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The activity regulation of the mitotic centromere-associated kinesin by Polo-like kinase 1.

Ritter A, Sanhaji M, Steinhäuser K, Roth S, Louwen F, Yuan J - Oncotarget (2015)

Bottom Line: The mitotic centromere-associated kinesin (MCAK), a potent microtubule depolymerase, is involved in regulating microtubule dynamics.The subsequent dephosphorylation of S632/S633 might be necessary to timely align the chromosomes onto the metaphase plate.Therefore, our studies suggest new mechanisms by which Plk1 regulates MCAK: the degradation of MCAK is controlled by Plk1 phosphorylation on S621, whereas its activity is modulated by Plk1 phosphorylation on S632/S633 in mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Gynecology and Obstetrics, School of Medicine, J. W. Goethe-University, 60590 Frankfurt, Germany.

ABSTRACT
The mitotic centromere-associated kinesin (MCAK), a potent microtubule depolymerase, is involved in regulating microtubule dynamics. The activity and subcellular localization of MCAK are tightly regulated by key mitotic kinases, such as Polo-like kinase 1 (Plk1) by phosphorylating multiple residues in MCAK. Since Plk1 phosphorylates very often different residues of substrates at different stages, we have dissected individual phosphorylation of MCAK by Plk1 and characterized its function in more depth. We have recently shown that S621 in MCAK is the major phosphorylation site of Plk1, which is responsible for regulating MCAK's degradation by promoting the association of MCAK with APC/CCdc20. In the present study, we have addressed another two residues phosphorylated by Plk1, namely S632/S633 in the C-terminus of MCAK. Our data suggest that Plk1 phosphorylates S632/S633 and regulates its catalytic activity in mitosis. This phosphorylation is required for proper spindle assembly during early phases of mitosis. The subsequent dephosphorylation of S632/S633 might be necessary to timely align the chromosomes onto the metaphase plate. Therefore, our studies suggest new mechanisms by which Plk1 regulates MCAK: the degradation of MCAK is controlled by Plk1 phosphorylation on S621, whereas its activity is modulated by Plk1 phosphorylation on S632/S633 in mitosis.

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Decreased k-fiber length in cells with MCAK SS/DD and unchanged localization of MCAK mutants(A) Experimental schedule for B and C. HeLa cells depleted of endogenous MCAK were rescued with wild type MCAK or its mutants. Cells were ice-cold treated for 12 min and stained for α-tubulin, pericentrin, ACA (anti-centromere antibody) and DNA. (B) The k-fiber length was evaluated by fluorescence microscopy using the software AxioVision SE64 Rel. 4.9. (n = 80 fibers from 15 metaphase cells for each condition). The results are presented as mean ± SD. ***p < 0.001. (C) Representative cells, in which the k-fiber length was measured, are shown. Scale bar: 5 μm. Insets are magnified three fold and presented aside. Hardly change in kinetochore region of MCAK mutants. (D and E) Unchanged localization of MCAK S632/S633 mutants in the kinetochore region. HeLa cells depleted of endogenous MCAK were rescued with wild type EGFP-MCAK or its mutants. Cells were synchronized to prometaphase with nocodazol, released for 1 h into fresh medium containing MG132 to keep cells in metaphase. Metaphase cells were then exposed to hypotonic buffer, centrifuged onto an object slide and stained for Hec1 and DNA, as detailed in Materials and Methods. (D) Quantification of colocalized EGFP-MCAK constructs with Hec1 was performed using the Pearson's coefficient with ImageJ software. The results are presented as mean ± SD (n = 120 kinetochore pairs). (E) Representatives are shown. Insets are magnified two fold presented aside with a highlighted example of the kinetochore/centromere region. Scale bar: 2.5 μm.
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Figure 4: Decreased k-fiber length in cells with MCAK SS/DD and unchanged localization of MCAK mutants(A) Experimental schedule for B and C. HeLa cells depleted of endogenous MCAK were rescued with wild type MCAK or its mutants. Cells were ice-cold treated for 12 min and stained for α-tubulin, pericentrin, ACA (anti-centromere antibody) and DNA. (B) The k-fiber length was evaluated by fluorescence microscopy using the software AxioVision SE64 Rel. 4.9. (n = 80 fibers from 15 metaphase cells for each condition). The results are presented as mean ± SD. ***p < 0.001. (C) Representative cells, in which the k-fiber length was measured, are shown. Scale bar: 5 μm. Insets are magnified three fold and presented aside. Hardly change in kinetochore region of MCAK mutants. (D and E) Unchanged localization of MCAK S632/S633 mutants in the kinetochore region. HeLa cells depleted of endogenous MCAK were rescued with wild type EGFP-MCAK or its mutants. Cells were synchronized to prometaphase with nocodazol, released for 1 h into fresh medium containing MG132 to keep cells in metaphase. Metaphase cells were then exposed to hypotonic buffer, centrifuged onto an object slide and stained for Hec1 and DNA, as detailed in Materials and Methods. (D) Quantification of colocalized EGFP-MCAK constructs with Hec1 was performed using the Pearson's coefficient with ImageJ software. The results are presented as mean ± SD (n = 120 kinetochore pairs). (E) Representatives are shown. Insets are magnified two fold presented aside with a highlighted example of the kinetochore/centromere region. Scale bar: 2.5 μm.

