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Objective Glycogen is a major energy reserve in liver and skeletal muscle. The master metabolic regulator AMP-activated protein kinase (AMPK) associates with glycogen via its regulatory β subunit carbohydrate-binding module (CBM). However, the physiological role of AMPK-glycogen binding in energy homeostasis has not been investigated in vivo. This study aimed to determine the physiological consequences of disrupting AMPK-glycogen interactions. Methods Glycogen binding was disrupted in mice via whole-body knock-in (KI) mutation of either the AMPK β1 (W100A) or β2 (W98A) isoform CBM. Systematic whole-body, tissue and molecular phenotyping was performed in KI and respective wild-type (WT) mice. Results While β1 W100A KI did not affect whole-body metabolism or exercise capacity, β2 W98A KI mice displayed increased adiposity and impairments in whole-body glucose handling and maximal exercise capacity relative to WT. These KI mutations resulted in reduced total AMPK protein and kinase activity in liver and skeletal muscle of β1 W100A and β2 W98A, respectively, versus WT mice. β1 W100A mice also displayed loss of fasting-induced liver AMPK total and α-specific kinase activation relative to WT. Destabilisation of AMPK was associated with increased fat deposition in β1 W100A liver and β2 W98A skeletal muscle versus WT. Conclusions These results demonstrate that glycogen binding plays critical roles in stabilising AMPK and maintaining cellular, tissue and whole-body energy homeostasis., Highlights • AMPK β subunit knock-in (KI) mice were generated to disrupt glycogen binding in vivo. • Loss of AMPK β2 glycogen binding impairs glucose handling and exercise capacity. • Loss of AMPK β2 glycogen binding increases adiposity. • AMPK β1 and β2 KI mice show increased liver and muscle fat deposition, respectively. • Loss of glycogen binding reduces cellular AMPK protein and kinase activity.

The AMP-activated protein kinase (AMPK), a central regulator of cellular energy balance and metabolism, binds glycogen via its β subunit. However, the physiological effects of disrupting AMPK-glycogen interactions remain incompletely understood. To chronically disrupt AMPK-glycogen binding, AMPK β double knock-in (DKI) mice were generated with mutations in residues critical for glycogen binding in both the β1 (W100A) and β2 (W98A) subunit isoforms. We examined the effects of this DKI mutation on whole-body substrate utilization, glucose homeostasis, and tissue glycogen dynamics. Body composition, metabolic caging, glucose and insulin tolerance, serum hormone and lipid profiles, and tissue glycogen and protein content were analyzed in chow-fed male DKI and age-matched wild-type (WT) mice. DKI mice displayed increased whole-body fat mass and glucose intolerance associated with reduced fat oxidation relative to WT. DKI mice had reduced liver glycogen content in the fed state concomitant with increased utilization and no repletion of skeletal muscle glycogen in response to fasting and refeeding, respectively, despite similar glycogen-associated protein content relative to WT. DKI liver and skeletal muscle displayed reductions in AMPK protein content versus WT. These findings identify phenotypic effects of the AMPK DKI mutation on whole-body metabolism and tissue AMPK content and glycogen dynamics.

The mammalian AMP-activated protein kinase (AMPK) is an obligatory αβγ heterotrimeric complex carrying a carbohydrate-binding module (CBM) in the β-subunit (AMPKβ) capable of attaching AMPK to glycogen. Nonetheless, AMPK localizes at many different cellular compartments, implying the existence of mechanisms that prevent AMPK from glycogen binding. Cell-free carbohydrate binding assays revealed that AMPK autophosphorylation abolished its carbohydrate-binding capacity. X-ray structural data of the CBM displays the central positioning of threonine 148 within the binding pocket. Substitution of Thr-148 for a phospho-mimicking aspartate (T148D) prevents AMPK from binding to carbohydrate. Overexpression of isolated CBM or β1-containing AMPK in cellular models revealed that wild type (WT) localizes to glycogen particles, whereas T148D shows a diffuse pattern. Pharmacological AMPK activation and glycogen degradation by glucose deprivation but not forskolin enhanced cellular Thr-148 phosphorylation. Cellular glycogen content was higher if pharmacological AMPK activation was combined with overexpression of T148D mutant relative to WT AMPK. In summary, these data show that glycogen-binding capacity of AMPKβ is regulated by Thr-148 autophosphorylation with likely implications in the regulation of glycogen turnover. The findings further raise the possibility of regulated carbohydrate-binding function in a wider variety of CBM-containing proteins., Journal of Biological Chemistry, 290 (18), ISSN:0021-9258, ISSN:1083-351X

SummaryAMP-activated protein kinase (AMPK) monitors cellular energy, regulates genes involved in ATP synthesis and consumption, and is allosterically activated by nucleotides and synthetic ligands. Analysis of the intact enzyme with hydrogen/deuterium exchange mass spectrometry reveals conformational perturbations of AMPK in response to binding of nucleotides, cyclodextrin, and a synthetic small molecule activator, A769662. Results from this analysis clearly show that binding of AMP leads to conformational changes primarily in the γ subunit of AMPK and subtle changes in the α and β subunits. In contrast, A769662 causes profound conformational changes in the glycogen binding module of the β subunit and in the kinase domain of the α subunit, suggesting that the molecular binding site of the latter resides between the α and β subunits. The distinct short- and long-range perturbations induced upon binding of AMP and A769662 suggest fundamentally different molecular mechanisms for activation of AMPK by these two ligands.

Glycogen synthase is a rate-limiting enzyme in the biosynthesis of glycogen and has an essential role in glucose homeostasis. The three-dimensional structures of yeast glycogen synthase (Gsy2p) complexed with maltooctaose identified four conserved maltodextrin-binding sites distributed across the surface of the enzyme. Site-1 is positioned on the N-terminal domain, site-2 and site-3 are present on the C-terminal domain, and site-4 is located in an interdomain cleft adjacent to the active site. Mutation of these surface sites decreased glycogen binding and catalytic efficiency toward glycogen. Mutations within site-1 and site-2 reduced the V(max)/S(0.5) for glycogen by 40- and 70-fold, respectively. Combined mutation of site-1 and site-2 decreased the V(max)/S(0.5) for glycogen by3000-fold. Consistent with the in vitro data, glycogen accumulation in glycogen synthase-deficient yeast cells (Δgsy1-gsy2) transformed with the site-1, site-2, combined site-1/site-2, or site-4 mutant form of Gsy2p was decreased by up to 40-fold. In contrast to the glycogen results, the ability to utilize maltooctaose as an in vitro substrate was unaffected in the site-2 mutant, moderately affected in the site-1 mutant, and almost completely abolished in the site-4 mutant. These data show that the ability to utilize maltooctaose as a substrate can be independent of the ability to utilize glycogen. Our data support the hypothesis that site-1 and site-2 provide a "toehold mechanism," keeping glycogen synthase tightly associated with the glycogen particle, whereas site-4 is more closely associated with positioning of the nonreducing end during catalysis.

