git1 regulates protusive activity and cell migration · 2002. 3. 6. · al., 2000). furthermore,...

IntroductionCell migration is essential in many biological processes,including embryogenesis, inflammation, wound healing andtumor metastasis (Lauffenburger and Horwitz, 1996). Themigratory process requires the coordinated activation andtargeting of both structural and signaling molecules. Asmigration is initiated, protrusions are extended at the leadingedge and new adhesions are formed. The assembly anddisassembly of actin is proposed to drive the formation andextension of the lamellipodia. Active myosin-based motorsmay generate the contractile forces necessary to translocate thecell body forward (Lauffenburger and Horwitz, 1996). At therear, adhesions must be released for the cell to move forward.Although many processes take place during cell migration,dynamic changes in polarized actin structures are believed tobe among the most crucial.

Members of the Rho family of small GTPases, includingRho, Rac and Cdc42, are key regulators of actin dynamics and

cell motility. Rho promotes the assembly of stress fibers andfocal adhesions (Ridley and Hall, 1992). Rac inducesmembrane ruffling and the formation of lamellipodia, whereasCdc42 regulates filopodial extension (Ridley and Hall, 1992;Nobes and Hall, 1995). These small GTPases cycle betweenan inactive (GDP-bound) state and an active (GTP-bound)state. The nucleotide state of these molecules is regulated byguanine nucleotide exchange factors (GEFs), which facilitatethe exchange of GDP for GTP, and GTPase-activating proteins(GAPs) that promote GTP hydrolysis. In the active state, theseproteins can interact with downstream targets or effectormolecules to elicit a biological response.

One of the best characterized effectors of the Rho familyof GTPases is p21-activated kinase (PAK). PAK is aserine/threonine kinase that binds to the active form of Racor Cdc42, resulting in a conformational change in PAK,autophosphorylation of the molecule at several sites andsubsequent activation (Manser et al., 1994; Lim et al., 1996;

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GIT1 is a multidomain protein that is thought to functionas an integrator of signaling pathways controlling vesicletrafficking, adhesion and cytoskeletal organization. Itregulates ARF GTPases and has binding domains forpaxillin and PIX, which is a PAK-binding protein and anexchange factor for Rac. We show that GIT1 cyclesbetween at least three distinct subcellular compartments,including adhesion-like structures, the leading edge andcytoplasmic complexes. The cytoplasmic structures, whichalso contain paxillin, PAK and PIX, do not detectablyco-localize with endosomal Golgi or membrane markers,suggesting that they represent a novel supramolecularcomplex. The GIT1 cytoplasmic complexes are motile andtended to move toward the cell periphery where they joinedexisting adhesions. In retracting regions of the cells, theGIT1 complexes moved away from the disassemblingadhesions toward the cell body. Using deletion mutants, wehave identified domains that target GIT1 to each of thecompartments. Localization to adhesions and the leadingedge requires the paxillin-binding domain, whichcomprises the C-terminal 140 residues (cGIT1), whereastargeting to the cytoplasmic complexes requires the central

region that contains ankyrin repeats and the PIX-bindingdomain. Expression of GIT1 or cGIT, but not nGIT1 inwhich the paxillin-binding domain is deleted, increases therate of migration and the size and number of protrusions.The latter are inhibited when GIT1 is co-expressed with akinase-dead PAK, suggesting that the GIT1 interactionwith PAK is required for enhanced migration andprotrusive activity. Furthermore, GIT1 targetsconstitutively activated PAK to adhesions and the leadingedge via its interaction with paxillin. Since expression ofcGIT targets endogenous GIT1 to the leading edge, itappears that the leading edge is the location of GIT1responsible for these activities. Thus, GIT1 is a componentof a motile, multimolecular complex that traffics a set ofsignaling components to specific locations in the cell wherethey regulate localized activities.

Movies available on-line

Key words: Cell adhesion, GFP, GIT1, p21 activated kinase (PAK),Rac, Paxillin

Summary

GIT1 functions in a motile, multi-molecular signalingcomplex that regulates protrusive activity and cellmigrationRi-ichiroh Manabe* ,‡, Mykola Kovalenko ‡, Donna J. Webb § and Alan Rick HorwitzDepartment of Cell Biology, University of Virginia, Charlottesville VA 22908 *Present address: Ri-chiroh Manabe, Japan Science and Technology Corporation, Sekiguchi Biomatrix Signaling Project, c/o Aichi Medical University, 21 Karimata-Yasago,Nagakute, Aichi 480-1195, Japan‡These authors contributed equally to this work§Author for correspondence (e-mail: [email protected])

Accepted 23 January 2002Journal of Cell Science 115, 1497-1510 (2002) © The Company of Biologists Ltd

Research Article

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Lei et al., 2000). The effects of PAK may be, at least partially,controlled by regulation of its subcellular localization. Inresting cells, PAK is distributed throughout the cytoplasm;however, when the cells are stimulated, PAK is targeted to focaladhesions and membrane ruffles (Dharmawardhane et al.,1997). Moreover, it has been demonstrated that it is theactivated fraction of PAK that localizes to the leading edge andfocal adhesions of migrating fibroblasts (Sells et al., 2000). AsPAK lacks a focal adhesion targeting domain, it is probablyrecruited to focal adhesions and membrane protrusions throughinteraction with binding partners. The PAK-binding protein,PIX, may be involved in the recruitment of PAK (Manser etal., 1998), alternatively, the adaptor protein, Nck, may functionin this capacity (Bagrodia and Cerione, 1999; Bokoch et al.,1996). However, the mechanisms that mediate PAK targetingto focal adhesions and membrane ruffles are not well defined.

There is a growing body of evidence implicating PAK inreorganization of the actin cytoskeleton. Although themechanism(s) by which PAK regulates actin dynamics is notwell understood, it has been suggested that PAK may mediatethese effects by phosphorylation of LIM kinase (Edwardset al., 1999). This results in the inactivation of the actindepolymerizing protein, cofilin, and stabilization of actinpolymers (Edwards et al., 1999). PAK can also alterthe phosphorylation state of myosin-II. Expression ofconstitutively activated PAK resulted in decreased activity ofmyosin light chain kinase and decreased phosphorylation of theregulatory myosin light chain (MLC) (Sanders et al., 1999).By contrast, in NIH3T3 cells, activated PAK promotedphosphorylation of MLC and localization of thephosphorylated protein to the lamellipodia (Sells et al., 1999).Finally, PAK may affect the actin cytoskeleton throughregulation of the Raf-Mek-ERK pathway (Bagrodia et al.,1995).

Paxillin is a multidomain scaffolding protein that functionsin the recruitment of both signaling and structural moleculesto focal adhesions (Turner, 2000). This protein comprisesmultiple SH3- and SH2-binding domains, four LIM domainsand five leucine-rich motifs (LD motifs) (Turner, 2000).Members of the ARF family of small GTPases, including GRKinteractor 1 (GIT1) and paxillin kinase linker (PKL), interactwith paxillin through its LD4 motif (West et al., 2001; Zhaoet al., 2000; Turner et al., 1999). Although the functionalsignificance of the GIT1-paxillin interaction is not welldefined, overexpression of GIT1 in fibroblasts has beenreported to sequester paxillin from focal complexes (Zhao etal., 2000). Furthermore, expression of another GIT1 familymember, PAG3, inhibited paxillin recruitment to focal contactin an ARF-GAP-dependent manner (Kondo et al., 2000). Theseproteins may function in linking PAK to paxillin through apaxillin-GIT1-PIX-PAK complex and in this manner localizePAK to paxillin-containing adhesions.

In this study, we show that GIT1 localizes to threedistinct subcellular compartments, including focal adhesions,cytoplasmic complexes and membrane protrusions. Paxillin,PIX and PAK all colocalized with GIT1 in these cytoplasmiccomplexes, suggesting that these structures might represent amultiprotein signaling module. The cytoplasmic complexes aremotile and appear to be involved in delivery of components toand from adhesions as they form and breakdown. Recruitmentof GIT1 to focal adhesions and the leading edge of the

lamellipodia requires the paxillin-binding domain. Expressionof GIT1 or its C-terminal 140 amino acids, which contains thepaxillin-binding site, increases the rate of migration andthe size and frequency of protrusions. The GIT1-inducedprotrusions are inhibited by expression of kinase-dead PAK,suggesting that PAK association with GIT1 is necessary forthe enhanced migration and protrusions. GIT1 targetsconstitutively activated PAK to focal adhesions and the leadingedge through its interaction with paxillin. Expression of cGIT1targets endogenous GIT1 to the leading edge. Thus, the leadingedge appears to be the location of GIT1 responsible for itseffects on protrusion and migration.

Materials and Methods ReagentsDulbecco’s modified media (DMEM) and lipofectamine transfectionreagent were from Gibco (Grand Island, NY). G418 was from Sigma(St Louis, MO). Enhanced chemiluminescene (ECL) detectionsystem was from Amersham Life Sciences (Buckinghamshire, UK).Effectene was from Qiagen (Valencia, CA) and Fugene 6 fromBoehringer Mannheim (Germany). Polyacrylamide was from Bio-Radlaboratories (Hercules, CA). Cy-3-labelled transferrin was fromJackson Laboratories (West Grove, PA), FM4-64 dye and BODIPY-Ceramide (Golgi marker) were from Molecular Probes (Eugene, OR).All PAK constructs were kindly provided by Jonathan Chernoff (FoxChase Cancer Center, Philadelphia, PA).

AntibodiesThe following antibodies were used for western blotting,immunoprecipitation and immunofluorescence. We prepared anaffinity-purified rabbit polyclonal antibody raised against the central140 amino acids of GIT1, the region that contains the least homologyto any known GIT1 family members. The peptide was cloned intopGEX-2T (Promega, Madison, WI). The GST fusion product waspurified from E. coli using the manufacturer’s protocol and used forimmunization. The antisera was produced by Covance (Denver, PA),and the antibody was purified by affinity chromatography aspreviously described (Koff et al., 1992) with our modifications. Theantibody did not recognize GIT1’s close homologue PKL in cellsstably expressing GFP-PKL. Transferrin receptor B65.3 mousemonoclocal antibody was a kind gift from Sam Green (U. of Virginia)and Ian Trowbridge (Salk Institute, LaJolla, CA). Anti-FAK 2A7 wasfrom Upstate Biotechnology (Lake Placid, NY). Monoclonalantibodies for paxillin, GIT1 and NCK were from TransductionLaboratories (Lexington, KY). HA monoclonal antibody was fromBoehringer Mannheim. Anti-c-Myc 9E10, anti-GFP, B2 and anti-phosphotyrosine PY20 were from Santa Cruz Biotechnology (SantaCruz, CA). FLAG M2 monoclonal antibody was from Stratagene(La Jolla, CA). Vinculin monoclonal antibody was from Sigma.Phosphopaxillin PY31 and PY118 antibodies were from BiosourceInternational (Camarillo, CA). Rhodamine-conjugated phalloidin andthe GFP polyclonal antibody A-11122 were from Molecular Probes.HRP-conjugated secondary sheep anti-mouse IgG and donkey anti-rabbit IgG antibodies were from Amersham. Rhodamine-conjugatedsheep anti-mouse IgG, FITC-conjugated sheep anti-mouse IgG andFITC-conjugated goat anti-rabbit IgG were from ICN BiochemicalDivision (Aurora, OH).

Isolation of GIT1 cDNAWe isolated a 1.5 kb cDNA encoding the C-terminal 140 amino acidsof GIT1 from a GFP-fusion cDNA library as previously described(Manabe et al., 2000). Briefly, a cDNA library derived from humanfibrosarcoma HT1080 cells was cloned into pCIneoEGFP (Fujii et al.,

Journal of Cell Science 115 (7)

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1999). Individual cDNAs were purified and transfected into REF52cells using Effectene according to the manufacturer’s protocol.

For isolation of full-length GIT1, we amplified the 5′ GIT1 cDNAfrom a human fetal brain library (Clontech) using the followingprimers: 5′-AAGGATCCGTCGACATGTCCCGAAAGGGGCCGCG-3′ (forward) and 5′-TTGAATTCGCGGCCGCCTACTCTTTGCCC-AGCTCTAGAAACC-3′ (reverse). The amplified cDNA wassequenced and cloned into pCIneoEGFP-cGIT1. A cDNA expressionvector for full-length GIT1 fused with FLAG at its C-terminus (Wt-GIT1-FLAG) was prepared as follows. A cDNA encoding C-terminalGIT1 without the stop codon was generated by PCR using thefollowing primers: 5′-AGGTTTCTAGAGCTGGGC-3′ (forward)and 5′-NotI-Stop-FLAG-SalI-CTGCTTCTTCTCTCGGG-3′ (reverse).The amplified cDNA was excised with XbaI and NotI and cloned intopEGFP-N1 (Clontech) with the sequence encoding the N-terminus ofGIT1. For the construction of the C-terminal deletion mutant of GIT1(nGIT1), the PCR-amplified 5′ GIT1 cDNA described above wascleaved with SalI and NotI and cloned into the SalI and NotI site ofpCIneohEGFP. For construction of a GFP-GIT1 chimera lacking acentral part of GIT1 (MiniGIT1), a HindIII fragment encoding thecentral part of GIT1 was excised and the resulting plasmid wasreligated.

Cell culture and transfectionCHO-K1, REF-52, HT1080 and A431 cells were maintained inDMEM supplemented with 10% FBS, 4 mM L-glutamine, 1 mMsodium pyruvate, non-essential amino acids, penicillin andstreptomycin. For most experiments involving microscopy, cells wereplated on either 12 mm glass cover slips or dishes prepared formicroscopic observation as previously described (Palecek et al.,1996). The coverslips and dishes were coated overnight at 4°C with2-10 µg/ml fibronectin (Fn). Cells were transfected with eitherLipofectamine (CHOK1 cells) or Fugene 6 (REF-52) and incubatedfor 24 to 72 hours. To prepare CHO-K1 cells stably expressing ourGIT1 constructs, cells were maintained in serum containing mediumwith 1 mg/ml G418 for 7 to 10 days after transfection. Cells wereFACS sorted by the level of expression, and those with a mediumexpression level were selected for experiments. As confirmed bywestern blotting, the GFP-fused GIT1 in stably expressing cells wasof the expected molecular mass indicating that it was not cleaved.

