3-phosphoinositide-dependent protein kinase 1, an Akt1 kinase, is involved in dephosphorylation of Thr-308 of Akt1 in Chinese hamster ovary cells

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the Akt1 phosphorylation state, wild-type (wt) PDK1 and its kinase dead (kd) mutant were expressed using an adenovirus gene transduction system in Chinese hamster ovary cells stably expressing insulin receptor. Immunoblotting using anti-phosphorylated Akt1 antibody revealed Thr-308 already to be maximally phosphorylated at 1 min but completely dephosphorylated at 5 min, with insulin stimulation, whereas insulin-induced Akt1 activation was maintained even after dephosphorylation of Thr-308. Overexpression of wt-PDK1 further increased insulin-stimulated phosphorylation of Thr-308, also followed by rapid dephosphorylation. The insulin-stimulated Akt1 activity was also enhanced by wt-PDK1 expression but was maintained even at 15 min. Thus, phosphorylation of Thr-308 is not essential for maintaining the Akt1 activity once it has been achieved. Interestingly, the insulin-stimulated phosphorylation state of Thr-308 was maintained even at 15 min in cells expressing kd-PDK1, suggesting that kd-PDK1 has a dominant negative effect on dephosphorylation of Thr-308 of Akt1. Calyculin A, an inhibitor of PP1 and PP2A, also prolonged the insulin-stimulated phosphorylation state of Thr-308. In addition, in vitro experiments revealed PP2A, but not PP1, to dephosphorylate completely Thr-308 of Akt1. These findings suggest that a novel pathway involving dephosphorylation of Akt1 at Thr-308 by a phosphatase, possibly PP2A, originally, identified as is regulated downstream from PDK1, an Akt1 kinase.     

Role of the PDK1-PKB-GSK3 pathway in regulating glycogen synthase and glucose uptake in the heart

In order to investigate the importance of the PDK1-PKB-GSK3 signalling network in regulating glycogen synthase (GS) in the heart, we have employed tissue specific conditional knockout mice lacking PDK1 in muscle (mPDK1-/-), as well as knockin mice in which the protein kinase B (PKB) phosphorylation site on glycogen synthase kinase-3alpha (GSK3alpha) (Ser21) and GSK3beta (Ser9) is changed to Ala. We demonstrate that in hearts from mPDK1-/- or double GSK3alpha/GSK3beta knockin mice, insulin failed to stimulate the activity of GS or induce its dephosphorylation at residues that are phosphorylated by GSK3. We also establish that in the heart, both GSK3 isoforms participate in the regulation of GS, with GSK3beta playing a more prominent role. This contrasts with skeletal muscle where GSK3beta is the major regulator of insulin-induced GS activity. Despite the inability of insulin to stimulate glycogen synthesis in hearts from the mPDK1-/- or double GSK3alpha/GSK3beta knockin mice, these animals possessed normal levels of cardiac glycogen, demonstrating that total glycogen levels are regulated independently of insulin's ability to stimulate GS in the heart and that mechanisms such as allosteric activation of GS by glucose-6-phosphate and/or activation of GS by muscle contraction, could operate to maintain normal glycogen levels in these mice. We also demonstrate that in cardiomyocytes derived from the mPDK1-/- hearts, although the levels of glucose transporter type 4 (GLUT4) are increased 2-fold, insulin failed to stimulate glucose uptake, providing genetic evidence that PDK1 plays a crucial role in enabling insulin to promote glucose uptake in cardiac muscle.     

Protein kinase Cdelta participates in insulin-induced activation of PKB via PDK1

PKCdelta has been shown to be activated by insulin and to interact with insulin receptor and IRS. PKB(Akt) plays an important role in glucose transport and glycogen synthesis. In this study, we investigated the possibility that PKCdelta may be involved in insulin-induced activation of PKB. Studies were conducted on primary cultures of rat skeletal muscle. PKB was activated by insulin stimulation within 5min and reached a peak by 15-30min. Insulin also increased the physical association between PKCdelta with PKB and with PDK1. The insulin-induced PKCdelta-PKB association was PI3K dependent. PKB-PKCdelta association was accounted for by the involvement of PDK1. Overexpression of dominant negative PKCdelta abrogated insulin-induced association of PKCdelta with both PKB and PDK1. Blockade of PKCdelta also decreased insulin-induced Thr308 PKB phosphorylation and PKB translocation. Moreover, PKCdelta inhibition reduced insulin-induced GSK3 phosphorylation. The results indicate that insulin-activated PKCdelta interacts with PDK1 to regulate PKB.     

Regulation of protein kinases in insulin,growth factor and Wnt signalling

Protein kinase cascades provide the regulatory mechanisms for many of the essential processes in eukaryotic cells. Recent structural and biochemical work has revealed the basis of phosphorylation regulation of three consecutive protein kinases - phosphoinositide-dependent kinase 1 (PDK1), protein kinase B (PKB)/Akt and glycogen synthase kinase 3beta (GSK3beta) - which transduce signals generated by insulin and/or growth factors binding to cell surface receptors. PDK1 and PKB are both AGC family kinases. Whereas PKB is positively regulated via its phosphorylated C-terminal hydrophobic motif, the activity and specificity of PDK1 are determined by equivalent hydrophobic motifs of substrate AGC kinases. In a contrasting mechanism, GSK3beta is negatively regulated by competitive autoinhibition by its phosphorylated N terminus. GSK3beta also functions in the developmental Wnt signalling pathway, but without cross-talk with the PDK1-PKB/Akt pathway. Structural studies of GSK3beta complexes are contributing to our understanding of the phosphorylation-independent mechanism that insulates the Wnt and insulin/growth factor pathways.     

Interaction of PDK1 with Phosphoinositides Is Essential for Neuronal Differentiation but Dispensable for Neuronal Survival

3-Phosphoinositide-dependent protein kinase 1 (PDK1) operates in cells in response to phosphoinositide 3-kinase activation and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P₃] production by activating a number of AGC kinases, including protein kinase B (PKB)/Akt. Both PDK1 and PKB contain pleckstrin homology (PH) domains that interact with the PtdIns(3,4,5)P₃ second messenger. Disrupting the interaction of the PDK1 PH domain with phosphoinositides by expressing the PDK1 K465E knock-in mutation resulted in mice with reduced PKB activation. We explored the physiological consequences of this biochemical lesion in the central nervous system. The PDK1 knock-in mice displayed a reduced brain size due to a reduction in neuronal cell size rather than cell number. Reduced BDNF-induced phosphorylation of PKB at Thr308, the PDK1 site, was observed in the mutant neurons, which was not rate limiting for the phosphorylation of those PKB substrates governing neuronal survival and apoptosis, such as FOXO1 or glycogen synthase kinase 3 (GSK3). Accordingly, the integrity of the PDK1 PH domain was not essential to support the survival of different embryonic neuronal populations analyzed. In contrast, PKB-mediated phosphorylation of PRAS40 and TSC2, allowing optimal mTORC1 activation and brain-specific kinase (BRSK) protein synthesis, was markedly reduced in the mutant mice, leading to impaired neuronal growth and differentiation.     

The Croonian Lecture 1998. Identification of a protein kinase cascade of major importance in insulin signal transduction

Diabetes affects 3% of the European population and 140 million people worldwide, and is largely a disease of insulin resistance in which the tissues fail to respond to this hormone. This emphasizes the importance of understanding how insulin signals to the cell's interior. We have recently dissected a protein kinase cascade that is triggered by the formation of the insulin 'second messenger' phosphatidylinositide (3,4,5) trisphosphate (PtdIns (3,4,5)P3) and which appears to mediate many of the metabolic actions of this hormone. The first enzyme in the cascade is termed 3-phosphoinositide-dependent protein kinase-1 (PDK1), because it only activates protein kinase B (PKB), the next enzyme in the pathway, in the presence of PtdIns (3,4,5)P3. PKB then inactivates glycogen synthase kinase-3 (GSK3). PDK1, PKB and GSK3 regulate many physiological events by phosphorylating a variety of intracellular proteins. In addition, PKB plays an important role in mediating protection against apoptosis by survival factors, such as insulin-like growth factor-1.     