AMPK beta subunits contain a conserved domain that causes association with glycogen. Although glycogen availability is known to affect AMPK regulation in vivo, the molecular mechanism for this has not been clear. We now show that AMPK is inhibited by glycogen, particularly preparations with high branching content. We synthesized a series of branched oligosaccharides and show that those with a single alpha1-->6 branch are allosteric inhibitors that also inhibit phosphorylation by upstream kinases. Removal of the outer chains of glycogen using phosphorylase, thus exposing the outer branches, renders inhibition of AMPK more potent. Inhibition by all carbohydrates tested was dependent on the glycogen-binding domain being abolished by mutation of residues required for carbohydrate binding. Our results suggest the hypothesis that AMPK, as well as monitoring immediate energy availability by sensing AMP/ATP, may also be able to sense the status of cellular energy reserves in the form of glycogen.

Protein phosphatase 1 (PP1) is widely distributed among tissues and species and acts as a regulator of many important cellular processes. By targeting the catalytic part of PP1 (PP1C) toward particular loci and substrates, regulatory subunits constitute key elements conferring specificity to the holoenzyme. Here, we report the identification of an (alpha/beta)8-barrel-like structure within the N-ter stretch of the human PP1 regulatory subunit hGM, which is part of the family of diverse proteins associated with glycogen metabolism. Protein homology modeling gave rise to a three-dimensional (3D) model for the 381 N-ter residue stretch of hGM, based on sequence similarity with Streptomyces olivochromogenes xylose isomerase, identified by using FASTA. The alignment was subsequently extended by using hydrophobic cluster analysis. The homology-derived model includes the putative glycogen binding area located within the 142-230 domain of hGM as well as a structural characterization of the PP1C interacting domain (segment 51-67). Refinement of the latter by molecular dynamics afforded a topology that is in agreement with previous X-ray studies (Egloff et al., 1997). Finite difference Poisson-Boltzmann calculations performed on the interacting domains of PP1C and hGM confirm the complementarity of the local electrostatic potentials of the two partners. This work highlights the presence of a conserved fold among distant species (mammalian, Caenorhabditis elegans, yeast) and, thus, emphasizes the involvement of PP1 in crucial basic cellular functions.

The SNF1/AMP-activated protein kinase (AMPK) family is required for adaptation to metabolic stress and energy homeostasis. The γ subunit of AMPK binds AMP and ATP, and mutations that affect binding cause human disease. We have here addressed the role of the Snf4 (γ) subunit in regulating SNF1 protein kinase in response to glucose availability in Saccharomyces cerevisiae. Previous studies of mutant cells lacking Snf4 suggested that Snf4 counteracts autoinhibition by the C-terminal sequence of the Snf1 catalytic subunit but is dispensable for glucose regulation, and AMP does not activate SNF1 in vitro. We first introduced substitutions at sites that, in AMPK, contribute to nucleotide binding and regulation. Mutations at several sites relieved glucose inhibition of SNF1, as judged by catalytic activity, phosphorylation of the activation-loop Thr-210, and growth assays, although analogs of the severe human mutations R531G/Q had little effect. We further showed that alterations of Snf4 residues that interact with the glycogen-binding domain (GBD) of the β subunit strongly relieved glucose inhibition. Finally, substitutions in the GBD of the Gal83 β subunit that are predicted to disrupt interactions with Snf4 and also complete deletion of the GBD similarly relieved glucose inhibition of SNF1. Analysis of mutant cells lacking glycogen synthase showed that regulation of SNF1 is normal in the absence of glycogen. These findings reveal novel roles for Snf4 and the GBD in regulation of SNF1.

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.

AMP-activated protein kinase (AMPK) coordinates cellular metabolism in response to energy demand as well as to a variety of stimuli. The AMPK beta subunit acts as a scaffold for the alpha catalytic and gamma regulatory subunits and targets the AMPK heterotrimer to glycogen. We have determined the structure of the AMPK beta glycogen binding domain in complex with beta-cyclodextrin. The structure reveals a carbohydrate binding pocket that consolidates all known aspects of carbohydrate binding observed in starch binding domains into one site, with extensive contact between several residues and five glucose units. beta-cyclodextrin is held in a pincer-like grasp with two tryptophan residues cradling two beta-cyclodextrin glucose units and a leucine residue piercing the beta-cyclodextrin ring. Mutation of key beta-cyclodextrin binding residues either partially or completely prevents the glycogen binding domain from binding glycogen. Modeling suggests that this binding pocket enables AMPK to interact with glycogen anywhere across the carbohydrate's helical surface.

AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolism in response to energy demand and supply by adjusting the ATP-generating and ATP-consuming pathways. AMPK potentially plays a critical role in diabetes and obesity as it is known to be activated by metforin and rosiglitazone, drugs used for the treatment of type II diabetes. AMPK is a heterotrimer composed of a catalytic alpha subunit and two regulatory subunits, beta and gamma. Mutations in the gamma subunit are known to cause glycogen accumulation, leading to cardiac arrhythmias. Recently, a functional glycogen-binding domain (GBD) has been identified in the beta subunit. Here, the crystallization of GBD in the presence of beta-cyclodextrin is reported together with preliminary X-ray data analysis allowing the determination of the structure by single isomorphous replacement and threefold averaging using in-house X-ray data collected from a selenomethionine-substituted protein.

The cysteine-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid) inactivates the Escherichia coli glycogen synthase (Holmes, E., and Preiss, J. (1982) Arch. Biochem. Biophys. 216, 736-740). To find the responsible residue, all cysteines, Cys(7), Cys(379), and Cys(408), were substituted combinatorially by Ser. 5,5'-Dithiobis(2-nitrobenzoic acid) modified and inactivated the enzyme if and only if Cys(379) was present and it was prevented by the substrate ADP-glucose (ADP-Glc). Mutations C379S and C379A increased the S(0.5) for ADP-Glc 40- and 77-fold, whereas the specific activity was decreased 5.8- and 4.3-fold, respectively. Studies of inhibition by glucose 1-phosphate and AMP indicated that Cys(379) was involved in the interaction of the enzyme with the phosphoglucose moiety of ADP-Glc. Other mutations, C379T, C379D, and C379L, indicated that this site is intolerant for bulkier side chains. Because Cys(379) is in a conserved region, other residues were scanned by mutagenesis. Replacement of Glu(377) by Ala and Gln decreased V(max) more than 10,000-fold without affecting the apparent affinity for ADP-Glc and glycogen binding. Mutation of Glu(377) by Asp decreased V(max) only 57-fold indicating that the negative charge of Glu(377) is essential for catalysis. The activity of the mutation E377C, on an enzyme form without other Cys, was chemically restored by carboxymethylation. Other conserved residues in the region, Ser(374) and Gln(383), were analyzed by mutagenesis but found not essential. Comparison with the crystal structure of other glycosyltransferases suggests that this conserved region is a loop that is part of the active site. The results of this work indicate that this region is critical for catalysis and substrate binding.

