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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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Analysis of the sites on human MKlp2 phosphorylated in vivo and in vitro by Plk1. (A) MKlp2, MKlp2 treated with Plk1 in vitro, and MKlp2 immune precipitated from 2.5 mg extract prepared from asynchronous cells (Int) and cells arrested with nocodazole and released for 2 h (Mit) were digested with trypsin and then analyzed by mass spectrometry. Note that as expected, MKlp2 is highly enriched in the mitotic cells. A region of the spectra showing phosphorylation of a peptide from 526 to 537 in Plk1-treated and in vivo MKlp2 is shown. Fragmentation of this peptide showed that the serine at 528 is phosphorylated by Plk1 and in vivo. (B) MKlp2 and MKlp2S528A (1 μg) were treated with the indicated amounts of Plk1 for 1 h at 30°C, and were then analyzed by gel electrophoresis and autoradiography. (C) Microtubule-binding assays were performed with 5 pmol MKlp2 and MKlp25528A treated with buffer or Plk1. Samples were then analyzed by Western blotting and detection with antibodies to the hexahistidine tag.
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fig4: Analysis of the sites on human MKlp2 phosphorylated in vivo and in vitro by Plk1. (A) MKlp2, MKlp2 treated with Plk1 in vitro, and MKlp2 immune precipitated from 2.5 mg extract prepared from asynchronous cells (Int) and cells arrested with nocodazole and released for 2 h (Mit) were digested with trypsin and then analyzed by mass spectrometry. Note that as expected, MKlp2 is highly enriched in the mitotic cells. A region of the spectra showing phosphorylation of a peptide from 526 to 537 in Plk1-treated and in vivo MKlp2 is shown. Fragmentation of this peptide showed that the serine at 528 is phosphorylated by Plk1 and in vivo. (B) MKlp2 and MKlp2S528A (1 μg) were treated with the indicated amounts of Plk1 for 1 h at 30°C, and were then analyzed by gel electrophoresis and autoradiography. (C) Microtubule-binding assays were performed with 5 pmol MKlp2 and MKlp25528A treated with buffer or Plk1. Samples were then analyzed by Western blotting and detection with antibodies to the hexahistidine tag.

Mentions: These observations raise a number of questions relating to Plk1 and MKlp2. Is Plk1 a direct and specific regulator of MKlp2 and its microtubule-binding properties, and do MKlp2 and microtubules control Plk1 activity? To answer these questions, the abilities of Plk1 and the cdk1–cyclin B mitotic kinase to phosphorylate MKlp2 and the known mitotic spindle phosphoproteins PRC1 and MKlp1 were tested (Fig. 3 D). Equimolar amounts of substrate were used in all kinase assays to allow a direct comparison of the different substrates. The cdk1–cyclin B mitotic kinase phosphorylated histone H1, PRC1, MKlp1, and to some extent MKlp2 (Fig. 3 D). In contrast, Plk1 strongly phosphorylated MKlp2, and weakly phosphorylated MKlp1, PRC1, and the model substrate casein (Fig. 3 D). The aurora-B kinase complex was unable to phosphorylate MKlp2 or MKlp1 (unpublished data). Therefore, Plk1 is a candidate kinase for MKlp2, which is specifically phosphorylated under the conditions used. The effect of microtubules on the ability of Plk1 to phosphorylate MKlp2 and the model substrate casein was then tested (Fig. 3 E). MKlp2 was phosphorylated by Plk1 in the absence of microtubules, and this was increased 10-fold by the addition microtubules (Fig. 3 E). No significant phosphorylation of MKlp2 was observed with microtubules alone, so this was most likely due to Plk1 and not a contaminating kinase. Under the same conditions, phosphorylation of casein was decreased, suggesting that this effect was not due to activation of Plk1 by microtubules toward all substrates. Therefore, microtubules regulate the phosphorylation of MKlp2 by Plk1, supporting the idea that Plk1 regulates MKlp2 at the central spindle. If Plk1 does regulate MKlp2 at the central spindle, then it should phosphorylate MKlp2 at the same sites in vitro and during mitosis in living cells. Mass spectrometric analysis of MKlp2 phosphorylated in vitro by Plk1 and precipitated from mitotic cells revealed that a peptide corresponding to amino acids 526–537 was phosphorylated at serine 528 in both cases (Fig. 4 A). Secondary phosphorylation sites at serines 62, 662, and 668 were also identified using this approach (unpublished data). Mutation of serine 528 to alanine resulted in a fivefold reduction in the amount of MKlp2 phosphorylation by Plk1 in vitro (Fig. 4 B). Therefore, MKlp2 is phosphorylated at serine 528 during mitosis, in vitro by Plk1, and this is a major site of Plk1 phosphorylation. This residue is in the neck-linker sequence of MKlp2, and by analogy with other kinesins, this region is likely to be critical for the control of microtubule binding and motor activity (Grummt et al., 1998; Rice et al., 1999; Mishima et al., 2002; Schafer et al., 2003). Thus, Plk1 is likely to be a physiological regulator of MKlp2, and the simplest explanation is that Plk1 regulates the binding of MKlp2 to microtubules. However, this was tested, and both MKlp2S528A and MKlp2 phosphorylated by Plk1 all showed similar microtubule-binding activity to MKlp2 control incubations (Fig. 4 C), and for this reason, other aspects of MKlp2 function were examined.


