Abstract
Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint. More recently, Wang et al. (2007) found that C53/LZAP may function as a tumor suppressor by way of inhibiting NF-κB signaling. We report here the identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexpression. Intriguingly, we found that C53 interacts with Chk1 and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell-cycle progression and DNA damage response.
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Acknowledgements
We thank Dr J Bartek (Danish Cancer Society) for Chk1-GFP-PACT construct. We are grateful for the insightful advice from Drs Junying Yuan (Harvard Medical School), Qingshen Gao, Jacek Topczewski, Kathy Randell, Bernard Mirkin (Northwestern University) and Marcus Peter (University of Chicago), and the help from Drs Francis Szele and Ed Kang (Children's Memorial Research Center and Northwestern University) on time-lapse videomicroscopy and William Goossens (Children's Memorial Research Center) on confocal microscopy. The project was supported by NIH grant R21 AG027840 and R01 GM081776 (to H Li), Children's Memorial Research Center and the Clarke family.
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Supplementary information
Supplementary information, Figure S1
C53 overexpression abrogated the G2/M arrest induced by etoposide. (PDF 168 kb)
Supplementary information, Figure S2
In vitro interaction between C53 and Chk1. (PDF 321 kb)
Supplementary information, Figure S3
Overexpression of C53 and its fragments alone in HeLa cells does not affect Cdk1 activation. (PDF 103 kb)
Supplementary information, Figure S4
Co-localization of C53 and Chk1 at the centrosome. (PDF 599 kb)
Supplementary information, Figure S5
Inactive Cdk1 (p-Y15-Cdk1) decorated centrosomes in C53-depleted U-2 OS cells. (PDF 485 kb)
Supplementary information, Figure S6
C53-GFP-PACT fusion protein targeted the centrosome. (PDF 664 kb)
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Jiang, H., Wu, J., He, C. et al. Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation. Cell Res 19, 458–468 (2009). https://doi.org/10.1038/cr.2009.14
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DOI: https://doi.org/10.1038/cr.2009.14
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