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¹ Undergraduate Program for Bioinformatics and Systems Biology, Graduate School of Information Science and Technology, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113–0033, Japan
² Department of Biophysics and Biochemistry, Graduate School of Science and Technology, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
³ Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, 277-8561, Japan
⁴ PRESTO, Japan Science and Technology Agency, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
⁵ Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, 67 Tsurumai, Showa, Nagoya, Aichi, 466-8550, Japan

	Abstract

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Sustained contraction of cells depends on sustained Rho-associated kinase (Rho-kinase) activation. We developed a computational model of the Rho-kinase pathway to understand the systems characteristics. Thrombin-dependent in vivo transient responses of Rho activation and Ca²⁺ increase could be reproduced in silico. Low and high thrombin stimulation induced transient and sustained phosphorylation, respectively, of myosin light chain (MLC) and myosin phosphatase targeting subunit 1 (MYPT1) in vivo. The transient phosphorylation of MLC and MYPT1 could be reproduced in silico, but their sustained phosphorylation could not. This discrepancy between in vivo and in silico in the sustained responses downstream of Rho-kinase indicates that a missing pathway(s) may be responsible for the sustained Rho-kinase activation. We found, experimentally, that the sustained phosphorylation of MLC and MYPT1 exhibit all-or-none responses. Bromoenol lactone, a specific inhibitor of Ca²⁺-independent phospholipase A2 (iPLA2), inhibited sustained phosphorylation of MLC and MYPT1, which indicates that sustained Rho-kinase activation requires iPLA2 activity. Thus, the systems analysis of the Rho-kinase pathway identified a novel iPLA2-dependent mechanism of the sustained Rho-kinase activation, which exhibits an all-or-none response.

	Introduction

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Endothelial cells form a dynamic barrier between blood and underlying connective tissues to regulate vascular homeostasis. Endothelial cell contraction, induced by various agonists (including thrombin), plays a significant role in various physiological and pathological processes (Bogatcheva et al. 2002). Vascular permeability is correlated with the endothelial cells’ barrier function, which is determined by the equilibrium between the contractile force produced by the actomyosin cytoskeleton and the tethering one from the cell–cell and cell–extracellular matrices (Dudek & Garcia 2001; Bogatcheva et al. 2002). The barrier dysfunction is an initial step of these reactions, and hence introduces secondary reactions such as tissue edema and leukocyte migration (Dudek & Garcia 2001). Similar to other non-muscle cells, endothelial cell contraction is triggered by a myosin light chain (MLC) phosphorylation-dependent actin-myosin interaction (Garcia et al. 1995). Thrombin-dependent MLC phosphorylation consists of transient and sustained phases (van Nieuw Amerongen et al. 1998). MLC phosphorylation is balanced by the kinases including Ca²⁺/calmodulin-dependent MLC kinase (MLCK) and Rho-associated kinase (Rho-kinase)/ROK/ROCKII (Birukova et al. 2004; Kawkitinarong et al. 2004), and by myosin phosphatase (Birukova et al. 2004). Thrombin-dependent transient Ca²⁺ increase leads to activation of MLCK in a Ca²⁺/calmodulin-dependent manner (Miyazaki et al. 2002), and thereby controls transient MLC phosphorylation and subsequent transient cell contraction (Verin et al. 1998; Sandoval et al. 2001). In contrast, thrombin-dependent Rho small GTPase activation leads to sustained Rho-kinase activation (Essler et al. 1998; van Nieuw Amerongen et al. 2000; Miyazaki et al. 2002). Activated Rho-kinase directly phosphorylates MLC (Amano et al. 1996). In addition, Rho-kinase phosphorylates myosin phosphatase targeting subunit 1 (MYPT1), a regulatory subunit of myosin phosphatase. This phosphorylation inhibits myosin phosphatase activity, which elevates MLC phosphorylation. The sustained Rho-kinase activation regulates sustained MLC phosphorylation and subsequent sustained cell contraction (Emmert et al. 2004). Protein kinase C (PKC)-potentiated protein phosphatase-1 inhibitory protein of 17 kDa (CPI-17) also inhibits myosin phosphatase activity in a PKC-mediated phosphorylation-dependent manner (Kitazawa et al. 1999); however, thrombin does not induce significant phosphorylation of CPI-17 in endothelial cells (Kolosova et al. 2004). Thus, transient and sustained MLC phosphorylation are regulated by activation of MLCK and Rho-kinase, respectively. However, the quantitative relationship between these pathways at the systems level remains to be explored.

Ca²⁺-independent phospholipase A2 (iPLA2) belongs to the phospholipase A2 superfamily, which hydrolyzes the sn-2 ester bond in phospholipids to release free fatty acids such as arachidonic acid (AA) and lysophospholipids (Winstead et al. 2000; Kudo & Murakami 2002; Akiba & Sato 2004; McHowat & Creer 2004). The iPLA2 has two isoforms: iPLA2-VIA and iPLA2-VIB (Winstead et al. 2000; Kudo & Murakami 2002; Akiba & Sato 2004). The iPLA2 plays key roles in homeostatic membrane phospholipid metabolism and in cellular signaling (Balsinde et al. 1995). The iPLA2 is required for agonist-induced Ca²⁺ sensitization of contractions in vascular smooth muscle cells (Guo et al. 2003). However, how iPLA2 regulates cell contraction is unknown.

We used an integrated approach of cell biological experiments and kinetic simulation model to explore the characteristics of the Rho-kinase pathway at the systems level (Bhalla & Iyengar 1999; Kuroda et al. 2001; Lukas 2004a,b; Sasagawa et al. 2005). We found a discrepancy between in vivo and in silico in the sustained responses downstream of Rho-kinase, and predicted that a missing pathway(s) may be responsible for the sustained Rho-kinase activation. We experimentally found that Ca²⁺-independent phospholipase A2 (iPLA2) is required for the sustained Rho-kinase activation. Furthermore, we demonstrated that the sustained Rho-kinase activation exhibits all-or-none response. Thus, the sustained Rho-kinase activation is regulated by iPLA2 in an all-or-none manner.

	Results

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A possible missing pathway required for the sustained phosphorylation of MLC underlies downstream of Rho small GTPase

We incorporated recent findings such as CPI-17 (Fig. 1, Supplementary Fig. S1, Supplementary Table S1) on the basis of our previous model to develop a biochemical simulation model of signaling networks that underlie MLC phosphorylation (Kuroda et al. 2001). We tried to reproduce in vivo dynamics of MLC phosphorylation together with Ca²⁺ increase, Rho activation and MYPT1 phosphorylation in human umbilical vein endothelial cells (HUVECs), in silico (Fig. 1 boxed). Because MLC monophosphorylation is essential to trigger actin-myosin interaction (Amano et al. 1996), and monophosphorylated and diphosophorylated two isoforms of MLC comigrated, we measured the non-phosphorylated and mono-/diphosphorylated MLC in vivo and in silico. Low thrombin stimulation (0.05 Unit/mL) induced transient MLC phosphorylation, whereas high thrombin stimulation (0.25 Unit/mL) induced transient and sustained MLC phosphorylation in vivo (Fig. 2A; see also Fig. 3). However, low and high thrombin stimulations induced only transient (but not sustained) MLC phosphorylation in silico (Fig. 2B).

View larger version (22K): [in this window] [in a new window]   Figure 1  Molecular framework of thrombin-dependent Rho-kinase activation and MLC phosphorylation. Schematic overview of MLC phosphorylation cascades by thrombin stimulation. Arrows and bars indicate stimulatory and inhibitory interactions, respectively. In vivo and in silico dynamics of the indicated molecules were measured (boxes) (Figure 2). All the biochemical reactions and parameters are provided in Supplementary Fig. S1 and Table S1, respectively.

View larger version (38K): [in this window] [in a new window]   Figure 2  Dynamics of thrombin-dependent MLC phosphorylation cascades in silico and in HUVECs. Dynamics of the indicated molecules in HUVECs (in vivo) (A,C,E,G) and in silico (B,D,F,H). (A,B), MLC phosphorylation; (C,D), Ca2+ elevation; (E,F), Rho activation and (G,H), MYPT1 phosphorylation. The in silico dynamics of the indicated molecules were plotted in response to 0.01 (dotted line), 0.05 (broken line), 0.25 (thin line) and 1.0 (thick line) Unit/mL of thrombin. (A) Serum starved HUVECs were stimulated with low (0.05 Unit/mL; dashed line) or high (0.25 Unit/mL; thin line) concentrations of thrombin for the indicated time and MLC phosphorylation were measured. (C) Ca2+ elevations in HUVECs were replotted from Sandoval et al. (2001). Used with permission from Sandoval et al. (2001) © 2001 The American Physiological Society. (E) Rho activation and (G) MYPT1 phosphorylation at Thr850 were measured by Rho pull-down assay (Ren et al. 1999) and by Western blotting against anti-phospho MYPT1(pT850) with normalization by total MYPT1 amount, respectively. Values are means +/– SEM. The results are representative of three independent experiments. Thrombin concentrations used in silico were derived from the standard conversion of 1 Unit/mL = 10 nM (Bahou et al. 1993). Insets were the corresponding original gel images of the Western blotting. A.U., arbitrary unit.

