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Updated: May 20, 2026
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
Forced activation of Cdk1 via wee1 inhibition impairs homologous recombination
M Krajewska1, A M Heijink, Y J W M Bisselink
1Department of Medical Oncology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Abstract:
In response to DNA breaks, the 'DNA damage response' provokes a cell cycle arrest to facilitate DNA repair. Recent findings have indicated that cells can respond to DNA damage throughout the cell cycle, except during mitosis. Specifically, various mitotic kinases, including Cdk1, Aurora A and Plk1, were shown to inactivate key DNA damage checkpoint proteins when cells enter mitosis. Aberrant activation of mitotic kinases during interphase could therefore modulate cellular responses to DNA damage. In this study, our aim was to determine how aberrant activation of Cdk1 affects the cellular responses to DNA damage. We used Wee1 inhibition, using MK-1775, to force Cdk1 activation, which did not cause cytotoxicity in non-transformed cells. Instead, it accelerated mitotic entry and caused radio sensitization in p53-defective cancer cells, but not in p53-proficient cancer cells. Interestingly, we showed that Wee1 inhibition leads to elevation of Cdk1 activity in interphase cells. When we subsequently analyzed DNA damage responses in cells with forced Cdk1 activation, we observed a marked reduction of 53BP1 at sites of DNA damage along with an increase in γ-H2AX staining after irradiation, indicative of defective DNA repair. Indeed, when DNA repair was analyzed using in vivo endonuclease-induced homologous recombination (HR) assays, compromised DNA repair after Wee1 inhibition was confirmed. This defect in HR was accompanied by increased phosphorylation of BRCA2 at the Cdk1 phosphorylation site S3291. Taken together, our results indicate that Wee1 inhibition leads to forced Cdk1 activation in interphase cells, which interferes with normal DNA damage responses.
Insights
Forcing cell cycle kinase Cdk1 activation during interphase impairs DNA damage repair. Wee1 inhibition radiosensitizes cancer cells by disrupting DNA repair, particularly in p53-defective cells.
Area of Science:
- Cellular Biology
- Molecular Oncology
Background:
- The DNA damage response (DDR) is crucial for maintaining genomic stability, typically involving cell cycle arrest to allow DNA repair.
- Mitotic kinases like Cdk1, Aurora A, and Plk1 are known to inactivate DDR proteins during mitosis.
- Aberrant activation of these kinases during interphase may disrupt normal DNA damage responses.
Purpose of the Study:
- To investigate the effects of aberrant Cyclin-dependent kinase 1 (Cdk1) activation on cellular responses to DNA damage.
- To determine if forced Cdk1 activation influences DNA repair mechanisms and radiosensitivity.
Main Methods:
- Utilized Wee1 inhibition with MK-1775 to induce Cdk1 activation in interphase cells.
- Assessed cytotoxicity, mitotic entry, and radiosensitivity in p53-proficient and p53-defective cancer cells.
- Analyzed DNA damage response markers (53BP1, γ-H2AX) and DNA repair via homologous recombination (HR) assays.
- Investigated phosphorylation of BRCA2 at the Cdk1 site S3291.
Main Results:
- Wee1 inhibition elevated Cdk1 activity in interphase cells, accelerating mitotic entry without causing cytotoxicity in non-transformed cells.
- Forced Cdk1 activation led to radiosensitization in p53-defective cancer cells but not in p53-proficient cells.
- Observed reduced 53BP1 recruitment and increased γ-H2AX staining, indicating defective DNA repair.
- Confirmed compromised homologous recombination repair and increased BRCA2 phosphorylation at S3291.
Conclusions:
- Forced Cdk1 activation in interphase by Wee1 inhibition disrupts DNA damage response pathways.
- This disruption compromises DNA repair, leading to radiosensitization, particularly in p53-defective cancer cells.
- Targeting Wee1 could be a therapeutic strategy to enhance cancer treatment efficacy by exploiting DDR defects.
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