.C. Nature. 1995; 373: 536-539Crossref PubMed Scopus (841) Google Scholar,61Pawson T. Nature. 1995; 373: 476-477Crossref PubMed Scopus (10) Google Scholar), Fyn could phosphorylate Vav at the very same tyrosine serving as an SH2-docking site to ensure further phosphorylation of Vav. Support for a kinase-substrate relationship between Fyn and Vav comes fromin vitro data (8Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (678) Google Scholar, 18Gulbins E. Coggeshall K.M. Baier G. Katzav S. Burn P. Altman A. Science. 1993; 260: 822-825Crossref PubMed Scopus (229) Google Scholar, 20Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 155-164Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) but also from in vivodata in T cells overexpressing wild-type or activated Fyn (62Fusaki N. Semba K. Katagiri T. Suzuki G. Matsuda S. Yamamoto T. Int. Immunol. 1994; 6: 1245-1255Crossref PubMed Scopus (34) Google Scholar) and in CD4/CD8 double negative T cells from lpr mice that presented elevated Fyn kinase activity and showed a constitutive tyrosine phosphorylation of Vav (63Mimura T. Minota S. Nojima Y. Morino N. Hamasaki K. Furuya H. Yazaki Y. J. Immunol. 1997; 158: 2977-2983PubMed Google Scholar). ZAP-70 may also contribute to Vav phosphorylation and may be necessary for fine-tuning the Vav function requested during TCR engagement. In this context, it will be interesting to ascertain in our system whether qualitative or quantitative differences exist in the phosphorylation pattern of Vav after CD28 or CD28 plus TCR triggering. If a sequential involvement of Syk and Src PTKs in Vav phosphorylation does exist as suggested byin vitro experiments (19Deckert M. Tartare-Deckert S. Couture C. Altman A. Immunity. 1996; 5: 591-604Abstract Full Text PDF PubMed Scopus (244) Google Scholar), this could apply to the TCR but not the CD28 pathway. Finally, we cannot exclude the possibility thatin vivo ZAP-70 exerts an indirect effect on Vav phosphorylation by favoring a better access of some substrates to activated Src PTKs during TCR ligation. By showing that Vav interacts with Fyn after physiological stimulation of TCR and CD28, our data may contribute to define better their function during T cell activation. Vav is a signaling protein expressed almost exclusively in cells of hematopoietic origin and is phosphorylated on tyrosine residues in response to a wide variety of stimuli (1Collins T.L. Deckert M. Altman A. Immunol. Today. 1997; 18: 221-225Abstract Full Text PDF PubMed Scopus (96) Google Scholar). Vav contains two SH31 domains spaced by an SH2 domain, a cysteine-rich zinc-binding domain, a pleckstrin homology domain, and a Dbl homology region characteristic of guanine nucleotide exchange factors for the small GTPases of the Rho family (1Collins T.L. Deckert M. Altman A. Immunol. Today. 1997; 18: 221-225Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 2Romero F. Fisher S. Cell. Signalling. 1996; 8: 545-553Crossref PubMed Scopus (47) Google Scholar). Recent studies have provided some clues as to the function of Vav. Thus, its role in positive regulation of lymphocyte activation is inferred from the severe defect in TCR and B cells antigen receptor-mediated activation in Vav-null mice (3Turner M.T. Mee J.P. Walters A.E. Quinn M.E. Mellor A.L. Zamoyska R. Tybulewicz V.L.J. Immunity. 1997; 7: 451-460Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 4Fischer K.-D. Zmuidzinas A. Gardner S. Barbacid M. Bernstein A. Guidos C. Nature. 1995; 374: 474-476Crossref PubMed Scopus (286) Google Scholar, 5Tarakhosky A. Turner M. Schaal S. Mee P.J. Duddy L.P. Rajewsky K. Tybulewicz V.L.J. Nature. 1995; 374: 467-469Crossref PubMed Scopus (389) Google Scholar, 6Zhang R. Alt F.W. Davidson L. Orkin S.H. Swat W. Nature. 