Smad molecules (Smad1-8) mediate BMP/TGF-β signaling and play important roles in various biological responses such as development and cancer progression. However, the mechanisms regulating stabilities of Smad proteins remain largely unknown. We found that Smad6 protein is more stable than other Smad family proteins, and revealed the molecular mechanisms that regulate Smad6 protein stability.
We first searched for the Smad6-interacting proteins that can affect Smad6 protein stability. The LC-MS/MS analysis revealed an ubiquitin ligase that directly interacts with Smad6. We found that the ubiquitin ligase polyubiquitinated Smad6, but unexpectedly promoted Smad6 protein stability. The ubiquitin assay using various Smad6 mutants identified the 7 lysine residues of Smad6 that are ubiquitinated by the ubiquitin ligase. Consistently, a Smad6 mutant in which the 7 lysine residues were replaced by arginine (Smad6 KR) did not undergo polyubiquitination by the ubiquitin ligase, and the protein stability of Smad6 KR was much lower than that of Smad6 WT. Smad6 was reported to worsen clinical condition of breast cancer by inhibiting BMP signaling. Interestingly, the tumorigenicity of Smad6 KR was much lower than that of Smad6 WT.