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.     

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.     

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.     

Glycogen synthase kinase 3beta is tyrosine phosphorylated by PYK2

Glycogen synthase kinase 3beta (GSK3beta) is a Ser/Thr kinase that is involved in numerous cellular activities. GSK3beta is activated by tyrosine phosphorylation. However, very little is known about the tyrosine kinases that are responsible for phosphorylating GSK3beta. In this report, we investigated the ability of the calcium-dependent tyrosine kinase, proline-rich tyrosine kinase 2 (PYK2) to tyrosine phosphorylate GSK3beta. In transfected CHO cells, it was demonstrated that PYK2 tyrosine phosphorylates GSK3beta in situ. The two kinases also coimmunoprecipitated. Furthermore, GSK3beta was tyrosine phosphorylated in vitro by an active, wild type PYK2, but not by the inactive, kinase dead form of PYK2. Therefore, this study is the first to demonstrate that GSK3beta is a substrate of PYK2 both in vitro and in situ.     

Axin negatively affects tau phosphorylation by glycogen synthase kinase 3beta

Glycogen synthase kinase 3beta (GSK3beta) is an essential protein kinase that regulates numerous functions within the cell. One critically important substrate of GSK3beta is the microtubule-associated protein tau. Phosphorylation of tau by GSK3beta decreases tau-microtubule interactions. In addition to phosphorylating tau, GSK3beta is a downstream regulator of the wnt signaling pathway, which maintains the levels of beta-catenin. Axin plays a central role in regulating beta-catenin levels by bringing together GSK3beta and beta-catenin and facilitating the phosphorylation of beta-catenin, targeting it for ubiquitination and degradation by the proteasome. Although axin clearly facilitates the phosphorylation of beta-catenin, its effects on the phosphorylation of other GSK3beta substrates are unclear. Therefore in this study the effects of axin on GSK3beta-mediated tau phosphorylation were examined. The results clearly demonstrate that axin is a negative regulator of tau phosphorylation by GSK3beta. This negative regulation of GSK3beta-mediated tau phosphorylation is due to the fact that axin efficiently binds GSK3beta but not tau and thus sequesters GSK3beta away from tau, as an axin mutant that does not bind GSK3beta did not inhibit tau phosphorylation by GSK3beta. This is the first demonstration that axin negatively affects the phosphorylation of a GSK3beta substrate, and provides a novel mechanism by which tau phosphorylation and function can be regulated within the cell.     

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.     

Structural basis for recruitment of glycogen synthase kinase 3beta to the axin-APC scaffold complex

Glycogen synthase kinase 3beta (GSK3beta) is a serine/threonine kinase involved in insulin, growth factor and Wnt signalling. In Wnt signalling, GSK3beta is recruited to a multiprotein complex via interaction with axin, where it hyperphosphorylates beta-catenin, marking it for ubiquitylation and destruction. We have now determined the crystal structure of GSK3beta in complex with a minimal GSK3beta-binding segment of axin, at 2.4 A resolution. The structure confirms the co-localization of the binding sites for axin and FRAT in the C-terminal domain of GSK3beta, but reveals significant differences in the interactions made by axin and FRAT, mediated by conformational plasticity of the 285-299 loop in GSK3beta. Detailed comparison of the axin and FRAT GSK3beta complexes allows the generation of highly specific mutations, which abrogate binding of one or the other. Quantitative analysis suggests that the interaction of GSK3beta with the axin scaffold enhances phosphorylation of beta-catenin by >20 000-fold.     

Glycogen synthase kinase (GSK) 3beta directly phosphorylates Serine 212 in the regulatory loop and inhibits microtubule affinity-regulating kinase (MARK) 2

MARK/Par-1, a kinase family with diverse functions particularly in inducing cell polarity, can phosphorylate microtubule-associated proteins in their repeat domain and cause their detachment from microtubules, and thereby microtubule destabilization. Because of its role in abnormal phosphorylation of the Tau protein in Alzheimer disease, we searched for regulatory kinases. MARK family kinases can be activated by phosphorylation of a conserved threonine (Thr-208 in MARK2), and inactivated by phosphorylation of a serine (Ser-212), both in the activation loop of the catalytic domain. Activation is achieved by the kinases MARKK/TAO1 or LKB1, although the inactivating kinase was unknown. We show here that GSK3beta serves the role of the inhibitory kinase. Because GSK3beta can also phosphorylate Tau at sites outside the repeat domain, the activation of GSK3beta, and concomitant inactivation of MARK can shift the pattern of pathological phosphorylation of Tau protein in Alzheimer disease.     

Structures of Escherichia coli branched-chain amino acid aminotransferase and its complexes with 4-methylvalerate and 2-methylleucine: induced fit and substrate recognition of the enzyme.

The following three-dimensional structures of three forms of Escherichia coli branched-chain amino acid aminotransferase (eBCAT) have been determined by the X-ray diffraction method: the unliganded pyridoxal 5'-phosphate (PLP) form at a 2.1 A resolution, and the two complexes with the substrate analogues, 4-methylvalerate (4-MeVA) as the Michaelis complex model and 2-methylleucine (2-MeLeu) as the external aldimine model at 2.4 A resolution. The enzyme is a trimer of dimers, and each subunit consists of small and large domains, and the interdomain loop. The active site is formed by the residues at the domain interface and those from two loops of the other subunit of the dimer unit, and binds one PLP with its re-face directed toward the protein side. Upon binding of a substrate, Arg40 changes its side-chain direction to interact with the interdomain loop, and the loop, which is disordered in the unliganded form, shows its ordered structure on the active-site cavity, interacts with the hydrophobic side chain of the substrate, and shields it from the solvent region. The substrate binds to the active-site pocket with its alpha-hydrogen toward the protein side, its side-chain on the side of O3 of PLP, and its alpha-carboxylate on the side of the phosphate group of PLP. The hydrophobic side-chain of the substrate is recognized by Phe36, Trp126, Tyr129, Tyr164, Tyr31*, and Val109*. The alpha-carboxylate of the substrate binds to the unique site constructed by three polar groups (two main-chain NH groups of the beta-turn at Thr257 and Ala258 and the hydroxy group of Tyr95) which are activated by the access of Arg40 to the main-chain C=O group of the beta-turn and the coordination of Arg97 to the hydroxy group. Since Arg40 is the only residue that significantly changes its side-chain conformation and directly interacts with the interdomain loop and the beta-turn, the residue plays important roles in the induced fit of the interdomain loop and the alpha-carboxylate recognition of the substrate.     

GSK3beta-Mediated Drp1 Phosphorylation Induced Elongated Mitochondrial Morphology against Oxidative Stress

Multiple phosphorylation sites of Drp1 have been characterized for their functional importance. However, the functional consequence of GSK3beta-mediated phosphorylation of Drp1 remains unclear. In this report, we pinpointed 11 Serine/Threonine sites spanning from residue 634∼736 of the GED domain and robustly confirmed Drp1 Ser693 as a novel GSK3beta phosphorylation site. Our results suggest that GSK3beta-mediated phosphorylation at Ser693 does cause a dramatic decrease of GTPase activity; in contrast, GSK3beta-mediated phosphorylation at Ser693 appears not to affect Drp1 inter-/intra-molecular interactions. After identifying Ser693 as a GSK3beta phosphorylation site, we also determined that K679 is crucial for GSK3beta-binding, which strongly suggests that Drp1 is a novel substrate for GSK3beta. Thereafter, we found that overexpressed S693D, but not S693A mutant, caused an elongated mitochondrial morphology which is similar to that of K38A, S637D and K679A mutants. Interestedly, using H89 and LiCl to inhibit PKA and GSK3beta signaling, respectively, it appears that a portion of the elongated mitochondria switched to a fragmented phenotype. In investigating the biofunctionality of phosphorylation sites within the GED domain, cells overexpressing Drp1 S693D and S637D, but not S693A, showed an acquired resistance to H₂O₂-induced mitochondrial fragmentation and ensuing apoptosis, which affected cytochrome c, capase-3, -7, and PARP, but not LC3B, Atg-5, Beclin-1 and Bcl2 expressions. These results also showed that the S693D group is more effective in protecting both non-neuronal and neuronal cells from apoptotic death than the S637D group. Altogether, our data suggest that GSK3beta-mediated phosphorylation at Ser693 of Drp1 may be associated with mitochondrial elongation via down-regulating apoptosis, but not autophagy upon H₂O₂ insult.     

