Mechanism of regulation of protein Kinase Aurora A in response to Mitotic DNA damage

[摘要] Mitosis is a highly ordered collection of events that ensures that the duplicatedgenome is distributed to the daughter cells equally. A failure to do so results in loss ofgenetic information leading to aneuploidy, a condition frequently associated withcancer. Several mitotic kinases are targets of the DNA damage checkpoint, amongwhich Cdk1, Aurora A and Plk1 are the most significant. Aurora A, the focus of studypresented in this thesis, was shown to be inactivated by DNA damage induced in theG2 phase, leading to cell cycle arrest in an ATM/ATR-Chk1 dependent manner.In the present study, we addressed the molecular mechanism leading to DNA damageinducedinhibition of Aurora A activity. We show that Aurora A is also a target of IRinducedDNA damage occurring in mitosis. We used a synchronization approach thatarrests cells in mitosis where Aurora A kinase activity is at the peak. As a measure ofits activity, we tested the phosphorylation of Aurora A at the T-loop residue, T288,using specific antibody. The results confirmed a decreased phospho-signal, indicativeof reduced kinase activity. We confirmed that protein phosphatase 1 (PP1) wasactivated by mitotic DNA damage by scoring the loss of phosphorylation at T320, aCdk1-dependent phosphosite in PP1, indicating with high probability that this is thephosphatase responsible for Aurora A T288 dephosphorylation.During mitosis, TPX2, a microtubule-associated protein, is the main regulator ofAurora A. TPX2 binds to Aurora A facilitating its localization to mitotic spindles and,in addition, activates it by protecting T288 from PP1-mediated dephosphorylation.This interaction ensures that the kinase is locked in an active conformation throughoutmitosis. Upon IR-induced mitotic damage, we observed inactivation of Aurora A in amanner that was directly linked to disruption of the Aurora A-TPX2 complex. This, inturn, was the result of decreased TPX2 protein level. By employing cycloheximide toprevent any nascent protein synthesis, we found that in response to mitotic DNAdamage, TPX2 became highly unstable, being degraded by the APC-Cdh1proteasome pathway with faster kinetic than in control cells. We showed that decreaseof the overall population of TPX2 was also significantly contributed by posttranscriptionalcontrol mechanisms, as the TPX2 mRNA level remained unvaried inthe presence of damage. An initial attempt to identify these pathways indicated a defect in the process of translation initiation, as seen by the reduced level of TPX2mRNA that was able to associate with the actively translating units, the polysomes.Collectively, our results indicate that upon mitotic DNA damage, increased TPX2protein instability and, particularly, lack of new TPX2 synthesis results in an overallunbalance of the existing protein pool in a manner that affects the Aurora A-TPX2complex. This, in turn, exposes the pT288 site to activated PP1 resulting ininactivation of Aurora A.

[发布机构] University of Zurich

[效力级别] 570 Life sciences

[关键词] Institute of Molecular Cancer Research;570 Life sciences;biology