Protein kinase A phosphorylation at serine-2808 of the cardiac Ca2+-release channel (ryanodine receptor) does not dissociate 12.6-kDa FK506-binding protein (FKBP12.6).
Dissociation of FKBP12.6 from the cardiac Ca2+-release channel (RyR2) as a consequence of protein kinase A (PKA) hyperphosphorylation of RyR2 at a single amino acid residue, serine-2808, has been proposed as an important mechanism underlying cardiac dysfunction in heart failure. However, the issue of whether PKA phosphorylation of RyR2 can dissociate FKBP12.6 from RyR2 is controversial. To additionally address this issue, we investigated the effect of PKA phosphorylation and mutations at serine-2808 of RyR2 on recombinant or native FKBP12.6-RyR2 interaction. Site-specific antibodies, which recognize the serine-2808 phosphorylated or nonphosphorylated form of RyR2, were used to unambiguously correlate the phosphorylation state of RyR2 at serine-2808 with its ability to bind FKBP12.6. We found that FKBP12.6 can bind to both the serine-2808 phosphorylated and nonphosphorylated forms of RyR2. The S2808D mutant thought to mimic constitutive phosphorylation also retained the ability to bind FKBP12.6. Complete phosphorylation at serine-2808 by exogenous PKA disrupted neither the recombinant nor native FKBP12.6-RyR2 complex. Furthermore, binding of site-specific antibodies to the serine-2808 phosphorylation site did not dissociate FKBP12.6 from or prevent FKBP12.6 from binding to RyR2. Taken together, our results do not support the notion that PKA phosphorylation at serine-2808 dissociates FKBP12.6 from RyR2.
Xiao
Bailong
B
Cardiovascular Research Group, Department of Biochemistry and Molecular Biology, University of Calgary, Alberta, Canada.
Sutherland
Cindy
C
Walsh
Michael P
MP
Chen
S R Wayne
SR
D019943:Amino Acid Substitution; D000818:Animals; D000937:Antigen-Antibody Reactions; D020013:Calcium Signaling / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004285:Dogs; D006801:Humans; D007668:Kidney; D046911:Macromolecular Substances; D051379:Mice; D020125:Mutation, Missense; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry; D017354:Point Mutation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology; D013329:Structure-Activity Relationship; D022021:Tacrolimus Binding Proteins / Q000378:metabolism*
D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D010768:Phosphoserine; D017868:Cyclic AMP-Dependent Protein Kinases; D022021:Tacrolimus Binding Proteins; C403484:tacrolimus binding protein 1B; D020123:Sirolimus