identification of novel raci-interacting protein involved

The EMBO Journal vol.15 no.15 pp.3778-3786, 1996

Identification of a novel Raci-interacting proteininvolved in membrane ruffling

Linda Van Aelst1, Tom Joneson2 andDafna Bar-Sagi2Cold Spring Harbor Laboratory, PO Box 100, Cold Spring Harbor,NY 11724 and 2Department of Molecular Genetics and Microbiology,SUNY, Stony Brook, NY 11794, USA

'Corresponding author

The Rac GTP binding proteins are implicated in actincytoskeleton-membrane interaction in mammaliancells. In fibroblast cells, Rac has been shown to mediategrowth factor-induced polymerization of actin to formmembrane ruffles and lamellipodia. We report herethe isolation of a novel Racl-interacting protein, PORL.POR1 binds directly to Racl, and the interaction ofPOR1 with Racl is GTP dependent. A mutation in theRacl effector binding loop shown to abolish membraneruffling also abolishes interaction with PORL. Trun-cated versions of POR1 inhibit the induction of mem-brane ruffling by an activated mutant of Racl,V12Racl, in quiescent rat embryonic fibroblast REF52cells. Furthermore, POR1 synergizes with an activatedmutant of Ras, V12Ras, in the induction of membraneruffling. These results suggest a potential role for POR1in Racl-mediated signaling pathways.Keywords: membrane ruffling/Racl/Ras/signaltransduction

IntroductionThe Rac proteins are members of the Rho subfamily ofRas-related proteins. This subfamily currently consists offive distinct proteins: Rho, Rac, Cdc42, TCIO and RhoG,having 50-55% homology with each other (Hall, 1994).Like all members of the Ras superfamily, Rac proteinsact as molecular switches cycling between an inactiveGDP-bound state and an active GTP-bound state andundergo carboxy-terminal processing (Didsbury et al.,1989; Hall, 1990; Kinsella et al., 1991). Rac proteins havebeen linked to the regulation of a wide spectrum of cellularprocesses. One function attributed to Rac is its involvementin the organization of the actin cytoskeleton in fibroblasts(Ridley et al., 1992). Microinjection of activated Rac(V12Rac) resulted in rapid actin polymerization at theplasma membrane to form membrane ruffles and lamel-lipodia, followed by the appearance of stress fibers in aRhoA-dependent manner (Ridley et al., 1992). Additionof growth factors or microinjecting activated Ras (V 12Ras)also led to the formation of membrane ruffles, and thisevent was prevented by a dominant negative mutant ofRac, N17Rac (Ridley et al., 1992). This suggested acascade in which growth factors and activated Ras inducethe activation of Rac, which in turn activates RhoA. The

activation of RhoA by Rac appears to involve synthesisof leukotrienes (Peppelenbosch et al., 1995). Furthermore,Cdc42, another member of the Rho subfamily, recently hasbeen implicated in the activation of Rac. Microinjection ofactivated Cdc42 resulted in the formation of filopodia,followed by lamellipodia and stress fibers, reflecting alsoa hierarchy of interactions with Cdc42 activating theRac/RhoA pathway (Kozma et al., 1995; Nobes andHall, 1995).A more specialized role for Rac has been identified in

phagocytic leukocytes. In these cells, Rac participates inthe activation of the multicomponent NADPH oxidase togenerate superoxide in response to microbial infection(Abo et al., 1991; Knaus et al., 1991). It has been possibleto reconstitute NADPH oxidase activity in a cell-freesystem using purified cytochrome b, p47phox, p67phoxand Rac in the GTP-bound form (Abo et al., 1992).

During the last year, Rac proteins have been shown tobe involved in additional biological activities. Rac wasfound to be an important mediator of oncogenic trans-formation by Ras (Khosravi-far et al., 1995; Qiu et al.,1995) and capable of transforming Ratl fibroblast cells toa malignant phenotype (Qiu et al., 1995). Recently, Racwas also shown to activate the transcription factors serumresponse factor (SRF) and Jun (Coso et al., 1995; Hillet al., 1995; Minden et al., 1995). The mechanism for theactivation of SRF is not known (Hill et al., 1995).However, the activation of Jun is shown to be mediatedby a Rac-mediated stimulation of JNK, a stress-activatedkinase that phosphorylates the transactivation domain ofJun (Coso et al., 1995; Minden et al., 1995). In addition,Rac has been implicated in invasion of the T-lymphomacells (Michiels et al., 1995). Finally, Rac appears to playan essential role in cell cycle progression through GI(Olson et al., 1995).An important step towards understanding the bio-

chemical mechanisms by which Rac proteins exert theirdiverse cellular effects is to identify and characterize Rac-interacting proteins that mediate the actions of Rac. Sofar, several Rac-interacting proteins have been identified(Miki et al., 1993; Diekmann et al., 1994; Horii et al.,1994; Lamarche and Hall, 1994; Manser et al., 1994;Zheng et al., 1994; Bagrodia et al., 1995a; Bokoch, 1995;Knaus et al., 1995; Martin et al., 1995; Michiels et al.,1995); however, only a few among them have been provento play a role in Rac-mediated cellular processes. TheTiaml protein, involved in T-cell invasion and the induc-tion of membrane ruffles, has been shown recently to bean exchange factor for Rac (Habets et al., 1994; Michielset al., 1995), while the serine/threonine kinase PAK, whichis stimulated by Rac in a GTP-dependent manner, wasreported recently to be required for JNK and p38 mitogen-activated protein kinase activation (Bagrodia et al., 1995b;Zhang et al., 1995). Whether these proteins are also

37) Oxford University Press3778

A novel Racl-interacting protein

20R1

1 ATGGTGTCAGGACCCAACCTCAATGAAACCAGCATTGTGTCTGGTGGCTATGGGGGCTCTGGTGATGGACTCATCCCCACAGGGTCTGGCCGCCATCCATCTCACAGC1 M V S G P N L N E T S I V S G G Y G G S G D G L I P T G S G R H P S H S

109 ACCACTCCTTC$GGCCCTGGAGATGAGGTGGCTCGGGGCATTGCTGGAGAAAAGTTTGACATCGTCAAGAAATGGGGCATCAACACCTATAAGTGCACAAAGCAACTG37 T T P S G P G D E V A R G I A G E K F D I V K K W G I N T Y K C T K 0 L

217 TTATCAGAACGATTTGGTCGAGGCTCACGGACTGTGGACCTGGAGCTAGAGCTGCAGATTGAGTTGCTGCGTGAGACGAAGCGCAAGTATGAGAGTGTCCTGCAGCTG73 L S E R F G R G S K T V D L E L E L 0 I E L L R E T K R K Y E S V L Q L

325 GGCCGGGCACTGACAGCCCACCTCTACAGCCTGCTGCAGACCCAGCATGCACTGGGTGATGCCTTTGCTGACCTCAGCCAGAAGTCCCCAGAGCTTCAGGAGGAATTT109 G R A L T A H L Y S L L Q T 0 H A L C D A F A D L S 0 K S P E L Q E E F

433 GGCTACAATGCAGAGACACAGAAACTACTATGCAAGAATGGGGAAACGCTGCTAGGAGCCGTGAACTTCTTTGTCTCTAGCATCAACACATTGGTCACCAAGACCATG145 G Y N A E T Q K L L C K N G E T L L G A V N F F V S S I N T L V T K T M

541 GAAGACACGCTCATGACTGTGAAACAGTATGAGGCTGCCAGGCTGGAATATGATGCCTACCGAACAGACTTAGAGGAGCTGAGTCTAGGCCCCCGGGATGCAGGGACA181 E D T L M T V K 0 Y E A A R L E Y D A Y R T D L E E L S L G P R D A G T

649 CGTGGTCGACTTGAGAGTGCCCAGGCCACTTTCCAGGCCCATCGGGACAAGTATGAGAAGCTGCGGGGAGATGTGGCCATCAAGCTCAAGTTCCTGGAAGAAAACAAG217 R G R L E S A Q A T F Q A H R D K Y E K L R G D V A I K L K F L E E N K

757 ATCAAGGTGATGCACAAGCAGCTGCTGCTCTTCCACAATGCTGTGTCCGCCTACTTTGCTGGGAACCAGAAACAGCTG AGCAGACCCTGCAGCAGTTCAACATCAAG253 I K V M H K Q L L L F H N A V S A Y F A G N Q K Q L E Q T L Q Q F N I K

8 65 CTGCGGCCTCCAGGAGCTGAGAAACCCTCCTGGCTAGAGGAGCAGTGA289 L R P P G A E K P S W L E E 0

Fig. 1. cDNA and predicted amino acid sequence of POR1. The putative leucine zipper is underlined. The nucleotide sequence has been deposited inthe EMBL Nucleotide Sequence Database under the accession No. X97567.

involved in other Rac-mediated activities remains to bedetermined.To gain more insight into the mode of action of Rac

proteins, we sought to isolate proteins able to interactwith Rac. Toward this goal, we used a two-hybrid screenusing V12 RacI as bait (Chien et al., 1991; Vojtek et al.,1993). We describe here the isolation of a novel RacI-interacting protein, designated PORI, that does not showany homology with previously identified Rac-interactingproteins. Biological data are presented suggesting a rolefor PORI in membrane ruffling.

ResultsIsolation of Racl-interacting clonesTo isolate Rac-interacting proteins, we employed a two-hybrid protein interaction screening procedure as described(see Materials and methods). Activated human RacI (V12Rac 1) was fused with the LexA DNA binding domainand used as target to screen a Jurkat complementary DNAlibrary fused with the GAL4 activation domain. The testerstrain for the screen, L40, contained two reporters, HIS3and LacZ. Primary positives were selected by identifyingcolonies that could grow in the absence of histidine andsubsequently were tested for P-galactosidase activity. Thefrequency of positive yeast clones was 1 in 60 000.Plasmids recovered from the blue His' colonies weretested for plasmid dependency. To date, three classes ofspecific, positive clones have been obtained. One classwas composed of a number of related library plasmids,all of which encoded fusions between the GAL4 activationdomain and a previously identified protein, D4. Thisprotein is 67% identical to the bovine rhoGDI (GDPdissociation inhibitor) protein and has been shown to haveGDI activity against Cdc42Hs and Rac proteins in vitro(Adra et al., 1993). A second class of positive clonesconsisted of multiple isolates of plasmids encoding fusionsbetween the GAL4 activation domain and a previouslyunknown protein. We called this protein PORI for partnerof Racl (see below). The third class of positive cloneswas represented by a single isolate. This library plasmidcontained a cDNA insert of -500 bp, and sequence analysis

showed it to be also a previously unidentified gene. Weare presently further investigating this clone.

