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The chromosomal passenger complex controls the function of endosomal sorting complex required for transport-III Snf7 proteins during cytokinesis.

Capalbo L, Montembault E, Takeda T, Bassi ZI, Glover DM, D'Avino PP - Open Biol (2012)

Bottom Line: Here, we show that the CPC subunit Borealin interacts directly with the Snf7 components of ESCRT-III in both Drosophila and human cells.Moreover, we find that the CPC's catalytic subunit, Aurora B kinase, phosphorylates one of the three human Snf7 paralogues-CHMP4C-in its C-terminal tail, a region known to regulate its ability to form polymers and associate with membranes.Phosphorylation at these sites appears essential for CHMP4C function because their mutation leads to cytokinesis defects.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.

ABSTRACT
Cytokinesis controls the proper segregation of nuclear and cytoplasmic materials at the end of cell division. The chromosomal passenger complex (CPC) has been proposed to monitor the final separation of the two daughter cells at the end of cytokinesis in order to prevent cell abscission in the presence of DNA at the cleavage site, but the precise molecular basis for this is unclear. Recent studies indicate that abscission could be mediated by the assembly of filaments comprising components of the endosomal sorting complex required for transport-III (ESCRT-III). Here, we show that the CPC subunit Borealin interacts directly with the Snf7 components of ESCRT-III in both Drosophila and human cells. Moreover, we find that the CPC's catalytic subunit, Aurora B kinase, phosphorylates one of the three human Snf7 paralogues-CHMP4C-in its C-terminal tail, a region known to regulate its ability to form polymers and associate with membranes. Phosphorylation at these sites appears essential for CHMP4C function because their mutation leads to cytokinesis defects. We propose that CPC controls abscission timing through inhibition of ESCRT-III Snf7 polymerization and membrane association using two concurrent mechanisms: interaction of its Borealin component with Snf7 proteins and phosphorylation of CHMP4C by Aurora B.

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Aurora B phosphorylates CHMP4C in vitro and in vivo. (a) GST-tagged CHMP4 proteins, GST alone or the positive control MBP (myelin basic protein) were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ-32P] ATP. The reactions were then separated by SDS-PAGE, and gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. Note that Aurora B is auto-phosphorylated and co-migrated with GST::CHMP4B. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. (b) GST-tagged full-length CHMP4C (FL), GST-tagged N-terminal CHMP4Cα12, GST-tagged C-terminal CHMP4Cα345, GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ-32P] ATP. Products of the reactions were then separated by SDS-PAGE and the gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. (c) GST-tagged wild-type CHMP4Cα345 (WT), the two GST:: CHMP4Cα345 variants containing S to A mutations at position 210 (S210A) or at position 210, 214 and 215 (StripleA), GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ-32P] ATP. Products of the reactions were then separated by SDS-PAGE and the gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. (d) HeLa cells were transfected with GFP::CHMP4C, synchronized in metaphase with thymidine/nocodazole block and then released into medium containing either ZM447439 or its solvent DMSO as control. Proteins were extracted, separated by SDS-PAGE, transferred onto PVDF membranes and analysed by Western blot to detect the variant of CHMP4C phosphorylated at serine 210, 214 and 215 (phospho-CHMP4C), cyclin B, Borealin, GFP::CHMP4C and tubulin as loading control. The numbers indicate the sizes (kDa) of the molecular mass marker.
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RSOB120070F5: Aurora B phosphorylates CHMP4C in vitro and in vivo. (a) GST-tagged CHMP4 proteins, GST alone or the positive control MBP (myelin basic protein) were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ-32P] ATP. The reactions were then separated by SDS-PAGE, and gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. Note that Aurora B is auto-phosphorylated and co-migrated with GST::CHMP4B. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. (b) GST-tagged full-length CHMP4C (FL), GST-tagged N-terminal CHMP4Cα12, GST-tagged C-terminal CHMP4Cα345, GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ-32P] ATP. Products of the reactions were then separated by SDS-PAGE and the gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. (c) GST-tagged wild-type CHMP4Cα345 (WT), the two GST:: CHMP4Cα345 variants containing S to A mutations at position 210 (S210A) or at position 210, 214 and 215 (StripleA), GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ-32P] ATP. Products of the reactions were then separated by SDS-PAGE and the gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. (d) HeLa cells were transfected with GFP::CHMP4C, synchronized in metaphase with thymidine/nocodazole block and then released into medium containing either ZM447439 or its solvent DMSO as control. Proteins were extracted, separated by SDS-PAGE, transferred onto PVDF membranes and analysed by Western blot to detect the variant of CHMP4C phosphorylated at serine 210, 214 and 215 (phospho-CHMP4C), cyclin B, Borealin, GFP::CHMP4C and tubulin as loading control. The numbers indicate the sizes (kDa) of the molecular mass marker.

Mentions: One obvious implication of the interaction between Borealin and CHMP4 proteins is that CPC could regulate ESCRT-III activity through phosphorylation of one or more of its components. To test this hypothesis, we investigated whether Aurora B could phosphorylate recombinant GST::CHMP4 proteins in an in vitro kinase assay. We found that all three GST::CHMP4 proteins were phosphorylated by Aurora B in vitro, although GST::CHMP4C seemed the best target (figure 5a). Therefore, we next focused on the identification of the CHMP4C residues phosphorylated by Aurora B using the same in vitro phosphorylation assay. Only the acidic C-terminal half of CHMP4C was strongly phosphorylated by Aurora B (figure 5b), and an in silico prediction analysis using the GPS 2.1 software [23] identified a serine located at position 210 as the best candidate for phosphorylation by Aurora B (table 3). The introduction of a serine (S) to alanine (A) mutation at this position (S210A) significantly reduced, but did not completely abolish, the level of Aurora B phosphorylation (figure 5c). To identify the other Aurora B phosphorylation sites, we mutagenized the majority of serines and threonines listed in table 3 to alanine and analysed the effect of these mutations, either alone or in combination with S210A, on Aurora B phosphorylation (see electronic supplementary material, figure S2). The introduction of the S214A and S215A mutations together with S210A almost completely abolished Aurora B phosphorylation (figure 5c and see electronic supplementary material, figure S2) indicating that these three residues are the major Aurora B phosphorylation sites in the C-terminal region of CHMP4C. Quite interestingly, these residues lie in a ‘linker’ region between the last two alpha-helices that has been shown to be important for the regulation of CHMP4 protein activity [24].Table 3.


The chromosomal passenger complex controls the function of endosomal sorting complex required for transport-III Snf7 proteins during cytokinesis.

Capalbo L, Montembault E, Takeda T, Bassi ZI, Glover DM, D'Avino PP - Open Biol (2012)

Aurora B phosphorylates CHMP4C in vitro and in vivo. (a) GST-tagged CHMP4 proteins, GST alone or the positive control MBP (myelin basic protein) were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ-32P] ATP. The reactions were then separated by SDS-PAGE, and gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. Note that Aurora B is auto-phosphorylated and co-migrated with GST::CHMP4B. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. (b) GST-tagged full-length CHMP4C (FL), GST-tagged N-terminal CHMP4Cα12, GST-tagged C-terminal CHMP4Cα345, GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ-32P] ATP. Products of the reactions were then separated by SDS-PAGE and the gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. (c) GST-tagged wild-type CHMP4Cα345 (WT), the two GST:: CHMP4Cα345 variants containing S to A mutations at position 210 (S210A) or at position 210, 214 and 215 (StripleA), GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ-32P] ATP. Products of the reactions were then separated by SDS-PAGE and the gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. (d) HeLa cells were transfected with GFP::CHMP4C, synchronized in metaphase with thymidine/nocodazole block and then released into medium containing either ZM447439 or its solvent DMSO as control. Proteins were extracted, separated by SDS-PAGE, transferred onto PVDF membranes and analysed by Western blot to detect the variant of CHMP4C phosphorylated at serine 210, 214 and 215 (phospho-CHMP4C), cyclin B, Borealin, GFP::CHMP4C and tubulin as loading control. The numbers indicate the sizes (kDa) of the molecular mass marker.
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Related In: Results  -  Collection

