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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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SPB localization of Cdk1 depends on Cdc15 kinase activity. (A and B) CDC15 and cdc15-as1 cells were synchronized with α-factor and released at 30°C into YPAD medium with the PP1 analogue 8 to inhibit activity of cdc15-as1 kinase (D’Aquino et al., 2005). Anaphase cells with a 4–10-µm spindle were examined for the Cdk1-GFP localization. n = 38 for CDC15, n = 43 for cdc15-as1. The arrows in A point toward the Cdk1-GFP signal at mSPBs. (C) Localization of Cdc15-GFP and cdc15-as1-GFP in anaphase. Two representative cells are shown for each strain grown in YPAD at 30°C in the presence of PP1 analogue 8. The first Cdc15-GFP and cdc15-as1-GFP pictures were taken under identical conditions. The second pictures in this row are linear enhancements of the first pictures. Bar, 5 µm. (D) Protein levels of Cdc15-GFP and cdc15-as1-GFP detected with anti-GFP antibody. Anti-Tub2 was used as loading control. (E) The Dbf2–Mob1 kinase complex is not required for SPB association of Cdk1 in anaphase. Synchronized wild-type, mob1-67, and dbf2-2 cells were grown in YPAD at 37°C after the release of the α-factor block. Anaphase cells were examined for SPB localization of Cdk1-GFP. (F) Quantification of E. n > 75 anaphase cells per strain. Bars, 5 µm.
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fig3: SPB localization of Cdk1 depends on Cdc15 kinase activity. (A and B) CDC15 and cdc15-as1 cells were synchronized with α-factor and released at 30°C into YPAD medium with the PP1 analogue 8 to inhibit activity of cdc15-as1 kinase (D’Aquino et al., 2005). Anaphase cells with a 4–10-µm spindle were examined for the Cdk1-GFP localization. n = 38 for CDC15, n = 43 for cdc15-as1. The arrows in A point toward the Cdk1-GFP signal at mSPBs. (C) Localization of Cdc15-GFP and cdc15-as1-GFP in anaphase. Two representative cells are shown for each strain grown in YPAD at 30°C in the presence of PP1 analogue 8. The first Cdc15-GFP and cdc15-as1-GFP pictures were taken under identical conditions. The second pictures in this row are linear enhancements of the first pictures. Bar, 5 µm. (D) Protein levels of Cdc15-GFP and cdc15-as1-GFP detected with anti-GFP antibody. Anti-Tub2 was used as loading control. (E) The Dbf2–Mob1 kinase complex is not required for SPB association of Cdk1 in anaphase. Synchronized wild-type, mob1-67, and dbf2-2 cells were grown in YPAD at 37°C after the release of the α-factor block. Anaphase cells were examined for SPB localization of Cdk1-GFP. (F) Quantification of E. n > 75 anaphase cells per strain. Bars, 5 µm.

Mentions: Our analysis already demonstrated the essential role of the GTPase Tem1 in recruiting Cdk1 to SPBs (Fig. 2 C). We now asked whether the MEN kinase Cdc15 was also required to promote the binding of Cdk1 to the dSPB. The role of CDC15 was first analyzed in cells expressing the cdc15-as1 allele that can be inhibited by the ATP analogue “PP1 analog 8” (D’Aquino et al., 2005). Inhibition of cdc15-as1 kinase activity strongly reduced the efficiency of Cdk1-GFP binding to the mSPB in any stage of anaphase (Fig. 3, A and B). This failure in Cdk1 recruitment was not caused by a deficiency in the binding of the cdc15-as1 protein to SPBs. On the contrary, cdc15-as1-GFP accumulated to a much higher level at SPBs than Cdc15-GFP (Fig. 3 C), even though the cellular levels of Cdc15 and cdc15-as1 were identical as shown by immunoblot (Fig. 3 D). The dependency of SPB localization upon Cdc15 activity was confirmed in cdc15-1 cells (Fig. S2, A and B).


