Sedgwick, Garry Gray (2010)
Ph.D. thesis, University of Birmingham.
The anaphase promoting complex /cyclosome (APC/C) is a multi-protein E3 ubiquitin ligase complex that regulates cellular proliferation through its ability to target essential cell cycle regulators such as cyclin A, cyclin B, securin and S-phase kinase-associated protein 2 (SKP2) for proteasomal-dependent degradation. APC/C substrates and coactivators are aberrantly expressed in many cancers. It is thought that the APC/C can also regulate cellular proliferation by controlling p21 and cell division cycle 6 homologue (CDC6) transcription. It is therefore of great interest to study other cellular proteins that interact with the APC/C as these proteins may also be important in cell cycle regulation and hence may enhance our understanding of the molecular basis of cancer. Therefore, the aim of my study was to identify novel APC/C interacting proteins through mass spectrometric analysis of APC/C subunit 7 (APC7) immunoprecipitates and go on to examine the functional significance of these interactions. I identified six novel APC7 interacting proteins, and decided to focus on two of these interactions. Initially I examined the interaction between APC7 and transcriptional intermediary factor 1\(\gamma\) (TIF1\(\gamma\)), a transcriptional repressor previously reported to be aberrantly expressed in cancer. Data presented in this study demonstrate that the APC/C and TIF1\(\gamma\) cooperate to regulate mitotic progression. Notably TIF1\(\gamma\) displays in vivo interactions with APC/C subunits 1-8, the APC/C‟s mitotic coactivator CDC20 and mitotic substrate cyclin A. Moreover, TIF1\(\gamma\) depletion by RNAi arrests cells in a metaphase-like state characterised by elevated levels of APC/C substrates, cyclin A, cyclin B and CDC20. In support of a role for TIF1\(\gamma\) in regulating mitotic progression by directly targeting the APC/C, cells treated with TIF1\(\gamma\)-specific siRNA exhibit reduced APC/C E3 ubiquitin ligase activity. This work defines TIF1\(\gamma\) as a novel mitotic regulatory protein essential for APC/C function and suggests that loss of TIF1\(\gamma\) may compromise genomic integrity by allowing the mis-segregation of chromosomes. I also investigated the functional relationship between APC7 and the nuclear factor 90/45 heterodimer (NF90/NF45), given that all of these proteins function as transcription factors. Results obtained demonstrate that APC5 and APC7 bind directly to NF90 in vitro and also form complexes with NF90 in vivo. It appears that APC/C interaction with NF90 is important in the regulation of interleukin-2 (IL-2) and tumour neucrosis factor (TNF) transcription, as exogenous APC5 and APC7 cooperate with NF90/NF45 to transactivate IL-2 and TNF promoter constructs. Lastly endogenous APC5 and APC7 bind to the IL-2 promoter in vivo while endogenous APC5 represses TNF transcription in vivo. Given that TNF and IL-2 stimulate cellular proliferation data presented here suggests that the APC/C might control cellular proliferation by regulating TNF and IL-2 transcription.
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