The POR1 proteinPORI was isolated from four different library plasmidsthat contained cDNAs of two different lengths. Sequenceanalysis revealed that all cDNA inserts contained an openreading frame (ORF), and inspection of the sequence ofthe longest cDNA revealed a potential initiator ATGcodon, which matches well the consensus sequences fortranslational initiation (Kozak, 1987). This clone spans anORF of 912 bp encoding a predicted protein of 33.8 kDa(Figure 1). Northern blot analysis detected a single mess-age of 1.35 kb (Figure 4a), suggesting that this cDNAclone represents the full-length gene. Furthermore, PORIoverexpressed protein had the same apparent size (34 kDa)in immunoblotting experiments using a polyclonal anti-body raised against GST-POR1 as the endogenous proteinin Jurkat cells (data not shown).The PORI predicted amino acid sequence was used to

search available databases using the BLAST programnetwork server. POR1 does not show homology withknown proteins. However, it does have homology (25%over its entire length) with a protein of unknown functionpredicted from nucleotide sequencing of the Caeno-rhabditis elegans genome. No conserved motifs besidesthe presence of a putative leucine zipper were found(Figure 1).

POR1 binds directly to the GTP-bound form ofRac1 in vitroTo exclude the possibility that the Racl-PORI interactionobserved in the two-hybrid system might be bridged byother factors within the yeast cell, we performed an in vitrobinding assay (Figure 2). Racl protein, purified as a GSTfusion protein from Escherichia coli, was loaded witheither GTPyS or GDP,3S and incubated with maltosebinding protein (MBP)-PORI fusion protein immobilizedon amylose resin. After washing the resin, the presenceof GST-Racl was determined by immunoblotting. Asshown in Figure 2, the interaction between Racl andPORI is direct. Furthermore, the binding of PORI to

3779

L.Van Aelst, T.Joneson and D.Bar-Sagi

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Fig. 2. In vitro binding of PORI to Racl. PORI protein, purified as anMBP fusion protein, was immobilized on amylose resin and incubatedwith GDP,BS-loaded Racl-GST (lane 1) or with an equal amount ofthe same Racl-GST loaded with GTPyS (lane 2). As control, MBP onamylose resin was incubated with the GST polypeptide (lane 3) andRacl-GST (lane 4). After 90 min tumbling, the amylose resin waswashed and analyzed for binding to RacI by SDS-PAGE, followed byimmunoblotting with anti-GST antibody. Lane 5 contains purifiedRac-GST.

POR1

VVt RaclV12Racl

V12A35Rac1A189Racl

Rho lA

Cdc42HsLamnin

Fig. 3. Interaction between POR 1 and members of the Rho family inthe two-hybrid system. PORI cDNA fused to the GAL4 DNA-activating domain (GAD) was transformed into the yeast reporterstrain L40 with LexA DNA binding domain fusions (LBD) containingWTRacl, V12Racl, V12A35Racl, A189Racl, RhoA, Cdc42Hs and,as negative control, lamin. For each transformation, four independentcolonies were picked and tested for growth on medium lackinghistidine.

Rac 1 is dependent on the nature of the guanyl nucleotidebound to Rac 1. PORI binds preferentially to the GTP-bound form of RacI.

Further characterization of the PORl-RaclinteractionTo define critical region(s) of Racl involved in POR1binding, modified forms of Rac 1 containing pointmutations were tested for their ability to interact withPOR1 in the two-hybrid system. One putative effectorbinding domain of Rac 1 spans residues 26-48. This regionshows -75% homology with the effector domain of Ras(Freeman et al., 1994). All members of the Ras superfamilycontain in this domain a highly conserved Thr residue atposition 35 which has been implicated in effector inter-action (Ridley et al., 1992; Self et al., 1993; Van Aelstet al., 1993; Diekmann et al., 1994). Substitution of Thrby Ala at position 35 ofV 12Rac 1 abolished POR I binding,indicating that the interaction of Racl with PORI ismediated, at least in part, by the effector binding domain(Figure 3 and Table I). This mutant Racl (T35A) did

Table I. Liquid 3-galactosidase assays of interactions between PORIand members of the Ras superfamily in the two-hybrid system

LBD GAD

PORI Raf

WTRacl 42 + 1.2 0.7 ± 0.8V12Racl 48 + 1.8 0.6 + 1.2V12A35Rac1 1.3 ± 1.1 0.9 + 0.8A189Racl 43 + 0.8 0.8 ± 0.9Cdc42 0.7 + 0.7 0.7 ± 0.6RhoA 1.8 + 0.8 0.9 + 0.7H-Ras 0.9 + 0.8 81 1 1.2Lamin 0.9 1 1.5 0.9 + 1.2

