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CENP-A is phosphorylated by Aurora B kinase and plays an unexpected role in completion of cytokinesis.

Zeitlin SG, Shelby RD, Sullivan KF - J. Cell Biol. (2001)

Bottom Line: The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1.Our data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell.These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Aurora B is a mitotic protein kinase that phosphorylates histone H3, behaves as a chromosomal passenger protein, and functions in cytokinesis. We investigated a role for Aurora B with respect to human centromere protein A (CENP-A), a centromeric histone H3 homologue. Aurora B concentrates at centromeres in early G2, associates with histone H3 and centromeres at the times when histone H3 and CENP-A are phosphorylated, and phosphorylates histone H3 and CENP-A in vitro at a similar target serine residue. Dominant negative phosphorylation site mutants of CENP-A result in a delay at the terminal stage of cytokinesis (cell separation). The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1. Our data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell. These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis.

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Aurora B and INCENP distribution in G2 and prophase. Aurora B kinase associates with centromeres during G2 and M. Immunofluorescence with anti–Aim-1 (Aim-1/Aurora B, green) and DAPI (DNA, blue). (a) Early G2 cells with hACA to detect centromeres (red), demonstrating that Aurora B associates with centromeres well before mitosis. (b) Mid G2 cells show clear colocalization of Aurora B and H3P-positive pericentric heterochromatin (red). (c) Late G2 cells with general distribution of Aurora B throughout the nuclei, at a time when H3P signal is present throughout the chromatin (red). (d) Prophase cells (with visible chromosome condensation judged by DAPI staining) with anti–CENP-A-Ser7P (red), showing that Aurora B associates with centromeres when CENP-A phosphorylation begins. Insets show that anti–CENP-A-Ser7P stains kinetochores, whereas Aurora B is detected in the inner centromere domain. (e) Early G2 cells show more Aurora B signals (green) than INCENP signals (red), with partial colocalization. (f) Mid G2 cells show more Aurora B signals, with an increase in INCENP signals and partial colocalization. (g) Late G2 cells with general distribution of Aurora B, and less INCENP signal in very few, larger foci. (h) Prophase cells with Aurora B distributed as in panel d, but INCENP does not colocalize with Aurora B concentrations at centromeres (yellow color would indicate overlap). Bar, 10 μm.
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fig1: Aurora B and INCENP distribution in G2 and prophase. Aurora B kinase associates with centromeres during G2 and M. Immunofluorescence with anti–Aim-1 (Aim-1/Aurora B, green) and DAPI (DNA, blue). (a) Early G2 cells with hACA to detect centromeres (red), demonstrating that Aurora B associates with centromeres well before mitosis. (b) Mid G2 cells show clear colocalization of Aurora B and H3P-positive pericentric heterochromatin (red). (c) Late G2 cells with general distribution of Aurora B throughout the nuclei, at a time when H3P signal is present throughout the chromatin (red). (d) Prophase cells (with visible chromosome condensation judged by DAPI staining) with anti–CENP-A-Ser7P (red), showing that Aurora B associates with centromeres when CENP-A phosphorylation begins. Insets show that anti–CENP-A-Ser7P stains kinetochores, whereas Aurora B is detected in the inner centromere domain. (e) Early G2 cells show more Aurora B signals (green) than INCENP signals (red), with partial colocalization. (f) Mid G2 cells show more Aurora B signals, with an increase in INCENP signals and partial colocalization. (g) Late G2 cells with general distribution of Aurora B, and less INCENP signal in very few, larger foci. (h) Prophase cells with Aurora B distributed as in panel d, but INCENP does not colocalize with Aurora B concentrations at centromeres (yellow color would indicate overlap). Bar, 10 μm.

Mentions: Using phosphohistone H3 antibodies to stage cells from early to late G2 and into M phase, we were able to track the progression of Aurora B localization as cells entered mitosis (Fig. 1). Aurora B is first detectable in the nuclei of cells before the onset of histone H3 phosphorylation, where it is associated with pericentric regions surrounding many but not all centromeres (Fig. 1 a). Aurora B becomes concentrated in large pericentric foci (Fig. 1 b) before and during histone H3 phosphorylation initiation in pericentric chromatin. As histone H3 phosphorylation spreads throughout the chromatin during late G2, Aurora B is more uniformly distributed throughout the nucleus (Fig. 1 c). During prophase, when chromosome condensation is extensive, and CENP-A phosphorylation is detectable, Aurora B is detected primarily at centromeres, between paired sister kinetochores (Fig. 1 d, inset). These observations show, for the first time, a concordant program of Aurora B localization and histone phosphorylation events that spans G2 and prophase in human cells, complementing previous demonstrations that Drosophila and Xenopus Aurora B homologues localize to centromeres during mitosis (Adams et al., 2000). These data suggest that the control of intracellular localization is an element of Aurora B regulation.


