Skip to main content
SpringerLink
Log in

Establishment of a Cell-Free System to Study the Activation of Chk2

  • Protocol
Checkpoint Controls and Cancer

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 280))

  • 1629 Accesses

Abstract

The checkpoint kinase Chk2 is activated in response to DNA damage through pathways requiring protein kinases ATM and/or ATR. The means by which Chk2 is activated by these kinases still remains to be addressed. Here we describe a cell-free system to study the activation of Chk2. Chk2 produced by a wheat germ extract in vitro transcription/translation system is inactive and can be activated by incubating with a rabbit reticulocyte lysate. This method will be useful for identification of cofactors required for activation of Chk2.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Blasina, A., Price, B. D., Turenne, G. A., and McGowan, C. H. (1999) Caffeine inhibits the checkpoint kinase ATM. Curr. Biol. 9, 1135–1138.

    Article  PubMed  CAS  Google Scholar 

  2. Chaturvedi, P., Eng, W. K., Zhu, Y., et al. (1999) Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Oncogene 18, 4047–4054.

    Article  PubMed  CAS  Google Scholar 

  3. Matsuoka, S., Huang, M., and Elledge, S. J. (1998) Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. Science 282, 1893–1897.

    Article  PubMed  CAS  Google Scholar 

  4. Ahn, J. Y., Schwarz, J. K., Piwnica-Worms, H., and Canman, C. E. (2000) Threonine 68 phosphorylation by ataxia telangiectasia mutated is required for efficient activation of Chk2 in response to ionizing radiation. Cancer Res. 60, 5934–5936.

    PubMed  CAS  Google Scholar 

  5. Matsuoka, S., Rotman, G., Ogawa, A., Shiloh, Y., Tamai, K., and Elledge, S. J. (2000) Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro. Proc. Natl. Acad. Sci. USA 97, 10389–10394.

    Article  PubMed  CAS  Google Scholar 

  6. Melchionna, R., Chen, X. B., Blasina, A., and McGowan, C. H. (2000) Threonine 68 is required for radiation-induced phosphorylation and activation of Cds1. Nat. Cell Biol. 2, 762–765.

    Article  PubMed  CAS  Google Scholar 

  7. Zhou, B. B., Chaturvedi, P., Spring, K., et al. (2000) Caffeine abolishes the mammalian G(2)/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity. J. Biol. Chem. 275, 10342–10348.

    Article  PubMed  CAS  Google Scholar 

  8. Xu, X., Tsvetkov, L. M., and Stern, D. F. (2002) Chk2 activation and phosphorylation-dependent oligomerization. Mol. Cell. Biol. 22, 4419–4432.

    Article  PubMed  CAS  Google Scholar 

  9. Ahn, J. Y., Li, X., Davis, H. L., and Canman, C. E. (2002) Phosphorylation of threonine 68 promotes oligomerization and autophosphorylation of the Chk2 protein kinase via the forkhead-associated domain. J. Biol. Chem. 277, 19,389–19,395.

    Article  PubMed  CAS  Google Scholar 

  10. Emili, A. (1998) MEC1-dependent phosphorylation of Rad9p in response to DNA damage. Mol. Cell 2, 183–189.

    Article  PubMed  CAS  Google Scholar 

  11. Sun, Z., Fay, D. S., Marini, F., Foiani, M., and Stern, D. F. (1996) Spk1/Rad53 is regulated by Mec1-dependent protein phosphorylation in DNA replication and damage checkpoint pathways. Genes Dev. 10, 395–406.

    Article  PubMed  CAS  Google Scholar 

  12. Sanchez, Y., Desany, B. A., Jones, W. J., Liu, Q., Wang, B., and Elledge, S. J. (1996) Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways. Science 271, 357–360.

    Article  PubMed  CAS  Google Scholar 

  13. Sun, Z., Hsiao, J., Fay, D. S., and Stern, D. F. (1998) Rad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpoint. Science 281, 272–274.

    Article  PubMed  CAS  Google Scholar 

  14. Vialard, J. E., Gilbert, C. S., Green, C. M., and Lowndes, N. F. (1998) The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage. EMBO J. 17, 5679–5688.

    Article  PubMed  CAS  Google Scholar 

  15. Gilbert, C. S., Green, C. M., and Lowndes, N. F. (2001) Budding yeast Rad9 is an ATP-dependent Rad53 activating machine. Mol. Cell 8, 129–136.

    Article  PubMed  CAS  Google Scholar 

  16. Sanchez, Y., Bachant, J., Wang, H., et al. (1999) Control of the DNA damage checkpoint by chk1 and rad53 protein kinases through distinct mechanisms. Science 286, 1166–1171.

    Article  PubMed  CAS  Google Scholar 

  17. Yarden, R. I., Pardo-Reoyo, S., Sgagias, M., Cowan, K. H., and Brody, L. C. (2002) BRCA1 regulates the G2/M checkpoint by activating Chk1 kinase upon DNA damage. Nat. Genet. 30, 285–289.

    Article  PubMed  Google Scholar 

  18. Schultz, L. B., Chehab, N. H., Malikzay, A., and Halazonetis, T. D. (2000) p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks. J. Cell Biol. 151, 1381–1390.

    Article  PubMed  CAS  Google Scholar 

  19. Rappold, I., Iwabuchi, K., Date, T., and Chen, J. (2001) Tumor suppressor p53 binding protein 1 (53BP1) is involved in DNA damage-signaling pathways. J. Cell Biol. 153, 613–620.

