Detection of a 60 kDa tyrosine-phosphorylated protein in insulin-stimulated hepatoma cells that associates with the SH2 domain of phosphatidylinositol 3-kinase.
Activation of the tyrosine kinase activity of the insulin receptor by autophosphorylation leads to phosphorylation of cellular substrates on tyrosine. Thus far, the best characterized is the insulin receptor substrate (IRS) 1, which has been proposed to serve as a docking protein for other molecules involved in signal transduction. A number of other proteins that become phosphorylated in response to insulin have been identified, some of which are reported to be tissue-specific. A 60 kDa phosphoprotein has been detected in adipocytes after insulin stimulation [Lavan and Lienhard (1993) J. Biol. Chem. 268, 5921-5928]. We have identified a protein of similar molecular mass in rat hepatoma cells transfected with the human insulin receptor. The 60 kDa protein in hepatoma cells is tyrosine-phosphorylated in response to insulin in a dose-dependent manner, with maximal phosphorylation occurring at 50 nM insulin. Although the dose-response of p60 phosphorylation mirrors that of IRS-1, the time course is slightly slower, with maximal phosphorylation observed 5 min after addition of insulin. Like the adipocyte protein, the 60 kDa protein detected in liver cells binds to the SH2 domain of the p85 regulatory subunit of phosphatidylinositol 3-kinase, but not to other SH2 domains. Binding of p60 to p85 is similar to the interaction between p85 and IRS-1 in that a tyrosine-phosphorylated peptide containing the YVXM motif can inhibit the association. The presence of this 60 kDa tyrosine-phosphorylated protein in adipocytes and hepatoma cells suggests that it represents another important intermediate in the insulin-receptor signal-transduction pathway.
Milarski
K L
KL
Department of Signal Transduction, Parke-Davis Pharmaceutical Research, Division of Warner Lambert Company, Ann Arbor, MI 48105, USA.
Lazar
D F
DF
Wiese
R J
RJ
Saltiel
A R
AR
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D007328:Insulin / Q000494:pharmacology*; D008099:Liver / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D019000:Phosphotyrosine; D011485:Protein Binding; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D007328:Insulin; D010455:Peptides; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011972:Receptor, Insulin