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Phosphorylation of Sli15 by Ipl1 is important for proper CPC localization and chromosome stability in Saccharomyces cerevisiae.

Makrantoni V, Corbishley SJ, Rachidi N, Morrice NA, Robinson DA, Stark MJ - PLoS ONE (2014)

Bottom Line: Consistent with these results, direct interaction of Sli15 with microtubules in vitro is greatly reduced either following phosphorylation by Ipl1 or when constitutive phosphorylation at the Ipl1-dependent phosphorylation sites is mimicked by aspartate or glutamate substitutions.Furthermore, we find that mimicking Ipl1 phosphorylation of Sli15 interferes with the 'tension checkpoint'--the CPC-dependent mechanism through which cells activate the spindle assembly checkpoint to delay anaphase in the absence of tension on kinetochore-microtubule attachments.Ipl1-dependent phosphorylation of Sli15 therefore inhibits its association with microtubules both in vivo and in vitro and may negatively regulate the tension checkpoint mechanism.

View Article: PubMed Central - PubMed

Affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Scotland, United Kingdom.

ABSTRACT
The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here we have identified multiple sites of CPC autophosphorylation on yeast Sli15 that are located within its central microtubule-binding domain and examined the functional significance of their phosphorylation by Ipl1 through mutation of these sites, either to non-phosphorylatable alanine (sli15-20A) or to acidic residues to mimic constitutive phosphorylation (sli15-20D). Both mutant sli15 alleles confer chromosome instability, but this is mediated neither by changes in the capacity of Sli15 to activate Ipl1 kinase nor by decreased efficiency of chromosome biorientation, a key process in cell division that requires CPC function. Instead, we find that mimicking constitutive phosphorylation of Sli15 on the Ipl1 phosphorylation sites causes delocalization of the CPC in metaphase, whereas blocking phosphorylation of Sli15 on the Ipl1 sites drives excessive localization of Sli15 to the mitotic spindle in pre-anaphase cells. Consistent with these results, direct interaction of Sli15 with microtubules in vitro is greatly reduced either following phosphorylation by Ipl1 or when constitutive phosphorylation at the Ipl1-dependent phosphorylation sites is mimicked by aspartate or glutamate substitutions. Furthermore, we find that mimicking Ipl1 phosphorylation of Sli15 interferes with the 'tension checkpoint'--the CPC-dependent mechanism through which cells activate the spindle assembly checkpoint to delay anaphase in the absence of tension on kinetochore-microtubule attachments. Ipl1-dependent phosphorylation of Sli15 therefore inhibits its association with microtubules both in vivo and in vitro and may negatively regulate the tension checkpoint mechanism.

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Ipl1-dependent Sli15 phosphorylation is dispensable for chromosome bi-orientation.Wild type SLI15 (VMY316), sli15-20A (VMY318) and sli15-20D (VMY320) cells containing CEN5-(tetO)336, tetR-GFP, Venus-TUB1 and pMET3-CDC20 were arrested in G1 with α-factor at 26°C and then released to a metaphase block in rich medium (containing 2 mM methionine to deplete Cdc20) for 2.5 h. (A) Representative stills from time-lapse images of live cells. Bi-oriented chromosomes show dynamic splitting and reassociation of sister CEN5s. Green, CEN5 labeled with tetR-GFP; red, Venus-tubulin. (B) Quantification of chromosome bi-orientation in metaphase-arrested cells from multiple time-lapse fields (n = number of cells scored in each category).
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pone-0089399-g004: Ipl1-dependent Sli15 phosphorylation is dispensable for chromosome bi-orientation.Wild type SLI15 (VMY316), sli15-20A (VMY318) and sli15-20D (VMY320) cells containing CEN5-(tetO)336, tetR-GFP, Venus-TUB1 and pMET3-CDC20 were arrested in G1 with α-factor at 26°C and then released to a metaphase block in rich medium (containing 2 mM methionine to deplete Cdc20) for 2.5 h. (A) Representative stills from time-lapse images of live cells. Bi-oriented chromosomes show dynamic splitting and reassociation of sister CEN5s. Green, CEN5 labeled with tetR-GFP; red, Venus-tubulin. (B) Quantification of chromosome bi-orientation in metaphase-arrested cells from multiple time-lapse fields (n = number of cells scored in each category).

