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 autophosphorylation sites phosphorylates Shc and retains mitogenic activity. In this report, we have shown that in these cells, in response to EGF, Ras is fully activated with formation of the tyrosine-phosphorylated Shc-Grb2-mSOS complex without the receptor. This pointed out the importance of Shc in EGF-induced Ras activation. To investigate the mechanism of tyrosine phosphorylation of Shc by EGF-R, we carried out in vitro kinase assays using immunoprecipitated EGF-R and bacterially-expressed Shc proteins as substrates. The EGF-R phosphorylated Shc, but not the Shc SH2 mutant, lacking binding ability for phosphotyrosine. This suggests that intact Shc SH2 is essential for the full-length Shc to become phosphorylated, probably by inducing a conformational change in Shc. Thus a Shc SH2 peptide may inhibit competitively Shc phosphorylation. We microinjected the Shc SH2 domain into NIH3T3 cells overexpressing the EGF-R. Microinjected Shc SH2 greatly suppressed EGF-induced DNA synthesis. But microinjection of neither the Shc SH2 mutant nor PLC-gamma 1 SH2 had any effect. This suppressing effect was rescued by comicroinjection of the full-length Shc, suggesting Shc SH2 specifically suppressed the Shc pathway. Thus we concluded Shc phosphorylation is crucial, whereas receptor autophosphorylation is dispensable, in EGF-induced mitogenesis.
Department of Genetics, University of Tokyo, Japan.
N
N
Gotoh
K
K
Muroya
S
S
Hattori
S
S
Nakamura
K
K
Chida
M
M
Shibuya
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis*; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000032:analysis; D016193:G1 Phase; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011506:Proteins / Q000502:physiology*; D016196:S Phase; D018909:src Homology Domains
D011506:Proteins; D004815:Epidermal Growth Factor; D004247:DNA; D066246:ErbB Receptors