therho-specificguaninenucleotideexchangefactordbs … · 2009-08-10 · dermal growth factor...

The Rho-specific Guanine Nucleotide Exchange Factor DbsRegulates Breast Cancer Cell Migration*

Received for publication, March 19, 2009 Published, JBC Papers in Press, April 14, 2009, DOI 10.1074/jbc.M901853200

Zhuoming Liu, Homer C. Adams III, and Ian P. Whitehead1

From the Department of Microbiology and Molecular Genetics and the New Jersey Medical School-University Hospital CancerCenter, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07101-1709

Dbs is a Rho-specific guanine nucleotide exchange factor(RhoGEF) that regulates neurotrophin-3-induced cell migra-tion in Schwann cells. Here we report that Dbs regulates cellmotility in tumor-derived, humanbreast epithelial cells throughactivation of Cdc42 and Rac1. Cdc42 and Rac1 are activated inT47D cells that stably express onco- or proto-Dbs, and activa-tion is dependent upon growth of the cells on collagen I. Tran-sient suppression of expression of Cdc42 or Rac1 by small inter-fering RNAs attenuates Dbs-enhancedmotility. Both onco- andproto-Dbs-enhanced motility correlates with an increase intyrosine phosphorylation of focal adhesion kinase on Tyr-397and p130Cas on Tyr-410 and an increase in the abundance of theCrk�p130Cas complex. Suppression of expression of Cdc42 or itseffector, Ack1, reduces tyrosine phosphorylation of focal adhe-sion kinase and p130Cas and disrupts the Crk�p130Cas complex.We further determined that suppressionof expressionofCdc42,Ack1, p130Cas, or Crk reduces Rac1 activation and cell motilityin Dbs-expressing cells to a level comparable with that in vectorcells. Therefore, a cascade of activation of Cdc42 and Rac1 byDbs through the Cdc42 effector Ack1 and the Crk�p130Cas com-plex is established. Suppressionof the expression of endogenousDbs reduces cell motility in both T47D cells andMDA-MB-231cells, which correlates with the down-regulation of Cdc42 activ-ity. This suggests that Dbs activates Cdc42 in these two humanbreast cancer cell lines and that the normal function of Dbsmaybe required to support cell movement.

Rho GTPases are a subfamily of the Ras superfamily of smallsignaling molecules that are widely expressed in mammaliancells (1). RhoA, Cdc42, and Rac1 are the most extensively stud-iedmembers of the RhoGTPase family, and each plays a prom-inent and discrete role in cell migration (2, 3). Cdc42 promotesthe formation of filopodia and is required to establish cell polar-ity (3–5); Rac1 promotes the formation of lamellipodia at theleading edge of motile cells (6), and RhoA promotes the forma-tion of stress fiberswhich generate the traction forces needed toretract the cell tail and move the cell body beyond the leadingedge (7, 8). Consistent with this important role in cell motility,RhoA, Cdc42, and Rac1 are often overexpressed in human

tumors including breast, lung, and colon (9), and overexpres-sion of constitutively active RhoA, Cdc42, or Rac1 increases cellmigration and invasion (2, 10, 11).The spatiotemporal regulation of Rho GTPase activity is

tightly controlled by three classes of proteins. Rho-specific gua-nine nucleotide exchange factors (RhoGEFs)2 activate Rho pro-teins by facilitating the exchange ofGDP forGTP; RhoGTPase-activating proteins (RhoGAPs) stimulate the intrinsic rate ofhydrolysis of Rho proteins, thus converting them into theirinactive state; Rho-specific guanine nucleotide dissociationinhibitors (RhoGDIs) compete with RhoGEFs for binding toGDP-bound Rho proteins and sequester Rho in the inactivestate (12).Dbs was identified in the screen for proteins whose overex-

pression causemalignant growth inmurine fibroblasts (13, 14).The full-length Dbs protein (proto-Dbs) is a RhoGEF familymember which contains multiple recognizable domains (Fig.1A) including a Sec14-like domain, spectrin-like repeats, a Rho-GEF domain (includes a DH and PH domain), and an SH3domain (13). The original oncogenic version of Dbs that wasidentified (amino acid residues 525–1097; designated onco-Dbs) contains the RhoGEF domain alone. When expressed inmurine fibroblasts, the transforming and catalytic activity ofDbs is subject to autoinhibition that is mediated by the NH2-terminal Sec14 domain (15). Although the endogenous func-tion of Dbs is not known, recent studies suggest that Dbs andthe Rac-specific exchange factor Tiam1 regulate neurotrophin-stimulated cell migration in Schwann cells through activationof Cdc42 and Rac1, respectively (16, 17).Conversion of Rho proteins to their active GTP-bound state

allows them to interact with effector signaling molecules. Ack1is a nonreceptor-tyrosine kinase that binds to active Cdc42 butnot Rac1 or RhoA (18, 19). Activated Ack1 is overexpressed inprimary tumors and cancer cell lines and has been implicated incancer metastasis (20). Recent studies have identified a signal-ing complex that regulates themotility of human breast epithe-lial cells that contains Cdc42, Ack1, p130Cas, andCrk (21). Ack1and p130Cas interact through their respective SH3 domains,and Ack1 phosphorylates p130Cas in a collagen I-dependentmanner. p130Cas was first identified as a hyperphosphorylatedadapter protein in cells transformed by v-Src and v-Crk (22, 23).Further studies showed that p130Cas is associated with bothcellular Src and Crk in a tyrosine phosphorylation-dependent* This work was supported, in whole or in part, by National Institutes of Health

Grant CA97066 (NCI, to the United States Public Health Service). This workwas also supported by American Cancer Society Research Scholar GrantRGS-04-199-01 (to I. P. W.).

