Mechanism of Activation of PKB/Akt by the Protein Phosphatase Inhibitor Calyculin A

The protein phosphatase inhibitor calyculin A activates PKB/Akt to ~50% of the activity induced by insulin-like growth factor 1 (IGF1) in HeLa cells promoting an evident increased phosphorylation of Ser473 despite the apparent lack of Thr308 phosphorylation of PKB. Nevertheless, calyculin A-induced activation of PKB seems to be dependent on basal levels of Thr308 phosphorylation, since a PDK1-dependent mechanism is required for calyculin A-dependent PKB activation by using embryonic stem cells derived from PDK1 wild-type and knockout mice. Data shown suggest that calyculin A-induced phosphorylation of Ser473 was largely blocked by LY294002 and SB-203580 inhibitors, indicating that both PI3-kinase/TORC2-dependent and SAPK2/p38-dependent protein kinases contributed to phosphorylation of Ser473 in calyculin A-treated cells. Additionally, our results suggest that calyculin A blocks the IGF1-dependent Thr308 phosphorylation and activation of PKB, likely due to an enhanced Ser612 phosphorylation of insulin receptor substrate 1 (IRS1), which can be inhibitory to its activation of PI3-kinase, a requirement for PDK1-induced Thr308 phosphorylation and IGF1-dependent activation of PKB. Our data suggest that PKB activity is most dependent on the level of Ser473 phosphorylation rather than Thr308, but basal levels of Thr308 phosphorylation are a requirement. Additionally, we suggest here that calyculin A regulates the IGF1-dependent PKB activation by controlling the PI3-kinase-associated IRS1 Ser/Thr phosphorylation levels.     

Adrenaline potentiates insulin-stimulated PKB activation via cAMP and Epac: implications for cross talk between insulin and adrenaline

Adrenaline and insulin are two of the most important hormones regulating a number of physiological processes in skeletal muscle. Insulin's effects are generally requiring PKB and adrenaline effects cAMP and PKA. Recent evidence indicates cAMP can regulate PKB in some cell types via Epac (Exchange protein directly activated by cAMP). This suggests possible crossover between insulin and adrenaline signalling in muscle. Here we find that adrenaline alone did not influence PKB activation, but adrenaline dramatically potentiated insulin-stimulated phosphorylation of PKB (both Ser473 and Thr308) and of PKBalpha and PKBbeta enzyme activities. These effects were inhibited by wortmannin but adrenaline did not increase insulin-stimulated p85alpha PI 3-kinase activity. Adrenaline effects occurred via beta-adrenergic receptors and accumulation of cAMP. Interestingly, the Epac specific cAMP analogue 8-(4-chlorophenylthio)-2'-O-methyl-cAMP potentiated insulin-stimulated PKB phosphorylation in a similar manner as adrenaline did without activating glycogen phosphorylase. Inhibition of PKA by H89 decreased adrenaline-stimulated glycogen phosphorylase activation but increased PKB activation, which further supports that adrenaline increases insulin-stimulated PKB phosphorylation via Epac. Further, while adrenaline and the Epac activator alone did not promote p70(S6K) Thr389 phosphorylation, they potentiated insulin effects. In conclusion, adrenaline potentiates insulin-stimulated activation of PKB and p70(S6K) via cAMP and Epac in skeletal muscle. Furthermore, the fact that adrenaline alone did not activate PKB or p70(S6K) suggests that a hormone can be a potent regulator of signalling despite no effects being seen when co-activators are lacking.     

IGF-I elicits growth of human intestinal smooth muscle cells by activation of PI3K,PDK-1, and p70S6 kinase

