INTRODUCTION

Rac GTPases regulate actin dynamics at the edge of migratingcells (Ridley, 1996; Hall, 1998), and there is now increasingevidence for a role played by the endocytic cycle in extendingthe front of eukaryotic cells (Bretscher and Aguado-Velasco,1998b). Recycling transferrin receptors in migrating fibroblastshave been found distributed over the surface of leading lamella(Hopkins et al., 1994), and a contribution of membranerecycling from the endocytic pathway in the formation of Rac-induced ruffles has been described (Bretscher and Aguado-Velasco, 1998a). Moreover, Rho family GTPases have beendirectly implicated in endocytic trafficking, and in coordinatingactin dynamics with trafficking at the cell periphery (Lamazeet al., 1996; Murphy et al., 1996). Progress in this directioncomes from studies on ADP-ribosylation factor 6 (ARF6), amember of the ARF family of GTPases. ARF6 is specificallylocalized in the endosomal compartment and at the plasmamembrane, and has been implicated in the regulation ofmembrane traffic between these compartments. The functionof ARF6 on membrane recycling is supported by the effects of

its overexpression on transferrin uptake and recycling to thecell surface (D’Souza-Schorey et al., 1995; Peters et al., 1995),and by the overlap of the ARF6-positive intracellularcompartment with the transferrin receptor-positive recyclingcompartment (D’Souza-Schorey et al., 1998). A functionalconnection between ARF6 and Rac comes from theobservation that the egress of recycled membrane to peripheralsites stimulated by ARF6 results in a localized polymerizationof cortical actin, protrusive activity, and an apparentstimulation of membrane turnover by macropinocytosis(Radhakrishna et al., 1996; D’Souza-Schorey et al., 1997).This functional connection is further supported by thecolocalization of Rac1 and ARF6 at the plasma membrane andon recycling endosomes, and by the block of Rac1-stimulatedruffling by the GTP binding-defective N27-ARF6 mutant(Radhakrishna et al., 1999), which has led to the suggestionthat the influence of ARF6 on Rac1-mediated lamellipodiadepends in part on the regulation of its trafficking to the plasmamembrane.

It is still unclear how ARF6-mediated vesicle recycling isincorporated into the extension process. Recent findings on

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Cell migration requires coordination between adhesion,actin organization and membrane traffic. Rac and ARF6have been shown to cooperate for the organization of actinat the cell surface. Recently, the GIT family of ARF-GAPshas been identified, which includes proteins that canfunctionally interact with both ARF and Rac GTPases. Thep95-APP1 protein is a member of this family, isolated aspart of a multi-molecular complex interacting with GTP-Rac. Our previous work has indicated that this protein maybe part of the machinery redirecting membrane recyclingtowards sites of protrusion during locomotion. Byanalyzing the distribution and the effects of truncatedforms of p95-APP1, we show here that the lack of theARF-GAP domain of p95-APP1 dramatically shifts itslocalization to large vesicles. The use of several markers ofthe endocytic pathway has revealed that the truncatedp95-APP1 localizes specifically to a Rab11-, transferrinreceptor-positive compartment. Other markers are

excluded from the p95-APP1-positive vesicles, while knowncomponents of the multi-molecular complex colocalize withtruncated p95-APP1 in this compartment. Coexpressionof a constitutively active form of Rac induces theredistribution of the truncated constructs and of theassociated PIX, PAK, and paxillin to peripheral sites ofRac-mediated actin organization, and the disassembly ofthe large Rab11-positive vesicles. Together, the datapresented indicate that p95-APP1 is part of a complex thatshuttles between the plasma membrane and the endocyticrecycling compartment, and suggest that the dynamicredistribution of the p95-APP1-containing complex ismediated both by the ARF-GAP domain, and by therecruitment of the complex at the cell surface at sites of Racactivation.

