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Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis.

Neef R, Preisinger C, Sutcliffe J, Kopajtich R, Nigg EA, Mayer TU, Barr FA - J. Cell Biol. (2003)

Bottom Line: We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1).An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells.We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Intracellular Protein Transport, Independent Junior Research Group, Max-Planck-Institute of Biochemistry, 82152 Martinsried, Germany.

ABSTRACT
We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1). MKlp2 is a target for Plk1, and phosphorylated MKlp2 binds to the polo box domain of Plk1. Plk1 also binds directly to microtubules and targets to the central spindle via its polo box domain, and this interaction controls the activity of Plk1 toward MKlp2. An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells. We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.

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Microtubules regulate the phosphorylation of MKlp2 by Plk1. (A) Microtubule-binding assays were performed with 5 pmol Plk1, MKlp2, and survivin. Samples were then analyzed by Western blotting and detection with antibodies to the hexahistidine tag, and on Coomassie brilliant blue–stained SDS-PAGE 10% minigels (right). (B) Microtubule-binding assays were performed with 5 pmol GST-tagged Plk1 amino acids 1–305 and 305–603. Samples were then analyzed by Western blotting and detection with antibodies to GST. (C) Constructs corresponding to the full-length myc-tagged human Plk1, amino acids 1–305, and 305–603 were expressed in HeLa S3 cells for 24 h using transient transfection. Cells were fixed with methanol and stained with antibodies to the myc-epitope and α-tubulin. The localization of the exogenously expressed Plk1 in telophase cells is shown; arrows mark the position of the microtubule bridge, central spindle structure. Bar, 10 μM. (D) Phosphorylation assays were performed with 1 pmol purified histone H1, PRC1, MKlp1, and MKlp2 using 50 ng recombinant cdk1–cyclin B kinase, and with 1 pmol purified casein, PRC1, MKlp1, and MKlp2 using 50 ng recombinant Plk1. Autoradiographs of samples separated by SDS-PAGE on 12% minigels are shown. The asterisk marks phosphorylated cyclin B. (E) Phosphorylation assays were performed with 1 pmol purified casein and MKlp2 using 10 ng recombinant Plk1 in the presence and absence of microtubules. Autoradiographs of samples separated by SDS-PAGE on 12% minigels are shown. The double asterisk marks a nonstoichiometric phosphorylation of tubulin by Plk1.
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fig3: Microtubules regulate the phosphorylation of MKlp2 by Plk1. (A) Microtubule-binding assays were performed with 5 pmol Plk1, MKlp2, and survivin. Samples were then analyzed by Western blotting and detection with antibodies to the hexahistidine tag, and on Coomassie brilliant blue–stained SDS-PAGE 10% minigels (right). (B) Microtubule-binding assays were performed with 5 pmol GST-tagged Plk1 amino acids 1–305 and 305–603. Samples were then analyzed by Western blotting and detection with antibodies to GST. (C) Constructs corresponding to the full-length myc-tagged human Plk1, amino acids 1–305, and 305–603 were expressed in HeLa S3 cells for 24 h using transient transfection. Cells were fixed with methanol and stained with antibodies to the myc-epitope and α-tubulin. The localization of the exogenously expressed Plk1 in telophase cells is shown; arrows mark the position of the microtubule bridge, central spindle structure. Bar, 10 μM. (D) Phosphorylation assays were performed with 1 pmol purified histone H1, PRC1, MKlp1, and MKlp2 using 50 ng recombinant cdk1–cyclin B kinase, and with 1 pmol purified casein, PRC1, MKlp1, and MKlp2 using 50 ng recombinant Plk1. Autoradiographs of samples separated by SDS-PAGE on 12% minigels are shown. The asterisk marks phosphorylated cyclin B. (E) Phosphorylation assays were performed with 1 pmol purified casein and MKlp2 using 10 ng recombinant Plk1 in the presence and absence of microtubules. Autoradiographs of samples separated by SDS-PAGE on 12% minigels are shown. The double asterisk marks a nonstoichiometric phosphorylation of tubulin by Plk1.

Mentions: To further characterize MKlp2 and its relationship with Plk1, microtubule-binding assays were performed with purified recombinant proteins and purified repolymerized bovine brain tubulin (Fig. 3). In this assay, both Plk1 and MKlp2 sedimented in the presence (but not in the absence) of microtubules, whereas survivin did not sediment under either condition (Fig. 3 A). Previous findings on MKlp2 showing that the COOH-terminal neck and stalk region contain a microtubule-binding domain could also be confirmed (Echard et al., 1998; unpublished data). The microtubule binding of Plk1 was mapped to the COOH-terminal polo box domain (Fig. 3 B), supporting the idea that this interaction reflects specific microtubule binding. Exogenously expressed full-length Plk1 and the COOH-terminal polo box domain both targeted to the central spindle in telophase, whereas the kinase domain displayed diffuse cytoplasmic fluorescence (Fig. 3 C) consistent with previous observations (Seong et al., 2002). These findings indicate that the polo box domain has the ability to target to a subset of microtubule structures.


Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis.

