INTRODUCTION

The small GTPases of the Rho family play key roles intransducing extracellular stimuli into distinct responsesincluding cell motility, adhesion, cell division andphagocytosis. The GTPases cycle between GTP-bound andGDP-bound forms and their activation requires the action ofguanine nucleotide exchange factors (GEFs) to promote theconversion of the GDP to the GTP state. Individual membersof the Rho GTPases are known to cause specific changes to theactin cytoskeleton of the cells. Active RhoA causes actin stressfibre formation, whereas dominant active Cdc42 and Rac1induce the formation of filopodia and lamellipodia,respectively. The rearrangement of the cytoskeletal structuresis pivotal to the outcome of the signal transduction eventsdownstream of the Rho GTPases (Lim et al., 1996; Van Aelstand D’Souza-Schorey, 1997).

Some of the downstream effectors of the Rho GTPases andthe pathways they regulate are well studied. In particular, theformation of stress fibres, which is downstream of RhoA-GTP,requires the function of Rho-kinase/ROK (Amano et al., 1997;Leung et al., 1995). ROK can phosphorylate and inactivate themyosin-binding subunit of the myosin light chain (MLC)phosphatase (Kimura et al., 1996). This results in an increasein phosphorylated MLC, which has enhanced actin binding andbundling activity, and hence an increase in stress fibreformation. The effector proteins downstream of Rac1 inlamellipodia formation are not as well characterized althoughPOR1 might be involved in this process (Van Aelst et al.,

1996). N-Wasp mediates the link between Cdc42 and theArp2/3 proteins in actin polymerization, which mightparticipate in the formation of filopodia (Miki et al., 1998;Rohatgi et al., 1999). The ROK-related target MRCK isdirectly involved in the formation of focal complexes (FCs) andfilopodia, as demonstrated by observations that a kinaseinactive mutant can block these processes downstream ofCdc42 (Leung et al., 1998).

A less well characterized morphological effect of RhoGTPases is microvillus formation. These apical membraneprotrusions, found on polarized epithelial cells, fibroblasts andlymphocytes, are important for the cells to sense extracellularsignals. At least one member of the ERM (ezrin, moesin andradixin) family of proteins is required to drive microvillusformation by specifically cross-linking actin filaments to theplasma membrane. The activation of ERM proteins is linkedto phosphorylation, phosphoinositide binding and RhoAsignalling pathways (Bretscher et al., 1997; Hirao et al., 1996;Matsui et al., 1999; Oshiro et al., 1998; Shaw et al., 1998;Tsukita and Yonemura, 1999). It has also been reported that,by activating Cdc42, RhoG causes the formation of microvilli(Gauthier-Rouviere et al., 1998).

PAK is an effector kinase of Cdc42 and Rac1 (Manser et al.,1994) in promoting the breakdown of Rho-dependent actinstress fibres and focal adhesion complexes (Manser et al.,1997; Sells et al., 1997). We have isolated a PAK-interactingexchange factor (PIX) that exhibits exchange activity towardsboth Cdc42 and Rac1 in vitro (Manser et al., 1998). Theidentification of PIX demonstrates a GTPase activator being

4239

PIX is a Rho-family guanine nucleotide exchange factorthat binds PAK. We previously described two isoforms ofPIX that differ in their N termini. Here, we report theidentification of a new splice variant of βPIX, designatedβ2PIX, that is the dominant species in brain and that lacksthe region of ~120 residues with predicted coiled-coilstructure at the C terminus of β1PIX. Instead, β2PIXcontains a serine-rich C terminus. To determine whetherthese splice variants differ in their cellular function, westudied the effect of expressing these proteins in HeLa cells.We found that the coiled-coil region plays a key role in the

localization of β1PIX to the cell periphery and is alsoresponsible for PIX dimerization. Overexpression of β1,but not β2PIX, drives formation of membrane ruffles andmicrovillus-like structures (via activation of Rac1 andCdc42, respectively), indicating that its function requireslocalized activation of these GTPases. Thus, β1PIX, likeother RhoGEFs, exerts specific morphological functionsthat are dependent on its intracellular location and aremediated by its C-terminal dimerization domain.

Key words: GEF, GTPase, Rho family, PIX, PAK

SUMMARY

β1PIX, the PAK-interacting exchange factor, requireslocalization via a coiled-coil region to promotemicrovillus-like structures and membrane rufflesCheng-Gee Koh 1,*, Ed Manser 1, Zhou-Shen Zhao 1, Chee-Peng Ng 2 and Louis Lim 1,3

1Glaxo-IMCB Group, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 1176092Membrane Biology Laboratory, Institute of Molecular and Cell Biology, Singapore 1176093Institute of Neurology, 1 Wakefield Street, London, WC1N 1PJ, UK*Author for correspondence (e-mail: mcbkohcg@imcb.nus.edu.sg)

Accepted 11 August 2001Journal of Cell Science 114, 4239-4251 (2001) © The Company of Biologists Ltd

RESEARCH ARTICLE

4240

directly coupled to an effector, thereby providing specificity tothe signalling pathway. In the case of T-cell receptor activationthe PIX-PAK interaction is indeed required for GTPase-mediated kinase activation (Ku et al., 2001). The complexmight also provide the link for cross-talk between Cdc42 andRac1 pathways because elevated levels of βPAK (which can berecruited by Cdc42) drive a Rac phenotype in PC12 cells(Obermeier et al., 1998; Sells et al., 1999). The PAK-PIXinteraction, mediated by the SH3 domain of PIX, plays a keyrole in these two cell systems.

Other identified domains of PIX include the calponinhomology (CH), Dbl homology (DH), pleckstrin homology(PH) and a GIT1 binding domain. Here, we demonstrate thata discrete coiled-coil C-terminal domain appears to regulatePIX function via intermolecular interactions. Although theuniversal pairing of DH and PH domains in Dbl-familyRhoGEFs suggests that the PH domain modulates the activitiesof the DH domain, the solution structure of β1PIX DH andPH domains does not reveal such a functional coupling(Aghazadeh et al., 1998).

The intracellular localization of RhoGEFs is often achievedby specific domains which associate with other proteins orphospholipids at the cell membrane. The PH domain of Dblmediates the oncogenic activities of the protein by targeting itto specific cytoskeletal components (Zheng et al., 1996). TheN-terminal PH domain of Tiam1 localizes it to the plasmamembrane allowing Rac-mediated membrane ruffling and JNKactivation (Michiels et al., 1997; Stam et al., 1997). The PHdomain of the Ras/Rac GEF Sos is involved in membranetargeting and is preferentially localized to the leading edge ofthe motile cells (Chen et al., 1997). By contrast, other Dbl-family proteins Lfc and GEF-H1 are localized to themicrotubule network (Glaven et al., 1999; Ren et al., 1998).The proper presentation of the exchange factors to theirrespective GTPases is thus critical to their biological activities.

In this paper, we report a βPIX splice variant that is enrichedin the brain. All PIX isoforms are substrates of PAK but theirexchange activities are not affected by the phosphorylation.β1PIX, but not β2PIX, translocates to the cell periphery, whereit drives formation of ruffles and microvillus-like structures.The coiled-coil region in β1PIX appears to be responsible forits localization to the cell periphery and for mediating itscellular activities.

MATERIALS AND METHODS

Isolation of β2PIX cDNA and two-hybrid screeningβ2PIX was isolated by screening a rat brain cDNA library in λZapIIfrom Stratagene, using [α32P]dCTP-labelled cDNA fragmentencompassing nucleotides 483-671 of αPIX. Various β2 cDNA wereassessed by restriction enzyme mapping, and a full length cDNAgenerated in the pXJ vectors by splicing the 3′ region of the β2 cDNAto the β1PIX sequence at the internal Kpn1 site.

Full length αPIX was cloned into pAS2-1 vector (Clontech) as thebait in a two-hybrid screen for PIX-interacting proteins. The N-terminal (1-360 bp) and C-terminal (2014-2331[stop-codon] bp) ofαPIX were amplified by PCR with restriction sites incorporated forthe convenience of cloning. Most of the cDNA fragment was clonedby insertion of the original cDNA from the internal BamHI (174 bp)to NcoI (2025 bp) sites. The cDNA library used was a human braincDNA Matchmaker library in pAct2 from Clontech. The bait

recombinant plasmid was first transformed into a reporter yeast strain(Y190), which contained the HIS3 and lacZ genes under the controlof a Gal4-responsive element. The matchmaker cDNA library wasthen transformed into Y190 containing the bait plasmid. The His3gene allowed a positive growth selection for clones that were thenscreened using the blue/white β-galactosidase (β-Gal) filter assay toconfirm the protein interactions. An estimated 1×106 transformantswere screened. After putative positive yeast clones were identified,recombinant plasmids in pAct2 were extracted and retransformed intoY190 containing the bait plasmid. The β-Gal filter assay was repeatedand positive clones were sequenced.

