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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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Related in: MedlinePlus

Elm1 phosphorylates C-terminal Kin4. In vitro kinase assays using purified GST-Elm1ΔC or GST-Elm1ΔC-KD and full-length GST-Kin4 and GST-Kin4-T209A (A) or the C-terminal domain of Kin4 (6His-Kin4-C; B). Autoradiographs and Coomassie-stained protein gels are shown. Note the autophosphorylation of Elm1. (C) Putative sites in 6His-Kin4-C phosphorylated by Elm1 in vitro, which were also detected in previously published in vivo studies (see text for details). Amino acid positions are indicated with numbers. N and C represent N and C termini, respectively. Domain positions shown are according to The UniProt Knowledgebase (UniProtKB).
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fig6: Elm1 phosphorylates C-terminal Kin4. In vitro kinase assays using purified GST-Elm1ΔC or GST-Elm1ΔC-KD and full-length GST-Kin4 and GST-Kin4-T209A (A) or the C-terminal domain of Kin4 (6His-Kin4-C; B). Autoradiographs and Coomassie-stained protein gels are shown. Note the autophosphorylation of Elm1. (C) Putative sites in 6His-Kin4-C phosphorylated by Elm1 in vitro, which were also detected in previously published in vivo studies (see text for details). Amino acid positions are indicated with numbers. N and C represent N and C termini, respectively. Domain positions shown are according to The UniProt Knowledgebase (UniProtKB).

Mentions: In vitro kinase assays using radioactive ATP, however, revealed that GST-Elm1ΔC was able to phosphorylate both bacterially expressed full-length GST-Kin4 and GST-Kin4-T209A (Fig. 6 A, lanes 2 and 3). Phosphorylation was caused by GST-Elm1ΔC, as no phosphorylation was observed when GST-Elm1ΔC kinase dead was used (GST-Elm1ΔC-KD; Fig. 6 A, lanes 5 and 6). Thus, Elm1 phosphorylates further sites in Kin4 in addition to T209.


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)

Elm1 phosphorylates C-terminal Kin4. In vitro kinase assays using purified GST-Elm1ΔC or GST-Elm1ΔC-KD and full-length GST-Kin4 and GST-Kin4-T209A (A) or the C-terminal domain of Kin4 (6His-Kin4-C; B). Autoradiographs and Coomassie-stained protein gels are shown. Note the autophosphorylation of Elm1. (C) Putative sites in 6His-Kin4-C phosphorylated by Elm1 in vitro, which were also detected in previously published in vivo studies (see text for details). Amino acid positions are indicated with numbers. N and C represent N and C termini, respectively. Domain positions shown are according to The UniProt Knowledgebase (UniProtKB).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig6: Elm1 phosphorylates C-terminal Kin4. In vitro kinase assays using purified GST-Elm1ΔC or GST-Elm1ΔC-KD and full-length GST-Kin4 and GST-Kin4-T209A (A) or the C-terminal domain of Kin4 (6His-Kin4-C; B). Autoradiographs and Coomassie-stained protein gels are shown. Note the autophosphorylation of Elm1. (C) Putative sites in 6His-Kin4-C phosphorylated by Elm1 in vitro, which were also detected in previously published in vivo studies (see text for details). Amino acid positions are indicated with numbers. N and C represent N and C termini, respectively. Domain positions shown are according to The UniProt Knowledgebase (UniProtKB).
Mentions: In vitro kinase assays using radioactive ATP, however, revealed that GST-Elm1ΔC was able to phosphorylate both bacterially expressed full-length GST-Kin4 and GST-Kin4-T209A (Fig. 6 A, lanes 2 and 3). Phosphorylation was caused by GST-Elm1ΔC, as no phosphorylation was observed when GST-Elm1ΔC kinase dead was used (GST-Elm1ΔC-KD; Fig. 6 A, lanes 5 and 6). Thus, Elm1 phosphorylates further sites in Kin4 in addition to T209.

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