Limits...
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.

Show MeSH

Related in: MedlinePlus

Generation of INCENP structural mutants to dissect INCENP–aurora B interactions. (A) Schematic representation of the main domains of INCENP with a sequence alignment of the INCENP IN box in several model organisms. Red triangles indicate key residues mutated in this study. Xl, Xenopus laevis; Gg, Gallus gallus; Hs, Homo sapiens; Mm, Mus musculus; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Sc, Saccharomyces cerevisiae. (B–D) Growth curves of DT40 wild-type (WT) and INCENPON/OFF cells expressing various forms of INCENP in the presence and absence of doxycycline. The expressed INCENP was TrAP-INCENPWT (B), TrAP-INCENPWT plus TrAP-INCENPW766G (C), and TrAP-INCENPWT plus TrAP-INCENPF802A (D). Growth of cultures expressing TrAP-INCENPWT was indistinguishable from wild-type cells. All cultures expressing only mutant INCENP died. (E–G) Immunoblots show levels of chromosomal passenger proteins in INCENPON/OFF cells (E) or INCENPON/OFF cells expressing TrAP-INCENPW766G (F) or TrAP-INCENPF802A (G). INCENPOFF cells expressing TrAP-INCENPWT are shown as controls. (E) Black circles indicate a proteolytic fragment of INCENP. At time 0, INCENP, which was driven by the tTA expressed from the KIF4A promoter, is ∼20× overexpressed, and the levels of the other passengers are correspondingly increased. All levels decrease to those shown in wild-type clone 18 cells after the addition of doxycycline provided that some form of INCENP was present. Levels of H3S10ph are shown as a measure of aurora B activity and α-tubulin as a loading control. White lines indicate that intervening lanes have been spliced out. (H) Immunoblots shows higher resolutions of INCENP and its fragment. TrAP-INCENP class I comigrate with INCENP class II. The red line indicates that the proteolytic fragments seen in all cells expressing TrAP-INCENP constructs are not INCENP class I. α-Tubulin was used as a loading control. (F–H) Black circles indicate the position of INCENP degradation fragments. Error bars indicate SD.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2806593&req=5

fig3: Generation of INCENP structural mutants to dissect INCENP–aurora B interactions. (A) Schematic representation of the main domains of INCENP with a sequence alignment of the INCENP IN box in several model organisms. Red triangles indicate key residues mutated in this study. Xl, Xenopus laevis; Gg, Gallus gallus; Hs, Homo sapiens; Mm, Mus musculus; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Sc, Saccharomyces cerevisiae. (B–D) Growth curves of DT40 wild-type (WT) and INCENPON/OFF cells expressing various forms of INCENP in the presence and absence of doxycycline. The expressed INCENP was TrAP-INCENPWT (B), TrAP-INCENPWT plus TrAP-INCENPW766G (C), and TrAP-INCENPWT plus TrAP-INCENPF802A (D). Growth of cultures expressing TrAP-INCENPWT was indistinguishable from wild-type cells. All cultures expressing only mutant INCENP died. (E–G) Immunoblots show levels of chromosomal passenger proteins in INCENPON/OFF cells (E) or INCENPON/OFF cells expressing TrAP-INCENPW766G (F) or TrAP-INCENPF802A (G). INCENPOFF cells expressing TrAP-INCENPWT are shown as controls. (E) Black circles indicate a proteolytic fragment of INCENP. At time 0, INCENP, which was driven by the tTA expressed from the KIF4A promoter, is ∼20× overexpressed, and the levels of the other passengers are correspondingly increased. All levels decrease to those shown in wild-type clone 18 cells after the addition of doxycycline provided that some form of INCENP was present. Levels of H3S10ph are shown as a measure of aurora B activity and α-tubulin as a loading control. White lines indicate that intervening lanes have been spliced out. (H) Immunoblots shows higher resolutions of INCENP and its fragment. TrAP-INCENP class I comigrate with INCENP class II. The red line indicates that the proteolytic fragments seen in all cells expressing TrAP-INCENP constructs are not INCENP class I. α-Tubulin was used as a loading control. (F–H) Black circles indicate the position of INCENP degradation fragments. Error bars indicate SD.

Mentions: Triple affinity purification (TrAP)–tagged INCENPWT class I (hereafter referred to as TrAP-INCENPWT) under control of an SV40 promoter that is insensitive to doxycycline repression efficiently rescued the life of the INCENPOFF cells. INCENPOFF/TrAP-INCENPWT cells grew with kinetics indistinguishable from wild-type or INCENPON cells (Fig. 3 B). The TrAP tag incorporates His, streptavidin-binding peptide (SBP), and S tags and can be monitored by immunoblotting and immunofluorescence using a monoclonal antibody recognizing the SBP tag (Hudson et al., 2008; Samejima et al., 2008). TrAP-INCENPWT class II could also support the growth of INCENPOFF cells (unpublished data). Thus, alternative splicing of INCENP is not essential for life in DT40 cells.


