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INCENP-aurora B interactions modulate kinase activity and chromosome passenger complex localization.

Xu Z, Ogawa H, Vagnarelli P, Bergmann JH, Hudson DF, Ruchaud S, Fukagawa T, Earnshaw WC, Samejima K - J. Cell Biol. (2009)

Bottom Line: Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone.This transfer requires significantly higher levels of aurora B activity.These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.

ABSTRACT
Dynamic localization of the chromosomal passenger complex (CPC) during mitosis is essential for its diverse functions. CPC targeting to centromeres involves interactions between Survivin, Borealin, and the inner centromere protein (CENP [INCENP]) N terminus. In this study, we investigate how interactions between the INCENP C terminus and aurora B set the level of kinase activity. Low levels of kinase activity, seen in INCENP-depleted cells or in cells expressing a mutant INCENP that cannot bind aurora B, are sufficient for a spindle checkpoint response when microtubules are absent but not against low dose taxol. Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone. This transfer requires significantly higher levels of aurora B activity. These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.

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In vivo analysis of CPC formation. (A and B) INCENPON/OFF cells stably expressing TrAP-tagged INCENP mutants were grown in doxycycline (Dox) to shut off expression of wild-type (WT) INCENP plus nocodazole (Noc; or not) to enrich for mitotic cells. (A) Immunoblots of streptavidin pull-downs with anti-SBP to reveal the proteins associated with INCENP. (B) Immunoblots of whole cell lysates with antibodies to INCENP (monoclonal antibody 3D3; Cooke et al., 1987) and the other passenger proteins. Equal numbers of cells were loaded per lane. α-Tubulin is used as a loading control. White lines indicate that intervening lanes have been spliced out. (C–H) Localization of exogenous TrAP-INCENP (red; panel 2) plus endogenous aurora B (green; C, E, G, and I [panel 3]) or CENP-A (green; D, F, H, and J [panel 3]) in INCENPOFF cells expressing TrAP-INCENPWT (C and D), TrAP-INCENPW766G (E and F), or TrAP-INCENPF802A (G and H). These images are of the nocodazole-treated cells used for the SBP pull-down experiment in A and B. (E) In all cases, the TrAP-INCENP localizes to centromeres, but only in INCENPOFF/TrAP-INCENPW766G cells is aurora B localization diffuse. Images were acquired using the same microscope settings for all experiments. Insets show magnified views of boxed regions. Bars, 5 µm.
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fig4: In vivo analysis of CPC formation. (A and B) INCENPON/OFF cells stably expressing TrAP-tagged INCENP mutants were grown in doxycycline (Dox) to shut off expression of wild-type (WT) INCENP plus nocodazole (Noc; or not) to enrich for mitotic cells. (A) Immunoblots of streptavidin pull-downs with anti-SBP to reveal the proteins associated with INCENP. (B) Immunoblots of whole cell lysates with antibodies to INCENP (monoclonal antibody 3D3; Cooke et al., 1987) and the other passenger proteins. Equal numbers of cells were loaded per lane. α-Tubulin is used as a loading control. White lines indicate that intervening lanes have been spliced out. (C–H) Localization of exogenous TrAP-INCENP (red; panel 2) plus endogenous aurora B (green; C, E, G, and I [panel 3]) or CENP-A (green; D, F, H, and J [panel 3]) in INCENPOFF cells expressing TrAP-INCENPWT (C and D), TrAP-INCENPW766G (E and F), or TrAP-INCENPF802A (G and H). These images are of the nocodazole-treated cells used for the SBP pull-down experiment in A and B. (E) In all cases, the TrAP-INCENP localizes to centromeres, but only in INCENPOFF/TrAP-INCENPW766G cells is aurora B localization diffuse. Images were acquired using the same microscope settings for all experiments. Insets show magnified views of boxed regions. Bars, 5 µm.

Mentions: As expected, TrAP-INCENPWT exhibited a typical CPC distribution pattern in INCENPOFF cells. It was concentrated in centromeres from prophase to metaphase, where it colocalized with aurora B, Borealin, and Survivin (Fig. S1, D–F). During anaphase, TrAP-INCENPWT accumulated in the cleavage furrow and midbody. TrAP-INCENPWT was also competent in CPC formation. INCENPOFF cells expressing TrAP-INCENPWT grown in doxycycline for 28 h were treated with nocodazole from hours 16–28 to enrich for mitotic cells. Subsequent streptavidin pull-downs confirmed that TrAP-INCENPWT was associated with endogenous aurora B, Borealin, and Survivin in vivo (Fig. 4, A and B, lane 1).


