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

Osmundson EC, Ray D, Moore FE, Gao Q, Thomsen GH, Kiyokawa H - J. Cell Biol. (2008)

Bottom Line: Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis.Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector.These data indicate that Smurf2 is a novel mitotic regulator.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

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.

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Expression of a catalytically inactive Smurf2 impairs the spindle assembly checkpoint with Mad2 destabilization. (a) Immunoblotting for mitotic regulators in HeLa cells transfected with Flag-tagged Smurf2(C716A) or a control plasmid. Cells were synchronized at S phase with thymidine treatment, released into a nocodazole-containing medium, and harvested after incubation at the indicated times after release (see Materials and methods). OE, overexpression. (b) Chromosomal misalignment at metaphase and lagging chromosomes at anaphase observed in cells expressing Smurf2(C716A). Immunofluorescence microscopy of cells stained with anti-Flag antibody for exogenously expressed Smurf2, ACA, and DAPI. Cells were harvested 8.5 h after release from the thymidine block. The second row shows misaligned chromosomes at metaphase, and the bottom row shows lagging chromosomes at anaphase in cells expressing Smurf2(C716A). Bars, 10 μm. (c) Percentages of cells with chromosomal defects in control cultures (open bars) and Smurf2(C716A)-transfected cultures (shaded bars). Data are means ± SEM (error bars) from three independent experiments.
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fig6: Expression of a catalytically inactive Smurf2 impairs the spindle assembly checkpoint with Mad2 destabilization. (a) Immunoblotting for mitotic regulators in HeLa cells transfected with Flag-tagged Smurf2(C716A) or a control plasmid. Cells were synchronized at S phase with thymidine treatment, released into a nocodazole-containing medium, and harvested after incubation at the indicated times after release (see Materials and methods). OE, overexpression. (b) Chromosomal misalignment at metaphase and lagging chromosomes at anaphase observed in cells expressing Smurf2(C716A). Immunofluorescence microscopy of cells stained with anti-Flag antibody for exogenously expressed Smurf2, ACA, and DAPI. Cells were harvested 8.5 h after release from the thymidine block. The second row shows misaligned chromosomes at metaphase, and the bottom row shows lagging chromosomes at anaphase in cells expressing Smurf2(C716A). Bars, 10 μm. (c) Percentages of cells with chromosomal defects in control cultures (open bars) and Smurf2(C716A)-transfected cultures (shaded bars). Data are means ± SEM (error bars) from three independent experiments.

Mentions: We next examined whether the E3 ligase activity of Smurf2 is important for its function to control the spindle assembly checkpoint. The cysteine-716 residue of Smurf2 is critical for its E3 ligase activity because Smurf2(C716A) protein is catalytically inactive (Kavsak et al., 2000). Thus, we transfected HeLa cells with an expression plasmid for Smurf2(C716A) and synchronized them at early S phase with double-thymidine treatments (Fig. 6 a). Cells were then released into a nocodazole-containing medium. Immunoblotting showed that cells transfected with Smurf2(C716A) failed to accumulate securin with significant reduction in Mad2 levels. Interestingly, cyclin B1 accumulation during G2/M progression was minimally affected, which is analogous to previous observations in Mad2 heterozygous cells (Michel et al., 2004). In contrast, transfection with wild-type Smurf2 affected none of these proteins (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200801049/DC1). This finding is consistent with the effects of Smurf2 siRNA, suggesting that Smurf2(C716A) can act as a dominant-negative mutant. Furthermore, cells transfected with Smurf2(C716A) showed a significant increase in cells displaying misaligned chromosomes, lagging chromosomes during mitotic exit, and multinucleation (Fig. 6, b and c). These data suggest that the E3 ligase activity of Smurf2 is required for proper control of the spindle assembly checkpoint.


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

Osmundson EC, Ray D, Moore FE, Gao Q, Thomsen GH, Kiyokawa H - J. Cell Biol. (2008)

Expression of a catalytically inactive Smurf2 impairs the spindle assembly checkpoint with Mad2 destabilization. (a) Immunoblotting for mitotic regulators in HeLa cells transfected with Flag-tagged Smurf2(C716A) or a control plasmid. Cells were synchronized at S phase with thymidine treatment, released into a nocodazole-containing medium, and harvested after incubation at the indicated times after release (see Materials and methods). OE, overexpression. (b) Chromosomal misalignment at metaphase and lagging chromosomes at anaphase observed in cells expressing Smurf2(C716A). Immunofluorescence microscopy of cells stained with anti-Flag antibody for exogenously expressed Smurf2, ACA, and DAPI. Cells were harvested 8.5 h after release from the thymidine block. The second row shows misaligned chromosomes at metaphase, and the bottom row shows lagging chromosomes at anaphase in cells expressing Smurf2(C716A). Bars, 10 μm. (c) Percentages of cells with chromosomal defects in control cultures (open bars) and Smurf2(C716A)-transfected cultures (shaded bars). Data are means ± SEM (error bars) from three independent experiments.
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fig6: Expression of a catalytically inactive Smurf2 impairs the spindle assembly checkpoint with Mad2 destabilization. (a) Immunoblotting for mitotic regulators in HeLa cells transfected with Flag-tagged Smurf2(C716A) or a control plasmid. Cells were synchronized at S phase with thymidine treatment, released into a nocodazole-containing medium, and harvested after incubation at the indicated times after release (see Materials and methods). OE, overexpression. (b) Chromosomal misalignment at metaphase and lagging chromosomes at anaphase observed in cells expressing Smurf2(C716A). Immunofluorescence microscopy of cells stained with anti-Flag antibody for exogenously expressed Smurf2, ACA, and DAPI. Cells were harvested 8.5 h after release from the thymidine block. The second row shows misaligned chromosomes at metaphase, and the bottom row shows lagging chromosomes at anaphase in cells expressing Smurf2(C716A). Bars, 10 μm. (c) Percentages of cells with chromosomal defects in control cultures (open bars) and Smurf2(C716A)-transfected cultures (shaded bars). Data are means ± SEM (error bars) from three independent experiments.
Mentions: We next examined whether the E3 ligase activity of Smurf2 is important for its function to control the spindle assembly checkpoint. The cysteine-716 residue of Smurf2 is critical for its E3 ligase activity because Smurf2(C716A) protein is catalytically inactive (Kavsak et al., 2000). Thus, we transfected HeLa cells with an expression plasmid for Smurf2(C716A) and synchronized them at early S phase with double-thymidine treatments (Fig. 6 a). Cells were then released into a nocodazole-containing medium. Immunoblotting showed that cells transfected with Smurf2(C716A) failed to accumulate securin with significant reduction in Mad2 levels. Interestingly, cyclin B1 accumulation during G2/M progression was minimally affected, which is analogous to previous observations in Mad2 heterozygous cells (Michel et al., 2004). In contrast, transfection with wild-type Smurf2 affected none of these proteins (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200801049/DC1). This finding is consistent with the effects of Smurf2 siRNA, suggesting that Smurf2(C716A) can act as a dominant-negative mutant. Furthermore, cells transfected with Smurf2(C716A) showed a significant increase in cells displaying misaligned chromosomes, lagging chromosomes during mitotic exit, and multinucleation (Fig. 6, b and c). These data suggest that the E3 ligase activity of Smurf2 is required for proper control of the spindle assembly checkpoint.

Bottom Line: Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis.Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector.These data indicate that Smurf2 is a novel mitotic regulator.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

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.

Show MeSH
Related in: MedlinePlus