Phosphorylation at tyrosine 527 of the proto-oncogene product, pp60()src, has been proposed to decrease the tyrosine kinase activity of the enzyme. We have investigated potential factors that might influence phosphorylation at this site by making mutant variants of the pp60"csrc protein. By effectively eliminating the site of N-terminal myristylation, we demonstrated that stable membrane association is not necessary for tyrosine 527 phosphorylation. Furthermore, mutational elimination of the enzymatic activity of this mutant pp60Csr protein did not alter the efficiency of phosphorylation at tyrosine 527. These data are consistent with the proposal that pp60csrc may be phosphorylated at tyrosine 527 by a cellular tyrosine kinase distinct from pp60)CSrC. In addition, using detergent-permeabilized celis, we established conditions that allow efficient phosphorylation of tyrosine 527 in vitro.The proto-oncogene product, pp6c-Jsrc' is the normal cellular homolog of the transforming protein of Rous sarcoma virus, pp6v-src (1). Although both of these proteins possess tyrosine-specific protein kinase activity, pp60v-src is a potent transforming protein, while pp60csrc does not induce oncogenic transformation even when expressed at levels comparable to those of pp6Ovsr, (20,21,28). Investigations of the fundamental properties that may be responsible for the difference in the oncogenic potential of these proteins have revealed several criteria that distinguish these proteins. (i) pp6Ov-src represents a mutated version of pp60c-src which contains several single-amino-acid differences throughout the protein and a major digression located in the extreme carboxy terminus, where the last 12 amino acids of pp6fv-src are unique and distinct from the last 19 amino acids of pp6Oc-src (35). This carboxy-terminal substitution (as well as some of the single-amino-acid changes) has been shown to be sufficient for activation of the oncogenic potential of pp60c src (20,33). (ii) The tyrosine-specific protein kinase activity of pp60v-src is 20-to 100-fold higher than the specific activity of pp6Ocsrc (13,19). (iii) pp60c-src and pp60v-src both undergo autophosphorylation in vitro at tyrosine 416 (29, 34); however, the in vivo site of tyrosine phosphorylation of pp6Ov-src is tyrosine 416, whereas pp6OC-src is phosphorylated in vivo at tyrosine 527 (8). Tyrosine residue 527 is not found in pp60v-src, and there is no tyrosine residue in the unique 12-amino-acid carboxy tail.Several lines of evidence suggest that phosphorylation at tyrosine 527 may regulate the tyrosine kinase activity of pp6c-src. (i) pp60csrc molecules isolated in the presence of sodium orthovanadate (a potent inhibitor of phosphotyrosine-specific phosphatases) possess a lower specific activity than pp60c src molecules isolated under conditions that allow hydrolysis of phosphate at this site (11). (ii) Enzymatic dephosphorylation of tyrosine 527 causes an activation of pp6fic-src tyrosine-specific protein kinase activity in vitro (10). (iii) pp60 c-src molecules that are bo...

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We have shown previously that the epidermal growth factor (EGF) receptor is phosphorylated at Ser-1002 and that this phosphorylation is associated with desensitization of the EGF receptor. Ser-1002 is followed immediately by Pro-1003, a residue that may promote the adoption of a specific conformation at this site or severe as a recognition element for the interaction of the EGF receptor with other proteins. To examine these possibilities, we have mutated Pro-1003 of the EGF receptor to a Gly residue and have analyzed the effect of this mutation on EGF-stimulated signaling. Cells expressing the P1003G EGF receptors exhibited higher EGF-stimulated autophosphorylation and synthetic peptide phosphorylation compared to cells expressing wild-type EGF receptors. In addition, the ability of EGF to stimulate PI 3-kinase activity and mitogen-activated protein kinase activity was enhanced in cells expressing the P1003G EGF receptor. Cells expressing P1003G receptors also demonstrated an increased ability to form colonies in soft agar in response to EGF. These results indicate that mutation of Pro-1003 leads to a potentiation of the biological effects of EGF. The findings are consistent with the hypothesis that Pro-1003 plays a role in a form of regulation that normally suppresses EGF receptor function.

Phosphorylation at tyrosine 527 of the proto-oncogene product, pp60c-src, has been proposed to decrease the tyrosine kinase activity of the enzyme. We have investigated potential factors that might influence phosphorylation at this site by making mutant variants of the pp60c-src protein. By effectively eliminating the site of N-terminal myristylation, we demonstrated that stable membrane association is not necessary for tyrosine 527 phosphorylation. Furthermore, mutational elimination of the enzymatic activity of this mutant pp60c-src protein did not alter the efficiency of phosphorylation at tyrosine 527. These data are consistent with the proposal that pp60c-src may be phosphorylated at tyrosine 527 by a cellular tyrosine kinase distinct from pp60c-src. In addition, using detergent-permeabilized cells, we established conditions that allow efficient phosphorylation of tyrosine 527 in vitro.