Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 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

doi:10.1074/jbc.m115.677898

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.

Highlights

The sodium (Naϩ)-calcium (Ca2ϩ) exchanger (NCX)3 is a bidirectional ion-transporting membrane protein, which exchanges three Naϩ for one Ca2ϩ across the plasma membrane
In an ongoing effort to understand regulation of cardiac NCX1 activity, we have investigated the molecular basis of PP1 catalytic subunit (PP1c) targeting to the NCX1 macromolecular complex, and we determined the functional consequences of this interaction
PP1c Anchors to the NCX1-KIFF/KVFF Motif in CBD1—phosphatase 1 (PP1) targeting is predominantly mediated by a short RVXF-docking motif, which is present in 90% of targeting proteins [31, 51]

Summary

Introduction

The sodium (Naϩ)-calcium (Ca2ϩ) exchanger (NCX) is a bidirectional ion-transporting membrane protein, which exchanges three Naϩ for one Ca2ϩ across the plasma membrane. Accumulating data indicate that PLM is one of these regulatory players This 72-amino acid transmembrane protein, belonging to the FXYD1 family of ion transporters [20], co-localizes and coimmunoprecipitates with NCX1 and has been shown to inhibit NCX1 activity when it is phosphorylated at serine 68 (pSer-68PLM) [21,22,23,24]. PSer-68-PLM is in turn regulated by PP1 [28] The latter is a ubiquitously expressed ϳ38.5-kDa serine/threonine phosphatase that counters the effects of serine/threonine kinases and has little intrinsic specificity for its substrates [29]. We investigated whether a direct and functional NCX1-PP1c interaction is a prerequisite for pSer-68-PLM dephosphorylation. We aimed to map the PP1ctargeting site on the NCX1 macromolecular complex and elucidate the biological effect of this interaction on NCX1 activity

Objectives

Results

Conclusion