Mentions: To corroborate these observations, four further assays were performed. First, the spindle length was measured in cells transfected with EGFP MCAK WT, EGFP MCAK SS/AA and EGFP MCAK SS/DD. Like EFGP MCAK WT, EGFP MCAK SS/DD and EGFP MCAK SS/AA were localized in spindle poles and the kinetochore/centromere region (Fig. 2F, EGFP). Whereas HeLa cells expressing MCAK SS/AA showed significantly longer spindles, overexpression of MCAK SS/DD led to the formation of shorter spindles relative to cells transfected with EFGP MCAK WT, indicating a higher depolymerization activity of this phosphomimetic mutant (Fig. 2F and G). Similar results were obtained in U2OS cells (Fig. S1C and D). Second, a well-established microtubule regrowth assay [34] was performed. HeLa cells depleted of endogenous MCAK were re-added with Flag-tagged wild type or its mutants (Fig. 3D). Cells were then cold-treated for 45 min to depolymerize their microtubules, re-warmed at 37°C for 0, 2 and 4 min for re-polymerization, stained for α-tubulin, pericentrin and DNA. The areas depicted by the red circles in metaphase cells on Figure 3C represent the regions where the microtubule intensity was quantified as described elsewhere [34]. Relative to control groups, control siRNA treated or rescued with wild type MCAK, cells depleted of endogenous MCAK displayed increased amounts of microtubule upon rewarming (Fig. 3B and C). While intensified microtubule signals were observed in MCAK SS/AA transfected cells, less polymerized microtubules were assembled in MCAK SS/DD rescued cells (Fig. 3B and C). Third, the length of the k-fibers was measured as described [35]. For that, cells depleted of endogenous MCAK were rescued with either wild type MCAK or its mutants, cold-treated for 12 min, to depolymerize all other types of microtubules but k-fibers, and stained for microscopic evaluation (Fig. 4A). The metaphase cells rescued with MCAK SS632/633DD showed significantly shorter k-fibers whereas longer k-fibers were observed in cells depleted of endogenous MCAK or rescued with MCAK WT and MCAK SS/AA (Fig. 4B and C). Finally, the intensity of whole microtubules was measured in cells depleted of endogenous MCAK and rescued with wild type MCAK or its mutants. Again, cells expressing MCAK SS/AA had more microtubules whereas cells transfected with MCAK SS/DD exhibited less amounts relative to wild type MCAK rescued cells (Fig. S2), suggesting that these mutations indeed affect spindle microtubule polymers.


