Cahu Julie, Olichon Aurelien, Hentrich Christian, Schek Henry, Drinjakovic Jovana, Zhang Cunjie, Doherty-Kirby Amanda, Lajoie Gilles, Surrey Thomas
European Molecular Biology Laboratory, Cell Biology and Biophysics Unit, Heidelberg, Germany.
PLoS One. 2008;3(12):e3936. doi: 10.1371/journal.pone.0003936. Epub 2008 Dec 15.
Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro.
METHODOLOGY/PRINCIPAL FINDINGS: We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation.
CONCLUSIONS/SIGNIFICANCE: These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein.
驱动蛋白-5亚家族的运动蛋白在大多数生物体的细胞分裂过程中对纺锤体组装起着至关重要的作用。这些马达蛋白使纺锤体中的微管交联并滑动。在有丝分裂期间，驱动蛋白-5马达蛋白在一个保守位点被细胞周期蛋白依赖性激酶1（Cdk1）磷酸化。非洲爪蟾的驱动蛋白-5在体外也被报道可被极光激酶A磷酸化。
方法/主要发现：我们在此研究这些磷酸化作用对非洲爪蟾驱动蛋白-5（称为Eg5）的影响。我们发现，Cdk1对Eg5 C末端尾巴上苏氨酸937的磷酸化不影响Eg5的速度，但强烈增加其与在缓冲液中组装的微管的结合。同样，这种磷酸化促进Eg5与非洲爪蟾卵提取物纺锤体中的微管结合。这种结合增强使纺锤体中Eg5的量升高到双极纺锤体形成所需的临界水平以上。我们还发现，极光激酶A对非洲爪蟾Eg5茎部丝氨酸543的磷酸化对于纺锤体形成不是必需的。
结论/意义：这些结果表明，Cdk1对Eg5的磷酸化对该马达蛋白与微管的相互作用有直接影响。在卵提取物中，Cdk1对Eg5的磷酸化确保纺锤体中Eg5的量高于纺锤体形成所需的水平。因此，这种对纺锤体的靶向增强似乎至少部分是Cdk1磷酸化后Eg5与微管结合增强的直接结果。这些发现推进了我们对这种重要的有丝分裂马达蛋白调控的理解。
