Purification and partial sequence analysis of pp185, the major cellular substrate of the insulin receptor tyrosine kinase.
Insulin stimulates the tyrosine phosphorylation of a 185-kDa putative cytosolic substrate protein (pp185) in diverse cell types. After intravenous insulin infusion into the live intact rat, pp185 and the 95-kDa insulin receptor beta-subunit were the major proteins that tyrosine phosphorylated in liver, skeletal muscle, and adipose tissue. Both proteins were maximally phosphorylated within 30 s, and both increased in phosphotyrosine content in parallel with increasing insulin dose. However, pp185 tyrosine phosphorylation was transient, with almost complete dephosphorylation within 2-3 min despite continued insulin stimulation. To identify pp185 directly, we purified pp185 from insulin-stimulated rat liver, using a denaturation-based extraction procedure that blocks endogenous protein phosphatases and thus allows a high yield, single step isolation of phosphotyrosyl proteins by anti-phosphotyrosine antibody immunoaffinity absorption. From 50 rat livers, 50-100 pmol of pp185 was isolated. Edman degradation of seven internal tryptic peptide fragments of pp185 yielded novel amino acid sequences, indicating that pp185 is a new protein. Antipeptide antibodies were raised which specifically recognize a single, 185-kDa insulin-stimulated phosphotyrosyl protein in liver, skeletal muscle, adipose tissue, and several cultured cell lines. These results indicate that pp185 is expressed in a variety of insulin-responsive tissues, is the major protein rapidly tyrosine phosphorylated under physiological conditions in the intact animal, and also provide a route for cloning the pp185 gene and elucidating the function of pp185 in insulin signal transduction.
Rothenberg
P L
PL
Research Division, Joslin Diabetes Center, Boston, Massachusetts.
Lane
W S
WS
Karasik
A
A
Backer
J
J
White
M
M
Kahn
C R
CR
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007328:Insulin / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000378:metabolism*
D007328:Insulin; D011506:Proteins; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin