THE 66-KDA SHC ISOFORM IS A NEGATIVE REGULATOR OF THE EPIDERMAL GROWTH FACTOR-STIMULATED MITOGEN-ACTIVATED PROTEIN-KINASE PATHWAY

In addition to tyrosine phosphorylation of the 66-, 52-, and 46-kDa Sh c isoforms, epidermal growth factor (EGF) treatment of Chinese hamster ovary cells expressing the human EGF receptor also resulted in the se rine/ threonine phosphorylation of approximately 50% of the 66-kDa Shc proteins. The serine/threonine phosphorylation occurred subsequent to tyrosine phosphorylation and was prevented by pretreatment of the cel ls with the MEK-specific inhibitor PD98059. Surprisingly, only the gel -shifted 66-kDa Shc isoform (serine/threonine phosphorylated) was tyro sine phosphorylated and associated with Grb2. In contrast, only the no n-serine/threonine-phosphorylated fraction of 66-kDa Shc was associate d with the EGF receptor. To assess the relationship between the three Shc isoforms in EGF-stimulated signaling, the cDNA encoding the 66-kDa Shc species was cloned from a 16-day-old mouse embryo library. Sequen ce alignment confirmed that the 66-kDa Shc cDNA resulted from alternat ive splicing of the primary Shc transcript generating a 110-amino acid extension at the amino terminus. Co-immunoprecipitation of Shc and Gr b2 from cells overexpressing the 52/46-kDa Shc isoforms versus the 66- kDa Shc species directly demonstrated a competition of binding for a l imited pool of Grb2 proteins. Furthermore, expression of the 66-kDa Sh c isoform markedly accelerated the inactivation of ERK following EGF s timulation. Together, these data indicate that the serine/threonine ph osphorylation of 66-kDa Shc impairs its ability to associate with the tyrosine-phosphorylated EGF receptor and can function in a dominant-in terfering manner by inhibiting EGF receptor downstream signaling pathw ays.