The genomic loci for tumor suppressor p53 and ARF are the most frequently mutated in all types of human cancer. On the other hand, the proto-oncoprotein MDM2, a principle negative regulator of p53 in the cell, is frequently overexpressed in many types of human cancer, ARF, MDM2, and p53 constitute an important tumor suppression pathway (ARF-MDM2-p53 pathway) that safeguards cells from aberrant, uncontrolled growth. Recent studies have indicated that the ARF-MDM2-p53 pathway is not strictly linear but branches out to other targets and that the pathway crosstalks with other cellular pathways, presumably through the interaction with other cellular proteins. The targets of the branched-out interaction and the mechanisms of the crosstalk regulation, however, are largely elusive. We have recently found that MDM2 interacts with the ribosomal protein L11 through its zinc finger region. L11 forms in vivo complexes with ARF, MDM2, and p53. Enforced expression of L11 prevents MDM2-mediated p53 ubiquitination and degradation, restores MDM2-suppressed p53 transactivation activity and induces a p53-dependent G1 cell cycle arrest. More importantly, studies have shown that human cancer-associated mutations in the MDM2 gene preferentially target the zinc finger domain disrupting L11 binding. One of the cancer-derived MDM2 mutants we have examined, MDM2 (C305F),which has a Cys-to-Phe substitution in the central zinc finger motif, exhibited several distinct characteristics including the disruption of L11 binding. MDM2(C305F) retained the E3 ligase activity to ubiquitinate p53 but did not promote p53 degradation. It showed a stronger ability than the wild type MDM2 in suppressing p53's transactivation activity and cells expressing MDM2(C305F) escaped from L11 overexpression-induced growth inhibition. MDM2(C305F) represents a useful mutant to dissect the mechanism of L11-regulated MDM2 and p53 function. Based on our preliminary data, we hypothesize that the L11-MDM2-p53 connection constitutes a pathway that monitors the rate of protein synthesis and coordinates cell growth with cell cycle progression. To understand the function and regulation of the LI 1-MDM2-p53 pathway, we propose the following aims:
Aim 1. To investigate the mechanism and regulation of the MDM2-L11 interaction.
Aim 2. To investigate the in vivo function of the MDM2-L11 interaction using a MDM2 (C305F) knockin.
Aim 3. To investigate the mechanism of ARF in the regulation of L11, MDM2, and p53.
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