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Coiled-coil domain containing protein 124 is a novel centrosome and midbody protein that interacts with the Ras-guanine nucleotide exchange factor 1B and is involved in cytokinesis.

Telkoparan P, Erkek S, Yaman E, Alotaibi H, Bayık D, Tazebay UH - PLoS ONE (2013)

Bottom Line: Here, we report the first characterization of coiled-coil domain-containing protein-124 (Ccdc124), a eukaryotic protein conserved from fungi-to-man, which we identified as a novel centrosomal and midbody protein.Knockdown of Ccdc124 in human HeLa cells leads to accumulation of enlarged and multinucleated cells; however, centrosome maturation was not affected.We found that Ccdc124 interacts with the Ras-guanine nucleotide exchange factor 1B (RasGEF1B), establishing a functional link between cytokinesis and activation of localized Rap2 signaling at the midbody.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, Ankara, Turkey.

ABSTRACT
Cytokinetic abscission is the cellular process leading to physical separation of two postmitotic sister cells by severing the intercellular bridge. The most noticeable structural component of the intercellular bridge is a transient organelle termed as midbody, localized at a central region marking the site of abscission. Despite its major role in completion of cytokinesis, our understanding of spatiotemporal regulation of midbody assembly is limited. Here, we report the first characterization of coiled-coil domain-containing protein-124 (Ccdc124), a eukaryotic protein conserved from fungi-to-man, which we identified as a novel centrosomal and midbody protein. Knockdown of Ccdc124 in human HeLa cells leads to accumulation of enlarged and multinucleated cells; however, centrosome maturation was not affected. We found that Ccdc124 interacts with the Ras-guanine nucleotide exchange factor 1B (RasGEF1B), establishing a functional link between cytokinesis and activation of localized Rap2 signaling at the midbody. Our data indicate that Ccdc124 is a novel factor operating both for proper progression of late cytokinetic stages in eukaryotes, and for establishment of Rap2 signaling dependent cellular functions proximal to the abscission site.

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Related in: MedlinePlus

RasGEF1B is an interaction partner of Ccdc124.(A) In vitro GST pull-down assay indicating a possible interaction between RasGEF1B and Ccdc124. GST-RasGEF1B protein were immobilized on GSH-beads, followed by incubation with empty PBS buffer control (lane 1), or with bacteria purified His-tagged Ccdc124 (lane 2). As controls, GSH-beads w/o RasGEF1B protein incubated with His-Ccdc124 to monitor the amount of His-Ccdc124 proteins binding to GSH-beads in the absence of a putative interaction partner (lane 3), or GSH-beads immobilized with GST protein and incubated with His-Ccdc124 to monitor interaction capacity of Ccdc124 with GST (lane 4). Lanes 5, 6, 7 are stainings of 100 ng bacteria purified GST-RasGEF1B, His-Ccdc124, and GST proteins, respectively, run in the same gel to monitor their corresponding sizes. Bands corresponding to His-Ccdc124 were marked with asterisks (*). (B) HEK-293 cells were either transfected with Flag-Ccdc124 or GFP-RasGEF1B expression vectors alone or with indicated control plasmids, or alternatively they were co-transfected with Flag-Ccdc124 and GFP-RasGEF1B together, followed by immunoprecipitations (IP) on cell lysates using protein-G beads with anti-Flag antibodies. Subsequently, immunoblots were done on IP or cell lysate samples using anti-GFP (monitoring GFP-RasGEF1B) or anti-Flag-HRP (to assess Flag-Ccdc124) antibodies.
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pone-0069289-g005: RasGEF1B is an interaction partner of Ccdc124.(A) In vitro GST pull-down assay indicating a possible interaction between RasGEF1B and Ccdc124. GST-RasGEF1B protein were immobilized on GSH-beads, followed by incubation with empty PBS buffer control (lane 1), or with bacteria purified His-tagged Ccdc124 (lane 2). As controls, GSH-beads w/o RasGEF1B protein incubated with His-Ccdc124 to monitor the amount of His-Ccdc124 proteins binding to GSH-beads in the absence of a putative interaction partner (lane 3), or GSH-beads immobilized with GST protein and incubated with His-Ccdc124 to monitor interaction capacity of Ccdc124 with GST (lane 4). Lanes 5, 6, 7 are stainings of 100 ng bacteria purified GST-RasGEF1B, His-Ccdc124, and GST proteins, respectively, run in the same gel to monitor their corresponding sizes. Bands corresponding to His-Ccdc124 were marked with asterisks (*). (B) HEK-293 cells were either transfected with Flag-Ccdc124 or GFP-RasGEF1B expression vectors alone or with indicated control plasmids, or alternatively they were co-transfected with Flag-Ccdc124 and GFP-RasGEF1B together, followed by immunoprecipitations (IP) on cell lysates using protein-G beads with anti-Flag antibodies. Subsequently, immunoblots were done on IP or cell lysate samples using anti-GFP (monitoring GFP-RasGEF1B) or anti-Flag-HRP (to assess Flag-Ccdc124) antibodies.