PORI and cRaf cDNAs fused to the GAL4 DNA-activating domain(GAD) were individually transformed into the yeast reporter strainL40 with LexA DNA binding domain fusions (LBD) containingWTRac1, V12Racl, V12A35Rac1, A189Racl, RhoA, CDC42Hs,H-Ras and, as negative control, lamin. Transformants were grown inselective synthetic medium, and ,B-galactosidase activity was assayedwith o-nitrophenyl 13-D-galactoside; values (mean ± SD of triplicatedeterminations) are given in Miller units (Miller, 1972).

interact strongly with D4 (data not shown). The Racproteins undergo carboxy-terminal processing which issignaled by C-terminal Cys-A-A-Leu (A = aliphaticamino acid) sequences (Kinsella et al., 1991). Substitutionof cysteine by alanine at position 189 of Rac 1, whichabolishes the isoprenylation site, did not disrupt theinteraction with PORI, indicating that carboxy-terminalprocessing of Racl is not essential for its interaction withPORI (Figure 3, Table I). We previously showed thatRAF and byr2 kinases, functional targets of Ras, are stillable to interact with Ras containing a mutation in theCAAX box (RasC186S) but not with Ras mutated in theeffector loop (RasT35A) using the LexA-based two-hybridsystem (Van Aelst et al., 1994).The smallest PORI cDNA clone isolated in the screen

lacked the first 207 residues (PORlANI), indicating thatthe first 207 bp of POR 1 are not required for Rac 1 binding.This POR1 N-terminal truncation mutant caused a 2-foldstronger activation of the LacZ reporter gene in a liquidP-galactosidase assay than full-length POR 1 (data notshown). However, deletion of the last 257 bp of PORI(PORlAC1) abolished the interaction with Racl (TableII). Interestingly, when PORlACI was used as a target toscreen a Jurkat cDNA library fused to the GAL1 activationdomain, the screen yielded so far invariably PORlANIand a smaller fragment, POR1AN2, containing base pairs571-912. The latter fragment was not able to interact withRacI (Table II).

Previously identified Rac-interacting proteins, such asect2 (Miki et al., 1993), P13 kinase (Zheng et al., 1994),PAK (Manser et al., 1994; Bagrodia et al., 1995a; Knauset al., 1995; Martin et al., 1995) and bc, (Lancaster et al.,1994), have been shown to interact not only with Rac butalso with other members of the Rho family. We thereforetested the possibility of whether POR1 would be able toassociate with RhoA or CDC42Hs. Whereas PORI wasable to interact with WTRac 1 and V 12 Rac 1, no interactioncould be observed with either RhoA or CDC42Hs usingthe two-hybrid system. We also did not see an interactionbetween Ras and PORI whereas, under the same condi-tions, interaction between c-Raf and Ras could be observed(Table I). c-Raf failed to interact with members of the

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Table II. Interaction between POR1 truncation mutants and various partners

GAD LBD

WTRacl V12Racl CDC42Hs RhoA PORlACI STE20 STEll

PORI + + - - + - -

PORlANI + + - - + -PORlACI - - - -

POR1AN2 - - - - + -WTRacl - - - -

V12Racl - - - - - +STEll - - - - _ _ +

The proteins to the left of the table were fused to GAD. The proteins at the top of the table were fused to LBD. Pairs were co-expressed in the yeastreporter strain L40. Values represent the presence of transformed colonies that expressed strong detectable j-galactosidase activity (+), weak (±) ornone (-). At least four transformants were tested for each determination. STEl 1 is a S.cerevisiae kinase that interacts with itself. STE20 is a yeastkinase with shows 70% identity with human p65PAK in the serine/threonine kinase domain. PORLANI; POR1AN2 and PORACI contain base pairs:208-912; 571-912 and 1-654 respectively.

Rho subfamily (Table I). Expression of LBD RhoA andCDC42Hs was confirmed by Western blot analysis usingLexA antibody (data not shown).

Expression and evolutionary conservation of POR1Since the Rac 1 gene has been shown to be expressed ina wide variety of tissues and cell lines (Moll et al.,1991), we were interested to see whether POR1 is alsoubiquitously expressed. We therefore examined byNorthern analysis its expression in eight different humantissues. As shown in Figure 4a, one single message in alltissues, with the highest expression in the pancreas, couldbe observed. Thus, similarly to Racl, PORI appears tobe ubiquitously expressed.To assess the extent of evolutionary conservation of

PORI, we hybridized, under low stringency conditions, azoo-blot containing genomic DNAs from nine eukaryoticspecies: human, monkey, rat, mouse, dog, cow, rabbit,chicken and yeast, with a PORI cDNA probe. As shownin Figure 4b, PORI appears to be highly conserved. Thesequence homology between PORI and a novel proteinpredicted from nucleotide sequencing of the C.elegansgenome suggests that a POR1 homolog also exists inC.elegans.

