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

Hafver, T. L. et al. (2016) Protein phosphatase 1c associated with the cardiac sodium calcium exchanger 1 regulates its activity by dephosphorylating serine 68-phosphorylated phospholemman. Journal of Biological Chemistry, 291(9), pp. 4561-4579. (doi: 10.1074/jbc.M115.677898) (PMID:26668322) (PMCID:PMC4813481)

Abstract

The sodium (Na+)-calcium (Ca2+) exchanger 1 (NCX1) is an important regulator of intracellular Ca2+ 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 Ca2+ 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.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Fuller, Professor Will
• Authors: Hafver, T. L., Hodne, K., Wanichawan, P., Aronsen, J. M., Dalhus, B., Lunde, P. K., Lunde, M., Martinsen, M., Enger, U. H., Fuller, W., Sjaastad, I., Louch, W. E., Sejersted, O. M., and Carlson, C. R.
• College/School: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Journal Name: Journal of Biological Chemistry
• Publisher: American Society for Biochemistry and Molecular Biology, Inc.
• ISSN: 0021-9258
• ISSN (Online): 1083-351X
• Published Online: 14 December 2015
• Copyright Holders: Copyright © 2016 The American Society for Biochemistry and Molecular Biology, Inc.
• First Published: First published in Journal of Biological Chemistry 291(9):4561-4579
• Publisher Policy: Reproduced in accordance with the copyright policy of the publisher