Ser-474 is the major target of insulin-mediated phosphorylation of protein kinase B β in primary rat adipocytes

The mechanism of activation for protein kinase B (PKB), an important target for insulin signaling, has been scarcely investigated in primary cells, In this study, we have characterized the insulin-induced phosphorylation and activation of PKB in primary rat adipocytes. Insulin stimulation resulted in a translocation of PKBbeta from cytosol to membranes, and phosphorylation and activation of PKBbeta. Phosphoamino acid analysis and phosphopeptide mapping demonstrated that the phosphorylation occurred mainly on serines, also when using calyculin A, and that these were localized within one major phosphopeptide. Radiosequencing showed that the radioactivity was released in Cycle No. 7. In addition, the peptide was specifically immunoprecipitated from a tryptic digest of PKBbeta using the anti-phospho-PKB (Ser-473) antibody. Taken together, these results show that rat adipocyte PKBbeta mainly is phosphorylated on Ser-474 in response to insulin stimulation, in contrast to previous studies in human embryonic kidney (HEK) 293 cells demonstrating, in addition, phosphorylation of Thr-309. (C) 2002 Elsevier Science Inc. All rights reserved.