Immunoprecipitation and western blottingPolyclonal GFP antibody was used to immunoprecipitate GFP-GIT1from the stable CHOK1 transfectants. Cells were grown to 80-90%confluency, washed with ice-cold PBS and then lysed with ice-cold10 mM Tris-HCl (pH 7.6), 50 mM NaCl, 1% NP-40 and 10% glycerol,1 mM DTT, 10 µg/ml aprotonin, 1 µg/ml pepstatin A, 1mMbenzamidine, 10 µg/ml leupeptin and 0.2 mM Pefabloc (BoehringerMannheim). Cell lysates were incubated on ice for 30 minutes andclarified by centrifugation (13,000 g for 15 minutes). Proteinconcentrations were determined by BCA assay, and equivalentamounts of the lysates were precleared with 6 µg of rabbit IgG for 30minutes followed by incubation with 6 µg of a GFP-specificpolyclonal antibody for 1 hour at 4°C. Complexes were incubatedwith protein A sepharose overnight and washed four times with ice-cold lysis buffer. The immunoprecipitates were subjected to SDS-PAGE on 10% slabs, transferred to nitrocellulose and detected bywestern blot analysis.

ImmunocytochemistryFor immunofluorescence, cells were grown to 50-70% confluency onglass coverslips or glass-bottomed 35 mm plates, washed with PBSonce, fixed with 2% formaldehyde for 15 minutes, incubated with 0.15M glycine for 10 minutes to stop fixation and permeabilized with

0.2% (v/v) Triton X-100 for 5 minutes at room temperature. Aftereach step, cells were washed with PBS two or three times. Cells werethen blocked with 5% normal goat serum in PBS for 1 hour at roomtemperature. This blocking buffer was also used for the antibodydilution. Primary antibodies or rhodamine-linked phalloidin wereapplied for 1 hour and FITC or rhodamine-conjugated secondaryantibody for 40 minutes. Slips were mounted on slides withVectashield mounting media (Vector Laboratories, Burlingame, CA)and visualized using rhodamine/Cy-3, EGFP or FITC filters.

Dye stainingFor staining with dyes, CHO-K1 cells were plated at 30-50%confluency on 35mm glass bottomed plates in complete DMEMmedium and allowed to spread overnight. All DMEM F-12 mediaused for staining (washes and dye dilution) was serum and phenol-red free. For transferrin staining, cells were washed three times withDMEM F-12 media and incubated for 7 minutes with 4 µg/ml Cy-3transferrin at 37°C. Cells were rinsed three times with PBS, fixed with2% formaldehyde and visualized. After fixation, untransfected cellswere stained with a GIT1-specific antibody followed by the FITC-conjugated rabbit IgG secondary antibody as described above andvisualized. For FM4-64, cells were washed three times with DMEMF-12 and incubated in DMEM F12 with 1 µg/ml FM4-64 for 10, 30,45, 60, 120, 180 minutes or overnight at 37°C. Cells were rinsed threetimes with PBS and used for microscopy. For BODIPY-Ceramidestaining, cells were incubated in DMEM F-12 containing the dye at1 µg/ml at 4°C for 30 minutes, washed three times and treated withdye free DMEM F-12 for another 30 minutes at 37°C and fixed.

Microscopy and image processingFor fluorescence, cells were visualized with a Nikon TE 300conventional fluorescence microscope (Melville, NY). For EGFPfluorescence, an Endow GFP filter cube (ex. HQ470/40, em.HQ525/50, Q495LP, dichroic mirror) was used (Chroma, Brattleboro,VT). Rhodamine, FM4-64, Cy3 Transferrin and BODIPY-Ceramidewere visualized using a rhodamin/TRITC cube (ex. BP520-550,barrier filter BA580IF, dichroic mirror DM565). AMCA fluorescence,for vinculin staining of PAK/GIT1 expressing cells, was visualizedusing a set of filters: ex. D360/40, em. D460/50. Both TRITC andAMCA filters were from Chroma. Individual images or z-stacks (fordeconvolution) were collected using a cool CCD camera (HamamatsuPhotonics, Bridgewater, NJ) and processed with Isee software(Inovision, Durham, NC). Confocal images were taken on ZeissAxiovert 100 confocal microscope (Carl Zeiss GmbH, Jena,Germany) with excitation and emission filters for FITC andTXRed/Cy3 and processed using Zeiss LSM Microsystem software.Images were analyzed using NIH Image software. For migrationspeed, the cell centroid was tracked. The average speed for the cellwas then determined by computing the average net displacement ofthe cell centroid divided by the time interval at each time point. Forprotrusiveness analysis, the cells were outlined at two time pointsseparated by 10 minutes; the two images were thresholded and thensubtracted to estimate the new area. The area measurements werecalibrated using a micrometer scale.

ResultsGIT1 localizes to distinct cellular compartmentsTo search for novel regulators of cell migration, cytoskeletalorganization and adhesion assembly, we subcloned a humancDNA library, derived from the HT1080 fibrosarcoma cell line,into a GFP-tagged expression plasmid, pCINeo-GFP, andscreened for clones that localize to focal adhesions orcytoskeletal structures (Fujii et al., 1999). One of the clones

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localized prominently in adhesion-like structures that co-stained with paxillin. It encoded the 140 C-terminal residues

of GIT1 (Fig. 1B,C), which had been previously identified asa regulator of β2-adrenergic receptor endocytosis (Premont et

al., 1998). To further examine the protein, wecloned its full-length cDNA into pCINeo-GFPand stably or transiently expressed theresulting GFP fusion protein in CHOK1 cells.Cells were sorted by flow cytometry into threeexpression categories: low, medium and highexpressors. Western blot analysis showed thatthe level of ectopic GIT1 in the mediumexpressors was comparable to that of theendogenous protein. Thus, the mediumexpressors were used for most studies.

In CHOK1 cells expressing medium levelsof the fusion protein, GIT1 localized inpaxillin- and vinculin-containing adhesionsand at the leading edge in membrane ruffles(Fig. 1E). When GIT1 was expressed in REF-52, WI38 and NIH3T3 cells, the localizationpatterns were comparable to those observed inCHOK1 cells. The majority of cells expressingectopic GIT1 showed cytoplasmic structures(Fig. 1E). GIT1 tagged with FLAG or fused tothe N-terminus of GFP showed localizationpatterns similar to GFP-GIT1, suggesting thatthe GFP fusion did not affect localization (datanot shown).

To examine the localization of theendogenous protein, we raised antibodiesagainst a GST fusion product consisting of thecentral 130 residues of GIT1 because thisregion showed the least homology to other GITfamily members. When lysates from CHOK1cells expressing GFP-GIT1 or GFP-PKL wereanalyzed on western blots using this antibody,a single prominent band was detected thatcorresponded to either endogenous GIT1 orGFP-GIT1 but not to the highly homologousGFP-PKL (data not shown). These resultssuggest that the antibody was specific for GIT1and does not cross-react with other GIT familymembers, such as PKL. Pre-incubation of


Fig. 1. Subcellular localization of GIT1.(A) Schematic representation of the GIT1 mutantsused in this study. (B) REF 52 cells were plated onfibronectin, fixed and stained for endogenouspaxillin. (C) GFP fluorescence localized toadhesion-like structures in REF52 cells transfectedwith GFP-cGIT1. (D) CHO K1 cells were stainedfor endogenous GIT1. The arrows indicatelocalization to the cytoplasmic complexes. CHOK1 cells expressing (E) wild-type GFP-GIT1,(F) GFP-nGIT1, and (G) GFP-mini-GIT1 wereviewed using fluorescence. Wild-type GIT1,cGIT1, and mini-GIT1, but not nGIT1, localized inadhesions, indicating that the adhesion-targetingdomain resides at the C-terminus of GIT1. Thelocalization of the wild-type GIT1 to the leadingedge of a lamellipodium is indicated by an arrowin panel (E). WI-38 cells were antibody stained for(H) endogenous GIT1 and (I) paxillin. GIT1 co-localized with paxillin in adhesions. Bar, 10 µm.


the antibody with the antigenic peptide inhibited theimmunoblotting (data not shown). The endogenous GIT1localized both to adhesion-like structures and small complexesthroughout the cytoplasm (Fig. 1D,H); neither of which wereobserved when the cells were stained with non-immune serumor antibody pre-incubated with the inhibitory peptide. Theseobservations suggest that the cytoplasmic localization of GFP-GIT1 is analogous to that of the endogenous protein and is nota result of overexpression at least at medium expression levels.

To examine the domains responsible for GIT1 localizationin various compartments, we prepared three GIT1 deletionmutants, N-, C- and ‘mini’-GIT1, tagged with GFP (Fig. 1A).nGIT1 comprises 630 residues beginning at the N-terminusand including the ARF-GAP domain, ankyrin repeats and aputative PIX-interaction domain. The cGIT1 mutant encodesthe C-terminal 130 residues, which contains the paxillin-

binding domain. Mini-GIT1 has the central 477 residuesdeleted, but it retains the ARF-GAP domain and paxillin-binding region (Zhao et al., 2000). Both wild-type GIT1 (Fig.1E), cGIT1 (Fig. 1C) and mini-GIT1 (Fig. 1G) localized toadhesions, whereas nGIT1 (Fig. 1F) did not, confirming thatthe adhesion targeting domain resides at the C-terminal end.In contrast, both nGIT1 and wild-type GIT1 localized incytoplasmic complexes, whereas c- and mini-GIT1 mutantsdid not. These observations point to the central region as thetargeting domain for the cytoplasmic complexes. Finally, bothcGIT1 and mini-GIT1 localized in ruffles whereas nGIT1 didnot, implicating the adhesion targeting domain, possiblythrough its interaction with paxillin, in the localization to theleading edge. Thus, localization of GIT1 to three distinctsubcellular compartments is mediated by discrete domainswithin the GIT1 protein.

Fig. 2. GIT1 cytoplasmic complexes do not colocalize with endosomal markers. CHO K1 cells expressing medium levels of GFP-GIT1 werefixed, permeabilized and stained for transferrin receptor and mannosidase-2 (a Golgi marker). In separate experiments, GIT1-expressing CHOK1 cells were incubated with Cy-3 transferrin for 7 minutes or FM4-64 dye for 24 hours. Cells were viewed in fluorescence using confocalmicroscopy. GIT1 did not co-localize with any of these markers. Bar, 10 µm.

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The nature of the GIT1 cytoplasmic complexesTo determine whether the GIT1-containing cytoplasmiccomplexes are vesicles, we probed CHO K1 cells expressingGFP-GIT1 as well as untransfected cells with severalmarkers for vesicular compartments. The markers included

(a) endosomal markers – Cy-3-labeled transferrin and atransferrin receptor antibody, (b) markers for bulk membraneinternalization – FM 4-64 dye and membrane-boundpalmitoylated GFP and (c) two Golgi markers – mannosidase-2 antibody and BODIPY-ceramide dye. After staining, the cellswere examined by confocal or deconvolution microscopy(Fig. 2).

As GIT1 and other ARF-GAP-containing proteins areinvolved in regulation of endocytosis (Moss and Vaughan,1998; Premont et al., 1998), we asked whether GFP-GIT1cytoplasmic complexes were of endocytic origin using Cy-3-labeled transferrin and transferrin receptor antibody asmarkers. While we observed some co-localization of GIT1with the transferrin receptor in the perinuclear region, it did notco-localize with the numerous small complexes that residedoutside this region. In addition, there was no detectable co-localization of GFP-GIT1 with transferrin.

ARF1, one of the ARFs for which GIT1 serves as a GAP, isreported to reside in the Golgi apparatus and regulate intra-Golgi and Golgi-ER trafficking (Moss and Vaughan, 1998). Inaddition, PAG3, a distant GIT1 relative, is also reported toreside in the Golgi apparatus (Kondo et al., 2000). Therefore,we probed for colocalization between GIT1 complexes and twoGolgi markers – BODIPY-ceramide and a mannosidase-2antibody. BODIPY-ceramide dye accumulates predominantlyin the Golgi, but also shows a secondary localization in otherorganelles such as lysosomes (Pagano et al., 1991), whereasthe mannosidase-2 antibody is highly Golgi specific (Baron etal., 1990). In spite of relatively strong labeling after a 30minute incubation with BODIPY-ceramide, GFP-GIT1complexes did not detectably co-localize with it. The absenceof clear co-localization was also observed using the anti-mannosidase-2 antibody marker, which produced a typicalperinuclear ring-like Golgi staining, but did not co-localizewith GFP-GIT1.

Since the GIT1 complexes did not colocalize robustly withany of the compartment specific markers, we compared theirlocalization with FM4-64 dye, a bulk membrane internalizationmarker. The cells were allowed to internalize the dye forvarious time intervals, from 5 minutes to overnight, to ensuresaturation labeling. Although the FM4-64 signal was strongthroughout the cell, there was no detectable co-localizationwith the GIT1 complexes. Furthermore, we could not detectco-localization of GIT1 cytoplasmic structures withmembrane-bound palmitoylated GFP, which indicated that theGIT1 complexes are not membrane enclosed.

These data suggest that the majority of the cytoplasmicstructures formed by GIT1 do not reside prominently in eitherendosomal or Golgi compartments. In addition, GFP-GIT1localization does not resemble that of aggresomes, which areaggregates of overexpressed protein, reportedly formed byseveral GFP-fused molecules. These aggregates are lessnumerous and morphologically different from what weobserved in the case of GIT1 (Garcia-Mata et al., 1999).

As the GIT1 containing structures did not appear to bevesicular, we asked whether these cytoplasmic complexescontain signaling or structural components. Paxillin, PIX andPAK co-localized with GIT1 in the cytoplasmic complexes(Fig. 3A,B,E-H). Although the majority of the GIT1 complexescontained PAK, we could not detect PAK in all of thesestructures. FAK, vinculin, α-actinin, ARF1, ARF6, Rac and the


Fig. 3. GIT1 cytoplasmic complexes contain paxillin and PAK. GFP-GIT1-expressing CHOK1 cells were either co-stained for paxillin orco-transfected with Myc-PAK and stained for Myc. (A) GFP-GIT1co-localized with (B) Myc-tagged PAK in the cytoplasmic structures(arrows). In GFP-GIT1-expressing cells co-transfected with a PIX-binding-deficient mutant of PAK, (C) GIT1 localized in cytoplasmiccomplexes (arrows) but (D) the mutant PAK was not detected inthese structures. Both (E) GFP-GIT1 and (G) endogenous GIT1 co-localized in cytoplasmic complexes with (F,H) endogenous paxillinas indicated by arrows. Bar, 10 µm.


Fig. 4. GIT1 cytoplasmic complexes move intoadhesions. CHOK1 cells stably expressing GFP-GIT1 were plated on 2 µg/ml fibronectin, andtheir movement captured using time-lapsefluorescence microscopy. (A) Cytoplasmiccomplexes containing GIT1 (two arrows show thepath of two separate complexes) move toward apre-existing adhesion and appear to fuse with it.(B,C) GFP-GIT1 (upper panels) complexes inadhesions co-localized with (B) paxillin and(C) vinculin (lower panels) in stably expressingCHOK1 cells. Bars, 10 µm. See Movie 1 atjcs.biologists.org.

Fig. 5. GIT complexes (the two arrows indicate twoseparate complexes) move from an adhesion at theperiphery toward a the cell cortex as the cell edgeretracted. Cells were plated on 2 µg/ml fibronectin andthe movement captured by time-lapse fluorescencemicroscopy. Bar, 10 µm. See Movie 2 atjcs.biologists.org.

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α5 integrin subunit were not detected in the GIT1 cytoplasmiccomplexes (data not shown).

To determine which domain of PAK was necessary for itslocalization in the cytoplasmic complexes, we co-expressedmutant forms of PAK with GIT1. Kinase-dead, Rac- andNck-binding-deficient PAK co-localized with GIT1 in thecomplexes (data not shown); however, PIX-binding-deficientPAK was not observed in the cytoplasmic complexes withGIT1 (Fig. 3C,D). This indicates that GIT1 interaction withPAK via PIX is essential for PAK localization in thecomplexes.

GIT1 cytoplasmic complexes are motile The presence of signaling components in the GIT1-containingcytoplasmic complexes suggested that they might serve asmotile signaling modules. This prompted an investigation ofthe dynamics of the small complexes that reside throughout thecell. When cells were viewed 2-4 hours after plating with time-lapse microscopy, GIT1 appeared in clusters of small discretecomplexes near the cell periphery (Fig. 4A). The GIT1complexes co-localized with paxillin and vinculin (Fig. 4B,C),which indicated their association with adhesions. The smallGIT1 complexes were motile and tended to move toward thecell periphery and join a group of similar structures (Fig. 4A)

(see Movie 1 at jcs.biologists.org). When a cell protrusionretracted, some GIT1 complexes would disassemble intodiscrete structures, which moved away from the retractinglamella (Fig. 5) (see Movie 2 at jcs.biologists.org). Thesecomplexes moved at an average rate of 30 µm/hour toward thecell center. In retracting regions of the cell, these GIT1complexes co-localized with paxillin in fibroblasts expressingCFP-GIT1 and YFP-paxillin (data not shown).

When cells were allowed to adhere for longer periods oftime, the number of GIT1 cytoplasmic structures decreasedsignificantly. A corresponding increase in GIT1 co-localizationwith paxillin or vinculin-containing adhesions was observed.We hypothesized that GIT1 may be cycling between the smallcytoplasmic complexes and adhesion-like structures. Duringcell detachment, GIT1 may move from the adhesions into thecytoplasm, whereas when it is needed in adhesions, it wouldthen be delivered there in the same manner. Consistent withour hypothesis, in migrating cells GIT1 emanated from the rearof the cell in complexes and moved toward the cell body (Fig.6) (see Movie 3 at jcs.biologists.org).

GIT1 localization affects cell migrationThe localization to adhesion-like structures and the leadingedge suggested that GIT1 might regulate cell migration. To


Fig. 6. GIT1 cytoplasmic complexes move from the rear toward the cell center. GFP-GIT1-expressing CHOK1 cells were filmed as described inFig. 5. GIT1 complexes moved from the retracting edge of the cell toward the perinuclear area (white arrows). In the top left panel, the path of aGIT1 complex was tracked (white line) and the arrows indicate the position of the complex at the beginning (white arrow) and end (gray arrow)of the observation period. Bar, 10 µm. See Movie 3 at jcs.biologists.org.


address this, we estimated migration speeds from time-lapseobservations of cells constitutively expressing GIT1 or itsmutants. Both wild-type GIT1 and cGIT1 increased themigration speeds of CHOK1 cells when compared with cells

expressing GFP alone (Fig. 7A), whereas nGIT1 did not affectmigration. For example, less than 40% of GFP or GFP-nGIT1transfected cells migrated at a rate of at least 25 µm/hour; thisfraction increased to over 80% for GIT1 expressors and almost

Fig. 7.Wild-type and cGIT1 enhancemigration and protrusion formation.CHO K1 cells expressing GFP alone,wild-type GIT1-GFP or GFP-cGIT1were plated on 2 µg/ml fibronectin,and their migration was captured usingtime-lapse microscopy (10 minuteintervals). (A) The fraction of cellsmigrating at various speeds wascalculated for each construct. Bothwild type and cGIT1 increased themigration speeds when compared tocells expressing GFP alone.(B) Protrusive activity was expressedas the net positive change in cell areaover time (10 minute). Cellsexpressing (D) wild-type GIT1 and(E) cGIT1 appeared much moreprotrusive when compared to cellsexpressing (C) GFP alone. The arrowsin (D) and (E) indicate protrusions.Cells expressing (F,G) GFP alone or(H,I) cGIT1-GFP were stained forendogenous GIT1. In GFP-expressingcells, endogenous GIT1 localized toadhesion-like structures as well as tocytoplasmic complexes, as indicatedby arrows in (F). In cells expressing(I) GFP-cGIT1, which localized inadhesion-like structures and at theleading edge, (H) endogenous GIT1was prominent in membrane ruffles atthe leading edge, arrows. Bar, 10 µm.

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60% for cGIT1 expressors. Accompanying the effects of GIT1on migration, we found that both wild-type and cGIT1 alsoincreased the size and rate of formation of protrusions relativeto GFP alone (Fig. 7B-E).

Thus, it appears that the C-terminal portion of GIT1, whichcontains targeting domains for adhesions and the leading edgeand binds to paxillin, is sufficient for promoting increasedmigration and protrusion formation. The remaining domains donot localize to adhesions or membrane ruffles and do not affectmigration. This observation is surprising since the adhesion-targeting domain has no known activities other than binding topaxillin, whereas the other domains have activities that includean ARF-GAP and PIX binding, both of which might affectmigration. It suggests, therefore, that ectopic expression ofcGIT1 might act by competing with endogenous GIT1 foradhesion-binding sites and promoting its relocalization to a sitewhere it is active. When cells were transfected with cGIT1, theendogenous GIT1 became more prominent in the membraneruffles at the leading edge compared with the cells expressingGFP alone (Fig. 7F-I).

GIT1 localizes PAK to focal adhesions and the leadingedge of lamellipodiaPAK, an effector of Rac, regulates migration through its effects

on the formation of protrusions. It also interacts with PIX, abinding partner of GIT1, and the active form localizes inadhesions and protrusions, suggesting an attractive mechanismfor the effects of GIT1 on migration. To determine whetherPAK mediates the effects of GIT1 on migration, we co-expressed GFP-GIT1 with a kinase-dead mutant of PAK inCHOK1 cells. When kinase-dead PAK was co-expressed withGIT1, the number and size of protrusions was dramaticallyreduced when compared with GIT1 alone (Fig. 8A,B). Whenthe number of protrusions was quantified, a three-fold decreasewas observed in cells expressing kinase-dead PAK and GIT1as compared with GIT1 alone (data not shown). These resultssuggest that the effects of GIT1 are mediated by PAK.

