title: Insulin stimulates tyrosine phosphorylation of multiple high molecular weight substrates in Fao hepatoma cells.
abstract: Insulin rapidly stimulates tyrosine phosphorylation of cellular proteins which migrate between 165 and 190 kDa during SDS-PAGE. These proteins, collectively called pp185, were originally found in anti-phosphotyrosine antibody (alpha PY) immunoprecipitates from insulin-stimulated Fao rat hepatoma cells. Recently, we purified and cloned IRS-1, one of the phosphoproteins that binds to alpha PY and migrates near 180 kDa following insulin stimulation of rat liver [Sun, X. J., et al. (1991) Nature 352, 73-77]. IRS-1 and pp185 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 pp185. The pp185 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 pp185 (HMW-pp185). Furthermore, the tryptic phosphopeptide map of IRS-1 was distinct from that of HMW-pp185, 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-pp185. Our results show that Fao cells contain at least two insulin receptor substrates, IRS-1 and HMW-pp185, which may play unique roles in insulin signal transmission.
authors.affiliation: Joslin Diabetes Center, Department of Medicine, Boston, Massachusetts 02215.
authors.forename: M
authors.initials: M
authors.lastname: Miralpeix
authors.forename: X J
authors.initials: XJ
authors.lastname: Sun
authors.forename: J M
authors.initials: JM
authors.lastname: Backer
authors.forename: M G
authors.initials: MG
authors.lastname: Myers
authors.forename: E
authors.initials: E
authors.lastname: Araki
authors.forename: M F
authors.initials: MF
authors.lastname: White
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology*; D008114:Liver Neoplasms, Experimental; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D007328:Insulin; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin