Glycogen synthase kinase 3beta phosphorylates p21WAF1/CIP1 for proteasomal degradation after UV irradiation

UV irradiation has been reported to induce p21(WAF1/CIP1) protein degradation through a ubiquitin-proteasome pathway, but the underlying biochemical mechanism remains to be elucidated. Here, we show that ser-114 phosphorylation of p21 protein by glycogen synthase kinase 3beta (GSK-3beta) is required for its degradation in response to UV irradiation and that GSK-3beta activation is a downstream event in the ATR signaling pathway triggered by UV. UV transiently increased GSK-3beta activity, and this increase could be blocked by caffeine or by ATR small interfering RNA, indicating ATR-dependent activation of GSK-3beta. ser-114, located within the putative GSK-3beta target sequence, was phosphorylated by GSK-3beta upon UV exposure. The nonphosphorylatable S114A mutant of p21 was protected from UV-induced destabilization. Degradation of p21 protein by UV irradiation was independent of p53 status and prevented by proteasome inhibitors. In contrast to the previous report, the proteasomal degradation of p21 appeared to be ubiquitination independent. These data show that GSK-3beta is activated by UV irradiation through the ATR signaling pathway and phosphorylates p21 at ser-114 for its degradation by the proteasome. To our knowledge, this is the first demonstration of GSK-3beta as the missing link between UV-induced ATR activation and p21 degradation.     

Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.     

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 3-dependent phosphorylation of Mdm2 regulates p53 abundance

The Mdm2 oncoprotein regulates abundance and activity of the p53 tumor suppressor protein. For efficient degradation of p53, Mdm2 needs to be phosphorylated at several contiguous residues within the central conserved domain. We show that glycogen synthase kinase 3 (GSK-3) phosphorylated the Mdm2 protein in vitro and in vivo in the central domain. Inhibition of GSK-3 rescued p53 from degradation in an Mdm2-dependent manner while its association with Mdm2 was not affected. Likewise, inhibition of GSK-3 did not alter localization of p53 and Mdm2 or the interaction of Mdm2 and MdmX. Ionizing radiation, which leads to p53 accumulation, directed phosphorylation of GSK-3 at serine 9, which preceded and overlapped with the increase in p53 levels. Moreover, expression of a GSK-3 mutant where serine 9 was replaced with an alanine reduced the accumulation of p53 and induction of its target p21(WAF-1). We therefore conclude that inhibition of GSK-3 contributes to hypophosphorylation of Mdm2 in response to ionizing rays, and in consequence to p53 stabilization.     

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.     

Glycogen synthase kinase 3beta phosphorylates Alzheimer's disease-specific Ser396 of microtubule-associated protein tau by a sequential mechanism

Phosphorylation of tau on S(396) was suggested to be a key step in the development of neurofibrillary pathology in Alzheimer's disease brain [Bramblett, G. T., Goedert, M., Jacks, R., Merrick, S. E., Trojanowski, J. Q., and Lee, V. M.-Y. (1993) Neuron 10, 1089-1099]. GSK3beta phosphorylates Ser(396) of tau in the brain by a mechanism which is not clear. In this study, when HEK-293 cells were cotransfected with tau and GSK3beta, GSK3beta co-immunoprecipitated with tau and phosphorylated tau on S(202), T(231), S(396), and S(400) but not on S(262), S(235), and S(404). Blocking phosphorylation on T(231), S(235), S(396), S(400), or S(404) did not prevent the subsequent phosphorylation on S(202) by GSK3beta. These data suggest that GSK3beta directly phosphorylates tau on S(202) (without requiring prephosphorylation). However, preventing phosphorylation on S(235), S(400), and S(404) prevented GSK3beta-dependent phosphorylation of T(231), S(396), and S(400), respectively. This indicates that phosphorylation of T(231), S(396), and S(400) by GSK3beta depends on a previous phosphorylation of S(235), S(400), and S(404), respectively. To examine S(396) phosphorylation, we analyzed phosphorylation of S(396), S(400), and S(404). Blocking phosphorylation of S(404) prevented the subsequent GSK3beta-dependent phosphorylation of both S(400) and S(396). When phosphorylation of S(404) was allowed but S(400) blocked, GSK3beta failed to phosphorylate S(396). Thus, GSK3beta phosphorylates S(396) by a two-step mechanism. In the first step, GSK3beta phosphorylates S(400) of previously S(404)-phosphorylated tau. This event primes tau for second-step phosphorylation of S(396) by GSK3beta. We conclude that GSK3beta phosphorylates tau directly at S(202) but requires the previous phosphorylation on S(235) to phosphorylate T(231). Phosphorylation of S(396), on the other hand, occurs sequentially. Once a priming kinase phosphorylates S(404), GSK3beta sequentially phosphorylates S(400) and then S(396).     

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.     

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.     

