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.     

Xenopus axin interacts with glycogen synthase kinase-3 beta and is expressed in the anterior midbrain

Axin is encoded by the fused locus in mice and is required for normal vertebrate axis formation. It has recently been shown that axin associates with the adenomatous polyposis coli gene product (APC), β-catenin and glycogen synthase kinase-3 (GSK-3) in a complex that appears to regulate the level of cytoplasmic β-catenin. We have identified the Xenopus homologue of axin through its interaction with GSK-3β. Xenopus axin (Xaxin) is expressed maternally and throughout early development with a low level of ubiquitous expression. Xaxin also shows remarkably high expression in the anterior mesencephalon adjacent to the forebrain–midbrain boundary.     

TCF-4 mediates cell type-specific regulation of proglucagon gene expression by beta-catenin and glycogen synthase kinase-3beta

The proglucagon gene (glu) encodes glucagon, expressed in pancreatic islets, and the insulinotropic hormone GLP-1, expressed in the intestines. These two hormones exert critical and opposite effects on blood glucose homeostasis. An intriguing question that remains to be answered is whether and how glu gene expression is regulated in a cell type-specific manner. We reported previously that the glu gene promoter in gut endocrine cell lines was stimulated by beta-catenin, the major effector of the Wnt signaling pathway, whereas glu mRNA expression and GLP-1 synthesis were activated via inhibition of glycogen synthase kinase-3beta, the major negative modulator of the Wnt pathway (Ni, Z., Anini, Y., Fang, X., Mills, G. B., Brubaker, P. L., & Jin, T. (2003) J. Biol. Chem. 278, 1380-1387). We now show that beta-catenin and the glycogen synthase kinase-3beta inhibitor lithium do not activate glu mRNA or glu promoter expression in pancreatic cell lines. In the intestinal GLUTag cell line, but not in the pancreatic InR1-G9 cell line, the glu promoter G2 enhancer-element was activated by lithium treatment via a TCF-binding motif. TCF-4 is abundantly expressed in the gut but not in pancreatic islets. Furthermore, both TCF-4 and beta-catenin bind to the glu gene promoter, as detected by chromatin immunoprecipitation. Finally, stable introduction of dominant-negative TCF-4 into the GLUTag cell line repressed basal glu mRNA expression and abolished the effect of lithium on glu mRNA expression and GLP-1 synthesis. We have therefore identified a unique mechanism that regulates glu expression in gut endocrine cells only. Tissue-specific expression of TCF factors thus may play a role in the diversity of the Wnt pathway.     

Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle: Phosphorylation of site 5 by glycogen synthase kinase-5 (casein kinase-II) is a prerequisite for phosphorylation of sites 3 by glycogen synthase kinase-3

Glycogen synthase kinase-5 (casein kinase-II) phosphorylates glycogen synthase on a serine termed site 5. This residue is just C-terminal to the 3 serines phosphorylated by glycogen synthase kinase-3, which are critical for the hormonal regulation of glycogen synthase in vivo. Although phosphorylation of site 5 does not affect the catalytic activity, it is demonstrated that this modification is a prerequisite for phosphorylation by glycogen synthase kinase-3. Since site 5 is almost fully phosphorylated in vivo under all conditions, the role of glycogen synthase kinase-5 would appear to be a novel one in forming the recognition site for another protein kinase     

Adenomatous polyposis coli (APC) regulates multiple signaling pathways by enhancing glycogen synthase kinase-3 (GSK-3) activity

Glycogen synthase kinase-3 (GSK-3) is essential for many signaling pathways and cellular processes. As Adenomatous Polyposis Coli (APC) functions in many of the same processes, we investigated a role for APC in the regulation of GSK-3-dependent signaling. We find that APC directly enhances GSK-3 activity. Furthermore, knockdown of APC mimics inhibition of GSK-3 by reducing phosphorylation of glycogen synthase and by activating mTOR, revealing novel roles for APC in the regulation of these enzymes. Wnt signaling inhibits GSK-3 through an unknown mechanism, and this results in both stabilization of β-catenin and activation of mTOR. We therefore hypothesized that Wnts may regulate GSK-3 by disrupting the interaction between APC and the Axin-GSK-3 complex. We find that Wnts rapidly induce APC dissociation from Axin, correlating with β-catenin stabilization. Furthermore, Axin interaction with the Wnt co-receptor LRP6 causes APC dissociation from Axin. We propose that APC regulates multiple signaling pathways by enhancing GSK-3 activity, and that Wnts induce APC dissociation from Axin to reduce GSK-3 activity and activate downstream signaling. APC regulation of GSK-3 also provides a novel mechanism for Wnt regulation of multiple downstream effectors, including β-catenin and mTOR.     

