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

Background: Smurf1 ubiquitinates Axin via K29 poly-ubiquitin chains, and interferes with Wnt/-catenin signaling. Results: The C2 domain of Smurf1 is responsible for interacting with Axin, and Smurf1-mediated Axin ubiquitination attenuates in the G2/M phase of cell cycle. Conclusion: Smurf1-Axin interaction follows a non-canonical pattern and is under the control of cell cycle. Significance: Further clarification of the characteristic of Smurf1-mediated K29-linked poly-ubiquitination of Axin.Previously, Smad ubiquitination regulatory factor 1 (Smurf1)-mediated Lys29 (K29)-linked poly-ubiquitination of Axin has been identified as a novel regulatory process in Wnt/-catenin signaling. In this work, we discovered that the C2 domain of Smurf1 is critical for targeting Axin for ubiquitination. We found that the C2 domain-mediated plasma membrane localization of Smurf1 is required for Axin ubiquitination, and interfering with that disturbs the co-localization of Smurf1 and Axin around the plasma membrane. Moreover, the C2 domain of Smurf1, rather than its WW domains, is involved in Smurf1's interaction with Axin; and the putative PPXY motifs (PY motif) of Axin are not essential for such an interaction, indicating that Smurf1 binds to Axin in a non-canonical way independent of WW-PY interaction. Further, we found that Smurf1-Axin interaction and Axin ubiquitination are attenuated in the G2/M phase of cell cycle, contributing to an increased cell response to Wnt stimulation at that stage. Collectively, we uncovered a dual role of Smurf1 C2 domain, recruiting Smurf1 to membrane for accessing Axin and mediating its interaction with Axin, and that Smurf1-mediated Axin ubiquitination is subjected to the regulation of cell cycle.