The HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly checkpoint

Abstract

Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 is critical for the metaphase–anaphase transition. APC/C-Cdc20 is required for polyubiquitination and degradation of securin and cyclin B at anaphase onset. The spindle assembly checkpoint delays APC/C-Cdc20 activation until all kinetochores attach to mitotic spindles. In this study, we demonstrate that a HECT (homologous to the E6-AP carboxyl terminus) ubiquitin ligase, Smurf2, is required for the spindle checkpoint. Smurf2 localizes to the centrosome, mitotic midbody, and centromeres. Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis. Smurf2 inactivation prevents nocodazole-treated cells from accumulating cyclin B and securin and prometaphase arrest. The silencing of Cdc20 in Smurf2-depleted cells restores mitotic accumulation of cyclin B and securin. Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector. Mad2 is mislocalized in Smurf2-depleted cells, suggesting that Smurf2 regulates the localization and stability of Mad2. These data indicate that Smurf2 is a novel mitotic regulator.

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

“…Specific short interfering RNA (siRNA) oligonucleotides against human Smurf1, Smurf2, and axin were synthesized by GenePharma. Their sequences were the following: Smurf1 siRNA-1, CC GACACUGUGAAAAACACTT (10, 24); Smurf1 siRNA-2, GCGUUUGG AUCUAUGCAAATT; Smurf2 siRNA, GAUGAGAACACUCCAAU UATT (25); and axin siRNA, CGAGAGCCAUCUACCGAAATT (26). The following commercial antibodies were used for Western blotting and immunoprecipitation: hemagglutinin (HA) and Myc (Covance); glutathione S-transferase (GST) and Flag (F1804) (Sigma-Aldrich); Smurf1, Smad7, ␤-actin, and glycogen synthase kinse 3␣/␤ (GSK3␣/␤; Santa Cruz Biotechnology); axin1 (C76H11), LRP6 (C5C7), LRP6 (C47H12), phospho-LRP6 (Ser1490), Smad1, and Flag (no.…”

Molecular functions of NEDD4 E3 ubiquitin ligases in cancer

“…The nuclear sequestration of Smurf2 appears to be important for tumor suppression, where it could control the stability of RNF20 and other cell growth-promoting molecules residing in the nucleus. These molecules include transcription factors KLF5 [171], ID1 and ID3 [172], and mitotic checkpoint complex component MAD2 [173]. In some types of tumors (e.g.…”

Ubiquitination of Tumor Necrosis Factor Receptor-associated Factor 4 (TRAF4) by Smad Ubiquitination Regulatory Factor 1 (Smurf1) Regulates Motility of Breast Epithelial and Cancer Cells

“…To date, the functions and the repertoires of known Smurf substrates have been expanded beyond TGF-␤/ BMP signaling. In in vitro cell culture systems and in mice, Smurf1 was reported to participate in the maintenance of bone homeostasis by targeting Runx2 and MEKK2, respectively (9 -11), whereas Smurf2 was implicated in cell cycle progression, senescence, DNA damage response, and genomic stability through a variety of protein targets (12)(13)(14). In addition to their respective unique functions, Smurf1 and Smurf2 also share a common function by controlling non-canonical Wnt output in setting up planar cell polarity (15), a process not only important during embryonic development, but also in tumor cell invasion (16).…”