Proc. Natl. Acad. Sci. USAVol. 93, pp. 5466-5471, May 1996Biochemistry

The multidomain protein Trio binds the LAR transmembranetyrosine phosphatase, contains a protein kinase domain, and hasseparate rac-specific and rho-specific guanine nucleotideexchange factor domains

(GTPase/immunoglobulin/pleckstrin homology/protein tyrosine phosphorylation/spectrin-like repeats)

ANNE DEBANT*tt, CARLES SERRA-PAGES*§, KATJA SEIPEL*t, STEPHEN O'BRIEN*, MAY TANG*, SANG-Ho PARKS,AND MICHEL STREULI*tII*Division of Tumor Immunology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115; Departments of tPathology and §Medicine, HarvardMedical School, Boston, MA 02115; and lWhitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology,9 Cambridge Center, Cambridge, MA 02142

Communicated by Charles Weissmann, Institut ifir Molekularbiologie, Zurich, Switzerland, January 24, 1996 (received for review October 23, 1995)

ABSTRACT rho-like GTP binding proteins play an essen-tial role in regulating cell growth and actin polymerization.These molecular switches are positively regulated by guaninenucleotide exchange factors (GEFs) that promote the ex-change of GDP for GTP. Using the interaction-trap assay toidentify candidate proteins that bind the cytoplasmic region ofthe LAR transmembrane protein tyrosine phosphatase (PT-Pase), we isolated a cDNA encoding a 2861-amino acid proteintermed Trio that contains three enzyme domains: two func-tional GEF domains and a protein serine/threonine kinase(PSK) domain. One of the Trio GEF domains (Trio GEF-Di)has rac-specific GEF activity, while the other Trio GEFdomain (Trio GEF-D2) has rho-specific activity. The C-terminal PSK domain is adjacent to an Ig-like domain and ismost similar to calcium/calmodulin-dependent kinases, suchas smooth muscle myosin light chain kinase which similarlycontains associated Ig-like domains. Near the N terminus,Trio has four spectrin-like repeats that may play a role inintracellular targeting. Northern blot analysis indicates thatTrio has a broad tissue distribution. Trio appears to bephosphorylated only on serine residues, suggesting that Triois not a LAR substrate, but rather that it forms a complex withLAR. As the LAR PTPase localizes to the ends of focaladhesions, we propose that LAR and the Trio GEF/PSK mayorchestrate cell-matrix and cytoskeletal rearrangements nec-essary for cell migration.

The ras-like GTP binding proteins cdc42, rac, and rho regulatediverse cellular processes including cell growth and actinremodeling associated with changes in cell morphology,growth, adhesion, and motility (1-4). In fibroblasts, cdc42regulates actin polymerization and focal complexes necessaryfor filopodia formation, rac mediates actin polymerization andfocal complex assembly within lamellipodia and membraneruffles, and rho induces actin stress fiber and focal adhesion(FA) complex formation (5). A hierarchical relationship existsamong cdc42, rac, and rho, whereby cdc42 regulates rac activityand rac regulates rho activity, suggesting that these proteinsmay orchestrate the spatial and temporal changes in the actincytoskeleton necessary for cell movement (5, 6). cdc42 and racalso regulate activation of the c-Jun N-terminal kinase/stress-activated kinase via a mitogen-activated protein (MAP) kinasepathway (7-9), and rac and rho are essential for ras transfor-mation (10, 11). cdc42, rho, and rac all appear to stimulate c-fostranscription (12), as well as cell cycle progression through GIand subsequent DNA synthesis (9). The activation state of

ras-like GTP binding proteins is positively regulated by gua-nine nucleotide exchange factors (GEFs) that promote theexchange of GDP for GTP, and negatively by GTPase acti-vating proteins (2). A number of putative GEFs for rho-likeGTPases have been identified by sequence comparison (2),and several of these demonstrate GEF activity in vitro. The dbland ost oncogene products have cdc42 and rho GEF activity(13-15); the lbc oncogene product has rho GEF activity (16);the invasion-inducing Tiaml gene product has cdc42, rho, andrac GEF activity (17); and the yeast CDC24 gene product hascdc42 GEF activity (18).LAR is a broadly expressed transmembrane protein tyrosine

phosphatase (PTPase) comprised of a cell adhesion-like ex-tracellular region and two intracellular PTPase domains (19-22). A role for LAR in regulating cell-matrix interactions wasproposed, as LAR colocalizes with a coiled-coil protein,termed LAR interacting protein 1 (LIP.1) at the ends of FAs(23), and LAR expression was observed at regions of associ-ation between cells and basement membrane in various tissues(19). To identify putative substrates and other proteins in-volved in LAR-mediated signal transduction, we screened forproteins that bind the LAR PTPase domains using the inter-action-trap assay and coimmunoprecipitation studies. A pro-tein thus isolated is a novel multidomain GEF we have namedTrio because it contains three enzyme domains: two GEFdomains, one of which has racl GEF activity and the other hasrhoA GEF activity, and a serine/threonine kinase (PSK)domain. In addition, Trio contains four N-terminal spectrin-like domains, two pleckstrin-like domains, and an Ig-likedomain. Because proteins with cdc42, rac, or rho GEF activityare generally involved in regulating cytoskeletal organization(1), it is likely that Trio in conjunction with LAR plays a keyrole in coordinating the cell-matrix interactions and cytoskel-etal rearrangements involved in cell movement.

