Since its discovery 10 years ago, the potential functions of protein kinase B (PKB)/AKT have been catalogued with increasing efficiency. The physiological relevance of some of the proposed mechanisms by which PKB/AKT mediates many of its effects has been questioned, and recent work using new reagents and approaches has revealed some cracks in our understanding of this important molecule, and also hinted that these effects may involve other players.

Over the past decade, protein kinase B (PKB, also termed Akt) has emerged as an important signaling mediator between extracellular cues and modulation of gene expression, metabolism, and cell survival. The enzyme is tightly controlled and consequences of its deregulation include loss of growth control and oncogenesis. Recent work has better characterized the mechanism of PKB activation, including upstream regulators and secondary binding partners. This minireview refreshes some old concepts with new twists and highlights current outstanding questions. ß

Phosphorylation of the Bcl-2 family protein Bad may represent an important bridge between survival signaling by growth factor receptors and the prevention of apoptosis. Bad phosphorylation was examined following cytokine stimulation, which revealed phosphorylation on a critical residue, serine 112, in a MEK-dependent manner. Furthermore, Bad phosphorylation also increased on several sites distinct from serine 112 but could not be detected on serine 136, previously thought to be a protein kinase B/Akt-targeted residue. Serine 112 phosphorylation was shown to be absolutely required for dissociation of Bad from Bcl-x L . These results demonstrate for the first time in mammalian cells the involvement of the Ras-MAPK pathway in the phosphorylation of Bad and the regulation of its function.

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 ...

The serine-threonine kinase Akt is a protooncogene involved in the regulation of cell proliferation and survival. Activation of Akt is initiated by binding to the phospholipid products of phosphoinositide 3-kinase at the inner leaflet of the plasma membranes followed by phosphorylation at Ser 473 and Thr 308 . We have found that Akt is activated by Salmonella enterica serovar Typhimurium in epithelial cells. A bacterial effector protein, SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation. In HeLa cells, wild type S. typhimurium induced translocation of Akt to membrane ruffles and phosphorylation at residues Thr 308 and Ser 473 and increased kinase activity. In contrast, infection with a SigD deletion mutant did not induce phosphorylation or activity although Akt was translocated to membrane ruffles. Complementation of the SigD deletion strain with a mutant containing a single Cys to Ser mutation (C462S), did not restore the Akt activation phenotype. This residue has previously been shown to be essential for inositol phosphatase activity of the SigD homologue, SopB. Our data indicate a novel mechanism of Akt activation in which the endogenous cellular pathway does not convert membrane-associated Akt into its active form. SigD is also the first bacterial effector to be identified as an activator of Akt.