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RSOB120070F5: Aurora B phosphorylates CHMP4C in vitro and in vivo. (a) GST-tagged CHMP4 proteins, GST alone or the positive control MBP (myelin basic protein) were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ-32P] ATP. The reactions were then separated by SDS-PAGE, and gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. Note that Aurora B is auto-phosphorylated and co-migrated with GST::CHMP4B. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. (b) GST-tagged full-length CHMP4C (FL), GST-tagged N-terminal CHMP4Cα12, GST-tagged C-terminal CHMP4Cα345, GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ-32P] ATP. Products of the reactions were then separated by SDS-PAGE and the gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. (c) GST-tagged wild-type CHMP4Cα345 (WT), the two GST:: CHMP4Cα345 variants containing S to A mutations at position 210 (S210A) or at position 210, 214 and 215 (StripleA), GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ-32P] ATP. Products of the reactions were then separated by SDS-PAGE and the gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. (d) HeLa cells were transfected with GFP::CHMP4C, synchronized in metaphase with thymidine/nocodazole block and then released into medium containing either ZM447439 or its solvent DMSO as control. Proteins were extracted, separated by SDS-PAGE, transferred onto PVDF membranes and analysed by Western blot to detect the variant of CHMP4C phosphorylated at serine 210, 214 and 215 (phospho-CHMP4C), cyclin B, Borealin, GFP::CHMP4C and tubulin as loading control. The numbers indicate the sizes (kDa) of the molecular mass marker.
Mentions: One obvious implication of the interaction between Borealin and CHMP4 proteins is that CPC could regulate ESCRT-III activity through phosphorylation of one or more of its components. To test this hypothesis, we investigated whether Aurora B could phosphorylate recombinant GST::CHMP4 proteins in an in vitro kinase assay. We found that all three GST::CHMP4 proteins were phosphorylated by Aurora B in vitro, although GST::CHMP4C seemed the best target (figure 5a). Therefore, we next focused on the identification of the CHMP4C residues phosphorylated by Aurora B using the same in vitro phosphorylation assay. Only the acidic C-terminal half of CHMP4C was strongly phosphorylated by Aurora B (figure 5b), and an in silico prediction analysis using the GPS 2.1 software [23] identified a serine located at position 210 as the best candidate for phosphorylation by Aurora B (table 3). The introduction of a serine (S) to alanine (A) mutation at this position (S210A) significantly reduced, but did not completely abolish, the level of Aurora B phosphorylation (figure 5c). To identify the other Aurora B phosphorylation sites, we mutagenized the majority of serines and threonines listed in table 3 to alanine and analysed the effect of these mutations, either alone or in combination with S210A, on Aurora B phosphorylation (see electronic supplementary material, figure S2). The introduction of the S214A and S215A mutations together with S210A almost completely abolished Aurora B phosphorylation (figure 5c and see electronic supplementary material, figure S2) indicating that these three residues are the major Aurora B phosphorylation sites in the C-terminal region of CHMP4C. Quite interestingly, these residues lie in a ‘linker’ region between the last two alpha-helices that has been shown to be important for the regulation of CHMP4 protein activity [24].Table 3.

Bottom Line: Here, we show that the CPC subunit Borealin interacts directly with the Snf7 components of ESCRT-III in both Drosophila and human cells.Moreover, we find that the CPC's catalytic subunit, Aurora B kinase, phosphorylates one of the three human Snf7 paralogues-CHMP4C-in its C-terminal tail, a region known to regulate its ability to form polymers and associate with membranes.Phosphorylation at these sites appears essential for CHMP4C function because their mutation leads to cytokinesis defects.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.

ABSTRACT
Cytokinesis controls the proper segregation of nuclear and cytoplasmic materials at the end of cell division. The chromosomal passenger complex (CPC) has been proposed to monitor the final separation of the two daughter cells at the end of cytokinesis in order to prevent cell abscission in the presence of DNA at the cleavage site, but the precise molecular basis for this is unclear. Recent studies indicate that abscission could be mediated by the assembly of filaments comprising components of the endosomal sorting complex required for transport-III (ESCRT-III). Here, we show that the CPC subunit Borealin interacts directly with the Snf7 components of ESCRT-III in both Drosophila and human cells. Moreover, we find that the CPC's catalytic subunit, Aurora B kinase, phosphorylates one of the three human Snf7 paralogues-CHMP4C-in its C-terminal tail, a region known to regulate its ability to form polymers and associate with membranes. Phosphorylation at these sites appears essential for CHMP4C function because their mutation leads to cytokinesis defects. We propose that CPC controls abscission timing through inhibition of ESCRT-III Snf7 polymerization and membrane association using two concurrent mechanisms: interaction of its Borealin component with Snf7 proteins and phosphorylation of CHMP4C by Aurora B.

Show MeSH
Related in: MedlinePlus