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Dbf2-Mob1 drives relocalization of protein phosphatase Cdc14 to the cytoplasm during exit from mitosis.

Mohl DA, Huddleston MJ, Collingwood TS, Annan RS, Deshaies RJ - J. Cell Biol. (2009)

Bottom Line: Throughout interphase, Cdc14 is sequestered in the nucleolus, but successful anaphase activates the mitotic exit network (MEN), which triggers dispersal of Cdc14 throughout the cell by a mechanism that has remained unknown.In this study, we show that a MEN component, protein kinase Dbf2-Mob1, promotes transfer of Cdc14 to the cytoplasm and consequent exit from mitosis by direct phosphorylation of Cdc14 on serine and threonine residues adjacent to a nuclear localization signal (NLS), thereby abrogating its NLS activity.Our results define a mechanism by which the MEN promotes exit from mitosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA. mohld@caltech.edu

ABSTRACT
Exit from mitosis is characterized by a precipitous decline in cyclin-dependent kinase (Cdk) activity, dissolution of mitotic structures, and cytokinesis. In Saccharomyces cerevisiae, mitotic exit is driven by a protein phosphatase, Cdc14, which is in part responsible for counteracting Cdk activity. Throughout interphase, Cdc14 is sequestered in the nucleolus, but successful anaphase activates the mitotic exit network (MEN), which triggers dispersal of Cdc14 throughout the cell by a mechanism that has remained unknown. In this study, we show that a MEN component, protein kinase Dbf2-Mob1, promotes transfer of Cdc14 to the cytoplasm and consequent exit from mitosis by direct phosphorylation of Cdc14 on serine and threonine residues adjacent to a nuclear localization signal (NLS), thereby abrogating its NLS activity. Our results define a mechanism by which the MEN promotes exit from mitosis.

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Phosphorylation sites within Cdc14’s NLS are required for its cell cycle regulation. (A) Mutation of Dbf2–Mob1 phosphorylation sites in NLS changes the nuclear/cytoplasmic distribution of NLS-GFP (green) reporter fusions. Wild-type (WT), PS1,2A, PS1,3A, PS1–3A, and PS1,2E NLS-GFP reporters are shown. Red fluorescent mCherry-Tub1 (red; γ adjusted) was used to identify interphase (short spindle) and late anaphase (long spindle) cells. The indicated NLS constructs were transiently expressed (3% galactose for 90 min) from the GAL1,10 promoter. Live cells were imaged. Wild-type NLS-GFP was concentrated in the nucleus of interphase cells (short spindle; arrows) and dispersed into the cytoplasm of late anaphase cells (long spindle; carrot). NLS-GFP alleles PS1,2A, PS1,3A, and PS1–3A do not exhibit cytoplasmic release of the reporter. In the table, the first column shows genotypes, the second column shows the number of cells for which the spindle was >5 µm long and the GFP signal was dispersed into the cytoplasm or concentrated in the nucleus, and the third column shows the statistical significance of the deviation from wild type (χ2 test). In contrast to the nonphosphorylatable mutants, PS1,2E, a putative phosphomimetic mutant, is defective for nuclear accumulation. The residues that comprise PS1–3 are specified in Fig. 3 B. (B) The PS mutant NLS-GFP is not released from the nucleus during late mitosis. Time-lapse microscopy was performed as described in Fig. 2 B. Representative time-lapse series are shown for wild-type and PS1,2A reporters. The wild-type NLS-GFP fusion was dispersed throughout the cell for approximately two time points (indicated by red numbers; n = 5). In contrast, mutant PS1,2A reporters did not redistribute to the cytoplasm for the duration of the time course (n = 5). Bars, 2 µm.
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fig4: Phosphorylation sites within Cdc14’s NLS are required for its cell cycle regulation. (A) Mutation of Dbf2–Mob1 phosphorylation sites in NLS changes the nuclear/cytoplasmic distribution of NLS-GFP (green) reporter fusions. Wild-type (WT), PS1,2A, PS1,3A, PS1–3A, and PS1,2E NLS-GFP reporters are shown. Red fluorescent mCherry-Tub1 (red; γ adjusted) was used to identify interphase (short spindle) and late anaphase (long spindle) cells. The indicated NLS constructs were transiently expressed (3% galactose for 90 min) from the GAL1,10 promoter. Live cells were imaged. Wild-type NLS-GFP was concentrated in the nucleus of interphase cells (short spindle; arrows) and dispersed into the cytoplasm of late anaphase cells (long spindle; carrot). NLS-GFP alleles PS1,2A, PS1,3A, and PS1–3A do not exhibit cytoplasmic release of the reporter. In the table, the first column shows genotypes, the second column shows the number of cells for which the spindle was >5 µm long and the GFP signal was dispersed into the cytoplasm or concentrated in the nucleus, and the third column shows the statistical significance of the deviation from wild type (χ2 test). In contrast to the nonphosphorylatable mutants, PS1,2E, a putative phosphomimetic mutant, is defective for nuclear accumulation. The residues that comprise PS1–3 are specified in Fig. 3 B. (B) The PS mutant NLS-GFP is not released from the nucleus during late mitosis. Time-lapse microscopy was performed as described in Fig. 2 B. Representative time-lapse series are shown for wild-type and PS1,2A reporters. The wild-type NLS-GFP fusion was dispersed throughout the cell for approximately two time points (indicated by red numbers; n = 5). In contrast, mutant PS1,2A reporters did not redistribute to the cytoplasm for the duration of the time course (n = 5). Bars, 2 µm.

