Aurora-B Regulates the Cleavage Furrow-specific Vimentin Phosphorylation in the Cytokinetic Process*

Hidemasa GotoDagger §, Yoshihiro YasuiDagger §, Aie KawajiriDagger §, Erich A. Nigg||, Yasuhiko Terada**, Masaaki TatsukaDagger Dagger , Koh-ichi NagataDagger , and Masaki InagakiDagger §§

From the Dagger  Division of Biochemistry, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Aichi 464-8681, Japan, the  Department of Pathology, Nagoya University School of Medicine, Nagoya, Aichi 466-8550, Japan, || Max-Planck Institute for Biochemistry, Department of Cell Biology, D-82152 Martinsried, Germany, the ** Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55455, and Dagger Dagger  Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan

Received for publication, October 24, 2002

    ABSTRACT
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Aurora-B is an evolutionally conserved protein kinase that regulates several mitotic events including cytokinesis. We previously demonstrated the possible existence of a protein kinase that phosphorylates at least Ser-72 on vimentin, the most widely expressed intermediate filament protein, in the cleavage furrow-specific manner. Here we showed that vimentin-Ser-72 phosphorylation occurred specifically at the border of the Aurora-B-localized area from anaphase to telophase. Expression of a dominant-negative mutant of Aurora-B led to a reduction of this vimentin-Ser-72 phosphorylation. In vitro analyses revealed that Aurora-B phosphorylates vimentin at ~2 mol phosphate/mol of substrate for 30 min and that this phosphorylation dramatically inhibits vimentin filament formation. We further identified eight Aurora-B phosphorylation sites, including Ser-72 on vimentin, and then constructed the mutant vimentin in which these identified sites are changed into Ala. Cells expressing this mutant formed an unusually long bridge-like intermediate filament structure between unseparated daughter cells. We then identified important phosphorylation sites for the bridge phenotype. Our findings indicate that Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation and controls vimentin filament segregation in cytokinetic process.

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Intermediate filaments (IFs),1 together with microtubules and actin filaments, form the cytoskeletal framework in the cytoplasm of various eukaryotic cells and are also present in nuclei as the major component of nuclear lamina. Unlike microtubules and actin filaments, the protein components of IFs vary in a cell-, tissue-, and differentiation-dependent manner. For example, glial fibrillary acidic protein (GFAP) and desmin, type III IF proteins, are expressed specifically in astroglial and muscular cells, respectively. On the other hand, vimentin, one of other type III IF proteins, is expressed in mesenchymal cells, in most types of cultured and tumor cells, and transiently in many cells during the early stages of development (1-3).

IFs are known to be dramatically reorganized during mitosis (4-6). This mitotic IF reorganization is known to be accompanied with increase in IF phosphorylation (7, 8). In vitro studies showed that the phosphorylation of IF proteins by various types of serine/threonine protein kinases induces disassembly of the filament structure (9). Using GFAP or vimentin mutated at mitosis-specific phosphorylation sites, we previously demonstrated that mitotic IF protein phosphorylation is required for proper IF segregation (10, 11). In the case of vimentin, at least three protein kinases are likely to participate in this process (11). The most likely candidate kinases are Rho-kinase, protein kinase C, and a protein kinase that phosphorylates at least Ser-72 on vimentin in the cleavage furrow-specific manner (11). However, little is known about the molecular identity and regulation of this vimentin-Ser-72 kinase.

Drosophila Aurora- and Saccharomyces cerevisiae Ipl1p-like protein kinases form a conserved family of enzymes from the budding yeast to mammals (12-14). Although the yeast genome encodes only one kinase, mammals have at least three subfamilies of Aurora/Ipl1p-related kinases (12-14). Among these kinases, Aurora-B (also called Aurora 1 or AIM-1) is associated with chromosomes from prophase to metaphase and with spindle midzone/midbody during the later mitotic phase (15, 16). Recent studies demonstrated that Aurora-B is involved in chromosome condensation/segregation and cytokinesis during mitotic progression (15-19). In the earlier mitotic phase (from prophase to metaphase), Aurora-B was reported to phosphorylate histone H3 (17-21) and CENP-A (22). H3 phosphorylation is thought to be required for correct chromosome condensation and subsequently chromosome segregation (17-19,23). However, much less is known about target substrates of Aurora-B in the cytokinetic process.

