Mechanism of reversal of phospholamban inhibition of the cardiac Ca 2+-ATPase by protein kinase A and by anti-phospholamban monoclonal antibody 2D12

Zhenhui Chen, Brandy L. Akin, Larry Jones

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Abstract

Our model of phospholamban (PLB) regulation of the cardiac Ca 2+-ATPase in sarcoplasmic reticulum (SERCA2a) states that PLB binds to the Ca2+-free, E2 conformation of SERCA2a and blocks it from transitioning from E2 to E1, the Ca2+-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 Ser16 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 Ca2+-ATPase activity and cross-linking to SERCA2a were monitored. In Ca2+-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 Ca2+ favoring E2. However, at a subsaturating Ca2+ concentration supporting some E1, cross-linking of phosphorylated PLB to SERCA2a was attenuated at all eight sites. KCa 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 crosslinking to SERCA2a at all eight sites regardless of Ca2+ concentration. Our results demonstrate that 2D12 restores maximal Ca2+-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 Ca2+ concentrations), only partially restoring Ca2+ affinity and Ca2+-ATPase activity.

Original languageEnglish
Pages (from-to)20968-20976
Number of pages9
JournalJournal of Biological Chemistry
Volume282
Issue number29
DOIs
StatePublished - Jul 20 2007

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Cyclic AMP-Dependent Protein Kinases
Adenosine Triphosphatases
Monoclonal Antibodies
Phosphorylation
Calcium-Transporting ATPases
phospholamban
Assays
Sarcoplasmic Reticulum Calcium-Transporting ATPases
Enzyme Activation
Crosslinking
Cysteine

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{d8e5fa1c58814df48054748a2daead08,
title = "Mechanism of reversal of phospholamban inhibition of the cardiac Ca 2+-ATPase by protein kinase A and by anti-phospholamban monoclonal antibody 2D12",
abstract = "Our model of phospholamban (PLB) regulation of the cardiac Ca 2+-ATPase in sarcoplasmic reticulum (SERCA2a) states that PLB binds to the Ca2+-free, E2 conformation of SERCA2a and blocks it from transitioning from E2 to E1, the Ca2+-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 Ser16 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 Ca2+-ATPase activity and cross-linking to SERCA2a were monitored. In Ca2+-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 Ca2+ favoring E2. However, at a subsaturating Ca2+ concentration supporting some E1, cross-linking of phosphorylated PLB to SERCA2a was attenuated at all eight sites. KCa 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 crosslinking to SERCA2a at all eight sites regardless of Ca2+ concentration. Our results demonstrate that 2D12 restores maximal Ca2+-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 Ca2+ concentrations), only partially restoring Ca2+ affinity and Ca2+-ATPase activity.",
author = "Zhenhui Chen and Akin, {Brandy L.} and Larry Jones",
year = "2007",
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TY - JOUR

T1 - Mechanism of reversal of phospholamban inhibition of the cardiac Ca 2+-ATPase by protein kinase A and by anti-phospholamban monoclonal antibody 2D12

AU - Chen, Zhenhui

AU - Akin, Brandy L.

AU - Jones, Larry

PY - 2007/7/20

Y1 - 2007/7/20

N2 - Our model of phospholamban (PLB) regulation of the cardiac Ca 2+-ATPase in sarcoplasmic reticulum (SERCA2a) states that PLB binds to the Ca2+-free, E2 conformation of SERCA2a and blocks it from transitioning from E2 to E1, the Ca2+-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 Ser16 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 Ca2+-ATPase activity and cross-linking to SERCA2a were monitored. In Ca2+-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 Ca2+ favoring E2. However, at a subsaturating Ca2+ concentration supporting some E1, cross-linking of phosphorylated PLB to SERCA2a was attenuated at all eight sites. KCa 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 crosslinking to SERCA2a at all eight sites regardless of Ca2+ concentration. Our results demonstrate that 2D12 restores maximal Ca2+-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 Ca2+ concentrations), only partially restoring Ca2+ affinity and Ca2+-ATPase activity.

AB - Our model of phospholamban (PLB) regulation of the cardiac Ca 2+-ATPase in sarcoplasmic reticulum (SERCA2a) states that PLB binds to the Ca2+-free, E2 conformation of SERCA2a and blocks it from transitioning from E2 to E1, the Ca2+-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 Ser16 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 Ca2+-ATPase activity and cross-linking to SERCA2a were monitored. In Ca2+-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 Ca2+ favoring E2. However, at a subsaturating Ca2+ concentration supporting some E1, cross-linking of phosphorylated PLB to SERCA2a was attenuated at all eight sites. KCa 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 crosslinking to SERCA2a at all eight sites regardless of Ca2+ concentration. Our results demonstrate that 2D12 restores maximal Ca2+-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 Ca2+ concentrations), only partially restoring Ca2+ affinity and Ca2+-ATPase activity.

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