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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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Cdk1 regulates kinase activity of Dbf2–Mob1. (A) Log-phase cells with the indicated phenotypes were serially diluted 10-fold and spotted onto YPD plates. Plates were incubated for 2 d at 23 or 37°C. (B) Active Dbf2–Mob1 complex was incubated without substrate (lane 1), GST (lane 2), and GST-C-Cdc14 (lane 3) in the presence of γ-[32P]ATP. Shown is an autoradiography. (C) Cdk1–Clb2 kinase inhibits the activation of Dbf2–Mob1 kinase by Cdc15 in vitro. GST-Mob1 in a complex with Dbf2 was incubated with Cdk1–Clb2 or Cdc15 in the first and second kinase reaction as indicated in the figure. After the second reaction, Mob1-Dbf2 kinase assays with GST-C-Cdc14 as substrate and anti-Mob1 immunoblots were performed. The top graph shows the specific Dbf2–Mob1 kinase activity. Shown is the outcome of one out of two independent experiments. Both results were identical. (D) Gal1-CLB2-ΔDB cells were arrested with α-factor in G1 phase in YPAR and released into a synchronized cell cycle at 30°C in YPAR. After ∼60 min, galactose (2%) was added. Cells in anaphase were used for immunoprecipitation of TAP-Dbf2 followed by kinase assays using GST-C-Cdc14 as substrate. Phosphorylation was determined by autoradiography and normalized to immunoprecipitated TAP-Dbf2 (immunoblot anti-TAP). Dbf2–Mob1 kinase activity is shown as mean ± SD of three experiments with the activity of wild-type cells set to 1.
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fig6: Cdk1 regulates kinase activity of Dbf2–Mob1. (A) Log-phase cells with the indicated phenotypes were serially diluted 10-fold and spotted onto YPD plates. Plates were incubated for 2 d at 23 or 37°C. (B) Active Dbf2–Mob1 complex was incubated without substrate (lane 1), GST (lane 2), and GST-C-Cdc14 (lane 3) in the presence of γ-[32P]ATP. Shown is an autoradiography. (C) Cdk1–Clb2 kinase inhibits the activation of Dbf2–Mob1 kinase by Cdc15 in vitro. GST-Mob1 in a complex with Dbf2 was incubated with Cdk1–Clb2 or Cdc15 in the first and second kinase reaction as indicated in the figure. After the second reaction, Mob1-Dbf2 kinase assays with GST-C-Cdc14 as substrate and anti-Mob1 immunoblots were performed. The top graph shows the specific Dbf2–Mob1 kinase activity. Shown is the outcome of one out of two independent experiments. Both results were identical. (D) Gal1-CLB2-ΔDB cells were arrested with α-factor in G1 phase in YPAR and released into a synchronized cell cycle at 30°C in YPAR. After ∼60 min, galactose (2%) was added. Cells in anaphase were used for immunoprecipitation of TAP-Dbf2 followed by kinase assays using GST-C-Cdc14 as substrate. Phosphorylation was determined by autoradiography and normalized to immunoprecipitated TAP-Dbf2 (immunoblot anti-TAP). Dbf2–Mob1 kinase activity is shown as mean ± SD of three experiments with the activity of wild-type cells set to 1.

Mentions: A genetic approach was used to address whether Cdk1 phosphorylation activates or inhibits Mob1 function. Conditional lethal dbf2-2 cells were combined with MOB1, MOB1-S36A, MOB1-T85A, MOB1-2A, or MOB1-7A alleles. All strains grew equally well at 23°C (Fig. 6 A). However, at 37°C MOB1-T85A, MOB1-2A, and MOB1-7A were clearly fitter than wild-type MOB1 cells, indicating that nonphosphorylated mutant Mob1 proteins possessed MEN-activating function. The overall impact of the mutations was insensitive to the additional deletion of DBF20 with the exception that dbf2-2 dbf20Δ cells were more temperature sensitive than dbf2-2 cells (Fig. S3 A). This indicates that the suppression of the dbf2-2 growth defect by the MOB1-2A allele does not require DBF20 function. Moreover, MOB1-2A did not suppress the growth defect of tem1-3, cdc15-1, and cdc14-1 cells (unpublished data). Thus, phosphorylation of Mob1 by Cdk1 inhibits the MEN at the level of the Dbf2–Mob1 complex.


