Coordinate phosphorylation of insulin-receptor kinase and its 175,000-Mr endogenous substrate in rat hepatocytes.
To investigate the early events in insulin signal transmission in liver, isolated rat hepatocytes were labeled with 32P, and proteins phosphorylated in response to insulin were detected by immunoprecipitation with anti-phosphotyrosine and anti-receptor antibodies and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and autoradiography. In these cells, insulin rapidly stimulated tyrosine phosphorylation of the 95,000-Mr beta-subunit of the insulin receptor and a 175,000-Mr phosphoprotein (pp175). Both proteins were precipitated by anti-phosphotyrosine antibody, whereas only the insulin receptor was recognized with anti-insulin-receptor antibody. In the insulin-stimulated state, both pp175 and the receptor beta-subunit were found to be phosphorylated on tyrosine and serine residues. Based on precipitation by the two antibodies, receptor phosphorylation was biphasic with an initial increase in tyrosine phosphorylation followed by a more gradual increase in serine phosphorylation over the first 30 min of stimulation. The time course of phosphorylation of pp175 was rapid and paralleled that of the beta-subunit of the insulin receptor. The pp175 was clearly distinguished from the insulin receptor, because it was detected only when boiling SDS was used to extract cellular phosphoproteins, whereas the insulin receptor was extracted with either Triton X-100 or SDS. In addition, the tryptic peptide maps of the two proteins were distinct. The dose-response curve for insulin stimulation was shifted slightly to the left of the insulin receptor, suggesting some signal amplification at this step. These data suggest that pp175 is a major endogenous substrate of the insulin receptor in liver and may be a cytoskeletal-associated protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Okamoto
M
M
Research Division, Joslin Diabetes Center, Boston, MA 02215.
Karasik
A
A
White
M F
MF
Kahn
C R
CR
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001345:Autoradiography; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D007328:Insulin / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000302:isolation & purification; D010750:Phosphoproteins / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000378:metabolism*
D000596:Amino Acids; D007328:Insulin; D046911:Macromolecular Substances; D010710:Phosphates; D010748:Phosphopeptides; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin