Glycogen is a branched polymer of glucose that acts as a store of energy in times of nutritional sufficiency for utilization in times of need. Its metabolism has been the subject of extensive investigation and much is known about its regulation by hormones such as insulin, glucagon and adrenaline (epinephrine). There has been debate over the relative importance of allosteric compared with covalent control of the key biosynthetic enzyme, glycogen synthase, as well as the relative importance of glucose entry into cells compared with glycogen synthase regulation in determining glycogen accumulation. Significant new developments in eukaryotic glycogen metabolism over the last decade or so include: (i) three-dimensional structures of the biosynthetic enzymes glycogenin and glycogen synthase, with associated implications for mechanism and control; (ii) analyses of several genetically engineered mice with altered glycogen metabolism that shed light on the mechanism of control; (iii) greater appreciation of the spatial aspects of glycogen metabolism, including more focus on the lysosomal degradation of glycogen; and (iv) glycogen phosphorylation and advances in the study of Lafora disease, which is emerging as a glycogen storage disease.

The protein kinase C (PKC) family of serine/threonine kinases functions downstream of nearly all membrane-associated signal transduction pathways. Here we identify PKC-alpha as a fundamental regulator of cardiac contractility and Ca(2+) handling in myocytes. Hearts of Prkca-deficient mice are hypercontractile, whereas those of transgenic mice overexpressing Prkca are hypocontractile. Adenoviral gene transfer of dominant-negative or wild-type PKC-alpha into cardiac myocytes enhances or reduces contractility, respectively. Mechanistically, modulation of PKC-alpha activity affects dephosphorylation of the sarcoplasmic reticulum Ca(2+) ATPase-2 (SERCA-2) pump inhibitory protein phospholamban (PLB), and alters sarcoplasmic reticulum Ca(2+) loading and the Ca(2+) transient. PKC-alpha directly phosphorylates protein phosphatase inhibitor-1 (I-1), altering the activity of protein phosphatase-1 (PP-1), which may account for the effects of PKC-alpha on PLB phosphorylation. Hypercontractility caused by Prkca deletion protects against heart failure induced by pressure overload, and against dilated cardiomyopathy induced by deleting the gene encoding muscle LIM protein (Csrp3). Deletion of Prkca also rescues cardiomyopathy associated with overexpression of PP-1. Thus, PKC-alpha functions as a nodal integrator of cardiac contractility by sensing intracellular Ca(2+) and signal transduction events, which can profoundly affect propensity toward heart failure.

Microscopic screening of a collection of cold-sensitive mutants of Saccharomyces cerevisiae led to the identification of a new gene, CDC55, which appears to be involved in the morphogenetic events of the cell cycle. CDC55 maps between CDC43 and CHC1 on the left arm of chromosome VII. At restrictive temperature, the original cdc55 mutant produces abnormally elongated buds and displays a delay or partial block of septation and/or cell separation. A cdc55 deletion mutant displays a cold-sensitive phenotype like that of the original isolate. Sequencing of CDC55 revealed that it encodes a protein of about 60 kDa, as confirmed by Western immunoblots using Cdc55p-specific antibodies. This protein has greater than 50% sequence identity to the B subunits of rabbit skeletal muscle type 2A protein phosphatase; the latter sequences were obtained by analysis of peptides derived from the purified protein, a polymerase chain reaction product, and cDNA clones. An extragenic suppressor of the cdc55 mutation lies in BEM2, a gene previously identified on the basis of an apparent role in bud emergence.

Increases in type 1 phosphatase (PP1) activity have been observed in end stage human heart failure, but the role of this enzyme in cardiac function is unknown. To elucidate the functional significance of increased PP1 activity, we generated models with (i) overexpression of the catalytic subunit of PP1 in murine hearts and (ii) ablation of the PP1-specific inhibitor. Overexpression of PP1 (threefold) was associated with depressed cardiac function, dilated cardiomyopathy, and premature mortality, consistent with heart failure. Ablation of the inhibitor was associated with moderate increases in PP1 activity (23%) and impaired ␤-adrenergic contractile responses. Extension of these findings to human heart failure indicated that the increased PP1 activity may be partially due to dephosphorylation or inactivation of its inhibitor. Indeed, expression of a constitutively active inhibitor was associated with rescue of ␤-adrenergic responsiveness in failing human myocytes. Thus, PP1 is an important regulator of cardiac function, and inhibition of its activity may represent a novel therapeutic target in heart failure.

