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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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Smurf2 is a novel regulator of the spindle assembly checkpoint. (a) Smurf2 localizes at centromeres when the spindle checkpoint is active. HeLa cells were synchronized by a double-thymidine protocol (see Materials and methods). Cells were then released into a fresh medium (time 0), and 3 h later nocodazole was added. Immunofluorescence microscopy after staining with ACA, anti-Smurf2 antibody, and DAPI for DNA. Cells were fixed 8.5 h after release from the thymidine in the presence of nocodazole. (b) Smurf2 depletion results in failed accumulation of APC/C-Cdc20 substrates during nocodazole-induced metaphase arrest. Immunoblotting was performed for mitotic regulators in HeLa cells transfected with Smurf2 siRNA #1 or nonspecific dsRNA (siNS). After release from the thymidine block, cells were incubated for the indicated hours in the presence of nocodazole. (c) Smurf2-depleted cells override nocodazole-induced spindle assembly checkpoint, resulting in tetraploidy. Control (siNS) and Smurf2-depleted cells were fixed at the indicated times and subjected to flow cytometry. (d) Cosilencing of Cdc20 restores mitotic accumulation of cyclin B1 and securin in Smurf2-depleted HeLa cells. Cells were synchronized and released into nocodazole-containing medium as described in b. (e and f) Smurf2 depletion results in loss of Mad2 from centromeres, whereas centromere localization of BubR1 is unaffected. Cells were fixed 8.5 h after release from the thymidine in the presence of nocodazole, and immunofluorescence microscopy was performed. (a, e, and f) The close-up images are shown with threefold higher magnification. Bars, 10 μm.
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fig4: Smurf2 is a novel regulator of the spindle assembly checkpoint. (a) Smurf2 localizes at centromeres when the spindle checkpoint is active. HeLa cells were synchronized by a double-thymidine protocol (see Materials and methods). Cells were then released into a fresh medium (time 0), and 3 h later nocodazole was added. Immunofluorescence microscopy after staining with ACA, anti-Smurf2 antibody, and DAPI for DNA. Cells were fixed 8.5 h after release from the thymidine in the presence of nocodazole. (b) Smurf2 depletion results in failed accumulation of APC/C-Cdc20 substrates during nocodazole-induced metaphase arrest. Immunoblotting was performed for mitotic regulators in HeLa cells transfected with Smurf2 siRNA #1 or nonspecific dsRNA (siNS). After release from the thymidine block, cells were incubated for the indicated hours in the presence of nocodazole. (c) Smurf2-depleted cells override nocodazole-induced spindle assembly checkpoint, resulting in tetraploidy. Control (siNS) and Smurf2-depleted cells were fixed at the indicated times and subjected to flow cytometry. (d) Cosilencing of Cdc20 restores mitotic accumulation of cyclin B1 and securin in Smurf2-depleted HeLa cells. Cells were synchronized and released into nocodazole-containing medium as described in b. (e and f) Smurf2 depletion results in loss of Mad2 from centromeres, whereas centromere localization of BubR1 is unaffected. Cells were fixed 8.5 h after release from the thymidine in the presence of nocodazole, and immunofluorescence microscopy was performed. (a, e, and f) The close-up images are shown with threefold higher magnification. Bars, 10 μm.