Key words: Cell motility, Actin organization, Recycling endosomes,GTP-binding proteins

SUMMARY

Molecular mechanisms regulating the subcellularlocalization of p95-APP1 between the endosomalrecycling compartment and sites of actin organizationat the cell surfaceVittoria Matafora, Simona Paris, Simona Dariozzi and Ivan de Curtis*Cell Adhesion Unit, DIBIT, S. Raffaele Scientific Institute, Via Olgettina 58, 20132 Milano, Italy*Author for correspondence (e-mail: decurtis.ivan@hsr.it)

Accepted 10 September 2001Journal of Cell Science 114, 4509-4520 (2001) © The Company of Biologists Ltd

RESEARCH ARTICLE

4510

proteins that share an ARF-specific GTPase activating protein(ARF-GAP) domain suggest that they are involved in thecoordination between membrane trafficking and actinreorganization during cell locomotion (de Curtis, 2001). Oneof these proteins, p95-APP1, has been recently identified in ourlaboratory as part of a multi-protein complex (p95-complex)interacting with GTP-bound Rac GTPases (Di Cesare et al.,2000). P95-APP1 is a member of a recently discovered familyof multi-domain proteins including GIT1 (Premont et al.,1998), p95PKL (Turner et al., 1999), and CAT2/GIT2(Bagrodia et al., 1999). These proteins are characterized by thepresence of an amino-terminal ARF-GAP domain, and by theability to interact directly via a Spa2 homology domain (SHD)with the Rac/Cdc42 exchanging factor PIX (Zhao et al., 2000),and with the focal adhesion protein paxillin, which binds to acarboxy-terminal paxillin binding domain (Turner et al., 1999).Our previous study on p95-APP1 has shown that both wildtypeand truncated p95-APP1 induce actin-rich protrusionsmediated by Rac and ARF6 (Di Cesare et al., 2000). Inparticular, we found that p95-C, the C-terminal portion of p95-APP1 including the paxillin binding domain, localizes at sitesof actin reorganization at the plasma membrane, stronglypromoting the formation of actin-rich protrusions. By contrast,the N-terminal portion of p95-APP1, including the ARF-GAPdomain and the three ankyrin repeats, colocalizes with N27-ARF6 in an endosomal compartment. By further dissectionof this multi-domain protein, we found that the truncatedp95-C2, including both PIX- and paxillin-binding domains,accumulates via PIX around large vesicles. These vesicles aredistinct from the smaller endocytic structures where the N-terminal truncated polypeptides including the ARF-GAPdomain and the ankyrin repeats accumulate by a PIX-independent mechanism. Together, these observations have ledus to hypothesize that the p95 complex is implicated in theregulation of membrane recycling between endosomes and theplasma membrane, and it is required to organize new integrin-mediated adhesions at sites of protrusion.

In this study, we have further analyzed the requirementsfor the subcellular distribution of the p95-complex byusing distinct p95-APP1-derived constructs, and we havecharacterized the endocytic compartments where distincttruncated forms of this multi-domain protein localize.Moreover, we have identified some of the mechanisms thatmay be involved in the regulation of the cycling of the p95complex between the endocytic compartment and the plasmamembrane. Our results show that both the ARF-GAP domainand Rac activation regulate the subcellular distribution of thep95-complex.

MATERIALS AND METHODS

Antibodies, plasmids and other reagentsThe polyclonal antibodies against Rac1B (Albertinazzi et al., 1998),PIX (Daniels et al., 1999), ARF6 (Gaschet and Hsu, 1999), Rab11(Sonnichsen et al., 2000), Early endosome antigen 1 (EEA1)(Simonsen et al., 1998), and the 6C4 mAb against lysobisphosphatidicacid (LBPA) (Kobayashi et al., 1998) have been previously described.Other antibodies included the anti-Flag mAb M5 (Kodak, New Haven,CT), a pAb anti-Flag (Santa Cruz Biotechnology Inc., Santa Cruz,CA), the anti-Myc mAb 9E10 (Sigma-Aldrich, Milan, Italy), theanti-HA-Tag mAb 12CA5, the anti-paxillin mAb (Transduction

Laboratories), the mAb against LEP100 (Fambrough et al., 1988), andthe anti-transferrin receptor mAb (Zymed). Secondary antibodies forimmunofluorescence were from Boehringer and Jackson-ImmunoResearch Laboratories, platelet-derived growth factor(PDGF) was from Upstate Biotechnology.

The pFlag-p95, pFlag-p95-C, pFlag-p95-C2, pFlag-p95-N4, pFlag-N17-Rac1B, pFlag-V12-Rac1B and pcDNA-N27-ARF6 plasmidswere described elsewhere (Di Cesare et al., 2000). The cDNAfragments corresponding to p95-C3, p95-C5 and p95-N5 were clonedinto the pFlag-CMV-2 vector (Kodak), to obtain the plasmids pFlag-p95-C3, p95-C5 and pFlag-p95-N5, respectively. The pFlag-p95-K39plasmid was obtained by site-directed mutagenesis with theQuickChangeTM site-directed mutagenesis kit (Stratagene GmbH,Heidelberg, Germany), starting from the pFlag-p95 plasmid, andusing the primers 5′-AGTGCTGCAGCGTGCACAAGAGCCTGG-GCCGCCACAT-3′ and 5′-ATGTGGCGGCCCAGGCTCTTGTGCA-CGCTGCAGCACT-3′. The pXJ40-HA-βPIX plasmid coding for theHA-tagged β-PIX polypeptide, and the pCMV6m/Pak1 plasmidcoding for Myc-tagged Pak1 have been described previously (Manseret al., 1998; Bernard et al., 1999).

The Pfu DNA polymerase was from Stratagene, Klenow fragmentof DNA polymerase was from Amersham Pharmacia Biotech, andrestriction enzymes were from Boehringer. [α-35S]dATP, 125I-anti-mouse Ig, and 125I-protein A were from Amersham PharmaciaBiotech. Other chemicals and FITC- and TRITC-conjugatedphalloidin were from Sigma-Aldrich.

Cell culture and transfectionsChicken embryo fibroblasts (CEFs) obtained from embryonic day 10chicken embryos were prepared and cultured as described(Albertinazzi et al., 1998). For immunofluorescence, CEFs grown oncoverslips were transfected either with the Ca2+ phosphate techniqueor with Dosper (Boehringer) as described (Albertinazzi et al., 1998).For biochemical analysis, transient expression of proteins wasachieved by transfection of CEFs by the Ca2+ phosphate technique.Cells were used for biochemical or morphological analysis 24 hoursafter transfection. For stimulation with PDGF, COS7 cells were grownin DMEM with 10% serum, transfected with FuGENE (Roche)according to manufacturer’s procedures, starved for 3-9 hours inmedium without serum, and incubated with 50 ng/ml PDGF. Treatedcells were immediately fixed and analyzed for immunofluorescenceas described below.

Immunoprecipitation and immunoblottingTransfected and non-transfected cells were extracted with lysis buffer(0.5% Triton X-100, 150 mM NaCl, 20 mM Tris-Cl, pH 7.5, 1 mMsodium-orthovanadate, 10 mM sodium fluoride and 10 µg ml–1 eachof antipain, chymostatin, leupeptin and pepstatin). Extracts wereclarified by centrifugation. 200-300 µg of protein from lysates oftransfected cells were pre-cleared by incubating them for 2 hours withrotation at 4°C with 25 µl of Protein-A Sepharose beads (AmershamPharmacia Biotech). Beads were washed four times with 1 ml of lysisbuffer. Unbound material was added to Protein A-Sepharose beadswith pre-adsorbed antibodies, and incubated for 2 hours at 4°C withrotation. Pellets from immunoprecipitations were washed four timeswith 1 ml of lysis buffer, and analysed by SDS-PAGE andimmunoblotting with the indicated antibodies. Filters were thenincubated with 0.2 µCi/ml of either 125I-protein A or 125I-anti-mouseIg (Amersham Pharmacia Biotech), and exposed to Hyperfilm-MP(Amersham Pharmacia Biotech).

ImmunofluorescenceTransfected cells were fixed with 3% paraformaldehyde and processedfor indirect immunofluorescence, as described (Cattelino et al., 1995).Fixed cells were incubated for 1 hour at room temperature withprimary antibodies. Cells were subsequently incubated for 40 minuteswith fluorescently-labelled secondary antibodies. Fluorescently-

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4511Regulation of p95-APP1 localization

labelled phalloidin was added during the incubation with thesecondary antibody. Samples were observed using a Zeiss-Axiophotor Zeiss-Axiovert microscope equipped with a 63× oil immersionobjective, and a Hamamatsu C4742-98 camera (HamamatsuPhotonics K. K.). Fluorescent images were collected using the Image-Pro® Plus software package (Media Cybernetics, L. P.), and processedusing Adobe PhotoShop 5.0. For the quantitation of the cytoplasmicprotrusions present in transfected and non-transfected cells, onlycytoplasmic protrusions longer than half the major axis of the cellbody were considered. In each experiments, three sets of 100 cellswere examined for each condition. Each value represents the averagefrom counts obtained from three independent experiments. For eachexperimental condition, a total of about 900 cells were examined.

RESULTS

Characterization of the localization of the p95-complex in fibroblastsSeveral constructs derived from the multi-domain protein p95-APP1 were used in this study to analyze the function of p95-APP1 in fibroblasts (Fig. 1A). The p95-C2 polypeptideincludes the SHD PIX-binding domain and the paxillin-binding domain (Fig. 1A). The specific interaction of p95-C2with paxillin was shown here by coimmunoprecipitation fromCEFs transfected with pFlag-p95-C2 (Fig. 1B, lanes 1-6).Immunoprecipitation with the anti-Flag antibody was used toshow the interaction of p95-C2 with β-PIX from CEFscotransfected with both pFlag-P95-C2 and pXJ40-HA-βPIX(Fig. 1B, lanes 7-9). These data indicate that p95-C2 can bindto both paxillin and β-PIX in these cells. As recently shown byus (Di Cesare et al., 2000), p95-C2 induced the formation oflarge vesicular structures in transfected CEFs, where both p95-C2 and β-PIX are localized (Fig. 2F,G). Coexpression of p95-C2 enhanced the recruitment of β-PIX around thesestructures, which were usually not as evident in CEFstransfected with pXJ40-HA-βPIX only. In this lattercase, β-PIX had a diffuse localization, withconcentration at the cell periphery (Fig. 2H), althoughlocalization of β-PIX at large vesicles was alsodetectable (Fig. 2N). Given the interaction of PAK1with β-PIX, we looked at the localization of PAK1 incells transfected with either β-PIX or p95-C2. In cells

cotransfected with pFlag-P95-C2 and pCMV6m/Pak1, PAKcolocalized with p95-C2 at large vesicles (Fig. 2A-D), incontrast to cells transfected with pCMV6m/Pak1 only, wherePAK1 showed a diffuse staining (Fig. 2E). Moreover, in CEFstransfected with pXJ40-HA-βPIX and pCMV6m/Pak1, Pak1and β-PIX colocalized both in ruffles at the periphery of thecells, and at large cytoplasmic vesicles (Fig. 2I,J). The largePIX-induced vesicles were distinct from the endocyticstructures positive for p95-N4, the truncated polypeptideincluding the N-terminal ARF-GAP domain and the firstankyrin repeat of p95-APP1 (Fig. 2M-O). In cells coexpressingPAK and p95-N4 we found no evident colocalization of PAK1with p95-N4-positive structures (Fig. 2K,L). Together thesedata indicate that the p95-C2/β-PIX/PAK1 complex localizesat large vesicles.

Identification of the compartment induced by p95-C2expressionTo characterize the compartment induced by the expressionof p95-C2 in fibroblasts, we compared by indirectimmunofluorescence the distribution of p95-C2 with that ofendogenous markers for distinct endosomal compartments.These markers included the Rab5 effector EEA1, specific forearly Rab5-positive endosomes (Simonsen et al., 1998), Rab11 (Ullrich et al., 1996) and the transferrin receptor (Dunn etal., 1989; Mukherjee et al., 1997) as markers for the recyclingendosomal compartment, LBPA for the late endosomes(Kobayashi et al., 1998), and LEP100 for the lysosomes(Fambrough et al., 1988). Interestingly, we found that p95-C2colocalized specifically only with markers for the recyclingcompartment (Fig. 3F,G,I,J), while no detectable colocalizationwas observed with earlier (Fig. 3A-E) or late endocyticmarkers (Fig. 3L-Q). By comparison with the distribution of