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)

Generation of INCENP structural mutants to dissect INCENP–aurora B interactions. (A) Schematic representation of the main domains of INCENP with a sequence alignment of the INCENP IN box in several model organisms. Red triangles indicate key residues mutated in this study. Xl, Xenopus laevis; Gg, Gallus gallus; Hs, Homo sapiens; Mm, Mus musculus; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Sc, Saccharomyces cerevisiae. (B–D) Growth curves of DT40 wild-type (WT) and INCENPON/OFF cells expressing various forms of INCENP in the presence and absence of doxycycline. The expressed INCENP was TrAP-INCENPWT (B), TrAP-INCENPWT plus TrAP-INCENPW766G (C), and TrAP-INCENPWT plus TrAP-INCENPF802A (D). Growth of cultures expressing TrAP-INCENPWT was indistinguishable from wild-type cells. All cultures expressing only mutant INCENP died. (E–G) Immunoblots show levels of chromosomal passenger proteins in INCENPON/OFF cells (E) or INCENPON/OFF cells expressing TrAP-INCENPW766G (F) or TrAP-INCENPF802A (G). INCENPOFF cells expressing TrAP-INCENPWT are shown as controls. (E) Black circles indicate a proteolytic fragment of INCENP. At time 0, INCENP, which was driven by the tTA expressed from the KIF4A promoter, is ∼20× overexpressed, and the levels of the other passengers are correspondingly increased. All levels decrease to those shown in wild-type clone 18 cells after the addition of doxycycline provided that some form of INCENP was present. Levels of H3S10ph are shown as a measure of aurora B activity and α-tubulin as a loading control. White lines indicate that intervening lanes have been spliced out. (H) Immunoblots shows higher resolutions of INCENP and its fragment. TrAP-INCENP class I comigrate with INCENP class II. The red line indicates that the proteolytic fragments seen in all cells expressing TrAP-INCENP constructs are not INCENP class I. α-Tubulin was used as a loading control. (F–H) Black circles indicate the position of INCENP degradation fragments. Error bars indicate SD.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2806593&req=5

fig3: Generation of INCENP structural mutants to dissect INCENP–aurora B interactions. (A) Schematic representation of the main domains of INCENP with a sequence alignment of the INCENP IN box in several model organisms. Red triangles indicate key residues mutated in this study. Xl, Xenopus laevis; Gg, Gallus gallus; Hs, Homo sapiens; Mm, Mus musculus; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Sc, Saccharomyces cerevisiae. (B–D) Growth curves of DT40 wild-type (WT) and INCENPON/OFF cells expressing various forms of INCENP in the presence and absence of doxycycline. The expressed INCENP was TrAP-INCENPWT (B), TrAP-INCENPWT plus TrAP-INCENPW766G (C), and TrAP-INCENPWT plus TrAP-INCENPF802A (D). Growth of cultures expressing TrAP-INCENPWT was indistinguishable from wild-type cells. All cultures expressing only mutant INCENP died. (E–G) Immunoblots show levels of chromosomal passenger proteins in INCENPON/OFF cells (E) or INCENPON/OFF cells expressing TrAP-INCENPW766G (F) or TrAP-INCENPF802A (G). INCENPOFF cells expressing TrAP-INCENPWT are shown as controls. (E) Black circles indicate a proteolytic fragment of INCENP. At time 0, INCENP, which was driven by the tTA expressed from the KIF4A promoter, is ∼20× overexpressed, and the levels of the other passengers are correspondingly increased. All levels decrease to those shown in wild-type clone 18 cells after the addition of doxycycline provided that some form of INCENP was present. Levels of H3S10ph are shown as a measure of aurora B activity and α-tubulin as a loading control. White lines indicate that intervening lanes have been spliced out. (H) Immunoblots shows higher resolutions of INCENP and its fragment. TrAP-INCENP class I comigrate with INCENP class II. The red line indicates that the proteolytic fragments seen in all cells expressing TrAP-INCENP constructs are not INCENP class I. α-Tubulin was used as a loading control. (F–H) Black circles indicate the position of INCENP degradation fragments. Error bars indicate SD.
Mentions: Triple affinity purification (TrAP)–tagged INCENPWT class I (hereafter referred to as TrAP-INCENPWT) under control of an SV40 promoter that is insensitive to doxycycline repression efficiently rescued the life of the INCENPOFF cells. INCENPOFF/TrAP-INCENPWT cells grew with kinetics indistinguishable from wild-type or INCENPON cells (Fig. 3 B). The TrAP tag incorporates His, streptavidin-binding peptide (SBP), and S tags and can be monitored by immunoblotting and immunofluorescence using a monoclonal antibody recognizing the SBP tag (Hudson et al., 2008; Samejima et al., 2008). TrAP-INCENPWT class II could also support the growth of INCENPOFF cells (unpublished data). Thus, alternative splicing of INCENP is not essential for life in DT40 cells.

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
Related in: MedlinePlus