PALMITOYLATION OF MULTIPLE SRC-FAMILY KINASES AT A HOMOLOGOUS N-TERMINAL MOTIF

We have recently identified a novel N-terminal cysteine-containing mot if which specifies the palmitoylation of several G-protein alpha-subun its [Parenti, Vigano, Newman, Milligan and Magee (1993) Biochem. J. 29 1, 349-353]. A related motif occurs at the N-terminus of members of th e Src family of protein tyrosine kinases except for Src itself and Blk . We have investigated whether the Src, Fyn, Yes and Lck gene products are palmitoylated. Src was not labelled with [H-3]palmitate when endo genously expressed in COS cells. In contrast, endogenous Yes immunopre cipitated from COS cells was palmitoylated. Fyn was palmitoylated in i nsect cells infected with a recombinant baculovirus and the palmitoyla tion was independent of protein synthesis, suggesting a dynamic turnov er of this lipid. Fatty acid analysis indicated that most of the label was incorporated as palmitate. Lck was palmitoylated when expressed b y transfection in COS cells. All of these protein tyrosine kinases wer e also detectably myristoylated in each of the systems tested. Experim ents performed with mutants of Lck expressed by transfection in COS ce lls indicated that cysteines at positions 3 and 5 were both palmitoyla tion sites and that myristoylation was required for palmitoylation. To confirm that palmitoylation was occurring on cysteines in the N-termi nal region of Fyn, site-directed mutagenesis was used to replace the c ysteines at positions 3 and 6 with alanine. The resulting protein was not palmitoylated but was still myristoylated when expressed in COS ce lls. A glycine to alanine mutant at position 2 was also not palmitoyla ted, showing that myristoylation is a prerequisite for palmitoylation. Our data indicate that Src family members containing the N-terminal c ysteine motif are indeed palmitoylated. By analogy with Ras, it is pos sible that palmitoylation may play an important role in the localizati on and function of Src family protein tyrosine kinases.