In this report, we show that the intracellular distribution of Aurora-B overlapped with that of vimentin phosphorylated at Ser-72. Inhibition of endogenous Aurora-B by the expression of its dominant-negative mutant reduced the vimentin-Ser-72 phosphorylation at undetectable levels. By identifying eight Aurora-B phosphorylation sites on vimentin in vitro, we have demonstrated that vimentin-Ser-72 is an in vitro phosphorylation site of Aurora-B. Mutations at Aurora-B phosphorylation sites on vimentin induced the impaired segregation of vimentin filaments in postmitotic cells. We then determined that Ser-72 is one of the important phosphorylation sites responsible for the vimentin IF-bridge phenotype formation.

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Materials-- Recombinant mouse vimentin (24) and GST-Aurora-B (20) were prepared from Escherichia coli. Anti-phosphoserine72 on vimentin was purified from the rabbit sera (11). Anti-Aurora-B or anti-Myc mouse monoclonal antibody was purchased from Transduction Laboratories (AIM-1) or Berkeley Antibody (9E10), respectively. The pcDNA3-myc vector (Invitrogen) carrying wild type (WT) or a kinase-inactive mutant (K/R; Ref. 15) of Aurora-B cDNA was prepared as described previously (20). The pDR2 (Clontech) vectors carrying several types of vimentin mutants were produced as described (11).

Phosphorylation of Vimentin-- The phosphorylation reaction for vimentin was performed at 25 °C in 100 µl of 25 mM Tris-Cl (pH 7.5), 2 mM MgCl2, 100 µM ATP, 0.1 µM calyculin A, and 150 µg/ml vimentin in the presence of either 10 µg/ml GST-Aurora-B WT or K/R, with or without [gamma -32P]ATP. The reaction was stopped by the addition of Laemmli's sample buffer and boiled.

Fragmentation of Phosphorylated Vimentin-- Vimentin (150 µg) was phosphorylated by GST-Aurora-B WT (10 µg) at 25 °C for 30 min in the reaction mixture (1 ml) as described above. The radioactive vimentin was fragmented by Lysyl-endopeptidase (Wako Pure Chemical Inc., Osaka, Japan) and then fractionated by reverse-phase HPLC on a Zorbax C8 column as described (25). All radioactivity loaded was recovered in a single peptide (~12-kDa polypeptides containing mainly the amino-terminal head domain; see also Fig. 3A). This phosphorylated vimentin fragment was treated with L-1-tosylamide-2-phenyl-ethyl chloromethyl ketone-treated trypsin (Sigma) and then refractionated by reverse-phase HPLC (25).

Determination of Phosphorylation Site(s) on Each Peptide-- Amino acid sequences of each phosphopeptide were analyzed using an ABI 476A gas-phase sequencer. Residue numbers of each phosphopeptide were determined by comparison with the reported sequence of mouse vimentin (26). Phosphorylated amino acid(s) were analyzed by phosphoamino acid analysis (27). The phosphorylation site(s) in each phosphopeptide were determined by detecting the inorganic phosphate (PI) after gas-phase sequencing (28). In brief, after the sequencing reached cycles corresponding to the phosphorylated residue, the inorganic phosphate (PI) and phosphopeptide were eluted from the membrane and then separated by electrophoresis at pH 3.5 on a cellulose thin-layer plate.

Transfection-- Using LipofectAMINE (Invitrogen) according to the manufacturer's protocol, HeLa cells or EBNA-expressing T24 cells (10) were transiently transfected with a pcDNA3-myc-Aurora-B (WT or K/R) or a pDR2 vector carrying each mutant vimentin, respectively.

    RESULTS AND DISCUSSION
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We previously demonstrated that expression of vimentin or GFAP in which putative phosphorylation sites are changed into Ala induced the formation of a long bridge-like IF structure (referred to as IF-bridge) between unseparated daughter cells (10, 11). This phenotype was induced by the site-specific mutation in the amino-terminal head domain of each IF protein. In the case of vimentin, mutation at Rho-kinase and protein kinase C phosphorylation sites and at Ser-72 is a prerequisite for a high percentage of postmitotic cells to show the IF-bridge phenotype (Fig. 1A; see also Ref. 11). Since few cells formed IF-bridge in postmitotic cells transfected with vimentin mutated at Rho-kinase and protein kinase C phosphorylation sites, vimentin-Ser-72 is one of the most essential mutation sites (Fig. 1A; see also Ref. 11). This site is phosphorylated specifically at the cleavage furrow from anaphase to cytokinesis (11). All these observations suggest that there may exist a protein kinase that by itself can induce vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation at least at Ser-72. However, this vimentin-Ser-72 kinase had not been identified previously.


