Protein Kinase B Activation Research Articles

The activation of protein kinase B by H2O2 or heat shock is mediated by phosphoinositide 3-kinase and not by mitogen-activated protein kinase-activated protein kinase-2.

Protein kinase B (PKB) isoforms became activated [and glycogen synthase kinase-3 (GSK3) became inhibited] when mouse Swiss 3T3 fibroblasts were exposed to oxidative stress (H2O2) or heat shock, but not when they were exposed to osmotic shock (0.5 M sorbitol or 0. 7 M NaCl), chemical stress (sodium arsenite), the protein-synthesis inhibitor anisomycin, or UV radiation. In contrast, all seven stimuli activated mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP-K2). The activation of MAPKAP-K2 was suppressed by the drug SB 203580, but not by inhibitors of phosphoinositide (phosphatidylinositide, PI) 3-kinase. In contrast, the activation of PKB isoforms and the inhibition of GSK3 by oxidative stress or heat shock were prevented by inhibitors of PI 3-kinase, but not by SB 203580. Thus the activation of PKB by oxidative stress or heat shock is mediated by PI 3-kinase and not by MAPKAP-K2. PKBalpha and PKBgamma were also activated by heat shock and oxidative stress in human embryonic kidney 293 cells and PKBgamma was activated by heat shock in NIH 3T3 cells; in each case activation was suppressed by inhibitors of PI 3-kinase. The activation of PKB isoforms by H2O2 may underlie some of the insulin-mimetic effects of this compound.

Involvement of the Pleckstrin Homology Domain in the Insulin-stimulated Activation of Protein Kinase B

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 (DeltaPH-PKB) did not impair the kinase activity; in contrast, the basal activity was elevated with respect to wild-type PKB. In addition, DeltaPH-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 DeltaPH-PKB was shown to be due to an elevation in the level of phosphorylation of this construct. In addition, the subcellular localization of DeltaPH-PKB, as determined by both immunofluorescence and fractionation, was predominately cytosolic, and DeltaPH-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.

Amino acid availability regulates p70 S6 kinase and multiple translation factors.

Incubation of Chinese hamster ovary cells without amino acids for up to 60 min caused a rapid marked decrease in p70 S6 kinase activity and increased binding of initiation factor eIF4E to its inhibitory regulator protein 4E-BP1. This was associated with dephosphorylation of 4E-BP1 and eIF4E and dissociation of eIF4E from eIF4G. All these effects were rapidly reversed by resupplying a mixture of amino acids and this was blocked by rapamycin and by inhibitors of phosphatidylinositol 3-kinase, implying a role for phosphatidylinositol 3-kinase in the signalling pathway linking amino acids with the control of p70 S6 kinase activity and the phosphorylation of these translation factors. Amino acid withdrawal also led to changes in the phosphorylation of other translation factors; phosphorylation of eIF4E decreased whereas elongation factor eEF2 became more heavily phosphorylated, each of these changes being associated with decreased activity of the factor in question. Earlier studies have suggested that protein kinase B (PKB) may act upstream of p70 S6 kinase. However, amino acids did not affect the activity of PKB, indicating that amino acids activate p70 S6 kinase through a pathway independent of this enzyme. Studies with individual amino acids suggested that the effects on p70 S6 kinase activity and translation-factor phosphorylation were independent of cell swelling. The data show that amino acid supply regulates multiple translation factors in mammalian cells.

Constitutive activation of protein kinase B alpha by membrane targeting promotes glucose and system A amino acid transport, protein synthesis, and inactivation of glycogen synthase kinase 3 in L6 muscle cells.

Phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated in the regulation of numerous cellular processes, including the insulin-induced regulation of glycogen synthase kinase 3 (GSK-3) and glucose transport. The hormonal-induced inactivation of GSK-3 is mediated by protein kinase B (PKB), a downstream target of PI 3-kinase, whose involvement in other insulin-stimulated responses remains poorly defined at present. In this study, we investigated whether the uptake of glucose, system A amino acid transport, and cellular protein synthesis are regulated by PKBalpha in L6 skeletal muscle cells. L6 cells stably overexpressing wild-type PKBalpha (wtPKBalpha) or a constitutively active membrane-targeted PKBalpha (mPKBalpha) showed a 3- and 15-fold increase in PKB activity, respectively. Both wtPKBalpha and mPKBalpha expression led to a significant increase in the basal uptake of glucose and methyl-aminoisobutyric acid (a substrate for the system A amino acid transporter), at least to a level seen in control cells treated with insulin. The stimulation in glucose transport was facilitated, in part, by the increased translocation of GLUT4 to the plasma membrane and also through an increase in the cellular synthesis of GLUT3. In the absence of insulin, only muscle cells expressing the constitutively active PKBalpha showed a significant increase in protein synthesis and an inhibition in GSK-3. Our results indicate that constitutive activation of PKBalpha in skeletal muscle stimulates the uptake of glucose, system A amino acids, and protein synthesis and promotes the inactivation of GSK-3. These observations imply that PKBalpha may have a role in the insulin-regulated control of these processes in skeletal muscle.

Translocation of PDK-1 to the plasma membrane is important in allowing PDK-1 to activate protein kinase B.

Protein kinase B (PKB) is involved in the regulation of apoptosis, protein synthesis and glycogen metabolism in mammalian cells. Phosphoinositide-dependent protein kinase (PDK-1) activates PKB in a manner dependent on phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), which is also needed for the translocation of PKB to the plasma membrane. It has been proposed that the amount of PKB activated is determined exclusively as a result of its translocation, and that a constitutively active pool of membrane-associated PDK-1 simply phosphorylates all the PKB made available. Here, we have investigated the effects of membrane localisation of PDK-1 on PKB activation. Ectopically expressed PDK-1 translocated to the plasma membrane in response to platelet-derived growth factor (PDGF) and translocation was sensitive to wortmannin, an inhibitor of phosphoinositide 3-kinase. Translocation of PDK-1 also occurred upon its co-expression with constitutively active phosphoinositide 3-kinase, but not with an inactive form. Overexpression of PDK-1 enhanced the ability of PDGF to activate PKB. PDK-1 disrupted in the pleckstrin homology (PH) domain which did not translocate to the membrane did not increase PKB activity in response to PDGF, whereas membrane-targeted PDK-1 activated PKB to the extent that it could not be activated further by PDGF. In response to PDGF, binding of Ptdlns (3,4,5)P3 and/or Ptdlns(3,4)P2 to the PH domain of PDK-1 causes its translocation to the plasma membrane where it co-localises with PKB, significantly contributing to the scale of PKB activation.

Protein Kinase B and Rac Are Activated in Parallel within a Phosphatidylinositide 3OH-kinase-controlled Signaling Pathway

The GTPase Rac and the protein kinase B (PKB) are downstream targets of phosphatidylinositide 3OH-kinase in platelet-derived growth factor-stimulated signaling pathways. We have generated PAE cell lines inducibly expressing mutants of Rac. Use of these cell lines suggests that Rac is involved in both platelet-derived growth factor-stimulated membrane ruffling and the activation of p70(S6K) but not in the activation of PKB. Furthermore, expression of constitutively active alleles of PKB in PAE cells suggests that PKB is able to regulate the activity of p70(S6K) but not the cytoskeletal changes underlying membrane ruffling. Thus, our results indicate that Rac and PKB are on separate pathways downstream of phosphatidylinositide 3OH-kinase in these cells but that both of these pathways are involved in the regulation of p70(S6K).

Essential Role for Protein Kinase B (PKB) in Insulin-induced Glycogen Synthase Kinase 3 Inactivation: CHARACTERIZATION OF DOMINANT-NEGATIVE MUTANT OF PKB

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.

Evidence against protein kinase B as a mediator of contraction-induced glucose transport and GLUT4 translocation in rat skeletal muscle

Both insulin and muscle contraction stimulate glucose transport activity. However, contraction stimulation does not involve the insulin signalling intermediate phosphatidylinositol 3-kinase (PI 3-kinase). Protein kinase B (PKB) has recently been identified as a direct downstream target of PI 3-kinase in the insulin signalling pathway. We have examined here whether the two stimuli share PKB as a convergent step in separate signalling pathways. Insulin stimulates both glucose transport, GLUT4 cell-surface content and PKB activity (by 4–6-fold above basal) in a wortmannin-sensitive manner in in vitro incubated rat soleus muscles. By contrast, muscle contraction, which stimulates glucose transport and the cell surface content of GLUT4 by 3-fold above basal levels, had no effect on PKB activity. These data demonstrate that PKB is not a mediator of contraction-induced glucose transport and GLUT4 translocation.

