Structure of GSK3beta reveals a primed phosphorylation mechanism.

GSK3beta was identified as the kinase that phosphorylates glycogen synthase but is now known to be involved in multiple signaling pathways. GSK3beta prefers prior phosphorylation of its substrates. We present the structure of unphosphorylated GSK3beta at 2.7 A. The orientation of the two domains and positioning of the activation loop of GSK3beta are similar to those observed in activated kinases. A phosphate ion held by Arg 96, Arg 180 and Lys 205 occupies the same position as the phosphate group of the phosphothreonine in activated p38gamma, CDK2 or ERK2. A loop from a neighboring molecule in the crystal occupies a portion of the substrate binding groove. The structure explains the unique primed phosphorylation mechanism of GSK3beta and how GSK3beta relies on a phosphoserine in the substrate for the alignment of the beta- and alpha-helical domains.     

Glycogen synthase kinase 3beta phosphorylates tau at both primed and unprimed sites. Differential impact on microtubule binding

Glycogen synthase kinase 3beta (GSK3beta) phosphorylates substrates, including the microtubule-associated protein tau, at both primed and unprimed epitopes. GSK3beta phosphorylation of tau negatively regulates tau-microtubule interactions; however the differential effects of phosphorylation at primed and unprimed epitopes on tau is unknown. To examine the phosphorylation of tau at primed and unprimed epitopes and how this impacts tau function, the R96A mutant of GSK3beta was used, a mutation that prevents phosphorylation of substrates at primed sites. Both GSK3beta and GSK3beta-R96A phosphorylated tau efficiently in situ. However, expression of GSK3beta-R96A resulted in significantly less phosphorylation of tau at primed sites compared with GSK3beta. Conversely, GSK3beta-R96A phosphorylated unprimed tau sites to a significantly greater extent than GSK3beta. Prephosphorylating tau with cdk5/p25 impaired the ability of GSK3beta-R96A to phosphorylate tau, whereas GSK3beta-R96A phosphorylated recombinant tau to a significantly greater extent than GSK3beta. Moreover, the amount of tau associated with microtubules was reduced by overexpression of GSK3beta but only when tau was phosphorylated at primed sites, as phosphorylation of tau by GSK3beta-R96A did not negatively regulate the association of tau with microtubules. These results demonstrate that GSK3beta-mediated phosphorylation of tau at primed sites plays a more significant role in regulating the interaction of tau with microtubules than phosphorylation at unprimed epitopes.     

Presenilin-1 is an unprimed glycogen synthase kinase-3beta substrate

Previously we described presenilin-1 (PS1) as a GSK-3beta substrate [Kirschenbaum, F., Hsu, S.C., Cordell, B. and McCarthy, J.V. (2001) Substitution of a glycogen synthase kinase-3beta phosphorylation site in presenilin 1 separates presenilin function from beta-catenin signalling. J. Biol. Chem. 276, 7366-7375; Kirschenbaum, F., Hsu, S.C., Cordell, B. and McCarthy, J.V. (2001) Glycogen synthase kinase-3beta regulates presenilin 1 C-terminal fragment levels. J. Biol. Chem. 276, 30701-30707], though it has not been determined whether PS1 is a primed or unprimed GSK-3beta substrate. A means of separating GSK-3beta activity toward primed and unprimed substrates was identified in the GSK-3beta-R96A phosphate binding pocket mutant [Frame, S., Cohen, P. and Biondi, R.M. (2001) A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation. Mol. Cell 7, 1321-1327], which is unable to phosphorylate primed but retains the ability to phosphorylate unprimed GSK-3beta substrates. By using wild type GSK-3beta, GSK-3beta-R96A, and a pharmacological modulator of GSK-3beta activity, we demonstrate that PS1 is an unprimed GSK-3beta substrate. These findings have important implications for regulation of PS1 function and the pathogenesis of Alzheimer's disease.     

FRAT-2 preferentially increases glycogen synthase kinase 3 beta-mediated phosphorylation of primed sites, which results in enhanced tau phosphorylation

Tau is a microtubule-associated protein found primarily in neurons, and its function is regulated by site-specific phosphorylation. Although it is well established that tau is phosphorylated at both primed and unprimed epitopes by glycogen synthase kinase 3 beta (GSK3 beta), how specific proteins that interact with GSK3 beta regulate tau phosphorylation has not been thoroughly examined. Members of the FRAT (frequently rearranged in advanced T-cell lymphoma) protein family have been shown to interact with GSK3 beta, and FRAT-1 has been shown to modulate the activity of GSK3 beta toward tau and other substrates. However, the effects of FRAT-2 on GSK3 beta activity and tau phosphorylation have not been examined. Therefore in this study the effects of FRAT-2 on GSK3 beta activity and tau phosphorylation were examined. In situ, FRAT-2 significantly increased GSK3 beta-mediated phosphorylation of tau at a primed epitope while not significantly affecting the phosphorylation of unprimed sites. Co-immunoprecipitation studies revealed that association of FRAT-2 with GSK3 beta resulted in a significant increase in phosphorylation of a primed substrate but did not alter phosphorylation of an unprimed substrate. Further, in vitro assays using recombinant proteins directly demonstrated that FRAT-2 enhances GSK3 beta-mediated phosphorylation of a primed substrate to a greater extent than an unprimed substrate. In addition, FRAT-2 is phosphorylated by GSK3 beta. This is the first demonstration of a protein differentially regulating the activity of GSK3 beta toward primed and unprimed epitopes.     

Structural basis for the complete loss of GSK3β catalytic activity due to R96 mutation investigated by molecular dynamics study

Many Ser/Thr protein kinases, to be fully activated, are obligated to introduce a phospho‐Ser/Thr in their activation loop. Presently, the similarity of activation loop between two crystal complexes, i.e. glycogen synthase kinase 3β (GSK3β)‐AMPNP and GSK3β‐sulfate ion complex, indicates that the activation segment of GSK3β is preformed requiring neither a phosphorylation event nor conformational changes. GSK3β, when participated in glycogen synthesis and Wnt signaling pathways, possesses a unique feature with the preference of such substrate with a priming phosphate. Experimental mutagenesis proved that the residue arginine at amino acid 96 mutations to lysine (R96K) or alanine (R96A) selectively abolish activity on the substrates involved in glycogen synthesis signaling pathway. Based on two solved crystal structures, wild type (WT) and two mutants (R96K and R96A) GSK3β‐ATP‐phospho‐Serine (pSer) complexes were modeled. Molecular dynamics simulations and energy analysis were employed to investigate the effect of pSer involvement on the GSK3β structure in WT, and the mechanisms of GSK3β deactivation due to R96K and R96A mutations. The results indicate that the introduction of pSer to WT GSK3β generates a slight lobe closure on GSK3β without any remarkable changes, which may illuminate the experimental conclusion, whereas the conformations of GSK3β and ATP undergo significant changes in two mutants. As to GSK3β, the affected positions distribute over activation loop, α‐helix, and glycine‐rich loop. Based on coupling among the mentioned positions, the allosteric mechanisms for distorted ATP were proposed. Energy decomposition on the residues of activation loop identified the important residues Arg96 and Arg180 in anchoring the phosphate group. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Primed phosphorylation of tau at Thr231 by glycogen synthase kinase 3beta (GSK3beta) plays a critical role in regulating tau's ability to bind and stabilize microtubules

Site-specific phosphorylation of tau negatively regulates its ability to bind and stabilize microtubule structure. Although tau is a substrate of glycogen synthase kinase 3beta (GSK3beta), the exact sites on tau that are phosphorylated by this kinase in situ have not yet been established, and the effect of these phosphorylation events on tau-microtubule interactions have not been fully elucidated. GSK3beta phosphorylates both primed and unprimed sites on tau, but only primed phosphorylation events significantly decrease the ability of tau to bind microtubules. The focus of the present study is on determining the importance of the GSK3beta-mediated phosphorylation of a specific primed site, Thr231, in regulating tau's function. Pre-phosphorylation of Ser235 primes tau for phosphorylation by GSK3beta at Thr231. Phosphorylation by GSK3beta of wild-type tau or tau with Ser235 mutated to Ala decreases tau-microtubule interactions. However, when Thr231 alone or Thr231 and Ser235 in tau were mutated to Ala, phosphorylation by GSK3beta did not decrease the association of tau with the cytoskeleton. Further, T231A tau was still able to efficiently bind microtubules after phosphorylation by GSK3beta. Expression of each tau construct alone increased tubulin acetylation, a marker of microtubule stability. However, when cells were cotransfected with wild-type tau and GSK3beta, the level of tubulin acetylation was decreased to vector-transfected levels. In contrast, coexpression of GSK3beta with mutated tau (T231A/S235A) did not significantly decrease the levels of acetylated tubulin. These results strongly indicate that phosphorylation of Thr231 in tau by GSK3beta plays a critical role in regulating tau's ability to bind and stabilize microtubules.     

