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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

CCDC124 mRNA is ubiquitously expressed in human tissues, and it encodes a 32 kDa protein.(A) Hybridization of part of the coding region of CCDC124 to an adult human multiple tissues Northern blot containing 2 µg of polyA-mRNA each lane. A single transcript of ∼1061 nucleotides was detectable in all human tissues analyzed, except the placenta with a second smaller transcript variant. The same blot was rehybridized with probes corresponding to two differentially expressed genes, β-actin and GAPDH, to monitor blotting quality. (B) Specific detection of ectopically expressed Ccdc124 by anti-Ccdc124 antibodies. HEK-293 cells, either non-transfected, or transfected with CMV-promoter controlled Ccdc124 were lysed, protein lysates were separated by SDS-PAGE, and immunoblot was performed either with anti-Ccdc124 antibodies alone, or same antibodies pre-incubated with 100 ng of competing peptide epitope corresponding to N-terminus 24mer peptide of Ccdc124. (C) Expression of Flag-tagged Ccdc124 protein was specifically detected by the anti-Ccdc124 or with anti-Flag antibodies, as indicated. Asterisk (*) indicates C-terminus flag-tag insertion dependent N-terminus cleaved form of Ccdc124. The expression of calnexin was confirmed in all cell lysates as an equal loading control.
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pone-0069289-g001: CCDC124 mRNA is ubiquitously expressed in human tissues, and it encodes a 32 kDa protein.(A) Hybridization of part of the coding region of CCDC124 to an adult human multiple tissues Northern blot containing 2 µg of polyA-mRNA each lane. A single transcript of ∼1061 nucleotides was detectable in all human tissues analyzed, except the placenta with a second smaller transcript variant. The same blot was rehybridized with probes corresponding to two differentially expressed genes, β-actin and GAPDH, to monitor blotting quality. (B) Specific detection of ectopically expressed Ccdc124 by anti-Ccdc124 antibodies. HEK-293 cells, either non-transfected, or transfected with CMV-promoter controlled Ccdc124 were lysed, protein lysates were separated by SDS-PAGE, and immunoblot was performed either with anti-Ccdc124 antibodies alone, or same antibodies pre-incubated with 100 ng of competing peptide epitope corresponding to N-terminus 24mer peptide of Ccdc124. (C) Expression of Flag-tagged Ccdc124 protein was specifically detected by the anti-Ccdc124 or with anti-Flag antibodies, as indicated. Asterisk (*) indicates C-terminus flag-tag insertion dependent N-terminus cleaved form of Ccdc124. The expression of calnexin was confirmed in all cell lysates as an equal loading control.

Mentions: Northern blot analysis have revealed that CCDC124 is ubiquitously expressed in all tested human tissues, and relatively high levels of expression were detected in the brain, placenta, liver, spleen, and prostate (Fig. 1A). In these analyses, a transcript of ∼1061 nucleotides was detectable in tested organs, in agreement with the predicted size of CCDC124 mRNA in the NCBI databases (http://genome.ucsc.edu), except in the placenta where we observed a second shorter mRNA species indicative of a transcript variant (Fig. 1A). CCDC124 cDNA would encode a protein of 223 amino acids with two putative coiled-coil domains between residues 18–82 in the N-terminal half of the protein as detected by the ELM (http://elm.eu.org) and COILS (www.ch.embnet.org/software/COILS_form.html) bioinformatics analysis platforms (Fig. S1). No significant homology to other proteins or domains were found.


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)

CCDC124 mRNA is ubiquitously expressed in human tissues, and it encodes a 32 kDa protein.(A) Hybridization of part of the coding region of CCDC124 to an adult human multiple tissues Northern blot containing 2 µg of polyA-mRNA each lane. A single transcript of ∼1061 nucleotides was detectable in all human tissues analyzed, except the placenta with a second smaller transcript variant. The same blot was rehybridized with probes corresponding to two differentially expressed genes, β-actin and GAPDH, to monitor blotting quality. (B) Specific detection of ectopically expressed Ccdc124 by anti-Ccdc124 antibodies. HEK-293 cells, either non-transfected, or transfected with CMV-promoter controlled Ccdc124 were lysed, protein lysates were separated by SDS-PAGE, and immunoblot was performed either with anti-Ccdc124 antibodies alone, or same antibodies pre-incubated with 100 ng of competing peptide epitope corresponding to N-terminus 24mer peptide of Ccdc124. (C) Expression of Flag-tagged Ccdc124 protein was specifically detected by the anti-Ccdc124 or with anti-Flag antibodies, as indicated. Asterisk (*) indicates C-terminus flag-tag insertion dependent N-terminus cleaved form of Ccdc124. The expression of calnexin was confirmed in all cell lysates as an equal loading control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3716640&req=5

pone-0069289-g001: CCDC124 mRNA is ubiquitously expressed in human tissues, and it encodes a 32 kDa protein.(A) Hybridization of part of the coding region of CCDC124 to an adult human multiple tissues Northern blot containing 2 µg of polyA-mRNA each lane. A single transcript of ∼1061 nucleotides was detectable in all human tissues analyzed, except the placenta with a second smaller transcript variant. The same blot was rehybridized with probes corresponding to two differentially expressed genes, β-actin and GAPDH, to monitor blotting quality. (B) Specific detection of ectopically expressed Ccdc124 by anti-Ccdc124 antibodies. HEK-293 cells, either non-transfected, or transfected with CMV-promoter controlled Ccdc124 were lysed, protein lysates were separated by SDS-PAGE, and immunoblot was performed either with anti-Ccdc124 antibodies alone, or same antibodies pre-incubated with 100 ng of competing peptide epitope corresponding to N-terminus 24mer peptide of Ccdc124. (C) Expression of Flag-tagged Ccdc124 protein was specifically detected by the anti-Ccdc124 or with anti-Flag antibodies, as indicated. Asterisk (*) indicates C-terminus flag-tag insertion dependent N-terminus cleaved form of Ccdc124. The expression of calnexin was confirmed in all cell lysates as an equal loading control.
Mentions: Northern blot analysis have revealed that CCDC124 is ubiquitously expressed in all tested human tissues, and relatively high levels of expression were detected in the brain, placenta, liver, spleen, and prostate (Fig. 1A). In these analyses, a transcript of ∼1061 nucleotides was detectable in tested organs, in agreement with the predicted size of CCDC124 mRNA in the NCBI databases (http://genome.ucsc.edu), except in the placenta where we observed a second shorter mRNA species indicative of a transcript variant (Fig. 1A). CCDC124 cDNA would encode a protein of 223 amino acids with two putative coiled-coil domains between residues 18–82 in the N-terminal half of the protein as detected by the ELM (http://elm.eu.org) and COILS (www.ch.embnet.org/software/COILS_form.html) bioinformatics analysis platforms (Fig. S1). No significant homology to other proteins or domains were found.

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