Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Xiaolu Yang

Abstract

p53 is a tumor suppressor that is widely mutated or deleted in cancer cells. Mdm2, an E3 ubiquitin ligase, is the master regulator of p53. It targets p53 for proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. There are complex regulatory mechanisms balancing the activity and stability of Mdm2 in a cell. Mdm2 has an extremely short half-life in the unstressed cell and its regulation is not well understood. Like most E3 ligases, Mdm2 can autoubiquitinate. Previously, the sole function of autoubiquitination was thought to be to signal Mdm2 degradation. Here I show that autoubiquitination of Mdm2 is an activating event. Mdm2 that has been conjugated with polyubiquitin chains exhibits substantially enhanced activity to polyubiquitinate p53. Mechanistically, autoubiquitination of Mdm2 facilitates the recruitment of the E2 ubiquitin-conjugating enzymes through non-covalent interactions between the ubiquitin chains on Mdm2 and the ubiquitin-binding domain on E2s. These results suggest a model in which polyubiquitin chains on an E3 increase the local concentration of E2 enzymes and permit the processivity of substrate ubiquitination. These results support the notion that autocatalysis may be a prevalent mode for turning on the activity of latent enzymes.

Mdm2 is a short-lived protein because it is ubiquitinated and targeted for proteasomal degradation. In the unstressed cell, a complex containing the adaptor protein Daxx and HAUSP help stabilize Mdm2 protein. Through a collaborative effort, we discover BRG1, an ATPase component of the human SWI/SNF chromatin remodeling complexes, as a novel component of the Mdm2-Daxx-HAUSP complex. We find that BRG1 interacts with and enhances the assembly of this complex. Interestingly, I find that BRG1 is essential for maintaining Mdm2 levels independently of its ATPase activity. Moreover, BRG1 controls cell proliferation, senescence, and transformation through the stabilization of Mdm2. These results define BRG1 as an essential component of the Mdm2-Daxx-HAUSP complex and reveal an activity of BRG1, beyond chromatin remodeling, that is required for cell survival. Altogether, these results provide novel insights into the intricate mechanisms regulating the activity and stability of the oncoprotein Mdm2, enabling its negative control of the tumor suppressor p53.

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