Identification and characterisation of GSK3b-phosphorylation dependent interactions of tumour suppressor gene Fbxw7


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Ranjithmeon Muraleedharan1, Bradley Spencer-Dene2, Abdolrahman S. Nateri1
1Cancer Genetics and Stem Cell Group, School of Clinical Sciences, University of Nottingham, Nottingham, UK, 2Experimental Pathology Laboratory, CR-UK London Research Institute, London, UK


The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated that Fbxw7 (F-box and WD repeat domain containing-7), the substrate recognition component of an SCF (complex of SKP1, CUL1 and F-box protein)-type E3 ubiquitin ligase, is a key regulator of cell differentiation and transformation (1, 2). However, the mechanisms underlying the decision of a stem cell to either differentiate or transform are incompletely understood.


We have recently established the yeast Ras Recruitment System (RRS) (3), to determine GSK3b-phosphorylation-dependent interactions of FBXW7:substrate(s) using human cDNA library. Among of several other Novel Fbxw7 Interacting Proteins (NFIPs), we focused on a transcriptional regulator that has a key role in the maintenance of stem cells, and we called NFIP9. The biochemical interaction of NFIP9 with Fbxw7 is analysed by immunoprecipitation and ubiquitination assays. Confocal microscopy analysis examined cellular localisation of the NFIP9. We determined the biological relevance of NFIP9 in human epithelial cancer cells with a highly proliferating capacity i.e. SW620, HT29 and HCT116. To explore the clinical significance of NFIP9 expression in colorectal cancer, we also studied primary and invasive cases from patients using a human colorectal cancer (CRC) tissue array.


Our data suggest that the NFIP9 as a novel target of Fbxw7. We show that SCFFbxw7 targets ubiquitinated phospho-NFIP9 for degradation in a GSK3-b-dependent manner. Heterogeneous NFIP9 expression pattern in CRCs and its correlation with clinicopathological variables shows statistically associated with more primary cases.


The molecular interacting network that maintains the biological properties of Fbxw7-targeted substrate degradation may open up potential treatment options for cancer therapy.