Conditional knock-out of integrin-linked kinase demonstrates an essential role in protein kinase B/Akt activation

Protein kinase B (PKB/Akt) plays a pivotal role in signaling pathways downstream of phosphatidylinositol 3-kinase, regulating fundamental processes such as cell survival, cell proliferation, differentiation, and metabolism. PKB/Akt activation is regulated by phosphoinositide phospholipid-mediated plasma membrane anchoring and by phosphorylation on Thr-308 and Ser-473. Whereas the Thr-308 site is phosphorylated by PDK-1, the identity of the Ser-473 kinase has remained unclear and controversial. The integrin-linked kinase (ILK) is a potential regulator of phosphorylation of PKB/Akt on Ser-473. Utilizing double-stranded RNA interference (siRNA) as well as conditional knock-out of ILK using the Cre-Lox system, we now demonstrate that ILK is essential for the regulation of PKB/Akt activity. ILK knock-out had no effect on phosphorylation of PKB/Akt on Thr-308 but resulted in almost complete inhibition of phosphorylation on Ser-473 and significant inhibition of PKB/Akt activity, accompanied by significant stimulation of apoptosis. The inhibition of PKB/Akt Ser-473 phosphorylation was rescued by kinase-active ILK but not by a kinase-deficient mutant of ILK, suggesting a role for the kinase activity of ILK in the stimulation of PKB/Akt phosphorylation. ILK knock-out also resulted in the suppression of phosphorylation of GSK-3beta on Ser-9 and cyclin D1 expression. These data establish ILK as an essential upstream regulator of PKB/Akt activation.     

Identification of a plasma membrane Raft-associated PKB Ser473 kinase activity that is distinct from ILK and PDK1

Protein kinase B (PKB/Akt) has been well established as an important signaling intermediate, and its deregulation has been implicated in the development of human cancer and diabetes (reviewed in). Full activation of PKB requires phosphorylation on residues Thr308 and Ser473. While the Thr308 kinase, named 3-phosphoinositide-dependent kinase-1 (PDK1), has been extensively characterized (reviewed in ), the identity of the Ser473 kinase remains unclear. We have focused our study on the plasma membrane (PM) fraction because membrane localization is sufficient to activate PKB, and this suggests that PKB upstream kinases are constitutively active at the membrane. Here, we report the identification of a constitutively active PKB Ser473 kinase activity enriched in buoyant, detergent-insoluble plasma membrane rafts that are distinct from the cytosolic distribution of PKB and PDK1. This Ser473 kinase activity was released from the membrane by high salt, and gel filtration analysis showed that the kinase responsible is present in a large complex of >500 kDa. Two major phosphoproteins and integrin-linked kinase (ILK) were detected in partially purified PKB Ser473 kinase preparations. In contrast to previous observations, however, ILK immunoprecipitates did not retain Ser473 kinase activity. Thus, we have identified a novel raft-associated PKB Ser473 kinase, implicating a role for lipid rafts in PKB signaling.     

Caffeine modulates phosphorylation of insulin receptor substrate-1 and impairs insulin signal transduction in rat skeletal muscle

