Insulin rapidly stimulates phosphorylation of a 46-kDa membrane protein on tyrosine residues as well as phosphorylation of several soluble proteins in intact fat cells.
It is speculated that the transmission of an insulin signal across the plasma membrane of cells occurs through activation of the tyrosine-specific receptor kinase, autophosphorylation of the receptor, and subsequent phosphorylation of unidentified substrates in the cell. In an attempt to identify possible substrates, we labeled intact rat fat cells with [32P]orthophosphate and used an antiphosphotyrosine antibody to identify proteins that become phosphorylated on tyrosine residues in an insulin-stimulated way. In the membrane fraction of the fat cells, we found, in addition to the 95-kDa beta-subunit of the receptor, a 46-kDa phosphoprotein that is phosphorylated exclusively on tyrosine residues. This protein is not immunoprecipitated by antibodies against different regions of the insulin receptor and its HPLC tryptic peptide map is different from the tryptic peptide map of the insulin receptor, suggesting that it is not derived from the receptor beta-subunit. Insulin stimulates the tyrosine phosphorylation of the 46-kDa protein within 150 sec in the intact cell 3- to 4-fold in a dose-dependent way at insulin concentrations between 0.5 nM and 100 nM. The insulin effect starts after 30 sec, is maximal at 150 sec, and declines to almost basal values by 5 min. Furthermore, the antiphosphotyrosine antibody precipitated at least five proteins in the soluble fraction of the fat cell. Insulin (0.5 nM, 100 nM) stimulated within 2 min the 32P incorporation into a 116-kDa band, a 62-kDa band, and three bands between 45 kDa and 50 kDa 2- to 10-fold. We suggest that the 46-kDa membrane protein and possibly also the soluble proteins are endogenous substrates of the receptor tyrosine kinase in fat cells and that their phosphorylation is an early step in insulin signal transmission.
D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D014357:Trypsin; D014443:Tyrosine
D007328:Insulin; D008565:Membrane Proteins; D010750:Phosphoproteins; D011506:Proteins; D014443:Tyrosine; D014357:Trypsin