Effect of phosphoinositide-dependent kinase 1 on protein kinase B translocation and its subsequent activation

In this report we investigated the function of phosphoinositide-dependent protein kinase 1 (PDK1) in protein kinase B (PKB) activation and translocation to the cell surface. Wild-type and PDK1 mutants were transfected into HeLa cells, and their subcellular localization was analyzed. PDK1 was found to translocate to the plasma membrane in response to insulin, and this process did not require a functional catalytic activity, since a catalytically inactive kinase mutant (Kd) of PDK1 was capable of translocating. The PDK1 presence at the cell surface was shown to be linked to phospholipids and therefore to serum-dependent phosphatidylinositol 3-kinase activity. Using confocal microscopy in HeLa cells we found that PDK1 colocalizes with PKB at the plasma membrane. Further, after cotransfection of PKB and a PDK1 mutant (Mut) unable to translocate to the plasma membrane, PKB was prevented from moving to the cell periphery after insulin stimulation. In response to insulin, a PKB mutant with its PH domain deleted (DeltaPH-PKB) retained the ability to translocate to the plasma membrane when coexpressed with PDK1. Finally, we found that DeltaPH-PKB was highly active independent of insulin stimulation when cotransfected with PDK1 mutants defective in their PH domain. These findings suggest that PDK1 brings PKB to the plasma membrane upon exposure of cells to insulin and that the PH domain of PDK1 acts as a negative regulator of its enzyme activity.     

Mechanism of phosphorylation of protein kinase B/Akt by a constitutively active 3-phosphoinositide-dependent protein kinase-1

Phosphorylation of Thr(308) in the activation loop and Ser(473) at the carboxyl terminus is essential for protein kinase B (PKB/Akt) activation. However, the biochemical mechanism of the phosphorylation remains to be characterized. Here we show that expression of a constitutively active mutant of mouse 3-phosphoinositide-dependent protein kinase-1 (PDK1(A280V)) in Chinese hamster ovary cells overexpressing the insulin receptor was sufficient to induce PKB phosphorylation at Thr(308) to approximately the same extent as insulin stimulation. Phosphorylation of PKB by PDK1(A280V) was not affected by treatment of cells with inhibitors of phosphatidylinositol 3-kinase or by deletion of the pleckstrin homology (PH) domain of PKB. C(2)-ceramide, a cell-permeable, indirect inhibitor of PKB phosphorylation, did not inhibit PDK1(A280V)-catalyzed PKB phosphorylation in cells and had no effect on PDK1 activity in vitro. On the other hand, co-expression of full-length protein kinase C-related kinase-1 (PRK1/PKN) or 2 (PRK2) inhibited PDK1(A280V)-mediated PKB phosphorylation. Replacing alanine at position 280 with valine or deletion of the PH domain enhanced PDK1 autophosphorylation in vitro. However, deletion of the PH domain of PDK1(A280V) significantly reduced PDK1(A280V)-mediated phosphorylation of PKB in cells. In resting cells, PDK1(A280V) localized in the cytosol and at the plasma membrane. However, PDK1(A280V) lacking the PH domain localized predominantly in the cytosol. Taken together, our findings suggest that the wild-type PDK1 may not be constitutively active in cells. In addition, activation of PDK1 is sufficient to phosphorylate PKB at Thr(308) in the cytosol. Furthermore, the PH domain of PDK1 may play both positive and negative roles in regulating the in vivo function of the enzyme. Finally, unlike the carboxyl-terminal fragment of PRK2, which has been shown to bind PDK1 and allow the enzyme to phosphorylate PKB at both Thr(308) and Ser(473), full-length PRK2 and its related kinase PRK1/PKN may both play negative roles in PKB-mediated downstream biological events.     

