Insulin stimulates increased catalytic activity of phosphoinositide-dependent kinase-1 by a phosphorylation-dependent mechanism

Phosphoinositide-dependent kinase-1 (PDK-1) is a serine-threonine kinase do wnstream from PI 3-kinase that phosphorylates and activates other important kinases such as Akt that are essential for cell survival and metabolism. P revious reports have suggested that PDK-1 has constitutive catalytic activi ty that is not regulated by stimulation of cells with growth factors. We no w show that insulin stimulation of NIH-3T3(IR) cells or rat adipose cells m ay significantly increase the intrinsic catalytic activity of PDK-1. Insuli n treatment of NIH-3T3(IR) fibroblasts overexpressing PDK-1 increased both phosphorylation of recombinant PDK-1 in intact cells and PDK-1 kinase activ ity in an immune-complex kinase assay. Insulin stimulation of rat adipose c ells also increased catalytic activity of endogenous PDK-1 immunoprecipitat ed from the cells. Both insulin-stimulated phosphorylation and activity of PDK-1 were inhibited by wortmannin and reversed by treatment with the phosp hatase PP-2A. A mutant PDK-1 with a disrupted PH domain (W538L) did not und ergo phosphorylation or demonstrate increased kinase activity in response t o insulin stimulation. Similarly, a PDK-1 phosphorylation site point mutant (S244A) had no increase in kinase activity in response to insulin stimulat ion. Thus, the insulin-stimulated increase in PDK-1 catalytic activity may involve PI 3-kinase- and phosphorylation-dependent mechanisms. We conclude that the basal constitutive catalytic activity of PDK-1 in NIH-3T3(IR) cell s and rat adipose cells can be significantly increased upon insulin stimula tion.