The Vav Binding Site (Y315) in ZAP-70 Is Critical for Antigen Receptor–mediated Signal Transduction

The NFAT luciferase reporter construct was a gift from Dr. G. Crabtree (Stanford University, Stanford, CA). The Vav plasmid (pCI115) was constructed by subcloning human Vav into pCIneo (Invitrogen, San Diego, CA). The parental plasmid for the ZAP-70 mutant was pCDNA3-ZAP-70. The Y315F mutant of ZAP-70 (ZAP70[Y315F]) was created by M13-based, oligonucleotide-directed, site-specific mutagenesis procedures (17). The myc epitope–tagged, wild-type ZAP-70 (pSXSRa-ZAP-myc) was provided by Dr. L. Samelson (National Institutes of Health, Bethesda, MD). DNA encoding wild-type rat Syk was subcloned into the mammalian expression vector pEFBOS. Glutathione S transferase (GST)- VavSH2 was provided by Dr. S. Katzav (Israel Air Force Aeromedical Center, Tel Hashomer, Israel). The human Shc plasmid and the FLAG epitope–tagged human SLP-76 cDNA were provided by Dr. M. Gishizky (Sugen Inc., Redwood City, CA) and Dr. G. Koretzky (University of Iowa, Iowa City, IA), respectively.

The mAb used for the stimulation of the BCR was M4 (provided by Drs. M. Cooper and C.L. Chen, University of Alabama, Birmingham, AL). Anti-Vav polyclonal Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antiphosphotyrosine mAb 4G10 was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Anti–ZAP-70 mAb (2F3.2) was described previously (18). The anti-myc epitope mAb (9E10) was provided by Dr. J.M. Bishop. The peptide used in this paper represents a biotinylated doubly phosphorylated version of the second ITAM of the TCR ζ chain (18).

Wild-type and various mutants of DT-40 cells were maintained and transfected transiently as previously described (17, 19). In brief, 30 μg of either an empty vector, wild-type ZAP-70 or ZAP-70(Y315F), and 20 μg of NFAT-Luc construct was used. Cells were then electroporated, processed, and assayed as described (17).

Cells were harvested, washed, were left either unstimulated or stimulated with M4 (2 μg/ml), and then lysed as previously described (13). Lysates were then immunoprecipitated with the indicated antibodies. When precipitated with GST fusion proteins, lysates were first precleared with GST alone (10 μg) before they were precipitated with the indicated GST fusion proteins (2–5 μg). Resulting immunoprecipitates or protein complexes were resolved by SDS-PAGE. Peptide binding and immunoblotting were carried out as previously described (18).

After transient transfection, wild-type and mutant ZAP-70 were immunoprecipitated and in vitro kinase assays were performed as previously described (17). Samples were then analyzed by SDS-PAGE, transferred to polyvinylidene difluoride membrane, treated with 1 M KOH for 1 h, and then subjected to autoradiography and immunoblotting.

To examine functional requirements of Y315 in ZAP-70 in antigen receptor–mediated signal transduction, we transfected the Syk-deficient DT-40 B cells with either wild-type or the mutated ZAP-70 (ZAP-70[Y315F]) along with a NFAT reporter construct (19). Consistent with the previous reports (19, 20), loss of syk in DT-40 resulted in a complete block in BCR-stimulated NFAT activation, a defect that could be rescued by expression of wild-type ZAP-70 (Fig. 1). In contrast, mutation of Y315 in ZAP-70 markedly impaired its ability to reconstitute BCR-induced NFAT activation (Fig. 1).

To determine whether tyrosine 315 within the YESP motif of ZAP-70 functions as the Vav binding site, we transiently transfected Syk-deficient DT-40 cells with either wild-type ZAP-70 or ZAP-70(Y315F) and examined their ability to interact with a GST fusion protein containing the Vav SH2 domain (GST-VavSH2). As shown in Fig. 2,A, GST-VavSH2 fusion protein selectively bound to wild-type ZAP-70 after BCR stimulation or by treatment with the protein tyrosine phosphatase inhibitor pervanadate (Fig. 2 A). In contrast, mutation of Y315 in ZAP70 markedly impaired its ability to bind to the Vav SH2 domain.

Lck also associates with ZAP-70 via its SH2 domain after TCR stimulation (21, 22). Interestingly, both wild-type ZAP-70 and ZAP-70(Y315F) from either BCR-stimulated or pervanadate-treated lysates could bind efficiently to the GST-LckSH2 domain (Fig. 2 B), indicating that Y315 in ZAP-70 is specifically required for its interaction with the Vav SH2 domain, but not Lck SH2 domain.

Although initial phosphopeptide mapping studies failed to identify Y315 as one of the major tyrosine phosphorylated residues in ZAP-70, it is important to note that not all of the phosphorylation sites observed by two-dimensional peptide mapping were identified (23, 24). In fact, the corresponding residue (Y348) in Syk has been shown to be a major in vitro autophosphorylation site and it serves as the binding site for the Vav SH2 domain (15, 25). Moreover, not only did mutation of Y315 in ZAP-70 abolish the Vav– ZAP-70 interaction, this interaction could also be completely disrupted by the presence of a ZAP-70 peptide encompassing phosphorylated Y315 (14). These observations strongly argue that Y315 in ZAP-70 does represent an in vivo phosphorylation site after antigen receptor stimulation.

To assess whether the Y315 mutation affects ZAP-70–mediated Vav tyrosine phosphorylation, we transiently coexpressed human Vav with empty vector, wild-type ZAP-70, ZAP70(Y315F), or wild-type Syk into Lyn and Syk doubledeficient DT-40 cells, in which BCR-induced Vav phosphorylation was completely absent (Fig. 3,A, data not shown, and reference 26). Coexpression of Vav with either wildtype ZAP-70 or Syk, but not ZAP-70(Y315F), led to Vav tyrosine phosphorylation, which was further induced by BCR stimulation (Fig. 3 A).

To further examine the impact of Y315 mutation on ZAP-70–mediated tyrosine phosphorylation of other downstream substrates, we analyzed the tyrosine phosphorylation status of SLP-76 and Shc. Coexpression of wild-type ZAP70 with either SLP-76 or Shc in Syk-deficient or Lyn and Syk double-deficient DT-40 cells resulted in BCR-stimulated SLP-76 or Shc phosphorylation (Fig. 3, B and C, and data not shown). Surprisingly, mutation of Y315 in ZAP70 substantially impaired its ability to mediate phosphorylation of SLP-76 and Shc (Fig. 3, B and C, and data not shown). In addition, mutation of Y315 also markedly reduced ZAP-70 tyrosine phosphorylation after antigen receptor stimulation in Syk-deficient cells and in Jurkat T cells (Fig. 3 D and data not shown). Taken together, Y315 of ZAP-70 is not only required for Vav tyrosine phosphorylation, but also for tyrosine phosphorylation of other downstream substrates such as SLP-76, Shc, and even for ZAP-70 itself.

One explanation for the global defects of ZAP-70(Y315F) could be that the Y315F mutation reduced ZAP-70 kinase activity. Myc epitope–tagged ZAP-70 or ZAP-70(Y315F) was expressed in Lyn and Syk double-deficient cells and the kinase activity of anti-myc epitope–tagged immunoprecipitates was measured as both autophosphorylation and phosphorylation of an exogenous substrate, band III. The in vitro kinase assay failed to reveal a substantial difference between wild-type ZAP-70 and ZAP-70(Y315F) in their abilities to phosphorylate band III, although there may be a modest reduction in autophosphorylation of ZAP-70(Y315F) (Fig. 4 A).

Another critical step for ZAP-70 phosphorylation and activation is binding of ZAP-70 to the ITAMs after receptor stimulation. We used a biotinylated doubly phosphorylated ITAM peptide to precipitate ZAP-70 from lysates of Syk-deficient DT-40 cells transfected with either wild-type ZAP-70 or ZAP-70(Y315F). Similar amounts of wild-type ZAP-70 and ZAP-70(Y315F) bound to the phosphorylated peptide (Fig. 4 B). In addition, similar amounts of tyrosinephosphorylated TCR ζ chains were found to co-immunoprecipitate with either form of ZAP-70 when analyzed in Jurkat T cells (data not shown). Taken together, these data demonstrate that mutation of Y315 in ZAP-70 did not dramatically affect its kinase activity or its binding to receptor ITAMs.

In summary, we demonstrate here that Y315 in ZAP-70 is required to interact with the Vav SH2 domain, and is critical for ZAP-70–mediated gene activation. Notably, the Y315-homologous residue in Syk is also required for its interaction with the Vav SH2 domain and for Vav phosphorylation (15). We provide evidence here that the Y315 mutation results in a global defect in ZAP-70–mediated signaling pathways, suggesting an important role of Y315 in regulating ZAP-70 function.

Antigen receptor stimulation results in the assembly of multiprotein complexes, a process likely to facilitate efficient tyrosine phosphorylation and/or activation of appropriate signaling molecules (2). The Vav–ZAP-70 binding via Y315 may be important in initiating the proper formation of such signaling complexes, as both proteins are able to interact with many other signaling molecules (2). Since Vav possesses a GEF domain for Rho/Rac/CDC42 (7, 8), the interaction between Vav and ZAP-70 may provide a mechanism by which ZAP-70 activates downstream Rho/ Rac/CDC42-mediated signaling events such as cytoskeletal rearrangement. Mutation of Y315 in ZAP-70 may result in an impaired recruitment, phosphorylation and/or activation of many proteins including ZAP-70, Vav, SLP-76, and Shc.

We thank Dr. G. Crabtree for the NFAT reporter plasmid, Dr. D. Cantrell (Imperial Cancer Research Fund, London, U.K.) for the pEF-BOS expression plasmid, Dr. S. Katzav for the Vav cDNAs and GSTVavSH2 construct, Dr. M. Gishizky for the Shc cDNA, Dr. Koretzky for the FLAG-SLP-76, and Drs. N. van Oers, T. Finkel and D. Yablonski (University of California, San Francisco, CA) for critical reading of the manuscript and for helpful discussions.