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

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

Cdk1 regulates kinase activity of Dbf2–Mob1. (A) Log-phase cells with the indicated phenotypes were serially diluted 10-fold and spotted onto YPD plates. Plates were incubated for 2 d at 23 or 37°C. (B) Active Dbf2–Mob1 complex was incubated without substrate (lane 1), GST (lane 2), and GST-C-Cdc14 (lane 3) in the presence of γ-[32P]ATP. Shown is an autoradiography. (C) Cdk1–Clb2 kinase inhibits the activation of Dbf2–Mob1 kinase by Cdc15 in vitro. GST-Mob1 in a complex with Dbf2 was incubated with Cdk1–Clb2 or Cdc15 in the first and second kinase reaction as indicated in the figure. After the second reaction, Mob1-Dbf2 kinase assays with GST-C-Cdc14 as substrate and anti-Mob1 immunoblots were performed. The top graph shows the specific Dbf2–Mob1 kinase activity. Shown is the outcome of one out of two independent experiments. Both results were identical. (D) Gal1-CLB2-ΔDB cells were arrested with α-factor in G1 phase in YPAR and released into a synchronized cell cycle at 30°C in YPAR. After ∼60 min, galactose (2%) was added. Cells in anaphase were used for immunoprecipitation of TAP-Dbf2 followed by kinase assays using GST-C-Cdc14 as substrate. Phosphorylation was determined by autoradiography and normalized to immunoprecipitated TAP-Dbf2 (immunoblot anti-TAP). Dbf2–Mob1 kinase activity is shown as mean ± SD of three experiments with the activity of wild-type cells set to 1.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2819678&req=5

fig6: Cdk1 regulates kinase activity of Dbf2–Mob1. (A) Log-phase cells with the indicated phenotypes were serially diluted 10-fold and spotted onto YPD plates. Plates were incubated for 2 d at 23 or 37°C. (B) Active Dbf2–Mob1 complex was incubated without substrate (lane 1), GST (lane 2), and GST-C-Cdc14 (lane 3) in the presence of γ-[32P]ATP. Shown is an autoradiography. (C) Cdk1–Clb2 kinase inhibits the activation of Dbf2–Mob1 kinase by Cdc15 in vitro. GST-Mob1 in a complex with Dbf2 was incubated with Cdk1–Clb2 or Cdc15 in the first and second kinase reaction as indicated in the figure. After the second reaction, Mob1-Dbf2 kinase assays with GST-C-Cdc14 as substrate and anti-Mob1 immunoblots were performed. The top graph shows the specific Dbf2–Mob1 kinase activity. Shown is the outcome of one out of two independent experiments. Both results were identical. (D) Gal1-CLB2-ΔDB cells were arrested with α-factor in G1 phase in YPAR and released into a synchronized cell cycle at 30°C in YPAR. After ∼60 min, galactose (2%) was added. Cells in anaphase were used for immunoprecipitation of TAP-Dbf2 followed by kinase assays using GST-C-Cdc14 as substrate. Phosphorylation was determined by autoradiography and normalized to immunoprecipitated TAP-Dbf2 (immunoblot anti-TAP). Dbf2–Mob1 kinase activity is shown as mean ± SD of three experiments with the activity of wild-type cells set to 1.
Mentions: A genetic approach was used to address whether Cdk1 phosphorylation activates or inhibits Mob1 function. Conditional lethal dbf2-2 cells were combined with MOB1, MOB1-S36A, MOB1-T85A, MOB1-2A, or MOB1-7A alleles. All strains grew equally well at 23°C (Fig. 6 A). However, at 37°C MOB1-T85A, MOB1-2A, and MOB1-7A were clearly fitter than wild-type MOB1 cells, indicating that nonphosphorylated mutant Mob1 proteins possessed MEN-activating function. The overall impact of the mutations was insensitive to the additional deletion of DBF20 with the exception that dbf2-2 dbf20Δ cells were more temperature sensitive than dbf2-2 cells (Fig. S3 A). This indicates that the suppression of the dbf2-2 growth defect by the MOB1-2A allele does not require DBF20 function. Moreover, MOB1-2A did not suppress the growth defect of tem1-3, cdc15-1, and cdc14-1 cells (unpublished data). Thus, phosphorylation of Mob1 by Cdk1 inhibits the MEN at the level of the Dbf2–Mob1 complex.

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