TheSmallGTPaseRhoARegulatestheContractionofSmooth ... · Activated GTP-bound RhoA or cdc42 were affinity-precipi-tated by glutathione beads and quantified by immunoblot. CellDissociationandImmunofluorescenceAnalysis—Freshly

The Small GTPase RhoA Regulates the Contraction of SmoothMuscle Tissues by Catalyzing the Assembly of CytoskeletalSignaling Complexes at Membrane Adhesion Sites*□S

Received for publication, April 9, 2012, and in revised form, August 9, 2012 Published, JBC Papers in Press, August 13, 2012, DOI 10.1074/jbc.M112.369603

Wenwu Zhang, Youliang Huang, and Susan J. Gunst1

From the Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202

Background: RhoA GTPase regulates airway smooth muscle (SM) contraction and actin polymerization by an unknownmechanism.Results: Agonist stimulation induces the RhoA-dependent recruitment of paxillin, vinculin, FAK, and cdc42 GEFs to adhe-somes, which catalyze cdc42 and N-WASP activation.Conclusion: RhoA regulates actin polymerization and SM contraction by regulating the adhesome assembly.Significance: Rho-mediated adhesome assembly is a novel mechanism for the regulation of agonist-induced SM contraction.

The activation of the small GTPase RhoA is necessary forACh-induced actin polymerization and airway smooth muscle(ASM) contraction, but the mechanism by which it regulatesthese events is unknown. Actin polymerization in ASM is cata-lyzed by the actin filament nucleation activator, N-WASp andthe polymerization catalyst, Arp2/3 complex. Activation of thesmall GTPase cdc42, a specific N-WASp activator, is alsorequired for actin polymerization and tension generation. Weassessed the mechanism by which RhoA regulates actin dynam-ics and smooth muscle contraction by expressing the dominantnegative mutants RhoA T19N and cdc42 T17N, and non-phos-phorylatable paxillin Y118/31F and paxillin �LD4 deletionmutants in SM tissues. Their effects were evaluated in muscletissue extracts and freshly dissociated SM cells. Protein interac-tions and cellular localization were analyzed using proximityligation assays (PLA), immunofluorescence, and GTPase andkinase assays. RhoA inhibition prevented ACh-induced cdc42activation, N-WASp activation and the interaction of N-WASpwith the Arp2/3 complex at the cell membrane. ACh inducedpaxillin phosphorylation and its association with the cdc42GEFS, DOCK180 and �/�PIX. Paxillin tyrosine phosphoryla-tion and its association with �PIX were RhoA-dependent, andwere required for cdc42 activation. The ACh-induced recruit-ment of paxillin and FAK to the cell membrane was dependenton RhoA. We conclude that RhoA regulates the contraction ofASM by catalyzing the assembly and activation of cytoskeletalsignaling modules at membrane adhesomes that initiate signal-ing cascades that regulate actin polymerization and tensiondevelopment in response to contractile agonist stimulation.Ourresults suggest that the RhoA-mediated assembly of adhesomecomplexes is a fundamental step in the signal transduction pro-cess in response to agonist -induced smoothmuscle contraction.

The small GTPase RhoA is widely recognized as an impor-tant regulator of cytoskeletal dynamics inmany cell types (1–5).RhoA activation can stimulate phosphorylation of the regula-tory light chain ofmyosin II through its inhibitory effects on thecatalytic activity of smooth muscle myosin light chain (MLC)2phosphatase or by activating kinases such as ROCK thatdirectly phosphorylate MLC (6–9). In smooth muscle, theRhoA-mediated effects on smooth muscle (SM) myosin II acti-vation have beenwidely recognized to play an important role inthe regulation of contractility and shortening (8, 9). However,in airway smoothmuscle tissues, the inhibition of RhoA activa-tion profoundly depresses agonist-induced tension develop-ment with little effect on SMMLCphosphorylation; thus RhoAappears to regulate the contractility of airway smooth muscleby a mechanism that is largely independent of its effects on SMmyosin II regulatory light chain phosphorylation (10).Actin polymerization and cytoskeletal reorganization play a

key role in the regulation of active tension in many smoothmuscle tissues and cells, including airway smooth muscle (11–15). The inhibition of actin polymerization depresses tensiondevelopment in response to contractile stimulation with littleor no effect on SM myosin II regulatory light chain phosphor-ylation and crossbridge cycling (11, 12, 16, 17). RhoA inactiva-tion dramatically depresses stimulus-induced actin polymeri-zation in airway smoothmuscle tissues, suggesting that the roleof RhoA in regulating active tension development in this tissueis primarily due to its effects on actin cytoskeletal dynamics.The objective of this study was to evaluate themolecularmech-anisms by which RhoA regulates cytoskeletal dynamics andtension development in airway smooth muscle.In airway smooth muscle, agonist-stimulated actin polymer-

ization requires activation of the actin nucleation promoting

* This work was supported, in whole or in part, by NHLBI, National Institutes ofHealth (NIH) Grants HL-29289 and HL074099; and an American Lung Asso-ciation and an NIH T32 postdoctoral fellowship (to W. Z.).

□S This article contains supplemental Figs. S1–S4.1 To whom correspondence should be addressed: Indiana University School

of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202. Tel.: 317-274-4108;E-mail: [email protected].

2 The abbreviations used are: MLC, smooth muscle myosin light chain; SM,smooth muscle; ASM, airway smooth muscle; PLA, proximity ligation assay;N-WASp, neuronal Wiskott-Aldrich syndrome protein; GEF, guanine nucle-otide exchange factor; PIX, PAK-interacting exchange factor; DOCK,Dedicator of Cytokinesis; ACh, acetylcholine; ROCK, Rho kinase; PAK, p21-activated kinase; GIT, G-protein receptor kinase-interacting tyrosinephosphorylated.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 41, pp. 33996 –34008, October 5, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

33996 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 41 • OCTOBER 5, 2012

by guest on March 8, 2020

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/cgi/content/full/M112.369603/DC1

http://www.jbc.org/

protein, N-WASp (neuronal Wiskott-Aldrich syndrome pro-tein) (17).WhenN-WASp is activated, it undergoes a change inconformation that enables it to bind to the actin-related proteincomplex (Arp2/3 complex), which forms a template for theaddition of monomeric actin (G-actin) to existing F-actin fila-ments resulting in formation of new F-actin (18–20). N-WASpactivation is directly and specifically regulated by the smallGTPase, cdc42, which binds to its CRIB domain (20–23). Nei-ther Rac nor Rho GTPases can bind directly to WASP familyproteins, thus RhoA cannot directly regulate N-WASp activity(24, 25). N-WASp is activated by cdc42 during the contractilestimulation of airway smooth muscle tissues, and cdc42 activa-tion is necessary for actin polymerization and active tensiondevelopment in this tissue (26).Proteins that localize to extracellular matrix-cytoskeletal

membrane junctions (adhesomes) have been implicated in theactivation of N-WASp and actin polymerization during thecontractile simulation of airway smooth muscle: The scaffold-ing/adaptor protein paxillin (27, 28) is recruited to the mem-brane and undergoes tyrosine phosphorylation in response toagonist stimulation (29, 30). Paxillin phosphorylation is re-quired for agonist-stimulated actin polymerization and con-traction in airway smooth muscle (31). The paxillin-bindingprotein vinculin (27) also localizes to adhesome complexes dur-ing the contractile stimulation airway smooth muscle and con-tributes to the regulation of actin polymerization (29, 32), asdoes focal adhesion kinase (FAK) (29), which induces the tyro-sine phosphorylation of paxillin (33, 34).Our present results provide evidence that RhoA regulates

tracheal smooth muscle contraction by catalyzing the recruit-ment of paxillin, vinculin, and FAK to an adhesion junctionassociated signaling complex, and that this promotes the asso-ciation of paxillin with the cdc42 guanine nucleotide exchangefactors (GEFs), PIX (PAK-interacting exchange factor) andDOCK180 (Dedicator of Cytokinesis) (35). This adhesome sig-naling complex mediates the activation cdc42, which regulatesactivation of the actin polymerization catalysts, N-WASp andthe Arp 2/3 complex and the polymerization of cortical actin.These results document a novel mechanism for the regulationof smooth muscle contraction by RhoA that is distinct from itspreviously documented role in regulating the phosphorylationof the regulatory light chain of SMmyosin II. Our observationssuggest that the RhoA-mediated assembly of adhesome signal-ing complexes is an essential step in excitation-contractioncoupling and tension development in smooth muscle tissuesduring agonist-induced contractile activation.

