Author

Xiaoxin Sun

Date

October 2007

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Molecular and Medical Genetics

Institution

Oregon Health & Science University

Abstract

The MDM2-p53 feedback loop is tightly controlled in cells to prevent errant p53 activation and at the same time to enable rapid stress responses. Different stress signals regulate this feedback loop through different mechanisms. This dissertation seeks to investigate the regulation of the MDM2-p53 feedback loop by a novel nucleolar protein nucleostemin, (NS), and three ribosomal proteins L5, L11, and L23. NS is essential for cell proliferation. Both downregulation and overexpression of the NS protein hinders cell proliferation, suggesting a physiological range of NS levels is important to maintain cellular homeostasis. However, the precise mechanism underlying this regulation is not yet known. Here I show that NS binds to the central acidic domain of MDM2 in the nucleoplasm and inhibits MDM2 E3 ubiquitin ligase activity, thus leading to the stabilization and activation of p53. Increases in NS levels in U2OS cells leads to higher expression of p21cip1/waf1 and G1 cell cycle arrest. MPA-mediated GTP depletion induces the translocation of NS from the nucleolus to the nucleoplasm and enhances its interaction with MDM2 in early time points. This interaction may contribute to the early stage activation of p53 upon MPA treatment. Downregulation of NS by either siRNA-mediated depletion or mycophenolic acid (MPA) also activates p53 and induces G1 arrest. These effects require ribosomal proteins L5 and L11 as depletion of NS enhanced their interaction with MDM2. Knockdown of either gene abrogates both the NS-depletion-induced and MPA-induced p53 activation and cell cycle arrest. Thus, my study uncovers the mechanisms by which aberrant NS levels activate p53 and induce cell cycle arrest. This occurs through modulation of the MDM2-p53 pathway by NS itself and certain ribosomal proteins. Previous studies have shown that three ribosomal proteins L5, L11 and L23 are important regulators of the MDM2-p53 feedback loop in response to nucleolar stress. Here we further investigate their role in response to nucleolar stress induced by MPA and 5-FU. I show that MPA treatment leads to nucleolar disruption and markedly increases the binding of L5 and L11 to MDM2 at later time points. Knockdown of either L11 or L5 by siRNA significantly decreased the MPA-induced stabilization and activation of p53. Similar to the cellular response to the MPA treatment, treatment with 5-FU increases the fraction of ribosome-free L5, L11, and L23 ribosomal proteins and their interaction with MDM2, leading to p53 activation and G1/S arrest. Conversely, individual knockdown of the expression of these ribosomal proteins by siRNA prevents the 5-FU-induced p53 activation and reverses the 5-FU-induced G1/S arrest. Taken together, these results suggest an important role for the nucleolus as a cellular stress sensor. In response to nucleolar stress induced by MPA, 5-FU or knockdown of endogenous nucleolar protein NS, nucleolar proteins, including NS and certain ribosomal proteins are released from the nucleolus to the nucleoplasm, where they associate with MDM2 to inhibit its activity and stabilize p53.

Identifier

doi:10.6083/M4639MQ0

School

School of Medicine

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