The activity regulation of the mitotic centromere-associated kinesin by Polo-like kinase 1.

Ritter A, Sanhaji M, Steinhäuser K, Roth S, Louwen F, Yuan J - Oncotarget (2015)

Decreased k-fiber length in cells with MCAK SS/DD and unchanged localization of MCAK mutants(A) Experimental schedule for B and C. HeLa cells depleted of endogenous MCAK were rescued with wild type MCAK or its mutants. Cells were ice-cold treated for 12 min and stained for α-tubulin, pericentrin, ACA (anti-centromere antibody) and DNA. (B) The k-fiber length was evaluated by fluorescence microscopy using the software AxioVision SE64 Rel. 4.9. (n = 80 fibers from 15 metaphase cells for each condition). The results are presented as mean ± SD. ***p < 0.001. (C) Representative cells, in which the k-fiber length was measured, are shown. Scale bar: 5 μm. Insets are magnified three fold and presented aside. Hardly change in kinetochore region of MCAK mutants. (D and E) Unchanged localization of MCAK S632/S633 mutants in the kinetochore region. HeLa cells depleted of endogenous MCAK were rescued with wild type EGFP-MCAK or its mutants. Cells were synchronized to prometaphase with nocodazol, released for 1 h into fresh medium containing MG132 to keep cells in metaphase. Metaphase cells were then exposed to hypotonic buffer, centrifuged onto an object slide and stained for Hec1 and DNA, as detailed in Materials and Methods. (D) Quantification of colocalized EGFP-MCAK constructs with Hec1 was performed using the Pearson's coefficient with ImageJ software. The results are presented as mean ± SD (n = 120 kinetochore pairs). (E) Representatives are shown. Insets are magnified two fold presented aside with a highlighted example of the kinetochore/centromere region. Scale bar: 2.5 μm.
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Figure 4: Decreased k-fiber length in cells with MCAK SS/DD and unchanged localization of MCAK mutants(A) Experimental schedule for B and C. HeLa cells depleted of endogenous MCAK were rescued with wild type MCAK or its mutants. Cells were ice-cold treated for 12 min and stained for α-tubulin, pericentrin, ACA (anti-centromere antibody) and DNA. (B) The k-fiber length was evaluated by fluorescence microscopy using the software AxioVision SE64 Rel. 4.9. (n = 80 fibers from 15 metaphase cells for each condition). The results are presented as mean ± SD. ***p < 0.001. (C) Representative cells, in which the k-fiber length was measured, are shown. Scale bar: 5 μm. Insets are magnified three fold and presented aside. Hardly change in kinetochore region of MCAK mutants. (D and E) Unchanged localization of MCAK S632/S633 mutants in the kinetochore region. HeLa cells depleted of endogenous MCAK were rescued with wild type EGFP-MCAK or its mutants. Cells were synchronized to prometaphase with nocodazol, released for 1 h into fresh medium containing MG132 to keep cells in metaphase. Metaphase cells were then exposed to hypotonic buffer, centrifuged onto an object slide and stained for Hec1 and DNA, as detailed in Materials and Methods. (D) Quantification of colocalized EGFP-MCAK constructs with Hec1 was performed using the Pearson's coefficient with ImageJ software. The results are presented as mean ± SD (n = 120 kinetochore pairs). (E) Representatives are shown. Insets are magnified two fold presented aside with a highlighted example of the kinetochore/centromere region. Scale bar: 2.5 μm.
Mentions: To corroborate these observations, four further assays were performed. First, the spindle length was measured in cells transfected with EGFP MCAK WT, EGFP MCAK SS/AA and EGFP MCAK SS/DD. Like EFGP MCAK WT, EGFP MCAK SS/DD and EGFP MCAK SS/AA were localized in spindle poles and the kinetochore/centromere region (Fig. 2F, EGFP). Whereas HeLa cells expressing MCAK SS/AA showed significantly longer spindles, overexpression of MCAK SS/DD led to the formation of shorter spindles relative to cells transfected with EFGP MCAK WT, indicating a higher depolymerization activity of this phosphomimetic mutant (Fig. 2F and G). Similar results were obtained in U2OS cells (Fig. S1C and D). Second, a well-established microtubule regrowth assay [34] was performed. HeLa cells depleted of endogenous MCAK were re-added with Flag-tagged wild type or its mutants (Fig. 3D). Cells were then cold-treated for 45 min to depolymerize their microtubules, re-warmed at 37°C for 0, 2 and 4 min for re-polymerization, stained for α-tubulin, pericentrin and DNA. The areas depicted by the red circles in metaphase cells on Figure 3C represent the regions where the microtubule intensity was quantified as described elsewhere [34]. Relative to control groups, control siRNA treated or rescued with wild type MCAK, cells depleted of endogenous MCAK displayed increased amounts of microtubule upon rewarming (Fig. 3B and C). While intensified microtubule signals were observed in MCAK SS/AA transfected cells, less polymerized microtubules were assembled in MCAK SS/DD rescued cells (Fig. 3B and C). Third, the length of the k-fibers was measured as described [35]. For that, cells depleted of endogenous MCAK were rescued with either wild type MCAK or its mutants, cold-treated for 12 min, to depolymerize all other types of microtubules but k-fibers, and stained for microscopic evaluation (Fig. 4A). The metaphase cells rescued with MCAK SS632/633DD showed significantly shorter k-fibers whereas longer k-fibers were observed in cells depleted of endogenous MCAK or rescued with MCAK WT and MCAK SS/AA (Fig. 4B and C). Finally, the intensity of whole microtubules was measured in cells depleted of endogenous MCAK and rescued with wild type MCAK or its mutants. Again, cells expressing MCAK SS/AA had more microtubules whereas cells transfected with MCAK SS/DD exhibited less amounts relative to wild type MCAK rescued cells (Fig. S2), suggesting that these mutations indeed affect spindle microtubule polymers.