EXPERIMENTAL PROCEDURES

Preparation of SmoothMuscle TissuesMeasurement of Force—Mongrel dogs (20–25 kg) were anesthetized with pentobarbitalsodium (30 mg/kg, iv.) and quickly exsanguinated in accord-ance with procedures approved by the Institutional AnimalCare and Use Committee (IACUC), Indiana University SchoolofMedicine. A tracheal segmentwas immediately removed andimmersed in physiological saline solution (PSS) (compositioninmM: 110NaCl, 3.4 KCl, 2.4 CaCl2, 0.8MgSO4, 25.8 NaHCO3,1.2 KH2PO4, and 5.6 glucose, bubbled with 95% O2, 5% CO2).Strips of tracheal smoothmuscle (1.0� 0.2–0.5� 15mm)were

dissected free of connective and epithelial tissues and main-tainedwithin a tissue bath in PSS at 37 °C. Contractile forcewasmeasured isometrically by attaching the tissues to Grass force-displacement transducers. Prior to the beginning of each exper-imental protocol, muscle length was increased to maintain apreload of �1 gm, and tissues were stimulated repeatedly withacetylcholine (ACh) until stable responses were obtained. Theforce of contraction to ACh was determined before and aftertreatment with plasmids or other reagents.Transfection of Smooth Muscle Tissues—The use of con-

structs for the hemagglutinin (HA) human RhoA Asn-19,human cdc42 Asn-17, and chicken paxillin Y118/31F in tra-cheal smooth muscle tissues have been previously described(10, 26, 31). Constructs for chicken paxillin �LD4 (36, 37),chicken paxillin Y118/31F were generously provided by Dr.Christopher Turner, SUNY Upstate Medical University. ThecDNAs encoding these constructs were subcloned into themammalian expression vector pcDNA3.1. Escherichia coli(Bluescript) transformedwith these plasmids were grown in LBmedium, and plasmids were purified by alkaline lysis with SDSusing a purification kit from Qiagen Inc.Plasmidswere introduced into tracheal smoothmuscle strips

by the method of reversible permeabilization (10, 17, 26, 31).After equilibration of the tissues and establishing a musclelength for the generation of maximal isometric force, musclestrips were attached tometal mounts tomaintain them isomet-rically at constant length. The strips were incubated succes-sively in each of the following solutions: Solution 1 (at 4 °C for120 min) containing (in mM): 10 EGTA, 5 Na2ATP, 120 KCl, 2MgCl2, and 20 N-Tris(hydroxymethyl)methyl-2-aminoethane-sulfonic acid (TES); Solution 2 (at 4 °C overnight) containing (inmM): 0.1 EGTA, 5 Na2ATP, 120 KCl, 2 MgCl2, 20 TES, and 10�g/ml plasmids. Solution 3 (at 4 °C for 30 min) containing (inmM): 0.1 EGTA, 5 Na2ATP, 120 KCl, 10 MgCl2, 20 TES; andSolution 4 (at 22 °C for 60 min) containing (in mM): 110 NaCl,3.4 KCl, 0.8 MgSO4, 25.8 NaHCO3, 1.2 KH2PO4, and 5.6 dex-trose. Solutions 1–3 were maintained at pH 7.1 and aeratedwith 100% O2. Solution 4 was maintained at pH 7.4 and wasaerated with 95%O2-5%CO2. After 30min in Solution 4, CaCl2was added gradually to reach a final concentration of 2.4 mM.The strips were then incubated in a CO2 incubator at 37 °C for2 days in serum-free DMEM containing 5 mM Na2ATP, 100units/ml penicillin, 100 �g/ml streptomycin, and 10 �g/mlplasmids.Assessment of RhoA and cdc42 Activation—The activation of

RhoA or cdc42was determined using pull-down assays for acti-vated RhoA or activated cdc42 (10, 26). Pulverized muscle tis-sues were mixed with lysis buffer (1% Np-40, 50 mM Tris-HCl,pH 7.4, 150 mM NaCl, 10% glycerol, 2 mM EDTA, phosphataseinhibitors (in mM: 2 sodium orthovanadate, 2 molybdate, and 2sodium pyrophosphate), and protease inhibitors (inmM: 2 ben-zamidine, 0.5 aprotinin, and 1 phenylmethylsulfonyl fluoride)for 2 h at 4 °C. For the analysis of RhoA activation, the extractedproteinswere reactedwith aGST-peptide for theRBD region ofrhotekin, which has a high affinity for GTP-Rho. For the anal-ysis of cdc42 activation, the extracted proteins were reactedwith GST-p21-activated kinase (PAK) binding domain (PBD).

RhoA Mediates Smooth Muscle Contractility by Regulating Adhesome Assembly

OCTOBER 5, 2012 • VOLUME 287 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 33997


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Activated GTP-bound RhoA or cdc42 were affinity-precipi-tated by glutathione beads and quantified by immunoblot.CellDissociation and ImmunofluorescenceAnalysis—Freshly

dissociated primary cells were used for these studies to avoidthe morphological changes in cytoskeletal organization andchanges in phenotype that occur during the culture of smoothmuscle cells. Smooth muscle cells were enzymatically dissoci-ated from tracheal muscle strips (17). For immunofluorescenceanalysis, freshly dissociated cells were plated onto glass cover-slips and allowed to adhere for 30–60 min, were stimulatedwith 10�5 M ACh or left unstimulated, and then fixed, permea-bilized, and incubated with primary antibodies against proteinsof interest.In Situ Proximity LigationAssay—DuolinkTM in situ proxim-

ity ligation assays (PLA) were performed to detect cellularinteractions betweenN-WASp and Arp2, vinculin and paxillin,FAK and paxillin, or PIX and paxillin (32, 38). PLA yields afluorescent signal when the target proteins are localized within40 nm of each other. Smooth muscle cells were freshly dissoci-ated from sham-treated or transfected canine tracheal smoothmuscle tissues as previously described (17, 29). Dissociatedsmooth muscle cells were stimulated with 10�5 M ACh or leftunstimulated and then fixed, permeabilized and incubatedwithprimary antibodies followed by secondary antibodies conju-gated to PLA probes. Duolink hybridization, ligation, amplifi-cation, and detection media were administered according tothe manufacturer’s instructions. Randomly selected cells fromboth unstimulated and ACh-stimulated groups were analyzedfor N-WASP-Arp2, vinculin, and paxillin, FAK and paxillin orpaxillin, and �-PIX interactions by visualizing Duolink fluores-cent spots using a Zeiss LSM 510 confocal microscope. Thetotal number of Duolink fluorescent spots for the N-WASp-Arp2 and paxillin-FAK interactions were counted using OlinkBioscience Image Tools software. The localization of the vincu-lin-paxillin complex was evaluated by determining the ratio ofpixel intensity between the cell periphery and the cell interiorusingMetamorph software (MolecularDevices, Inc. Sunnyvale,CA) (see supplemental Fig. S1 for details).Immunoblots—Pulverized muscle strips were mixed with

extraction buffer containing: 20 mM Tris-HCl at pH 7.4, 2%TritonX-100, 0.2%SDS, 2mMEDTA, phosphatase inhibitors (2mM sodium orthovanadate, 2mMmolybdate, and 2mM sodiumpyrophosphate), and protease inhibitors (2 mM benzamidine, 0.5mM aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Eachsample was centrifuged, and the supernatant was then boiled insample buffer (1.5% dithiothreitol, 2% SDS, 80 mM Tris-HCl, pH6.8, 10% glycerol, and 0.01% bromphenol blue) for 5min. Proteinswere separated by SDS-PAGE and transferred to nitrocellulose.The nitrocellulose membrane was blocked with 5% milk for 1 hand probed with primary antibodies against proteins of interestfollowed by horseradish peroxidase-conjugated IgG. Proteinswere visualized by enhanced chemiluminescence (ECL).Immunoprecipitation of Proteins—Pulverized muscle tissues

were mixed with extraction lysis buffer (see “Assessment ofRhoA and cdc42 Activation”). Each sample was centrifuged(14,000 � g) for 30 min for the collection of supernatant. Mus-cle extracts containing equal amounts of protein were pre-cleared for 30 min with 30 �l of 10% protein A/G-Sepharose

and then incubated overnightwith primary antibodies. Sampleswere then incubated for 2 h with 50 �l of a 10% suspensionof protein A/G-Sepharose beads. Immunocomplexes werewashed three times in a buffer containing 50 mM Tris HCl, pH7.6, 150 mM NaCl, and 0.1% Triton X-100. All procedures ofimmunoprecipitation were performed at 4 °C.Analysis of F-actin and G-actin—The relative proportions of

F-actin and G-actin in smooth muscle tissues were analyzedusing an assay kit from Cytoskeleton as previously described(10, 17, 26, 39). Briefly, each of the tracheal smooth musclestrips was homogenized in 200 �l of F-actin stabilization buffer(50mM PIPES, pH 6.9, 5 0mMNaCl, 5mMMgCl2, 5 mM EGTA,5% glycerol, 0.1% Triton X-100, 0.1% Nonidet P-40, 0.1%Tween-20, 0.1% �-mercaptoethanol, 0.001% antifoam, 1 mM

ATP, 1�g/ml pepstatin, 1�g/ml leupeptin, 10�g/ml benzami-dine, and 500 �g/ml tosyl arginine methyl ester). Supernatantsof the protein extracts were collected after centrifugation at150,000 � g for 60 min at 37 °C. The pellets were resuspendedin 200�l of ice-coldwater containing 10�M cytochalasinD andthen incubated on ice for 1 h to depolymerize F-actin. Theresuspended pellets were gently mixed every 15 min. Fourmicroliters of supernatant (G-actin) and pellet (F-actin) frac-tions were subjected to immunoblot analysis using anti-actinantibody (cloneAC-40; Sigma). The relative amounts of F-actinand G-actin were determined using densitometry.Reagents and Antibodies—RhoA and cdc42 activation assay

kits were obtained from Cytoskeleton (Denver, CO). ROCKinhibitors, H-1152P and Y27632 were obtained from Calbio-chem. The ROCK activity assay kit was obtained fromCell Bio-labs (San Diego, CA). The FAK inhibitor FP 573228 wasobtained from Tocris Bioscience (Bristol, UK). The DuolinkTMin situ proximity ligation assay kit was purchased from OlinkBioscience (Uppsala, Sweden). All other reagentswere obtainedfrom Sigma.Sources of antibodies are as follows: monoclonal RhoA,

Cytoskeleton; monoclonal paxillin and monoclonal cdc42,monoclonal anti-CrkII, BD Transduction; polyclonal paxillintyrosine 118, BIOSOURCE; monoclonal N-WASp, polyclonalArp2, N-WASp tyrosine 256, Abcam; polyclonal anti-FAK,polyclonal anti-�-PIX and anti-�-PIX, polyclonal anti-GIT1and GIT2, polyclonal anti-DOCK180, polyclonal anti-PAK1,Cell Signaling; monoclonal anti-FAK and monoclonal anti-FAK Y397, BD Biosciences; monocloncal anti-ELMO1, horse-radish peroxidase-conjugated IgG, Amersham Biosciences;polyclonal vinculin antibody (against canine cardiac vinculin)was custom made by BABCO (Richmond, CA).Statistical Analysis—Comparisons between two groups were

performed using paired or unpaired Student’s t tests. Compar-isons among multiple groups were performed using one-wayANOVA. Values refer to the number of cells or tissue stripsused to obtain mean values. p � 0.05 was considered statisti-cally significant.

RESULTS

RhoA Inactivation Inhibits ACh-inducedN-WASpActivationin Smooth Muscle Tissues—We expressed RhoA T19N proteinin tracheal muscle tissues to determine whether RhoA activa-tion is necessary for the ACh-induced activation of N-WASp,




http://ww

w.jbc.org/

Dow

nloaded from


http://www.jbc.org/

which is required for ACh-induced actin polymerization andtension development (17). The expression of the dominant-negative RhoAT19N protein in tracheal muscle tissues inhibitsendogenous RhoA activity and suppresses actin polymerizationand active tension generation in response to stimulation withACh with little effect on SM myosin light chain phosphoryla-tion (10).When N-WASp undergoes activation, it localizes at the

cell membrane and undergoes a conformational change thatenables it to bind to the Arp2/3 complex and undergo tyro-sine phosphorylation at residue 256 (19, 20, 40). To assessN-WASp activation in tissue extracts, we measuredN-WASp tyrosine 256 phosphorylation and evaluated theinteraction of N-WASp with the Arp2/3 complex. Our

results show that expression of RhoA T19N inhibits theACh-induced increase in N-WASp tyrosine 256 phosphory-lation (Fig. 1A), and that this inhibition of N-WASp activa-tion is associated with a marked depression of ACh-inducedactive tension development (Fig. 1B). The inhibition of actinpolymerization by RhoA T19N was confirmed in a separateset of tissues (Fig. 1C). Co-immunoprecipitation analysis dem-onstrated that the RhoA T19 mutant also inhibited theincreased interaction of Arp2 and N-WASp that occurs inresponse to ACh stimulation (Fig. 1D). The ACh-inducedincrease in N-WASp Y256 phosphorylation, the association ofN-WASp with the Arp2/3 complex, actin polymerization andactive tension developmentwere unaffected by sham treatmentof the tissues and/or by the expression of WT RhoA.

FIGURE 1. RhoA inactivation inhibits ACh induced N-WASp activation in smooth muscle tissues. A, N-WASp tyrosine 256 phosphorylation measured byimmunoblot in extracts of 6 muscle tissues transfected with wild type RhoA (WT), RhoA T19N, or sham-treated. RhoA T19N significantly inhibited ACh-inducedN-WASp phosphorylation relative to WT RhoA or sham-treated tissues (n � 5). B, RhoA T19N significantly inhibited ACh-induced tension development relativeto WT RhoA or sham-treated tissues (n � 10). (All force measurements normalized to sham response.) C, immunoblot of soluble (G, globular) and insoluble(F, filamentous) actin in fractions from extracts of unstimulated or ACh-stimulated muscle tissues treated with RhoA T19N or with no treatment (sham). RhoAT19N significantly inhibited the increase in the F actin to G-actin ratio in response to 10�5

M ACh (n � 4). D, ACh induced a significant increase in Arp2coimmunoprecipitation with N-WASp in sham-treated tissues but not in RhoA T19N-treated tissues (n � 4). IP: immunoprecipitate; IB, immunoblot. E, duolinkin situ PLA shows the interaction of N-WASp and Arp2 in freshly dissociated differentiated canine tracheal smooth muscle cells stimulated with ACh, but not inunstimulated cells. Fluorescence and phase contrast images are shown for each cell. F, in cells from sham-treated tissues, mean DuoLink PLA spots weresignificantly higher in ACh-stimulated cells than in unstimulated cells (n � 27, 25). In cells from RhoA T19N-treated tissues, the mean number of DuoLink PLAspots was very small and did not change significantly with ACh-stimulation (n � 24, 32 respectively). (Cells dissociated from tissues obtained from threeseparate experiments.) All tissues and cells were incubated with ACh for 5 min. All values are means � S.E. *, significantly different (p � 0.05).




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

A Duolink in situ PLA using probes targeted to N-WASp,and Arp2 was also used to assess the interaction of N-WASpand Arp2 in freshly dissociated tracheal smooth muscle cellsfrom untreated tissues and tissues treated with RhoAT19N(Fig. 1, E and F and supplemental Fig. S2). In sham-treated

cells, stimulation with ACh resulted in a dramatic increase inthe number of fluorescent spots along the membrane, indi-cating many interactions between N-WASp and the Arp2/3complex. In contrast, ACh-stimulated cells expressing RhoA

FIGURE 2. RhoA inactivation inhibits the ACh-induced activation of cdc42in canine tracheal smooth muscle tissues. A, activated cdc42 (cdc42-GTP)was affinity-precipitated from extracts of 3 unstimulated and 3 ACh-stimu-lated muscle strips, and the amount of activated cdc42 precipitated fromeach extract was quantified by immunoblot. Activated cdc42 was signifi-cantly higher in extracts from 10�5

M ACh stimulated sham-treated tissuesthan from ACh stimulated tissues expressing RhoA T19N or cdc42 T17N (n �8). Blot shows non-contiguous lanes from a single gel. B, RhoA T19N andcdc42 T17N significantly inhibited tension development in response to ACh(n � 16). C, activated GTP-bound RhoA (RhoA-GTP) was affinity-precipitatedfrom extracts of 6 unstimulated or ACh stimulated muscle strips and quanti-fitated by immunoblot. RhoA activation was significantly inhibited in tissuesexpressing RhoA T19N but was unaffected by the expression of cdc42 T17N(n � 6). Blot shows non-contiguous lanes from a single gel. All values aremeans � S.E. *, significantly different, p � 0.05.

FIGURE 3. Rho regulates cdc42 and N-WASp activation by regulating pax-illin phosphorylation. A, paxillin Tyr-118 phosphorylation in response to10�5

M ACh stimulation was significantly inhibited in tissues expressing RhoAT19N, but not in tissues expressing cdc42 T17N (n � 10). Blot shows non-contiguous lanes from a single gel. B, Thr-696 phosphorylation of MYPT1, asubstrate of activated ROCK, was measured by immunobot in tissue extractsfrom ACh stimulated tissues. MYPT1 phosphorylation was significantly lowerin tissues treated with the ROCK inhibitor, H-1152P (1 �M) (n � 4). C, there areno significant effects of the ROCK inhibitor on paxillin phosphorylation inACh-stimulated or unstimulated tissues (n � 4). All values are means � S.E.*, significantly different, p � 0.05.




http://ww

w.jbc.org/

Dow

nloaded from


http://www.jbc.org/

T19N showed few fluorescent spots. These data provide fur-ther evidence that RhoA activation is required for the ACh-induced activation of N-WASp in airway smooth muscle.

FIGURE 4. Paxillin tyrosine phosphorylation is required for cdc42 andN-WASP activation, but not for RhoA activation. A, increase of paxillin Tyr-118 phosphorylation in response to 10�5

M ACh was significantly lower intissues expressing paxillin Y118/Y31F (n � 5). Blot shows non-contiguouslanes from a single gel. Paxillin Y118/31F significantly inhibited tension devel-opment relative to sham-treated tissues (n � 8). B, expression of paxillinY118/31F (n � 4) or cdc42 T17N (n � 5) significantly inhibited N-WASp tyro-sine 256 phosphorylation in response to ACh stimulation. C, increase in co-immunoprecipitation of Arp2 with N-WASp in response to ACh stimulationwas significantly inhibited in tissues expressing paxillin Y118/31F (n � 3). Blotshow non-contiguous lanes from a single gel. D, immunoblots against acti-vated GTP-bound RhoA or GTP-bound cdc42 affinity-precipitated fromextracts of 4 sham or 4 paxillin Y118/31F-treated muscle strips. Expression ofpaxillin Y118/31F did not significantly affect RhoA activation in response to10�5

M ACh (n � 6), but it significantly inhibited cdc42 activation (n � 4). Allvalues are means � S.E. *, significantly different (p � 0.05).