蛋白激酶B及其在磷脂酰肌醇3-激酶介导的信号转导中的作用

蛋白激酶Ｂ（ＰＫＢ）是原癌基因ｃ－ａｋｔ的表达产物,它参与由生长因子激活的经磷脂磷肌醇３－激酶（ＰＩ３Ｋ）介导的信号转导过程。与许多蛋白激酶相似,ＰＫＢ分子具有一特殊的ＡＨ／ＰＨ结构域（ＡＨ／ＰＨｄｏｍａｉｎ）,后者能介导信号分子间的相互作用。ＰＫＢ是ＰＩ３Ｋ直接的靶蛋白。ＰＩ３Ｋ产生的脂类第二信使ＰＩ－３,４,Ｐ２和ＰＩ－３,４,５－Ｐ３等均能与ＰＫＢ和磷酸肌醇依赖性蛋白激酶（ＰＤＫ）的ＡＨ／Ｐ     

Evidence that 3-phosphoinositide-dependent protein kinase-1 mediates phosphorylation of p70 S6 kinase in vivo at Thr-412 as well as Thr-252

Protein kinase B and p70 S6 kinase are members of the cyclic AMP-dependent/cyclic GMP-dependent/protein kinase C subfamily of protein kinases and are activated by a phosphatidylinositol 3-kinase-dependent pathway when cells are stimulated with insulin or growth factors. Both of these kinases are activated in cells by phosphorylation of a conserved residue in the kinase domain (Thr-308 of protein kinase B (PKB) and Thr-252 of p70 S6 kinase) and another conserved residue located C-terminal to the kinase domain (Ser-473 of PKB and Thr-412 of p70 S6 kinase). Thr-308 of PKBalpha and Thr-252 of p70 S6 kinase are phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) in vitro. Recent work has shown that PDK1 interacts with a region of protein kinase C-related kinase-2, termed the PDK1 interacting fragment (PIF). Interaction with PIF converts PDK1 from a form that phosphorylates PKB at Thr-308 alone to a species capable of phosphorylating Ser-473 as well as Thr-308. This suggests that PDK1 may be the enzyme that phosphorylates both residues in vivo. Here we demonstrate that PDK1 is capable of phosphorylating p70 S6 kinase at Thr-412 in vitro. We study the effect of PIF on the ability of PDK1 to phosphorylate p70 S6 kinase. Surprisingly, we find that PDK1 bound to PIF is no longer able to interact with or phosphorylate p70 S6 kinase in vitro at either Thr-252 or Thr-412. The expression of PIF in cells prevents insulin-like growth factor 1 from inducing the activation of the p70 S6 kinase and its phosphorylation at Thr-412. Overexpression of PDK1 in cells induces the phosphorylation of p70 S6 kinase at Thr-412 in unstimulated cells, and a catalytically inactive mutant of PDK1 prevents the phosphorylation of p70 S6K at Thr-412 in insulin-like growth factor 1-stimulated cells. These observations indicate that PDK1 regulates the activation of p70 S6 kinase and provides evidence that PDK1 mediates the phosphorylation of p70 S6 kinase at Thr-412.     

In vivo role of the PIF-binding docking site of PDK1 defined by knock-in mutation

PKB/Akt, S6K, SGK and RSK are mediators of responses triggered by insulin and growth factors and are activated following phosphorylation by 3-phosphoinositide-dependent protein kinase-1 (PDK1). To investigate the importance of a substrate-docking site in the kinase domain of PDK1 termed the 'PIF-pocket', we generated embryonic stem (ES) cells in which both copies of the PDK1 gene were altered by knock-in mutation to express a form of PDK1 retaining catalytic activity, in which the PIF-pocket site was disrupted. The knock-in ES cells were viable, mutant PDK1 was expressed at normal levels and insulin-like growth factor 1 induced normal activation of PKB and phosphorylation of the PKB substrates GSK3 and FKHR. In contrast, S6K, RSK and SGK were not activated, nor were physiological substrates of S6K and RSK phosphorylated. These experiments establish the importance of the PIF-pocket in governing the activation of S6K, RSK, SGK, but not PKB, in vivo. They also illustrate the power of knock-in technology to probe the physiological roles of docking interactions in regulating the specificity of signal transduction pathways.     