Endogenous IGF-I regulates growth of human intestinal smooth muscle cells by jointly activating phosphatidylinositol 3-kinase (PI3K) and ERK1/2. The 70-kDa ribosomal S6 kinase (p70S6 kinase) is a key regulator of cell growth activated by several independently regulated kinases. The present study characterized the role of p70S6 kinase in IGF-I-induced growth of human intestinal smooth muscle cells and identified the mechanisms of p70S6 kinase activation. IGF-I-induced growth elicited via either the PI3K or ERK1/2 pathway required activation of p70S6 kinase. IGF-I elicited concentration-dependent activation of PI3K, 3-phosphoinositide-dependent kinase-1 (PDK-1), and p70S6 kinase that was sequential and followed similar time courses. IGF-I caused time-dependent and concentration-dependent phosphorylation of p70S6 kinase on Thr(421)/Ser(424), Thr(389), and Thr(229) that paralleled p70S6 kinase activation. p70S6 kinase(Thr(421)/Ser(424)) phosphorylation was PI3K dependent and PDK-1 independent, whereas p70S6 kinase(Thr(389)) and p70S6 kinase(Thr(229)) phosphorylation and p70S6 kinase activation were PI3K dependent and PDK-1 dependent. IGF-I elicited sequential Akt(Ser(308)), Akt(Ser(473)), and mammalian target of rapamycin(Ser(2448)) phosphorylation; however, transfection of muscle cells with kinase-inactive Akt1(K179M) showed that these events were not required for IGF-I to activate p70S6 kinase and stimulate proliferation of human intestinal muscle cells.     

Differential activation of protein kinase B and p70(S6)K by glucose and insulin-like growth factor 1 in pancreatic beta-cells (INS-1)

It has been shown that IGF-1-induced pancreatic beta-cell proliferation is glucose-dependent; however, the mechanisms responsible for this glucose dependence are not known. Adenoviral mediated expression of constitutively active phosphatidylinositol 3-kinase (PI3K) in the pancreatic beta-cells, INS-1, suggested that PI3K was not necessary for glucose-induced beta-cell proliferation but was required for IGF-1-induced mitogenesis. Examination of the signaling components downstream of PI3K, 3-phosphoinositide-dependent kinase 1, protein kinase B (PKB), glycogen synthase kinase-3, and p70-kDa-S6-kinase (p70(S6K)), suggested that a major part of glucose-dependent beta-cell proliferation requires activation of mammalian target of rapamycin/p70(S6K), independent of phosphoinositide-dependent kinase 1/PKB activation. Adenoviral expression of the kinase-dead form of PKB in INS-1 cells decreased IGF-1-induced beta-cell proliferation. However, a surprisingly similar decrease was also observed in adenoviral wild type and constitutively active PKB-infected cells. Upon analysis of extracellular signal-regulated protein kinase 1 and 2 (ERK1/ERK2), an increase in ERK1/ERK2 phosphorylation activation by glucose and IGF-1 was observed in kinase-dead PKB-infected cells, but this phosphorylation activation was inhibited in the constitutively active PKB-infected cells. Hence, there is a requirement for the activation of both ERK1/ERK2 and mammalian target of rapamycin/p70(S6K) signal transduction pathways for a full commitment to glucose-induced pancreatic beta-cell mitogenesis. However, for IGF-1-induced activation, these pathways must be carefully balanced, because chronic activation of one (PI3K/PKB) can lead to dampening of the other (ERK1/2), reducing the mitogenic response.     

Dual regulation of platelet protein kinase B

Protein kinase B (PKB) is a serine/threonine kinase that is activated by growth hormones and implicated in prevention of apoptosis, glycogen metabolism, and glucose uptake. A key enzyme in PKB activation is phosphatidylinositide 3-kinase (PI-3K), which triggers the dual phosphorylation of PKB by phosphatidylinositol-dependent kinases (PDKs). Here we report that the major PKB subtype in platelets is PKBalpha, which is activated by phosphorylation of Thr(308) and Ser(473) and has a constitutively phosphorylated Thr(450) that does not contribute to PKB activation. alpha-Thrombin and thrombopoietin activate PKBalpha via PI-3K and trigger the concurrent phosphorylation of Thr(308) (via PDK1) and Ser(473) (via a not yet identified PDK2). In addition, alpha-thrombin activates a PI-3K-independent pathway involving phospholipase Cbeta and calcium-dependent protein kinase C subtypes (PKCalpha/beta). This route is specific for phosphorylation of Ser(473) and can be initiated by direct PKC activation with phorbol ester or purified active PKC catalytic fragment in platelet lysate. Different degrees of Ser(473) and Thr(308) phosphorylation correlate with different degrees of enzyme activity. These data reveal a PI-3K-independent PKB activation in which PKCalpha/beta regulates the phosphorylation of Ser(473) in PKBalpha. The independent control of the two phosphorylation sites may contribute to fine regulation of PKBalpha activity.     