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Fig. 1.   Regulation of the cleavage furrow-specific vimentin-Ser-72 phosphorylation by Aurora-B. As shown in A, vimentin with (+) or without (-) Ser-72 mutation in addition to mutations of sites phosphorylated by Rho-kinase and protein kinase C (Ser to Ala) was expressed in T24 cells, as described (11). Nuclei and vimentin were stained with propidium iodide (red) and anti-vimentin (green), respectively (11). B and C, spatio-temporal distribution of vimentin-Ser-72 phosphorylation (Vimentin-P-Ser-72; green) and Aurora-B (red) in Swiss 3T3 cells by immunofluorescence. Cells growing on type I collagen-coated glass coverslips were fixed with 3.7% formaldehyde in phosphate-buffered saline for 10 min at 37 °C and then permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 10 min at room temperature. Confocal images of anaphase, telophase, and cytokinetic cells represent single horizontal sections (B). Confocal images on four serial focal planes of an anaphase cell were obtained at 2-µm intervals (C). D, phosphorylation of vimentin-Ser-72 in HeLa cells expressing WT or K/R of Myc-tagged Aurora-B. 24-48 h after transfection, cells were doubly stained with anti-phosphoserine72 on vimentin (Vimentin-P-Ser-72; green) and anti-Myc (red), and then DNAs were stained with 0.5 µg/ml 4'6-diamidine-2-phenylindole-dihydrochloride (DAPI) (blue). Fluorescently labeled cells were examined using an Olympus BH2-RFCA microscope. Bars, 10 µm.

Since Aurora-B is involved in cytokinesis (15, 16), we compared the subcellular distribution of Aurora-B with that of vimentin-Ser-72 phosphorylation. Vimentin phosphorylated at Ser-72 is associated with the cleavage furrow to form a ring-like structure, and it is distributed very close to the border of the Aurora-B-containing area from anaphase to telophase (Fig. 1, B and C). During cytokinesis, this phosphorylated vimentin is separated into two populations at the midzone (Fig. 1B, arrow). To determine whether Aurora-B is involved in vimentin-Ser-72 phosphorylation, we transfected cells with pcDNA3-myc-Aurora-B (K/R), a construct with a mutation at the ATP-binding site (15, 20). This mutant is known to disperse endogenous Aurora-B in the spindle midzone and then to block its function in a dominant-negative manner (15, 29). In Aurora-B (K/R)-expressing cells, we often observe abnormal mitoses, such as abnormal chromosome segregation including a chromatin bridge (Fig. 1D). Similar observations were noted in studies done to deplete Aurora-B using RNA-mediated interference in Caenorhabditis elegans and Drosophila (16-19). Vimentin phosphorylation at Ser-72 was not observed in such cells (Fig. 1D), which suggested that vimentin phosphorylation at Ser-72 is governed by Aurora-B.

To determine whether vimentin can serve as an excellent substrate for Aurora-B, we carried out the in vitro studies. Aurora-B can phosphorylate vimentin (Fig. 2A); this phosphorylation level increases in a time-dependent manner and reaches about 2 mol of phosphate/mol of protein at 30 min (Fig. 2B). However, Aurora-B (K/R) cannot induce vimentin phosphorylation (Fig. 2A). Next, we examined the effect of vimentin phosphorylation by Aurora-B on filament-forming ability. Analyses by centrifugation (Fig. 2C) and by electron microscopy (Fig. 2D) revealed that the phosphorylation of vimentin by Aurora-B dramatically inhibits filament formation.


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Fig. 2.   Phosphorylation of vimentin by Aurora-B. As shown in A, vimentin was phosphorylated by K/R (lane 1) or WT (lane 2) of Aurora-B. After SDS-PAGE, the gel was stained with Coomassie Brilliant Blue (CBB) and then subjected to autoradiography (32P). B, time course of vimentin phosphorylation by Aurora-B WT. As shown in C and D, vimentin (Vim.) treated with K/R (lanes 1 and 2) or WT (lanes 3 and 4) of Aurora-B for 60 min was incubated with 150 mM NaCl at 37 °C for further 60 min. After incubation, samples were divided into two. One was subjected to high speed centrifugation (12,000 × g). The supernatant (lanes 1 and 3) and the precipitate (lanes 2 and 4) were subjected to SDS-PAGE and, the gel was stained with Coomassie Brilliant Blue (C). The other was observed using electron microscopy (25) (D). Bar, 200 nm.

