title: Characterization and quantitation of phospholamban and its phosphorylation state using antibodies.
abstract: Quantitative immunoassays to discriminate and quantitate phospholamban and its phosphorylation states in heart homogenates were developed using known amounts of protein determined by amino acid analysis. Synthetic 1-52 phospholamban, the hydrophilic 1-25 peptide, and 1-25 phosphopeptides containing P-Ser(16), P-Thr(17), and dually phosphorylated (P-Ser(16), P-Thr(17)) were used to calibrate immunoblot systems. In addition, synthetic 1-52 peptide was phosphorylated using cAMP-dependent protein kinase (P-Ser(16)) or Ca(2+)-calmodulin protein kinase (P-Thr(17)) and then separated from unphosphorylated 1-52 by HPLC prior to quantitation. Further, canine cardiac sarcoplasmic reticulum was phosphorylated in vitro using [gamma-(32)P]-ATP with cAMP-dependent protein kinase and/or Ca(2+)-calmodulin-dependent protein kinase as well as sequential phosphorylation in both orders to assess the veracity of antibody recognition of phosphorylated forms. Western blots proved useful in characterizing the reactivity of the different antibodies to phospholamban and phosphorylated phospholamban, but were inefficient for accurate quantitation and problems with antibody recognition of dually phosphorylated phospholamban were found. mAb 1D11 recognized all forms of phospholamban, polyclonal antibodies 285 and PS-16 were highly selective for P-Ser(16) phospholamban but had diminished reactivity to diphosphorylated (P-Ser(16), P-Thr(17)) phospholamban, and polyclonal antibody PT-17, although selective for P-Thr(17) phospholamban, generated very weak signals on Western blots and reacted poorly with diphosphorylated phospholamban. Results in quantitative immunodot blot experiments were even more compelling. None of the phosphorylation specific antibodies reacted with the diphospho 1-25 phospholamban peptide. Transgenic mouse hearts expressing varying levels of PLB and ferret heart biopsy samples taken before and after isoproterenol perfusion were analyzed. In all samples containing phospholamban, a basal level of Ser(16) phosphorylation (about 4% of the total PLB population) and a lesser amount of Thr(17) phosphorylation was observed. Upon isoproterenol perfusion, Ser(16) phosphorylation increased only to 17% of the total phospholamban population with a similar change in Thr(17) phosphorylation. This suggests that phospholamban phosphorylation may serve as an electrostatic switch that dissociates inactive calcium pump complexes into catalytically active units. Thus, direct correlations between phospholamban phosphorylation state and contractile parameters may not be valid.
authors.affiliation: Department of Pharmacology, Merck Research Laboratories, West Point, Pennsylvania, 19486, USA.
authors.forename: E J
authors.initials: EJ
authors.lastname: Mayer
authors.forename: W
authors.initials: W
authors.lastname: Huckle
authors.forename: R G
authors.initials: RG
authors.lastname: Johnson
authors.forename: E
authors.initials: E
authors.lastname: McKenna
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004285:Dogs; D005289:Ferrets; D051379:Mice; D009206:Myocardium / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D000099211:Phospholamban
chemical_list: D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D010446:Peptide Fragments; D010748:Phosphopeptides; D010768:Phosphoserine; D010769:Phosphothreonine; D000099211:Phospholamban