St-Denis Nicole, Gupta Gagan D, Lin Zhen Yuan, Gonzalez-Badillo Beatriz, Pelletier Laurence, Gingras Anne-Claude
From the ‡Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada;
From the ‡Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada; §Department of Molecular Genetics, University of Toronto, Toronto ON M5S 1A8, Canada.
Mol Cell Proteomics. 2015 Apr;14(4):946-60. doi: 10.1074/mcp.M114.046086. Epub 2015 Feb 6.
The myotubularins are a family of phosphatases that dephosphorylate the phosphatidylinositols phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-phosphate. Several family members are mutated in disease, yet the biological functions of the majority of myotubularins remain unknown. To gain insight into the roles of the individual enzymes, we have used affinity purification coupled to mass spectrometry to identify protein-protein interactions for the myotubularins. The myotubularin interactome comprises 66 high confidence (false discovery rate ≤1%) interactions, including 18 pairwise interactions between individual myotubularins. The results reveal a number of potential signaling contexts for this family of enzymes, including an intriguing, novel role for myotubularin-related protein 3 and myotubularin-related protein 4 in the regulation of abscission, the final step of mitosis in which the membrane bridge remaining between two daughter cells is cleaved. Both depletion and overexpression of either myotubularin-related protein 3 or myotubularin-related protein 4 result in abnormal midbody morphology and cytokinesis failure. Interestingly, myotubularin-related protein 3 and myotubularin-related protein 4 do not exert their effects through lipid regulation at the midbody, but regulate abscission during early mitosis, by interacting with the mitotic kinase polo-like kinase 1, and with centrosomal protein of 55 kDa (CEP55), an important regulator of abscission. Structure-function analysis reveals that, consistent with known intramyotubularin interactions, myotubularin-related protein 3 and myotubularin-related protein 4 interact through their respective coiled coil domains. The interaction between myotubularin-related protein 3 and polo-like kinase 1 relies on the divergent, nonlipid binding Fab1, YOTB, Vac1, and EEA1 domain of myotubularin-related protein 3, and myotubularin-related protein 4 interacts with CEP55 through a short GPPXXXY motif, analogous to endosomal sorting complex required for transport-I components. Disruption of any of these interactions results in abscission failure, by disrupting the proper recruitment of CEP55, and subsequently, of endosomal sorting complex required for transport-I, to the midbody. Our data suggest that myotubularin-related protein 3 and myotubularin-related protein 4 may act as a bridge between CEP55 and polo-like kinase 1, ensuring proper CEP55 phosphorylation and regulating CEP55 recruitment to the midbody. This work provides a novel role for myotubularin-related protein 3/4 heterodimers, and highlights the temporal and spatial complexity of the regulation of cytokinesis.
肌管素是一类磷酸酶，可使磷脂酰肌醇-3-磷酸和磷脂酰肌醇-3,5-磷酸去磷酸化。该家族的几个成员在疾病中发生了突变，但大多数肌管素的生物学功能仍不清楚。为了深入了解各个酶的作用，我们使用了亲和纯化结合质谱法来鉴定肌管素的蛋白质-蛋白质相互作用。肌管素相互作用组包含66种高可信度（错误发现率≤1%）的相互作用，包括各个肌管素之间的18种成对相互作用。结果揭示了该酶家族的一些潜在信号传导背景，包括肌管素相关蛋白3和肌管素相关蛋白4在分裂后期调控中的一个有趣的新作用，分裂后期是有丝分裂的最后一步，此时两个子细胞之间残留的膜桥被切断。肌管素相关蛋白3或肌管素相关蛋白4的缺失和过表达都会导致中体形态异常和胞质分裂失败。有趣的是，肌管素相关蛋白3和肌管素相关蛋白4并非通过对中体脂质的调节发挥作用，而是在有丝分裂早期通过与有丝分裂激酶polo样激酶1以及55 kDa的中心体蛋白（CEP55，一种重要的分裂后期调节因子）相互作用来调控分裂后期。结构-功能分析表明，与已知的肌管素内部相互作用一致，肌管素相关蛋白3和肌管素相关蛋白4通过它们各自的卷曲螺旋结构域相互作用。肌管素相关蛋白3与polo样激酶1之间的相互作用依赖于肌管素相关蛋白3中不同的、非脂质结合的Fab1、YOTB、Vac1和EEA1结构域，而肌管素相关蛋白4通过一个短的GPPXXXY基序与CEP55相互作用，类似于运输所需的内体分选复合体-I的组分。这些相互作用中的任何一个被破坏都会导致分裂后期失败，因为它会破坏CEP55以及随后运输所需的内体分选复合体-I向中体的正确募集。我们的数据表明，肌管素相关蛋白3和肌管素相关蛋白4可能作为CEP55和polo样激酶1之间的桥梁，确保CEP55的正确磷酸化并调节CEP55向中体的募集。这项工作揭示了肌管素相关蛋白3/4异二聚体的新作用，并突出了胞质分裂调控在时间和空间上的复杂性。
