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Investigation of the role of UBE4B ubiquitin ligase in p53 regulation in response to DNA damage in ATM-proficient and ATM-deficient cells
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- Author / Creator
- Al Yousef, Habib Saleh M
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The tumour suppressor protein p53 maintains genomic integrity by coordinating the DNA damage response, which includes growth arrest, DNA damage repair, and apoptotic cell death. p53 is mutated in more than 50% of human cancer, which reflects its importance. The complex mechanism that controls the regulation of p53 remains only partially understood. p53 is mainly regulated by post-translational modifications such as phosphorylation and ubiquitination. Following exposure to DNA-damaging agents, protein kinases such as ATM and ATR activate and stabilize p53 via phosphorylation. In unstressed cells, ubiquitin ligases bind and assist to degrade p53, keeping p53 at the basal level to enable the cell’s normal function. The MDM2 ubiquitin ligase represents the most extensively studied negative regulator of p53. Previous studies with unstressed cells have shown that UBE4B, a p53 ubiqutin ligase, is essential for MDM2-mediated p53 polyubiquitination and degradation both in vitro and in vivo. However, the role of UBE4B in regulating p53 in response to DNA damage remains unknown. This dissertation hypothesized that ATM or ATR phosphorylates p53, thus affecting its level and disrupting UBE4B-p53 interactions in response to DNA damage. Also, UBE4B constitutes a critical p53 negative regulator in the ATM- or ATR-dependent pathway. To test this hypotheses, the main objectives of this study aimed to (i) determine the levels of UBE4B, MDM2, and p53 following DNA damage; (ii) explore the phosphorylation status of p53 following DNA damage; (iii) examine the interactions of UBE4B and MDM2 with p53; (iv) determine the effects of UBE4B and MDM2 on p53 degradation and (v) analyze the cytoplasmic as well as nuclear localization of p53, UBE4B, and MDM2. Most experiments involved human cells that express wild-type p53, with a few experiments including p53-null cells. Non-cancerous ATM-proficient and ATM-deficient EBV-transformed lymphoblastoid cell lines that grow in suspension were employed. In addition, some studies were performed with cancerous (MCF7 and H1299) and non-cancerous (MCF10) cell lines that grow as adherent cultures. Western blot analysis was performed to detect the protein levels and co-immunoprecipitation was utilized to explore the protein-protein interactions. Moreover, DNA extraction and transformation were used to prepare various constructs. Cellular fractionation analysis was utilized to investigate the subcellular localization of proteins and flow cytometric analysis was used to examine cell cycle distributions. The findings with lymphoblastoid cells suggested that induced UBE4B levels and interactions with p53 are ATM-independent. However, it is unknown whether ATR signalling or some other protein kinase p53 activator may constitute the predominant pathway operating in AT cells. Consistent with previous reports, the data in this study revealed that UBE4B binds to and promotes the degradation of phosphorylated forms of p53, such as Ser15 or Ser392, after exposure to ionizing radiation (IR); furthermore, this investigation found that this downregulation remains independent of MDM2. In conclusion, these research findings provide new insights into the potential role of UBE4B in p53 regulation via the ATM or ATR pathway following response to DNA damage. Collectively, the data revealed that the pattern of UBE4B induction or its interactions with p53 in ATM positive wild-type cells differs from that in AT cells, supporting the previous studies which reported that UBE4B may negatively regulate phosphorylated p53 in response to ionizing radiation. Further investigations are needed to explore the role of UBE4B in p53 regulation, especially within various contexts of the DNA damage response. Such studies will assist to better understand the ever-expanding complexity of p53 regulation, which eventually may contribute towards developing novel p53-based therapeutic cancer approaches.
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- Graduation date
- Fall 2017
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.