FIGURE 5. The association between paxillin and vinculin is unaffected byACh stimulation or RhoA inhibition. A and B, vinculin was immunoprecipi-tated from extracts from sham-treated and RhoA-treated muscles and immu-noprecipitates were blotted for vinculin, paxillin, and paxillin Tyr-118. Therewere no significant differences between unstimulated and ACh-stimulatedtissues, or between sham and RhoA T19N-treated tissues in the amount ofpaxillin that coprecipitated with vinculin (n � 4). Phospho-paxillin at Tyr-118increased significantly in vinculin immunoprecipitates from 10�5

M ACh stim-ulated in sham-treated tissues, but not in RhoA T19N-treated tissues. All val-ues are means � S.E. *, significantly different (p � 0.05).

FIGURE 6. Rho regulates the paxillin-vinculin complex recruitment to thecell membrane in response to contractile stimulation. A, Duolink PLA wasused to evaluate the interaction of paxillin and vinculin in freshly dissociated cellsstimulated with 10�4

M ACh for 5 min or left unstimulated. DuoLink spots wereobserved throughout the cytoplasm of unstimulated cells but primarily at the cellperiphery of ACh stimulated cells from sham treated tissues. In cells from RhoAT19N-treated tissues, DuoLink spots were distributed throughout the cytoplasmof both unstimulated and ACh-stimulated cells. B, localization of paxillin-vinculincomplex was quantified by calculating ratio of fluorescence intensity betweenthe cell periphery and the cell interior. (See supplemental Fig. S1 for details.) Incells from sham-treated tissues, ACh stimulation significantly increased themembrane to cytoplasm ratio. A significant increase in the membrane to cyto-plasm ratio in response to ACh stimulation was not observed in cells treated withRhoA T19N (n � 14–23). C, there were no significant differences in the total cel-lular fluorescence intensity of paxillin-vinculin complexes in cells from sham-treated tissues and RhoA T19N-treated tissues with or without ACh (n � 14–23).All values are means � S.E. *, significantly different, p � 0.05.




http://ww

w.jbc.org/

Dow

nloaded from


http://www.jbc.org/

RhoA Inactivation Inhibits the ACh-induced Activation ofcdc42 in Canine Tracheal Smooth Muscle Tissues—Activationof the actin regulatory protein N-WASp is directly and specif-ically regulated by the small GTPase, cdc42 (20–23). Neitherrac nor Rho GTPases can bind directly to WASP family pro-teins, thus RhoA cannot directly regulate N-WASp activity (24,25). We therefore questioned whether RhoA might mediateN-WASp activation in trachealmuscle tissues by regulating theactivation of cdc42.We found that the inhibition of RhoA activity by RhoAT19N

or cell permeable C3 exoenzyme inhibited the increase in cdc42activation that occurs in response to ACh stimulation, and thatit also inhibited tension development (Fig. 2, A, B, and supple-mental Fig. S3A). In contrast, when cdc42 was inactivated bythe expression of the inactive cdc42mutant, cdc42T17N,RhoAactivationwas not inhibited (Fig. 2C). The inactivation of eithercdc42 or RhoA inhibited the increase in tension stimulated byACh. Our results demonstrate that RhoA activation is prereq-

uisite to the activation of cdc42 by ACh in tracheal smoothmuscle, but that cdc42 does not regulate RhoA activity.RhoA Regulates cdc42 andN-WASp Activation by Regulating

Paxillin Phosphorylation—We next evaluated the mechanismby which RhoA regulates the activity of cdc42 and N-WASp. Intracheal smooth muscle, the activation of N-WASp and cdc42are mediated by the interaction of N-WASp with the SH2/SH3adaptor protein, CrkII, which binds to tyrosine-phosphorylatedpaxillin via its SH2 domain and to N-WASp via its SH3 domain(34, 39, 41). Paxillin undergoes tyrosine phosphorylation attyrosine 31 and tyrosine 118 in response to the contractile stim-ulation of tracheal smooth muscle (31, 42). The phosphoryla-tion of paxillin at these sites is necessary for actin polymeriza-tion and tension development (31).We first assessed the roles of RhoA and cdc42 in the regula-

tion of paxillin tyrosine phosphorylation. The inactivation ofRhoA by RhoA T19N or cell permeable C3 exoenzyme inhib-ited the tyrosine phosphorylation of paxillin induced by ACh,

FIGURE 7. The phosphorylation of paxillin occurs at the cell membrane and requires the RhoA-dependent recruitment of FAK. A, cells freshly dissociatedfrom sham-treated or RhoA T19N treated muscle tissues were stimulated with 10�4

M ACh or left unstimulated, fixed, and double-stained for FAK and paxillin.Both proteins were distributed throughout the cytoplasm of unstimulated cells and localized to the cell membrane of ACh-stimulated cells. Neither FAK norpaxillin significantly increased at the cell membrane in response to ACh stimulation in cells dissociated from RhoA T19N-treated tissues. B, FAK phosphorylationat tyrosine 397 was significantly increased by ACh in sham-treated tissues but not RhoA T19N-inhibited tissues (n � 11). C, Tyr-118 paxillin was observed at thecell membrane of both unstimulated and ACh-stimulated cells, but the intensity of paxillin Tyr-118 phosphorylation was higher in ACh-stimulated cells. D andE, interaction of paxillin and FAK in freshly dissociated tracheal smooth muscle cells was detected using the Duolink PLA. In cells from sham-treated tissues, fewor no spots are detected in unstimulated cells; whereas many spots are seen at the membrane of the ACh-stimulated cell. The total number of DuoLink spotswas significantly higher in ACh-stimulated smooth muscle cells than in unstimulated cells (cells from 5 separate experiments, n � 34, 30). In cells from RhoAT19N-treated smooth muscle tissues, few or no spots were detected in unstimulated cells or ACh-stimulated cells. Cells from three separate experiments, (n �27, 15). All values are means � S.E. *, significantly different (p � 0.05).




http://ww

w.jbc.org/

Dow

nloaded from



http://www.jbc.org/

whereas the inactivation of cdc42 had no effect on paxillinphosphorylation (Fig. 3A and supplemental Fig. S3B); thus theactivation of RhoA but not cdc42 activation is prerequisite forpaxillin tyrosine phosphorylation.We then investigated the possibility that Rho kinase (ROCK)

might be an effector of RhoA-mediated paxillin phosphoryla-tion. The ROCK inhibitor, H-1152P, effectively inhibited phos-phorylation of the ROCK substrateMYPT1 at threonine 696 intracheal tissues (Fig. 3B), and also inhibited the in vitro phos-phorylation of a synthetic MYPT peptide (data not shown).ACh stimulation of canine tracheal smooth muscle tissuesincreased ROCK activation (Fig. 3B). However, ROCK inhibi-tion did not inhibit ACh-induced paxillin phosphorylation (Fig.3C). Similar results were obtained using the ROCK inhibitor,Y27632 (data not shown). Thus, RhoA activation is necessaryfor paxillin tyrosine phosphorylation in response to ACh, butRhoA mediated paxillin phosphorylation is not regulated byROCK.Paxillin Tyrosine Phosphorylation Is Required for cdc42 and

N-WASP Activation—We evaluated the role of paxillin phos-phorylation in regulating the ACh-induced activation of cdc42and N-WASp by expressing the non-phosphorylatable paxillinmutant, paxillin Y118/31F, in tracheal muscle tissues (31). Theexpression of paxillin Y118/31F prevented the tyrosine phos-phorylation of endogenous paxillin and tension development inresponse to ACh (Fig. 4A). The suppression of paxillin phos-phorylation by paxillin Y118/31F inhibited N-WASp phos-phorylation and its association with the Arp2/3 complex inresponse to ACh (Fig. 4, B and C). As expected, cdc42 inactiva-tion also inhibited the activation of N-WASp (Fig. 4B). Theinhibition of paxillin phosphorylation also suppressed the acti-vation of cdc42, but it did not affect the activation of RhoA (Fig.4D). We conclude that paxillin phosphorylation is prerequisitefor cdc42 and N-WASp activation, but that paxillin does notregulate RhoA activity.RhoA Regulates the Recruitment of Paxillin to the Cell Mem-

brane inResponse toContractile Stimulation—Paxillin can bindto vinculin (27) and it co-localizes with vinculin in differenti-ated airway smoothmuscle cells (29). The stimulation of airwaysmooth muscle cells increases the localization of both paxillinand vinculin at the membrane (29). We therefore investigatedthe possibility that RhoA regulates the interaction of paxillinwith vinculin or the recruitment of paxillin to the membrane.The effect of contractile stimulation and RhoA inactivation

on the interaction between paxillin and vinculin was assessedby co-immunoprecipitation analysis (Fig. 5). Paxillin co-precip-itated with vinculin from unstimulated and ACh-stimulatedtissues at the same ratio regardless of the phosphorylation stateof paxillin, which suggests that paxillin and vinculin form astable molecular complex that is unaffected by ACh stimula-tion. RhoA inactivation by RhoA T19N had no effect on theco-immunoprecipitation of paxillin with vinculin, indicatingthat the interaction of paxillin with vinculin is not RhoAdependent.Duolink PLA was used to determine the effect of RhoA acti-

vation on the cellular localization of paxillin/vinculin com-plexes (Fig. 6, A and B and supplemental Fig. S4). In sham-treated tissues, paxillin/vinculin complexes were observed

throughout the cytoplasm of unstimulated cells but localizedpredominantly at the membrane of ACh-stimulated cells. Incells expressing RhoAT19N, paxillin/vinculin complexes didnot localize at the membrane of ACh-stimulated cells; a diffusedistribution throughout the cytoplasm was observed in bothstimulated and unstimulated cells. Neither ACh stimulationnor RhoA inactivation significantly affected the total fluores-cence intensity of paxillin-vinculin complexes in the smoothmuscle cells, indicating that the association between paxillinand vinculin is unaffected by ACh stimulation or RhoA activa-tion (Fig. 6C). We conclude that RhoA regulates the recruit-ment of a protein complex containing both paxillin and vincu-lin to membrane adhesion junctions in response to stimulationwith ACh.RhoA Regulates the Association of Paxillin with FAK at the