Abbreviations: PTPase, protein tyrosine phosphatase; GEF, guaninenucleotide exchange factor; PSK, protein serine/threonine kinase;MAP kinase, mitogen-activated protein kinase; LIP.1, LAR-interacting protein 1; GST, glutathione S-transferase; HA, hemagglu-tinin; FA, focal adhesion; FCS, fetal calf serum; PMA, phorbol12-myristate 13-acetate; PH, pleckstrin homology; MLC, myosin lightchain; MLCK, MLC kinase.Data deposition: The sequence reported in this paper has beendeposited in the GenBank data base (accession no. U42390).*Present address: Centre de Recherches en Biochimie Macromolecu-laire, Centre National de la Recherche Scientifique, BP5051, 1919Route de Mende, F 34033, Montpellier cedex 1, France.'To whom reprint request should be addressed at: Division of TumorImmunology, Dana-Farber Cancer Institute, 44 Binney Street, Bos-ton, MA 02115.

5466

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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MATERIALS AND METHODSInteraction-Trap Assay. Plasmid DNAs and yeast strains

used for the interaction-trap assay were provided by RogerBrent and colleagues (Massachusetts General Hospital, Bos-ton) and used essentially as described (24). The human fibro-blast cell WI-38 (ATCC CCL 75) cDNA library used for theinteraction-trap assay was kindly provided by Claude Sardet(Whitehead Institute, Cambridge, MA). The various LAR (25)and CD45 (26) regions fused to the LexA peptide are shownin Fig. 1A.Northern Blot Analysis. Northern blot analysis was done

using a human multiple tissue Northern blot (Clontech), whichcontains 2 gg of poly(A)+-selected RNA from different hu-man tissues per lane, and was hybridized with a random primed[a-32P]dCTP labeled Trio cDNA probe (encoding aa 2249-2861 plus 140 bp of 3' nontranslated sequence) according tothe manufacturer's instructions.

Antibodies. To generate anti-Trio mAbs, mice were immu-nized with Escherichia coli-derived glutathione S-transferase(GST)-TrioAl fusion protein. To this end, Trio cDNA se-

quences encoding Trio aa 2450-2861 were cloned into thepGEX.2T expression vector (Pharmacia); E. coli NM522 cellswere transfected with the plasmid, and GST-TrioAl fusionprotein was purified from bacterial lysates by glutathioneSepharose 4B (Pharmacia) chromatography using standardmethods, and then used as immunogen. Hypoxanthine/aminopterin/thymidine-resistant hybridomas derived fromGST-TrioAl immunized mice were initially selected by usingELISA and then by immunoprecipitation studies. Anti-TriomAbs thus obtained were termed a-Trio.56 (IgGl) anda-Trio.68 (IgGl). The anti-hemagglutinin (HA) mAb 12CA5was obtained from the Harvard University mAb Facility(Cambridge, MA), and anti-LAR sera was isolated fromrabbits immunized with E. coli-derived LAR intracellularregion protein (aa 1275-1881).

Cells and Transfections. Simian COS-7 cells, human breastadenocarcinoma MCF7 cells, and HeLa cells were cultured as

described (23). COS-7 cell transient transfections were done bythe diethylaminoethyl-dextran/dimethyl sulfoxide method(27).

Cell Labeling and Protein Analysis. Cell proteins were

metabolically labeled with [35S]methionine or [32P]orthophos-phate as described (23), except that for the [32P]orthophos-phate labeling, cells were preincubated in media lacking fetalcalf serum (FCS) for 15 h before labeling. Preparation of cellextracts and immunoprecipitations were done as described(23) using -2 gg.a-Trio.56 or a-Trio.68 mAb, 2 ,ug controlisotype-matched mAb, or 1 ,ul anti-HA mAb 12CA5 ascitesfluid. Immunoprecipitated proteins were analyzed usingSDS/6% PAGE analysis with reducing conditions followed byautoradiography (18-72 h). The relative amounts of 32p_labeled Trio were determined by densitometric scanning ofautoradiographs. Phosphoamino acid analysis was performedessentially as described (28).Plasmid Constructions and DNA Sequencing. cDNA clones

encoding Trio were isolated from the human WI-38 cDNAlibrary, as well as from fetal brain and heart cDNA libraries(Clontech) by using standard techniques and sequenced usingthe dideoxy method of sequencing. The pMT.HA.Cl.1G0 (Trioaa 2450-2861), pMT.HA.TrioA2 (Trio aa 1118-1919), andpMT.HA.TrioA3 (Trio aa 1849-2451) plasmids were con-

structed by inserting appropriate cDNA fragments into thepMT.HAtag expression vector (23). TrioA&.2 and TrioA.3contain 2 and 17 aa derived from vector sequences at theirC-termini, respectively. pMT.cytoLAR encodes the LAR cy-

toplasmic region (aa 1275-1881) fused to the 30 N-terminalresidues of DHFR encoded in the pMT.2 expression vector.Exchange Assays. The TrioA2 and TrioA3 proteins used for

exchange assays were produced in COS-7 cells using pMT.