View larger version (44K): [in this window] [in a new window]   Figure 3  Thrombin-dose dependency of MLC and MYPT1 phosphorylation in HUVECs. Transient and sustained MLC phosphorylation were measured in HUVECs at (A) 2 min and (B) 30 min after the thrombin stimulation, respectively. The apparent Hill coefficients, nH, and EC50s were also indicated. Unimodal distribution of the transient (C; 2 min) and sustained (D; 30 min) MLC phosphorylation in individual HUVECs. Thrombin-stimulated HUVECs were fixed and stained with anti-diphospho MLC antibody (T18, S19). Immunofluorescent intensities in individual cells were plotted in the histogram with Gaussian-fitting curves, and the mean intensities were indicated. (E) Thrombin-dose dependent MYPT1 phosphorylation (pT850) was measured at 30 min after the thrombin stimulation.

These results indicate high consistency between in vivo and in silico dynamics from thrombin to Ca²⁺ or Rho. Also, constant thrombin stimulation triggered only transient response of Ca²⁺ or Rho because of Ca²⁺ depletion from the internal Ca²⁺ store and the thrombin receptor degradation, respectively, in silico. We examined in vivo and in silico dynamics of phosphorylation of MYPT1, downstream of Rho-kinase. The low thrombin stimulation induced transient MYPT1 phosphorylation, whereas the high thrombin stimulation induced transient and sustained MYPT1 phosphorylation in vivo in Threonine 850 (Fig. 2G) and in Threonine 697 (data not shown), both of which have been shown to be phosphorylated by Rho-kinase (Feng et al. 1999; Birukova et al. 2004). In contrast, the low and high thrombin stimulations induced only transient MYPT1 phosphorylation in silico (Fig. 2H), which raises a possibility that a missing pathway underlies downstream of Rho. In addition, the sustained MLC and MYPT1 phosphorylation required the sustained Rho-kinase activation, rather than their slow dephosphorylation (Birukova et al. 2004) (see Figs 4 and 5).

View larger version (26K): [in this window] [in a new window]   Figure 4  iPLA2 activity is required for the sustained MLC phosphorylation. (A) The effects of the indicated inhibitors on the sustained MLC phosphorylation in HUVECs. The indicated inhibitors (10 µM, except for cycloheximide (50 µg/mL)) were added 30 min before the thrombin stimulation (0.25 Unit/mL) and the sustained MLC phosphorylation were measured at 30 min after the stimulation. (B) The effects of BEL (10 µM) and Y27632 (10 µM) on the thrombin (0.25 Unit/mL)-dependent MLC phosphorylation. (C) The dose-dependency of BEL and Y27632 on the thrombin (0.25 Unit/mL)-dependent sustained MLC phosphorylation (30 min). The apparent IC50s were indicated. (D) Requirement of continuous Rho-kinase and iPLA2 activities on the sustained MLC phosphorylation. Y27632 (10 µM) and BEL (10 µM) were added at 30 min before (–30') or 10 min after (10') thrombin stimulation (0.25 Unit/mL), and the sustained MLC phosphorylation was examined at 30 min after thrombin stimulation. Abbreviations: BEL, bromoenol lactone; NDGA, nordihydroguaiaretic acid.