Crystal structure of glycogen synthase kinase 3 beta: structural basis for phosphate-primed substrate specificity and autoinhibition.

Glycogen synthase kinase 3 beta (GSK3 beta) plays a key role in insulin and Wnt signaling, phosphorylating downstream targets by default, and becoming inhibited following the extracellular signaling event. The crystal structure of human GSK3 beta shows a catalytically active conformation in the absence of activation-segment phosphorylation, with the sulphonate of a buffer molecule bridging the activation-segment and N-terminal domain in the same way as the phosphate group of the activation-segment phospho-Ser/Thr in other kinases. The location of this oxyanion binding site in the substrate binding cleft indicates direct coupling of P+4 phosphate-primed substrate binding and catalytic activation, explains the ability of GSK3 beta to processively hyperphosphorylate substrates with Ser/Thr pentad-repeats, and suggests a mechanism for autoinhibition in which the phosphorylated N terminus binds as a competitive pseudosubstrate with phospho-Ser 9 occupying the P+4 site.     

Regulation of human cytidine triphosphate synthetase 1 by glycogen synthase kinase 3

Cytidine triphosphate synthetase (CTPS) catalyzes the rate-limiting step in the de novo synthesis of CTP, and both the yeast and human enzymes have been reported to be regulated by protein kinase A or protein kinase C phosphorylation. Here, we provide evidence that stimulation or inhibition of protein kinase A and protein kinase C does not alter the phosphorylation of endogenous human CTPS1 in human embryonic kidney 293 cells under the conditions tested. Unexpectedly, we found that low serum conditions increased phosphorylation of endogenous CTPS1 and this phosphorylation was inhibited by the glycogen synthase kinase 3 (GSK3) inhibitor indirubin-3'-monoxime and GSK3beta short interfering RNAs, demonstrating the involvement of GSK3 in phosphorylation of endogenous human CTPS1. Separating tryptic peptides from [(32)P]orthophosphate-labeled cells and analyzing the phosphopeptides by mass spectrometry identified Ser-574 and Ser-575 as phosphorylated residues. Mutation of Ser-571 demonstrated that Ser-571 was the major site phosphorylated by GSK3 in intact human embryonic kidney 293 cells by GSK3 in vitro. Furthermore, mutation of Ser-575 prevented the phosphorylation of Ser-571, suggesting that phosphorylation of Ser-575 was necessary for priming the GSK3 phosphorylation of Ser-571. Low serum was found to decrease CTPS1 activity, and incubation with the GSK3 inhibitor indirubin-3'-monoxime protected against this decrease in activity. Incubation with an alkaline phosphatase increased CTPS1 activity in a time-dependent manner, demonstrating that phosphorylation inhibits CTPS1 activity. This is the first study to investigate the phosphorylation and regulation of human CTPS1 in human cells and suggests that GSK3 is a novel regulator of CTPS activity.     

Mechanism of kinase inactivation and nonbinding of FRATide to GSK3β due to K85M mutation: molecular dynamics simulation and normal mode analysis

