PDF(Pubmed)
Abstract：
Myosin motors perform many fundamental functions in eukaryotic cells by providing force generation, transport or tethering capacity. Motor activity control within the cell involves on/off switches, however, few examples are known of how myosins regulate speed or processivity and fine-tune their activity to a specific cellular task. Here, we describe a phosphorylation event for myosins of class VI (MYO6) in the motor domain, which accelerates its ATPase activity leading to a 4-fold increase in motor speed determined by actin-gliding assays, single molecule mechanics and stopped flow kinetics. We demonstrate that the serine/threonine kinase DYRK2 phosphorylates MYO6 at S267 in vitro. Single-molecule optical-tweezers studies at low load reveal that S267-phosphorylation results in faster nucleotide-exchange kinetics without change in the working stroke of the motor. The selective increase in stiffness of the acto-MYO6 complex when proceeding load-dependently into the nucleotide-free rigor state demonstrates that S267-phosphorylation turns MYO6 into a stronger motor. Finally, molecular dynamic simulations of the nucleotide-free motor reveal an alternative interaction network within insert-1 upon phosphorylation, suggesting a molecular mechanism, which regulates insert-1 positioning, turning the S267-phosphorylated MYO6 into a faster motor.
摘要：
肌球蛋白马达通过产生力在真核细胞中执行许多基本功能，运输或系留能力。细胞内的运动活动控制包括开/关开关，然而,关于肌球蛋白如何调节速度或持续性，并根据特定的细胞任务微调其活动的例子很少。这里,我们描述了运动域中VI类肌球蛋白（MYO6）的磷酸化事件，加速其ATPase活性，导致肌动蛋白滑动测定确定的运动速度增加4倍，单分子力学和停流动力学。我们证明丝氨酸/苏氨酸激酶DYRK2在体外在S267处磷酸化MYO6。在低负荷下的单分子光学镊子研究表明，S267磷酸化会导致更快的核苷酸交换动力学，而不会改变电动机的工作行程。当负载依赖性地进入无核苷酸的严格状态时，acto-MYO6复合物的刚度选择性增加表明S267磷酸化将MYO6转变为更强的马达。最后,无核苷酸运动的分子动力学模拟揭示了插入-1磷酸化后的替代相互作用网络，暗示了一种分子机制，调节插入件-1的定位，将S267磷酸化的MYO6变成更快的马达。
