Cyclin A-Cdk2 Phosphorylates BH3 only Protein Bad in vitro and in vivo

Increasing evidence suggests that Cyclin A-Cdk2 activity is required in the apoptosis process induced by various stimuli. To determine a specific substrate of Cyclin A-Cdk2 for apoptosis, in this study, we carried out an in vitro kinase assay using immunoprecipitated complex Cyclin A-Cdk2 as an enzyme source, and recombinant protein GST-Bad as a substrate. Our study showed that Bad was clearly phosphorylated by Cyclin A-Cdk2 in vitro. To examine whether protein Bad can also be phosphorylated by Cyclin A-Cdk2 kinase in vivo, we transiently overexpressed protein Bad with Cyclin A or Cdk2-dn, a dominant negative version of Cdk2, in Hela cells and determined the phosphorylation status of protein Bad. The test showed that protein Bad was clearly phosphorylated in Cyclin A overexpressed cells, but not in Cdk2-dn or mock transfectent. Moreover, etoposide also caused the phosphorylation of endogenetic Bad. In conclusion, here we provide first time evidence that protein Bad can be a substrate of Cyclin A-Cdk2 apoptosis for in vitro and in vivo.