    Article  PubMed  CAS  Google Scholar 

  20. Anderson, L., Henderson, C., and Adachi, Y. (2001) Phosphorylation and rapid relocalization of 53BP1 to nuclear foci upon DNA damage. Mol. Cell. Biol. 21, 1719–1729.

    Article  PubMed  CAS  Google Scholar 

  21. Wang, B., Matsuoka, S., Carpenter, P. B., and Elledge, S. J. (2002) 53BP1, a mediator of the DNA damage checkpoint. Science 298, 1435–1438.

    Article  PubMed  CAS  Google Scholar 

  22. DiTullio, R. A., Jr., Mochan, T. A., Venere, M., et al. (2002) 53BP1 functions in an ATM-dependent checkpoint pathway that is constitutively activated in human cancer. Nat. Cell Biol. 4, 998–1002.

    Article  PubMed  CAS  Google Scholar 

  23. Goldberg, M., Stucki, M., Falck, J., et al. (2003) MDC1 is required for the intra-Sphase DNA damage checkpoint. Nature 421, 952–956.

    Article  PubMed  CAS  Google Scholar 

  24. Lou, Z., Minter-Dykhouse, K., Wu, X., and Chen, J. (2003) MDC1 is coupled to activated CHK2 in mammalian DNA damage response pathways. Nature 421, 957–961.

    Article  PubMed  CAS  Google Scholar 

  25. Peng, A. and Chen, P. L. (2003) NFBD1, like 53BP1, is an early and redundant transducer mediating Chk2 phosphorylation in response to DNA damage. J. Biol. Chem. 278, 8873–8876.

    Article  PubMed  CAS  Google Scholar 

  26. Shang, Y. L., Bodero, A. J., and Chen, P. L. (2003) NFBD1, a novel nuclear protein with signature motifs of FHA and BRCT, and an internal 41-amino acid repeat sequence, is an early participant in DNA damage response. J. Biol. Chem. 278, 6323–6329.

    Article  PubMed  CAS  Google Scholar 

  27. Stewart, G. S., Wang, B., Bignell, C. R., Taylor, A. M., and Elledge, S. J. (2003) MDC1 is a mediator of the mammalian DNA damage checkpoint. Nature 421, 961–966.

    Article  PubMed  CAS  Google Scholar 

  28. Xu, X. and Stern, D. F. (2003) NFBD1/KIAA0170 is a chromatin-associated protein involved in DNA damage signaling pathways. J. Biol. Chem. 278, 8795–8803.

    Article  PubMed  CAS  Google Scholar 

  29. Xu, X. and Stern, D. F. (2003) NFBD1/MDC1 regulates ionizing radiation-induced focus formation of DNA checkpoint signaling and repair factors. FASEB J. 18,1842–1848.

    Article  Google Scholar 

  30. Chehab, N. H., Malikzay, A., Appel, M., and Halazonetis, T. D. (2000) Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53. Genes Dev. 14, 278–288.

    PubMed  CAS  Google Scholar 

  31. Hirao, A., Kong, Y. Y., Matsuoka, S., et al. (2000) DNA damage-induced activation of p53 by the checkpoint kinase Chk2. Science 287, 1824–1827.

    Article  PubMed  CAS  Google Scholar 

  32. Shieh, S. Y., Ahn, J., Tamai, K., Taya, Y., and Prives, C. (2000) The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. Genes Dev. 14, 289–300.

    PubMed  CAS  Google Scholar 

  33. Lee, J. S., Collins, K. M., Brown, A. L., Lee, C. H., and Chung, J. H. (2000) hCds1-mediated phosphorylation of BRCA1 regulates the DNA damage response. Nature 404, 201–204.

    Article  PubMed  CAS  Google Scholar 

  34. Falck, J., Mailand, N., Syljuasen, R. G., Bartek, J., and Lukas, J. (2001) The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis. Nature 410, 842–847.

    Article  PubMed  CAS  Google Scholar 

  35. Brown, A. L., Lee, C. H., Schwarz, J. K., Mitiku, N., Piwnica-Worms, H., and Chung, J. H. (1999) A human Cds1-related kinase that functions downstream of ATM protein in the cellular response to DNA damage. Proc. Natl. Acad. Sci. USA 96, 3745–3750.

    Article  PubMed  CAS  Google Scholar 

  36. Xu, X., Liao, J., Creek, K. E., and Pirisi, L. (1999) Human keratinocytes and tumor-derived cell lines express alternatively spliced forms of transforming growth factor-alpha mRNA, encoding precursors lacking carboxyl-terminal valine residues. Oncogene 18, 5554–5562.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pathology, Yale University School of Medicine, New Haven, CT

    Xingzhi Xu

  2. Department of Pathology, School of Medicine, Yale University, New Haven, CT

    David F. Stern

Authors
  1. Xingzhi Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David F. Stern
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Molecular Microbiology and Immunology and K. Norris Jr. Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA

    Axel H. Schönthal PhD

Rights and permissions

Reprints and permissions

Copyright information

© 2004 Humana Press Inc.

About this protocol

Cite this protocol

Xu, X., Stern, D.F. (2004). Establishment of a Cell-Free System to Study the Activation of Chk2. In: Schönthal, A.H. (eds) Checkpoint Controls and Cancer. Methods in Molecular Biology™, vol 280. Humana Press. https://doi.org/10.1385/1-59259-788-2:165

Download citation

  • DOI: https://doi.org/10.1385/1-59259-788-2:165

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-214-8

  • Online ISBN: 978-1-59259-788-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation