Title: Malin Decreases Glycogen Accumulation by Promoting the Degradation of Protein Targeting to Glycogen (PTG)
Abstract: Lafora disease (LD) is an autosomal recessive neurodegenerative disease that results in progressive myoclonus epilepsy and death. LD is caused by mutations in either the E3 ubiquitin ligase malin or the dual specificity phosphatase laforin. A hallmark of LD is the accumulation of insoluble glycogen in the cytoplasm of cells from most tissues. Glycogen metabolism is regulated by phosphorylation of key metabolic enzymes. One regulator of this phosphorylation is protein targeting to glycogen (PTG/R5), a scaffold protein that binds both glycogen and many of the enzymes involved in glycogen synthesis, including protein phosphatase 1 (PP1), glycogen synthase, phosphorylase, and laforin. Overexpression of PTG markedly increases glycogen accumulation, and decreased PTG expression decreases glycogen stores. To investigate if malin and laforin play a role in glycogen metabolism, we overexpressed PTG, malin, and laforin in tissue culture cells. We found that expression of malin or laforin decreased PTG-stimulated glycogen accumulation by 25%, and co-expression of malin and laforin abolished PTG-stimulated glycogen accumulation. Consistent with this result, we found that malin ubiquitinates PTG in a laforin-dependent manner, both in vivo and in vitro, and targets PTG for proteasome-dependent degradation. These results suggest an additional mechanism, involving laforin and malin, in regulating glycogen metabolism. Lafora disease (LD) is an autosomal recessive neurodegenerative disease that results in progressive myoclonus epilepsy and death. LD is caused by mutations in either the E3 ubiquitin ligase malin or the dual specificity phosphatase laforin. A hallmark of LD is the accumulation of insoluble glycogen in the cytoplasm of cells from most tissues. Glycogen metabolism is regulated by phosphorylation of key metabolic enzymes. One regulator of this phosphorylation is protein targeting to glycogen (PTG/R5), a scaffold protein that binds both glycogen and many of the enzymes involved in glycogen synthesis, including protein phosphatase 1 (PP1), glycogen synthase, phosphorylase, and laforin. Overexpression of PTG markedly increases glycogen accumulation, and decreased PTG expression decreases glycogen stores. To investigate if malin and laforin play a role in glycogen metabolism, we overexpressed PTG, malin, and laforin in tissue culture cells. We found that expression of malin or laforin decreased PTG-stimulated glycogen accumulation by 25%, and co-expression of malin and laforin abolished PTG-stimulated glycogen accumulation. Consistent with this result, we found that malin ubiquitinates PTG in a laforin-dependent manner, both in vivo and in vitro, and targets PTG for proteasome-dependent degradation. These results suggest an additional mechanism, involving laforin and malin, in regulating glycogen metabolism. Lafora disease (LD) 3The abbreviations used are: LDLafora diseaseLBLafora bodiesIPimmunoprecipitationCBMcarbohydrate binding modulesPP1protein phosphatase 1CHOChinese hamster ovaryHRPhorseradish peroxidaseGSTglutathione S-transferasePTGprotein targeting to glycogenEPM2Aepilepsy myoclonus gene 2A. (OMIM number 254780) is an autosomal recessive disease resulting in severe neurodegeneration, epilepsy, and death (1Lafora G.R. Gluck B. Z. Ges. Neurol. Psychiatr. 1911; 6: 1-14Crossref Scopus (164) Google Scholar, 2Van Heycop Ten Ham M.W. Vinken P.J. Bruyn G.W. Handbook of Clinical Neurology. North Holland Publishing Company, Holland, Amsterdam1975: 647-666Google Scholar). It is one of five major progressive myoclonus epilepsies and is characterized by myoclonus, tonic seizures, and progressive neurological deterioration (3Berkovic S.F. So N.K. Andermann F. J. Clin. 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Lafora disease Lafora bodies immunoprecipitation carbohydrate binding modules protein phosphatase 1 Chinese hamster ovary horseradish peroxidase glutathione S-transferase protein targeting to glycogen epilepsy myoclonus gene 2A. LBs develop in the cytoplasm of cells from multiple tissues, including brain, kidney, skin, liver, and cardiac and skeletal muscle (1Lafora G.R. Gluck B. Z. Ges. Neurol. Psychiatr. 1911; 6: 1-14Crossref Scopus (164) Google Scholar, 7Busard H.L. Gabreels-Festen A.A. Renier W.O. Gabreels F.J. Stadhouders A.M. Ann. Neurol. 1987; 21: 599-601Crossref PubMed Scopus (55) Google Scholar, 8Schwarz G.A. Yanoff M. Arch. Neurol. 1965; 12: 172-188Crossref PubMed Scopus (91) Google Scholar, 9Lafora G.R. Virchows Arch. f. Pathol. Anat. 1911; 205: 295Crossref Scopus (103) Google Scholar). Whereas animals normally store carbohydrates as soluble glycogen, LBs are insoluble accumulations of a "foreign" carbohydrate. Like glycogen, LBs are composed of α1,4-glycosidic linkages between glucose residues with α1,6 branches. However, unlike glycogen the branching in LBs is not as ordered and branches occur far less frequently, every 15–30 glucose monomers versus every 12–14 for glycogen (10Sakai M. Austin J. Witmer F. Trueb L. Neurology. 1970; 20: 160-176Crossref PubMed Google Scholar, 11Yokoi S. Austin J. Witmer F. J. Neuropathol. Exp. Neurol. 1967; 26: 125-127PubMed Google Scholar). Another naturally occurring carbohydrate that is similar to LBs is plant amylopectin, the major component of plant starch. Amylopectin is also composed of α1,4-glycosidic linkages between glucose residues with α1,6 branches occurring every 12–20 residues. Accordingly, multiple studies have found that the biochemical properties of LBs resemble amylopectin (10Sakai M. Austin J. Witmer F. Trueb L. Neurology. 1970; 20: 160-176Crossref PubMed Google Scholar, 11Yokoi S. Austin J. Witmer F. J. Neuropathol. Exp. Neurol. 1967; 26: 125-127PubMed Google Scholar, 12Yokoi S. Austin J. Witmer F. Sakai M. Arch. Neurol. 1968; 19: 15-33Crossref PubMed Scopus (116) Google Scholar). Recessive mutations in the EPM2A (epilepsy myoclonus gene 2A) locus are responsible for ∼48% of LD cases (13Chan E.M. Young E.J. Ianzano L. Munteanu I. Zhao X. Christopoulos C.C. Avanzini G. Elia M. Ackerley C.A. Jovic N.J. Bohlega S. Andermann E. Rouleau G.A. Delgado-Escueta A.V. Minassian B.A. Scherer S.W. Nat. Genet. 2003; 35: 125-127Crossref PubMed Scopus (267) Google Scholar, 14Ianzano L. Zhang J. Chan E.M. Zhao X.C. Lohi H. Scherer S.W. Minassian B.A. Hum. Mutat. 2005; 26: 397Crossref PubMed Scopus (54) Google Scholar, 15Chan E.M. Omer S. Ahmed M. Bridges L.R. Bennett C. Scherer S.W. Minassian B.A. Neurology. 2004; 63: 565-567Crossref PubMed Scopus (81) Google Scholar, 16Serratosa J.M. Gomez-Garre P. Gallardo M.E. Anta B. de Bernabe D.B. Lindhout D. Augustijn P.B. Tassinari C.A. Malafosse R.M. Topcu M. Grid D. Dravet C. Berkovic S.F. de Cordoba S.R. Hum. Mol. Genet. 