Isoform Specificity of the Na/K-ATPase Association and Regulation by Phospholemman

Bossuyt, Julie; Despa, Sandra; Han, Fei; Hou, Zhanjia; Robia, Seth L.; Lingrel, Jerry B.; Bers, Donald M.

doi:10.1074/jbc.m109.047357

Cited by 68 publications

(113 citation statements)

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“…binding. As recently published, the actual regulatory effect in cardiac myocytes is caused by phosphorylation of FXYD1 (Bibert et al 2008;Bossuyt et al 2009;Despa et al 2005;Fuller et al 2004Fuller et al , 2009Pavlovic et al 2007;Silverman et al 2005). This concept is supported by the phosphorylation experiments shown in Figs.…”

Section: Fxyd1 Phosphorylationsupporting

confidence: 73%

“…ions for activation of the pump (Figs. 2a, 3, 4), similar to what has been detected in intact cells in the presence of unphosphorylated FXYD1 (Bibert et al 2008;Bossuyt et al 2009;Crambert et al 2002b;Despa et al 2005;Han et al 2009Han et al , 2010Lifshitz et al 2006). These results demonstrate, first, that FXYD1 associates correctly with the enzyme during in vitro reconstitution since the behavior resembles that found in native cell membranes.…”

Section: Discussionsupporting

confidence: 54%

“…Several studies have approved that unphosphorylated FXYD1 reduces the apparent affinity of the Na,K-ATPase for intracellular Na ? ions in both cardiac myocytes (Bossuyt et al 2009;Despa et al 2005;Han et al 2009) and heterologous expression systems (Bibert et al 2008;Crambert et al 2002a;Han et al 2010;Lifshitz et al 2006). In the bulk of the plasma membrane of human cardiac myocytes the total content of negatively charged lipid head groups has been estimated to be on the order of 18 mol% with a fraction of *3 mol% phosphatidylserine (Gloster and Harris 1969).…”

Section: Discussionmentioning

confidence: 99%

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Surface Charges of the Membrane Crucially Affect Regulation of Na,K-ATPase by Phospholemman (FXYD1)

et al. 2013

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The human a 1 /His 10 -b 1 isoform of Na,K-ATPase has been reconstituted as a complex with and without FXYD1 into proteoliposomes of various lipid compositions in order to study the effect of the regulatory subunit on the half-saturating Na ? concentration (K 1/2 ) of Na ? ions for activation of the ion pump. It has been shown that the fraction of negatively charged lipid in the bilayer crucially affects the regulatory properties. At low concentrations of the negatively charged lipid DOPS (\10 %), FXYD1 increases K 1/2 of Na ? ions for activation of the ion pump. Phosphorylation of FXYD1 by protein kinase A at Ser68 abrogates this effect. Conversely, for proteoliposomes made with high concentrations of DOPS ([10 %), little or no effect of FXYD1 on the K 1/2 of Na ? ions is observed. Depending on ionic strength and lipid composition of the proteoliposomes, FXYD1 can alter the K 1/2 of Na ? ions by up to twofold. We propose possible molecular mechanisms to explain the regulatory effects of FXYD1 and the influence of charged lipid and protein phosphorylation. In particular, the positively charged C-terminal helix of FXYD1 appears to be highly mobile and may interact with the cytoplasmic N domain of the a-subunit, the interaction being strongly affected by phosphorylation at Ser68 and the surface charge of the membrane.

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Section: Fxyd1 Phosphorylationsupporting

confidence: 73%

Section: Discussionsupporting

confidence: 54%

Section: Discussionmentioning

confidence: 99%

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Surface Charges of the Membrane Crucially Affect Regulation of Na,K-ATPase by Phospholemman (FXYD1)

et al. 2013

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“…ϩ -K ϩ pump inhibition mediated by cAMP-dependent pathways in cardiac myocytes was reported in early studies (8,9), but many more recent studies have reported that these pathways mediate pump stimulation either by increasing maximal pump rate or by increasing pump affinity for intracellular Na ϩ (7,(11)(12)(13)(14)(15)(16)30). In our study, forskolin induced glutathionylation of the Na ϩ -K ϩ pump ␤ 1 subunit.…”

Section: Nasupporting

confidence: 47%

Activation of cAMP-dependent Signaling Induces Oxidative Modification of the Cardiac Na+-K+ Pump and Inhibits Its Activity

White

Liu

García

et al. 2010

Journal of Biological Chemistry

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Cellular signaling can inhibit the membrane Na؉ -K ؉ pump via protein kinase C (PKC)-dependent activation of NADPH oxidase and a downstream oxidative modification, glutathionylation, of the ␤ 1 subunit of the pump ␣/␤ heterodimer. It is firmly established that cAMP-dependent signaling also regulates the pump, and we have now examined the hypothesis that such regulation can be mediated by glutathionylation. Exposure of rabbit cardiac myocytes to the adenylyl cyclase activator forskolin increased the co-immunoprecipitation of NADPH oxidase subunits p47 phox and p22 phox , required for its activation, and increased superoxide-sensitive fluorescence. Forskolin also increased glutathionylation of the Na ؉ -K ؉ pump ␤ 1 subunit and decreased its co-immunoprecipitation with the ␣ 1 subunit, findings similar to those already established for PKC-dependent signaling. The decrease in co-immunoprecipitation indicates a decrease in the ␣ 1 /␤ 1 subunit interaction known to be critical for pump function. In agreement with this, forskolin decreased ouabain-sensitive electrogenic Na ؉ -K ؉ pump current (arising from the 3:2 Na ؉ :K ؉ exchange ratio) of voltage-clamped, internally perfused myocytes. The decrease was abolished by the inclusion of superoxide dismutase, the inhibitory peptide for the ⑀-isoform of PKC or inhibitory peptide for NADPH oxidase in patch pipette solutions that perfuse the intracellular compartment. Pump inhibition was also abolished by inhibitors of protein kinase A and phospholipase C. We conclude that cAMPand PKC-dependent inhibition of the cardiac Na ؉ -K ؉ pump occurs via a shared downstream oxidative signaling pathway involving NADPH oxidase activation and glutathionylation of the pump ␤ 1 subunit.