As a serine/threonine protein kinase, glycogen synthase kinase 3β (GSK3β) is an essential component of several cellular processes, including insulin, growth factor, and Wnt signaling. The conserved K85 is important to GSK3β activity and FRATide binding. To elucidate the mechanisms concerning kinase inactivation and nonbinding of FRATide to GSK3β, molecular dynamics (MD) simulation, molecular mechanics generalized Born/surface area (MM_GBSA) calculation, and normal mode analysis (NMA) were performed on both the wild-type (WT) and the K85M mutation of the GSK3β-FRATide complex. The results revealed that the periodic open-closed conformational change of the G loop, together with the compact conformation of the RD pocket, was disturbed in the K85M mutant, in contrast to those in the WT. This in turn caused inhibition of GSK3β. Specifically, the correct folding pattern of GSK3β was disrupted in the K85M mutant, resulting in the loss of two key hydrogen bonds between K214 of FRATide and E290 and K292 of GSK3β, respectively. Furthermore, MM_GBSA calculations indicated that the K85M mutation could lead to a less energy-favorable GSK3β-FRATide complex. In addition, NMA demonstrated that the "rocking" of the N- and C-terminal domains of GSK3β, which coordinates the mutual movement of both lobes, inducing the opening and closing of the active site of GSK3β, which may assist the entry of ATP into the ATP binding site and the release of the ADP product. Strikingly, this phenomenon was not clearly observed in the K85M mutation. This study provides a structural basis for the effect of the K85M mutation on the GSK3β-FRATide complex.     

Regulation of glycogen synthase kinase-3beta by products of lipid peroxidation in human neuroblastoma cells

The potential role of 4-hydroxynonenal (HNE), a major product of membrane lipid peroxidation, in regulating glycogen synthase kinase-3beta (GSK3beta) activity was examined in human neuroblastoma IMR-32 cells. The inhibition of GSK3beta activity by HNE was observed by in vitro kinase assays with two substrates, the synthetic glycogen synthase peptide-2 and the human recombinant tau. GSK3beta activity is regulated by Ser9 (inhibitory) and Tyr216 (stimulatory) phosphorylation. By using specific activity-dependent phospho-antibodies, immunoblot analysis revealed that HNE induces an increase in phosphorylation of GSK3beta in Ser9, enhancing basal phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase 2 (ERK2) signalling pathways. Ser9-GSK3beta phosphorylation induced by HNE was abolished by treatment with LY294002 or U0126, two inhibitors of PI3K/AKT and ERK pathways, respectively. These experiments provide evidence for a crucial role of the PI3K/AKT and ERK2 pathways as intracellular targets of HNE that mediate the inhibition of GSK3beta activity in regulating cellular response to HNE in viable cells under conditions in which membrane lipid peroxidation occurs. These data support a key role for GSK3beta as a mediator of the signalling pathways activated by oxidative stress, and therefore it may be included among the redox-sensitive enzymes.     

The binding and phosphorylation of Thr231 is critical for Tau's hyperphosphorylation and functional regulation by glycogen synthase kinase 3beta

Neuropathological hallmarks of Alzheimer's disease are extracellular senile plaques and intracellular neurofibrillary lesions. The neurofibrillary lesions mainly consist of the hyperphosphorylated microtubule-associated protein Tau predominantly expressed in the axon of CNS neurons. Hyperphosphorylation of Tau negatively affects its binding to tubulin and decreases the capacity to promote microtubule assembly. Among a number of proline-directed kinases capable of phosphorylating paired helical filament-Tau, glycogen synthase kinase 3beta (GSK3beta) was first identified as a Tau protein kinase I and has been demonstrated to phosphorylate Tau both in vivo and in vitro. However, the phosphorylation mechanism of Tau by GSK3beta remained unclear. In this study, we show that the T231 is the primary phosphorylation site for GSK3beta and the Tau227-237 (AVVRTPPKSPS) derived from Tau containing T231P232 motif is identified as the GSK3beta binding site with high affinity of a Kd value 0.82 +/- 0.16 mumol/L. Our results suggest that direct binding and phosphorylation of T231P232 motif by GSK3beta induces conformational change of Tau and consequentially alters the inhibitory activity of its N-terminus that allows the phosphorylation of C-terminus of Tau by GSK3beta. Furthermore, hyperphosphorylation reduces Tau's ability to promote tubulin assembly and to form bundles in N18 cells. T231A mutant completely abolishes Tau phosphorylation by GSK3beta and retains the ability to promote tubulin polymerization and bundle formation. Taken together, these results suggest that phosphorylation of T231 by GSK3beta may play an important role in Tau's hyperphosphorylation and functional regulation.     