1999; 8: 345-352Crossref PubMed Scopus (198) Google Scholar). EPM2A encodes a bi-modular protein, called laforin, that contains a carbohydrate binding module type 20 (CBM 20) at the amino terminus and a dual specificity phosphatase domain at the carboxyl terminus (16Serratosa J.M. Gomez-Garre P. Gallardo M.E. Anta B. de Bernabe D.B. Lindhout D. Augustijn P.B. Tassinari C.A. Malafosse R.M. Topcu M. Grid D. Dravet C. Berkovic S.F. de Cordoba S.R. Hum. Mol. Genet. 1999; 8: 345-352Crossref PubMed Scopus (198) Google Scholar, 17Minassian B.A. Lee J.R. Herbrick J.A. Huizenga J. Soder S. Mungall A.J. Dunham I. Gardner R. Fong C.Y. Carpenter S. Jardim L. Satishchandra P. Andermann E. Snead O.C. Lopes-Cendes 3rd, I. Tsui L.C. Delgado-Escueta A.V. Rouleau G.A. Scherer S.W. Nat. Genet. 1998; 20: 171-174Crossref PubMed Scopus (418) Google Scholar, 18Wang J. Stuckey J.A. Wishart M.J. Dixon J.E. J. Biol. Chem. 2002; 277: 2377-2380Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Laforin is conserved in all vertebrates and a subclass of protists (19Gentry M.S. Dowen R.H. Worby 3rd, C.A. Mattoo S. Ecker J.R. Dixon J.E. J. Cell Biol. 2007; 178: 477-488Crossref PubMed Scopus (124) Google Scholar). Intriguingly, of the 128 human phosphatases laforin is the only phosphatase that possesses a CBM of any type. CBMs typically target a protein to a carbohydrate and the enzymatic portion of the protein modifies the carbohydrate in some manner (e.g. α-amylase). Consistent with this precedence, we demonstrated that laforin and a laforin-like protein from plants, called SEX4 (20Niittyla T. Comparot-Moss S. Lue W.-L. Messerli G. Trevisan M. Seymour M.D.J. Gatehouse J.A. Villadsen D. Smith S.M. Chen J. Zeeman S.C. Smith A.M. J. Biol. Chem. 2006; 281: 11815-11818Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), release phosphate from complex carbohydrates (19Gentry M.S. Dowen R.H. Worby 3rd, C.A. Mattoo S. Ecker J.R. Dixon J.E. J. Cell Biol. 2007; 178: 477-488Crossref PubMed Scopus (124) Google Scholar, 21Worby C.A. Gentry M.S. Dixon J.E. J. Biol. Chem. 2006; 281: 30412-30418Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Alternatively, GSK3β is also reported to be a substrate of laforin (22Liu Y. Wang Y. Wu C. Liu Y. Zheng P. J. Biol. Chem. 2006; 281: 34768-34774Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 23Lohi H. Ianzano L. Zhao X.-C. Chan E.M. Turnbull J. Scherer S.W. Ackerley C.A. Minassian B.A. Hum. Mol. Genet. 2005; 14: 2727-2736Crossref PubMed Scopus (133) Google Scholar, 24Wang Y. Liu Y. Wu C. Zhang H. Zheng X. Zheng Z. Geiger T.L. Nuovo G.J. Liu Y. Zheng P. Cancer Cell. 2006; 10: 179-190Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), but we and others have been unable to observe this result (21Worby C.A. Gentry M.S. Dixon J.E. J. Biol. Chem. 2006; 281: 30412-30418Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 25Wang W. Lohi H. Skurat A.V. Depaoli-Roach A.A. Minassian B.A. Roach P.J. Arch. Biochem. Biophys. 2007; 457: 264-269Crossref PubMed Scopus (30) Google Scholar). Therefore, we purposed that laforin and SEX4 are carbohydrate phosphatases (19Gentry M.S. Dowen R.H. Worby 3rd, C.A. Mattoo S. Ecker J.R. Dixon J.E. J. Cell Biol. 2007; 178: 477-488Crossref PubMed Scopus (124) Google Scholar, 21Worby C.A. Gentry M.S. Dixon J.E. J. Biol. Chem. 2006; 281: 30412-30418Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Approximately 40% of LD patients have recessive mutations in EPM2B (epilepsy myoclonus gene 2B), which encodes the protein malin (13Chan E.M. Young E.J. Ianzano L. Munteanu I. Zhao X. Christopoulos C.C. Avanzini G. Elia M. Ackerley C.A. Jovic N.J. Bohlega S. Andermann E. Rouleau G.A. Delgado-Escueta A.V. Minassian B.A. Scherer S.W. Nat. Genet. 