Limits...
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.

Show MeSH

Related in: MedlinePlus

Smurf2 is a cell cycle–regulated protein localizing specifically in the centrosome, mitotic midzone, and midbody. (a) HeLa cells were synchronized by a double-thymidine protocol. At the indicated hours after release from the block, cells were analyzed by immunoblotting for the proteins shown. (b) Immunofluorescence microscopy for γ-tubulin (green) and Smurf2 (red) in HeLa cells at interphase and metaphase. Polyclonal anti-Smurf2 antibody was used for Smurf2 staining. DAPI was used to stain chromosomal DNA (blue). The close-up images are shown with threefold higher magnification. (c) Immunofluorescent staining for α-tubulin (green) and Smurf2 (red) in human mammary epithelial MCF-10A cells during mitosis. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2568023&req=5

fig1: Smurf2 is a cell cycle–regulated protein localizing specifically in the centrosome, mitotic midzone, and midbody. (a) HeLa cells were synchronized by a double-thymidine protocol. At the indicated hours after release from the block, cells were analyzed by immunoblotting for the proteins shown. (b) Immunofluorescence microscopy for γ-tubulin (green) and Smurf2 (red) in HeLa cells at interphase and metaphase. Polyclonal anti-Smurf2 antibody was used for Smurf2 staining. DAPI was used to stain chromosomal DNA (blue). The close-up images are shown with threefold higher magnification. (c) Immunofluorescent staining for α-tubulin (green) and Smurf2 (red) in human mammary epithelial MCF-10A cells during mitosis. Bars, 10 μm.

Mentions: Pub1p, a fission yeast HECT family E3 ligase, regulates G2/M cell cycle progression by ubiquitinating Cdc25p (Nefsky and Beach, 1996). The mammalian HECT family includes Smurf1, Smurf2, AIP4/Itch, and Nedd4 (Kee and Huibregtse, 2007). Our recent study on the TGF-β regulation of Cdc25A ubiquitination (Ray et al., 2005) led us to examine whether Smurf proteins, which are known to modulate TGF-β signaling (Zhu et al., 1999; Kavsak et al., 2000), were involved in mitotic regulation. We first examined the levels of Smurf2 protein in HeLa cells synchronized by a thymidine-aphidicolin protocol (Fig. 1 a). The expression of Smurf2 protein was highest 6–8 h after release, which slightly preceded the peaks of cyclin B1 and Cdc25A expression around G2/M transition. Smurf1 expression was constant throughout the cell cycle (unpublished data). Thus, Smurf2 is a cell cycle–regulated protein that accumulates during late G2 through early mitosis. We then examined the subcellular localization of Smurf2, which revealed concentrated localization of Smurf2 at centrosomes in HeLa cells (Fig. 1 b). The specificity of Smurf2 polyclonal antibody used for immunofluorescence microscopy was verified by immunoblotting with Smurf2-depleted cells (Fig. S1 a, available at http://www.jcb.org/cgi/content/full/jcb.200801049/DC1). Smurf2 localized at perinuclear centrosomes during interphase as well as at centrosomes aligned bipolarly in metaphase cells, demonstrating colocalization with a centrosomal marker, γ-tubulin. Similar centrosomal localization of Smurf2 was observed in HeLa cells using Smurf2 monoclonal antibody and pericentrin antibody, another centrosomal marker, as well as in U2OS cells transfected with Flag-tagged Smurf2 (Fig. S1, b and c). We then examined Smurf2 localization in MCF-10A cells undergoing mitosis and cytokinesis in which the protein exhibited a dynamic relocalization pattern. Smurf2 localized predominantly at centrosomes during metaphase, whereas focal signals for Smurf2 were also observed in noncentrosomal structures in the cytoplasm (Fig. 1 c). During anaphase, a portion of Smurf2 apparently relocalized to the center of the spindle midzone, which is rich in microtubules stained with α-tubulin antibody. During telophase, Smurf2 was found concentrated at the midbody in the intercellular bridge. Similar Smurf2 relocalization during mitosis was observed in HeLa cells (unpublished data).