The AMP-activated protein kinase (AMPK) is an alphabetagamma heterotrimer that is activated by low cellular energy status and affects a switch away from energy-requiring processes and toward catabolism. While it is primarily regulated by AMP and ATP, high muscle glycogen has also been shown to repress its activation. Mutations in the gamma2 and gamma3 subunit isoforms lead to arrhythmias associated with abnormal glycogen storage in human heart and elevated glycogen in pig muscle, respectively. A putative glycogen binding domain (GBD) has now been identified in the beta subunits. Coexpression of truncated beta subunits lacking the GBD with alpha and gamma subunits yielded complexes that were active and normally regulated. However, coexpression of alpha and gamma with full-length beta caused accumulation of AMPK in large cytoplasmic inclusions that could be counterstained with anti-glycogen or anti-glycogen synthase antibodies. These inclusions were not affected by mutations that increased or abolished the kinase activity and were not observed by using truncated beta subunits lacking the GBD. Our results suggest that the GBD binds glycogen and can lead to abnormal glycogen-containing inclusions when the kinase is overexpressed. These may be related to the abnormal glycogen storage bodies seen in heart disease patients with gamma2 mutations.

Lafora disease (progressive myoclonus epilepsy of Lafora type) is an autosomal recessive neurodegenerative disorder resulting from defects in the EPM2A gene. EPM2A encodes a 331-amino acid protein containing a carboxyl-terminal phosphatase catalytic domain. We demonstrate that the EPM2A gene product also contains an amino-terminal carbohydrate binding domain (CBD) and that the CBD is critical for association with glycogen both in vitro and in vivo. The CBD domain localizes the phosphatase to specific subcellular compartments that correspond to the expression pattern of glycogen processing enzyme, glycogen synthase. Mutations in the CBD result in mis-localization of the phosphatase and thereby suggest that the CBD targets laforin to intracellular glycogen particles where it is likely to function. Thus naturally occurring mutations within the CBD of laforin likely result in progressive myoclonus epilepsy due to mis-localization of phosphatase expression.

Specialized carbohydrate-binding domains, the Starch-Binding Domain (SBD) and the Glycogen Binding Domain (GBD), are motifs of approximately 100 amino acids directly or indirectly associated with starch or glycogen metabolism. Members of the regulatory β subunit of the heterotrimeric complex AMPK/SNF1/SnRK1 contain an SBD or GBD. In Arabidopsis thaliana, the β regulatory subunit AKINβ2 and a γ-type subunit, AKINβγ, also have an SBD. In this work, we compared the SBD of AKINβ2 and AKINβγ with the GBD present in rat AMPKβ1 and demonstrated that they conserved the same overall topology. The majority of the amino acids identified in the protein-carbohydrate interactions in the rat AMPKβ1 are conserved in the two plant proteins. In AKINβγ, there is an insertion of three amino acids that creates a loop adjacent to one of the conserved tryptophan residues. Functionally, the SBD from AKINβγ and AKINβ2 could bind starch, but there was an important difference in the association when an amylose/amylopectin (A/A) mixture was used. The physiological relevance of binding to starch was clear for AKINβγ, because immunolocalization experiments identified this protein inside the chloroplast. SnRK1 activity was not affected by the addition of A/A to the reaction mixture. However, addition of starch inhibited the activity 85%. Furthermore, proteins associated with A/A and starch in an in vitro-binding assay accounted for 10 to 20% of total SnRK1 kinase activity. Interestingly, the identification of the SnRK1 subunits associated to the protein-carbohydrate complex indicated that only the catalytic subunits, AKIN10 and AKIN11, and the regulatory subunit AKINβγ were present. These results suggest that a dimer formed between either catalytic subunit and AKINβγ could be associated with the A/A mixture in its active form but the same subunits are inactivated when binding to starch.

Mutations in human laforin lead to an autosomal neurodegenerative disorder Lafora disease. In N-terminal carbohydrate binding domain of laforin, two mutations W32G and K87A are reported as highly disease causing laforin mutants. Experimental studies reported that mutations are responsible for the abolishment of glycogen binding which is a critical function of laforin. Our current computational study focused on the role of conformational changes in human laforin structure due to existing single mutation W32G and prepared double mutation W32G/K87A related to loss of glycogen binding. We performed 10 ns molecular dynamics (MD) simulation studies in the Gromacs package for both mutations and analyzed the trajectories. From the results, the global properties like root mean square deviation, root mean square fluctuation, radius of gyration, solvent accessible surface area and hydrogen bonds showed structural changes in atomic level observed in W32G and W32G/K87A laforin mutants. The conformational change induced by mutants influenced the loss of the overall stability of the native laforin. Moreover, the change in overall motion of protein was analyzed by principal component analysis and results showed protein clusters expanded more than native and also change in direction in case of double mutant in conformational space. Overall, our report provides theoretical information on loss of structure-function relationship due to flexible nature of laforin mutants. In conclusion, comparative MD simulation studies support the experimental data on W32G and W32G/K87A related to the lafora disease mechanism on glycogen binding.

The product of the CG9238 gene that we termed glycogen binding subunit 70E (Gbs-70E) was characterized by biochemical and molecular genetics methods. The interaction between Gbs-70E and all catalytic subunits of protein phosphatase 1 (Pp1-87B, Pp1-9C, Pp1-96A and Pp1-13C) of Drosophila melanogaster was confirmed by pairwise yeast two-hybrid tests, co-immunoprecipitation and pull down experiments. The binding of Gbs-70E to glycogen was demonstrated by sedimentation analysis. With RT-PCR we found that the mRNAs coding for the longer Gbs-70E PB/PC protein were expressed in all developmental stages of the fruit flies while the mRNA for the shorter Gbs-70E PA was restricted to the eggs and the ovaries of the adult females. The development specific expression of the shorter splice variant was not conserved in different Drosophila species. The expression level of the gene was manipulated by P-element insertions and gene deletion to analyze the functions of the gene product. A small or moderate reduction in the gene expression resulted in no significant changes, however, a deletion mutant expressing very low level of the transcript lived shorter and exhibited reduced glycogen content in the imagos. In addition, the gene deletion decreased the fertility of the fruit flies. Our results prove that Gbs-70E functions as the glycogen binding subunit of protein phosphatase 1 that regulates glycogen content and plays a role in the development of eggs in D. melanogaster.

