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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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Activity of the MEN regulates binding of Cdk1 to SPBs. (A) Cdk1 does not associate with SPBs in cells with a misaligned anaphase spindle. KAR9 CDK1-3GFP SPC42-eqFP611 and kar9Δ CDK1-3GFP SPC42-eqFP611 cells grown in YPAD were synchronized with α-factor at 30°C and released at 37°C. The pictures in the right corner show enlargements of SPB signals. (B) Quantification of anaphase cells of experiment A with correctly or misaligned spindles. n > 200 cells per strain. (C) Cdc5 and Tem1 are required for Cdk1 localization to SPBs. α-Factor–synchronized wild-type, Gal1-CDC5, and Gal1-UPL-TEM1 cells were grown in YPD medium at 30°C to deplete Cdc5 and Upl-Tem1. Anaphase cells were analyzed for SPB localization of Cdk1-3GFP. n > 75 cells for each strain. (D) α-Factor–synchronized BFA1 CDK1-3GFP SPC42-eqFP611 and bfa1Δ CDK1-3GFP SPC42-eqFP611 cells were grown in YPAD medium at 30°C and examined in anaphase for colocalization of Cdk1-3GFP and the SPB marker Spc42-eqFP611. The pictures in the right corner show enlargements of SPB signals. (E) Quantification of D as illustrated in the figure. n > 80 anaphase cells were analyzed. Bars, 5 µm.
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fig2: Activity of the MEN regulates binding of Cdk1 to SPBs. (A) Cdk1 does not associate with SPBs in cells with a misaligned anaphase spindle. KAR9 CDK1-3GFP SPC42-eqFP611 and kar9Δ CDK1-3GFP SPC42-eqFP611 cells grown in YPAD were synchronized with α-factor at 30°C and released at 37°C. The pictures in the right corner show enlargements of SPB signals. (B) Quantification of anaphase cells of experiment A with correctly or misaligned spindles. n > 200 cells per strain. (C) Cdc5 and Tem1 are required for Cdk1 localization to SPBs. α-Factor–synchronized wild-type, Gal1-CDC5, and Gal1-UPL-TEM1 cells were grown in YPD medium at 30°C to deplete Cdc5 and Upl-Tem1. Anaphase cells were analyzed for SPB localization of Cdk1-3GFP. n > 75 cells for each strain. (D) α-Factor–synchronized BFA1 CDK1-3GFP SPC42-eqFP611 and bfa1Δ CDK1-3GFP SPC42-eqFP611 cells were grown in YPAD medium at 30°C and examined in anaphase for colocalization of Cdk1-3GFP and the SPB marker Spc42-eqFP611. The pictures in the right corner show enlargements of SPB signals. (E) Quantification of D as illustrated in the figure. n > 80 anaphase cells were analyzed. Bars, 5 µm.

Mentions: The MEN is an SPB-associated signaling cascade that becomes active in anaphase (Stegmeier and Amon, 2004). MEN activation could promote the recruitment of Cdk1 to SPBs. To test this hypothesis, we analyzed the localization of Cdk1 in cells lacking either the KAR9 or DYN1 genes. In kar9Δ or dyn1Δ cells, ∼10–20% of anaphase spindles become misaligned in the mother cell body (Li et al., 1993; Miller and Rose, 1998). The MEN of kar9Δ and dyn1Δ cells is inactive when the anaphase spindle is mispositioned, but active when the spindle is correctly aligned along the mother–bud axis (Bardin et al., 2000; Pereira et al., 2000). In kar9Δ cells with a correctly aligned spindle, Cdk1-3GFP bound in early anaphase to the mSPB (Fig. 2 A, kar9Δ, enlargement 4; and Fig. 2 B) in the same way as it did in wild-type cells (Fig. 2 A, KAR9, enlargement 1; and Fig. 2 B). In contrast, Cdk1 was not detectable at either of the two SPBs in kar9Δ cells with a misaligned spindle (Fig. 2 A, kar9Δ, asterisks and enlargements 2 and 3; and Fig. 2 B). In these cells Cdk1 was distributed throughout the nucleus. Similar data were obtained for dyn1Δ cells (Fig. S1, A and B). Thus, MEN activity may regulate the SPB recruitment of Cdk1.