Multiple phosphoinositide 3-kinase-dependent steps in activation of protein kinase B

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.     

Thyroid hormone induces rapid activation of Akt/protein kinase B-mammalian target of rapamycin-p70S6K cascade through phosphatidylinositol 3-kinase in human fibroblasts

We have demonstrated that T3 increases the expression of ZAKI-4alpha, an endogenous calcineurin inhibitor. In this study we characterized a T3-dependent signaling cascade leading to ZAKI-4alpha expression in human skin fibroblasts. We found that T3-dependent increase in ZAKI-4alpha was greatly attenuated by rapamycin, a specific inhibitor of a protein kinase, mammalian target of rapamycin (mTOR), suggesting the requirement of mTOR activation by T3. Indeed, T3 activated mTOR rapidly through S2448 phosphorylation, leading to the phosphorylation of p70(S6K), a substrate of mTOR. This mTOR activation is mediated through phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) signaling cascade because T3 induced Akt/PKB phosphorylation more rapidly than that of mTOR, and these T3-dependent phosphorylations were blocked by both PI3K inhibitors and by expression of a dominant negative PI3K (Deltap85alpha). Furthermore, the association between thyroid hormone receptor beta1 (TRbeta1) and PI3K-regulatory subunit p85alpha, and the inhibition of T3-induced PI3K activation and mTOR phosphorylation by a dominant negative TR (G345R) demonstrated the involvement of TR in this T3 action. The liganded TR induces the activation of PI3K and Akt/PKB, leading to the nuclear translocation of the latter, which subsequently phosphorylates nuclear mTOR. The rapid activation of PI3K-Akt/PKB-mTOR-p70(S6K) cascade by T3 provides a new molecular mechanism for thyroid hormone action.     

Ceramide inhibits protein kinase B/Akt by promoting dephosphorylation of serine 473

The second messenger ceramide (N-alkylsphingosine) has been implicated in a host of cellular processes including growth arrest and apoptosis. Ceramide has been reported to have effects on both protein kinases and phosphatases and may constitute an important component of stress response in various tissues. We have examined in detail the relationship between ceramide signaling and the activation of an important signaling pathway, phosphatidylinositol (PI) 3-kinase and its downstream target, protein kinase B (PKB). PKB activation was observed following stimulation of cells with the cytokine granulocyte-macrophage colony-stimulating factor. Addition of cell-permeable ceramide analogs, C(2)- or C(6)-ceramide, caused a partial loss (50-60%) of PKB activation. This reduction was not a result of decreased PI(3,4,5)P(3) or PI(3,4)P(2) generation by PI 3-kinase. Two residues of PKB (threonine 308 and serine 473) require phosphorylation for maximal PKB activation. Serine 473 phosphorylation was consistently reduced by treatment with ceramide, whereas threonine 308 phosphorylation remained unaffected. In further experiments, ceramide appeared to accelerate serine 473 dephosphorylation, suggesting the activation of a phosphatase. Consistent with this, the reduction in serine 473 phosphorylation was inhibited by the phosphatase inhibitors okadaic acid and calyculin A. Surprisingly, threonine 308 phosphorylation was abolished in cells treated with these inhibitors, revealing a novel mechanism of regulation of threonine 308 phosphorylation. These results demonstrate that PI 3-kinase-dependent kinase 2-catalyzed phosphorylation of serine 473 is the principal target of a ceramide-activated phosphatase.     

Inhibition of PDK-1 activity causes a reduction in cell proliferation and survival