We report two novel mutations in two Japanese patients with glycogen storage disease type IIIa (GSD IIIa). In addition, we review the literature on mutations in GSD III to understand better the molecular basis of GSD III. In our first case, the homozygous A-to-C mutation at the acceptor site of intron 5 (IVS5–2A>C) was identified. This leads to the skipping of exon 6 and the predicted mutant protein was found to be 68 amino acids shorter than normal. This is the first report of skipping exon 6, which encodes one of the putative active sites, resulting in a profoundly deleterious effect on debrancher activity. In our second case, the homozygous deletion of an A at position 4234 (4234delA) was identified; this induces a frameshift resulting in the appearance of a stop codon at amino acid position 1276 (1276X). In patients with GSD IIIa, several mutations of the debrancher gene located in the C-terminal region containing putative glycogen binding domains have been identified as well as 4234delA in our second case. On the other hand, specific localization of the mutations within exon 3 was proposed in patients with GSD IIIb.

Glycogen storage disease type III (GSD III) is an autosomal recessive disease caused by the deficiency of glycogen debranching enzyme (AGL). We report the finding of two new mutations in a GSD IIIa Ashkenazi Jewish patient. Both mutations are insertion of an adenine into a stretch of 8 adenines towards the 3' end of the coding region, one at position 3904 (3904insA) in exon 30, the second at position 4214 (4214insA) in exon 32. The mutations cause frameshifts and premature terminations of the glycogen debranching enzyme, the first causing a frameshift at amino acid 1304, the second causing a frameshift at amino acid 1408 of the total of 1532. These mutations demonstrate the importance of the 125 amino acids at the carboxy-terminus of the debrancher enzyme for its activity and support the suggestion that the putative glycogen binding domain is located in the carboxy-terminus of the AGL. The mutations cause distinctive single-strand conformation polymorphism (SSCP) patterns enabling easy detection.

Lafora disease (LD) is an autosomal recessive, progressive form of myoclonus epilepsy which affects worldwide. LD occurs mainly in countries like southern Europe, northern Africa, South India, and in the Middle East. LD occurs with its onset mainly in teenagers and leads to decline and death within 2 to 10 years. The genes EPM2A and EPM2B are commonly involved in 90 % of LD cases. EPM2A codes for protein laforin which contains an amino terminal carbohydrate binding module (CBM) belonging to the CBM20 family and a carboxy terminal dual specificity phosphatase domain. Mutations in laforin are found to abolish glycogen binding and have been reported in wet lab methods. In order to investigate on structural insights on laforin mutation K81A, we performed molecular dynamics (MD) simulation studies for native and mutant protein. MD simulation results showed loss of stability due to mutation K87A which confirmed the structural reason for conformational changes observed in laforin. The conformational change of mutant laforin was confirmed by analysis using root mean square deviation, root mean square fluctuation, solvent accessibility surface area, radius of gyration, hydrogen bond, and principle component analysis. Our results identified that the flexibility of K87A mutated laforin structure, with replacement of acidic amino acid to aliphatic amino acid in functional CBM domain, have more impact in abolishing glycogen binding that favors LD.

Uncontrolled elongation of glycogen chains, not adequately balanced by their branching, leads to the formation of an insoluble, presumably neurotoxic, form of glycogen called polyglucosan. To test the suspected pathogenicity of polyglucosans in neurological glycogenoses, we have modeled the typical glycogenosis Adult Polyglucosan Body Disease (APBD) by suppressing glycogen branching enzyme 1 (GBE1, EC 2.4.1.18) expression using lentiviruses harboring short hairpin RNA (shRNA). GBE1 suppression in embryonic cortical neurons led to polyglucosan accumulation and associated apoptosis, which were reversible by rapamycin or starvation treatments. Further analysis revealed that rapamycin and starvation led to phosphorylation and inactivation of glycogen synthase (GS, EC 2.4.1.11), dephosphorylated and activated in the GBE1-suppressed neurons. These protective effects of rapamycin and starvation were reversed by overexpression of phosphorylation site mutant GS only if its glycogen binding site was intact. While rapamycin and starvation induce autophagy, autophagic maturation was not required for their corrective effects, which prevailed even if autophagic flux was inhibited by vinblastine. Furthermore, polyglucosans were not observed in any compartment along the autophagic pathway. Our data suggest that glycogen branching enzyme repression in glycogenoses can cause pathogenic polyglucosan buildup, which might be corrected by GS inhibition. Knockdown of glycogen branching enzyme in neurons led to accumulation of an insoluble form of glycogen called polyglucosan, to apoptosis and to activation of glycogen synthase. These effects were reversed by glycogen synthase inhibition through starvation and rapamycin treatments, suggesting a potential therapeutic value of glycogen synthase inhibition for treating glycogen storage disorders.

In this study we demonstrate that recombinant rabbit muscle protein phosphatase-1 immobilized on CH-Sepharose is an efficient affinity chromatography support for the isolation of subunits of phosphatase-1. The support was used to isolate the glycogen binding subunit of phosphatase-1 from muscle and nonmuscle rat tissues. Examination of the affinity-purified material from rat heart and liver showed that the major component was a 160-kDa polypeptide on SDS-PAGE. The identity of the purified liver 160-kDa polypeptide as the glycogen binding subunit was confirmed by the demonstration that it is capable of binding to glycogen, and is phosphorylated by the catalytic subunit of PKA. The novel observation was made that the phosphorylation was dependent on the presence of glycogen. Examination of the material from heart, lung, liver, kidney, and brain showed a similar phenomenon. Our studies show that this subunit is widely distributed in tissues. The affinity support was also efficient in the isolation of the NIPP-1 (nuclear inhibitor of protein phosphatase-1) proteins from calf thymus. Examination of heat-treated extracts of rat liver led to the isolation of a novel 19-kDa inhibitory protein which could also be phosphorylated by PKA and may represent the rat liver homolog of calf thymus NIPP-1.

In rabbit muscle, analyzed by Western blot, the glycogen-bound protein phosphatase-1 (PP-1G) is composed of a 37-kilodalton (kDa) catalytic subunit complexed to a 160-kDa glycogen-binding subunit (G-subunit) responsible for the interaction of PP-1G with glycogen. PP-1G has not been characterized in humans. In the present study, G-subunit was identified in human muscle extracts by Western blot using an antibody raised against a sequence (the phosphoregulatory domain) of the rabbit muscle G-subunit. The human G-subunit was also a 160-kDa protein by Western blot. When the G-subunit content of skeletal muscle was quantitated in 17 Pima Indians with a wide range of insulin sensitivities determined during euglycemic clamps, there was a significant negative correlation (r = -0.55; P = 0.02) between the G-subunit content and in vivo insulin-mediated glucose disposal rates. The results suggest that insulin resistance is associated with an increased content and/or immunoreactivity of G-subunit in human muscle.

Glycogen synthase, a central enzyme in glucose metabolism, catalyzes the successive addition of α-1,4-linked glucose residues to the non-reducing end of a growing glycogen molecule. A non-catalytic glycogen-binding site, identified by x-ray crystallography on the surface of the glycogen synthase from the archaeon Pyrococcus abyssi, has been found to be functionally conserved in the eukaryotic enzymes. The disruption of this binding site in both the archaeal and the human muscle glycogen synthases has a large impact when glycogen is the acceptor substrate. Instead, the catalytic efficiency remains essentially unchanged when small oligosaccharides are used as substrates. Mutants of the human muscle enzyme with reduced affinity for glycogen also show an altered intracellular distribution and a marked decrease in their capacity to drive glycogen accumulation in vivo. The presence of a high affinity glycogen-binding site away from the active center explains not only the long-recognized strong binding of glycogen synthase to glycogen but also the processivity and the intracellular localization of the enzyme. These observations demonstrate that the glycogen-binding site is a critical regulatory element responsible for the in vivo catalytic efficiency of GS. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc., This work was supported by the Dirección General de Investigación, Ministerio de Ciencia y Tecnología, Spain Grants BFU2009-09268 (to I. F.), CTQ2006-00743 (to J. C. F.), and BFU2008-00769 (to J. J. G.), a grant from the Generalitat de Catalunya AGAUR 2009SGR-1176 (to J. J. G.), a grant from the Fundación Marcelino Botín, and the CIBER de Diabetes y Enfermedades Metabólicas Asociadas.

The in vivo phosphorylation stoichiometries of 4 serines on the glycogen-binding (G)-subunit of protein phosphatase 1 (PP1) have been determined. In fed rabbits injected with propranolol stoichiometries (mol/mol) were: site 1 (0.67 +/- 0.09), site 2 (0.20 +/- 0.07), site 3a (0.23 +/- 0.01) and site 3b (0). After injection with adrenalin they became: site 1 (0.90 +/- 0.02), site 2 (0.72 +/- 0.01), site 3a (0.23 +/- 0.02) and site 3b (0). These results, together with other data, establish that site 2 phosphorylation by cyclic AMP-dependent protein kinase triggers dissociation of PP1 from the G-subunit in vivo. They also demonstrate that a residue phosphorylated in vitro by glycogen synthase kinase 3 (site 3a) is phosphorylated in vivo.

The esdC (early sexual development) gene was isolated by using an expressed sequence tag (EST) as a probe from a genomic library of the early sexual developmental stage mycelia of Aspergillus nidulans. The sequence analysis revealed that the esdC gene contains a 59bp intron and encodes a 266 amino acid polypeptide with a calculated molecular weight of 29.4kDa. The EsdC protein is conserved among filamentous fungi and has a domain with similarity to a glycogen binding domain conserved in the beta subunit of the AMP-activated protein kinase (AMPK) complex. Although the esdD gene was expressed during asexual development, the expression reached its maximum at 10h and decreased thereafter up to 50h after the end of the induction of sexual development. In an esdC-null mutant under a veA(+) background, no sexual structures were formed at any condition examined. However, esdC overexpression did not lead to an induction of sexual development. In addition, to the effect of the esdC mutation on the sexual development, more conidiophores were formed in the esdC-null mutant than in a wild type. These results indicate that the esdC gene is necessary for sexual structure formation but its overexpression is not sufficient to enhance this process. Expression of the esdC gene throughout development was positively regulated by the veA gene. In addition, very little and no esdC transcript, respectively, was observed in an flbA-null mutant and in a fadA(G42R) mutant, and the esdC transcript level was higher in a fadA-null mutant and in a sfaD-null mutant than in a wild type, indicating that inactivation of FadA is necessary for positive regulation of esdC expression.

Lafora disease, a progressive myoclonus epilepsy, is an autosomal recessive disease caused in approximately 80% of cases by mutation of the EPM2A gene, which encodes a dual specificity protein phosphatase called laforin. In addition to its phosphatase domain, laforin contains an N-terminal carbohydrate-binding domain (CBD). Mouse laforin was expressed as an N-terminally polyHis tagged protein in Escherichia coli and purified close to homogeneity. The enzyme was active towards p-nitrophenylphosphate (50-80mmol/min/mg, K(m) 4.5mM) with maximal activity at pH 4.5. Laforin binds to glycogen, as previously shown, and caused potent inhibition, half maximally at approximately 1mug/ml. Less branched glucose polymers, amylopectin and amylose, were even more potent, with half maximal inhibition at 10 and 100ng/ml, respectively. With all polysaccharides, however, inhibition was incomplete and laforin retained 20-30% of its native activity at high polysaccharide concentrations. Glucose and short oligosaccharides did not affect activity. Substitution of Trp32 in the CBD by Gly, a mutation found in a patient, caused only a 30% decrease in laforin activity but abolished binding to and inhibition by glycogen, indicating that impaired glycogen binding is sufficient to cause Lafora disease.

The yeast Snf1 kinase and its mammalian ortholog, AMP-activated protein kinase (AMPK), regulate responses to metabolic stress. Previous studies identified a glycogen-binding domain in the AMPK beta1 subunit, and the sequence is conserved in the Snf1 kinase beta subunits Gal83 and Sip2. Here we use genetic analysis to assess the role of this domain in vivo. Alteration of Gal83 at residues that are important for glycogen binding of AMPK beta1 abolished glycogen binding in vitro and caused diverse phenotypes in vivo. Various Snf1/Gal83-dependent processes were upregulated, including glycogen accumulation, expression of RNAs encoding glycogen synthase, haploid invasive growth, the transcriptional activator function of Sip4, and activation of the carbon source-responsive promoter element. Moreover, the glycogen-binding domain mutations conferred transcriptional regulatory phenotypes even in the absence of glycogen, as determined by analysis of a mutant strain lacking glycogen synthase. Thus, mutation of the glycogen-binding domain of Gal83 positively affects Snf1/Gal83 kinase function by a mechanism that is independent of glycogen binding.

