Skip to main content

Measurement of Changes in Apoptosis and Cell Cycle Regulatory Kinase Cdk2

  • Protocol
Apoptosis Methods and Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 282))

Abstract

Many cell cycle regulatory proteins have been shown to be able to regulate cell death. Activation of Cdk2 has been shown to be necessary for the apoptosis of quiescent cells such as thymocytes, neurons, and endothelial cells. This activation is stimulus-specific because it occurs in glucocorticoid and DNA damage-induced but not in CD95-induced apoptosis in thymocytes. Apoptotic Cdk2 activation is controlled by apoptosis regulatory proteins like p53 and Bcl2 family members and correlates with degradation of its inhibitors p21Cip1 and p27Kip1 by activated caspases. Methods for measuring Cdk2 changes in activity in quiescent cells undergoing apoptosis are detailed in this chapter.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Guo, M. and Hay, B. A. (1999) Cell proliferation and apoptosis. Curr. Opin. Cell Biol. 11, 745–752.

    Article  PubMed  CAS  Google Scholar 

  2. Tsai, L. H., Delalle, I., Caviness, V. S., Jr., Chae, T., and Harlow, E. (1994) p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5. Nature 371, 419–423.

    Article  PubMed  CAS  Google Scholar 

  3. Norbury, C. and Nurse, P. (1992) Animal cell cycles and their control. Annu.Rev.Biochem. 61, 441–470.

    Article  PubMed  CAS  Google Scholar 

  4. Sherr, C. J. and Roberts, J. M. (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 13, 1501–1512.

    Article  PubMed  CAS  Google Scholar 

  5. Morgan, D. O. (1995) Principles of CDK regulation. Nature 374, 131–134.

    Article  PubMed  CAS  Google Scholar 

  6. Strasser, A., Huang, D. C., and Vaux, D. L. (1997) The role of the bcl-2/ced-9 gene family in cancer and general implications of defects in cell death control for tumourigenesis and resistance to chemotherapy. Biochim. Biophys. Acta, 1333, F151–F178.

    PubMed  CAS  Google Scholar 

  7. Kwiatkowska, K. and Sobota, A. (1999) Signaling pathways in phagocytosis. Bioessays 21, 422–431.

    Article  PubMed  CAS  Google Scholar 

  8. Fadok, V. A., Bratton, D. L., Rose, D. M., Pearson, A., Ezekewitz, R. A., and Henson, P. M. (2000) A receptor for phosphatidylserinespecific clearance of apoptotic cells. Nature 405, 85–90.

    Article  PubMed  CAS  Google Scholar 

  9. Levine, A. J. (1997) p53, the cellular gatekeeper for growth and division. Cell 88, 323–331.

    Article  PubMed  CAS  Google Scholar 

  10. Brady, H. J., Gil-Gomez, G., Kirberg, J., and Berns, A. J. (1996) Bax alpha perturbs T cell development and affects cell cycle entry of T cells. EMBO J. 15, 6991–7001.

    PubMed  CAS  Google Scholar 

  11. Wallach, D., Varfolomeev, E. E., Malinin, N. L., Goltsev, Y. V., Kovalenko, A. V., and Boldin, M. P. (1999) Tumor necrosis factor receptor and Fas signaling mechanisms. Annu.Rev.Immunol. 17, 331–367.

    Article  PubMed  CAS  Google Scholar 

  12. Chao, D. T. and Korsmeyer, S. J. (1998) BCL-2 family: regulators of cell death. Annu. Rev. Immunol. 16, 395–419.

    Article  PubMed  CAS  Google Scholar 

  13. Earnshaw, W. C., Martins, L. M., and Kaufmann, S. H. (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem. 68, 383–424.

    Article  PubMed  CAS  Google Scholar 

  14. Brady, H. J. and Gil-Gomez, G. (1999) The cell cycle and apoptosis. Results Probl. Cell Differ. 23, 127–144.

    PubMed  CAS  Google Scholar 

  15. Gil-Gomez, G., Berns, A., and Brady, H. J. (1998) A link between cell cycle and cell death: Bax and Bcl-2 modulate Cdk2 activation during thymocyte apoptosis. EMBO J. 17, 7209–7218.

    Article  PubMed  CAS  Google Scholar 

  16. Levkau, B., Koyama, H., Raines, E. W., Clurman, B. E., Herren, B., Orth, K., Roberts, J. M., and Ross, R. (1998) Cleavage of p21Cip1/Waf1 and p27Kip1 mediates apoptosis in endothelial cells through activation of Cdk2: role of a caspase cascade. Mol. Cell 1, 553–563.

    Article  PubMed  CAS  Google Scholar 

  17. Sheaff, R. J. (1997) Regulation of mammalian cyclin-dependent kinase 2. Methods Enzymol. 283, 173–193.

    Article  PubMed  CAS  Google Scholar 

  18. Phelps, D. E. and Xiong, Y. (1997) Assay for activity of mammalian cyclin D-dependent kinases CDK4 and CDK6. Methods Enzymol. 283, 194–205.

    Article  PubMed  CAS  Google Scholar 

  19. Azzi, L., Meijer, L., Ostvold, A. C., Lew, J., and Wang, J. H. (1994) Purification of a 15-kDa cdk4-and cdk5-binding protein. J. Biol. Chem. 269, 13,279–13,288.

    PubMed  CAS  Google Scholar 

  20. Zhao, J., Dynlacht, B., Imai, T., Hori, T., and Harlow, E. (1998) Expression of NPAT, a novel substrate of cyclin E-CDK2, promotes S-phase entry. Genes Dev. 12(4), 456–461.

    Article  PubMed  CAS  Google Scholar 

  21. Jeffrey, P. D., Russo, A. A., Polyak, K., Gibbs, E., Hurwitz, J., Massague, J., and Pavletich, N. P. (1995) Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex. Nature 376, 313–320.

    Article  PubMed  CAS  Google Scholar 

  22. Lauper, N., Beck, A. R., Cariou, S., Richman, L., Hofmann, K., Reith, W., et al. (1998) Cyclin E2: a novel CDK2 partner in the late G1 and S phases of the mammalian cell cycle. Oncogene 17, 2637–2643.

    Article  PubMed  CAS  Google Scholar 

  23. Mok, C. L., Gil-Gomez, G., Williams, O., Coles, M., Taga, S., Tolaini, M., et al. (1999) Bad can act as a key regulator of T cell apoptosis and T cell development. J. Exp. Med. 189, 575–586.

    Article  PubMed  CAS  Google Scholar 

  24. Norbury, C., MacFarlane, M., Fearnhead, H., and Cohen, G. M. (1994) Cdc2 activation is not required for thymocyte apoptosis. Biochem. Biophys. Res. Commun. 202, 1400–1406.

    Article  PubMed  CAS  Google Scholar 

  25. Gordon, J. A. (1991) Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods Enzymol. 201, 477–482.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2004 Humana Press Inc.

About this protocol

Cite this protocol

Gil-Gómez, G. (2004). Measurement of Changes in Apoptosis and Cell Cycle Regulatory Kinase Cdk2. In: Brady, H.J.M. (eds) Apoptosis Methods and Protocols. Methods in Molecular Biology, vol 282. Humana Press. https://doi.org/10.1385/1-59259-812-9:131

Download citation

  • DOI: https://doi.org/10.1385/1-59259-812-9:131

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-873-8

  • Online ISBN: 978-1-59259-812-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics