Regulation of mitotic spindle orientation by phosphorylation of end binding protein 1
Regulation of mitotic spindle orientation by phosphorylation of end binding protein 1
复制标题
通过末端结合蛋白 1 磷酸化调节有丝分裂纺锤体方向
DOI：
10.1016/j.yexcr.2019.111618
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发表时间：
2019-11-01
影响因子：
3.7
通讯作者：
Liu, Min
中科院分区：
文献类型：
--
作者：
Chen, Miao;Cao, Yu;Liu, Min
末端结合蛋白1（EB1）是微管动力学的关键调节因子，它在微管正端协调分层相互作用网络，以控制细胞的正常分裂。已知EB1的活性受丝氨酸/苏氨酸磷酸化调控；然而，酪氨酸磷酸化如何影响EB1的活性仍知之甚少。在本研究中，我们绘制了同步化细胞中EB1的酪氨酸磷酸化图谱，并在有丝分裂细胞中鉴定出两个酪氨酸磷酸化位点（Y217和Y247）。利用磷酸化缺陷型（Y/F）和磷酸化模拟型（Y/D）突变体，我们发现Y247而非Y217对星体微管的稳定性至关重要。Y247D突变体导致纺锤体角度增大，表明纺锤体定向存在缺陷。延时显微镜观察显示，Y247D突变体通过延长前中期和中期的持续时间，显著延缓了有丝分裂进程。结构分析表明，Y247突变体导致EB同源（EBH）结构域中疏水腔的不稳定，从而影响其与p150（glued）的相互作用，而p150（glued）是Gαi/LGN/NuMA复合物捕获所必需的一种蛋白质。这些发现揭示了EB1磷酸化在有丝分裂纺锤体定向调控和细胞分裂中的关键作用。
End binding protein 1 (EB1) is a key regulator of microtubule dynamics that orchestrates hierarchical interaction networks at microtubule plus ends to control proper cell division. EB1 activity is known to be regulated by serine/threonine phosphorylation; however, how tyrosine phosphorylation affects EB1 activity remains poorly understood. In this study, we mapped the tyrosine phosphorylation pattern of EB1 in synchronized cells and identified two tyrosine phosphorylation sites (Y217 and Y247) in mitotic cells. Using phospho-deficient (Y/F) and phospho-mimic (Y/D) mutants, we revealed that Y247, but not Y217, is critical for astral microtubule stability. The Y247D mutant contributed to increased spindle angle, indicative of defects in spindle orientation. Time-lapse microscopy revealed that the Y247D mutant significantly delayed mitotic progression by increasing the duration times of prometaphase and metaphase. Structural analysis suggests that Y247 mutants lead to instability of the hydrophobic cavity in the EB homology (EBH) domain, thereby affecting its interactions with p150(glued), a protein essential for G alpha i/LGN/NuMA complex capture. These findings uncover a crucial role for EB1 phosphorylation in the regulation of mitotic spindle orientation and cell division.