Caffeine decreases insulin sensitivity and insulin-stimulated glucose transport in skeletal muscle; however, the precise mechanism responsible for this deleterious effect is not understood fully. We investigated the effects of incubation with caffeine on insulin signaling in rat epitrochlearis muscle. Caffeine (≥1 mM, ≥15 min) suppressed insulin-stimulated insulin receptor substrate (IRS)-1 Tyr(612) phosphorylation in a dose- and time-dependent manner. These responses were associated with inhibition of the insulin-stimulated phosphorylation of phosphatidylinositol 3-kinase (PI3K) Tyr(458), Akt Ser(473), and glycogen synthase kinase-3β Ser(9) and with inhibition of insulin-stimulated 3-O-methyl-d-glucose (3MG) transport but not with inhibition of the phosphorylation of insulin receptor-β Tyr(1158/62/63). Furthermore, caffeine enhanced phosphorylation of IRS-1 Ser(307) and an IRS-1 Ser(307) kinase, inhibitor-κB kinase (IKK)-α/β Ser(176/180). Blockade of IKK/IRS-1 Ser(307) by caffeic acid ameliorated the caffeine-induced downregulation of IRS-1 Tyr(612) phosphorylation and 3MG transport. Caffeine also increased the phosphorylation of IRS-1 Ser(789) and an IRS-1 Ser(789) kinase, 5'-AMP-activated protein kinase (AMPK). However, inhibition of IRS-1 Ser(789) and AMPK phosphorylation by dantrolene did not rescue the caffeine-induced downregulation of IRS-1 Tyr(612) phosphorylation or 3MG transport. In addition, caffeine suppressed the phosphorylation of insulin-stimulated IRS-1 Ser(636/639) and upstream kinases, including the mammalian target of rapamycin and p70S6 kinase. Intravenous injection of caffeine at a physiological dose (5 mg/kg) in rats inhibited the phosphorylation of insulin-stimulated IRS-1 Tyr(612) and Akt Ser(473) in epitrochlearis muscle. Our results indicate that caffeine inhibits insulin signaling partly through the IKK/IRS-1 Ser(307) pathway, via a Ca(2+)- and AMPK-independent mechanism in skeletal muscle.     

Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase

Full activation of protein kinase B (PKB)/Akt requires phosphorylation on Thr-308 and Ser-473 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 kinase (S473K), respectively. Although PDK1 has been well characterized, the identification of the S473K remains controversial. A major PKB Ser-473 kinase activity was purified from the membrane fraction of HEK293 cells and found to be DNA-dependent protein kinase (DNA-PK). DNA-PK co-localized and associated with PKB at the plasma membrane. In vitro, DNA-PK phosphorylated PKB on Ser-473, resulting in a approximately 10-fold enhancement of PKB activity. Knockdown of DNA-PK by small interfering RNA inhibited Ser-473 phosphorylation induced by insulin and pervanadate. DNA-PK-deficient glioblastoma cells did not respond to insulin at the level of Ser-473 phosphorylation; this effect was restored by complementation with the human PRKDC gene. We conclude that DNA-PK is a long sought after kinase responsible for the Ser-473 phosphorylation step in the activation of PKB.     

Integrin-linked kinase is a positive mediator of L6 myoblast differentiation

Overexpression of ILK in L6 myoblasts results in increased ILK kinase activity, stimulating myotube formation and induction of biochemical differentiation markers. Expression of a dominant negative ILK mutant, ILK(E359K), inhibits endogenous ILK activation and L6 differentiation. Cell cycle analysis of ILK(E359K) cells cultured in serum-free conditions indicates significant apoptosis (11-19% sub-diploid peak) which is not seen in insulin treated cells. Expression of ILK variants does not have significant effects on S-phase transit, however. Known targets of ILK, PKB/Akt or glycogen synthase kinase 3beta are not obviously involved in ILK-induced L6 differentiation. Insulin-stimulated phosphorylation of PKB at Ser473 is unimpaired in the ILK(E359K) cells, suggesting that PKB is not a myogenic target of ILK. Inhibition of GSK3beta by LiCl blocks L6 myogenesis, indicating that ILK-mediated inhibition of GSK3beta is not sufficient for differentiation. Our data do suggest that a LiCl-sensitive interaction of ILK is important in L6 myoblast differentiation.     

Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343

Protein kinase B (PKB/Akt) is a regulator of cell survival and apoptosis. To become fully activated, PKB/Akt requires phosphorylation at two sites, threonine 308 and serine 473, in a phosphatidylinositol (PI) 3-kinase-dependent manner. The kinase responsible for phosphorylation of threonine 308 is the PI 3-kinase-dependent kinase-1 (PDK-1), whereas phosphorylation of serine 473 has been suggested to be regulated by PKB/Akt autophosphorylation in a PDK-1-dependent manner. However, the integrin-linked kinase (ILK) has also been shown to regulate phosphorylation of serine 473 in a PI 3-kinase-dependent manner. Whether ILK phosphorylates this site directly or functions as an adapter molecule has been debated. We now show by in-gel kinase assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry that biochemically purified ILK can phosphorylate PKB/Akt directly. Co-immunoprecipitation analysis of cell extracts demonstrates that ILK can complex with PKB/Akt as well as PDK-1 and that ILK can disrupt PDK-1/PKB association. The amino acid residue serine 343 of ILK within the activation loop is required for kinase activity as well as for its interaction with PKB/Akt. Mutational analysis of ILK further shows a crucial role for arginine 211 of ILK within the phosphoinositide phospholipid binding domain in the regulation of PKB- serine 473 phosphorylation. A highly selective small molecule inhibitor of ILK activity also inhibits the ability of ILK to phosphorylate PKB/Akt in vitro and in intact cells. These data demonstrate that ILK is an important upstream kinase for the regulation of PKB/Akt.     

Integrin-linked kinase stabilizes myotendinous junctions and protects muscle from stress-induced damage

Skeletal muscle expresses high levels of integrin-linked kinase (ILK), predominantly at myotendinous junctions (MTJs) and costameres. ILK binds the cytoplasmic domain of beta1 integrin and mediates phosphorylation of protein kinase B (PKB)/Akt, which in turn plays a central role during skeletal muscle regeneration. We show that mice with a skeletal muscle-restricted deletion of ILK develop a mild progressive muscular dystrophy mainly restricted to the MTJs with detachment of basement membranes and accumulation of extracellular matrix. Endurance exercise training enhances the defects at MTJs, leads to disturbed subsarcolemmal myofiber architecture, and abrogates phosphorylation of Ser473 as well as phosphorylation of Thr308 of PKB/Akt. The reduction in PKB/Akt activation is accompanied by an impaired insulin-like growth factor 1 receptor (IGF-1R) activation. Coimmunoprecipitation experiments reveal that the beta1 integrin subunit is associated with the IGF-1R in muscle cells. Our data identify the beta1 integrin-ILK complex as an important component of IGF-1R/insulin receptor substrate signaling to PKB/Akt during mechanical stress in skeletal muscle.     

Protein kinase B is regulated in platelets by the collagen receptor glycoprotein VI

Phosphoinositide 3-kinase (PI3K) is a critical component of the signaling pathways that control the activation of platelets. Here we have examined the regulation of protein kinase B (PKB), a downstream effector of PI3K, by the platelet collagen receptor glycoprotein (GP) VI and thrombin receptors. Stimulation of platelets with collagen or convulxin (a selective GPVI agonist) resulted in PI3K-dependent, and aggregation independent, Ser(473) and Thr(308) phosphorylation of PKBalpha, which results in PKB activation. This was accompanied by translocation of PKB to cell membranes. The phosphoinositide-dependent kinase PDK1 is known to phosphorylate PKBalpha on Thr(308), although the identity of the kinase responsible for Ser(473) phosphorylation is less clear. One candidate that has been implicated as being responsible for Ser(473) phosphorylation, either directly or indirectly, is the integrin-linked kinase (ILK). In this study we have examined the interactions of PKB, PDK1, and ILK in resting and stimulated platelets. We demonstrate that in platelets PKB is physically associated with PDK1 and ILK. Furthermore, the association of PDK1 and ILK increases upon platelet stimulation. It would therefore appear that formation of a tertiary complex between PDK1, ILK, and PKB may be necessary for phosphorylation of PKB. These observations indicate that PKB participates in cell signaling downstream of the platelet collagen receptor GPVI. The role of PKB in collagen- and thrombin-stimulated platelets remains to be determined.     

