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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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Passenger and motor protein localization in CENP-A mutant cell lines. (a–c) Immunofluorescence to detect DNA (blue), Aurora B (green), and INCENP (red). In both wild-type (C4) and S7A cells, Aurora B and INCENP are localized only at the midzone during anaphase, while in S7E cells Aurora B and INCENP are also detected on chromosomes (bottom row, third panel). (d–i) DNA is shown in blue (DAPI) in all panels. d, MKLP-1 (red); e, CENP-E (red, with tubulin in green) at the midzone. f, survivin (red, with tubulin in green); g, INCENP (red) at the midbody.
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fig5: Passenger and motor protein localization in CENP-A mutant cell lines. (a–c) Immunofluorescence to detect DNA (blue), Aurora B (green), and INCENP (red). In both wild-type (C4) and S7A cells, Aurora B and INCENP are localized only at the midzone during anaphase, while in S7E cells Aurora B and INCENP are also detected on chromosomes (bottom row, third panel). (d–i) DNA is shown in blue (DAPI) in all panels. d, MKLP-1 (red); e, CENP-E (red, with tubulin in green) at the midzone. f, survivin (red, with tubulin in green); g, INCENP (red) at the midbody.

Mentions: Aurora B itself has recently been shown to exist in a complex with INCENP and survivin, in addition to having a known functional role in cytokinesis (Tatsuka et al., 1998; Terada et al., 1998; Adams et al., 2000). We observed a qualitative difference in Aurora B and INCENP localization during anaphase. Aurora B and INCENP normally transfer completely to the midzone at anaphase (Fig. 5 a). In CENP-A–S7A cells, both proteins have normal anaphase midzone localization (Fig. 5 b). In contrast, in CENP-A–S7E cells, a detectable portion of both Aurora B and INCENP is bound to anaphase chromosomes, while a portion of both proteins are localized to the midzone (Fig. 5 c). Thus, the S7E mutation interferes with Aurora B and INCENP dynamics at the stage when passenger proteins transfer to the midzone. This is not a general defect in anaphase midzone assembly, since MKLP-1 (Fig. 5 d) and CENP-E (Fig. 5 e) are localized normally during this stage. The passenger proteins ultimately adopt a normal localization in the midbody at the end of telophase (survivin, Fig. 5 f; INCENP, Fig. 5 g; Aurora B, Fig. 6). Together, CENP-A Ser7 mutations appear to alter the dynamics of Aurora B and INCENP partitioning between chromosomes and the midzone during anaphase, but do not interfere with the localization of passenger proteins at midbodies during cytokinesis.


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)

Passenger and motor protein localization in CENP-A mutant cell lines. (a–c) Immunofluorescence to detect DNA (blue), Aurora B (green), and INCENP (red). In both wild-type (C4) and S7A cells, Aurora B and INCENP are localized only at the midzone during anaphase, while in S7E cells Aurora B and INCENP are also detected on chromosomes (bottom row, third panel). (d–i) DNA is shown in blue (DAPI) in all panels. d, MKLP-1 (red); e, CENP-E (red, with tubulin in green) at the midzone. f, survivin (red, with tubulin in green); g, INCENP (red) at the midbody.
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Related In: Results  -  Collection

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fig5: Passenger and motor protein localization in CENP-A mutant cell lines. (a–c) Immunofluorescence to detect DNA (blue), Aurora B (green), and INCENP (red). In both wild-type (C4) and S7A cells, Aurora B and INCENP are localized only at the midzone during anaphase, while in S7E cells Aurora B and INCENP are also detected on chromosomes (bottom row, third panel). (d–i) DNA is shown in blue (DAPI) in all panels. d, MKLP-1 (red); e, CENP-E (red, with tubulin in green) at the midzone. f, survivin (red, with tubulin in green); g, INCENP (red) at the midbody.
Mentions: Aurora B itself has recently been shown to exist in a complex with INCENP and survivin, in addition to having a known functional role in cytokinesis (Tatsuka et al., 1998; Terada et al., 1998; Adams et al., 2000). We observed a qualitative difference in Aurora B and INCENP localization during anaphase. Aurora B and INCENP normally transfer completely to the midzone at anaphase (Fig. 5 a). In CENP-A–S7A cells, both proteins have normal anaphase midzone localization (Fig. 5 b). In contrast, in CENP-A–S7E cells, a detectable portion of both Aurora B and INCENP is bound to anaphase chromosomes, while a portion of both proteins are localized to the midzone (Fig. 5 c). Thus, the S7E mutation interferes with Aurora B and INCENP dynamics at the stage when passenger proteins transfer to the midzone. This is not a general defect in anaphase midzone assembly, since MKLP-1 (Fig. 5 d) and CENP-E (Fig. 5 e) are localized normally during this stage. The passenger proteins ultimately adopt a normal localization in the midbody at the end of telophase (survivin, Fig. 5 f; INCENP, Fig. 5 g; Aurora B, Fig. 6). Together, CENP-A Ser7 mutations appear to alter the dynamics of Aurora B and INCENP partitioning between chromosomes and the midzone during anaphase, but do not interfere with the localization of passenger proteins at midbodies during cytokinesis.

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