title: Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman.
abstract: The sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na(+)/K(+)-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca(2+) binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X5-8Φ1Φ2-X8-9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation.
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0450 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0316 Oslo, Norway.
authors.forename: Tandekile Lubelwana
authors.initials: TL
authors.lastname: Hafver
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0450 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0316 Oslo, Norway, the Department of Basic Sciences and Aquatic Medicine, Norwegian University of Life Sciences (NMBU), 0454 Oslo, Norway.
authors.forename: Kjetil
authors.initials: K
authors.lastname: Hodne
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0450 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0316 Oslo, Norway.
authors.forename: Pimthanya
authors.initials: P
authors.lastname: Wanichawan
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0450 Oslo, Norway, the Bjørknes College, Oslo, Norway.
authors.forename: Jan Magnus
authors.initials: JM
authors.lastname: Aronsen
authors.affiliation: the Department of Microbiology, Oslo University Hospital, Rikshospitalet, 0424 Oslo, Norway, the Department of Medical Biochemistry, Institute for Clinical Medicine, University of Oslo, 0424 Oslo, Norway and.
authors.forename: Bjørn
authors.initials: B
authors.lastname: Dalhus
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0450 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0316 Oslo, Norway.
authors.forename: Per Kristian
authors.initials: PK
authors.lastname: Lunde
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0450 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0316 Oslo, Norway.
authors.forename: Marianne
authors.initials: M
authors.lastname: Lunde
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0450 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0316 Oslo, Norway.
authors.forename: Marita
authors.initials: M
authors.lastname: Martinsen
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0450 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0316 Oslo, Norway.
authors.forename: Ulla Helene
authors.initials: UH
authors.lastname: Enger
authors.affiliation: the Cardiovascular and Diabetes Medicine, School of Medicine, University of Dundee, Dundee, Scotland, United Kingdom DD1 9SY.
authors.forename: William
authors.initials: W
authors.lastname: Fuller
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0450 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0316 Oslo, Norway.
authors.forename: Ivar
authors.initials: I
authors.lastname: Sjaastad
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0450 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0316 Oslo, Norway.
authors.forename: William Edward
authors.initials: WE
authors.lastname: Louch
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0450 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0316 Oslo, Norway.
authors.forename: Ole Mathias
authors.initials: OM
authors.lastname: Sejersted
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0450 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0316 Oslo, Norway, c.r.carlson@medisin.uio.no.
authors.forename: Cathrine Rein
authors.initials: CR
authors.lastname: Carlson
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D019295:Computational Biology; D004195:Disease Models, Animal; D057809:HEK293 Cells; D006333:Heart Failure / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D054645:Protein Phosphatase 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D008565:Membrane Proteins; D050505:Mutant Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; C069141:phospholemman; D012694:Serine; C517104:Ppp1cc protein, rat; D054645:Protein Phosphatase 1
keywords: animal model; computer modeling; electrophysiology; heart failure; ion channel; peptide array; phosphoprotein phosphatase 1 (PP1); protein motif; protein-protein interaction; sodium-calcium exchange