Cyclin dependent kinase 5 and its interacting proteins in cell death induced in vivo by cyclophosphamide in...

Activation or inactivation of members of the cyclin-dependent kinase family is important during cell cycle progression. However, Cdk5, a member of this family that was originally identified because of its high structural homology to Cdc2, is activated during cell differentiation and cell death but not during cell cycle progression. We previously demonstrated a correlation between the up-regulation of Cdk5 protein and kinase activity and cell death during development and pathogenesis. We report here that cyclophosphamide (CP) induces massive apoptotic cell death in mouse embryos and that Cdk5 is expressed in apoptotic cells displaying fragmented DNA. During CP-induced cell death, Cdk5 protein expression is substantially increased as detected by immunohistochemistry but not by Western blot, while its mRNA level remains the same as control, and its kinase activity is markedly elevated. The up-regulation of Cdk5 during CP-induced cell death is not due to de novo protein synthesis. We also examined p35, a regulatory protein of Cdk5 in neuronal differentiation. Using a yeast two-hybrid system, we isolated p35, a neuronal differentiation specific protein, as a protein that interacts with Cdk5 in CP-treated embryos. p35 mRNA level does not change, but the protein expression of p25, a truncated form of p35, is elevated during cell death in vivo, as established here, as well as during cell death in vitro. Our results suggest a role for Cdk5 and its regulatory proteins during CP induced cell death. These results further support the view that Cdk5 and its regulation may be key players in the execution of cell death regardless of how the cell dies, whether through biological mechanisms, disease states such as Alzheimer's disease, or induction by CP.