A novel regulator of cell cycle and cell death

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DESCRIPTION (provided by applicant): Our long-term goal is to elucidate the roles of a novel protein C53 in regulation of cell cycle progression and DNA damage response, and to explore potential exploitation of this protein as a novel therapeutic approach for cancer treatment. Regulation of cell cycle and cellular response to DNA damage is critical for animal development and ageing, and the pathogenesis of human diseases like cancer. Defects in cell cycle regulation and DNA damage response may lead to genome instability and oncogenesis. Cells utilize the so-called checkpoint mechanisms to ensure the completion of a particular phase before the next phase is initiated, thereby maintaining truthful transmission of genetic material. One of the key checkpoints is so-called the G2/M checkpoint that regulates mitotic entry and activation of cyclin-dependent kinase 1 (Cdk1), a driving force for progression of cell division. We have recently identified Cdk5 activator p35 binding protein C53 as a novel regulator of cell cycle and DNA damage response. Our study demonstrated that C53 depletion leads to defective cell cycle and resistance to apoptosis induced by chemotherapeutic agents. Additionally, C53 overexpression abolished the G2/M checkpoint-mediated Cdk1 inactivation, thereby sensitizing cancer cells to various DNA damage agents. We further found that C53 depletion results in inactivation of Cdk1 and delayed mitotic entry, while C53 overexpression antagonizes Chk1-mediated Cdk1 inactivation. Importantly, we found that a fraction of endogenous C53 protein localizes at the centrosome, an organelle that is emerging as a pivotal multi- functional platform that integrates many important signal pathways. Based upon our preliminary study, we hypothesize that C53 plays a crucial role in unperturbed cell cycle progression. By antagonizing the activity of centrosome-associated Chk1, C53 acts as a positive promoter of initial activation of cyclin B1/Cdk1 kinase at the centrosome. To test our hypothesis, we aim: 1) To Investigate the centrosomal association of C53. In this aim, by using GFP-centrin stable cells and immunofluorescence and biochemical analyses, we will investigate the centrosomal association of C53 and its docking mechanism. Moreover, We will make a centrosome-targeted C53 to test whether C53 centrosomal localization is important for its function. 2) To characterize the role of C53 in initial activation of Cdk1/cyclin B1 during the G2/M transition. In this aim, we will use synchronized cells and immunofluorescence assays to examine the effect of C53 on initial activation of Cdk1/cyclin B1 at the centrosome. Furthermore, we will test whether C53 exerts its function through affecting the activity of centrosomal Chk1 and other centrosome-associated mitotic regulators. This study will provide insight on a novel regulatory mechanism for cell cycle control and checkpoint response. It will lay a foundation for exploitation of this protein as a novel therapeutic target in cancer treatment.