Localization studies utilizing T7-tagged PORI micro-injected into the nucleus of REF-52 or cultured Ratlfibroblast cells have suggested that PORI can be foundin both the cytoplasm and the plasma membrane (data notshown). The Racl protein has been shown also to belocated both in the cytoplasm and along the surface ofthe plasma membrane (Ridley et al., 1992). To investigatefurther the subcellular localization ofPORI, we transfectedRatl fibroblast lines expressing V12Rac 1 under controlof a tetracycline-repressible promotor (Qiu et al., 1995)with T7-epitope tagged POR1. T7-epitope tagged vector(pCGT) was used as negative control. As shown in Figure4c, we could detect expression of PORI in membraneruffles induced by V12Rac 1. No staining of membraneruffles could be observed when pCGT was transfected inthese cells (data not shown).

Role of POR1 in membrane rufflingTo investigate whether POR1 might have a role in mem-brane ruffling, a microinjection assay was used. In thisassay, expression plasmids were introduced into the nucleiof quiescent REF-52 cells and membrane ruffling was

monitored 3 h after injection. As shown in Figure 6,microinjection of an expression vector encoding V 1 2Rac 1at a concentration of 5 ,ug/ml induced extensive membraneruffling. In comparison, injection of PORI expressionvector at concentrations of up to 100 ig/ml had only aslight effect on membrane ruffling (not shown). Twoapproaches were undertaken to investigate further a pos-sible involvement of PORI in membrane ruffling.Rac proteins have been shown to be involved in the

induction of membrane ruffling by V12Ras (Ridley et al.,1992). Moreover, it has been shown recently that cellsco-expressing an activated form of Ras (Ras6lL) and anactivated form of Rac1 (RacI151) displayed significantlymore membrane ruffling activity than did cells transformedby either protein alone (Khosravi-far et al., 1995). Wereasoned that if PORI is a component of the Rac 1signaling pathway then it might synergize with V12Rasin a membrane ruffling assay. We first determined theplasmid concentrations for V 12Ras and PORI at whichthey do not induce membrane ruffles when microinjectedindividually. As shown in Figure 5, no membrane rufflingwas observed following injection of V12Ras at a concen-tration of 1 ,ug/ml or PORI at a concentration of 50 gg/ml.However, co-injection of both V12Ras and PORI at theseplasmid concentrations resulted in extensive membraneruffling (Figure 5). Surprisingly, we were unable to detectsynergistic interaction between either V12Racl orWTRacl and PORI.We next assessed the ability of the N- and C-terminal

truncated versions of PORI, PORIANI and PORlACI,to block V12Racl-induced membrane ruffling. We firsttested whether these fragments when microinjected separ-ately were able to induce membrane ruffling. No membraneruffling could be observed with either of the PORIfragments. Co-injection of expression plasmids encodingVl2Racl (5 ,ug/ml) and POR/AN1 (80 gg/ml) into REF-52 cells resulted in the inhibition of membrane ruffling(Figure 6). The inhibitory effect of PORIANI was dosedependent. This observation was not surprising sincePORlANI was found to bind Racl in the two-hybridsystem. Interestingly, inhibition of V1 2Rac 1-inducedruffling was also observed with PORlACI (Figure 6).This result was unexpected since POR1AC1 does notinteract with Racl in the two-hybrid system (Table II).This effect, however, appears to be specific, since co-injection of the full-length PORI at the same concentration

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Fig. 4. (a) Northern analysis of poly(A)+ RNAs from various human tissues. The blot (purchased from Clontech) was hybridized with (top) PORIcDNA or (bottom) P-actin cDNA according to the manufacturer's recommendations. Locations of molecular size marker are shown in kb.(b) Evolutionary conservation of PORI. A zoo-blot containing 4 tg of EcoRI-digested genomic DNAs from nine eukaryotic species, human,monkey, rat, mouse, dog, cow, rabbit, chicken and yeast, was hybridized under low stringent conditions with PORl cDNA probe. All genomiccDNA was isolated from kidney tissue except for chicken (liver tissue) and human (placental tissue). (c) Localization of PORI protein in Ratlfibroblast lines expressing V12Rac. Ratl fibroblast cells expressing V12Racl under control of a tetracycline-repressible promoter were transfectedwith I pg of T7 epitope-tagged PORI. After 16 h, cells were fixed in 3.7% formaldehyde and stained with anti-T7 antibody to identify PORl-expressing cells (top). The antibodies were visualized with rhodamine-conjugated goat anti-mouse secondary antibodies. Filamentous actin was

visualized using FITC-phalloidin (bottom).

does not interfere with Vi12Rac 1-induced ruffling (Figure6). Interestingly, PORlACI does interact with full-lengthPORI and PORlANI (Table II). The level of V12Raclexpression was comparable when co-expressed with eachof the above three plasmids (data not shown). The domin-ant inhibitory effects of POR1 truncations on V12Racl-induced membrane ruffling are consistent with a role forPOR1 in Racl-induced membrane ruffling.