Mentions: Defective chromosome alignment/segregation and premature anaphase onset in Smurf2-depleted cells are reminiscent of the phenotypes of impaired spindle assembly checkpoint. To examine whether Smurf2 is involved in the checkpoint, cells progressing synchronously through G2 toward M were treated with the microtubule polymerization inhibitor nocodazole. For examinations of Smurf2 localization, cells were fixed as they progressed into prometaphase arrest (Fig. 4 a). Interestingly, Smurf2 localized in a focal fashion and was closely associated with the immunoreactivities against human anticentromere antibodies (ACAs), which recognize several centromere proteins (Earnshaw et al., 1986). The localization of Smurf2 at centromeres was observed only transiently in cells undergoing nocodazole-induced arrest at prometaphase. This localization pattern is consistent with a putative role for Smurf2 in the spindle assembly checkpoint. We then determined cellular levels of mitosis regulatory proteins in Smurf2-depleted and control cells synchronously progressing into mitosis in the presence of nocodazole (Fig. 4 b). Cellular levels of Smurf2 and the APC/C-Cdc20 substrates securin, cyclin B1, and aurora B (Fang et al., 1999) accumulated as control cells progressed through the G2/M transition 8–10 h after release from the thymidine block. Cyclin A2 was markedly down-regulated during nocodazole-induced prometaphase arrest as expected (Fry and Yamano, 2006). Strikingly, securin, cyclin B1, and aurora B failed to accumulate in Smurf2-depleted cells. In contrast, Smurf2 depletion affected minimally or only modestly the levels of Emi1, cyclin A2, and the APC/C-Cdh1 substrates Plk1 and Cdc20. These proteins are not direct targets of APC/C-Cdc20, whereas cyclin B1, securin, and aurora B are well-characterized substrates. These data suggest that Smurf2 depletion results in premature activation of APC/C-Cdc20. Flow cytometry further demonstrated that control cells were arrested with 4N DNA content 14 h after release from the thymidine block, whereas 17% of Smurf2-depleted cells had 8N DNA, which is consistent with binucleation (Fig. 4 c). 26 h after release, 63% of Smurf2-depleted cells exhibited 8N DNA. These data suggest that Smurf2 depletion resulted in continued cell cycle progression in the presence of nocodazole without successful cytokinesis. To confirm that Smurf2 depletion permitted inappropriate APC/C-Cdc20 activity in the presence of nocodazole, we attempted to silence Cdc20 in Smurf2-depleted cells (Fig. 4 d). siRNA-mediated codepletion of Cdc20 and Smurf2 resulted in restored mitotic accumulation of cyclin B1 and securin (but not Mad2; Fig. 4 d, fourth row) in synchronized cultures of nocodazole-treated HeLa cells. Aurora B is also up-regulated modestly by the codepletion. Importantly, the addition of Cdc20 siRNA did not significantly affect the silencing efficacy of the Smurf2 siRNA or the levels of cyclin A2 and Plk1. These data suggest that Smurf2 is required for the spindle assembly checkpoint–mediated inhibition of APC/C-Cdc20 and mitotic accumulation of securin and cyclin B1.


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)

Smurf2 is a novel regulator of the spindle assembly checkpoint. (a) Smurf2 localizes at centromeres when the spindle checkpoint is active. HeLa cells were synchronized by a double-thymidine protocol (see Materials and methods). Cells were then released into a fresh medium (time 0), and 3 h later nocodazole was added. Immunofluorescence microscopy after staining with ACA, anti-Smurf2 antibody, and DAPI for DNA. Cells were fixed 8.5 h after release from the thymidine in the presence of nocodazole. (b) Smurf2 depletion results in failed accumulation of APC/C-Cdc20 substrates during nocodazole-induced metaphase arrest. Immunoblotting was performed for mitotic regulators in HeLa cells transfected with Smurf2 siRNA #1 or nonspecific dsRNA (siNS). After release from the thymidine block, cells were incubated for the indicated hours in the presence of nocodazole. (c) Smurf2-depleted cells override nocodazole-induced spindle assembly checkpoint, resulting in tetraploidy. Control (siNS) and Smurf2-depleted cells were fixed at the indicated times and subjected to flow cytometry. (d) Cosilencing of Cdc20 restores mitotic accumulation of cyclin B1 and securin in Smurf2-depleted HeLa cells. Cells were synchronized and released into nocodazole-containing medium as described in b. (e and f) Smurf2 depletion results in loss of Mad2 from centromeres, whereas centromere localization of BubR1 is unaffected. Cells were fixed 8.5 h after release from the thymidine in the presence of nocodazole, and immunofluorescence microscopy was performed. (a, e, and f) The close-up images are shown with threefold higher magnification. Bars, 10 μm.
