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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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Antibodies to the neck and tail region of MKlp2 prevent phosphorylation by Plk1 and cytokinesis. (A) HeLa cells were injected with affinity-purified antibodies to the MKlp2 neck region and control affinity-purified antibodies to GFP. Cells were fixed with methanol 24 h after injection and then stained with a CY2-conjugated secondary antibody to detect the injected antibody (green) and with DAPI for the DNA (blue). The number of binucleated cells was counted and is expressed as a percentage of total injected cells detected (n = 3). Bar, 10 μM. (B) 1 μg MKlp2 preincubated with buffer, affinity-purified antibodies to MKlp2 or GFP was treated for 1 h at 30°C with 125 ng Plk1, itself preincubated with affinity-purified antibodies to Plk1 or GFP, then analyzed by gel electrophoresis and autoradiography. (C) 1 μg MKlp2 preincubated with buffer, affinity-purified antibodies to MKlp2 or GFP was treated with 250 ng Plk1, and then was used as a target in a ligand blot with Plk1 amino acids 305–603 as the probe. Two strips containing MKlp2 phosphorylated by Plk1 were incubated with 2 μg affinity-purified antibodies to MKlp2 or GFP before probing with the Plk1 polo box domain. (D) Microtubule-binding assays were performed with 5 pmol MKlp2 and MKlp2 preincubated on ice for 60 min with 1 μg affinity-purified antibodies to MKlp2 or GFP. Samples were then analyzed by Western blotting and detection with antibodies to the hexahistidine tag. (E) Microtubules labeled with fluorescein were incubated with buffer alone, MKlp2, or MKlp2 preincubated for 60 min on ice with 1 μg affinity-purified antibodies to MKlp2 or GFP. Exposure time was 1,000 msec for all images, and representative images from five independent experiments are shown. Samples were renumbered and scored for bundling activity by two different people. Bar, 10 μM.
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fig8: Antibodies to the neck and tail region of MKlp2 prevent phosphorylation by Plk1 and cytokinesis. (A) HeLa cells were injected with affinity-purified antibodies to the MKlp2 neck region and control affinity-purified antibodies to GFP. Cells were fixed with methanol 24 h after injection and then stained with a CY2-conjugated secondary antibody to detect the injected antibody (green) and with DAPI for the DNA (blue). The number of binucleated cells was counted and is expressed as a percentage of total injected cells detected (n = 3). Bar, 10 μM. (B) 1 μg MKlp2 preincubated with buffer, affinity-purified antibodies to MKlp2 or GFP was treated for 1 h at 30°C with 125 ng Plk1, itself preincubated with affinity-purified antibodies to Plk1 or GFP, then analyzed by gel electrophoresis and autoradiography. (C) 1 μg MKlp2 preincubated with buffer, affinity-purified antibodies to MKlp2 or GFP was treated with 250 ng Plk1, and then was used as a target in a ligand blot with Plk1 amino acids 305–603 as the probe. Two strips containing MKlp2 phosphorylated by Plk1 were incubated with 2 μg affinity-purified antibodies to MKlp2 or GFP before probing with the Plk1 polo box domain. (D) Microtubule-binding assays were performed with 5 pmol MKlp2 and MKlp2 preincubated on ice for 60 min with 1 μg affinity-purified antibodies to MKlp2 or GFP. Samples were then analyzed by Western blotting and detection with antibodies to the hexahistidine tag. (E) Microtubules labeled with fluorescein were incubated with buffer alone, MKlp2, or MKlp2 preincubated for 60 min on ice with 1 μg affinity-purified antibodies to MKlp2 or GFP. Exposure time was 1,000 msec for all images, and representative images from five independent experiments are shown. Samples were renumbered and scored for bundling activity by two different people. Bar, 10 μM.

Mentions: Antibodies to the neck and stalk region of MKlp2 can block cytokinesis when microinjected into HeLa cells (Hill et al., 2000), and we decided to reinvestigate these findings in the light of the data presented in the current paper on Plk1 regulation of MKlp2. HeLa cells microinjected with affinity-purified antibodies to the neck region of MKlp2 failed to complete cytokinesis, whereas cells injected with a control antibody divided normally (Fig. 8 A). When MKlp2 was preincubated with these antibodies before use as a substrate in Plk1 kinase assays, a 10-fold reduction in the phosphorylation of MKlp2 was observed relative to the control conditions (Fig. 8 B). Incubation of the Plk1 used in these assays with antibodies to the polo box domain also prevented phosphorylation of MKlp2 (Fig. 8 B), indicating the importance of this domain for Plk1 function. Pre-incubation of MKlp2 with specific antibodies not only reduced its phosphorylation by Plk1, but this MKlp2 showed a reduced ability to bind Plk1 on ligand blots (Fig. 8 C). However, when the MKlp2 antibodies were added to the blocking reaction of the ligand blot, they were unable to prevent binding of Plk1 to phosphorylated MKlp2 (Fig. 8 C). The effect of these antibodies on the behavior of MKlp2 in microtubule binding was then tested, but no obvious differences were seen between buffer, MKlp2, and control antibody incubations (Fig. 8 D). In bundling assays, the MKlp2 antibodies caused a slight reduction in the length of the microtubule bundles compared with control incubations with MKlp2 alone or with GFP antibodies without affecting the number or morphology of the bundles formed (Fig. 8 E). The failure of cytokinesis in HeLa cells injected with antibodies to the MKlp2 neck and stalk region could therefore be due to a failure of Plk1 to phosphorylate MKlp2 because antibody binding has little effect on the other properties of the kinesin investigated here. These observations support the idea that phosphorylation of MKlp2 by Plk1 is necessary for MKlp2 function in Plk1 localization to the central spindle, and for the control of cytokinesis in human cells.


