Structural and functional effects of myosin-binding protein-C phosphorylation in heart muscle are not mimicked by serine-to-aspartate substitutions.
Structural and functional effects of myosin-binding protein-C phosphorylation in heart muscle are not mimicked by serine-to-aspartate substitutions.
复制标题
DOI：
10.1074/jbc.ac118.004816
复制
发表时间：
2018-09-14
期刊：
影响因子：
--
通讯作者：
Irving M
中科院分区：
文献类型：
--
作者：
Kampourakis T;Ponnam S;Sun YB;Sevrieva I;Irving M
肌球蛋白结合蛋白C（MyBP-C）是心肌收缩性的重要调节因子，其调节功能由其m结构域的多个丝氨酸磷酸化调控。在体内和体外研究中，m结构域磷酸化的结构和功能效应通常从相应的丝氨酸-天冬氨酸（Ser-Asp）取代中推断出来。在这里，使用体外结合试验和原位结构和功能测定在大鼠心脏和cMyBP-C的表达C1 mC 2区域，包含由结构域C1和C2侧翼的m-结构域的心室骨小梁的组合，我们测试这些取代是否实际上模拟磷酸化的影响。分别从附着于肌钙蛋白C或肌球蛋白调节轻链的双功能探针的偏振荧光的变化确定细和粗肌丝结构的原位变化。我们发现，外源性C1 mC 2激活收缩的情况下，钙和伴随的变化细丝结构的作用被废除的m-域的三磷酸化，但不受相应的Ser-Asp取代。后者在粗丝结构中产生了中间变化。三磷酸化和Ser-Asp取代都在体外消除了C1 mC 2与肌球蛋白亚片段2（肌球蛋白S2）之间的相互作用，但对细丝结合产生了不同的影响。这些结果表明，一些先前的推论cMyBP-C中的Ser-Asp取代的影响，应重新考虑和三磷酸化和Ser-Asp取代对cMyBP-C的不同影响可能为未来的研究提供了有用的基础。
Myosin-binding protein-C (cMyBP-C) is a key regulator of contractility in heart muscle, and its regulatory function is controlled in turn by phosphorylation of multiple serines in its m-domain. The structural and functional effects of m-domain phosphorylation have often been inferred from those of the corresponding serine-to-aspartate (Ser–Asp) substitutions, in both in vivo and in vitro studies. Here, using a combination of in vitro binding assays and in situ structural and functional assays in ventricular trabeculae of rat heart and the expressed C1mC2 region of cMyBP-C, containing the m-domain flanked by domains C1 and C2, we tested whether these substitutions do in fact mimic the effects of phosphorylation. In situ changes in thin and thick filament structure were determined from changes in polarized fluorescence from bifunctional probes attached to troponin C or myosin regulatory light chain, respectively. We show that both the action of exogenous C1mC2 to activate contraction in the absence of calcium and the accompanying change in thin filament structure are abolished by tris-phosphorylation of the m-domain, but unaffected by the corresponding Ser–Asp substitutions. The latter produced an intermediate change in thick filament structure. Both tris-phosphorylation and Ser–Asp substitutions abolished the interaction between C1mC2 and myosin sub-fragment 2 (myosin S2) in vitro, but yielded different effects on thin filament binding. These results suggest that some previous inferences from the effects of Ser–Asp substitutions in cMyBP-C should be reconsidered and that the distinct effects of tris-phosphorylation and Ser–Asp substitutions on cMyBP-C may provide a useful basis for future studies.