Progressive myoclonus epilepsy of Lafora type (LD, MIM 254780) is a fatal autosomal recessive disorder characterized by the presence of progressive neurological deterioration, myoclonus, epilepsy and polyglucosan intracellular inclusion bodies, called Lafora bodies. Lafora bodies resemble glycogen with reduced branching, suggesting an alteration in glycogen metabolism. Linkage analysis and homozygosity mapping localized EPM2A, a major gene for LD, to chromosome 6q24. EPM2A encodes a protein of 331 amino acids (named laforin) with two domains, a dual-specificity phosphatase domain and a carbohydrate binding domain. Here we show that, in addition, laforin interacts with itself and with the glycogen targeting regulatory subunit R5 of protein phosphatase 1 (PP1). R5 is the human homolog of the murine Protein Targeting to Glycogen, a protein that also acts as a molecular scaffold assembling PP1 with its substrate, glycogen synthase, at the intracellular glycogen particles. The laforin-R5 interaction was confirmed by pull-down and co-localization experiments. Full-length laforin is required for the interaction. However, a minimal central region of R5 (amino acids 116-238), including the binding sites for glycogen and for glycogen synthase, is sufficient to interact with laforin. Point-mutagenesis of the glycogen synthase-binding site completely blocked the interaction with laforin. The majority of the EPM2A missense mutations found in LD patients result in lack of phosphatase activity, absence of binding to glycogen and lack of interaction with R5. Interestingly, we have found that the LD-associated EPM2A missense mutation G240S has no effect on the phosphatase or glycogen binding activities of laforin but disrupts the interaction with R5, suggesting that binding to R5 is critical for the laforin function. These results place laforin in the context of a multiprotein complex associated with intracellular glycogen particles, reinforcing the concept that laforin is involved in the regulation of glycogen metabolism., This work was supported by grants from the Asociación Lafora España, the Spanish CICYT (SAF2001-0613) and the Instituto Carlos III (PI02/0536, G03/054 and G03/011). K.E.H. was supported by a Marie Curie postdoctoral fellowship (HPMD-CT-2001-01259) from the European Commission and P.G.-G. by a fellowship from the Asociación Lafora España and by the Centro de Investigación en Enfermedades Neurológicas

The skeletal muscle glycogen-binding subunit (GM) of protein phosphatase-1 (PP1) is the founding member of a family of proteins that tether the PP1 catalytic subunit (PP1C) to glycogen and promote the dephosphorylation of glycogen synthase. A hydrophobic sequence (called here the VFV motif) is conserved among GM, the liver subunit GL, and the widely expressed subunits, PTG, R5 and U5. This study analyzed the role of this VFV motif in binding to glycogen and PP1C. Glutathione S-transferase (GST) fusions with the N-terminal domain of GM (GST-GM(1–240)) and with the full length R5 protein (GST-R5) both bound to glycogen in a co-sedimentation assay. In contrast, GST itself did not bind to glycogen. A single residue substitution in GST-GM(1–240), F155A, reduced glycogen binding by 40%. Double residue substitutions V150A/F155A and F155A/V159A resulted in greater reductions (60–70%) in glycogen binding, showing these hydrophobic residues influenced the protein-glycogen interaction. The wild type and V150A/F155A fusion proteins were digested by trypsin into the same sized fragments at the same rate. Furthermore, the wild type and mutated GST-GM proteins as well as GST-R5 bound equivalent amounts of PP1C, in either pull-down or far-Western assays. These results demonstrated retention of overall tertiary structure by the mutated fusion proteins, and indicated that glycogen and PP1C binding are independent of one another. A 68 residue segment of R5 encompassing the VFV motif was sufficient to produce glycogen binding when fused to GST. This motif, that is in bacterial and fungal starch metabolizing enzymes, probably has been conserved during evolution as a functional domain for binding glycogen and starch.

A yeast two-hybrid screen of a chicken gizzard cDNA library detected the interaction of the catalytic subunit of protein phosphatase type 1 with a novel subunit. Subsequent characterization established similarity (58%) to the rat liver glycogen-binding subunit. Northern analyses showed expression in a wide range of tissues.

Mitogen activated protein kinases (MAPKs) are thought to function as key enzymes in signalling processes stimulated by growth factors and differentiating factors [I]. Two forms of MAPK with molecular weights of 42 and 44 kDa have been purified from a number of cell types [2]. MAPK requires phosphorylation of threonine and tyrosine residues for activity [3]. Possible in vivo substrates for MAPK include transcription factors c-myc and c-jun [4] and two protein kinases, S6 kinase I1 [5] and MAP kinase activated protein kinase-2 (MAPKAP-2) [6], all of which are activated by MAPK in vifro. MAPK has been implicated in having an important role both in the stimulation of cell proliferation by growth factors [7] and in the stimulation of glycogen synthesis by insulin [5] . In rabbit skeletal muscle S6 kinase I1 activates glycogen synthase and inactivates phosphorylase kinase via phosphorylation of a single serine residue (site 1) of the glycogen binding subunit of PP-IG and stimulation of its phosphatase activity [5]. Increased phosphorylation of PP-IG at site 1 is observed in skeletal muscle of rabbits injected with insulin [5] . The physiological roles of MAPKAP-2 are not yet known, although it phosphorylates a number of substrates in virro, including glycogen synthase [6]. Activation of MAPK by growth factors and insulin has been demonstrated in a number of cell lines [8], however few studies have examined the activity of MAPK in liver. In freshly isolated hepatocytes from rat MAPK activity was stimulated by approximately 20% following a 10 min incubation with a supraphysiological concentration of insulin [9]. A sequential activation of MAPK and S6 kinase I1 was observed in intact rat liver following insulin injection in vivo [lo]. In this study we investigated the possible role of MAPK in the regulation of glycogen synthesis in hepatocyte cultures. MAPK activity was determined in hepatocyte cultures exposed to epidermal growth factor (EGF), which inhibits glycogenesis and is a potent mitogen, and insulin, which stimulates glycogenesis and is not a mitogen [I I].

The AMP-activated protein kinase (AMPK) is the critical component of a highly conserved signalling pathway found in all eukaryotes that plays a key role in regulating metabolic processes in response to variations in energy supply and demand. AMPK protects cells from stresses that decrease cellular energy charge (i.e increase the AMP:ATP ratio) by initiating a shift in metabolism towards the generation of ATP while simultaneously down regulating pathways that consume ATP. The role of AMPK as an energy sensor extends beyond the cell and it is now apparent that it is a key regulator of whole-body energy homeostasis. These functions have stimulated considerable interest in AMPK as a promising target to treat metabolic disorders such as obesity and Type 2 diabetes. Recently, crystal structures of heterotrimeric core fragments and individual domains of AMPK from mammals, Schizosaccharomyces pombe and Saccharomyces cerevisiae have been solved. Together they provide an impressive insight into the molecular interactions involved in regulating kinase activity, heterotrimeric assembly, glycogen binding, and binding of the regulatory nucleotides AMP and ATP.

