Glycogen synthase kinases. Classification of a rabbit liver casein and glycogen synthase kinase (casein kinase-1) as a distinct enzyme

A protein kinase, able to phosphorylate casein, phosvitin, and glycogen synthase, was purified approximately 9000-fold from rabbit liver, and appeared analogous to an enzyme studied by Itarte and Huang (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057). This enzyme, designated here casein kinase-1, was shown to be a distinct glycogen synthase kinase and in particular to be different from the protein kinase GSK-3 (Hemmings, B.A., Yellowlees, D., Kernohan, J.C., and Cohen, P. (1981) Eur. J. Biochem. 119, 443-451). Casein kinase-1 had native molecular weight of 30,000 as judged by gel filtration. The enzyme phosphorylated beta-casein A or B better than kappa-casein or alpha s1-casein, and modified only serine residues in beta-casein B and phosvitin. The apparent Km for ATP was 11 microM, and GTP was ineffective as a phosphoryl donor. The phosphorylation of glycogen synthase by casein kinase-1 was inhibited by glycogen, half-maximally at 2 mg/ml, and by heparin, half-maximally at 0.5-1.0 microgram/ml, but was unaffected by Ca2+ and/or calmodulin, or by cyclic AMP. Phosphorylation of muscle glycogen synthase proceeded to a stoichiometry of at least 6 phosphates/subunit with reduction in the +/- glucose-6-P activity ratio to less than 0.4. Phosphate was introduced into both a COOH-terminal CNBr fragment (CB-2) as well as a NH2-terminal fragment (CB-1). At a phosphorylation stoichiometry of 6 phosphates/subunit, 84% of the phosphate was associated with CB-2 and 6.5% with CB-1. The remainder of the phosphate was introduced into another CNBr fragment of apparent molecular weight 16,500. Phosphorylation by casein kinase-1 correlated with reduced electrophoretic mobilities, as analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate, of the intact glycogen synthase subunit, as well as the CNBr fragments CB-1 and CB-2.     

Phosphorylation of calcineurin by glycogen synthase (casein) kinase-1

A previous study demonstrated that calcineurin preparations contain variable amounts of endogenous phosphate. This observation suggests that calcineurin may be regulated by protein phosphorylation. In this study we have used calcineurin as a potential substrate for eight different protein kinases and significant phosphorylation was observed only with glycogen synthase (casein) kinase-1 (CK-1). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that only subunit A of calcineurin was phosphorylated. The incorporation of 32P into calcineurin catalyzed by CK-1 ranged from 0.4 to 1.5 mol, depending on the preparation of the substrate used. Peptide mapping revealed that two major sites on calcineurin were phosphorylated. No change in calcineurin activity was observed as a result of phosphorylation. The results of this study suggest that CK-1 may be responsible for phosphorylating calcineurin in vivo.     

Inhibition of glycogen synthase (casein) kinase-1 by heparin

Previous reports have shown that heparin is an inhibitor of casein kinase-2 (CK-2). It is unclear whether heparin is also an inhibitor of glycogen synthase (casein) kinase-1 (CK-1), a type 1 casein kinase. In this study it is shown that CK-1 is potently inhibited by heparin when phosvitin or calcineurin are used as substrates. With casein as a substrate, however, the kinase is insensitive to inhibition by heparin. Using phosvitin as a substrate half-maximal inhibition of CK-1 was observed with 0.14 microgram/ml heparin. Kinetic analyses indicate that at a constant concentration (0.10 mM) of ATP the Km of CK-1 for phosvitin is increased eightfold in the presence of 0.9 microgram/ml heparin; the Vmax is unchanged with or without heparin. At a constant concentration of phosvitin (4 mg/ml) heparin (0.9 microgram/ml) decreased the Vmax for ATP by 57%; the Km is unchanged with or without heparin. The inhibition of CK-1 by heparin can be reversed by KCl (greater than 100 mM). These results indicate that heparin is a potent inhibitor not only of CK-2 but also of CK-1. Hence heparin inhibition can no longer be arbitrarily used as a criterion to discriminate between these kinases.     