CENP-A is phosphorylated by Aurora B kinase and plays an unexpected role in completion of cytokinesis.

Zeitlin SG, Shelby RD, Sullivan KF - J. Cell Biol. (2001)

Aurora B and INCENP distribution in G2 and prophase. Aurora B kinase associates with centromeres during G2 and M. Immunofluorescence with anti–Aim-1 (Aim-1/Aurora B, green) and DAPI (DNA, blue). (a) Early G2 cells with hACA to detect centromeres (red), demonstrating that Aurora B associates with centromeres well before mitosis. (b) Mid G2 cells show clear colocalization of Aurora B and H3P-positive pericentric heterochromatin (red). (c) Late G2 cells with general distribution of Aurora B throughout the nuclei, at a time when H3P signal is present throughout the chromatin (red). (d) Prophase cells (with visible chromosome condensation judged by DAPI staining) with anti–CENP-A-Ser7P (red), showing that Aurora B associates with centromeres when CENP-A phosphorylation begins. Insets show that anti–CENP-A-Ser7P stains kinetochores, whereas Aurora B is detected in the inner centromere domain. (e) Early G2 cells show more Aurora B signals (green) than INCENP signals (red), with partial colocalization. (f) Mid G2 cells show more Aurora B signals, with an increase in INCENP signals and partial colocalization. (g) Late G2 cells with general distribution of Aurora B, and less INCENP signal in very few, larger foci. (h) Prophase cells with Aurora B distributed as in panel d, but INCENP does not colocalize with Aurora B concentrations at centromeres (yellow color would indicate overlap). Bar, 10 μm.
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fig1: Aurora B and INCENP distribution in G2 and prophase. Aurora B kinase associates with centromeres during G2 and M. Immunofluorescence with anti–Aim-1 (Aim-1/Aurora B, green) and DAPI (DNA, blue). (a) Early G2 cells with hACA to detect centromeres (red), demonstrating that Aurora B associates with centromeres well before mitosis. (b) Mid G2 cells show clear colocalization of Aurora B and H3P-positive pericentric heterochromatin (red). (c) Late G2 cells with general distribution of Aurora B throughout the nuclei, at a time when H3P signal is present throughout the chromatin (red). (d) Prophase cells (with visible chromosome condensation judged by DAPI staining) with anti–CENP-A-Ser7P (red), showing that Aurora B associates with centromeres when CENP-A phosphorylation begins. Insets show that anti–CENP-A-Ser7P stains kinetochores, whereas Aurora B is detected in the inner centromere domain. (e) Early G2 cells show more Aurora B signals (green) than INCENP signals (red), with partial colocalization. (f) Mid G2 cells show more Aurora B signals, with an increase in INCENP signals and partial colocalization. (g) Late G2 cells with general distribution of Aurora B, and less INCENP signal in very few, larger foci. (h) Prophase cells with Aurora B distributed as in panel d, but INCENP does not colocalize with Aurora B concentrations at centromeres (yellow color would indicate overlap). Bar, 10 μm.
Mentions: Using phosphohistone H3 antibodies to stage cells from early to late G2 and into M phase, we were able to track the progression of Aurora B localization as cells entered mitosis (Fig. 1). Aurora B is first detectable in the nuclei of cells before the onset of histone H3 phosphorylation, where it is associated with pericentric regions surrounding many but not all centromeres (Fig. 1 a). Aurora B becomes concentrated in large pericentric foci (Fig. 1 b) before and during histone H3 phosphorylation initiation in pericentric chromatin. As histone H3 phosphorylation spreads throughout the chromatin during late G2, Aurora B is more uniformly distributed throughout the nucleus (Fig. 1 c). During prophase, when chromosome condensation is extensive, and CENP-A phosphorylation is detectable, Aurora B is detected primarily at centromeres, between paired sister kinetochores (Fig. 1 d, inset). These observations show, for the first time, a concordant program of Aurora B localization and histone phosphorylation events that spans G2 and prophase in human cells, complementing previous demonstrations that Drosophila and Xenopus Aurora B homologues localize to centromeres during mitosis (Adams et al., 2000). These data suggest that the control of intracellular localization is an element of Aurora B regulation.

Bottom Line: The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1.Our data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell.These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Aurora B is a mitotic protein kinase that phosphorylates histone H3, behaves as a chromosomal passenger protein, and functions in cytokinesis. We investigated a role for Aurora B with respect to human centromere protein A (CENP-A), a centromeric histone H3 homologue. Aurora B concentrates at centromeres in early G2, associates with histone H3 and centromeres at the times when histone H3 and CENP-A are phosphorylated, and phosphorylates histone H3 and CENP-A in vitro at a similar target serine residue. Dominant negative phosphorylation site mutants of CENP-A result in a delay at the terminal stage of cytokinesis (cell separation). The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1. Our data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell. These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis.

Show MeSH
Related in: MedlinePlus