Reaction of four of the eight sulfhydryl groups per subunit of rat liver glycogen synthetase D through mixed disulfide formation with oxidized glutathione leads to inactivation of the enzyme and its dissociation from glycogen. The differential protection offered by glucose 6-phosphate against inactivation and dissociation permits determination of the number of sulfhydryl groups involved in each process. Modification of the first sulfhydryl group had no effect on enzyme activity or glycogen binding. Reaction of the second sulfhydryl group led to complete inactivation of the enzyme and was accompanied by an increase in the Ka for glucose-6-P. Modification of two more sulfhydryl groups per subunit resulted in release of the enzyme from glycogen. Gel filtration experiments revealed that disruption of the glycogen-glycogen synthetase D complex was a consequence of dissociation of the enzyme itself into a subunit (mol wt 86,000) which could not bind glycogen. The enzyme-glycogen complex could be reconstituted from glycogen and glycogen-free, inactive glycogen synthetase D by treatment with glucose-6-P. Reduction with dithiothreitol, which by itself failed to restore the complex, significantly lowered the concentration of glucose-6-P required to fully achieve reconstitution. Treatment with glucose-6-P, but not reduction with dithiothreitol, reassociated the inactive subunit into a high molecular weight species which is the form capable of binding glycogen. These results suggest that glucose-6-P plays an important role in maintaining the glycogen-glycogen synthetase D complex.

The glycogen-bound form of protein phosphatase-1 (termed protein phosphatase-1G) is composed of the catalytic (C) subunit complexed to a glycogen-binding (G) subunit that anchors the enzyme to glycogen [Strålfors et al. (1985) Eur. J. Biochem. 149, 295-303]. Incubation of purified protein phosphatase-1G with cyclic-AMP-dependent protein kinase and MgATP, which leads to stoichiometric phosphorylation of the G-subunit [Caudwell et al. (1986) FEBS Lett. 194, 85-90], was found to promote the release of the phosphatase from glycogen; similar observations were made using glycogen-protein particle preparations. An intravenous injection of adrenaline decreased protein phosphatase-1 activity associated with the glycogen-protein particles by 50% with a corresponding increase in the amount present in the cytosol. By contrast, adrenaline did not affect the distribution of glycogen synthase or glycogen phosphorylase which remained entirely bound to glycogen in these experiments. The specific release of protein phosphatase-1 from glycogen may facilitate its inactivation by inhibitor-1 in the cytosol, thereby preventing dephosphorylation of the glycogen metabolising enzymes. Translocation of protein phosphatase-1 may represent a novel mechanism for the activation of glycogenolysis and inhibition of glycogen synthesis by adrenaline.

Crystallographic binding studies of various metabolites to phosphorylase b in the presence of 2 m m -IMP have been carried out at low resolution (8.7 A) with three-dimensional data and at high resolution (3 a) with two-dimensional data. From correlation of peaks observed in difference Fourier syntheses based on these two sets of data, the following binding sites have been identified: (1) the “active” site to which the substrate, glucose 1-phosphate, and the substrate analogues, maltotriose and arsenate, bind and which is close to the subunit-subunit interface of the phosphorylase dimer; (2) the allosteric adenine-nucleotide binding site to which the allosteric activator AMP and the allosteric inhibitor ATP bind and which is very close to the active site; (3) the inhibitor binding site for glucose 6-phosphate, which is also close to the active site. Glucose 6-phosphate causes extensive conformational changes in the protein, which are the largest observed for all the metabolites studied so far; (4) a glycogen binding site on the surface of the molecule to which maltotriose binds. The distance over the surface of the phosphorylase molecule from this site to the active site is 50 to 60 A; (5) a second glucose 1-phosphate binding site situated in the interior of the molecule. The significance of this site is not yet understood but its position in the centre of the molecule suggests that it may have a key role in the control and catalysis of phosphorylase.

The glycogen-bound form of protein phosphatase-1 (PP-1G) was previously purified as a heterodimer composed of a 37-kDa catalytic (C) subunit and a proteolytically sensitive 103-kDa glycogen-binding (G) subunit [Strahlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295-303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161-kDa protein, and that the 103-kDa species (now termed G') is itself a product of proteolysis. A second phosphorylation site for cAMP-dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP-1 at a slow rate, whereas site 1 was resistant to autodephosphorylation. PP-1G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen-protein particles during washing with a variety of solvents. The PP-1G holoenzyme was released from glycogen-protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP-1G. Binding experiments with purified PP-1G and glycogen indicated a bimolecular process with Kapp values corresponding to about 8 nM glycogen and 4 nM PP-1G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high-affinity glycogen-binding domain on the G subunit, and indicate that a physiological concentrations of phosphatase and glycogen, PP-1G should be almost entirely bound to glycogen.

Meyer et al. [1 ] have described the preparation of a particulate suspension from rabbit muscle containing glycogen and a number of enzymes. They suggest that the glycogen binding enzymes are attached to the large glycogen molecules in the suspension and they term these aggregates 'glycogen particles'. The enzymes present include phosphorylase b, phosphorylase b kinase, phosphorylase phosphatase and other enzymes of glycogen metabolism, trace glycolytic enzymes and some of the enzymes concerned with nucleotide interconversions. Studies by the same group have shown that some of these enzymes have anomalous properties when bound to the glycogen particles and they postulate specific interactions either between the enzymes themselves or with the carbohydrate matrix to account for this. They also suggest that these protein-glycogen complexes are the glycogen granules seen in sections of muscle by electron microscopy and that the 'glycogen particle' is therefore a cellular oganelle [2 -5 ] . Transient production of phosphorylase a could be generated in this glycogen particle rich fraction by the addition of ATP, calcium and magnesium ions. This 'flash activation', was completely calcium dependent because of the calcium requirement of phosphorylase b kinase, and was terminated by the action of phosphorylase phosphatase on the phosphorylase a after the ATP had been used up [2]. Using spin-labelled phosphorylase b it has been possible to monitor the conformational changes which occur both when ligands bind to the enzyme and when it is activated by conversion to phosphorylase a [6,7]. This clearly offers a way of studying the behaviour of phosphorylase attached to the glycogen particle and in this paper we describe the use of spin labelled phosphorylase b to monitor flash activation and the interactions of ligands with phosphorylase during this process.

