Cathrine R Carlson1, Jan Magnus Aronsen1,2,3, Anna Bergan-Dahl1,4, Marie Christine Moutty5, Marianne Lunde1,4, Per Kristian Lunde1,4, Hilde Jarstadmarken1, Pimthanya Wanichawan1, Laetitia Pereira6, Terje R S Kolstad1,4, Bjørn Dalhus7,8, Hariharan Subramanian9,10, Susanne Hille10,11, Geir Christensen1,4, Oliver J Müller10,11, Viacheslav Nikolaev9,10, Donald M Bers6, Ivar Sjaastad1,4, Xin Shen1,4, William E Louch1,4, Enno Klussmann5,12, Ole M Sejersted1,4. 1. Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Norway (C.R.C., J.M.A., A.B.-D., M.L., P.K.L., H.J., P.W., T.R.S.K., G.C., I.S., X.S., W.E.L., O.M.S.). 2. Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo Norway (J.M.A.). 3. Department of Pharmacology, Oslo University Hospital, Norway (J.M.A.). 4. The KG Jebsen Cardiac Research Center, University of Oslo, Norway (A.B.-D., M.L., P.K.L., T.R.S.K., G.C., I.S., X.S., W.E.L., O.M.S.). 5. Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany (M.C.M., E.K.). 6. Department of Pharmacology, University of California at Davis (L.P., D.M.B.). 7. Department of Microbiology, Oslo University Hospital, Norway (B.D.). 8. Department of Medical Biochemistry, Institute for Clinical Medicine, University of Oslo, Norway (B.D.). 9. Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany (H.S., V.N.). 10. German Centre for Cardiovascular Research, Partner Site Hamburg/Kiel/Lübeck, Germany (H.S., S.H., O.J.M., V.N.). 11. Department of Internal Medicine III, University of Kiel, Kiel, Germany (S.H., O.J.M.). 12. German Centre for Cardiovascular Research (DZHK), Partner Site Berlin, Germany (E.K.).
Abstract
BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+-ATPase 2 (SERCA2) mediates Ca2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca2+ release from SR and triggers contraction. Ca2+/CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. METHODS: A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology. RESULTS: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca2+ reuptake by SERCA2 and Ca2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca2+-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. CONCLUSIONS: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.
BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+-ATPase 2 (SERCA2) mediates Ca2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca2+ release from SR and triggers contraction. Ca2+/CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. METHODS: A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology. RESULTS: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca2+ reuptake by SERCA2 and Ca2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca2+-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. CONCLUSIONS: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.
Entities:
Keywords:
calcium-calmodulin-dependent protein kinase type 2; calmodulin; myocytes, cardiac; phospholamban; ryanodine receptor; sarcoplasmic reticulum calcium-transporting ATPases
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