Modulation of integrin signal transduction by ILKAP, a protein phosphatase 2C associating with the integrin-linked kinase, ILK1

ILKAP, a protein serine/threonine (S/T) phosphatase of the PP2C family, was isolated in a yeast two-hybrid screen baited with integrin-linked kinase, ILK1. Association of ILK1 and ILKAP was independent of the catalytic activity of either partner, as assayed in co-precipitation and two-hybrid experiments. Condi tional expression of ILKAP in HEK 293 cells resulted in selective inhibition of ECM- and growth factor-stimulated ILK1 activity, but did not inhibit Raf-1 kinase activity. A catalytic mutant of ILKAP, H154D, did not inhibit ILK1 kinase activity. Two cellular targets of ILK1, glycogen synthase kinase 3 beta (GSK3beta) and protein kinase B (PKB)/AKT, were differentially affected by ILKAP-mediated inhibition of ILK1. Catalytically active, but not mutant ILKAP, strongly inhibited insulin-like growth factor-1-stimulated GSK3beta phosphorylation on Ser9, but did not affect phosphorylation of PKB on Ser473, suggesting that ILKAP selectively affects ILK-mediated GSK3beta signalling. Consistent with this, active, but not H154D mutant or the related PP2Calpha, selectively inhibited transactivation of a Tcf/Lef reporter gene, TOPFlash, in 293 cells. We propose that ILKAP regulates ILK1 activity, targeting ILK1 signalling of Wnt pathway components via modulation of GSK3beta phosphorylation.     

G protein-coupled receptor kinase 2 mediates endothelin-1-induced insulin resistance via the inhibition of both Galphaq/11 and insulin receptor substrate-1 pathways in 3T3-L1 adipocytes

G protein-coupled receptor kinases (GRKs) regulate seven-transmembrane receptors (7TMRs) by phosphorylating agonist-activated 7TMRs. Recently, we have reported that GRK2 can function as a negative regulator of insulin action by interfering with G protein-q/11 alpha-subunit (Galphaq/11) signaling, causing decreased glucose transporter 4 (GLUT4) translocation. We have also reported that chronic endothelin-1 (ET-1) treatment leads to heterologous desensitization of insulin signaling with decreased tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and Galphaq/11, and decreased insulin-stimulated glucose transport in 3T3-L1 adipocytes. In the current study, we have investigated the role of GRK2 in chronic ET-1-induced insulin resistance. Insulin-induced GLUT4 translocation was inhibited by pretreatment with ET-1 for 24 h, and we found that this inhibitory effect was rescued by microinjection of anti-GRK2 antibody or GRK2 short interfering RNA. We further found that GRK2 mediates the inhibitory effects of ET-1 by two distinct mechanisms. Firstly, adenovirus-mediated overexpression of either wild-type (WT)- or kinase-deficient (KD)-GRK2 inhibited Galphaq/11 signaling, including tyrosine phosphorylation of Galphaq/11 and cdc42-associated phosphatidylinositol 3-kinase activity. Secondly, ET-1 treatment caused Ser/Thr phosphorylation of IRS-1 and IRS-1 protein degradation. Overexpression of KD-GRK2, but not WT-GRK2, inhibited ET-1-induced serine 612 phosphorylation of IRS-1 and restored activation of this pathway. Taken together, these results suggest that GRK2 mediates ET-1-induced insulin resistance by 1) inhibition of Galphaq/11 activation, and this effect is independent of GRK2 kinase activity, and 2) GRK2 kinase activity-mediated IRS-1 serine phosphorylation and degradation.     

PKB inhibition prevents the stimulatory effect of insulin on glucose transport and protein translocation but not the antilipolytic effect in rat adipocytes

We identified 1-(5 chloronaphthalenesulfonyl)-1H-hexahydro-1, 4-diazepine, also known as ML-9, as a powerful inhibitor of PKB activity in different cells as well as of recombinant PKB. It also inhibits other downstream serine/threonine kinases, such as PKA and p90 S6 kinase, but not upstream tyrosine phosphorylation or PI3-kinase activation in response to insulin. We compared the effects of ML-9 and wortmannin on several insulin-stimulated effects in isolated rat fat cells. Both ML-9 and wortmannin inhibited glucose transport and GLUT4/IGF II receptor translocation to the plasma membrane. In contrast, only wortmannin inhibited the antilipolytic effect and PDE3B activation by insulin. Thus, ML-9 inhibits PKB but not PI3-kinase activation in response to insulin and is useful to differentiate between these effects. Both PI3-kinase and PKB are important for glucose transport and intracellular protein translocation while PKB does not appear to play an important role for the antilipolytic effect or activation of PDE3B in response to insulin.     

