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A mechanism of repression of TGFbeta/ Smad signaling by oncogenic Ras
M Kretzschmar1, J Doody, I Timokhina
1Cell Biology Program and Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 USA.
Abstract:
TGFbeta can override the proliferative effects of EGF and other Ras-activating mitogens in normal epithelial cells. However, epithelial cells harboring oncogenic Ras mutations often show a loss of TGFbeta antimitogenic responses. Here we report that oncogenic Ras inhibits TGFbeta signaling in mammary and lung epithelial cells by negatively regulating the TGFbeta mediators Smad2 and Smad3. Oncogenically activated Ras inhibits the TGFbeta-induced nuclear accumulation of Smad2 and Smad3 and Smad-dependent transcription. Ras acting via Erk MAP kinases causes phosphorylation of Smad2 and Smad3 at specific sites in the region linking the DNA-binding domain and the transcriptional activation domain. These sites are separate from the TGFbeta receptor phosphorylation sites that activate Smad nuclear translocation. Mutation of these MAP kinase sites in Smad3 yields a Ras-resistant form that can rescue the growth inhibitory response to TGFbeta in Ras-transformed cells. EGF, which is weaker than oncogenic mutations at activating Ras, induces a less extensive phosphorylation and cytoplasmic retention of Smad2 and Smad3. Our results suggest a mechanism for the counterbalanced regulation of Smad2/Smad3 by TGFbeta and Ras signals in normal cells, and for the silencing of antimitogenic TGFbeta functions by hyperactive Ras in cancer cells.
Insights
Oncogenic Ras mutations disrupt normal epithelial cell growth by inhibiting TGF-beta signaling. This Ras-mediated inhibition of Smad2 and Smad3 proteins prevents anti-proliferative responses, contributing to cancer development.
Area of Science:
- Cellular Biology
- Molecular Oncology
- Signal Transduction
Background:
- Transforming Growth Factor-beta (TGFbeta) normally inhibits epithelial cell proliferation.
- Oncogenic Ras mutations are frequently found in cancers and can lead to loss of TGFbeta's growth-inhibitory effects.
Purpose of the Study:
- To investigate the mechanism by which oncogenic Ras inhibits TGFbeta signaling in epithelial cells.
- To identify the specific mediators and pathways involved in this Ras-mediated suppression.
Main Methods:
- Studied TGFbeta signaling in mammary and lung epithelial cells with and without oncogenic Ras.
- Utilized techniques to assess Smad2 and Smad3 nuclear accumulation and Smad-dependent transcription.
- Investigated the role of Erk MAP kinases and specific phosphorylation sites on Smad proteins.
- Employed site-directed mutagenesis to create Ras-resistant Smad3 variants.
Main Results:
- Oncogenic Ras inhibits TGFbeta signaling by negatively regulating Smad2 and Smad3.
- Ras, via Erk MAP kinases, phosphorylates Smad2/Smad3 at unique sites, preventing nuclear translocation and transcriptional activity.
- Mutating these specific Ras-sensitive sites in Smad3 restored TGFbeta's growth inhibitory function in Ras-transformed cells.
- Epidermal Growth Factor (EGF) showed a weaker inhibitory effect on Smad2/Smad3 compared to oncogenic Ras.
Conclusions:
- Ras-MAPK signaling antagonizes TGFbeta-Smad signaling by phosphorylating Smad2/Smad3, leading to cytoplasmic retention.
- This mechanism explains how hyperactive Ras silences TGFbeta's anti-proliferative functions in cancer cells.
- Understanding this crosstalk is crucial for developing targeted cancer therapies.