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Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis.

Yamaguchi T, Goto H, Yokoyama T, Silljé H, Hanisch A, Uldschmid A, Takai Y, Oguri T, Nigg EA, Inagaki M - J. Cell Biol. (2005)

Bottom Line: This elevation followed the Cdk1-induced vimentin-Ser55 phosphorylation, and was impaired by Plk1 depletion.Mutational analyses revealed that Plk1-induced vimentin-Ser82 phosphorylation plays an important role in vimentin filaments segregation, coordinately with Rho-kinase and Aurora-B.Taken together, these results indicated a novel mechanism that Cdk1 regulated mitotic vimentin phosphorylation via not only a direct enzyme reaction but also Plk1 recruitment to vimentin.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Aichi Cancer Center Research Institute, Aichi 464-8681, Japan.

ABSTRACT
Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its significance has remained unknown. Here we demonstrated a direct interaction between Plk1 and vimentin-Ser55 phosphorylated by Cdk1, an event that led to Plk1 activation and further vimentin phosphorylation. Plk1 phosphorylated vimentin at approximately 1 mol phosphate/mol substrate, which partly inhibited its filament forming ability, in vitro. Plk1 induced the phosphorylation of vimentin-Ser82, which was elevated from metaphase and maintained until the end of mitosis. This elevation followed the Cdk1-induced vimentin-Ser55 phosphorylation, and was impaired by Plk1 depletion. Mutational analyses revealed that Plk1-induced vimentin-Ser82 phosphorylation plays an important role in vimentin filaments segregation, coordinately with Rho-kinase and Aurora-B. Taken together, these results indicated a novel mechanism that Cdk1 regulated mitotic vimentin phosphorylation via not only a direct enzyme reaction but also Plk1 recruitment to vimentin.

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The significance of Plk1-induced vimentin phosphorylation on vimentin filament segregation. (A) T24 cells were transfected with pDR2 carrying vimentin WT or S82E. 48 h after transfection, cells were stained with anti-vimentin antibody (green) and propidium iodide (red). Magnified images were also indicated in the right panel. Cells were analyzed by laser-scanning confocal microscopy. (B) Vimentin IF bridge formation and multinucleate cell in T24 cells expressing vimentin mutants (right). Cells were stained with anti-vimentin antibody (green) and propidium iodide (red). The percentage of vimentin IF-bridge or multinucleate cells were scored as described (Yasui et al., 2001). Data represents means ± SEM in at least three independent experiments. (C) Blue, green, yellow, or red color represents Plk1, Cdk1, cyclin B, and phosphates within residues on vimentin, respectively. The red arrow indicates phosphorylation reaction.
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fig5: The significance of Plk1-induced vimentin phosphorylation on vimentin filament segregation. (A) T24 cells were transfected with pDR2 carrying vimentin WT or S82E. 48 h after transfection, cells were stained with anti-vimentin antibody (green) and propidium iodide (red). Magnified images were also indicated in the right panel. Cells were analyzed by laser-scanning confocal microscopy. (B) Vimentin IF bridge formation and multinucleate cell in T24 cells expressing vimentin mutants (right). Cells were stained with anti-vimentin antibody (green) and propidium iodide (red). The percentage of vimentin IF-bridge or multinucleate cells were scored as described (Yasui et al., 2001). Data represents means ± SEM in at least three independent experiments. (C) Blue, green, yellow, or red color represents Plk1, Cdk1, cyclin B, and phosphates within residues on vimentin, respectively. The red arrow indicates phosphorylation reaction.

Mentions: We constructed a mutant vimentin in which Ser82 is changed to Glu (S82E), which is assumed to mimic Ser82 phosphorylation. Exogenous WT expression induced the formation of a filamentous IF network in T24 cells (expressing no endogenous vimentin), similar to the network observed in cells expressing endogenous vimentin (Fig. 5 A). On the other hand, S82E mutant no longer formed a clear filamentous IF network: instead, the dot-like IF structures and short filaments were distributed diffusely in the cytoplasm. Together with in vitro data (Fig. 3 D), these results indicated that Plk1, as well as Aurora-B and Rho-kinase, may participate in the reorganization of vimentin filaments through vimentin phosphorylation.


Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis.