To identify the sites on vimentin phosphorylated by Aurora-B, vimentin (150 µg) was phosphorylated by Aurora-B in the presence of [gamma -32P]ATP to about 2.0 mol of phosphate/mol of protein for 30 min, and then this phosphorylated vimentin was digested with a lysine-specific protease. Since vimentin has no lysine residues in the head domain (see Fig. 3D) but many in the rod or tail domain, this procedure generates a 12-kDa fragment consisting of the head domain and much smaller polypeptides (26). SDS-PAGE analysis revealed that almost all radioactivities in the phosphorylated vimentin were recovered in this 12-kDa fragment (Fig. 3A), thus indicating that the phosphorylation sites were restricted to only the head domain of vimentin. This phosphorylated head domain separated by reverse-phase HPLC was treated with trypsin, and the resulting peptides were subjected to reverse-phase HPLC again. Consequently, six radioactive peptides were obtained (Fig. 3B); each amino acid sequence is also shown in Fig. 3D. All these peptides were phosphorylated at serine residues, as determined by phosphoamino acid analysis (Fig. 3C). The sites of phosphorylation in each phosphopeptide (P1-P6) were determined to be Ser-64/Ser-65, Ser-6, Ser-24, Ser-72, Ser-38/Ser-46, and Ser-86, respectively (data not shown; see also Fig. 3D and Ref. 28). These findings indicate that Aurora-B can directly phosphorylate vimentin at Ser-72.


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Fig. 3.   Identification of Aurora-B phosphorylation sites on vimentin (Vim.) in vitro. As shown in A, vimentin phosphorylated by Aurora-B WT was treated with (lane 2) or without (lane 1) lysyl-endopeptidase. After SDS-PAGE, radiolabeled bands were visualized autoradiographically. As shown in B, the radioactive amino-terminal head domain of vimentin (A) was digested with trypsin and fractionated by reverse-phase HPLC, as described (25). The radioactivity of each fraction (0.8 ml) was measured in a 32P Beckman liquid scintillation counter. As shown in C, six radioactive peaks (P1-P6) were subjected to phosphoamino acid analysis. The positions of phosphoserine (P-Ser), phosphothreonine (P-Thr), and phosphotyrosine (P-Tyr) are indicated. D, a map of the vimentin molecule showing phosphorylation sites by Cdc2 kinase, cAMP-dependent protein kinase (PKA), protein kinase C (PKC), p21-activated kinase (PAK), CaM kinase II, Rho-kinase (25, 30, 31), or Aurora-B. Each determined amino acid sequence of P1-P6 (B) is also underlined. The phosphorylation sites are indicated by P within a circle. E, vimentin (a and c), S71E (b), and S72E (e) were phosphorylated by Aurora-B (a and b) or Rho-kinase (c and d), digested with trypsin, fractionated by reverse-phase HPLC, and analyzed as described in B. The arrows show phosphorylated peptides. Determination of phosphorylation sites on each peptide was performed as described under "Experimental Procedures."

Rho-kinase phosphorylates Ser-71, a neighboring site of Ser-72, on vimentin (25). If vimentin is phosphorylated on Ser-71 by Rho-kinase, does Aurora-B still phosphorylate Ser-72 on vimentin? To address this, we made two vimentin mutants, S71E and S72E, in which Ser-71 or Ser-72 is changed to Glu. Since Ser-71 and Ser-72 are specific phosphorylation sites for Rho-kinase and Aurora-B, respectively, S71E and S72E mimic vimentin phosphorylated by either kinase. As shown in Fig. 3E, S71E was phosphorylated in vitro by Aurora-B at Ser-72, and S72E was also phosphorylated by Rho-kinase at Ser-71. These results strongly suggest that when vimentin is phosphorylated at Ser-71 by Rho-kinase or at Ser-72 by Aurora-B, Aurora-B could phosphorylate vimentin at Ser-72, and Rho-kinase also phosphorylates vimentin at Ser-71. These results also suggest the possibility that Ser-71 and Ser-72 could be phosphorylated on the same vimentin molecule in cells undergoing cytokinesis.

