MICROSCOPY RESEARCH AND TECHNIQUE 49:173–182 (2000) Regulation of Intermediate Filament Organization During Cytokinesis: Possible Roles of Rho-associated Kinase HIDEMASA GOTO, HIDETAKA KOSAKO, AND MASAKI INAGAKI* Laboratory of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi 464-8681, Japan KEY WORDS: intermediate filament (IF); cleavage furrow; cytokinesis; Rho; Rho-associated kinase (Rho-kinase) ABSTRACT Intermediate filaments (IFs), which form the structural framework of cytoskeleton, have been found to be dramatically reorganized during mitosis. Some protein kinases activated in mitosis are thought to control spatial and temporal IF reorganization through phosphorylation of IF proteins. Rho-associated kinase (Rho-kinase), one of the putative targets of the small GTPase Rho, does phosphorylate IF proteins, specifically at the cleavage furrow during cytokinesis. This cleavage furrow-specific phosphorylation plays an important role in the local IF breakdown and efficient separation of IF networks. Recent studies on Rho signaling pathways have introduced new models about the molecular mechanism of rearrangements of cytoskeletons including IFs during cytokinesis. Microsc. Res. Tech. 49:173–182, 2000. © 2000 Wiley-Liss, Inc. INTRODUCTION Intermediate filaments (IFs), 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 cell-, tissue-, and differentiation-dependent manner and are divided into six groups (type I through type VI). 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, other type III IF protein, is expressed in mesenchymal cells, in most types of cultured and tumor cells, and transiently in many cells during development (Eriksson et al., 1992; Fuchs and Weber, 1994; Steinert and Roop, 1988). IFs were thought to be relatively stable in comparison with microtubules and actin filaments in the past. However, IFs are far more dynamic than has been previously considered. There is increasing evidence that site-specific phosphorylation of IF proteins alters their filament structures (Inagaki et al., 1987, 1996). Recent detailed analyses on sitespecific IF phosphorylation in defined subcellular locations at various stages of mitosis have brought new insights into the molecular mechanisms involved in the mitotic IF reorganization (Foisner, 1997; Inagaki et al., 1994, 1997; Ku et al., 1998; Nishizawa et al., 1991). The small GTPase Rho is implicated in a wide spectrum of cellular functions, including cytoskeletal rearrangements, transcriptional activation, and smooth muscle contraction (Hall, 1998; Takai et al., 1995). Rho is also known to play important roles in cytokinesis (Drechsel et al., 1996; Kishi et al., 1993; Mabuchi et al., 1993; O’Connell et al., 1999). Upon stimulation with certain signals, GDP-bound inactive form of Rho is converted to GTP-bound active form, which presumably binds to specific targets and thereby exerts its biological functions (Ren et al., 1999). Several groups have succeeded in identifying putative targets of Rho; © 2000 WILEY-LISS, INC. protein kinase N (PKN), rhophilin, rhotekin, citron kinase/citron-N, the myosin binding subunit (MBS) of myosin phosphatase, mDia (mammalian homolog of Drosophila diaphanous), phospholipase D, and Rhoassociated kinase (Rho-kinase)/ROK/ROCK (Lim et al., 1996; Narumiya et al., 1997; Van Aelst and D’souzaSchorey, 1997). Among such effectors, Rho-kinase (Matsui et al., 1996) /ROK (Leung et al., 1995, 1996) /ROCK (Ishizaki et al., 1996; Nakagawa et al., 1996) was identified as a serine/threonine kinase with a molecular mass of about 160 kDa. GTP-bound active Rho interacts with Rho-kinase and thereby elevates its kinase activity (Matsui et al., 1996; Ishizaki et al., 1996). Rho-kinase is shown to mediate some biological effects of Rho: stress fiber and focal adhesion formation, smooth muscle contraction, neurite retraction, microvilli formation, and cell migration (Kaibuchi et al., 1999). In this review, we will give an overview of spatiotemporal distribution of site-specific IF phosphorylation during mitosis (especially cytokinesis), and discuss the relationship between the cleavage furrow-specific IF phosphorylation and Rho-kinase. We will also speculate on the molecular mechanism of cytoskeletal rearrangements through Rho and its specific targets (especially Rho-kinase) during cytokinesis. CLEAVAGE FURROW-SPECIFIC PHOSPHORYLATION OF INTERMEDIATE FILAMENT (IF) PROTEINS DURING CYTOKINESIS In eukaryotes, mitosis is characterized by the