Analysis of the role of CDK and ubiquitin E3 ligase activity in regulation of CIZ1 proteostasis

Caprani, Louise and Copeland, Nikki and Allinson, Sarah (2022) Analysis of the role of CDK and ubiquitin E3 ligase activity in regulation of CIZ1 proteostasis. Masters thesis, Lancaster University.

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Abstract

Cip1 interacting Zinc finger protein 1 (CIZ1) is a nuclear matrix protein and coordinates the activity of cyclin A- cyclin dependent kinase 2 (CDK2) for efficient initiation of DNA replication. The overexpression of CIZ1 has been associated with tumorigenesis in several common cancers including breast, prostate, colorectal, gall bladder and hepatocellular carcinoma, this suggests that CIZ1 may be a viable target for therapeutic intervention in CIZ1-dependent cancers. The working model for CIZ1 regulation suggests that CIZ1 protein levels are regulated by DDK/CDK2 mediated phosphorylation which protects from ubiquitin proteasome system (UPS) mediated degradation. Furthermore, inhibition of DDK/ CDK2 may facilitate the UPS- mediated degradation of CIZ1. The UPS-mediated degradation of CIZ1 would be dependent on a functional UPS, therefore the E3 ligases that target CIZ1 may represent biomarkers for patient stratification when determining which patients would respond to CDK inhibitors as a way of reducing CIZ1 levels. Further characterisation of the molecular pathways that regulate CIZ1 levels could provide potential avenues for reducing CIZ1 in cancer. Here, the efficacy of small molecule CDK/DDK inhibitors to reduce CIZ1 levels was evaluated in murine fibroblasts. Furthermore, three E3 ligases (UBR5, FBXO38 and UBE2O) that potentially target CIZ1 were transfected into 3T3 cells in order to characterise their potential role in regulating CIZ1 protein levels. In this study, inhibition of CDK4/6, DDK and CDK2 resulted in a non-significant reduction in CIZ1 levels during G1/S. Furthermore, inhibition of CDK1 resulted in a non- significant reduction in CIZ1 levels later in the cell cycle. Inhibition of DDK/CDK2 potentially facilitates the UPS mediated degradation of CIZ1, inhibition of the proteasome using MG132 prevented CIZ1 degradation with concomitant inhibition of DDK or CDK2. For E3 transfection experiments, there was difficulty validating E3 expression in the 3T3 cells. Nevertheless, there was a non-significant reduction in CIZ1 levels in cells transfected with UBR5-GFP or FBXO38- FLAG. Together the findings suggest that manipulation of CIZ1 regulators could provide an avenue to reduce CIZ1 levels, however the findings are preliminary and further validation of the findings is required in future work.