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Measurement of changes in Cdk2 and cyclin o-associated kinase activity in apoptosis

Ramon Roset1, Gabriel Gil-Gómez

  • 1Institut Municipal d'Investigació Mèdica, Barcelona, Spain.

Methods in Molecular Biology (Clifton, N.J.)
|July 18, 2009
PubMed

Insights

Cyclin O controls the activation of Cdk2 (cyclin-dependent kinase 2) during apoptosis in lymphoid cells. This study details methods to measure this kinase activity, aiding research into cell death regulation.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Cell cycle regulatory proteins play a role in cell death.
  • Cdk2 activation is crucial for apoptosis in quiescent cells like thymocytes, neurons, and endothelial cells.
  • This activation is stimulus-dependent, occurring with glucocorticoids and DNA damage but not CD95-induced apoptosis.

Purpose of the Study:

  • To describe methods for measuring changes in Cdk2 activity during apoptosis.
  • To detail the generation of an antibody for immunoprecipitating cyclin O complexes.
  • To demonstrate the use of this antibody for measuring kinase activity associated with cyclin O.

Main Methods:

  • Measurement of Cdk2 activity during apoptosis.
  • Generation of a specific antibody against cyclin O.
  • Immunoprecipitation of cyclin O complexes from apoptotic cells under native conditions.
  • Assay of kinase activity associated with immunoprecipitated cyclin O.

Main Results:

  • Apoptotic Cdk2 activation in lymphoid cells is regulated by cyclin O.
  • Cyclin O activity is modulated by p53 and Bcl-2 family proteins.
  • Methods were established to quantify Cdk2 activity linked to cyclin O during apoptosis.

Conclusions:

  • Cyclin O is a key regulator of Cdk2 activity in apoptosis.
  • The developed antibody and methods enable the study of proapoptotic cyclin O kinase activity.
  • This research contributes to understanding the mechanisms of stimulus-specific apoptosis.

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