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. 2011 Oct 14;286(41):36020-36031.
doi: 10.1074/jbc.M111.282145. Epub 2011 Aug 25.

The inhibitory effect of phospholemman on the sodium pump requires its palmitoylation

Lindsay B Tulloch  1 , Jacqueline Howie  1 , Krzysztof J Wypijewski  1 , Catherine R Wilson  1 , William G Bernard  1 , Michael J Shattock  2 , William Fuller  3
Affiliations

The inhibitory effect of phospholemman on the sodium pump requires its palmitoylation

Lindsay B Tulloch et al. J Biol Chem. .

Abstract

Phospholemman (PLM), the principal sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. We investigated post-translational modifications of PLM additional to phosphorylation in adult rat ventricular myocytes (ARVM). LC-MS/MS of tryptically digested PLM immunoprecipitated from ARVM identified cysteine 40 as palmitoylated in some peptides, but no information was obtained regarding the palmitoylation status of cysteine 42. PLM palmitoylation was confirmed by immunoprecipitating PLM from ARVM loaded with [(3)H]palmitic acid and immunoblotting following streptavidin affinity purification from ARVM lysates subjected to fatty acyl biotin exchange. Mutagenesis identified both Cys-40 and Cys-42 of PLM as palmitoylated. Phosphorylation of PLM at serine 68 by PKA in ARVM or transiently transfected HEK cells increased its palmitoylation, but PKA activation did not increase the palmitoylation of S68A PLM-YFP in HEK cells. Wild type and unpalmitoylatable PLM-YFP were all correctly targeted to the cell surface membrane, but the half-life of unpalmitoylatable PLM was reduced compared with wild type. In cells stably expressing inducible PLM, PLM expression inhibited the sodium pump, but PLM did not inhibit the sodium pump when palmitoylation was inhibited. Hence, palmitoylation of PLM controls its turnover, and palmitoylated PLM inhibits the sodium pump. Surprisingly, phosphorylation of PLM enhances its palmitoylation, probably through the enhanced mobility of the phosphorylated intracellular domain increasing the accessibility of cysteines for the palmitoylating enzyme, with interesting theoretical implications. All FXYD proteins have conserved intracellular cysteines, so FXYD protein palmitoylation may be a universal means to regulate the sodium pump.