Protein kinase B activation and lamellipodium formation are independent phosphoinositide 3-kinase-mediated events differentially regulated by endogenous Ras.

Regulation of phosphoinositide 3-kinase (PI 3-kinase) can occur by binding of the regulatory p85 subunit to tyrosine-phosphorylated proteins and by binding of the p110 catalytic subunit to activated Ras. However, the way in which these regulatory mechanisms act to regulate PI 3-kinase in vivo is unclear. Here we show that several growth factors (basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], and epidermal growth factor [EGF; to activate an EGF receptor-Ret chimeric receptor]) all activate PI 3-kinase in vivo in the neuroectoderm-derived cell line SKF5. However, these growth factors differ in their ability to activate PI 3-kinase-dependent signaling. PDGF and EGF(Ret) treatment induced PI 3-kinase-dependent lamellipodium formation and protein kinase B (PKB) activation. In contrast, bFGF did not induce lamellipodium formation but activated PKB, albeit to a small extent. PDGF and EGF(Ret) stimulation resulted in binding of p85 to tyrosine-phosphorylated proteins and strong Ras activation. bFGF, however, induced only strong activation of Ras. In addition, while RasAsn17 abolished bFGF activation of PKB, PDGF- and EGF(Ret)-induced PKB activation was only partially inhibited and lamellipodium formation was unaffected. Interestingly, in contrast to activation of only endogenous Ras (bFGF), ectopic expression of activated Ras did result in lamellipodium formation. From this we conclude that, in vivo, p85 and Ras synergize to activate PI 3-kinase and that strong activation of only endogenous Ras exerts a small effect on PI 3-kinase activity, sufficient for PKB activation but not lamellipodium formation. This differential sensitivity to PI 3-kinase activation could be explained by our finding that PKB activation and lamellipodium formation are independent PI 3-kinase-induced events.

Mechanism of activation and function of protein kinase B

The past year has seen significant advances in our understanding of how protein kinase B (PKB) is activated and of the central role it plays in insulin signalling and in mediating the protective effects of survival factors against apoptosis. The highlights include the discovery of a protein kinase required for the 3-phosphoinositide-dependent activation of PKB and the identification of several physiological substrates for PKB.

Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B.

Protein kinase B (PKB) is activated in response to phosphoinositide 3-kinases and their lipid products phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P3] and PtdIns(3,4)P2 in the signaling pathways used by a wide variety of growth factors, antigens, and inflammatory stimuli. PKB is a direct target of these lipids, but this regulation is complex. The lipids can bind to the pleckstrin homologous domain of PKB, causing its translocation to the membrane, and also enable upstream, Thr308-directed kinases to phosphorylate and activate PKB. Four isoforms of these PKB kinases were purified from sheep brain. They bound PtdIns(3,4,5)P3 and associated with lipid vesicles containing it. These kinases contain an NH2-terminal catalytic domain and a COOH-terminal pleckstrin homologous domain, and their heterologous expression augments receptor activation of PKB, which suggests they are the primary signal transducers that enable PtdIns(3,4,5)P3 or PtdIns- (3,4)P2 to activate PKB and hence to control signaling pathways regulating cell survival, glucose uptake, and glycogen metabolism.

Phosphatidylinositol 3-Kinase Couples the Interleukin-2 Receptor to the Cell Cycle Regulator E2F

Cell cycle progression initiated by interleukin-2 (IL-2) in T cells is critical for lymphoproliferation and an immune response. Phosphatidyl inositol 3-kinase (PI3K) is activated by IL-2. However, nuclear targets for PI3K are not known. Here we identify the cell cycle regulator E2F as an IL-2 target in T lymphocytes and PI3K as the critical signaling pathway. We eliminate both Stat5 and Raf/MEK pathways from E2F regulation. Protein kinase B (PKB) is activated by IL-2 via PI3K. The expression of an active PKB is sufficient to induce E2F activity. Inhibition of PI3K inhibits phosphorylation of Rb, induction of cyclin D3, and degradation of p27 kip1 . These results establish a crucial PI3K/PKB–mediated link between the IL-2 receptor and the cell cycle machinery.