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)

Antibodies to the neck and tail region of MKlp2 prevent phosphorylation by Plk1 and cytokinesis. (A) HeLa cells were injected with affinity-purified antibodies to the MKlp2 neck region and control affinity-purified antibodies to GFP. Cells were fixed with methanol 24 h after injection and then stained with a CY2-conjugated secondary antibody to detect the injected antibody (green) and with DAPI for the DNA (blue). The number of binucleated cells was counted and is expressed as a percentage of total injected cells detected (n = 3). Bar, 10 μM. (B) 1 μg MKlp2 preincubated with buffer, affinity-purified antibodies to MKlp2 or GFP was treated for 1 h at 30°C with 125 ng Plk1, itself preincubated with affinity-purified antibodies to Plk1 or GFP, then analyzed by gel electrophoresis and autoradiography. (C) 1 μg MKlp2 preincubated with buffer, affinity-purified antibodies to MKlp2 or GFP was treated with 250 ng Plk1, and then was used as a target in a ligand blot with Plk1 amino acids 305–603 as the probe. Two strips containing MKlp2 phosphorylated by Plk1 were incubated with 2 μg affinity-purified antibodies to MKlp2 or GFP before probing with the Plk1 polo box domain. (D) Microtubule-binding assays were performed with 5 pmol MKlp2 and MKlp2 preincubated on ice for 60 min with 1 μg affinity-purified antibodies to MKlp2 or GFP. Samples were then analyzed by Western blotting and detection with antibodies to the hexahistidine tag. (E) Microtubules labeled with fluorescein were incubated with buffer alone, MKlp2, or MKlp2 preincubated for 60 min on ice with 1 μg affinity-purified antibodies to MKlp2 or GFP. Exposure time was 1,000 msec for all images, and representative images from five independent experiments are shown. Samples were renumbered and scored for bundling activity by two different people. Bar, 10 μM.
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Related In: Results  -  Collection

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fig8: Antibodies to the neck and tail region of MKlp2 prevent phosphorylation by Plk1 and cytokinesis. (A) HeLa cells were injected with affinity-purified antibodies to the MKlp2 neck region and control affinity-purified antibodies to GFP. Cells were fixed with methanol 24 h after injection and then stained with a CY2-conjugated secondary antibody to detect the injected antibody (green) and with DAPI for the DNA (blue). The number of binucleated cells was counted and is expressed as a percentage of total injected cells detected (n = 3). Bar, 10 μM. (B) 1 μg MKlp2 preincubated with buffer, affinity-purified antibodies to MKlp2 or GFP was treated for 1 h at 30°C with 125 ng Plk1, itself preincubated with affinity-purified antibodies to Plk1 or GFP, then analyzed by gel electrophoresis and autoradiography. (C) 1 μg MKlp2 preincubated with buffer, affinity-purified antibodies to MKlp2 or GFP was treated with 250 ng Plk1, and then was used as a target in a ligand blot with Plk1 amino acids 305–603 as the probe. Two strips containing MKlp2 phosphorylated by Plk1 were incubated with 2 μg affinity-purified antibodies to MKlp2 or GFP before probing with the Plk1 polo box domain. (D) Microtubule-binding assays were performed with 5 pmol MKlp2 and MKlp2 preincubated on ice for 60 min with 1 μg affinity-purified antibodies to MKlp2 or GFP. Samples were then analyzed by Western blotting and detection with antibodies to the hexahistidine tag. (E) Microtubules labeled with fluorescein were incubated with buffer alone, MKlp2, or MKlp2 preincubated for 60 min on ice with 1 μg affinity-purified antibodies to MKlp2 or GFP. Exposure time was 1,000 msec for all images, and representative images from five independent experiments are shown. Samples were renumbered and scored for bundling activity by two different people. Bar, 10 μM.
Mentions: Antibodies to the neck and stalk region of MKlp2 can block cytokinesis when microinjected into HeLa cells (Hill et al., 2000), and we decided to reinvestigate these findings in the light of the data presented in the current paper on Plk1 regulation of MKlp2. HeLa cells microinjected with affinity-purified antibodies to the neck region of MKlp2 failed to complete cytokinesis, whereas cells injected with a control antibody divided normally (Fig. 8 A). When MKlp2 was preincubated with these antibodies before use as a substrate in Plk1 kinase assays, a 10-fold reduction in the phosphorylation of MKlp2 was observed relative to the control conditions (Fig. 8 B). Incubation of the Plk1 used in these assays with antibodies to the polo box domain also prevented phosphorylation of MKlp2 (Fig. 8 B), indicating the importance of this domain for Plk1 function. Pre-incubation of MKlp2 with specific antibodies not only reduced its phosphorylation by Plk1, but this MKlp2 showed a reduced ability to bind Plk1 on ligand blots (Fig. 8 C). However, when the MKlp2 antibodies were added to the blocking reaction of the ligand blot, they were unable to prevent binding of Plk1 to phosphorylated MKlp2 (Fig. 8 C). The effect of these antibodies on the behavior of MKlp2 in microtubule binding was then tested, but no obvious differences were seen between buffer, MKlp2, and control antibody incubations (Fig. 8 D). In bundling assays, the MKlp2 antibodies caused a slight reduction in the length of the microtubule bundles compared with control incubations with MKlp2 alone or with GFP antibodies without affecting the number or morphology of the bundles formed (Fig. 8 E). The failure of cytokinesis in HeLa cells injected with antibodies to the MKlp2 neck and stalk region could therefore be due to a failure of Plk1 to phosphorylate MKlp2 because antibody binding has little effect on the other properties of the kinesin investigated here. These observations support the idea that phosphorylation of MKlp2 by Plk1 is necessary for MKlp2 function in Plk1 localization to the central spindle, and for the control of cytokinesis in human cells.

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