Fig. 1. (A) P95-APP1-derived constructs used in this study.Zn, zinc finger; GAP, ARF-GAP domain; ANK, ankyrinrepeats; SHD, Spa2 homology 1 domain; COIL, coiled-coilregion; PBS, paxillin binding subdomain; K39 indicates theamino acid substitution in the ARF-GAP domain of thep95-K39 construct. (B) Coimmunoprecipitation of p95-C2with paxillin and PIX. Aliquots of a lysate from CEFstransfected with pFlag-p95-C2 (lanes 1-6) wereimmunoprecipitated with the mAb anti-Flag and blottedwith anti-Flag to detect p95-C2 (lanes 1-3) or with anti-paxillin (lanes 4-6). Lysate from CEFs cotransfected withpFlag-p95-C2 and pXJ40-HA-βPIX (lanes 7-9) wereimmunoprecipitated with the anti-Flag mAb.Immunoprecipitates were blotted with the anti-Flag mAb todetect p95-C2 (bottom panel) and with the anti-HA mAb todetect the coprecipitating β-PIX polypeptide (top panel).Pre, beads from pre-clearing; IP, immunoprecipitates; nb,material not bound to beads. Molecular weight markers areindicated to the left of panels.

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Rab11 (Fig. 3H) and of the transferrin receptor (Fig. 3K)in non-transfected cells, it was evident that p95-C2expression induced an alteration of the morphologyof the recycling compartment. Therefore, the largevesicles observed by p95-C2 expression correspond tomorphologically altered recycling compartment.

We then compared the distribution of the markers of theendocytic pathway with the endocytic structures identifiedby the expression of the N-terminal fragment p95-N4 (Fig.1A). No overlap was detected between the small scatteredstructures positive for p95-N4 and the early EEA1-positivecompartment (Fig. 4A-C), nor with the later LBPA-positive compartment (Fig. 4L-N), or the LEP100-positivelysosomes (Fig. 4O-Q). In contrast to p95-C2, no obviousoverlap was visible between p95-N4 and the endogenous

transferrin receptor (Fig. 4J-K). In the case ofRab11, colocalization with p95-N4 was sporadic(Fig. 4D-I), indicating that the p95-N4-positivecompartment is largely distinct from Rab11-positive recycling endosomes. Comparison withnon-transfected cells showed that p95-C2 andp95-N4 did not alter the localization of EEA1,LEP100 or LBPA (not shown). Together, ourobservations suggest that p95-N4 and p95-C2accumulate at distinct intracellular locations,which may correspond to different stages of theendocytic cycle.

To look at the connection between the enlarged,Rab11-positive structures and ARF6, we have

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Fig. 2.Subcellular localization of p95-C2, β-PIX, PAK1 andp95-N4. (A-J) Localization of p95-C2, β-PIX and PAK1 atlarge vesicles. Cells were cotransfected with pFlag-p95-C2(A,C) and pCMV6m/Pak1 (B,D), or with pFlag-p95-C2 (F) andpXJ40-HA-βPIX (G). (E) Cell transfected with CMV6m/Pak1only. (H) Cell transfected with pXJ40-HA-βPIX only. In (I,J)cells were cotransfected with pXJ40-HA-βPIX (I) andpCMV6m/Pak1 (J). (K-O) Localization of p95-N4 at smallvesicles. Cells were cotransfected with pFlag-p95-N4 (K) andpCMV6m/Pak1 (L), and with pFlag-p95-N4 (M) and pXJ40-HA-βPIX (N). In (O), merged signals for p95-N4 (shown inred) and β-PIX (shown in green) are shown. Cells were fixedone day after transfection and processed for indirectimmunofluorescence as described in Materials and Methods.Bar, 10 µm.

Fig. 3.Specific colocalization of p95-C2 with markersof the endocytic recycling compartment. In cellstransfected with pFlag-p95-C2 the distribution of thep95-C2 polypeptide was compared with thedistribution of the early endocytic marker EEA1 (A-E), with the markers for the recycling compartmentRab11 (F,G) and transferrin receptor (I,J), with themarker for late endosomal compartment LBPA (L-N),and with the lysosomal marker LEP100 (O-Q). H andK show the distribution in non-transfected cells ofRab11 and transferrin receptor, respectively. Cellswere fixed and processed for indirectimmunofluorescence one day after transfection. Bars,10 µm. In (C), p95-C2 is shown in red, EEA1 is shownin green. In (N) and (Q), p95-C2 is shown in green,endocytic markers are shown in red.

