Limits...
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.

Show MeSH

Related in: MedlinePlus

Plk1 depletion impaired the elevation of vimentin-Ser82 phosphorylation in mitosis. (A) Cell cycle–dependent phosphorylation of vimentin-Ser55 and vimentin-Ser82 during mitosis in U251 cells. Green color represents vimentin phosphorylated at Ser55 (4A4) or at Ser82 (MO82). DNA was stained with propidium iodide (red). (B) Interphase (I) or early mitotic (M) U251 cell lysates were subjected to immunoblotting with 4A4 or MO82 antibody. (C) T24 cells were transfected with pDR2 carrying vimentin WT or S55A. 48 h after transfection, cells were stained with anti-vimentin antibody (green) and MO82 (red) and DAPI (blue). (D) 44 h transfection, cells were synchronized at the prometaphase by the addition of nocodazole. After 4 h (48 h after transfection), interphase or early mitotic cell lysate were subjected to immunoblotting analysis with MO82 or anti-vimentin antibody. (E) 48 h after transfection with or without Plk1 siRNA duplex, U251 cells were stained with anti-Plk1, MO82, 4A4, or anti-vimentin antibody (green), and DAPI (blue). (F) 36 h transfection, cells were synchronized at the prometaphase by the addition of nocodazole. After 12 h (48 h after transfection), interphase or early mitotic cell lysate were subjected to immunoblotting analysis with anti-Plk1, MO82, 4A4, or anti-vimentin antibody.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171270&req=5

fig4: Plk1 depletion impaired the elevation of vimentin-Ser82 phosphorylation in mitosis. (A) Cell cycle–dependent phosphorylation of vimentin-Ser55 and vimentin-Ser82 during mitosis in U251 cells. Green color represents vimentin phosphorylated at Ser55 (4A4) or at Ser82 (MO82). DNA was stained with propidium iodide (red). (B) Interphase (I) or early mitotic (M) U251 cell lysates were subjected to immunoblotting with 4A4 or MO82 antibody. (C) T24 cells were transfected with pDR2 carrying vimentin WT or S55A. 48 h after transfection, cells were stained with anti-vimentin antibody (green) and MO82 (red) and DAPI (blue). (D) 44 h transfection, cells were synchronized at the prometaphase by the addition of nocodazole. After 4 h (48 h after transfection), interphase or early mitotic cell lysate were subjected to immunoblotting analysis with MO82 or anti-vimentin antibody. (E) 48 h after transfection with or without Plk1 siRNA duplex, U251 cells were stained with anti-Plk1, MO82, 4A4, or anti-vimentin antibody (green), and DAPI (blue). (F) 36 h transfection, cells were synchronized at the prometaphase by the addition of nocodazole. After 12 h (48 h after transfection), interphase or early mitotic cell lysate were subjected to immunoblotting analysis with anti-Plk1, MO82, 4A4, or anti-vimentin antibody.

Mentions: We investigated the temporal and spatial distribution of vimentin-Ser55 and vimentin-Ser82 phosphorylation at each stage of mitosis. As shown in Fig. 4, A and B, Ser55 phosphorylation occurred from prometaphase to metaphase, as reported previously (Tsujimura et al., 1994). Although Ser82 phosphorylation was weakly observed even in interphase, the elevation of Ser82 phosphorylation occurred from metaphase (from prometaphase in some cells) and was maintained until the end of mitosis. These results indicated that the elevation of vimentin-Ser82 phosphorylation occurred just after the initiation of vimentin-Ser55 phosphorylation by Cdk1.


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 depletion impaired the elevation of vimentin-Ser82 phosphorylation in mitosis. (A) Cell cycle–dependent phosphorylation of vimentin-Ser55 and vimentin-Ser82 during mitosis in U251 cells. Green color represents vimentin phosphorylated at Ser55 (4A4) or at Ser82 (MO82). DNA was stained with propidium iodide (red). (B) Interphase (I) or early mitotic (M) U251 cell lysates were subjected to immunoblotting with 4A4 or MO82 antibody. (C) T24 cells were transfected with pDR2 carrying vimentin WT or S55A. 48 h after transfection, cells were stained with anti-vimentin antibody (green) and MO82 (red) and DAPI (blue). (D) 44 h transfection, cells were synchronized at the prometaphase by the addition of nocodazole. After 4 h (48 h after transfection), interphase or early mitotic cell lysate were subjected to immunoblotting analysis with MO82 or anti-vimentin antibody. (E) 48 h after transfection with or without Plk1 siRNA duplex, U251 cells were stained with anti-Plk1, MO82, 4A4, or anti-vimentin antibody (green), and DAPI (blue). (F) 36 h transfection, cells were synchronized at the prometaphase by the addition of nocodazole. After 12 h (48 h after transfection), interphase or early mitotic cell lysate were subjected to immunoblotting analysis with anti-Plk1, MO82, 4A4, or anti-vimentin antibody.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Plk1 depletion impaired the elevation of vimentin-Ser82 phosphorylation in mitosis. (A) Cell cycle–dependent phosphorylation of vimentin-Ser55 and vimentin-Ser82 during mitosis in U251 cells. Green color represents vimentin phosphorylated at Ser55 (4A4) or at Ser82 (MO82). DNA was stained with propidium iodide (red). (B) Interphase (I) or early mitotic (M) U251 cell lysates were subjected to immunoblotting with 4A4 or MO82 antibody. (C) T24 cells were transfected with pDR2 carrying vimentin WT or S55A. 48 h after transfection, cells were stained with anti-vimentin antibody (green) and MO82 (red) and DAPI (blue). (D) 44 h transfection, cells were synchronized at the prometaphase by the addition of nocodazole. After 4 h (48 h after transfection), interphase or early mitotic cell lysate were subjected to immunoblotting analysis with MO82 or anti-vimentin antibody. (E) 48 h after transfection with or without Plk1 siRNA duplex, U251 cells were stained with anti-Plk1, MO82, 4A4, or anti-vimentin antibody (green), and DAPI (blue). (F) 36 h transfection, cells were synchronized at the prometaphase by the addition of nocodazole. After 12 h (48 h after transfection), interphase or early mitotic cell lysate were subjected to immunoblotting analysis with anti-Plk1, MO82, 4A4, or anti-vimentin antibody.
Mentions: We investigated the temporal and spatial distribution of vimentin-Ser55 and vimentin-Ser82 phosphorylation at each stage of mitosis. As shown in Fig. 4, A and B, Ser55 phosphorylation occurred from prometaphase to metaphase, as reported previously (Tsujimura et al., 1994). Although Ser82 phosphorylation was weakly observed even in interphase, the elevation of Ser82 phosphorylation occurred from metaphase (from prometaphase in some cells) and was maintained until the end of mitosis. These results indicated that the elevation of vimentin-Ser82 phosphorylation occurred just after the initiation of vimentin-Ser55 phosphorylation by Cdk1.

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