Rho-kinase/ROCK is involved in cytokinesis through the ... - Nature

Oncogene (2000) 19, 6059 ± 6064 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc

Rho-kinase/ROCK is involved in cytokinesis through the phosphorylation of myosin light chain and not ezrin/radixin/moesin proteins at the cleavage furrow Hidetaka Kosako*,1,2, Toshimichi Yoshida3, Fumio Matsumura4, Toshimasa Ishizaki5, Shuh Narumiya5 and Masaki Inagaki1 1

Laboratory of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, 464-8681, Japan; 2Department of Neurobiology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan; 3Department of Pathology, Mie University School of Medicine, Tsu 514-8507, Japan; 4Department of Molecular Biology and Biochemistry, Rutgers University, New Jersey, NJ 08855, USA; 5Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto 606-8315, Japan

The small GTPase Rho and one of its targets, Rhokinase (also termed ROK or ROCK), are implicated in various cellular functions including stress ®ber formation, smooth muscle contraction, tumor cell invasion and cell motility. We have previously reported that Rho-kinase accumulates at the cleavage furrow during cytokinesis in several cultured cells. Here, using Rho-kinase inhibitors, Y-27632 and HA1077, we found that Rho-kinase is responsible for the phosphorylation of myosin regulatory light chain at Ser19 in the cleavage furrow during cytokinesis. On the other hand, phosphorylation of ezrin/radixin/moesin (ERM) proteins at the cleavage furrow was enhanced by the addition of the above Rhokinase inhibitors. Treatment with Y-27632 strongly enhanced the accumulation of Rho-kinase but not RhoA and citron kinase at the cleavage furrow. Furthermore, the furrow ingression in cytokinesis was signi®cantly prolonged in the presence of Y-27632. These results suggest that Rho-kinase is involved in the progression of cytokinesis through the phosphorylation of several proteins including myosin light chain at the cleavage furrow. Oncogene (2000) 19, 6059 ± 6064. Keywords: cell cycle; cytokinesis; Rho-kinase; myosin light chain; ERM proteins Introduction In animal cells, cytokinesis involves an actomyosinbased contractile ring that forms and constricts at the division plane. Important new information has emerged about spatial and temporal regulation of cytokinesis, such as the function of spindle midzone and the control by GTPase systems (reviewed in Hales et al., 1999). The small GTPase Rho is known to play important roles in various cellular functions such as stress ®ber formation, transcriptional activation, smooth muscle contraction, tumor cell invasion, cell morphology, cell motility and cytokinesis (reviewed in Takai et al., 1995; Van Aelst and D'Souza-Schorey, 1997; Hall, 1998).

Rho-kinase/ROK/ROCK is one of targets for Rho and is involved in some of the above cellular functions downstream of Rho (reviewed in Lim et al., 1996; Narumiya et al., 1997; Kaibuchi et al., 1999). So far, several actomyosin-associated proteins including myosin binding subunit of myosin phosphatase (Kimura et al., 1996), myosin light chain (MLC; Amano et al., 1996), ezrin/radixin/moesin (ERM) proteins (Matsui et al., 1998; Fukata et al., 1998) and adducin (Kimura et al., 1998) have been reported as the putative physiological substrates for Rho-kinase. Phosphorylation of ERM proteins at their C-terminal threonine residue (CP-ERMs) interferes with their head-to-tail association, leading to the activation as F-actin/plasma membrane cross-linkers (reviewed in Mangeat et al., 1999). CP-ERMs concentrate at the cleavage furrow during cytokinesis (reviewed in Tsukita and Yonemura, 1999). MLC phosphorylation at Ser19 is believed to promote the contractility of actomyosin in cells (reviewed in Huttenlocher et al., 1995). Using site- and phosphorylation state-speci®c antibodies, Ser19-phosphorylated MLC has been reported to accumulate at the cleavage furrow during cytokinesis (Matsumura et al., 1998). We have previously reported that Rho-kinase also accumulates at the cleavage furrow during cytokinesis (Kosako et al., 1999). Furthermore, a dominant negative form of Rho-kinase was reported to increase mutinuclei in mammalian cultured cells (Yasui et al., 1998). However, the molecular functions of Rho-kinase at the cleavage furrow remain elusive. Here we show that Rho-kinase is responsible for the phosphorylation of myosin light chain at the cleavage furrow by using Rho-kinase inhibitors, Y-27632 and HA1077 (Uehata et al., 1997). Phosphorylation of ERM proteins at the cleavage furrow was not inhibited by these inhibitors. Furthermore, speci®c overaccumulation of Rho-kinase was observed in the presence of these inhibitors. Ingression of the cleavage furrow was prolonged by the addition of Y-27632, suggesting the involvement of Rho-kinase in cytokinesis.

