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. 2005 Nov 7;171(3):431-6.
doi: 10.1083/jcb.200504091. Epub 2005 Oct 31.

Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis

Tomoya Yamaguchi  1 , Hidemasa Goto, Tomoya Yokoyama, Herman Silljé, Anja Hanisch, Andreas Uldschmid, Yasushi Takai, Takashi Oguri, Erich A Nigg, Masaki Inagaki
Affiliations

Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis

Tomoya Yamaguchi et al. J Cell Biol. .

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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Figures

Figure 1.
Figure 1.
Plk1 binds to vimentin phosphorylated by Cdk1. (A) Site-specific phosphorylation of vimentin during metaphase/anaphase in U251 cells. (B) Metaphase or postmitotic T24 cells expressing vimentin WT or vimentin mutated at Cdk1 (mutant I: Ser41 and Ser55) or Rho-kinase + Aurora-B (mutant II: Ser6, Ser24, Ser38, Ser46, Ser64, Ser65, Ser71, Ser72, and Ser86) phosphorylation sites (Yasui et al., 2001). Green color represents the staining with anti-vimentin (A and B) or the site- and phosphorylation state–specific antibody for Ser55, Ser71, or Ser72 on vimentin (A). DNA was also stained with propidium iodide (red). (C) Specific interaction of Plk1 with vimentin phosphorylated by Cdk1. Vimentin was incubated with (Cdk1) or without (Control) Cdk1. Each sample was then subjected to immunoblotting with 4A4 (P-ser55) or Far Western blotting using GST-PBD. (D) Site specificity of plk1 determined by competition assay. After Far Western blotting (C and D), transferred membrane was stained with Coomassie brilliant blue (CBB). (E) GST pull-down assay using GST-PBD. Each sample was prepared as described in Materials and methods, and then subjected to immunoblotting with 4A4 (P-Ser55) and anti-vimentin antibody.
Figure 2.
Figure 2.
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.
Figure 3.
Figure 3.
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.
Figure 4.
Figure 4.
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.
Figure 5.
Figure 5.
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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