formation of two distinct cytoskeletal structures. A bipolar Contract grant sponsor: Ministry of Education, Science, Sports, and Culture of Japan; Contract grant sponsor: Japan Society of the Promotion of Science Research for the Future; Contract grant sponsor: Bristol-Myers-Squibb. *Correspondence to: Masaki Inagaki, Laboratory of Biochemistry, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya, Aichi 4648681, Japan. E-mail: [email protected] Received 1 September 1999; accepted in revised form 2 December 1999 174 H. GOTO ET AL. Fig. 1. The site-specific phosphorylation of GFAP (A) and vimentin (B) during mitosis in U251 human astroglial cells. A: Metaphase or anaphase cells are stained with the antibody MO389 (anti-GFAP), YC10 (anti-phosphoSer8 on GFAP), TMG7 (anti-phosphoThr7 on GFAP), KT13 (anti-phosphoSer13 on GFAP), or KT34 (anti-phosphoSer38 on GFAP; green). DNAs are stained with propidium iodide (red). Modified with the permission from Kosako H, Amano M, Yanagida M, Tanabe K, Nishi Y, Kaibuchi K, Inagaki M. 1997. Phosphorylation of glial fibrillary acidic protein at the same sites by cleavage furrow kinase and Rho-associated kinase. J Biol Chem 272:10333–10336, and Matsuzawa K, Kosako H, Azuma I, Inagaki N, Inagaki M. 1998. Possible regulation of intermediate filament proteins by Rho-binding kinases. In: Hermann H, Hariis JR, editors. Intermediate filament subcellular biochemistry, vol. 31. New York: Plenum Press, p 423– 435. B: Anaphase cells are doubly stained with anti-vimentin (red) and TM38 (anti-phosphoSer38 on vimentin) or TM71 (anti-phosphoSer71 on vimentin; green). DNAs are stained with DAPI (blue). Bars ⫽ 10 mm. Modified with the permission from Kosako H, Goto H, Yanagida M, Matsuzawa K, Fujita M, Tomono Y, Okigaki T, Odai H, Kaibuchi K, Inagaki M. 1999. Specific accumulation of Rho-associated kinase at the cleavage furrow: cleavage furrow-specific phosphorylation of intermediate filaments. Oncogene 18:2783–2788. mitotic spindle is composed of microtubules and their associated proteins, and play a central role in nuclear division. A contractile ring, composed of actin filaments and myosin II just beneath the plasma membrane, appears after nuclear division and partitions the cell surface and cellular components by pulling the membrane inward (Mabuchi, 1986; Rappaport, 1986). IFs are also found to be reorganized dramatically during mitosis but the degree of reorganization differs depending on cell types. During early mitosis (prophase/metaphase), the extent of remodeling varies between a disassembly into soluble oligomers in Xenopus oocytes (Klymkowsky et al., 1991), a reorganization to nonfilamentous insoluble aggregates (Franke et al., 1982; 175 RHO-ASSOCIATED KINASE DURING CYTOKINESIS Figure 1. (Continued) Rosevear et al., 1990), and a collapse of IF systems into a cage-like structure around the mitotic spindle without any indications of physical breakage (Blose, 1979; Jones et al., 1985; Lane et al., 1982). During cytokinesis (late mitosis), in many types of cells, IFs appear to be interrupted as intact bundles on the plane of the cleavage furrow (Blose, 1979; Franke et al., 1983; Jones et al., 1985; Lane et al., 1982). The mitotic IF reorganization is known to be accompanied with increase in IF phosphorylation (Ce- lis et al., 1983; Evans and Flik, 1982). The first direct evidence supporting IF regulation by phosphorylation was obtained for vimentin by in vitro study: treatment of in vitro polymerized vimentin with purified protein kinase A caused their filament disassembly (Inagaki et al., 1987). Accumulating in vitro data suggest that the phosphorylation of IF proteins by various types of protein kinases induces disassembly of the filament structure (Inagaki et al., 1996). These observations lead to the notion that IF reor- TABLE 1. In vivo (CF kinase) and in vitro (Cdc2 kinase, A kinase, C kinase, CaM kinase II, and Rho-kinase) phosphorylation sites on GFAP and vimentin, and antibodies that recognize site-specific phosphorylation* GFAP Vimentin Antibody site TMG7 Thr7 YC10 Ser8 KT13 Ser13 KT34 Ser38 MO6 Ser6 YT33 Ser33 TM38 Ser38 TM50 Ser50 4A4 Ser55 TM71 Ser71 MO82 Ser82 Cdc2 kinase A kinase C kinase CaM kinase II Rho-kinase CF kinase ⫺ ⫹⫹ ⫺ ⫺ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹ ⫺ ⫺ ⫺ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫺ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫺ ⫹⫹⫹ ⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫺ ⫹⫹⫹ ⫹⫹⫹ ⫺ ⫺ ⫺ ⫹⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹ ⫹⫹⫹ ⫺ ⫾ ⫺ ⫹⫹⫹ ⫺ ⫺ *CF kinase is a protein kinase that phosphorylates type III IF proteins specifically at the cleavage furrow during cytokinesis (Nishizawa et al., 1991; Matsuoka et al., 1992; Sekimata et al., 1996). Rho-associated kinase (Matui et al., 1996), one of the serine/threonine kinases downstream of Rho. It is also called ROK (Leung et al., 1995; 1996) or ROCK (Ishizaki et al., 1995; Nakagawa et al., 1996). 176 H. GOTO ET AL. Fig. 2. Accumulation of Rho-kinase at the cleavage furrow. A: Distribution of Rho-kinase in MDBK cells. Interphase, metaphase, and telophase cells are stained with anti-Rho-kinase (green; Kosako et al., 1999). DNAs are stained with propidium iodide (red). Bars ⫽ 10 mm. B: Colocalization of Rho-kinase and vimentin phosphorylated at Ser71 during cytokinesis. Telophase Swiss 3T3 or NIH3T3 cell is stained with anti-Rhokinase (green) and TM71 (red; Kosako et al., 1999). ganization may be regulated by phosphorylation of IF proteins during mitosis. Site- and phosphorylation state-specific antibodies that can recognize a phosphorylated residue and its flanking sequence are of great use in order to analyze spatiotemporal distribution of site-specific IF phosphorylation at each stage of mitosis. We first established a method to produce site- and phosphorylation state-specific antibodies, using phosphorylated peptides or synthetic phosphopeptides as antigens for immunization. This method has an advantage in that one can predesign a phosphorylated residue as an epitope. Now, these site- and phosphorylation state-specific antibodies are widely utilized to analyze the phosphorylation of various proteins in cellular events (Inagaki et al., 1994, 1997). Detailed studies by using site- and phosphorylation state-specific antibodies revealed that mitotic IF phosphorylation is spatio-temporally regulated by distinct protein kinase(s) (Foisner, 1997; Inagaki et al., 1994, 1996, 1997; Ku et al., 1998). The first evidence was obtained with the analysis using these antibodies for four distinct sites on GFAP (Matsuoka et al., 1992; Nishizawa et al., 1991). Ser8 phosphorylation of GFAP in glial cells began in the prometaphase, remained until metaphase, and declined gradually thereafter. The phosphorylation was observed diffusely throughout cytoplasm (Fig. 1A). On the other hand, phosphorylation of residues Thr7, Ser13, and Ser38 were spatially and temporally distinct, increasing in anaphase, maintained until telophase, and decreasing at the exit of mitosis, and this phosphorylation was localized specifically at the cleavage furrow (Fig. 1A). These observations suggested that GFAP is phosphorylated by at least two different kinases. It is notable that these phosphorylation patterns correlate well with the observation concerning IF reorganization during mitosis (see above). Therefore, these phosphorylations may play some important roles in mitotic IF reorganization. Identifying protein kinases responsible for mitotic IF phosphorylation is of great importance in order to understand how IF reorganization is regulated during RHO-ASSOCIATED KINASE DURING CYTOKINESIS Figure 2. mitosis. Site- and phosphorylation state-specific antibodies also provide useful information to identify in vivo IF kinase(s) (Inagaki et al., 1994, 1996, 1997). The protein kinase activated during early mitosis phosphorylates GFAP only at Ser8 but not at Thr7, Ser13, and Ser38 (Fig. 1). Cdc2 kinase is known to be activated specifically during early mitosis (Murray and Hunt, 1993; Norbury and Nurse, 1992) and phosphorylate GFAP only at Ser8 in vitro (Tsujimura et al., 1994a). Since vimentin-Ser55 was phosphorylated specifically by Cdc2 kinase among known IF kinases in vitro (Chou et al., 1991; Kusubata et al., 1992), we further produced a site- and phosphorylation statespecific antibody for this site. Ser55 on vimentin was also phosphorylated in various types of cells only during early mitotic phase and biochemical analysis of mitotic cell lysates revealed that Ser55 phosphorylating activity and Cdc2 protein was coeluted as a single peak (Tsujimura et al., 1994b). Together with data obtained by tryptic peptide analysis (Chou et al., 1990), these observations strongly suggest that Cdc2 kinase is responsible for in vivo IF phosphorylation specifically during early mitosis. On the other hand, little was elucidated about the molecular identity and regulation of a protein