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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 phosphorylates the NH2 termini of histone H3 and CENP-A in vitro. Arrows indicate the migration of CENP-A–GST. (a) Immunoprecipitated Aurora B phosphorylates both H3 and CENP-A NH2 termini expressed as GST fusions. Top, representative Coomassie stain of SDS-PAGE. Lower bands are GST alone or fused to the NH2 terminus of either H3 or CENP-A; upper bands are antibody light chain, either anti–Aim-1 or control isotype-matched mouse IgG1. Also shown are mock reactions where GST fusions were mixed with the protein G sepharose beads used in immunoprecipitation. Bottom, autoradiogram of the same gel. Aurora B clearly phosphorylates the H3 and CENP-A NH2 termini. No signal is seen in either of the negative controls when the Aurora B antibody is not present. (b) Aurora B immunoprecipitation significantly concentrates the Aurora B kinase activity. Whole extract mixed with GST fusions does not result in phosphorylation of CENP-A or H3 NH2 termini (extract), and the Aurora B antibody alone contains no kinase activity (antibody). Whole extract alone allowed to autophosphorylate does not reveal CENP-A or H3 bands (auto). (c) Ser7 is a target Aurora B phosphorylation site in the CENP-A NH2 terminus. Aurora B immunoprecipitations were mixed with the CENP-A NH2-terminal GST fusion (WT), or site-directed mutants of this construct (S7A and S7E). CENP-A phosphorylation is reduced 50% by mutation of Ser7; however, it is not abolished. Immunoprecipitation with an irrelevant antibody (mouse IgG1) is negative. (d) Mutation of CENP-A Ser7 abolishes reactivity with the anti–CENP-A-Ser7P antibody. GST fusion proteins were incubated with immunoprecipitated Aurora B in the presence of cold ATP before Western blotting. Top, ponceau stain; bottom, Western blot. Only wild-type CENP-A reacts positively with the Ser7P antibody. There is no cross-reaction with GST or the NH2 terminus of H3, and mutation of Ser7 to alanine (S7A) or glutamate (S7E; unpublished data) abolishes reactivity.
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fig2: Aurora B phosphorylates the NH2 termini of histone H3 and CENP-A in vitro. Arrows indicate the migration of CENP-A–GST. (a) Immunoprecipitated Aurora B phosphorylates both H3 and CENP-A NH2 termini expressed as GST fusions. Top, representative Coomassie stain of SDS-PAGE. Lower bands are GST alone or fused to the NH2 terminus of either H3 or CENP-A; upper bands are antibody light chain, either anti–Aim-1 or control isotype-matched mouse IgG1. Also shown are mock reactions where GST fusions were mixed with the protein G sepharose beads used in immunoprecipitation. Bottom, autoradiogram of the same gel. Aurora B clearly phosphorylates the H3 and CENP-A NH2 termini. No signal is seen in either of the negative controls when the Aurora B antibody is not present. (b) Aurora B immunoprecipitation significantly concentrates the Aurora B kinase activity. Whole extract mixed with GST fusions does not result in phosphorylation of CENP-A or H3 NH2 termini (extract), and the Aurora B antibody alone contains no kinase activity (antibody). Whole extract alone allowed to autophosphorylate does not reveal CENP-A or H3 bands (auto). (c) Ser7 is a target Aurora B phosphorylation site in the CENP-A NH2 terminus. Aurora B immunoprecipitations were mixed with the CENP-A NH2-terminal GST fusion (WT), or site-directed mutants of this construct (S7A and S7E). CENP-A phosphorylation is reduced 50% by mutation of Ser7; however, it is not abolished. Immunoprecipitation with an irrelevant antibody (mouse IgG1) is negative. (d) Mutation of CENP-A Ser7 abolishes reactivity with the anti–CENP-A-Ser7P antibody. GST fusion proteins were incubated with immunoprecipitated Aurora B in the presence of cold ATP before Western blotting. Top, ponceau stain; bottom, Western blot. Only wild-type CENP-A reacts positively with the Ser7P antibody. There is no cross-reaction with GST or the NH2 terminus of H3, and mutation of Ser7 to alanine (S7A) or glutamate (S7E; unpublished data) abolishes reactivity.