The functional specificity of type 1 protein phosphatases (PP1) depends on the associated regulatory/targeting and inhibitory subunits. To gain insights into the mechanism of PP1 regulation by inhibitor-2, an ancient and intrinsically disordered regulator, we solved the crystal structure of the complex to 2.5Å resolution. Our studies show that, when complexed with PP1c, I-2 acquires three regions of order: site 1, residues 12-17, binds adjacent to a region recognized by many PP1 regulators; site 2, amino acids 44 -56, interacts along the RVXF binding groove through an unsuspected sequence, KSQKW; and site 3, residues 130 -169, forms ␣-helical regions that lie across the substratebinding cleft. Specifically, residues 148 -151 interact at the catalytic center, displacing essential metal ions, accounting for both rapid inhibition and slower inactivation of PP1c. Thus, our structure provides novel insights into the mechanism of PP1 inhibition and subsequent reactivation, has broad implications for the physiological regulation of PP1, and highlights common inhibitory interactions among phosphoprotein phosphatase family members.

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

Cyclin G2, together with cyclin G1 and cyclin I, defines a novel cyclin family expressed in terminally differentiated tissues including brain and muscle. Cyclin G2 expression is up-regulated as cells undergo cell cycle arrest or apoptosis in response to inhibitory stimuli independent of p53 (Horne, M., Donaldson, K., Goolsby, G., Tran, D., Mulheisen, M., Hell, J. and Wahl, A. (1997) J. Biol. Chem. 272, 12650 -12661). We tested the hypothesis that cyclin G2 may be a negative regulator of cell cycle progression and found that ectopic expression of cyclin G2 induces the formation of aberrant nuclei and cell cycle arrest in HEK293 and Chinese hamster ovary cells. Cyclin G2 is primarily partitioned to a detergentresistant compartment, suggesting an association with cytoskeletal elements. We determined that cyclin G2 and its homolog cyclin G1 directly interact with the catalytic subunit of protein phosphatase 2A (PP2A). An okadaic acid-sensitive (<2 nM) phosphatase activity coprecipitates with endogenous and ectopic cyclin G2. We found that cyclin G2 also associates with various PP2A B regulatory subunits, as previously shown for cyclin G1. The PP2A/A subunit is not detectable in cyclin G2-PP2A-B-C complexes. Notably, cyclin G2 colocalizes with both PP2A/C and B subunits in detergent-resistant cellular compartments, suggesting that these complexes form in living cells. The ability of cyclin G2 to inhibit cell cycle progression correlates with its ability to bind PP2A/B and C subunits. Together, our findings suggest that cyclin G2-PP2A complexes inhibit cell cycle progression.

Lafora disease is a progressive myoclonus epilepsy with onset in the teenage years followed by neurodegeneration and death within 10 years. A characteristic is the widespread formation of poorly branched, insoluble glycogen-like polymers (polyglucosan) known as Lafora bodies, which accumulate 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 is able to release the small amount of covalent phosphate normally present in glycogen. In studies of Epm2a ؊/؊ mice that lack laforin, we observed a progressive change in the properties and structure of glycogen that paralleled the formation of Lafora bodies. At three months, glycogen metabolism remained essentially normal, even though the phosphorylation of glycogen has increased 4-fold and causes altered physical properties of the polysaccharide. By 9 months, the glycogen has overaccumulated by 3-fold, has become somewhat more phosphorylated, but, more notably, is now poorly branched, is insoluble in water, and has acquired an abnormal morphology visible by electron microscopy. These glycogen molecules have a tendency to aggregate and can be recovered in the pellet after low speed centrifugation of tissue extracts. The aggregation requires the phosphorylation of glycogen. The aggregrated glycogen sequesters glycogen synthase but not other glycogen metabolizing enzymes. We propose that laforin functions to suppress excessive glycogen phosphorylation and is an essential component of the metabolism of normally structured glycogen.Glycogen is a branched polymer of glucose that acts as a repository of energy used in times of need (1). Liver and skeletal muscle contain the two largest deposits in mammals, but heart, brain, adipose, as well as many other tissues synthesize the polymer. Polymerization occurs via ␣-1,4-glycosidic linkages between glucose residues, with branch points introduced by ␣-1,6-glycosidic linkages. The frequency of branching determines the topology of glycogen and distinguishes it from the carbohydrate moiety of starch (2). A unique three-dimensional structure of glycogen cannot be determined experimentally because of the polydispersity of the molecule, but a widely accepted model of its structure has been proposed (3). Glycogen is composed of successive layers, or tiers, of glucose residues; a full size molecule consists of 12 tiers, with M r ϭ ϳ10 7 and a diameter of ϳ40 nm (4). Glycogen has been reported to contain small amounts of covalently linked phosphate, on the order of 0.064% by weight or 0.121% mol/mol (5-7). The mechanism by which the phosphate is introduced remains unclear. One suggestion (5) has been the existence of a glucose-1-phosphate transferase that would form C1-C6 bridging phosphodiesters, but this enzyme has not been further defined at the molecular level. In plant amylopectin, C1 and C3 phosphomonoesters have been shown to be formed by dikinase enzymes (8), but...