Generation of anti-PIX antibodiesPolyclonal anti-PIX SH3 antibodies were raised by injection ofglutathione-S-transferase (GST) fusion protein of αPIX (amino acids155-545) into rabbit. The rabbit antisera obtained were tested forspecificity by western blot analysis using protein lysate of rat tissuesand cell lysates containing transfected Flag-tagged PIX isoforms. Itwas found that the antisera was able to recognize specific bands (usedat 1:500 dilution). Only a single band was observed with the Flag-PIX transfected cell lysates. The antibodies were affinity purifiedusing MBP-PIX-SH3 column. A similar method was used to purifyantibodies against the serine-rich C terminus of β2PIX. The peptideused was amino acids 556-625 of β2PIX.

Cell fractionationCOS-7 cells were lysed in hypotonic buffer (50 mM HEPES (pH 7.3),1 mM MgCl2) without any detergent. NaCl and PMSF were added to0.3 M and 1 mM, respectively, to the cell lysates. The cell lysates werespun at 100,000 g to separate the S100 soluble fraction and P100pellets. The P100 pellets were then extracted with buffer containingTritonX-100 (50 mM HEPES (pH 7.3), 1 mM MgCl2, 0.3 M NaCl,1% Triton X-100 and 1 mM PMSF) to extract the membraneembedded proteins. After centrifugation at 100,000 g, the pellets wereextracted with buffer containing 1% SDS to extract the cytoskeletalproteins. Extracts from different fraction were resolved on SDS-PAGEgels and analysed by western hybridization.

Guanine nucleotide exchange assayThe guanine nucleotide exchange activity was measured using Rac1assay as previously described (Manser et al., 1998). The GEFactivity was determined by the incorporation of [35S]GTPγS (NEN)into Escherichia coliexpressed and purified GST-Rac1. The PIXproteins used in these assays were purified from transientlytransfected COS-7 cells using anti-Flag M2 beads (Sigma) andquantified by Coomassie staining of duplicates. Bacteriallyexpressed GST-PAK was used to phosphorylate a set of theimmunoprecipitated PIX (~2 µg) prior to the exchange assays. 8 µgof GST-PAK protein was used per reaction, which also contained500 µM ATP in the kinase buffer (50 mM Hepes pH 7.3, 10 mMMgCl2, 2 mM MnCl2). The reaction was incubated at 30°C for 30minutes. The reactions were stopped by addition of EDTA. TheFlag-beads were washed twice with PBS buffer before proceedingwith the exchange assay. In each exchange assay about 2 µg of Flag-PIX fusion protein and 5 µg of GST-Rac1 were used. The reactionmixture was incubated at 30°C. Bound [35S]GTPγS was assessed byabsorption of protein onto nitrocellulose membranes and liquidscintillation counting. In each experiment, two aliquots were takenat each time. Each experiment was repeated.

In vitro kinase assayImmunoprecipitated Flag-PAK or GST-PAK was washed with kinasebuffer and then incubated in 30 µl of the same buffer containing 500µM ATP and 10 µCi [γ-33P]ATP (Amersham) and 10 µg of myelinbasic protein as substrate. The reaction mixtures were incubated at30°C for 30 minutes and the reactions were stopped by the additionof the SDS sample buffer.

JOURNAL OF CELL SCIENCE 114 (23)

4241PIX dimerization induces ruffles and microvilli

Cell transfection and microinjectionRelevant cDNAs were cloned into the pXJ-Flag vector formammalian cell expression (Manser et al., 1997). pXJ-Flag-αPIXwas cloned similarly as described above. pXJ-Flag-β2PIX wascloned replacing the C terminus of β1PIX by a PCR fragment at theinternal KpnI (962 bp) site. The N-terminal deletion mutant, pXJ-Flag-∆N80β1PIX, was generated by splicing a PCR fragmentcovering 241 bp to the KpnI site (962 bp) of β1PIX to vectorcontaining DNA fragment C-terminal to the KpnI site. The C-terminal truncation mutant pXJ-Flag-β1PIX1-555 was generated bysplicing a PCR fragment containing the truncated C terminus to the

N terminus of β1PIX at the internal KpnI site. pXJ-GST-β1PIX-C-ter was constructed by cloning a PCR fragment containingnucleotides 1612-1941 of β1PIX. The cDNA constructs weretransfected into COS-7 or HeLa cells using DOSPER (BoehringerMannheim) or SuperFect (Qiagen) using the supplier’s protocol. Inmost cases, 8 µg of plasmid DNA was used per 100 mm dish of 80%confluent cells and 1.5 µg of plasmid DNA was used for 20×20 mmtwo-well chamber slide (Nalge Nunc International). Cell staining wasdone as previously described (Manser et al., 1997).

HeLa cells were seeded onto 20×20 mm glass cover slips andinjected using an Eppendorf microinjector (no number given on the

776

∆CH SH3 DH PH

αPIX

coiled coilGIT1 Binding

647417 448 555

β1PIX I

serine-rich625β2PIX III

630β1PIX -c II

705β1PIX -b I II

90% 81% 72% 79%89%

A

B

----------SAHS SFSSTGQPRGPLEPPQIIKPWSLSCL

LTPAYHTLPHPSHHGTPHTTISWGPLEPPKTPKPWSLSCL

565

438 β1/β2PIXαPIX

RPAPPLRPSAALGYKERMSYILKESSKSPKTMKKFLHKRK

RPAPPLRPSAALCYKEDLS-------RSPKTMKKLLPKRK

595

478

αPIXβ1/β2PIX

TERKPSEEEYVIRKSTAALEEDAQILKVIEAYCTSANFQQ

PERKPSDEEFAVRKSTAALEEDAQILKVIEAYCTSAKTRQ

635

512

αPIXβ1/β2PIX

GHGSSTRKDSIPQVLLPEEEKLIIEETRSNGQTIMEEKSL

TLNSSSRKESAPQVLLPEEEKIIVEETKSNGQTVIEEKSLTLNSTWQ---GTDLM---HNHVLADDDQSSLDSLGRRSSL

675

551

551

αPIXβ1PIX

VDTVYALKDEVRELKQENKRMKQCLEEELKSRRDLEKLVRVDTVYALKDEVQELRQDNKKMKKSLEEEQRARKDLEKLVR

SRLV------PSDLSEDSEY--DSLWTAHSYR--MGSASR

715

591

585

αPIXβ1PIXβ2PIX

RLLKQTDECIRGESSSKTSILP

KVLKNMND PAWDETNLK SCCSSISHQN

765

631

615

αPIXβ1PIXβ2PIX

coiled-coil

β2PIX

GIT1 binding * *

GIT1 binding

Fig. 1.Properties of PIX. (A) Different isoforms of PIX. Abbreviations: CH, calponin homology domain; SH3, Src homology 3; DH, Dblhomology domain; PH, pleckstrin homology domain; inverted triangle, deletion in αPIX; I, insert sequence of ~30 amino acids inβ1PIX andβ2PIX not present in αPIX; II, insert sequence of ~60 amino acids present in β2PIX, β1PIX-b and β1PIX-c. The numbers represent thepercentage similarity between the domains of αPIX and β1PIX. β1PIX-b, β1PIX-c and β2PIX are all splice variants of β1PIX. (B) Alignmentof the C termini of αPIX, β1PIX and β2PIX. The C termini of αPIX and β1PIX contain a coiled-coil structure, whereas that of β2PIX is serinerich. The consensus sequences are boxed. The inverted arrowheads mark the start of the coiled-coil regions of αPIX and β1PIX, and also theserine-rich region of β2PIX. The major PAK phosphorylation sites are marked with asterisks. The GIT1 binding region is from residues 460 to555 of β1PIX. (C) Western analysis of cell lysates of different tissues from rat and also several cell lines. The membranes were probed withanti-PIX-SH3 antibodies (top and middle), which recognized all isoforms of PIX, and with anti-β2PIX-C-terminus antibodies (bottom), whichonly recognized the β2 isoforms. Abbreviations for the rat tissue protein lysates: B, brain; K, kidney; Li, liver; Lu, lung; S, spleen; Te, testis;Th, thymus. Abbreviations for the cell line protein lysates (middle): C, COS-7; H, HeLa; N, NIH 3T3; K, kidney protein lysates; F, pXJ-Flag-β1PIX transfected COS-7 cell lysate.

4242

apparatus) and a Zeiss axiovert microscope. Plasmid (50 ng ml–1)encoding green fluorescent protein (GFP) was injected with the cDNAof interest (50 ng ml–1). The injected cells were returned to theincubator for 2-4 hours prior to fixation in 3% paraformaldehyde asdescribed previously (Manser et al., 1997).

ImmunoprecipitationRelevant cDNAs were cloned in pXJ-Flag or pXJ-GST mammalianexpression vectors (Manser et al., 1997). The cells were harvested inprotein lysate buffer (50 mM Hepes (pH7.5), 0.3 M NaCl, 1 mMMgCl2, 1 mM EGTA, 10 mM β-glycerophosphate, 10 mM NaF, 1 mMsodium vanadate, 5% glycerol, 5 mM DTT, 0.5% Triton X-100). Thecell lysates were passed through a 30G syringe (×3) and were clearedby centrifugation at 10,000 g for 5 minutes. The Flag-tag proteinswere isolated using anti-Flag Mab M2-beads (Sigma) and GSTfusions with glutathione-Sepharose beads (Pharmacia). Proteincomplexes were dissociated from the beads by heating to 100°C in 1×SDS buffer for 3 minutes.

Scanning electron microscopyHeLa cells were first plated on glass coverslips and microinjected withthe DNA constructs of interest in pXJ-Flag or pXJ-HA expressionvectors (Manser et al., 1997) together with pXJ-GFP plasmid DNAfor identifying the injected cells. Injected cells were incubated for 2-4 hours to allow protein expression. Cells that failed to express theGFP marker were removed and remaining cells expressing the proteinof interest were fixed with 1% glutaraldehyde. Samples were thengradually dehydrated using increasing ethanol, followed by criticalpoint drying and gold sputtering. The samples were analysed with aPhillips XL30-FEG scanning electron microscope. About 30-50 cellswere examined for each experiment.

RESULTS

Characterization of a new βPIX isoformPIX are GEFs that contain an N-terminal SH3 domain thatspecifically binds PAK, and are encoded by the αPIX and βPIX(p85SPR, p85COOL-1) genes (Bagrodia et al., 1998; Manser etal., 1998; Oh et al., 1997). It appears that the p78 βPIX speciesis ubiquitous. cDNA coding for a shorter variant of βPIX(p50COOL-1) and two other cDNA species (β1PIX-band β1PIX-c) have been reported, although the corresponding proteinshave yet to be identified (Bagrodia et al., 1998; Kim et al.,2000). We have now isolated another cDNA splice variantencoding a protein (β2PIX) in which a shorter serine-richdomain replaces the C-terminal coiled-coil sequences (residues556-646) that are present in the original cDNA product that wenow designate β1PIX. β2PIX is 625 amino acids long (Fig. 1A)with a predicted molecular mass of 68 kDa. A sequencecomparison of the different C-terminal domains of αPIX,β1PIX and β2PIX is shown in Fig. 1B. The predicted coiled-coil regions of αPIX and β1PIX are well conserved, and aremost similar to sequences found in the Myo2 protein. An insert(I) of 31 amino acid residues (417-448) in β1PIX and β2PIXfollowing the PH domain is not present in αPIX.

To characterize β2PIX, antibodies were raised against theserine-rich C-terminal domain. Western analysis revealed thatthe p70 β2PIX was most abundant in brain (from which thecDNA was isolated) and was also present in testis and thymus(Fig. 1C). Interestingly, the antibody detected a larger (p80)

JOURNAL OF CELL SCIENCE 114 (23)

Fig. 2. Cellular distribution of PIX and PAK. (A) Distribution of PIX andPAK by cell fractionation. COS-7 cells transfected with pXJ-Flag-β1PIX andpXJ-Flag-β2PIX were fractionated, analysed by SDS-PAGE and probed withanti-Flag antibodies. Untransfected COS-7 cell fractions were also analysedby SDS-PAGE and probed with anti-PIX-SH3 or anti-α/β-PAK (PAK1 andPak3) antibodies. The fractions collected are: T, total protein; S, water-soluble

proteins (S100 fraction); Tx, Triton-X-100-soluble proteins (P100); P, SDS-soluble proteins of the pellet from Triton-X-100 extraction, whichcontain cytoskeletal proteins. (B) Cellular localization of PIX isoforms and mutants. Various PIX constructs were transiently transfected intoHeLa cells. The DNA constructs used were pXJ-Flag-αPIX, pXJ-Flag-β1PIX, pXJ-Flag-β2PIX, pXJ-Flag-β1PIX1-555, pXJ-Flag-∆N80β1PIXand pXJ-GST-β1PIX-C-ter. The proteins expressed were immunostained using anti-Flag or anti-GST antibodies. Constructs that containedthe coiled-coil C terminus were localized at the cell periphery, whereas β2PIX and the C-terminally truncated mutant β1PIX1-555 showedcytoplasmic and nuclear staining.

4243PIX dimerization induces ruffles and microvilli

species that was recognized by antibodies raisedagainst the SH3 domain (which is common to allPIX). This might represent a protein species withadditional insert sequences flanking the PHdomain, as reported by Kim et al. (Kim et al., 2000)or with an N-terminal CH domain that is alsopresent in βPIX (Z.-S.Z., unpublished). Thus, p70β2PIX shows a more restricted expression patternthan the β1PIX. Western analysis has found β1PIXto be the predominant isoform present in COS-7,HeLa and NIH 3T3 cell lines (Fig. 1C, middle).

PIX isoforms are associated with differentcellular fractionsDNA constructs of Flag-β1PIX and Flag-β2PIXwere transfected into COS-7 cells to determine thedistribution of these two isoforms in the cell. Fromthe analysis of fractionated cell extracts, most Flag-β1PIX was present in the Triton-X-100-solublefraction (Fig. 2A). Some Flag-β1PIX was alsofound in the water-soluble extract and in the SDS-soluble fraction. However, most of the Flag-β2PIXwas found in the water-soluble fraction (Fig. 2A).

Fig. 3. PIX forms homodimers. (A) The C terminus ofβ1PIX is required for PIX dimerization. GST-β1PIX-C-ter was transfected with various Flag-PIX constructsinto COS-7 cells except for the last lane where fulllength β1PIX constructs were used. The GST-taggedprotein complex was isolated using glutathione-Sepharose beads (GST pull down), separated by SDS-PAGE and subjected to western blot analysis with anti-Flag antibodies. Only constructs that contained the Cterminus could form complexes with GST-β1PIX-C-ter.(B) The coiled-coil but not the serine-rich C-terminusof PIX is responsible for dimerization. Differentlytagged β1PIX and β2PIX were transfected into COS-7cells with: 1, pXJ-GST-β1PIX and pXJ-Flag-β1PIX; 2,pXJ-GST-β1PIX and pXJ-Flag-β2PIX; 3, pXJ-GST-β2PIX and pXJ-Flag-β2PIX. The expressed protein wasisolated using glutathione or Flag beads and subjectedto western blot analysis with anti-Flag or anti-GSTantibodies, respectively. The asterisk marks the GST-β1PIX fusion protein that was found in the Flag-immunoprecipitated complex. (C) PAK and PIX canform a multimeric complex. COS-7 cells weretransiently transfected with (1) GST-PAK andFlag-∆Ν80β1PIX, (2) GST-PAK, HA-PIX and Flag-∆Ν80β1PIX, (3) GST-PAK, HA-PAK and Flag-β1PIX1-459, (4) GST-PAK, HA-PAK and Flag-∆Ν80β1PIX, (5) GST-PAK, HA-PAK and Flag-β1PIX.GST fusion proteins were isolated from the cell lysatesusing glutathione beads and subjected to western blotanalysis. Flag-∆N80β1PIX could be found in theisolated GST-PAK complex only when dimerized withfull-length HA-β1PIX (lane 2). HA-PAK was detectedin the isolated complex of (5), implying that PAK-PIX-PIX-PAK tetramer could be formed (lane 5). Theβ1PIX dimer is required to bring GST-PAK and HA-PAK together into the same complex; a mutant ofβ1PIX (β1PIX1-459) unable to dimerize could not do so(lane 3). The asterisks mark the HA-tagged proteins,the hashes mark the Flag-tagged proteins.

4244 JOURNAL OF CELL SCIENCE 114 (23)

β1PIX

0 10 20 30 40 500

10

20

30

40

50

60

Time/ mincp

m (x

1000

)

αPIX

0

10

20

30

40

50

60

cpm

(x10

00)

0 10 20 30 40 50

Time/ min

β2PIX

cpm

(x10

00)

Time/ min

0

10

20

30

40

50

60

0 10 20 30 40 50

GST

PIX

PIX+PAK

B

Fig. 4.Phosphorylation by PAK did not affect the exchange activitiesof PIX. (A) PAK phosphorylates PIX between residues 496 and 555of β1PIX. Various Flag-tagged PIX isoforms and mutants weretransfected into COS-7 cells and immunoprecipitated. Theprecipitated proteins were subjected to in vitro phosphorylation byGST-PAK with [γ-33P]ATP in the reaction buffer. The phosphorylatedproduct was analysed on SDS-PAGE gel and exposed to X-ray film.Top, the Coomassie-Blue-stained gel. Bottom, the autoradiograph.The asterisks mark the bands of interest. The Flag-tagged proteinsare: lane 1, β2PIX; lane 2, β1PIX; lane 3, β1PIX1-459; lane 4,β1PIX1-555; lane 5, ∆N80β1PIX; lane 6, β1PIX∆pro (deletion ofamino acids 460-495 of β1PIX); lane 7, GST-β1PIX-C-ter; lane 8,GST-PAK alone. (B) Various Flag-tagged isoforms of PIX weretransiently transfected into COS-7 cells. The immunoprecipitatedPIX was assayed for exchange activity using GST-Rac1 as substratewith and without prior in vitro phosphorylation by GST-PAK (thekinase prepared as a GST fusion protein from E. coli is constitutivelyactive). Each exchange assay was repeated with four determinants foreach time point and the average is shown. (C) GST-PAK wastransfected with wild-type Cdc42 and various PIX DNA constructsinto COS-7 cells. GST-PAK fusion protein from the different

transfection cell lysates was isolated by glutathione-Sepharose beads. Kinase activity was then measured by in vitro kinase assay using [γ-33P]ATP and myelin basic protein as substrates. The figure shows results from three different sets of transfection experiments and kinase assays.Top, western blot probed with anti-GST antibody to show the amount of GST-PAK from the different transfection cell lysates isolated byglutathione beads. Middle, autoradiograph showing the corresponding MBP phosphorylation by the GST-PAK pulled down in top panel.Bottom, normalized kinase activity of the GST-PAK from the corresponding transfection cell lysates. There was mobility shift of the GST-PAKband when PAK was transfected with wild-type Cdc42 and active Cdc42.

4245PIX dimerization induces ruffles and microvilli

Fig. 5.αPIX and β1PIX induced cellruffling via Rac1. (A) αPIX and β1PIXpromote the formation of phase-darkruffle-like structures. HeLa cells weremicroinjected with DNA constructs ofthe various PIX and PIX mutants inpXJ-40 vector. The cells were thenviewed by phase-contrast microscopy.The injected cells are marked byarrowheads. The DNA construct usedwere pXJ-Flag-αPIX, pXJ-Flagβ1PIX,pXJ-Flag-β2PIX, pXJ-Flag-∆N80β1PIX, pXJ-Flag-β1PIX1-555,pXJ-GST-β1PIX-C-ter, pXJ-Flag-β1PIX-DHm and pXJ-GST vector.(B) The formation of these ruffles couldbe blocked by Rac1N17 but notCdc42N17. HeLa cells weremicroinjected with pXJ-40 DNAconstruct of β1PIX together withRac1N17 or Cdc42N17. The injected cellsare marked by arrow heads. (C) β1PIXand β2PIX could induce filopodia andcell rounding when wild-type Cdc42was also overexpressed. HeLa cellswere microinjected with the plasmidDNA constructs in pXJ-40 vector andthe cells were observed 2-2.5 hoursafter microinjection. The plasmid DNAinjected are: 1, hPem2; 2, Cdc42V12; 3,β1PIX and Cdc42 wild type (w/t); 4,β2PIX and Cdc42 w/t; 5, Cdc42 w/talone.

4246

Very little if any was found in the Triton-X-100-soluble fraction or in the SDSfraction.

Endogenous PIX and PAK proteins wereanalysed in fractionated COS-7 cellextracts. Western blotting with anti-PIX(SH3) or anti-α/βPAK (PAK1 and PAK3)antibodies revealed that ~70% of PIX(probably p78 β1PIX) and essentially allPAK were recovered in the cytosolicfraction (Fig. 2A). The remaining 30% ofPIX appeared mostly in the detergent-soluble (membrane fraction), with somefound in the detergent-insoluble (oftendefined as the cytoskeletal) fraction.

The coiled-coil domain affectsβ1PIX localizationTo determine the intracellular distributionof the various PIX isoforms and thepotential role of their various domains,cDNAs encoding different Flag-taggedPIX proteins were transiently transfectedinto HeLa cells (Fig. 2B). Both αPIX andβ1PIX were distributed in the cytoplasmbut prominent at the cell periphery,whereas β2PIX was found primarily incytoplasm and nucleus. When the coiled-coil domain of β1PIX was deleted, theβ1PIX1-555 protein showed cytoplasmicand nuclear localization similar to that ofβ2PIX. By contrast, removal of N-terminalSH3 domain (∆Ν80β1PIX) did not affectthe peripheral membrane localization ofβ1PIX. By itself, the coiled-coil C-terminus of β1PIX could localize GST to

JOURNAL OF CELL SCIENCE 114 (23)

Fig. 6.β1PIX induced the formation ofmicrovillus-like structures by activatingCdc42. (A) The formation of microvillus-likestructures was blocked by Cdc42N17 but notRac1N17. Scanning electron micrographs(SEMs) of HeLa cells microinjected withvarious plasmid DNA. The DNAs were pXJ-GST vector, pXJ-Flag-β1PIX, pXJ-Flag-β2PIX, pXJ-HA-CDC42V12, pXJ-Flag-β1PIXplus pXJ-Flag-RacN17 and pXJ-Flag-β1PIXplus pXJ-Flag-Cdc42N17. The apical surfacestructures induced by β1PIX are unlike thefilopodia induced by Cdc42V12. (B) PIXmutants did not induce microvillus-likestructures. SEMs of HeLa cells microinjectedwith plasmid constructs of the PIX mutantconstructs pXJ-Flag-∆N80β1PIX, pXJ-Flag-β1PIX-DHm and pXJ-Flag-β1PIX1-555.(C) The morphological effect of localizedactivation of Rac1 and Cdc42. Apicalprotrusions induced by β1PIX are acombination of microvillus-like structures andlamellipodia (marked by arrowheads), seenhere as higher-magnification SEM. Scale bar,10 µm.

4247PIX dimerization induces ruffles and microvilli

the cell periphery. These results indicated that the coiled-coildomain is important for the peripheral localization of αPIX andβ1PIX, and might represent the key targeting sequence forcertain αPIX and β1PIX proteins.

β1PIX but not β2PIX can form homodimers via thecoiled-coil domainIn a two-hybrid screen for PIX partners, full-length αPIX wasfound to interact with a construct containing a C-terminalportion of αPIX (residues 662-776). This C-terminal region ofαPIX also interacted with β1PIX, indicating that PIX homo-and heterodimers can be formed (data not shown). Becauseβ1PIX coiled-coil domain could dimerize but did not interactwith β1PIX1-555 (Fig. 3A), we conclude that β1PIX does notinteract in a head-to-tail manner. When GST-β1PIX538-646

(β1PIX-C-ter) was used to pull down various Flag-β1PIXconstructs, only those constructs containing thecomplementary C terminus of β1PIX were precipitated (Fig.3A). Full-length Flag-β1PIX also brought down GST-β1PIX.By contrast, no co-precipitation was observed between GST-β1PIX and Flag-β2PIX or between GST-β2PIX and Flag-β2PIX (Fig. 3B). Thus, the coiled-coil region is implicated indimerization. β2PIX appears to be monomeric because itbehaves differently from the other two isoforms.

PIX and PAK form multimeric complexesBecause PIX can form dimers and also binds tightly to PAKvia its SH3 domain, we investigated whether PIX and PAK canexist as multimeric complexes in the cell. GST-PAK, HA-β1PIX and Flag-∆Ν80β1PIX were transfected together intoCOS-7 cells. Flag-∆Ν80β1PIX could be detected in the GST-PAK complex. The results indicated that PAK binds to PIX indimeric form (Fig. 3C, lane 2). GST-PAK itself does notprecipitate with ∆N80β1PIX because the SH3 domain thatbinds PAK is missing in this mutant (Fig. 3C, lane 1). WhenDNA constructs of GST-PAK, HA-PAK and Flag-β1PIX1-459

(lacking the dimerization domain) were transfected togetherinto COS-7 cells, Flag-β1PIX1-459 was found in the complexbut HA-PAK could not be detected in the GST-PAK complex(Fig. 3C, lane 3). Neither could HA-PAK be recovered fromthe GST-PAK complex (∆N80β1PIX was included to drivenon-productive dimers with endogenous β1PIX) (Fig. 3C, lane4). However, GST-PAK could complex to HA-PAK whenβ1PIX (wild type) was present. The results suggest that a GST-PAK-(Flag-PIX)2-HA-PAK tetramer could be formed (Fig. 3C,lane 5). Hence, PAK and PIX proteins associate as multimericcomplexes.

Phosphorylation of PIX has no effect on its GEFactivityPIX was first identified as a protein that both binds to andis phosphorylated by PAK (Manser et al., 1998). Weimmunoprecipitated various Flag-tagged isoforms and mutantsof PIX from transfected COS-7 cells and subjected them to invitro phosphorylation by GST-PAK. We found that a majorPAK phosphorylation site(s) was located between residues459 and 555 of β1PIX (Fig. 4A, lanes 3,4). A deletionmutant termed β1PIX∆pro (deletion of 460-495) was stillphosphorylated by PAK (Fig. 4A, lane 6), suggesting that aprominent phosphorylation site(s) resides in β1PIX 496-555.We have mapped the major phosphorylation sites to S525 and

T526 of β1PIX (data not shown). This region is conserved inαPIX and therefore phosphorylation might regulate a commonactivity among PIX proteins. One testable function isregulation of Rac1 or Cdc42 GEF activity.

Given the differences in the in vivo behaviour of the twoβPIX splice variants (see next section), we assayed the guaninenucleotide exchange activities of the three PIX isoformstowards Rac1. Immunoprecipitated Flag-αPIX, β1PIX, andβ2PIX were quantified by Coomassie staining (not shown) andalso analysed for their GEF activities after in vitrophosphorylation by PAK. The Rac1-GTP exchange assay (Fig.4B) indicated that the exchange activities of all three isoformsare similar in vitro and that PAK phosphorylation of αPIX,β1PIX or β2PIX neither enhances nor inhibits their GEFactivities.

βPIX can negatively regulate αPAKAlthough a truncated form of βPIX suppresses PAK activation(Bagrodia et al., 1998) a truncated αPIX was reported toenhance PAK activity (Daniels et al., 1999). We thereforecompared the effects of βPIX isoforms on PAK activity in vivo.PIX constructs were expressed with GST-αPAK (Pak1) andCdc42 in COS-7 cells. Wild-type Cdc42 stimulates PAKactivity to much more limited degree than Cdc42V12 (Fig. 4C)and allows an assessment of potential activation andsuppression. GST-PAK was isolated on glutathione-Sepharosebeads and assayed using myelin basic protein (MBP) assubstrate (Fig. 4C). Consistent with previous observations,αPAK was inactive in the absence of Cdc42 (~2% of that inthe presence of Cdc42V12) (Manser et al., 1997) and β1PIX had

Rac1

ruffles

Cdc42

filopodia

PIX PIX

dimerization

PIX PIX

PAKGIT-1FAK

sensory & motility functions

PAK

PIX

PAKPAK

GIT-1 paxill in

PAKP

assembly

microvill i

Cell periphery

FC

Fig. 7.A proposed scheme for the morphological changes elicited bythe PAK-PIX-GIT1 complex. PIX is localized at the cell periphery,possibly by the localization signal found in the PIX coiled-coil Cterminus or via formation of PIX dimers that interact with otherproteins or phospholipids found at the plasma membrane-cytoskeleton interface. PAK is recruited to the focal complexes (FCs)through its binding to membrane-bound PIX, which also binds toGIT1, which in turn binds paxillin (Turner et al., 1999; Zhao et al.,2000a). Active PIX could then induce membrane ruffles by activatingRac1 as well as the formation of filopodium- and microvillus-likestructures by activating Cdc42. Upon autophosphorylation, PAKdissociates from the PIX-GIT1 complex, whereas PIX-GIT1 remainsat the cell periphery.

4248

no activating effect on PAK in these cells (data not shown).However, with Cdc42, β1PIX inhibited αPAK activity. Similarinhibition did not occur with the β1PIX1-555mutant lacking thecoiled-coil domain, and β2PIX showed an intermediate effect(Fig. 4C). Because inhibition was also observed in a β1PIXmutant (∆Ν80) lacking the PAK-binding SH3 domain, it seemsthese effects are not mediated by the direct binding of PIX toPAK.

β1PIX but not β2PIX drives the formation ofmembrane rufflesWe previously demonstrated that αPIX causes morphologicalchanges in HeLa cells consistent with Rac1 activation.However, these are somewhat different from the morphologyof Rac1V12-producing cells (Manser et al., 1998).Microinjection of αPIX and β1PIX plasmid DNA causedsimilar phase-dark ruffles at the cell periphery (Fig. 5A). Thismorphological change was not observed in cells microinjectedwith plasmids encoding β2PIX, β1PIX1-555, ∆Ν80β1PIX,β1PIX-DHm (an exchange-activity-deficient mutant) or theβ1PIX C-terminal domain. Thus, the ability of β1PIX togenerate ruffles was dependent on the integrity of domainsinvolved with PAK binding as well as dimerization. Theseobservations are consistent with the dependence oflamellipodia formation on PAK-PIX interactions in PC12 cells(Obermeier et al., 1998). The formation of these ruffles wasblocked by co-injection with dominant negative Rac1N17 butnot with dominant negative Cdc42N17 (Fig. 5B). Rac1N17 byitself did not result in any obvious change in the cellmorphology (data not shown). It has been shown that PIX hasexchange activity towards Cdc42 in vitro (Manser et al., 1998)and in vivo (Yoshii et al., 1999). However, β1PIX neitherinduces filopodium-like peripheral structures nor drives the cellrounding that is characteristic of other Cdc42 GEFs, such ashPem2 (Reid et al., 1999) (Fig. 5C). Co-injection of β1PIXwith wild-type Cdc42 gave a phenotype not seen with Cdc42alone, and more similar to cells overexpressing Cdc42V12 (Fig.5C). Co-injection of β1PIX with wild-type Rac1 elicited ruffle-like structures and enhanced cell spreading, a phenotypeassociated with overexpression of Rac1V12 but not wild-typeRac1 in these cells (not shown). Similarly, co-injection ofβ2PIX with Cdc42 (Fig. 5C) produced an activated Cdc42phenotype. A Rac1V12 phenotype was observed when β2PIXwas co-injected with wild-type Rac1 (data not shown). Thus,overexpression of β1PIX or β2PIX elicits phenotypesassociated with activated Rac1 and Cdc42 only when levels ofthe wild-type GTPase are increased, unlike with Tiam1 orhPEM2. This activity was apparent even though a proportionof β2PIX was targeted to the nucleus.

Scanning electron microscopy reveals PIX-inducedmicrovillus-like structuresBecause we were unable to observe morphological changesother than the phase-dark ruffles by light microscopy, scanningelectron microscopy (SEM) was used to investigate changes atthe cell surface. HeLa cells were microinjected with β1PIXcDNA and fixed after 2 hours for imaging by SEM. The β1PIXexpressing cells exhibited numerous membrane protrusions ontheir surface compared with uninjected cells, although theperipheral ruffles seen by light microscopy were notparticularly evident by this technique (Fig. 6A). These surface

protrusions resembled microvilli and were not induced byβ2PIX. By contrast, Cdc42V12 produced apical structures thatwere significantly longer than the PIX-induced microvillus-like structures, which explains why these structures cannot beseen by light microscopy. The β1PIX induced structures wereblocked by co-injection with Cdc42N17 but not with Rac1N17

(Fig. 6A, bottom) implying that the formation of microvillus-like structures was indeed downstream of Cdc42. Cells injectedwith vector DNA alone exhibited a smooth plasma membranesurface (Fig. 6A).