Results *Correspondence: H Kosako, Department of Neurobiology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Received 20 April 2000; revised 28 September 2000; accepted 3 October 2000

Disruption of actin stress fibers by Rho-kinase inhibitors Rho-kinase is well known to be involved in the formation of actin stress ®bers. To con®rm the eects

Involvement of Rho-kinase in cytokinesis H Kosako et al

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of two types of Rho-kinase inhibitors, Y-27632 and HA1077, we added 10 mM Y-27632 or HA1077 to U251 human glioma cells for 60 min and then stained with rhodamine-phalloidin. As shown in Figure 1, Factin stress ®bers were dramatically disrupted by both inhibitors, while the eect of HA1077 seemed to be somewhat weaker than that of Y-27632. Inhibition of myosin light chain phosphorylation at the cleavage furrow by Rho-kinase inhibitors We examined the eects of Rho-kinase inhibitors on the phosphorylation of myosin light chain (MLC) at the cleavage furrow during cytokinesis. Y-27632 was added to U251 cells for 60 min and then stained with a site- and phosphorylation state-speci®c antibody for Ser19-phosphorylated MLC (Matsumura et al., 1998). As shown in Figure 2a, phosphorylation of MLC-Ser19 at the cleavage furrow was strongly inhibited by Y27632. Similar results were obtained with MDBK bovine epithelial cells and HeLa cells (Figure 2b). HA1077 also inhibited the phosphorylation of MLCSer19 at the cleavage furrow of MDBK cells (Figure 3). Thus phosphorylation of MLC-Ser19 at the cleavage furrow during cytokinesis was inhibited by both Rhokinase inhibitors.

Figure 1 Disruption of actin stress ®bers by Rho-kinase inhibitors. U251 cells were treated with 10 mM Y-27632 or 10 mM HA1077 for 60 min at 378C and then stained with rhodamine-phalloidin Oncogene

Effects of Rho-kinase inhibitors on CP-ERMs at the cleavage furrow We then examined the eects of Y-27632 and HA1077 on ERM proteins phosphorylated at their C-terminal threonine residue (CP-ERMs) at the cleavage furrow. Y-27632 or HA1077 was added to U251 cells for 60 min and then stained with a site- and phosphorylation state-speci®c antibody for CP-ERMs (Matsui et al., 1998). It was surprising that CP-ERMs were increased by both Rho-kinase inhibitors as shown in Figure 4. Thus Rho-kinase appears not to be responsible for the phosphorylation of ERM proteins at the cleavage furrow. Effects of Rho-kinase inhibitors on the distribution of Rho, citron kinase, mDia and Rho-kinase during cytokinesis RhoA was previously reported to accumulate at the cleavage furrow (Takaishi et al., 1995) and we con®rmed clear accumulation of RhoA at the cleavage furrow by the TCA ®xation method (Hayashi et al.,

Figure 2 Phosphorylation of myosin regulatory light chain (MLC) at Ser19 in the cleavage furrow was inhibited by Y27632. (a) U251, and (b)MDBK and HeLa cells were treated with 0, 10 or 30 mM Y-27632 for 60 min and then stained with pp2b (anti-phosphorylated ML C-Ser19 antibody). Scale bars, 10 mm


(cytokinesis) prolonged about threefold by Y-27632 (Figure 6b). Although each time may not be clear in Figure 6a, we measured each time by observing cells at 10 s intervals. Thus the contractile process during cytokinesis was prolonged signi®cantly by the inhibition of Rho-kinase.