cAMP-dependent protein kinase or p21-activated kinase was also reported to phosphorylate vimentin at Ser-72 in vitro (Fig. 3D, see also Refs. 30 and 31). So there is the possibility that other kinases, such as cAMP-dependent protein kinase or p21-activated kinase, may phosphorylate vimentin at Ser-72 in the cleavage furrow-specific manner. Since vimentin is distributed diffusely at the cytoplasm in the outside of mitotic spindle during mitosis (5, 6), localization of vimentin-Ser-72 kinase is likely to be restricted to the cleavage furrow area. However, neither the cAMP-dependent protein kinase active form nor p21-activated kinase was localized at the cleavage furrow area during mitosis; both kinases are diffusely distributed in the cytoplasm during mitosis.2 On the other hand, Aurora-B is localized in the spindle midzone (15, 16), at the border of which vimentin-Ser-72 phosphorylation occurs (Fig. 1, B and C). Expression of a dominant-negative mutant of Aurora-B led to a reduction of this vimentin-Ser-72 phosphorylation (Fig. 1D). Together with in vitro data (Figs. 2 and 3), these observations lead us to the notion that Aurora-B regulates the cleavage furrow-specific vimentin-Ser-72 phosphorylation, likely through the direct enzyme-substrate reaction.

Aurora-B phosphorylates vimentin at Ser-72 but not at Ser-71 (Fig. 3). On the other hand, Rho-kinase phosphorylates vimentin at Ser-71 but not at Ser-72 in vitro (Fig. 3D; see also Ref. 25). Since Rho-kinase accumulates at the cleavage furrow (32), it may be responsible for vimentin-Ser-71 phosphorylation. Rho-kinase also phosphorylates GFAP, an IF protein expressed specifically in astroglial cells, at Thr-7, Ser-13, and Ser-38 in vitro (33). Phosphorylation at these three sites occurs specifically at the cleavage furrow (34). Recently, we have obtained the data that Aurora-B can phosphorylate GFAP at the same three sites.3 Taken together, the cleavage furrow-specific IF phosphorylation may be regulated by both Rho-kinase and Aurora-B.

To analyze the functional significance of the cleavage furrow-specific vimentin phosphorylation, we constructed a set of vimentin mutants in which sites phosphorylated by Rho-kinase, Aurora-B, or both are changed to Ala. When these constructs were transfected in T24 cells, IF-bridge formation was observed in cells expressing the mutants (Fig. 4A and data not shown). As shown in Fig. 4B, the bridge phenotype was frequently observed in cells expressing vimentin with mutations in Aurora-B sites (~30%) as compared with wild type vimentin (0%), whereas mutations in Rho-kinase sites displayed more moderate effects (~2%). Mutations at both Aurora-B and Rho-kinase sites showed synergistic effects (~51%) in the IF-bridge formation (Fig. 4B). The expression of the above vimentin mutants had no effects on T24 cell shape or on the vimentin filament structure in interphase cells (Fig. 4D). These mutational analyses reveal that both Aurora-B and Rho-kinase may play important roles in the correct separation of vimentin filaments.


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Fig. 4.   Effects of mutations in the Aurora-B and/or Rho-kinase phosphorylation sites on the formation of bridge-like structure of vimentin filaments. A, vimentin IF-bridge-like structure in T24 cells expressing vimentin with mutations at Aurora-B- and Rho-kinase-phosphorylation sites. The green color is vimentin immunoreactivity, whereas the red color shows chromosomes (11). Bar, 40 µm. B, quantification of IF-bridge formation induced by vimentin without (WT) or with mutations at phosphorylation sites by Rho-kinase (Rho-K), Aurora-B, or both. 48 h after transfection of the vimentin mutants, the cells were fixed and stained for vimentin and chromosomes. The percentage of vimentin bridge-positive cells during mitosis was scored. Data are means ±S.E. of at least triplicate determinations. At least 200 cells per each sample were counted, and at least three independent experiments were carried out. C, quantification of IF-bridge formation induced by the vimentins mutated at all Aurora-B sites but one. The unaltered residues in respective constructs are indicated below the histogram. 48 h after transfection of the vimentin mutants, the cells were fixed and stained for vimentin and chromosomes. Vimentin IF-bridge-positive cells induced by the vimentin mutants were counted and expressed as relative percentage to the vimentin mutated at all Aurora-B phosphorylation sites (Control). Data are means ±S.E. of at least triplicate determinations. At least 200 cells per each sample were counted, and at least three independent experiments were carried out. D, normal filament formation of vimentin mutants expressed in T24 cells. The same samples as in panel B were immunostained for vimentin mutants expressed in interphase cells. Bar, 40 µm. E, a scheme showing the cleavage furrow-specific IF phosphorylation by Aurora-B or Rho-kinase. MT; microtubules.

