T cell activation stimulates p56(lck), p59(fyn), ZAP-70, Vav-SLP-76 binding, and IL-2 transcription. Major questions concern the tyrosine-kinase and relevant site(s) needed for Vav-SLP-76 complex formation and its role in IL-2 production. Here, we show that of the three kinases, only ZAP-70 phosphorylates SLP-76 at specific sites that allow Vav SH2 domain binding. Therefore, while p56(lck) regulates proximal events, ZAP-70 acts downstream on targets such as SLP-76. We also show by in vitro and in vivo analysis that two SLP-76 pYESP motifs (Y113 and Y128) mediate binding, the first being more efficient. A third pYEPP motif (Y145) failed to bind. Finally, TCR zeta CD3 ligation of T cell hybridoma DC27.10 induces IL-2 production without detectable Vav-SLP-76 binding. Therefore, despite effects of Vav-SLP-76 on IL-2 expression, Vav-SLP-76 binding per se is not essential for IL-2 production in all T cells.

TcR ͞CD3 ligation initiates a signaling cascade involving CD4͞CD8-p56 lck , p59 fyn , and ZAP-70, as well as lymphoid downstream proteins VAV, SLP-76, and FYB͞SLAP. A current question concerns the nature of the downstream binding partner(s) of FYB in T cells. In this study, using a two-hybrid screen with FYB as bait, we have identified eight clones, four of which correspond to the recently published lymphoid protein SKAP55, and two which correspond to a related protein with some 44% homology to SKAP55 (termed SKAP55-related protein, SKAP55R). The SKAP55 clones showed only minor differences (two substitutions and one residue deletion) from SKAP55. SKAP55R has the same overall structure as SKAP55 except for the presence of a unique N terminus with a well-defined coiled-coil domain. Both SKAP55 and SKAP55R were found to bind FYB through their SH3 domains and to act as substrates for the FYN kinase in T cells. Furthermore, immunof luorescence confocal microscopy showed that FYB and SKAP55 colocalize in the perinuclear region of cells. SKAP55 also colocalizes with another FYB binding protein, SLP-76. Taken together, these observations demonstrate that FYB is part of an interactive matrix with SKAP55 and a SKAP55-related protein.Ligation of CD4͞CD8-p56 lck and the T cell receptor complex (TcR ͞CD3) activates src protein-tyrosine kinases (PTKs) p56 lck and p59 fyn (1), leading to phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the TcR and CD3 chains (2-4). The phosphorylated ITAM recruits ZAP-70 by means of tandem SH2 domain binding (5, 6). CD4-p56 lck can further up-regulate ZAP-70 catalytic activity by phosphorylation of residue Y-493 (7). The importance of ZAP-70 in T cell signaling has been shown by defects in early signaling events and interleukin 2 (IL-2) transcription in ZAP-70-negative Jurkat cells (8). The Lck-SH2 domain can also bind to ZAP-70, thereby consolidating the CD4͞CD8-p56 lck and TcR ͞CD3 aggregate (9). Consistent with this capability, p56 lck has been found associated with the TcR ͞ CD3 complex (10). p56 lck may also play roles in signaling by other systems by virtue of its ability to phosphorylate other surface receptors such as CD5 and CD28 (11-13).Recent advances have identified downstream targets of the TcR͞CD3 and CD4͞CD8-p56 lck complexes that include hematopoietic proteins VAV, SLP-76, and FYB. VAV possesses several domains, including a guanine nucleotide exchange factor (GEF) domain for the Rho and Rac small GTP-binding proteins (14-16). In turn, VAV binds to another hematopoietic protein, , which possesses a C-terminal SH2 domain and proline-rich motifs (20). ZAP-70 is the key kinase that phosphorylates 22), allowing the SH2 domain to bind to two pYESP motifs (Y-113 and Y-128) within the protein (22). VAV and SLP-76 cooperate to augment TcR ͞CD3 induced IL-2 transcription (17, 18). VAV-SLP-76 complex formation, however, is not essential for TcR-induced IL-2 production in all T cells (22). Instead, SLP-76 requires an intact SLP-76 SH2 domain in its pote...

lck is then needed to activate the kinase, an event that leads to ZAP-70-mediated phosphorylation of its targets (6). p56 lck SH2 domain binding to ZAP-70 further consolidates CD4-p56 lck or CD8-p56 lck in the TcR/CD3 aggregate (7).In addition to proximal kinases, recent studies have identified an array of immune cell-specific adaptor proteins in T-cells that act as substrates for kinases and play important roles in T-cell function. These include LAT (linker for activation of T-cell) as well as the cytosolic intracellular proteins SLP-76 (SH2-domain-containing leukocyte protein of 76 kDa) and FYB/