title: Substrate recognition by the cell surface palmitoyl transferase DHHC5.
abstract: The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, but requires a ∼ 120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme-substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipid interactions that inhibit the Na pump.
authors.affiliation: Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, College of Medicine, Dentistry and Nursing, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, United Kingdom;
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Howie
authors.affiliation: Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, College of Medicine, Dentistry and Nursing, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, United Kingdom;
authors.forename: Louise
authors.initials: L
authors.lastname: Reilly
authors.affiliation: Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, College of Medicine, Dentistry and Nursing, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, United Kingdom;
authors.forename: Niall J
authors.initials: NJ
authors.lastname: Fraser
authors.affiliation: Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, College of Medicine, Dentistry and Nursing, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, United Kingdom;
authors.forename: Julia M
authors.initials: JM
authors.lastname: Vlachaki Walker
authors.affiliation: Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, College of Medicine, Dentistry and Nursing, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, United Kingdom;
authors.forename: Krzysztof J
authors.initials: KJ
authors.lastname: Wypijewski
authors.affiliation: Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, College of Medicine, Dentistry and Nursing, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, United Kingdom;
authors.forename: Michael L J
authors.initials: ML
authors.lastname: Ashford
authors.affiliation: School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom;
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Calaghan
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom; and.
authors.forename: Heather
authors.initials: H
authors.lastname: McClafferty
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom; and.
authors.forename: Lijun
authors.initials: L
authors.lastname: Tian
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom; and.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Shipston
authors.affiliation: Cardiovascular Division, The Rayne Institute, St Thomas' Hospital, King's College London, London SE1 7EH, United Kingdom.
authors.forename: Andrii
authors.initials: A
authors.lastname: Boguslavskyi
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: Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, College of Medicine, Dentistry and Nursing, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, United Kingdom; w.fuller@dundee.ac.uk.
authors.forename: William
authors.initials: W
authors.lastname: Fuller
mesh_terms: D000217:Acyltransferases; D020816:Amino Acid Motifs; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D004285:Dogs; D004705:Endocytosis; D020869:Gene Expression Profiling; D057809:HEK293 Cells; D006801:Humans; D054878:Lipoylation; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology*; D051379:Mice; D009206:Myocardium / Q000378:metabolism; D009473:Neuronal Plasticity; D010743:Phospholipids / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000737:chemistry; D013379:Substrate Specificity; D013569:Synapses
chemical_list: D008565:Membrane Proteins; D010743:Phospholipids; D010750:Phosphoproteins; D034741:RNA, Small Interfering; C069141:phospholemman; D012964:Sodium; D000217:Acyltransferases; C549503:DHHC5 protein, mouse
keywords: DHHC; ion transport; palmitoylation; phospholemman; sodium pump