Phosphoregulation of the titin-cap protein telethonin in cardiac myocytes.
Phosphoregulation of the titin-cap protein telethonin in cardiac myocytes.
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DOI：
10.1074/jbc.m113.479030
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发表时间：
2014-01-17
期刊：
影响因子：
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通讯作者：
Avkiran M
中科院分区：
文献类型：
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作者：
Candasamy AJ;Haworth RS;Cuello F;Ibrahim M;Aravamudhan S;Krüger M;Holt MR;Terracciano CM;Mayr M;Gautel M;Avkiran M
背景：Telethonin突变通过未知机制与心肌病相关。结果如下：Telethonin是CaMK家族激酶的底物，并以双磷酸化状态存在于心肌细胞中，其中非磷酸化Telethonin破坏横小管组织和细胞内钙瞬变。结论：Telethonin磷酸化对维持正常心肌细胞形态和钙处理至关重要。意义：磷酸化端粒酶功能的破坏可能有助于心肌病的发病机制。Telethonin（也称为titin-cap或t-cap）是一种肌肉特异性蛋白质，其突变通过未知机制与心脏和骨骼肌病相关。我们以前的工作确定了心脏telethonin作为蛋白激酶D催化结构域的相互作用伙伴。在这项研究中，激酶测定结合MS和定点诱变证实telethonin作为底物的蛋白激酶D和钙/钙调蛋白依赖性激酶II在体外，并确定Ser-157和Ser-161的磷酸化位点。磷酸盐亲和电泳和MS显示内源性telethonin存在于一个组成型双磷酸化的形式在分离的成年大鼠心室肌细胞和小鼠和大鼠心室肌。通过腺病毒基因转移在肌细胞中异源表达后，野生型telethonin变为双磷酸化，而S157 A/S161 A telethonin保持非磷酸化。然而，这两种蛋白质主要定位于肌节Z盘，在那里它们部分取代内源性telethonin。S157 A/S161 A telethonin的这种部分替代破坏了横小管组织，延长了细胞内Ca 2+瞬变的峰值时间，并增加了其方差。这些数据揭示，第一次，心脏telethonin是组成性的双磷酸化，并表明这种磷酸化是至关重要的正常telethonin功能，这可能包括维持横小管组织和细胞内Ca 2+瞬变。
Background: Telethonin mutations are associated with cardiomyopathy through unknown mechanisms. Results: Telethonin is a substrate for CaMK family kinases and exists in a bis-phosphorylated state in cardiomyocytes, in which non-phosphorylated telethonin disrupts transverse tubule organization and intracellular calcium transients. Conclusion: Telethonin phosphorylation is critical for the maintenance of normal cardiomyocyte morphology and calcium handling. Significance: Disruption of phospho-telethonin functions may contribute to pathogenesis in cardiomyopathy. Telethonin (also known as titin-cap or t-cap) is a muscle-specific protein whose mutation is associated with cardiac and skeletal myopathies through unknown mechanisms. Our previous work identified cardiac telethonin as an interaction partner for the protein kinase D catalytic domain. In this study, kinase assays used in conjunction with MS and site-directed mutagenesis confirmed telethonin as a substrate for protein kinase D and Ca2+/calmodulin-dependent kinase II in vitro and identified Ser-157 and Ser-161 as the phosphorylation sites. Phosphate affinity electrophoresis and MS revealed endogenous telethonin to exist in a constitutively bis-phosphorylated form in isolated adult rat ventricular myocytes and in mouse and rat ventricular myocardium. Following heterologous expression in myocytes by adenoviral gene transfer, wild-type telethonin became bis-phosphorylated, whereas S157A/S161A telethonin remained non-phosphorylated. Nevertheless, both proteins localized predominantly to the sarcomeric Z-disc, where they partially replaced endogenous telethonin. Such partial replacement with S157A/S161A telethonin disrupted transverse tubule organization and prolonged the time to peak of the intracellular Ca2+ transient and increased its variance. These data reveal, for the first time, that cardiac telethonin is constitutively bis-phosphorylated and suggest that such phosphorylation is critical for normal telethonin function, which may include maintenance of transverse tubule organization and intracellular Ca2+ transients.