Central role of glycogen synthase kinase-3beta in endoplasmic reticulum stress-induced caspase-3 activation

Stress of the endoplasmic reticulum (ER), which is associated with many neurodegenerative conditions, can lead to the elimination of affected cells by apoptosis through only partially understood mechanisms. Thapsigargin, which causes ER stress by inhibiting the ER Ca(2+)-ATPase, was found to not only activate the apoptosis effector caspase-3 but also to cause a large and prolonged increase in the activity of glycogen synthase kinase-3beta (GSK3beta). Activation of GSK3beta was obligatory for thapsigargin-induced activation of caspase-3, because inhibition of GSK3beta by expression of dominant-negative GSK3beta or by the GSK3beta inhibitor lithium blocked caspase-3 activation. Thapsigargin treatment activated GSK3beta by inducing dephosphorylation of phospho-Ser-9 of GSK3beta, a phosphorylation that normally maintains GSK3beta inactivated. Caspase-3 activation induced by thapsigargin was blocked by increasing the phosphorylation of Ser-9-GSK3beta with insulin-like growth factor-1 or with the phosphatase inhibitors okadaic acid and calyculin A, but the calcineurin inhibitors FK506 and cyclosporin A were ineffective. Insulin-like growth factor-1, okadaic acid, calyculin A, and lithium also protected cells from two other inducers of ER stress, tunicamycin and brefeldin A. Thus, ER stress activates GSK3beta through dephosphorylation of phospho-Ser-9, a prerequisite for caspase-3 activation, and this process is amenable to pharmacological intervention.     

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.     

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.     

The PPLA motif of glycogen synthase kinase 3beta is required for interaction with Fe65

Glycogen synthase kinase 3beta (GSK 3 beta) is a serine/ threonine kinase that phosphorylates substrates such as beta-catenin and is involved in a variety of biological processes, including embryonic development, metabolism, tumorigenesis, and cell death. Here, we present evidence that human GSK 3beta is associated with Fe65, which has the characteristics of an adaptor protein, possessing a WW domain, and two phosphotyrosine interaction domains, PID1 and PID2. The GSK 3beta catalytic domain also contains a putative WW domain binding motif ((371)PPLA(374)), and we observed, using a pull down approach and co-immuno-precipitation, that it interacts physically with Fe65 via this motif. In addition, we detected co-localization of GSK 3beta and Fe65 by confocal microscopy, and this co-localization was disrupted by mutation of the putative WW domain binding motif of GSK 3beta.Finally, in transient transfection assays interaction of GSK 3 beta (wt) with Fe65 induced substantial cell apoptosis, whereas interaction with the GSK 3beta AALA mutant ((371)AALA(374)) did not, and we noted that phosphorylation of the Tyr 216 residue of the GSK 3beta AALA mutant was significantly reduced compared to that of GSK 3beta wild type. Thus, our observations indicate that GSK 3beta binds to Fe65 through its (371)PPLA(374) motif and that this interaction regulates apoptosis and phosphorylation of Tyr 216 of GSK 3beta.     

Crystal structure of N-acetyl-gamma-glutamyl-phosphate reductase from Mycobacterium tuberculosis in complex with NADP(+)