1995; 374: 470-473Crossref PubMed Scopus (374) Google Scholar) and from studies showing that Vav synergizes with TCR stimulation for IL-2 gene transcription (7Wu J. Katzav S. Weiss A. Mol. Cell. Biol. 1995; 15: 4337-4346Crossref PubMed Scopus (166) Google Scholar). Moreover, Vav has been reported to associate with PTKs, adapter proteins such as Grb2, SLP-76, Nck, Crk, as well as cytoskeletal proteins (1Collins T.L. Deckert M. Altman A. Immunol. Today. 1997; 18: 221-225Abstract Full Text PDF PubMed Scopus (96) Google Scholar) suggesting its potential implication in different signaling pathways. Tyrosine phosphorylation of Vav results in an augmentation of its GDP/GTP exchange activity for the Rho family GTPases, Rac, CDC42, and RhoA (8Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (678) Google Scholar, 9Han J. Das B. Wei W. Van Aelst L. Mosteller R.D. Khosravi-Far R. Westwick J.K. Der C.J. Broek D. Mol. Cell. Biol. 1997; 17: 1346-1353Crossref PubMed Scopus (276) Google Scholar). These proteins regulate signaling leading to actin cytoskeleton changes (10Machesky L.M. Hall A. Trends Cell Biol. 1996; 6: 304-310Abstract Full Text PDF PubMed Scopus (253) Google Scholar), and interestingly, T cells lacking Vav are defective in actin cap formation induced by TCR triggering (11Holsinger L.J. Graef I.A. Swat W. Chi T. Bautista D.M. Davidson L. Lewis R.S. Alt F.W. Crabtree G.R. Curr. Biol. 1998; 8: 563-572Abstract Full Text Full Text PDF PubMed Google Scholar, 12Fischer K.-D. Kong Y.-Y. Nishina H. Tedford K. Marengère L.E.M. Kozieradski I. Sasaki T. Starr M. Chan G. Gardener S. Nghiem M.P. Bouchard D. Barbacid M. Bernstein A. Penninger J.M. Curr. Biol. 1998; 8: 554-562Abstract Full Text Full Text PDF PubMed Google Scholar). In addition, Vav may be implicated, via the Rho family of GTPases, in the activation of c-Jun N-terminal kinase (8Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (678) Google Scholar,13Olson M. Pasteris N.G. Gorski J.L. Hall A. Curr. Biol. 1996; 6: 1628-1633Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). In T cells, Vav becomes tyrosine-phosphorylated upon stimulation of the TCR by antibodies (14Margolis B. Hu P. Katzav S. Oliver J.M. Ullrich A. Weiss A. Schlessinger J. Nature. 1992; 356: 71-74Crossref PubMed Scopus (302) Google Scholar) or of the co-stimulatory receptor CD28 by its natural ligands B7-1 (CD80) or B7-2 (CD86) and by specific antibodies (15Nunès J.A. Truneh A. Olive D. Cantrell D.A. J. Biol. Chem. 1996; 271: 1591-1598Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Why Vav is one of the few PTK substrates whose phosphorylation is directed by the TCR and CD28 (15Nunès J.A. Truneh A. Olive D. Cantrell D.A. J. Biol. Chem. 1996; 271: 1591-1598Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) is unclear. However, this dual control on Vav may reflect an important regulatory mechanism in lymphocyte activation. Indeed, TCR ligation alone by antigen-major histocompatibility complexes is usually insufficient to trigger full T cell activation and requires co-engagement of CD28 (16Lenschow D. Walunas T.L. Bluestone J.A. Annu. Rev. Immunol. 1996; 14: 233-258Crossref PubMed Scopus (2338) Google Scholar). Moreover, also in B cells, Vav phosphorylation is controlled by both the antigen receptor and co-stimulatory molecules (1Collins T.L. Deckert M. Altman A. Immunol. Today. 1997; 18: 221-225Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 17Tedder T.F. Inaoki M. Sato S. Immunity. 1997; 6: 107-118Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Thus, establishing how these receptors control Vav activation is important for understanding its specific function in the context of lymphocyte activation. The identity of the PTKs that phosphorylate Vav during T cell activation, that is the PTKs connecting the TCR and CD28 to Vav, remains unclear as little effort has been made to address this question in a physiological setting. Co-expression experime