title: Inhibition of phospholamban phosphorylation by O-GlcNAcylation: implications for diabetic cardiomyopathy.
abstract: Cardiac-type sarco(endo)plasmic reticulum Ca(2)-ATPase (SERCA2a) plays a major role in cardiac muscle contractility. Phospholamban (PLN) regulates the function of SERCA2a via its Ser(16)-phosphorylation. Since it has been proposed that the Ser/Thr residues on cytoplasmic and nuclear proteins are modified by O-linked N-acetylglucosamine (O-GlcNAc), we examined the effect of O-GlcNAcylation on PLN function in rat adult cardiomyocytes. Studies using enzymatic labeling and co-immunoprecipitation of wild type and a series of mutants of PLN showed that PLN was O-GlcNAcylated and Ser(16) of PLN might be the site for O-GlcNAcylation. In cardiomyocytes treated with O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), the O-GlcNAcylation was significantly increased compared to non-treated cells. Simultaneously, Ser(16)-phosphorylation of PLN was reduced. In Chinese hamster ovary cells where PLN cDNA and O-GlcNAc transferase siRNA were co-transfected, the Ser(16)-phosphorylation of PLN was significantly increased compared to controls. The same results were observed in heart homogenates from diabetic rats. In a co-immunoprecipitation of PLN with SERCA2a, the physical interaction between the two proteins was increased in PUGNAc-treated cardiomyocytes. Unlike non-treated cells, the activity of SERCA2a and the profiles of calcium transients in PUGNAc-treated cardiomyocytes were not significantly changed even after treatment with catecholamine. These data suggest that PLN is O-GlcNAcylated to induce the inhibition of its phosphorylation, which correlates to the deterioration of cardiac function. This might define a novel mechanism by which PLN regulation of SERCA2a is altered under conditions where O-GlcNAcylation is increased, such as those occurring in diabetes.
authors.affiliation: Department of Biochemistry, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo, Japan.
authors.forename: Shunichi
authors.initials: S
authors.lastname: Yokoe
authors.forename: Michio
authors.initials: M
authors.lastname: Asahi
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Takeda
authors.forename: Kinya
authors.initials: K
authors.lastname: Otsu
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Taniguchi
authors.forename: Eiji
authors.initials: E
authors.lastname: Miyoshi
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Suzuki
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D000215:Acylation; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002395:Catecholamines / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000150:complications* / Q000378:metabolism / Q000473:pathology; D058065:Diabetic Cardiomyopathies / Q000209:etiology* / Q000378:metabolism / Q000473:pathology; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010091:Oximes / Q000494:pharmacology; D048448:Phenylcarbamates / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors; D000099211:Phospholamban
chemical_list: D000117:Acetylglucosamine; D002118:Calcium; D002135:Calcium-Binding Proteins; D002395:Catecholamines; D017868:Cyclic AMP-Dependent Protein Kinases; D017351:N-Acetylglucosaminyltransferases; D010091:Oximes; D048448:Phenylcarbamates; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D001619:beta-N-Acetylhexosaminidases; D000099211:Phospholamban; C068836:N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime; C449880:O-GlcNAc transferase