Activation of ROCK and MLCK tunes regional stress fiber formation and mechanics via preferential myosin light chain phosphorylation.
Activation of ROCK and MLCK tunes regional stress fiber formation and mechanics via preferential myosin light chain phosphorylation.
复制标题
DOI：
10.1091/mbc.e17-06-0401
复制
发表时间：
2017-12-15
影响因子：
3.3
通讯作者：
Kumar S
中科院分区：
文献类型：
--
作者：
Kassianidou E;Hughes JH;Kumar S
采用分级诱导调节性轻链(RLC)激活剂MLCK和ROCK，探讨RLC磷酸化与肌球蛋白应激纤维粘弹性的关系。MLCK通过RLC的单磷酸化控制外周应力纤维的力学，而ROCK通过二磷酸化作用于中枢应力纤维。肌球蛋白应激纤维(SFS)的组装和机制依赖于肌球蛋白调节轻链(RLC)的磷酸化，肌球蛋白轻链激酶(MLCK)和Rho相关激酶(ROCK)驱动肌球蛋白调节轻链(RLC)的磷酸化。尽管以前的工作表明MLCK和ROCK控制着细胞内不同的SFS池，但这些激酶在调节RLC磷酸化方面有何不同，或磷酸化如何影响单个SF的机制仍不清楚。在这里，我们结合遗传方法和生物物理工具来探索激酶活性、RLC磷酸化、SF定位和SF机制之间的关系。我们发现，MLCK的过度表达以一种分级的方式增加了RLC的单磷酸化(p-RLC)，并且这种p-RLC定位于外周SFS。相反，分级的ROCK过度表达优先增加RLC二磷酸化(pp-RLC)，pp-RLC定位于中心SFS。亚细胞激光消融单个SFs的结果表明，MLCK和ROCK分别定量地调节了外周和中央型SFs的粘弹性性质。MLCK和ROCK对单SF机制的影响可能相应地通过单磷酸化和双磷酰化RLC突变体的过度表达而表现出来。我们的结果指出了一个模型，在该模型中，MLCK和ROCK通过RLC的单磷酸化和二磷酸化来调节外周和中枢SF的粘弹性，提供了激酶活性、RLC磷酸化和SF粘弹性之间的新的定量联系。
Graded induction of regulatory light chain (RLC) activators MLCK and ROCK were used to explore the relationship between RLC phosphorylation and actin-myosin stress fiber viscoelasticity. MLCK controls peripheral stress fiber mechanics by monophosphorylation of RLC, whereas ROCK acts on central stress fibers via diphosphorylation. The assembly and mechanics of actomyosin stress fibers (SFs) depend on myosin regulatory light chain (RLC) phosphorylation, which is driven by myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK). Although previous work suggests that MLCK and ROCK control distinct pools of cellular SFs, it remains unclear how these kinases differ in their regulation of RLC phosphorylation or how phosphorylation influences individual SF mechanics. Here, we combine genetic approaches with biophysical tools to explore relationships between kinase activity, RLC phosphorylation, SF localization, and SF mechanics. We show that graded MLCK overexpression increases RLC monophosphorylation (p-RLC) in a graded manner and that this p-RLC localizes to peripheral SFs. Conversely, graded ROCK overexpression preferentially increases RLC diphosphorylation (pp-RLC), with pp-RLC localizing to central SFs. Interrogation of single SFs with subcellular laser ablation reveals that MLCK and ROCK quantitatively regulate the viscoelastic properties of peripheral and central SFs, respectively. The effects of MLCK and ROCK on single-SF mechanics may be correspondingly phenocopied by overexpression of mono- and diphosphomimetic RLC mutants. Our results point to a model in which MLCK and ROCK regulate peripheral and central SF viscoelastic properties through mono- and diphosphorylation of RLC, offering new quantitative connections between kinase activity, RLC phosphorylation, and SF viscoelasticity.