SPEG Controls Calcium Reuptake Into the Sarcoplasmic Reticulum Through Regulating SERCA2a by Its Second Kinase-Domain
SPEG Controls Calcium Reuptake Into the Sarcoplasmic Reticulum Through Regulating SERCA2a by Its Second Kinase-Domain
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SPEG 通过其第二激酶结构域调节 SERCA2a 来控制肌浆网的钙再摄取
DOI：
10.1161/circresaha.118.313916
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发表时间：
2019-03-01
影响因子：
20.1
通讯作者：
Chen, Shuai
中科院分区：
文献类型：
--
作者：
Quan, Chao;Li, Min;Chen, Shuai
理论基础：SpeG(横纹肌优先表达的蛋白激酶)有两个激活域，对心脏发育和功能至关重要。然而，目前还不清楚这两个激酶域是如何维持心脏功能的。目的：确定SpeG蛋白2个激动域的分子功能。方法和结果：用蛋白质组学方法鉴定SERCA2a(肌浆/内质网钙ATPase 2a)是一种与SpeG的第二个激活区相互作用的蛋白质，而不是第一个激动区。此外，在培养细胞和原代新生大鼠心肌细胞中，SpeG的第二个激动域可以使SERCA2a上的Thr484磷酸化，促进其寡聚化，并增加肌浆/内质网对钙的重新摄取。SpeG对SERCA2a的磷酸化增强了其钙转运活性，而不影响其ATPase活性。去除新生大鼠心肌细胞SpeG可抑制SERCA2a-Thr484磷酸化和肌浆网钙重摄取。此外，SERCA2aThr484Ala突变蛋白的过表达也减缓了新生大鼠心肌细胞肌浆网钙的重摄取。相反，结构域图和磷酸化分析表明，SpeG的第一个激酶结构域相互作用并磷酸化其最近确定的底物JPH2(JECTOHLIN-2)。为了在体内进一步研究这种SpeG-SERCA2a信号通路，建立了一个可诱导的心脏特异性SpeG基因敲除小鼠模型。可诱导的SpeG缺失可降低心脏中SERCA2a-Thr484的磷酸化及其寡聚化。重要的是，可诱导的SpeG缺失抑制了SERCA2a钙转运活动，并损害了心肌细胞钙重新摄取到肌浆网，这在心脏的形态和功能改变之前，最终导致成年小鼠的心力衰竭。结论：我们的研究结果表明，SpeG的两个激活区可能在心功能调节中起着不同的作用。SpeG的第二个激酶结构域是SERCA2a的关键调节因子。我们的发现表明，SpeG可能作为一个新的靶点来调节SERCA2a的激活，以治疗钙稳态受损的心脏病。
Rationale: SPEG (Striated muscle preferentially expressed protein kinase) has 2 kinase-domains and is critical for cardiac development and function. However, it is not clear how these 2 kinase-domains function to maintain cardiac performance. Objective: To determine the molecular functions of the 2 kinase-domains of SPEG. Methods and Results: A proteomics approach identified SERCA2a (sarcoplasmic/endoplasmic reticulum calcium ATPase 2a) as a protein interacting with the second kinase-domain but not the first kinase-domain of SPEG. Furthermore, the second kinase-domain of SPEG could phosphorylate Thr484 on SERCA2a, promote its oligomerization and increase calcium reuptake into the sarcoplasmic/endoplasmic reticulum in culture cells and primary neonatal rat cardiomyocytes. Phosphorylation of SERCA2a by SPEG enhanced its calcium-transporting activity without affecting its ATPase activity. Depletion of Speg in neonatal rat cardiomyocytes inhibited SERCA2a-Thr484 phosphorylation and sarcoplasmic reticulum calcium reuptake. Moreover, overexpression of SERCA2aThr484Ala mutant protein also slowed sarcoplasmic reticulum calcium reuptake in neonatal rat cardiomyocytes. In contrast, domain mapping and phosphorylation analysis revealed that the first kinase-domain of SPEG interacted and phosphorylated its recently identified substrate JPH2 (junctophilin-2). An inducible heart-specific Speg knockout mouse model was generated to further study this SPEG-SERCA2a signal nexus in vivo. Inducible deletion of Speg decreased SERCA2a-Thr484 phosphorylation and its oligomerization in the heart. Importantly, inducible deletion of Speg inhibited SERCA2a calcium-transporting activity and impaired calcium reuptake into the sarcoplasmic reticulum in cardiomyocytes, which preceded morphological and functional alterations of the heart and eventually led to heart failure in adult mice. Conclusions: Our data demonstrate that the 2 kinase-domains of SPEG may play distinct roles to regulate cardiac function. The second kinase-domain of SPEG is a critical regulator for SERCA2a. Our findings suggest that SPEG may serve as a new target to modulate SERCA2a activation for treatment of heart diseases with impaired calcium homeostasis.