Insulin receptor tyrosine kinase-catalyzed phosphorylation of 422(aP2) protein. Substrate activation by long-chain fatty acid.
It was established previously that the 15-kDa protein phosphorylated in 3T3-L1 adipocytes treated with insulin and phenylarsine oxide is O-phospho-Tyr19 422(aP2) protein, a fatty acid-binding protein. To assess its capacity to serve as substrate of the insulin receptor tyrosine kinase in vitro, native 422(aP2) protein was isolated from 3T3-L1 adipocytes and purified to homogeneity. Receptor-catalyzed phosphorylation of 422(aP2) protein on Tyr19 was markedly activated when long-chain fatty acid, e.g. oleic acid, is bound to the protein. Fatty acid had no effect on autophosphorylation of the insulin receptor by its intrinsic tyrosine kinase. Both saturated (C14, C16, and C18) and unsaturated (all cis-delta 9 C16, -delta 9 C18, and -delta 9,12 C18, -delta 9,12,15 C18, and -delta 5,8,11,14 C20) fatty acids caused substrate activation. The Km for 422(aP2) protein was greatly reduced (from 170 to 3 microM) by oleic acid with little or no effect on Vmax. Upon binding fatty acid to 422(aP2) protein the susceptibility of Tyr19 and Tyr128 to iodination by the lactoperoxidase method increased greatly. These results indicate that upon binding fatty acid, 422(aP2) protein undergoes a conformational change whereby Tyr19, which lies within a consensus-type sequence for tyrosine kinase substrates, becomes accessible for phosphorylation by the insulin receptor tyrosine kinase and to iodination by lactoperoxidase.
Hresko
R C
RC
Department of Biological Chemistry, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
Hoffman
R D
RD
Flores-Riveros
J R
JR
Lane
M D
MD
D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008970:Molecular Weight; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology; D010766:Phosphorylation; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin
D001152:Arsenicals; D002352:Carrier Proteins; C082667:Fabp5 protein, mouse; C086936:Fabp7 protein, mouse; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D007328:Insulin; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D009829:Oleic Acids; C029341:oxophenylarsine; D019301:Oleic Acid; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin