Regulation of protein kinase B in rat adipocytes by insulin, vanadate, and peroxovanadate. Membrane translocation in response to peroxovanadate.
Protein kinase B (PKB) (also referred to as RAC/Akt kinase) has been shown to be controlled by various growth factors, including insulin, using cell lines and transfected cells. However, information is so far scarce regarding its regulation in primary insulin-responsive cells. We have therefore used isolated rat adipocytes to examine the mechanisms, including membrane translocation, whereby insulin and the insulin-mimicking agents vanadate and peroxovanadate control PKB. Stimulation of adipocytes with insulin, vanadate, or peroxovanadate caused decreased PKB mobility on sodium dodecyl sulfate-polyacrylamide gels, indicative of increased phosphorylation, which correlated with an increase in kinase activity detected with the peptide KKRNRTLTK. This peptide was found to detect activated PKB selectively in crude cytosol and partially purified cytosol fractions from insulin-stimulated adipocytes. The decrease in electrophoretic mobility and activation of PKB induced by insulin was reversed both in vitro by treatment of the enzyme with alkaline phosphatase and in the intact adipocyte upon removal of insulin or addition of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Significant translocation of PKB to membranes could not be demonstrated after insulin stimulation, but peroxovanadate, which appeared to activate PI 3-kinase to a higher extent than insulin, induced substantial translocation. The translocation was prevented by wortmannin, suggesting that PI 3-kinase and/or the 3-phosphorylated phosphoinositides generated by PI 3-kinase are indeed involved in the membrane targeting of PKB.
Wijkander
J
J
Section for Molecular Signaling, Department of Cell and Molecular Biology, Lund University, S-221 00 Lund, Sweden. jonny wijkander@medkem.lu.se
Holst
L S
LS
Rahn
T
T
Resjö
S
S
Castan
I
I
Manganiello
V
V
Belfrage
P
P
Degerman
E
E
D017667:Adipocytes / Q000201:enzymology*; D000818:Animals; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D007328:Insulin / Q000502:physiology*; D008297:Male; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017207:Rats, Sprague-Dawley; D014638:Vanadates / Q000494:pharmacology*
D007328:Insulin; D011518:Proto-Oncogene Proteins; C069276:peroxovanadate; D014638:Vanadates; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt