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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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Bypass of MEN by inactivation of Cdc14’s C-terminal NLS compromises the checkpoint that monitors spindle position. (A) Inactivation of Cdc14’s C-terminal NLS bypasses the essential function of the MEN kinase Cdc15. Cdc15Δ yeast carrying plasmid-borne copies of the indicated CDC14 alleles and kept alive by a CDC15 URA3 plasmid were serially diluted and plated on 5-fluoroorotic acid to select for transformants that were able to grow without the CDC15 URA3 plasmid. (B) Growth of cdc15Δ and CDC15+ cells harboring the endogenous CDC14 gene and plasmid (pRS315)-borne alleles. Growth was measured by threefold serial dilution of yeast cells that were spotted onto synthetic media lacking leucine and incubated at 30°C. Mutant PS1,2E and BP1,2A alleles bypass cdc15Δ but grow more slowly than wild-type (WT) cells, suggesting that NLS mutations do not fully restore CDC15’s essential functions. (C) Model for MEN regulation of Cdc14. (i) MEN activity promotes release of Cdc14 from nucleolar Net1, and some of this Cdc14 makes its way to the cytoplasm. MEN also prevents reuptake of this cytoplasmic Cdc14 into the nucleus by inactivating the C-terminal NLS, allowing Cdc14 to accumulate in the cytoplasm. (ii) In a MEN mutant (e.g., cdc15Δ), release of Cdc14 from the nucleolus is inhibited. Those few molecules that are released and translocated to the cytoplasm fail to accumulate there because the C-terminal NLS remains active. Consequently, both the nuclear and cytoplasmic pools of Cdc14 are very low. (iii) The same as in ii is shown except that the C-terminal NLS is mutated. Consequently, the small number of Cdc14 molecules that leak through to the cytoplasm can accumulate there, and thereby enable suppression of cdc15Δ. (D) Constitutive inactivation of Cdc14’s C-terminal NLS compromises growth of dyn1Δ cells. Threefold serial dilutions of strains carrying the indicated CDC14 NLS mutant alleles on a low copy centromeric plasmid in either CDC14 dyn1Δ or CDC14 DYN1 backgrounds are shown. (E, left) DIC and DAPI fluorescence images of dyn1Δ cells harboring the indicated CDC14 alleles on a low copy centromeric plasmid. Yeast cultures were grown at 25°C. Cells with multiple nuclei are indicated by arrows. (right) Graph indicating the frequency with which dyn1Δ cells accumulated multiple nuclei in the presence of various CDC14 alleles (error bars indicate SD of three replicate experiments; n > 350 for each strain). VC, vector control. Bars, 2 µm.
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fig7: Bypass of MEN by inactivation of Cdc14’s C-terminal NLS compromises the checkpoint that monitors spindle position. (A) Inactivation of Cdc14’s C-terminal NLS bypasses the essential function of the MEN kinase Cdc15. Cdc15Δ yeast carrying plasmid-borne copies of the indicated CDC14 alleles and kept alive by a CDC15 URA3 plasmid were serially diluted and plated on 5-fluoroorotic acid to select for transformants that were able to grow without the CDC15 URA3 plasmid. (B) Growth of cdc15Δ and CDC15+ cells harboring the endogenous CDC14 gene and plasmid (pRS315)-borne alleles. Growth was measured by threefold serial dilution of yeast cells that were spotted onto synthetic media lacking leucine and incubated at 30°C. Mutant PS1,2E and BP1,2A alleles bypass cdc15Δ but grow more slowly than wild-type (WT) cells, suggesting that NLS mutations do not fully restore CDC15’s essential functions. (C) Model for MEN regulation of Cdc14. (i) MEN activity promotes release of Cdc14 from nucleolar Net1, and some of this Cdc14 makes its way to the cytoplasm. MEN also prevents reuptake of this cytoplasmic Cdc14 into the nucleus by inactivating the C-terminal NLS, allowing Cdc14 to accumulate in the cytoplasm. (ii) In a MEN mutant (e.g., cdc15Δ), release of Cdc14 from the nucleolus is inhibited. Those few molecules that are released and translocated to the cytoplasm fail to accumulate there because the C-terminal NLS remains active. Consequently, both the nuclear and cytoplasmic pools of Cdc14 are very low. (iii) The same as in ii is shown except that the C-terminal NLS is mutated. Consequently, the small number of Cdc14 molecules that leak through to the cytoplasm can accumulate there, and thereby enable suppression of cdc15Δ. (D) Constitutive inactivation of Cdc14’s C-terminal NLS compromises growth of dyn1Δ cells. Threefold serial dilutions of strains carrying the indicated CDC14 NLS mutant alleles on a low copy centromeric plasmid in either CDC14 dyn1Δ or CDC14 DYN1 backgrounds are shown. (E, left) DIC and DAPI fluorescence images of dyn1Δ cells harboring the indicated CDC14 alleles on a low copy centromeric plasmid. Yeast cultures were grown at 25°C. Cells with multiple nuclei are indicated by arrows. (right) Graph indicating the frequency with which dyn1Δ cells accumulated multiple nuclei in the presence of various CDC14 alleles (error bars indicate SD of three replicate experiments; n > 350 for each strain). VC, vector control. Bars, 2 µm.