HA.TrioA2 and pMT.HA.TrioA&3 plasmid DNAs. As control,cells were transfected with a control pMT.HA plasmid. Fol-lowing transfection, -2 x 108 cells were resuspended in 2 mlof ice-cold suspension buffer [20 mM Tris HCl, pH 8.0/1 mMEDTA/1 mM dithiothreitol (DTT)/0.5 mM phenylmethylsul-fonyl fluoride/10 ,ug of leupeptin per ml/10 ,g of aprotininper ml], and lysates were prepared using a Dounce homoge-nizer. Insoluble material was removed by centrifugation in amicrofuge for 15 min at 4°C. cdc42, rac, rho, and ras werepurified from baculovirus infected cells as described (29) andwere provided by Marc Symons (Onyx Pharmaceuticals, Rich-mond, CA). [3H]GDP loaded GTP binding proteins andexchange assays were performed essentially as described (30).Briefly, [3H]GDP loaded GTP binding proteins were preparedby incubating the purified proteins (0.5 jig) in 90 j,l exchangebuffer [50mM Tris HCl, pH 7.5/50mM NaCl/5 mM EDTA/1mM DTT/1 mg of bovine serum albumin per ml] containing7 ACi [3H]GDP (29.2 Ci/mmol; 1 Ci = 37 GBq; NEN/DuPont) for 20 min at 25°C. Following incubation, the reactionwas quenched with 90 Al of stop exchange buffer (50 mMTrisHCl, pH 7.5/10mM MgCl2/1 mM DTT), and then dilutedwith 1.5 ml of reaction stop buffer (50 mM Tris HCl, pH 7.5/1mM GTP/2 mM MgCl2). COS cell lysate (10 ,l) was added to80 ,lI of [3H]GDP loaded GTP binding proteins and incubatedat 25°C for the indicated times. The reactions were quenchedby adding 0.5 ml of stop buffer (50 mM Tris HCl, pH 7.5/10mM MgCl2) and immediately filtered through a nitrocellulosefilter (BA85, 0.45 ,um; Schleicher & Schuell); filters were thenwashed with stop buffer and the amount of radioactivity on thefilters was determined.

RESULTS

The LAR-D2 PTPase Domain Binds a Broadly ExpressedPhosphoserine Protein. To isolate candidate proteins thatinteract with the LAR PTPase domains, a human WI-38fibroblast cDNA library was screened using the interaction-trap assay and the cytoplasmic LAR-D1D2 bait (aa 1275-1881) as described (23, 24). Two cDNA clones thus isolated,Cl.1GO and Cl.2G0, were independent isolates derived fromthe same gene as determined by DNA sequence analysis. Inaddition to binding the LAR-D1D2 bait, the Cl.1GO peptidealso bound the LAR deletion bait, LAR-D2 (aa 1530-1881)that contains only the LAR-D2 PTPase domain, but did notbind the LAR deletion LAR-Dl bait (aa 1275-1715) or theCD45 bait (aa 584-1281) (Fig. 1A). Thus, the CL.1GO fusionpeptide specifically interacts with the LAR-D2 PTPase region.To determine if the Cl.1GO peptide also binds LAR inmammalian cells, a HA-tagged Cl.1GO peptide was transientlyexpressed in COS cells (HA-Cl.1GO) together with the cyto-plasmic region of LAR (cytoLAR; residues 1275-1881). Inaddition to immunoprecipitating the 44-kDa HA-Cl.1GO pep-tide, the anti-HA mAb coimmunoprecipitated a 68-kDa pro-tein that exactly comigrated with cytoLAR immunoprecipi-tated using an anti-LAR sera (Fig. 1B). Furthermore, westernblot analysis using the anti-LAR sera confirmed that the68-kDa protein present in the immunoprecipitates from theHA-Cl.1GO plus cytoLAR cotranfectants was cytoLAR (datanot shown). Thus, the Cl.1GO peptide binds cytoLAR inmammalian cells, as well as in the yeast interaction-trap assay.Two mAbs, termed anti-Trio.56 and anti-Trio.68, raised

against a GST-Cl.1GO fusion protein, both immunoprecipi-tated a large protein (>250 kDa) from [35S]methionine-labeled MCF7 cell lysates (Fig. 24). This protein was termedTrio (see below) and the Cl.1GO peptide was redesignatedTrioAl. Coimmunoprecipitation studies of endogenous LARand Trio could not demonstrate an association of theseproteins, and the anti-Trio mAbs did not detect Trio byimmunofluorescence. Thus, the in vivo association betweenLAR and Trio remains to be established. Immunoprecipitation

Biochemistry: Debant et al.

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5468 Biochemistry: Debant et al.

Af-gal units using theBaits Cl.IGO interactor

LAR-DID2 (1275-1881) 1 1"