2003; 35: 125-127Crossref PubMed Scopus (267) Google Scholar). Malin is also a bi-modular protein, containing a RING domain followed by six NHL domains. Two laboratories have shown that the RING domain of malin functions as an E3 ubiquitin ligase (23Lohi H. Ianzano L. Zhao X.-C. Chan E.M. Turnbull J. Scherer S.W. Ackerley C.A. Minassian B.A. Hum. Mol. Genet. 2005; 14: 2727-2736Crossref PubMed Scopus (133) Google Scholar, 26Gentry M.S. Worby C.A. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8501-8506Crossref PubMed Scopus (195) Google Scholar) and the NHL domains are predicted to form a β-propeller type protein interaction domain (27Edwards T.A. Wilkinson B.D. Wharton R.P. Aggarwal A.K. Genes Dev. 2003; 17: 2508-2513Crossref PubMed Scopus (91) Google Scholar, 28Slack F.J. Ruvkun G. Trends Biochem. Sci. 1998; 23: 474-475Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Additionally, we found that malin directly binds and ubiquitinates laforin, and targets laforin for degradation (26Gentry M.S. Worby C.A. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8501-8506Crossref PubMed Scopus (195) Google Scholar). Malin was recently shown to ubiquitinate glycogen debranching enzyme (AGL/GDE) and target it for degradation as well (29Cheng A. Zhang M. Gentry M.S. Worby C.A. Dixon J.E. Saltiel A.R. Genes Dev. 2007; 21: 2399-2409Crossref PubMed Scopus (81) Google Scholar). Herein, we demonstrate that malin also ubiquitinates protein targeting to glycogen (PTG/R5), a regulatory subunit of protein phosphatase 1 (PP1). This ubiquitination both targets PTG for degradation and inhibits glycogen accumulation. Additionally, PTG ubiquitination, both in vivo and in vitro, is dependent on the presence of laforin. Despite the ubiquitination of PTG by malin, we were unable to detect a direct interaction between malin and PTG and postulate that laforin acts as a scaffold protein to facilitate PTG ubiquitination by malin. These results suggest that malin degrades multiple enzymes involved in glycogen metabolism to tightly control this process. Thus, when malin is defective glycogen metabolism proceeds aberrantly and LBs are produced. Plasmids and Proteins–Laforin and malin constructs and protein purification were described previously (18Wang J. Stuckey J.A. Wishart M.J. Dixon J.E. J. Biol. Chem. 2002; 277: 2377-2380Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 19Gentry M.S. Dowen R.H. Worby 3rd, C.A. Mattoo S. Ecker J.R. Dixon J.E. J. Cell Biol. 2007; 178: 477-488Crossref PubMed Scopus (124) Google Scholar, 21Worby C.A. Gentry M.S. Dixon J.E. J. Biol. Chem. 2006; 281: 30412-30418Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 26Gentry M.S. Worby C.A. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8501-8506Crossref PubMed Scopus (195) Google Scholar). PTG family members were amplified from expressed sequence tag clones and inserted into the pcDNA3.1 FLAG (30Taylor G.S. Maehama T. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8910-8915Crossref PubMed Scopus (285) Google Scholar) and pcDNA3.1-V5 (Invitrogen) eukaryotic expression vectors, although malin was inserted into pcDNA3.1/myc-His and into the pcDNA3.1 FLAG as previously described (26Gentry M.S. Worby C.A. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8501-8506Crossref PubMed Scopus (195) Google Scholar). pcDNA3.1 MDM2 and pcDNA3.1 PIRH2 were a gift from Dr. Michael Karin. Cell Culture and Transfection–Chinese hamster ovary (CHO) cells stably transformed with the insulin receptor (CHO-IR) were maintained at 37 °C with 5% CO2 in Earle's minimal essential media (Invitrogen) containing 10% fetal bovine serum, 50 units/ml penicillin/streptomycin, and 50 mg/ml Geneticin (Invitrogen). Subconfluent cultures of CHO-IR cells were transfected with FuGENE (Roche Applied Sciences) according to the manufacturer's protocol. Each transfection was performed with 10 μg of total DNA, and supplemented with pcDNA3.1 when necessary. Transfected cells were allowed to recover 24 h prior to harvest for protein expression and 48 h prior to harvest for glycogen measurements. Immunoprecipitations (IPs)–Cell lysates were prepared as described (21Worby C.A. Gentry M.S. Dixon J.E. J. Biol. Chem. 2006; 281: 30412-30418Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The supernatants were mixed with anti-FLAG M2 affinity gel (Sigma) or anti-myc agarose (Sigma) for 2–4 h at 4 °C with constant agitation. The resins were pelleted by centrifugation at 500 × g for 1 min and washed three times with 1 ml of lysis buffer. The beads were resuspended in 30 μl of 4× NuPage sample buffer (Invitrogen) and subjected to Western analyses. Denaturing IPs were performed as described (29Cheng A. Zhang M. Gentry M.S. Worby C.A. Dixon J.E. Saltiel A.R. Genes Dev. 2007; 21: 2399-2409Crossref PubMed Scopus (81) Google Scholar). Western blots were probed with the following antibodies: α-FLAG (Sigma), α-myc 9E10 (Sigma), α-V5 (Invitrogen), α-ubiquitin (Covance), avidin-HRP (Boston Biochem), α-laforin, α-malin, or α-PTG. The α-laforin, α-malin, and α-PTG antibodies were generated by immunizing rabbits (Cocalico) with recombinant laforin, a fusion protein of GST and the RING domain of malin, or PTG, respectively, and antibodies were affinity purified from the serum with a HiTrap NHS-activated HP affinity column (GE Healthcare) and/or IgG purified with a Montage™ Antibody Purification kit (Millipore). Goat α-mouse HRP or donkey α-rabbit HRP (GE Healthcare) were used as needed. The HRP signal was detected using SuperSignal West Pico (Pierce). Glycogen Measurements–Glycogen measurements were performed as previously described with the following modifications (31Fong N.M. Jensen T.C. Shah A.S. Parekh N.N. Saltiel A.R. Brady M.J. J. Biol. Chem. 2000; 275: 35034-35039Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). CHO-IR cells were grown in 100-mm dishes and transfected with the appropriate expression construct(s). Each transfection was performed with 10 μg of total DNA, and supplemented with pcDNA3.1 when necessary. All experimental points were done in triplicate or quadruplicate. Cells were washed three times with phosphate-buffered saline, frozen at -80 °C for 30 min, and lysed by three alternate cycles of freezing (dry ice) and thawing (37 °C). Cells were harvested in 400 μl of 0.2 m sodium acetate, pH 4.8. Glycogen was hydrolyzed to glucose with 500 μl of 250 milliunits/ml amyloglucosidase (Sigma) and incubated at 37 °C for 2–8 h with constant rocking. Samples were neutralized with 125 μl of 0.5 m NaOH and cleared by centrifugation for 5 min at 3000 × g. Glucose concentrations were determined using the Roche Applied Science d-glucose determination kit according to the manufacturer's instructions. 50–200 μl of cell lysate was added to a hexokinase/glucose-6-phosphate dehydrogenase reaction, and the resulting NADPH was measured at 340 nm. In Vitro Ubiquitination Assay–In vitro ubiquitin assays were performed as previously described (26Gentry M.S. Worby C.A. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8501-8506Crossref PubMed Scopus (195) Google Scholar) with the following changes. PTG was used as a substrate instead of laforin. 35S-Labeled PTG was made in vitro using the TnT T7-coupled reticulocyte lysate system (Promega), it was then immunoprecipitated out of the reaction mixture with anti-FLAG M2 affinity gel (Sigma), washed three times with ubiquitin assay buffer, and eluted into 50 μl of assay buffer with 10 μg of FLAG peptide (Sigma). 6 μl of immunopurified 35S-PTG was used in each reaction. The reaction mixture was incubated at 37 °C for 1 h. Reaction products were separated, transferred, and subjected to Western analysis using avidin-HRP (Boston Biochem) to detect biotinylated ubiquitin. After detection, blots were washed in Tris-buffered saline, 0.1% NaN3 to abolish the HRP signal, dried, and exposed on film to detect 35S-PTG. Because LD patients develop cytoplasmic accumulations of insoluble carbohydrate, the disease likely involves the mis-regulation of glycogen metabolism. The enzymes regulating glycogen metabolism, as well as detailed mechanisms of how these enzymes are modulated, have been elucidated over the past 40 years (32Roach P.J. Curr. Mol. Med. 2002; 2: 101-120Crossref PubMed Scopus (357) Google Scholar, 33Roach P.J. Skurat A.V. Harris R.A. Jefferson L.S. Cherrington A.D. The Endocrine Pancreas and Regulation of Metabolism. Oxford University Press, Inc., New York2001: 609-647Google Scholar, 34Ferrer J.C. Favre C. Gomis R.R. Fernandez-Novell J.M. Garcia-Rocha M. de la Iglesia N. Cid E. Guinovart J.J. FEBS Lett. 2003; 546: 127-132Crossref PubMed Scopus (180) Google Scholar, 35Cid E. Geremia R.A. Guinovart J.J. Ferrer J.C. FEBS Lett. 2002; 528: 5-11Crossref PubMed Scopus (22) Google Scholar). Glycogen metabolism is subject to multiple levels of regulation all of which impinge on glycogen synthase, the rate-limiting enzyme in synthesis, and/or phosphorylase, the enzyme that catalyzes the phosphorolysis of the α1,4-glycosidic linkages. One regulator of these enzymes is PTG (gene PPP1R3C), a regulatory subunit of PP1. PTG is expressed in all tissues except testis, and is most abundant in skeletal muscle, heart, and liver (36Armstrong C.G. Browne G.J. Cohen P. Cohen P.T. FEBS Lett. 1997; 418: 210-214Crossref PubMed Scopus (86) Google Scholar, 37Printen J.A. Brady M.J. Saltiel A.R. Science. 1997; 275: 1475-1478Crossref PubMed Scopus (245) Google Scholar). PTG binds both glycogen and the primary enzymes involved in regulating glycogen metabolism, phosphorylase, and glycogen synthase (37Printen J.A. Brady M.J. Saltiel A.R. Science. 1997; 275: 1475-1478Crossref PubMed Scopus (245) Google Scholar). Two groups have shown that disruption of the PTG gene product substantially decreases glycogen stores in tissue culture cells and a mouse model (38Crosson S.M. Khan A. Printen J. Pessin J.E. Saltiel A.R. J. Clin. Investig. 2003; 111: 1423-1432Crossref PubMed Scopus (86) Google Scholar, 39Greenberg C.C. Danos A.M. Brady M.J. Mol. Cell. Biol. 2006; 26: 334-342Crossref PubMed Scopus (27) Google Scholar). In addition, PTG directly interacts with laforin (40Fernandez-Sanchez M.E. Criado-Garcia O. Heath K.E. Garcia-Fojeda B. Medrano-Fernandez I. Gomez-Garre P. Sanz P. Serratosa J.M. Rodriguez de Cordoba S. Hum. Mol. Genet. 