Involvement of glycogen synthase kinase-3beta and tau phosphorylation in neuronal Golgi disassembly

The dissociation of the neuronal Golgi complex is a classical feature observed in neurodegenerative disorders including Alzheimer's disease. The goal of this study is to determine if the phosphorylation of tau protein is involved in neuronal Golgi disassembly. Primary cortical cultures were exposed to two Golgi toxins, brefeldin A (BFA) or nordihydroguaiaretic acid (NDGA). Immunocytochemical studies using the anti58 k antibody revealed that Golgi disassembly started in exposed neurons a few minutes after treatment. BFA and NDGA induced a rapid and transient increase in tau phosphorylation in a site-specific manner on immunoblots. In addition, the increase in tau phosphorylation directly correlated with a transient dissociation of tau from the cytoskeleton and a decrease of the acetylated tubulin. Furthermore, the activity of glycogen synthase kinase-3beta (GSK-3beta) increased transiently, as demonstrated by the kinase activity assay and by immunoblottings of serine-9 and tyrosine-216 phosphorylated of GSK-3beta. A decrease of the Akt phosphorylated form was also shown. The increase in tau phosphorylation was inhibited by the GSK-3beta inhibitor, lithium. Finally, morphometric studies showed that lithium partially blocked the Golgi disassembly caused by BFA or NDGA. Together these findings indicate that GSK-3beta activity and tau phosphorylation state are involved in the maintenance of the neuronal Golgi organization.     

Phosphorylation of axin, a Wnt signal negative regulator, by glycogen synthase kinase-3beta regulates its stability

Axin forms a complex with glycogen synthase kinase-3beta (GSK-3beta) and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin, thereby stimulating the degradation of beta-catenin. Because GSK-3beta also phosphorylates Axin in the complex, the physiological significance of the phosphorylation of Axin was examined. Treatment of COS cells with LiCl, a GSK-3beta inhibitor, and okadaic acid, a protein phosphatase inhibitor, decreased and increased, respectively, the cellular protein level of Axin. Pulse-chase analyses showed that the phosphorylated form of Axin was more stable than the unphosphorylated form and that an Axin mutant, in which the possible phosphorylation sites for GSK-3beta were mutated, exhibited a shorter half-life than wild type Axin. Dvl-1, which was genetically shown to function upstream of GSK-3beta, inhibited the phosphorylation of Axin by GSK-3beta in vitro. Furthermore, Wnt-3a-containing conditioned medium down-regulated Axin and accumulated beta-catenin in L cells and expression of Dvl-1(DeltaPDZ), in which the PDZ domain was deleted, suppressed this action of Wnt-3a. These results suggest that the phosphorylation of Axin is important for the regulation of its stability and that Wnt down-regulates Axin through Dvl.     

The glamour and gloom of glycogen synthase kinase-3

Glycogen synthase kinase-3 (GSK3) is now recognized as a key component of a surprisingly large number of cellular processes and diseases. Several mechanisms play a part in controlling the actions of GSK3, including phosphorylation, protein complex formation, and subcellular distribution. These are used to control and direct the far-reaching influences of GSK3 on cellular structure, growth, motility and apoptosis. Dysregulation of GSK3 is linked to several prevalent pathological conditions, such as diabetes and/or insulin resistance, and Alzheimer's disease. Therefore, much effort is currently directed towards understanding the functions and control of GSK3, and identifying methods capable of diminishing the deleterious impact of GSK3 in pathological conditions.     