In the next set of experiments, we determined the Aurora-B phosphorylation sites important for vimentin IF-bridge formation since the kinase phosphorylates eight different sites. As shown in Fig. 4C, when Ser-6, Ser-24, Ser-38, or Ser-72 was left unaltered, the number of the cells with IF-bridge was considerably decreased as compared with vimentin mutated at all Aurora-B sites. The vimentin mutants without alteration at Ser-46, Ser-65, or Ser-86 showed moderate IF-bridge-forming ability, and conservation of Ser-64 had no effects on the ability. Since Ser-38 is a common phosphorylation site to Aurora-B and Rho-kinase, the IF-bridge-forming ability of the vimentin mutant with unaltered Ser-38 was much less as compared with other mutants tested. Together, Ser-6, Ser-24, Ser-38, Ser-46, Ser-65, Ser-72, and Ser 86 were suggested to be pertinent in vivo phosphorylation sites by Aurora-B and are contributing to IF-bridge formation. To obtain vimentin IF-bridge phenotype efficiently, mutations at many Aurora-B phosphorylation sites are required. Conversely, phosphorylation at only one or two sites out of eight Aurora-B sites is enough for proper vimentin filament segregation in vivo. This hypothesis is well consistent with the in vitro observation that phosphorylation level at 1-2 mol phosphate/mol of vimentin induces efficient vimentin filament disruption.

In the present study, we observed two characteristic phenotypes by blocking Aurora-B function: the chromosome bridge (Fig. 1D) and the IF-bridge (Fig. 4). The former was induced by kinase-negative Aurora-B at the late mitotic phase, and the cell no longer completes cytokinesis. It is notable that, in the experiments using the K/R mutant, cell cycle is arrested at the late mitotic phase, and the possible effects of the mutant on vimentin after cytokinesis cannot be analyzed. To overcome this problem and analyze the function of Aurora-B on vimentin, we used vimentin mutated at Aurora-B phosphorylation sites and detected the IF-bridge after cytokinesis. The functional relationship between the chromatin-bridge and the IF-bridge is an issue for future study.

We propose the following mechanisms regulating the cleavage furrow-specific vimentin phosphorylation (Fig. 4E). Continuous furrow ingression may increase vimentin accessibility not only to Rho-kinase in the plasma membrane of the cleavage furrow (32) but also to Aurora-B in the spindle midzone (15, 16). These localized interactions induce cleavage furrow-specific vimentin phosphorylation by both kinases. Although Aurora-B more than Rho-kinase may contribute to the localized elevation of the vimentin phosphorylation level (Fig. 4B), these kinases are most likely to function in cooperation and induce the break/looseness of the vimentin filament network, specifically at the cleavage furrow.

In summary, we show that Aurora-B governs the cleavage furrow-specific phosphorylation of vimentin-Ser-72. Vimentin phosphorylation by Aurora-B may play an important role in the segregation of vimentin filaments. Our findings also support the notion that Aurora-B regulates cytokinesis partly through the cleavage furrow-specific phosphorylation of cytoskeletal proteins including vimentin.

    ACKNOWLEDGEMENTS

We thank Dr. F. Matsumura (Rutgers University) for critique of the manuscript. We also thank Dr. Y. Fukami (Kobe Univiversity) for helpful comments. M. Ohara (Fukuoka, Japan) provided language assistance.

    FOOTNOTES

* This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Technology, Sports, and Culture of Japan and by a grant-in-aid for the Second Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labour and Welfare, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ The first three authors equally contributed to this work.

§§ To whom correspondence should be addressed. Tel.: 81-52-762-6111 (ext. 7020); Fax: 81-52-763-5233; E-mail: minagaki@aichi-cc.jp.

Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M210892200

2 A. Kawajiri, Y. Fukami, and M. Inagaki, unpublished observations.

3 A. Kawajiri and M. Inagaki, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: IF, intermediate filament; GFAP, glial fibrillary acidic protein; IF-bridge, a long bridge-like IF structure; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; WT, wild type; K/R, kinase-inactive.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMETAL PROCEDURE
RESULTS AND DISCUSSION
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