Insulin receptor substrate-2 phosphorylation is necessary for protein kinase C zeta activation by insulin in L6hIR cells

We have investigated glycogen synthase (GS) activation in L6hIR cells expressing a peptide corresponding to the kinase regulatory loop binding domain of insulin receptor substrate-2 (IRS-2) (KRLB). In several clones of these cells (B2, F4), insulin-dependent binding of the KRLB to insulin receptors was accompanied by a block of IRS-2, but not IRS-1, phosphorylation, and insulin receptor binding. GS activation by insulin was also inhibited by >70% in these cells (p < 0.001). The impairment of GS activation was paralleled by a similarly sized inhibition of glycogen synthase kinase 3 alpha (GSK3 alpha) and GSK3 beta inactivation by insulin with no change in protein phosphatase 1 activity. PDK1 (a phosphatidylinositol trisphosphate-dependent kinase) and Akt/protein kinase B (PKB) activation by insulin showed no difference in B2, F4, and in control L6hIR cells. At variance, insulin did not activate PKC zeta in B2 and F4 cells. In L6hIR, inhibition of PKC zeta activity by either a PKC zeta antisense or a dominant negative mutant also reduced by 75% insulin inactivation of GSK3 alpha and -beta (p < 0.001) and insulin stimulation of GS (p < 0.002), similar to Akt/PKB inhibition. In L6hIR, insulin induced protein kinase C zeta (PKC zeta) co-precipitation with GSK3 alpha and beta. PKC zeta also phosphorylated GSK3 alpha and -beta. Alone, these events did not significantly affect GSK3 alpha and -beta activities. Inhibition of PKC zeta activity, however, reduced Akt/PKB phosphorylation of the key serine sites on GSK3 alpha and -beta by >80% (p < 0.001) and prevented full GSK3 inactivation by insulin. Thus, IRS-2, not IRS-1, signals insulin activation of GS in the L6hIR skeletal muscle cells. In these cells, insulin inhibition of GSK3 alpha and -beta requires dual phosphorylation by both Akt/PKB and PKC zeta.     

Phosphoinositide 3-kinase-mediated reduction of insulin receptor substrate-1/2 protein expression via different mechanisms contributes to the insulin-induced desensitization of its signaling pathways in L6 muscle cells

Impaired glucose tolerance precedes type 2 diabetes and is characterized by hyperinsulinemia, which develops to balance peripheral insulin resistance. To gain insight into the deleterious effects of hyperinsulinemia on skeletal muscle, we studied the consequences of prolonged insulin treatment of L6 myoblasts on insulin-dependent signaling pathways. A 24-h long insulin treatment desensitized the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) and p42/p44 MAPK pathways toward a second stimulation with insulin or insulin-like growth factor-1 and led to decreased insulin-induced glucose uptake. Desensitization was correlated to a reduction in insulin receptor substrate (IRS)-1 and IRS-2 protein levels, which was reversed by the PI3K inhibitor LY294002. Co-treatment of cells with insulin and LY294002, while reducing total IRS-1 phosphorylation, increased its phosphotyrosine content, enhancing IRS-1/PI3K association. PDK1, mTOR, and MAPK inhibitors did not block insulin-induced reduction of IRS-1, suggesting that the PI3K serine-kinase activity causes IRS-1 serine phosphorylation and its commitment to proteasomal degradation. Contrarily, insulin-induced IRS-2 down-regulation occurred via a PI3K/mTOR pathway. Suppression of IRS-1/2 down-regulation by LY294002 rescued the responsiveness of PKB and MAPK toward acute insulin stimulation. Conversely, adenoviral-driven expression of constitutively active PI3K induced an insulin-independent reduction in IRS-1/2 protein levels. IRS-2 appears to be the chief molecule responsible for MAPK and PKB activation by insulin, as knockdown of IRS-2 (but not IRS-1) by RNA interference severely impaired activation of both kinases. In summary, (i) PI3K mediates insulin-induced reduction of IRS-1 by phosphorylating it while a PI3K/mTOR pathway controls insulin-induced reduction of IRS-2, (ii) in L6 cells, IRS-2 is the major adapter molecule linking the insulin receptor to activation of PKB and MAPK, (iii) the mechanism of IRS-1/2 down-regulation is different in L6 cells compared with 3T3-L1 adipocytes. In conclusion, the reduction in IRS proteins via different PI3K-mediated mechanisms contributes to the development of an insulin-resistant state in L6 myoblasts.     