Mentions: If dispersion of Cdc14 throughout the cell contributes to the exit from mitosis, we reasoned that constitutive inactivation of Cdc14’s C-terminal NLS might reduce the requirement for MEN. To address this question, we transformed a cdc15Δ strain kept alive by a CDC15 URA3 plasmid with CEN plasmid-borne CDC14 alleles and selected for loss of the CDC15 vector by plating cells on 5FOA. BP mutant CDC14-BP1,2A allowed bypass of cdc15Δ, whereas wild-type CDC14 did not (Fig. 7 A). Moreover, the CDC14-PS1,2E phosphomimetic allele also enabled bypass of cdc15Δ, although their growth relative to cells with a fully functional CDC15 gene was compromised (Fig. 7, A and B). This experiment, together with the experiment shown in Fig. 6, provides strong evidence that phosphorylation of Cdc14’s C-terminal NLS by Dbf2–Mob1 is one (but not the only) mechanism by which MEN promotes exit from mitosis.


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)

Bypass of MEN by inactivation of Cdc14’s C-terminal NLS compromises the checkpoint that monitors spindle position. (A) Inactivation of Cdc14’s C-terminal NLS bypasses the essential function of the MEN kinase Cdc15. Cdc15Δ yeast carrying plasmid-borne copies of the indicated CDC14 alleles and kept alive by a CDC15 URA3 plasmid were serially diluted and plated on 5-fluoroorotic acid to select for transformants that were able to grow without the CDC15 URA3 plasmid. (B) Growth of cdc15Δ and CDC15+ cells harboring the endogenous CDC14 gene and plasmid (pRS315)-borne alleles. Growth was measured by threefold serial dilution of yeast cells that were spotted onto synthetic media lacking leucine and incubated at 30°C. Mutant PS1,2E and BP1,2A alleles bypass cdc15Δ but grow more slowly than wild-type (WT) cells, suggesting that NLS mutations do not fully restore CDC15’s essential functions. (C) Model for MEN regulation of Cdc14. (i) MEN activity promotes release of Cdc14 from nucleolar Net1, and some of this Cdc14 makes its way to the cytoplasm. MEN also prevents reuptake of this cytoplasmic Cdc14 into the nucleus by inactivating the C-terminal NLS, allowing Cdc14 to accumulate in the cytoplasm. (ii) In a MEN mutant (e.g., cdc15Δ), release of Cdc14 from the nucleolus is inhibited. Those few molecules that are released and translocated to the cytoplasm fail to accumulate there because the C-terminal NLS remains active. Consequently, both the nuclear and cytoplasmic pools of Cdc14 are very low. (iii) The same as in ii is shown except that the C-terminal NLS is mutated. Consequently, the small number of Cdc14 molecules that leak through to the cytoplasm can accumulate there, and thereby enable suppression of cdc15Δ. (D) Constitutive inactivation of Cdc14’s C-terminal NLS compromises growth of dyn1Δ cells. Threefold serial dilutions of strains carrying the indicated CDC14 NLS mutant alleles on a low copy centromeric plasmid in either CDC14 dyn1Δ or CDC14 DYN1 backgrounds are shown. (E, left) DIC and DAPI fluorescence images of dyn1Δ cells harboring the indicated CDC14 alleles on a low copy centromeric plasmid. Yeast cultures were grown at 25°C. Cells with multiple nuclei are indicated by arrows. (right) Graph indicating the frequency with which dyn1Δ cells accumulated multiple nuclei in the presence of various CDC14 alleles (error bars indicate SD of three replicate experiments; n > 350 for each strain). VC, vector control. Bars, 2 µm.