Glycogen synthase kinase-3 beta regulates NF-kappa B1/p105 stability

A number of different kinases have been implicated in NF-kappa B regulation and survival function. Here we investigated the molecular cross-talk between glycogen synthase kinase-3 beta (GSK-3 beta) and the p105 precursor of the NF-kappa B p50 subunit. GSK-3 beta forms an in vivo complex with and specifically phosphorylates NF-kappa B1/p105 at Ser-903 and Ser-907 in vitro. In addition, the p105 phosphorylation level is reduced in fibroblasts lacking GSK-3 beta as compared with wild-type cells. GSK-3 beta has a dual effect on p105: it stabilizes p105 under resting conditions and primes p105 for degradation upon tumor necrosis factor (TNF)-alpha treatment. Indeed, constitutive processing of p105 to p50 occurs at a higher rate in cells lacking GSK-3 beta with respect to wild-type cells and can be reduced upon reintroduction of GSK-3 beta by transfection. Moreover, p105 degradation in response to TNF-alpha is prevented in GSK-3 beta-/- fibroblasts and by a Ser to Ala point mutation on p105 at positions 903 or 907. Interestingly, the increased sensitiveness to TNF-alpha-induced death occurring in GSK-3 beta-/- fibroblasts, which is coupled to a perturbation of p50/105 ratio, can be reproduced by p105 silencing in wild-type fibroblasts.     

Glycogen synthase kinase 3alpha-specific regulation of murine hepatic glycogen metabolism

Glycogen synthase kinase 3 comprises two isoforms (GSK-3alpha and GSK-3beta) that are implicated in type II diabetes, neurodegeneration, and cancer. GSK-3 activity is elevated in human and rodent models of diabetes, and various GSK-3 inhibitors improve glucose tolerance and insulin sensitivity in rodent models of obesity and diabetes. Here, we report the generation of mice lacking GSK-3alpha. Unlike GSK-3beta mutants, which die before birth, GSK-3alpha knockout (GSK-3alpha KO) animals are viable but display enhanced glucose and insulin sensitivity accompanied by reduced fat mass. Fasted and glucose-stimulated hepatic glycogen content was enhanced in GSK-3alpha KO mice, whereas muscle glycogen was unaltered. Insulin-stimulated protein kinase B (PKB/Akt) and GSK-3beta phosphorylation was higher in GSK-3alpha KO livers compared to wild-type littermates, and IRS-1 expression was markedly increased. We conclude that GSK-3 isoforms exhibit tissue-specific physiological functions and that GSK-3alpha KO mice are insulin sensitive, reinforcing the potential of GSK-3 as a therapeutic target for type II diabetes.     

Glycogen synthase kinase 3 beta induces caspase-cleaved tau aggregation in situ

Tau is a substrate of caspases, and caspase-cleaved tau has been detected in Alzheimer's disease brain but not in control brain. Furthermore, in vitro studies have revealed that caspase-cleaved tau is more fibrillogenic than full-length tau. Considering these previous findings, the purpose of this study was to determine how the caspase cleavage of tau affected tau function and aggregation in a cell model system. The effects of glycogen synthase kinase 3 beta (GSK3 beta), a well established tau kinase, on these processes also were examined. Tau or tau that had been truncated at Asp-421 to mimic caspase cleavage (Tau-D421) was transfected into cells with or without GSK3 beta, and phosphorylation, microtubule binding, and tau aggregation were examined. Tau-D421 was not as efficiently phosphorylated by GSK3 beta as full-length tau. Tau-D421 efficiently bound microtubules, and in contrast to the full-length tau, co-expression with GSK3 beta did not result in a reduction in the ability of Tau-D421 to bind microtubules. In the absence of GSK3 beta, neither Tau-D421 nor full-length tau formed Sarkosyl-insoluble inclusions. However, in the presence of GSK3 beta, Tau-D421, but not full-length tau, was present in the Sarkosyl-insoluble fraction and formed thioflavin-S-positive inclusions in the cell. Nonetheless, co-expression of GSK3 beta and Tau-D421 did not result in an enhancement of cell death. These data suggest that a combination of phosphorylation events and caspase activation contribute to the tau oligomerization process in Alzheimer's disease, with GSK3 beta-mediated tau phosphorylation preceding caspase cleavage.     

Glycogen synthase kinase 3: a drug target for CNS therapies

Abstract Glycogen synthase kinase3 (GSK3) is emerging as a prominent drug target in the CNS. The most exciting of the possibilities of GSK3 lies within the treatment of Alzheimer's disease (AD) where abnormal increases in GSK3 levels and activity have been associated with neuronal death, paired helical filament tau formation and neurite retraction as well as a decline in cognitive performance. Abnormal activity of GSK3 is also implicated in stroke. Lithium, a widely used drug for affective disorders, inhibits GSK3 at therapeutically relevant concentrations. Thus while the rationale remains testable, pharmaceutical companies are investing in finding a selective inhibitor of GSK3. In the present review, we summarize the properties of GSK3, and discuss the potential for such a therapy in AD, and other CNS disorders.