We then asked whether GIT1 was involved in the targetingof PAK, which has been reported to localize to adhesions andthe leading edge (Zhao et al., 2000). When expressedectopically, a constitutively active mutant of PAK localized toboth adhesions and the leading edge. This localization wassignificantly enhanced by co-expression with GIT1 (Fig. 8C-E). In cells co-expressing GIT1 and constitutively active PAK,approximately 90% of the cells showed prominent PAKlocalization to adhesions and the leading edge, whereas thisprominent localization was observed in only 30% of the cellsco-expressing active PAK and GFP. Wild-type PAK, when co-expressed with GIT1, also localized to adhesions and the


Fig. 8.GIT1 targets PAK to adhesions and themembrane at the leading edge of the lamellipodium.GFP-GIT1-expressing cells were co-transfected with(A) Myc tagged-kinase-dead or (C-E) constitutivelyactive PAK. Although a cell expressing (B) GFP-GIT1alone showed lamellipodia-like protrusions (arrows),they were significantly reduced when (A) kinase-deadPAK was co-expressed. (D) Constitutively active PAKlocalized prominently to adhesions and the leading edge(arrows) when co-expressed with (C) GIT1. (E) Cellswere co-stained for vinculin to verify localization ofadhesions. Enhanced localization of constitutivelyactive PAK to adhesions and the leading edge was notobserved when PAK was co-expressed with either (F-H) n-GIT1 or (I-K) mini-GIT1, indicating the need forboth the C- terminus of GIT1 and its central PIX-binding domain. (F-H) GFP-nGIT1 and (I-K) GFP-mini-GIT1 expressing cells were co-stained for(G,J) constitutively active PAK and (H,K) vinculin. Bar,20 µm.


leading edge, although not as prominently as the constitutivelyactive form. In addition, the kinase-dead form of PAK did notlocalize to adhesions or the leading edge irrespective of thepresence of ectopic GIT1. When PAK is co-expressed withnGIT1 (Fig. 8F-H) or mini-GIT1 (Fig. 8I-K), increased PAKlocalization to vinculin-containing adhesions or the leadingedge was not observed. This suggests that GIT1 targeting ofactive PAK to these regions is dependent on its interactionswith both paxillin and PIX.

Rac controls GIT1 localizationRac is another well known regulator of protrusion formationand migration (Hall, 1998) that might mediate the effects ofGIT1. Rac is regulated by PIX, a putative exchange factor thatalso binds to GIT1. Consonant with this hypothesis, we foundthat GIT1-induced protrusions were significantly reduced byco-expression of a dominant-negative mutant of Rac (N17-Rac) (Fig. 9). When cells were transfected with an activatedmutant of Rac, V12-Rac, we observed a dramatic increase inGIT1 localization to adhesions and to the edge of lamellipodiaand a decrease in the number of GIT1 cytoplasmic complexes

(Fig. 9). This effect could be seen in most cellsco-expressing GIT1, either constitutively ortransiently, and the observations with V12-Racwere especially obvious in CHO-K1 cellsbecause they do not normally form largesubstrate adhesions. When we expressed V12-Rac in parental CHO-K1 cells and probed forendogenous GIT1, we saw similar results, that is,the effect was detectable in greater than 90% ofthe transfected cells. Interestingly, an increase inthe number, and to a lesser extent, the size, ofvinculin containing adhesion was also observedin cells expressing V12-Rac (Fig. 9). Theseobservations suggest that the intracellulardistribution of GIT1 between cytoplasmicstructures and adhesions is regulated by Rac. Inaddition, Rac is necessary for the formation ofGIT1-induced protrusions. The localization ofnGIT1, a mutant lacking the paxillin-bindingdomain, in cytoplasmic complexes, but not inadhesions, was unaffected by V12-Rac,suggesting that the paxillin-binding domain wasessential for adhesion targeting of GIT1 by V12-Rac.

DiscussionGIT1 is rapidly emerging as a scaffolding proteinthat has multiple binding domains for severaldifferent molecules, including ARFs, PIX andFAK (Turner et al., 2001). Despite a growinginterest in the GIT family of proteins, its functionin integrating signaling pathways that control celladhesion and migration is not well defined.The goal of our study was to determine themechanisms by which GIT1 regulates thesecellular processes. Our results show that GIT1-containing cytoplasmic complexes can cyclebetween at least three distinct intracellular

compartments including adhesions, the membrane at theleading edge and cytoplasmic complexes. The central region ofGIT1 determines targeting to cytoplasmic complexes andinteraction with PAK, whereas the C-terminus, which binds topaxillin, is necessary for localization to adhesive complexesand the leading edge. These cytoplasmic structures function asmodules that deliver signaling molecules, such as PAK, toadhesions and the leading edge and as a pool of GIT1complexes derived from the breakdown of adhesions. GIT1localization appears to determine its functional effects. Forexample, GIT1 enhances migration and protrusive activitywhen it resides at the leading edge of the cell.

A GIT1-related protein, p95-APP1, has been reported to co-localize with the transferrin receptor and an endosomal marker,Lucifer yellow (DiCesare, 2000). In contrast, we were unableto detect significant co-localization of GIT1 complexes witheither transferrin or its receptor. Furthermore, ARF6 and α5integrin, which reside in endosomal vesicles, did not co-localize with GIT1 complexes. The experimental methods ofDiCesare et al. differed from ours in several respects. First,their study utilized conventional fluorescence microscopy,while we used deconvolution and confocal microscopy to

Fig. 9.Constitutively active Rac enhances GIT1 localization to adhesions anddecreases its presence in the cytoplasmic complexes. GFP-GIT1-expressing ornon-transfected CHOK1 cells were transfected with Myc-tagged V12-Rac or N17-Rac, fixed and stained with an anti-Myc antibody. Cells in the bottom panels wereco-stained for endogenous GIT1. V12-Rac induced strong localization of GFP-GIT1 (top two panels) and endogenous GIT1 (bottom two panels) in adhesions,dramatically diminishing its localization in cytoplasmic complexes. Unlike V12-Rac, N17-Rac (middle panels) did not have an effect. Bar, 20 µm.

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remove out of focus light. Second, p95-APP1 was expressedtransiently, while we selected stably expressing cells in whichectopic GIT1 levels were comparable to that of the endogenousprotein. However, a more likely explanation for thediscrepancy is that GIT1-related proteins can localizedifferently. This is supported by a study (Kondo et al., 2000)demonstrating that PAG3, a GIT1-related protein, localizes inthe Golgi, a site in which neither GIT1 nor p95-APP1 reside.These differences in location will probaby be critical to thefunctions of the GIT1-related proteins once they become moreapparent.

