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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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Localization and activity of Kin4 in elm1Δ cells. (A) Kin4-GFP localization in nocodazole-arrested cells. Spc42-eqFP served as an SPB marker. Images were taken without fixation. Note that the two SPBs collapse, forming a large single eqFP signal, due to depolymerization of the microtubules by nocodazole. Bars, 3 µm. (B) Quantification of A showing one representative experiment out of three; 100–150 cells were counted per sample. (C) In vitro kinase assay using immunoprecipitated Kin4-6HA (lanes 2, 3, 5, and 6) or Kin4-T209A-6HA (lane 4) from cycling cultures of wild type (WT), rts1Δ, or elm1Δ cells as indicated. Nontagged Kin4 (lane 1) was used as a control for contaminating kinases. Kin4-6HA was detected by immunoblotting (anti-HA). MBP-Bfa1 was used as a substrate (Coomassie staining). Incorporation of the 32P isotope was determined by autoradiography. Note that a 66-kD degradation product of MBP-Bfa1 was phosphorylated efficiently by Kin4-6HA as described in Maekawa et al. (2007). Plus and minus represent the presence and the absence of MBP-Bfa1. Kin4 did not phosphorylate MBP alone (Maekawa et al., 2007; not depicted).
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fig4: Localization and activity of Kin4 in elm1Δ cells. (A) Kin4-GFP localization in nocodazole-arrested cells. Spc42-eqFP served as an SPB marker. Images were taken without fixation. Note that the two SPBs collapse, forming a large single eqFP signal, due to depolymerization of the microtubules by nocodazole. Bars, 3 µm. (B) Quantification of A showing one representative experiment out of three; 100–150 cells were counted per sample. (C) In vitro kinase assay using immunoprecipitated Kin4-6HA (lanes 2, 3, 5, and 6) or Kin4-T209A-6HA (lane 4) from cycling cultures of wild type (WT), rts1Δ, or elm1Δ cells as indicated. Nontagged Kin4 (lane 1) was used as a control for contaminating kinases. Kin4-6HA was detected by immunoblotting (anti-HA). MBP-Bfa1 was used as a substrate (Coomassie staining). Incorporation of the 32P isotope was determined by autoradiography. Note that a 66-kD degradation product of MBP-Bfa1 was phosphorylated efficiently by Kin4-6HA as described in Maekawa et al. (2007). Plus and minus represent the presence and the absence of MBP-Bfa1. Kin4 did not phosphorylate MBP alone (Maekawa et al., 2007; not depicted).

Mentions: Binding of Kin4 to the cortex and SPB is important for SPOC function (Maekawa et al., 2007). We therefore asked whether Elm1 affected this aspect of Kin4 function. The localization of Kin4-GFP was monitored after treatment with nocodazole to increase the proportion of cells with Kin4 at SPBs (Pereira and Schiebel, 2005). Strains also carried the SPB protein Spc42 (Donaldson and Kilmartin, 1996), tagged with the red fluorescent protein eqFP. Kin4-GFP localized to the mother cell cortex and SPBs of wild-type cells (Fig. 4, A and B). As reported previously, deletion of RTS1 drastically reduced the recruitment of Kin4-GFP to both the SPB and cortex (Fig. 4 A; Chan and Amon, 2009). Interestingly, a higher percentage of elm1Δ cells showed reduced cortical Kin4-GFP, whereas Kin4-GFP association with SPBs resembled that seen in wild-type controls. Furthermore, in 80% of elm1Δ cells showing diminished association of Kin4-GFP with the cortex, the Kin4-GFP that did associate was more concentrated at the tip of the cell. This type of localization was likely caused by hyperactivation of Swe1, as SWE1 deletion eliminated this heterogeneity of Kin4-GFP association with the cortex in elm1Δ cells (unpublished data). Thus, Elm1 is required for the proper recruitment of Kin4 to the cortex.


