AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR

Background: The sarcoplasmic reticulum (SR) Ca 2+ -ATPase 2 (SERCA2) mediates a 2+ -reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates a 2+ -release from SR and triggers contraction. a 2+ /calmodulin (CaM)-dependent protein kinase II (CaMKII) regulates activities of SERCA2 through phosphorylation of phospholamban (PLN) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local a 2+ -signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. Methods: A role for A-kinase anchoring protein 18δ (AKAP18δ) 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, adeno-associated 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 two distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to natural CaMKII inhibitor peptide and 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 a 2+ -threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster a 2+ -reuptake by SERCA2 and a 2+ -release through RYR, AKAP18δ-N had opposite effects. We propose a model where the two unique AKAP18δ regions fine-tune a 2+ -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 reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.