LAR-D1 (1275-1715) .-F -IJLAR-D2 (1530-1881) I,,l-JCD45 (584-1281) _

DI D2

207<10118<10

a-HA a-LAR

8 8!Lysate:

g < 0c) = = +

kDa I I I I97 -

kD

20

iO° so B control a-Trio.68

. . 1. 1 1

kDa

200-

69 - qi - - cytoLAR P-Ser

P-ThrP-Tyr

45 - _ _ .- HA.CI.1GO

1 2 3 4

FIG. 1. Identification of a LAR PTPase interacting protein,Gl.1GO. (A) Mapping of sequences required for LAR and Cl.GObinding using the interaction-trap assay. Schematically shown are theregions of the LAR and CD45 cytoplasmic PTPase domains used asbaits. Numbers in brackets indicate the amino acid residues includedin the various fusion proteins. The Dl and D2 PTPase domains areindicated by open rectangles. Measurements of 03-galactosidase (13-gal)levels in liquid cultures were done in duplicate from two independentisolates, and the average values of ,B-gal units are shown. All of thefusion bait proteins, as well as the Cl.1G0 interactor protein wereefficiently expressed in yeast as determined by immunoblotting ex-periments (data not shown). (B) Coimmunoprecipitation ofHA.Cl.GO and LAR. Shown is a SDS/PAGE analysis of proteinsimmunoprecipitated with a-HA mAb, 12CA5, or an a-LAR sera.COS-7 cells were transfected with the pMT.cytoLAR expressionvector (lanes 1 and 4), pMT.HA.Cl.1GO (lane 2), or with a mix of boththe pMT.cytoLAR and pMT.HA.Cl.1G0 expression plasmids (lane 3).Eighteen hours after transfection, cell proteins were metabolicallylabeled with [35S]methionine for 4 h. Following labeling, cell extractswere prepared and then immunoprecipitation analysis was performedusing the anti-HA mAb (lanes 1-3) or anti-LAR sera (lane 4).Molecular mass standards in kDa are shown at the left. At the right ofthe figure are indicated the positions of the cytoLAR and theHA.Cl.1GO proteins.

analysis of Trio from [32P]orthophosphate-labeled HeLa celllysates demonstrated that Trio is a phosphoprotein. Additionof the protein kinase C activator PMA, or the PTPase inhibitorpervanadate caused modest increases (3.3- and 2.8-fold, re-spectively) in the amount of 32P-labeled Trio protein, as wellas a slight decrease in the migration of Trio isolated from thePMA-treated cells (Fig. 2B). Phosphoamino acid analysis of32P-labeled Trio protein indicated that phosphorylation isexclusively on serine residues even when cells were treated withpervanadate (Fig. 2B Inset, and data not shown). The absenceof tyrosine phosphorylation suggests that Trio is not a sub-strate for the LAR PTPase. Trio also did not significantlyaffect in vitro LAR PTPase activity (data not shown).Northern blot analysis using a Trio cDNA probe demon-

strated that an -10.5 kb Trio mRNA was present in all eighthuman tissue samples tested (Fig. 3). Thus, Trio appears to bea broadly expressed phosphoserine protein that binds theLAR-D2 PTPase domain.

FIG. 2. Biochemical characterization of Trio. (A) SDS/PAGEanalysis of a-Trio.56, a-Trio.68 mAb, and isotype matched controlmAb immunoprecipitated protein from [35S]methionine-labeled hu-man breast adenocarcinoma MCF7 cell lysates. Molecular mass stan-dards in kDa are shown at the left. The position of the >250-kDa Trioprotein is indicated by an asterisk (*). (B) SDS/PAGE analysis ofa-Trio.68 mAb and isotype-matched control mAb immunoprecipi-tated protein from [32P]orthophosphate-labeled HeLa cell lysates.Lysates were prepared from cells that were treated for 15 min withmedium containing 10% FCS (media), 100 ng of phorbol 12-myristate13-acetate (PMA) per ml plus 10% FCS (+PMA), or 100 ,AM sodiumpervanadate (PV) plus 10% FCS (+PV). On the bottom of the figureis shown the phosphoamino acid analysis of the 32P-labeled proteinimmunoprecipitated by the a-Trio.68 mAb from medium-treated cells.The positions of the control, nonradiolabeled phosphorylated aminoacids phosphoserine (P-Ser), phosphothreonine (P-Thr), and phos-photyrosine (P-Tyr) are indicated by ovals.

Trio Is a Multidomain Protein with Three Putative Enzy-matic Functions. The primary structure of Trio was deducedby isolating and sequencing a series of overlapping TriocDNAs. A composite cDNA of 10.4 kb thus isolated containsan open reading frame encoding a protein of 2861 aa with a

calculated molecular mass of 323,897 (Fig. 4A), which is in

a,i 3

kb

9.5 -7.5 -

4.4 -

2.4 -

____..... _ ... _.s sw .w.

:.

FIG. 3. Trio mRNA expression. Northern blot analysis of 2 jig ofpoly(A)+ RNA isolated from the human tissues indicated at the top ofthe figure using a radiolabeled Trio cDNA probe. Size markers in kbare shown at the left. Audioradiography with an intensifying screenwas for 2 days.

A

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Proc. Natl. Acad. Sci. USA 93 (1996) 5469

