ACTIVATION OF P56(LCK) BY P72(SYK) THROUGH PHYSICAL ASSOCIATION AND N-TERMINAL TYROSINE PHOSPHORYLATION

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 approximate to 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(lck) 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.