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Elm1 kinase activates the spindle position checkpoint kinase Kin4.

Caydasi AK, Kurtulmus B, Orrico MI, Hofmann A, Ibrahim B, Pereira G - J. Cell Biol. (2010)

Bottom Line: How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established.Here, we show that the bud neck-associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4.Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment.

View Article: PubMed Central - HTML - PubMed

Affiliation: German Cancer Research Center, DKFZ-ZMBH Alliance, Molecular Biology of Centrosomes and Cilia, 69120 Heidelberg, Germany.

ABSTRACT
Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck-associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4.

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T209 phosphorylation increases during metaphase independently of SPOC activation. (A) Cells were arrested at G1 phase, S phase, metaphase (meta), and late anaphase (ana) by α-factor, hydroxyurea, Gal1-CDC20 depletion, and clb2ΔDB overexpression, respectively. Kin4-6HA was immunoprecipitated from cell lysates and probed with anti-HA and anti–T209-P antibodies. Quantifications show the ratio of anti–T209-P to anti-HA signals. The graph represents the mean of five independent experiments. Error bars show the standard deviation. The asterisk indicates the group significantly different from the others (P < 0.05, t test). (B) KIN4-6HA Gal1-CDC20 cells arrested in metaphase by Cdc20 depletion were treated with DMSO or nocodazole. Samples were analyzed and quantified as in A. The graph represents the mean of three independent experiments.
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fig8: T209 phosphorylation increases during metaphase independently of SPOC activation. (A) Cells were arrested at G1 phase, S phase, metaphase (meta), and late anaphase (ana) by α-factor, hydroxyurea, Gal1-CDC20 depletion, and clb2ΔDB overexpression, respectively. Kin4-6HA was immunoprecipitated from cell lysates and probed with anti-HA and anti–T209-P antibodies. Quantifications show the ratio of anti–T209-P to anti-HA signals. The graph represents the mean of five independent experiments. Error bars show the standard deviation. The asterisk indicates the group significantly different from the others (P < 0.05, t test). (B) KIN4-6HA Gal1-CDC20 cells arrested in metaphase by Cdc20 depletion were treated with DMSO or nocodazole. Samples were analyzed and quantified as in A. The graph represents the mean of three independent experiments.

Mentions: Next, we asked whether the phosphorylation of Kin4 at T209 by Elm1 is cell cycle regulated. Cells were arrested in G1, S phase, metaphase, or late anaphase, and Kin4-6HA was enriched by immunoprecipitation (Fig. 8 A). Monitoring the ratio of Kin4 phosphorylated at T209 to the total Kin4 amount showed that T209 phosphorylation peaked in metaphase (Fig. 8 A). We next asked whether the levels of Kin4 T209 phosphorylation changed in response to microtubule defects. We arrested cells in metaphase by depletion of CDC20 before adding nocodazole to the culture (Fig. 8 B). T209 phosphorylation of these metaphase-arrested cells was not significantly increased after the depolymerization of microtubules (Fig. 8 B). This indicates that phosphorylation of T209 does not increase in response to SPOC activation; rather, it suggests that Elm1 “licenses” Kin4 to be competent to act in the SPOC.


Elm1 kinase activates the spindle position checkpoint kinase Kin4.

Caydasi AK, Kurtulmus B, Orrico MI, Hofmann A, Ibrahim B, Pereira G - J. Cell Biol. (2010)

T209 phosphorylation increases during metaphase independently of SPOC activation. (A) Cells were arrested at G1 phase, S phase, metaphase (meta), and late anaphase (ana) by α-factor, hydroxyurea, Gal1-CDC20 depletion, and clb2ΔDB overexpression, respectively. Kin4-6HA was immunoprecipitated from cell lysates and probed with anti-HA and anti–T209-P antibodies. Quantifications show the ratio of anti–T209-P to anti-HA signals. The graph represents the mean of five independent experiments. Error bars show the standard deviation. The asterisk indicates the group significantly different from the others (P < 0.05, t test). (B) KIN4-6HA Gal1-CDC20 cells arrested in metaphase by Cdc20 depletion were treated with DMSO or nocodazole. Samples were analyzed and quantified as in A. The graph represents the mean of three independent experiments.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig8: T209 phosphorylation increases during metaphase independently of SPOC activation. (A) Cells were arrested at G1 phase, S phase, metaphase (meta), and late anaphase (ana) by α-factor, hydroxyurea, Gal1-CDC20 depletion, and clb2ΔDB overexpression, respectively. Kin4-6HA was immunoprecipitated from cell lysates and probed with anti-HA and anti–T209-P antibodies. Quantifications show the ratio of anti–T209-P to anti-HA signals. The graph represents the mean of five independent experiments. Error bars show the standard deviation. The asterisk indicates the group significantly different from the others (P < 0.05, t test). (B) KIN4-6HA Gal1-CDC20 cells arrested in metaphase by Cdc20 depletion were treated with DMSO or nocodazole. Samples were analyzed and quantified as in A. The graph represents the mean of three independent experiments.
Mentions: Next, we asked whether the phosphorylation of Kin4 at T209 by Elm1 is cell cycle regulated. Cells were arrested in G1, S phase, metaphase, or late anaphase, and Kin4-6HA was enriched by immunoprecipitation (Fig. 8 A). Monitoring the ratio of Kin4 phosphorylated at T209 to the total Kin4 amount showed that T209 phosphorylation peaked in metaphase (Fig. 8 A). We next asked whether the levels of Kin4 T209 phosphorylation changed in response to microtubule defects. We arrested cells in metaphase by depletion of CDC20 before adding nocodazole to the culture (Fig. 8 B). T209 phosphorylation of these metaphase-arrested cells was not significantly increased after the depolymerization of microtubules (Fig. 8 B). This indicates that phosphorylation of T209 does not increase in response to SPOC activation; rather, it suggests that Elm1 “licenses” Kin4 to be competent to act in the SPOC.

Bottom Line: How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established.Here, we show that the bud neck-associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4.Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment.

View Article: PubMed Central - HTML - PubMed

Affiliation: German Cancer Research Center, DKFZ-ZMBH Alliance, Molecular Biology of Centrosomes and Cilia, 69120 Heidelberg, Germany.

ABSTRACT
Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck-associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4.

Show MeSH
Related in: MedlinePlus