kinase, which phosphorylate Thr7, Ser13, and Ser38 on GFAP specifically at the cleavage furrow during cytokinesis, in the past (Fig. 1A). Since this kinase seemed to be activated specifically at the cleavage furrow during 177 (Continued) cytokinesis, we tentatively named it cleavage furrow (CF) kinase (Matsuoka et al., 1992; Nishizawa et al., 1991). This CF kinase activity was observed not only in astroglial cells but also in several cultured cells in which GFAP was ectopically expressed (Sekimata et al., 1996). These findings indicated that the activation of CF kinase occurs in a wide range of cell types, suggesting its important role in cytokinesis. Considering that Rho is implicated in cytokinesis (Drechsel et al., 1996; Kishi et al., 1993; Mabuchi et al., 1993; O’Connell et al., 1999), we postulated the possible involvement of Rho in the regulation of CF kinase activity. Recently, Rho-kinase, one of Rho targets (also see Introduction), was revealed to phosphorylate GFAP at Thr7, Ser13, and Ser38 but not at Ser8 in vitro (Kosako et al., 1997). The in vitro Rho-kinase phosphorylation sites (Ser38 and Ser71) on vimentin were also shown to be phosphorylated at the cleavage furrow during cytokinesis (Goto et al., 1998; Kosako et al., 1999; Fig. 1B). Table 1 summarizes known phosphorylation sites of GFAP and vimentin. Furthermore, we obtained evidence that Rho-kinase phosphorylated desmin, the other III IF protein, at Thr16, Thr75, and Thr76 in vitro (Inada et al., 1998), which were phosphorylated specifically at the cleavage furrow (Inada et al., 1999). All these observations indicate that Rho-kinase has the substrate specificity extremely similar to CF kinase, which phosphorylates at least type III IF proteins during cytokinesis. 178 H. GOTO ET AL. ACCUMULATION OF RHO AND RHO-KINASE AT THE CLEAVAGE FURROW DURING CYTOKINESIS All accumulating data on the substrate specificity are consistent with the idea that Rho-kinase is responsible for CF kinase activity (Table 1). These observations raise the question whether Rho-kinase is activated during cytokinesis. Rho-kinase is thought to be activated by forming a complex with GTP-bound Rho (Ishizaki et al., 1996; Matsui et al., 1996). Thus, information on spatiotemporal distribution of Rho and Rho-kinase in the late mitotic cell is of great importance in order to speculate where Rho-kinase is activated during cytokinesis. Rho has been reported to concentrate into the cleavage furrow during cytokinesis (Takaishi et al., 1995). Recently, Rho-kinase was also shown to accumulate into the cleavage furrow (Fig. 2A). This cleavage furrowspecific accumulation first appears at late anaphase and is maintained until telophase, then gradually decreases at the exit of mitosis (Kosako et al., 1999). This subcellular localization of Rho-kinase is very similar to that of Rho, indicating that Rho-kinase binds to Rho and thereby is activated at the cleavage furrow. Rho is thought to participate in cytokinesis since inhibition of endogenous Rho by botulium ADP-ribosyltransferase C3 blocked cytokinesis in Xenopus embryos (Kishi et al., 1993) and sand dollar eggs (Mabuchi et al., 1993). Rho may be involved in the assembly of actin filaments and proper constriction of the contractile ring, which play important roles in defining the spatiotemporal pattern of cleavage (Drechsel et al., 1996; O’Connell et al., 1999). Recently, Rho-kinase was reported to mediate some biological effects of Rho during cytokinesis, i.e., expression of the dominant-negative form of Rho-kinase inhibits cytokinesis in Xenopus embryos and mammalian cells, resulted in multinucleation (Yasui et al., 1998). This suggests that Rho-kinase may be activated and phosphorylate multiple proteins during cytokinesis. GFAP, vimentin, and desmin serve as excellent substrates for Rho-kinase in vitro (Goto et al., 1998; Inada et al., 1998; Kosako et al., 1997). The in vitro phosphorylation sites of these type III IF proteins are identical to the in vivo sites, occurring at the cleavage furrow (Goto et al., 1998; Inada et al., 1999; Kosako et al., 1999; Fig. 1 and Table 1). In addition, this cleavage furrow-specific IF phosphorylation occurs near the area where Rho-kinase accumulates during cytokinesis (Kosako et al., 1999; Fig. 2B). This evidence led us to suggest that Rho-kinase phosphorylates at least type III IF proteins specifically at the cleavage furrow during cytokinesis. BIOLOGICAL SIGNIFICANCE OF CLEAVAGE FURROW-SPECIFIC