SnoN and TGFbeta Signaling

这项建议的长期目标是了解SnoN癌蛋白与Smad蛋白相互作用调节细胞生长和分化的机制，以及这种相互作用的放松如何有助于癌症的发展。转化生长因子β是一种强有力的上皮细胞生长抑制因子。在哺乳动物细胞中，TGFbeta信号主要由Smad蛋白介导。转化生长因子β受体和Smad蛋白都是肿瘤抑制因子。转化生长因子β受体或Smad蛋白的失活已被证明与人类癌症的发生和发展有关。本研究的目的是对SnoN和Smad蛋白之间的相互作用进行详细的分子表征，并研究这种相互作用在调节上皮细胞分化和转化中的作用。TGFbeta1与TGFbeta受体结合后，受体激酶激活，R-Smads、Smad2和Smad3磷酸化。然后，磷酸化的R-Smads与Smad4结合，积聚在细胞核中，调节TGFbeta反应基因的转录。P.I.最近发现了一种核癌蛋白SnoN，它是一种与SMADS相关的蛋白。SnoN与Smad2和Smad4在TGFbeta反应的DNA元件上相互作用，并通过招募转录共抑制因子N-COR来抑制它们激活转录的能力。在TGFbeta刺激后，SnoN被Srnad3的核积聚迅速降解，从而解除了对Smad2/Smad4介导的靶基因反式激活的抑制。两小时后，TGFbeta诱导SnoN水平显著升高，导致Smad介导的转录激活终止。在这些研究的基础上，P.I.提出SnoN是TGFbeta信号的关键负调控因子。本研究旨在扩展这些观察，并对SnoN和Smad蛋白之间的相互作用进行详细的结构和功能分析，并开发一个分化和癌变模型系统来研究这种相互作用在上皮细胞分化和转化调控中的作用。这些研究的具体目的是：1)分析SnoN-SnoN相互作用对抑制TGFbeta信号的作用；2)分析SnoN和SnoN-Smad相互作用在乳腺上皮细胞分化和乳腺癌进展中的作用；3)鉴定和纯化SnoN相关蛋白；4)分析Smad3诱导SnoN降解的机制。这些研究将有助于更好地了解SnoN癌蛋白的生理功能，以及SnoN/Smad相互作用在上皮癌变中的作用。

The long term objective of this proposal is to understand the mechanism by which the SnoN oncoprotein interacts with the Smad proteins to regulate cell growth and differentiation, and how deregulation of this interaction contributes to the development of cancer. TGFbeta is a potent inhibitor of epithelial cell growth. In mammalian cells, TGFbeta signaling is mainly mediated by the Smad proteins. Both TGFbeta receptors and Smad proteins are tumor suppressors. Inactivation of TGFbeta receptors or Smad proteins has been shown to contribute to the cause and progression of human cancers. This proposal is designed to carry out a detailed molecular characterization of the interaction between SnoN and the Smad proteins and to investigate the role of this interaction in the regulation of epithelial cell differentiation and transformation. Binding of TGFbeta1 to the TGFbeta receptors results in activation of the receptor kinases and subsequent phosphorylation of the R-Smads, Smad2 and Smad3. The phosphorylated R-Smads then associate with Smad4, accumulate in the nucleus and regulate transcription of TGFbeta responsive genes. The P.I. has recently identified a nuclear oncoprotein, SnoN, as a Smads-associated protein. SnoN interacts with Smad2 and Smad4 on a TGFbeta-responsive DNA element and repress their abilities to activate transcription through recruitment of the transcriptional co- repressor N-CoR. Immediately following TGFbeta stimulation, SnoN is rapidly degraded by the nuclear accumulation of Srnad3, thus relieving the inhibition on Smad2/Smad4-mediated transactivation of target genes. By two hours, TGFbeta induces a marked increase in SnoN level, resulting in the termination of Smad-mediated transcriptional activation. Based on these studies, the P.I. proposes that SnoN is a key negative regulator of TGFbeta signaling. This proposal is designed to extend these observations and carry out a detailed structural and functional analyses of the interaction between SnoN and the Smad proteins and to develop a differentiation and carcinogenesis model system to investigate the role of this interaction in the regulation of epithelial cell differentiation and transformation. The specific aims are: 1) Analysis of the role of Smad- SnoN interaction to repression of TGFbeta signaling; 2) Analysis of the role of SnoN and SnoN-Smad interaction in mammary epithelial cell differentiation and breast cancer progression; 3) Identification and purification of SnoN associated proteins; 4) Analysis of the mechanism of Smad3-induced degradation of SnoN. These studies will allow a better understanding of the physiological function of the SnoN oncoprotein, and the role of the SnoN/Smad interaction in epithelial carcinogenesis.