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12/2010; , ISBN: 9780470650714
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ABSTRACT: Stbd1 is a protein of previously unknown function that is most prevalent in liver and muscle, the major sites for storage of the energy reserve glycogen. The protein is predicted to contain a hydrophobic N terminus and a C-terminal CBM20 glycan binding domain. Here, we show that Stbd1 binds to glycogen in vitro and that endogenous Stbd1 locates to perinuclear compartments in cultured mouse FL83B or Rat1 cells. When overexpressed in COSM9 cells, Stbd1 concentrated at enlarged perinuclear structures, co-localized with glycogen, the late endosomal/lysosomal marker LAMP1 and the autophagy protein GABARAPL1. Mutant Stbd1 lacking the N-terminal hydrophobic segment had a diffuse distribution throughout the cell. Point mutations in the CBM20 domain did not change the perinuclear localization of Stbd1, but glycogen was no longer concentrated in this compartment. Stable overexpression of glycogen synthase in Rat1WT4 cells resulted in accumulation of glycogen as massive perinuclear deposits, where a large fraction of the detectable Stbd1 co-localized. Starvation of Rat1WT4 cells for glucose resulted in dissipation of the massive glycogen stores into numerous and much smaller glycogen deposits that retained Stbd1. In vitro, in cells, and in animal models, Stbd1 consistently tracked with glycogen. We conclude that Stbd1 is involved in glycogen metabolism by binding to glycogen and anchoring it to membranes, thereby affecting its cellular localization and its intracellular trafficking to lysosomes.
Journal of Biological Chemistry 11/2010; 285(45):34960-71. · 4.77 Impact Factor
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ABSTRACT: Lafora disease is a progressive myoclonus epilepsy with onset typically in the second decade of life and death within 10 years. Lafora bodies, deposits of abnormally branched, insoluble glycogen-like polymers, form in neurons, muscle, liver, and other tissues. Approximately half of the cases of Lafora disease result from mutations in the EPM2A gene, which encodes laforin, a member of the dual-specificity protein phosphatase family that additionally contains a glycogen binding domain. The molecular basis for the formation of Lafora bodies is completely unknown. Glycogen, a branched polymer of glucose, contains a small amount of covalently linked phosphate whose origin and function are obscure. We report here that recombinant laforin is able to release this phosphate in vitro, in a time-dependent reaction with an apparent K(m) for glycogen of 4.5 mg/ml. Mutations of laforin that disable the glycogen binding domain also eliminate its ability to dephosphorylate glycogen. We have also analyzed glycogen from a mouse model of Lafora disease, Epm2a(-/-) mice, which develop Lafora bodies in several tissues. Glycogen isolated from these mice had a 40% increase in the covalent phosphate content in liver and a 4-fold elevation in muscle. We propose that excessive phosphorylation of glycogen leads to aberrant branching and Lafora body formation. This study provides a molecular link between an observed biochemical property of laforin and the phenotype of a mouse model of Lafora disease. The results also have important implications for glycogen metabolism generally.
Proceedings of the National Academy of Sciences 01/2008; 104(49):19262-6. · 9.68 Impact Factor
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ABSTRACT: Laforin, encoded by the EPM2A gene, by sequence is a member of the dual specificity protein phosphatase family. Mutations in the EPM2A gene account for around half of the cases of Lafora disease, an autosomal recessive neurodegenerative disorder, characterized by progressive myoclonus epilepsy. The hallmark of the disease is the presence of Lafora bodies, which contain polyglucosan, a poorly branched form of glycogen, in neurons, muscle and other tissues. Glycogen metabolizing enzymes were analyzed in a transgenic mouse over-expressing a dominant negative form of laforin that accumulates Lafora bodies in several tissues. Skeletal muscle glycogen was increased 2-fold as was the total glycogen synthase protein. However, the -/+glucose-6-P activity of glycogen synthase was decreased from 0.29 to 0.16. Branching enzyme activity was increased by 30%. Glycogen phosphorylase activity was unchanged. In whole brain, no differences in glycogen synthase or branching enzyme activities were found. Although there were significant differences in enzyme activities in muscle, the results do not support the hypothesis that Lafora body formation is caused by a major change in the balance between glycogen elongation and branching activities.
Archives of Biochemistry and Biophysics 02/2007; 457(2):264-9. · 2.93 Impact Factor
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ABSTRACT: Glycogen synthase plays a key role in regulating glycogen metabolism. In a search for regulators of glycogen synthase, a yeast two-hybrid study was performed. Two glycogen synthase-interacting proteins were identified in human skeletal muscle, glycogenin-1, and nebulin. The interaction with glycogenin was found to be mediated by the region of glycogenin which contains the 33 COOH-terminal amino acid residues. The regions in glycogen synthase containing both NH2- and COOH-terminal phosphorylation sites are not involved in the interaction. The core segment of glycogen synthase from Glu21 to Gly503 does not bind COOH-terminal fragment of glycogenin. However, this region of glycogen synthase binds full-length glycogenin indicating that glycogenin contains at least one additional interacting site for glycogen synthase besides the COOH-terminus. We demonstrate that the COOH-terminal fragment of glycogenin can be used as an effective high affinity reagent for the purification of glycogen synthase from skeletal muscle and liver.