DiscussionThe Racl proteins are members of the Ras superfamilyof small guanine nucleotide binding proteins. This groupof GTPases has attracted much interest because of itsinvolvement in fundamental cellular processes including

cell motility, transcriptional regulation and cell prolifera-tion (reviewed by Symons, 1995). The biochemical basisof most of the biological activities associated with Raclis not known. In this study, we report the identificationand characterization of a novel Racl-interacting protein,PORI.PORI encodes a protein of predicted mol. wt 33.8 kDa

that does not show sequence homology with any of thepreviously described Racl-interacting proteins includingRhoGAP, BcrGAP (Lamarche and Hall, 1994), Tiaml(Michiels et al., 1995), Ost (Horii et al., 1994), p67phox(Diekmann et al., 1994); P13 kinase (Zheng et al., 1994)and PAK (Manser et al., 1994; Bagrodia et al., 1995a;Knaus et al., 1995; Martin et al., 1995) or any othercharacterized proteins in the database. The functional

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Fig. 5. Synergy between V12Ras and PORI for induction of membrane ruffles. Quiescent REF-52 cells were microinjected with (A) empty vector(50 ,g/ml), (B) pDCR V12Ras (50 pg/ml), (C) pDCR V12Ras (1 p,g/ml), (D) pcDNA3 PORI (50 pg/ml) and (E) pDCR Vl2Ras (1 p,g/ml) andpcDNA3 PORI (50 pug/mi). At 3 h after injection, cells were fixed and stained with rhodamine-labeled phalloidin to show ruffles.

significance of POR1 is suggested by the fact that POR1is highly conserved in evolution. Similarly, Rac 1 is highlyconserved in evolution. It has been found in differentmetazoans such as C.elegans, Drosophila, mice and human(Moll et al., 1991; Chen et al., 1993; Harden et al., 1995).Interestingly, the PORI gene appeared also to be expressedin the yeast Saccaromyces cerevisiae. To date, no homologfor human Rac 1 in yeast has been identified. This mightindicate that either the Rac 1 gene in yeast has not beenidentified, or that CDC42 S.c. or a related protein replacesRac 1 function in yeast. Alternatively, POR1 might haveadditional Racl-independent functions in yeast.Among the primary biological readouts for Racl func-

tion is the induction of membrane ruffling. The observationthat expression of POR\AN 1, a truncated version of POR 1that retains the ability to interact with V12Rac 1, resultsin the inhibition of V12Racl-induced membrane rufflingprovides evidence for an in vivo interaction betweenRacl and POR1. Furthermore, the observation that theexpression of a truncated fragment of POR1, PORlAC1which does not interact with Rac 1, interferes withV12Racl-induced membrane ruffling is consistent with apotential role for PORI in cellular processes controllingmembrane ruffling. Given the ability of POR lAC 1 to forma complex with PORI, this inhibitory effect could resultfrom the sequestering of endogenous PORI molecules. Itis, however, also possible that the biological effects of thePORI truncation mutants result from their non-productiveinteraction with other regulatory components crucial formembrane ruffling. The findings that PORI synergizeswith an activated mutant of Ras for the induction ofmembrane ruffles and that POR1 localizes to membraneruffles induced by V12Racl lend further support to thenotion that POR1 is involved in signaling events that leadto membrane ruffling.POR 1 interacts directly and preferentially with the GTP-

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Fig. 6. Effects of POR1 truncation mutants on Vi 2Rac I-inducedmembrane ruffling. Quiescent REF-52 cells were microinjected with(A) pcDNA3 V12Racl (5 pg/ml) and empty vector (80 pg/ml),(B) pcDNA3 V12Racl (5 pg/ml) and pcDNA3 PORI (80 pg/ml),(C) pcDNA3 V12Racl (5 pg/ml) and pcDNA3 PORIANI (80 pg/ml)and (D) pcDNA3 V12Racl (5 pg/ml) and pcDNA3 PORlACL(80 pg/ml). At 3 h after injection, membrane ruffles were recorded.Similar results were obtained using a T7 epitope-tagged V12Racl. Thearrowheads indicate membrane ruffles.

bound form of Rac 1. Furthermore, POR1 does not bindRacl mutated in the effector loop Racl (T35A). Similarbinding profiles were observed previously for severalGTPases and their well characterized downstream effectors(Vojtek et al., 1993; Manser et al., 1994; Van Aelst et al.,1994). Thus, based on these findings, it is enticing tospeculate that POR1 is a downstream effector of Rac 1.However, at this stage, it cannot be excluded that thefunctional position of POR1 in the Rac 1 signaling cascadeis more complex. The fact that POR 1 does not synergizewith V12Rac 1 or WTRac 1 suggests that the function of

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POR 1 cannot be explained in terms of a simple linearpathway. There is growing evidence for complex inter-actions between proteins involved in the signaling cascadesmediated by Rho-like proteins. For example, in S.cere-visiae and Schizosaccharomyces pombe, Rho-like proteinsare required for normal polarized cell growth and cellshape (Johnson and Pringle, 1990; Chenevert, 1994; Millerand Johnson, 1994). In both yeasts, the Rho-like protein,Cdc42, has been shown to be part of a multimolecularcomplex. In S.pombe, cdc42 has been shown to interactwith scdl and scd2, homologs of S.cerevisiae CDC24 andBEMI respectively, and shkl (pakl +), a homolog ofS.cerevisiae STE20 and mammalian p65PAK (Chang et al.,1994; Marcus et al., 1995; Ottilie et al., 1995). InS.cerevisiae, CDC42 has been shown to interact withCDC24 and STE20, which in turn interact with BEM1and STE5 (Leeuw et al., 1995; Zheng et al., 1995). Byanalogy, POR 1 might be a component of a multimolecularcomplex mediating Racl-induced signals in mammaliancells. More experiments defining the biochemical role ofPOR1 are required to determine the physiological functionof this protein.