AMKCAMDVLP LKEKVAYLSGGRDKRGGPI LTFPARSNHORI RQEDLRRLI SYLACIPSEVCKRGFVIVDMRGSWDS IKPLKILESFPCCIHVALIIKPDNFWQKRTNFGSSKFEETNMVSLEGLTKVVDPSQLTPEDGCLEYN 150HEEWIEIRVAFEDYISNATHMLSRLEELQDILAKKELPQDLEGARNMIEEHSQLKKKVIKAPIEDLDLEGQKLLQRIQSSESFPKKNSGSGNADLQNLLPKVSTMLDRLHSTRQHLHQWHVRKLKLDQCFQLRLFEQDAEKMFDWITN 300KGLFLNSYTE IGTSHPHAMELQ12HNHFAMNCMNVYVN INR IMSVANRLVESGHYASQQI RQIASQLEQEWKAFAAALDERSTLLDMSSIFHQKAEKYMSDSWCKACGEVDLPSEQDLEDAI HHHQGIYEHITLAYSEVSQDGKSLL 450DKLQRPLTPGSSDSLTASANYSKAVHVLDVIHEVLHHQRHRTIWQHRKVRLHQR QLCVFQQEVQQVLDWIENHGEAFLSKH7UVGKSLHRARALQKRHEDEEVAQNTYTNADKLLEAAEQLAQTGECDPEEIYQAAHQLEDRIQDF 600VRRVEQRKILLDMSVSHTHVKELWTWLEEIKE LLDDVYAESVEAVQDLI KRFGQQQTTLQVTVNVIKEGEDLIQQLRDSAISSNKTPHNSSINIETVQLDEAQSQMEELFQERKI KLELFLHVRIFERDAI DI ISDLESWNDEL 750SQQMNDFDTEDLTIAEQRLQHHADKALTMNNLTFDVIHQGQDLLQYVNEVQASCVELLCDRDVDMATRVQDLLEFLHEKQQELDLAAEQHRKHLE CVQLRHLQAEVKQVLGWIRNGESMLNAGLITASSLQEAEQLQREHEQFQHAIEK 900THQSALQVQKAEAMLQANHYDMDMI RDCAEKVASHWQQLMLKMEDRLKLVNASVAFYKTSEQVCSVLESLEQEYKREEDWCGGADKL PNSETDHVTPMI SKHLEQKEALKACTLARRNADVFLKYLHRNSVNMPGVTH IKAPEQV 1050KN LNELFQRENRVLHYWTMRKRRLDQCiYVVFERSAKQALEWIHDNGEYLSTHTSGSS IQHTQELLKEHEEFQITAKQTKERVKLLIQLADGFCEKGHAHAAEIKKCVTAVDKRYRDFSLRMEKYRTSLEKALGISSDSNKSSKSL 1200QLDI IPASIPGSEVKLRDJHELNEEKRKSARRKEFIMAELIQTEKAYVRDLRECMDTYLWEMTSCVEEIPPGIVNKELI IFONMQEIYEFHNNIFLKELEKYEQLPEDVGHCFVTWADKFQMYVTYOCKFPDSTOLILEHAGSYFDEIQ 1350QRHGLANSIOSSYLIFKPVQRI TKYQLLLKELLTCCEEGKGEI KLEVLSVPKRNDAHLSMLEGFDENIESQGELI0ESFQVWDPKTLIRKGRERHLFLFEMSLVSKEKDSSGRSKYLYKSK0TSELGVE0KEDPCKFAL 01500ORTPTSDNKIVLKASSIENKQDWIKHIREVIQERTI HLKGALKEPIHI P1TAPATRQKGRRDGEDLDS60SSQPDISIASRTSQNTLDSDKLSGGCELIHDFTACNSELTIRRGQLERPHDKPDWCLVRTTDRSPG 1650LVPCGSLCIAHSRSSMEMEGIFNHKDSLSVSSNDAS PPASVASLQPFMIGAQSSP PKRPGNTLKWLTSPVRRLSSGADHKLAHKKSRDRKSADAGSQKDSDDSATPDETEERGNEG LSSGTLSKSSSSGMQSCGEEE 1800GEEGADAVPLPPPMAIQHSLLQPFSQDDASSRLLVRPTSSETPSAELVSAIEELVKSLEDRPSSLLVDWDSSSPSFPSDNSLLSSSSPIDEMEERKSSSVKRRHYVLQELVETERDYVRDLYVEYMLMEDGVPDDMK 1950CKDKIVFGNIHQIYDWHRDFFLGELEKCLEDPEKGSLFV21ERRLHMY0IAYCQNPKSEHIVSEYIDTFFEDLKQRL0HRLQLTDLLIKPVQRIMKYQLLLKDFLKYSSLDTSELERAVEMCIVPRRCNDM WGRFDKIV 2100AQGKLLL"2DTFLVTDDAGLLPRCRERRIFLEQIVIFSEPLDKKGFSMPGFLFKNS IKVSCLCLEENVENDPCKFALTSRTGDWVETFIHSSS PSVRQ'IWIHEI NQILENQRNNALTSPIEYQRNHSGGZGGGGSGAkAGVGAAA 2250AAG PPVAAAATVAAPAAAAAPPARAGAG PPGS PSLSDTTPPCWSPLQPRARQRQTR CQSESSSSSNISTLVTHDYTAKEDEINVYQGEVVQILASNQNMLVFRTDCPAAEGWIPGFVLGHTSAVIVENPDGTKKSTSWHTAL 2400RLRKKSEKKI:KEDGKREGKLENGYRKSREGLSNKVSVKLLN PNYIYDVPPEFV I PLSEVI ETGETVVLRCRVCGRhPKASI TWKGPEHNTLNNDGHYSISYSDLGEATLK IVGVTTEDDG IYI C IAVNDMGSASSSASLRVLGPGMDG IMVJ 2 5 5 0IWKDNFDSFYSEVAELGRGRFSVVKKCDQKGTKRAVATKFtVNKKLMKRDQVTHiELG ILQSU2HPLLVG LLDTFETPTSYI LVLEMADQGRLLDCVVRWGSLTEGKI RAHLGEVLEAVRYLHNCRIAHLDLKPENI LVDESI AKPTIKLAD 2 70 0FGDAVQLNIIYYIHQLLGNPEFAA PE:I ILNPVS LTSDrIWSVGVLTYVLLSGVS PFLDDSVFEETCLNI CRLDFSFPDDYFKGVSiJKAKEFwVCFLLQEDPAKR PSAALALS2EN2WIAGNGRS';VL,DTSRLTSF IERRKtHiNDVRP IRSI R 285 0NFLQSRLLPRV 2861