View larger version (24K): [in this window] [in a new window]   Figure 5  iPLA2 activity is required for the sustained MYPT1 phosphorylation. (A) The effects of BEL (10 µM) and Y27632 (10 µM) on the thrombin (0.25 Unit/mL)-dependent MYPT1 phosphorylation. (B) The dose-dependency of BEL and Y27632 on the thrombin (0.25 Unit/mL)-dependent sustained MYPT1 phosphorylation (30 min). The apparent IC50s were indicated. (C) Requirement of continuous Rho-kinase and iPLA2 activities on the sustained MYPT1 phosphorylation. Y27632 (10 µM) and BEL (10 µM) were added at 30 min before (–30') or 10 min after (10') thrombin stimulation (0.25 Unit/mL), and the sustained MYPT1 phosphorylation was examined at 30 min after thrombin stimulation. (D) Effect of Y27632 and BEL on direct phosphorylation of MLC by Rho-kinase was examined in vitro. MLC phosphorylation for 2 min incubation (at the initial linear velocity) was plotted in the presence or absence of the indicated concentration of the inhibitors. (E) Ten minutes after thrombin stimulation, the indicated iPLA2 products were added with BEL simultaneously. AA (50 µM), LPA (10 µM), LPC (20 µM), PAF (10 nM). MLC phosphorylation was examined at 30 min after BEL and iPLA2 products addition.

All-or-none responses of sustained MLC and MYPT1 phosphorylation

We examined thrombin dose–dependency of transient and sustained MLC phosphorylation to explore the characteristics of the missing pathway that is responsible for the sustained MLC and MYPT1 phosphorylation. The transient MLC phosphorylation exhibited graded response, whereas the sustained MLC phosphorylation exhibited all-or-none response (Fig. 3A,B). EC₅₀s, which gave 50% of the maximal responses of the transient and sustained MLC phosphorylation, were 0.061 and 0.063 Unit/mL with Hill coefficients (n_H) of 1.8 and 6.9, respectively (Fig. 3A,B). The sustained MLC phosphorylation always exhibited an all-or-none response, even though the threshold of the response varied between 0.05 and 0.1 Unit/mL or between 0.1 and 0.25 Unit/mL (data not shown). We further confirmed the different characteristics between the transient and sustained MLC phosphorylation in individual cells. The transient MLC phosphorylation in individual cells showed a unimodal distribution and gradual shift in a thrombin-dose dependent manner (Fig. 3C). In contrast, the sustained MLC phosphorylation in individual cells also showed a unimodal distribution, but exhibited an all-or-none shift (Fig. 3D). In individual cells the intermediate level of sustained MLC phosphorylation, which can be seen in graded response (Ferrell & Machleder 1998; Ferrell 2002), was not seen around the threshold (0.05 and 0.25 Unit/mL of thrombin).

These results indicate that the transient and sustained MLC phosphorylation exhibited graded and all-or-none responses, respectively. Similarly, the sustained MYPT1 phosphorylation exhibited all-or-none response (Fig. 3E). The EC₅₀ of the sustained MYPT1 phosphorylation was 0.071 Unit/mL, with Hill coefficient of 45.7. Such high Hill coefficient may reflect a highly non-linear system for the sustained MYPT1 phosphorylation, such as the positive feedback loop systems seen in MAP kinase activation in Xenopus oocyte maturation (Bagowski & Ferrell 2001). The similarities between the sustained MLC and MYPT1 phosphorylation of EC₅₀s and high Hill coefficients suggest that both phosphorylation depend on the sustained Rho-kinase activation. The all-or-none responses of the sustained MLC and MYPT1 phosphorylation indicate a highly non-linear property of a missing pathway(s) responsible for the sustained Rho-kinase activation.

Ca²⁺-independent PLA2 activity is required for sustained Rho-kinase activation and MLC phosphorylation

We examined experimentally the effects of inhibitors of various molecules, which are reportedly involved in the regulation of MLC phosphorylation (Borbiev et al. 2003; Guo et al. 2003; Xiao et al. 2004, 2005), on the sustained MLC phosphorylation to identify a molecule(s) involved in a missing pathway that is responsible for the sustained Rho-kinase activation. Y27632, a specific inhibitor of Rho-kinase, inhibited the sustained MLC phosphorylation (Fig. 4A) as previously reported (Uehata et al. 1997; Missy et al. 2001). We found that bromoenol lactone (BEL), a specific iPLA2 inhibitor, also inhibited the sustained MLC phosphorylation (Fig. 4A), which agrees with the involvement of iPLA2 in Ca²⁺ sensitization in smooth muscle cells (Guo et al. 2003). ONO-RS-082, a general PLA2 inhibitor (Banga et al. 1986), showed a similar effect (Fig. 4A). In contrast, other inhibitors such as AACOCF3 (cytosolic PLA2 inhibitor; Bartoli et al. 1994), scalaradial (secretory PLA2 inhibitor; Marshall et al. 1994), indomethacin (general cyclooxygenase inhibitor; Futaki et al. 1994), nordihydroguaiaretic acid (lipoxygenase inhibitor; Forstermann et al. 1988), GF109203X (PKC inhibitor; Toullec et al. 1991) and U0126 (MEK inhibitor; Favata et al. 1998) did not affect the sustained MLC phosphorylation (Fig. 4A). Furthermore, cycloheximide, a protein synthesis inhibitor, did not affect the MLC phosphorylation (Fig. 4A), which indicates that the sustained MLC phosphorylation does not require de novo protein synthesis. BEL and Y27632 specifically inhibited the sustained MLC phosphorylation (Fig. 4B), which indicates that both inhibitors specifically inhibited Rho-kinase activation, rather than MLCK activation, which is responsible for the transient MLC phosphorylation (Verin et al. 1998). BEL and Y27632 decreased the basal level of MLC phosphorylation, but did not seem to significantly inhibit the transient MLC phosphorylation (Fig. 4B), the latter of which depends on MLCK (Verin et al. 1998). This also suggests that the basal level of MLC phosphorylation depends on the basal activity of Rho-kinase, which also requires iPLA2 activity. The apparent IC₅₀s of BEL and Y27632 for the sustained MLC phosphorylation, which gave 50% of the maximal inhibition, were 6.4 and 0.5 µM, respectively (Fig. 4C). The IC₅₀ of BEL was within the similar range of that for iPLA2 inhibition (Balsinde et al. 1995). This is consistent with the idea that BEL inhibits iPLA2, and subsequently inhibits Rho-kinase activation.

We next asked whether the continuous Rho-kinase and iPLA2 activities at the sustained phase are needed for the sustained MLC phosphorylation. Addition of Y27632 or BEL 10 min after the stimulation inhibited MLC phosphorylation (Fig. 4D), which indicates that the sustained MLC phosphorylation depends on the continuous Rho-kinase and iPLA2 activities at the sustained phase, and suggests that sustained MLC phosphorylation is not due to slow dephosphorylation.

We examined whether the sustained MYPT1 phosphorylation is also inhibited by these inhibitors, and found that Y27632 and BEL similarly inhibited the sustained MYPT1 phosphorylation (Fig. 5A). The apparent IC₅₀s of BEL and Y27632 for the sustained MYPT1 phosphorylation were 7.6 and 0.7 µM, respectively (Fig. 5B), both of which are similar to those for the sustained MLC phosphorylation (Fig. 4C). We also examined whether the continuous Rho-kinase and iPLA2 activities at the sustained phase are needed for the sustained MYPT1 phosphorylation. Addition of Y27632 or BEL 10 min after the stimulation inhibited MYPT1 phosphorylation (Fig. 5C). We also confirmed that Y27632, but not BEL, inhibited Rho-kinase-dependent MLC phosphorylation in vitro (Fig. 5D), indicating that the effect of BEL is not due to the inhibition of Rho-kinase activity. Taken together, these results indicate that iPLA2 activity is required for the sustained Rho-kinase activation, and suggest that the sustained MLC and MYPT1 phosphorylation are not due to slow dephosphorylation.

It is possible that some specific irreversible distribution of molecules can be responsible for the sustained MYPT1 and MLC phosphorylation. However, our result of the requirement of continuous activity of Rho-kinase for sustained phosphorylation (Figs 4D and 5C) indicates that the sustained phosphorylation is a reversible reaction. Therefore, this possibility seems to be less likely.

We further examined whether some products of iPLA2, such as AA, lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), or platelet-activating factor (PAF), can rescue the inhibitory action of BEL on the sustained MLC phosphorylation (Fig. 5E). However, none of the products could rescue the inhibitory action of BEL. This result suggests that BEL may affect additional molecules other than iPLA2, or that other products of iPLA2, those that have not been tested in this study, may be responsible for the sustained Rho-kinase activation.

We detected the expression of iPLA2-VIA protein, but not iPLA2-VIB protein, in HUVECs (data not shown). We tried to use siRNA to Rho-kinase or iPLA2 to examine whether reduction of these proteins inhibits sustained MLC phosphorylation, but we could reduce each protein expression only by 17% and 11%, respectively, when lamin A/C reduced its protein by 74% (data not shown). We did not further examine the role of both proteins with siRNAs in the sustained Rho-kinase activation.

