Defense Date

2006

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biochemistry

First Advisor

Dr. Swati Palit Deb

Abstract

MDM2 has been shown to induce G0-Gl/S phase arrest. To determine the cell cycle step targeted by MDM2, flow cytometry was employed to detect induction of events during the G1-S phase transition in MDM2-arrested cells. MDM2 overexpression does not prevent expression of cyclin D, cyclin D-CDK mediated phosphorylation of Rb or cyclin E in normal, immortal or tumor-derived cells. However, MDM2 down-regulates cyclin A expression specifically in normal cells, which is associated with G1 arrest. The domain of MDM2 capable of this function is located within its N-terminal 58-109 amino acids. To down-regulate cyclin A, MDM2 requires a functional pl6/Brg 1 pathway, as silencing of either of these proteins disables this function of MDM2. Bromodeoxyuridine incorporation studies suggest that another inhibitory domain, ID2, inhibits DNA replication, while an MDM2 deletion mutant containing the N-terminal 1-220 amino acids including inhibitory domain ID1 does not effectively prevent BrdU incorporation in an immortal cell line that is non-responsive to growth arrest by the cyclin A inhibitory domain. This suggests that induction of MDM2 leads to G1 arrest by at least two independent mechanisms, and multiple genetic damages are necessary to overcome MDM2-mediated growth arrest. To determine novel interacting partners of MDM2, proteomic analysis of MDM2 overexpressed in tumor-derived H1299 cells was carried out. This analysis revealed interaction of MDM2 with the translation elongation factor efl-α, and was validated by immunoprecipitation and Western blotting and shown to colocalize with MDM2 in the cytoplasm. To interact with efl-α, MDM2 was determined to require two domains, one of which is located within amino acids 221-325 and another within the N-terminal 58 amino acids of MDM2.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

June 2008

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