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 [γ- 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.