Chemical genetics reveals the requirement for Polo-like kinase 1 activity in positioning RhoA and triggering cytokinesis in human cells
Chemical genetics reveals the requirement for Polo-like kinase 1 activity in positioning RhoA and triggering cytokinesis in human cells
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DOI：
10.1073/pnas.0701140104
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发表时间：
2007-03-13
影响因子：
11.1
通讯作者：
Jallepalli, Prasad V.
中科院分区：
文献类型：
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作者：
Burkard, Mark E.;Randall, Catherine L.;Jallepalli, Prasad V.
Polo样激酶（Plks）在有丝分裂和细胞分裂中起重要作用。而低等真核生物通常含有一个单一的Plk，哺乳动物细胞表达几个密切相关，但功能不同的Plk。我们在这里描述了一个化学遗传系统，其中一个单一的Plk家族成员，Plk 1，可以通过使用等位基因特异性，小分子抑制剂，以及该系统的应用程序，以解剖Plk 1的胞质分裂的作用，以高选择性和时间分辨率失活。为此，我们通过同源重组破坏了人类细胞中PLK 1基因座的两个拷贝，然后通过使用野生型激酶（Plk 1（wt））或突变型激酶（其催化口袋已扩大以容纳庞大的嘌呤类似物（Pik 1（as）重建Plk 1活性。当在这些类似物的存在下培养时，Plk 1（as）细胞在前中期积累，具有与PLK 1（Delta/Delta）细胞中发现的缺陷平行的缺陷。此外，急性治疗的Plk 1（作为）细胞在后期防止招聘Plk 1本身和Rho鸟嘌呤核苷酸交换因子（RhoGEF）Ect 2的中央纺锤体，废除RhoA GTdR定位到赤道皮质，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Plk 1的晚期有丝分裂功能，虽然很难或不可能在Plk 1缺失的细胞中检测到，但很容易用化学遗传学揭示。
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 (Pik1(as)). When cultured in the presence of these analogs, Plk1(as) cells accumulate in prometaphase with defects that parallel those found in PLK1(Delta/Delta) 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.