Surface structures were either absent or sparse in cellexpressing β1PIX mutants (β1PIX-DHm, β1PIX1-555 or∆Ν80β1PIX) (Fig. 6B). Constitutively active Rac1V12 causedcell spreading but no filopodium- or microvillus-like structureswere observed (not shown). At higher magnification, weobserved that the β1PIX-induced microvillus-like structuresarose from ruffles (Fig. 6C, arrowheads), indicating that thesemight be hybrid structures. These observations suggesting thatPIX can activate both Cdc42 and Rac1 in vivo are in agreementwith previous reports (Daniels et al., 1999; Manser et al., 1998;Yoshii et al., 1999) and we now show these activities to bedependent upon the coiled-coil domain at the C-terminus.

DISCUSSION

A role for PIX in localization of its partnersIn this paper, we report an alternate spliced isoform of βPIXwith distinct properties and tissue distribution. The cellulardistributions of β1PIX and β2PIX differ in that most β1PIX ismembrane bound but β2PIX is mainly cytosolic (Fig. 2A). Asmall proportion of PIX might bind either to lipid rafts or thecortical cytoskeleton because it cannot be extracted bynonionic detergent. The predicted coiled-coil C-terminaldomain of αPIX and β1PIX drives both dimerization andtargeting to the membrane. One possibility is that dimerizationpotentiates a relatively weak membrane-binding activity of thePIX monomer (e.g. through the PH domain). Constructs thatlack the coiled-coil C-termini are distributed primarily in thecytoplasm and nucleus. However, because the coiled-coilsequence alone localizes GST protein to the cell periphery(Fig. 2B), it appears that this property is intrinsic to thisdomain.

αPIX contains an additional CH domain at its N terminusthat is not present in βPIX. Although the CH domain is foundin many actin-binding proteins and signalling molecules, asingle CH domain is not sufficient to bind actin (Gimona andMital, 1998). It has been proposed that actin binding requirestwo CH domains (Stradal et al., 1998), and so dimerizationmight confer actin binding on αPIX.

PIX isoforms containing coiled-coil C-termini might formheterodimers. None of the cell lines we have tested containmultiple PIX isoforms (Fig. 1C), although both αPIX andβ1PIX are present in Jurkat cells (Ku et al., 2001). Membranelocalization could certainly facilitate dimer formation, as in thecase of Ras (Inouye et al., 2000), where dimerization of theGTPase is essential for the activation of Raf-1. Yet anotherexample is the N terminus of amphiphysin II, which containssequences responsible for both plasma membrane targeting anddimerization (Ramjaun et al., 1999). It has been reportedrecently that the DH domain of the Dbl oncoprotein forms

JOURNAL OF CELL SCIENCE 114 (23)

4249PIX dimerization induces ruffles and microvilli

oligomers and that oligomerization is essential for Dbl-inducedtransformation (Zhu et al., 2001). It was suggested thatoligomerization of Dbl could result in a signalling complex thatfurther augments and co-ordinates the GEF activities of Dbl.Clearly, dimerization of PIX provides the possibility offorming multimeric complexes with PAK, GIT1/p95PKL(Bagrodia et al., 1999; Turner et al., 1999; Zhao et al., 2000a)and associated proteins (Fig. 7). Interestingly, PAK has alsorecently been reported to form dimers via the Cdc42/Rac1-binding domain (Lei et al., 2000).

The importance of PIX for PAK function is demonstrated inT-cell receptor signalling. Efficient αPAK (PAK1) activationrequires its binding sites for Rho GTPases and for PIX.Overexpression of β1PIX that either cannot bind PAK or lacksGEF function prevents PAK1 activation (Ku et al., 2001); it isalso suggested that the kinase needs to be localized by GIT1.We have previously shown that PAK requires PIX to localizeto FCs (Manser et al., 1998). Significantly, PIX binding toGIT1 links it to the central FC components paxillin and FAK.This protein complex promotes focal adhesion turnover andRac1-dependent motility (Turner et al., 1999; Zhao et al.,2000a). Although PAK activation and autophosphorylationlead to dissociation from its partner, PIX (Zhao et al., 2000b),the ability of PIX to inhibit kinase activity (Fig. 4C) potentiallyincreases the lifetime of the complex.

Although PAK can bind PIX tightly, their subcellularlocation confirms our previous observations of a dynamicassociation between the pair. Thus, essentially all of the αPAKand βPAK are found in the cytosol, whereas ~30% of PIX ismembrane associated and also present in a detergent-insolublefraction (Fig. 2A). Because active (autophosphorylated) PAKdissociates from PIX (Zhao et al., 2000b), PAK will onlytransiently complex to PIX at the membrane owing to thepresence of kinase activators at this site (Lu et al., 1997).However, introducing a PAK inhibitor into cells allows PAK tobe stabilized within FCs (Zhao et al., 2000a) (where it isusually not visible). Thus, we propose a model in which PAKcan be recruited to the membrane and FCs through binding tomembrane-associated PIX (Fig. 7). Indeed addition of a CAAXbox to PAK has a similar effect of driving αPAK to FCs(Manser et al., 1997). This localization is further enhanced byPIX binding to GIT1, which unmasks the cryptic paxillinbinding site in the GIT1 C-terminal region (Zhao et al., 2000a).Upon activation, PAK is immediately released back to thecytosol while PIX-GIT1 remains at the membrane (and FCs).This might explain why most of PAK fractionates into thecytosolic fraction, whereas PIX is distributed between thecytosol and the membrane-cytoskeleton fraction.

Localized morphological changes produced by PIXThe interactions that lead to PIX-mediated cell shape changesare complex. Although the in vitro and in vivo GEF activitiesof PIX are very low compared with other Dbl family members(Manser et al., 1998), PIX can apparently still causemorphological changes but in a more restricted manner. Thisrestriction reflects PIX’s localization to the membrane, wherebound phosphatidylinositol-3-kinase reportedly co-operateswith αPIX in activating Cdc42/Rac1 (Yoshii et al., 1999).Here, we observe that only PIX isoforms that dimerize causeruffle and microvillus-like structure formation. Nonetheless,the binding of PAK to PIX is important because the SH3

deletion mutant (∆Ν80) is ineffective. This is consistent withprevious data showing that PAK-induced lamellipodiumformation is dependent upon PIX binding (Obermeier et al.,1998). We suggest that the β2PIX isoform would becomeactive by recruitment through as yet unidentified membrane-associated partners.

The induction of phase dark ruffles by full-length β1PIX wasnot observed with β2PIX, the exchange deficient mutantβ1PIX-DHm or β1PIX lacking either the SH3 domain or thecoiled-coil region. These results suggest that the C-terminalsequences target PIX to the cell periphery, where its exchangeactivity is closely linked to PAK association. These ruffleswere unlike those induced by microinjection of a more activeRac1-specific GEF, Tiam1 (Michiels et al., 1995) (data notshown), which gives a similar phenotype to cells injected withRac1V12, as reported previously (Manser et al., 1997). Thissuggests that β1PIX drives a more localized production ofRac1-GTP, although co-injection of β1PIX with Rac1 didresult in cell spreading, resembling that generated byexpression of Rac1V12 (data not shown). The phase-dark rufflesare not blocked by Cdc42N17, indicating that Rac1 activationis direct.

We show here for the first time that PIX also promoteslocalized changes on the cell membrane resembling microvilli.Microvilli and filopodia do share many similarities andcomponents. Although villin has been recognized as a tissue-specific component, these structures can form in its absence,which explains why microvilli are seen in epithelial cells andfibroblasts that do not express villin. The structures induced byPIX are microvillus-like, based on their position, number andultrastructure by SEM (i.e. they are not visible by lightmicroscopy). Such short microvillus-like structures have beenreported with RhoA and RhoG, although, with the latter, theyare thought to occur indirectly via activation of Cdc42(Gauthier-Rouviere et al., 1998; Shaw et al., 1998). Consistentwith this, β1PIX-induced microvillus-like structures areblocked by Cdc42N17 but not Rac1N17. That β2PIX and β1PIXderivatives (β1PIX-DHm, β1PIX1-555, ∆Ν80) do not inducemicrovillus-like structures confirms that the localization of(functional) PIX is important in its induction of microvillus-like structures, which is potentiated by an association withPAK.