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Figures

FIGURE 1.
FIGURE 1.
PLM is palmitoylated in ARVM. A, PLM was immunoprecipitated from ARVM using antibodies specific for unphosphorylated (C2) or Ser-63- or Ser-68-phosphorylated PLM. Immunoprecipitation reactions were separated on SDS-PAGE, and gels were stained with Coomassie Brilliant Blue. PLM (marked with an arrow) and co-purifying proteins were identified by LC-MS/MS, and a coverage map of PLM was generated (numbering refers to the mature processed form of PLM). The primary sequence of rat PLM is shown. Residues in boldface type were identified in the coverage map from immunoprecipitation C2. Cys-40 (C40) was found palmitoylated in some peptides, but no information was obtained regarding the palmitoylation status of Cys-42 (C42). B, ARVM were loaded with [3H]palmitic acid, and PLM was immunoprecipitated. Immunoprecipitation starting material (lysate), material not immunoprecipitated (not IP-ed) and immunoprecipitated material (IP fraction) are shown. A 3H-labeled protein with an apparent molecular mass of 15 kDa enriched in the immunoprecipitation (autorad, top) is PLM (immunoblot, bottom). C, PLM was immunoprecipitated with antibodies specific for unphosphorylated (C2) and serine 68-phosphorylated (S68) PLM, and the immunoprecipitated (IP) fraction was subjected to fatty acyl biotin exchange. PLM was detected by immunoblotting (top) and using streptavidin-HRP (bottom). Biotinylated PLM was only detected in immunoprecipitations treated with hydroxylamine (ha), not Tris, confirming PLM palmitoylation.
FIGURE 2.
FIGURE 2.
PLM is substoichiometrically palmitoylated in ARVM lysates. A, FAE schematic (top); dotted lines indicate the points at which samples are routinely taken to monitor the reaction. A typical purification for duplicate samples immunoblotted for PLM and phospholamban (negative control) is shown (bottom, showing the substantial enrichment of PLM in the FAE-hydroxylamine (ha) fraction compared with unfractionated lysate). UF, unfractionated lysates; SM, starting material before streptavidin affinity purification; UB, material not streptavidin affinity-purified. B, proteins purified by FAE were analyzed alongside an equal proportion of their unfractionated starting materials. PLM palmitoylation stoichiometry was estimated by comparison with the stoichiometrically palmitoylated protein caveolin 3. FAE purifies 11 ± 2% of PLM and 66 ± 3% of caveolin 3 (n = 9) from the starting material (filled bars). Using the assumption that caveolin 3 is 100% palmitoylated, this yields an estimate of 16 ± 2% as the palmitoylation stoichiometry of PLM in ARVM (unfilled bars). Error bars, S.E.
FIGURE 3.
FIGURE 3.
PLM is palmitoylated at both cysteine 40 and cysteine 42. A, wild type, C40S, C42S, and C40S/C42S PLM-YFP fusion proteins were transfected into HEK cells. Subcellular localization of PLM-YFP was measured using confocal microscopy (top) and cell-impermeable biotinylation reagents (cell surface fractions only, immunoblotted as indicated; bottom). Wild type and all mutant proteins are expressed at the cell surface. B, FAE of wild type and mutant PLM-YFPs expressed in HEK cells, detected with anti-GFP. Mutants C40S and C42S both cause a reduction in the amount of PLM purified by FAE; however, only double mutant C40S/C42S is not purified by FAE, indicating that both cysteine 40 and cysteine 42 in PLM are palmitoylated (2-BP: 100 μm, 4 h). ha, hydroxylamine.
FIGURE 4.
FIGURE 4.
Relationship between PLM palmitoylation and phosphorylation. A, freshly isolated ARVM were treated with the β-adrenergic agonist isoprenaline (Iso) or the adenylate cyclase activator forskolin (Forsk) to activate protein kinase A and induce serine 68 phosphorylation of PLM. Palmitoylated proteins were purified by FAE and immunoblotted as shown. Purification of PLM relative to caveolin 3 (normalized to PLM purified by FAE from control cells) was significantly enhanced following PKA activation, suggesting a link between PLM phosphorylation at serine 68 and PLM palmitoylation (n = 8). B, wild type and S68A and S68E mutants of a PLM-YFP fusion protein were transfected into HEK cells, and the effect of PKA activation with forskolin on PLM palmitoylation was investigated. PLM-YFP was detected with anti-GFP. Forskolin increases purification of PLM-YFP by FAE (normalized to PLM-YFP purified by FAE from cells transfected with WT PLM-YFP) but is without effect on purification of PLM-S68A-YFP or PLM-S68E-YFP, indicating that phosphorylation of Ser-68 on PLM primes PLM for palmitoylation (n = 10). C, HEK cells transfected with wild type and C40S/C42S PLM-YFP were treated with forskolin (10 μm) or the phorbol ester PMA (300 nm) for 10 min at 37 °C and immunoblotted with antibodies phosphospecific for PLM phosphorylated at Ser-63, Ser-68, and Thr-69. Palmitoylation-defective PLM is phosphorylated normally in HEK cells. *, p < 0.05; Kruskal-Wallis analysis followed by Dunn's multiple comparisons test. ha, hydroxylamine. Error bars, S.E.
FIGURE 5.
FIGURE 5.
Palmitoylation influences the degradation rate of PLM but not the sodium pump α1 subunit in HEK cells. A, HEK cells transfected with wild type and C40S/C42S PLM-YFP were briefly incubated with sulfo-NHS-SS-biotin and then incubated for the times indicated (h) before cell lysis. Biotinylated proteins that had not been degraded were purified by streptavidin affinity chromatography and immunoblotted for PLM-YFP (with anti-GFP) and sodium pump α1 subunit alongside unfractionated cell lysates. B, the degradation rate of PLM-YFP follows simple first order kinetics. The natural log of streptavidin-purified protein plotted against time is linear; the half-life of the protein was derived from the gradient of the straight line. In the example immunoblots shown in A, the half-life of wild type PLM-YFP is 4.0 h, and the half-life of unpalmitoylatable PLM-YFP is 1.7 h. C, the half-life of biotinylated unpalmitoylatable PLM-YFP is significantly reduced compared with wild type PLM-YFP, indicating that palmitoylation influences the degradation rate of PLM (n = 4). There is no difference in the degradation rate of the sodium pump α1 subunit in the same cells. *, p < 0.05. Error bars, S.E.
FIGURE 6.
FIGURE 6.
Influence of PLM palmitoylation on sodium pump α1 in FT-293 cells stably expressing tetracycline-inducible PLM-YFP. PLM-YFP expression (detected with anti-GFP) was induced with tetracycline (tet; 1 μg/ml) for 48 h. A, palmitoylation stoichiometry of PLM-YFP. Proteins purified by FAE were analyzed alongside an equal proportion of their unfractionated starting materials. FAE purifies 29 ± 4% of PLM-YFP (filled bars; n = 7). Using the assumption that FAE purifies 66% of palmitoylated proteins, the palmitoylation stoichiometry of PLM-YFP in this cell line is 45 ± 6% (unfilled bars). B, effect of 2-BP (100 μm) treatment for 24 h on PLM-YFP palmitoylation. FAE indicates an ∼60% reduction in PLM-YFP palmitoylation (n = 5). C, PLM-YFP was immunoprecipitated, and samples were immunoblotted as shown. Palmitoylation does not influence the association of PLM-YFP with the sodium pump α1 subunit. D, cell surface biotinylation experiments indicate identical cell surface expression of the sodium pump α1 subunit following induction of PLM-YFP and a minor effect of 2-BP on the cell surface expression of PLM-YFP but not the sodium pump α1 subunit. E, sodium pump activity (ouabain-sensitive 86Rb uptake) measurements following induction of PLM-YFP expression in the presence and absence of 2-BP (n = 10). PLM-YFP inhibits sodium pump activity but not if cells are also treated with 2-BP, indicating that palmitoylated PLM inhibits the sodium pump but unpalmitoylated PLM does not. *, p < 0.05; analysis of variance followed by Student-Newman-Keuls post hoc analysis. ha, hydroxylamine; Error bars, S.E.
FIGURE 7.
FIGURE 7.
Molecular models of PLM (green) and the sodium pump α subunit (red and blue). Amino acid labels are italicized, and PLM helices H1–H4 and sodium pump α subunit transmembrane domains M1–M10 are in boldface type. A, NMR structure of PLM, indicating the positions of the palmitoylated cysteines (C40 and C42) in helix 3 (H3), also highlighting the positions of the amino acids in the transmembrane helix (helix 2; H2) proposed to interact with the sodium pump α subunit (36) and the intracellular helix containing the phosphorylation site Ser-68 (helix 4; H4). The proposed position of the membrane (based on hydropathy analysis (5)) is indicated by dotted lines. The 16-carbon saturated fatty acid palmitic acid would probably reach as much as half way across the lipid bilayer. B, the protein sequences of human PLM (FXYD1; PDB code 2JO1) and porcine sodium pump γ chain (PDB code 3B8E) were aligned with Clustal. The NMR structure of PLM was superimposed over the crystal structure of sodium pump γ chain using a pairwise structural alignment of C-αs spanning the transmembrane helix region only (PLM residues 16–36 and γ subunit residues 26–46). Three PLM side chains (Phe-28, Arg-38, and Arg-41) were rotated to occupy the same positions as those in the γ subunit structure. Regions of the α subunit that may be influenced by palmitoylation of PLM are colored red. C, proposed positions of the sodium binding sites (magenta viewed from the intracellular side), positioned according to Refs. , –.
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