REQUIREMENT FOR C-SRC CATALYTIC ACTIVITY AND THE SH3 DOMAIN IN PLATELET-DERIVED GROWTH-FACTOR BB AND EPIDERMAL GROWTH-FACTOR MITOGENIC SIGNALING

The Src family protein-tyrosine kinases are required for mitogenic sig naling from the platelet-derived growth factor (PDGF), colony stimulat ing factor-1, and epidermal growth factor (EGF) receptor protein-tyros ine kinases (RPTK) (Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7696-7 700; Roche, S., Koegl, M., Barone, M. V., Roussel, M. F., and Courtnei dge, S. A. (1995) Mel. Cell. Biol. 15, 1102-1109). In NIH3T3 fibroblas ts, c-Src, Fyn, and c-Yes associate with the activated PDGF receptor, are substrates for receptor phosphorylation, and are themselves activa ted. Src family catalytic function is required for RPTK mitogenic sign aling as evidenced by the SH2-dependent dominant negative phenotype ex hibited by kinase-inactive Src and Fyn mutants (Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7696-7700). Here, we have generated clonal Src(- ) murine fibroblast cell lines overexpressing various murine c-Src mut ants and studied the effect of these mutant Src proteins on PDGF- and EGF-induced mitogenesis. Two c-Src SH3 domain mutants, Y133F and Y138F , each inhibited PDGF BB- and EGF-induced DNA synthesis in quiescent c ells. This demonstrates an involvement of the Src SH3 domain in PDGF b eta and EGF receptor mitogenic signaling. Since both Tyr-133 and Tyr-1 38 are located on the ligand binding surface of the SH3 domain, these results suggest that the c-Src SH3 domain is required for PDGF and EGF mitogenic signaling. The dominant negative effect of either single mu tant on PDGF receptor signaling was reversed by a second SH2-inactivat ing mutation. We conclude that the c-Src SH3 domain function requires the SH2 domain in the case of the PDGF receptor, presumably because bi nding of c-Src to the receptor via its SH2 domain is a prerequisite fo r the SH3 domain function. In contrast, SH2 function is apparently not essential for the SH3 function in EGF receptor signaling.