Effect of double mutations K214/A–E215/Q of FRATide on GSK3β: insights from molecular dynamics simulation and normal mode analysis

Glycogen synthase kinase 3β (GSK3β) is a multifunctional serine/threonine protein kinase that is involved in several biological processes including insulin and Wnt signaling pathways. The Wnt signaling via FRAT-mediated displacement of axin inhibits GSK3β activity toward non-primed substrates without affecting its activity toward primed substrates. Herein, molecular dynamics simulation, molecular mechanics generalized Born/surface area (MM_GBSA) calculation, and normal mode analysis are performed to explore the structural influence of the double mutations K214/A-E215/Q of FRATide on the GSK3β-FRATide complex. The results reveal that the priming phosphate-binding site, the primed substrate-binding site, the alignment of the critical active site residues in the ATP-binding site, as well as the periodic open-closed conformational change of the ATP-binding site, which are critical for the catalytic activity of GSK3β, are negligibly influenced in the mutated system compared with the wild-type (WT) system. This indicates that FRATide does not inhibit the GSK3β activity toward primed substrates. Additionally, MM_GBSA calculation indicates that the less energy-favorable GSK3β-FRATide complex is observed in the mutant than in the WT complex.     

Sequential phosphorylation of insulin receptor substrate-2 by glycogen synthase kinase-3 and c-Jun NH2-terminal kinase plays a role in hepatic insulin signaling

Serine phosphorylation of insulin receptor substrate (IRS) proteins is a potential inhibitory mechanism in insulin signaling. Here we show that IRS-2 is phosphorylated by glycogen synthase kinase (GSK)-3. Phosphorylation by GSK-3 requires prior phosphorylation of its substrates, prompting us to identify the "priming kinase." It was found that the stress activator anisomycin enhanced the ability of GSK-3 to phosphorylate IRS-2. Use of a selective c-Jun NH(2)-terminal kinase (JNK) inhibitor and cells overexpressing JNK implicated JNK as the priming kinase. This allowed us to narrow down the number of potential GSK-3 phosphorylation sites within IRS-2 to four regions that follow the motif SXXXSP. IRS-2 deletion mutants enabled us to localize the GSK-3 and JNK phosphorylation sites to serines 484 and 488, respectively. Mutation at serine 488 reduced JNK phosphorylation of IRS-2, and mutation of each site separately abolished GSK-3 phosphorylation of IRS-2. Treatment of H4IIE liver cells with anisomycin inhibited insulin-induced tyrosine phosphorylation of IRS-2; inhibition was reversed by pretreatment with the JNK and GSK-3 inhibitors. Moreover, overexpression of JNK and GSK-3 in H4IIE cells reduced insulin-induced tyrosine phosphorylation of IRS-2 and its association with the p85 regulatory subunit of phosphatidylinositol 3-kinase. Finally, both GSK-3 and JNK are abnormally upregulated in the diabetic livers of ob/ob mice. Together, our data indicate that IRS-2 is sequentially phosphorylated by JNK and GSK-3 at serines 484/488 and provide evidence for their inhibitory role in hepatic insulin signaling.     

The Mood‐Stabilizing Agent Valproate Inhibits the Activity of Glycogen Synthase Kinase‐3

Abstract : Valproic acid (VPA) is a potent broad‐spectrum anti‐epileptic with demonstrated efficacy in the treatment of bipolar affective disorder. It has previously been demonstrated that both VPA and lithium increase activator protein‐1 (AP‐1) DNA binding activity, but the mechanisms underlying these effects have not been elucidated. However, it is known that phosphorylation of c‐jun by glycogen synthase kinase (GSK)‐3β inhibits AP‐1 DNA binding activity, and lithium has recently been demonstrated to inhibit GSK‐3β. These results suggest that lithium may increase AP‐1 DNA binding activity by inhibiting GSK‐3β. In the present study, we sought to determine if VPA, like lithium, regulates GSK‐3. We have found that VPA concentration‐dependently inhibits both GSK‐3α and ‐3β, with significant effects observed at concentrations of VPA similar to those attained clinically. Incubation of intact human neuroblastoma SH‐SY5Y cells with VPA results in an increase in the subsequent in vitro recombinant GSK‐3β‐mediated ³²P incorporation into two putative GSK‐3 substrates (~85 and 200 kDa), compatible with inhibition of endogenous GSK‐3β by VPA. Consistent with GSK‐3β inhibition, incubation of SH‐SY5Y cells with VPA results in a significant time‐dependent increase in both cytosolic and nuclear β‐catenin levels. GSK‐3β plays a critical role in the CNS by regulating various cytoskeletal processes as well as long‐term nuclear events and is a common target for both lithium and VPA ; inhibition of GSK‐3β in the CNS may thus underlie some of the long‐term therapeutic effects of mood‐stabilizing agents.     

