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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

The guanine nucleotide exchange factor RasGEF1B controls cellular Rap2.GTP status.HEK-293 cells were transfected either with RasGEF1B specific shRNA expressing vector (Sh-D, Figure S4), scrambled shRNA expressing vector, or empty vector (negative control), 48 hrs later cells were lysed as described at Materials and Methods, they were cleared by centrifugation, and active Rap was precipitated with a glutathione S-transferase fusion protein of the Ras-binding domain of RalGDS precoupled to glutathione-Sepharose beads. Rap2 activation assay were carried-out by using anti-Rap2 monoclonal Abs. Results confirmed that shRNA mediated down-regulation of RasGEF1B expression effectively block generation of Rap2.GTP, although total cellular Rap2 was not affected. Calnexin expression was monitored as loading control.
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pone-0069289-g007: The guanine nucleotide exchange factor RasGEF1B controls cellular Rap2.GTP status.HEK-293 cells were transfected either with RasGEF1B specific shRNA expressing vector (Sh-D, Figure S4), scrambled shRNA expressing vector, or empty vector (negative control), 48 hrs later cells were lysed as described at Materials and Methods, they were cleared by centrifugation, and active Rap was precipitated with a glutathione S-transferase fusion protein of the Ras-binding domain of RalGDS precoupled to glutathione-Sepharose beads. Rap2 activation assay were carried-out by using anti-Rap2 monoclonal Abs. Results confirmed that shRNA mediated down-regulation of RasGEF1B expression effectively block generation of Rap2.GTP, although total cellular Rap2 was not affected. Calnexin expression was monitored as loading control.

Mentions: Activation cycle of small G proteins is regulated by guanine nucleotide exchange factors (GEFs), which induce dissociation of bound GDP and its replacement by the more abundant GTP, and the resulting conformational change allows the binding of effector proteins and thereby stimulation of downstream signaling. Previous functional studies by Yaman et al. [24] indicated that under in vitro conditions RasGEF1B specifically activates Rap2 by stimulating guanine nucleotide exchange only of this small G protein, whereas it does not activate even its close family member, Rap1, or other members of Ras family. We decided to take these in vitro studies to one level up, and confirm the stimulatory effect of RasGEF1B on Rap2 GDP/GTP nucleotide exchange by knocking-down this GEF in HEK-293 cells, followed by assessing GTP-bound active Rap2 (Rap2.GTP) status in these cells. For this, we used a Rap-activity assay method based on immunoprecipitation of active Rap proteins by the Rap-binding domain of RalGDS (RBD-RalGDS), as previously described [31]. When RasGEF1B is knocked-down to nearly 55% in HEK-293 cells by specific down-regulatory shRNA-containing vectors (Fig. S4), significantly less Rap2.GTP were present in cells, even though total Rap2 amounts were not affected (Fig. 7). This indicated a functional link between RasGEF1B and Rap2 GTP-binding protein activation in this cell system. These results were consistent with our previous in vitro studies that established Rap2 as the sole RasGEF1B activated Ras-family GTP-binding protein [24].


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)

The guanine nucleotide exchange factor RasGEF1B controls cellular Rap2.GTP status.HEK-293 cells were transfected either with RasGEF1B specific shRNA expressing vector (Sh-D, Figure S4), scrambled shRNA expressing vector, or empty vector (negative control), 48 hrs later cells were lysed as described at Materials and Methods, they were cleared by centrifugation, and active Rap was precipitated with a glutathione S-transferase fusion protein of the Ras-binding domain of RalGDS precoupled to glutathione-Sepharose beads. Rap2 activation assay were carried-out by using anti-Rap2 monoclonal Abs. Results confirmed that shRNA mediated down-regulation of RasGEF1B expression effectively block generation of Rap2.GTP, although total cellular Rap2 was not affected. Calnexin expression was monitored as loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3716640&req=5

pone-0069289-g007: The guanine nucleotide exchange factor RasGEF1B controls cellular Rap2.GTP status.HEK-293 cells were transfected either with RasGEF1B specific shRNA expressing vector (Sh-D, Figure S4), scrambled shRNA expressing vector, or empty vector (negative control), 48 hrs later cells were lysed as described at Materials and Methods, they were cleared by centrifugation, and active Rap was precipitated with a glutathione S-transferase fusion protein of the Ras-binding domain of RalGDS precoupled to glutathione-Sepharose beads. Rap2 activation assay were carried-out by using anti-Rap2 monoclonal Abs. Results confirmed that shRNA mediated down-regulation of RasGEF1B expression effectively block generation of Rap2.GTP, although total cellular Rap2 was not affected. Calnexin expression was monitored as loading control.
Mentions: Activation cycle of small G proteins is regulated by guanine nucleotide exchange factors (GEFs), which induce dissociation of bound GDP and its replacement by the more abundant GTP, and the resulting conformational change allows the binding of effector proteins and thereby stimulation of downstream signaling. Previous functional studies by Yaman et al. [24] indicated that under in vitro conditions RasGEF1B specifically activates Rap2 by stimulating guanine nucleotide exchange only of this small G protein, whereas it does not activate even its close family member, Rap1, or other members of Ras family. We decided to take these in vitro studies to one level up, and confirm the stimulatory effect of RasGEF1B on Rap2 GDP/GTP nucleotide exchange by knocking-down this GEF in HEK-293 cells, followed by assessing GTP-bound active Rap2 (Rap2.GTP) status in these cells. For this, we used a Rap-activity assay method based on immunoprecipitation of active Rap proteins by the Rap-binding domain of RalGDS (RBD-RalGDS), as previously described [31]. When RasGEF1B is knocked-down to nearly 55% in HEK-293 cells by specific down-regulatory shRNA-containing vectors (Fig. S4), significantly less Rap2.GTP were present in cells, even though total Rap2 amounts were not affected (Fig. 7). This indicated a functional link between RasGEF1B and Rap2 GTP-binding protein activation in this cell system. These results were consistent with our previous in vitro studies that established Rap2 as the sole RasGEF1B activated Ras-family GTP-binding protein [24].

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