title: Phosphorylation of phospholamban by cAMP-dependent protein kinase enhances interactions between Ca-ATPase polypeptide chains in cardiac sarcoplasmic reticulum membranes.
abstract: We have used spin-label EPR spectroscopy to examine possible alterations in protein-protein interactions that accompany the activation of the cardiac sarcoplasmic reticulum (SR) Ca-ATPase following the phosphorylation of phospholamban (PLB). Using a radioactive derivative of a maleimide spin label (MSL), we have developed conditions for the selective spin-labeling of the Ca-ATPase in both native cardiac and skeletal sarcoplasmic reticulum membranes. The rotational dynamics of the cardiac and skeletal Ca-ATPase isoforms in native SR membranes were measured using saturation transfer EPR. We report that the phosphorylation of PLB in cardiac SR results in a (1.8 +/- 0.2)-fold reduction in the overall rotational mobility of the Ca-ATPase. The alteration in the rotational dynamics of the Ca-ATPase is the direct result of the phosphorylation of PLB, and is not related to the phosphorylation of the Ca-ATPase or any other SR proteins since no alteration in the ST-EPR spectrum is observed as a result of conditions that phosphorylate the cardiac Ca-ATPase with ATP. Neither do the use of conditions that activate the Ca-ATPase in cardiac SR result in the alteration of the rotational dynamics or catalytic properties of the Ca-ATPase in skeletal SR where PLB is not expressed. Measurements of the rotational dynamics of stearic acid spin labels (SASL) incorporated into cardiac SR membranes with a nitroxide at the 5- and 12-positions using conventional EPR indicate that there is virtually no difference in the lipid acyl chain dynamics in cardiac SR membranes upon the phosphorylation of PLB. These results indicate that the decrease in the rotational dynamics of the Ca-ATPase in cardiac SR membranes associated with the phosphorylation of PLB is related to enhanced interactions between individual Ca-ATPase polypeptide chains due to (i) an alteration in the spatial arrangement of cardiac Ca-ATPase polypeptide chains within a defined oligomeric state or (ii) increased protein-protein associations. We suggest that altered interactions between Ca-ATPase polypeptide chains and PLB serves to modulate the activation barrier associated with calcium activation of the Ca-ATPase in cardiac SR membranes.
authors.affiliation: Department of Biochemistry, University of Kansas, Lawrence 66045-2106, USA.
authors.forename: S
authors.initials: S
authors.lastname: Negash
authors.forename: L T
authors.initials: LT
authors.lastname: Chen
authors.forename: D J
authors.initials: DJ
authors.lastname: Bigelow
authors.forename: T C
authors.initials: TC
authors.lastname: Squier
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000378:metabolism; D050356:Lipid Metabolism; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013113:Spin Labels; D013229:Stearic Acids / Q000378:metabolism; D013816:Thermodynamics; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D017868:Cyclic AMP-Dependent Protein Kinases; D003545:Cysteine; D005033:Ethylmaleimide; D013113:Spin Labels; D013229:Stearic Acids; D000099211:Phospholamban; C031183:stearic acid