Activation of protein kinase B (PKB) by growth factors and hormones has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase).In this report, we show that PKB can also be activated by PKA (cyclic AMP [cAMP]-dependent protein kinase) through a PI3-kinase-independent pathway. Although this activation required phosphorylation of PKB, PKB is not likely to be a physiological substrate of PKA since a mutation in the sole PKA consensus phosphorylation site of PKB did not abolish PKA-induced activation of PKB. In addition, mechanistically, this activation was different from that of growth factors since it did not require phosphorylation of the S473 residue, which is essential for full PKB activation induced by insulin. These data were supported by the fact that mutation of residue S473 of PKB to alanine did not prevent it from being activated by forskolin. Moreover, phosphopeptide maps of overexpressed PKB from COS cells showed differences between insulin-and forskolin-stimulated cells that pointed to distinct activation mechanisms of PKB depending on whether insulin or cAMP was used. We looked at events downstream of PKB and found that PKA activation of PKB led to the phosphorylation and inhibition of glycogen synthase kinase-3 (GSK-3) activity, a known in vivo substrate of PKB. Overexpression of a dominant negative PKB led to the loss of inhibition of GSK-3 in both insulin-and forskolin-treated cells, demonstrating that PKB was responsible for this inhibition in both cases. Finally, we show by confocal microscopy that forskolin, similar to insulin, was able to induce translocation of PKB to the plasma membrane. This process was inhibited by high concentrations of wortmannin (300 nM), suggesting that forskolin-induced PKB movement may require phospholipids, which are probably not generated by class I or class III PI3-kinase. However, high concentrations of wortmannin did not abolish PKB activation, which demonstrates that translocation per se is not important for PKA-induced PKB activation.

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Both MAPK and protein kinase C (PKC) signaling pathways promote cell survival and protect against cell death. Here, we show that 12-O-tetradecanoylphorbol-13-acetate (TPA) prevents Fas-induced apoptosis in T lymphocytes. The effect of TPA was specifically abolished by the PKC inhibitor GF109203X and by dominant negative PKC, PKC⑀, and PKC␣, suggesting that novel and conventional PKC isoforms mediate phorbol ester action. Moreover, TPA stimulated phosphorylation of BAD at serine 112, an effect abrogated by GF109203X but not by the MEK inhibitor PD98059. Expression of constitutively active PKC increased the phosphorylation of BAD at serine 112 but not at serine 136. Additionally, Fas-mediated cell death was enhanced by overexpression of a catalytically inactive form of p90Rsk (Rsk2-KN). Finally, Rsk2-KN abolished the protective effect of constitutively active PKC and totally blocked phosphorylation of BAD on serine 112. Thus, novel PKC and PKC⑀ rescue T lymphocytes from Fas-mediated apoptosis via a p90Rsk-dependent phosphorylation and inactivation of BAD.In several cell lines, apoptosis is antagonized by growth factors and hormones and, more generally, by stimuli that promote cell survival. Interleukin 3 and insulin-like growth factor 1 exert their antiapoptotic effect through activation of phosphatidylinositol 3-kinase, which, in turn, leads to activation of the serine/threonine protein kinase B (PKB/Akt), which promotes cell survival by phosphorylating BAD at Ser 136 (1-6). Interestingly, interleukin 3, through activation of a mitochondrial membrane based protein kinase A, also stimulates phosphorylation of BAD at Ser 112 (6, 7 (8,9). Brain-derived neurotrophic factor exerts its antiapoptotic effect in a mitogen-activated protein kinase (MAPK)/ extracellular signal-regulated kinase pathway (8). The mechanism of action of TPA remains unclear, although MAPKdependent (10 -13) and -independent pathways have been described (9). TPA is a tumor promoter that binds and activates members of a family of serine/threonine protein kinases termed protein kinase C (PKC). PKC comprised at least 12 isotypes that have been classified into three groups according to their structure and cofactor requirement: (a) conventional PKCs (PKC␣, PKC␤I, PKC␤II, and PKC␥) are diacylglycerol-and calcium-dependent, (b) novel PKCs (PKC␦, PKC⑀, PKC, PKC, and PKC) are diacylglycerol-dependent but calciumindependent, and (c) atypical PKCs (PKC, PKC, and PKC) are not activated by phorbol esters but can bind diacylglycerol (14, 15). Overexpression of PKC⑀, PKC␣, or PKC increases the resistance of cells to apoptosis, and PKC inhibitors are known to sensitize cells to apoptosis (16 -20). Additionally, Fas ligation-induced apoptosis in Jurkat T cells resulted in a blockade of cellular PKC activity, suggesting a link between the two events (21). Although involvement of PKC in the suppression of apoptosis has been demonstrated recently, the mechanisms by which PKC promotes cell survival remain to be elucidated. EXPERIMENTAL PROCEDURESReporter Plasmids, Tr...