Mentions: To determine whether Aurora B possesses a CENP-A kinase activity, we asked whether immunoprecipitated Aurora B can utilize the NH2-terminal tail of CENP-A as a substrate. Aurora B was immunoprecipitated from nocodazole-arrested HeLa cells and incubated with CENP-A and H3 NH2 termini fused to glutathione S-transferase (GST) in an in vitro kinase reaction. Aurora B phosphorylated histone H3-GST, as expected from studies in other species (Hsu et al., 2000; Adams et al., 2001b; Giet and Glover, 2001; Murnion et al., 2001). CENP-A–GST was also used as a substrate with a somewhat lower efficiency (69% of the phosphate incorporated by H3-GST), while Aurora B did not phosphorylate GST alone (Fig. 2 a). This CENP-A kinase activity is highly concentrated by the immunoprecipitation procedure, since whole cell extracts do not have detectable kinase activity against these substrates, and therefore the immunoprecipitated activity is unlikely to represent a contaminating mitotic kinase (Fig. 2 b). To determine whether Ser7 of CENP-A is a substrate residue for Aurora B phosphorylation we performed two experiments. CENP-A–GST constructs were prepared in which Ser7 was mutated to alanine or glutamic acid. Phosphorylation of these proteins by Aurora B was reduced by 50%, demonstrating that Ser7 is a kinase substrate (Fig. 2 c). However, Aurora B is able to phosphorylate one or more of the other eight Ser residues in the NH2 terminus of CENP-A in vitro. In a second experiment, GST fusion proteins phosphorylated by Aurora B were probed by Western blot using an antibody specific for CENP-A phosphorylated at Ser7, demonstrating formation of the Ser7 phosphoepitope (Fig. 2 d). Together, the dynamic subcellular localization of Aurora B and in vitro kinase activity strongly suggest that Aurora B functions both as a CENP-A Ser7 kinase in mitosis as well as an H3 kinase in G2.


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 phosphorylates the NH2 termini of histone H3 and CENP-A in vitro. Arrows indicate the migration of CENP-A–GST. (a) Immunoprecipitated Aurora B phosphorylates both H3 and CENP-A NH2 termini expressed as GST fusions. Top, representative Coomassie stain of SDS-PAGE. Lower bands are GST alone or fused to the NH2 terminus of either H3 or CENP-A; upper bands are antibody light chain, either anti–Aim-1 or control isotype-matched mouse IgG1. Also shown are mock reactions where GST fusions were mixed with the protein G sepharose beads used in immunoprecipitation. Bottom, autoradiogram of the same gel. Aurora B clearly phosphorylates the H3 and CENP-A NH2 termini. No signal is seen in either of the negative controls when the Aurora B antibody is not present. (b) Aurora B immunoprecipitation significantly concentrates the Aurora B kinase activity. Whole extract mixed with GST fusions does not result in phosphorylation of CENP-A or H3 NH2 termini (extract), and the Aurora B antibody alone contains no kinase activity (antibody). Whole extract alone allowed to autophosphorylate does not reveal CENP-A or H3 bands (auto). (c) Ser7 is a target Aurora B phosphorylation site in the CENP-A NH2 terminus. Aurora B immunoprecipitations were mixed with the CENP-A NH2-terminal GST fusion (WT), or site-directed mutants of this construct (S7A and S7E). CENP-A phosphorylation is reduced 50% by mutation of Ser7; however, it is not abolished. Immunoprecipitation with an irrelevant antibody (mouse IgG1) is negative. (d) Mutation of CENP-A Ser7 abolishes reactivity with the anti–CENP-A-Ser7P antibody. GST fusion proteins were incubated with immunoprecipitated Aurora B in the presence of cold ATP before Western blotting. Top, ponceau stain; bottom, Western blot. Only wild-type CENP-A reacts positively with the Ser7P antibody. There is no cross-reaction with GST or the NH2 terminus of H3, and mutation of Ser7 to alanine (S7A) or glutamate (S7E; unpublished data) abolishes reactivity.