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fig4: Smurf2 is a novel regulator of the spindle assembly checkpoint. (a) Smurf2 localizes at centromeres when the spindle checkpoint is active. HeLa cells were synchronized by a double-thymidine protocol (see Materials and methods). Cells were then released into a fresh medium (time 0), and 3 h later nocodazole was added. Immunofluorescence microscopy after staining with ACA, anti-Smurf2 antibody, and DAPI for DNA. Cells were fixed 8.5 h after release from the thymidine in the presence of nocodazole. (b) Smurf2 depletion results in failed accumulation of APC/C-Cdc20 substrates during nocodazole-induced metaphase arrest. Immunoblotting was performed for mitotic regulators in HeLa cells transfected with Smurf2 siRNA #1 or nonspecific dsRNA (siNS). After release from the thymidine block, cells were incubated for the indicated hours in the presence of nocodazole. (c) Smurf2-depleted cells override nocodazole-induced spindle assembly checkpoint, resulting in tetraploidy. Control (siNS) and Smurf2-depleted cells were fixed at the indicated times and subjected to flow cytometry. (d) Cosilencing of Cdc20 restores mitotic accumulation of cyclin B1 and securin in Smurf2-depleted HeLa cells. Cells were synchronized and released into nocodazole-containing medium as described in b. (e and f) Smurf2 depletion results in loss of Mad2 from centromeres, whereas centromere localization of BubR1 is unaffected. Cells were fixed 8.5 h after release from the thymidine in the presence of nocodazole, and immunofluorescence microscopy was performed. (a, e, and f) The close-up images are shown with threefold higher magnification. Bars, 10 μm.
Mentions: Defective chromosome alignment/segregation and premature anaphase onset in Smurf2-depleted cells are reminiscent of the phenotypes of impaired spindle assembly checkpoint. To examine whether Smurf2 is involved in the checkpoint, cells progressing synchronously through G2 toward M were treated with the microtubule polymerization inhibitor nocodazole. For examinations of Smurf2 localization, cells were fixed as they progressed into prometaphase arrest (Fig. 4 a). Interestingly, Smurf2 localized in a focal fashion and was closely associated with the immunoreactivities against human anticentromere antibodies (ACAs), which recognize several centromere proteins (Earnshaw et al., 1986). The localization of Smurf2 at centromeres was observed only transiently in cells undergoing nocodazole-induced arrest at prometaphase. This localization pattern is consistent with a putative role for Smurf2 in the spindle assembly checkpoint. We then determined cellular levels of mitosis regulatory proteins in Smurf2-depleted and control cells synchronously progressing into mitosis in the presence of nocodazole (Fig. 4 b). Cellular levels of Smurf2 and the APC/C-Cdc20 substrates securin, cyclin B1, and aurora B (Fang et al., 1999) accumulated as control cells progressed through the G2/M transition 8–10 h after release from the thymidine block. Cyclin A2 was markedly down-regulated during nocodazole-induced prometaphase arrest as expected (Fry and Yamano, 2006). Strikingly, securin, cyclin B1, and aurora B failed to accumulate in Smurf2-depleted cells. In contrast, Smurf2 depletion affected minimally or only modestly the levels of Emi1, cyclin A2, and the APC/C-Cdh1 substrates Plk1 and Cdc20. These proteins are not direct targets of APC/C-Cdc20, whereas cyclin B1, securin, and aurora B are well-characterized substrates. These data suggest that Smurf2 depletion results in premature activation of APC/C-Cdc20. Flow cytometry further demonstrated that control cells were arrested with 4N DNA content 14 h after release from the thymidine block, whereas 17% of Smurf2-depleted cells had 8N DNA, which is consistent with binucleation (Fig. 4 c). 26 h after release, 63% of Smurf2-depleted cells exhibited 8N DNA. These data suggest that Smurf2 depletion resulted in continued cell cycle progression in the presence of nocodazole without successful cytokinesis. To confirm that Smurf2 depletion permitted inappropriate APC/C-Cdc20 activity in the presence of nocodazole, we attempted to silence Cdc20 in Smurf2-depleted cells (Fig. 4 d). siRNA-mediated codepletion of Cdc20 and Smurf2 resulted in restored mitotic accumulation of cyclin B1 and securin (but not Mad2; Fig. 4 d, fourth row) in synchronized cultures of nocodazole-treated HeLa cells. Aurora B is also up-regulated modestly by the codepletion. Importantly, the addition of Cdc20 siRNA did not significantly affect the silencing efficacy of the Smurf2 siRNA or the levels of cyclin A2 and Plk1. These data suggest that Smurf2 is required for the spindle assembly checkpoint–mediated inhibition of APC/C-Cdc20 and mitotic accumulation of securin and cyclin B1.

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