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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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Plk1 phosphorylates vimentin at Ser82. (A) Vimentin was incubated with GST-Plk1-WT or -K82R (a kinase dead mutant). The incorporation of radioactivity into vimentin was analyzed by autoradiography and the total amount of vimentin was determined by CBB. Each sample was also subjected to immunoblotting with each site- and phosphorylation state–specific antibody. The following were used as a positive control for each antibody (phosphorylated vimentin, PV): vimentin phosphorylated by protein kinase A (PKA) for MO6 (P-Ser6) and TM28 (P-Ser28), by PKC for YT33 (P-Ser33) and TM50 (P-Ser50), by Ca2+/calmodulin-dependent protein kinase II for TM38 (P-Ser38) and MO82 (P-Ser82), by Cdk1 for 4A4 (P-Ser55), by Rho-kinase for TM71 (P-Ser71), and by Aurora-B for YG72 (P-Ser72), respectively. As a negative control, nonphosphorylated vimentin (V) was also immunoblotted. (B) COS-7 cells were transfected with myc-Plk1-T210D or -K82R. Green, red, or blue color represents vimentin-phosphoSer82 (MO82), the expression of Plk1 mutants (Myc), or DNA, respectively. (C) Time course of vimentin phosphorylation by Plk1. (D) Vimentin was preincubated with or without Plk1, and then incubated in the polymerization buffer (Goto et al., 1998) at 37°C for 60 min further. After incubation, each sample was centrifuged at 12,000 g. The supernatant (s) and the precipitate (p) fractions were analyzed using SDS-PAGE, then, the gel was subjected to staining with CBB or the immunoblotting with MO82 (P-Ser82). (E) The sequence of residues 76–85 on vimentin and of various vimentin mutant peptides. (F) Plk1 activity toward each vimentin peptide indicated in E was quantified as a percentage of the radioactivity of each peptide relative to that of WT peptide.
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fig3: Plk1 phosphorylates vimentin at Ser82. (A) Vimentin was incubated with GST-Plk1-WT or -K82R (a kinase dead mutant). The incorporation of radioactivity into vimentin was analyzed by autoradiography and the total amount of vimentin was determined by CBB. Each sample was also subjected to immunoblotting with each site- and phosphorylation state–specific antibody. The following were used as a positive control for each antibody (phosphorylated vimentin, PV): vimentin phosphorylated by protein kinase A (PKA) for MO6 (P-Ser6) and TM28 (P-Ser28), by PKC for YT33 (P-Ser33) and TM50 (P-Ser50), by Ca2+/calmodulin-dependent protein kinase II for TM38 (P-Ser38) and MO82 (P-Ser82), by Cdk1 for 4A4 (P-Ser55), by Rho-kinase for TM71 (P-Ser71), and by Aurora-B for YG72 (P-Ser72), respectively. As a negative control, nonphosphorylated vimentin (V) was also immunoblotted. (B) COS-7 cells were transfected with myc-Plk1-T210D or -K82R. Green, red, or blue color represents vimentin-phosphoSer82 (MO82), the expression of Plk1 mutants (Myc), or DNA, respectively. (C) Time course of vimentin phosphorylation by Plk1. (D) Vimentin was preincubated with or without Plk1, and then incubated in the polymerization buffer (Goto et al., 1998) at 37°C for 60 min further. After incubation, each sample was centrifuged at 12,000 g. The supernatant (s) and the precipitate (p) fractions were analyzed using SDS-PAGE, then, the gel was subjected to staining with CBB or the immunoblotting with MO82 (P-Ser82). (E) The sequence of residues 76–85 on vimentin and of various vimentin mutant peptides. (F) Plk1 activity toward each vimentin peptide indicated in E was quantified as a percentage of the radioactivity of each peptide relative to that of WT peptide.

Mentions: We next examined whether vimentin is a good substrate for Plk1. Vimentin was phosphorylated by GST-Plk1-WT, but not by -K82R (Fig. 3 A): the phosphorylation level increased in a time-dependent manner and reached ∼1 mol of phosphate/mol of protein at 120 min (Fig. 3 C). Immunological analyses revealed that Plk1 phosphorylated Ser82, but not Ser6, Ser28, Ser33, Ser38, Ser50, Ser55, Ser71, and Ser72 on vimentin in vitro (Fig. 3 A). In addition, Ser82 phosphorylation was observed in COS-7 cells expressing Plk1 T210D (a constitutively active mutant) but not K82R (Fig. 3 B). These observations suggested that Plk1 induced vimentin phosphorylation at Ser82 likely through a direct enzyme–substrate reaction.