IF PHOSPHORYLATION During cytokinesis, the extent of IF remodeling varies in cell types, but IFs appear to be interrupted on the plane of the cleavage furrow (Blose, 1979; Franke et al., 1983; Jones et al., 1985; Lane et al., 1982). This localized IF reorganization is highly correlated with the cleavage furrow-specific IF phosphorylation. In vitro studies revealed that the phosphorylation of type III IF proteins by Rho-kinase led to disassembly of their fil- ament structures (Goto et al., 1998; Inada et al., 1998; Kosako et al., 1997). Recently, we produced a mutant GFAP in which Rho-kinase/CF kinase phosphorylation sites (Thr7, Ser13, and Ser38) are changed to Ala residues and introduced this mutant GFAP into type III IF-negative T24 cells (Yasui et al., 1998). The expression of this mutant GFAP impaired cytokinetic segregation of GFAP filaments, resulting in the formation of an unusually long bridge-like structure between unseparated daughter cells (Fig. 3). Wild type GFAP and mutants in which three Ser/Thr different from Rhokinase/CF kinase sites were altered showed no remarkable phenotype (Yasui et al., 1998). These observations suggest that the phosphorylation of IF proteins by Rhokinase/CF kinase is required for the local IF breakdown and the separation of IFs into daughter cells. POSSIBLE ROLES OF RHO AND RHO-KINASE IN CYTOKINESIS Rho is thought to play an important role in the formation and constriction of the contractile ring (Drechsel et al., 1996; Kishi et al., 1993; Mabuchi et al., 1993; O’Connell et al., 1999; Takaishi et al., 1995). It is very likely that Rho binds to its targets specifically at the cleavage furrow and exerts its biological functions during cytokinesis. We propose a model for the possible pathways downstream of Rho during cytokinesis in Figure 4. Rho-kinase is one of the targets mediating significant biological effects of Rho during cytokinesis, i.e., expression of the dominant-negative form of Rhokinase inhibited the cytokinesis, resulting in the production of multinuclei (Yasui et al., 1998). Rho-kinase is shown to regulate the phosphorylation of regulatory light chain of myosin II (RMLC) at Ser19 by direct phosphorylation of RMLC and by inactivation of myosin phosphatase through MBS phosphorylation (Amano et al., 1996; Chihara et al., 1997; Kimura et al., 1996). During cytokinesis, RMLC phosphorylation at Ser19 occurred specifically at the cleavage furrow (Matsumura et al., 1998) and near the area where Rho-kinase accumulated (Kosako et al., 1999). Since RMLC phosphorylation at Ser19 is believed to promote the contractility of actomyosin in the cells (Huttenlocher et al., 1995), these observations indicate that Rho-kinase may play important roles not only in proper IF separation into daughter cells but also in the formation and constriction of the contractile ring. Citron kinase, a Rho target with the structural similarity to Rho-kinase, was recently reported to localize to the cleavage furrow (Madaule et al., 1998). Overexpression of its mutants results in production of multinuclei and abnormal contraction during cytokinesis (Madaule et al., 1998). These observations suggest that citron kinase may play an important role in the contractile process of cytokinesis. However, the putative substrate(s) have not be identified yet, at least, type III IF proteins might not be phosphorylated by citron kinase (Inagaki, unpublished observation). Thus, little is known about the molecular mechanism by which citron kinase regulates the contractile process of cytokinesis. A mammalian homolog of Drosophila diaphanous (mDia), one of the putative targets of Rho, was re- RHO-ASSOCIATED KINASE DURING CYTOKINESIS 179 Fig. 3. GFAP bridge-like structures in T24 cells expressing a mutant GFAP with mutations in Rho-kinase phosphorylation sites (Thr7, Ser13, and Ser38 on GFAP are changed into Ala). Green and red colors represent GFAP stained with MO389 and propidium iodide, respectively. Bar ⫽ 10 mm. Modified with the permission from Yasui Y, Amano M, Nagata K, Inagaki N, Nakamura H, Saya H, Kaibuchi K, Inagaki M. 1998. Roles of Rho-associated kinase in cytokinesis; mutations in Rho-associated kinase phosphorylation sites impair cytokinetic segregation of glial filaments. J Cell Biol 143:1249 –1258. ported to concentrate weakly into the cleavage furrow in some mitotic cells (Watanabe et al., 1997). This study also showed that Rho regulated actin polymerization by targeting profilin via