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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 increases its catalytic activity via its binding to vimentin phosphorylated at Ser55 by Cdk1. (A) After GST-Plk1-WT was preincubated with PV55, PV41, or V55 (Tsujimura et al., 1994) for 5 min at RT, casein was phosphorylated by above Plk1. The SDS-PAGE gel was stained with CBB and then subjected to autoradiography (32P). (B) Time course of Plk1 kinase activity with or without PV55. (C) After vimentin was preincubated with or without Cdk1, each sample was further incubated with or without Plk1. The total amount of vimentin was determined by CBB. The phosphorylation of vimentin at Ser55 was detected by immunoblotting using 4A4. The incorporation of radioactivity was analyzed by autoradiography. (D) Quantification of Plk1 kinase activity indicated in C.
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fig2: Plk1 increases its catalytic activity via its binding to vimentin phosphorylated at Ser55 by Cdk1. (A) After GST-Plk1-WT was preincubated with PV55, PV41, or V55 (Tsujimura et al., 1994) for 5 min at RT, casein was phosphorylated by above Plk1. The SDS-PAGE gel was stained with CBB and then subjected to autoradiography (32P). (B) Time course of Plk1 kinase activity with or without PV55. (C) After vimentin was preincubated with or without Cdk1, each sample was further incubated with or without Plk1. The total amount of vimentin was determined by CBB. The phosphorylation of vimentin at Ser55 was detected by immunoblotting using 4A4. The incorporation of radioactivity was analyzed by autoradiography. (D) Quantification of Plk1 kinase activity indicated in C.

Mentions: The above specific binding led us to examine its effect on the catalytic activity of Plk1. First, we used casein as a control substrate. As shown in Fig. 2, A and B, the preincubation of Plk1 with PV55 increased its kinase activity toward casein about three times over that without peptide. However, only marginal stimulation was observed in the case of V55 or PV41, confirming the peptide specificity of the Plk1 activation (Fig. 2 A).


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 increases its catalytic activity via its binding to vimentin phosphorylated at Ser55 by Cdk1. (A) After GST-Plk1-WT was preincubated with PV55, PV41, or V55 (Tsujimura et al., 1994) for 5 min at RT, casein was phosphorylated by above Plk1. The SDS-PAGE gel was stained with CBB and then subjected to autoradiography (32P). (B) Time course of Plk1 kinase activity with or without PV55. (C) After vimentin was preincubated with or without Cdk1, each sample was further incubated with or without Plk1. The total amount of vimentin was determined by CBB. The phosphorylation of vimentin at Ser55 was detected by immunoblotting using 4A4. The incorporation of radioactivity was analyzed by autoradiography. (D) Quantification of Plk1 kinase activity indicated in C.
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Related In: Results  -  Collection

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

fig2: Plk1 increases its catalytic activity via its binding to vimentin phosphorylated at Ser55 by Cdk1. (A) After GST-Plk1-WT was preincubated with PV55, PV41, or V55 (Tsujimura et al., 1994) for 5 min at RT, casein was phosphorylated by above Plk1. The SDS-PAGE gel was stained with CBB and then subjected to autoradiography (32P). (B) Time course of Plk1 kinase activity with or without PV55. (C) After vimentin was preincubated with or without Cdk1, each sample was further incubated with or without Plk1. The total amount of vimentin was determined by CBB. The phosphorylation of vimentin at Ser55 was detected by immunoblotting using 4A4. The incorporation of radioactivity was analyzed by autoradiography. (D) Quantification of Plk1 kinase activity indicated in C.
Mentions: The above specific binding led us to examine its effect on the catalytic activity of Plk1. First, we used casein as a control substrate. As shown in Fig. 2, A and B, the preincubation of Plk1 with PV55 increased its kinase activity toward casein about three times over that without peptide. However, only marginal stimulation was observed in the case of V55 or PV41, confirming the peptide specificity of the Plk1 activation (Fig. 2 A).

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