2003; 12: 3161-3171Crossref PubMed Scopus (91) Google Scholar). For these reasons, we hypothesized that laforin and/or malin might inhibit the activity of PTG, either directly or indirectly. Co-expression of Malin and Laforin Reduces Glycogen Accumulation–Overexpression of PTG leads to an increase in glycogen accumulation in tissue culture cells and ex vivo organ models (41Gasa R. Jensen P.B. Berman H.K. Brady M.J. DePaoli-Roach A.A. Newgard C.B. J. Biol. Chem. 2000; 275: 26396-26403Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 42Greenberg C.C. Meredith K.N. Yan L. Brady M.J. J. Biol. Chem. 2003; 278: 30835-30842Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 43Lerin C. Montell E. Berman H.K. Newgard C.B. Gomez-Foix A.M. J. Biol. Chem. 2000; 275: 39991-39995Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 44O'Doherty R.M. Jensen P.B. Anderson P. Jones J.G. Berman H.K. Kearney D. Newgard C.B. J. Clin. Investig. 2000; 105: 479-488Crossref PubMed Scopus (71) Google Scholar). We sought to test the hypothesis that malin and laforin oppose the action of PTG. To gain insights into the mechanistic nature that malin and laforin play in glycogen metabolism, we measured glycogen accumulation in tissue culture cells expressing various combinations of PTG, laforin, and malin. We utilized CHO-IR cells because they do not express PTG and produce a minimal amount of glycogen under normal tissue culture conditions (Fig. 1, A and B) (37Printen J.A. Brady M.J. Saltiel A.R. Science. 1997; 275: 1475-1478Crossref PubMed Scopus (245) Google Scholar). However, they exhibit a 7-fold increase in glycogen production when transfected with PTG (Fig. 1, A and B) (37Printen J.A. Brady M.J. Saltiel A.R. Science. 1997; 275: 1475-1478Crossref PubMed Scopus (245) Google Scholar). We transfected CHO-IR cells with a combination of PTG, laforin, and/or malin to determine how laforin and malin affect glycogen stores. Cells transfected with PTG and either laforin or malin yielded a 25% reduction in glycogen amounts compared with cells transfected with PTG and a vector control (Fig. 1A). This reduction was not dependent on the phosphatase activity of laforin, because transfection with PTG and the catalytically inactive laforinC266S yielded a similar reduction in glycogen stores (Fig. 1A). However, the reduction was dependent on the protein-protein interaction domain of malin. Cells transfected with PTG and a LD disease mutation in the NHL domain of malin, malinE280K, contained similar glycogen stores as vector control cells (Fig. 1A). The effect of malin and laforin in concert was quite striking. Cells transfected with PTG, malin, and laforin contained a similar amount of glycogen as cells transfected with a vector control (Fig. 1A). Therefore, malin and laforin each independently decreased PTG-stimulated glycogen accumulation, and cumulatively, they essentially eliminated PTG-stimulated glycogen accumulation. To determine whether this result was a nonspecific affect of overexpressing an E3 ubiquitin ligase, we transfected cells with PTG and MDM2, the E3 ligase that negatively regulates p53 (45Fuchs S.Y. Adler V. Buschmann T. Wu X. Ronai Z. Oncogene. 