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

Author:
  • Olga Göransson
  • Svante Resjö
  • Lars Rönnstrand
  • Vincent Manganiello
  • Eva Degerman
Publishing year: 2002
Language: English
Pages: 175-182
Publication/Series: Cellular Signalling
Volume: 14
Issue: 2
Document type: Journal article
Publisher: Elsevier

Abstract 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.

Keywords

  • Microbiology
  • 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

Other

Published
  • Insulin Signal Transduction
  • ISSN: 1873-3913
Eva Degerman
E-mail: eva.degerman [at] med.lu.se

Professor

Insulin Signal Transduction

+46 46 222 85 83

+46 70 885 83 62

BMC C1121b

66

Lund University Diabetes Centre, CRC, SUS Malmö, Entrance 72, House 91:12. SE-205 02 Malmö. Telephone: +46 40 39 10 00