Regulation of MDM2 by the ribosomal protein L11

描述（由申请人提供）：肿瘤抑制基因p53和ARF的基因组位点在所有类型的人类癌症中是最常突变的。另一方面，原癌蛋白MDM 2是细胞中p53的主要负调节因子，在许多类型的人类癌症中经常过表达，ARF、MDM 2和p53构成了重要的肿瘤抑制途径（ARF-MDM 2-p53途径），其保护细胞免于异常、不受控制的生长。最近的研究表明，ARF-MDM 2-p53通路不是严格的线性，而是分支到其他靶点，并且该通路可能通过与其他细胞蛋白质的相互作用与其他细胞通路交叉。然而，分支相互作用的目标和串扰调节的机制在很大程度上是难以捉摸的。我们最近发现MDM 2通过其锌指区与核糖体蛋白L11相互作用。L11在体内与ARF、MDM 2和p53形成复合物。L11的增强表达阻止MDM 2介导的p53泛素化和降解，恢复MDM 2抑制的p53反式激活活性，并诱导p53依赖性G1细胞周期停滞。更重要的是，研究表明，MDM 2基因中的人类癌症相关突变优先靶向破坏L11结合的锌指结构域。我们研究的癌症衍生的MDM 2突变体之一，MDM 2（C305 F），其在中心锌指基序中具有Cys到Phe的取代，表现出几个不同的特征，包括破坏L11结合。MDM 2（C305 F）保留了E3连接酶活性以泛素化p53，但不促进p53降解。结果表明，与野生型MDM 2相比，表达MDM 2的细胞（C305 F）具有更强的抑制p53反式激活活性的能力，并且能够逃脱L11过表达诱导的生长抑制。MDM 2（C305 F）是一种有用的突变体，可用于分析L11调节MDM 2和p53功能的机制。基于我们的初步数据，我们假设L11-MDM 2-p53连接构成了一个监测蛋白质合成速率并协调细胞生长与细胞周期进程的途径。为了解L11-MDM 2-p53通路的功能和调控，我们提出以下目的：目的1.研究MDM 2-L11相互作用的机制和调控。目标2.使用MDM 2（C305 F）敲入研究MDM 2-L11相互作用的体内功能。目标3.探讨ARF对L11、MDM 2和p53的调控机制。

DESCRIPTION (provided by applicant): 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.