Downregulation of Ski and SnoN co-repressors by anisomycin

Proteasome pathway regulates TGF-beta signaling; degradation of activated Smad2/3 and receptors turns TGF-beta signal off, while degradation of negative modulators such as Ski and SnoN maintains the signal. We have found that anisomycin is able to downregulate Ski and SnoN via proteasome as TGF-beta does, but through a mechanism independent of Smad activation. The mechanism used by anisomycin to downregulate Ski and SnoN is also independent of MAPK activation and protein synthesis inhibition. TGF-beta signal was the only pathway described causing Ski and SnoN degradation, thus this new effect of anisomycin on endogenous Ski and SnoN proteins suggests alternative processes to downregulate these negative modulators of TGF-beta signaling.     

SnoN co-repressor binds and represses smad7 gene promoter

SnoN and Ski oncoproteins are co-repressors for Smad proteins and repress TGF-beta-responsive gene expression. The smad7 gene is a TGF-beta target induced by Smad signaling, and its promoter contains the Smad-binding element (SBE) required for a positive regulation by the TGF-beta/Smad pathway. SnoN and Ski co-repressors also bind SBE but regulate negatively smad7 gene. Ski along with Smad4 binds and represses the smad7 promoter, whereas the repression mechanism by SnoN is not clear. Ski and SnoN overexpression inhibits smad7 reporter expression induced through TGF-beta signaling. Using chromatin immunoprecipitation assays, we found that SnoN binds smad7 promoter at the basal condition, whereas after a short TGF-beta treatment for 15-30 min SnoN is downregulated and no longer bound smad7 promoter. Interestingly, after a prolonged TGF-beta treatment SnoN is upregulated and returns to its position on the smad7 promoter, functioning probably as a negative feedback control. Thus, SnoN also seems to regulate negatively the TGF-beta-responsive smad7 gene by binding and repressing its promoter in a similar way to Ski.     

AIP4 restricts transforming growth factor-beta signaling through a ubiquitination-independent mechanism

Smad7 functions as an intracellular antagonist in transforming growth factor-beta (TGF-beta) signaling. In addition to interacting stably with the activated TGF-beta type I receptor (TbetaRI) to prevent phosphorylation of the receptor-regulated Smads (Smad2 and Smad3), Smad7 also induces degradation of the activated TbetaRI through association with different E3 ubiquitin ligases. Using the two-hybrid screen, we identified atrophin 1-interacting protein 4 (AIP4) as an E3 ubiquitin ligase that specifically targets Smad7 for ubiquitin-dependent degradation without affecting the turnover of the activated TbetaRI. Surprisingly, we found that despite the ability to degrade Smad7, AIP4 can inhibit TGF-beta signaling, presumably by enhancing the association of Smad7 with the activated TbetaRI. Consistent with this notion, expression of a catalytic mutant of AIP4, which is unable to induce ubiquitination and degradation of Smad7, also stabilizes the TbetaRI.Smad7 complex, resulting in inhibition of TGF-beta signaling. The ability of AIP4 to enhance the inhibitory function of Smad7 independent of its ubiquitin ligase activity reveals a new mechanism by which E3 ubiquitin ligases may function to turn off TGF-beta signaling.     

Atrophin-1-interacting protein 4/human Itch is a ubiquitin E3 ligase for human enhancer of filamentation 1 in transforming growth factor-beta signaling pathways

Atrophin-1-interacting protein 4 (AIP4) is the human homolog of the mouse Itch protein (hItch), an E3 ligase for Notch and JunB. Human enhancer of filamentation 1 (HEF1) has been implicated in signaling pathways such as those mediated by integrin, T cell receptor, and B cell receptor and functions as a multidomain docking protein. Recent studies suggest that HEF1 is also involved in the transforming growth factor-beta (TGF-beta) signaling pathways, by interacting with Smad3, a key signal transducer downstream of the TGF-beta type I receptor. The interaction of Smad3 with HEF1 induces HEF1 proteasomal degradation, which was further enhanced by TGF-beta stimulation. The detailed molecular mechanisms of HEF1 degradation regulated by Smad3 were poorly understood. Here we report our studies that demonstrate the function of AIP4 as an ubiquitin E3 ligase for HEF1. AIP4 forms a complex with both Smad3 and HEF1 through its WW domains in a TGF-beta-independent manner and regulates HEF1 ubiquitination and degradation, which can be enhanced by TGF-beta stimulation. These findings reveal a new mechanism for Smad3-regulated proteasomal degradation events and also broaden the network of cross-talk between the TGF-beta signaling pathway and those involving HEF1 and AIP4.