Novel Role for p90 Ribosomal S6 Kinase in the Regulation of Cardiac Myofilament Phosphorylation
Novel Role for p90 Ribosomal S6 Kinase in the Regulation of Cardiac Myofilament Phosphorylation
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DOI：
10.1074/jbc.m110.202713
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发表时间：
2011-02-18
影响因子：
4.8
通讯作者：
Avkiran, Metin
中科院分区：
文献类型：
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作者：
Cuello, Friederike;Bardswell, Sonya C.;Avkiran, Metin
在心肌中，90 kDa核糖体S6激酶（RSK）被各种刺激激活，并通过直接磷酸化调节肌膜Na+/H+交换。关于其他心脏RSK底物和功能的信息有限。我们评估了心肌肌球蛋白结合蛋白C（cMyBP-C），一种肌节调节磷蛋白，作为潜在的RSK底物。在大鼠心室肌细胞中，内皮素1（ET 1）激活RSK可增加cMyBP-C在Ser（282）处的磷酸化，这可被选择性RSK抑制剂D1870抑制。ET 1和D1870均不影响Ser（273）或Ser（302）的磷酸化状态，cMyBP-C残基另外被cAMP依赖性蛋白激酶（PKA）靶向。通过腺病毒表达野生型或激酶失活的RSK同种型进行的互补遗传获得和功能丧失实验证实了RSK介导的cMyBP-C丝氨酸磷酸化（282）。利用野生型或突变型（S273 A、S282 A、S302 A）重组cMyBP-C片段作为底物的激酶测定显示RSK直接和选择性的Ser（282）磷酸化。Ser（P）（282）抗体免疫标记和共聚焦荧光显微镜显示RSK介导的cMyBP-C磷酸化跨越肌节A带的C区。在化学透化的小鼠心室肌中，活性RSK再次诱导cMyBP-C中的选择性Ser（282）磷酸化，伴随着力发展的Ca 2+敏感性显著降低和跨桥循环动力学显著加速，与肌钙蛋白I在Ser（22）/Ser（23）的磷酸化无关。这些RSK诱导的变化幅度与PKA诱导的变化幅度相当，PKA在Ser（273）和Ser（302）处额外磷酸化cMyBP-C。结论cMyBP-C中的Ser（282）是一种新的心肌RSK底物，其选择性磷酸化可能调节心肌肌丝功能。
In myocardium, the 90-kDa ribosomal S6 kinase (RSK) is activated by diverse stimuli and regulates the sarcolemmal Na+/H+ exchanger through direct phosphorylation. Only limited information is available on other cardiac RSK substrates and functions. We evaluated cardiac myosin-binding protein C (cMyBP-C), a sarcomeric regulatory phosphoprotein, as a potential RSK substrate. In rat ventricular myocytes, RSK activation by endothelin 1 (ET1) increased cMyBP-C phosphorylation at Ser(282), which was inhibited by the selective RSK inhibitor D1870. Neither ET1 nor D1870 affected the phosphorylation status of Ser(273) or Ser(302), cMyBP-C residues additionally targeted by cAMP-dependent protein kinase (PKA). Complementary genetic gain- and loss-of-function experiments, through the adenoviral expression of wild-type or kinase-inactive RSK isoforms, confirmed RSK-mediated phosphorylation of cMyBP-C at Ser(282). Kinase assays utilizing as substrate wild-type or mutated (S273A, S282A, S302A) recombinant cMyBP-C fragments revealed direct and selective Ser(282) phosphorylation by RSK. Immunolabeling with a Ser(P)(282) antibody and confocal fluorescence microscopy showed RSK-mediated phosphorylation of cMyBP-C across the C-zones of sarcomeric A-bands. In chemically permeabilized mouse ventricular muscles, active RSK again induced selective Ser(282) phosphorylation in cMyBP-C, accompanied by significant reduction in Ca2+ sensitivity of force development and significant acceleration of cross-bridge cycle kinetics, independently of troponin I phosphorylation at Ser(22)/Ser(23). The magnitudes of these RSK-induced changes were comparable with those induced by PKA, which phosphorylated cMyBP-C additionally at Ser(273) and Ser(302). We conclude that Ser(282) in cMyBP-C is a novel cardiac RSK substrate and its selective phosphorylation appears to regulate cardiac myofilament function.