Insulin Stimulates Tyrosine Phosphorylation of Multiple High Molecular Weight Substrates in Fao Hepatoma Cells

Montserrat Miralpeix, Xiao Jian Sun, Jonathan M. Backer, Eichii Araki, Martin G. Myers, Morris F. White

Research output: Contribution to journalArticle

30 Scopus citations

Abstract

Insulin rapidly stimulates tyrosine phosphorylation of cellular proteins which migrate between 165 and 190 kDa during SDS-PAGE. These proteins, collectively called ppl85, were originally found in anti-phosphotyrosine antibody (αPY) immunoprecipitates from insulin-stimulated Fao rat hepatoma cells. Recently, we purified and cloned IRS-1, one of the phosphoproteins that binds to aPY and migrates near 180 kDa following insulin stimulation of rat liver [Sun, X.J., et al. (1991) Nature 352, 73-77]. IRS-1 and ppl85 undergo tyrosine phosphorylation immediately after insulin stimulation and show an insulin dose response similar to that of insulin receptor autophosphorylation. However, IRS-1 was consistently 10 kDa smaller than the apparent molecular mass of ppl85. The ppl85 contained some immunoblottable IRS-1; however, cell lysates depleted of IRS-1 with anti-IRS-1 antibody still contained the high molecular weight forms of pp 185 (HMW-pp 185). Furthermore, the tryptic phosphopeptide map of IRS-1 was distinct from that of HMW-ppl85, suggesting that at least two substrates migrate in this region during SDS-PAGE. Moreover, the phosphatidylinositol 3'-kinase and its 85-kDa associated protein (p85) bound to IRS-1 in Fao cells, but weakly or not at all to HMW-ppl85. Our results show that Fao cells contain at least two insulin receptor substrates, IRS-1 and HMW-ppl85, which may play unique roles in insulin signal transmission.

Original languageEnglish (US)
Pages (from-to)9031-9039
Number of pages9
JournalBiochemistry
Volume31
Issue number37
DOIs
StatePublished - Feb 1 1992
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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