Contraction activates glucose uptake and glycogen synthase normally in muscles from dexamethasone-treated rats

Glucocorticoids cause insulin resistance in skeletal muscle. The aims of the present study were to investigate the effects of contraction on glucose uptake, insulin signaling, and regulation of glycogen synthesis in skeletal muscles from rats treated with the glucocorticoid analog dexamethasone (1 mg x kg(-1) x day(-1) ip for 12 days). Insulin resistance in dexamethasone-treated rats was confirmed by reduced insulin-stimulated glucose uptake (approximately 35%), glycogen synthesis (approximately 70%), glycogen synthase activation (approximately 80%), and PKB Ser(473) phosphorylation (approximately 40%). Chronic dexamethasone treatment did not impair glucose uptake during contraction in soleus or epitrochlearis muscles. In epitrochlearis (but not in soleus), the presence of insulin during contraction enhanced glucose uptake to similar levels in control and dexamethasone-treated rats. Contraction also increased glycogen synthase fractional activity and dephosphorylated glycogen synthase at Ser(645), Ser(649), Ser(653), and Ser(657) normally in muscles from dexamethasone-treated rats. After contraction, insulin-stimulated glycogen synthesis was completely restored in epitrochlearis and improved in soleus from dexamethasone-treated rats. Contraction did not increase insulin-stimulated PKB Ser(473) or glycogen synthase kinase-3 (GSK-3) phosphorylation. Instead, contraction increased GSK-3beta Ser(9) phosphorylation in epitrochlearis (but not in soleus) in muscles from control and dexamethasone-treated rats. In conclusion, contraction stimulates glucose uptake normally in dexamethasone-induced insulin resistant muscles. After contraction, insulin's ability to stimulate glycogen synthesis was completely restored in epitrochlearis and improved in soleus from dexamethasone-treated rats.     

A novel PKB/Akt inhibitor, MK-2206, effectively inhibits insulin-stimulated glucose metabolism and protein synthesis in isolated rat skeletal muscle

PKB (protein kinase B), also known as Akt, is a key component of insulin signalling. Defects in PKB activation lead to insulin resistance and metabolic disorders, whereas PKB overactivation has been linked to tumour growth. Small-molecule PKB inhibitors have thus been developed for cancer treatment, but also represent useful tools to probe the roles of PKB in insulin action. In the present study, we examined the acute effects of two allosteric PKB inhibitors, MK-2206 and Akti 1/2 (Akti) on PKB signalling in incubated rat soleus muscles. We also assessed the effects of the compounds on insulin-stimulated glucose uptake, glycogen and protein synthesis. MK-2206 dose-dependently inhibited insulin-stimulated PKB phosphorylation, PKBβ activity and phosphorylation of PKB downstream targets (including glycogen synthase kinase-3α/β, proline-rich Akt substrate of 40?kDa and Akt substrate of 160?kDa). Insulin-stimulated glucose uptake, glycogen synthesis and glycogen synthase activity were also decreased by MK-2206?in a dose-dependent manner. Incubation with high doses of MK-2206 (10?μM) inhibited insulin-induced p70 ribosomal protein S6 kinase and 4E-BP1 (eukaryotic initiation factor 4E-binding protein-1) phosphorylation associated with increased eEF2 (eukaryotic elongation factor 2) phosphorylation. In contrast, Akti only modestly inhibited insulin-induced PKB and mTOR (mammalian target of rapamycin) signalling, with little or no effect on glucose uptake and protein synthesis. MK-2206, rather than Akti, would thus be the tool of choice for studying the role of PKB in insulin action in skeletal muscle. The results point to a key role for PKB in mediating insulin-stimulated glucose uptake, glycogen synthesis and protein synthesis in skeletal muscle.     