Archives of Biochemistry and Biophysics 01/2007; 456(1):93-7. · 2.93 Impact Factor
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ABSTRACT: Laforin, encoded by the EPM2A gene, is a dual specificity protein phosphatase that has a functional glycogen-binding domain. Mutations in the EPM2A gene account for around half of the cases of Lafora disease, an autosomal recessive neurodegenerative disorder, characterized by progressive myoclonus epilepsy. The hallmark of the disease is the presence of Lafora bodies, which contain polyglucosan, a poorly branched form of glycogen, in neurons and other tissues. We examined the level of laforin protein in several mouse models in which muscle glycogen accumulation has been altered genetically. Mice with elevated muscle glycogen have increased laforin as judged by Western analysis. Mice completely lacking muscle glycogen or with 10% normal muscle glycogen had reduced laforin. Mice defective in the GAA gene encoding lysosomal alpha-glucosidase (acid maltase) overaccumulate glycogen in the lysosome but did not have elevated laforin. We propose, therefore, that laforin senses cytosolic glycogen accumulation which in turn determines the level of laforin protein.
Biochemical and Biophysical Research Communications 12/2006; 350(3):588-92. · 2.48 Impact Factor
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ABSTRACT: The regulation of glycogen metabolism is critical for the maintenance of glucose and energy homeostasis in mammals. Glycogen synthase, the enzyme responsible for glycogen production, is regulated by multisite phosphorylation in yeast and mammals. We have previously identified PAS kinase as a physiological regulator of glycogen synthase in Saccharomyces cerevisiae. We provide evidence here that PAS kinase is an important regulator of mammalian glycogen synthase. Glycogen synthase is efficiently phosphorylated by PAS kinase in vitro at Ser-640, a known regulatory phosphosite. Efficient phosphorylation requires a region of PAS kinase outside the catalytic domain. This region appears to mediate a direct interaction between glycogen synthase and PAS kinase, thereby targeting kinase activity to this substrate specifically. This interaction is regulated by the PAS kinase PAS domain, raising the possibility that this interaction (and phosphorylation event) is modulated by the cellular metabolic state. This mode of regulation provides a mechanism for metabolic status to impinge directly on the cellular decision of whether to store or use available energy.
Proceedings of the National Academy of Sciences 12/2005; 102(46):16596-601. · 9.68 Impact Factor
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ABSTRACT: Glycogenin is a self-glucosylating protein that initiates glycogen biosynthesis. We recently identified a family of proteins, GNIPs, that interact with glycogenin and stimulate its self-glucosylating activity [J. Biol. Chem. 277 (2002) 19331]. The GNIP gene (also called TRIM7) encodes at least four distinct isoforms of GNIP, three of which (GNIP1, GNIP2, and GNIP3) have in common a COOH-terminal B30.2 domain and predicted coiled-coil regions. Based on Western blot analysis, the GNIP1 protein is widely distributed in tissues. From analysis of a series of deletion mutants of GNIP2 using the yeast two-hybrid system, the B30.2 domain was found to be responsible for the interaction with glycogenin. A truncated form of recombinant GNIP2, lacking the NH2-terminal coiled-coil region, was cross-linked to glycogenin by glutaraldehyde treatment, supporting the idea that the B30.2 domain was sufficient for the interaction. In the course of this study, GNIP2 was also found to interact with itself, via the coiled-coil domain. Heterologous interactions between GNIP1 and GNIP2 were also detected. Since glycogenin is also a dimer, higher order multimeric complexes between glycogenin and GNIPs would be possible.
Archives of Biochemistry and Biophysics 02/2004; 421(2):236-42. · 2.93 Impact Factor
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ABSTRACT: Glycogen synthase, a key enzyme in the regulation of glycogen synthesis by insulin, is controlled by multisite phosphorylation. Glycogen synthase kinase-3 (GSK-3) phosphorylates four serine residues in the COOH terminus of glycogen synthase. Phosphorylation of one of these residues, Ser(640) (site 3a), causes strong inactivation of glycogen synthase. In previous work, we demonstrated in cell models that site 3a can be phosphorylated by an as yet unidentified protein kinase (3a-kinase) distinct from GSK-3. In the present study, we purified the 3a-kinase from rabbit skeletal muscle and identified one constituent polypeptide as HAN11, a WD40 domain protein with unknown function. Another polypeptide was identified as DYRK1A, a member of the dual-specificity tyrosine phosphorylated and regulated protein kinase (DYRK) family. Two isoforms of DYRK, DYRK1A and DYRK1B, co-immunoprecipitate with HAN11 when coexpressed in COS cells indicating that the proteins interact in mammalian cells. Co-expression of DYRK1A, DYRK1B, or DYRK2 with a series of glycogen synthase mutants with Ser/Ala substitutions at the phosphorylation sites in COS cells revealed that protein kinases cause phosphorylation of site 3a in glycogen synthase. To confirm that DYRKs directly phosphorylate glycogen synthase, recombinant DYRK1A, DYRK2, and glycogen synthase were produced in bacterial cells. In the presence of Mg-ATP, both DYRKs inactivated glycogen synthase by more than 10-fold. The inactivation correlated with phosphorylation of site 3a in glycogen synthase. These results indicate that protein kinase(s) from the DYRK family may be involved in a new mechanism for the regulation of glycogen synthesis.
