Smurf1-Mediated Lys29-Linked Nonproteolytic Polyubiquitination of Axin Negatively Regulates Wnt/β-Catenin Signaling

Abstract

Ubiquitination plays important and diverse roles in modulating protein functions. As a C2-WW-HECT-type ubiquitin ligase, Smad ubiquitination regulatory factor 1 (Smurf1) commonly serves to regulate ubiquitin-dependent protein degradation in a number of signaling pathways. Here, we report a novel function of Smurf1 in regulating Wnt/β-catenin signaling through targeting axin for nonproteolytic ubiquitination. Our data unambiguously demonstrate that Smurf1 ubiquitinates axin through Lys 29 (K29)-linked polyubiquitin chains. Unexpectedly, Smurf1-mediated axin ubiquitination does not lead to its degradation but instead disrupts its interaction with the Wnt coreceptors LRP5/6, which subsequently attenuates Wnt-stimulated LRP6 phosphorylation and represses Wnt/β-catenin signaling. The inhibitory function of Smurf1 on Wnt/β-catenin signaling is further evidenced by analysis with Smurf1 knockout murine embryonic fibroblasts. We next identified K789 and K821 in axin as the ubiquitination sites by Smurf1. Consistently, Smurf1 could neither disrupt the interaction of an axin^K789/821R double mutant with LRP5/6 nor attenuate the phosphorylation of LRP6 in axin^K789/821R-expressing cells. Collectively, our studies uncover Smurf1 as a new regulator for the Wnt/β-catenin signaling pathway via modulating the activity of axin.

ACKNOWLEDGMENTS

We thank Ying E. Zhang for generously providing Smurf1^−/− MEFs, Rong-Gui Hu for series of E3 ligase plasmids, Joan Massagué (Memorial Sloan-Kettering Cancer Center, USA) for Smurf1-HA and Smurf1-CA-HA plasmids, Di Chen (University of Rochester) for the Smurf1-Myc construct, Xi He (Children's Hospital Boston) for the plasmids of LRP6 and MESD, Ye-Guang Chen (Tsinghua University, China) for Smad7-Flag, and Long Yu for the plasmids of Ub-WT, Ub-K0, Ub-K48, and Ub-K63. We are grateful to Dang-Sheng Li and Xiao-Min Song for critical reading of the manuscript.

This work was supported by a Ministry of Science and Technology of China grant (2010CB912100) and National Natural Science Foundation of China grants (31230044, 30930052, and 91213304).