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

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

SPB localization of Cdk1 depends on Cdc15 kinase activity. (A and B) CDC15 and cdc15-as1 cells were synchronized with α-factor and released at 30°C into YPAD medium with the PP1 analogue 8 to inhibit activity of cdc15-as1 kinase (D’Aquino et al., 2005). Anaphase cells with a 4–10-µm spindle were examined for the Cdk1-GFP localization. n = 38 for CDC15, n = 43 for cdc15-as1. The arrows in A point toward the Cdk1-GFP signal at mSPBs. (C) Localization of Cdc15-GFP and cdc15-as1-GFP in anaphase. Two representative cells are shown for each strain grown in YPAD at 30°C in the presence of PP1 analogue 8. The first Cdc15-GFP and cdc15-as1-GFP pictures were taken under identical conditions. The second pictures in this row are linear enhancements of the first pictures. Bar, 5 µm. (D) Protein levels of Cdc15-GFP and cdc15-as1-GFP detected with anti-GFP antibody. Anti-Tub2 was used as loading control. (E) The Dbf2–Mob1 kinase complex is not required for SPB association of Cdk1 in anaphase. Synchronized wild-type, mob1-67, and dbf2-2 cells were grown in YPAD at 37°C after the release of the α-factor block. Anaphase cells were examined for SPB localization of Cdk1-GFP. (F) Quantification of E. n > 75 anaphase cells per strain. Bars, 5 µm.
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Related In: Results  -  Collection

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Show All Figures
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fig3: SPB localization of Cdk1 depends on Cdc15 kinase activity. (A and B) CDC15 and cdc15-as1 cells were synchronized with α-factor and released at 30°C into YPAD medium with the PP1 analogue 8 to inhibit activity of cdc15-as1 kinase (D’Aquino et al., 2005). Anaphase cells with a 4–10-µm spindle were examined for the Cdk1-GFP localization. n = 38 for CDC15, n = 43 for cdc15-as1. The arrows in A point toward the Cdk1-GFP signal at mSPBs. (C) Localization of Cdc15-GFP and cdc15-as1-GFP in anaphase. Two representative cells are shown for each strain grown in YPAD at 30°C in the presence of PP1 analogue 8. The first Cdc15-GFP and cdc15-as1-GFP pictures were taken under identical conditions. The second pictures in this row are linear enhancements of the first pictures. Bar, 5 µm. (D) Protein levels of Cdc15-GFP and cdc15-as1-GFP detected with anti-GFP antibody. Anti-Tub2 was used as loading control. (E) The Dbf2–Mob1 kinase complex is not required for SPB association of Cdk1 in anaphase. Synchronized wild-type, mob1-67, and dbf2-2 cells were grown in YPAD at 37°C after the release of the α-factor block. Anaphase cells were examined for SPB localization of Cdk1-GFP. (F) Quantification of E. n > 75 anaphase cells per strain. Bars, 5 µm.
Mentions: Our analysis already demonstrated the essential role of the GTPase Tem1 in recruiting Cdk1 to SPBs (Fig. 2 C). We now asked whether the MEN kinase Cdc15 was also required to promote the binding of Cdk1 to the dSPB. The role of CDC15 was first analyzed in cells expressing the cdc15-as1 allele that can be inhibited by the ATP analogue “PP1 analog 8” (D’Aquino et al., 2005). Inhibition of cdc15-as1 kinase activity strongly reduced the efficiency of Cdk1-GFP binding to the mSPB in any stage of anaphase (Fig. 3, A and B). This failure in Cdk1 recruitment was not caused by a deficiency in the binding of the cdc15-as1 protein to SPBs. On the contrary, cdc15-as1-GFP accumulated to a much higher level at SPBs than Cdc15-GFP (Fig. 3 C), even though the cellular levels of Cdc15 and cdc15-as1 were identical as shown by immunoblot (Fig. 3 D). The dependency of SPB localization upon Cdc15 activity was confirmed in cdc15-1 cells (Fig. S2, A and B).

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