THE SH2 DOMAIN OF SHC SUPPRESSES EGF-INDUCED MITOGENESIS IN A DOMINANT-NEGATIVE MANNER

Recently, we have shown that an EGF-R-mutant lacking the autophosphory lation sites phosphorylates She and retains mitogenic activity, In thi s report, we have shown that in these cells, in response to EGF, Ras i s fully activated with formation of the tyrosine-phosphorylated Shc-Gr b2-mSOS complex without the receptor. This pointed out the importance of She in EGF-induced Ras activation, To investigate the mechanism of tyrosine phosphorylation of She by EGF-R, we carried out in vitro kina se assays using immunoprecipitated EGF-R and bacterially-expressed She proteins as substrates, The EGF-R phosphorylated She, but not the She SH2 mutant, lacking binding ability for phosphotyrosine, This suggest s that intact She SH2 is essential for the full-length She to become p hosphorylated, probably by inducing a conformational change in She. Th us a She SH2 peptide may inhibit competitively She phosphorylation. We microinjected the She SH2 domain into NIH3T3 cells overexpressing the EGF-R. Microinjected She SH2 greatly suppressed EGF-induced DNA synth esis. But microinjection of neither the She SH2 mutant nor PLC-gamma 1 SH2 had any effect, This suppressing effect was rescued by comicroinj ection of the full-length She, suggesting She SH2 specifically suppres sed the She pathway. Thus we concluded She phosphorylation is crucial, whereas receptor autophosphorylation is dispensable, in EGF-induced m itogenesis.