INCENP-aurora B interactions modulate kinase activity and chromosome passenger complex localization.

Xu Z, Ogawa H, Vagnarelli P, Bergmann JH, Hudson DF, Ruchaud S, Fukagawa T, Earnshaw WC, Samejima K - J. Cell Biol. (2009)

In vivo analysis of CPC formation. (A and B) INCENPON/OFF cells stably expressing TrAP-tagged INCENP mutants were grown in doxycycline (Dox) to shut off expression of wild-type (WT) INCENP plus nocodazole (Noc; or not) to enrich for mitotic cells. (A) Immunoblots of streptavidin pull-downs with anti-SBP to reveal the proteins associated with INCENP. (B) Immunoblots of whole cell lysates with antibodies to INCENP (monoclonal antibody 3D3; Cooke et al., 1987) and the other passenger proteins. Equal numbers of cells were loaded per lane. α-Tubulin is used as a loading control. White lines indicate that intervening lanes have been spliced out. (C–H) Localization of exogenous TrAP-INCENP (red; panel 2) plus endogenous aurora B (green; C, E, G, and I [panel 3]) or CENP-A (green; D, F, H, and J [panel 3]) in INCENPOFF cells expressing TrAP-INCENPWT (C and D), TrAP-INCENPW766G (E and F), or TrAP-INCENPF802A (G and H). These images are of the nocodazole-treated cells used for the SBP pull-down experiment in A and B. (E) In all cases, the TrAP-INCENP localizes to centromeres, but only in INCENPOFF/TrAP-INCENPW766G cells is aurora B localization diffuse. Images were acquired using the same microscope settings for all experiments. Insets show magnified views of boxed regions. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig4: In vivo analysis of CPC formation. (A and B) INCENPON/OFF cells stably expressing TrAP-tagged INCENP mutants were grown in doxycycline (Dox) to shut off expression of wild-type (WT) INCENP plus nocodazole (Noc; or not) to enrich for mitotic cells. (A) Immunoblots of streptavidin pull-downs with anti-SBP to reveal the proteins associated with INCENP. (B) Immunoblots of whole cell lysates with antibodies to INCENP (monoclonal antibody 3D3; Cooke et al., 1987) and the other passenger proteins. Equal numbers of cells were loaded per lane. α-Tubulin is used as a loading control. White lines indicate that intervening lanes have been spliced out. (C–H) Localization of exogenous TrAP-INCENP (red; panel 2) plus endogenous aurora B (green; C, E, G, and I [panel 3]) or CENP-A (green; D, F, H, and J [panel 3]) in INCENPOFF cells expressing TrAP-INCENPWT (C and D), TrAP-INCENPW766G (E and F), or TrAP-INCENPF802A (G and H). These images are of the nocodazole-treated cells used for the SBP pull-down experiment in A and B. (E) In all cases, the TrAP-INCENP localizes to centromeres, but only in INCENPOFF/TrAP-INCENPW766G cells is aurora B localization diffuse. Images were acquired using the same microscope settings for all experiments. Insets show magnified views of boxed regions. Bars, 5 µm.
Mentions: As expected, TrAP-INCENPWT exhibited a typical CPC distribution pattern in INCENPOFF cells. It was concentrated in centromeres from prophase to metaphase, where it colocalized with aurora B, Borealin, and Survivin (Fig. S1, D–F). During anaphase, TrAP-INCENPWT accumulated in the cleavage furrow and midbody. TrAP-INCENPWT was also competent in CPC formation. INCENPOFF cells expressing TrAP-INCENPWT grown in doxycycline for 28 h were treated with nocodazole from hours 16–28 to enrich for mitotic cells. Subsequent streptavidin pull-downs confirmed that TrAP-INCENPWT was associated with endogenous aurora B, Borealin, and Survivin in vivo (Fig. 4, A and B, lane 1).

Bottom Line: Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone.This transfer requires significantly higher levels of aurora B activity.These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.

ABSTRACT
Dynamic localization of the chromosomal passenger complex (CPC) during mitosis is essential for its diverse functions. CPC targeting to centromeres involves interactions between Survivin, Borealin, and the inner centromere protein (CENP [INCENP]) N terminus. In this study, we investigate how interactions between the INCENP C terminus and aurora B set the level of kinase activity. Low levels of kinase activity, seen in INCENP-depleted cells or in cells expressing a mutant INCENP that cannot bind aurora B, are sufficient for a spindle checkpoint response when microtubules are absent but not against low dose taxol. Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone. This transfer requires significantly higher levels of aurora B activity. These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.

Show MeSH