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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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Strains lacking three distinct mechanisms for controlling the interaction of the CPC with spindle microtubules fail to show an additive growth defect.Equivalent 10-fold serial dilutions of the indicated strains were spotted onto YPAD medium and YPAD medium containing the indicated concentrations of benomyl and then incubated for 2 days at 26°C. ipl1-2A and ipl1-2E are alleles in which Cdc28 phosphorylation sites Ser-50 and Ser-76 in Ipl1 have been substituted by either alanine or glutamate residues respectively, while sli15-335A and sli15-335D have either alanine or aspartate substituted for the key serine residue in Sli15 that is phosphorylated by Cdc28 [18].
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pone-0089399-g009: Strains lacking three distinct mechanisms for controlling the interaction of the CPC with spindle microtubules fail to show an additive growth defect.Equivalent 10-fold serial dilutions of the indicated strains were spotted onto YPAD medium and YPAD medium containing the indicated concentrations of benomyl and then incubated for 2 days at 26°C. ipl1-2A and ipl1-2E are alleles in which Cdc28 phosphorylation sites Ser-50 and Ser-76 in Ipl1 have been substituted by either alanine or glutamate residues respectively, while sli15-335A and sli15-335D have either alanine or aspartate substituted for the key serine residue in Sli15 that is phosphorylated by Cdc28 [18].

Mentions: Recent work has indicated that several mechanisms influence the interaction of yeast CPC with microtubules including a weak, direct interaction between microtubules and Ipl1 itself [23], [45] that we have confirmed in our work, binding via Bim1 that is antagonized by Cdc28 phosphorylation on Ser-50 and Ser-76 in Ipl1 [45], Cdc28 phosphorylation of Sli15 (principally on Ser-335) that is antagonized by Cdc14 phosphatase as cells enter anaphase [18] and phosphorylation of Sli15 by Ipl1 [26] as shown here. Given the highly conserved nature of CPC relocalization to the spindle in anaphase, interfering with each of these pathways individually has surprisingly little impact on cell viability or proliferation, indicating that these mechanisms might function in a redundant manner to regulate CPC localization. We therefore attempted to generate strains containing different combinations of either alanine or phosphomimic substitution mutations affecting the three phosphorylation-dependent mechanisms. However, all combinations of mutations could be generated without any signs of synthetic negative genetic interactions, both when the mutations would be expected to drive premature microtubule binding or to interfere with it (Figure 9). Thus while each of these mechanisms leads to detectable phenotypes when perturbed, even in combination such perturbations still have limited consequences for proliferation.


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)

Strains lacking three distinct mechanisms for controlling the interaction of the CPC with spindle microtubules fail to show an additive growth defect.Equivalent 10-fold serial dilutions of the indicated strains were spotted onto YPAD medium and YPAD medium containing the indicated concentrations of benomyl and then incubated for 2 days at 26°C. ipl1-2A and ipl1-2E are alleles in which Cdc28 phosphorylation sites Ser-50 and Ser-76 in Ipl1 have been substituted by either alanine or glutamate residues respectively, while sli15-335A and sli15-335D have either alanine or aspartate substituted for the key serine residue in Sli15 that is phosphorylated by Cdc28 [18].
© Copyright Policy
Related In: Results  -  Collection

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

pone-0089399-g009: Strains lacking three distinct mechanisms for controlling the interaction of the CPC with spindle microtubules fail to show an additive growth defect.Equivalent 10-fold serial dilutions of the indicated strains were spotted onto YPAD medium and YPAD medium containing the indicated concentrations of benomyl and then incubated for 2 days at 26°C. ipl1-2A and ipl1-2E are alleles in which Cdc28 phosphorylation sites Ser-50 and Ser-76 in Ipl1 have been substituted by either alanine or glutamate residues respectively, while sli15-335A and sli15-335D have either alanine or aspartate substituted for the key serine residue in Sli15 that is phosphorylated by Cdc28 [18].
Mentions: Recent work has indicated that several mechanisms influence the interaction of yeast CPC with microtubules including a weak, direct interaction between microtubules and Ipl1 itself [23], [45] that we have confirmed in our work, binding via Bim1 that is antagonized by Cdc28 phosphorylation on Ser-50 and Ser-76 in Ipl1 [45], Cdc28 phosphorylation of Sli15 (principally on Ser-335) that is antagonized by Cdc14 phosphatase as cells enter anaphase [18] and phosphorylation of Sli15 by Ipl1 [26] as shown here. Given the highly conserved nature of CPC relocalization to the spindle in anaphase, interfering with each of these pathways individually has surprisingly little impact on cell viability or proliferation, indicating that these mechanisms might function in a redundant manner to regulate CPC localization. We therefore attempted to generate strains containing different combinations of either alanine or phosphomimic substitution mutations affecting the three phosphorylation-dependent mechanisms. However, all combinations of mutations could be generated without any signs of synthetic negative genetic interactions, both when the mutations would be expected to drive premature microtubule binding or to interfere with it (Figure 9). Thus while each of these mechanisms leads to detectable phenotypes when perturbed, even in combination such perturbations still have limited consequences for proliferation.

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