Differential contribution of troponin I phosphorylation sites to the endothelin-modulated contractile response
Differential contribution of troponin I phosphorylation sites to the endothelin-modulated contractile response
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DOI：
10.1074/jbc.m506043200
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发表时间：
2005-12-16
影响因子：
4.8
通讯作者：
Robinson, DA
中科院分区：
文献类型：
--
作者：
Westfall, MV;Lee, AM;Robinson, DA
心肌肌钙蛋白I是内皮素激活的蛋白激酶C的磷酸化靶标。早期在心肌细胞中表达非磷酸化慢骨骼肌钙蛋白I的工作提供了蛋白激酶C介导的心肌肌钙蛋白I磷酸化加速松弛的证据。然而，用慢速骨骼异构体替代也会改变肌丝的pH反应和钙瞬变，这可能会影响内皮素介导的松弛。这里，钙瞬变的差异不能解释表达心肌肌钙蛋白I和慢骨骼肌钙蛋白I的心肌细胞对内皮素的不同松弛反应，也不能解释Na+/H+交换的激活。心肌肌钙蛋白I中的三个独立簇被蛋白激酶C磷酸化，我们开始确定Thr(144)和Ser(23)/Ser(24)簇对内皮素介导的收缩反应的贡献。与对照组相比，用含有苏氨酸(144)的心肌肌钙蛋白I替代慢速骨骼肌钙蛋白I中发现的Pro残基的心肌细胞可以延长对急性内皮素的松弛反应。Ser(23)/Ser(24)也是纯化的心肌肌钙蛋白I的蛋白激酶C磷酸化的靶点，尽管这一簇在完整的心肌细胞中没有明显的磷酸化，但在加入内皮素后1小时内就发生了显著的磷酸化。用ALA取代Ser(23)/Ser(24)表明，在更长时间的内皮素刺激过程中，这一簇对松弛有显著作用。总体而言，这些突变体的结果提供了证据，证明Thr(144)在急性松弛加速中发挥重要作用，而Ser(23)/Ser(24)在更长时间内内皮素激活蛋白激酶C的过程中起到松弛作用。
Cardiac troponin I is a phosphorylation target for endothelin-activated protein kinase C. Earlier work in cardiac myocytes expressing nonphosphorylatable slow skeletal troponin I provided evidence that protein kinase C-mediated cardiac troponin I phosphorylation accelerates relaxation. However, replacement with the slow skeletal isoform also alters the myofilament pH response and the Ca2+ transient, which could influence endothelin-mediated relaxation. Here, differences in the Ca2+ transient could not explain the divergent relaxation response to endothelin in myocytes expressing cardiac versus slow skeletal troponin I nor could activation of Na+/H+ exchange. Three separate clusters within cardiac troponin I are phosphorylated by protein kinase C, and we set out to determine the contribution of the Thr(144) and Ser(23)/Ser(24) clusters to the endothelin-mediated contractile response. Myocyte replacement with a cardiac troponin I containing a Thr(144) substituted with the Pro residue found in slow skeletal troponin I resulted in prolonged relaxation in response to acute endothelin compared with control myocytes. Ser(23)/Ser(24) also is a target for protein kinase C phosphorylation of purified cardiac troponin I, and although this cluster was not acutely phosphorylated in intact myocytes, significant phosphorylation developed within 1 h after adding endothelin. Replacement of Ser(23)/Ser(24) with Ala indicated that this cluster contributes significantly to relaxation during more prolonged endothelin stimulation. Overall, results with these mutants provide evidence that Thr(144) plays an important role in the acute acceleration of relaxation, whereas Ser(23)/Ser(24) contributes to relaxation during more prolonged activation of protein kinase C by endothelin.