Recent genetic evidence implicates the large GEF Trio ina pathway that includes DPak and the Drosophila Nckhomologue Dock. (Newsome et al., 2000). The N-terminalGEF domain of Trio, TrioGEF1, can activate both RhoG andRac1 (Bellanger et al., 1998; Blangy et al., 2000; Debant et al.,1996). Whether Trio and RhoG lie upstream of PIX remains tobe addressed. We have not ruled out the possibility that PIXactivates other Rho GTPases such as TC10 (Neudauer et al.,1998). Apart from the induction of long filopodia, TC10 alsocaused the formation of microvilli (Vignal et al., 2000).Although TCL is very similar to TC10, it elicits differenteffects on cell morphology, including long, thin extension atthe cell periphery and large dorsal protrusions (Vignal et al.,2000). Hence, it is unlikely that PIX preferentially activatesTCL.

Ezrin/radixin/moesin (ERM) proteins are essential formicrovillus formation and their connection to PIX signallingis of interest. Antisense phosphorothioate oligonucleotidemixtures against ERM mRNAs induce disappearance of

4250

microvilli in epithelial cells (Takeuchi et al., 1994). Thebreakdown of microvilli is commonly observed in the earlystage of apoptosis, when the ERM proteins are found totranslocate from the microvilli to the cytoplasm (Kondo et al.,1997). Moesin has been reported to be phosphorylated by theRho kinase and the related myotonic-dystrophy-kinase-relatedCdc42-binding kinase (MRCK), which is an effector of Cdc42(Leung et al., 1998; Oshiro et al., 1998). Thus, PIX at least canplay a role in the activation of the ERM proteins by recruitmentof MRCK.

In conclusion, we show that a C-terminal domain of αPIXand β1PIX plays a key role in the dimerization and localizationof PIX, which are required to drive changes in cell morphology.A proposed model of how the PAK, PIX and GIT1 function(Fig. 7) suggests that the FC provides an important dockingsite, which is consistent with PAK playing a key role in thesestructures (Manser et al., 1997). When PAK is activated byCdc42-GTP or Rac1-GTP, subsequent phosphorylation of PIXand GIT1 must modulate some as-yet-unidentified function.PIX isoforms are substrates of PAK but their in vitro exchangeactivity was unaffected by phosphorylation. By contrast, theRac1 GEF Vav1 is potently activated upon tyrosinephosphorylation by Src-family members (Crespo et al., 1997).Although β1PIX associates with the membrane via the coiled-coil domain, we do detect a significant amount of solubleβ1PIX, suggesting this process is regulated. As membraneassociation is critical for PIX to drive the local formation ofmicrovillus-like structures and membrane ruffles, it will beimportant to determine what other molecular interactions playa role in this process.

This work is supported by the Glaxo Singapore Research Fund. TheGenBank accession number for β2PIX is AY034823. While we wererevising the manuscript, Kim et al. published similar observations thatβPIX could form homodimers (Kim, S., Lee, S. H. and Park, D.(2001). Leucine zipper-mediated homodimerization of the p21-activated kinase-interacting factor, beta Pix. Implication for a role incytoskeletal reorganization. J. Biol. Chem. 276, 10581-10584).

REFERENCES

Aghazadeh, B., Zhu, K., Kubiseski, T. J., Liu, G. A., Pawson, T., Zheng,Y. and Rosen, M. K. (1998). Structure and mutagenesis of the Dblhomology domain. Nat. Struct. Biol. 5, 1098-1107.

Amano, M., Chihara, K., Kimura, K., Fukata, Y., Nakamura, N.,Matsuura, Y. and Kaibuchi, K. (1997). Formation of actin stress fibers andfocal adhesions enhanced by Rho-kinase. Science275, 1308-1311.

Bagrodia, S., Taylor, S. J., Jordon, K. A., Van Aelst, L. and Cerione, R. A.(1998). A novel regulator of p21-activated kinases. J. Biol. Chem. 273,23633-23636.

Bagrodia, S., Bailey, D., Lenard, Z., Hart, M., Guan, J. L., Premont, R.T., Taylor, S. J. and Cerione, R. A. (1999). A tyrosine-phosphorylatedprotein that binds to an important regulatory region on the cool family ofp21-activated kinase-binding proteins. J. Biol. Chem. 274, 22393-22400.

Bellanger, J. M., Lazaro, J. B., Diriong, S., Fernandez, A., Lamb, N. andDebant, A. (1998). The two guanine nucleotide exchange factor domainsof Trio link the Rac1 and the RhoA pathways in vivo. Oncogene16, 147-152.

Blangy, A., Vignal, E., Schmidt, S., Debant, A., Gauthier-Rouviere, C. andFort, P. (2000). TrioGEF1 controls Rac- and Cdc42-dependent cellstructures through the direct activation of rhoG. J. Cell Sci. 113, 729-739.

Bretscher, A., Reczek, D. and Berryman, M. (1997). Ezrin: a protein requiringconformational activation to link microfilaments to the plasma membrane inthe assembly of cell surface structures. J. Cell Sci. 110, 3011-3018.

Chen, R. H., Corbalan-Garcia, S. and Bar-Sagi, D. (1997). The role of the

PH domain in the signal-dependent membrane targeting of Sos. EMBO J.16, 1351-1359.

Crespo, P., Schuebel, K. E., Ostrom, A. A., Gutkind, J. S. and Bustelo, X.R. (1997). Phosphotyrosine-dependent activation of Rac-1 GDP/GTPexchange by the Vav proto-oncogene product. Nature385, 169-172.

Daniels, R. H., Zenke, F. T. and Bokoch, G. M. (1999). alphaPix stimulatesp21-activated kinase activity through exchange factor-dependent and -independent mechanisms. J Biol Chem274, 6047-6050 [erratum: J. Biol.Chem. 274, 15292].

Debant, A., Serra-Pages, C., Seipel, K., O’Brien, S., Tang, M., Park, S. H.and Streuli, M. (1996). The multidomain protein Trio binds the LARtransmembrane tyrosine phosphatase, contains a protein kinase domain, andhas separate Rac-specific and Rho-specific guanine nucleotide exchangefactor domains. Proc. Natl. Acad. Sci. USA93, 5466-5471.

Gauthier-Rouviere, C., Vignal, E., Meriane, M., Roux, P., Montcourier, P.and Fort, P. (1998). RhoG GTPase controls a pathway that independentlyactivates Rac1 and Cdc42Hs. Mol. Biol. Cell9, 1379-1394.

Gimona, M. and Mital, R. (1998). The single CH domain of calponin isneither sufficient nor necessary for F-actin binding. J. Cell Sci. 111, 1813-1821.

Glaven, J. A., Whitehead, I., Bagrodia, S., Kay, R. and Cerione, R. A.(1999). The Dbl-related protein, Lfc, localizes to microtubules and mediatesthe activation of Rac signaling pathways in cells. J. Biol. Chem. 274, 2279-2285.

Hirao, M., Sato, N., Kondo, T., Yonemura, S., Monden, M., Sasaki, T.,Takai, Y. and Tsukita, S. (1996). Regulation mechanism of ERM(ezrin/radixin/moesin) protein/plasma membrane association: possibleinvolvement of phosphatidylinositol turnover and Rho-dependent signalingpathway. J. Cell Biol. 135, 37-51.

Inouye, K., Mizutani, S., Koide, H. and Kaziro, Y. (2000). Formation of theRas dimer is essential for Raf-1 activation. J. Biol. Chem. 275, 3737-3740.

Kim, S., Kim, T., Lee, D., Park, S. H., Kim, H. and Park, D. (2000).Molecular cloning of neuronally expressed mouse betaPix isoforms.Biochem. Biophys. Res. Commun. 272, 721-725.

Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M.,Yamamori, B., Feng, J., Nakano, T., Okawa, K. et al. (1996). Regulationof myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase).Science273, 245-248.

Kondo, T., Takeuchi, K., Doi, Y., Yonemura, S., Nagata, S. and Tsukita,S. (1997). ERM (ezrin/radixin/moesin)-based molecular mechanism ofmicrovillar breakdown at an early stage of apoptosis. J. Cell Biol. 139, 749-758.

Ku, G. M., Yablonski, D., Manser, E., Lim, L. and Weiss, A. (2001). APAK1-PIX-PKL complex is activated by the T-cell receptor independent ofNck, Slp-76 and LAT. EMBO J. 20, 457-465.

Lei, M., Lu, W., Meng, W., Parrini, M. C., Eck, M. J., Mayer, B. J. andHarrison, S. C. (2000). Structure of PAK1 in an autoinhibited conformationreveals a multistage activation switch. Cell 102, 387-397.

Leung, T., Manser, E., Tan, L. and Lim, L. (1995). A novel serine/threoninekinase binding the Ras-related RhoA GTPase which translocates the kinaseto peripheral membranes. J. Biol. Chem. 270, 29051-29054.

