Abstract
Effector kinase Chk1 is an evolutionarily conserved protein kinase. It is a key mediator linking the mechanisms that monitor DNA integrity to components of the cell cycle engine. In this study, recombinant vectors pEGFP-C1-Chk1/CΔ288/CΔ334/CΔ368 were constructed and transfected into HeLa cells to study the effect of the Chk1 regulatory domain on the regulation of subcellular Chk1 location in response to DNA damage. We found that DNA damage-induced nuclear accumulation is regulated by 34 amino acids (334–368) in the C-terminal regulatory domain. Recombinant vectors pXJ41-Chk1/CΔ288/CΔ334/CΔ368 were co-transfected with reporter plasmid pEGFP-N2 into HeLa cells to study the repair abilities of the different human Chk1 truncation mutants. In addition, recombinant vectors were transfected into HeLa cells to study the effects of the different truncation mutants on the cell cycle. Furthermore, to study the kinase activity of the different truncation mutants, Ser216 phosphorylation of Cdc25C was studied by Western blot analysis. We found that the enzymatic activity of CΔ368, missing the 108 C-terminal amino acids (368–476), was higher than that of full-length Chk1, and CΔ368 delayed the cell cycle progression. The enzymatic activity of CΔ334, missing the 142 C-terminal amino acids (334–476), was equivalent to that of full-length Chk1. CΔ288, missing the 188 C-terminal amino acids (288–476), had almost no enzymatic activity, suggesting that the regulatory domain contains both inhibitory and regulatory elements. This study provides useful information for further research on Chk1 function.
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References
Zhou B B, Elledge S J. The DNA damage response: Putting checkpoints in perspective. Nature, 2000, 408: 433–439
Zachos G, Rainey M D, Gillespie D A. Chk1-deficient tumour cells are viable but exhibit multiple checkpoint and survival defects. EMBO J, 2003, 22: 713–723
Merry C, Fu K, Wang J, et al. Targeting the checkpoint kinase Chk1 in cancer therapy. Cell Cycle, 2010, 9: 279–283
Li X F, Ward T, Yao X B, et al. Chk1 prevents abnormal mitosis of S-phase HeLa cells containing DNA damage. Chinese Sci Bull, 2009, 54: 4205–4213
Ng C P, Lee H C, Ho C W, et al. Differential mode of regulation of the checkpoint kinases CHK1 and CHK2 by their regulatory domains. J Biol Chem, 2004, 279: 8808–8819
Sweeney G. Leptin signalling. Cell Signal, 2002, 14: 655–663
Bolderson E, Richard D J, Zhou B B, et al. Recent advances in cancer therapy targeting proteins involved in DNA double-strand break repair. Clin Cancer Res, 2009, 15: 6314–6320
Carrassa L, Sanchez Y, Erba E, et al. U2OS cells lacking Chk1 undergo aberrant mitosis and fail to activate the spindle check-point. J Cell Mol Med, 2009, 13: 1565–1576
Karlsson-Rosenthal C, Millar J B. Cdc25: Mechanisms of checkpoint inhibition and recovery. Trends Cell Biol, 2006, 16: 285–292
Pan Y, Ren K H, He H W, et al. Knockdown of Chk1 sensitizes human colon carcinoma HCT116 cells in a p53-dependent manner to lidamycin through abrogation of a G2/M checkpoint and induct ion of apoptosis. Cancer Biol Ther, 2009, 8: 1559–1566
McNeely S, Conti C, Sheikh T, et al. Chk1 inhibition after replicative stress activates a double strand break response mediated by ATM and DNA-dependent protein kinase. Cell Cycle, 2010, 9: 995–1004
Xiao Z, Xue J, Gu W Z, et al. Cyclin B1 is an efficacy-predicting biomarker for Chk1 inhibitors. Biomarkers, 2008, 13: 579–596
Nigg E A. Mitotic kinases as regulators of cell division and its checkpoints. Nat Rev Mol Cell Biol, 2001, 2: 21–32
Ling H, Wen L, Ji X X, et al. Growth inhibitory effect and Chk1-dependent signaling involved in G2/M arrest on human gastric cancer cells induced by diallyl disulfide. Braz J Med Biol Res, 2010, 43: 271–278
Wilsker D, Petermann E, Helleday T, et al. Essential function of Chk1 can be uncoupled from DNA damage checkpoint and replication control. Proc Natl Acad Sci USA, 2008, 105: 20752–20757
Liu Q, Guntuku S, Cui X S, et al. Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev, 2000, 14: 1448–1459
Zhao H, Watkins J L, Piwnica-Worms H. Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints. Proc Natl Acad Sci USA, 2002, 99: 14795–14800
Lopez-Girona A, Tanaka K, Chen X B, et al. Serine-345 is required for Rad3-dependent phosphorylation and function of checkpoint kinase Chk1 in fission yeast. Proc Natl Acad Sci USA, 2001, 98: 11289–11294
Guo Z, Kumagai A, Wang S X, et al. Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts. Genes Dev, 2000, 14: 2745–2756
Gatei M, Sloper K, Sorensen C, et al. Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation. J Biol Chem, 2003, 278: 14806–14811
Jiang K, Pereira E, Maxfield M, et al. Regulation of Chk1 includes chromatin association and 14-3-3 binding following phosphorylation on Ser-345. J Biol Chem, 2003, 278: 25207–25217
Dunaway S, Liu H Y, Walworth N C. Interaction of 14-3-3 protein with Chk1 affects localization and checkpoint function. J Cell Sci, 2004, 118: 39–50
Kim E M, Burke D J. DNA damage activates the SAC in an ATM/ATR-dependent manner, independently of the kinetochore. PLoS Genet, 2008, 4: e1000015
Chen L, Liu T H, Walworth N C. Association of Chk1 with 14-3-3 proteins is stimulated by DNA damage. Genes Dev, 1999, 13: 675–685
Seimiya H, Sawada H, Muramatsu Y, et al. Involvement of 14-3-3 proteins in nuclear localization of telomerase. EMBO J, 2000, 19: 2652–2661
Muslin A J, Tanner J W, Allen P M, et al. Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell, 1996, 84: 889–897
Zeng Y, Piwnica-Worms H. DNA damage and replication checkpoints in fission yeast require nuclear exclusion of the Cdc25 phosphatase via 14-3-3 binding. Mol Cell Biol, 1999, 19: 7410–7419
Atanassov B S, Ninova P D, Anachkova B B, et al. Relationship between DNA repair capacity and resistance to genotoxins in four human cell lines. Cancer Detect Prev, 2003, 27: 24–29
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Ning, L., Wang, H., San, D. et al. C-terminal domain of Chk1 regulates its subcellular location and kinase activity for DNA repair. Chin. Sci. Bull. 56, 3138–3147 (2011). https://doi.org/10.1007/s11434-011-4538-4
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DOI: https://doi.org/10.1007/s11434-011-4538-4