Protein phosphatase 1c associated with the cardiac sodium calcium exchanger1 regulates its activity by dephosphorylating serine 68 phosphorylated phospholemman

Tandekile Lubelwana Hafver, Kjetil Hodne, Pimthanya Wanichawan, Jan Magnus Aronsen, Bjørn Dalhus, Per Kristian Lunde, Marianne Lunde, Marita Martinsen, Ulla Helene Enger, William Fuller, Ivar Sjaastad, William Edward Louch, Ole Mathias Sejersted, Cathrine Rein Carlson (Lead / Corresponding author)

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    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 the present 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, pull-down experiments and peptide arrays constrained PP1c anchoring to the K-I/V-F-F motif in the first Ca(2+) binding domain (CBD1) in NCX1. This binding site is also partially in agreement with the extended PP1 binding motif; K-I/V-F-F-X5-8-Φ1Φ2-X8-9-R. The cytosolic loop of NCX1, containing the K-I/V-F-F 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-F-F 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-F-F motif) resulted in NCX1 current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation.

    Original languageEnglish
    Pages (from-to)4561-4579
    Number of pages8
    JournalJournal of Biological Chemistry
    Volume291
    Issue number9
    Early online date14 Dec 2015
    DOIs
    Publication statusPublished - 26 Feb 2016

    Keywords

    • animal model
    • computer modeling
    • electrophysiology
    • heart failure
    • ion channel
    • peptide array
    • phosphoprotein phosphatase 1 (PP1)
    • protein motif
    • protein-protein interaction
    • sodium-calcium exchange

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