Leung, T., Chen, X. Q., Tan, I., Manser, E. and Lim, L. (1998). Myotonicdystrophy kinase-related Cdc42-binding kinase acts as a Cdc42 effector inpromoting cytoskeletal reorganization. Mol. Cell. Biol. 18, 130-140.

Lim, L., Manser, E., Leung, T. and Hall, C. (1996). Regulation ofphosphorylation pathways by p21 GTPases. The p21 Ras-related Rhosubfamily and its role in phosphorylation signalling pathways. Eur. J.Biochem. 242, 171-185.

Lu, W., Katz, S., Gupta, R. and Mayer, B. J. (1997). Activation of Pak bymembrane localization mediated by an SH3 domain from the adaptor proteinNck. Curr. Biol. 7, 85-94.

Manser, E., Leung, T., Salihuddin, H., Zhao, Z. S. and Lim, L. (1994). Abrain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature367, 40-46.

Manser, E., Huang, H. Y., Loo, T. H., Chen, X. Q., Dong, J. M., Leung, T.and Lim, L. (1997). Expression of constitutively active alpha-PAK revealseffects of the kinase on actin and focal complexes. Mol. Cell. Biol. 17, 1129-1143.

Manser, E., Loo, T. H., Koh, C. G., Zhao, Z. S., Chen, X. Q., Tan, L., Tan,I., Leung, T. and Lim, L. (1998). PAK kinases are directly coupled to thePIX family of nucleotide exchange factors. Mol. Cell 1, 183-192.

Matsui, T., Yonemura, S. and Tsukita, S. (1999). Activation of ERM proteinsin vivo by Rho involves phosphatidyl-inositol 4-phosphate 5-kinase and notROCK kinases. Curr. Biol. 9, 1259-1262.

JOURNAL OF CELL SCIENCE 114 (23)

4251PIX dimerization induces ruffles and microvilli

Michiels, F., Habets, G. G., Stam, J. C., van der Kammen, R. A. andCollard, J. G. (1995). A role for Rac in Tiam1-induced membrane rufflingand invasion. Nature375, 338-340.

Michiels, F., Stam, J. C., Hordijk, P. L., van der Kammen, R. A., Ruuls-Van Stalle, L., Feltkamp, C. A. and Collard, J. G. (1997). Regulatedmembrane localization of Tiam1, mediated by the NH2-terminal pleckstrinhomology domain, is required for Rac-dependent membrane ruffling and C-Jun NH2-terminal kinase activation. J. Cell Biol. 137, 387-398.

Miki, H., Sasaki, T., Takai, Y. and Takenawa, T. (1998). Induction offilopodium formation by a WASP-related actin-depolymerizing protein N-WASP. Nature391, 93-96.

Neudauer, C. L., Joberty, G., Tatsis, N. and Macara, I. G. (1998). Distinctcellular effects and interactions of the Rho-family GTPase TC10. Curr. Biol.8, 1151-1160.

Newsome, T. P., Schmidt, S., Dietzl, G., Keleman, K., Asling, B., Debant, A.and Dickson, B. J. (2000). Trio combines with dock to regulate Pak activityduring photoreceptor axon pathfinding in Drosophila. Cell 101, 283-294.

Obermeier, A., Ahmed, S., Manser, E., Yen, S. C., Hall, C. and Lim, L.(1998). PAK promotes morphological changes by acting upstream of Rac.EMBO J. 17, 4328-4339.

Oh, W. K., Yoo, J. C., Jo, D., Song, Y. H., Kim, M. G. and Park, D. (1997).Cloning of a SH3 domain-containing proline-rich protein, p85SPR, and itslocalization in focal adhesion. Biochem. Biophys. Res. Commun. 235, 794-798.

Oshiro, N., Fukata, Y. and Kaibuchi, K. (1998). Phosphorylation of moesinby Rho-associated kinase (Rho-kinase) plays a crucial role in the formationof microvillus-like structures-like structures. J. Biol. Chem. 273, 34663-34666.

Ramjaun, A. R., Philie, J., de Heuvel, E. and McPherson, P. S. (1999). TheN terminus of amphiphysin II mediates dimerization and plasma membranetargeting. J. Biol. Chem. 274, 19785-19791.

Reid, T., Bathoorn, A., Ahmadian, M. R. and Collard, J. G. (1999).Identification and characterization of hPEM-2, a guanine nucleotideexchange factor specific for Cdc42. J. Biol. Chem. 274, 33587-33593.

Ren, Y., Li, R., Zheng, Y. and Busch, H. (1998). Cloning and characterizationof GEF-H1, a microtubule-associated guanine nucleotide exchange factorfor Rac and Rho GTPases. J. Biol. Chem. 273, 34954-34960.

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 links Cdc42-dependent signals to actin assembly. Cell 97,221-231.

Sells, M. A., Knaus, U. G., Bagrodia, S., Ambrose, D. M., Bokoch, G. M.and Chernoff, J. (1997). Human p21-activated kinase (Pak1) regulates actinorganization in mammalian cells. Curr. Biol. 7, 202-210.

Sells, M. A., Boyd, J. T. and Chernoff, J. (1999). p21-activated kinase 1(Pak1) regulates cell motility in mammalian fibroblasts. J. Cell Biol. 145,837-849.

Shaw, R. J., Henry, M., Solomon, F. and Jacks, T. (1998). RhoA-dependentphosphorylation and relocalization of ERM proteins into apicalmembrane/actin protrusions in fibroblasts. Mol. Biol. Cell9, 403-419.

Stam, J. C., Sander, E. E., Michiels, F., van Leeuwen, F. N., Kain, H. E.,van der Kammen, R. A. and Collard, J. G. (1997). Targeting of Tiam1 tothe plasma membrane requires the cooperative function of the N-terminalpleckstrin homology domain and an adjacent protein interaction domain. J.Biol. Chem. 272, 28447-28454.

Stradal, T., Kranewitter, W., Winder, S. J. and Gimona, M. (1998). CHdomains revisited. FEBS Lett. 431, 134-137.

Takeuchi, K., Sato, N., Kasahara, H., Funayama, N., Nagafuchi, A.,Yonemura, S. and Tsukita, S. (1994). Perturbation of cell adhesion andmicrovillus-like structures formation by antisense oligonucleotides to ERMfamily members. J. Cell Biol. 125, 1371-1384.

Tsukita, S. and Yonemura, S. (1999). Cortical actin organization: lessonsfrom ERM (ezrin/radixin/moesin) proteins. J. Biol. Chem. 274, 34507-34510.

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. CellBiol. 145, 851-863.

Van Aelst, L. and D’Souza-Schorey, C. (1997). Rho GTPases and signalingnetworks. Genes Dev. 11, 2295-2322.

Van Aelst, L., Joneson, T. and Bar-Sagi, D. (1996). Identification of a novelRac1-interacting protein involved in membrane ruffling. EMBO J. 15, 3778-3786.

Vignal, E., De Toledo, M., Comunale, F., Ladopoulou, A., Gauthier-Rouviere, C., Blangy, A. and Fort, P. (2000). Characterization of TCL, anew GTPase of the Rho family related to TC10 andCcdc42. J. Biol. Chem.275, 36457-36464.

Yoshii, S., Tanaka, M., Otsuki, Y., Wang, D. Y., Guo, R. J., Zhu, Y., Takeda,R., Hanai, H., Kaneko, E. and Sugimura, H. (1999). alphaPIX nucleotideexchange factor is activated by interaction with phosphatidylinositol 3-kinase. Oncogene18, 5680-5690.

Zhao, Z., Manser, E., Loo, T. H. and Lim, L. (2000a). Coupling of PAK-interacting exchange factor PIX to GIT1 promotes focal complexdisassembly. Mol. Cell. Biol. 20, 6354-6363.

Zhao, Z. S., Manser, E. and Lim, L. (2000b). Interaction between PAK andNck: a template for Nck targets and role of PAK autophosphorylation. Mol.Cell. Biol. 20, 3906-3917.

Zheng, Y., Zangrilli, D., Cerione, R. A. and Eva, A. (1996). The pleckstrinhomology domain mediates transformation by oncogenic Dbl throughspecific intracellular targeting. J. Biol. Chem. 271, 19017-19020.

Zhu, K., Debreceni, B., Bi, F. and Zheng, Y. (2001). Oligomerization of DHdomain is essential for Dbl-induced transformation. Mol. Cell. Biol. 21, 425-437.