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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Chemical genetics reveals the requirement for Polo-like kinase 1 activity in positioning RhoA and triggering cytokinesis in human cells
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Chemical genetics reveals the requirement for Polo-like kinase 1 activity in positioning RhoA and triggering cytokinesis in human cells

机译：化学遗传学揭示了在定位RhoA和触发人类细胞胞质分裂中需要Polo样激酶1活性

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摘要

Polo-like kinases (Plks) play crucial roles in mitosis and cell division. Whereas lower eukaryotes typically contain a single Plk, mammalian cells express several closely related but functionally distinct Plks. We describe here a chemical genetic system in which a single Plk family member, Plk1, can be inactivated with high selectivity and temporal resolution by using an allele-specific, small-molecule inhibitor, as well as the application of this system to dissect Plk1's role in cytokinesis. To do this, we disrupted both copies of the PLK1 locus in human cells through homologous recombination and then reconstituted Plk1 activity by using either the wild-type kinase (Plk1~(wt)) or a mutant version whose catalytic pocket has been enlarged to accommodate bulky purine analogs (Plk1~(as)). When cultured in the presence of these analogs, Plk1~(as) cells accumulate in prometaphase with defects that parallel those found in PLK1~(Δ/Δ) cells. In addition, acute treatment of Plk1~(as) cells during anaphase prevents recruitment of both Plk1 itself and the Rho guanine nucleotide exchange factor (RhoGEF) Ect2 to the central spindle, abolishes RhoA GTPase localization to the equatorial cortex, and suppresses cleavage furrow formation and cell division. Our studies define and illuminate a late mitotic function of Plk1 that, although difficult or impossible to detect in Plk1-depleted cells, is readily revealed with chemical genetics.
机译：Polo样激酶（Plk）在有丝分裂和细胞分裂中起关键作用。低等真核生物通常仅包含一个Plk，而哺乳动物细胞则表达几个紧密相关但功能不同的Plk。我们在这里描述一个化学遗传系统，其中单个Plk家族成员Plk1可通过使用等位基因特异性小分子抑制剂以高选择性和时间分辨率失活，以及该系统在研究Plk1的作用中的应用在胞质分裂中。为此，我们通过同源重组破坏了人类细胞中PLK1基因座的两个拷贝，然后通过使用野生型激酶（Plk1〜（wt））或突变型（其催化口袋已扩大以容纳）来重建Plk1活性。笨重的嘌呤类似物（Plk1〜（as））。当在这些类似物的存在下培养时，Plk1〜（as）细胞在前中期积累，其缺陷与PLK1〜（Δ/Δ）细胞中的缺陷相似。此外，后期对Plk1〜（as）细胞进行急性处理可防止Plk1本身和Rho鸟嘌呤核苷酸交换因子（RhoGEF）Ect2募集到中心纺锤体，消除RhoA GTPase在赤道皮质的定位，并抑制卵裂沟的形成。和细胞分裂。我们的研究定义并阐明了Plk1的晚期有丝分裂功能，尽管在Plk1缺失的细胞中很难检测或不可能检测到，但化学遗传学很容易揭示这一功能。

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