Cysteine residues 244 and 458–459 within the catalytic subunit of Na,K-ATPase control the enzyme's hydrolytic and signaling function under hypoxic conditions

Petrushanko, I. Y. et al. (2017) Cysteine residues 244 and 458–459 within the catalytic subunit of Na,K-ATPase control the enzyme's hydrolytic and signaling function under hypoxic conditions. Redox Biology, 13, pp. 310-319. (doi: 10.1016/j.redox.2017.05.021) (PMID:28601781) (PMCID:PMC5470536)

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Our previous findings suggested that reversible thiol modifications of cysteine residues within the actuator (AD) and nucleotide binding domain (NBD) of the Na,K-ATPase may represent a powerful regulatory mechanism conveying redox- and oxygen-sensitivity of this multifunctional enzyme. S-glutathionylation of Cys244 in the AD and Cys 454-458-459 in the NBD inhibited the enzyme and protected cysteines’ thiol groups from irreversible oxidation under hypoxic conditions. In this study mutagenesis approach was used to assess the role these cysteines play in regulation of the Na,K-ATPase hydrolytic and signaling functions. Several constructs of mouse α1 subunit of the Na,K-ATPase were produced in which Cys244, Cys 454-458-459 or Cys 244-454-458-459 were replaced by alanine. These constructs were expressed in human HEK293 cells. Non-transfected cells and those expressing murine α1 subunit were exposed to hypoxia or treated with oxidized glutathione (GSSG). Both conditions induced inhibition of the wild type Na,K-ATPase. Enzymes containing mutated mouse α1 lacking Cys244 or all four cysteines (Cys 244-454-458-459) were insensitive to hypoxia. Inhibitory effect of GSSG was observed for wild type murine Na,K-ATPase, but was less pronounced in Cys454-458-459Ala mutant and completely absent in the Cys244Ala and Cys 244-454-458-459Ala mutants. In cells, expressing wild type enzyme, ouabain induced activation of Src and Erk kinases under normoxic conditions, whereas under hypoxic conditions this effect was inversed. Cys454-458-459Ala substitution abolished Src kinase activation in response to ouabain treatment, uncoupled Src from Erk signaling, and interfered with O2-sensitivity of Na,K-ATPase signaling function. Moreover, modeling predicted that S-glutathionylation of Cys 458 and 459 should prevent inhibitory binding of Src to NBD. Our data indicate for the first time that cysteine residues within the AD and NBD influence hydrolytic as well as receptor function of the Na,K-ATPase and alter responses of the enzyme to hypoxia or upon treatment with cardiotonic steroids.

Item Type:Articles
Additional Information:This study was supported by the Swiss National Science Foundation (SNF) (#IZK0Z3_157269) (for Figs. 1, 2A, 3) and Russian Science Foundation (# 14-14-01152) (for Figs. 2B, 4, 5, 6, Supplementary materials).
Glasgow Author(s) Enlighten ID:Fuller, Dr Will
Authors: Petrushanko, I. Y., Mitkevich, V. A., Lakunina, V. A., Anashkina, A. A., Spirin, P. V., Rubtsov, P. M., Prassolov, V. S., Bogdanov, N. B., Hänggi, P., Fuller, W., Makarov, A. A., and Bogdanova, A.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
Journal Name:Redox Biology
ISSN (Online):2213-2317
Published Online:31 May 2017
Copyright Holders:Copyright © 2017 The Authors
First Published:First published in Redox Biology 13:310-319
Publisher Policy:Reproduced under a Creative Commons License

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