AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR.
AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR.
复制标题
AKAP 18 δ介导并调节Phospholamban-SERCA 2和RYR处的CaMK II活性。
DOI：
10.1161/circresaha.120.317976
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发表时间：
2022-01-07
影响因子：
20.1
通讯作者：
Sejersted OM
中科院分区：
文献类型：
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作者：
Carlson CR;Aronsen JM;Bergan-Dahl A;Moutty MC;Lunde M;Lunde PK;Jarstadmarken H;Wanichawan P;Pereira L;Kolstad TRS;Dalhus B;Subramanian H;Hille S;Christensen G;Müller OJ;Nikolaev V;Bers DM;Sjaastad I;Shen X;Louch WE;Klussmann E;Sejersted OM
肌浆网Ca ~（2+）-ATP酶2（SERCA ~ 2）介导Ca ~（2+）重摄取进入肌浆网，从而促进心肌细胞舒张，而兰尼碱受体（RYR）介导Ca ~（2+）从肌浆网释放，触发收缩。Ca 2 +/钙调素（CaM）依赖性蛋白激酶II（CaMKII）通过直接磷酸化受磷蛋白（PLN）和RYR来调节SERCA 2的活性。然而，CaMKIIδ锚定到SERCA 2-PLN和RYR的机制及其通过局部Ca 2+信号的调节仍然是难以捉摸的。本研究的目的是研究在SERCA 2-PLN和RYR的CaMKIIδ锚定和调节。通过生物信息学、肽阵列、细胞渗透肽技术、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、表面等离子体共振和α筛选技术。我们的研究结果表明，AKAP 18 δ锚定并直接调节SERCA 2-PLN和RYR的CaMK II δ活性，通过两个不同的AKAP 18 δ区域。N-末端区域（AKAP 18 δ-N）通过与天然CaMK II抑制剂肽同源的区域和Thr 17-PLN区域结合来抑制CaMK II δ。AKAP 18 δ-N也结合CaM，引入第二水平的控制。相反，AKAP 18 δ-C与神经元CaMKIIα激活肽（N2 B-s）具有同源性，通过降低激酶激活的表观Ca 2+阈值和诱导CaM捕获来激活CaMKIIδ。AKAP 18 δ-C促进SERCA 2对Ca 2+的快速再摄取和RYR对Ca 2+的快速释放，而AKAP 18 δ-N则具有相反的作用。我们提出了一个模型，其中两个独特的AKAP 18 δ区域微调SERCA 2-PLN和RYR处CaMKIIδ的Ca 2+频率依赖性激活。AKAP 18 δ锚定并功能性调节PLN-SERCA 2和RYR处的CaMKII活性，表明AKAP 18 δ在调节心跳中的关键作用。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CaMKII同种型的AKAP，将AKAP 18 δ也定义为CaM-KAP。
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+/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 Ca2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. 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. 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 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 two unique AKAP18δ regions fine-tune Ca2+-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 reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.