B Trio GEF-D1 1237 IMAELIQTEKAYVRDLRECMDTYLWEMTSGV EEIPFPIVNKELIIFGNMQEIYEFHNNIFLKELERYEQLPEDVGHCFVTW4ADKFQMYVTYCKNKPDSTQLILEHATrio GEF-D2 1914 VLQELVETERDYVRDLGYVVEGYHALMKE DOVPDDMKGKDKIVFGNI4HQIYDWHRDFFLGELEKCLEDPEKRLSLFVKHERRLHMYIAYCQNFPKSEHIVSEYIdbi GEF 499V9 ELIQERVYRELYVLLGYREMDNPEMDLMPPLLRNKDILFGIYEHNDIFLSSLENCHAPERVGPCFLERKDDFQMYAKYCQNKPRSETIWRKYSost GEF 446 VMNELLDTERAYVEELLCVLEGYAAEMDNPLMAHLISTGLQNKKNILFGNMEEIYHFHNRIFLRELESCIDCPELVGRCFLEREEQIYEKYCQNPRSESLWRQCSConsensus0EF cons.

v EL TEr YVr L v gY aeMvl e tE Yv L 1I. SCRi...

nK I FGNm eIY fHn iFL LE c PE vG cF fqMY YCqNKP SeiF n h f 1 F Y

I. SCR2...

G SYFDEIQQRHGLANSISSYLIKPVQRITKYQLLLKELLTCCE E GKGEIRDGLEVMLSVPKRANDD TFFEDLKQRLCHRLQLTDLLIKPVQRIMKYQLLLKDFLKYSKKASLDTSELERAVEVMCIVPRRCNDECAFFQECQRKLKHRLRLDSYLLKPVQRITKYQLLLKELLKYSKDCE GSALLKKALDAMLDLLKSVNDDCPFFQECQKKLDHKLSLDSYLLKPVQRITKYQLLLKEMLKYSKHCE GAEDLQEALSSILCILKAVND

fF e q 1 h 1 1 syL PFVQRItKYQLLLKe Lkysk e g L al 1 k ND1 L Pv r y L L EL1 t

I.... SCR3 ... ..I

YSEVAELGRGRFSVVKKCDQKGTRRAVATRFVNKKLMKRDQVTH

14072085674621

ELCILQSLQHPLLVGLLDTFETPTSYILVLEHADQGRLLDCVVRWOSLTE GRIRAHLGEVCRIALDLKLPENIL

8 YEFEEELGRGAFSIVYLGENKQTKQRYAIKVINKSELGKDYEKNL KMEVDILRKVNHPNIIALKELFDTPEKLYLVMELVTGGELFDKIVEKGSYSEADAANLVEKIVSAVGYLHGLNIVHRDLKPENLLY ELG G F V K T Y K K E IL HP L E G L D S E H DLKPEN

G G V K E D K N

VDESOLAPTIKLAOFGDAVQLNTTYYIHQLLGNPEFAAPEI ILGNFPVSLTSDTWOSVGVLTYVLLSVSPFLDDOVEETCLNICRLDFSFPDDYFKGVSQKAKEFVCFLLQEDPFARPSAALALQEQWLQA 2816LLDRNVPKPRIKIIDFGNEFKNIF GTPEFVAPEIVNYEPLGLEADMWS2IGVITILLSGASPFLGDT9QETANVSAVNYEFEDYFSNSALKDFIRRLLKDPKRMTIQDSLQHPWIKP 269LKSKENHLEVAIADFGLSK IIGQTLVrMQTACGTPSYVAPEVNATGYDKEVDWSSIGVIT LLCGFPPFYGDTVPEIFEQIMDEVNYEPEEYWGGISKEAKDFIGKLLVVDVSKRLNATNALNHPWLS 267

DFG G P APE D WS V TY LL G PF D E F Y S K F LL D KR L WLD G PE D 0 R

SP-D3 SP-D4 GEF-Dl PH-DI GEF-D2 PH-D2rMr- Xs

Ig-like PSK

FIG. 4. Trio contains multiple domains. (A) The Trio 2861-aa sequence deduced from cDNA cloning is shown using the standard one letteramino acid code. Numbers at the right indicate amino acid residues. (B) The two Trio GEF domains (Trio GEF-Di and -D2) are aligned withthe dbl GEF (31) and ost GEF (15) domain sequences. Consensus invariant amino acids are in uppercase letters and consensus amino acids presentin dbl and ost and one of the two Trio GEF sequences are in lowercase letters. The GEF consensus sequence and the extent of the GEF structurallyconserved regions (SCR) 1-3 are adapted from ref 2. Numbers indicate amino acid residues used for the alignment. (C) The Trio PSK domainsequence is aligned with the kinase domains of the death-associated protein (DAP) PSK (32) and Dictyostelium MLCK (33). Consensus invariantamino acids are in uppercase letters. The PSK consensus sequences is from ref. 34. (D) The overall structure of Trio is schematically shown withthe relative length and positions of the four Trio spectrin (SP)-like domains, two GEF domains, two pleckstrin homology (PH) domains, Ig-likedomain, and the PSK domain. The horizontal line below the Trio schematic indicates the relative length and position of the ClIGO peptide.

