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 inactivation of glycogen synthase kinase-3 (GSK-3) and the activation of the serine/threonine protein kinase B (PKB). GSK-3 inactivation occurs through phosphorylation 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-negative function (inhibition of growth factor-induced activation 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 insulin-induced GSK-3 regulation. Furthermore, it is shown that PKB and GSK-3 co-immunoprecipitate, indicating 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 necessary and sufficient for insulin-induced GSK-3 inactivation and establish a linear pathway from insulin receptor to GSK-3. Regulation of GSK-3 by PKB is likely through direct interaction, as both proteins co-immunoprecipitate. This interaction also resulted in a translocation of GSK-3 to the membrane in cells expressing transforming gagPKB.Activation of membrane-bound receptors of both the serpentine and tyrosine kinase classes often results in the activation of the lipid kinase PI 1 3-kinase, of which several different isoforms have been described (1). Activation of PI 3-kinase results in the formation of 3Ј-phosphorylated phosphatidylinositol (PI-3P) lipids (i.e. PI 3,4-P 2 and PI 3,4,5-P 3 ). These lipids were long suspected to perform a second messenger function. However, until recently the nature of the PI-3P binding proteins and their role in cellular signaling remained unsolved.

Insulin signalling in skeletal muscle of subjects with or without Type II-diabetes and first degree relatives of patients with the disease

“…Glycogen synthase is stimulated by insulin, and there are several indications that PI3′-kinase activity and potentially also Akt activation is necessary for this activation [51,52]. In this study, we did not detect a reduced activity at the clamp insulin concentration in the diabetic patients and FDR subjects.…”

Anchorage-independent multi-cellular spheroids as an in vitro model of growth signaling in Ewing tumors

“…We have observed that the down-regulation of cyclin D1 in response to PI3K inhibition in ET cells is also post-transcriptional. It is known that GSK3b enhances cyclin D1 protein phosphorylation and degradation (Diehl et al, 1998;Shao et al, 2000), and that activation of AKT inhibits this function by phosphorylating GSK3b at serine residue 9 (Cross et al, 1995;van Weeren et al, 1998). We found no di erences in the phosphorylation status of GSK3b(Ser9) in ET spheroids and monolayers, even when cyclin D1 levels were dramatically di erent.…”

Akt–PDK1 Complex Mediates Epidermal Growth Factor-induced Membrane Protrusion through Ral Activation

“…In the same study, however, requirement of Akt was negated because kinase activity of PDK1 was dispensable for Ral activation and because Ral could not be activated by Akt-CAAX, an Akt mutant that contains the C-terminal CAAX motif of Ki-Ras and thereby localizes constitutively at the plasma membrane. However, in one study, it has been reported that this Akt-CAAX mutant lacks kinase activity and inhibits insulin-induced GSK3 activation by endogenous Akt (van Weeren et al, 1998). Therefore, we used another active Akt mutant, mDPH-Akt, and found that this active Akt elevated basal and EGFstimulated Ral activity (Figure 6).…”