Neef R, Preisinger C, Sutcliffe J, Kopajtich R, Nigg EA, Mayer TU, Barr FA - J. Cell Biol. (2003)

Microtubules regulate the phosphorylation of MKlp2 by Plk1. (A) Microtubule-binding assays were performed with 5 pmol Plk1, MKlp2, and survivin. Samples were then analyzed by Western blotting and detection with antibodies to the hexahistidine tag, and on Coomassie brilliant blue–stained SDS-PAGE 10% minigels (right). (B) Microtubule-binding assays were performed with 5 pmol GST-tagged Plk1 amino acids 1–305 and 305–603. Samples were then analyzed by Western blotting and detection with antibodies to GST. (C) Constructs corresponding to the full-length myc-tagged human Plk1, amino acids 1–305, and 305–603 were expressed in HeLa S3 cells for 24 h using transient transfection. Cells were fixed with methanol and stained with antibodies to the myc-epitope and α-tubulin. The localization of the exogenously expressed Plk1 in telophase cells is shown; arrows mark the position of the microtubule bridge, central spindle structure. Bar, 10 μM. (D) Phosphorylation assays were performed with 1 pmol purified histone H1, PRC1, MKlp1, and MKlp2 using 50 ng recombinant cdk1–cyclin B kinase, and with 1 pmol purified casein, PRC1, MKlp1, and MKlp2 using 50 ng recombinant Plk1. Autoradiographs of samples separated by SDS-PAGE on 12% minigels are shown. The asterisk marks phosphorylated cyclin B. (E) Phosphorylation assays were performed with 1 pmol purified casein and MKlp2 using 10 ng recombinant Plk1 in the presence and absence of microtubules. Autoradiographs of samples separated by SDS-PAGE on 12% minigels are shown. The double asterisk marks a nonstoichiometric phosphorylation of tubulin by Plk1.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172827&req=5

fig3: Microtubules regulate the phosphorylation of MKlp2 by Plk1. (A) Microtubule-binding assays were performed with 5 pmol Plk1, MKlp2, and survivin. Samples were then analyzed by Western blotting and detection with antibodies to the hexahistidine tag, and on Coomassie brilliant blue–stained SDS-PAGE 10% minigels (right). (B) Microtubule-binding assays were performed with 5 pmol GST-tagged Plk1 amino acids 1–305 and 305–603. Samples were then analyzed by Western blotting and detection with antibodies to GST. (C) Constructs corresponding to the full-length myc-tagged human Plk1, amino acids 1–305, and 305–603 were expressed in HeLa S3 cells for 24 h using transient transfection. Cells were fixed with methanol and stained with antibodies to the myc-epitope and α-tubulin. The localization of the exogenously expressed Plk1 in telophase cells is shown; arrows mark the position of the microtubule bridge, central spindle structure. Bar, 10 μM. (D) Phosphorylation assays were performed with 1 pmol purified histone H1, PRC1, MKlp1, and MKlp2 using 50 ng recombinant cdk1–cyclin B kinase, and with 1 pmol purified casein, PRC1, MKlp1, and MKlp2 using 50 ng recombinant Plk1. Autoradiographs of samples separated by SDS-PAGE on 12% minigels are shown. The asterisk marks phosphorylated cyclin B. (E) Phosphorylation assays were performed with 1 pmol purified casein and MKlp2 using 10 ng recombinant Plk1 in the presence and absence of microtubules. Autoradiographs of samples separated by SDS-PAGE on 12% minigels are shown. The double asterisk marks a nonstoichiometric phosphorylation of tubulin by Plk1.
Mentions: To further characterize MKlp2 and its relationship with Plk1, microtubule-binding assays were performed with purified recombinant proteins and purified repolymerized bovine brain tubulin (Fig. 3). In this assay, both Plk1 and MKlp2 sedimented in the presence (but not in the absence) of microtubules, whereas survivin did not sediment under either condition (Fig. 3 A). Previous findings on MKlp2 showing that the COOH-terminal neck and stalk region contain a microtubule-binding domain could also be confirmed (Echard et al., 1998; unpublished data). The microtubule binding of Plk1 was mapped to the COOH-terminal polo box domain (Fig. 3 B), supporting the idea that this interaction reflects specific microtubule binding. Exogenously expressed full-length Plk1 and the COOH-terminal polo box domain both targeted to the central spindle in telophase, whereas the kinase domain displayed diffuse cytoplasmic fluorescence (Fig. 3 C) consistent with previous observations (Seong et al., 2002). These findings indicate that the polo box domain has the ability to target to a subset of microtubule structures.

Bottom Line: We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1).An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells.We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Intracellular Protein Transport, Independent Junior Research Group, Max-Planck-Institute of Biochemistry, 82152 Martinsried, Germany.

ABSTRACT
We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1). MKlp2 is a target for Plk1, and phosphorylated MKlp2 binds to the polo box domain of Plk1. Plk1 also binds directly to microtubules and targets to the central spindle via its polo box domain, and this interaction controls the activity of Plk1 toward MKlp2. An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells. We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.

Show MeSH
Related in: MedlinePlus