High glucose inhibits insulin-stimulated nitric oxide production without reducing endothelial nitric-oxide synthase Ser1177 phosphorylation in human aortic endothelial cells

Recent studies have indicated that insulin activates endothelial nitric-oxide synthase (eNOS) by protein kinase B (PKB)-mediated phosphorylation at Ser1177 in endothelial cells. Because hyperglycemia contributes to endothelial dysfunction and decreased NO availability in types 1 and 2 diabetes mellitus, we have studied the effects of high glucose (25 mM, 48 h) on insulin signaling pathways that regulate NO production in human aortic endothelial cells. High glucose inhibited insulin-stimulated NO synthesis but was without effect on NO synthesis stimulated by increasing intracellular Ca2+ concentration. This was accompanied by reduced expression of IRS-2 and attenuated insulin-stimulated recruitment of PI3K to IRS-1 and IRS-2, yet insulin-stimulated PKB activity and phosphorylation of eNOS at Ser1177 were unaffected. Inhibition of insulin-stimulated NO synthesis by high glucose was unaffected by an inhibitor of PKC. Furthermore, high glucose down-regulated the expression of CAP and Cbl, and insulin-stimulated Cbl phosphorylation, components of an insulin signaling cascade previously characterized in adipocytes. These data suggest that high glucose specifically inhibits insulin-stimulated NO synthesis and down-regulates some aspects of insulin signaling, including the CAP-Cbl signaling pathway, yet this is not a result of reduced PKB-mediated eNOS phosphorylation at Ser1177. Therefore, we propose that phosphorylation of eNOS at Ser1177 is not sufficient to stimulate NO production in cells cultured at 25 mM glucose.     

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.     

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.     

Muscle-specific deletion of rictor impairs insulin-stimulated glucose transport and enhances Basal glycogen synthase activity

Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity.     

The phosphorylation of Ser318 of insulin receptor substrate 1 is not per se inhibitory in skeletal muscle cells but is necessary to trigger the attenuation of the insulin-stimulated signal

The Ser/Thr phosphorylation of insulin receptor substrate 1 (IRS) is one key mechanism to stimulate and/or attenuate insulin signal transduction. Using a phospho-specific polyclonal antibody directed against phosphorylated Ser(318) of IRS-1, we found a rapid and transient insulin-stimulated phosphorylation of Ser(318) in human and rodent skeletal muscle cell models and in muscle tissue of insulin-treated mice. None of the investigated insulin resistance-associated factors (e.g. high glucose, tumor necrosis factor-alpha, adrenaline) stimulated the phosphorylation of Ser(318). Studying the function of this phosphorylation, we found that replacing Ser(318) by alanine completely prevented both the attenuation of insulin-stimulated Akt/protein kinase B Ser(473) phosphorylation and glucose uptake after 60 min of insulin stimulation. Unexpectedly, after acute insulin stimulation, we observed that phosphorylation of Ser(318) is not inhibitory but rather enhances insulin signal transduction because introduction of Ala(318) led to a reduction of the insulin-stimulated Akt/protein kinase B phosphorylation. Furthermore, replacing Ser(318) by glutamate, i.e. mimicking phosphorylation, improved glucose uptake after acute insulin stimulation. These data suggest that phosphorylation of Ser(318) is not per se inhibitory but is necessary to trigger the attenuation of the insulin-stimulated signal in skeletal muscle cells. Investigating the molecular mechanism of insulin-stimulated Ser(318) phosphorylation, we found that phosphatidylinositol 3-kinase-mediated activation of atypical protein kinase C-zeta and recruitment of protein kinase C-zeta to IRS-1 was responsible for this phosphorylation. We conclude that Ser(318) phosphorylation of IRS-1 is an early physiological event in insulin-stimulated signal transduction, which attenuates the continuing action of insulin.     