4513Regulation of p95-APP1 localization

compared the distribution of the N27-ARF6mutant, known to localize into intracellularendosomal structures, with the distribution ofdistinct p95 mutants. We found that N27-ARF6 largely overlapped with p95-N4 (Fig.5A,B), the N-terminal portion of p95including the ARF-GAP domain and the firstankyrin repeat, which only sporadicallycolocalized with Rab11 (Fig. 4D-I). N27-ARF6 localized also at large p95-C2-positivevesicles (Fig. 5C,D), while in cells withsmaller p95-C2-positive vesicles, N27-ARF6converged into distinct elongated structures,where no detectable p95-C2 was observed(Fig. 5E-G). These results indicate that theexpression of p95-C2 protein influenced boththe Rab11- and ARF6-positive endocyticcompartments, and led to coalescence of thetwo compartments once large vesicles have formed as aconsequence of p95-C2 expression. Given the proposed role ofboth ARF6 and Rab11 into membrane recycling, these datapoint to a functional connection between these two types ofGTP-binding proteins during vesicular transport to the cellmembrane.

Alteration of the ARF-GAP domain of p95-APP1affects the intracellular distribution of thepolypeptideThe finding that ablation of the N-terminal portion of p95-

APP1 induced the redistribution of the resulting p95-C2protein to large endocytic vesicles suggests that the ARF-GAPdomain of p95-APP1 regulates the intracellular distribution ofthe protein. To test this hypothesis we looked at the effects ofthe expression of the p95-C3 polypeptide (Fig. 1A) in whichjust the ARF-GAP domain was deleted. As for p95-C2, theexpression of p95-C3 induced the formation of large vacuolesin most transfected cells, which often appeared as clusters ofcoalescent vesicles (Fig. 6Aa). In some cases these vesiclescovered a large area of the cytoplasm (Fig. 6Ac). As for p95-C2, both PAK (Fig. 6Ae-f) and β-PIX (Fig. 6Ag) colocalized

around the large vesicles with p95-C3 (notshown). The characterization of the largevesicles indicated that the p95-C3-labelledstructures corresponded to a transferrin receptor-positive (not shown), Rab11-positive (Fig. 6Ah-i) endosomal compartment. The accumulation ofp95-C3 around this compartment was specific,

Fig. 4.Comparison of the distribution of p95-N4with markers of the endocytic compartment. Incells transfected with pFlag-p95-N4 thedistribution of the p95-N4 polypeptide wascompared with the distribution of the earlyendocytic marker EEA1 (A-C), with the recyclingendosomal compartment markers Rab11 (D-I) andtransferrin receptor (J, K), with the marker for lateendosomal compartment LBPA (L-N), and withthe lysosomal marker LEP100 (O-Q). Bars, 10 µm(A-F,J-Q); 5 µm (G-I). In C, p95-N4 is shown inred and EEA1 is shown in green. In F and I, p95-N4 is shown in green, and Rab11 is shown in red.In N and Q, p95-N4 is shown in green, andendocytic markers are shown in red.

Fig. 5. Comparison of the distribution of N27-Arf6with that of distinct p95-APP1-derived polypeptides.Cells were cotransfected with pFlag-p95-N4 (A) andpcDNA-N27-ARF6 (B), with pFlag-p95-C2 (C,E)and pcDNA-N27-ARF6 (D,F). One day aftertransfection cells were fixed and processed forindirect immunofluorescence. In G the mergedsignals of p95-C2 (red) and N27-ARF6 (green) areshown. Bars, 10 µm.

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Fig. 6.Characterization of the effects of p95-C3 andp95-K39 expression in fibroblasts. (A) Cellstransfected with pFlag-p95-C3 were stained for p95-C3 (a,c) and F-actin (b,d). The same cells are shownin a,b and in c,d, respectively. Cells cotransfectedwith pFlag-p95-C3 and pCMV6m/Pak1 (e,f), andwith pFlag-p95-C3 and pXJ40-HA-βPIX (g) wereimmunostained to detect p95-C3 (e), PAK1 (f) andβ-PIX (g). Colocalization of p95-C3 (h) with Rab11(i). In (j), the distribution of p95-C3 (green) and thatof EEA1 (red) are compared. Bars, 10 µm.(B) Identification of the β-PIX/p95-K39/paxillincomplex. Fibroblasts cotransfected with pFlag-p95-K39 and pXJ40-HA-βPIX were lysed and incubatedfirst with control Protein-A-Sepharose beads (leftlane), and then with beads coated with the anti-FlagmAb (right lane). After SDS-PAGE and blotting,filters were incubated with anti-Flag mAb (top),anti-PIX pAb (middle), and anti-paxillin mAb(bottom). Molecular weight markers are indicated tothe left of the blot. (C) Cells transfected with pFlag-p95-K39 were analyzed for the distribution of p95-K39 (a), and F-actin (b); c shows the localization β-PIX at large vesicles in a cell cotransfected withpFlag-p95-K39 and pXJ40-HA-βPIX; d shows thelocalization at large vesicles of PAK in a cellcotransfected with pFlag-p95-K39 andpCMV6m/Pak1. Bars, 10 µm.

Fig. 7.Effects of p95-C, p95-N5, andp95-C5 on cell morphology. Fibroblaststransfected with pFlag-p95-C, pFlag-p95-N5, or pFlag-p95-C5 were used forimmunofluorescence. (A,B) The effectsof each construct on the protrusiveactivity of the transfected cells wasquantified as described in Materials andMethods, and compared to the protrusiveactivity of non-transfected cells(Control). (A) Percentage of cells withprotrusions. (B) Number of protrusionsper cell. For each value, threeindependent experiments were examined;bars represent the s.e.m. (C) Cells weretransfected with pFlag-p95-N5 (a,c,f,h,j),with pFlag-p95-C (b), or with pFlag-p95-C5 (l) and treated forimmunofluorescence one day aftertransfection. The distribution of p95-N5was compared with the distribution ofendogenous Rab11 (d,i,k), and ofendogenous EEA1 (g). In (e) the signalfor p95-N5 is shown in green, Rab11 isshown in red. Bars, 10 µm (a-i,l,m); 5 µm(j,k). Arrows point to corresponding areasof the cytoplasm in j and k.

4515Regulation of p95-APP1 localization

since the subcellular distribution of both theearly endocytic marker EEA1 (Fig. 6Aj), andof the late endosomal marker LBPA (notshown) did not show any evident overlap withthe p95-C3-positive structures.

All proteins known to have ARF-GAPactivity contain an arginine five residues afterthe fourth cysteine constituting the zinc fingerwhich is critical for ARF-GAP activity.ASAP1, PAP, ARF-GAP1 and ACAPscontaining a mutation in this residue, havelittle or no detectable GAP activity (Mandiyanet al., 1999; Randazzo et al., 2000; Szafer etal., 2000; Jackson et al., 2000). This argininecorresponds to Arg-39 in p95-APP1. To testwhether the effects observed upon p95-C3expression could be attributed to inactivationof the ARF-GAP activity, and not to theeffects of the ablation of a portion of theprotein, we expressed the epitope-tagged p95-K39 protein, in which Arg-39 was substituted by a lysine. Thep95-K39 protein was still able to interact with β-PIX andpaxillin, as shown by coimmunoprecipitation from transfectedfibroblasts (Fig. 6B), and induced a phenotype very similar tothat obtained with the p95-C3 protein in a fraction of thetransfected cells (Fig. 6C). In fact, several transfected cellsshowed the localization of p95-K39 at large vesicles (Fig.6Ca), which were Rab11-positive (not shown). Both PIX (Fig.6Cc) and PAK (Fig. 6Cd) colocalized with p95-K39 around thelarge vesicles, indicating that the p95-K39/β-PIX/PAKcomplex was concentrated at this morphologically alteredrecycling compartment. Together, these data implicate theARF-GAP domain of p95-APP1 in the regulation of thesubcellular distribution of the p95-APP1/β-PIX/PAK complex.