Materials and methodsTwo-hybrid screenFor two-hybrid screening, the complete ORF of human V12Rac 1 wasinserted in the vector pVJLII, which was derived from pBTM 116 (Vojteket al., 1993). This vector directs the expression of a fusion between theDNA-binding domain of LexA and the entire V12Racl protein from anADH promoter. This plasmid, which contains a TRP marker, was co-transformed with a Jurkat cDNA library (LEU2 marker) (Benichou et al.,1994) into the yeast strain L40 (Vojtek et al., 1993).

Transformants were plated on yeast drop-out medium lacking leucine,tryptophan and histidine, thereby selecting for plasmids encoding proteinscapable of two-hybrid interaction as evidenced by trans-activation of aGALJ-HIS3 reporter gene and histidine prototrophy. After 10 days ofgrowth, His+ colonies were scored for 1-galactosidase as describedpreviously (Van Aelst et al., 1993). Positive colonies showed blue colorin from 30 min to several hours. Plasmid DNA was recovered frompositive colonies and introduced by electroporation into Ecoli MH4.MH4 is leuB- and this defect can be complemented by the LEU2 genein the library plasmid. Primary positives were tested for target specificityby re-transforming into the tester strain (L40) in conjunction with anumber of different LexA DNA binding domain-target fusions (fusionswith V12Racl, RAF, byr2, SNF4 and lamin). Only library plasmids thatdid activate marker expression in the presence of V12Racl but not inthe presence of the others were analyzed further.

Plasmid constructionsFor two-hybrid constructs, fusions to the transcription activation domainof GAL4 (GAD) were constructed using pGADGH (Van Aelst et al.,1993); fusions to the DNA binding domain of LexA (LBD) were madein pVJLII, which was derived from pBTM116. WTRacl, V12Racl,RhoA and CDC42 were amplified by PCR from cDNA clones (generousgifts of Alan Hall and Adra Chaker) and subcloned as a BaniHI-SalIfragment in pVJLII. Al89Rac I and V12A35Rac1 mutants were createdby PCR site-directed mutagenesis. Expression of the correspondingproteins was confirmed by Western blot analysis using LexA antibody(generous gift of Erica Golemis). LBD STE20 was constructed bycloning a BamHI-SalI fragment of STE20 (from HPSTE20 provided byS.Marcus) in pVJLII; pGAD1318 PORI and pGAD1318 POR1ANIwere isolated in the two-hybrid screen. pGAD PORIAC 1 and LBDPORlAC1 were constructed by subcloning a BamHI-SalI fragment ofPORI (from pGADGH PORI) in the vectors pGADGH and pVLJIIrespectively. The Jurkat cDNA library was constructed in pGAD1318,a derivative of pGADGH (kindly provided by J.Camonis, INSERMU-148, Paris, France) and is described in Benichou et al. (1994).pcDNA3, pDCR and pCGT were used as mammalian expression

vectors. pcDNA3 was obtained from Invitrogen. pDCR (generous giftof C.Nicolette, CSHL) is a mammalian expression vector containing the

cytomegalovirus promoter followed by unique Sall and BamHI sites andthe rabbit 3-globin terminator and splice sequence. This region and theneor gene are flanked by Moloney murine leukemia retrovirus 5' and 3'long terminal repeats. pCGT is derived from pCGN (Tanaka and Herr,1990) with a replacement of the Lerner epitope by the T7 epitope(generous gift from Angus Wilson, CSHL). pDCR V12Ras contains thefull-length Ras mutant inserted as a SalI-BamHI fragment in the Sall-BamHI sites of pDCR. pCGT PORI and pCGT Vl2Racl contain full-length genes inserted as XbaI-XhoI and XbaI-SalI fragments in theXbaI-SalI site of pCGT. pcDNA3 V12 Racl, pcDNA3 PORlACI andpcDNA3 PORl, pcDNA3 PORlANI constructs are obtained by insertingthe corresponding cDNAs as BamHI-SalI and BamHI-XhoI fragmentsrespectively in the BamHI-XhoI site of pcDNA3. GST-Racl containswild-type Rac I inserted as a BamHI-SalI fragment in the vector pRP259,which is a derivative of pGEX-2T (Pharmacia) with a polylinker insertedand was a generous gift from M.Gebbink (The Netherlands CancerInstitute). MBP-PORI contains full-length PORI inserted as an Xbal-XhoI fragment in the XbaI-Sall sites of the vector pMAL-c2 (NewEngland Biolabs). The sequences of all PCR products were verified bydideoxy sequencing.