Differential regulation of NHE1 phosphorylation and glucose uptake by inhibitors of the ERK pathway and p90RSK in 3T3-L1 adipocytes

Insulin stimulates trafficking of GLUT4 to the cell surface for glucose uptake into target cells, and phosphorylation of Ser703 of the Na⁺/H⁺ exchanger NHE1, which activates proton efflux. The latter has been proposed to facilitate optimal glucose uptake into cardiomyocytes. We found that the insulin-stimulated phosphorylation of Ser703 of NHE1 is mediated by p90RSK but not directly coupled to glucose uptake in 3T3-L1 adipocytes in the short-term. Inhibiting Erk1/2 activation prevented NHE1 phosphorylation but not glucose uptake in 3T3-L1 adipocytes. In contrast, both NHE1 phosphorylation and insulin-stimulated uptake of glucose into 3T3-L1 adipocytes were blocked by inhibitors of the N-terminal kinase domain of p90RSK, namely BI-D1870 and SL0101, but not the FMK inhibitor of the C-terminal kinase domain of p90RSK, though in our hands FMK did not inhibit p90RSK in 3T3-L1 adipocytes. Further experiments were consistent with phosphorylation of AS160 by PKB/Akt mediating insulin-stimulated trafficking of GLUT4 to the plasma membrane. BI-D1870 and SL0101 however, inhibited glucose uptake without blocking GLUT4 translocation. While BI-D1870 partially inhibited insulin-stimulated PKB activation in these cells, this only partially inhibited AS160 phosphorylation and did not block GLUT4 trafficking, suggesting that p90RSK might regulate glucose transport after GLUT4 translocation. Moreover, BI-D1870 also prevented PMA-induced glucose transport in 3T3-L1 adipocytes further suggesting a role for p90RSK in regulating uptake of glucose into the cells. Kinetic experiments are consistent with SL0101 being a direct competitor of 2-deoxyglucose entry into cells, and this compound might also inhibit uptake of glucose into cells via inhibiting p90RSK, as revealed by comparison with the inactive form of the inhibitor. Taken together, we propose that BI-D1870 and SL0101 might exert their inhibitory effects on glucose uptake in 3T3-L1 adipocytes at least partially through a p90RSK dependent step after GLUT4 becomes associated with the plasma membrane.     

Thiazolidinedione treatment enhances insulin effects on protein kinase C-zeta /lambda activation and glucose transport in adipocytes of nondiabetic and Goto-Kakizaki type II diabetic rats

We evaluated effects of the thiazolidinedione, rosiglitazone, on insulin-induced activation of protein kinase C (PKC)-zeta/lambda and glucose transport in adipocytes of Goto-Kakizaki (GK)-diabetic and nondiabetic rats. Insulin effects on PKC-zeta/lambda and 2-deoxyglucose uptake were diminished by approximately 50% in GK adipocytes, as compared with control adipocytes. This defect in insulin-induced PKC-zeta/lambda activation was associated with diminished activation of IRS-1-dependent phosphatidylinositol (PI) 3-kinase, and was accompanied by diminished phosphorylation of threonine 410 in the activation loop of PKC-zeta; in contrast, protein kinase B (PKB) activation and phosphorylation were not significantly altered. Rosiglitazone completely reversed defects in insulin-stimulated 2-deoxyglucose uptake, PKCzeta/lambda enzyme activity and PKC-zeta threonine 410 phosphorylation, but had no effect on PI 3-kinase activation or PKB activation/phosphorylation in GK adipocytes. Similarly, in adipocytes of nondiabetic rats, rosiglitazone provoked increases in insulin-stimulated 2-deoxyglucose uptake, PKC-zeta/lambda enzyme activity and phosphorylation of both threonine 410 activation loop and threonine 560 autophosphorylation sites in PKC-zeta, but had no effect on PI 3-kinase activation or PKB activation/phosphorylation. Our findings suggest that (a) decreased effects of insulin on glucose transport in adipocytes of GK-diabetic rats are due at least in part to diminished phosphorylation/activation of PKC-zeta/lambda, and (b) thiazolidinediones enhance glucose transport responses to insulin in adipocytes of both diabetic and nondiabetic rats through increases in phosphorylation/activation of PKC-zeta/lambda.