TY - GEN

T1 - Regulation of Ciz1 by Cyclin A-Cyclin Dependent Kinase 2 (CDK2) mediated phosphorylation

AU - Baxter, Diana

PY - 2016

Y1 - 2016

N2 - The progression of the cell cycle is a tightly regulated process, regulated by the activities of cyclin dependent kinases. Phosphorylation of target proteins regulates key transitions in the cell cycle contributing to accurate DNA replication and cell division. Here we evaluate the regulation of the protein Ciz1 by cyclin A-CDK2. Ciz1 promotes localisation of cyclin A-CDK2 to chromatin sites at the G1/S phase transition and this ternary complex promotes initiation of DNA replication. However, the mechanisms by which Ciz1 is regulated are not fully understood. Here, Ciz1 protein levels are shown to be reduced after inhibition of CDK activity, suggesting that CDK-mediated phosphorylation of Ciz1 could contribute to increased protein stability. As Ciz1 is known to be a substrate of cyclin A-CDK2, enzyme kinetic studies were performed to evaluate whether there is preferential phosphorylation at specific sites within Ciz1. We characterise new phospho-specific antibodies that will be of benefit in future in in vivo studies evaluating the role of phosphorylation at specific sites in regulation of Ciz1 function. Using His-ubiquitin, we show that Ciz1 is covalently linked to ubiquitin. These complexes are stabilised after inhibition of the proteasome, consistent with degradation by the proteasomal. Further evidence presented demonstrates that an E3 ligase (Cdh1) interacts with Ciz1 via a conserved destruction box in the N-terminus. Taken together, the data presented here suggest that Ciz1 is regulated at multiple levels post-transitionally that contribute to ensuring it accumulates precisely at the G1/S phase transition. This may have important implications for its role as a driver mutation in cancer biology.

AB - The progression of the cell cycle is a tightly regulated process, regulated by the activities of cyclin dependent kinases. Phosphorylation of target proteins regulates key transitions in the cell cycle contributing to accurate DNA replication and cell division. Here we evaluate the regulation of the protein Ciz1 by cyclin A-CDK2. Ciz1 promotes localisation of cyclin A-CDK2 to chromatin sites at the G1/S phase transition and this ternary complex promotes initiation of DNA replication. However, the mechanisms by which Ciz1 is regulated are not fully understood. Here, Ciz1 protein levels are shown to be reduced after inhibition of CDK activity, suggesting that CDK-mediated phosphorylation of Ciz1 could contribute to increased protein stability. As Ciz1 is known to be a substrate of cyclin A-CDK2, enzyme kinetic studies were performed to evaluate whether there is preferential phosphorylation at specific sites within Ciz1. We characterise new phospho-specific antibodies that will be of benefit in future in in vivo studies evaluating the role of phosphorylation at specific sites in regulation of Ciz1 function. Using His-ubiquitin, we show that Ciz1 is covalently linked to ubiquitin. These complexes are stabilised after inhibition of the proteasome, consistent with degradation by the proteasomal. Further evidence presented demonstrates that an E3 ligase (Cdh1) interacts with Ciz1 via a conserved destruction box in the N-terminus. Taken together, the data presented here suggest that Ciz1 is regulated at multiple levels post-transitionally that contribute to ensuring it accumulates precisely at the G1/S phase transition. This may have important implications for its role as a driver mutation in cancer biology.

KW - Ciz1

KW - Cyclin A

KW - Cyclin-Dependent Kinase 2

KW - Cell cycle

M3 - Master's Thesis

PB - Lancaster University

ER -