RAPID T-CELL RECEPTOR-MEDIATED TYROSINE PHOSPHORYLATION OF P120, AN FYN LCK SRC HOMOLOGY-3 DOMAIN-BINDING PROTEIN/

Tyrosine phosphorylation of cellular proteins is the earliest identifi able event following T-cell antigen receptor (TCR) stimulation and is essential for activating downstream signaling machinery. Two Src-famil y protein-tyrosine kinases, the TCR-associated p59(fyn) (Fyn) and the CD4/8-associated p56(lck) (Lck), have emerged as the likely mediators of early tyrosine phosphorylation in T cells. Here, we show direct bin ding of a 120-kDa TCR-induced phosphotyrosyl polypeptide, p120, to glu tathione S-transferase fusion proteins of the Src homology 3 (SH3) dom ains of Fyn, Lck, and p60(Src) (Src) but not other proteins. While bin ding of p120 to Fyn SH2 domain was phosphotyrosine-dependent as expect ed, its binding to the SH3 domain was independent of tyrosine phosphor ylation, as shown by lack of competition with a phosphotyrosyl competi tor peptide. In contrast, an SH3-specific proline-rich peptide complet ely abolished p120 binding to SH3. p120 was tyrosine-phosphorylated wi thin 10 sec following stimulation of Jurkat cells with anti-CD3 monocl onal antibody, with maximal phosphorylation at 30 sec. Importantly, p1 20 was found associated with Fyn and Lck proteins in unstimulated Jurk at cells and served as an in vitro substrate for these kinases. These results provide evidence for a role of the SH3 domains of Fyn and Lck in the recruitment of early tyrosine-phosphorylation substrates to the TCR-associated tyrosine kinases.