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

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 CaMKII delta anchoring to SERCA2-PLN and RYR and its regulation by local Ca2+ signals remain elusive. The objective of this study was to investigate CaMKII delta anchoring and regulation at SERCA2-PLN and RYR. Methods: A role for AKAP18 delta (A-kinase anchoring protein 18 delta) in CaMKII delta 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 AKAP18 delta anchors and directly regulates CaMKII delta activity at SERCA2-PLN and RYR, via 2 distinct AKAP18 delta regions. An N-terminal region (AKAP18 delta-N) inhibited CaMKII delta through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18 delta-N also bound CaM, introducing a second level of control. Conversely, AKAP18 delta-C, which shares homology to neuronal CaMKII alpha activator peptide (N2B-s), activated CaMKII delta by lowering the apparent Ca2+ threshold for kinase activation and inducing CaM trapping. While AKAP18 delta-C facilitated faster Ca2+ reuptake by SERCA2 and Ca2+ release through RYR, AKAP18 delta-N had opposite effects. We propose a model where the 2 unique AKAP18 delta regions fine-tune Ca2+-frequency-dependent activation of CaMKII delta at SERCA2-PLN and RYR. Conclusions: AKAP18 delta anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18 delta 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 AKAP18 delta also as a CaM-KAP.