Cell Membrane and Paxillin Tyrosine Phosphorylation inResponse toContractile Stimulation—FAKbinds to paxillin andcan induce the phosphorylation of paxillin at tyrosines 31and 118 (33, 34). We used immunofluorescence analysis andDuolink PLA to evaluate the effect of RhoA on FAK activationand on the interaction between FAK and paxillin. RhoA inacti-vation inhibited the recruitment of both FAK and paxillin tocell membrane and prevented the phosphorylation and activa-

FIGURE 8. The inhibition of FAK prevents ACh-induced cdc42 activationand actin polymerization. A and B, treatment of tracheal smooth muscletissues with 30 �M FAK inhibitor, FP 573228, inhibits phosphorylation of theFAK activation site Tyr-397 and paxillin phosphorylation at Tyr-118 inresponse to 10�5

M ACh (n � 4). Blot of paxillin phosphorylation shows non-contiguous lanes from a single gel C, FAK inhibitor, FP 573228, significantlyinhibited the increase in the F actin to G-actin ratio in response to 10�5

M ACh(n � 4). Immunoblot of soluble (G, globular) and insoluble (F, filamentous)actin in fractions from extracts of unstimulated or ACh-stimulated muscletissues treated with FAK inhibitor or with no inhibitor. D, treatment with FAKinhibitor, FP 573228 inhibits ACh- induced cdc42 activation (n � 3). For eachassay, activated cdc42 (cdc42-GTP) was affinity-precipitated from extracts of2 unstimulated and 2 ACh-stimulated muscle strips, and the amount of acti-vated cdc42 precipitated from each extract was quantified by immunoblot.




http://ww

w.jbc.org/

Dow

nloaded from



http://www.jbc.org/

tion of FAK in response to ACh stimulation (Fig. 7, A and B).Tyrosine-phosphorylated paxillin was observed only at themembrane in both unstimulated and ACh-stimulated smoothmuscle cells (Fig. 7C).We assessed the cellular location of the interaction between

FAK and paxillin using Duolink PLA and evaluated whetherRhoA regulated the interaction of FAK and paxillin. In sham-treated muscles, FAK interacted with paxillin in ACh-stimu-lated cells but not in unstimulated cells, and the interactionbetween FAK and paxillin occurred at the cell membrane (Fig.7, D and E).The Inhibition of FAK Kinase Activity Suppresses ACh-

induced Cdc42 Activation and Actin Polymerization—Weassessed the effect of inhibiting FAK activation on the regula-tion of paxillin phosphorylation, cdc42 activation and actinpolymerization by treating trachealmuscle tissueswith the syn-thetic FAK inhibitor, FP 573228 (43). We first confirmed theeffectiveness of FP 573228 at inhibiting FAK activation in tra-cheal muscle tissues by measuring its effect on the ACh-in-duced phosphorylation of FAK at its activation site, Tyr-397.Treatment of the tissues with 30 �M FP 573228 prevented theincrease in FAKTyr-397 phosphorylation induced byACh, andalso suppressed theACh-induced increase inpaxillinphosphor-ylation at Tyr-118 (Fig. 8, A and B; n � 4, p � 0.05). The inhi-bition of FAK activation inhibited cdc42 activation and actinpolymerization induced by stimulation with ACh (Fig. 8, C andD). The inhibition of FAK also suppressed force by more than50% (data not shown).

These results suggest that FAK and paxillin are indepen-dently recruited to themembrane by a RhoA-dependentmech-anism, and that after FAK is recruited to the membrane, itundergoes activation and induces the phosphorylation of pax-illin. Thus, RhoA appears to regulate paxillin phosphorylationindirectly by mediating the recruitment of both FAK and pax-illin to membrane complexes where they can interact.The Association of Paxillin with cdc42 GEFs Is Increased by

ACh Stimulation and Inhibited by RhoA Inhibition—We eval-uated several GEFs as potential mediators of cdc42 activationby paxillin during contractile stimulation. The PIX proteinsbind to PAK and have GEF activity toward both cdc42 and rac(35, 44). PIX proteins form a complex with GIT1 (G-proteinreceptor kinase-interacting tyrosine phosphorylated) andGIT2(also called paxillin kinase linker or PKL), which are ArfGAPSthat also regulate cdc42 and rac (45). During the adhesion ofcultured cells to a substrate, the localization of PAX-GIT com-plexes to focal adhesion sites is mediated by the regulated bind-ing of GIT molecules to the LD4 domain of paxillin (36, 37).DOCK (Dedicator of Cytokinesis) family proteins also have

GEF activity toward rac and cdc42 (35). DOCK180 interactswith Crk, a paxillin-binding adaptor protein (39, 41) and part-ners with ELMO (engulfment and cell motility protein) to cat-alyze nuclear exchange (46, 47).We analyzedmuscle tissue extracts by co-immunoprecipita-

tion and found that the association of paxillin, CrkII, andELMO1 with DOCK180 increases in muscles after contractilestimulation with ACh (Fig. 9A). The association of PIX pro-

FIGURE 9. RhoA activation regulates the association of paxillin with cdc42 GEF. A, co-immunoprecipitation of ELMO1, paxillin, and CrkII with DOCK180increased in ACh-stimulated tissues. Result typical of six independent experiments. B, co-immunoprecipitation of �-PIX, PAK1 and CrkII with paxillin increasedafter contractile stimulation with ACh. Result typical of four independent experiments. C, co-immunoprecipitation of GIT1 and �-PIX with paxillin increased inACh-stimulated tissues and was significantly inhibited in tissues expressing RhoA T19N (n � 4). D, Duolink PLA reveals interaction between �-PIX and paxillinat the membrane of muscle cells after contractile stimulation, whereas they are not observed in unstimulated muscle cells. RhoA T19N inhibited the formationof paxillin/�-PIX complexes. E, in sham-treated tissues, the mean number of DuoLink PLA spots was significantly higher in ACh-stimulated cells than inunstimulated cells. (Cells dissociated from tissues from 3 separate experiments (n � 19, 18 cells.) In cells from RhoA T19N-treated tissues, the mean number ofDuoLink PLA spots was small in both unstimulated and ACh-stimulated cells and was not significantly different (n � 18). All values are means � S.E.*, significantly different (p � 0.05).




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

teins, GIT1, CrkII, and Pak with paxillin also increased in cellsactivated with ACh (Fig. 9, B and C). Similar analysis usingDuolink PLA technology revealed extensive complexesbetween �-PIX and paxillin at the membrane of muscle cellsafter contractile stimulation, that were observed only mini-mally in unstimulatedmuscle cells (Fig. 9,D and E). The forma-tion of membrane complexes between �-PIX, GIT1, and paxil-lin was inhibited by the inactivation of RhoA (Fig. 9, C–E).We confirmed the role of the paxillin/GIT/PIX complex in

the activation of cdc42 by expressing a paxillin mutant with adeletion of the LD4 domain in the muscle tissues (paxillin�LD4) (36, 37). The LD4 domain of paxillin regulates its bind-ing to GIT proteins and is required for the localization of GITandPIXproteins tomembrane adhesomes (36, 37). The expres-sion of paxillin �LD4 in the muscle tissues prevented the inter-action of paxillin with GIT1 and �-PIX in response to stimula-tion with ACh, as indicated by co-immunoprecipitationanalysis of muscle tissue extracts and Duolink PLA assays infreshly dissociated cells (Fig. 10, A–C). Expression of paxillin�LD4 also inhibited the ACh-induced activation of cdc42 andforce development (Fig. 10,D andE). These results indicate thatcontractile activation stimulates the recruitment of GEF signal-ing complexes to adhesomes via a RhoA-dependent mecha-nism where they couple paxillin to the activation of cdc42.

DISCUSSION

Our studies demonstrate that the activation of RhoAGTPaseduring the contractile stimulation of airway smoothmuscle tis-sues catalyzes the assembly of signaling complexes at integrinadhesion sites that are necessary for the regulation of actinpolymerization and tension development (summarized in Fig.11). The adaptor/scaffolding protein complex that is assembledin response to RhoA activation includes paxillin and vinculin,FAK, which induces paxillin tyrosine phosphorylation, and the�/�-PIX andDOCK180GEF signalingmodules, which can reg-ulate the activity of cdc42 and rac. We show that paxillin cou-ples to theseGEF signalingmodules and regulates the activity ofcdc42, which activates N-WASp to catalyze actin polymeriza-tion by the Arp2/3 complex. The association of paxillin withthese GEFs is also RhoA-dependent. Thus, RhoA activation isprerequisite to all of the steps in the signaling pathway thatregulates N-WASp activation in response to contractile stimu-lation in airway smooth muscle. These studies describe a novelmechanism for the regulation of smoothmuscle contraction byRhoA that is independent of its well-known role in regulatingthe phosphorylation of the regulatory light chain of smoothmuscle myosin II. We demonstrate that RhoA-mediated adhe-some assembly plays a critical role in the process of excitation-contraction coupling during agonist-induced smooth musclecontraction.The regulation of airway smooth muscle contraction by

RhoA has been ascribed to its inhibitory effects on the catalyticactivity of smoothmuscleMLC phosphatase (48, 49). The inhi-bition ofMLCphosphatase can augment agonist-inducedMLCphosphorylation, which increases smooth muscle contractility(8, 9). However, our results demonstrate that this is not theprimary mechanism by which RhoA regulates the contractilityof airway smooth muscle.

