This project will explore the regulatory system that governs progression through the stages of the eukaryotic cell division cycle, with an emphasis on the control of chromosome segregation in mitosis. Cell-cycle progression is governed by an important class of protein kinases called the cyclin-dependent kinases or Cdks, whose activity depends on association with cyclin regulatory subunits. In the proposed studies, biochemical and molecular genetic approaches will be used in the budding yeast Saccharomyces cerevisiae to address the mechanisms by which Cdks control progression through the cell cycle in general and through mitosis in particular. As in the previous funding period, much of the proposed work will focus on the identification and characterization of the protein substrates that are phosphorylated by Cdks in the cell. In the first aim, innovative mass-spectrometry-based approaches will be used to identify new Cdk targets, after which selected targets will be analyzed in detail to assess their function in the cell cycle and the mechanisms by which Cdks influence that function. The work proposed in the second aim is directed toward the study of how changes in the phosphorylation state of Cdk substrates help govern the separation and segregation of the duplicated chromosomes in anaphase. Preliminary studies suggest that the phosphoregulation of one Cdk substrate, securin, enhances the switchlike properties of the metaphase-to-anaphase transition, and the proposed experiments will address this possibility through the development of novel methods to analyze this transition. Other experiments will pursue the possibility that the efficient segregation of repetitive DNA regions in anaphase depends on the dephosphorylation of specific Cdk substrates. Finally, the experiments in the third aim will be directed toward the identification and characterization of substrates for a phosphatase, Cdc14, that is known to dephosphorylate many Cdk substrates in late mitosis. The information gained from these studies will provide important new insights into the control of cell-cycle progression and thereby enhance our understanding of diseases, such as cancer, in which cell-cycle control is defective.
When a cell reproduces, the chromosomes are first duplicated and then segregated into a pair of daughter cells. Errors in these processes can result in uncontrolled cell proliferation, genetic damage or defects in chromosome number, any of which can accelerate cancer progression or cause developmental defects. The proposed studies focus on enzymes called the cyclin-dependent kinases, which are key regulators of cell division in all eukaryotes. These studies will lead to a better understanding of how errors in cell division and chromosome segregation can arise in human disease.
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|Lopez, Michael S; Choy, Jonathan W; Peters, Ulf et al. (2013) Staurosporine-derived inhibitors broaden the scope of analog-sensitive kinase technology. J Am Chem Soc 135:18153-9|
|Lyons, Nicholas A; Fonslow, Bryan R; Diedrich, Jolene K et al. (2013) Sequential primed kinases create a damage-responsive phosphodegron on Eco1. Nat Struct Mol Biol 20:194-201|
|Kõivomägi, Mardo; Valk, Ervin; Venta, Rainis et al. (2011) Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase. Nature 480:128-31|
|Lyons, Nicholas A; Morgan, David O (2011) Cdk1-dependent destruction of Eco1 prevents cohesion establishment after S phase. Mol Cell 42:378-89|
|Kõivomägi, Mardo; Valk, Ervin; Venta, Rainis et al. (2011) Dynamics of Cdk1 substrate specificity during the cell cycle. Mol Cell 42:610-23|
|Holt, Liam J; Tuch, Brian B; Villén, Judit et al. (2009) Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution. Science 325:1682-6|
|Holt, Liam J; Krutchinsky, Andrew N; Morgan, David O (2008) Positive feedback sharpens the anaphase switch. Nature 454:353-7|
|Blethrow, Justin D; Glavy, Joseph S; Morgan, David O et al. (2008) Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates. Proc Natl Acad Sci U S A 105:1442-7|
|Goga, Andrei; Yang, Dun; Tward, Aaron D et al. (2007) Inhibition of CDK1 as a potential therapy for tumors over-expressing MYC. Nat Med 13:820-7|
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