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Molecular basis for the binding and selective dephosphorylation of Na+/H+ exchanger 1 by calcineurin.

ABSTRACT: Very little is known about how Ser/Thr protein phosphatases specifically recruit and dephosphorylate substrates. Here, we identify how the Na+/H+-exchanger 1 (NHE1), a key regulator of cellular pH homeostasis, is regulated by the Ser/Thr phosphatase calcineurin (CN). NHE1 activity is increased by phosphorylation of NHE1 residue T779, which is specifically dephosphorylated by CN. While it is known that Ser/Thr protein phosphatases prefer pThr over pSer, we show that this preference is not key to this exquisite CN selectivity. Rather a combination of molecular mechanisms, including recognition motifs, dynamic charge-charge interactions and a substrate interaction pocket lead to selective dephosphorylation of pT779. Our data identify T779 as a site regulating NHE1-mediated cellular acid extrusion and provides a molecular understanding of NHE1 substrate selection by CN, specifically, and how phosphatases recruit specific substrates, generally.

SUBMITTER: Hendus-Altenburger R

PROVIDER: S-EPMC6677818 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Publications

Molecular basis for the binding and selective dephosphorylation of Na+/H+ exchanger 1 by calcineurin.

Hendus-Altenburger Ruth R Wang Xinru X Sjøgaard-Frich Lise M LM Pedraz-Cuesta Elena E Sheftic Sarah R SR Bendsøe Anne H AH Page Rebecca R Kragelund Birthe B BB Pedersen Stine F SF Peti Wolfgang W

Nature communications 20190802 1

Very little is known about how Ser/Thr protein phosphatases specifically recruit and dephosphorylate substrates. Here, we identify how the Na+/H+-exchanger 1 (NHE1), a key regulator of cellular pH homeostasis, is regulated by the Ser/Thr phosphatase calcineurin (CN). NHE1 activity is increased by phosphorylation of NHE1 residue T779, which is specifically dephosphorylated by CN. While it is known that Ser/Thr protein phosphatases prefer pThr over pSer, we show that this pre ...[more]

PMID: 31375679

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Project description:Fully-activated Na+/H+ exchanger-1 (NHE1) generates the cardiomyocyte's largest trans-membrane extrusion of H+ ions for an equimolar influx of Na+ ions. This has the desirable effect of clearing excess intracellular acidity, but comes at a large energetic premium because the exchanged Na+ ions must ultimately be extruded by the sodium pump, a process that consumes the majority of the heart's non-contractile ATP. We hypothesize that the state of NHE1 activation depends on metabolic resources, which become limiting in periods of myocardial hypoxia. To test this functionally, NHE1 activity was measured in response to in vitro and in vivo hypoxic treatments. NHE1 flux was interrogated as a function of intracellular pH by fluorescence imaging of rodent ventricular myocytes loaded with pH-sensitive dyes BCECF or cSNARF1. Anoxic superfusates promptly inhibited NHE1, tracking the time-course of mitochondrial depolarization. Mass spectrometry of NHE1 immuno-precipitated from Langendorff-perfused anoxic hearts identified Tyr-581 dephosphorylation and Tyr-561 phosphorylation. The latter residue is part of the domain that interacts with phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane lipid that becomes depleted under metabolic inhibition. Tyr-561 phosphorylation is expected to electrostatically weaken this activatory interaction. To test if a period of hypoxia produces a persistent inhibition of NHE1, measurements under normoxia were performed on myocytes that had been incubated in 2% O2 for 4 h. NHE1 activity remained inhibited, but the effect was ablated in the presence of Dasatinib, an inhibitor of Abl/Src-family tyrosine kinases. Chronic tissue hypoxia in vivo, attained in a mouse model of anemic hypoxia, also resulted in persistently slower NHE1. In summary, we show that NHE1 responds to oxygen, a physiologically-relevant metabolic regulator, ostensibly to divert ATP for contraction. We describe a novel mechanism of NHE1 inhibition that may be relevant in cardiac disorders featuring altered oxygen metabolism, such as myocardial ischemia and reperfusion injury.

Dataset Information

Molecular basis for the binding and selective dephosphorylation of Na+/H+ exchanger 1 by calcineurin.

Publications

Molecular basis for the binding and selective dephosphorylation of Na<sup>+</sup>/H<sup>+</sup> exchanger 1 by calcineurin.

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