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.

中文翻译：

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AKAP18δ 锚定并调节受磷蛋白-SERCA2 和 RYR 的 CaMKII 活性

背景：肌浆网 (SR) Ca ²⁺ -ATPase 2 (SERCA2) 介导 Ca ²⁺再摄取进入 SR，从而促进心肌细胞松弛，而兰尼碱受体 (RYR) 介导SR 释放Ca ^{2+并触发收缩。}Ca ²⁺ /CaMKII（CaM [钙调蛋白] 依赖性蛋白激酶 II）通过直接磷酸化 PLN（受磷蛋白）和 RYR 的磷酸化来调节 SERCA2 的活性。然而，CaMKIIδ 锚定到 SERCA2-PLN 和 RYR 的机制及其受局部 Ca ^2+的调节信号仍然难以捉摸。本研究的目的是研究 SERCA2-PLN 和 RYR 的 CaMKIIδ 锚定和调节。技术、免疫沉淀、下拉、转染、免疫印迹、邻近连接、基于 FRET 的 CaMKII 活性和基于 ELISA 的测定、全细胞和 SR 囊泡荧光成像、高分辨率显微镜、腺病毒转导、腺相关病毒注射、结构建模、表面等离子体结果：我们的结果表明，AKAP18δ 通过 2 个不同的 AKAP18δ 区域锚定并直接调节 SERCA2-PLN 和 RYR 的 CaMKIIδ 活性。N 末端区域 (AKAP18δ-N) 通过结合与天然 CaMKII 抑制肽同源的区域和 Thr17-PLN 区域来抑制 CaMKIIδ。AKAP18δ-N 还结合了 CaM，引入了二级控制。相反，与神经元 CaMKIIα 激活肽 (N2B-s) 具有同源性的 AKAP18δ-C 通过降低表观 Ca 激活 CaMKIIδ²⁺激酶激活和诱导 CaM 捕获的阈值。虽然 AKAP18δ-C 促进SERCA2 更快地再摄取Ca ²⁺并通过 RYR 释放Ca ^{2+ ，但 AKAP18δ-N 具有相反的作用。}我们提出了一个模型，其中 2 个独特的 AKAP18δ 区域微调 Ca ²⁺ -SERCA2-PLN 和 RYR 处 CaMKIIδ 的频率依赖性激活。结论：AKAP18δ 锚定并在功能上调节 PLN-SERCA2 和 RYR 处的 CaMKII 活性，表明其具有关键作用AKAP18δ 在心跳调节中的作用。据我们所知，这是第一个显示增强心脏 CaMKII 活性的蛋白质，也是第一个被报道锚定 CaMKII 同种型的 AKAP（A 激酶锚定蛋白），将 AKAP18δ 定义为 CaM-KAP。