A role for phosphorylation of glycogen synthase kinase-3alpha in bovine sperm motility regulation

The long-term goal of our work is to understand biochemical mechanisms underlying sperm motility and fertility. In a recent study we showed that tyrosine phosphorylation of a 55-kDa protein varied in direct proportion to motility. Tyrosine phosphorylation of the protein was low in immotile compared to motile epididymal sperm. Inhibition or stimulation of motility by high calcium levels or cAMP, respectively, results in a corresponding decrease or increase in tyrosine phosphorylation of the 55-kDa protein. Here we report purification and identification of this motility-associated protein. Soluble extracts from bovine caudal epididymal sperm were subjected to DEAE-cellulose, Affi-Gel blue, and cellulose phosphate chromatography. Tyrosine phosphate immunoreactive fractions contained glycogen synthase kinase-3 (GSK-3) activity, suggesting a possible correspondence between these proteins. This suggestion was verified by Western blot analyses following one-dimensional and two-dimensional gel electrophoresis of the purified protein using monoclonal and affinity-purified polyclonal antibodies against the catalytic amino-terminus and carboxy-terminus regions of GSK-3. Further confirmation of the identity of these proteins came from Western blot analysis using antibodies specific to the tyrosine phosphorylated GSK-3. Using this antibody, we also showed that GSK-3 tyrosine phosphorylation was high in motile compared to immotile sperm. Immunocytochemistry revealed that GSK-3 is present in the flagellum and the anterior portion of the sperm head. These data suggest that GSK-3, regulated by phosphorylation, could be a key element underlying motility initiation in the epididymis and regulation of mature sperm function.     

The phosphorylation of rabbit skeletal muscle glycogen synthase by glycogen synthase kinase-2 and adenosine-3':5'-monophosphate-dependent protein kinase.

Purified glycogen synthase is contaminated with traces of two protein kinases that can phosphorylate the enzyme. One is protein kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) and the second is an activity termed glycogen synthase kinase-2 [Nimmo, H.G. and Cohen P, (1974)]. Glycogen synthase kinase-2 has been found to be localized relatively specifically in the protein-glycogen complex. It has been purified 4000-fold by two procedures, both of which involve disruption of the complex, followed by the DEAE-cellulose and phosphocellulose chromatographies. However the salt concentration at which glycogen synthase kinase-2 is eluted from DEAE-cellulose depends on the method that is used to disrupt the complex. The results indicate that glycogen synthase kinase-2 is firmly attached to a protein component of the complex. The isolation procedures separate glycogen synthase kinase-2 from phosphorylase kinase, cyclic AMP-dependent protein kinase and other glycogen-metabolising enzymes. Glycogen synthase kinase-2 is the major phosvitin kinase in skeletal muscle, although glycogen synthase is a six to eight-fold better substrate than phosvitin under the standard assay conditions. Phosphorylase kinase and phosphorylase b are not substrates for glycogen synthase kinase 2. Following incubation with cyclic-AMP-dependent protein kinase, cyclic AMP and Mg-ATP, the phosphorylation of glycogen synthase reaches a plateau at 1.0 molecules of phosphate incorporated per subunit and the activity ratio measured in the absence and presence of glucose 6-phosphate falls from 0.8 to a plateau of 0.18. The Ka for glucose 6-phosphate of this phosphorylated species, termed glycogen synthase b1, is the 0.6 mM. Following incubation with glycogen synthase kinase-2 and Mg-ATP, the phosphorylation reaches a plateau of 0.92 molecules of phosphate incorporated per subunit and the activity ratio decreases to a plateau of 0.08. The Ka for glucose 6-phosphate of this phosphorylated species, termed glycogen synthetase b2, is 4 mM. In the presence of both cyclic-AMP-dependent protein kinase and glycogen synthase kinase-2, the phosphorylation of glycogen synthase reaches a plateau when 1.95 molecules of phoshophate have been incorporated per subunit. The activity ratio is 0.01 and the Ka for glucose 6-phosphate is 10 mM. The results indicate that glycogen synthase can be regulated by two distinct phosphorylation-dephosphorylation cycles. The implication of these findings for the regulation of glycogen synthase in vivo are discussed.