Cyclin E, in conjunction with its catalytic partner CDK2, regulates diverse aspects of cell division. Normal cells tightly regulate cyclin E, whereas cancer cells often exhibit abnormal cyclin E activity and this directly contributes to genetic instability and tumorigenesis. The research proposed in this application uses a combined approach of biochemistry, animal models, human gene targeting, and proteomics, to understand novel aspects of cyclin E regulation and function. One critical mechanism of cyclin E regulation that is often disrupted in cancer cells is its degradation by the Fbw7 ubiquitin ligase. The interactions of cyclin E and Fbw7 are complex and regulated by two cyclin E phosphodegrons that bind to Fbw7, and the relationships between cyclin E and Fbw7 will be studied in Aim 1. The first subaim will determine the physiologic significance of the N-terminal degron through the development of a mouse knockin strain in which this degron is mutated. In the second subaim we will test the hypothesis that variations in CDK2 specific activity regulate cyclin E accessibility to Fbw7 during the cell cycle, and that this involves both CDK2 and cyclin E phosphorylation. Finally, in the third subaim we will use purified components to test the hypothesis that both cyclin E degrons can simultaneously engage an Fbw7 dimer, and determine the feasibility of a structural analysis of cyclin E bound to an Fbw7 dimer. Robust mouse models are needed for mechanistic and therapeutic studies of cyclin E- associated cancer, and these will be developed in Aim 2. In the first subaim, we will combine cyclin E degron mutations with the disruption of two tumor suppressor genes that normally restrain cyclin E: p53 and p27. The goal of the second subaim is to identify genes that cooperate with cyclin E during tumorigenesis and the pathways that suppress cyclin E-driven hyperproliferation in vivo. The approach that we will take is to use a genetic screen employing the "Sleeping Beauty" transposon system to identify cooperating genes and pathways in mice bearing cyclin E degron mutations. Approximately a dozen cyclin E-CDK2 substrates are known, and these have wide ranging cell cycle functions. Studies in yeast have revealed more than 200 CDK substrates and it is likely that many cyclin E-CDK2 substrates are unknown. We have developed a kinase engineering/mass spectrometry-based approach that efficiently identifies candidate CDK2 substrates. The goal of this aim is to utilize these methods to identify CDK2 substrates, and then to use biochemical and gene targeting methods to study the functions of a subgroup of validated substrates. These latter studies are critical, because they will determine the physiologic significance of endogenous substrates.

Public Health Relevance

The research in this proposal focuses on a protein, called cyclin E, that plays a central role in cell division and cancer. The goals of this research are to understand the functions and regulation of cyclin E in normal cells, and why loss of these normal controls causes cancer. This research may increase our understanding of why cancer develops and lead to new cancer treatment strategies.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA102742-09
Application #
8204660
Study Section
Special Emphasis Panel (ZRG1-CB-N (02))
Program Officer
Hildesheim, Jeffrey
Project Start
2003-07-01
Project End
2013-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
9
Fiscal Year
2012
Total Cost
$370,973
Indirect Cost
$160,193
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
078200995
City
Seattle
State
WA
Country
United States
Zip Code
98109
Davis, Ryan J; Welcker, Markus; Clurman, Bruce E (2014) Tumor suppression by the Fbw7 ubiquitin ligase: mechanisms and opportunities. Cancer Cell 26:455-64
Hughes, Bridget T; Sidorova, Julia; Swanger, Jherek et al. (2013) Essential role for Cdk2 inhibitory phosphorylation during replication stress revealed by a human Cdk2 knockin mutation. Proc Natl Acad Sci U S A 110:8954-9
Davis, Michael A; Larimore, Elizabeth A; Fissel, Brian M et al. (2013) The SCF-Fbw7 ubiquitin ligase degrades MED13 and MED13L and regulates CDK8 module association with Mediator. Genes Dev 27:151-6
Hizli, Asli A; Chi, Yong; Swanger, Jherek et al. (2013) Phosphorylation of eukaryotic elongation factor 2 (eEF2) by cyclin A-cyclin-dependent kinase 2 regulates its inhibition by eEF2 kinase. Mol Cell Biol 33:596-604
Grim, Jonathan E; Knoblaugh, Sue E; Guthrie, Katherine A et al. (2012) Fbw7 and p53 cooperatively suppress advanced and chromosomally unstable intestinal cancer. Mol Cell Biol 32:2160-7
Sengupta, Tanushri; Abraham, Gathi; Xu, Yanfei et al. (2011) Hypoxia-inducible factor 1 is activated by dysregulated cyclin E during mammary epithelial morphogenesis. Mol Cell Biol 31:3885-95
Minella, Alex C; Loeb, Keith R; Knecht, Andrea et al. (2008) Cyclin E phosphorylation regulates cell proliferation in hematopoietic and epithelial lineages in vivo. Genes Dev 22:1677-89
Minella, A C; Grim, J E; Welcker, M et al. (2007) p53 and SCFFbw7 cooperatively restrain cyclin E-associated genome instability. Oncogene 26:6948-53
O'Neil, Jennifer; Grim, Jonathan; Strack, Peter et al. (2007) FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to gamma-secretase inhibitors. J Exp Med 204:1813-24
Geng, Yan; Lee, Young-Mi; Welcker, Markus et al. (2007) Kinase-independent function of cyclin E. Mol Cell 25:127-39

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