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 cell cycle–regulated protein localizing specifically in the centrosome, mitotic midzone, and midbody. (a) HeLa cells were synchronized by a double-thymidine protocol. At the indicated hours after release from the block, cells were analyzed by immunoblotting for the proteins shown. (b) Immunofluorescence microscopy for γ-tubulin (green) and Smurf2 (red) in HeLa cells at interphase and metaphase. Polyclonal anti-Smurf2 antibody was used for Smurf2 staining. DAPI was used to stain chromosomal DNA (blue). The close-up images are shown with threefold higher magnification. (c) Immunofluorescent staining for α-tubulin (green) and Smurf2 (red) in human mammary epithelial MCF-10A cells during mitosis. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2568023&req=5

fig1: Smurf2 is a cell cycle–regulated protein localizing specifically in the centrosome, mitotic midzone, and midbody. (a) HeLa cells were synchronized by a double-thymidine protocol. At the indicated hours after release from the block, cells were analyzed by immunoblotting for the proteins shown. (b) Immunofluorescence microscopy for γ-tubulin (green) and Smurf2 (red) in HeLa cells at interphase and metaphase. Polyclonal anti-Smurf2 antibody was used for Smurf2 staining. DAPI was used to stain chromosomal DNA (blue). The close-up images are shown with threefold higher magnification. (c) Immunofluorescent staining for α-tubulin (green) and Smurf2 (red) in human mammary epithelial MCF-10A cells during mitosis. Bars, 10 μm.
Mentions: Pub1p, a fission yeast HECT family E3 ligase, regulates G2/M cell cycle progression by ubiquitinating Cdc25p (Nefsky and Beach, 1996). The mammalian HECT family includes Smurf1, Smurf2, AIP4/Itch, and Nedd4 (Kee and Huibregtse, 2007). Our recent study on the TGF-β regulation of Cdc25A ubiquitination (Ray et al., 2005) led us to examine whether Smurf proteins, which are known to modulate TGF-β signaling (Zhu et al., 1999; Kavsak et al., 2000), were involved in mitotic regulation. We first examined the levels of Smurf2 protein in HeLa cells synchronized by a thymidine-aphidicolin protocol (Fig. 1 a). The expression of Smurf2 protein was highest 6–8 h after release, which slightly preceded the peaks of cyclin B1 and Cdc25A expression around G2/M transition. Smurf1 expression was constant throughout the cell cycle (unpublished data). Thus, Smurf2 is a cell cycle–regulated protein that accumulates during late G2 through early mitosis. We then examined the subcellular localization of Smurf2, which revealed concentrated localization of Smurf2 at centrosomes in HeLa cells (Fig. 1 b). The specificity of Smurf2 polyclonal antibody used for immunofluorescence microscopy was verified by immunoblotting with Smurf2-depleted cells (Fig. S1 a, available at http://www.jcb.org/cgi/content/full/jcb.200801049/DC1). Smurf2 localized at perinuclear centrosomes during interphase as well as at centrosomes aligned bipolarly in metaphase cells, demonstrating colocalization with a centrosomal marker, γ-tubulin. Similar centrosomal localization of Smurf2 was observed in HeLa cells using Smurf2 monoclonal antibody and pericentrin antibody, another centrosomal marker, as well as in U2OS cells transfected with Flag-tagged Smurf2 (Fig. S1, b and c). We then examined Smurf2 localization in MCF-10A cells undergoing mitosis and cytokinesis in which the protein exhibited a dynamic relocalization pattern. Smurf2 localized predominantly at centrosomes during metaphase, whereas focal signals for Smurf2 were also observed in noncentrosomal structures in the cytoplasm (Fig. 1 c). During anaphase, a portion of Smurf2 apparently relocalized to the center of the spindle midzone, which is rich in microtubules stained with α-tubulin antibody. During telophase, Smurf2 was found concentrated at the midbody in the intercellular bridge. Similar Smurf2 relocalization during mitosis was observed in HeLa cells (unpublished data).

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