mDia beneath the plasma membranes. Since disruption of diaphanous or profilin gene results in cytokinesisdefective phenotype (Balasubramanian et al., 1994; Castrillon and Wasserman, 1994; Haugwitz et al., 1994; Verheyen and Cooley, 1994), mDia is thought to associate with profilin and regulate the assembly of actin filaments at the cleavage furrow. FUTURE PROSPECTS Accumulating evidence suggests that site-specific phosphorylation of IF proteins alters their filament structure. Recent analyses on site-specific IF phosphorylation in defined subcellular locations at various mitotic stages demonstrates that several protein kinases 180 H. GOTO ET AL. Fig. 4. A scheme showing possible signaling pathways from Rho during cytokinesis. phosphorylate IF proteins in a spatio-temporally regulated manner. Mutational analysis has revealed that cleavage furrow-specific IF phosphorylation by CF kinase/Rho-kinase plays an important role in efficient separation of IFs to daughter cells. Recent analyses on Rho signalling pathways have promoted our understanding of how Rho regulates cytoskeletal rearrangements during cytokinesis. Rho-kinase may be essential, not only for separation of IFs into daughter cells, but also for the formation and constriction of contractile ring. On the other hand, little is known about the effects of IF phosphorylation by other mitotic kinases on IF structure and cellular behavior. We speculate that IF phosphorylation during early mitosis may be necessary for mitotic morphological change, together with rearrangements of other cytoskeletal filament systems and regulation of interaction of IFs and other cytoskeletal filaments. These possibilities need to be addressed in future studies. ACKNOWLEDGMENTS We thank Dr. K. Nagata for critically reading the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research and Cancer Re- search from the Ministry of Education, Science, Sports, and Culture of Japan, Japan. REFERENCES Amano M, Ito M, Kimura K, Fukata Y, Chihara K, Nakano T, Matsuura Y, Kaibuchi K. 1996. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J Biol Chem 271: 20246 –20249. Balasubramanian MK, Hirani BR, Burke JD, Gould KL. 1994. The Schizosaccharomyces pombe cdc3⫹ gene encodes a profilin essential for cytokinesis. J Cell Biol 125:1289 –1301. Blose SH. 1979. Ten-nanometer filaments and mitosis: maintenance of structural continuity in dividing endothelial cells. Proc Natl Acad Sci USA 76:3372–3376. Castrillon DH, Wasserman SA. 1994. diaphanous is required for cytokinesis in Drosophila and shares domains of similarity with the products of the limb deformity gene. Development 120:3367–3377. Celis JE, Lasen PM, Fey SJ, Celis A. 1983. Phosphorylation of keratin and vimentin polypeptides in normal and transformed mitotic human epithelial amnion cells: behavior of keratin and vimentin filaments during mitosis. J Cell Biol 97:1429 –1434. Chihara K, Amano M, Nakamura N, Yano T, Shibata M, Tokui T, Ichikawa H, Ikebe R, Ikebe M, Kaibuchi K. 1997. Cytoskeletal rearrangements and transcriptional activation of c-fos serum responce element by Rho-kinase. J Biol Chem 272:25121–25127. Chou Y-H, Bischoff JR, Beach D, Goldman RD. 1990. Intermediate filament reorganization during mitosis is mediated by p34cdc2 kinase phosphorylation of vimentin. Cell 62:1063–1071. RHO-ASSOCIATED KINASE DURING CYTOKINESIS Chou Y-H, Ngai K-L, Goldman RD. 1991. The regulation of intermediate filament reorganization in mitosis. J Biol Chem 266:7325– 7328. Drechsel DN, Hyman AA, Hall A, Glotzer M. 1996. A requirement for Rho and Cdc42 during cytokinesis in Xenopus embryos. Curr Biol 7:12–23. Eriksson JE, Opal P, Goldman RD. 1992. Intermediate filament dynamics. Curr Opin Cell Biol 4:99 –104. Evans RM, Flik LM. 1982. An alteration in the phosphorylation of vimentin-type intermediate filaments is associated with mitosis in cultured mammalian cells. Cell 29:43–52. Foisner R. 1997. Dynamic organisation of intermediate filaments and associated proteins during the cell cycle. BioEssays 19:297–305. Franke WW, Schmid E, Grund C, Geiger B 1982. Intermediate filament proteins in nonfilamentous structures: transient disintegration and inclusion of subunit proteins in granular aggregates. Cell 30:103–113. Franke WW, Schmid E, Wellsteed J, Grund C, Gigi O, Geiger B. 1983. Change of cytokeratin filament organization during the cell cycle: selective masking of an immunologic determinant in interphase Ptk2 cells. J Cell Biol 97:1255–1260. Fuchs