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)

Plk1 phosphorylates vimentin at Ser82. (A) Vimentin was incubated with GST-Plk1-WT or -K82R (a kinase dead mutant). The incorporation of radioactivity into vimentin was analyzed by autoradiography and the total amount of vimentin was determined by CBB. Each sample was also subjected to immunoblotting with each site- and phosphorylation state–specific antibody. The following were used as a positive control for each antibody (phosphorylated vimentin, PV): vimentin phosphorylated by protein kinase A (PKA) for MO6 (P-Ser6) and TM28 (P-Ser28), by PKC for YT33 (P-Ser33) and TM50 (P-Ser50), by Ca2+/calmodulin-dependent protein kinase II for TM38 (P-Ser38) and MO82 (P-Ser82), by Cdk1 for 4A4 (P-Ser55), by Rho-kinase for TM71 (P-Ser71), and by Aurora-B for YG72 (P-Ser72), respectively. As a negative control, nonphosphorylated vimentin (V) was also immunoblotted. (B) COS-7 cells were transfected with myc-Plk1-T210D or -K82R. Green, red, or blue color represents vimentin-phosphoSer82 (MO82), the expression of Plk1 mutants (Myc), or DNA, respectively. (C) Time course of vimentin phosphorylation by Plk1. (D) Vimentin was preincubated with or without Plk1, and then incubated in the polymerization buffer (Goto et al., 1998) at 37°C for 60 min further. After incubation, each sample was centrifuged at 12,000 g. The supernatant (s) and the precipitate (p) fractions were analyzed using SDS-PAGE, then, the gel was subjected to staining with CBB or the immunoblotting with MO82 (P-Ser82). (E) The sequence of residues 76–85 on vimentin and of various vimentin mutant peptides. (F) Plk1 activity toward each vimentin peptide indicated in E was quantified as a percentage of the radioactivity of each peptide relative to that of WT peptide.
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Related In: Results  -  Collection

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

fig3: Plk1 phosphorylates vimentin at Ser82. (A) Vimentin was incubated with GST-Plk1-WT or -K82R (a kinase dead mutant). The incorporation of radioactivity into vimentin was analyzed by autoradiography and the total amount of vimentin was determined by CBB. Each sample was also subjected to immunoblotting with each site- and phosphorylation state–specific antibody. The following were used as a positive control for each antibody (phosphorylated vimentin, PV): vimentin phosphorylated by protein kinase A (PKA) for MO6 (P-Ser6) and TM28 (P-Ser28), by PKC for YT33 (P-Ser33) and TM50 (P-Ser50), by Ca2+/calmodulin-dependent protein kinase II for TM38 (P-Ser38) and MO82 (P-Ser82), by Cdk1 for 4A4 (P-Ser55), by Rho-kinase for TM71 (P-Ser71), and by Aurora-B for YG72 (P-Ser72), respectively. As a negative control, nonphosphorylated vimentin (V) was also immunoblotted. (B) COS-7 cells were transfected with myc-Plk1-T210D or -K82R. Green, red, or blue color represents vimentin-phosphoSer82 (MO82), the expression of Plk1 mutants (Myc), or DNA, respectively. (C) Time course of vimentin phosphorylation by Plk1. (D) Vimentin was preincubated with or without Plk1, and then incubated in the polymerization buffer (Goto et al., 1998) at 37°C for 60 min further. After incubation, each sample was centrifuged at 12,000 g. The supernatant (s) and the precipitate (p) fractions were analyzed using SDS-PAGE, then, the gel was subjected to staining with CBB or the immunoblotting with MO82 (P-Ser82). (E) The sequence of residues 76–85 on vimentin and of various vimentin mutant peptides. (F) Plk1 activity toward each vimentin peptide indicated in E was quantified as a percentage of the radioactivity of each peptide relative to that of WT peptide.
Mentions: We next examined whether vimentin is a good substrate for Plk1. Vimentin was phosphorylated by GST-Plk1-WT, but not by -K82R (Fig. 3 A): the phosphorylation level increased in a time-dependent manner and reached ∼1 mol of phosphate/mol of protein at 120 min (Fig. 3 C). Immunological analyses revealed that Plk1 phosphorylated Ser82, but not Ser6, Ser28, Ser33, Ser38, Ser50, Ser55, Ser71, and Ser72 on vimentin in vitro (Fig. 3 A). In addition, Ser82 phosphorylation was observed in COS-7 cells expressing Plk1 T210D (a constitutively active mutant) but not K82R (Fig. 3 B). These observations suggested that Plk1 induced vimentin phosphorylation at Ser82 likely through a direct enzyme–substrate reaction.

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