G-Protein-coupled Receptors and Fcγ-receptors Mediate Activation of Akt/Protein Kinase B in Human Phagocytes

Activation of the serine/threonine kinase Akt, also called protein kinase B (PKB), was investigated in human neutrophils. Stimulation of the cells with the chemoattractant fMet-Leu-Phe or the chemokines IL-8 and GROalpha leads to the rapid and transient activation of PKB. Maximum PKB activation correlates with the well documented kinetics of respiratory burst and exocytosis. Wortmannin, a selective inhibitor of phosphoinositide 3-kinases (PI 3-kinases) in neutrophils, abrogates PKB activation. Similarly homo and heterotypic cross-linking of FcgammaIIA and FcgammaIIIB causes a transient activation of PKB that is sensitive to wortmannin treatment. Kinase activity measurements in immunoprecipitates from lysates of the myelocytic GM-1 cells or GM-1/CXCR1 cells, which are transfected with the IL-8 receptor 1, confirmed the transient activation of PKB observed in neutrophils. Stimulation of human monocytes with the CC chemokine RANTES (regulated on activation normal T cell expressed and secreted) also results in the activation of PKB. Preincubation of monocytes and neutrophils with Bordetella pertussis toxin inhibits fMet-Leu-Phe and RANTES-stimulated PKB activation, demonstrating that coupling of the receptors to heterotrimeric Gi-protein is required. The data show, that activation of PKB by Gi-protein-coupled receptors is mediated by PI 3-kinase and suggest that PKB is a constituent of neutrophil activating pathways.

Regulation of Protein Kinase B in Rat Adipocytes by Insulin, Vanadate, and Peroxovanadate: MEMBRANE TRANSLOCATION IN RESPONSE TO PEROXOVANADATE

Protein kinase B (PKB) (also referred to as RAC/Akt kinase) has been shown to be controlled by various growth factors, including insulin, using cell lines and transfected cells. However, information is so far scarce regarding its regulation in primary insulin-responsive cells. We have therefore used isolated rat adipocytes to examine the mechanisms, including membrane translocation, whereby insulin and the insulin-mimicking agents vanadate and peroxovanadate control PKB. Stimulation of adipocytes with insulin, vanadate, or peroxovanadate caused decreased PKB mobility on sodium dodecyl sulfate-polyacrylamide gels, indicative of increased phosphorylation, which correlated with an increase in kinase activity detected with the peptide KKRNRTLTK. This peptide was found to detect activated PKB selectively in crude cytosol and partially purified cytosol fractions from insulin-stimulated adipocytes. The decrease in electrophoretic mobility and activation of PKB induced by insulin was reversed both in vitro by treatment of the enzyme with alkaline phosphatase and in the intact adipocyte upon removal of insulin or addition of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Significant translocation of PKB to membranes could not be demonstrated after insulin stimulation, but peroxovanadate, which appeared to activate PI 3-kinase to a higher extent than insulin, induced substantial translocation. The translocation was prevented by wortmannin, suggesting that PI 3-kinase and/or the 3-phosphorylated phosphoinositides generated by PI 3-kinase are indeed involved in the membrane targeting of PKB.

Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B.

Protein kinase B (PKB) is a proto-oncogene that is activated in signaling pathways initiated by phosphoinositide 3-kinase. Chromatographic separation of brain cytosol revealed a kinase activity that phosphorylated and activated PKB only in the presence of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3]. Phosphorylation occurred exclusively on threonine-308, a residue implicated in activation of PKB in vivo. PtdIns(3,4,5)P3 was determined to have a dual role: Its binding to the pleckstrin homology domain of PKB was required to allow phosphorylation by the upstream kinase and it directly activated the upstream kinase.

CAMP stimulates protein kinase B in a Wortmannin-insensitive manner

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.

