Insulin stimulates the tyrosine phosphorylation of a 61-kilodalton protein in rat adipocytes.
Insulin stimulated the tyrosine phosphorylation of a 61-kilodalton (kDa) protein in rat adipocytes prelabeled for 2 h with [32P]orthophosphate. Tyrosine phosphorylation of this 61-kDa protein displayed very similar insulin concentration dependency to receptor autophosphorylation and tyrosine phosphorylation of a high molecular mass receptor substrate of 160 kDa. Phosphorylation of the 61-kDa protein was very rapid with maximum labeling attained at 30 sec, paralleling that of the other two proteins. Phosphoamino acid analysis revealed that each of the insulin-responsive phosphoproteins contained phosphoserine as well as phosphotyrosine, though the ratio of two phosphoamino acids recovered from each protein differed. The 61-kDa protein yielded relatively equal proportions of phosphoserine and phosphotyrosine. In contrast, the insulin receptor yielded relatively more label on phosphotyrosine than phosphoserine, whereas label incorporated into the 160-kDa protein was recovered primarily on phosphoserine. Cleveland peptide maps using either Staphylococcus aureus V8 proteinase or chymotrypsin revealed no similarities between the 61-kDa protein and the other tyrosine phosphorylated proteins. With subcellular fractionation, the 160-kDa protein was found in equal proportions in the high speed pellet (100,000 g) and supernatant. The 61-kDa protein had a similar distribution to that of the 160-kDa protein but was also detected in the low speed pellet (10,000 g). The insulin receptor was localized to the low speed pellet. In summary, rat adipocytes contain an insulin-dependent phosphotyrosyl protein of 61 kDa which is distinct from the more prominent high molecular mass receptor substrate. This 61-kDa protein has characteristics consistent with it being a substrate for the insulin receptor tyrosine kinase.
Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, New York 14642.
R A
RA
Mooney
K L
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Bordwell
D000273:Adipose Tissue / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D002458:Cell Fractionation; D015151:Immunoblotting; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis / Q000378:metabolism; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism*
D007328:Insulin; D010761:Phosphorus Radioisotopes; D011506:Proteins; D010768:Phosphoserine; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin