Ser-474 is the major target of insulin-mediated phosphorylation of protein kinase B beta 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 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.
Göransson
Olga
O
Section for Molecular Signalling, Department of Cell and Molecular Biology, Lund University, BMC, C11, S-221 84 Lund, Sweden. olga.goransson@medkem.lu.se
Resjö
Svante
S
Rönnstrand
Lars
L
Manganiello
Vincent
V
Degerman
Eva
E
D017667:Adipocytes / Q000187:drug effects / Q000201:enzymology*; D000818:Animals; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000201:enzymology; D008297:Male; D008387:Marine Toxins; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017207:Rats, Sprague-Dawley
D004791:Enzyme Inhibitors; D007328:Insulin; D008387:Marine Toxins; D010080:Oxazoles; D011518:Proto-Oncogene Proteins; D010768:Phosphoserine; C059041:calyculin A; C494923:Akt2 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D010749:Phosphoprotein Phosphatases