Mentions: Our data so far indicate that the C-terminal region of Cdc14 contained NLS activity, was relocalized to the cytoplasm in late mitosis in a DBF2-dependent manner, and was directly phosphorylated by Dbf2–Mob1. Collectively, these observations suggested that Dbf2 phosphorylates residues adjacent to NLS in late mitosis, which inactivates it and results in redistribution of NLS-GFP to the cytoplasm. To test this hypothesis, we individually mutated phosphorylation sites within our NLS-GFP reporter and assayed the effects of these mutations on nuclear localization (Fig. 4 A). Wild-type NLS-GFP expressed for 90 min from the GAL1,10 promoter was concentrated in the nucleus in interphase (short spindle) cells. In late mitotic (long spindle) cells, the same construct was largely dispersed throughout the cell. In contrast, the double mutants PS1,2A and PS1,3A, as well as a mutant lacking all six sites (PS1–3A) were tightly restricted to the nucleus in both interphase and late mitotic cells (Fig. 4 A).


Dbf2-Mob1 drives relocalization of protein phosphatase Cdc14 to the cytoplasm during exit from mitosis.

Mohl DA, Huddleston MJ, Collingwood TS, Annan RS, Deshaies RJ - J. Cell Biol. (2009)

Phosphorylation sites within Cdc14’s NLS are required for its cell cycle regulation. (A) Mutation of Dbf2–Mob1 phosphorylation sites in NLS changes the nuclear/cytoplasmic distribution of NLS-GFP (green) reporter fusions. Wild-type (WT), PS1,2A, PS1,3A, PS1–3A, and PS1,2E NLS-GFP reporters are shown. Red fluorescent mCherry-Tub1 (red; γ adjusted) was used to identify interphase (short spindle) and late anaphase (long spindle) cells. The indicated NLS constructs were transiently expressed (3% galactose for 90 min) from the GAL1,10 promoter. Live cells were imaged. Wild-type NLS-GFP was concentrated in the nucleus of interphase cells (short spindle; arrows) and dispersed into the cytoplasm of late anaphase cells (long spindle; carrot). NLS-GFP alleles PS1,2A, PS1,3A, and PS1–3A do not exhibit cytoplasmic release of the reporter. In the table, the first column shows genotypes, the second column shows the number of cells for which the spindle was >5 µm long and the GFP signal was dispersed into the cytoplasm or concentrated in the nucleus, and the third column shows the statistical significance of the deviation from wild type (χ2 test). In contrast to the nonphosphorylatable mutants, PS1,2E, a putative phosphomimetic mutant, is defective for nuclear accumulation. The residues that comprise PS1–3 are specified in Fig. 3 B. (B) The PS mutant NLS-GFP is not released from the nucleus during late mitosis. Time-lapse microscopy was performed as described in Fig. 2 B. Representative time-lapse series are shown for wild-type and PS1,2A reporters. The wild-type NLS-GFP fusion was dispersed throughout the cell for approximately two time points (indicated by red numbers; n = 5). In contrast, mutant PS1,2A reporters did not redistribute to the cytoplasm for the duration of the time course (n = 5). Bars, 2 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654127&req=5