1 To whom correspondence should be addressed: Cancer Center H level, 205South Orange Ave., Newark, NJ 07101-1709. Fax: 973-972-2668; E-mail:[email protected].

2 The abbreviations used are: RhoGEF, Rho-specific guanine nucleotideexchange factor; FAK, focal adhesion kinase; HA, hemagglutinin; GST, glutathi-one S-transferase; siRNA, small interfering RNA; PAK, p21-activated kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 23, pp. 15771–15780, June 5, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

JUNE 5, 2009 • VOLUME 284 • NUMBER 23 JOURNAL OF BIOLOGICAL CHEMISTRY 15771

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manner (24, 25). Focal adhesion kinase (FAK) binds to the NH2terminus of p130Cas and phosphorylates the COOH terminusin a region that is involved in p130Cas binding to Src (26). Thebinding of Crk to p130Cas recruits binding partners to the SH3domain of Crk, including C3G and DOCK180, which activateRap1 and Rac1, respectively (27–31). Thus, formation of theCrk�p130Cas complex is considered to be a molecular switchthat can induce cell migration by activating Rac1 (32).Here we show that both proto-Dbs and onco-Dbs increase

cell migration in human breast adenocarcinoma cells in a col-lagen I-dependent manner. Increased motility is dependentupon the activation of Rac1 and Cdc42 and is mediated by theassembly of Crk�p130Cas complexes. Suppression of endoge-nous Dbs expression in human tumor-derived breast epithelialcells limits cell motility, suggesting that Dbs may be a criticalregulator of cell behavior in breast cancer.

EXPERIMENTAL PROCEDURES

Molecular Constructs—The pCTV3H mammalian expres-sion vector has been described previously (13). pCTV3H-dbs-HA6 and pCTV3H-dbs-FL contain cDNA fragments thatencode a transforming derivative of Dbs (onco-Dbs) and full-length Dbs (proto-Dbs), respectively (33). Both proteins arefused to a hemagglutinin (HA) epitope tag.Cell Culture and Transfection—T47D, MDA-MB-231, M14,

MDA-MB-453, and ZR-57–1 cells were cultured at 37 °C in 5%CO2 in RPMI 1640 medium supplemented with 10% fetalbovine serum (Benchmark). MCF-10A cells were cultured at37 °C in 5% CO2 in Dulbecco’s modified Eagle’s medium/F-12supplemented with 5% horse serum (Invitrogen), 20 ng/ml epi-dermal growth factor (Sigma), 0.5 mg/ml hydrocortisone(Sigma), and 10�g/ml insulin (Invitrogen). Allmedia were sup-plemented with 100 unit/ml penicillin and 100 �g/ml strepto-mycin. T47D cells stably expressing proto-Dbs, onco-Dbs, ortheir cognate vector were generated as described previously(13). Briefly, cells were transfected by Lipofectamine reagent(Invitrogen) according to the manufacturer’s recommendedprotocol. Cells were maintained in growth medium supple-mentedwith hygromycin B (200�g/ml, Invitrogen) for 14 days.For each individual stable cell line, more than 200 drug-resis-tant colonies were combined to generate a polyclonal stable cellline. Three independent cell lines were generated and tested foreach construct.Cell Growth Assays—T47D cells stably expressing onco-Dbs,

proto-Dbs, or cognate vector were resuspended in growthmedium. 5 � 103 cells were seeded into each well of a 48-welltissue culture dish. Cells were cultured either on plastic or plas-tic precoated with collagen I (50 �g/ml). Cells were trypsinized(3 wells per day) and counted using a hemocytometer for 10consecutive days.RhoA, Cdc42, and Rac1 Activation Assay—Affinity precipi-

tation assays to measure the levels of endogenous GTP-boundRhoA, Cdc42, and Rac1 were performed using the Rho bindingdomain of Rhotekin (GST-C21) and the Cdc42/Rac1 bindingdomain of PAK (GST-PAK) as previously described (34). 2 �106 cells were cultured on 10-cm tissue culture dishes pre-coated with collagen I (50 �g/ml; BD Biosciences) or poly-L-lysine (0.01%; Sigma). Cells weremaintained in growthmedium

for 48 h and then serum-starved for 18 h in RPMI 1640mediumsupplementedwith 0.5% fetal bovine serum.Cells were subcon-fluent when cell lysates were prepared.Transwell Motility Assays—Assays were performed as previ-

ously described (11). Subconfluent cells were detached by0.05% trypsin, and equal numbers were loaded into the topchamber of transwells coated from the underside with collagenI and allowed to migrate for 16 h. Where indicated, cell assayswere performed 48 h after transfectionwith siRNAs. In all casessufficient numbers of cells were loaded such that we observedgreater than 50 cells/field passing through the filter in the vec-tor controls. Cells that migrated to the underside of the mem-branes were fixed and stained with Diff-Quik (Dade Behring)and counted. All experiments were performed in triplicate, and8–12 fields of view for each transwell were counted. The aver-age amount of cells per field was then calculated. Results wereexpressed as -fold changes relative to controls. Where indi-cated, the Rac-specific inhibitor, NSC23766 (C24H38Cl3N7, 50�M, Calbiochem), was added to the medium in the bottomchamber and the medium that was used to resuspend the cells30 min before loading the transwell.In Vitro Wound Healing Assay—Assays were performed as

previously described by others (35). T47D cells stably express-ing proto-Dbs, onco-Dbs, or their cognate vector were culturedon 35-mm tissue culture dishes. Where indicated, dishes wereprecoated with poly-L-lysine (0.01%) or collagen I (50 �g/ml).Confluent monolayers of cells were starved in RPMI 1640media supplemented with 0.5% fetal bovine serum overnight.The surface of the monolayer was scraped using a pipette tip,and cells alongside the wound were allowed to migrate towardthe opening for 18 h under serum-deprived conditions (0.5%fetal bovine serum). Closure was monitored and photographedat multiple sites, and representative images are provided.Where indicated, cells were transiently transfected withsiRNAs 48 h before serum starvation.siRNAs—Cells at 70% confluencewere transfected by siRNAs

from different manufacturers with SiLentFectTM lipid reagent(Bio-Rad) according to the manufacturer’s protocols. Minimalconcentrations of siRNAs that can reduce the expression of thetarget proteins by 70% was determined byWestern blot and anOdyssey IR Imager. Concentrations of individual siRNAs thatwere used are 100 nM Cdc42 (Santa Cruz sc-29256 or Ambions2765), 150 nM Rac1 (Santa Cruz sc-36351 or Ambion s11713),150 nM p130Cas (Santa Cruz sc-44317 or Ambion s225161), 100nM Crk (Santa Cruz sc-37072 or Ambion s3521), 150 nM Ack(Santa Cruz sc-29632 or Ambion s19851), 150 nM Ost (SantaCruz sc-41728 or Dharmacon DR3773961), 150 nM Bcr (SantaCruz sc-29795 or Dharmacon L-003875-00), and 150 nMTiam1 (Santa Cruz sc-36669 or Ambion s14138). ScrambledsiRNAs (Santa Cruz sc-37007 or Ambion AM4611) wereincluded in all experiments as negative controls. ScrambledsiRNAs were also used to bring the total amount of siRNAs upto the same concentration when needed. The viability of cellstransfected by siRNAs was determined by the trypan blueexclusion method as previously described (36).Immunoprecipitation and Western Blot Analysis—Immuno-

precipitation andWestern blot analysis were performed as pre-viously described (37). Antibodies used for Western blots

Dbs Regulates Breast Cancer Cell Migration

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include: anti-HA (Berkeley Antibody Co., Inc.), anti-Crk (BDTransduction Laboratories), anti-p-Crk (pY207, Cell Signal-ing), anti-p130Cas (BD Transduction), anti-phospho-p130Cas(pY410, Cell Signaling), anti-FAK (Santa Cruz, sc-557), anti-phospho-FAK (pY397, BIOSOURCE), anti-Ack1 (Santa Cruz,sc-323), anti-Cdc42 (Santa Cruz, sc-8401), anti-Rac1 (BDTransduction Laboratories), anti-RhoA (Santa Cruz, sc-418),anti-Src (Santa Cruz, sc-18), anti-phospho-Src (pY418, Calbio-chem), anti-Bcr (Cell Signaling), anti-Tiam1 (Santa Cruz,sc-872), anti-Ost (Santa Cruz, sc-901), and anti-actin (SantaCruz, sc-8432).Statistics—Statistical analyses were performed by one-way

analysis of variance followed by the Tukey HSD (honestly sig-nificant difference) test for multiple comparisons using JMPstatistical software. p values of �0.05 were considered signifi-cant. Once the statistical analysis was complete, the data wereconverted to -fold activations to facilitate ease of comparisonbetween the figures.

RESULTS

IncreasedCellMotility inT47DCells That Stably ExpressDbs—T47D is a well differentiated human breast carcinoma cell linethat has been previously established as a useful system to studythe effects of deregulated Rho protein signaling on cell migra-

tion and invasion (11, 38, 39). To examine the role of Dbs intumor cell motility, we generated T47D cell lines that stablyexpress HA-tagged onco-Dbs, proto-Dbs, and cognate vector(Fig. 1A). To control for clonal differences in cell behavior,three independent sets of cell lines were generated and com-pared. Expression of the HA-tagged proteins was confirmed byWestern blot (Fig. 1B). When examined in a transwell motilityassay, the cell lines expressing onco-Dbs or proto-Dbs showedincreased cell motility (�2- and 3-fold, respectively) whencompared with vector control cells (Fig. 1C). To confirm theincreased motility, the cell lines were also compared in an invitro wound healing assay. Because transwell motility assayswere performed on filters coated with collagen I, the woundhealing assays were performed on plates coated with eitherpoly-L-lysine or collagen I.When cultured on plates pre-coatedwith collagen I, cells stably expressing onco-Dbs or proto-Dbsboth showed enhanced cell motility in the wound healing assay(Fig. 1D). However, when cultured on tissue culture dishes pre-coated with poly-L-lysine, no differences in motility wereobserved. No significant enhancement ofmotility was observedwhen cells were cultured on uncoated plastic, collagen IV,fibronectin, or laminin (not shown). To confirm that theincreased migration could not be attributed to an accelerated

FIGURE 1. Onco-Dbs and proto-Dbs induce cell migration in tumor-derived breast epithelial cells. A, domain structure of the onco-Dbs and proto-Dbsproteins (Sec14 � Sec14-like domain; Spec � Spectrin-like repeats; DH � Dbl homology domain; PH � pleckstrin homology domain; SH3 � Src homology 3domain). B, stable expression of HA-epitope-tagged onco-Dbs (Mr � 65) and proto-Dbs (Mr � 129 kDa) was confirmed by Western blot using an anti-HAantibody. Three independent sets of cell lines were generated. C, T47D cells stably expressing vector (Vec), onco-Dbs, or proto-Dbs were compared in atranswell motility assay on filters pre-coated with collagen I. The motility of cells stably expressing onco-Dbs or proto-Dbs is expressed relative to that of cellsstably expressing vector. Data are represented as the mean � S.D. of three independent experiments performed in triplicate. D, T47D cells stably expressingvector, onco-Dbs, or proto-Dbs were cultured to monolayer on dishes coated with poly-L-lysine or collagen I, as indicated. Cells were serum-starved overnight,and then the surface of the plate was scraped. Migration of cells at the wound edge was monitored and photographed at 18 h. Representative images areshown. E, growth curves of T47D cells stably expressing vector, onco-Dbs, or proto-Dbs. Cells were cultured in triplicate on poly-L-lysine (filled symbols) or ondishes pre-coated with collagen I (open symbols) and counted on the indicated days. Data shown are representative of three independent experiments.




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rate of cell division, the growth rates of all cell lines was com-pared on both collagen I and poly-L-lysine (Fig. 1E). Althoughcells grown on collagen I grew faster than those grown on poly-L-lysine, no differences were observed between vector andDbs-expressing cells under either culture condition.DbsActivates Endogenous Cdc42 and Rac1 in T47DCells in a

Collagen I-dependentManner—Because Dbs regulates motilityin Schwann cells through activation of Cdc42, we determined ifDbs induces T47D cell motility by activating this GTPase. Rac1and RhoA were included in the analysis for comparison. Whenplated on poly-L-lysine, no differences were observed in RhoA-GTP, Cdc42-GTP, or Rac1-GTP levels in the cell lines that sta-bly express onco-Dbs, proto-Dbs, or vector (Fig. 2, A and B).However, when cells were cultured on dishes pre-coated withcollagen I, elevated levels of Cdc42-GTP and Rac1-GTP wereobserved in the cells that express onco-Dbs or proto-Dbs (Fig.2,C andD). Consistent activation of RhoAwas also observed incells that express onco-Dbs but not proto-Dbs. This suggeststhat Cdc42 and Rac1, but not RhoA, may contribute to proto-Dbs-mediated cell motility.Endogenous Cdc42 and Rac1 Are Required for Dbs-induced

Motility in T47D Cells—Next we determined whether Rac1 orCdc42 activation is required to support Dbs-mediatedmotility.Using siRNAs,we suppressed the expression ofCdc42 andRac1in T47D cells stably expressing onco-Dbs, proto-Dbs, or cog-nate vector. Scrambled siRNAs were included in the analysis asnegative controls. For comparison, Rac was also inhibited witha specific pharmacological inhibitor (NSC23766). The siRNAstargeting Cdc42 (Fig. 3A) or Rac1 (Fig. 3B) suppressed theexpression level of their target proteins significantly, whichreduced the levels of endogenous Cdc42-GTP and Rac-GTP toa level that was lower than cells transfected with scrambledcontrols. In parallel transfections, siRNAs targeting Cdc42 andRac1 reduced the motility of cells stably expressing onco-Dbsor proto-Dbs to a level equivalent to that of cells stably express-ing cognate vector (Fig. 3, C and D). A similar reduction inmotility was observed in cells treated with the Rac-specificinhibitor (Fig. 3, E–G). In all cases the reduced motility couldnot be attributed to increased cell death, as more than 95% oftreated cells were viable as determined by trypan blue exclusion(data not shown). Collectively, these results suggest that Dbsregulates cell motility in T47D cells in a Cdc42- and Rac1-de-pendent manner.

Ack1 Is Required for Dbs-mediated Motility in T47D Cells—Ack1 is a non-receptor tyrosine kinase that is an effector forCdc42 (40). To determine whetherAck1mediates Dbs-inducedmotility, we transiently suppressed the expression of Ack1 inT47D cells that stably express onco-Dbs, proto-Dbs, or cognatevector. siRNAs targeting Ack1 significantly suppressed theexpression of Ack1 (70%; Fig. 4A) in all three cell lines andreduced the motility of cells stably expressing onco-Dbs orproto-Dbs to a level similar to that seen in cells stably express-ing vector (Fig. 4, B and C). The reduced cell motility was notattributable to increased cell death as measured by trypan blueexclusion (data not shown).Dbs Increases the Phosphorylation of p130Cas and Its Associ-

ation with Crk in a Cdc42- and Ack1-dependentManner—Pre-vious studies have demonstrated that phosphorylation ofp130Cas on Tyr-410 creates a docking site for Crk and supportsthe formation of the Crk�p130Cas complex (41). Although theassociation betweenp130Cas andCrk canbe regulated byCdc42through Ack1, it is unclear whether Ack1 phosphorylatesp130Cas directly or through other kinases that phosphorylatep130Cas, such as Src andFAK (21).WedeterminedwhetherDbsregulatesmotility in T47D cells by promoting the association ofp130Cas and Crk. Although the overall expression level ofp130Cas in T47D cells that stably express onco-Dbs or proto-Dbswas not significantly different from vector control cells, thelevel of Tyr-410 phosphorylation was increased by 2–3-fold(Fig. 5A). Consistent with this, a 2–3-fold increase in theamount of Crk that co-immunoprecipitates with p130Cas wasalso observed in the cells that stably express onco- or proto-Dbs(Fig. 5A).The association of Crk with its binding partners is negatively

regulated by an intramolecular interaction between its SH2domain and phosphorylated Tyr-221 (42). To determinewhether the increased association of Crk with p130Cas couldalso be attributed to reduced phosphorylation of Crk on Tyr-221, we also examined the levels of total and Tyr-221-phospho-rylated Crk in T47D cells that stably express onco-Dbs orproto-Dbs (Fig. 5A). No differences were observed in the levelsof phosphorylated or total Crk when compared with vectorcontrol cells, suggesting that Dbs does not regulate the phos-phorylation of Crk.To determine whether the phosphorylation of p130Cas and

its association with Crk is mediated by Cdc42 and Ack1, we

VectorOnco-DbsProto-Dbs

Rac1

FIGURE 2. Dbs activates Rac1 and Cdc42 in a collagen I-dependent manner. T47D cells stably expressing vector, onco-Dbs, or proto-Dbs were cultured ontissue culture dishes pre-coated with poly-L-lysine (A and B) or collagen I (C and D). A and C, cell lysates were collected and examined by Western blot forexpression of total Cdc42, RhoA, and Rac1. Lysates were subjected to affinity purification with immobilized GST-PAK or GST-Rhotekin, and active GTP-boundCdc42, RhoA, or Rac1 was visualized by Western blot. B and D, quantifications were performed using an Odyssey IR imager. Levels of active Rho were firstnormalized against total Rho and then expressed as -fold activation relative to vector controls. Data are represented as the mean � S.D. of three independentexperiments.




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transiently suppressed the expression of Cdc42 or Ack1 inT47D cells stably expressing onco-Dbs, proto-Dbs, or cognatevector (Fig. 5B). Although reduced expression of Cdc42 andAck1 does not change the overall expression level of p130Cas(Fig. 5C) in both the onco- and proto-Dbs-expressing cells, thelevel of p130Cas that was phosphorylated on Tyr-410 was either

equivalent to or below the levels in the vector control cells.Similarly, the increased association of p130Cas with Crk in thecells expressing onco-Dbs and proto-Dbs was also found to bedependent upon the expression of Cdc42 and Ack1. Therefore,the phosphorylation of p130Cas and its association with Crk isregulated by Dbs in a Cdc42- and Ack1-dependent manner.

FIGURE 3. Endogenous Cdc42 and Rac1 are required for Dbs-induced motility in T47D cells. T47D cells stably expressing vector, onco-Dbs, or proto-Dbswere cultured on dishes pre-coated with collagen I and then transfected by siRNAs (si) targeting Cdc42 (A) and Rac1 (B). Scrambled siRNAs (Ctrl) were includedas negative controls. A, total expression of Cdc42 (top panel) was determined by Western blot using an anti-Cdc42 antibody. The active level of Cdc42 (middlepanel) was determined by affinity purification with immobilized GST-PAK. Vec, vector. B, total expression of Rac1 (top panel) was determined by Western blotusing an anti-Rac1 antibody. The active level of Rac1 (middle panel) was determined by affinity purification with immobilized GST-PAK. In parallel experimentsthe cells were examined in transwell motility (C) or wound healing (D) assays performed on collagen I. All experiments were performed in triplicate, and dataare presented as in Fig. 1. Rho activation (E), transwell motility (F), and wound healing (G) assays were also performed in the presence (NSC23766) or absence(mock) of a Rac specific inhibitor. E, the active level of RhoA was determined by affinity purification with immobilized GST-C21.

FIGURE 4. Ack1 is required for Dbs-mediated motility in T47D cells. T47D cells stably expressing vector (Vec) or onco- or proto-Dbs were cultured on dishespre-coated with collagen I and transfected by siRNAs targeting Ack1 (si Ack1). Scrambled siRNAs (si Ctrl) were included as negative controls. A, lysates werecollected, and the expression level of Ack1 was determined by Western blot using an anti-Ack1 antibody. In parallel transfections the cells were examined intranswell motility (B) and wound healing (C) assays. Assays were performed and quantitated as described in Fig. 1. B, data are represented as the mean � S.D.of three independent experiments.




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Dbs Increases FAKPhosphorylation in aCdc42- andAck1-de-pendent Manner—FAK and Src have been shown to phospho-rylate the substrate domain of p130Cas (26, 43–46), and bothare themselves activated by phosphorylation on tyrosine withintheir respective activation loops (Tyr-397 for FAK and Tyr-418for Src) (47). To determine whether Src or FAK are activated inT47D cells that stably express onco-Dbs or proto-Dbs, we uti-lized antibodies that specifically recognize the activated ver-sions of both kinases. No differences were observed in theoverall level of total and activated Src in the onco-Dbs- orproto-Dbs-expressing cells relative to the vector control cells(Fig. 6A). However, elevated levels of activated FAK weredetected in cells that express onco- and proto-Dbs relative tovector control cells.To determine whether FAK phosphorylation is dependent

on Cdc42 or Ack1, levels of total and activated FAK weremeasured in the lysates in which the expression of Cdc42 orAck1 was suppressed (Fig. 5B). Although the siRNAs thattarget Cdc42 or Ack1 did not change the overall expressionlevel of FAK, the level of activated FAK in the cells thatexpress onco-Dbs or proto-Dbs was reduced to the levelsseen in the vector controls (Fig. 6B). This suggests that Dbsactivates FAK in an Ack1-dependent manner and provides amechanism through which Ack can promote the assembly ofthe Crk�p130Cas complexes.

p130Cas and Crk Are Required forDbs-induced Cell Migration andRac Activation—Next, we deter-mined if p130Cas and Crk arerequired for Dbs-mediated motilityin T47D cells. Using siRNAs, wetransiently suppressed expressionof p130Cas, Crk, Ack1, or Cdc42 inT47D cells stably expressing onco-Dbs, proto-Dbs, or cognate vector(Figs. 5B and 7A). siRNAs that indi-vidually target p130Cas or Crkreduced the motility of cells stablyexpressing onco-Dbs or proto-Dbsto a level equivalent to that seen in

vector control cells (Fig. 7,B andC). Because others have shownthat the p130Cas�Crk complex promotes motility by stimulat-ing Rac activation, we also performed affinity precipitationassays to determine whether Rac activation in cells that stablyexpress onco- or proto-Dbs is dependent upon expression ofp130Cas, Crk, Ack1, or Cdc42. Reduced expression of p130Cas,Crk, Ack1, or Cdc42 reduced the level of Rac activation in boththe onco-Dbs- and proto-Dbs-expressing cells to the levels seenin the vector control cells (Fig. 7D). The latter observation sug-gests that Dbs activation of Rac1 in T47D cells is indirect andmediated by Cdc42.EndogenousDbs Supports CellMotility inHumanBreast Epi-

thelial Cells—Because overexpression of proto-Dbs increasesthemotility of T47D cells, we determined whether endogenousDbs supports cell motility in this same cell type. To examinethis, we transiently suppressed the expression of endogenousDbs as well as two additional RhoGEF family members, Tiam1and Bcr. Bcr is a RhoGEF that is active toward RhoA, Cdc42,and Rac1 (48) and is the fusion partner for Abl in chronicmyelogenous leukemia (49). Tiam1 is a Rac-specific exchangefactor that is mutated in human renal cancers (50) and cooper-ates withDbs to regulatemotility in Schwann cells (17). siRNAstargeting Dbs, Bcr, and Tiam1 significantly reduced the expres-sion of their respective target proteins (Fig. 8A). The identity of

FIGURE 5. Dbs increases the phosphorylation of p130Cas and its association with Crk in a Cdc42- and Ack1-dependent manner. A, T47D cells stablyexpressing onco-Dbs, proto-Dbs, or cognate vector were cultured on collagen I. Lysates were prepared and separated into two aliquots. The first aliquot wassubject to immunoprecipitation (IP) using an anti-p130Cas antibody. The immunoprecipitate was then examined by Western blot (WB) for the presence ofp130Cas and Crk. The second aliquot was then examined by Western blot for levels of p130Cas phosphorylated on Tyr-410 (p-p130Cas), total p130Cas, Crkphosphorylated on Tyr-221 (p-Crk), and total Crk, as indicated. B and C, the stable cell lines were cultured on collagen I and transfected with siRNAs (si) targetingCdc42 and Ack1. Scrambled siRNAs were included as controls (si Ctrl). Lysates were collected, and one aliquot was examined by Western blot for expression ofCdc42 and Ack1 (B). C, an additional aliquot of the lysate was then examined for levels of phosphorylated (p-p130Cas) and total p130Cas. A third aliquot wassubject to immunoprecipitation, and the immunoprecipitate was examined by Western blot with an anti-Crk antibody.

FIGURE 6. Dbs increases FAK phosphorylation in a Cdc42- and Ack1-dependent manner. A, T47D cells thatstably express vector (Vec), onco-Dbs, or proto-Dbs were cultured on collagen I. Lysates were prepared andexamined by Western blot for levels of total Src, Src phosphorylated on Tyr-418 (p-Src), total FAK, and FAKphosphorylated on Tyr-397 (p-FAK). B, the cells were also transfected with siRNAs (si) targeting Cdc42 and Ack1as indicated. Cell lysates were then collected and examined by Western blot for levels of total and phospho-rylated (Tyr-397; p-FAK) FAK.