Inhibition of glycogen synthase (casein) kinase-1 by divalent metal ions

The specificity of glycogen synthase (casein) kinase-1 (CK-1) for different divalent metal ions was explored in this study. Of nine metal ions (Mg2+, Mn2+, Zn2+, Cu2+, Ca2+, Ba2+, Ni2+, Co2+, Fe2+) tested, only Mg2+ supported significant kinase activity. Several of the other metals, however, inhibited the Mg2+-stimulated kinase activity. Half-maximal inhibitions by Mn2+, Zn2+, Co2+, Fe2+, and Ni2+ were observed at 55, 65, 110, 125, and 284 microM, respectively. Kinetic analyses indicate that the metal ions are acting as competitive inhibitors of CK-1 with respect to the protein substrate (casein) and as noncompetitive inhibitors with respect to the nucleotide substrate (ATP). The inhibition of CK-1 by the different metal ions can be reversed by EGTA.     

Molecular imaging of glycogen synthase kinase-3β and casein kinase-1α kinases

Glycogen synthase kinase-3β (GSK3β) and casein kinase-1α (CK1α) are multifunctional kinases that play critical roles in the regulation of a number of cellular processes. In spite of their importance, molecular imaging tools for noninvasive and real-time monitoring of their kinase activities have not been devised. Here we report development of the bioluminescent GSK3β and CK1α reporter (BGCR) based on firefly luciferase complementation. Treatment of SW620 cells stably expressing the reporter with inhibitors of GSK3β (SB415286 and LiCl) or CK1α (CKI-7) resulted in dose- and time-dependent increases in BGCR activity that were validated using Western blotting. No increase in bioluminescence was observed in the case of S37A mutant (GSK3β inhibitors) or S45A mutant (CKI-7), demonstrating the specificity of the reporter. Imaging of mice tumor xenograft generated with BGCR-expressing SW620 cells following treatment with LiCl showed unique oscillations in GSK3β activity that were corroborated by phosphorylated GSK3β immunoblotting. Taken together, the BGCR is a novel molecular imaging tool that reveals unique insight into GSK3β and CK1α kinase activities and may provide a powerful tool in experimental therapeutics for rapid optimization of dose and schedule of targeted therapies and for monitoring therapeutic response.     

Tissue distribution and subcellular localization of hyaluronan synthase isoenzymes

Hyaluronan synthases (HAS) are unique plasma membrane glycosyltransferases secreting this glycosaminoglycan directly to the extracellular space. The three HAS isoenzymes (HAS1, HAS2, and HAS3) expressed in mammalian cells differ in their enzymatic properties and regulation by external stimuli, but clearly distinct functions have not been established. To overview the expression of different HAS isoenzymes during embryonic development and their subcellular localization, we immunostained mouse embryonic samples and cultured cells with HAS antibodies, correlating their distribution to hyaluronan staining. Their subcellular localization was further studied by GFP–HAS fusion proteins. Intense hyaluronan staining was observed throughout the development in the tissues of mesodermal origin, like heart and cartilages, but also for example during the maturation of kidneys and stratified epithelia. In general, staining for one or several HASs correlated with hyaluronan staining. The staining of HAS2 was most widespread, both spatially and temporally, correlating with hyaluronan staining especially in early mesenchymal tissues and heart. While epithelial cells were mostly negative for HASs, stratified epithelia became HAS positive during differentiation. All HAS isoenzymes showed cytoplasmic immunoreactivity, both in tissue sections and cultured cells, while plasma membrane staining was also detected, often in cellular extensions. HAS1 had brightest signal in Golgi, HAS3 in Golgi and microvillous protrusions, whereas most of the endogenous HAS2 immunoreactivity was localized in the ER. This differential pattern was also observed with transfected GFP–HASs. The large proportion of intracellular HASs suggests that HAS forms a reserve that is transported to the plasma membrane for rapid activation of hyaluronan synthesis.     

