Understanding the impact of Fbw7 mutations on substrate function and cancer cell biology

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The FBXW7 tumor suppressor gene encodes a substrate-recognition subunit of the SCFFbw7 ubiqui-tin ligase and targets for degradation a number of oncogenic proteins, such as c-Myc, c-Jun, Notch, and cyclin E. Heterozygous missense mutations in the substrate-binding domain are the most com-mon genetic alteration in Fbw7 and are found in T-cell leukemias, ovarian, endometrial and gastroin-testinal cancers. These mutants are partially compromised in substrate degradation and are consid-ered to be “null” alleles, but their impact on substrate function and cell biology remains largely unclear. Strikingly, we find that although tumor-derived Fbw7 mutants do not efficiently direct protein degradation, they retain the ability to bind and ubiquitinate at least some substrates. Moreover, certain functions of Fbw7 substrates are impaired in Fbw7-null cells, arguing that Fbw7 promotes some of their activities. Based on preliminary data, we suggest that the impact of heterozygous Fbw7 mutations extends beyond compromising substrate turnover and can have additional non-proteolytic effects on substrate function. We propose to rigorously analyze how ubiquitination by the cancer-derived Fbw7 mutants affects the activity of multiple substrates, their downstream pathways and cancer cell physiology. We will directly test the impact of mutations on substrate recognition and the catalytic activity of Fbw7 using in vitro assays and analyze their impact on substrate function and cell biology using tissue culture and mouse xenograft models.