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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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Expression of CENP-A Ser7 mutants in HeLa cells. (a) Western blot with sequential detection using two antibodies: 12CA5 to detect the epitope tag (HA), followed by hACA to detect endogenous CENP-A. C4 cells are epitope-tagged wild-type; S7A-8 and S7E-4 are representative mutant cell lines. Cells were collected either uninduced (”un”) or induced for 24 h by removal of tetracycline (”in”). (b) CENP-A mutant cell lines have normal kinetochore and spindle structure. Interphase (top) and mitotic cells (second row) with DNA (DAPI, blue), epitope tag (anti-HA, green), and centromeres (hACA, red). Bottom two rows, anti–CENP-C (red) and anti–CENP-E (red), respectively, with DAPI (DNA, blue) and antitubulin (green). (c) CENP-A mutant cell lines have normal cell-cycle profiles. DNA was detected with propidium iodide and analyzed by flow cytometry (representative profile shown as inset). TTA is the parental cell line with no epitope tag, C4 is wild-type epitope–tagged CENP-A, and S7A and S7E are each averaged values for two independent cell lines (S7A-8, S7A-18, S7E-4, and S7E-12). All cell lines appear to be similar within the standard deviation of five independent experiments (error bars). Bar, 10 μm.
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fig3: Expression of CENP-A Ser7 mutants in HeLa cells. (a) Western blot with sequential detection using two antibodies: 12CA5 to detect the epitope tag (HA), followed by hACA to detect endogenous CENP-A. C4 cells are epitope-tagged wild-type; S7A-8 and S7E-4 are representative mutant cell lines. Cells were collected either uninduced (”un”) or induced for 24 h by removal of tetracycline (”in”). (b) CENP-A mutant cell lines have normal kinetochore and spindle structure. Interphase (top) and mitotic cells (second row) with DNA (DAPI, blue), epitope tag (anti-HA, green), and centromeres (hACA, red). Bottom two rows, anti–CENP-C (red) and anti–CENP-E (red), respectively, with DAPI (DNA, blue) and antitubulin (green). (c) CENP-A mutant cell lines have normal cell-cycle profiles. DNA was detected with propidium iodide and analyzed by flow cytometry (representative profile shown as inset). TTA is the parental cell line with no epitope tag, C4 is wild-type epitope–tagged CENP-A, and S7A and S7E are each averaged values for two independent cell lines (S7A-8, S7A-18, S7E-4, and S7E-12). All cell lines appear to be similar within the standard deviation of five independent experiments (error bars). Bar, 10 μm.

Mentions: Several individual colonies were selected for each mutation based on immunofluorescence screening with anti-HA to detect the epitope-tagged proteins (see Materials and methods). Colonies that exhibited uniform expression within the population were further examined by Western blot with a human anticentromere antiserum which recognizes both the tagged and endogenous proteins (Fig. 3 a). For further study, two independent cell lines were chosen for each mutant which exhibited protein expression levels comparable to endogenous CENP-A. All experiments were performed using cells induced for 24 h.


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)

Expression of CENP-A Ser7 mutants in HeLa cells. (a) Western blot with sequential detection using two antibodies: 12CA5 to detect the epitope tag (HA), followed by hACA to detect endogenous CENP-A. C4 cells are epitope-tagged wild-type; S7A-8 and S7E-4 are representative mutant cell lines. Cells were collected either uninduced (”un”) or induced for 24 h by removal of tetracycline (”in”). (b) CENP-A mutant cell lines have normal kinetochore and spindle structure. Interphase (top) and mitotic cells (second row) with DNA (DAPI, blue), epitope tag (anti-HA, green), and centromeres (hACA, red). Bottom two rows, anti–CENP-C (red) and anti–CENP-E (red), respectively, with DAPI (DNA, blue) and antitubulin (green). (c) CENP-A mutant cell lines have normal cell-cycle profiles. DNA was detected with propidium iodide and analyzed by flow cytometry (representative profile shown as inset). TTA is the parental cell line with no epitope tag, C4 is wild-type epitope–tagged CENP-A, and S7A and S7E are each averaged values for two independent cell lines (S7A-8, S7A-18, S7E-4, and S7E-12). All cell lines appear to be similar within the standard deviation of five independent experiments (error bars). Bar, 10 μm.
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Related In: Results  -  Collection

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fig3: Expression of CENP-A Ser7 mutants in HeLa cells. (a) Western blot with sequential detection using two antibodies: 12CA5 to detect the epitope tag (HA), followed by hACA to detect endogenous CENP-A. C4 cells are epitope-tagged wild-type; S7A-8 and S7E-4 are representative mutant cell lines. Cells were collected either uninduced (”un”) or induced for 24 h by removal of tetracycline (”in”). (b) CENP-A mutant cell lines have normal kinetochore and spindle structure. Interphase (top) and mitotic cells (second row) with DNA (DAPI, blue), epitope tag (anti-HA, green), and centromeres (hACA, red). Bottom two rows, anti–CENP-C (red) and anti–CENP-E (red), respectively, with DAPI (DNA, blue) and antitubulin (green). (c) CENP-A mutant cell lines have normal cell-cycle profiles. DNA was detected with propidium iodide and analyzed by flow cytometry (representative profile shown as inset). TTA is the parental cell line with no epitope tag, C4 is wild-type epitope–tagged CENP-A, and S7A and S7E are each averaged values for two independent cell lines (S7A-8, S7A-18, S7E-4, and S7E-12). All cell lines appear to be similar within the standard deviation of five independent experiments (error bars). Bar, 10 μm.
Mentions: Several individual colonies were selected for each mutation based on immunofluorescence screening with anti-HA to detect the epitope-tagged proteins (see Materials and methods). Colonies that exhibited uniform expression within the population were further examined by Western blot with a human anticentromere antiserum which recognizes both the tagged and endogenous proteins (Fig. 3 a). For further study, two independent cell lines were chosen for each mutant which exhibited protein expression levels comparable to endogenous CENP-A. All experiments were performed using cells induced for 24 h.

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