Activation of protein kinase B (PKB) by growth factors has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). Here, we show that agents which raise intracellular cAMP can also stimulate PKB. However, this effect is not sensitive to wortmannin, indicating that it is PI3-kinase independent. This activation does not appear to result from direct phosphorylation by protein kinase A (PKA) since GST-PKB is not an effective PKA substrate. In addition, the activation pathway of PKB by cAMP seems to be linked to that of growth factors, albeit downstream of PI3-kinase. Evidence for this is that a constitutive active PKB, T308D, S473D, containing activating mutations in the serine and threonine residues which are phosphorylated subsequent to PI3-kinase activation, cannot be further stimulated by cAMP elevations. Hence, these data suggest that, in addition to growth factors, cAMP can also lead to activation of PKB. This cAMP stimulatory action appears to require phosphorylation of T308 and S473, and hence would indicate that cAMP modulates the phosphorylation event of these PKB regulatory sites.

In this report we investigated the function of phosphoinositide-dependent protein kinase 1 (PDK1) in protein kinase B (PKB) activation and translocation to the cell surface. Wild-type and PDK1 mutants were transfected into HeLa cells, and their subcellular localization was analyzed. PDK1 was found to translocate to the plasma membrane in response to insulin, and this process did not require a functional catalytic activity, since a catalytically inactive kinase mutant (Kd) of PDK1 was capable of translocating. The PDK1 presence at the cell surface was shown to be linked to phospholipids and therefore to serum-dependent phosphatidylinositol 3-kinase activity. Using confocal microscopy in HeLa cells we found that PDK1 colocalizes with PKB at the plasma membrane. Further, after cotransfection of PKB and a PDK1 mutant (Mut) unable to translocate to the plasma membrane, PKB was prevented from moving to the cell periphery after insulin stimulation. In response to insulin, a PKB mutant with its PH domain deleted (⌬PH-PKB) retained the ability to translocate to the plasma membrane when coexpressed with PDK1. Finally, we found that ⌬PH-PKB was highly active independent of insulin stimulation when cotransfected with PDK1 mutants defective in their PH domain. These findings suggest that PDK1 brings PKB to the plasma membrane upon exposure of cells to insulin and that the PH domain of PDK1 acts as a negative regulator of its enzyme activity.Protein kinase B␣ (PKB␣), also known as c-Akt or RAC (related to protein kinase A and C), is composed of a Nterminal pleckstrin homology (PH) domain followed by a kinase catalytic domain which shares homology with the A and C protein kinases. Two other isoforms of PKB (termed PKB␤ and PKB␥) have been identified and are expressed in ovarian, pancreatic, and breast cancer cells (10,11). This serine/threonine kinase is rapidly activated in response to stimulation of tyrosine kinase receptors such as those for platelet-derived growth factor (PDGF), insulin, basic fibroblast growth factor, and epidermal growth factor (9,15,18). PKB stimulation by insulin or growth factors is thought to be dependent on phosphatidylinositol 3Ј-kinase (PI 3-kinase) for the following reasons: (i) it is sensitive to pharmacological inhibitors of PI 3-kinase (18), (ii) dominant-negative PI 3-kinase mutants block PKB activation (24), and (iii) constitutively active PI 3-kinase mutants stimulate PKB (9).A model has been proposed to explain activation of PKB in response to hormones and growth factors (2). According to this model, stimulation of cell surface receptors leads to an increase in the level of phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P 3 ) and PtdIns-3,4-P 2 via PI 3-kinase. Although it was initially reported that phospholipids could directly activate PKB by interacting with its PH domain (16), more recently it has been shown that this process most likely fulfills other and/or additional functions. Indeed, it may facilitate PKB localization to the plasma membrane. This view stems from the observ...