Mentions: Following the yeast two-hybrid assays, we were able to confirm the interaction between Ccdc124 and RasGEF1B, first by in vitro GST pull-down methods (Fig. 5A), and then by analyzing their association in mammalian HEK-293 cells transfected with flag-Ccdc124 and GFP-RasGEF1B containing expression vectors (Fig. 5B). Coimmunoprecipitation of GFP-RasGEF1B and flag-Ccdc124 in transfected cells could indicate a functional interaction between these two proteins. In parallel to functional studies to establish cellular roles of Ccdc124 and RasGEF1B, we sought to determine whether the subcellular localizations of these two proteins were comparable throughout the cell cycle. In fact, in a previous study, RasGEF1B was shown to localize in endosomal vesicles when fluorescent protein-fused versions (YFP-RasGEF1B or mRFP-RasGEF1B) were ectopically expressed in asynchronous CHO cells [29]. However, the subcellular localization of RasGEF1B was not previously addressed in cell cycle-synchronized cells. Identification of an endosomal vesicle factor such as RasGEF1B as an interaction partner of centrosomal and/or midbody localized Ccdc124 is particularly interesting, as critical roles of endosomes in physical separation of cells during cytokinetic abscission are well established in recent studies (for a review, see [30]). Subsequently, we generated a rabbit polyclonal antibody recognizing the C-terminal 19 amino acids epitope of the Zebrafish orthologue of RasGEF1B, and by immunoblotting analysis we first established that it also recognizes the human RasGEF1B protein (Fig. S3). Then, by using this antibody, we observed that even though in HeLa cells at interphase and prophase it displays characteristics of previously suggested cytoplasmic/early endosome localization [29], in metaphase cells RasGEF1B was located at a pericentrosomal/centrosomal position, as assessed by its co-localization with γ-tubulin, a subcellular localization similar to that Ccdc124 at metaphase cells (Fig. 6A, compare with Fig. 2).


Coiled-coil domain containing protein 124 is a novel centrosome and midbody protein that interacts with the Ras-guanine nucleotide exchange factor 1B and is involved in cytokinesis.

Telkoparan P, Erkek S, Yaman E, Alotaibi H, Bayık D, Tazebay UH - PLoS ONE (2013)

RasGEF1B is an interaction partner of Ccdc124.(A) In vitro GST pull-down assay indicating a possible interaction between RasGEF1B and Ccdc124. GST-RasGEF1B protein were immobilized on GSH-beads, followed by incubation with empty PBS buffer control (lane 1), or with bacteria purified His-tagged Ccdc124 (lane 2). As controls, GSH-beads w/o RasGEF1B protein incubated with His-Ccdc124 to monitor the amount of His-Ccdc124 proteins binding to GSH-beads in the absence of a putative interaction partner (lane 3), or GSH-beads immobilized with GST protein and incubated with His-Ccdc124 to monitor interaction capacity of Ccdc124 with GST (lane 4). Lanes 5, 6, 7 are stainings of 100 ng bacteria purified GST-RasGEF1B, His-Ccdc124, and GST proteins, respectively, run in the same gel to monitor their corresponding sizes. Bands corresponding to His-Ccdc124 were marked with asterisks (*). (B) HEK-293 cells were either transfected with Flag-Ccdc124 or GFP-RasGEF1B expression vectors alone or with indicated control plasmids, or alternatively they were co-transfected with Flag-Ccdc124 and GFP-RasGEF1B together, followed by immunoprecipitations (IP) on cell lysates using protein-G beads with anti-Flag antibodies. Subsequently, immunoblots were done on IP or cell lysate samples using anti-GFP (monitoring GFP-RasGEF1B) or anti-Flag-HRP (to assess Flag-Ccdc124) antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3716640&req=5

