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Smad4-Ubiquitination by opposing E3 and DUB activities: a central control element in TGF-beta signaling

Final Report Summary - ENZYMES FOR SMAD4-UB (Smad4-Ubiquitination by opposing E3 and DUB activities: a central control element in TGF-beta signaling)

At the time of submission our main objectives were:
1) defining new principles in the regulation of Smad signaling by ubiquitination, in particular monoubiquitination and regulative ubiquitination.
2) Identification and characterization of novel enzymes involved in Smad regulation by ubiquitination
To address these issues, we carried out a screen for new DUBs involved in TGFb and BMP signaling. During growth factor stimulation, cells must tightly hold in check the activity of signal transducers. For TGFβ this is critical, as strength and duration of the signal can instruct different cell fates. We found that cycles of Smad monoubiquitination and deubiquitination are critical to set TGFβ responsiveness. We found that Smads are monoubiquitinated and that monoubiquitination occurs on residues that are essential for Smad transcriptional functions. The study reveals the existence of novel enzymatic toolboxes in TGFβ signal transduction.
In the case of Smad4, monoubiquitination serves to disrupt the Smad2/Smad4 complex. In the case of Receptor Smads, monoubiquitination affects the DNA-binding surface.
At difference with Smad4, R-Smad ubiquitination and deubiquitination is a mean to regulate their DNA binding. R-Smads are modified by inhibitory E3 ubiquitin ligases, notably by several members of the HECT family of ubiquitin ligases, such as Smurfs, NEDD-4, NEDD4-like, Itch and others. The effects of these ligases have so far been investigated in the context of R-Smads polyubiquitination, leading, in some cellular contexts, to R-Smad degradation. However, it is well-established that the ubiquitin system can effectively regulate protein function independently from protein turnover. The main appeal of regulative ubiquitination is that, just like phosphorylation, it is a reversible modification. Whether regulative ubiquitination also impinges on R-Smads was unknown. By identifying a new DUB (SADE in our screen), here we provide evidences that regulative, yet reversible, R-Smads ubiquitination is physiologically relevant to dissociate the Smad complex from DNA.

Potential impact:
This work breaks new ground in our understanding of how the ubiquitin system inhibits and terminates signaling from TGFβ superfamily ligands. Many E3 ubiquitin ligases are known as relevant inhibitors of R-Smad, controlling TGFβ/BMP gene responses in vivo. So far, the study of the mechanisms of this inhibition is largely based on overexpression of E3s or of specific RSmad point-mutants. Through this new enzyme, here we investigated how physiological ubiquitination impacts on R-Smad activity, and reveal the reversibility of these events. Indeed, we found regulative ubiquitinations in R-Smads. This is an important novelty in the present scenario that only envisions RSmad polyubiquitination.

PROJECT IMPLICATIONS (wider societal implications): TGFb and BMP are tumor suppressors and metastasis inducers. Identifying a new set of enzymes for their regulation opens the possibility to target this pathway with new small-molecule inhibitors and thus new therapeutic perspectives.