Multiple Phosphoinositide 3-Kinase-Dependent Steps in Activation of Protein Kinase B

Abstract

The protein kinase B (PKB)/Akt family of serine kinases is rapidly activated following agonist-induced stimulation of phosphoinositide 3-kinase (PI3K). To probe the molecular events important for the activation process, we employed two distinct models of posttranslational inducible activation and membrane recruitment. PKB induction requires phosphorylation of two critical residues, threonine 308 in the activation loop and serine 473 near the carboxyl terminus. Membrane localization of PKB was found to be a primary determinant of serine 473 phosphorylation. PI3K activity was equally important for promoting phosphorylation of serine 473, but this was separable from membrane localization. PDK1 phosphorylation of threonine 308 was primarily dependent upon prior serine 473 phosphorylation and, to a lesser extent, localization to the plasma membrane. Mutation of serine 473 to alanine or aspartic acid modulated the degree of threonine 308 phosphorylation in both models, while a point mutation in the substrate-binding region of PDK1 (L155E) rendered PDK1 incapable of phosphorylating PKB. Together, these results suggest a mechanism in which 3 phosphoinositide lipid-dependent translocation of PKB to the plasma membrane promotes serine 473 phosphorylation, which is, in turn, necessary for PDK1-mediated phosphorylation of threonine 308 and, consequentially, full PKB activation.Protein kinase B (PKB), also termed Akt, has been the subject of intense study due to its role in transducing signals from phosphoinositide 3-kinase (PI3K) that regulate cell survival and intermediary metabolism. Several protooncogene products modulate the activation of PI3K and, as a consequence, PKB has been shown to play roles in many of the cellular functions that are altered during oncogenesis and other diseases (reviewed in reference 12). Interference with PKB activation may therefore have therapeutic value.Activation of PKB entails a complex series of events involving additional proteins. First, the PI3K-generated lipid products PI(3,4,5)P 3 and PI(3,4)P 2 recruit PKB to the plasma membrane through their affinity for the PH domain of PKB (14,20,21). Once membrane proximal, at least two residues of PKB are rapidly phosphorylated, including threonine 308 (T308) and serine 473 (S473) (1). T308 lies within the kinase T loop, and its phosphorylation is presumed to generate a conformational change that permits access to the substrates, analogous to T-loop phosphorylation in other protein kinases. In the case of PKB, this reaction is catalyzed by another 3Ј phosphoinositide-regulated kinase termed PDK1 (2, 33). S473 is located within a hydrophobic region close to the carboxyl terminus of PKB and is also phosphorylated during activation (1), but the mechanism of its phosphorylation and the role it serves in activating PKB are incompletely understood.Several lines of evidence suggest that S473 is autophosphorylated. For example, catalytically inactive mutants of PKB do not undergo S473 phosphorylation (34). There is also evidence for an autonomous S473 ...

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