Centro de Biología Estructural, Instituto Venezolano de Investigaciones Científicas (IVIC), Apdo. 20632, Caracas, 1020A, Venezuela.
Biochem Biophys Res Commun. 2020 Mar 26;524(1):198-204. doi: 10.1016/j.bbrc.2020.01.044. Epub 2020 Jan 23.
Thick filaments from some striated muscles are regulated by phosphorylation of myosin regulatory light chains (RLCs). A tarantula thick filament quasi-atomic model achieved by cryo-electron microscopy has advanced our understanding on how this regulation occurs. In native thick filaments, an asymmetric intramolecular interaction between the actin-binding region of one myosin head ("blocked") and the converter region of the other head ("free") switches both heads off, establishing the myosin interacting-heads motif (IHM). This structural finding, together with motility assays, sequence analysis, and mass spectrometry (MS) observations have suggested a cooperative phosphorylation activation (CPA) mechanism for thick filament activation. In the CPA mechanism, some myosin free heads are phosphorylated constitutively in Ser35 by protein kinase C (PKC) and -under Ca control - others (free or blocked) heads temporally on Ser45 by myosin light chain kinase (MLCK), in a way that explains both force development and post-tetanic potentiation in tarantula striated muscle. We tested this model using MS to verify if Ca-activation phosphorylates de novo un-phosphorylated Ser35 heads. For this purpose, we standardized an approach based on O isotopic ATP labeling to accurately detect by MS-MS the RLC phosphorylation under Ca-activation. MS spectra showed de novoO incorporation only on Ser45 but not on Ser35. As the constitutive Ser35 phosphorylation cannot be dephosphorylated, this result suggests that the number of RLCs on free heads with constitutively phosphorylated Ser35 does remain constant on Ca-activation supporting that the myosin has a basal activation and force modulation or potentiation is controlled by MLCK Ser45 phosphorylation.
来自某些横纹肌的粗丝受肌球蛋白调节轻链（RLC）的磷酸化调节。通过低温电子显微镜获得的狼蛛粗丝准原子模型提高了我们对这种调节发生方式的理解。在天然粗丝中，一个肌球蛋白头部的肌动蛋白结合区（“封闭”）和另一个头部的转换器区之间的非对称分子内相互作用会使两个头部都失活，从而建立肌球蛋白相互作用头部基序（IHM）。这一结构发现，以及运动分析、序列分析和质谱（MS）观察，都表明了粗丝激活的协同磷酸化激活（CPA）机制。在 CPA 机制中，一些肌球蛋白自由头部在 Ser35 处被蛋白激酶 C（PKC）持续磷酸化，而在 Ca 控制下，其他（自由或封闭）头部在 Ser45 处被肌球蛋白轻链激酶（MLCK）暂时磷酸化，这种方式解释了狼蛛横纹肌中的力发展和后抽搐增强。我们使用 MS 来测试这个模型，以验证 Ca 激活是否会磷酸化新的未磷酸化的 Ser35 头部。为此，我们基于 O 同位素 ATP 标记标准化了一种方法，以通过 MS-MS 准确检测 Ca 激活下的 RLC 磷酸化。MS 谱显示只有 Ser45 有新的 O 掺入，而 Ser35 没有。由于组成性 Ser35 磷酸化不能去磷酸化，这一结果表明，具有组成性 Ser35 磷酸化的自由头部上的 RLC 数量在 Ca 激活时保持不变，这支持肌球蛋白具有基础激活和力调节或增强是由 MLCK Ser45 磷酸化控制的。