The diverse biological actions of insulin and insulin-like growth factor I (IGF-I) are initiated by binding of the polypeptides to their respective cell surface tyrosine kinase receptors. These activated receptors phosphorylate a series of endogenous substrates on tyrosine, amongst which the insulin receptor substrate (IRS) proteins are the best characterized. Their phosphotyrosine-containing motifs become binding sites for Src homology 2 (SH2) domains on proteins such as SH2 domain-containing protein-tyrosine-phosphatase (SHP)-2/Syp, growth factor receptor bound-2 protien, (Grb-2), and phosphatidyl inositol 3 kinase (PI3 kinase), which participate in activation of specific signaling cascades. However, the IRS molecules are not only platforms for signaling molecules, they also orchestrate the generation of signal specificity, integration of signals induced by several extracellular stimuli, and signal termination and modulation. An extensive review is beyond the scope of the present article, which will be centered on our own contribution and reflect our biases.

Involvement of the pleckstrin homology (PH) domain in the insulin-stimulated activation of protein kinase B (PKB) was investigated in human embryonic kidney 293 cells. Different PKB constructs that contain mutations or deletions in the PH domain were transfected into cells, and the results on the basal and insulin-induced kinase activities were analyzed. Deletion of the entire PH domain (⌬PH-PKB) did not impair the kinase activity; in contrast, the basal activity was elevated with respect to wild-type PKB. In addition, ⌬PH-PKB was responsive to insulin, and as for wild-type PKB, this was dependent on phosphoinositide 3-kinase. By contrast, a point mutation within the PH domain that impairs phospholipid binding (R25C) resulted in a construct that was not responsive to insulin. However, this defect was overcome by mutations that mimic the phosphorylation state of the active kinase. The increase in the basal activity of ⌬PH-PKB was shown to be due to an elevation in the level of phosphorylation of this construct. In addition, the subcellular localization of ⌬PH-PKB, as determined by both immunofluorescence and fractionation, was predominately cytosolic, and ⌬PH-PKB was present in the plasma membrane at much lower levels compared with wild-type PKB. These data show that phosphorylation is the major factor regulating the activity of PKB and that either removal of the PH domain or binding of phospholipids is required to permit this phosphorylation. In addition, membrane localization does not appear to be required for the activation process, but instead, binding of PKB to membrane phospholipids permits a conformational change in the molecule that allows for phosphorylation. Protein kinase B (PKB),1 also referred to as Akt or RAC kinase, is a serine/threonine protein kinase that was cloned by virtue of its homology to protein kinases A and C and is the cellular homolog of the product of the v-akt oncogene (1-4). The kinase is activated in response to treatment of cells with agonists that bind to receptors possessing tyrosine kinase activity such as those of platelet-derived growth factor and insulin (5-7). The activity of PKB in response to these agents is strictly dependent on the activity of phosphoinositide 3-kinase (PI3K) since (i) it is sensitive to wortmannin, an inhibitor of PI3K; (ii) mutants of the platelet-derived growth factor receptor that cannot interact with PI3K are incapable of PKB activation; and (iii) constitutively active forms of PI3K are able to activate PKB in intact cells (5-7). The effect of 3Ј-phosphoinositides is believed to be mediated in part by the N-terminal pleckstrin homology (PH) domain of PKB. In addition, PI3K-independent activation of PKB has been demonstrated in response to heat shock, ␤-adrenergic receptor activation, and cAMP (8 -10).To date, PKB has been implicated in physiological processes regulating both cellular growth and metabolism. The first identified substrate of PKB was glycogen-synthase kinase-3, which is inhibited in vivo after phosphorylation by PKB (11,12). In additi...

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