fig4: Phosphorylation sites within Cdc14’s NLS are required for its cell cycle regulation. (A) Mutation of Dbf2–Mob1 phosphorylation sites in NLS changes the nuclear/cytoplasmic distribution of NLS-GFP (green) reporter fusions. Wild-type (WT), PS1,2A, PS1,3A, PS1–3A, and PS1,2E NLS-GFP reporters are shown. Red fluorescent mCherry-Tub1 (red; γ adjusted) was used to identify interphase (short spindle) and late anaphase (long spindle) cells. The indicated NLS constructs were transiently expressed (3% galactose for 90 min) from the GAL1,10 promoter. Live cells were imaged. Wild-type NLS-GFP was concentrated in the nucleus of interphase cells (short spindle; arrows) and dispersed into the cytoplasm of late anaphase cells (long spindle; carrot). NLS-GFP alleles PS1,2A, PS1,3A, and PS1–3A do not exhibit cytoplasmic release of the reporter. In the table, the first column shows genotypes, the second column shows the number of cells for which the spindle was >5 µm long and the GFP signal was dispersed into the cytoplasm or concentrated in the nucleus, and the third column shows the statistical significance of the deviation from wild type (χ2 test). In contrast to the nonphosphorylatable mutants, PS1,2E, a putative phosphomimetic mutant, is defective for nuclear accumulation. The residues that comprise PS1–3 are specified in Fig. 3 B. (B) The PS mutant NLS-GFP is not released from the nucleus during late mitosis. Time-lapse microscopy was performed as described in Fig. 2 B. Representative time-lapse series are shown for wild-type and PS1,2A reporters. The wild-type NLS-GFP fusion was dispersed throughout the cell for approximately two time points (indicated by red numbers; n = 5). In contrast, mutant PS1,2A reporters did not redistribute to the cytoplasm for the duration of the time course (n = 5). Bars, 2 µm.
Mentions: Our data so far indicate that the C-terminal region of Cdc14 contained NLS activity, was relocalized to the cytoplasm in late mitosis in a DBF2-dependent manner, and was directly phosphorylated by Dbf2–Mob1. Collectively, these observations suggested that Dbf2 phosphorylates residues adjacent to NLS in late mitosis, which inactivates it and results in redistribution of NLS-GFP to the cytoplasm. To test this hypothesis, we individually mutated phosphorylation sites within our NLS-GFP reporter and assayed the effects of these mutations on nuclear localization (Fig. 4 A). Wild-type NLS-GFP expressed for 90 min from the GAL1,10 promoter was concentrated in the nucleus in interphase (short spindle) cells. In late mitotic (long spindle) cells, the same construct was largely dispersed throughout the cell. In contrast, the double mutants PS1,2A and PS1,3A, as well as a mutant lacking all six sites (PS1–3A) were tightly restricted to the nucleus in both interphase and late mitotic cells (Fig. 4 A).

Bottom Line: Throughout interphase, Cdc14 is sequestered in the nucleolus, but successful anaphase activates the mitotic exit network (MEN), which triggers dispersal of Cdc14 throughout the cell by a mechanism that has remained unknown.In this study, we show that a MEN component, protein kinase Dbf2-Mob1, promotes transfer of Cdc14 to the cytoplasm and consequent exit from mitosis by direct phosphorylation of Cdc14 on serine and threonine residues adjacent to a nuclear localization signal (NLS), thereby abrogating its NLS activity.Our results define a mechanism by which the MEN promotes exit from mitosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA. mohld@caltech.edu

ABSTRACT
Exit from mitosis is characterized by a precipitous decline in cyclin-dependent kinase (Cdk) activity, dissolution of mitotic structures, and cytokinesis. In Saccharomyces cerevisiae, mitotic exit is driven by a protein phosphatase, Cdc14, which is in part responsible for counteracting Cdk activity. Throughout interphase, Cdc14 is sequestered in the nucleolus, but successful anaphase activates the mitotic exit network (MEN), which triggers dispersal of Cdc14 throughout the cell by a mechanism that has remained unknown. In this study, we show that a MEN component, protein kinase Dbf2-Mob1, promotes transfer of Cdc14 to the cytoplasm and consequent exit from mitosis by direct phosphorylation of Cdc14 on serine and threonine residues adjacent to a nuclear localization signal (NLS), thereby abrogating its NLS activity. Our results define a mechanism by which the MEN promotes exit from mitosis.

Show MeSH