Journal of Biological Chemistry 02/2004; 279(4):2490-8. · 4.77 Impact Factor
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ABSTRACT: Glycogen, a branched polymer of glucose, serves as an energy reserve in many organisms. The degree of branching likely reflects the balance between the activities of glycogen synthase and branching enzyme. Mice overexpressing constitutively active glycogen synthase in skeletal muscle (GSL30) have elevated muscle glycogen. To test whether excess glycogen synthase activity affected glycogen branching, we examined the glycogen from skeletal muscle of GSL30 mice. The absorption spectrum of muscle glycogen determined in the presence of iodine was shifted to higher wavelengths in the GSL30 animals, consistent with a decrease in the degree of branching. As judged by Western blotting, the levels of glycogenin and the branching enzyme were also elevated. Branching enzyme activity also increased approximately threefold. However, this compared with an increase in glycogen synthase of some 50-fold, so that the increase in branching enzyme in response to overexpression of glycogen synthase was insufficient to synthesize normally branched glycogen.
Biochemical and Biophysical Research Communications 07/2003; 305(4):826-30. · 2.48 Impact Factor
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ABSTRACT: Glycogenin is a self-glucosylating protein involved in the initiation of glycogen biosynthesis. Self-glucosylation leads to the formation of an oligosaccharide chain, which, when long enough, supports the action of glycogen synthase to elongate it and form a mature glycogen molecule. To identify possible regulators of glycogenin, the yeast two-hybrid strategy was employed. By using rabbit skeletal muscle glycogenin as a bait, cDNAs encoding three different proteins were isolated from the human skeletal muscle cDNA library. Two of the cDNAs encoded glycogenin and glycogen synthase, respectively, proteins known to be interactors. The third cDNA encoded a polypeptide of unknown function and was designated GNIP (glycogenin interacting protein). Northern blot analysis revealed that GNIP mRNA is highly expressed in skeletal muscle. The gene for GNIP generates at least four isoforms by alternative splicing. The largest isoform GNIP1 contains, from NH(2)- to COOH-terminal, a RING finger, a B box, a putative coiled-coil region, and a B30.2-like motif. The previously identified protein TRIM7 (tripartite motif containing protein 7) is also derived from the GNIP gene and is composed of the RING finger, B box, and coiled-coil regions. The GNIP2 and GNIP3 isoforms consist of the coiled-coil region and B30.2-like domain. Physical interaction between GNIP2 and glycogenin was confirmed by co-immunoprecipitation, and in addition GNIP2 was shown to stimulate glycogenin self-glucosylation 3-4-fold. GNIPs may represent a novel participant in the initiation of glycogen synthesis.
Journal of Biological Chemistry 06/2002; 277(22):19331-8. · 4.77 Impact Factor
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ABSTRACT: Glycogen synthase kinase-3 inactivates rabbit muscle glycogen synthase by sequential phosphorylation of four COOH-terminal
residues Ser (site 4), Ser (site 3c), Ser (site 3b), and Ser (site 3a). Effective recognition of glycogen synthase by glycogen synthase kinase-3 occurs only after the phosphorylation
of Ser (site 5) catalyzed by casein kinase II. The present study addresses specifically the role of sites 3a and 3b in the regulation
of glycogen synthase expressed in COS cells. Simultaneous Ser Ala substitutions at sites 3 a, b and c, 4, and 5 in the same protein molecule eliminated P labeling in the proteolytic fragment Arg-Lys, which contains these sites. This mutant enzyme (which also had a Ser Ala substitution at site 2 in the NH2 terminus) had a -/+ glucose-6-P activity ratio of 0.8, similar to that of totally dephosphorylated enzyme. Reinstating serine residues at either site 3a or site 3b restored
labeling in the Arg-Lys peptide and caused a decrease in the activity ratio to 0.4-0.6. When both sites 3a and 3b were reintroduced, there was complete
inactivation of the enzyme. Thus, sites 3a and 3b are sufficient for the inactivation of glycogen synthase and act synergistically
to control activity. This investigation demonstrates the existence of an alternate mechanism for the phosphorylation of sites
3a and 3b that does not depend on prior phosphorylation of site 5.
Journal of Biological Chemistry 05/1995; 270(21):12491-12497. · 4.77 Impact Factor
