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.).
Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo Norway (J.M.A.).
Circ Res. 2022 Jan 7;130(1):27-44. doi: 10.1161/CIRCRESAHA.120.317976. Epub 2021 Nov 24.
The sarcoplasmic reticulum (SR) Ca-ATPase 2 (SERCA2) mediates Ca reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca release from SR and triggers contraction. Ca/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 Ca signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR.
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.
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 Ca threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca reuptake by SERCA2 and Ca release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR.
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.
肌浆网 Ca-ATPase 2（SERCA2）介导 Ca 重摄取进入肌浆网，从而促进心肌细胞松弛，而兰尼碱受体（RYR）介导 Ca 从肌浆网释放并触发收缩。Ca/CaMKII（CaM [钙调蛋白]-依赖性蛋白激酶 II）通过磷酸化 PLN（磷酸化肌球蛋白轻链结合蛋白）和 RYR 通过直接磷酸化来调节 SERCA2 的活性。然而，CaMKIIδ 锚定到 SERCA2-PLN 和 RYR 的机制及其受局部 Ca 信号的调节仍不清楚。本研究的目的是研究 SERCA2-PLN 和 RYR 上的 CaMKIIδ 锚定和调节。
通过生物信息学、肽阵列、细胞通透肽技术、免疫沉淀、下拉、转染、免疫印迹、邻近连接、FRET 基 CaMKII 活性和 ELISA 基测定、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、表面等离子体共振和 alpha 筛选技术分析 AKAP18δ（蛋白激酶 A 锚定蛋白 18δ）在 CaMKIIδ 锚定和调节中的作用。
我们的结果表明，AKAP18δ 通过 2 个不同的 AKAP18δ 区域将 CaMKIIδ 锚定并直接调节 SERCA2-PLN 和 RYR 的活性。N 端区域（AKAP18δ-N）通过与天然 CaMKII 抑制剂肽和 Thr17-PLN 区域同源的区域结合抑制 CaMKIIδ。AKAP18δ-N 还结合了 CaM，引入了第二级控制。相反，与神经元 CaMKIIα 激活肽（N2B-s）具有同源性的 AKAP18δ-C 通过降低激酶激活的表观 Ca 阈值并诱导 CaM 捕获来激活 CaMKIIδ。虽然 AKAP18δ-C 促进了 SERCA2 的更快 Ca 重摄取和通过 RYR 的 Ca 释放，但 AKAP18δ-N 则产生相反的效果。我们提出了一个模型，其中 2 个独特的 AKAP18δ 区域精细调节 SERCA2-PLN 和 RYR 上 Ca 频率依赖性的 CaMKIIδ 激活。
AKAP18δ 将 CaMKII 活性锚定并在 PLN-SERCA2 和 RYR 上进行功能调节，表明 AKAP18δ 在调节心跳方面起着至关重要的作用。据我们所知，这是第一个显示增强心脏中 CaMKII 活性的蛋白质，也是第一个报道锚定 CaMKII 同工型的 AKAP（蛋白激酶 A 锚定蛋白），将 AKAP18δ 定义为 CaM-KAP。