E, Weber K. 1994. Intermediate filaments: structure, dynamics, function, and disease. Ann Rev Biochem 63:345–382. Goto H, Kosako H, Tanabe K, Yanagida M, Sakurai M, Amano M, Kaibuchi K, Inagaki M. 1998. Phosphorylation of vimentin by Rhoassociated kinase at a unique amino-terminal site that is specifically phosphorylated during cytokinesis. J Biol Chem 273:11728 – 11736. Hall A. 1998. Rho GTPase and the actin cytoskeleton. Science 279: 509 –514. Haugwitz M, Noegel AA, Karakesisoglou J, Schleicher M. 1994. Dictyostelium amoebae that lack G-actin-sequestering profilins show defects in F-actin content, cytokinesis and development. Cell 79: 303–314. Huttenlocher A, Sandborg RR, Horwitz AF. 1995. Adhesion in cell migration. Curr Opin Cell Biol 7:697–706. Inada H, Goto H, Tanabe K, Nishi Y, Kaibuchi K, Inagaki M. 1998. Rho-associated kinase phosphorylates desmin, the myogenic intermediate filament protein, at unique amino-terminal sites. Biochem Biophys Res Commun 253:21–25. Inada H, Togashi H, Nakamura Y, Kaibuchi K, Nagata K, Inagaki M. 1999. Balance between activities of Rho-kinase and protein phosphatase 1 modulates turnover of phosphorylation and dynamics of desmin/vimentin filaments. J Biol Chem 274:34932–34939. Inagaki M, Nishi Y, Nishizawa K, Matsuyama M, Sato C. 1987. Site-specific phosphorylation induces disassembly of vimentin filaments in vitro. Nature 328:649 – 652. Inagaki M, Matsuoka Y, Tsujimura K, Ando S, Tokui T, Takahashi T, Inagaki N. 1996. Dynamic property of intermediate filaments; regulation by phosphorylation. BioEssays 18:481– 487. Inagaki M, Inagaki N, Takahashi T, Takai Y. 1997. Phosphorylationdependent control of structures of intermediate filaments: a novel approach using site- and phosphorylation state-specific antibodies. J Biochem 121:407– 414. Inagaki N, Ito M, Nakano T, Inagaki M. 1994. Spatiotemporal distribution of protein kinase and phosphatase activities. Trends Biochem Sci 19:448 – 452. Ishizaki T, Maekawa M, Fujisawa K, Okawa K, Iwamatsu A, Fujita A, Watanabe N, Saito Y, Kakizuka A, Morii N, Narumiya S. 1996. The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. EMBO J 15:1885–1893. Jones JCR, Goldman AE, Yang H-Y, Goldman RD. 1985. The organizational fate of intermediate filament networks in two epithelial cell types during mitosis. J Cell Biol 100:93–102. Kaibuchi K, Kuroda S, Amano M. 1999. Regulation of the cytoskeleton and cell adhesion by Rho family GTPases in mammalian cells. Ann Rev Biochem 68:459 – 486. Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K. 1996. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273:245–248. Kishi K, Sasaki T, Kuroda S, Itoh T, Takai Y. 1993. Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI). J Cell Biol 120:1187– 1195. Klymkowsky M W, Maynell L A, Nislow C. 1991. Cytokeratin phosphorylation, cytokeratin filament severing and the solubilization of the maternal mRNA Vg1. J Cell Biol 114:787–797. Kosako H, Amano M, Yanagida M, Tanabe K, Nishi Y, Kaibuchi K, Inagaki M. 1997. Phosphorylation of glial fibrillary acidic protein at 181 the same sites by cleavage furrow kinase and Rho-associated kinase. J Biol Chem 272:10333–10336. Kosako H, Goto H, Yanagida M, Matsuzawa K, Fujita M, Tomono Y, Okigaki T, Odai H, Kaibuchi K, Inagaki M. 1999. Specific accumulation of Rho-associated kinase at the cleavage furrow: cleavage furrow-specific phosphorylation of intermediate filaments. Oncogene 18:2783–2788. Ku NO, Liao J, Chou CF, Omary MB. 1998. Implications of intermediate filament protein phosphorylation. Cancer Metast Rev 15:429 – 444. Kusubata M, Tokui T, Matsuoka Y, Okumura E, Tachibana K, Hisanaga S, Kishimoto T, Yasuda H, Kamijo M, Ohba Y, Tsujimura K, Yatani R, Inagaki M. 1992. p13suc1 suppresses the catalytic function of p34cdc2 kinase for intermediate filament protein, in vitro. J Biol Chem 267:20937–20942. Lane EB, Goodman SL, Trejdosiewics LK. 1982. Disruption of the keratin filament network during epithelial cell division. EMBO J 1:1365–1372. Leung T, Manser E, Tan L, Lim L. 1995. A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J Biol Chem 270:29051– 29054. Leung T, Chen X-Q, Manser E, Lim L. 1996. The p160 RhoA-binding kinase ROKa is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol Cell Biol 16:5313–5327. Lim L, Manser E, Leung T, Hall C. 1996. Regulation of phosphorylation pathways by p21 GTPase: The p21 Ras-related Rho subfamily and its role in phosphorylation signalling pathways. Eur J Biochem 242:171–185. Mabuchi I. 1986. Biochemical aspects of cytokinesis. Int Rev Cytol 101:175–213. Mabuchi I, Hamaguchi Y, Fujimoto H, Morii N, Mishima M, Narumiya S. 1993. A rho-like protein is involved in the organisation of the contractile ring in dividing sand dollar eggs. Zygote 1:325–331. Madaule P, Eda M, Watanabe N, Fujisawa K, Matsuoka T, Bito H, Ishizaki T, Narumiya S. 1998. Role of citron kinase as a target of the small GTPase Rho in cytokinesis. Nature 394:491– 494. Matsui T, Amano M, Yamamoto T, Chihara K, Nakafuku M, Ito M, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K. 1996. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J 15:2208 –2216. Matsumura F, Ono S, Yamakita Y, Totsukawa G, Yamashiro S. 1998. Specific localization of serine 19 phosphorylated myosin II during cell locomotion and mitosis of cultured cells. J Cell Biol 140:119 – 129. Matsuoka Y, Nishizawa K, Yano T, Shibata M, Ando S, Takahashi T, Inagaki M 1992. Two different protein kinases act on a different time schedule as glial filament kinases during mitosis. EMBO J 11:2895–2902. Matsuzawa K, Kosako H, Azuma I, Inagaki N, Inagaki M. 1998. Possible regulation of intermediate filament proteins by Rho-binding kinases. In: Hermann H, Hariis JR, editors. Intermediate filament subcellular biochemistry, vol. 31. New York: Plenum Press, p 423– 435. Murray A, Hunt T. 1993. The cell cycle: an introduction. Oxford: Oxford University Press, p 42–56. Nakagawa O, Fujisawa K, Ishizaki T, Saito Y, Nakao K, Narumiya S. 1996. ROCK-I and ROCK-II, two isoforms of Rho-associated coiledcoil forming protein serine/threonine kinase in mice. FEBS Lett 392:189 –193. Narumiya S, Ishizaki T, Watanabe N. 1997. Rho effectors and reorganization of actin cytoskeleton. FEBS Lett 410:68 –72. Nishizawa K, Yano T, Shibata M, Ando S, Saga S, Takahashi T, Inagaki M. 1991. Specific localization of phosphointermediate filament protein in the constricted area of dividing cells. J Biol Chem 266:3074 –3079. Norbury C, Nurse P. 1992. Animal cell cycles and their control. Ann Rev Biochem 61:441– 470. O’Connell CB, Wheatley SP, Ahmed S, Wang Y. 1999. The small GTP-binding protein Rho regulates cortical activities in cultured cells during division. J Cell Biol 144:305–313. Rappaport R. 1986. Establishment of the mechanism of cytokinesis in animal cells. Int Rev Cytol 105:245–281. Ren X-D, Kiosses WB, Schwartz MA. 1999. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J 18:578 –585. Rosevear ER, McReynolds M, Goldman RD. 1990. Dynamic properties of intermediate filaments: disassembly and reassembly during mitosis in baby hamster kidney cells. Cell Motil Cytoskeleton 17:150 – 166. 182 H. GOTO ET AL. Sekimata M, Tsujimura K, Tanaka J, Takeuchi Y, Inagaki N, Inagaki M. 1996. Detection of protein kinase activity specifically activated at metaphase-anaphase transition. J Cell Biol 132:635– 641. Steinert PM, Roop DR. 1988. Molecular and cellular biology of intermediate filaments. Ann Rev Biochem 57:593– 625. Takai Y, Sasaki T, Tanaka K, Nakanishi H. 1995. Rho as a regulator of the cytoskeleton. Trends Biochem Sci 20:227–231. Takaishi K, Sasaki T, Kameyama T, Tsukita S, Tsukita S, Takai Y. 1995. Translocation of activated Rho from the cytoplasm to membrane ruffling area, cell-cell adhesion sites and cleavage furrows. Oncogene 11:39 – 48. Tsujimura K, Tanaka J, o S, Matsuoka Y, Kusubata M, Sugiura H, Yamauchi T, Inagaki M 1994a) Identification of phosphorylation sites on glial fibrillary acidic protein for cdc2 kinase and Ca2⫹calmodulin-dependent protein kinase II. J Biochem 116:426 – 434. Tsujimura K, Ogawara M, Takeuchi Y, Imajoh-Ohmi S, Ha MH, Inagaki M. 1994b. Visualization and function of vimentin phosphor- ylation by cdc2 kinase during mitosis. J Biol Chem 269:31097– 31106. Verheyen EM, Cooley L. 1994. Profilin mutations disrupt multiple actin-dependent processes during Drosophila development. Development 120:717–728. Van Aelst L, D’souza-Schorey C. 1997. RhoGTPase and signaling networks. Genes Dev 11:2295–2322. Watanabe N, Madaule P, Reid T, Ishizaki T, Watanabe G Kakizuka A, Saito Y, Nakao K, Jockusch BM, Narumiya S. 1997. p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin. EMBO J 16:3044 – 3056. Yasui Y, Amano M, Nagata K, Inagaki N, Nakamura H, Saya H, Kaibuchi K, Inagaki M. 1998. Roles of Rho-associated kinase in cytokinesis; mutations in Rho-associated kinase phosphorylation sites impair cytokinetic segregation of glial filaments. J Cell Biol 143:1249 –1258.