Insulin stimulates tyrosine phosphorylation of multiple high molecular weight substrates in Fao hepatoma cells.
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.
Miralpeix
M
M
Joslin Diabetes Center, Department of Medicine, Boston, Massachusetts 02215.
Sun
X J
XJ
Backer
J M
JM
Myers
M G
MG
Araki
E
E
White
M F
MF
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
D007328:Insulin; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin