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/␤-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/␤-catenin signaling. The inhibitory function of Smurf1 on Wnt/␤-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/␤-catenin signaling pathway via modulating the activity of axin.

Smurf1-mediated Axin Ubiquitination Requires Smurf1 C2 Domain and Is Cell Cycle-dependent

“…Our previous study has demonstrated that Smurf1 ubiquitinates Axin through K29-linked poly-ubiquitination, which interrupts Axin-LRP5/6 association and then inhibits Wnt/␤-catenin signaling (14). In the present work, we showed that the C2 domain of Smurf1 was crucial for its interaction with Axin, suggesting a non-canonical WW-PY-independent interaction for the two proteins.…”

“…Point mutations of Axin and Smurf1 were generated using a Stratagene QuikChange site-directed mutagenesis kit. Other plasmids have been applied previously (14). Specific siRNA oligos against human Smurf1 and Axin were synthesized in Genepharma.…”

Ubiquitin chain diversity at a glance

“…Wnt ligands lead to inactivation of this destruction complex, allowing β-catenin to accumulate, translocate to the nucleus, and, together with its partner TCF/LEF-family member, activate a transcriptional program (Clevers and Nusse, 2012). Recently, the ubiquitin E3 ligase Smad ubiquitylation regulatory factor 1 (Smurf1) has been shown to modify Axin with nondegradable K29-linked ubiquitin polymers (Fei et al, 2013). K29-polyubiquitylation of Axin disrupts its interaction with the Wnt coreceptors LRP5 and LRP6 (LRP5/6), which subsequently attenuates Wnt-stimulated LRP6 phosphorylation and represses Wnt/β-catenin signaling (Fei et al, 2013).…”

“…Recently, the ubiquitin E3 ligase Smad ubiquitylation regulatory factor 1 (Smurf1) has been shown to modify Axin with nondegradable K29-linked ubiquitin polymers (Fei et al, 2013). K29-polyubiquitylation of Axin disrupts its interaction with the Wnt coreceptors LRP5 and LRP6 (LRP5/6), which subsequently attenuates Wnt-stimulated LRP6 phosphorylation and represses Wnt/β-catenin signaling (Fei et al, 2013). Interestingly, the OTU family DUB Trabid (also known in mammals as ZRANB1), which is a positive regulator of Wnt-induced transcription, shows a strong preference for K29 and K33 linkages (Kristariyanto et al, 2015a;Licchesi et al, 2012;Mevissen et al, 2013;Michel et al, 2015;Virdee et al, 2010).…”

Rapid development of proteomics in China: from the perspective of the Human Liver Proteome Project and technology development

“…With the exception of substrate specificity, the specific ubiquitin chains also play regulatory roles in the biological functions of their modified protein substrates. Using an MS-based approach, Li Lin and his colleagues [55] demonstrated that Smurf1-mediated Lys29-linked polyubiquitination of axin negatively regulates Wnt/β-catenin signaling without proteolytic activity.…”