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 s hown to be controlled by various growth factors, including insulin, us ing cell lines and transfected cells, However, information is so far s carce 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-mi micking agents vanadate and peroxovanadate control PKB. Stimulation of adipocytes with insulin, vanadate, or peroxovanadate caused decreased PKB mobility on sodium dodecyl sulfate-polyacrylamide gels, indicativ e of increased phosphorylation, which correlated with an increase in k inase activity detected with the peptide KKRNRTLTK. This peptide was f ound to detect activated PKB selectively in crude cytosol and partiall y purified cytosol fractions from insulin-stimulated adipocytes. The d ecrease in electrophoretic mobility and activation of PKB induced by i nsulin was reversed both in vitro by treatment of the enzyme with alka line phosphatase and in the intact adipocyte upon removal of insulin o r addition of the phosphatidylinositol 3-kinase (PI I-kinase) inhibito r wortmannin. Significant translocation of PKB to membranes could not Its demonstrated after insulin stimulation, but peroxovanadate, which appeared to activate PI 3-kinase to a higher extent than insulin, indu ced substantial translocation, The translocation was prevented by wort mannin, suggesting that PI 3-kinase and/or the 3-phosphorylated phosph oinositides generated by PI 3-kinase are indeed involved in the membra ne targeting of PKB.