The Protein Stability of Axin, a Negative Regulator of Wnt Signaling, Is Regulated by Smad Ubiquitination Regulatory Factor 2 (Smurf2)

Eek-hoon Jho,Sewoon Kim

doi:10.1074/jbc.m110.137471

Abstract

Axin is a negative regulator of Wnt/β-catenin signaling via regulating the level of β-catenin, which is a key effector molecule. Therefore, controlling the level of Axin is a critical step for the regulation of Wnt/β-catenin signaling. It has been shown that ubiquitination-mediated proteasomal degradation may play a critical role in the regulation of Axin; however, the E3 ubiquitin ligase(s), which attaches ubiquitin to a target protein in combination with an E2 ubiquitin-conjugating enzyme, for Axin has not yet been identified. Here, we show that Smurf2 is an E3 ubiquitin ligase for Axin. Transient expression of Smurf2 down-regulated the level of Axin and increased the ubiquitination of Axin. Conversely, shRNA specific to Smurf2 blocked Axin ubiquitination. Essential domains of Axin responsible for Smurf2 interaction as well as Smurf2-mediated down-regulation and ubiquitination were identified. In vitro ubiquitination assays followed by analysis using mass spectroscopy revealed that Smurf2 specifically ubiquitinylated Lys(505) of Axin and that the Axin(K505R) mutant resisted degradation. Knockdown of endogenous Smurf2 increased the level of endogenous Axin and resulted in reduced β-catenin/Tcf reporter activity. Overall, our data strongly suggest that Smurf2 is a genuine E3 ligase for Axin.

Highlights

Smurf2 (Smad ubiquitination regulatory factor 2) is a HECT domain E3 ligase involved in the ubiquitination of several proteins, including Smad proteins, which are involved in TGF-␤ signaling [13]
Knockdown of Smurf2 in HeLa and Caco-2 cells, but not in SNU475 cells that do not express Axin due to mutation [10], resulted in reduced reporter activity (Fig. 4E). These results suggest that the inhibition of ␤-catenin/T cell factor (Tcf) reporter activity by Smurf2 shRNA is mediated by the regulation of Axin
The ubiquitin/proteasomal pathway is involved in Axin regulation; the E3 ligase responsible for this process has not been identified due to the extremely low expression and instability of Axin

Summary

Introduction

Immunoprecipitation analysis showed that both exogenously expressed and endogenous Axin interacted with Smurf2 (Fig. 1, A and B). The supernatant was collected and diluted 10 times in RIPA buffer, followed by immunoprecipitation with anti-Myc antibody and immunoblotting to detect Axin ubiquitination. When Smurf2, but not the Smurf2(C716G) mutant, with ubiquitin, Axin⌬228 –354 and Axin⌬508 –711 was transfected with E1, and UbcH5A produced polyubiquitination signals (Fig. 3B, Smurf2, down-regulation and polyubiquitination of Axin were lanes 6 and 7).

Results

Conclusion