Endurance exercise training increases APPL1 expression and improves insulin signaling in the hepatic tissue of diet-induced obese mice, independently of weight loss

Hepatic insulin resistance is the major contributor to fasting hyperglycemia in type 2 diabetes. The protein kinase Akt plays a central role in the suppression of gluconeogenesis involving forkhead box O1 (Foxo1) and peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), and in the control of glycogen synthesis involving the glycogen synthase kinase beta (GSK3β) in the liver. It has been demonstrated that endosomal adaptor protein APPL1 interacts with Akt and blocks the association of Akt with its endogenous inhibitor, tribbles-related protein 3 (TRB3), improving the action of insulin in the liver. Here, we demonstrated that chronic exercise increased the basal levels and insulin-induced Akt serine phosphorylation in the liver of diet-induced obese mice. Endurance training was able to increase APPL1 expression and the interaction between APPL1 and Akt. Conversely, training reduced both TRB3 expression and TRB3 and Akt association. The positive effects of exercise on insulin action are reinforced by our findings that showed that trained mice presented an increase in Foxo1 phosphorylation and Foxo1/PGC-1α association, which was accompanied by a reduction in gluconeogenic gene expressions (PEPCK and G6Pase). Finally, exercised animals demonstrated increased at basal and insulin-induced GSK3β phosphorylation levels and glycogen content at 24 h after the last session of exercise. Our findings demonstrate that exercise increases insulin action, at least in part, through the enhancement of APPL1 and the reduction of TRB3 expression in the liver of obese mice, independently of weight loss.     

Protein kinase Cδ but not PKCα is involved in insulin-induced glucose metabolism in hepatocytes

The liver is a major insulin‐responsive tissue responsible for glucose regulation. One important mechanism in this phenomenon is insulin‐induced glycogen synthesis. Studies in our laboratory have shown that protein kinase Cs delta (PKCδ) and alpha (α) have important roles in insulin‐induced glucose transport in skeletal muscle, and that their expression and activity are regulated by insulin. Their importance in glucose regulation in liver cells is unclear. In this study we investigated the possibility that these isoforms are involved in the mediation of insulin‐induced glycogen synthesis in hepatocytes. Studies were done on rat hepatocytes in primary culture and on the AML‐12 (alpha mouse liver) cell line. Insulin increased activity and tyrosine phosphorylation of PKCδ within 5 min. In contrast, activity and tyrosine phosphorylation of PKCα were not increased by insulin. PKCδ was constitutively associated with IR, and this was increased by insulin stimulation. Suppression of PKCδ expression by transfection with RNAi, or overexpression of kinase dead (dominant negative) PKCδ reduced both the insulin‐induced activation of PKB/Akt and the phosphorylation of glycogen synthase kinase 3 (GSK3) and reduced significantly insulin‐induced glucose uptake. In addition, treatment of primary rat hepatocytes with rottlerin abrogated insulin‐induced increase in glycogen synthesis. Neither overexpression nor inhibition of PKCα appeared to alter activation of PKB, phosphorylation of GSK3 or glucose uptake in response to insulin. We conclude that PKCδ, but not PKCα, plays an essential role in insulin‐induced glucose uptake and glycogenesis in hepatocytes. J. Cell. Biochem. 113: 2064–2076, 2012. © 2012 Wiley Periodicals, Inc.     

Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1

PDK1 (3-phosphoinositide-dependent protein kinase 1) activates a group of protein kinases belonging to the AGC [PKA (protein kinase A)/PKG (protein kinase G)/PKC (protein kinase C)]-kinase family that play important roles in mediating diverse biological processes. Many cancer-driving mutations induce activation of PDK1 targets including Akt, S6K (p70 ribosomal S6 kinase) and SGK (serum- and glucocorticoid-induced protein kinase). In the present paper, we describe the small molecule GSK2334470, which inhibits PDK1 with an IC?? of ~10 nM, but does not suppress the activity of 93 other protein kinases including 13 AGC-kinases most related to PDK1 at 500-fold higher concentrations. Addition of GSK2334470 to HEK (human embryonic kidney)-293, U87 or MEF (mouse embryonic fibroblast) cells ablated T-loop residue phosphorylation and activation of SGK isoforms and S6K1 induced by serum or IGF1 (insulin-like growth factor 1). GSK2334470 also inhibited T-loop phosphorylation and activation of Akt, but was more efficient at inhibiting Akt in response to stimuli such as serum that activated the PI3K (phosphoinositide 3-kinase) pathway weakly. GSK2334470 inhibited activation of an Akt1 mutant lacking the PH domain (pleckstrin homology domain) more potently than full-length Akt1, suggesting that GSK2334470 is more effective at inhibiting PDK1 substrates that are activated in the cytosol rather than at the plasma membrane. Consistent with this, GSK2334470 inhibited Akt activation in knock-in embryonic stem cells expressing a mutant of PDK1 that is unable to interact with phosphoinositides more potently than in wild-type cells. GSK2334470 also suppressed T-loop phosphorylation and activation of RSK2 (p90 ribosomal S6 kinase 2), another PDK1 target activated by the ERK (extracellular-signal-regulated kinase) pathway. However, prolonged treatment of cells with inhibitor was required to observe inhibition of RSK2, indicating that PDK1 substrates possess distinct T-loop dephosphorylation kinetics. Our data define how PDK1 inhibitors affect AGC signalling pathways and suggest that GSK2334470 will be a useful tool for delineating the roles of PDK1 in biological processes.     

Potential role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in insulin-stimulated glucose transporter 4 translocation in adipocytes

Insulin stimulation of Glut 4 translocation requires the activation of phosphatidylinositol 3-kinase (PI 3-kinase) but the downstream pathway remains ill-defined. We demonstrated that the overexpression of PDK1 (3-phosphoinositide-dependent protein kinase 1), a downstream effector of PI 3-kinase, stimulated Glut 4 translocation in adipocytes. This effect does not require the PH domain of PDK1, but expression of the pleckstrin homology domain-deleted PDK1 inhibits the effect of insulin, but not okadaic acid, on Glut 4 translocation. These results support a role of the PDK1 pathway in the transmission of insulin signal to Glut translocation.     

The role of glycogen synthase kinase 3beta in insulin-stimulated glucose metabolism.

To characterize the contribution of glycogen synthase kinase 3beta (GSK3beta) inactivation to insulin-stimulated glucose metabolism, wild-type (WT-GSK), catalytically inactive (KM-GSK), and uninhibitable (S9A-GSK) forms of GSK3beta were expressed in insulin-responsive 3T3-L1 adipocytes using adenovirus technology. WT-GSK, but not KM-GSK, reduced basal and insulin-stimulated glycogen synthase activity without affecting the -fold stimulation of the enzyme by insulin. S9A-GSK similarly decreased cellular glycogen synthase activity, but also partially blocked insulin stimulation of the enzyme. S9A-GSK expression also markedly inhibited insulin stimulation of IRS-1-associated phosphatidylinositol 3-kinase activity, but only weakly inhibited insulin-stimulated Akt/PKB phosphorylation and glucose uptake, with no effect on GLUT4 translocation. To further evaluate the role of GSK3beta in insulin signaling, the GSK3beta inhibitor lithium was used to mimic the consequences of insulin-stimulated GSK3beta inactivation. Although lithium stimulated the incorporation of glucose into glycogen and glycogen synthase enzyme activity, the inhibitor was without effect on GLUT4 translocation and pp70 S6 kinase. Lithium stimulation of glycogen synthesis was insensitive to wortmannin, which is consistent with its acting directly on GSK3beta downstream of phosphatidylinositol 3-kinase. These data support the hypothesis that GSK3beta contributes to insulin regulation of glycogen synthesis, but is not responsible for the increase in glucose transport.