agreement with the apparent size of the protein (Fig. 2).However, because the most N-terminal, in-frame methioninecodon is not preceded by a termination codon, the numberingof the deduced Trio amino acids is provisional. A search ofsequence data bases revealed that Trio is a novel proteincomprised of several domains with sequence similarity to otherproteins. The Trio N-terminal region is similar to the N-terminal regions of the dbs (35), dbl (31), and ost (15) GEFs.For example, the Trio region spanning aa 3-208 is -31%identical to the dbs region spanning aa 69-281, and Trio aa126-208 are 36% identical to ost aa 10-93. Following thisN-terminal region of unknown function there are four domains(designated Trio SP-D1 to -D4; aa 252-359, 479-585, 819-925,and 1050-1157, respectively) that are 25-32% identical to thechicken a-spectrin 5 and 13 repeat sequences (36). Thesequences between these domains are weakly related to spec-trin-repeats, suggesting that there might be eight tandemlyarranged spectrin-like repeats between residues 252 and 1157.C-terminal to these repeats, there are two 170 aa regions (aa1237-1407 and 1914-2085) that are most similar to rho familyGEF domains (2). For instance, the Trio GEF domain se-quences (designated Trio GEF-Di and Trio GEF-D2) are44-49% identical with the dbl (31) and ost GEF (15) domainsequences (Fig. 4B). Furthermore, Trio GEF-Di and -D2contain essentially all of the conserved residues that define thethree structurally conserved regions (Fig. 4B) in GEFs (2).

C-terminal to each GEF domain there are regions withsequence similarity to pleckstrin homology (PH) domains,which are found in diverse signal transduction molecules (37).The Trio-PH1 (aa 1435-1534) and -PH2 (aa 2113-2214)domains are 25-37% identical to the PH domains foundC-terminal to the dbl and ost GEF domains.At the Trio C-terminal end (aa 2560-2816) there is a region

that has all the sequence hallmarks of a PSK domain (34). Ofthe 15 invariant or nearly invariant aa present in PSKs (34), allare conserved in Trio (Fig. 4C). The Trio PSK domain is mostsimilar to calcium/calmodulin-dependent kinases and is 44%and 37% identical to the DAP kinase (32) and Dictyosteliummyosin light chain (MLC) PSK domains (33), respectively (Fig.4C). Adjacent to the kinase domain there is an Ig-like domain(aa 2448-2541) that is 34% identical with an Ig-like domainpresent N-terminal of the chicken smooth muscle MLC kinase(MLCK) (38). Thus, the overall structure of Trio (Fig. 4D)consists of four N-terminal spectrin-like domains followed bytwo repeats of GEF/PH domains followed by an Ig-likedomain and a PSK domain. The arrangement of at least threeputative enzymatic domains, two GEF domains and a PSKdomain, is the basis for the name Trio.The Two Trio GEF Domains Have Distinct Substrate Spec-

ificities for rac and rho. To determine whether the TrioGEF-Di and -D2 domains are functional GEFs, deletionmutants termed TrioA2 (aa 1118-1919) and TrioA3 (aa 1849-

Trio GEF-DiTrio GEF-D2dbl GEFoat GEFConsensusCEF cons.

6013

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Trio PSK 2S5DAP PSK 1MLCK PSKConsensusPSK cons

Trio PSKDAP PSKMLCK PSKConsensusPSK cons.

SP-D1 SP-D2_j7777L__rw.w7"

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2451) were generated that encode either the Trio GEF-Dldomain with the adjacent PH-D1 domain or the Trio GEF-D2domain with its adjacent PH-D2 domain (Fig. SA). TrioA2 andTrioA3 were produced in COS cells as HA-fusion proteins.Lysates prepared from TrioA2, TrioA3, or control transfectedcells were then incubated together with either [3H]GDPloaded racl, rhoA, cdc42, or ras, and then the amount of[3H]GDP-bound protein remaining after 20 min was deter-mined using a filter binding assay (13, 30). TrioA2 had signif-icant GEF activity for racl (79% release compared to control),and limited or no activity (19% to -8%) with rhoA, cdc42, andras (Fig. SB). In contrast, TrioA3 had significant activity withrhoA (82% release), and limited or no activity (3% to -1%)with racl, cdc42, or ras (Fig. SB). A time course analysis of[3H]GDP release using the TrioA2 or TrioA3 lysates with racland rhoA substantiated the observation that TrioA2 hasrac-specific GEF activity, and TrioA3 has rhoA-specific GEFactivity (Fig. 5 C and D). These results strongly suggest that theGEF-Dl and -D2 domains are functional GEF domains andthat the GEF-Dl is a rac-specific GEF, and that GEF-D2 is arho-specific GEF.