PERSPECTIVES AND SUMMARY .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HISTORICAL INTRODUCTION .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiological Role and Metabolic Regulation of Activity . . . .... ... . . .... . . .. . . .. .. . . .. . Subunit Relationships . Chemical Studies . Kinetics and Mechanism . ..... . . . . . . .. .. . .. . . . . . . . . . . . .. . . .. . . .. .. .. . . . . .. . . .. . . . Allosteric Conformational Transitions and Physicochemical Probes .. Amino Acid Sequences . THE PRIMARY STRUCTURE OF PHOSPHORYLASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Strategic Approach to the Research Problem ..... . ...... ...... . The Amino Acid Sequence and Location of Special Residues ......... . . . . . .. . . .. ... . . . . Sequence Homologies . . . . . .. . . .. . . . . .... . . . . ....... .... . ....... . THE THREE-DIMENSIONAL STRUCTURE OF PHOSPHORYLASE a . X-ray and Electron Microscope Studies of Quaternary Structure and Subunit Shape .... . Crystal Forms of Phosphorylase . ... . . . . . . . ..... . . . . . . . . . ....... . . . . . . . . . ..... . . . Description of the Molecule . . . . . . .. . . . . . . .. .. .. . . . . ........ . . . . . . . . . . . . . . Pyridoxal Phosphate . . . . . . . . . ..... . .... . . . .... .... . ... . ... . . . Ser-14 Phosphate . FUNCTIONAL AND BIOLOGICAL SIGNIFICANCE OF THE STRUCTURE Ligand Binding Sites .... . . Glycogen binding . . . . . . . . . .. . . .. ... . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . +12034

Single molecules of glycogen phosphorylase b exhibit images in the electron microscope which are similar in shape and dimension to those derived from X-ray crystallography. Phosphorylase a exhibits tetramers but shows dimers in the presence of glucose. Glycogen debranching enzyme appears as a monomer with an unusual crescent or shrimp-like shape, with occasional isologous aggregation to circular dimers. The longest dimension of the monomer is very similar to that of the phosphorylase dimer, 11.5 nm. Strong binding of the debranching enzyme to glycogen is readily visualized in the electron microscope. It is suggested that the distinctive shape of the debranching enzyme may be related to its catalytic function.

A full-length cDNA encoding a novel human protein phosphatase 1 (PP1) binding subunit of molecular mass 36 kDa, termed PPP1R5, was sequenced. PPP1R5 shows 42% identity to the glycogen binding subunit (G(L)) of PP1 from rat liver and 28% identity to the N-terminal region of the glycogen binding subunit (G(M)) of PP1 from human skeletal muscle. Like G(L), PPP1R5 modulates the specificity of PP1, but it differs from G(L) in being present in a wide variety of tissues, besides liver. The amino acid sequence and properties of PPP1R5 indicate that it is not subject to the same modes of covalent and allosteric regulation by hormones as are G(M) and G(L).

The glycogen-binding (G) subunit of protein phosphatase-1G is phosphorylated stoichiometrically by glycogen synthase kinase-3 (GSK3), and with a greater catalytic efficiency than glycogen synthase, but only after prior phosphorylation by cyclic AMP-dependent protein kinase (A-kinase) at site 1. The residues phosphorylated are the first two serines in the sequence AIFKPGFSPQPSRRGS-, while the C-terminal serine (site 1) is one of the two residues phosphorylated by A-kinase. These findings demonstrate that (i) the G subunit undergoes multisite phosphorylation in vitro; (ii) phosphorylation by GSK3 requires the presence of a C-terminal phosphoserine residue; (iii) GSK3 can synergise with protein kinases other than casein kinase-2.

Prior phosphorylation of its substrate has been shown to be important for substrate recognition by the protein kinase glycogen synthase kinase-3 (GSK-3). Phosphorylation of glycogen synthase by GSK-3 is known to be enhanced by the previous action of casein kinase II and the sequence -SXXXS(P)- was proposed as the minimal recognition determinant for GSK-3. The glycogen binding subunit of type 1 phosphoprotein phosphatase has been shown to be phosphorylated by cyclic AMP-dependent protein kinase at serine-13 in the sequence KPGFS(5)PQPS(9)RRGS(13)ESSEEVYV (F.B. Caudwell, A. Hiraga, and P. Cohen (1986) FEBS Lett. 194, 85-89). Inspection of the sequence revealed potential GSK-3 sites at residues 5 and 9. Using a synthetic peptide with the above sequence, we found that phosphorylation of serine-13 by cyclic AMP-dependent protein kinase permitted the recognition of serine-9 and serine-5 by GSK-3. The work provides another example of a substrate for GSK-3 and demonstrates that the action of GSK-3 is linked to the presence of phosphate in the substrate and not the action of any particular protein kinase. In the course of the analyses, a novel feature of trypsin cleavage of phosphopeptides was noted. In the sequence -SRRGS(P)- trypsin acted uniquely after the first arginine whereas in the sequence -S(P)RRGS(P)- it cleaved randomly at either arginine residue. The fact that GSK-3 could phosphorylate a peptide derived from a phosphatase subunit also raises the possibility that GSK-3 might be involved in controlling glycogen-associated type 1 phosphatase and, more generally, in mediating cyclic AMP control of protein phosphorylation in cells.

A glycogen-adipoyldihydrazide-Sepharose 4B column has been prepared for the analysis of glycogen-binding protein components of rat tissues. Glycogen-metabolizing enzymes; glycogen synthase, phosphorylase, branching enzyme, and debranching enzyme of skeletal muscle and liver have been adsorbed to the column, while those of brain showed very low affinities to it. On SDS gel electrophoresis of the glycogen-binding protein fractions, at least five and nine additional protein components have been detected in skeletal muscle and liver, respectively.

Properties characteristic of the structure and function of dimeric concanavalin A have been studied as a function of pH in the acid pH range using preparations comprising intact subunits or enriched in fragmented chains. For intact subunits, the glycogen binding ability falls to zero with a midpoint of pH 4.7, the release of Mn+2, Ca+2 and the fluorescent ligand 4-methylumbelliferyl-alpha-D-mannopyranoside from the lectin coincides over a pH range centered at pH 3.9, and the CD spectra of the aromatic amino acid residues increase sharply in amplitude between pH 4.0 and 1.5. Nevertheless, the sedimentation coefficient and peptide CD spectrum change insignificantly in the pH range 5 to 2, indicating that dimeric concanavalin A retains its secondary structure and overall hydrodynamic shape essentially unchanged upon acidification. The behavior of concanavalin comprising primarily fragmented chains is not significantly different from that of intact subunits, although it precipitates glycogen less efficiently. It is concluded that dimeric concanavalin A does not undergo a concerted change in structure upon acidification, but rather that it passes through a series of states differing from one another in their local conformations. The distinction in binding between the monosaccharide and the polysaccharide is attributed to participation of a secondary binding site in the latter case. A change in optical activity at 283 nm in the pH range 5-6 is ascribed to disruption of intersubunit interactions of Tyr 67 as the protein undergoes the dimer-tetramer equilibrium.

The glycogen-binding (G) subunit of protein phosphatase-1 is phosphorylated in vivo. In rabbits injected with propranolol the serine residue termed site-1 was phosphorylated in 56% of the molecules isolated, and phosphorylation increased to 82% after administration of adrenalin. It is concluded that the G-subunit is a physiological substrate for cyclic AMP-dependent protein kinase. The G-subunit remained largely bound to glycogen even after injection of adrenalin, whereas half of the protein phosphatase-1 activity associated with glycogen was released into the cytosol. The results indicate that adrenalin induces dissociation of the catalytic subunit from the G-subunit in vivo.