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

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

Activity of the MEN regulates binding of Cdk1 to SPBs. (A) Cdk1 does not associate with SPBs in cells with a misaligned anaphase spindle. KAR9 CDK1-3GFP SPC42-eqFP611 and kar9Δ CDK1-3GFP SPC42-eqFP611 cells grown in YPAD were synchronized with α-factor at 30°C and released at 37°C. The pictures in the right corner show enlargements of SPB signals. (B) Quantification of anaphase cells of experiment A with correctly or misaligned spindles. n > 200 cells per strain. (C) Cdc5 and Tem1 are required for Cdk1 localization to SPBs. α-Factor–synchronized wild-type, Gal1-CDC5, and Gal1-UPL-TEM1 cells were grown in YPD medium at 30°C to deplete Cdc5 and Upl-Tem1. Anaphase cells were analyzed for SPB localization of Cdk1-3GFP. n > 75 cells for each strain. (D) α-Factor–synchronized BFA1 CDK1-3GFP SPC42-eqFP611 and bfa1Δ CDK1-3GFP SPC42-eqFP611 cells were grown in YPAD medium at 30°C and examined in anaphase for colocalization of Cdk1-3GFP and the SPB marker Spc42-eqFP611. The pictures in the right corner show enlargements of SPB signals. (E) Quantification of D as illustrated in the figure. n > 80 anaphase cells were analyzed. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
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fig2: Activity of the MEN regulates binding of Cdk1 to SPBs. (A) Cdk1 does not associate with SPBs in cells with a misaligned anaphase spindle. KAR9 CDK1-3GFP SPC42-eqFP611 and kar9Δ CDK1-3GFP SPC42-eqFP611 cells grown in YPAD were synchronized with α-factor at 30°C and released at 37°C. The pictures in the right corner show enlargements of SPB signals. (B) Quantification of anaphase cells of experiment A with correctly or misaligned spindles. n > 200 cells per strain. (C) Cdc5 and Tem1 are required for Cdk1 localization to SPBs. α-Factor–synchronized wild-type, Gal1-CDC5, and Gal1-UPL-TEM1 cells were grown in YPD medium at 30°C to deplete Cdc5 and Upl-Tem1. Anaphase cells were analyzed for SPB localization of Cdk1-3GFP. n > 75 cells for each strain. (D) α-Factor–synchronized BFA1 CDK1-3GFP SPC42-eqFP611 and bfa1Δ CDK1-3GFP SPC42-eqFP611 cells were grown in YPAD medium at 30°C and examined in anaphase for colocalization of Cdk1-3GFP and the SPB marker Spc42-eqFP611. The pictures in the right corner show enlargements of SPB signals. (E) Quantification of D as illustrated in the figure. n > 80 anaphase cells were analyzed. Bars, 5 µm.
Mentions: The MEN is an SPB-associated signaling cascade that becomes active in anaphase (Stegmeier and Amon, 2004). MEN activation could promote the recruitment of Cdk1 to SPBs. To test this hypothesis, we analyzed the localization of Cdk1 in cells lacking either the KAR9 or DYN1 genes. In kar9Δ or dyn1Δ cells, ∼10–20% of anaphase spindles become misaligned in the mother cell body (Li et al., 1993; Miller and Rose, 1998). The MEN of kar9Δ and dyn1Δ cells is inactive when the anaphase spindle is mispositioned, but active when the spindle is correctly aligned along the mother–bud axis (Bardin et al., 2000; Pereira et al., 2000). In kar9Δ cells with a correctly aligned spindle, Cdk1-3GFP bound in early anaphase to the mSPB (Fig. 2 A, kar9Δ, enlargement 4; and Fig. 2 B) in the same way as it did in wild-type cells (Fig. 2 A, KAR9, enlargement 1; and Fig. 2 B). In contrast, Cdk1 was not detectable at either of the two SPBs in kar9Δ cells with a misaligned spindle (Fig. 2 A, kar9Δ, asterisks and enlargements 2 and 3; and Fig. 2 B). In these cells Cdk1 was distributed throughout the nucleus. Similar data were obtained for dyn1Δ cells (Fig. S1, A and B). Thus, MEN activity may regulate the SPB recruitment of Cdk1.

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