TGF‐β signaling involves a wide array of signaling molecules and multiple controlling events. Scaffold proteins create a functional proximity of signaling molecules and control the specificity of signal transduction. While many components involved in the TGF‐β pathway have been elucidated, little is known about how those components are coordinated by scaffold proteins. Here, we show that Axin activates TGF‐β signaling by forming a multimeric complex consisting of Smad7 and ubiquitin E3 ligase Arkadia. Axin depends on Arkadia to facilitate TGF‐β signaling, as their small interfering RNAs reciprocally abolished the stimulatory effect on TGF‐β signaling. Specific knockdown of Axin or Arkadia revealed that Axin and Arkadia cooperate with each other in promoting Smad7 ubiquitination. Pulse‐chase experiments further illustrated that Axin significantly decreased the half‐life of Smad7. Axin also induces nuclear export of Smad7. Interestingly, Axin associates with Arkadia and Smad7 independently of TGF‐β signal, in contrast to its transient association with inactive Smad3. However, coexpression of Wnt‐1 reduced Smad7 ubiquitination by downregulating Axin levels, underscoring the importance of Axin as an intrinsic regulator in TGF‐β signaling.

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Axin is a multifunctional protein, regulating Wnt signaling and the c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK) pathway as well as tumorigenesis. In the present study, we found that Axin interacts with three SUMO-1 (small ubiquitin-related modifier) conjugating enzymes 3 (E3), PIAS1, PIASx␤, and PIASy. The extreme C-terminal six amino acid residues of Axin are critical for the Axin/E3 interaction as deletion of the six residues (Axin⌬C6) completely abolished the ability of Axin to interact with E3 enzymes. Axin⌬C6 also failed to activate JNK, although it was intact in both its interaction with MEKK1 and homodimerization. Consistent with the presence of a doublet of the KV(E/D) sumoylation consensus motif at the C-terminal end (KVEKVD), we found that Axin is heavily sumoylated. Deletion of the C-terminal six amino acids drastically reduced sumoylation, indicating that the C-terminal six amino acids stretch is the main sumoylation site for Axin. Sumoylation-defective mutants failed to activate JNK but effectively destabilized ␤-catenin and attenuated LEF1 transcriptional activity. In addition, we show that dominant negative Axin mutants blocked PIAS-mediated JNK activation, in accordance with the requirement of sumoylation for Axin-mediated JNK activation.Taken together, we demonstrate that sumoylation plays a role for Axin to function in the JNK pathway.