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Discussion

Figure 3 Phosphorylation of MLC at Ser19 in the cleavage furrow was inhibited by HA1077. MDBK cells were treated with 0, 10 or 30 mM HA1077 for 60 min and then stained with pp2b (green). DNAs were stained with propidium iodide (red). Scale bar, 10 mM

1999; Figure 5). Citron kinase, another Rho eector (Madaule et al., 1998), also accumulated at the cleavage furrow but mDial (another Rho eector; Watanabe et al., 1997) did not accumulate at the cleavage furrow in our experimental conditions. Immunostaining patterns of RhoA, citron kinase and mDial did not change signi®cantly by Y-27632, although accumulation of RhoA appeared to be enhanced slightly (Figure 5). In contrast, concentration of Rho-kinase at the cleavage furrow was strongly enhanced by the treatment with Y-27632 (Figure 5) and HA1077 (data not shown). These results indicate that Rho-kinase speci®cally overaccumulates at the cleavage furrow by the Rho-kinase inhibitors. Effects of Y-27632 on the progression of cytokinesis To examine the eects of Y-27632 on the progression of cytokinesis, 30 mM Y-27632 was added to U251 cells, and then mitotic cells were analysed by time-lapse videomicroscopy. Formation of the cleavage furrow was initiated about 5 or 6 min after the start of chromosome segregation in control or Y-27632-treated cells, respectively (Figure 6a). Although furrow ingression apparently ®nished within 3 min in control cells, furrow ingression takes about 9 min in Y-27632-treated cells (Figure 6a). Time from the start of chromosome segregation to furrow appearance (anaphase) prolonged slightly by Y-27632 (Figure 6b). In contrast, time from furrow appearance to the stop of ingression

In interphase cells, Rho is suggested to regulate MLC phosphorylation through its two eectors, Rho-kinase and myosin binding subunit of myosin phosphatase (reviewed in Kaibuchi et al., 1999). In this study, we showed that Rho-kinase is also implicated in MLC phosphorylation at the cleavage furrow during cytokinesis. Rho-kinase accumulating at the cleavage furrow might directly phosphorylate MLC and/or phosphorylate and inactivate myosin phosphatase in cytokinetic cells. Whether the phosphorylation state of myosin phosphatase in cytokinetic cells is aected by the Rhokinase inhibitors is currently under investigation. Inhibition of MLC phosphorylation at the cleavage furrow by the Rho-kinase inhibitors could be interpreted in several ways including the above possibilities. MLC might be dislocated in other areas, or the actinmyosin complexes might be disassembled at the cleavage furrow. It is necessary to compare precise distributions of Rho and Rho-kinase with those of actin, MLC, MLC kinase, myosin heavy chain and myosin phosphatase at the cleavage furrow in the future work. Rho-kinase can phosphorylate ERM proteins at their C-terminal threonine in vitro (Matsui et al., 1998; Fukata et al., 1998). However, Rho-kinase was recently reported not to phosphorylate ERM proteins in vivo (Matsui et al., 1999), a result that appears to con¯ict with another report that Rho-kinase can induce moesin phosphorylation in COS-7 cells (Oshiro et al., 1998). Here we showed that phosphorylated ERM proteins were increased at the cleavage furrow by both Rho-kinase inhibitors, suggesting that Rho-kinase does not phosphorylate ERM proteins at the cleavage furrow during cytokinesis. Phosphatidylinositol 4phosphate 5-kinase, another eector of Rho, might be involved in the ERM phosphorylation at the cleavage furrow as suggested by Matsui et al. (1999). We found that Rho-kinase speci®cally overaccumulated at the cleavage furrow by the Rho-kinase inhibitors, while localization patterns of RhoA, citron kinase and mDial did not change signi®cantly. There may exist feedback regulation trying to maintain Rhokinase activity at the cleavage furrow. Although the molecular mechanism of Rho-kinase overaccumulation in the presence of its inhibitors is unclear, this phenomenon suggests that Rho-kinase plays some roles at the cleavage furrow in cytokinesis. In the present study, ingression of the cleavage furrow was prolonged by Y-27632, indicating that Rho-kinase is required for the normal progression of cytokinesis. Although cytokinetic process was prolonged by Y-27632, cytokinesis was completed in the apparent absence of myosin light chain phosphorylation which indicates the lack of the functional actomyosin ring. This may be due to the undetectable low level of myosin light chain phosphorylation. Oncogene