As our study indicated that the GIT1-containing cytoplasmicstructures were not vesicular, we hypothesized that GIT1 mightserve as a scaffold to bring together molecules to form asignaling module. Structural and signaling molecules,including FAK, vinculin, α-actinin, ARF1, AFR6, Rac and α5integrin, were not detected in the cytoplasmic GIT complexes.However, paxillin, PIX and PAK were present in thesecytoplasmic complexes. The localization of PAK with GIT1complexes and the enhanced adhesion targeting of thismolecule by GIT1 suggests that these cytoplasmic structuresmay target PAK to adhesions. Since PAK lacks an adhesion-targeting domain, its binding partners probably function in itsrecruitment to adhesions. The guanine nucleotide exchangefactor PIX could be involved in PAK localization to adhesion;alternatively, the adaptor protein Nck could function in thiscapacity (Sells et al., 2000; Bagrodia and Cerione, 1999;Bokoch et al., 1996). Our results show that PIX co-localizeswith GIT1 in the cytoplasmic structures; however, we did notobserve Nck in these complexes. In addition, a PAK mutantdeficient in PIX binding could not be detected in GIT1complexes. Thus, PIX most likely recruits PAK to thesesignaling complexes, which are targeted to adhesions and themembrane at the leading edge of the lamellipodia.

Our results show that expression of wild-type GIT1significantly increased migration and the rate of protrusionformation in CHOK1 cells. We speculated that GIT1 mediatesthese effects by targeting PAK to adhesions and the leadingedge of the lamellipodia. Active PAK may direct cellularmovement by regulating reorganization of the actincytoskeleton at the leading edge of the cell (Dharmawardhaneet al., 1997; Sells et al., 2000; Sells et al., 1997; Kiosses et al.,1999). Furthermore, PAK could regulate cell migration byaltering the disassembly of actin stress fibers and adhesions(Sells et al., 1997; Manser et al., 1997; Zhao et al., 2000).Consistent with our hypothesis, when kinase-dead PAK wasco-expressed with GIT1, the number and size of protrusionswas significantly reduced, suggesting that the effects of GIT1are dependent upon PAK.

Interestingly, cGIT1 promoted migration, whereas nGIT,which contained the ARF-GAP and PIX-binding domain, hadno effect. A previous study reported that the C-terminus ofGIT1 had no effect on cell movement; however, the C-terminusof GIT1, which was targeted to the membrane bymyristoylation, increased migration (Zhao et al., 2000). Thisimplies that membrane targeting of C-terminal GIT1 isnecessary to stimulate migration. By contrast, our studysuggests that expression of the C-terminus of GIT1, in theabsence of myristoylation, can promote migration. As cGIT1neither co-immunoprecipitated with PAK nor contains aputative PIX-PAK interaction domain, the enhanced migration

is probably not due to the targeting of PAK. To try to addressthe mechanism, we asked whether ectopic expression of cGIT1affects the distribution of endogenous GIT1. When cells weretransfected with cGIT1 and stained with the GIT1 antibody, weobserved increased localization of endogenous GIT1 at theleading edge of the lamellipodia. This result suggested thatcGIT1 competes with endogenous GIT1 for adhesion-bindingsites and promotes its localization to the leading edge of thecells.

GIT1 co-localized with paxillin in adhesions andcytoplasmic complexes. As small GTP-binding proteins, suchas ARF1, participate in recruitment of paxillin to adhesions(Norman et al., 1998), we asked whether GIT1 may beinvolved in cycling of paxillin between these twocompartments. Two members of the GIT family, PAG3/PAPαand GIT2-short, appear to mediate the subcellular localizationof paxillin to perinuclear areas through the ARF-GAP domain(Kondo et al., 2000; Mazaki et al., 2001). In our study, whenectopic GIT1 was expressed in CHO K1 cells, we did notobserve an increase in paxillin in adhesions. Rather GIT1decreased the localization of paxillin to adhesions andincreased its recruitment to the GIT1-containing cytoplasmiccomplexes. When we stained the GIT1-expressing cells withphospho-paxillin antibody, the active form of paxillin(tyrosine-phosphorylated) was detected in adhesions, but notin the cytoplasmic complexes. This suggests that GIT1complexes sequester paxillin in its inactive conformation in thecytoplasmic pool. Once paxillin is recruited to adhesions, itmay function to target GIT1 to this subcellular compartment.Consistent with our hypothesis, the kinetics of GIT1localization indicated that paxillin was recruited to adhesionsbefore GIT1 (R.-I.M. and A.F.H., unpublished). In addition,when paxillin was ectopically expressed, we observed anincrease in GIT1 in adhesions. Furthermore, the N-terminus ofGIT1 was not recruited to adhesions, indicating that thepaxillin-binding domain, but not the ARF-GAP domain, isnecessary for its localization.

The small GTPase Rac induces the formation oflamellipodia and initiates the development of focal complexesin this region (Rottner et al., 1999; Hall, 1998). In our study,GIT1-induced protrusions were decreased in cells expressingdominant-negative Rac. This indicates that the formation ofGIT1 protrusions is regulated by the Rac pathway. In cellsexpressing constitutively active Rac, we observed a significantincrease in GIT1 localization to adhesions and the leading edgeof the lamellipodia and a decrease in cytoplasmic structures.However, Rac did not localize to the GIT1 cytoplasmiccomplexes. Thus, Rac can regulate the intracellular distributionof GIT1, but its localization to the cytoplasmic complexes isnot necessary for the targeting of GIT1. As localization ofnGIT1 was unaffected by expression of constitutively activeRac, paxillin may be involved in Rac-induced localization ofGIT1 to adhesions.

On the basis of our observations, we propose the followingworking model. GIT1 complexes cycle between cytoplasmicpools, adhesions and the leading edge. The paxillin-bindingdomain is necessary for GIT1 localization to adhesions,suggesting that paxillin recruits GIT1 to these structures.Consistent with this, paxillin localizes to adhesions prior toGIT1 recruitment (R.-I.M. and A.F.H., unpublished). GIT1may then deliver PAK and PIX to adhesions and membrane



protrusions. At the leading edge, PAK can regulate actinpolymerization. Additionally, PAK targeting to adhesions mayregulate their turnover and thereby modulate cell migration.PIX, in this location, might also serve to activate Rac owing toits activity as an exchange factor. As adhesions break down atthe rear of a migrating cell, the GIT1 cytoplasmic complexesmove from the adhesions toward the cell body. These GIT1signaling modules are then available for targeting to adhesionsand the membrane at the leading edge of the lamellipodia. Inthis manner, GIT1, through its function as an adaptor protein,can regulate protrusive activity and cell migration. It hasrecently been shown that GIT1 is tyrosine phosphorylated inan adhesion-dependent manner (Bagrodia et al., 1999). It istempting to speculate that phosphorylation of GIT1 may serveas a regulatory mechanism, perhaps by altering theconformation of this protein, and thus affecting its subcellularlocalization and/or binding partners. Rac may also play a rolein regulating the distribution of this molecule among thecompartments.

This work was supported by NIH Grant GM23244 and theUniversity of Virginia Cancer Center. D.J.W. was supported byNational Institutes of Health postdoctoral training grant HD07528-01.We would like to thank Jonathan Chernoff and Chris Turner forreagents and suggestions. We are especially grateful to JamesCasanova for helpful discussion and advice. We would also like toextend our gratitude to Karen Donais for excellent help with thefigures.

ReferencesBagrodia, S. and Cerione, R. (1999). PAK to the future. Trends Cell Biol. 9,

350-355.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 familyof p21-activated kinase-binding proteins. J. Biol. Chem. 274, 22393-22400.

