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

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.

SUBMITTER: Tulloch LB

PROVIDER: S-EPMC3195638 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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

Tulloch Lindsay B LB Howie Jacqueline J Wypijewski Krzysztof J KJ Wilson Catherine R CR Bernard William G WG Shattock Michael J MJ Fuller William W

The Journal of biological chemistry 20110825 41

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 confirm ...[more]

PMID: 21868384

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Project description:BackgroundPhospholemman regulates the plasmalemmal sodium pump in excitable tissues.ResultsIn cardiac muscle, a subpopulation of phospholemman with a unique phosphorylation signature associates with other phospholemman molecules but not with the pump.ConclusionPhospholemman oligomers exist in cardiac muscle.SignificanceMuch like phospholamban regulation of SERCA, phospholemman exists as both a sodium pump inhibiting monomer and an unassociated oligomer. Phospholemman (PLM), the principal quantitative sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. Much like phospholamban, which regulates the related ATPase SERCA, PLM is reported to oligomerize. We investigated subpopulations of PLM in adult rat ventricular myocytes based on phosphorylation status. Co-immunoprecipitation identified two pools of PLM: one not associated with the sodium pump phosphorylated at Ser(63) and one associated with the pump, both phosphorylated at Ser(68) and unphosphorylated. Phosphorylation of PLM at Ser(63) following activation of PKC did not abrogate association of PLM with the pump, so its failure to associate with the pump was not due to phosphorylation at this site. All pools of PLM co-localized to cell surface caveolin-enriched microdomains with sodium pump α subunits, despite the lack of caveolin-binding motif in PLM. Mass spectrometry analysis of phosphospecific immunoprecipitation reactions revealed no unique protein interactions for Ser(63)-phosphorylated PLM, and cross-linking reagents also failed to identify any partner proteins for this pool. In lysates from hearts of heterozygous transgenic animals expressing wild type and unphosphorylatable PLM, Ser(63)-phosphorylated PLM co-immunoprecipitated unphosphorylatable PLM, confirming the existence of PLM multimers. Dephosphorylation of the PLM multimer does not change sodium pump activity. Hence like phospholamban, PLM exists as a pump-inhibiting monomer and an unassociated oligomer. The distribution of different PLM phosphorylation states to different pools may be explained by their differential proximity to protein phosphatases rather than a direct effect of phosphorylation on PLM association with the pump.

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

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

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