Novel procedure to investigate the effect of phosphorylation on protein complex formation in vitro and in cells

The identification of phosphorylation state-dependent interacting proteins provides clues as to the function of the phosphorylation. Techniques such as yeast two hybrid and co-immunoprecipitation do not employ a single species of fully phosphorylated proteins. This is a particular problem for substrates of glycogen synthase kinase-3 (GSK3), where multiple Ser/Thr residues can be targeted, almost always subsequent to a priming phosphorylation by an alternative kinase. We previously identified the brain enriched collapsin response mediator proteins (CRMP2 and CRMP4) as physiological substrates of GSK3. Cdk5 phosphorylates CRMP2 at Ser522, priming for subsequent phosphorylation at three residues by GSK3 in vitro and in vivo. It is clear that phosphorylation of CRMP2 influences axonal growth; however, the molecular processes underlying this action are not fully established. In addition, the role of phosphorylation in other actions of CRMPs has not been elucidated. We developed a novel procedure to isolate CRMP2 and CRMP4 fully phosphorylated at four sites, namely, Ser522 (by CDK5), Ser518, Thr514, and Thr509 (by GSK3). These phosphoproteins were then used to identify binding partners in rat brain lysates in direct comparison with the non-phosphorylated isoforms. We validated the approach by confirming that a previously reported interaction with tubulin-beta is regulated by phosphorylation. We also show that CRMPs (CRMP1, CRMP2, and CRMP4) form heteromers and found that these complexes may also be regulated by phosphorylation. We identified DYRK and Pin1 as novel CRMP4 binding proteins with DYRK interacting preferentially with dephospho-CRMP4 and Pin1 with phospho-CRMP4. Finally, we used this approach to identify the mitochondrial protein ANT as a novel CRMP2 and CRMP4 binding protein. We believe that this approach could be applied generally to the study of phosphorylation-dependent interactions.     

A GSK3-binding peptide from FRAT1 selectively inhibits the GSK3-catalysed phosphorylation of axin and beta-catenin.

The Axin-dependent phosphorylation of beta-catenin catalysed by glycogen synthase kinase-3 (GSK3) is inhibited during embryogenesis. This protects beta-catenin against ubiquitin-dependent proteolysis, leading to its accumulation in the nucleus, where it controls the expression of genes important for development. Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) is a mammalian homologue of a GSK3-binding protein (GBP), which appears to play a key role in the correct establishment of the dorsal-ventral axis in Xenopus laevis. Here, we demonstrate that FRATtide (a peptide corresponding to residues 188-226 of FRAT1) binds to GSK3 and prevents GSK3 from interacting with Axin. FRATtide also blocks the GSK3-catalysed phosphorylation of Axin and beta-catenin, suggesting a potential mechanism by which GBP could trigger axis formation. In contrast, FRATtide does not suppress GSK3 activity towards other substrates, such as glycogen synthase and eIF2B, whose phosphorylation is independent of Axin but dependent on a 'priming' phosphorylation. This may explain how the essential cellular functions of GSK3 can continue, despite the suppression of beta-catenin phosphorylation.     

PTEN is destabilized by phosphorylation on Thr366

Although PTEN (phosphatase and tensin homologue deleted on chromosome 10) is one of the most commonly mutated tumour suppressors in human cancers, loss of PTEN expression in the absence of mutation appears to occur in an even greater number of tumours. PTEN is phosphorylated in vitro on Thr366 and Ser370 by GSK3 (glycogen synthase kinase 3) and CK2 (casein kinase 2) respectively, and specific inhibitors of these kinases block these phosphorylation events in cultured cells. Although mutation of these phosphorylation sites did not alter the phosphatase activity of PTEN in vitro or in cells, blocking phosphorylation of Thr366 by either mutation or GSK3 inhibition in glioblastoma cell lines led to a stabilization of the PTEN protein. Our data support a model in which the phosphorylation of Thr366 plays a role in destabilizing the PTEN protein.     

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.