Mentions: To investigate whether Ipl1-dependent phosphorylation of Sli15 plays a role in chromosome stability, chromosome loss rates were measured using a colony-sectoring assay in wild-type, sli15-20A and sli15-20D strains. As shown in Table 5, both sli15-20A and sli15-20D led to an approximately 10-fold increase in chromosome loss rate. Thus either blocking or constitutively mimicking phosphorylation reduced the fidelity of chromosome transmission, indicating that both mutant proteins are functionally compromised in some way and suggesting that phosphorylation of Sli15 by Ipl1 plays a role in ensuring accurate chromosome segregation. A key role of the CPC in yeast and other eukaryotes is in the establishment of chromosome bi-orientation, and both ipl1 and sli15 temperature-sensitive mutants show high levels of mono-oriented chromosomes at their restrictive temperatures [2]. Thus elevated chromosome loss in the sli15 mutants could result from failure of the CPC to promote chromosome biorientation. We therefore generated strains containing the sli15-20A or sli15-20D alleles in which we could monitor chromosome biorientation through the behavior of GFP-labelled sister CEN5s in cells arrested in metaphase by Cdc20 depletion. Bioriented chromosomes show dynamic splitting and reassociation of GFP-labelled sister centromeres in metaphase-arrested cells, whereas mono-oriented chromosomes show a single, unresolved GFP locus at one end of the metaphase spindle [31]. As shown in Figure 4, chromosome biorientation was unaffected by either sli15-20A or sli15-20D, occurring with the same efficiency as in the SLI15 wild-type control strain. Thus inefficient chromosome biorientation cannot account for the elevated rate of chromosome loss conferred by either the sli15-20A or the sli15-20D allele.


Phosphorylation of Sli15 by Ipl1 is important for proper CPC localization and chromosome stability in Saccharomyces cerevisiae.

Makrantoni V, Corbishley SJ, Rachidi N, Morrice NA, Robinson DA, Stark MJ - PLoS ONE (2014)

Ipl1-dependent Sli15 phosphorylation is dispensable for chromosome bi-orientation.Wild type SLI15 (VMY316), sli15-20A (VMY318) and sli15-20D (VMY320) cells containing CEN5-(tetO)336, tetR-GFP, Venus-TUB1 and pMET3-CDC20 were arrested in G1 with α-factor at 26°C and then released to a metaphase block in rich medium (containing 2 mM methionine to deplete Cdc20) for 2.5 h. (A) Representative stills from time-lapse images of live cells. Bi-oriented chromosomes show dynamic splitting and reassociation of sister CEN5s. Green, CEN5 labeled with tetR-GFP; red, Venus-tubulin. (B) Quantification of chromosome bi-orientation in metaphase-arrested cells from multiple time-lapse fields (n = number of cells scored in each category).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928436&req=5

