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


Bhatia, P. Mechanism of regulation of protein Kinase Aurora A in response to Mitotic DNA damage. 2010, University of Zurich, Faculty of Science.

Abstract

Mitosis is a highly ordered collection of events that ensures that the duplicated
genome is distributed to the daughter cells equally. A failure to do so results in loss of
genetic information leading to aneuploidy, a condition frequently associated with
cancer. Several mitotic kinases are targets of the DNA damage checkpoint, among
which Cdk1, Aurora A and Plk1 are the most significant. Aurora A, the focus of study
presented in this thesis, was shown to be inactivated by DNA damage induced in the
G2 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 damageinduced
inhibition of Aurora A activity. We show that Aurora A is also a target of IRinduced
DNA damage occurring in mitosis. We used a synchronization approach that
arrests cells in mitosis where Aurora A kinase activity is at the peak. As a measure of
its activity, we tested the phosphorylation of Aurora A at the T-loop residue, T288,
using specific antibody. The results confirmed a decreased phospho-signal, indicative
of reduced kinase activity. We confirmed that protein phosphatase 1 (PP1) was
activated by mitotic DNA damage by scoring the loss of phosphorylation at T320, a
Cdk1-dependent phosphosite in PP1, indicating with high probability that this is the
phosphatase responsible for Aurora A T288 dephosphorylation.
During mitosis, TPX2, a microtubule-associated protein, is the main regulator of
Aurora 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 throughout
mitosis. Upon IR-induced mitotic damage, we observed inactivation of Aurora A in a
manner that was directly linked to disruption of the Aurora A-TPX2 complex. This, in
turn, was the result of decreased TPX2 protein level. By employing cycloheximide to
prevent any nascent protein synthesis, we found that in response to mitotic DNA
damage, TPX2 became highly unstable, being degraded by the APC-Cdh1
proteasome pathway with faster kinetic than in control cells. We showed that decrease
of the overall population of TPX2 was also significantly contributed by posttranscriptional
control mechanisms, as the TPX2 mRNA level remained unvaried in
the 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 TPX2
mRNA that was able to associate with the actively translating units, the polysomes.
Collectively, our results indicate that upon mitotic DNA damage, increased TPX2
protein instability and, particularly, lack of new TPX2 synthesis results in an overall
unbalance of the existing protein pool in a manner that affects the Aurora A-TPX2
complex. This, in turn, exposes the pT288 site to activated PP1 resulting in
inactivation of Aurora A.

Abstract

Mitosis is a highly ordered collection of events that ensures that the duplicated
genome is distributed to the daughter cells equally. A failure to do so results in loss of
genetic information leading to aneuploidy, a condition frequently associated with
cancer. Several mitotic kinases are targets of the DNA damage checkpoint, among
which Cdk1, Aurora A and Plk1 are the most significant. Aurora A, the focus of study
presented in this thesis, was shown to be inactivated by DNA damage induced in the
G2 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 damageinduced
inhibition of Aurora A activity. We show that Aurora A is also a target of IRinduced
DNA damage occurring in mitosis. We used a synchronization approach that
arrests cells in mitosis where Aurora A kinase activity is at the peak. As a measure of
its activity, we tested the phosphorylation of Aurora A at the T-loop residue, T288,
using specific antibody. The results confirmed a decreased phospho-signal, indicative
of reduced kinase activity. We confirmed that protein phosphatase 1 (PP1) was
activated by mitotic DNA damage by scoring the loss of phosphorylation at T320, a
Cdk1-dependent phosphosite in PP1, indicating with high probability that this is the
phosphatase responsible for Aurora A T288 dephosphorylation.
During mitosis, TPX2, a microtubule-associated protein, is the main regulator of
Aurora 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 throughout
mitosis. Upon IR-induced mitotic damage, we observed inactivation of Aurora A in a
manner that was directly linked to disruption of the Aurora A-TPX2 complex. This, in
turn, was the result of decreased TPX2 protein level. By employing cycloheximide to
prevent any nascent protein synthesis, we found that in response to mitotic DNA
damage, TPX2 became highly unstable, being degraded by the APC-Cdh1
proteasome pathway with faster kinetic than in control cells. We showed that decrease
of the overall population of TPX2 was also significantly contributed by posttranscriptional
control mechanisms, as the TPX2 mRNA level remained unvaried in
the 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 TPX2
mRNA that was able to associate with the actively translating units, the polysomes.
Collectively, our results indicate that upon mitotic DNA damage, increased TPX2
protein instability and, particularly, lack of new TPX2 synthesis results in an overall
unbalance of the existing protein pool in a manner that affects the Aurora A-TPX2
complex. This, in turn, exposes the pT288 site to activated PP1 resulting in
inactivation of Aurora A.

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Additional indexing

Item Type:Dissertation
Referees:Ferrari S, Jiricny J, Meraldi P, Simanis V
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2010
Deposited On:31 Oct 2011 13:18
Last Modified:26 Jan 2017 08:49
Number of Pages:206
Related URLs:http://opac.nebis.ch/F/?local_base=NEBIS&con_lng=GER&func=find-b&find_code=SYS&request=006630962

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