1998; 17: 2543-2547Crossref PubMed Scopus (213) Google Scholar, 46Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3790) Google Scholar, 47Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2880) Google Scholar). Whereas cells transfected with wild-type malin decreased PTG-stimulated glycogen stores, MDM2 did not reduce PTG-stimulated glycogen stores (Fig. 1B). In a similar manner, we wished to ensure that simply overexpression of another gene or the addition of another plasmid did not reduce PTG-stimulated glycogen stores. Therefore, we transiently expressed PTG and either DJ-1, a protein involved in Parkinson disease, or pEB6 (empty vector). In each case the transfected cells accumulated a similar amount of PTG-stimulated glycogen stores as PTG alone (Fig. 1B). Therefore, the decrease in PTG-stimulated glycogen stores was not an artifact generated by a second vector, another gene being expressed, or expression of another E3 ubiquitin ligase. PTG is one of five regulatory subunits that target PP1 to glycogen and modulate glycogen accumulation (36Armstrong C.G. Browne G.J. Cohen P. Cohen P.T. FEBS Lett. 1997; 418: 210-214Crossref PubMed Scopus (86) Google Scholar, 37Printen J.A. Brady M.J. Saltiel A.R. Science. 1997; 275: 1475-1478Crossref PubMed Scopus (245) Google Scholar, 41Gasa R. Jensen P.B. Berman H.K. Brady M.J. DePaoli-Roach A.A. 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We investigated if malin and laforin specifically inhibit PTG-stimulated glycogen stores, or if they inhibit stimulated glycogen stores of other PTG family members. PTG and R6 (gene PPP1R3D) are expressed in a wide range of human tissues, including the brain (36Armstrong C.G. Browne G.J. Cohen P. Cohen P.T. FEBS Lett. 1997; 418: 210-214Crossref PubMed Scopus (86) Google Scholar, 37Printen J.A. Brady M.J. Saltiel A.R. Science. 1997; 275: 1475-1478Crossref PubMed Scopus (245) Google Scholar). The other three PP1 targeting subunits display tissue-specific expression patterns in skeletal muscle, and/or heart and liver tissue (48Doherty M.J. Moorhead G. Morrice N. Cohen P. Cohen P.T. FEBS Lett. 1995; 375: 294-298Crossref PubMed Scopus (140) Google Scholar, 49Munro S. Ceulemans H. Bollen M. Diplexcito J. Cohen P.T. FEBS J. 2005; 272: 1478-1489Crossref PubMed Scopus (59) Google Scholar, 50Munro S. Cuthbertson D.J. Cunningham J. Sales M. Cohen P.T. Diabetes. 2002; 51: 591-598Crossref PubMed Scopus (48) Google Scholar). One such subunit is GL (gene PPP1R3B), which is expressed in muscle and liver tissue (48Doherty M.J. Moorhead G. Morrice N. Cohen P. Cohen P.T. FEBS Lett. 1995; 375: 294-298Crossref PubMed Scopus (140) Google Scholar, 50Munro S. Cuthbertson D.J. Cunningham J. Sales M. Cohen P.T. Diabetes. 2002; 51: 591-598Crossref PubMed Scopus (48) Google Scholar). To test our hypothesis, we transfected cells with PTG, R6, or GL alone, and along with malin and laforin. As expected, PTG, R6, and GL all stimulated increased glycogen accumulation (Fig. 1C). Malin and laforin inhibited R6-stimulated glycogen accumulation, but they did not inhibit GL-stimulated glycogen accumulation (Fig. 1C). Therefore, malin and laforin inhibit PP1 regulatory subunit-stimulated glycogen accumulation of some PP1 regulatory subunits, PTG/R5 and R6, but not all of them. Interestingly these are the only two regulatory subunits that exhibit a wide expression pattern and are expressed in brain tissue (36Armstrong C.G. Browne G.J. Cohen P. Cohen P.T. FEBS Lett. 1997; 418: 210-214Crossref PubMed Scopus (86) Google Scholar, 37Printen J.A. Brady M.J. Saltiel A.R. Science. 1997; 275: 1475-1478Crossref PubMed Scopus (245) Google Scholar). PTG Is Ubiquitinated and Targeted for Degradation in a Laforin- and Malin-dependent Manner–We previously reported that malin promotes the ubiquitination and degradation of laforin and stated that this result, whereas correct, is in conflict with our understanding of LD