Abstract
Our model of phospholamban (PLB) regulation of the cardiac Ca(2+)-ATPase in sarcoplasmic reticulum (SERCA2a) states that PLB binds to the Ca(2+)-free, E2 conformation of SERCA2a and blocks it from transitioning from E2 to E1, the Ca(2+)-bound state. PLB and Ca(2+) binding to SERCA2a are mutually exclusive, and PLB inhibition of SERCA2a is manifested as a decreased apparent affinity of SERCA2a for Ca(2+). Here we extend this model to explain the reversal of SERCA2a inhibition that occurs after phosphorylation of PLB at Ser(16) by protein kinase A (PKA) and after binding of the anti-PLB monoclonal antibody 2D12, which recognizes residues 7-13 of PLB. Site-specific cysteine variants of PLB were co-expressed with SERCA2a, and the effects of PKA phosphorylation and 2D12 on Ca(2+)-ATPase activity and cross-linking to SERCA2a were monitored. In Ca(2+)-ATPase assays, PKA phosphorylation and 2D12 partially and completely reversed SERCA2a inhibition by decreasing K(Ca) values for enzyme activation, respectively. In cross-linking assays, cross-linking of PKA-phosphorylated PLB to SERCA2a was inhibited at only two of eight sites when conducted in the absence of Ca(2+) favoring E2. However, at a subsaturating Ca(2+) concentration supporting some E1, cross-linking of phosphorylated PLB to SERCA2a was attenuated at all eight sites. K(Ca) values for cross-linking inhibition were decreased nearly 2-fold at all sites by PLB phosphorylation, demonstrating that phosphorylated PLB binds more weakly to SERCA2a than dephosphorylated PLB. In parallel assays, 2D12 blocked PLB cross-linking to SERCA2a at all eight sites regardless of Ca(2+) concentration. Our results demonstrate that 2D12 restores maximal Ca(2+)-ATPase activity by physically disrupting the binding interaction between PLB and SERCA2a. Phosphorylation of PLB by PKA weakens the binding interaction between PLB and SERCA2a (yielding more PLB-free SERCA2a molecules at intermediate Ca(2+) concentrations), only partially restoring Ca(2+) affinity and Ca(2+)-ATPase activity.
Highlights
Anti-PLB monoclonal antibodies targeted near the phosphorylation site of PLB reverse SERCA2a inhibition, but more completely than protein kinase A (PKA) phosphorylation of PLB (10 –12), and mimic the contractile effects of -adrenergic stimulation of the heart when injected into cardiac myocytes [11]
We examined the effects of PKA phosphorylation of PLB and 2D12 on the cross-linking of PLB to SERCA2a at both cytoplasmic and transmembrane residues, and we correlated these results with Ca2ϩ activation of ATPase activity
2D12 almost completely reversed N30C-PLB inhibition of the Ca2ϩ-ATPase (Fig. 1, closed triangles), lowering the KCa value to 0.16 M, which is similar to the KCa value obtained when SERCA2a was expressed by itself
Summary
Materials—The cross-linking agents BMH, EMCS, and KMUS were purchased from Pierce. bBBr was from Molecular Probes. Phosphorylation of PLB in Sf21 membranes was performed in a master mix containing 80 units of dialyzed catalytic subunit of PKA with 110 g of microsomal protein in 100 l of 40 mM MOPS (pH 7.0), 3.2 mM MgCl2, 75 mM KCl, and 3 mM ATP. Phosphorylation was conducted for 10 min at 30 °C, and 11-g aliquots of pre-phosphorylated or control (minus catalytic subunit) membranes were diluted into concentrated Ca2ϩ/EGTA buffers to yield the final concentrations below for cross-linking experiments or for Ca2ϩ-ATPase assays. Reactions were conducted with g of microsomal protein pre-phosphorylated in the presence or absence of catalytic subunit of PKA as described above in l of 40 mM MOPS (pH 7.0), 3.2 mM MgCl2, 75 mM KCl, and 3 mM ATP supplemented with Ca2ϩ/ EGTA.
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