Site-specific phosphorylation of myosin binding protein-C coordinates thin and thick filament activation in cardiac muscle
Site-specific phosphorylation of myosin binding protein-C coordinates thin and thick filament activation in cardiac muscle
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DOI：
10.1073/pnas.1903033116
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发表时间：
2019-07-30
影响因子：
11.1
通讯作者：
Kampourakis, Thomas
中科院分区：
文献类型：
--
作者：
Ponnam, Saraswathi;Sevrieva, Ivanka;Kampourakis, Thomas
心脏对身体不同需求的反应是由激活蛋白激酶的信号通路调节的，蛋白激酶使肌节蛋白磷酸化。虽然磷酸化心肌肌球蛋白结合蛋白-C（cMyBP-C）已被认为是心肌收缩力的关键调节因子，但对其作用机制知之甚少。在这里，我们使用蛋白激酶A（PKA）和C激酶（PKC β），以及核糖体S6激酶II（RSK 2），它们对cMyBP-C的多个磷酸化位点具有不同的特异性，以表明单个位点不是独立的，cMyBP-C的磷酸化是由这些位点之间的正调控偶联和负调控偶联控制的。PKA磷酸化cMyBP-C的N端的3个保守的丝氨酸残基是分级的，并拮抗磷酸化的PKC β，反之亦然。相反，cMyBP-C的RSK 2磷酸化加速PKA磷酸化。我们使用cMyBP-C的调节N-末端结构域在定义的磷酸化状态下与分离的心脏天然细丝和心脏肌球蛋白的S2结构域进行蛋白质-蛋白质相互作用研究，以显示cMyBP-C的该区域的位点特异性磷酸化控制其与含肌动蛋白的细丝和含肌球蛋白的粗细丝的相互作用。我们还使用了荧光探针上的肌球蛋白相关的调节轻链在粗丝和肌钙蛋白C在细丝监测结构的变化，在完整的心肌细胞的肌丝与激活心肌收缩的N-末端区域的cMyBP-C在其不同的磷酸化状态。我们的研究结果表明，cMyBP-C作为一个多个信号通路，决定下游的生理功能的肌节整合。
The heart's response to varying demands of the body is regulated by signaling pathways that activate protein kinases which phosphorylate sarcomeric proteins. Although phosphorylation of cardiac myosin binding protein-C (cMyBP-C) has been recognized as a key regulator of myocardial contractility, little is known about its mechanism of action. Here, we used protein kinase A (PKA) and C epsilon (PKC epsilon), as well as ribosomal S6 kinase II (RSK2), which have different specificities for cMyBP-C's multiple phosphorylation sites, to show that individual sites are not independent, and that phosphorylation of cMyBP-C is controlled by positive and negative regulatory coupling between those sites. PKA phosphorylation of cMyBP-C's N terminus on 3 conserved serine residues is hierarchical and antagonizes phosphorylation by PKC epsilon, and vice versa. In contrast, RSK2 phosphorylation of cMyBP-C accelerates PKA phosphorylation. We used cMyBP-C's regulatory N-terminal domains in defined phosphorylation states for protein-protein interaction studies with isolated cardiac native thin filaments and the S2 domain of cardiac myosin to show that site-specific phosphorylation of this region of cMyBP-C controls its interaction with both the actin-containing thin and myosin-containing thick filaments. We also used fluorescence probes on the myosin-associated regulatory light chain in the thick filaments and on troponin C in the thin filaments to monitor structural changes in the myofilaments of intact heart muscle cells associated with activation of myocardial contraction by the N-terminal region of cMyBP-C in its different phosphorylation states. Our results suggest that cMyBP-C acts as a sarcomeric integrator of multiple signaling pathways that determines downstream physiological function.