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endogenous Dbs was confirmed using two independent com-mercially available siRNAmixes (not shown). Lysates were alsoexamined for levels of activated Cdc42 and Rac1 (Fig. 8B). Thereduced expression of Dbs (but not Tiam1 or Bcr) was associ-ated with reduced levels of activated Cdc42 in T47D cells, indi-cating that Dbs normally regulates Cdc42 in this cell type.Reduced expression of Dbs had no effect on RhoA-GTP levels,indicating that RhoA is not a normal substrate for Dbs in thiscell type. Activated Rac levels were reduced substantially whenTiam1 expression was suppressed, to a more limited extentwhen Dbs expression was suppressed and not at all when Bcrexpression was suppressed. In parallel experiments, the trans-fected cells were examined in transwell motility (Fig. 8C) and invitro wound healing (Fig. 8D) assays. In the transwell motilityassays, siRNAs targeting Dbs and Tiam1 reduced cell motility

by about 50 and 90%, respectively (Fig. 8C), whereas siRNAstargeting Bcr had no effect. Similarly, cell motility is dependentupon Tiam1 and Dbs expression, but not Bcr expression, in thewound healing assay (Fig. 8D).When tissue culture dishes werepre-coated with poly-L-lysine, siRNAs targeting Bcr, Dbs, orTiam1 had no significant effect on T47D cell migration (notshown).To confirm that Dbs-mediated motility is not restricted to

T47D cells, we examined a panel of human breast tumor-de-rived cell lines forDbs expression (Fig. 9A). For comparison, thepanel also included the highly motile M14 melanoma cell lineandMCF10A, which are derived from benign breast tissue. Dbsexpression was observed in all cell lines examined, with thelowest expression in theMCF10A and T47D cells. The identityof Dbs was confirmed using a second commercially available

0

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FIGURE 7. p130Cas and Crk are required for Dbs-induced cell migration and Rac activation. A, T47D cells stably expressing vector (Vec), onco-Dbs, orproto-Dbs were cultured on collagen I and then transfected with siRNAs (si) targeting p130Cas, Crk, or Ack1. Scrambled siRNAs (si Ctrl) were included as anegative control. Cell lysates were collected and examined by Western blot for expression of Crk, p130Cas, and Ack1. Cells transfected with siRNAs targetingp130Cas and Crk were also examined in transwell motility (B) and wound healing (C) assays. Data for both assays was collected, quantitated, and representedas described in Fig. 1. D, lysates from all transfections was also examined by affinity purification assays for levels of active Rac1 (Rac1 GTP).

FIGURE 8. Endogenous Dbs and Tiam1 support cell motility in T47D cells. T47D cells were cultured on collagen I and transfected with siRNAs (si) targetingDbs, Bcr, and Tiam1. Scrambled siRNAs (si Ctrl) were included as controls. A, cell lysates were prepared and examined by Western blot for expression of Dbs, Bcr,and Tiam1 as indicated. B, lysates were also examined by affinity precipitation assays for levels of activated Cdc42 (Cdc42 GTP), Rac1 (Rac1 GTP), and RhoA (RhoAGTP). Cells transfected with siRNAs for Dbs, Bcr, and Tiam1 were also examined in transwell motility (C) and wound healing (D) assays. Both assays wereperformed on collagen I, and data were collected and presented as in Fig. 1.




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antibody (not shown). MDA-MB-231 is the most highly motileand invasive human breast cancer epithelial cell line on ourpanel.We suppressed the expression of endogenous Dbs in thiscell line (Fig. 9B) and examined the levels of Cdc42 activity andcell motility. Similar to what we observed in the T47D cells, thereduced expression of Dbs correlated with the reduced levels ofactivated Cdc42 (Fig. 9B) and cell motility (Fig. 9C) in a dose-dependent manner.

DISCUSSION

In the current study we have determined that stable expres-sion of both onco- and proto-Dbs enhances the motility ofhuman tumor-derived breast epithelial cells. Although Dbsenhances motility when cultured on collagen I, no enhance-ment was observed when cells were cultured on plastic, poly-L-lysine, collagen IV, fibronectin, or laminin. Activation of Cdc42and Rac1 was also only observed when cells were cultured oncollagen I, and siRNAs targeting endogenous Dbs in T47D andMDA-MB-231 cells only suppress motility if cells are culturedon collagen I. This suggests that endogenous and overexpressedDbs may regulate motility through similar mechanisms. Thisspecificity for collagen I may have particular significance forbreast cancer progression. During metastasis, breast cancercells must transverse the stromal extracellular matrix, whichcontains collagen I (51), and excessive expression of collagen I isa predisposing factor in breast and pancreatic cancer (52–54).In addition, culturing on collagen I can trigger epithelial-to-mesenchymal transition in pancreatic cancer cells, lung cancercells, and mouse mammary epithelial cells (55–57).Although several reports have indicated that specific mem-

bers of the RhoGEF familymay bemutated in human tumors, itis not clear that overexpression of a RhoGEF is sufficient tostimulate Rho activation or transformation (58). RhoGEFs nor-mally exist in the cell in an inactive state where they are subjectto tight autoinhibitory regulation (58). In NIH 3T3 cells thecatalytic and transforming activity of Dbs is autoinhibited bythe NH2-terminal Sec14 domain (15). However, in T47D cellswe observed that proto-Dbs stimulates motility to a similarextent as onco-Dbs, suggesting that proto-Dbsmay not be sub-ject to autoinhibition in this cell type. It also suggests that sim-

ply increasing the level of expression of a RhoGEF in a tumorcell line can be sufficient to increase the motile phenotype.Although it is possible that collagen I supports the effects ofDbson motility by relieving the autoinhibitory mechanism, thisseems unlikely, as onco-Dbs, which lacks the Sec14 domain,also requires collagen I to support motility.Both onco- and proto-Dbs activate Cdc42 and Rac1 in T47D

