Activation of p56^lck by p72^syk through Physical Association and N-Terminal Tyrosine Phosphorylation†

Abstract

The p56^lck and p59^fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72^syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56^lck. Here we examine the regulation of p72^syk and its interaction with p56^lck in transfected COS-1 cells. p72^syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by ~90%. Coexpression of lck did not alter the catalytic activity of p72^syk, but the expressed p56^lck was much more active in the presence of p72^syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56^lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56^lck was no longer phosphorylated by p72^syk. Phosphate was also detected in p56^lck at Tyr-192 in lymphoid cells. These findings suggest that p56^lck is positively regulated by the p72^syk kinase.