Abstract
In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G(2)/M transition. We find that activation of Aurora A first occurs at centrosomes at late G(2) and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.
Highlights
Linked to many types of human cancer [1]
RNAi-mediated inactivation of Aurora A kinase in the randomly growing, non-synchronized HeLa cells did not arrest in G2 as would be expected for a role in Cdk1 activation, but instead the cells were arrested in mitosis with monopolar spindles [11,12,13]
This study focuses on elucidating the relationship between Cdk1 and block, and the level of phosphorylated histone H1 was markedly Aurora A kinase during G2/M transition of human cells
Summary
Linked to many types of human cancer [1]. how these mitotic kinases interact and coordinate with each other is a long standing question, especially in mammalian cells. The Aurora A kinase is shown to play a role in the activation of Cdk and entry into mitosis in Xenopus oocyte cycling extracts [7]. Contrary to Xenopus oocyte cycling extract, depletion of Plk by RNAi in human cells resulted in activation of Cdk and a mitotic arrest [8], suggesting that Plk is not essential for Cdk activation of human cells. Depletion of Aurora A kinase in synchronized HeLa cells by RNAi was reported to prevent Cdk activation and entry into mitosis [10]. Inactivation of Cdk kinase prevents entry into mitosis and mitotic activation of Aurora A, suggesting that Cdk acts upstream of Aurora A kinase during G2/M transition. We further show that Aurora A and Plk have redundant functions in feedback activation of Cdk to promote timely entry into mitosis
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