Phosphorylation and inactivation of rabbit skeletal muscle glycogen synthase: distinction between kinase Fa-, phosphorylase kinase-, and glycogen synthase (casein) kinase-1-catalyzed reactions

Rabbit skeletal muscle glycogen synthase was phosphorylated by kinase Fa, phosphorylase kinase, and cAMP-independent synthase (casein) kinase-1 to determine the differences among these kinase-catalyzed reactions. The stoichiometry of phosphate incorporation, the extent of inactivation, and the sites of phosphorylation were compared. Synthase (casein) kinase-1 catalyzes the highest level of synthase phosphorylation (4 mol/subunit) and inactivation (reduction of the activity ratio to below 0.05). The sites, defined by characteristic tryptic peptides, phosphorylated by synthase (casein) kinase-1 are distinguishable from those by kinase Fa and phosphorylase kinase. In addition, synthase (casein) kinase-1, unlike kinase Fa, does not activate ATP X Mg2+-dependent protein phosphatase. These results demonstrate that synthase (casein) kinase-1 is a distinct glycogen synthase kinase.     

Glycogen synthase kinase-3beta regulates presenilin 1 C-terminal fragment levels

The majority of familial Alzheimer's disease cases have been attributed to mutations in the presenilin 1 (PS1) gene. PS1 is synthesized as an inactive holoprotein that undergoes endoproteolytic processing to generate a functional N- and C-terminal heterodimer (NTF and CTF, respectively). We identified a single residue in PS1, Ser(397), which regulates the CTF levels in a population of dimer that has a rapid turnover. This residue is part of a highly conserved glycogen synthase kinase-3beta (GSK-3beta) consensus phosphorylation site within the loop domain of PS1. Site-directed mutagenesis at the Ser(397) position increased levels of PS1 CTF but not NTF or holoprotein. Similar increases in only CTF levels were seen when cells expressing wild type PS1 were treated with lithium chloride, an inhibitor of GSK-3beta. Both wild type and PS1 S397A CTF displayed a biphasic turnover, reflecting rapidly degraded and stable populations. Rapid turnover was delayed for mutant PS1 S397A, causing increased CTF. These data demonstrate that PS1 NTF.CTF endoproteolytic fragments are generated in excess, that phosphorylation at Ser(397) by GSK-3beta regulates the discard of excess CTF, and that the disposal of surplus NTF is mediated by an independent mechanism. Overall, the results indicate that production of active NTF.CTF dimer is more complex than limited endoproteolysis of PS1 holoprotein and instead involves additional regulatory events.     

Ribofuranosyl-benzimidazole derivatives as inhibitors of casein kinase-2 and casein kinase-1

5,6-Dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DiCl-RB) is a powerful inhibitor of casein kinase-2 (CK-2) [Zandomeni, R. et al. (1986) J. Biol. Chem. 261, 3414-3420]. Here a series of 17 analogues of DiCl-RB has been employed for studying the specificity and the mode of action of this family of CK-2 inhibitors. The two halogen substituents on the benzene ring are shown to play a prominent role in inhibition, the 5,6-dibromo derivative (DiBr-RB) being fivefold more effective than DiCl-RB (Ki = 2 microM, with GTP as substrate), whereas the difluoro derivative (DiF-RB) is nearly as ineffective as unsubstituted 1-(beta-D-ribofuranosyl)benzimidazole. On the other hand, although some modifications of the ribose group significantly decrease the inhibitory efficiency, the sugar moiety is not strictly required, since dichlorobenzimidazole itself (DiCl-Bz) is an inhibitor almost as good as DiCl-RB. Inhibition of CK-2 by DiCl-RB and by its analogues, DiCl-Bz included, is of the competitive type with respect to the nucleotide substrate, the Ki values being lower with GTP than with ATP. The Ki values of the most potent inhibitor, DiBr-RB, with ATP and GTP, are 6 microM and 2 microM, respectively, denoting an affinity for the enzyme higher than that of the physiological substrates, ATP and GTP. DiBr-RB has been assayed for its inhibitory capacity toward several protein kinase other than CK-2. Protein kinase-C, cAMP-dependent protein kinase, the Ser/Thr protein kinase expressed by Pseudorabies virus, and four different tyrosine protein kinases from spleen, proved insensitive to DiBr-RB concentrations capable of almost entirely suppressing the activity of rat liver and maize seedling CK-2. Casein kinase-1 however is nearly as sensitive as CK-2 to DiBr-RB. Inhibition of CK-1 is also of the competitive type with respect to ATP (Ki = 14 microM). Although the inhibitory spectrum of CK-1 by the various analogues is reminiscent of that observed with CK-2, a remarkable difference is revealed by 5'-phosphorylation of ribose which increases the Ki with CK-2 while decreasing that with CK-1.     

