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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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A phosphorylation site mutant of MKlp2 fails to rescue targeting of Plk1 to the central spindle in MKlp2-depleted cells. HeLa S3 cells were treated with the MKlp2 siRNA duplex for 18 h and were then transfected with siRNA-resistant GFP-tagged MKlp2 or a phosphorylation site mutant for 12 h. Cells were fixed with methanol and then stained with antibodies to Plk1, GFP was directly visualized, and DNA was stained with DAPI. Bar, 10 μM.
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fig7: A phosphorylation site mutant of MKlp2 fails to rescue targeting of Plk1 to the central spindle in MKlp2-depleted cells. HeLa S3 cells were treated with the MKlp2 siRNA duplex for 18 h and were then transfected with siRNA-resistant GFP-tagged MKlp2 or a phosphorylation site mutant for 12 h. Cells were fixed with methanol and then stained with antibodies to Plk1, GFP was directly visualized, and DNA was stained with DAPI. Bar, 10 μM.

Mentions: To show that the interaction of Plk1 with phosphorylated MKlp2 is relevant for the targeting of Plk1 to the central spindle, MKlp2 and a phosphorylation site mutant of MKlp2 were expressed in cells depleted of MKlp2 using siRNA (Fig. 7). Wild-type MKlp2 expressed in MKlp2-depleted cells localized to the central spindle, and in addition, rescued Plk1 targeting to the central spindle (Fig. 7). In contrast, the phosphorylation site mutant of MKlp2, although localized to the central spindle, did not rescue Plk1 targeting to this structure (Fig. 7). Therefore, MKlp2 targeting to the central spindle does not require phosphorylation by Plk1, but phosphorylation is needed for the recruitment of Plk1 to the central spindle.


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)

A phosphorylation site mutant of MKlp2 fails to rescue targeting of Plk1 to the central spindle in MKlp2-depleted cells. HeLa S3 cells were treated with the MKlp2 siRNA duplex for 18 h and were then transfected with siRNA-resistant GFP-tagged MKlp2 or a phosphorylation site mutant for 12 h. Cells were fixed with methanol and then stained with antibodies to Plk1, GFP was directly visualized, and DNA was stained with DAPI. Bar, 10 μM.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: A phosphorylation site mutant of MKlp2 fails to rescue targeting of Plk1 to the central spindle in MKlp2-depleted cells. HeLa S3 cells were treated with the MKlp2 siRNA duplex for 18 h and were then transfected with siRNA-resistant GFP-tagged MKlp2 or a phosphorylation site mutant for 12 h. Cells were fixed with methanol and then stained with antibodies to Plk1, GFP was directly visualized, and DNA was stained with DAPI. Bar, 10 μM.
Mentions: To show that the interaction of Plk1 with phosphorylated MKlp2 is relevant for the targeting of Plk1 to the central spindle, MKlp2 and a phosphorylation site mutant of MKlp2 were expressed in cells depleted of MKlp2 using siRNA (Fig. 7). Wild-type MKlp2 expressed in MKlp2-depleted cells localized to the central spindle, and in addition, rescued Plk1 targeting to the central spindle (Fig. 7). In contrast, the phosphorylation site mutant of MKlp2, although localized to the central spindle, did not rescue Plk1 targeting to this structure (Fig. 7). Therefore, MKlp2 targeting to the central spindle does not require phosphorylation by Plk1, but phosphorylation is needed for the recruitment of Plk1 to the central spindle.

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