Activation of p56^lck through Mutation of a Regulatory CarboxyTerminal Tyrosine Residue Requires Intact Sites of Autophosphorylation and Myristylation

Abstract

Mutation of the major site of in vivo tyrosine phosphorylation of p56^lck (tyrosine 505) to a phenylalanine constitutively enhances the p56^lck-associated tyrosine-specific protein kinase activity. The mutant polypeptide is extensively phosphorylated in vivo at the site of in vitro Lck autophosphorylation (tyrosine 394) and is capable of oncogenic transformation of rodent fibroblasts. These observations have suggested that phosphorylation at Tyr-505 down regulates the tyrosine protein kinase activity of p56^lck. Herein we have attempted to examine whether other posttranslational modifications may be involved in regulation of the enzymatic function of p56^lck. The results indicated that activation of p56^lck by mutation of Tyr-505 was prevented by a tyrosine-to-phenylalanine substitution at position 394. Furthermore, activation of p56^lck by mutation of the carboxy-terminal tyrosine residue was rendered less efficient by substituting an alanine residue for the amino-terminal glycine. This second mutation prevented p56^lck myristylation and stable membrane association and was associated with decreased in vivo phosphorylation at Tyr-394. Taken together, these findings imply that lack of phosphorylation at Tyr-505 may be insufficient for enhancement of the p56^lck-associated tyrosine protein kinase activity. Our data suggest that activation of p56^lck may be dependent on phosphorylation at Tyr-394 and that this process may be facilitated by myristylation, membrane association, or both.