title: Synthetic phosphopeptides enable quantitation of the content and function of the four phosphorylation states of phospholamban in cardiac muscle.
abstract: We have studied the differential effects of phospholamban (PLB) phosphorylation states on the activity of the sarcoplasmic reticulum Ca-ATPase (SERCA). It has been shown that unphosphorylated PLB (U-PLB) inhibits SERCA and that phosphorylation of PLB at Ser-16 or Thr-17 relieves this inhibition in cardiac sarcoplasmic reticulum. However, the levels of the four phosphorylation states of PLB (U-PLB, P16-PLB, P17-PLB, and doubly phosphorylated 2P-PLB) have not been measured quantitatively in cardiac tissue, and their functional effects on SERCA have not been determined directly. We have solved both problems through the chemical synthesis of all four PLB species. We first used the synthetic PLB as standards for a quantitative immunoblot assay, to determine the concentrations of all four PLB phosphorylation states in pig cardiac tissue, with and without left ventricular hypertrophy (LVH) induced by aortic banding. In both LVH and sham hearts, all phosphorylation states were significantly populated, but LVH hearts showed a significant decrease in U-PLB, with a corresponding increase in the ratio of total phosphorylated PLB to U-PLB. To determine directly the functional effects of each PLB species, we co-reconstituted each of the synthetic peptides in phospholipid membranes with SERCA and measured calcium-dependent ATPase activity. SERCA inhibition was maximally relieved by P16-PLB (the most highly populated PLB state in cardiac tissue homogenates), followed by 2P-PLB, then P17-PLB. These results show that each PLB phosphorylation state uniquely alters Ca(2+) homeostasis, with important implications for cardiac health, disease, and therapy.
authors.affiliation: From the Departments of Biochemistry, Molecular Biology and Biophysics and.
authors.forename: Naa-Adjeley D
authors.initials: NA
authors.lastname: Ablorh
authors.affiliation: From the Departments of Biochemistry, Molecular Biology and Biophysics and.
authors.forename: Xiaoqiong
authors.initials: X
authors.lastname: Dong
authors.affiliation: From the Departments of Biochemistry, Molecular Biology and Biophysics and.
authors.forename: Zachary M
authors.initials: ZM
authors.lastname: James
authors.affiliation: Medicine, University of Minnesota, Minneapolis, Minnesota 55455.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Xiong
authors.affiliation: Medicine, University of Minnesota, Minneapolis, Minnesota 55455.
authors.forename: Jianyi
authors.initials: J
authors.lastname: Zhang
authors.affiliation: From the Departments of Biochemistry, Molecular Biology and Biophysics and.
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
authors.affiliation: From the Departments of Biochemistry, Molecular Biology and Biophysics and karim002@umn.edu.
authors.forename: Christine B
authors.initials: CB
authors.lastname: Karim
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004195:Disease Models, Animal; D006333:Heart Failure / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D010748:Phosphopeptides / Q000737:chemistry*; D010766:Phosphorylation; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D013552:Swine; D000099211:Phospholamban
chemical_list: D000251:Adenosine Triphosphatases; D002118:Calcium; D002135:Calcium-Binding Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017868:Cyclic AMP-Dependent Protein Kinases; D010748:Phosphopeptides; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban
keywords: Antibody; Ca-ATPase; Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII); Cardiac Muscle; Heart Failure; PKA; Phospholamban; Protein Kinase A (PKA); Protein Phosphorylation; Reconstitution Of Membrane Proteins