A mechanism of repression of TGFβ/Smad signaling by oncogenic Ras

TGF beta can override the proliferative effects of EGF and other Pas-activating mitogens in normal epithelial cells. However, epithelial cells harboring oncogenic Pas mutations often show a loss of TGF beta antimitogenic responses. Here we report that oncogenic Pas inhibits TGF beta signaling in mammary and lung epithelial cells by negatively regulating the TGF beta mediators Smad2 and Smad3. Oncogenically activated Ras inhibits the TGF beta-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 TGF beta receptor phosphorylation sites that activate Smad nuclear translocation. Mutation of these MAP kinase sites in Smad3 yields a Pas-resistant form that can rescue the growth inhibitory response to TGF beta in Ras-transformed cells. EGF, which is weaker than oncogenic mutations at activating Rns, 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 TGF beta and Pas signals in normal cells, and for the silencing of antimitogenic TGF beta functions by hyperactive Pas in cancer cells.