Limits...
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.

Show MeSH
The Plk1 polo box domain binds to phosphorylated MKlp2. (A) HeLa S3 cells grown in suspension were arrested with 90 ng/ml nocodazole for 20 h, and then released for 2 h. Immune precipitations with affinity-purified antibodies to MKlp2, Plk1, and the nonspecific control GFP were performed from 100-μg soluble extracts prepared from these cells. Western blot analysis was performed on 20 μg cell extract, and one fifth of the immune precipitated material. (B) Ligand blots were performed with Plk1 amino acids 305–603 as the probe, and Plk1, cdk1/cyclin B, PRC1, MKlp2, treated with Plk1 or cdk1/cyclin B as targets. (C) Ligand blots were performed with Plk1 amino acids 305–603 as the probe, and MKlp2 or MKlp2S528A treated with Plk1 as targets. The asterisks mark a truncation product in the MKlp2 preparations.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172827&req=5

fig6: The Plk1 polo box domain binds to phosphorylated MKlp2. (A) HeLa S3 cells grown in suspension were arrested with 90 ng/ml nocodazole for 20 h, and then released for 2 h. Immune precipitations with affinity-purified antibodies to MKlp2, Plk1, and the nonspecific control GFP were performed from 100-μg soluble extracts prepared from these cells. Western blot analysis was performed on 20 μg cell extract, and one fifth of the immune precipitated material. (B) Ligand blots were performed with Plk1 amino acids 305–603 as the probe, and Plk1, cdk1/cyclin B, PRC1, MKlp2, treated with Plk1 or cdk1/cyclin B as targets. (C) Ligand blots were performed with Plk1 amino acids 305–603 as the probe, and MKlp2 or MKlp2S528A treated with Plk1 as targets. The asterisks mark a truncation product in the MKlp2 preparations.

Mentions: In MKlp2-depleted cells, Plk1 fails to localize to the central spindle region, despite the fact that other components such as PRC1 and MKlp1 are still able to do so. The simplest explanation for this observation is that Plk1 binds to MKlp2. To test this, Plk1 and MKlp2 were precipitated from cells arrested in mitosis with nocodazole, and were then released for 2 h to allow the cells to enter anaphase. Plk1 immune precipitates contain MKlp2, and the converse was also true, whereas a control antibody did not precipitate either protein (Fig. 6 A). MKlp1 was not precipitated by any of the antibodies used (Fig. 6 A). Therefore, Plk1 and MKlp2 are in a complex in mitotic cells, as they coprecipitate under conditions where another central spindle protein does not. Because the Plk1 polo box domain is a phospho-specific adaptor (Elia et al., 2003), in addition to the microtubule-binding properties described in Fig. 3 A, it was possible that it might bind directly to phosphorylated MKlp2. The Plk1 polo box domain strongly bound to MKlp2 phosphorylated by Plk1 on ligand blots, whereas untreated MKlp2 gave a weak signal and the control protein PRC1 did not bind either before or after cdk1–cyclin B1 phosphorylation (Fig. 6 B). This recognition was dependent on phosphorylation of serine 528 because MKlp2S528A showed a fivefold reduction in binding to the polo box domain (Fig. 6 C). Plk1 purified from insect cells shows extensive autophosphorylation (unpublished data) and cyclin B1 is phosphorylated by Plk1; however, these phosphorylations were not recognized by the polo box domain. The Plk1 kinase domain did not bind to any of the proteins under these conditions (Fig. S4). Therefore, Plk1 exists in a complex with MKlp2 in mitotic cells, and binds directly to phosphorylated MKlp2 by its polo box domain.


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)

The Plk1 polo box domain binds to phosphorylated MKlp2. (A) HeLa S3 cells grown in suspension were arrested with 90 ng/ml nocodazole for 20 h, and then released for 2 h. Immune precipitations with affinity-purified antibodies to MKlp2, Plk1, and the nonspecific control GFP were performed from 100-μg soluble extracts prepared from these cells. Western blot analysis was performed on 20 μg cell extract, and one fifth of the immune precipitated material. (B) Ligand blots were performed with Plk1 amino acids 305–603 as the probe, and Plk1, cdk1/cyclin B, PRC1, MKlp2, treated with Plk1 or cdk1/cyclin B as targets. (C) Ligand blots were performed with Plk1 amino acids 305–603 as the probe, and MKlp2 or MKlp2S528A treated with Plk1 as targets. The asterisks mark a truncation product in the MKlp2 preparations.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: The Plk1 polo box domain binds to phosphorylated MKlp2. (A) HeLa S3 cells grown in suspension were arrested with 90 ng/ml nocodazole for 20 h, and then released for 2 h. Immune precipitations with affinity-purified antibodies to MKlp2, Plk1, and the nonspecific control GFP were performed from 100-μg soluble extracts prepared from these cells. Western blot analysis was performed on 20 μg cell extract, and one fifth of the immune precipitated material. (B) Ligand blots were performed with Plk1 amino acids 305–603 as the probe, and Plk1, cdk1/cyclin B, PRC1, MKlp2, treated with Plk1 or cdk1/cyclin B as targets. (C) Ligand blots were performed with Plk1 amino acids 305–603 as the probe, and MKlp2 or MKlp2S528A treated with Plk1 as targets. The asterisks mark a truncation product in the MKlp2 preparations.
Mentions: In MKlp2-depleted cells, Plk1 fails to localize to the central spindle region, despite the fact that other components such as PRC1 and MKlp1 are still able to do so. The simplest explanation for this observation is that Plk1 binds to MKlp2. To test this, Plk1 and MKlp2 were precipitated from cells arrested in mitosis with nocodazole, and were then released for 2 h to allow the cells to enter anaphase. Plk1 immune precipitates contain MKlp2, and the converse was also true, whereas a control antibody did not precipitate either protein (Fig. 6 A). MKlp1 was not precipitated by any of the antibodies used (Fig. 6 A). Therefore, Plk1 and MKlp2 are in a complex in mitotic cells, as they coprecipitate under conditions where another central spindle protein does not. Because the Plk1 polo box domain is a phospho-specific adaptor (Elia et al., 2003), in addition to the microtubule-binding properties described in Fig. 3 A, it was possible that it might bind directly to phosphorylated MKlp2. The Plk1 polo box domain strongly bound to MKlp2 phosphorylated by Plk1 on ligand blots, whereas untreated MKlp2 gave a weak signal and the control protein PRC1 did not bind either before or after cdk1–cyclin B1 phosphorylation (Fig. 6 B). This recognition was dependent on phosphorylation of serine 528 because MKlp2S528A showed a fivefold reduction in binding to the polo box domain (Fig. 6 C). Plk1 purified from insect cells shows extensive autophosphorylation (unpublished data) and cyclin B1 is phosphorylated by Plk1; however, these phosphorylations were not recognized by the polo box domain. The Plk1 kinase domain did not bind to any of the proteins under these conditions (Fig. S4). Therefore, Plk1 exists in a complex with MKlp2 in mitotic cells, and binds directly to phosphorylated MKlp2 by its polo box domain.

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