Glycogen synthase kinase-3 signaling in Alzheimer's disease

Alzheimer's disease (AD) is the most common form of neurodegenerative disorder with dementia, accounting for approximately 70% of the all cases. Currently, 5.8 million people in the U.S. are living with AD and by 2050 this number is expected to double resulting in a significant socio-economic burden. Despite intensive research, the exact mechanisms that trigger AD are still not known and at the present there is no cure for it. In recent years, many signaling pathways associated with AD neuropathology have been explored as possible candidate targets for the treatment of this condition including glycogen synthase kinase-3β (GSK3-β). GSK3-β is considered a key player in AD pathophysiology since dysregulation of this kinase influences all the major hallmarks of the disease including: tau phosphorylation, amyloid-β production, memory, neurogenesis and synaptic function. The present review summarizes the current understanding of the GSK3-β neurobiology with particular emphasis on its effects on specific signaling pathways associated with AD pathophysiology. Moreover, it discusses the feasibility of targeting GSK3-β for AD treatment and provides a summary of the current research effort to develop GSK3-β inhibitors in preclinical and clinical studies.     

Molecular identification, tissue distribution and subcellular localization of mST3GalV/GM3 synthase

A molecular screen for a mouse homologue of a Drosophila carbohydrate binding protein, called Gliolectin, yielded a cDNA encoding mST3GalV/GM3 synthase (CMP-NeuAc: lactosylceramide alpha2, 3-sialyltransferase). By in situ hybridization and immunohistochemistry, mST3GalV exhibits differential expression in neural and non-neural tissues. Although expressed by all neurons in the central nervous system, neuronal populations that contribute their axons to myelinated efferent projections, such as cerebellar Purkinje cells and spinal motorneurons, demonstrate the highest ST3GalV expression. When stained with anti-mST3GalV antiserum (designated CS2), subpopulations of neurons display an elaborate Golgi apparatus, frequently extending into one or more dendritic processes. The extended spatial distribution of the neuronal Golgi apparatus, particularly in spinal motorneurons, allowed the confocal immunohistochemical colocalization of mST3GalV with markers for medial/trans-Golgi but not the cis-Golgi or trans-Golgi network, consistent with previous observations suggesting that ganglioside glycosyltransferases are enriched in late Golgi compartments. Among non-neural tissues, liver and testes demonstrate cell-type specific CS2 staining. In liver, endothelial cells lining a ring of sinusoids, concentric with the central vein, express mST3GalV. Kupffer cells are also stained with CS2 antiserum but hepatocyte expression is undetectable. In the seminiferous tubules of the testes, ST3GalV is found in somatic (Leydig, Sertoli) and early germline cells (spermatogonia and primary spermatocytes); the epididymal epithelium exhibits intense ST3GalV expression. Since GM3 is a precursor for the synthesis of a- and b-series gangliosides, the range of mST3GalV/GM3 synthase expression among various cell populations indicates that certain cell types possess greater reliance on ganglioside function than others.     

