Ciz1 accumulation is regulated by opposing cyclin dependent kinase (CDK) and ubiquitin-proteasome system (UPS) activities


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Tekle Pauzaite1
1Lancaster University

Abstract

Background

The cell cycle is regulated by the concerted activities of cyclin dependent kinase and Dbf4 dependent kinases. Cyclin A-CDK2 cooperates with Ciz1 to promote exit from G1 phase and initiation of DNA replication. Overexpression of Ciz1 has been associated with tumourigenesis and defects in X-chromosome inactivation, demonstrating that a better understanding of the regulation of Ciz1 accumulation is required. Here we show that Ciz1 protein accumulates in G1 phase and that Ciz1 levels correlate with cyclin E, cyclin A and Dbf4 expression. CDK and DDK levels rise during G1 phase to promote the G1/S transition. Differential phosphorylation of Ciz1 increases with rising CDK/DDK activities suggesting that Ciz1 is stabilised by CDK mediated phosphorylation.

Method

Embryonic mice fibroblasts and human cancer cell lines research.

Chemical and genetic kinase inhibition.

Protein purification using various chromatography steps.

Ciz1 ubiquitylation assays.

Results

Chemical and genetic inhibition of CDK and DDK activity reduces Ciz1 levels, which is reversed by proteasomal inhibition. In addition, polyubiquitylated Ciz1 was also detected, consistent with ubiquitin proteasome system (UPS) mediated degradation of Ciz1. Therefore, Ciz1 is regulated by opposing CDK activity, which promotes accumulation, and UPS activity that promotes Ciz1 degradation in low CDK environments. This leads to a model that suggests that CDK inhibition may reduce Ciz1 levels in Ciz1 dependent tumours. The efficacy of CDK2 inhibition was determined in the Ciz1 dependent prostate cancer cell line, PC3. Finally, the repurposing of CDK2 small molecule inhibitors effectively reduce Ciz1 levels and proliferation in PC3 cells, demonstrating the efficacy of this approach.

Conclusion

The data suggest that targeting of CDK networks may have clinical benefits in Ciz1 dependent tumours. It may provide benefit of increased sensitivity to chemotherapy and stratification of therapies.