3-Phosphoinositide-dependent kinase-1 (PDK-1) was identified by its ability to phosphorylate and activate protein kinase B (PKB) in vitro [1,2] and can phosphorylate and activate additional protein kinases in the AGC family in vitro [3-6]. Its role in vivo has, however, only begun to be addressed. We used antisense oligonucleotides directed against PDK-1 expression to explore the role of PDK-1 in human glioblastoma cells (U87-MG), which express a mutant PTEN allele. Reduction in PDK-1 levels resulted in inhibition of PKB activity, and a reduction in phosphorylation on Thr308 and Ser473 of PKB. p70 S6 kinase (p70(S6K)) activity was also reduced. Cell proliferation was dramatically inhibited following treatment with PDK-1 antisense oligonucleotides, due to a combination of decreased cell doubling and an increase in apoptosis. This is in contrast to direct inhibition of phosphoinositide 3-OH kinase (PI 3-kinase), which results in G1 arrest with no effect on apoptosis. This study confirms both PKB and p70(S6K) as in vivo substrates for PDK-1. The effect of acute PDK-1 loss on cell proliferation and survival suggests the involvement of PI 3-kinase dependent and independent signaling events, and implicates PDK-1 as a potential therapeutic target for human neoplasms.     

Domain Swapping Used To Investigate the Mechanism of Protein Kinase B Regulation by 3-Phosphoinositide-Dependent Protein Kinase 1 and Ser473 Kinase

Protein kinase B (PKB or Akt), a downstream effector of phosphoinositide 3-kinase (PI 3-kinase), has been implicated in insulin signaling and cell survival. PKB is regulated by phosphorylation on Thr308 by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and on Ser473 by an unidentified kinase. We have used chimeric molecules of PKB to define different steps in the activation mechanism. A chimera which allows inducible membrane translocation by lipid second messengers that activate in vivo protein kinase C and not PKB was created. Following membrane attachment, the PKB fusion protein was rapidly activated and phosphorylated at the two key regulatory sites, Ser473 and Thr308, in the absence of further cell stimulation. This finding indicated that both PDK1 and the Ser473 kinase may be localized at the membrane of unstimulated cells, which was confirmed for PDK1 by immunofluorescence studies. Significantly, PI 3-kinase inhibitors prevent the phosphorylation of both regulatory sites of the membrane-targeted PKB chimera. Furthermore, we show that PKB activated at the membrane was rapidly dephosphorylated following inhibition of PI 3-kinase, with Ser473 being a better substrate for protein phosphatase. Overall, the results demonstrate that PKB is stringently regulated by signaling pathways that control both phosphorylation/activation and dephosphorylation/inactivation of this pivotal protein kinase.     

Akt2, a novel functional link between p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in myogenesis

Activation of either the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt or the p38 mitogen-activated protein kinase (MAPK) signaling pathways accelerates myogenesis but only when the reciprocal pathway is functional. We therefore examined the hypothesis that cross-activation between these signaling cascades occurs to orchestrate myogenesis. We reveal a novel and reciprocal cross-talk and activation between the PI 3-kinase/Akt and p38 MAPK pathways that is essential for efficient myoblast differentiation. During myoblast differentiation, Akt kinase activity correlated with S473 but not T308 phosphorylation and occurred 24 h after p38 activation. Inhibition or activation of p38 with SB203580, dominant-negative p38, or MKK6EE regulated Akt kinase activity. Analysis of Akt isoforms revealed a specific increase in Akt2 protein levels that coincided with AktS473 phosphorylation during myogenesis and an enrichment of S473-phosphorylated Akt2. Akt2 promoter activity and protein levels were regulated by p38 activation, thus providing a mechanism for communication. Subsequent Akt activation by S473 phosphorylation was PI 3-kinase dependent and specific for Akt2 rather than Akt1. Complementary to p38-mediated transactivation of Akt, activation or inhibition of PI 3-kinase regulated p38 activity upstream of MKK6, demonstrating reciprocal communication and positive feedback characteristic of myogenic regulation. Our findings have identified novel communication between p38 MAPK and PI 3-kinase/Akt via Akt2.     

The putative morphogen, DIF-1, of Dictyostelium discoideum activates Akt/PKB in human leukemia K562 cells.

The differentiation-inducing factor-1 (DIF-1) is a putative morphogen that induces stalk-cell formation in the lower eukaryote Dictyostelium discoideum. This molecule has been shown to inhibit cell growth and induce erythroid differentiation in human leukemia K562 cells. In the present study, to clarify the mechanism of the actions of DIF-1, we examined the effect of DIF-1 on Akt/protein kinase B (PKB) in K562 cells. Akt/PKB is a serine/threonine kinase that plays a pivotal role in the regulation of cell survival and differentiation in a variety of cells. A nonphosphorylated (inactive) form of Akt/PKB was ordinarily expressed in K562 cells. However, Akt/PKB was phosphorylated and potently activated within several hours of incubation with 5-30 microM DIF-1, and this activation was inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase). Calcium-increasing agents thapsigargin and A23187 also activated Akt/PKB slightly, which was inhibited by wortmannin. By contrast, calcium-reducing agents TMB-8 and EGTA together with A23187 inhibited the DIF-1-induced activation of Akt/PKB. PMA (PKC activator) also activated Akt/PKB but this activation was not inhibited by wortmannin. DIF-1 exhibited no marked effect on the activation of PKCalpha, beta, and gamma, which were activated by PMA. These results indicate that DIF-1 activates Akt/PKB possibly via cytosolic calcium and subsequent activation of PI3-kinase and also that PMA activates Akt/PKB in a PI3-kinase-independent manner.     

Mechanism of protein kinase B activation by cyclic AMP-dependent protein kinase.

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.     

Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway

The S/T-protein kinases activated by phosphoinositide 3-kinase (PI3K) regulate a myriad of cellular processes. Here, we show that an approach using a combination of biochemistry and bioinformatics can identify substrates of these kinases. This approach identifies the tuberous sclerosis complex-2 gene product, tuberin, as a potential target of Akt/PKB. We demonstrate that, upon activation of PI3K, tuberin is phosphorylated on consensus recognition sites for PI3K-dependent S/T kinases. Moreover, Akt/PKB can phosphorylate tuberin in vitro and in vivo. We also show that S939 and T1462 of tuberin are PI3K-regulated phosphorylation sites and that T1462 is constitutively phosphorylated in PTEN(-/-) tumor-derived cell lines. Finally, we find that a tuberin mutant lacking the major PI3K-dependent phosphorylation sites can block the activation of S6K1, suggesting a means by which the PI3K-Akt pathway regulates S6K1 activity.     

Stimulation of glycogen synthesis by insulin requires S6 kinase and phosphatidylinositol-3-kinase in HTC-IR cells

In order to study the role of phosphatidylinositol-3-kinase (PI3K), PKB, FRAP, S6 kinase, and MAP kinase in insulin-stimulated glycogen synthesis, we used a specific inhibitor of PI3K, LY294002, the immunosuppressant inhibitor of FRAP, rapamycin, and the inhibitor of MAPK kinase (MEK)/MAPK, PD98059, in rat HTC hepatoma cells overexpressing human insulin receptors. The PI3K inhibitor LY294002 completely blocks insulin-stimulated glycogen synthesis by inhibiting glycogen synthase, PKB (Akt-1), and FRAP (RAFT) autophosphorylation, as well as p70 S6 kinase activation, whereas insulin receptor substrates tyrosine phosphorylation and MEK activity were not affected. However, rapamycin only partially blocks insulin-stimulated glycogen synthesis by partial inhibition of glycogen synthase, whereas it completely blocks S6 kinase activation and FRAP autophosphorylation, but does not affect either PKB autophosphorylation, MEK activity, or insulin receptor tyrosine phosphorylation. Insulin-stimulated glycogen synthesis and glycogen synthase were not affected by the MEK/MAPK inhibitor PD98059. These data suggest that the PI3K, and not the MAPK pathway plays an important role in the insulin-stimulated glycogen synthesis in the hepatocyte, partly mediated by FRAP and S6 kinase activation. However, the inhibition of FRAP and S6 kinase activation is not sufficient to block insulin-stimulated glycogen synthesis, suggesting an important role of a branching pathway upstream of S6 kinase and downstream of PI3K, which is probably mediated by PKB in the signaling of the insulin receptor in hepatoma HTC cells.     

