RESUMO
The regulation of cell growth and differentiation by transforming growth factor-beta (TGF-beta) is mediated by the Smad proteins. In the nucleus, the Smad proteins are negatively regulated by two closely related nuclear proto-oncoproteins, Ski and SnoN. When overexpressed, Ski and SnoN induce oncogenic transformation of chicken embryo fibroblasts. However, the mechanism of transformation by Ski and SnoN has not been defined. We have previously reported that Ski and SnoN interact directly with Smad2, Smad3, and Smad4 and repress their ability to activate TGF-beta target genes through multiple mechanisms. Because Smad proteins are tumor suppressors, we hypothesized that the ability of Ski and SnoN to inactivate Smad function may be responsible for their transforming activity. Here, we show that the receptor regulated Smad proteins (Smad2 and Smad3) and common mediator Smad (Smad4) bind to different regions in Ski and SnoN. Mutation of both regions, but not each region alone, markedly impaired the ability of Ski and SnoN to repress TGF-beta-induced transcriptional activation and cell cycle arrest. Moreover, when expressed in chicken embryo fibroblasts, mutant Ski or SnoN defective in binding to the Smad proteins failed to induce oncogenic transformation. These results suggest that the ability of Ski and SnoN to repress the growth inhibitory function of the Smad proteins is required for their transforming activity. This may account for the resistance to TGF-beta-induced growth arrest in some human cancer cell lines that express high levels of Ski or SnoN.
Assuntos
Transformação Celular Neoplásica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Animais , Sítios de Ligação , Carcinoma Hepatocelular , Divisão Celular/fisiologia , Embrião de Galinha , Galinhas , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Hepáticas , Mutagênese , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteína Smad2 , Proteína Smad3 , Proteína Smad4 , Supressão Genética , Ativação Transcricional , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Células Tumorais CultivadasRESUMO
The Ski family of nuclear oncoproteins represses TGF-beta signaling through interactions with the Smad proteins. The crystal structure of the Smad4 binding domain of human c-Ski in complex with the MH2 domain of Smad4 reveals specific recognition of the Smad4 L3 loop region by a highly conserved interaction loop (I loop) from Ski. The Ski binding surface on Smad4 significantly overlaps with that required for binding of the R-Smads. Indeed, Ski disrupts the formation of a functional complex between the Co- and R-Smads, explaining how it could lead to repression of TGF-beta, activin, and BMP responses. Intriguingly, the structure of the Ski fragment, stabilized by a bound zinc atom, resembles the SAND domain, in which the corresponding I loop is responsible for DNA binding.