Abstract：
Myosin phosphatase targeting subunit1 (MYPT1) is a critical subunit of myosin phosphatase (MP), which brings PP1Cδ phosphatase and its substrate together. We previously showed that MYPT1 depletion resulted in oblique chromatid segregation. Therefore, we hypothesized that MYPT1 may control microtubule-dependent motor activity. Dynein, a minus-end microtubule motor, is known to be involved in mitotic spindle assembly. We thus examined whether MYPT1 and dynein may interact. Proximity ligation assay and co-immunoprecipitation revealed that MYPT1 and dynein intermediate chain (DIC) were associated. We found that DIC phosphorylation is increased in MYPT1-depleted cells in vivo, and that MP was able to dephosphorylate DIC in vitro. MYPT1 depletion also altered the localization and motility of Rab7-containing vesicles. MYPT1-depletion dispersed the perinuclear Rab7 localization to the peripheral in interphase cells. The dispersed Rab7 localization was rescued by microinjection of a constitutively active, truncated MYPT1 mutant, supporting that MP is responsible for the altered Rab7 localization. Analyses of Rab7 vesicle trafficking also revealed that minus-end transport was reduced in MYPT1-depleted cells. These results suggest an unexpected role of MP: MP controls dynein activity in both mitotic and interphase cells, possibly by dephosphorylating dynein subunits including DIC.
摘要：
肌球蛋白磷酸酶靶向亚单位1（MYPT1）是肌球蛋白磷酸酶（MP）的关键亚基，将PP1Cδ磷酸酶和它的底物结合在一起。我们先前表明MYPT1耗竭导致斜染色单体分离。因此,我们假设MYPT1可能控制微管依赖性运动活动.Dynein,一个负端微管马达，已知参与有丝分裂纺锤体的组装。因此，我们检查了MYPT1和动力蛋白是否可能相互作用。邻近连接测定和共免疫沉淀显示MYPT1和动力蛋白中间链（DIC）相关。我们发现DIC磷酸化在体内MYPT1耗尽细胞中增加，并且MP能够在体外使DIC去磷酸化。MYPT1耗竭也改变了含Rab7的囊泡的定位和运动性。MYPT1耗尽将核周Rab7定位分散到间期细胞中的外周。分散的Rab7定位是通过显微注射组成型活性，截短的MYPT1突变体，支持MP负责Rab7本地化的改变。对Rab7囊泡运输的分析还显示，在MYPT1耗尽的细胞中，负端转运减少。这些结果表明，MP：MP控制有丝分裂和间期细胞中的动力蛋白活性，可能通过脱磷酸化动力蛋白亚基，包括DIC。