cells, and siRNAs or pharmacological inhibitors that targettheseGTPases block cell motility. Because previous in vitro andin vivo nucleotide exchange analyses have shown that Dbs isactive toward RhoA and Cdc42, but not Rac1 (14, 59), we pre-sume that Dbs activates Rac1 indirectly in T47D cells. In a pre-vious study it has been shown that collagen I promotes theassociation of Cdc42 with a complex that contains Ack1,p130Cas, and Crk and that this complex in turn promotes themotility of breast cancer cell lines (21). Because theCrk�p130Cascomplex has been shown to promote Rac activation, this pro-vides a mechanism through which Dbs could activate Rac1 in aCdc42-dependentmanner. In the current study we have shownthat onco-Dbs and proto-Dbs can activate Cdc42, increase thephosphorylation of p130Cas, and increase its association withCrk in a Cdc42- andAck1-dependentmanner. Additionally, wehave shown that siRNAs targeting p130Cas, Crk, or Cdc42reduce Rac1 activation. Thus, in our model we propose thatcollagen I stimulates the association of p130Cas with Cdc42 aspreviously demonstrated (21). Activation of Cdc42 by Dbswould then recruit Ack1 and promote the assembly and stabi-lization of Crk�p130Cas complexes, which would then recruitand activate exchange factors for Rac.Our previous observationthat Rac binds directly to the PH domain of Dbs also provides amechanism for recruiting Rac to the complex and would allowCdc42 and Rac to be assembled into a regulatory cascade (59).Although it is not clear how Ack1 promotes the assembly of

Crk�p130Cas complexes, both FAK and Src can phosphorylatep130Cas, and both are known to regulate cellmotility (60). Phos-phorylation on Tyr-397 in the activation loop of FAK and Tyr-418 in the activation loop of Src activate their respective kinaseactivities (61, 62). By using antibodies that recognize the phos-phorylated state of these residues, we discovered that the level

FIGURE 9. Endogenous Dbs supports cell motility in MBA-MD-231 cells. A, cell lysates were collected from the indicated cell lines and examined by Westernblot for expression of endogenous Dbs. Tubulin expression was also examined as a control for equivalent loading. B, MBA-MD-231 cells were cultured oncollagen I and transfected with siRNAs (si) targeting Dbs at the indicated concentrations. Scrambled siRNAs (si Ctrl) were included as controls. Cell lysates wereexamined by Western blot for expression of Dbs as indicated. Lysates were also examined by affinity precipitation assays for levels of activated Cdc42 (Cdc42GTP). C, MBA-MD-231 cells transfected with siRNAs for Dbs were also examined in transwell motility assays. Assays were performed on collagen I, and data werecollected and presented as in Fig. 1.




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of phospho-Src is unchanged, but the level of phospho-FAK isincreased in T47D cells that express Dbs. Furthermore, siRNAstargeting Cdc42 or Ack1 reduce the level of phosphorylatedFAK to control levels, suggesting that FAK is downstream ofCdc42 and Ack1. Therefore, Ack1may phosphorylate and acti-vate FAK, which in turn may phosphorylate p130Cas. However,we cannot exclude the possibility that Ack1may phosphorylatep130Cas directly.

EndogenousDbswas detected in all breast epithelial cell linesexamined, and suppression of Dbs expression reduces themotility of T47D and MDA-MB-231 cells, which correlateswith a reduction in the level of active Cdc42. These resultsimply that endogenousDbs utilizes Cdc42 as a substrate in bothof these cell types and is required for their motility. The speci-ficity of this observation was confirmed in parallel experimentsin which we showed that a second known Cdc42 activator, Bcr,does not control Cdc42 activation ormotility in these cell lines.Our observations are consistent with a previous study in whichsiRNA-mediated suppression of endogenous Dbs reducedCdc42-GTP levels and neurotrophin-3-mediated cell migra-tion in Schwann cells (16).Our previous studies have shown that onco-Dbs mediates

transformation in fibroblasts through regulation of MLC2 in aRhoA/ROCK-dependent manner (37). Consistent with this,onco-Dbs activates RhoA when overexpressed in T47D cells.However, proto-Dbs does not activate RhoA in this cell type,and suppression of endogenous Dbs expression does not alterRhoA-GTP levels. Thus, proto-Dbs does not appear to be cou-pled to RhoA activation in T47D cells.Dbs was also detected in MCF10A cells, suggesting that it

may have other functions in breast epithelial cells and that itspresence is not sufficient to induce a motile phenotype.Interestingly, the Rac1-specific exchange factor Tiam1 isalso required for Rac activation and cell motility in bothSchwann cells (17) and breast epithelial cells (current study),and we observe a precise positive correlation between Tiam1expression and motility in tumor-derived breast epithelialcells.3 This supports our model that Dbs and Tiam1 may infact be key regulatory components of a signaling complexthat is required to support normal cell motility during devel-opment and aberrant motility during tumorigenesis. Ourresults suggest that in the absence of either exchange factor,motility is impaired.

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Zhuoming Liu, Homer C. Adams III and Ian P. WhiteheadCancer Cell Migration

The Rho-specific Guanine Nucleotide Exchange Factor Dbs Regulates Breast

doi: 10.1074/jbc.M901853200 originally published online April 14, 20092009, 284:15771-15780.J. Biol. Chem.

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