Abstract 21298: Phospholemman Palmitoylation: a Novel Means of Sodium Pump Regulation
Phospholemman (PLM), the principal sarcolemmal substrate for protein kinases A and C (PKA and PKC) in the heart, is a key regulator of the cardiac sodium pump. We investigated post-translational modifications of PLM additional to phosphorylation in freshly isolated adult rat ventricular myocytes (ARVM) and transiently transfected HEK cells. LC-MS/MS of tryptically digested PLM immunoprecipitated from ARVM identified cysteine 40 (C40) as palmitoylated in some peptides, but no information was obtained regarding the palmitoylation status of cysteine 42 (C 42). To confirm that PLM is palmitoylated, ARVM were loaded with 3H-palmitic acid, lysed, and PLM immunoprecipitated; autoradiography indicated incorporation of 3H-palmitate into immunoprecipitated PLM. We also confirmed PLM palmitoylation by performing acyl-biotin exchange of immunoprecipitated PLM, and by immunoblotting following streptavidin affinity purification from ARVM lysates that had been subjected to acyl-biotin exchange. In all cases, substitution of hydroxylamine with TRIS prior to cysteine biotinylation confirmed the specificity of the acyl-biotin exchange for thioester bonds. Recombinant intracellular region of PLM was palmitoylated spontaneously following incubation with 3H-palmitoyl-CoA in vitro ; MALDI analysis of this material suggested a single palmitoylation site is present in PLM. In order to identify the site(s) of palmitoylation in PLM and the functional effect of palmitoylation on the sodium pump, HEK cells were transiently transfected with PLM fused with yellow fluorescent protein (YFP) and with the point mutants C40S, C42S and the double mutant C40/42S. Wild type and mutant PLM-YFPs were all correctly targeted to the cell surface membrane and co-immunoprecipitated with the sodium pump alpha-1 subunit. In summary, we have identified a new post-translational modification of the cardiac phosphoprotein PLM. Palmitoylation is a dynamic, reversible post-translational modification that can control protein subcellular localisation, tertiary and quarternary structure. Palmitoylation of phospholemman may provide a means of regulating the cardiac sodium pump independent of phosphorylation.
- © 2010 by American Heart Association, Inc.