title: The inhibitory effect of phospholemman on the sodium pump requires its palmitoylation.
abstract: Phospholemman (PLM), the principal sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. We investigated post-translational modifications of PLM additional to phosphorylation in adult rat ventricular myocytes (ARVM). LC-MS/MS of tryptically digested PLM immunoprecipitated from ARVM identified cysteine 40 as palmitoylated in some peptides, but no information was obtained regarding the palmitoylation status of cysteine 42. PLM palmitoylation was confirmed by immunoprecipitating PLM from ARVM loaded with [(3)H]palmitic acid and immunoblotting following streptavidin affinity purification from ARVM lysates subjected to fatty acyl biotin exchange. Mutagenesis identified both Cys-40 and Cys-42 of PLM as palmitoylated. Phosphorylation of PLM at serine 68 by PKA in ARVM or transiently transfected HEK cells increased its palmitoylation, but PKA activation did not increase the palmitoylation of S68A PLM-YFP in HEK cells. Wild type and unpalmitoylatable PLM-YFP were all correctly targeted to the cell surface membrane, but the half-life of unpalmitoylatable PLM was reduced compared with wild type. In cells stably expressing inducible PLM, PLM expression inhibited the sodium pump, but PLM did not inhibit the sodium pump when palmitoylation was inhibited. Hence, palmitoylation of PLM controls its turnover, and palmitoylated PLM inhibits the sodium pump. Surprisingly, phosphorylation of PLM enhances its palmitoylation, probably through the enhanced mobility of the phosphorylated intracellular domain increasing the accessibility of cysteines for the palmitoylating enzyme, with interesting theoretical implications. All FXYD proteins have conserved intracellular cysteines, so FXYD protein palmitoylation may be a universal means to regulate the sodium pump.
authors.affiliation: Centre for Cardiovascular and Lung Biology, Division of Medical Sciences, College of Medicine Dentistry & Nursing, University of Dundee, Dundee DD1 9SY, United Kingdom.
authors.forename: Lindsay B
authors.initials: LB
authors.lastname: Tulloch
authors.affiliation: Centre for Cardiovascular and Lung Biology, Division of Medical Sciences, College of Medicine Dentistry & Nursing, University of Dundee, Dundee DD1 9SY, United Kingdom.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Howie
authors.affiliation: Centre for Cardiovascular and Lung Biology, Division of Medical Sciences, College of Medicine Dentistry & Nursing, University of Dundee, Dundee DD1 9SY, United Kingdom.
authors.forename: Krzysztof J
authors.initials: KJ
authors.lastname: Wypijewski
authors.affiliation: Centre for Cardiovascular and Lung Biology, Division of Medical Sciences, College of Medicine Dentistry & Nursing, University of Dundee, Dundee DD1 9SY, United Kingdom.
authors.forename: Catherine R
authors.initials: CR
authors.lastname: Wilson
authors.affiliation: Centre for Cardiovascular and Lung Biology, Division of Medical Sciences, College of Medicine Dentistry & Nursing, University of Dundee, Dundee DD1 9SY, United Kingdom.
authors.forename: William G
authors.initials: WG
authors.lastname: Bernard
authors.affiliation: Cardiovascular Division, The Rayne Institute, St. Thomas' Hospital, King's College London, London SE1 7EH, United Kingdom.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Shattock
authors.affiliation: Centre for Cardiovascular and Lung Biology, Division of Medical Sciences, College of Medicine Dentistry & Nursing, University of Dundee, Dundee DD1 9SY, United Kingdom. Electronic address: w.fuller@dundee.ac.uk.
authors.forename: William
authors.initials: W
authors.lastname: Fuller
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D057809:HEK293 Cells; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D054878:Lipoylation / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D020125:Mutation, Missense; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D017434:Protein Structure, Tertiary; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D010750:Phosphoproteins; C069141:phospholemman; D000254:Sodium-Potassium-Exchanging ATPase