Mechanisms and Functions of SMAD2 Degradation

转化生长因子-β(TFG-β)超家族成员是后生动物体内控制多种细胞反应的多功能生长因子。转化生长因子-β1抑制大部分上皮细胞和造血细胞的生长，并调节间充质细胞产生细胞外基质。失去对转化生长因子-β的抗增殖反应通常被认为是肿瘤进展的重要一步。这项应用的长期目标是了解转化生长因子-β抗增殖信号通路的改变如何导致人类疾病中生长控制的放松调控的分子基础。这一应用的一般策略是关注泛素/蛋白酶体依赖的肿瘤抑制因子Smad2的降解机制。Smad2是一种重要的转化生长因子-β信号转导分子，通过转录依赖机制抑制细胞增殖。然而，Smad2的精确调控仍然是个谜。这一提议的统一假设是泛素/蛋白酶体途径通过特定的泛素E3连接酶靶向Smad2进行降解。为了验证这一假说，我们将研究一种新发现的泛素E3连接酶S-MURF2降解Smad2的机制，并探讨SMURF2活性解除调控在Smad2降解中的生理后果。提出了三个具体的目标：1.明确S-MURF-2介导的Smad2降解的分子机制。将确定S-2和Smad2之间的物理相互作用、相互作用所需的结构域以及底物选择性的分子基础。2.研究SMurf2介导的Smad2降解在转化生长因子-β信号转导中的生理作用。它将测试在转化生长因子-β诱导的细胞周期停滞和非洲爪哇早期胚胎发育过程中，SMurf2介导的Smad2降解如何影响Smad2信号转导。3.检测SMurf2-Smad2在上皮细胞中的降解规律。将使用分子和生化方法来确定SMurf2介导的Smad2降解的细胞机制，并分析正常细胞和癌细胞中Smad2的差异降解。这些研究将有助于建立泛素化介导的Smad2在转化生长因子-β诱导的生长抑制过程中转化的工作理论，并有助于理解Smad2在人类癌症恶性转化和进展中的作用机制。研究结果可为合理设计癌症防治新的治疗方法提供依据。

Members of transforming growth factor-beta (TFG-beta) superfamily are multifunctional growth factors that control a broad range of cellular responses in metazoan organism. TGF- betas inhibit the growth of most epithelial and hematopoietic cells and regulates the production of extracellular matrix by mesenchymal cells. Loss of the antiproliferative responsiveness to TGF-beta is often considered as a major step in tumor progression. The long-term objective of this application is to understand the molecular basis of how alterations in TGF-beta antiproliferative signaling pathways lead to deregulation of growth control in human diseases. The general strategy of this application is to focus on the mechanism of ubiquitin/proteasome- dependent degradation of tumor suppressor Smad2. Smad2 is an important TGF-beta signal transducer that inhibits cell proliferation though transcription-dependent mechanisms. However, the precise regulation of Smad2 remains enigmatic. The unifying hypothesis of this proposal is that ubiquitin/proteasome pathway targets Smad2 for degradation through a specific ubiquitin E3 ligase. To test this hypothesis, we will study the mechanism of Smad2 degradation by a newly identified ubiquitin E3 ligase called Smurf2 and investigate the physiological consequences of deregulation of Smurf2 activity in Smad2 degradation. Three Specific Aims are proposed: 1. Define the molecular mechanism of Smurf2-mediated Smad2 degradation. The physical interaction between Smurf2and Smad2, the structural domains required for the interaction, and the molecular basis for substrate selectivity will be determined. 2. Investigate the physiological impact of Smurf2-mediated Smad2 degradation in TGF- beta signaling. It will be tested how Smurf2-mediated Smad2 degradation affects Smad2 signaling during TGF-beta induced cell cycle arrest and Xenopus early embryonic development. 3. Examine the regulation of Smurf2-Smad2 degradation in epithelial cells. Molecular and biochemical approaches will be used to define the cellular mechanism of Smurf2-mediated Smad2 degradation and to analyze differential Smad2 degradation in normal and cancer cells. The proposed studies should help to establish a working theory for ubiquitination-mediated Smad2 turnover during TGF- beta-induced growth inhibition and to understand the mechanisms of Smad actions in malignant transformation and progression of human cancers. Finally, the result may provide a foundation of the rational design of novel therapeutic approaches for cancer prevention and treatment.