Phosphorylation of cardiac troponin I by mammalian sterile 20-like kinase 1.
Phosphorylation of cardiac troponin I by mammalian sterile 20-like kinase 1.
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DOI：
10.1042/bj20081340
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发表时间：
2009-02-15
期刊：
影响因子：
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通讯作者：
Sun J
中科院分区：
文献类型：
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作者：
You B;Yan G;Zhang Z;Yan L;Li J;Ge Q;Jin JP;Sun J
哺乳动物不育20样激酶1 （Mst1）是一种普遍表达的丝氨酸/苏氨酸激酶，其在心脏中的激活可导致心肌细胞凋亡和扩张性心肌病。然而，其心肌底物仍不清楚。在以显性阴性Mst1 （K59R）为诱饵的人心脏cDNA文库酵母双杂交筛选中，心肌肌钙蛋白I （cTnI）被鉴定为与Mst1相互作用的蛋白。在共转染的HEK293细胞和天然心肌细胞中，通过共免疫沉淀证实了cTnI与Mst1的相互作用，其中cTnI与全长Mst1相互作用，但不与其n端激酶片段相互作用。体外磷酸化实验表明，cTnI是游离形式或重组肌钙蛋白复合物中Mst1的敏感底物。相比之下，心肌TnT仅在与肌钙蛋白复合物结合时才被Mst1磷酸化。质谱分析表明，Mst1磷酸化cTnI的Thr31、Thr51、Thr129和Thr143位点。用Ala替代Thr31可使Mst1介导的cTnI磷酸化降低约90%，而用Ala替代Thr51或thr129或Thr143可使Mst1催化的cTnI磷酸化降低约60%，这表明Thr31是Mst1的优先磷酸化位点。蛋白表位分析和结合实验表明，Mst1介导的磷酸化调节了cTnI的分子构象及其与TnT和TnC的结合亲和力，具有一定的功能意义。我们的研究结果表明，Mst1是心脏中cTnI和/或cTnT磷酸化的新介质，可能有助于在各种生理和病理生理条件下调节肌丝功能。
Mammalian sterile 20—like kinase 1 (Mst1) is a ubiquitously expressed serine/threonine kinase and its activation in the heart causes cardiomyocyte apoptosis and dilated cardiomyopathy. Its myocardial substrates, however, remain unknown. In a yeast two-hybrid screen of human heart cDNA library with a dominant negative Mst1 (K59R) as bait, cardiac troponin I (cTnI) was identified as an Mst1-interacting protein. The interaction of cTnI with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and native cardiac myocytes, in which cTnI interacted with full length Mst1, but not with its N-terminal kinase fragment. In vitro phosphorylation assays demonstrated that cTnI is a sensitive substrate for Mst1 in its free form or in reconstituted troponin complex. In contrast, cardiac TnT was phosphorylated by Mst1 only when incorporated in the troponin complex. Mass spectrometric analysis indicated that Mst1 phosphorylates cTnI at Thr31, Thr51, Thr129, and Thr143. Substitution of Thr31 with Ala substantially reduced Mst1-mediated cTnI phosphorylation by approximately 90%, while replacement of either Thr51 orThr129, or Thr143 with Ala reduced Mst1-catalyzed cTnI phosphorylation by approximately 60%, suggesting that Thr31 is a preferential phosphorylation site for Mst1. Protein epitope analysis and binding assays showed that Mst1 mediated phosphorylation modulates the molecular conformation of cTnI and its binding affinity to TnT and TnC, thus indicating functional significances. Our results suggest that Mst1 is a novel mediator of cTnI and/or cTnT phosphorylation in the heart and may contribute to the modulation of myofilament function under a variety of physiological and pathophysiological conditions.