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 Km 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.polyglucosan ͉ Lafora bodies ͉ Epm2a ͉ phosphate

Glycogen synthase kinase-3 inactivates rabbit muscle glycogen synthase by sequential phosphorylation of four COOH-terminal residues Ser652 (site 4), Ser648 (site 3c), Ser644 (site 3b), and Ser640 (site 3a). Effective recognition of glycogen synthase by glycogen synthase kinase-3 occurs only after the phosphorylation of Ser656 (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 32P labeling in the proteolytic fragment Arg634-Lys682, 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 approximately 0.8, similar to that of totally dephosphorylated enzyme. Reinstating serine residues at either site 3a or site 3b restored labeling in the Arg634-Lys682 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.

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.

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.

To investigate the role of glycogen synthase in controlling glycogen accumulation, we generated three lines of transgenic mice in which the enzyme was overexpressed in skeletal muscle by using promoter-enhancer elements derived from the mouse muscle creatine kinase gene. In all three lines, expression was highest in muscles composed primarily of fast-twitch fibers, such as the gastrocnemius and anterior tibialis. In these muscles, glycogen synthase activity was increased by as much as 10-fold, with concomitant increases (up to 5-fold) in the glycogen content. The uridine diphosphoglucose concentrations were markedly decreased, consistent with the increase in glycogen synthase activity. Levels of glycogen phosphorylase in these muscles increased (up to 3-fold), whereas the amount of the insulin-sensitive glucose transporter 4 either remained unchanged or decreased. The observation that increasing glycogen synthase enhances glycogen accumulation supports the conclusion that the activation of glycogen synthase, as well as glucose transport, contributes to the accumulation of glycogen in response to insulin in skeletal muscle.