The 66-kDa Shc isoform is a negative regulator of the epidermal growth factor-stimulated mitogen-activated protein kinase pathway

Shuichi Okada, Aimee W. Kao, Brian P. Ceresa, Pamela Blaikie, Ben Margolis, Jeffrey E. Pessin

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Abstract

In addition to tyrosine phosphorylation of the 66-, 52-, and 46-kDa Shc isoforms, epidermal growth factor (EGF) treatment of Chinese hamster ovary cells expressing the human EGF receptor also resulted in the serine/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 cells with the MEK- specific inhibitor PD98059. Surprisingly, only the gel-shifted 66-kDa Shc isoform (serine/threonine phosphorylated) was tyrosine phosphorylated and associated with Grb2. In contrast, only the non-serine/threonine- phosphorylated fraction of 66-kDa Shc was associated 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. Sequence alignment confirmed that the 66-kDa Shc cDNA resulted from alternative splicing of the primary Shc transcript generating a 110-amino acid extension at the amino terminus. Co- immunoprecipitation of Shc and Grb2 from cells overexpressing the 52/46-kDa Shc isoforms versus the 66-kDa Shc species directly demonstrated a competition of binding for a limited pool of Grb2 proteins. Furthermore, expression of the 66-kDa Shc isoform markedly accelerated the inactivation of ERK following EGF stimulation. Together, these data indicate that the serine/threonine phosphorylation of 66-kDa Shc impairs its ability to associate with the tyrosine-phosphorylated EGF receptor and can function in a dominant-interfering manner by inhibiting EGF receptor downstream signaling pathways.

Original languageEnglish (US)
Pages (from-to)28042-28049
Number of pages8
JournalJournal of Biological Chemistry
Volume272
Issue number44
DOIs
StatePublished - Oct 31 1997

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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