Protein Kinase B/Akt Participates in GLUT4 Translocation by Insulin in L6 Myoblasts

L6 myoblasts stably transfected with a GLUT4 cDNA harboring an exofacial myc epitope tag (L6-GLUT4myc myoblasts) were used to study the role of protein kinase B alpha (PKBalpha)/Akt1 in the insulin-induced translocation of GLUT4 to the cell surface. Surface GLUT4myc was detected by immunofluorescent labeling of the myc epitope in nonpermeabilized cells. Insulin induced a marked translocation of GLUT4myc to the plasma membrane within 20 min. This was prevented by transient transfection of a dominant inhibitory construct of phosphatidylinositol (PI) 3-kinase (Deltap85alpha). Transiently transfected cells were identified by cotransfection of green fluorescent protein. A constitutively active PKBalpha, created by fusion of a viral Gag protein at its N terminus (GagPKB), increased the cell surface density of GLUT4myc compared to that of neighboring nontransfected cells. A kinase-inactive, phosphorylation-deficient PKBalpha/Akt1 construct with the mutations K179A (substitution of alanine for the lysine at position 179), T308A, and S473A (AAA-PKB) behaved as a dominant-negative inhibitor of insulin-dependent activation of cotransfected wild-type hemagglutinin (HA)-tagged PKB. Furthermore, AAA-PKB markedly inhibited the insulin-induced phosphorylation of cotransfected BAD, demonstrating inhibition of the endogenous PKB/Akt. Under the same conditions, AAA-PKB almost entirely blocked the insulin-dependent increase in surface GLUT4myc. PKBalpha with alanine substitutions T308A and S473A (AA-PKB) or K179A (A-PKB) alone was a less potent inhibitor of insulin-dependent activation of wild-type HA-PKB or GLUT4myc translocation than was AAA-PKB. Cotransfection of AAA-PKB with a fourfold DNA excess of HA-PKB rescued insulin-stimulated GLUT4myc translocation. AAA-PKB did not prevent actin bundling (membrane ruffling), though this response was PI 3-kinase dependent. Therefore, it is unlikely that AAA-PKB acted by inhibiting PI 3-kinase signaling. These results outline an important role for PKBalpha/Akt1 in the stimulation of glucose transport by insulin in muscle cells in culture.     

Intracellular signaling of angiotensin II-induced p70 S6 kinase phosphorylation at Ser(411) in vascular smooth muscle cells. Possible requirement of epidermal growth factor receptor, Ras, extracellular signal-regulated kinase, and Akt

Activation of p70 S6 kinase (p70(S6K)) by growth factors requires multiple signal inputs involving phosphoinositide 3-kinase (PI3K), its effector Akt, and an unidentified kinase that phosphorylates Ser/Thr residues (Ser(411), Ser(418), Ser(424), and Thr(421)) clustered at its autoinhibitory domain. However, the mechanism by which G protein-coupled receptors activate p70(S6K) remains largely uncertain. By using vascular smooth muscle cells in which we have demonstrated Ras/extracellular signal-regulated kinase (ERK) activation through Ca(2+)-dependent, epidermal growth factor (EGF) receptor transactivation by G(q)-coupled angiotensin II (Ang II) receptor, we present a unique cross-talk required for Ser(411) phosphorylation of p70(S6K) by Ang II. Both p70(S6K) Ser(411) and Akt Ser(473) phosphorylation by Ang II appear to involve EGF receptor transactivation and were inhibited by dominant-negative Ras, whereas the phosphorylation of p70(S6K) and ERK but not Akt was sensitive to the MEK inhibitor. By contrast, the phosphorylation of p70(S6K) and Akt but not ERK was sensitive to PI3K inhibitors. Similar inhibitory pattern on these phosphorylation sites by EGF but not insulin was observed. Taken together with the inhibition of Ang II-induced p70(S6K) activation by dominant-negative Ras and the MEK inhibitor, we conclude that Ang II-initiated activation of p70(S6K) requires both ERK cascade and PI3K/Akt cascade that bifurcate at the point of EGF receptor-dependent Ras activation.     

