Functional Analysis of the F-box protein Fbxl17
University of Cambridge
Doctor of Philosophy (PhD)
MetadataShow full item record
Mason, B. J. (2020). Functional Analysis of the F-box protein Fbxl17 (Doctoral thesis). https://doi.org/10.17863/CAM.51955
Advances in DNA sequencing technology have allowed detailed characterisation of cancer genomes and has highlighted the contribution of somatic structural variations to the mutational landscape of epithelial tumours. However, our understanding of the functional consequences of such genome rearrangements remains rudimentary. By surveying the METABRIC dataset, consisting of segmented array-CGH copy number data, and paired-end whole-genome DNA and RNA sequencing data from primary breast tumours, we found that the F-box protein encoded by FBXL17 is frequently rearranged in breast cancer. F-box proteins are the substrate-recognition components of Skp1-cullin 1-F-box protein (SCF) E3 ligases. As essential components of the ubiquitin proteasome system (UPS) they are responsible for directing target proteins for ubiquitination. Fbxl17 is a relatively understudied member of the FBXL family of F-box proteins and, in breast cancers, is disrupted in the region of the gene that encodes its substrate-binding leucine rich repeat (LRR) domain. Truncating Fbxl17 LRRs impaired its association with the other SCF holoenzyme subunits Skp1, Cul1 and Rbx1, and decreased its ubiquitination activity. Loss of the LRRs also affected Fbxl17 binding to its targets. Thus, genomic rearrangements in FBXL17 are likely to disrupt SCFFbxl17-regulated networks in cancer cells. To investigate the functional effect of these rearrangements, we performed a yeast two-hybrid screen to identify Fbxl17-interacting proteins. Among the 37 binding partners Uap1, an enzyme involved in O-GlcNAcylation of proteins was identified most frequently. We demonstrate that Fbxl17 binds to UAP1 directly and inhibits its phosphorylation, which we propose regulates UAP1 activity. Knockdown of Fbxl17 expression elevated O-GlcNAcylation in breast cancer cells, arguing for a functional role for Fbxl17 in this metabolic pathway. To identify further interacting partners of Fbxl17, we performed a mass spectrometry analysis of purified Fbxl17 SCF E3 ubiquitin ligases. Co-immunoprecipitates were enriched for DNA damage/ DNA repair proteins suggesting a novel role for Fbxl17 in the DNA damage response (DDR). We have demonstrated that Fbxl17 is recruited to DNA damage sites rapidly upon double-stand break (DSB) induction and knockdown of Fbxl17 protein expression sensitises cells to the DNA damaging agent Camptothecin. Furthermore, Fbxl17 can ubiquitinate the tandem BRCT domain of the well-known DDR protein 53BP1, which we propose targets 53BP1 for proteasomal degradation. In conclusion, we have identified two regulatory networks of Fbxl17 which provide an insight into the role of Fbxl17 in breast cancer pathogenesis. These pathways may be amenable to therapeutic targeting in the future for the treatment of breast cancers rearranged in FBXL17.
Breast cancer, FBXL17, Genome rearrangements, O-GlcNAcylation, Uap1, DNA damage, 53BP1, Ubiquitin, E3 ligase
Breast Cancer Now Funded (2013NovPhD172)
Embargo Lift Date
This record's DOI: https://doi.org/10.17863/CAM.51955
All rights reserved, Figure 1.7 permission to reprint obtained from Springer Nature, license number: 4818320962719 Figures 1.4,1.5 and 1.10 from open access papers and so can be used so long as cited.