Department of Molecular and Cell Biology, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
Institute of Structural and Molecular Biology, Birkbeck College, Malet Street, London WC1E 7HX, UK.
Sci Signal. 2019 Aug 13;12(594):eaaw2939. doi: 10.1126/scisignal.aaw2939.
EML4 is a microtubule-associated protein that promotes microtubule stability. We investigated its regulation across the cell cycle and found that EML4 was distributed as punctate foci along the microtubule lattice in interphase but exhibited reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo-electron microscopy with 3D reconstruction revealed that the basic N-terminal domain of EML4 mediated its binding to the acidic C-terminal tails of α- and β-tubulin on the microtubule surface. The mitotic kinases NEK6 and NEK7 phosphorylated the EML4 N-terminal domain at Ser and Ser in vitro, and depletion of these kinases in cells led to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only bound inappropriately to mitotic microtubules but also increased their stability and interfered with chromosome congression. In addition, constitutive activation of NEK6 or NEK7 reduced the association of EML4 with interphase microtubules. Together, these data support a model in which NEK6- and NEK7-dependent phosphorylation promotes the dissociation of EML4 from microtubules in mitosis in a manner that is required for efficient chromosome congression.
EML4 是一种微管相关蛋白，可促进微管稳定性。我们研究了其在细胞周期中的调控，发现 EML4 在间期呈点状焦点分布在微管晶格上，但在有丝分裂中与纺锤体微管的结合减少。微管沉淀和带有 3D 重建的冷冻电子显微镜显示，EML4 的碱性 N 端结构域介导其与微管表面的α-和β-微管蛋白的酸性 C 端尾巴结合。有丝分裂激酶 NEK6 和 NEK7 在体外将 EML4 的 N 端结构域磷酸化 Ser 和 Ser，细胞中这些激酶的耗竭导致 EML4 在有丝分裂中与微管的结合增加。S144A-S146A 双突变体不仅不恰当地与有丝分裂微管结合，而且增加了它们的稳定性并干扰了染色体向动粒的移动。此外，NEK6 或 NEK7 的组成性激活减少了 EML4 与间期微管的结合。总之，这些数据支持了一种模型，即 NEK6 和 NEK7 依赖性磷酸化促进 EML4 在有丝分裂中从微管解离，这是染色体向动粒移动所必需的。