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fig2: Aurora B phosphorylates the NH2 termini of histone H3 and CENP-A in vitro. Arrows indicate the migration of CENP-A–GST. (a) Immunoprecipitated Aurora B phosphorylates both H3 and CENP-A NH2 termini expressed as GST fusions. Top, representative Coomassie stain of SDS-PAGE. Lower bands are GST alone or fused to the NH2 terminus of either H3 or CENP-A; upper bands are antibody light chain, either anti–Aim-1 or control isotype-matched mouse IgG1. Also shown are mock reactions where GST fusions were mixed with the protein G sepharose beads used in immunoprecipitation. Bottom, autoradiogram of the same gel. Aurora B clearly phosphorylates the H3 and CENP-A NH2 termini. No signal is seen in either of the negative controls when the Aurora B antibody is not present. (b) Aurora B immunoprecipitation significantly concentrates the Aurora B kinase activity. Whole extract mixed with GST fusions does not result in phosphorylation of CENP-A or H3 NH2 termini (extract), and the Aurora B antibody alone contains no kinase activity (antibody). Whole extract alone allowed to autophosphorylate does not reveal CENP-A or H3 bands (auto). (c) Ser7 is a target Aurora B phosphorylation site in the CENP-A NH2 terminus. Aurora B immunoprecipitations were mixed with the CENP-A NH2-terminal GST fusion (WT), or site-directed mutants of this construct (S7A and S7E). CENP-A phosphorylation is reduced 50% by mutation of Ser7; however, it is not abolished. Immunoprecipitation with an irrelevant antibody (mouse IgG1) is negative. (d) Mutation of CENP-A Ser7 abolishes reactivity with the anti–CENP-A-Ser7P antibody. GST fusion proteins were incubated with immunoprecipitated Aurora B in the presence of cold ATP before Western blotting. Top, ponceau stain; bottom, Western blot. Only wild-type CENP-A reacts positively with the Ser7P antibody. There is no cross-reaction with GST or the NH2 terminus of H3, and mutation of Ser7 to alanine (S7A) or glutamate (S7E; unpublished data) abolishes reactivity.
Mentions: To determine whether Aurora B possesses a CENP-A kinase activity, we asked whether immunoprecipitated Aurora B can utilize the NH2-terminal tail of CENP-A as a substrate. Aurora B was immunoprecipitated from nocodazole-arrested HeLa cells and incubated with CENP-A and H3 NH2 termini fused to glutathione S-transferase (GST) in an in vitro kinase reaction. Aurora B phosphorylated histone H3-GST, as expected from studies in other species (Hsu et al., 2000; Adams et al., 2001b; Giet and Glover, 2001; Murnion et al., 2001). CENP-A–GST was also used as a substrate with a somewhat lower efficiency (69% of the phosphate incorporated by H3-GST), while Aurora B did not phosphorylate GST alone (Fig. 2 a). This CENP-A kinase activity is highly concentrated by the immunoprecipitation procedure, since whole cell extracts do not have detectable kinase activity against these substrates, and therefore the immunoprecipitated activity is unlikely to represent a contaminating mitotic kinase (Fig. 2 b). To determine whether Ser7 of CENP-A is a substrate residue for Aurora B phosphorylation we performed two experiments. CENP-A–GST constructs were prepared in which Ser7 was mutated to alanine or glutamic acid. Phosphorylation of these proteins by Aurora B was reduced by 50%, demonstrating that Ser7 is a kinase substrate (Fig. 2 c). However, Aurora B is able to phosphorylate one or more of the other eight Ser residues in the NH2 terminus of CENP-A in vitro. In a second experiment, GST fusion proteins phosphorylated by Aurora B were probed by Western blot using an antibody specific for CENP-A phosphorylated at Ser7, demonstrating formation of the Ser7 phosphoepitope (Fig. 2 d). Together, the dynamic subcellular localization of Aurora B and in vitro kinase activity strongly suggest that Aurora B functions both as a CENP-A Ser7 kinase in mitosis as well as an H3 kinase in G2.

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