The enzyme N-acetyl-gamma-glutamyl-phosphate reductase (AGPR) catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductive dephosphorylation of N-acetyl-gamma-glutamyl-phosphate to N-acetylglutamate-gamma-semialdehyde. This reaction is part of the arginine biosynthetic pathway that is essential for some microorganisms and plants, in particular, for Mycobacterium tuberculosis (Mtb). The structures of apo MtbAGPR in the space groups P2(1)2(1)2(1) and C2 and the structure of MtbAGPR bound to the cofactor NADP(+) have been solved and analyzed. Each MtbAGPR subunit consists of alpha/beta and alpha+beta domains; NADP(+) is bound in the cleft between them. The hydrogen bonds and hydrophobic contacts between the enzyme and cofactor have been examined. Comparison of the apo and the bound enzyme structures has revealed a conformational change in MtbAGPR upon NADP(+) binding. Namely, a loop (Leu88 to His92) moves more than 5 A to confine sterically the cofactor's adenine moiety in a hydrophobic pocket. To identify the catalytically important residues in MtbAGPR, a docking of the substrate to the enzyme has been performed using the present structure of the MtbAGPR/NADP(+) complex. It reveals that residues His217 and His219 could form hydrogen bonds with the docked substrate. In addition, an ion pair could form between the substrate phosphate group and the guanidinium group of Arg114. These interactions optimally place and orient the substrate for subsequent nucleophilic attack by Cys158 on the substrate gamma-carboxyl group. His219 is the most probable general base to accept a proton from Cys158 and an adjacent ion pair interaction with the side-chain carboxyl group of Glu222 could help to stabilize the resulting positive charge on His219. For this catalytic triad to function efficiently it requires a small conformational change of the order of 1 A in the loop containing His217 and His219; this could easily result from the substrate binding.     

Glycogen synthase kinase 3beta interacts with and phosphorylates the spindle-associated protein astrin

Emerging evidence shows that glycogen synthase kinase 3beta (GSK3beta) is involved in mitotic division and that inhibiting of GSK3beta kinase activity causes defects in spindle microtubule length and chromosome alignment. However, the purpose of GSK3beta involvement in spindle microtubule assembly and accurate chromosome segregation remains obscure. Here, we report that GSK3beta interacts with the spindle-associated protein Astrin both in vitro and in vivo. Additionally, Astrin acts as a substrate for GSK3beta and is phosphorylated at Thr-111, Thr-937 ((S/T)P motif) and Ser-974/Thr-978 ((S/T)XXX(S/T)-p motif; p is a phosphorylatable residue). Inhibition of GSK3beta impairs spindle and kinetochore accumulation of Astrin and spindle formation at mitosis, suggesting that Astrin association with the spindle microtubule and kinetochore may be dependent on phosphorylation by GSK3beta. Conversely, depletion of Astrin by small interfering RNA has no detectable influence on the localization of GSK3beta. Interestingly, in vitro assays demonstrated that Astrin enhances GSK3beta-mediated phosphorylation of other substrates. Moreover, we showed that coexpression of Astrin and GSK3beta differentially increases GSK3beta-mediated Tau phosphorylation on an unprimed site. Collectively, these data indicate that GSK3beta interacts with and phosphorylates the spindle-associated protein Astrin, resulting in targeting Astrin to the spindle microtubules and kinetochores. In turn, the GSK3beta-Astrin complex may also facilitate further physiological and pathological phosphorylation.     

Glycogen synthase kinase 3beta together with 14-3-3 protein regulates diabetic cardiomyopathy: effect of losartan and tempol

Glycogen synthase kinase (GSK) 3beta is a multifunctional protein that positively regulates myocardial apoptosis and negatively regulates hypertrophy. However, the role of GSK3beta in the diabetic myocardium is largely unknown. We found that GSK3beta became more active (less phosphorylated at serine 9) via decreased Akt phosphorylation, in parallel to c-Jun NH2 terminal kinase activation, which correlated with increased activated caspase 3 and myocardial apoptosis 3 days after streptozotocin (STZ) injection in mice. However, 28 days after STZ injection, GSK3beta became inactive, which correlated with the enhanced protein kinase C beta2 and p38 mitogen activated protein kinase expression, nuclear translocation of nuclear factor of activated T cells c3, cardiac hypertrophy and fibrosis. All of the above parameters were exacerbated in dominant-negative 14-3-3 transgenic mice. Our results suggest that GSK3beta together with 14-3-3 protein plays essential roles in the signaling of diabetic cardiomyopathy, and treatment with either losartan or tempol prevents these changes.