Studies of adhesome assembly during substrate adhesion incultured cell lines have documented an important role forRhoA in the assembly andmaturation of focal adhesions (2, 50,51). These studies suggest that RhoA activation induces phos-phorylation of the light chain of non-musclemyosin II, and thatthis is prerequisite to the formation of focal adhesions andstress fibers (52). In these cells, inhibiting non-musclemyosin IIactivation disrupts the formation of Rho-induced stress fibers

FIGURE 10. The association of paxillin with �-PIX regulates cdc42 activa-tion. A, co-immunoprecipitation of GIT1 and �-PIX with paxillin was signifi-cantly inhibited in tissues expressing paxillin �LD4 (n � 4). B, Duolink PLAreveals interaction between �-PIX and paxillin. Paxillin �LD4 inhibited theformation of paxillin/�-PIX complexes. C, in sham-treated tissues, the meannumber of DuoLink PLA spots was significantly higher in ACh-stimulated cellsthan in unstimulated cells. (Cells dissociated from tissues from 3 separateexperiments (n � 19, 18 cells.)) In cells from paxillin �LD4-treated tissues, themean number of DuoLink PLA spots was small in both unstimulated andACh-stimulated cells and was not significantly different (n � 18). All values aremeans � S.E. *, significantly different (p � 0.05). D, expression of paxillin �LD4inhibits ACh-induced cdc42 activation. (n � 4). For each assay, activatedcdc42 (cdc42-GTP) was affinity-precipitated from extracts of 2 unstimulatedand 2 ACh-stimulated muscle strips. E, paxillin �LD4 significantly inhibitedtension development relative to sham-treated tissues (n � 8). All values aremeans � S.E. *, significantly different (p � 0.05).




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

and results in focal adhesion disassembly. It is possible thatRhoA regulates adhesome assembly and excitation contractioncoupling during the contraction of airway smooth muscle tis-sues by an analogous mechanism.In airway smooth muscle, adhesion protein activation, and

actin polymerization are independent of the phosphorylation ofSM myosin light chain by myosin light chain kinase (MLCK)(12, 17, 32). TheMLC kinase inhibitor, ML-7, suppresses myo-sin light chain phosphorylation and tension development inresponse to contractile stimulation, but it has no effect onACh-induced actin polymerization (10). The inhibition of MLCKalso does not prevent paxillin phosphorylation in response toACh (32). Thus, if myosin II is involved in the recruitment ofproteins to adhesomes in airway smooth muscle, its activationmust occur independently of MLCK. One possibility is thatnon-muscle rather than smooth muscle isoforms of myosin IIare involved inmediating adhesome assembly in airway smoothmuscle, and that non-muscle myosin II activation but notsmooth muscle myosin II activation occurs via a RhoA-depen-dent mechanism. If so, our results would suggest that theRhoA-mediated phosphorylation of non-musclemyosin II lightchain would be critical for excitation-contraction couplingand smooth muscle contraction. Non-muscle myosin II isexpressed in cultured human airway smooth muscle cells (53)and has also been documented in vascular smooth muscle tis-sues (54).Studies of other cell types have implicated RhoA in other

mechanisms that contribute to the regulation of cytoskeletaland actin dynamics. RhoA can activate LIM kinase, whichphosphorylates and inactivates the actin-dynamizing proteincofilin (2, 55). However, it seems unlikely that RhoA activationcould regulate actin dynamics in airway smooth muscle by reg-ulating LIM kinase, because in this tissue contractile stimula-tion causes the dephosphorylation and activation of cofilin, andcofilin activation is necessary for actin polymerization and con-traction (56).RhoA is also known to regulate activity of the formins, actin

nucleating proteins that associate with the fast-growing(barbed) ends of actin filaments and are involved in the forma-tion of stress fibers in adhesive cells (57, 58). The role of forminsin agonist-induced actin polymerization and contraction insmooth muscle is unknown. The possibility that RhoA-medi-ated formin activation plays a role in actin polymerization andcontraction in smooth muscle cannot be ruled out.

Focal adhesion kinase can induce the phosphorylation ofpaxillin in a src-dependent manner (59, 60). We therefore con-sidered the possibility that Rho might regulate paxillin phos-phorylation and actin dynamics in airway smooth muscle cellsby regulating the activity of FAK.Tyrosine phosphorylated pax-illin increased in response to ACh stimulation and was foundonly at themembrane. FAKwas also recruited to themembranein response to ACh stimulation, and this recruitment wasinhibited by RhoA inactivation (Fig. 7, B and C). Duolink PLAanalysis demonstrated that paxillin and FAKonly interact at themembrane of stimulated airway smooth muscle cells, and thatthe interaction between paxillin and FAK is inhibited by RhoAinactivation. The inhibition of FAK activation prevented ACh-induced paxillin phosphorylation, cdc42 activation, and actinpolymerization. Thus, we conclude that FAK is recruited to themembrane by a RhoA-dependentmechanism independently ofpaxillin, and that FAK forms a complex with paxillin at theadhesome and induces its phosphorylation.The Rho effector, ROCK, has also been implicated in the

regulation of paxillin phosphorylation in several studies of non-muscle cell lines (61–63). However, in airway tissues the inhi-bition of ROCKdid not suppressACh-induced paxillin tyrosinephosphorylation, indicating that ROCK is unlikely to play a rolein the regulation of paxillin phosphorylation in this tissue.Studies of adhesion and spreading in several substrate-ad-

herent non-muscle cell lines have demonstrated that the acti-vation of cdc42 and rac can cause a transient suppression ofRhoA activity, and that this suppression of RhoA activity isfollowed by RhoA activation and cell contraction. In culturedNMuMG cells, phosphorylated paxillin inhibits RhoA activa-tion following integrin engagement by regulating the activity ofp190RhoGAP, which suppresses RhoA activity during celladhesion (64). Arthur and DeMali have proposed that cdc42and RhoA are activated sequentially in response to integrinengagement, with cdc42 activation occurring initially inde-pendently of RhoA activity (65). Our observations clearly dem-onstrate that this paradigm is not applicable to airway smoothmuscle during contractile stimulation, as we found that RhoAinhibition suppresses the ACh-induced activation of cdc42(Fig. 2).Our observations that contractile stimulation does not affect

the ratio of co-immunoprecipitation of paxillin and vinculin orthe number of paxillin/vinculin complexes reported by PLAanalysis suggests that these proteins are stably associated in

FIGURE 11. Model for proposed mechanism for the regulation of ACh induced RhoA activation on the assembly of an adhesome signaling complex inASM. 1, ACh stimulation activates RhoA, which induces the independent recruitment of paxillin-vinculin complexes and FAK to cell adhesomes; 2, FAK andpaxillin interact at the adhesome and activated FAK induces the phosphorylation of paxillin, which remains bound to activated vinculin; 3, phosphorylation ofpaxillin facilitates the formation of a complex containing paxillin and Crk II with DOCK180 and PIX GEFs. This complex induces the activation of cdc42. 4, cdc42activation catalyzes the activation of N-WASp, which interacts with the Arp2/3 complex to induce actin polymerization in the cortical region of the smoothmuscle cell. This enables tension generation by the smooth muscle contractile apparatus.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

airway smooth muscle. PLA analysis revealed paxillin/vinculincomplexes to be localized throughout the cytoplasm ofunstimulated cells but concentrated at the membrane of thestimulated cells, suggesting that these proteins are recruited tothe membrane as a stable complex in response to ACh stimu-lation. In previous studies using immunofluorescence, wefound that paxillin and vinculin colocalized in airway smoothmuscle cells under all conditions (29). In the present study,RhoA inhibition did not affect the interaction of paxillin andvinculin, but it prevented the recruitment of the paxillin/vincu-lin complexes to themembrane. Thus RhoA activation appearsto be required for the process that regulates the movement ofthe paxillin-vinculin complex.The large multiprotein “adhesome” complexes that connect

cells within tissues to the extracellular matrix at adhesion junc-tions are widely known to play critical functions in cells thatextend far beyond their structural role. While they providemechanical coupling between cells and their matrix environ-ment, they also enable cells to sense and respond to changes inthe properties of their surrounding milieu (66, 67). Adhesomeconstituents and their associated cytoskeletal networks canmediate cellular responses to a variety of stimuli, includinggrowth factors, inflammatory mediators, and mechanicalforces (15, 67, 68).Our current observations suggest that RhoA-mediated adhe-

some complex assembly is a fundamental step in the process ofsignal transduction initiated by the contractile agonist stimula-tion of airway smooth muscle. Both actin polymerization andcontraction depend on the RhoA-mediated recruitment of pax-illin-vinculin complexes to adhesome junctions. Paxillin hasbeen implicated in mediating signal transduction pathwaysactivated by growth hormones and inflammatory mediators inmultiple cell types (66), and may also be important forresponses to thesemediators in airway smoothmuscle. Thus, inairway smooth muscle, RhoA mediated assembly of adhesomecomplexes may be a fundamental step in the process of signaltransduction in response to diverse extracellular stimuli.