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)

Analysis of the sites on human MKlp2 phosphorylated in vivo and in vitro by Plk1. (A) MKlp2, MKlp2 treated with Plk1 in vitro, and MKlp2 immune precipitated from 2.5 mg extract prepared from asynchronous cells (Int) and cells arrested with nocodazole and released for 2 h (Mit) were digested with trypsin and then analyzed by mass spectrometry. Note that as expected, MKlp2 is highly enriched in the mitotic cells. A region of the spectra showing phosphorylation of a peptide from 526 to 537 in Plk1-treated and in vivo MKlp2 is shown. Fragmentation of this peptide showed that the serine at 528 is phosphorylated by Plk1 and in vivo. (B) MKlp2 and MKlp2S528A (1 μg) were treated with the indicated amounts of Plk1 for 1 h at 30°C, and were then analyzed by gel electrophoresis and autoradiography. (C) Microtubule-binding assays were performed with 5 pmol MKlp2 and MKlp25528A treated with buffer or Plk1. Samples were then analyzed by Western blotting and detection with antibodies to the hexahistidine tag.
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Related In: Results  -  Collection

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fig4: Analysis of the sites on human MKlp2 phosphorylated in vivo and in vitro by Plk1. (A) MKlp2, MKlp2 treated with Plk1 in vitro, and MKlp2 immune precipitated from 2.5 mg extract prepared from asynchronous cells (Int) and cells arrested with nocodazole and released for 2 h (Mit) were digested with trypsin and then analyzed by mass spectrometry. Note that as expected, MKlp2 is highly enriched in the mitotic cells. A region of the spectra showing phosphorylation of a peptide from 526 to 537 in Plk1-treated and in vivo MKlp2 is shown. Fragmentation of this peptide showed that the serine at 528 is phosphorylated by Plk1 and in vivo. (B) MKlp2 and MKlp2S528A (1 μg) were treated with the indicated amounts of Plk1 for 1 h at 30°C, and were then analyzed by gel electrophoresis and autoradiography. (C) Microtubule-binding assays were performed with 5 pmol MKlp2 and MKlp25528A treated with buffer or Plk1. Samples were then analyzed by Western blotting and detection with antibodies to the hexahistidine tag.
Mentions: These observations raise a number of questions relating to Plk1 and MKlp2. Is Plk1 a direct and specific regulator of MKlp2 and its microtubule-binding properties, and do MKlp2 and microtubules control Plk1 activity? To answer these questions, the abilities of Plk1 and the cdk1–cyclin B mitotic kinase to phosphorylate MKlp2 and the known mitotic spindle phosphoproteins PRC1 and MKlp1 were tested (Fig. 3 D). Equimolar amounts of substrate were used in all kinase assays to allow a direct comparison of the different substrates. The cdk1–cyclin B mitotic kinase phosphorylated histone H1, PRC1, MKlp1, and to some extent MKlp2 (Fig. 3 D). In contrast, Plk1 strongly phosphorylated MKlp2, and weakly phosphorylated MKlp1, PRC1, and the model substrate casein (Fig. 3 D). The aurora-B kinase complex was unable to phosphorylate MKlp2 or MKlp1 (unpublished data). Therefore, Plk1 is a candidate kinase for MKlp2, which is specifically phosphorylated under the conditions used. The effect of microtubules on the ability of Plk1 to phosphorylate MKlp2 and the model substrate casein was then tested (Fig. 3 E). MKlp2 was phosphorylated by Plk1 in the absence of microtubules, and this was increased 10-fold by the addition microtubules (Fig. 3 E). No significant phosphorylation of MKlp2 was observed with microtubules alone, so this was most likely due to Plk1 and not a contaminating kinase. Under the same conditions, phosphorylation of casein was decreased, suggesting that this effect was not due to activation of Plk1 by microtubules toward all substrates. Therefore, microtubules regulate the phosphorylation of MKlp2 by Plk1, supporting the idea that Plk1 regulates MKlp2 at the central spindle. If Plk1 does regulate MKlp2 at the central spindle, then it should phosphorylate MKlp2 at the same sites in vitro and during mitosis in living cells. Mass spectrometric analysis of MKlp2 phosphorylated in vitro by Plk1 and precipitated from mitotic cells revealed that a peptide corresponding to amino acids 526–537 was phosphorylated at serine 528 in both cases (Fig. 4 A). Secondary phosphorylation sites at serines 62, 662, and 668 were also identified using this approach (unpublished data). Mutation of serine 528 to alanine resulted in a fivefold reduction in the amount of MKlp2 phosphorylation by Plk1 in vitro (Fig. 4 B). Therefore, MKlp2 is phosphorylated at serine 528 during mitosis, in vitro by Plk1, and this is a major site of Plk1 phosphorylation. This residue is in the neck-linker sequence of MKlp2, and by analogy with other kinesins, this region is likely to be critical for the control of microtubule binding and motor activity (Grummt et al., 1998; Rice et al., 1999; Mishima et al., 2002; Schafer et al., 2003). Thus, Plk1 is likely to be a physiological regulator of MKlp2, and the simplest explanation is that Plk1 regulates the binding of MKlp2 to microtubules. However, this was tested, and both MKlp2S528A and MKlp2 phosphorylated by Plk1 all showed similar microtubule-binding activity to MKlp2 control incubations (Fig. 4 C), and for this reason, other aspects of MKlp2 function were examined.

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