Multisite phosphorylation of glycogen synthase. Molecular basis for the substrate specificity of glycogen synthase kinase-3 and casein kinase-II (glycogen synthase kinase-5)

Glycogen synthase kinase-3 was isolated from rabbit skeletal muscle by an improved procedure. The purification was estimated to be 67000-fold and 0.2 mg of enzyme was isolated from 5000 g muscle, corresponding to an overall yield of 7%. The preparation was homogeneous by ultracentrifugal and electrophoretic criteria. The enzyme had a relative molecular mass of 47 kDa by sedimentation equilibrium centrifugation and 51 kDa by SDS-polyacrylamide gel electrophoresis. These values demonstrate that glycogen synthase kinase-3 is monomeric. The Stokes radius of 37 nm suggests the molecule to be asymmetric. The activating factor of the Mg-ATP dependent form of protein phosphatase-1 coeluted with glycogen synthase kinase-3 activity at the final step, establishing that these two activities reside in the same protein. Glycogen synthase kinase-3 phosphorylates glycogen synthase at sites-3, while casein kinase-II phosphorylates site-5, just C-terminal to sites-3 (Picton, C., Aitken, A., Bilham, T. and Cohen, P. (1982) Eur. J. Biochem. 124, 37-45). The basis for the substrate specificities of these protein kinases was investigated using chymotryptic peptides that contain the sites phosphorylated by each enzyme. These studies showed that efficient phosphorylation of sites-3, required the presence of phosphate in site-5 and a region of polypeptide more than 20 residues C-terminal to site-5. In contrast, efficient phosphorylation by casein kinase-II does not require this C-terminal region, and the results are consistent with the view that the enzyme recognises acidic residues immediately C-terminal to site-5.     

Light-dependent subcellular localization of nucleoside diphosphate kinase-1 in Neurospora crassa

Nucleoside diphosphate kinase (NDK) is a housekeeping enzyme localized in cellular organelles and distributed in various organs in prokaryotes and eukaryotes. In Neurospora crassa, NDK-1 is suggested to control catalases in response to heat, oxidative stress and light. In this study, we identified the presence of NDK-1 during most developmental stages in submerged mycelia, aerial hyphae, asexual conidia and perithecia, and the localization of it in soluble, mitochondrial, nuclear and membrane fractions in the mycelial cell. A light-dependent localization of NDK-1 was shown by Western blotting and immunohistochemical analysis using anti-NDK-1 antibody. In the mycelia, NDK-1 was compartmentalized on the plasma membrane in darkness, while it was relocated in the cytoplasm under light. These results suggest that NDK-1 protein was translocated from the plasma membrane to cytoplasm in response to light, and may interact with catalase.     

Polyamines stimulate the activity of glycogen synthase (casein) kinase-1 from bovine kidney and different rat tissues

Previous studies have established that casein kinase-2 (CK-2) is stimulated by polyamines. In this study it is shown that glycogen synthase (casein) kinase-1 (CK-1) can be activated similarly. Using casein as the substrate, bovine kidney CK-1 was stimulated 7-, 2-, and 0.5-fold by spermine, spermidine, and putrescine, respectively. Half-maximal activation of CK-1 by these polyamines was observed at 0.25, 0.70, and 0.50 mM, respectively. CK-1 was optimally activated by spermine at low ionic strength and low Mg2+ concentrations (1-3 mM). Using phosvitin as the substrate, CK-1 was stimulated at low concentrations (0-0.8 mM) and inhibited at higher concentrations of spermine. By contrast CK-2 was inhibited at all concentrations of spermine when phosvitin was used as substrate. Using calcineurin (not a substrate for CK-2) as a substrate, CK-1 from bovine kidney or from three rat tissues (liver, kidney, and testis) was stimulated greater than 2-fold by spermine. It is further shown that heparin inhibits CK-1 and this inhibition can be reversed by spermine. The Vmax of CK-1 for both casein and ATP is increased by spermine while the Km remains unchanged by the polyamine. These studies indicate that CK-1, like CK-2, is a heparin-inhibited and polyamine-activated protein kinase. The results also suggest that CK-1 may be activated by spermine in vivo.     

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.