Regulation of MDM2 by the ribosomal protein L11

描述(申请人提供)：肿瘤抑制基因P53和ARF的基因组基因座在所有类型的人类癌症中都是最频繁突变的。另一方面，原癌蛋白MDM2是细胞中主要的P53负调控因子，在许多类型的人类癌症中经常过度表达，ARF、MDM2和P53构成了重要的肿瘤抑制途径(ARF-MDM2-P53途径)，保护细胞免受异常和失控的生长。最近的研究表明，ARF-MDM2-P53通路并不是严格意义上的线性，而是分支到其他靶点，该通路可能通过与其他细胞蛋白的相互作用而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MDM2通过其锌指区与核糖体蛋白L11相互作用。L11在体内与ARF、MDM2和P53形成复合体。L11的增强表达可阻止MDM2介导的P53泛素化和降解，恢复MDM2抑制的P53反式激活活性，并诱导依赖于P53的G1细胞周期停滞。更重要的是，研究表明，人类癌症相关的MDM2基因突变优先针对破坏L11结合的锌指结构域。我们研究的MDM2突变体之一，MDM2(C305F)，它在中心锌指基序中具有Cys-to-Phe替换，表现出几个明显的特征，包括L11结合的中断。MDM2(C305F)保留了泛素化P53的E3连接酶活性，但不促进P53的降解。与野生型mdm2相比，mdm2具有更强的抑制P53‘S反式激活活性的能力，表达mdm2的C305F细胞可逃脱L11过表达诱导的生长抑制。MDM2(C305F)是一个有用的突变体，可以用来剖析L11调控MDM2和P53功能的机制。根据我们的初步数据，我们假设L11-MDM2-P53连接构成了一条监控蛋白质合成速度并协调细胞生长和细胞周期进展的途径。为了了解LI1-MDM2-P53通路的功能和调控，我们提出了以下目标：目的1.探讨MDM2-L11相互作用的机制和调控。目的2.用MDM2(C305F)敲击研究MDM2-L11相互作用的体内功能。目的3.探讨ARF对L11、MDM2和P53基因表达的调节机制。

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.