Bagrodia, S., Derijard, B., Davis, R. J. and Cerione, R. A.(1995). Cdc42and PAK-mediated signaling leads to Jun kinase and p38 mitogen-activatedprotein kinase activation. J. Biol. Chem. 270, 27995-27998.

Baron, M. D. and Garoff, H. (1990). Mannosidase-2 and the 135-kDa Golgi-specific antigen recognized monoclonal antibody 53FC3 are the samedimeric protein. J. Biol. Chem. 265, 19928-19931.

Bokoch, G. M., Wang, Y., Bohl, B. P., Sells, M. A., Quilliam, L. A. andKnaus, U. G. (1996). Interaction of the Nck adapter protein with p21-activated kinase (PAK1). J. Biol. Chem. 271, 25746-25749.

Daniels, H. and Bokoch, G. M. (1999). P21-activated protein kinase: a crucialcomponent in morphological signaling? Trends Biochem. Sci. 24, 350-355.

Dharmawardhane, S., Sanders, L. C., Martin, S. S., Daniels, R. H. andBokoch, G. M. (1997). Localization of p21-activated kinase (PAK1) topinocytic vesicles and cortical actin structures in stimulated cells. J. CellBiol. 138, 1265-1278.

DiCesare, A., Paris, S., Albertinazzi, C., Dariozzi, S., Andersen, J., Mann,M., Longhi, R. and DeCurtis, I. (2000). p95-APP1 links membranetransport to Rac-mediated reorganization of actin. Nat. Cell Biol. 2, 521-530.

Edwards, D. C., Sanders, L. C., Bokoch, G. M. and Gill, G. N.(1999).Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signaling toactin cytoskeletal dynamics. Nat. Cell Biol. 1, 253-259.

Fujii, G., Tsuchiya, R., Ezoe, E. and Hirohashi, S. (1999). Analysis ofnuclear localization signals using a green fluorescent protein-fusion proteinlibrary. Exp. Cell Res. 251, 299-306.

Garcia-Mata, R., Bebok, Z., Soscher, E. J. and Sztul, E. S. (1999).Characterization and dynamics of aggresome formation by a cytosolic GFP-chimera. J. Cell Biol. 146, 1239-1254.

Hall, A. (1998). Rho family GTPases and the actin cytoskeleton. Science279,509-514.

Kiosses, W. B., Daniels, H. R., Otey, C., Bokoch, G. M. and Schwartz, M.A. (1999). A role for p-21 activated kinase in endothelial cell migration. J.Cell Biol. 147, 831-843.

Kondo, A., Hashimoto, S., Yano, H., Nagayama, K., Mazaki, Y. andHisataka, S. (2000). A new paxillin-binding protein, PAG3/PAPα/KIAA0,bearing an ADP-ribosylation factor GTP-ase activating protein activity isinvolved in paxillin recruitment to focal adhesions and cell migration. Mol.Biol. Cell 11, 1315-1327.

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.

Lauffenburger, D. A. and Horwitz, A. F. (1996) Cell migration: a physicallyintegrated molecular process. Cell 84, 359-369.

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.

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.

Manabe, R., Kovalenko, M., Turner, C., Chernoff, J. and Horwitz, A. F.(2000). GIT1 targets active PAK to focal adhesions via interactions withpaxillin. Mol. Biol. Cell (Abstr.) 11, 183a.

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., Zhao, X. Q., Chen,L., Tan, I., Tan, T. L. and Lim, L. (1998). PAK kinases are directlycoupled to the PIX family of nucleotide exchange factors. Mol. Cell 1,183-192.

Mazaki, Y., Hashimoto, S., Okawa, K., Tsubouchi, A., Nakamura, K.,Yagi, R., Yano, H., Kondo, A., Iwamatsu, A., Mizoguchi, A. and Sabe,H. (2001). An ADP-ribosylation factor GTP-ase activating protein GIT2-short/KIA0148 in involved in subcellular localization of paxillin and actincytoskeletal organization. Mol. Biol. Cell12, 645-662.

Moss, J. and Lefkowitz, R. J.(1998). β2-Adrenergic receptor regulation byGIT1, a G-protein coupled receptor kinase-associated ADP ribosylationfactor GTPase activating protein. Proc. Natl. Acad. Sci. USA95, 14082-14087.

Moss, J. and Vaughan, M.(1998). Molecules in the ARF Orbit. J. Biol. Chem.273, 21431-21434.

Nobes, C. D. and Hall, A.(1995). Rho, Rac and Cdc42 GTPases regulate theassembly of multimolecular focal complexes associated with actin stressfibers, lamellipodia and filopodia. Cell 81, 53-62.

Norman, J. C., Jones, D., Barry, S. T., Holt, M. R., Cockroft, S. andCritchley, D. R. (1998). ARF1 mediates paxillin recruitment to focaladhesions and potentiates Rho-stimulated stress fiber formation in intact andpermeabilized Swiss 3T3 fibroblasts. J. Cell Biol. 143, 949-990.

Pagano, R. E., Martin, O. C., Kang, H. C. and Haugland, R. P.(1991). Anovel fluorescent ceramide analogue for studying membrane traffic inanimal cells: accumulation at the Golgi apparatus results in altered spectralproperties of the sphingolipid precursor. J. Cell Biol. 113, 1267-1279.

Palecek, S. P., Schmidt, C. E., Lauffenburger, D. A. and Horwitz, A. F.(1996). Integrin dynamics on the tail region of migrating fibroblasts. J. CellSci. 109, 941-952.

Premont, R. T., Claing, A., Vitale, N., Freeman, J. L. R., Pitcher, J. A.,Patton, W. A., Ridley, A. J. and Hall, A.(1992). The small GTP-bindingprotein Rho regulates the assembly of focal adhesions and actin stress fibersin response to growth factors. Cell 70, 389-399.

Rottner, K., Hall, A. and Small, J. V.(1999). Interplay between Rac and Rhoin the control of substrate contact dynamics. Curr. Biol. 9, 640-648.

Sanders, L. C., Matsumura, F., Bokoch, G. M. and de Lanerolle, P.(1999).Inhibition of myosin light chain kinase by p21-activated kinase. Science283,2083-2085.

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., Pfaff, A. and Chernoff, J. (2000). Temporal and spacialdistribution of activated PAK1 in fibroblasts. J. Cell Biol. 151, 449-458.

Thomas, S. and Leventhal, P. S. (1999). Paxillin LD4 motif binds PAK and

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PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: a role incytoskeletal remodeling. J. Cell Biol. 145, 851-863.

Turner, C. E. (2000). Paxillin and focal adhesion signaling. Nat. Cell Biol. 2,E231-E236.

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.

Turner, C. E., West, K. A. and Brown, M. C. (2001). Paxillin-ARF GAPsignaling and the cytoskeleton. Curr. Opin. Cell Biol. 13, 593-599.

West, K. A., Zhang, H., Brown, M. C., Nikolopoulos, S. N., Riedy, M. C.,Horwitz, A. F. and Turner, C. E. (2001). The LD4 motif of paxillinregulates cell spreading and motility through an interaction with paxillinkinase linker (PKL). J. Cell Biol. 154, 161-176.

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


git1 regulates protusive activity and cell migration · 2002. 3. 6. · al., 2000). furthermore,...

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