Related Experiment Videos
Cell-cycle-dependent and ATM-independent expression of human Chk1 kinase
Y S Kaneko1, N Watanabe, H Morisaki
1Department of Geriatric Research, National Institute for Longevity Sciences, Obu, Aichi, Japan.
Abstract:
Checkpoint genes cause cell cycle arrest when DNA is damaged or DNA replication is blocked. Although a human homolog of Chk1 (hChk1) has recently been reported to be involved in the DNA damage checkpoint through phosphorylation of Cdc25A, B, and C, it is not known at which phase(s) of the cell cycle hChk1 functions and how hChk1 causes cell cycle arrest in response to DNA damage. In the present study, we demonstrate that in normal human fibroblasts (MJ90), hChk1 is expressed specifically at the S to M phase of the cell cycle at both the RNA and protein levels and that it is localized to the nucleus at this time. hChk1 activity, as determined by phosphorylation of Cdc25C, is readily detected at the S to M phase of the cell cycle, and DNA damage induced by UV or ionizing radiation does not enhance the expression of hChk1 or its activity. Furthermore, hChk1 exists in an active form at the S to M phase in fibroblasts derived from patients with ataxia telangiectasia (AT) which lack the functional AT mutated (ATM) gene product, suggesting that hChk1 expression is independent of functional ATM. Taken together with the findings that phosphorylation of Cdc25C on serine 216 is increased at the S to M phase, it is suggested that at this particular phase of the cell cycle, even in the absence of DNA damage, hChk1 phosphorylates Cdc25C on serine 216, which is considered to be a prerequisite for the G2/M checkpoint. Thus, hChk1 may play an important role in keeping Cdc25C prepared for responding to DNA damage by phosphorylating its serine residue at 216 during the S to M phase.
Insights
Human Checkpoint kinase 1 (hChk1) functions during the S to M phase, preparing Cdc25C for the G2/M checkpoint. Its activity is independent of DNA damage and functional ATM, suggesting a role in maintaining cell cycle readiness.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Checkpoint genes induce cell cycle arrest in response to DNA damage or replication blocks.
- Human Checkpoint kinase 1 (hChk1) is implicated in DNA damage checkpoints via Cdc25A, B, and C phosphorylation.
- The specific cell cycle phases and mechanisms of hChk1-mediated arrest remain unclear.
Purpose of the Study:
- To determine the cell cycle phase(s) of hChk1 function.
- To elucidate how hChk1 contributes to cell cycle arrest following DNA damage.
- To investigate the relationship between hChk1 activity, cell cycle phase, and DNA damage response.
Main Methods:
- Analysis of hChk1 expression (RNA and protein) and localization in human fibroblasts.
- Assay of hChk1 activity through Cdc25C phosphorylation.
- Examination of hChk1 function in normal fibroblasts and those from ataxia telangiectasia patients.
- Investigation of hChk1 response to UV or ionizing radiation-induced DNA damage.
Main Results:
- hChk1 is expressed and active specifically during the S to M phase of the cell cycle.
- DNA damage does not enhance hChk1 expression or activity.
- hChk1 activity during S to M phase is independent of the ATM gene product.
- Phosphorylation of Cdc25C on serine 216 increases during S to M phase, correlating with hChk1 activity.
Conclusions:
- hChk1 functions during the S to M phase to phosphorylate Cdc25C at serine 216, a prerequisite for the G2/M checkpoint.
- hChk1 activity is constitutive during S to M phase and independent of DNA damage or ATM.
- hChk1 likely maintains Cdc25C in a state of readiness to respond to DNA damage during the S to M phase.