Biomedical Sciences ETDs

Publication Date

12-1-2010

Abstract

Cyclin-Cdk complexes positively regulate cell cycle progression. Cyclins are regulatory subunits that bind to and activate cyclin-dependent kinases or Cdks. Cyclin E associates with Cdk2 to mediate G1/S phase transition of the cell cycle. Cyclin E is overexpressed in breast, lung, skin, gastrointestinal, cervical, and ovarian cancers. Its overexpression correlates with poor patient prognosis and is involved in the etiology of breast cancer. We have been studying how this protein is downregulated during development in order to determine if these mechanisms are disrupted during tumorigenesis, leading to its overexpression. Using Xenopus laevis embryos as a model, we have shown previously that during the first 12 embryonic cell cycles Cyclin E levels remain constant yet Cdk2 activity oscillates twice per cell cycle. Cyclin E is abruptly destabilized by an undefined mechanism after the 12th cell cycle, which corresponds to the midblastula transition (MBT). Based on work our work and work by others, we have hypothesized that differential phosphorylation and a change in localization result in Cyclin E degradation by the 26S proteasome at the MBT. To test this, we generated a series of point mutations in conserved threonine/serine residues implicated in degradation of human Cyclin E. Using Western blot analysis, we show that similarly to human Cyclin E, mutation of these residues to unphosphorylatable alanine stabilizes Cyclin E past the MBT when they are expressed in vivo. Cyclin E localization was studied by immunofluorescence analysis of endogenous and exogenous protein in pre-MBT, MBT, and post-MBT embryos. In addition, we developed a novel method of conjugating recombinant His6-tagged Cyclin E to fluorescent (CdSe)ZnS nanoparticles (quantum dots) capped with dihydrolipoic acid. Confocal microscopy was used to visualize His6Cyclin E-quantum dot complexes inside embryo cells in real time. We found that re-localization at the MBT from the cytoplasm to the nucleus precedes Cyclin E degradation. Mutations in phosphorylation sites did not abrogate nuclear accumulation of Cyclin E at the MBT. Inhibition of nuclear import and proteasome function resulted in accumulation of Cyclin E in the cytoplasm. We also show that Cyclin E is ubiquitinated at the MBT corresponding to its downregulation. These results suggest conservation of mechanism of degradation between human and Xenopus Cyclin E. In addition, our results show that the nuclear presence of Cyclin E is necessary for its downregulation at the MBT. Taken together with the fact that Cyclin E is often mislocalized to the cytoplasm in human cancers that overexpress it, our work suggests that a similar nuclear presence may be necessary for human Cyclin E degradation, and that this process could be disrupted during the process of tumorigenesis.

Keywords

Xenopus laevis, development, Cyclin E, degradation, ubiquitination, 26S proteasome

Sponsors

National Institutes of Health National Science Foundation

Document Type

Dissertation

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Doctoral

Department Name

Biomedical Sciences Graduate Program

First Committee Member (Chair)

Perrone-Bizzozero, Nora

Second Committee Member

Bear, David

Third Committee Member

Osinski, Marek

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