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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 NLS are required for efficient release from a late mitotic block. (A) Mutation of NLS phosphorylation sites impaired mitotic exit after reversal of cell cycle arrest. CDC14 and cdc14-(PS1,2A) cultures were placed at 37°C for 2 h to achieve a late mitotic cdc15-2 arrest and returned to permissive temperature (25°C). Clb2p and Cdc28 levels were followed by immunoblot analysis. (B) Cell aliquots from A were stained with DAPI and evaluated by fluorescence microscopy. Combined DIC and DAPI fluorescence images are shown. (C, left) Clb2 protein detected in A was quantified and normalized to Cdc28 levels. Relative units of fluorescence intensity were plotted for each time point. (right) The frequency of large-budded cells (n > 200) with segregated nuclei (B) was plotted for each time point. WT, wild type. Bars, 2 µm.
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fig6: Phosphorylation sites within NLS are required for efficient release from a late mitotic block. (A) Mutation of NLS phosphorylation sites impaired mitotic exit after reversal of cell cycle arrest. CDC14 and cdc14-(PS1,2A) cultures were placed at 37°C for 2 h to achieve a late mitotic cdc15-2 arrest and returned to permissive temperature (25°C). Clb2p and Cdc28 levels were followed by immunoblot analysis. (B) Cell aliquots from A were stained with DAPI and evaluated by fluorescence microscopy. Combined DIC and DAPI fluorescence images are shown. (C, left) Clb2 protein detected in A was quantified and normalized to Cdc28 levels. Relative units of fluorescence intensity were plotted for each time point. (right) The frequency of large-budded cells (n > 200) with segregated nuclei (B) was plotted for each time point. WT, wild type. Bars, 2 µm.

Mentions: The robust growth of cells sustained by Cdc14-PS1,2A suggested that Dbf2–Mob1 needn’t inactivate the C-terminal NLS region of Cdc14 for cells to exit from mitosis. To reconcile the profound effect these mutations have on NLS function with their ability to complement cdc14Δ, we sought to determine whether these mutants were at least partially impaired by using a more sensitive assay. The temperature-sensitive cdc15-2 allele was introduced into CDC14 and cdc14-PS1,2A strains, and the cells were synchronized in late anaphase by shifting them to the nonpermissive temperature. Once the cultures had arrested at the cdc15-2 block, they were returned to the permissive temperature to restore Cdc15 function, and we tracked two hallmarks of mitotic exit: degradation of mitotic cyclin Clb2 and the completion of cytokinesis. CDC14 cells efficiently escaped mitotic arrest after reversal of the cdc15-2 block. Clb2 protein levels began to drop between 40 and 50 min after shifting the culture to 25°C (Fig. 6, A and C), and cells executed cytokinesis, resulting in a sharp reduction of the number of large-budded cells with segregated nuclei (Fig. 6, B and C). In contrast, cdc14-PS1,2A cells failed to resume cell division after reversal of the cdc15-2 block, resulting in high levels of Clb2 protein for the duration of the experiment and a failure to undergo cytokinesis (Fig. 6, A–C). The cytokinetic defect is consistent with the behavior of a Cdc14 NES mutant that also fails to accumulate in the cytoplasm (Bembenek et al., 2005). Thus, MEN-dependent inactivation of Cdc14’s C-terminal NLS is important for exit from mitosis when the activity of MEN has been compromised by the cdc15-2 mutation.


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 NLS are required for efficient release from a late mitotic block. (A) Mutation of NLS phosphorylation sites impaired mitotic exit after reversal of cell cycle arrest. CDC14 and cdc14-(PS1,2A) cultures were placed at 37°C for 2 h to achieve a late mitotic cdc15-2 arrest and returned to permissive temperature (25°C). Clb2p and Cdc28 levels were followed by immunoblot analysis. (B) Cell aliquots from A were stained with DAPI and evaluated by fluorescence microscopy. Combined DIC and DAPI fluorescence images are shown. (C, left) Clb2 protein detected in A was quantified and normalized to Cdc28 levels. Relative units of fluorescence intensity were plotted for each time point. (right) The frequency of large-budded cells (n > 200) with segregated nuclei (B) was plotted for each time point. WT, wild type. Bars, 2 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig6: Phosphorylation sites within NLS are required for efficient release from a late mitotic block. (A) Mutation of NLS phosphorylation sites impaired mitotic exit after reversal of cell cycle arrest. CDC14 and cdc14-(PS1,2A) cultures were placed at 37°C for 2 h to achieve a late mitotic cdc15-2 arrest and returned to permissive temperature (25°C). Clb2p and Cdc28 levels were followed by immunoblot analysis. (B) Cell aliquots from A were stained with DAPI and evaluated by fluorescence microscopy. Combined DIC and DAPI fluorescence images are shown. (C, left) Clb2 protein detected in A was quantified and normalized to Cdc28 levels. Relative units of fluorescence intensity were plotted for each time point. (right) The frequency of large-budded cells (n > 200) with segregated nuclei (B) was plotted for each time point. WT, wild type. Bars, 2 µm.
Mentions: The robust growth of cells sustained by Cdc14-PS1,2A suggested that Dbf2–Mob1 needn’t inactivate the C-terminal NLS region of Cdc14 for cells to exit from mitosis. To reconcile the profound effect these mutations have on NLS function with their ability to complement cdc14Δ, we sought to determine whether these mutants were at least partially impaired by using a more sensitive assay. The temperature-sensitive cdc15-2 allele was introduced into CDC14 and cdc14-PS1,2A strains, and the cells were synchronized in late anaphase by shifting them to the nonpermissive temperature. Once the cultures had arrested at the cdc15-2 block, they were returned to the permissive temperature to restore Cdc15 function, and we tracked two hallmarks of mitotic exit: degradation of mitotic cyclin Clb2 and the completion of cytokinesis. CDC14 cells efficiently escaped mitotic arrest after reversal of the cdc15-2 block. Clb2 protein levels began to drop between 40 and 50 min after shifting the culture to 25°C (Fig. 6, A and C), and cells executed cytokinesis, resulting in a sharp reduction of the number of large-budded cells with segregated nuclei (Fig. 6, B and C). In contrast, cdc14-PS1,2A cells failed to resume cell division after reversal of the cdc15-2 block, resulting in high levels of Clb2 protein for the duration of the experiment and a failure to undergo cytokinesis (Fig. 6, A–C). The cytokinetic defect is consistent with the behavior of a Cdc14 NES mutant that also fails to accumulate in the cytoplasm (Bembenek et al., 2005). Thus, MEN-dependent inactivation of Cdc14’s C-terminal NLS is important for exit from mitosis when the activity of MEN has been compromised by the cdc15-2 mutation.

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