REFERENCES1. Allen, W. E., Jones, G. E., Pollard, J. W., and Ridley, A. J. (1997) Rho, Rac,

and Cdc42 regulate actin organization and cell adhesion in macrophages.J. Cell Sci. 110, 707–720

2. Burridge, K., and Wennerberg, K. (2004) Rho and Rac take center stage.Cell 116, 167–179

3. DeMali, K. A., Wennerberg, K., and Burridge, K. (2003) Integrin signalingto the actin cytoskeleton. Curr. Opin. Cell Biol. 15, 572–582

4. Ridley, A. J. (1999) Rho family proteins and regulation of the actin cyto-skeleton. Prog. Mol. Subcellul. Biol. 22, 1–22

5. Ridley, A. J. (2006) Rho GTPases and actin dynamics in membrane pro-trusions and vesicle trafficking. Trends Cell Biol. 16, 522–529

6. Amano, M., Ito, M., Kimura, K., Fukata, Y., Chihara, K., Nakano, T., Mat-suura, Y., and Kaibuchi, K. (1996) Phosphorylation and activation ofmyosin by Rho-associated kinase (Rho-kinase). J. Biol. Chem. 271,20246–20249

7. Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M.,Yamamori, B., Feng, J., Nakano, T., Okawa, K., Iwamatsu, A., and Kaibu-chi, K. (1996) Regulation of myosin phosphatase by Rho and Rho-associ-ated kinase (Rho-kinase) Science 273, 245–248

8. Puetz, S., Lubomirov, L. T., and Pfitzer, G. (2009) Regulation of smoothmuscle contraction by small GTPases. Physiology 24, 342–356

9. Somlyo, A. P., and Somlyo, A. V. (2003) Ca2� sensitivity of smoothmuscle

and nonmuscle myosin II: modulated by G proteins, kinases, and myosinphosphatase. Physiol. Rev. 83, 1325–1358

10. Zhang, W., Du, L., and Gunst, S. J. (2010) The effects of the small GTPaseRhoA on themuscarinic contraction of airway smoothmuscle result fromits role in regulating actin polymerization. Am. J. Physiol. 299,C298–C306

11. Corteling, R. L., Brett, S. E., Yin, H., Zheng, X. L.,Walsh,M. P., andWelsh,D. G. (2007) The functional consequence of RhoA knockdown by RNAinterference in rat cerebral arteries. Am. J. Physiol. Heart Circ. Physiol.293, H440–H447

12. Gunst, S. J., and Zhang, W. (2008) Actin cytoskeletal dynamics in smoothmuscle: a new paradigm for the regulation of smooth muscle contraction.Am. J. Physiol. Cell Physiol. 295, C576–C587

13. Kim, H. R., Gallant, C., Leavis, P. C., Gunst, S. J., andMorgan, K. G. (2008)Cytoskeletal remodeling in differentiated vascular smooth muscle is actinisoform dependent and stimulus dependent. Am. J. Physiol. Cell Physiol.295, C768–C778

14. Rembold, C. M., Tejani, A. D., Ripley, M. L., and Han, S. (2007) Paxillinphosphorylation, actin polymerization, noise temperature, and the sus-tained phase of swine carotid artery contraction. Am. J. Physiol. CellPhysiol. 293, C993–C1002

15. Zhang, W., and Gunst, S. J. (2008) Interactions of airway smooth musclecells with their tissue matrix: implications for contraction. Proc. Am. Tho-rac. Soc. 5, 32–39

16. Mehta, D., and Gunst, S. J. (1999) Actin polymerization stimulated bycontractile activation regulates force development in canine trachealsmooth muscle. J. Physiol. 519, 829–840

17. Zhang, W., Wu, Y., Du, L., Tang, D. D., and Gunst, S. J. (2005) Activationof the Arp2/3 complex by N-WASp is required for actin polymerizationand contraction in smooth muscle. Am. J. Physiol. Cell Physiol. 288,C1145–C1160

18. Higgs, H. N., and Pollard, T. D. (2001) Regulation of actin filament net-work formation through ARP2/3 complex: activation by a diverse array ofproteins. Annu. Rev. Biochem. 70, 649–676

19. Mullins, R.D. (2000)HowWASP-family proteins and theArp2/3 complexconvert intracellular signals into cytoskeletal structures. Curr. Opin. CellBiol. 12, 91–96

20. Rohatgi, R., Ma, L., Miki, H., Lopez, M., Kirchhausen, T., Takenawa, T.,and Kirschner, M. W. (1999) The interaction between N-WASP and theArp2/3 complex linksCdc42-dependent signals to actin assembly.Cell 97,221–231

21. Carlier, M. F., Ducruix, A., and Pantaloni, D. (1999) Signaling to actin: theCdc42-N-WASP-Arp2/3 connection. Chem. Biol. 6, R235–R240

22. Higgs, H. N., and Pollard, T. D. (2000) Activation by Cdc42 and PIP(2) ofWiskott-Aldrich syndrome protein (WASp) stimulates actin nucleationby Arp2/3 complex. J. Cell Biol. 150, 1311–1320

23. Rohatgi, R., Ho, H. Y., and Kirschner, M. W. (2000) Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4, 5-bisphosphate.J. Cell Biol. 150, 1299–1310

24. Li, R., Debreceni, B., Jia, B., Gao, Y., Tigyi, G., and Zheng, Y. (1999) Local-ization of the PAK1-, WASP-, and IQGAP1-specifying regions of Cdc42.J. Biol. Chem. 274, 29648–29654

25. Suzuki, T., Mimuro, H., Miki, H., Takenawa, T., Sasaki, T., Nakanishi, H.,Takai, Y., and Sasakawa, C. (2000) Rho family GTPase Cdc42 is essentialfor the actin-based motility of Shigella in mammalian cells. J. Exp. Med.191, 1905–1920

26. Tang, D. D., and Gunst, S. J. (2004) The small GTPase Cdc42 regulatesactin polymerization and tension development during contractile stimu-lation of smooth muscle. J. Biol. Chem. 279, 51722–51728

27. Turner, C. E., Glenney, J. R., Jr., and Burridge, K. (1990) Paxillin: a newvinculin-binding protein present in focal adhesions. J. Cell Biol. 111,1059–1068

28. Turner, C. E., Kramarcy, N., Sealock, R., and Burridge, K. (1991) Localiza-tion of paxillin, a focal adhesion protein, to smoothmuscle dense plaques,and the myotendinous and neuromuscular junctions of skeletal muscle.Exp. Cell Res. 192, 651–655

29. Opazo Saez, A., Zhang,W.,Wu, Y., Turner, C. E., Tang, D. D., and Gunst,S. J. (2004)Tension development during contractile stimulation of smooth




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

muscle requires recruitment of paxillin and vinculin to the membrane.Am. J. Physiol. Cell Physiol. 286, C433–C447

30. Wang, Z., Pavalko, F.M., andGunst, S. J. (1996) Tyrosine phosphorylationof the dense plaque protein paxillin is regulated during smooth musclecontraction. Am. J. Physiol. 271, C1594–C602

31. Tang, D. D., Turner, C. E., and Gunst, S. J. (2003) Expression of non-phosphorylatable paxillin mutants in canine tracheal smooth muscle in-hibits tension development. J. Physiol. 553, 21–35

32. Huang, Y., Zhang, W., and Gunst, S. J. (2011) Activation of vinculin in-duced by cholinergic stimulation regulates contraction of tracheal smoothmuscle tissue. J. Biol. Chem. 286, 3630–3644

33. Bellis, S. L., Miller, J. T., and Turner, C. E. (1995) Characterization oftyrosine phosphorylation of paxillin in vitro by focal adhesion kinase.J. Biol. Chem. 270, 17437–17441

34. Schaller, M. D., and Parsons, J. T. (1995) pp125FAK-dependent tyrosinephosphorylation of paxillin creates a high-affinity binding site for Crk.Mol. Cell Biol. 15, 2635–2645

35. Sinha, S., and Yang, W. (2008) Cellular signaling for activation of RhoGTPase Cdc42. Cell Signal. 20, 1927–1934

36. West, K. A., Zhang, H., Brown, M. C., Nikolopoulos, S. N., Riedy, M. C.,Horwitz, A. F., andTurner, C. E. (2001)The LD4motif of paxillin regulatescell spreading and motility through an interaction with paxillin kinaselinker (PKL). J. Cell Biol. 154, 161–176

37. Turner, C. E., Brown, M. C., Perrotta, J. A., Riedy, M. C., Nikolopoulos,S. N., McDonald, A. R., Bagrodia, S., Thomas, S., and Leventhal, P. S.(1999) Paxillin LD4 motif binds PAK and PIX through a novel 95-kDankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling.J. Cell Biol. 145, 851–863

