P95(VAV) ASSOCIATES WITH TYROSINE-PHOSPHORYLATED SLP-76 IN ANTIGEN-STIMULATED T-CELLS

p95(vav), the product of the vav protooncogene, has been implicated in the T cell receptor (TCR)-mediated signaling cascade. p95(vav) is pho sphorylated on tyrosine residues after TCR stimulation by anti-TCR/CD3 antibodies and possesses a number of landmark features of signaling m olecules such as a putative guanine nucleotide exchange factor domain, a pleckstrin homology domain, and an Src homology (SH) 2 and two SH3 domains, which provide the capacity to form multimeric signaling compl exes. However, the precise role of p95(vav) in TCR signaling remains u nclear. In this work we show that physiological stimulation of T cell hybridomas with antigen presented by major histocompatibility complex class II molecules leads to a strong tyrosine phosphorylation of p95(v av) and its association with tyrosine-phosphorylated SLP-76. SLP-76 is a newly described SH2-containing protein that has been previously fou nd to bind to the adapter molecule Grb2. Moreover, we provide evidence that p95(vav)-SLP-76 association is SH2-mediated by demonstrating tha t this interaction can be inhibited by a phosphopeptide containing a p utative p95(vav)-SH2-binding motif (pYESP) present in SLP-76. Furtherm ore, in vitro experiments show that after antigen stimulation, phospho rylated p95(vav)-SLP-76 can bind to Grb2 in a complex that contains pp 36/38 and pp116 proteins. Our data provide a clue to explain recent in dependent observations that overexpression of p95(vav) or SLP-76 enhan ces TCR-mediated gene activation.