pone-0089399-g004: Ipl1-dependent Sli15 phosphorylation is dispensable for chromosome bi-orientation.Wild type SLI15 (VMY316), sli15-20A (VMY318) and sli15-20D (VMY320) cells containing CEN5-(tetO)336, tetR-GFP, Venus-TUB1 and pMET3-CDC20 were arrested in G1 with α-factor at 26°C and then released to a metaphase block in rich medium (containing 2 mM methionine to deplete Cdc20) for 2.5 h. (A) Representative stills from time-lapse images of live cells. Bi-oriented chromosomes show dynamic splitting and reassociation of sister CEN5s. Green, CEN5 labeled with tetR-GFP; red, Venus-tubulin. (B) Quantification of chromosome bi-orientation in metaphase-arrested cells from multiple time-lapse fields (n = number of cells scored in each category).
Mentions: To investigate whether Ipl1-dependent phosphorylation of Sli15 plays a role in chromosome stability, chromosome loss rates were measured using a colony-sectoring assay in wild-type, sli15-20A and sli15-20D strains. As shown in Table 5, both sli15-20A and sli15-20D led to an approximately 10-fold increase in chromosome loss rate. Thus either blocking or constitutively mimicking phosphorylation reduced the fidelity of chromosome transmission, indicating that both mutant proteins are functionally compromised in some way and suggesting that phosphorylation of Sli15 by Ipl1 plays a role in ensuring accurate chromosome segregation. A key role of the CPC in yeast and other eukaryotes is in the establishment of chromosome bi-orientation, and both ipl1 and sli15 temperature-sensitive mutants show high levels of mono-oriented chromosomes at their restrictive temperatures [2]. Thus elevated chromosome loss in the sli15 mutants could result from failure of the CPC to promote chromosome biorientation. We therefore generated strains containing the sli15-20A or sli15-20D alleles in which we could monitor chromosome biorientation through the behavior of GFP-labelled sister CEN5s in cells arrested in metaphase by Cdc20 depletion. Bioriented chromosomes show dynamic splitting and reassociation of GFP-labelled sister centromeres in metaphase-arrested cells, whereas mono-oriented chromosomes show a single, unresolved GFP locus at one end of the metaphase spindle [31]. As shown in Figure 4, chromosome biorientation was unaffected by either sli15-20A or sli15-20D, occurring with the same efficiency as in the SLI15 wild-type control strain. Thus inefficient chromosome biorientation cannot account for the elevated rate of chromosome loss conferred by either the sli15-20A or the sli15-20D allele.

Bottom Line: Consistent with these results, direct interaction of Sli15 with microtubules in vitro is greatly reduced either following phosphorylation by Ipl1 or when constitutive phosphorylation at the Ipl1-dependent phosphorylation sites is mimicked by aspartate or glutamate substitutions.Furthermore, we find that mimicking Ipl1 phosphorylation of Sli15 interferes with the 'tension checkpoint'--the CPC-dependent mechanism through which cells activate the spindle assembly checkpoint to delay anaphase in the absence of tension on kinetochore-microtubule attachments.Ipl1-dependent phosphorylation of Sli15 therefore inhibits its association with microtubules both in vivo and in vitro and may negatively regulate the tension checkpoint mechanism.

View Article: PubMed Central - PubMed

Affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Scotland, United Kingdom.

ABSTRACT
The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here we have identified multiple sites of CPC autophosphorylation on yeast Sli15 that are located within its central microtubule-binding domain and examined the functional significance of their phosphorylation by Ipl1 through mutation of these sites, either to non-phosphorylatable alanine (sli15-20A) or to acidic residues to mimic constitutive phosphorylation (sli15-20D). Both mutant sli15 alleles confer chromosome instability, but this is mediated neither by changes in the capacity of Sli15 to activate Ipl1 kinase nor by decreased efficiency of chromosome biorientation, a key process in cell division that requires CPC function. Instead, we find that mimicking constitutive phosphorylation of Sli15 on the Ipl1 phosphorylation sites causes delocalization of the CPC in metaphase, whereas blocking phosphorylation of Sli15 on the Ipl1 sites drives excessive localization of Sli15 to the mitotic spindle in pre-anaphase cells. Consistent with these results, direct interaction of Sli15 with microtubules in vitro is greatly reduced either following phosphorylation by Ipl1 or when constitutive phosphorylation at the Ipl1-dependent phosphorylation sites is mimicked by aspartate or glutamate substitutions. Furthermore, we find that mimicking Ipl1 phosphorylation of Sli15 interferes with the 'tension checkpoint'--the CPC-dependent mechanism through which cells activate the spindle assembly checkpoint to delay anaphase in the absence of tension on kinetochore-microtubule attachments. Ipl1-dependent phosphorylation of Sli15 therefore inhibits its association with microtubules both in vivo and in vitro and may negatively regulate the tension checkpoint mechanism.

Show MeSH
Related in: MedlinePlus