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.
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