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Mutual regulation of cyclin-dependent kinase and the mitotic exit network.

König C, Maekawa H, Schiebel E - J. Cell Biol. (2010)

Bottom Line: The mitotic exit network (MEN) is a spindle pole body (SPB)-associated, GTPase-driven signaling cascade that controls mitotic exit.Our data revise the understanding of the spatial regulation of the MEN.Consistent with this model, only triple mutants that lack BUB2 and the Cdk1 phosphorylation sites in Mob1 and Cdc15 show mitotic exit defects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), ZMBH-DKFZ Alliance, 69120 Heidelberg, Germany.

ABSTRACT
The mitotic exit network (MEN) is a spindle pole body (SPB)-associated, GTPase-driven signaling cascade that controls mitotic exit. The inhibitory Bfa1-Bub2 GTPase-activating protein (GAP) only associates with the daughter SPB (dSPB), raising the question as to how the MEN is regulated on the mother SPB (mSPB). Here, we show mutual regulation of cyclin-dependent kinase 1 (Cdk1) and the MEN. In early anaphase Cdk1 becomes recruited to the mSPB depending on the activity of the MEN kinase Cdc15. Conversely, Cdk1 negatively regulates binding of Cdc15 to the mSPB. In addition, Cdk1 phosphorylates the Mob1 protein to inhibit the activity of Dbf2-Mob1 kinase that regulates Cdc14 phosphatase. Our data revise the understanding of the spatial regulation of the MEN. Although MEN activity in the daughter cells is controlled by Bfa1-Bub2, Cdk1 inhibits MEN activity at the mSPB. Consistent with this model, only triple mutants that lack BUB2 and the Cdk1 phosphorylation sites in Mob1 and Cdc15 show mitotic exit defects.

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Cdc15 and Cdk1 show mutual regulation at the mSPB. (A) CDC14 and td-cdc14 cells harboring CDC15-GFP mCherry-TUB1 were examined for Cdc15-GFP localization in anaphase. The arrows highlight Cdc15-GFP at SPBs. Bar, 5 µm. (B and C) Anaphase cells of CDC14 CDC15-GFP, td-cdc14 CDC15-GFP, and td-cdc14 CDC15-7A-GFP were grown in YPAD and analyzed for GFP signal at SPBs. Quantified relative fluorescent intensities are summarized in box-and-whisker plots: boxes span between the 25th and 75th percentile with a line at the median; whiskers extend from the 10th to 90th percentile. P-values were calculated using unpaired t tests and indicate significant differences between * or ** marked bars. (B) n > 50 anaphase cells per strain. (C) n > 50 for CDC15-GFP cells and n = 24 for CDC15-7A-GFP cells. (D) CDC15 and CDC15-7A cells were grown in SC medium. Cells in anaphase were examined for Cdk1-GFP localization to SPBs. Bar, 5 µm. (E) Quantification of Cdk1-GFP signal at the mSPB. Relative fluorescent intensities in box-and-whisker plots as in B and C. n > 50 cells were analyzed per strain. (F) Cdk1-GFP protein levels measured with anti-GFP antibody and actin as loading control.
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fig4: Cdc15 and Cdk1 show mutual regulation at the mSPB. (A) CDC14 and td-cdc14 cells harboring CDC15-GFP mCherry-TUB1 were examined for Cdc15-GFP localization in anaphase. The arrows highlight Cdc15-GFP at SPBs. Bar, 5 µm. (B and C) Anaphase cells of CDC14 CDC15-GFP, td-cdc14 CDC15-GFP, and td-cdc14 CDC15-7A-GFP were grown in YPAD and analyzed for GFP signal at SPBs. Quantified relative fluorescent intensities are summarized in box-and-whisker plots: boxes span between the 25th and 75th percentile with a line at the median; whiskers extend from the 10th to 90th percentile. P-values were calculated using unpaired t tests and indicate significant differences between * or ** marked bars. (B) n > 50 anaphase cells per strain. (C) n > 50 for CDC15-GFP cells and n = 24 for CDC15-7A-GFP cells. (D) CDC15 and CDC15-7A cells were grown in SC medium. Cells in anaphase were examined for Cdk1-GFP localization to SPBs. Bar, 5 µm. (E) Quantification of Cdk1-GFP signal at the mSPB. Relative fluorescent intensities in box-and-whisker plots as in B and C. n > 50 cells were analyzed per strain. (F) Cdk1-GFP protein levels measured with anti-GFP antibody and actin as loading control.

Mentions: In synchronized CDC15-GFP cells, Cdc15 showed a weak association with both the mSPB and dSPB in early anaphase (Fig. 4 B, yellow bar, 4–6 µm) and, as reported (Visintin and Amon, 2001), this association was enhanced in mid- to late anaphase (Fig. 4, A and B, blue bar, >6 µm). Importantly, Cdk1 regulated SPB binding of Cdc15 as shown in td-cdc14 degron cells. In td-cdc14 cells the hyperphosphorylated Cdc15-GFP did not bind strongly to SPBs, even after the spindle had extended to lengths exceeding 6 µm (Fig. 4, A and B, red bars). Cdk1 phosphorylation of Cdc15 may therefore inhibit SPB association. To confirm this, we quantified SPB binding of the nonphosphorylated Cdc15-7A-GFP in td-cdc14 cells. Cdc15-7A-GFP bound strongly to SPBs nearly independently of anaphase progression (Fig. 4 C, red bars).


Mutual regulation of cyclin-dependent kinase and the mitotic exit network.

König C, Maekawa H, Schiebel E - J. Cell Biol. (2010)

Cdc15 and Cdk1 show mutual regulation at the mSPB. (A) CDC14 and td-cdc14 cells harboring CDC15-GFP mCherry-TUB1 were examined for Cdc15-GFP localization in anaphase. The arrows highlight Cdc15-GFP at SPBs. Bar, 5 µm. (B and C) Anaphase cells of CDC14 CDC15-GFP, td-cdc14 CDC15-GFP, and td-cdc14 CDC15-7A-GFP were grown in YPAD and analyzed for GFP signal at SPBs. Quantified relative fluorescent intensities are summarized in box-and-whisker plots: boxes span between the 25th and 75th percentile with a line at the median; whiskers extend from the 10th to 90th percentile. P-values were calculated using unpaired t tests and indicate significant differences between * or ** marked bars. (B) n > 50 anaphase cells per strain. (C) n > 50 for CDC15-GFP cells and n = 24 for CDC15-7A-GFP cells. (D) CDC15 and CDC15-7A cells were grown in SC medium. Cells in anaphase were examined for Cdk1-GFP localization to SPBs. Bar, 5 µm. (E) Quantification of Cdk1-GFP signal at the mSPB. Relative fluorescent intensities in box-and-whisker plots as in B and C. n > 50 cells were analyzed per strain. (F) Cdk1-GFP protein levels measured with anti-GFP antibody and actin as loading control.
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fig4: Cdc15 and Cdk1 show mutual regulation at the mSPB. (A) CDC14 and td-cdc14 cells harboring CDC15-GFP mCherry-TUB1 were examined for Cdc15-GFP localization in anaphase. The arrows highlight Cdc15-GFP at SPBs. Bar, 5 µm. (B and C) Anaphase cells of CDC14 CDC15-GFP, td-cdc14 CDC15-GFP, and td-cdc14 CDC15-7A-GFP were grown in YPAD and analyzed for GFP signal at SPBs. Quantified relative fluorescent intensities are summarized in box-and-whisker plots: boxes span between the 25th and 75th percentile with a line at the median; whiskers extend from the 10th to 90th percentile. P-values were calculated using unpaired t tests and indicate significant differences between * or ** marked bars. (B) n > 50 anaphase cells per strain. (C) n > 50 for CDC15-GFP cells and n = 24 for CDC15-7A-GFP cells. (D) CDC15 and CDC15-7A cells were grown in SC medium. Cells in anaphase were examined for Cdk1-GFP localization to SPBs. Bar, 5 µm. (E) Quantification of Cdk1-GFP signal at the mSPB. Relative fluorescent intensities in box-and-whisker plots as in B and C. n > 50 cells were analyzed per strain. (F) Cdk1-GFP protein levels measured with anti-GFP antibody and actin as loading control.
Mentions: In synchronized CDC15-GFP cells, Cdc15 showed a weak association with both the mSPB and dSPB in early anaphase (Fig. 4 B, yellow bar, 4–6 µm) and, as reported (Visintin and Amon, 2001), this association was enhanced in mid- to late anaphase (Fig. 4, A and B, blue bar, >6 µm). Importantly, Cdk1 regulated SPB binding of Cdc15 as shown in td-cdc14 degron cells. In td-cdc14 cells the hyperphosphorylated Cdc15-GFP did not bind strongly to SPBs, even after the spindle had extended to lengths exceeding 6 µm (Fig. 4, A and B, red bars). Cdk1 phosphorylation of Cdc15 may therefore inhibit SPB association. To confirm this, we quantified SPB binding of the nonphosphorylated Cdc15-7A-GFP in td-cdc14 cells. Cdc15-7A-GFP bound strongly to SPBs nearly independently of anaphase progression (Fig. 4 C, red bars).

Bottom Line: The mitotic exit network (MEN) is a spindle pole body (SPB)-associated, GTPase-driven signaling cascade that controls mitotic exit.Our data revise the understanding of the spatial regulation of the MEN.Consistent with this model, only triple mutants that lack BUB2 and the Cdk1 phosphorylation sites in Mob1 and Cdc15 show mitotic exit defects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), ZMBH-DKFZ Alliance, 69120 Heidelberg, Germany.

ABSTRACT
The mitotic exit network (MEN) is a spindle pole body (SPB)-associated, GTPase-driven signaling cascade that controls mitotic exit. The inhibitory Bfa1-Bub2 GTPase-activating protein (GAP) only associates with the daughter SPB (dSPB), raising the question as to how the MEN is regulated on the mother SPB (mSPB). Here, we show mutual regulation of cyclin-dependent kinase 1 (Cdk1) and the MEN. In early anaphase Cdk1 becomes recruited to the mSPB depending on the activity of the MEN kinase Cdc15. Conversely, Cdk1 negatively regulates binding of Cdc15 to the mSPB. In addition, Cdk1 phosphorylates the Mob1 protein to inhibit the activity of Dbf2-Mob1 kinase that regulates Cdc14 phosphatase. Our data revise the understanding of the spatial regulation of the MEN. Although MEN activity in the daughter cells is controlled by Bfa1-Bub2, Cdk1 inhibits MEN activity at the mSPB. Consistent with this model, only triple mutants that lack BUB2 and the Cdk1 phosphorylation sites in Mob1 and Cdc15 show mitotic exit defects.

Show MeSH
Related in: MedlinePlus