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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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Binding kinetic analysis of wild type and mutant versions of Sli15 to immobilized microtubules by biolayer interferometry.A comparison of the binding abilities between recombinant GST-Sli15-His6 and its mutants to 5 µM taxol-stabilized, biotinylated polymerized tubulin, immobilized to streptavidin-coated biosensors is shown. (A) Binding results for five different concentrations of wild-type and mutant Sli15 (see inset) with equivalent concentrations of recombinant GST protein used as a control. Data shown are representative of three separate experiments. (B) Binding of wild type Sli15 and Ipl1 alone or in combination to taxol-stabilized, biotinylated polymerized tubulin with or without Sli15 phosphorylation by Ipl1 (+ATP); see inset for key. Data shown are representative of two separate experiments.
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pone-0089399-g007: Binding kinetic analysis of wild type and mutant versions of Sli15 to immobilized microtubules by biolayer interferometry.A comparison of the binding abilities between recombinant GST-Sli15-His6 and its mutants to 5 µM taxol-stabilized, biotinylated polymerized tubulin, immobilized to streptavidin-coated biosensors is shown. (A) Binding results for five different concentrations of wild-type and mutant Sli15 (see inset) with equivalent concentrations of recombinant GST protein used as a control. Data shown are representative of three separate experiments. (B) Binding of wild type Sli15 and Ipl1 alone or in combination to taxol-stabilized, biotinylated polymerized tubulin with or without Sli15 phosphorylation by Ipl1 (+ATP); see inset for key. Data shown are representative of two separate experiments.

Mentions: Since the behavior of the mutant Sli15 proteins in vivo strongly suggested that interaction of Sli15 with microtubules is affected by Ipl1 phosphorylation, we next investigated the direct binding of recombinant wild-type and mutant Sli15 proteins, prepared using GST/His6 tandem affinity purification, to taxol-stabilized microtubules. Using a novel assay based on biolayer interferometry, both GST-Sli15 and GST-Sli15-20A bound to microtubules, with GST-Sli15-20A showing greater binding over a range of concentrations (Figure 7). Thus even in the absence of phosphorylation, the wild-type protein binds microtubules less well than the alanine substitution mutant, showing that removal of the 20 polar side chains in the Sli15-20A microtubule domain can enhance its affinity for microtubules in comparison with the wild-type, non-phosphorylated protein. Most notably, however, binding of Sli15-20D was virtually undetectable even at the highest protein concentration used (500 nM). Thus the in vitro microtubule binding properties of the recombinant Sli15 proteins mirrored their behavior in vivo in metaphase-arrested cells and imply that the addition of multiple, negatively charged groups to the Sli15 microtubule-binding domain disrupts its ability to bind microtubules.


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)

Binding kinetic analysis of wild type and mutant versions of Sli15 to immobilized microtubules by biolayer interferometry.A comparison of the binding abilities between recombinant GST-Sli15-His6 and its mutants to 5 µM taxol-stabilized, biotinylated polymerized tubulin, immobilized to streptavidin-coated biosensors is shown. (A) Binding results for five different concentrations of wild-type and mutant Sli15 (see inset) with equivalent concentrations of recombinant GST protein used as a control. Data shown are representative of three separate experiments. (B) Binding of wild type Sli15 and Ipl1 alone or in combination to taxol-stabilized, biotinylated polymerized tubulin with or without Sli15 phosphorylation by Ipl1 (+ATP); see inset for key. Data shown are representative of two separate experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0089399-g007: Binding kinetic analysis of wild type and mutant versions of Sli15 to immobilized microtubules by biolayer interferometry.A comparison of the binding abilities between recombinant GST-Sli15-His6 and its mutants to 5 µM taxol-stabilized, biotinylated polymerized tubulin, immobilized to streptavidin-coated biosensors is shown. (A) Binding results for five different concentrations of wild-type and mutant Sli15 (see inset) with equivalent concentrations of recombinant GST protein used as a control. Data shown are representative of three separate experiments. (B) Binding of wild type Sli15 and Ipl1 alone or in combination to taxol-stabilized, biotinylated polymerized tubulin with or without Sli15 phosphorylation by Ipl1 (+ATP); see inset for key. Data shown are representative of two separate experiments.
Mentions: Since the behavior of the mutant Sli15 proteins in vivo strongly suggested that interaction of Sli15 with microtubules is affected by Ipl1 phosphorylation, we next investigated the direct binding of recombinant wild-type and mutant Sli15 proteins, prepared using GST/His6 tandem affinity purification, to taxol-stabilized microtubules. Using a novel assay based on biolayer interferometry, both GST-Sli15 and GST-Sli15-20A bound to microtubules, with GST-Sli15-20A showing greater binding over a range of concentrations (Figure 7). Thus even in the absence of phosphorylation, the wild-type protein binds microtubules less well than the alanine substitution mutant, showing that removal of the 20 polar side chains in the Sli15-20A microtubule domain can enhance its affinity for microtubules in comparison with the wild-type, non-phosphorylated protein. Most notably, however, binding of Sli15-20D was virtually undetectable even at the highest protein concentration used (500 nM). Thus the in vitro microtubule binding properties of the recombinant Sli15 proteins mirrored their behavior in vivo in metaphase-arrested cells and imply that the addition of multiple, negatively charged groups to the Sli15 microtubule-binding domain disrupts its ability to bind microtubules.

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