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

Abstract

) 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 ...

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

“…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.…”

“…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).…”

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

“…PKC has been reported to increase Na + /K + -ATPase activity in other cell types including arterial smooth muscle [53] and cardiac myocytes [54]. However, unlike with SERCA, there is no evidence for phosphorylation of this protein in the resting platelet phosphoproteome [47], although given the need for activation with a strong platelet activator for the Na + /K + -ATPase to be influenced by PKC, phosphorylation of this protein might not be expected under resting conditions.…”

Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman

“…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-68-PLM) (21)(22)(23)(24). Interestingly, pSer-68-PLM relieves inhibition of the Na ϩ /K ϩ -ATPase (NKA), causing an increase in NKA activity (25,26), suggesting that PLM may serve as a regulator of both NCX (24) and NKA (27) depending on its phosphorylation status. pSer-68-PLM is in turn regulated by PP1 (28).…”