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“…We (6,11) have previously shown that PLM reduces the NKA activity, mainly by reducing the apparent pump affinity for internal Na ϩ , in intact cardiac myocytes. PLM phosphorylation relieves this inhibition and thus mediates the PKA/PKCdependent stimulation of NKA (2,6,11). Biochemical studies (10) indicate that PKA phosphorylates PLM mostly at the Ser68 site (although some phosphorylation also occurs at the Ser63 site) and PKC phosphorylates both Ser63 and Ser68 sites.…”

mentioning

confidence: 99%

Role of phospholemman phosphorylation sites in mediating kinase-dependent regulation of the Na⁺-K⁺-ATPase

Han

Bossuyt

Martin

et al. 2010

American Journal of Physiology-Cell Physiology

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) is a major target for phosphorylation mediated by both PKA (at Ser68) and PKC (at both Ser63 and Ser68) in the heart. In intact cardiac myocytes, PLM associates with and inhibits Na ϩ -K ϩ -ATPase (NKA), mainly by reducing its affinity for internal Na ϩ . The inhibition is relieved upon PLM phosphorylation by PKA or PKC. The aim here was to distinguish the role of the Ser63 and Ser68 PLM phosphorylation sites in mediating kinase-induced modulation of NKA function. We expressed wild-type (WT) PLM and S63A, S68A, and AA (Ser63 and Ser68 to alanine double mutant) PLM mutants in HeLa cells that stably express rat NKA-␣ 1 and we measured the effect of PKA and PKC activation on NKA-mediated intracellular Na ϩ concentration decline. PLM expression (WT or mutant) significantly decreased the apparent NKA affinity for internal Na ϩ and had no significant effect on the maximum pump rate (V max). PKA activation with forskolin (20 M) restored NKA Na ϩ affinity in cells expressing WT but not AA PLM and did not affect V max in either case. Similarly, PKC activation with 300 nM phorbol 12,13-dibutyrate increased NKA Na ϩ affinity in cells expressing WT, S63A, and S68A PLM and had no effect in cells expressing AA PLM. Neither forskolin nor phorbol 12,13-dibutyrate affected NKA function in the absence of PLM. We conclude that PLM phosphorylation at either Ser63 or Ser68 is both necessary and sufficient for completely relieving the PLM-induced NKA inhibition.FXYD; apparent Na affinity; PKA; PKC PHOSPHOLEMMAN (PLM) WAS FIRST REPORTED as an important phosphorylation substrate for both PKA and PKC (13,17,18) in the heart; however, its physiological role was poorly understood. More recent studies (4, 5, 21) indicate that PLM belongs to a family of proteins (FXYD gene family) that bind specifically to and regulate the Na ϩ -K ϩ -ATPase (NKA) in various tissues. PLM is the only FXYD protein highly expressed in the heart and is unique among the FXYD proteins in that it has multiple phosphorylation sites in the cytoplasmic domain.PLM associates specifically and stably with rat NKA ␣ 1 -, ␣ 2 -, and ␣ 3 -isoforms and reduces their apparent affinity for intracellular Na ϩ and extracellular K ϩ when coexpressed in oocytes (4). We (6, 11) have previously shown that PLM reduces the NKA activity, mainly by reducing the apparent pump affinity for internal Na ϩ , in intact cardiac myocytes. PLM phosphorylation relieves this inhibition and thus mediates the PKA/PKCdependent stimulation of NKA (2, 6, 11). Biochemical studies (10) indicate that PKA phosphorylates PLM mostly at the Ser68 site (although some phosphorylation also occurs at the Ser63 site) and PKC phosphorylates both Ser63 and Ser68 sites. Removal of these phosphorylation sites in the shark PLM-like protein restores the apparent Na ϩ affinity of the NKA (14, 15). NKA is strongly stimulated in sarcolemmal membranes isolated from ischemic rat hearts compared with the controls, and this is accompanied by activation of PKA and PKC and subsequent phosphorylation of PLM (but not of the NKA ...

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Isoform Specificity of the Na/K-ATPase Association and Regulation by Phospholemman

Cited by 68 publications

References 49 publications

Surface Charges of the Membrane Crucially Affect Regulation of Na,K-ATPase by Phospholemman (FXYD1)

Surface Charges of the Membrane Crucially Affect Regulation of Na,K-ATPase by Phospholemman (FXYD1)

Activation of cAMP-dependent Signaling Induces Oxidative Modification of the Cardiac Na+-K+ Pump and Inhibits Its Activity

Role of phospholemman phosphorylation sites in mediating kinase-dependent regulation of the Na⁺-K⁺-ATPase

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Isoform Specificity of the Na/K-ATPase Association and Regulation by Phospholemman

Cited by 68 publications

References 49 publications

Surface Charges of the Membrane Crucially Affect Regulation of Na,K-ATPase by Phospholemman (FXYD1)

Surface Charges of the Membrane Crucially Affect Regulation of Na,K-ATPase by Phospholemman (FXYD1)

Activation of cAMP-dependent Signaling Induces Oxidative Modification of the Cardiac Na+-K+ Pump and Inhibits Its Activity

Role of phospholemman phosphorylation sites in mediating kinase-dependent regulation of the Na+-K+-ATPase

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Role of phospholemman phosphorylation sites in mediating kinase-dependent regulation of the Na⁺-K⁺-ATPase