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)

Localization and activity of Kin4 in elm1Δ cells. (A) Kin4-GFP localization in nocodazole-arrested cells. Spc42-eqFP served as an SPB marker. Images were taken without fixation. Note that the two SPBs collapse, forming a large single eqFP signal, due to depolymerization of the microtubules by nocodazole. Bars, 3 µm. (B) Quantification of A showing one representative experiment out of three; 100–150 cells were counted per sample. (C) In vitro kinase assay using immunoprecipitated Kin4-6HA (lanes 2, 3, 5, and 6) or Kin4-T209A-6HA (lane 4) from cycling cultures of wild type (WT), rts1Δ, or elm1Δ cells as indicated. Nontagged Kin4 (lane 1) was used as a control for contaminating kinases. Kin4-6HA was detected by immunoblotting (anti-HA). MBP-Bfa1 was used as a substrate (Coomassie staining). Incorporation of the 32P isotope was determined by autoradiography. Note that a 66-kD degradation product of MBP-Bfa1 was phosphorylated efficiently by Kin4-6HA as described in Maekawa et al. (2007). Plus and minus represent the presence and the absence of MBP-Bfa1. Kin4 did not phosphorylate MBP alone (Maekawa et al., 2007; not depicted).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3101594&req=5

fig4: Localization and activity of Kin4 in elm1Δ cells. (A) Kin4-GFP localization in nocodazole-arrested cells. Spc42-eqFP served as an SPB marker. Images were taken without fixation. Note that the two SPBs collapse, forming a large single eqFP signal, due to depolymerization of the microtubules by nocodazole. Bars, 3 µm. (B) Quantification of A showing one representative experiment out of three; 100–150 cells were counted per sample. (C) In vitro kinase assay using immunoprecipitated Kin4-6HA (lanes 2, 3, 5, and 6) or Kin4-T209A-6HA (lane 4) from cycling cultures of wild type (WT), rts1Δ, or elm1Δ cells as indicated. Nontagged Kin4 (lane 1) was used as a control for contaminating kinases. Kin4-6HA was detected by immunoblotting (anti-HA). MBP-Bfa1 was used as a substrate (Coomassie staining). Incorporation of the 32P isotope was determined by autoradiography. Note that a 66-kD degradation product of MBP-Bfa1 was phosphorylated efficiently by Kin4-6HA as described in Maekawa et al. (2007). Plus and minus represent the presence and the absence of MBP-Bfa1. Kin4 did not phosphorylate MBP alone (Maekawa et al., 2007; not depicted).
Mentions: Binding of Kin4 to the cortex and SPB is important for SPOC function (Maekawa et al., 2007). We therefore asked whether Elm1 affected this aspect of Kin4 function. The localization of Kin4-GFP was monitored after treatment with nocodazole to increase the proportion of cells with Kin4 at SPBs (Pereira and Schiebel, 2005). Strains also carried the SPB protein Spc42 (Donaldson and Kilmartin, 1996), tagged with the red fluorescent protein eqFP. Kin4-GFP localized to the mother cell cortex and SPBs of wild-type cells (Fig. 4, A and B). As reported previously, deletion of RTS1 drastically reduced the recruitment of Kin4-GFP to both the SPB and cortex (Fig. 4 A; Chan and Amon, 2009). Interestingly, a higher percentage of elm1Δ cells showed reduced cortical Kin4-GFP, whereas Kin4-GFP association with SPBs resembled that seen in wild-type controls. Furthermore, in 80% of elm1Δ cells showing diminished association of Kin4-GFP with the cortex, the Kin4-GFP that did associate was more concentrated at the tip of the cell. This type of localization was likely caused by hyperactivation of Swe1, as SWE1 deletion eliminated this heterogeneity of Kin4-GFP association with the cortex in elm1Δ cells (unpublished data). Thus, Elm1 is required for the proper recruitment of Kin4 to the cortex.

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