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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.

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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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Phosphorylation of Mob1 by Cdk1 kinase inhibits the MEN. (A) Distribution of the two full consensus sites [S/T-P-x-K/R] (bold) and the five minimal consensus sites [S/T-P] in the Mob1 protein. (B) Phosphorylation of Mob1-2A and Mob1-7A by Cdk1–Clb2 is strongly reduced. Purified GST-Mob1, GST-Mob1-2A, and GST-Mob1-7A were incubated with Cdk1–Clb2 in the presence of γ-[32P]ATP. 32P-Mob1 was determined by autoradiography. The two asterisks indicate a protein band in the Cdk1–Clb2 preparation that is phosphorylated by Cdk1–Clb2. CBB: Coomassie brilliant blue–stained gel. (C) Immunoprecipitated Mob1-6HA was incubated as indicated. Immunoblot with anti-HA antibodies. (D) MOB1-6HA and MOB1-2A-6HA cells were arrested with α-factor in G1 phase (t = 0) and released into a synchronized cell cycle at 30°C. Cells were analyzed for Mob1 phosphorylation and Clb2 and Sic1 protein levels by immunoblotting. Budding index and timing of anaphase are shown below the graphs.
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fig5: Phosphorylation of Mob1 by Cdk1 kinase inhibits the MEN. (A) Distribution of the two full consensus sites [S/T-P-x-K/R] (bold) and the five minimal consensus sites [S/T-P] in the Mob1 protein. (B) Phosphorylation of Mob1-2A and Mob1-7A by Cdk1–Clb2 is strongly reduced. Purified GST-Mob1, GST-Mob1-2A, and GST-Mob1-7A were incubated with Cdk1–Clb2 in the presence of γ-[32P]ATP. 32P-Mob1 was determined by autoradiography. The two asterisks indicate a protein band in the Cdk1–Clb2 preparation that is phosphorylated by Cdk1–Clb2. CBB: Coomassie brilliant blue–stained gel. (C) Immunoprecipitated Mob1-6HA was incubated as indicated. Immunoblot with anti-HA antibodies. (D) MOB1-6HA and MOB1-2A-6HA cells were arrested with α-factor in G1 phase (t = 0) and released into a synchronized cell cycle at 30°C. Cells were analyzed for Mob1 phosphorylation and Clb2 and Sic1 protein levels by immunoblotting. Budding index and timing of anaphase are shown below the graphs.

Mentions: CDC15-7A cells do not have a mitotic exit defect (Jaspersen and Morgan, 2000). Cdk1 may regulate additional MEN proteins. Mob1 is a good candidate because it is phosphorylated by Cdk1 in vitro (Holt et al., 2007). Analysis of the amino acid sequence of Mob1 identified two full Cdk1 consensus sites (Fig. 5 A; S/T-P-x-K/R). Mutagenesis of these two sites, S36 and T85, to alanine reduced the phosphorylation of Mob1-2A by Cdk1–Clb2 to 11% of wild-type Mob1 (Fig. 5 B). Mutagenesis of the five remaining S/T-P sites (Mob1-7A) further diminished the ability of Cdk1–Clb2 to phosphorylate Mob1 (Fig. 5 B). Thus, Cdk1–Clb2 phosphorylates Mob1 in vitro predominantly at two sites, S36 and T85.


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

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

Phosphorylation of Mob1 by Cdk1 kinase inhibits the MEN. (A) Distribution of the two full consensus sites [S/T-P-x-K/R] (bold) and the five minimal consensus sites [S/T-P] in the Mob1 protein. (B) Phosphorylation of Mob1-2A and Mob1-7A by Cdk1–Clb2 is strongly reduced. Purified GST-Mob1, GST-Mob1-2A, and GST-Mob1-7A were incubated with Cdk1–Clb2 in the presence of γ-[32P]ATP. 32P-Mob1 was determined by autoradiography. The two asterisks indicate a protein band in the Cdk1–Clb2 preparation that is phosphorylated by Cdk1–Clb2. CBB: Coomassie brilliant blue–stained gel. (C) Immunoprecipitated Mob1-6HA was incubated as indicated. Immunoblot with anti-HA antibodies. (D) MOB1-6HA and MOB1-2A-6HA cells were arrested with α-factor in G1 phase (t = 0) and released into a synchronized cell cycle at 30°C. Cells were analyzed for Mob1 phosphorylation and Clb2 and Sic1 protein levels by immunoblotting. Budding index and timing of anaphase are shown below the graphs.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
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fig5: Phosphorylation of Mob1 by Cdk1 kinase inhibits the MEN. (A) Distribution of the two full consensus sites [S/T-P-x-K/R] (bold) and the five minimal consensus sites [S/T-P] in the Mob1 protein. (B) Phosphorylation of Mob1-2A and Mob1-7A by Cdk1–Clb2 is strongly reduced. Purified GST-Mob1, GST-Mob1-2A, and GST-Mob1-7A were incubated with Cdk1–Clb2 in the presence of γ-[32P]ATP. 32P-Mob1 was determined by autoradiography. The two asterisks indicate a protein band in the Cdk1–Clb2 preparation that is phosphorylated by Cdk1–Clb2. CBB: Coomassie brilliant blue–stained gel. (C) Immunoprecipitated Mob1-6HA was incubated as indicated. Immunoblot with anti-HA antibodies. (D) MOB1-6HA and MOB1-2A-6HA cells were arrested with α-factor in G1 phase (t = 0) and released into a synchronized cell cycle at 30°C. Cells were analyzed for Mob1 phosphorylation and Clb2 and Sic1 protein levels by immunoblotting. Budding index and timing of anaphase are shown below the graphs.
Mentions: CDC15-7A cells do not have a mitotic exit defect (Jaspersen and Morgan, 2000). Cdk1 may regulate additional MEN proteins. Mob1 is a good candidate because it is phosphorylated by Cdk1 in vitro (Holt et al., 2007). Analysis of the amino acid sequence of Mob1 identified two full Cdk1 consensus sites (Fig. 5 A; S/T-P-x-K/R). Mutagenesis of these two sites, S36 and T85, to alanine reduced the phosphorylation of Mob1-2A by Cdk1–Clb2 to 11% of wild-type Mob1 (Fig. 5 B). Mutagenesis of the five remaining S/T-P sites (Mob1-7A) further diminished the ability of Cdk1–Clb2 to phosphorylate Mob1 (Fig. 5 B). Thus, Cdk1–Clb2 phosphorylates Mob1 in vitro predominantly at two sites, S36 and T85.

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