Phosphatidylinositol 3-Kinase Links the Interleukin-2 Receptor to Protein Kinase B and p70 S6 Kinase

Phosphatidylinositol 3-kinase (PI 3-kinase) is activated by the cytokine interleukin-2 (IL-2). We have used a constitutively active PI 3-kinase to identify IL-2-mediated signal transduction pathways directly regulated by PI 3-kinase in lymphoid cells. The serine/threonine protein kinase B (PKB)/Akt can act as a powerful oncogene in T cells, but its positioning in normal T cell responses has not been explored. Herein, we demonstrate that PKB is activated by IL-2 in a PI 3-kinase-dependent fashion. Importantly, PI 3-kinase signals are sufficient for PKB activation in IL-2-dependent T cells, and PKB is a target for PI 3-kinase signals in IL-2 activation pathways. The present study establishes also that PI 3-kinase signals or PKB signals are sufficient for activation of p70 S6 kinase in T cells. PI 3-kinase can contribute to, but is not sufficient for, activation of extracellular signal-regulated kinases (Erks) and Erk effector pathways. Therefore, PI 3-kinase is a selective regulator of serine/threonine kinase signal transduction pathways in T lymphocytes, and this enzyme provides a crucial link between the interleukin-2 receptor, the protooncogene PKB, and p70 S6 kinase.

Expression of a constitutively activated form of protein kinase B (c-Akt) in 3T3-L1 preadipose cells causes spontaneous differentiation.

Insulin/IGF-1 is required for differentiation of 3T3-L1 adipose cells. Downstream targets of insulin/IGF-1 that lead to adipocyte differentiation appear to include Ras, phosphatidylinositol (PI) 3-kinase, Raf, and mitogen-activated protein kinase. We have tested whether protein kinase B (PKB), a serine/threonine kinase activated by PI 3-kinase, is sufficient for 3T3-L1 preadipose cell differentiation. A plasmid vector encoding a version of PKB that is constitutively activated (Gag-PKB) was expressed in 3T3-L1 preadipose cells (Gag-PKB cells). Spontaneous morphological changes indicative of adipocyte differentiation were observed in Gag-PKB cells. The cells assumed a spherical shape and they acquired characteristic lipid droplets that stained positively for Oil Red O. Northern blot analysis detected upregulation of LPL and aP2 mRNA, specific indicators of adipocyte differentiation. Our data demonstrate that constitutive activation of PKB is sufficient to trigger adipocyte differentiation.

Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction.

A serine/threonine kinase, named protein kinase B (PKB) for its sequence homology to both protein kinase A and C, has previously been isolated. PKB, which is identical to the kinase Rac, was later found to be the cellular homologue of the transforming v-Akt. Here we show that PKB is activated by stimuli such as insulin, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Activation of PKB was inhibited by the phosphatidylinositol-3-OH kinase (PI(3)K) inhibitor wortmannin and by coexpression of a dominant-negative mutant of PI(3)K. PDGF receptor mutants that lack detectable associated PI(3)K activity also fail to induce PKB activation, PKB kinase activity is correlated with phosphorylation of PKB on serine. Finally, we show that a constructed Gag-PKB fusion protein, homologous to the v-akt oncogene, displays significantly increased ligand-independent kinase activity. Furthermore, this activity is sufficient to activate the p70 S6-kinase (p70S6K). These results suggest a role for PKB in PI(3)K-mediated signal transduction.

Reiter's Disease

Background: Head and neck squamous cell carcinoma (HNSCC) has a 50% relapse rate. The tumor microenvironment has been linked to resistance of cancer cells to chemotherapy. We hypothesized that the tumor matrix proteins collagen and fibronectin play protective roles in HNSCC. Materials and Methods: We investigated the effects of collagen I, collagen IV and fibronectin on growth, 2-D and 3-D clonogenic potential, resistance to paclitaxel, apoptosis and activation of phosphoinositol-3 kinase (PI3K) in MD-1483 HNSCC cells. Results: Collagen I, collagen IV and fibronectin specifically increased the efficiency of 2-D colony formation through binding integrins α2β1 and α5β1, respectively, and provided resistance to paclitaxel-induced colony elimination and apoptosis. Collagen I, but not fibronectin, increased the efficiency of 3-D colony formation and induced resistance to paclitaxel. Activation of protein kinase-B by collagen I was necessary for the protective effect. Conclusion: These data support the potential contribution of fibronectin and collagen to chemotherapy resistance in HNSCC, with effects of collagen mediated by PI3K.