38. Söderberg, O., Leuchowius, K. J., Gullberg, M., Jarvius, M., Weibrecht, I.,Larsson, L. G., and Landegren, U. (2008) Characterizing proteins and theirinteractions in cells and tissues using the in situ proximity ligation assay.Methods 45, 227–232

39. Tang, D. D., Zhang, W., and Gunst, S. J. (2005) The adapter protein CrkIIregulates neuronal Wiskott-Aldrich syndrome protein, actin polymeriza-tion, and tension development during contractile stimulation of smoothmuscle. J. Biol. Chem. 280, 23380–23389

40. Torres, E., and Rosen, M. K. (2006) Protein-tyrosine kinase and GTPasesignals cooperate to phosphorylate and activate Wiskott-Aldrich syn-drome protein (WASP)/neuronal WASP. J. Biol. Chem. 281, 3513–3520

41. Petit, V., Boyer, B., Lentz, D., Turner, C. E., Thiery, J. P., and Vallés, A. M.(2000) Phosphorylation of tyrosine residues 31 and 118 on paxillin regu-lates cell migration through an association with CRK in NBT-II cells.J. Cell Biol. 148, 957–970

42. Tang, D., Mehta, D., and Gunst, S. J. (1999) Mechanosensitive tyrosinephosphorylation of paxillin and focal adhesion kinase in tracheal smoothmuscle. Am. J. Physiol. 276, C250–8

43. Slack-Davis, J. K., Martin, K. H., Tilghman, R. W., Iwanicki, M., Ung, E. J.,Autry, C., Luzzio, M. J., Cooper, B., Kath, J. C., Roberts, W. G., and Par-sons, J. T. (2007) Cellular characterization of a novel focal adhesion kinaseinhibitor. J. Biol. Chem. 282, 14845–14852

44. Rosenberger, G., Jantke, I., Gal, A., and Kutsche, K. (2003) Interaction ofalphaPIX (ARHGEF6) with�-parvin (PARVB) suggests an involvement ofalphaPIX in integrin-mediated signaling. Hum. Mol. Genet. 12, 155–167

45. Lynch, E. A., Stall, J., Schmidt, G., Chavrier, P., and D’Souza-Schorey, C.(2006) Proteasome-mediated degradation of Rac1-GTP during epithelialcell scattering.Mol. Biol. Cell 17, 2236–2242

46. Côté, J. F., and Vuori, K. (2002) Identification of an evolutionarily con-served superfamily of DOCK180-related proteinswith guanine nucleotideexchange activity. J. Cell Sci. 115, 4901–4913

47. Gumienny, T. L., Brugnera, E., Tosello-Trampont, A. C., Kinchen, J. M.,Haney, L. B., Nishiwaki, K., Walk, S. F., Nemergut, M. E., Macara, I. G.,Francis, R., Schedl, T., Qin, Y., Van Aelst, L., Hengartner, M. O., andRavichandran, K. S. (2001) CED-12/ELMO, a novel member of the CrkII/Dock180/Rac pathway, is required for phagocytosis and cell migration.Cell 107, 27–41

48. Bai, Y., and Sanderson, M. J. (2006) Modulation of the Ca2� sensitivity ofairway smoothmuscle cells inmurine lung slices.Am. J. Physiol. Lung Cell

Mol. Physiol. 291, L208–L22149. Sanderson, M. J., Delmotte, P., Bai, Y., and Perez-Zogbhi, J. F. (2008) Reg-

ulation of airway smooth muscle cell contractility by Ca2� signaling andsensitivity. Proc. Am. Thorac. Soc. 5, 23–31

50. Chrzanowska-Wodnicka, M., and Burridge, K. (1996) Rho-stimulatedcontractility drives the formation of stress fibers and focal adhesions.J. Cell Biol. 133, 1403–1415

51. Pasapera, A. M., Schneider, I. C., Rericha, E., Schlaepfer, D. D., and Wa-terman, C. M. (2010) Myosin II activity regulates vinculin recruitment tofocal adhesions through FAK-mediated paxillin phosphorylation. J. CellBiol. 188, 877–890

52. Vicente-Manzanares, M., Ma, X., Adelstein, R. S., and Horwitz, A. R.(2009) Non-muscle myosin II takes centre stage in cell adhesion and mi-gration. Nat. Rev. Mol. Cell Biol. 10, 778–790

53. Milton, D. L., Schneck, A. N., Ziech, D. A., Ba, M., Facemyer, K. C., Ha-layko, A. J., Baker, J. E., Gerthoffer, W. T., and Cremo, C. R. (2011) Directevidence for functional smooth muscle myosin II in the 10S self-inhibitedmonomeric conformation in airway smooth muscle cells. Proc. Natl.Acad. Sci. U.S.A. 108, 1421–1426

54. Yuen, S. L., Ogut, O., and Brozovich, F. V. (2009) Nonmuscle myosin isregulated during smooth muscle contraction. Am. J. Physiol. Heart Circ.Physiol. 297, H191–H199

55. Maekawa,M., Ishizaki, T., Boku, S.,Watanabe,N., Fujita, A., Iwamatsu, A.,Obinata, T., Ohashi, K., Mizuno, K., and Narumiya, S. (1999) Signalingfrom Rho to the actin cytoskeleton through protein kinases ROCK andLIM-kinase. Science 285, 895–898

56. Zhao, R., Du, L., Huang, Y., Wu, Y., and Gunst, S. J. (2008) Actin depoly-merization factor/cofilin activation regulates actin polymerization andtension development in canine tracheal smooth muscle. J. Biol. Chem.283, 36522–36531

57. Zigmond, S. H. (2004) Formin-induced nucleation of actin filaments.Curr. Opin. Cell Biol. 16, 99–105

58. Kovar, D. R. (2006) Molecular details of formin-mediated actin assembly.Curr. Opin. Cell Biol. 18, 11–17

59. Klinghoffer, R. A., Sachsenmaier, C., Cooper, J. A., and Soriano, P. (1999)Src family kinases are required for integrin but not PDGFR signal trans-duction. EMBO J. 18, 2459–2471

60. Richardson, A., and Parsons, T. (1996) A mechanism for regulation of theadhesion-associated protein-tyrosine kinase pp125FAK. Nature 380,538–540

61. Sinnett-Smith, J., Lunn, J. A., Leopoldt, D., and Rozengurt, E. (2001)Y-27632, an inhibitor of Rho-associated kinases, prevents tyrosine phos-phorylation of focal adhesion kinase and paxillin induced by bombesin:dissociation from tyrosine phosphorylation of p130(CAS). Exp. Cell Res.266, 292–302

62. Tsuji, T., Ishizaki, T., Okamoto, M., Higashida, C., Kimura, K., Fu-ruyashiki, T., Arakawa, Y., Birge, R. B., Nakamoto, T., Hirai, H., and Na-rumiya, S. (2002) ROCK and mDia1 antagonize in Rho-dependent Racactivation in Swiss 3T3 fibroblasts. J. Cell Biol. 157, 819–830

63. Worthylake, R. A., and Burridge, K. (2003) RhoA and ROCK promotemigration by limiting membrane protrusions. J. Biol. Chem. 278,13578–13584

64. Tsubouchi, A., Sakakura, J., Yagi, R.,Mazaki, Y., Schaefer, E., Yano, H., andSabe, H. (2002) Localized suppression of RhoA activity by Tyr31/118-phosphorylated paxillin in cell adhesion and migration. J. Cell Biol. 159,673–683

65. Arthur, W. T., Noren, N. K., and Burridge, K. (2002) Regulation of Rhofamily GTPases by cell-cell and cell-matrix adhesion. Biol. Res. 35,239–246

66. Zaidel-Bar, R., Itzkovitz, S., Ma’ayan, A., Iyengar, R., and Geiger, B. (2007)Functional atlas of the integrin adhesome. Nat. Cell Biol. 9, 858–867

67. Zaidel-Bar, R., and Geiger, B. (2010) The switchable integrin adhesome.J. Cell Sci. 123, 1385–1388

68. Weber, G. F., Bjerke, M. A., and DeSimone, D. W. (2011) Integrins andcadherins join forces to form adhesive networks. J. Cell Sci. 124,1183–1193




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Wenwu Zhang, Youliang Huang and Susan J. GunstAdhesion Sites

Catalyzing the Assembly of Cytoskeletal Signaling Complexes at Membrane The Small GTPase RhoA Regulates the Contraction of Smooth Muscle Tissues by

doi: 10.1074/jbc.M112.369603 originally published online August 13, 20122012, 287:33996-34008.J. Biol. Chem.

10.1074/jbc.M112.369603Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

http://www.jbc.org/content/suppl/2012/08/13/M112.369603.DC1

http://www.jbc.org/content/287/41/33996.full.html#ref-list-1

This article cites 68 references, 31 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M112.369603

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;287/41/33996&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/287/41/33996

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=287/41/33996&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/287/41/33996

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/suppl/2012/08/13/M112.369603.DC1

http://www.jbc.org/content/287/41/33996.full.html#ref-list-1

http://www.jbc.org/

Documents

TheSmallGTPaseRhoARegulatestheContractionofSmooth ... · Activated GTP-bound RhoA or cdc42 were affinity-precipi-tated by glutathione beads and quantified by immunoblot. CellDissociationandImmunofluorescenceAnalysis—Freshly