Doctor of Philosophy
Dr. Fred Dick
The retinoblastoma tumor suppressor protein (pRB) functions to maintain proliferative control and act as a barrier to tumorigenesis. pRB is capable of regulating E2F transcription factors to mediate control of proliferation through transcriptional regulation of S-phase target gene expression. In addition, pRB can stabilize the CDK inhibitor p27 through an interaction with two ubiquitin ligase complexes. Further, pRB is capable of forming a unique interaction with E2F1 termed the ‘specific’ interaction that is capable of blocking E2F1 induced apoptosis. These functions of pRB are mediated by distinct binding interfaces and their contributions to the overall functionality of pRB are not well defined. In this thesis multiple experimental approaches are employed to study the function of the distinct binding sites in isolation to better define their functional roles. As described in chapter 2 the E2F1 ‘specific site’ is capable of maintaining and interaction with hyperphosphorylated pRB while the E2F ‘general site’ is disrupted by phosphorylation. This suggests that pRB can function beyond the G1 phase of the cell cycle to regulate E2F1 through the ‘specific site’. Using a series of novel synthetic mutations of pRB we found that multiple binding sites contribute in a redundant manner to the overall cell cycle arrest ability of pRB. While, the ‘general site’ appears to play a critical role in the regulation of cell cycle arrest through the regulation of E2F transcription factors, the LXCXE binding cleft and the ‘specific site’ can function redundantly to control proliferation. A gene-targeted mouse model was developed that disrupted the ‘general site’ while leaving other binding sites on pRB intact. Strikingly, these mice are unable to regulate E2F target gene expression yet they maintain appropriate proliferative control in multiple cellular contexts. The maintained proliferative control by pRB appears to be largely due to the activity of p27 as disruption of E2F regulation and p27 deficiency results in loss of proliferative control and subsequent tumorigenesis. Taken together, this work defines the contribution of the distinct binding sites to the overall functionality of pRB and provides insight into the disruption of pRB in human cancer.
Cecchini, Matthew J., "Dissecting the molecular role of distinct binding interfaces on the retinoblastoma tumor suppressor in growth control and tumorigenesis." (2011). Electronic Thesis and Dissertation Repository. 170.