Bottom Line: The mitotic centromere-associated kinesin (MCAK), a potent microtubule depolymerase, is involved in regulating microtubule dynamics.The subsequent dephosphorylation of S632/S633 might be necessary to timely align the chromosomes onto the metaphase plate.Therefore, our studies suggest new mechanisms by which Plk1 regulates MCAK: the degradation of MCAK is controlled by Plk1 phosphorylation on S621, whereas its activity is modulated by Plk1 phosphorylation on S632/S633 in mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Gynecology and Obstetrics, School of Medicine, J. W. Goethe-University, 60590 Frankfurt, Germany.

ABSTRACT
The mitotic centromere-associated kinesin (MCAK), a potent microtubule depolymerase, is involved in regulating microtubule dynamics. The activity and subcellular localization of MCAK are tightly regulated by key mitotic kinases, such as Polo-like kinase 1 (Plk1) by phosphorylating multiple residues in MCAK. Since Plk1 phosphorylates very often different residues of substrates at different stages, we have dissected individual phosphorylation of MCAK by Plk1 and characterized its function in more depth. We have recently shown that S621 in MCAK is the major phosphorylation site of Plk1, which is responsible for regulating MCAK's degradation by promoting the association of MCAK with APC/CCdc20. In the present study, we have addressed another two residues phosphorylated by Plk1, namely S632/S633 in the C-terminus of MCAK. Our data suggest that Plk1 phosphorylates S632/S633 and regulates its catalytic activity in mitosis. This phosphorylation is required for proper spindle assembly during early phases of mitosis. The subsequent dephosphorylation of S632/S633 might be necessary to timely align the chromosomes onto the metaphase plate. Therefore, our studies suggest new mechanisms by which Plk1 regulates MCAK: the degradation of MCAK is controlled by Plk1 phosphorylation on S621, whereas its activity is modulated by Plk1 phosphorylation on S632/S633 in mitosis.

Show MeSH
Related in: MedlinePlus