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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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Bud neck localization of Elm1 is not necessary for Kin4 activity. (A) Still images of live KIN4-GFP ELM1-3mCherry cells. Bars, 3 µm. (B) Radioactive kinase assay of immunoprecipitated Kin4-6HA from Gal1-clb2ΔDB–overexpressing cells arrested with nocodazole with a bud neck (lane 1) and without a bud neck (lanes 2 and 3). The percentages of budded and nonbudded cells are indicated. See Materials and methods for details. (C) Both Gal1-clb2ΔDB and Gal1-clb2ΔDB elm1Δ cells were arrested with α-factor (G1-phase, t0) and forced to enter mitosis without bud formation upon clb2ΔDB overexpression in the presence of nocodazole. Samples were taken every hour and analyzed by immunoblotting. The asterisk marks Kin4-hyperphosphorylated forms. (D) cdc12-6 and cdc12-6 elm1Δ cells were arrested at 23°C with α-factor and released at 37°C in nocodazole-containing medium. In vitro kinase assays were performed using immunoprecipitated Kin4-6HA (anti-HA blot) and MBP-Bfa1 (Coomassie). Incorporation of the 32P isotope in MBP-Bfa1 was measured by autoradiography.
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fig9: Bud neck localization of Elm1 is not necessary for Kin4 activity. (A) Still images of live KIN4-GFP ELM1-3mCherry cells. Bars, 3 µm. (B) Radioactive kinase assay of immunoprecipitated Kin4-6HA from Gal1-clb2ΔDB–overexpressing cells arrested with nocodazole with a bud neck (lane 1) and without a bud neck (lanes 2 and 3). The percentages of budded and nonbudded cells are indicated. See Materials and methods for details. (C) Both Gal1-clb2ΔDB and Gal1-clb2ΔDB elm1Δ cells were arrested with α-factor (G1-phase, t0) and forced to enter mitosis without bud formation upon clb2ΔDB overexpression in the presence of nocodazole. Samples were taken every hour and analyzed by immunoblotting. The asterisk marks Kin4-hyperphosphorylated forms. (D) cdc12-6 and cdc12-6 elm1Δ cells were arrested at 23°C with α-factor and released at 37°C in nocodazole-containing medium. In vitro kinase assays were performed using immunoprecipitated Kin4-6HA (anti-HA blot) and MBP-Bfa1 (Coomassie). Incorporation of the 32P isotope in MBP-Bfa1 was measured by autoradiography.

Mentions: To clarify this point, we analyzed colocalization of Elm1 and Kin4 in the same cells. As the fluorescent signals from 3mCherry-tagged versions of Kin4 and Elm1 were too weak for time-lapse imaging, we performed population analysis of Kin4-GFP Elm1-3mCherry cultures without fixation. No colocalization of Kin4-GFP and Elm1-3mCherry could be detected in any anaphase cells (n = 200; Fig. 9 A). Furthermore, FRAP analysis of Elm1-GFP showed that the binding of Elm1-GFP to the bud neck was dynamic, with a half-life of 15 ± 6 s (Fig. S4 E). We therefore postulated that Elm1 and Kin4 do not need to interact at the bud neck for Elm1 to activate Kin4. This conclusion is consistent with the finding that Kin4 is already phosphorylated by Elm1 earlier in mitosis when Kin4 only associates with the mother cell cortex and not the bud neck.


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)

Bud neck localization of Elm1 is not necessary for Kin4 activity. (A) Still images of live KIN4-GFP ELM1-3mCherry cells. Bars, 3 µm. (B) Radioactive kinase assay of immunoprecipitated Kin4-6HA from Gal1-clb2ΔDB–overexpressing cells arrested with nocodazole with a bud neck (lane 1) and without a bud neck (lanes 2 and 3). The percentages of budded and nonbudded cells are indicated. See Materials and methods for details. (C) Both Gal1-clb2ΔDB and Gal1-clb2ΔDB elm1Δ cells were arrested with α-factor (G1-phase, t0) and forced to enter mitosis without bud formation upon clb2ΔDB overexpression in the presence of nocodazole. Samples were taken every hour and analyzed by immunoblotting. The asterisk marks Kin4-hyperphosphorylated forms. (D) cdc12-6 and cdc12-6 elm1Δ cells were arrested at 23°C with α-factor and released at 37°C in nocodazole-containing medium. In vitro kinase assays were performed using immunoprecipitated Kin4-6HA (anti-HA blot) and MBP-Bfa1 (Coomassie). Incorporation of the 32P isotope in MBP-Bfa1 was measured by autoradiography.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101594&req=5

fig9: Bud neck localization of Elm1 is not necessary for Kin4 activity. (A) Still images of live KIN4-GFP ELM1-3mCherry cells. Bars, 3 µm. (B) Radioactive kinase assay of immunoprecipitated Kin4-6HA from Gal1-clb2ΔDB–overexpressing cells arrested with nocodazole with a bud neck (lane 1) and without a bud neck (lanes 2 and 3). The percentages of budded and nonbudded cells are indicated. See Materials and methods for details. (C) Both Gal1-clb2ΔDB and Gal1-clb2ΔDB elm1Δ cells were arrested with α-factor (G1-phase, t0) and forced to enter mitosis without bud formation upon clb2ΔDB overexpression in the presence of nocodazole. Samples were taken every hour and analyzed by immunoblotting. The asterisk marks Kin4-hyperphosphorylated forms. (D) cdc12-6 and cdc12-6 elm1Δ cells were arrested at 23°C with α-factor and released at 37°C in nocodazole-containing medium. In vitro kinase assays were performed using immunoprecipitated Kin4-6HA (anti-HA blot) and MBP-Bfa1 (Coomassie). Incorporation of the 32P isotope in MBP-Bfa1 was measured by autoradiography.
Mentions: To clarify this point, we analyzed colocalization of Elm1 and Kin4 in the same cells. As the fluorescent signals from 3mCherry-tagged versions of Kin4 and Elm1 were too weak for time-lapse imaging, we performed population analysis of Kin4-GFP Elm1-3mCherry cultures without fixation. No colocalization of Kin4-GFP and Elm1-3mCherry could be detected in any anaphase cells (n = 200; Fig. 9 A). Furthermore, FRAP analysis of Elm1-GFP showed that the binding of Elm1-GFP to the bud neck was dynamic, with a half-life of 15 ± 6 s (Fig. S4 E). We therefore postulated that Elm1 and Kin4 do not need to interact at the bud neck for Elm1 to activate Kin4. This conclusion is consistent with the finding that Kin4 is already phosphorylated by Elm1 earlier in mitosis when Kin4 only associates with the mother cell cortex and not the bud neck.

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