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

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Author

Summary, in English

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 beta in primary rat adipocytes. Insulin stimulation resulted in a translocation of PKB beta from cytosol to membranes, and phosphorylation and activation of PKB beta. 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 PKB beta using the anti-phospho-PKB (Ser-473) antibody. Taken together, these results show that rat adipocyte PKB beta 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.

Publishing year

2002

Language

English

Pages

175-182

Publication/Series

Cellular Signalling

Volume

14

Issue

2

Document type

Journal article

Publisher

Elsevier

Topic

  • Microbiology

Keywords

  • Intracellular Membranes : enzymology
  • Oxazoles : pharmacology
  • Phosphoprotein Phosphatase : antagonists & inhibitors
  • Phosphorylation
  • Phosphoserine : metabolism
  • Protein Transport
  • Proto-Oncogene Proteins : chemistry : metabolism
  • Rats
  • Support Non-U.S. Gov't
  • Rats Sprague-Dawley
  • Insulin : pharmacology
  • Male
  • Enzyme Inhibitors : pharmacology
  • Cells Cultured
  • Electrophoresis Gel Two-Dimensional
  • Animal
  • Adipocytes : drug effects : enzymology

Status

Published

Research group

  • Insulin Signal Transduction

ISBN/ISSN/Other

  • ISSN: 1873-3913