PI(3,4,5)P3 and PI(3,4)P2 levels correlate with PKB/akt phosphorylation at Thr308 and Ser473, respectively; PI(3,4)P2 levels determine PKB activity

The PI3K-PKB pathway is an important and widely studied pathway in cell signaling. The enzyme activity of PI3K produces D-3 phosphoinositides, including the lipid second messengers PI(3,4,5)P3 and PI(3,4)P2. PI(3,4,5)P3 has been deemed to be the most important second messenger for triggering PKB phosphorylation. PKB has two regulatory phosphorylation sites, Thr308 and Ser473, both of which contribute to its full activity. The direct relationship between PI3K lipid products and PKB phosphorylation is still not entirely clear. Our previous study showed that PI(3,4)P2 has a specific role in contributing to PKB phosphorylation on Ser473 sites in mast cells. In this study, we used two strategies to further elucidate this question in a well-established B cell system. First, by SHIP overexpression, we examined PKB activation under conditions where PI(3,4,5)P3 accumulation is largely suppressed. Second, we used dose response of different forms of B-cell receptor ligands to manipulate the relative levels of PI(3,4,5)P3 and PI(3,4)P2. Our results demonstrate a close relationship between PI(3,4,5)P3 levels and Thr308 phosphorylation levels, and PI(3,4)P2 levels and Ser473 phosphorylation levels, respectively. Furthermore, overall PKB activity, primarily consisting of cytosolic enzyme, was dependent upon levels of PI(3,4)P2, while only membrane-associated PKB activity was dependent upon PI(3,4,5)P3 levels. We conclude that PI(3,4,5)P3 and PI(3,4)P2 have distinct roles in determining PKB phosphorylation and activity. Thus, when investigating PI3K-PKB pathways, the importance of both lipids must be considered.     

PKCalpha reduces the lipid kinase activity of the p110alpha/p85alpha PI3K through the phosphorylation of the catalytic subunit

The modulation of phosphoinositide 3-kinase (PI3K) activity influences the quality of cellular responses triggered by various receptor tyrosine kinases. Protein kinase C (PKC) has been reported to phosphorylate signalling molecules upstream of PI3K and thereby it may affect the activation of PI3K. Here, we provide the first evidence for a direct effect of a PKC isoenzyme on the activity of PI3K. PKCalpha but not PKCepsilon phosphorylated the catalytic subunit of the p110alpha/p85alpha PI3K in vitro in a manner inhibited by the PKC inhibitor bisindolylmaleimide I (BIM I). The incubation of PI3K with active PKCalpha resulted in a significant decrease in its lipid kinase activity and this effect was also attenuated by BIM I. We conclude that PKCalpha is able to modulate negatively the lipid kinase activity of the p110alpha/p85alpha PI3K through the phosphorylation of the catalytic subunit.     

Activation of phosphoinositide 3-kinase/PKB pathway by CB(1) and CB(2) cannabinoid receptors expressed in prostate PC-3 cells. Involvement in Raf-1 stimulation and NGF induction

Cannabinoids exert a variety of physiological and pharmacological responses in humans through interaction with specific cannabinoid receptors. Cannabinoid receptors described to date belong to the seven-transmembrane-domain receptor superfamily and are coupled through the inhibitory G(i) protein to adenylyl cyclase inhibition. However, downstream signal transduction mechanisms triggered by cannabinoids are poorly understood. We examined here the involvement of the phosphoinositide 3-kinase (PI3K)/PKB pathway in the mechanism of action of cannabinoids in human prostate epithelial PC-3 cells. Cannabinoid receptors CB(1) and CB(2) are expressed in these cells, as shown by RT-PCR, Western blot and immunofluorescence techniques. Treatment of PC-3 cells with either Delta(9)-tetrahydrocannabinol (THC), the major psychoactive ingredient of marijuana, or R-(+)-methanandamide (MET), an analogue of the endogenous cannabinoid anandamide, increased phosphorylation of PKB in Thr308 and Ser473. The stimulation of PKB induced by cannabinoids was blocked by the two cannabinoid receptor antagonists, SR 141716 and SR 144528, and by the PI3K inhibitor LY 294002. These results indicate that activation of cannabinoid receptors in PC-3 cells stimulate the PI3K/PKB pathway. We further investigated the involvement of Raf-1/Erk activation in the mechanism of action of cannabinoid receptors. THC and MET induced translocation of Raf-1 to the membrane and phosphorylation of p44/42 Erk kinase, which was reversed by cannabinoid receptor antagonists and PI3K inhibitor. These results point to a sequential connection between cannabinoid receptors/PI3K/PKB pathway and Raf-1/Erk in prostate PC-3 cells. We also show that this pathway is involved in the mechanism of NGF induction exerted by cannabinoids in PC-3 cells.