	Discussion

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Here we show that the sustained Rho-kinase activation requires continuous iPLA2 activity, and exhibits all-or-none response in sustained MLC phosphorylation. We tried to examine the involvement of iPLA2 products and their derivatives, such as AA, LPA, LPC or PAF. Addition of these products to HUVECs did not rescue the inhibitory effects of BEL on thrombin-induced MLC phosphorylation (Fig. 5E). These results suggest that iPLA2 is not a direct upstream molecule of Rho-kinase, but that iPLA2 is needed to activate unidentified upstream molecules of Rho-kinase, which induce the sustained Rho-kinase activation. Alternatively, BEL may affect unknown molecules other than iPLA2, and thereby inhibit sustained Rho-kinase activation. Further study is necessary to address this issue.

Sustained Rho-kinase activation exhibits an all-or-none response, which indicates a highly non-linear property of iPLA2-dependent sustained Rho-kinase activation. Although how iPLA2 regulates sustained Rho-kinase activation is unknown, in principle, the all-or-none response at the steady state can arise from a monostable, ultrasensitive system with a high Hill coefficient or from a bistable system (Ferrell 2002). Therefore, the sustained Rho-kinase activation is regulated by the iPLA2-dependent sustained pathway, which alternatively depends on a monostable or bistable system (Fig. 6). In HUVECs, the high thrombin stimulation induced transient Rho activation and sustained Rho-kinase activation, which clearly indicates the uncorrelated activation of Rho and Rho-kinase. On the other hand, Rho activation is required in many cells for Rho-kinase activation (Kawano et al. 1999; Anderson et al. 2004; Kawkitinarong et al. 2004; van Nieuw Amerongen et al. 2004). In a bistable system such as a positive feedback loop, a suprathreshold stimulation (even a transient one) can induce the transition of the system from the lower to the upper stable point. Considering the behavior of the bistable system, the transient Rho activation, induced by the high thrombin stimulation, may be regarded as a suprathreshold stimulation. It may trigger the transition from the lower to the upper stable point of the bistable system and result in sustained Rho-kinase activation. Therefore, transient Rho activation may be required for sustained Rho-kinase activation. We experimentally tried to show more direct evidence of bistable systems; hysteresis, which results in a different dose-response curve in response to thrombin with prior or subsequent addition of BEL (Supplementary Fig. S2). If the system is bistable, the dose-response curve of subsequent addition shifts to the right. However, the dose-response curves are similar with prior and subsequent BEL addition, suggesting that the sustained MLC phosphorylation is a monostable system. However, further study is necessary to address whether iPLA2-dependent Rho-kinase activation depends on a monostable or bistable system.

View larger version (15K): [in this window] [in a new window]   Figure 6  Possible pathways responsible for the sustained Rho-kinase activation.The sustained Rho-kinase activation exhibits all-or-none response that may depend on a monostable ultrasensitive system or a bistable system in an iPLA2-dependent manner.

	Experimental procedures

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Reagents

All inhibitors were obtained from commercial sources. Y27632, AACOCF3, Scalaradial and indomethacin were from Calbiochem (San Diego, CA, USA). BEL and NDGA were from Cayman (Ann Arbor, MI, USA). ONO-RS-082 and GF109203X were from Biomol (Plymouth Meeting, PA, USA). U0126 was from Promega (Madison, WI, USA). Cycloheximide was from Sigma (St. Louis, MO, USA). [-³²P]ATP was from Amersham (Little Chalfont, UK). The plasmid for glutathione-S-transferase (GST)-Rhotekin-Rho-binding domain (RBD) was kindly provided by Martin A. Schwartz at the University of Virginia (Charlottesville, VA, USA).

Block diagram and simulation of MLC phosphorylation

All reactions were represented by molecule-molecule interactions and enzymatic reactions (Bhalla & Iyengar 1999). We used a GENESIS simulator (version 2.2) with a Kinetikit (version 8) interface to solve the ordinary differential equations with a time step of 10 ms, and simulation was run for 3600 s. The model consisted of 28 molecules and 68 rate constants. The rate constants consisted of 45 and 23 rate constants for molecule-molecule interactions and enzymatic reactions, respectively. The biochemical reactions and the rate constants used in the study are shown in Supplementary Fig. S1 and Supplementary Table S1, respectively. We combined mono- and di-phosphorylated MLC as phosphorylated MLC for comparison with the experimental data. The GENESIS script of our in silico model is also available as a text file on our website (http://www.kurodalab.org/info/myosin.g). The model in SBML format can be also obtained on our website (http://www.kurodalab.org/info/myosin.xml).

