Normal and Neoplastic Regulation of Cyclin E

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DESCRIPTION (provided by applicant): The cyclin E protein controls the transition from the G1 to the S-phase of the cell cycle. Deregulated cyclin E activity causes abnormal cell division, and mutations leading to aberrant cyclin E-cdk2 regulation are found in most human cancers. The research described in this proposal seeks to understand the mechanisms that govern cyclin E in normal cells and tumors. We are particularly interested in the regulation and function of cyclin E phosphorylation in cell cycle control and tumorigenesis. Multiple site-specific phosphorylations control cyclin E, but the role of specific mitogenic and signal transduction pathways in regulating these phosphorylations is unknown. The goal of the first aim is to characterize the regulation of cyclin E phosphorylation in normal cells and in tumor cells. We will develop antibodies that recognize phosphorylated forms of cyclin E, and these reagents will be used to examine cyclin E phosphorylation in normal cells, and to determine if it is abnormal in tumor cells. We are particularly interested in the relationships between phosphorylated cyclin E and the Fbw7 ubiquitin ligase. Because most studies on cyclin E phosphorylation have utilized overexpressed cyclin E, it has been difficult to distinguish the role of phosphorylation from the consequences of overexpression. We will thus use "knock-in" models in which phosphorylation sites are mutated in the context of the endogenous cyclin E locus to study the function of specific phosphorylations. These studies will use homologous recombination techniques in mice, as well as develop new methods based on adeno-associated virus vectors in human cells. The latter method may be broadly applicable to studies of protein phosphorylation in systems where mouse models are unnecessary or undesirable. The mechanisms of cyclin E-associated tumorigenesis are largely unknown. The overall goal of this aim is to use cyclin E transgenic and knock-in strains to develop models of cyclin E-associated cancer. Because we have discovered a homeostatic p53-dependent response that restrains excess cyclin E activity, we will specifically test the hypothesis that loss of p53 function is an integral step in the development of tumors with deregulated cyclin E expression. These models will be used to study the mechanisms of cyclin E-associated tumorigenesis and may facilitate the development of new therapeutic strategies.