Essential Role for Protein Kinase B (PKB) in Insulin-induced Glycogen Synthase Kinase 3 Inactivation

Abstract

Activation of phosphatidylinositide 3؅-OH kinase (PI 3-kinase) is implicated in mediating a variety of growth factor-induced responses, among which are the inacti-vation of glycogen synthase kinase-3 (GSK-3) and the activation of the serine/threonine protein kinase B (PKB). GSK-3 inactivation occurs through phosphoryla-tion of Ser-9, and several kinases, such as protein kinase C, mitogen-activated protein kinase-activated protein kinase-1 (p90 Rsk), p70 S6kinase , and also PKB have been shown to phosphorylate this site in vitro. In the light of the many candidates to mediate insulin-induced GSK-3 inactivation we have investigated the role of PKB by constructing a PKB mutant that exhibits dominant-neg-ative function (inhibition of growth factor-induced acti-vation of PKB at expression levels similar to wild-type PKB), as currently no such mutant has been reported. We observed that the PKB mutant (PKB-CAAX) acts as an efficient inhibitor of PKB activation and also of in-sulin-induced GSK-3 regulation. Furthermore, it is shown that PKB and GSK-3 co-immunoprecipitate, indi-cating a direct interaction between GSK-3 and PKB. An additional functional consequence of this interaction is implicated by the observation that the oncogenic form of PKB, gagPKB induces a cellular relocalization of GSK-3 from the cytosolic to the membrane fraction. Our results demonstrate that PKB activation is both neces-sary and sufficient for insulin-induced GSK-3 inactiva-tion and establish a linear pathway from insulin recep-tor to GSK-3. Regulation of GSK-3 by PKB is likely through direct interaction, as both proteins co-immuno-precipitate. This interaction also resulted in a translo-cation of GSK-3 to the membrane in cells expressing transforming gagPKB.