Cell culture and thrombin stimulation

HUVECs and culture media (HuMedia-EG2) were obtained from a commercial source (Kurabo, Osaka, Japan). HUVECs at the 6th passage were seeded on to 35-mm collagen-coated dishes at 2.5 x 10⁵ cells. One day later, HUVECs were starved for serum for 16 h and then stimulated with thrombin (Sigma). For the experiments with inhibitors, HUVECs were pretreated with inhibitors 30 min before thrombin stimulation unless otherwise specified.

MLC and MYPT1 phosphorylation

After thrombin stimulation, the reaction was terminated with 10% (wt/vol) trichloroacetic acid. For MLC phosphorylation, the resulting cell precipitates were solubilized with urea sample buffer and subjected to urea/glycerol gels (Garcia et al. 1995). Cell lysates were transferred onto nitrocellulose membrane and subjected to immunoblotting with anti-MLC antibody (1 : 400; Sigma). For MYPT1 phosphorylation, the cell precipitates were solubilized with SDS sample buffer, and subjected to immunoblotting with anti-phospho MYPT1 (T850) antibody (1 : 500; Upstate, Lake Placid, NY, USA). The membranes were stripped with stripping buffer (0.2 M glycine, pH 2.5; 0.1% TritonX-100) and reprobed with anti-MYPT1 antibody (1 : 500; BD Biosciences, Franklin Lakes, NJ, USA) for normalization. Horseradish preoxidase (HRP)-conjugated secondary antibodies (Amersham) were used at 1: 2000 and an enhanced chemiluminescence (ECL) detection kit (Amersham) was used for HRP detection.

GTP-Rho pull-down assay

The activation of RhoA was determined by pull-down assay with GST-Rhotekin-RBD as described (Ren et al. 1999). Briefly, the GTP-Rho bound to beads was subjected to SDS-PAGE followed by immunoblotting with anti-Rho antibody (1 : 100; Santa Cruz, Santa Cruz, CA, USA).

Immunofluorescence analysis

HUVECs were serum starved for 16 h and stimulated with various concentrations of thrombin. After the stimulation, cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) for 10 min and permeabilized with 0.2% TritonX-100 in PBS for 10 min. Phosphorylated MLC was stained with anti-diphospho MLC antibody (T18, S19) (1 : 200; Cell Signaling, Beverly, MA, USA), and then labeled with FITC-conjugated anti-rabbit IgG antibody (1 : 200; Amersham). ß-catenin was doubly stained with anti-ß-catenin antibody to identify the cell margin (1 : 100; BD Biosciences), followed by label with Cy3-conjugated anti-mouse IgG antibody (1 : 200; Amersham). Images were taken by fluorescent microscope (BX51, Olympus, Tokyo, Japan) and intensities per cells were analyzed by AQUA-Lite (version 1.2, Hamamatsu, Shizuoka, Japan) software. Immunofluorescent intensities were plotted in the histogram and overlaid with Gaussian-fitting curves.

In vitro MLC phosphorylation assay

The phosphorylation assay was performed as previously described (Amano et al. 1996) with minor modification. The kinase reaction for Rho-kinase was carried out in 50 µL of a reaction mixture (50 mM Tris/HCl at pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 5 mM MgCl₂, 100 µM [-³²P]ATP (7.4 GBq/mmol) and 2 nM GST-Rho-kinase-CAT). GST-MLC (1 µM) was incubated as a substrate in the presence of BEL or Y27632 for 2 min at 30 °C. The reaction was terminated by addition of SDS sample buffer and boiling. The samples were subjected to SDS-PAGE and analyzed by an image analyzer (Fujifilm, Kanagawa, Japan).

	Acknowledgements

 
We thank Dr M. Amano for critically reading this manuscript and H. Kobayashi for establishment of the simulation model in SBML format. This work was supported by KAKENHI (grant-in-aid for scientific research) on Priority Areas ‘Systems Genomics’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by a grant-in-aid from the Nakajima Foundation.

	Footnotes

 
Communicated by: Noriko Osumi

^aPresent address: Neurobiology Sector, SISSA—ISAS, via Beirut 2–4, 34014 Trieste, Italy

^* Correspondence: E-mail: skuroda{at}bi.s.u-tokyo.ac.jp

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Received: 23 February 2006
Accepted: 11 June 2006

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