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fig7: Bypass of MEN by inactivation of Cdc14’s C-terminal NLS compromises the checkpoint that monitors spindle position. (A) Inactivation of Cdc14’s C-terminal NLS bypasses the essential function of the MEN kinase Cdc15. Cdc15Δ yeast carrying plasmid-borne copies of the indicated CDC14 alleles and kept alive by a CDC15 URA3 plasmid were serially diluted and plated on 5-fluoroorotic acid to select for transformants that were able to grow without the CDC15 URA3 plasmid. (B) Growth of cdc15Δ and CDC15+ cells harboring the endogenous CDC14 gene and plasmid (pRS315)-borne alleles. Growth was measured by threefold serial dilution of yeast cells that were spotted onto synthetic media lacking leucine and incubated at 30°C. Mutant PS1,2E and BP1,2A alleles bypass cdc15Δ but grow more slowly than wild-type (WT) cells, suggesting that NLS mutations do not fully restore CDC15’s essential functions. (C) Model for MEN regulation of Cdc14. (i) MEN activity promotes release of Cdc14 from nucleolar Net1, and some of this Cdc14 makes its way to the cytoplasm. MEN also prevents reuptake of this cytoplasmic Cdc14 into the nucleus by inactivating the C-terminal NLS, allowing Cdc14 to accumulate in the cytoplasm. (ii) In a MEN mutant (e.g., cdc15Δ), release of Cdc14 from the nucleolus is inhibited. Those few molecules that are released and translocated to the cytoplasm fail to accumulate there because the C-terminal NLS remains active. Consequently, both the nuclear and cytoplasmic pools of Cdc14 are very low. (iii) The same as in ii is shown except that the C-terminal NLS is mutated. Consequently, the small number of Cdc14 molecules that leak through to the cytoplasm can accumulate there, and thereby enable suppression of cdc15Δ. (D) Constitutive inactivation of Cdc14’s C-terminal NLS compromises growth of dyn1Δ cells. Threefold serial dilutions of strains carrying the indicated CDC14 NLS mutant alleles on a low copy centromeric plasmid in either CDC14 dyn1Δ or CDC14 DYN1 backgrounds are shown. (E, left) DIC and DAPI fluorescence images of dyn1Δ cells harboring the indicated CDC14 alleles on a low copy centromeric plasmid. Yeast cultures were grown at 25°C. Cells with multiple nuclei are indicated by arrows. (right) Graph indicating the frequency with which dyn1Δ cells accumulated multiple nuclei in the presence of various CDC14 alleles (error bars indicate SD of three replicate experiments; n > 350 for each strain). VC, vector control. Bars, 2 µm.
Mentions: If dispersion of Cdc14 throughout the cell contributes to the exit from mitosis, we reasoned that constitutive inactivation of Cdc14’s C-terminal NLS might reduce the requirement for MEN. To address this question, we transformed a cdc15Δ strain kept alive by a CDC15 URA3 plasmid with CEN plasmid-borne CDC14 alleles and selected for loss of the CDC15 vector by plating cells on 5FOA. BP mutant CDC14-BP1,2A allowed bypass of cdc15Δ, whereas wild-type CDC14 did not (Fig. 7 A). Moreover, the CDC14-PS1,2E phosphomimetic allele also enabled bypass of cdc15Δ, although their growth relative to cells with a fully functional CDC15 gene was compromised (Fig. 7, A and B). This experiment, together with the experiment shown in Fig. 6, provides strong evidence that phosphorylation of Cdc14’s C-terminal NLS by Dbf2–Mob1 is one (but not the only) mechanism by which MEN promotes exit from mitosis.

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
Related in: MedlinePlus