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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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Plk1 regulates the microtubule-bundling activity of MKlp2. Microtubules labeled with fluorescein were incubated with buffer alone, MKlp2, or MKlp2S528A in the presence and absence of 1 mM ATP, Plk1, MKlp2 or MKlp2S528A treated with Plk1 in the presence of 1 mM ATP, and PRC1 treated with 1 mM ATP or Plk1 and 1 mM ATP. Bundling was observed in the presence of MKlp2 and PRC1, but not Plk1 or with microtubules alone. Exposure time was 500 msec for all images, and representative images from 14 independent experiments are shown. Bar, 10 μM.
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fig5: Plk1 regulates the microtubule-bundling activity of MKlp2. Microtubules labeled with fluorescein were incubated with buffer alone, MKlp2, or MKlp2S528A in the presence and absence of 1 mM ATP, Plk1, MKlp2 or MKlp2S528A treated with Plk1 in the presence of 1 mM ATP, and PRC1 treated with 1 mM ATP or Plk1 and 1 mM ATP. Bundling was observed in the presence of MKlp2 and PRC1, but not Plk1 or with microtubules alone. Exposure time was 500 msec for all images, and representative images from 14 independent experiments are shown. Bar, 10 μM.

Mentions: To test the hypothesis that MKlp2 and Plk1 cooperate to control microtubule organization, the effects of purified MKlp2 on microtubules in vitro were examined (Fig. 5) because full-length MKlp2 (but not the motor or neck and stalk domains alone) can bundle microtubules when expressed in cells (Fig. S3). Fluorescently labeled microtubules were incubated alone, or with MKlp2 and Plk1, in the presence and absence of ATP. MKlp2-bundled microtubules in the absence and addition of ATP resulted in the formation of loops from these bundles and fraying at the bundle ends, whereas Plk1, although able to bind microtubules, caused no microtubule bundling. MKlp2 treated with Plk1 did not form the regular microtubule bundles seen with MKlp2 only; many single microtubules were seen instead, and the bundles that were formed were loose parallel arrays rather than the dense bundles seen with untreated MKlp2. Therefore, Plk1 negatively regulates the microtubule-bundling properties of MKlp2. MKlp2S528A bundled microtubules even after Plk1 treatment, indicating that serine 528 is a regulatory site. Microtubule bundling by PRC1, which is not a substrate of Plk1, was unaffected by Plk1 treatment. The effects of Plk1 treatment on MKlp2-mediated microtubule bundling are therefore unlikely to be due to direct interference of Plk1 with the assay, and these results therefore support the proposal that MKlp2 function on central spindle microtubules involves regulation by Plk1.


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)

Plk1 regulates the microtubule-bundling activity of MKlp2. Microtubules labeled with fluorescein were incubated with buffer alone, MKlp2, or MKlp2S528A in the presence and absence of 1 mM ATP, Plk1, MKlp2 or MKlp2S528A treated with Plk1 in the presence of 1 mM ATP, and PRC1 treated with 1 mM ATP or Plk1 and 1 mM ATP. Bundling was observed in the presence of MKlp2 and PRC1, but not Plk1 or with microtubules alone. Exposure time was 500 msec for all images, and representative images from 14 independent experiments are shown. Bar, 10 μM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172827&req=5

fig5: Plk1 regulates the microtubule-bundling activity of MKlp2. Microtubules labeled with fluorescein were incubated with buffer alone, MKlp2, or MKlp2S528A in the presence and absence of 1 mM ATP, Plk1, MKlp2 or MKlp2S528A treated with Plk1 in the presence of 1 mM ATP, and PRC1 treated with 1 mM ATP or Plk1 and 1 mM ATP. Bundling was observed in the presence of MKlp2 and PRC1, but not Plk1 or with microtubules alone. Exposure time was 500 msec for all images, and representative images from 14 independent experiments are shown. Bar, 10 μM.
Mentions: To test the hypothesis that MKlp2 and Plk1 cooperate to control microtubule organization, the effects of purified MKlp2 on microtubules in vitro were examined (Fig. 5) because full-length MKlp2 (but not the motor or neck and stalk domains alone) can bundle microtubules when expressed in cells (Fig. S3). Fluorescently labeled microtubules were incubated alone, or with MKlp2 and Plk1, in the presence and absence of ATP. MKlp2-bundled microtubules in the absence and addition of ATP resulted in the formation of loops from these bundles and fraying at the bundle ends, whereas Plk1, although able to bind microtubules, caused no microtubule bundling. MKlp2 treated with Plk1 did not form the regular microtubule bundles seen with MKlp2 only; many single microtubules were seen instead, and the bundles that were formed were loose parallel arrays rather than the dense bundles seen with untreated MKlp2. Therefore, Plk1 negatively regulates the microtubule-bundling properties of MKlp2. MKlp2S528A bundled microtubules even after Plk1 treatment, indicating that serine 528 is a regulatory site. Microtubule bundling by PRC1, which is not a substrate of Plk1, was unaffected by Plk1 treatment. The effects of Plk1 treatment on MKlp2-mediated microtubule bundling are therefore unlikely to be due to direct interference of Plk1 with the assay, and these results therefore support the proposal that MKlp2 function on central spindle microtubules involves regulation by Plk1.

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