In vitro binding assayRacl protein was purified as a glutathione-S-transferase fusion protein(GST-Racl) from Ecoli. The PORI protein was purified as an MBPfusion protein from Ecoli. GST-Racl was eluted from the agarosebeads with three bed volumes of 10 mM reduced glutathione in 50 mMTris-HCl (pH 7.1) for -20 min at room temperature. The eluate wasdialyzed with three washes of phosphate-buffered saline (PBS; pH 7.1)with a Centricon-10 Column. The GST fusion proteins were loaded withguanine nucleotides by incubation with 200 ,uM GDP,BS or GTPyS inloading buffer containing 20 mM Tris pH 7.4, 1 mM dithiothreitol(DTT), 5 mM EDTA. The reaction was stopped by addition of MgCl2to a final concentration of 10 mM. For the binding experiments, GSTor GST-Racl were incubated with the amylose resin-bound MBP orMBP-PORI using 5 ,tg of each protein. The binding reactions contained20 mM Tris pH 7.4, 1 mM DTT, 100 mM NaCl, 1 mM EDTA, 0.1%Triton X-100, 10% glycerol. After 90 min at 4°C, the resin wassedimented and then washed three times with 1 ml of the bindingreaction buffer. The proteins were removed from the amylose resin withSDS sample buffer at 100°C for 10 min, separated by electrophoresison 12.5% SDS-PAGE and then transferred to nitrocellulose. The GSTor GST-Racl fusion proteins were detected by immunoblotting with aGST monoclonal antibody (Santa Cruz Biotechnology) followed by ECLdetection (Amersham).

Microinjection assayREF-52 cells were plated onto glass coverslips and cultured in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% fetal calfserum (FCS). The cells were grown to confluence, then placed instarvation medium (DMEM with 0.5% FCS) for 24 h before microinjec-tion. A plasmid mixture containing the indicated plasmids in microinjec-tion buffer [50 mM HEPES (pH 7.2), 100 mM KCl and 5 mM NaHPO4]was microinjected into cell nuclei. Three hours after injection, membraneruffles were recorded using a CCD camera (Hitachi KP-MIU). Theimages were processed using Image-Pro Plus (V 1.3) software (MediaCybernetics) (Figure 6). To show membrane ruffles in Figure 5, cells oncoverslips were fixed in 3.7% formaldehyde in PBS for 1 h at roomtemperature. Subsequently, the cells on coverslips were incubated for1 h with 0.1 mg/ml rhodamine-labeled phalloidin (Molecular Probes,Inc.) dissolved in methanol in a humidified chamber. The coverslipswere mounted and pictures taken.

Transfection assayRatl fibroblast cells and Ratl fibroblast cells expressing V12 Racl weregrown to 80% confluence in DMEM with 10% fetal bovine serum andtransfected with I .tg of total DNA per 60 mm dish for 16 h asdescribed by Gimona et al. (1995). After 16 h, cells were prepared forimmunofluorescence.

Localization studiesFor localization of PORl, complementary DNA encoding PORl wascloned in the T7-tagged vector pCGT and injected or transfected inREF-52 or RatI fibroblast cells and RatI fibroblast cells expressing V12Racl cells. After 16 h, the cells were fixed, permeabilized with 0.1%Triton X-100 in PBS for 3 min and incubated with mouse monoclonalanti-T7 antibody (Novogen). This was followed by incubation withrhodamine-conjugated goat anti-mouse secondary antibody (Molecular

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Probes) for I h at 37°C in a humidified chamber. Filamentous actin wasvisualized using FITC-phalloidin. Fluorescent images were photographedon a Zeis Axiophot microscope using a X63 oil-immersion lens andKodak P3200 T-max film.

Preparation of anti-POR1 antibodiesA plasmid containing a fusion between GST and the ORF of PORI wasintroduced into E.coli BL21. Fusion proteins were expressed and purifiedby established methods. using glutathione-Sepharose (Pharmacia). Poly-clonal antisera to this fusion protein were generated by Pocono RabbitFarm and Laboratory.

Northern blot and zoo-blot analysisThe Northern and zoo-blots were purchased from Clontech and hybridizedwith [Vx-32P]PORI (and 3-actin cDNA for the Northern blot) accordingto the manufacturer's recommendations. Full-length PORI cDNA usedas probe was obtained by PCR.

AcknowledgementsWe thank M.H.Wigler for his support and useful discussions: MarioGimona for his help and advice on the localization studies, M.Symonsfor providing Ratl fibroblasts lines expressing V12Racl under thecontrol of a tetracycline-repressible promotor; A.Chakar. J.Camonis.S.Marcus. A.Hall and A.Wilson for kindly providing plasmids andreagents; Susan Kaplan, L.Rodgers. M.Riggs, K.Farina and G.Asoulinefor technical assistance: and the Belgian National Fund for ScientificResearch. This study is supported by grants from the American CancerSociety and the National Cancer Institute to M.Wigler, and NIH grantsCA55360 and CA28146 to D.B.S.

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Received on? December 15, 1995; revised on1 Apr-il 23, 1996

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identification of novel raci-interacting protein involved

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