PLK1-mediated phosphorylation of WDR62/MCPH2 ensures proper mitotic spindle orientation
PLK1-mediated phosphorylation of WDR62/MCPH2 ensures proper mitotic spindle orientation
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DOI：
10.1093/hmg/ddx330
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发表时间：
2017-11-15
影响因子：
3.5
通讯作者：
Matsuura, Shinya
中科院分区：
文献类型：
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作者：
Miyamoto, Tatsuo;Akutsu, Silvia Natsuko;Matsuura, Shinya
原发性小头畸形(MCPH)是一种常染色体隐性遗传性疾病，以先天性头围减少为特征。在这里，我们用全外显子组测序的方法在一个小头畸形日本家庭的两个兄弟姐妹中发现了编码有丝分裂中心体蛋白WDR62/MCPH2基因c.731C>；T(p.Ser244Leu)和c.2413G>；T(p.Glu 805X)的复合杂合突变。然而，WDR62/MCPH2突变引起的小头畸形的分子和细胞病理学尚不清楚。为了阐明WDR62的生理作用，我们使用CRISPR/Cas9系统和单链寡核苷酸作为点突变靶向供体，建立了WDR62/MCPH2 c.731 C>；T(p.Ser244Leu)错义突变的人细胞系。在正常中期，有丝分裂的纺锤体平行于底物形成，以确保细胞的对称分裂，而WDR62/MCPH2突变的细胞由于星形微管组装受损而呈现随机的纺锤体取向。研究表明，有丝分裂蛋白1(Polo-like kinase1，PLK1)是星体微管发育过程中维持纺锤体定向所必需的。在这项研究中，我们证明了WDR62是一种PLK1底物，在Ser897处被磷酸化，并且这种在纺锤体极点的磷酸化促进了星形微管的组装，从而稳定了纺锤体的取向。我们的发现为PLK1-WDR62通路在维持正确的纺锤体定向中的作用提供了深入的见解。
Primary microcephaly (MCPH) is an autosomal recessive disorder characterized by congenital reduction of head circumference. Here, we identified compound heterozygous mutations c.731 C > T (p.Ser 244 Leu) and c.2413 G > T (p.Glu 805 X) in the WDR62/MCPH2 gene, which encodes the mitotic centrosomal protein WDR62, in two siblings in a Japanese family with microcephaly using whole-exome sequencing. However, the molecular and cellular pathology of microcephaly caused by WDR62/MCPH2 mutation remains unclear. To clarify the physiological role of WDR62, we used the CRISPR/Cas9 system and single-stranded oligonucleotides as a point-mutation-targeting donor to generate human cell lines with knock-in of WDR62/MCPH2 c.731 C > T (p.Ser 244 Leu) missense mutation. In normal metaphase, the mitotic spindle forms parallel to the substratum to ensure symmetric cell division, while WDR62/MCPH2-mutated cells exhibited a randomized spindle orientation caused by the impaired astral microtubule assembly. It was shown that a mitotic kinase, Polo-like kinase 1 (PLK1), is required for the maintenance of spindle orientation through astral microtubule development. In this study, we demonstrated that WDR62 is a PLK1 substrate that is phosphorylated at Ser 897, and that this phosphorylation at the spindle poles promotes astral microtubule assembly to stabilize spindle orientation. Our findings provide insights into the role of the PLK1-WDR62 pathway in the maintenance of proper spindle orientation.