pone-0069289-g005: RasGEF1B is an interaction partner of Ccdc124.(A) In vitro GST pull-down assay indicating a possible interaction between RasGEF1B and Ccdc124. GST-RasGEF1B protein were immobilized on GSH-beads, followed by incubation with empty PBS buffer control (lane 1), or with bacteria purified His-tagged Ccdc124 (lane 2). As controls, GSH-beads w/o RasGEF1B protein incubated with His-Ccdc124 to monitor the amount of His-Ccdc124 proteins binding to GSH-beads in the absence of a putative interaction partner (lane 3), or GSH-beads immobilized with GST protein and incubated with His-Ccdc124 to monitor interaction capacity of Ccdc124 with GST (lane 4). Lanes 5, 6, 7 are stainings of 100 ng bacteria purified GST-RasGEF1B, His-Ccdc124, and GST proteins, respectively, run in the same gel to monitor their corresponding sizes. Bands corresponding to His-Ccdc124 were marked with asterisks (*). (B) HEK-293 cells were either transfected with Flag-Ccdc124 or GFP-RasGEF1B expression vectors alone or with indicated control plasmids, or alternatively they were co-transfected with Flag-Ccdc124 and GFP-RasGEF1B together, followed by immunoprecipitations (IP) on cell lysates using protein-G beads with anti-Flag antibodies. Subsequently, immunoblots were done on IP or cell lysate samples using anti-GFP (monitoring GFP-RasGEF1B) or anti-Flag-HRP (to assess Flag-Ccdc124) antibodies.
Mentions: Following the yeast two-hybrid assays, we were able to confirm the interaction between Ccdc124 and RasGEF1B, first by in vitro GST pull-down methods (Fig. 5A), and then by analyzing their association in mammalian HEK-293 cells transfected with flag-Ccdc124 and GFP-RasGEF1B containing expression vectors (Fig. 5B). Coimmunoprecipitation of GFP-RasGEF1B and flag-Ccdc124 in transfected cells could indicate a functional interaction between these two proteins. In parallel to functional studies to establish cellular roles of Ccdc124 and RasGEF1B, we sought to determine whether the subcellular localizations of these two proteins were comparable throughout the cell cycle. In fact, in a previous study, RasGEF1B was shown to localize in endosomal vesicles when fluorescent protein-fused versions (YFP-RasGEF1B or mRFP-RasGEF1B) were ectopically expressed in asynchronous CHO cells [29]. However, the subcellular localization of RasGEF1B was not previously addressed in cell cycle-synchronized cells. Identification of an endosomal vesicle factor such as RasGEF1B as an interaction partner of centrosomal and/or midbody localized Ccdc124 is particularly interesting, as critical roles of endosomes in physical separation of cells during cytokinetic abscission are well established in recent studies (for a review, see [30]). Subsequently, we generated a rabbit polyclonal antibody recognizing the C-terminal 19 amino acids epitope of the Zebrafish orthologue of RasGEF1B, and by immunoblotting analysis we first established that it also recognizes the human RasGEF1B protein (Fig. S3). Then, by using this antibody, we observed that even though in HeLa cells at interphase and prophase it displays characteristics of previously suggested cytoplasmic/early endosome localization [29], in metaphase cells RasGEF1B was located at a pericentrosomal/centrosomal position, as assessed by its co-localization with γ-tubulin, a subcellular localization similar to that Ccdc124 at metaphase cells (Fig. 6A, compare with Fig. 2).

Bottom Line: Here, we report the first characterization of coiled-coil domain-containing protein-124 (Ccdc124), a eukaryotic protein conserved from fungi-to-man, which we identified as a novel centrosomal and midbody protein.Knockdown of Ccdc124 in human HeLa cells leads to accumulation of enlarged and multinucleated cells; however, centrosome maturation was not affected.We found that Ccdc124 interacts with the Ras-guanine nucleotide exchange factor 1B (RasGEF1B), establishing a functional link between cytokinesis and activation of localized Rap2 signaling at the midbody.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, Ankara, Turkey.

ABSTRACT
Cytokinetic abscission is the cellular process leading to physical separation of two postmitotic sister cells by severing the intercellular bridge. The most noticeable structural component of the intercellular bridge is a transient organelle termed as midbody, localized at a central region marking the site of abscission. Despite its major role in completion of cytokinesis, our understanding of spatiotemporal regulation of midbody assembly is limited. Here, we report the first characterization of coiled-coil domain-containing protein-124 (Ccdc124), a eukaryotic protein conserved from fungi-to-man, which we identified as a novel centrosomal and midbody protein. Knockdown of Ccdc124 in human HeLa cells leads to accumulation of enlarged and multinucleated cells; however, centrosome maturation was not affected. We found that Ccdc124 interacts with the Ras-guanine nucleotide exchange factor 1B (RasGEF1B), establishing a functional link between cytokinesis and activation of localized Rap2 signaling at the midbody. Our data indicate that Ccdc124 is a novel factor operating both for proper progression of late cytokinetic stages in eukaryotes, and for establishment of Rap2 signaling dependent cellular functions proximal to the abscission site.

Show MeSH
Related in: MedlinePlus