Indirubin derivatives modulate TGFβ/BMP signaling at different levels and trigger ubiquitin-mediated depletion of nonactivated R-Smads.

Regulatory Smads (R-Smads), Smad1/5/8 and Smad2/3, are the central mediators of TGFβ and BMP signaling pathways. Here, we screened indirubin derivatives, known kinase inhibitors, and observed strong interference with BMP signaling. We found that indirubin derivative E738 inhibited both TGFβ and BMP pathways through ubiquitin-proteasome-mediated depletion of total R-Smad pools, although phospho-R-Smad levels were initially stabilized by GSK3β and cyclin-dependent kinase inhibition. E738 also enhanced p38 and JNK phosphorylation, involved in Smad-independent TGFβ/BMP signaling. Additionally, using a small siRNA screen, we showed that depletion of ubiquitin proteases USP9x and USP34 significantly reduced total R-Smad levels, mimicking E738 treatment. In fact, both USP9x and USP34 levels were significantly reduced in E738-treated cells. Our findings not only describe the complex activity profile of the indirubin derivative E738, but also reveal a mechanism for controlling TGFβ/BMP signaling, the control of R-Smad protein levels through deubiquitination.