A SD-D4 GEF-DI PH-D1 GEF-D2 PH-D2

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DISCUSSIONWe have characterized a novel 2861-aa phosphoproteintermed Trio that is comprised of two GEF domains, each withan adjacent PH domain, a PSK domain with an adjacent Ig-likedomain, and four spectrin-like repeats (see Fig. 4D). The TrioGEF-Dl domain has racl GEF activity, and the Trio GEF-D2domain has rhoA GEF activity. Thus, Trio is a unique memberof the growing family of rho/rac GEFs (2, 39) that contains twofunctional GEF domains. Given the role of rac and rho inregulating actin remodeling associated with changes in cellmorphology, adhesion, and motility (1), as well as cell growthcontrol (3, 9), it is likely that Trio is also involved in one ormore of these processes as a upstream positive regulator.PH domains are found adjacent to all functional rho/rac

GEF domains, as well as in a number of other signal trans-duction proteins, and are likely to play a role in protein-protein interactions and/or membrane localization (37, 40).Sequence comparison of the Trio GEF and PH domains andother rho-like GEFs with known in vitro GEF activities doesnot reveal any immediate basis for the distinct rho and racspecificities of the two Trio GEF/PH domains. The functionalrole of the Trio spectrin-like domains is not known. Spectrinrepeats are '106 aa long and found in a number of proteins,including the actin binding proteins spectrin, fodrin, a-actinin,and dystrophin (41). These domains may play a role inconnecting functional domains and/or mediate protein-protein interactions and thus are potentially important fortargeting Trio to the cytoskeleton and/or plasma membrane.The Trio PSK domain has all the sequence hallmarks of afunctional PSK (34), suggesting that Trio has kinase activity.Adjacent to the Trio PSK domain there is an Ig-like domain,which may also play a role in protein-protein interactions.Other intracellular proteins, including smooth muscle MLCK(38) and the Caenorhabditis elegans twitchin kinase (42), alsocontain Ig-like domains. The Trio PSK domain has the highestdegree of sequence similarity with calcium/calmodulin-dependent PSKs, although it is not yet known whether TrioPSK activity requires calmodulin. Trio purified by immuno-precipitation had neither detectable autophosphorylation ac-tivity nor kinase activity toward a number of artificial sub-strates (including cytoLAR), even in the presence of calmod-ulin (data not shown), suggesting that Trio is a highly regulatedenzyme and/or has a strict substrate specificity.While the physiological relevance of the interaction between

the LAR PTPase and the Trio GEF/PSK remains to beestablished, a role of a LAR-Trio complex in coordinatingcytoskeletal rearrangements is consistent with their enzymatic

-20C

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0

00

'e

1.0

0.8

0.6

0.4

0.2

0

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0 10 20 30Time (min)

0 10 20 30Time (miin)

FIG. 5. Trio GEF domains have distinct substrate specificity for racand rho in vitro. (A) At the top of the figure is schematically shown theregion of Trio containing the two GEF domains, and below are shownthe extent of the Trio deletion mutants, TrioA2 (GEF-Dl) and TrioA&3(GEF-D2), produced in COS cells to assay GEF activity. (B) Activityof COS cell lysates containing TrioA&2 (solid bars) or TrioA3 (stripedbars) to catalyze the release of [3H]GDP from racl, rhoA, cdc42, orras. The activity is expressed as the percent [3H]GDP released fromeach GTP binding protein after 20 min. Background values obtainedusing lysates prepared from vector-only transfected COS cells weresubtracted. GEF activity was determined as described, and the averagevalues of duplicate samples are given. The experiment shown isrepresentative of two independent GEF activity assays. Kinetics ofracl (C) and rhoA (D) GEF activity present in COS lysates containingTrioA2 (-), TrioA3 (0), or vector-only control (El). The activity ispresented as the relative amount of bound [3H]GDP remaining atvarious times to the amount bound at time 0. The relatively high degreeof spontaneous release of [3H]GDP from rac was previously observedby others (17).

activities. Protein tyrosine phosphorylation plays an essentialrole in regulating cell-matrix interactions, including rho-induced stress fiber formation (43) and the LAR PTPase has

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been localized to the ends of FAs, suggesting that LAR mayregulate FA disassembly (23). The Trio rac GEF activity maypromote actin polymerization and focal complex formationwithin lamellipodia and membrane ruffles, whereas rho GEFactivity could induce actin stress fiber and FA formation. Themultidomain structure ofTrio suggests that it coordinates actinpolymerization with FA complex formation and links themwith tyrosine and serine/threonine phosphorylation. Takentogether, we propose that LAR and Trio integrate diversesignals involved in cell migration, a process that requirescomplex spatial and temporal organization of cytoskeletalrearrangements.

We thank Drs. Nancy Kedersha and Haruo Saito for critical reviewof the manuscript and advice, and Drs. Stuart F. Schlossman andRobert A. Weinberg for encouragement and support, Dr. MarcSymons (Onyx Pharmaceuticals) for kindly providing the GTP bindingproteins used in this study, and Dr. Quintus Medley, Dr. TimothyErnst, and Herlinde Gerrits for help. K.S. was supported by postdoc-toral fellowship from the Swiss National Science Foundation. Thiswork was supported by Grant CA55547 from the National Institutes ofHealth, a Dana-Farber Cancer Institute Barr Program grant, and aPew Scholar in the Biomedical Sciences Award to M.S.

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