The Fbw7 ubiquitin ligase network: normal and neoplastic functions

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This application focuses on Fbw7, an E3 ubiquitin ligase and tumor suppressor, and on its substrate c-Myc (hereafter, Myc), an oncogenic transcription factor widely implicated in human cancers. E3 ligases mark protein substrates for degradation through ubiquitin conjugation. Fbw7 recognizes a network of proteins with crucial roles in proliferation, differentiation, metabolism, and apoptosis. Fbw7 substrates include important oncoproteins (e.g., cyclin E, Notch, Myc, Jun) and thus Fbw7 mutations promote tumorigenesis by deregulating its oncogenic substrates. The Fbw7 pathway therefore has broad implications for cancer biology and for the development of new therapeutic strategies. We will address important and unresolved aspects of this pathway in two general areas. The first involves how phosphorylation and Fbw7 control Myc stability and activity, in both normal cells and in cancers. The second area will address new paradigms in the ways that Fbw7 recognizes substrates and how these interactions may underlie Fbw7 mutations in cancers. This proposal may thus impact many areas of research related to Myc and the Fbw7 pathway. Fbw7 binds substrates after they are phosphorylated within motifs termed degrons, that typically contain two phosphorylated residues that interact with Fbw7. The first two Aims are focused on Myc regulation by phosphorylation of a newly discovered Myc T244 degron that acts in concert with the canonical Myc T58 degron to bind Fbw7 dimers. Aim 1 will study how T244 degron phosphorylation is regulated in normal and tumor cells and whether hierarchical Myc T244 degron phosphorylation controls Myc stability, as well as how the T58 and T244 degrons are coordinately regulated by mitogenic and oncogenic signaling pathways. Aim 2 will study the functions of the T244 degron in normal cells and tumorigenesis and how it cooperates with the T58 degron. This will be accomplished through physiologic knockin models, in human cells and mice, to create engineered Myc mutations that ablate Myc degron phosphorylations. Aim 3 will study how Fbw7 dimers interact with substrates and how these interactions underlie Fbw7 mutations and their functions in cancers. This includes determining the extent to which two separate degrons are required for degradation across the Fbw7 substrate network. Identifying these new degrons may lead to entirely new pathways that control the degradation of critical substrates and that may be abnormal in cancer cells. Tumors often have heterozygous Fbw7 missense mutations that dimerize with wt-Fbw7, and the hypothesis that these mutations specifically stabilize oncogenic substrates that require two degrons will be tested. Finally, the Myc T244 degron binds Fbw7 through a novel mode that involves Fbw7 R689, a tumor hotspot, and the role of R689 in dimer-dependent selective substrate recognition will be studied, as well as similar possible functions for other Fbw7 missense mutations.