Yamaguchi T, Goto H, Yokoyama T, Silljé H, Hanisch A, Uldschmid A, Takai Y, Oguri T, Nigg EA, Inagaki M - J. Cell Biol. (2005)

The significance of Plk1-induced vimentin phosphorylation on vimentin filament segregation. (A) T24 cells were transfected with pDR2 carrying vimentin WT or S82E. 48 h after transfection, cells were stained with anti-vimentin antibody (green) and propidium iodide (red). Magnified images were also indicated in the right panel. Cells were analyzed by laser-scanning confocal microscopy. (B) Vimentin IF bridge formation and multinucleate cell in T24 cells expressing vimentin mutants (right). Cells were stained with anti-vimentin antibody (green) and propidium iodide (red). The percentage of vimentin IF-bridge or multinucleate cells were scored as described (Yasui et al., 2001). Data represents means ± SEM in at least three independent experiments. (C) Blue, green, yellow, or red color represents Plk1, Cdk1, cyclin B, and phosphates within residues on vimentin, respectively. The red arrow indicates phosphorylation reaction.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171270&req=5

fig5: The significance of Plk1-induced vimentin phosphorylation on vimentin filament segregation. (A) T24 cells were transfected with pDR2 carrying vimentin WT or S82E. 48 h after transfection, cells were stained with anti-vimentin antibody (green) and propidium iodide (red). Magnified images were also indicated in the right panel. Cells were analyzed by laser-scanning confocal microscopy. (B) Vimentin IF bridge formation and multinucleate cell in T24 cells expressing vimentin mutants (right). Cells were stained with anti-vimentin antibody (green) and propidium iodide (red). The percentage of vimentin IF-bridge or multinucleate cells were scored as described (Yasui et al., 2001). Data represents means ± SEM in at least three independent experiments. (C) Blue, green, yellow, or red color represents Plk1, Cdk1, cyclin B, and phosphates within residues on vimentin, respectively. The red arrow indicates phosphorylation reaction.
Mentions: We constructed a mutant vimentin in which Ser82 is changed to Glu (S82E), which is assumed to mimic Ser82 phosphorylation. Exogenous WT expression induced the formation of a filamentous IF network in T24 cells (expressing no endogenous vimentin), similar to the network observed in cells expressing endogenous vimentin (Fig. 5 A). On the other hand, S82E mutant no longer formed a clear filamentous IF network: instead, the dot-like IF structures and short filaments were distributed diffusely in the cytoplasm. Together with in vitro data (Fig. 3 D), these results indicated that Plk1, as well as Aurora-B and Rho-kinase, may participate in the reorganization of vimentin filaments through vimentin phosphorylation.

Bottom Line: This elevation followed the Cdk1-induced vimentin-Ser55 phosphorylation, and was impaired by Plk1 depletion.Mutational analyses revealed that Plk1-induced vimentin-Ser82 phosphorylation plays an important role in vimentin filaments segregation, coordinately with Rho-kinase and Aurora-B.Taken together, these results indicated a novel mechanism that Cdk1 regulated mitotic vimentin phosphorylation via not only a direct enzyme reaction but also Plk1 recruitment to vimentin.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Aichi Cancer Center Research Institute, Aichi 464-8681, Japan.

ABSTRACT
Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its significance has remained unknown. Here we demonstrated a direct interaction between Plk1 and vimentin-Ser55 phosphorylated by Cdk1, an event that led to Plk1 activation and further vimentin phosphorylation. Plk1 phosphorylated vimentin at approximately 1 mol phosphate/mol substrate, which partly inhibited its filament forming ability, in vitro. Plk1 induced the phosphorylation of vimentin-Ser82, which was elevated from metaphase and maintained until the end of mitosis. This elevation followed the Cdk1-induced vimentin-Ser55 phosphorylation, and was impaired by Plk1 depletion. Mutational analyses revealed that Plk1-induced vimentin-Ser82 phosphorylation plays an important role in vimentin filaments segregation, coordinately with Rho-kinase and Aurora-B. Taken together, these results indicated a novel mechanism that Cdk1 regulated mitotic vimentin phosphorylation via not only a direct enzyme reaction but also Plk1 recruitment to vimentin.

Show MeSH
Related in: MedlinePlus