Regulation of Substrate Degradation and Cancer Metabolism by the Fbw7 Ubiquitin Ligase

Thesis (Ph.D.)--University of Washington, 2017-03Fbw7 is the substrate binding subunit of an SCF E3 ubiquitin ligase, which catalyzes the polyubiquitylation of substrates, initiating their recognition and degradation by the proteasome. The affinity of Fbw7 for its substrates is substantially increased following substrate phosphorylation at specific amino acids within short, linear sequences termed Cdc4 phosphodegrons (CPDs). Thus, regulation of substrate phosphorylation is a significant determinant of the contexts in which Fbw7 substrates will be degraded. Fbw7 is also a bona-fide tumor suppressor that is frequently mutated in human cancers; accordingly, many of Fbw7’s substrates are potent oncoproteins. It is through the deregulation of substrate degradation that Fbw7 mutations exert their oncogenic effects. For my dissertation, I studied two aspects of Fbw7 biology: the regulation of substrate CPD phosphorylation and the biologic consequences of Fbw7 inactivation in colorectal cancers. Cyclin E-CDK2 is an important regulator of the G1/S transition in the cell cycle, and cyclin E is also an Fbw7 substrate. Phosphorylation of cyclin E at serine 384 (S384) is the critical switch that enables high-affinity binding of Fbw7. Interestingly, S384 can only be autophosphorylated by the bound CDK2 molecule in cis; thus, cyclin E instigates its own degradation. We found that the PP2A-B56 phosphatase specifically opposes autophosphorylation of cyclin E at S384, and this dephosphorylation stabilizes catalytically active cyclin E-CDK2 complexes at the G1/S transition. siRNA-mediated depletion of PP2A-B56 is sufficient to decrease cyclin E kinase activity and shorten its half-life, which is consistent with the increased degradation of the catalytically active pool of cyclin E. Moreover, the phosphatase activity towards S384 is high in interphase but low in prometaphase. This likely provides a failsafe mechanism to ensure the complete degradation of cyclin E in mitosis, which is necessary for error-free chromosome segregation. Therefore, the dephosphorylation of cyclin E specifically at S384 is a critical regulator of its degradation by Fbw7 during the cell cycle. A comprehensive understanding of the changes in cellular function that result from Fbw7 mutations in tumors is an ongoing area of investigation. This is due in part to Fbw7’s large network of substrates that together regulate many biological functions. To better understand the consequences of Fbw7 mutations in human colorectal cancers, we applied novel computational approaches to analyze colorectal cancer gene expression datasets. These analyses identified altered cellular metabolism and mitochondrial function as important and conserved consequences of Fbw7 mutations. This computational prediction was validated using targeted gene expression assays and functional studies of cellular metabolism, and also revealed deregulated nucleotide biosynthesis in Fbw7-mutant cells. Accordingly, Fbw7-/- cells were hypersensitive to both genetic and small-molecule inhibition of DHODH, an enzyme in the pyrimidine biosynthetic pathway. These experiments suggest that the metabolic deregulation present in Fbw7-mutant cancers could lead to specific vulnerabilities that can be therapeutically targeted. Together, these studies have provided new and important insights into Fbw7 biology. Fbw7 has critical functions in normal cells, including organismal development, differentiation, and stem cell maintenance; conversely, inactivating mutations in Fbw7 are frequently selected for in neoplastic cells. A complete understanding of the Fbw7 pathway—from the regulation of its molecular interactions to the changes in cellular processes observed in cancers—will be critical to fully understand the importance of Fbw7 in cellular physiology, with the ultimate goal of developing new approaches to treating human disease