SCF Ubiquitin Ligases in Cell Cycle Control and Chromosome Stability

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PROJECT SUMMARY ABSTRACT Dynamic oscillations in the abundance and activity of key proteins drives cell cycle progression. This is typified by cyclins and cyclin kinase inhibitors (CKIs), which oscillate during cell cycle progression and determine the activation kinetics of Cyclin Dependent Kinases, which propel the cell cycle forward. Defining the pathways, networks and mechanisms underlying dynamics in protein abundance during normal cell cycles is essential to understanding proliferative control. Ubiquitin is the major regulator of protein degradation in eukaryotes and plays an essential and highly conserved role in cell cycle progression. The Skp1-Cul1-F-box (SCF) family of E3 ubiquitin ligases are major regulators of cell cycle progression and sculpt the protein landscape post- translationally to facilitate proliferation. SCF ligases engage a set of ~70 interchangeable substrate receptors termed F-box proteins, which dictate SCF target selection. The eponymous Cyclin F is the founding member of the F-box family and has a cyclin homology domain like that found in the canonical CDK activators. Rather than activate a CDK to promote cell cycle via phosphorylation, cyclin F binds to the SCF, promoting cell cycle through ubiquitination. Cyclin F mRNA and protein levels oscillate significantly during the cell cycle and to a greater extent than other F-box encoding genes. Cyclin F is required for viability in mice and essential for growth/survival in many human cell lines. Further, it is overexpressed in cancer, and its mutation is linked to the neurodegenerative disease amyotrophic lateral sclerosis. Nevertheless, there remain significant knowledge gaps related to what pathways cyclin F controls and how it is regulated. Defining substrates and mechanisms of cyclin F is significant to a fundamental understanding of cell cycle, as well as human health. During the previous funding cycle, our lab identified several cyclin F substrates, highlighting its importance in cell cycle control. Here, we build on that work, defining a new substrate with important roles in normal cell cycle control and with relevance to disease, while also beginning to dissect the complex mechanisms by which cyclin F is controlled. Borne out of innovative, unbiased, computational, and proteomic strategies, our data suggest new, critical roles for cyclin F in cell cycle, and undescribed mechanisms underlying its regulation. In aim 1, we examine the role of cyclin F in G1/S progression through regulation of a tumor suppressor in the retinoblastoma (RB)-pocket protein family, RBL2/p130. We will determine the mechanisms by which cyclin F controls the RB-pathway and how this contributes to gene expression, cell cycle, and proliferative control. In aims 2 and 3, we turn our attention towards mechanisms that converge on cyclin F to regulate its abundance and potentially activity. Aim 2 is focused on the regulation of cyclin F by kinases and how they determine cyclin F stability. In aim 3 we extend this analysis of post-translational regulation of cyclin F, determining how it is regulated by deubiquitinases. Together, these studies will provide a comprehensive analysis for how cyclin F is itself regulated and its contributions to cell cycle in humans.