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Figure 4 ERM proteins phosphorylated at C-terminal threonine residue (CP-ERMs) were increased by Y-27632 and HA1077 during cytokinesis. U251 cells were treated with 0, 10 or 30 mM Y-27632 (a) or HA1077 (b) for 60 min, and then stained with 297S anti-CP-ERMs mAb (green). DNAs were stained with propidium iodide (red). Scale bars, 10 mM

Figure 5 Speci®c overaccumulation of Rho-kinase at the cleavage furrow by Y-27632. U251 cells were treated with 0, 10 or 30 mM Y-27632 for 60 min, and then stained with anti-RhoA, anti-citron kinase, anti-mDial or anti-Rho-kinase antibody. Scale bar 10 mM

However, this observation is consistent with the recent report that C3-injected mammalian cultured cells lacking the myosin ring can complete cytokinesis (O'Connell et al., 1999). Furthermore, myosin II is not required for cytokinesis in Dictyostelium cells grown on a solid substrate (reviewed in Gerisch and Weber, 2000). Thus cytokinesis may occur without myosin activation in substrate-anchored cells. Oncogene

Materials and methods Materials and chemicals Y-27632 was kindly provided by Wel®de Corporation (Osaka, Japan). HA1077 was purchased from Asahi Chemical Industries Ltd. Rhodamine-phalloidin and Alexa Fluor 488-conjugated anti-rabbit, anti-rat and anti-mouse IgG were purchased from Molecular Probes. Propidium


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Figure 6 Contractile process during cytokinesis was prolonged by Y-27632. (a) U251 cells were treated with or without 30 mM Y27632. Then metaphase cells were observed at 1 min intervals. (b) U251 cells treated with or without 30 mM Y-27632 were observed at 10 s intervals and the time of anaphase (from the start of chromosome segregation to furrow appearance) and cytokinesis (from furrow appearance to the stop of ingression) was scored

iodide was purchased from Sigma. Anti-RhoA mouse mAb (26C4) was purchased from Santa Cruz Biotechnology. Cell culture U251 cells, MDBK cells and HeLa cells were cultured at 378C in Dulbecco's modi®ed Eagle's medium supplemented with 10% fetal bovine serum. Immunofluorescence microscopy Cells growing on glass coverslips were ®xed with ice-cold 10% trichloroacetic acid for 15 min and then permeabilized with 0.1% Triton X-100 in PBS for 10 min as described by Hayashi et al. (1999). For staining with rhodamine-phalloidin and pp2b (anti-phosphorylated MLC-Ser19), cells were ®xed with 3.7% formaldehyde in PBS for 10 min at room temperature and then permeabilized with 0.1% Triton X-

100 in PBS for 10 min at room temperature. Incubation with primary antibodies diluted with PBS containing 1% sucrose and 1% bovine serum albumin was for 2 h at 378C. After three washes with PBS, cells were incubated for 1 h with appropriate secondary antibodies diluted 1 : 400 and subsequently washed with PBS. Then DNAs were stained with 0.25 mg/ml propidium iodide for 5 min at room temperature. The following antibodies were used for indirect immuno¯uorescence microscopy: pp2b rabbit pAb (Matsumura et al., 1998) diluted 1 : 50; 297S (anti-CP-ERMs) rat mAb (Matsui et al., 1998) diluted 1 : 3; anti-RhoA (26C4) mouse mAb diluted 1 : 100; anti-citron kinase rabbit pAb (Madaule et al., 1998) diluted 1 : 300; anti-mDial rabbit pAb (Watanabe et al., 1997) diluted 1 : 300; anti-Rho-kinase rabbit pAb (anti-CAT; Kosako et al., 1999) diluted 1 : 450; Alexa Fluor 488-conjugated goat anti-rabbit, anti-rat and anti-mouse IgG diluted 1 : 400. Fluorescently labeled cells were examined with an Olympus LSM-GB200 confocal microscope. Oncogene


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Acknowledgments We are grateful to Drs S Yonemura, Sa Tsukita and Sh Tsukita for providing us with anti-CP-ERMs mAb (297S). H Kosako thanks Dr M Nakafuku for encouragement and support. This work was supported in part by Grants-in-Aid

for Scienti®c Research from the Ministry of Education, Science, Sports and Culture of Japan, Japan Society of the Promotion of Science Research for the Future and a grant from Bristol-Myers-Squibb.

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