Role of p95-APP1 in paxillin localizationWe have previously reported that the expression of the p95-Cpolypeptide, including the C-terminal paxillin-binding domain,but not the SHD PIX-binding domain (Fig. 1A) inducesprotrusions in fibroblasts (Di Cesare et al., 2000). This effectis accompanied by the redistribution of paxillin away fromfocal adhesions, which are still detectable by using anti-integrin antibodies (Di Cesare et al., 2000). To test therequirement of paxillin-binding for enhanced protrusiveactivity, we have prepared the pFlag-p95-N5 plasmid codingfor the p95-N5 polypeptide, in which the C-terminal paxillin-binding domain was deleted (Fig. 1A). In transfected cells,p95-N5 was often observed associated to cytoplasmic vesicles

(Fig. 7Ca), not visible in cells transfected with p95-C (Fig.7Cb). In cells transfected with p95-N5 the morphology of theRab11 compartment was clearly affected (Fig.7Ch,i), althoughthe colocalization of Rab11 with p95-N5-positive vesicles wasonly partial, and not as striking as in cells transfected with theARF-GAP mutants. In several cases, rather than a completecolocalization of the two proteins around the vesicles (Fig.7Cc-e), several Rab11-positive spots were observed around thelarge p95-N5 positive vesicles (Fig. 7Cj,k). However, stainingwith anti-EEA1 antibodies indicated lack of colocalization ofp95-N5 with EEA1-positive vesicles (Fig. 7Cf,g). In general,cells expressing p95-N5 had less prominent protrusionscompared with cells expressing p95-C (Fig. 7Ca,b,respectively). Quantitation of the percentage of cells withprotrusions (Fig. 7A), and of the number of protrusions per cell(Fig. 7B) indicated a significant decrease in the protrusiveactivity of p95-N5-transfected cells, thus implicating therequirement of paxillin binding for the stimulation of theprotrusive activity. Similar findings were observed by theexpression of the p95-C5 mutant (Fig. 1A), derived from p95-C from which the paxillin-binding region had been deleted(Fig. 7Cl,m): also in this case no enhancement of protrusiveactivity could be observed in the transfected cells (Fig. 7A,B).

We therefore analyzed the effects of the p95-APP1-derivedconstructs on paxillin distribution (Fig. 8). As previouslyshown by us, p95-C induced the redistribution of paxillin fromfocal adhesion into a diffuse cytoplasmic staining withconcentration at protrusions (Fig. 8A,B). By contrast, we

Fig. 8.Distribution of paxillin in fibroblastsexpressing distinct p95-APP1-derivedpolypeptides. Cells expressing the indicatedconstructs were treated for immunofluorescenceone day after transfection. P95-APP1-derivedconstructs were detected with an anti-Flagantibody (A,C,E,G,I), β-PIX was detected with theanti-PIX pAb (L), and paxillin was detected withan anti-paxillin mAb (B,D,F,H,J,K,M). In K, at thecenter of the field the distribution of paxillin in acell transfected with p95-K39 is shown. The samefield is shown in (A,B), (C,D), (E,F), (G,H), (I,J),and (L,M). Bars, 10 µm.

4516

observed no effects on paxillindistribution by p95-N5 (Fig. 8E,F). Inparticular, paxillin could not be detectedaround the p95-N5-positive vesiclesobserved in the transfected cells. Asexpected, the expression of the p95-C5mutant did not alter the distribution ofpaxillin, which remained in focaladhesions (Fig. 8G,H). However, asalready shown for p95-C2 (Di Cesare etal., 2000), both p95-C3 (Fig. 8C,D) andp95-K39 (Fig. 8I-K) could induce adramatic redistribution of paxillin fromfocal adhesions to the large p95-positivevesicles. In these cells, paxillin washardly detectable in focal adhesionswhen compared with the neighbouring non-transfected cells.In the case of β-PIX overexpression, an intermediatephenotype could be observed (Fig. 8L,M). In fact, somepaxillin recruitment together with PIX could be observed in thecells showing PIX-positive vesicles, although a clear signal ofpaxillin in focal adhesions could still be detected.

These data show that p95-APP1 is able to relocate paxillinwithin the cell, and that the ability of the different p95-derivedconstructs to do so correlates with the presence of the C-terminal paxillin-binding site.

Rac activation affects p95-C2 localizationThe p95-APP1 protein was first identified by us as an indirectinteractor of activated Rac GTPases. To test the hypothesis thatactivation of Rac at the cell surface may regulate thedistribution of p95-APP1 within the cell, we coexpressed thetruncated p95-C2 protein with constitutively activated Rac. Incontrast to the strong concentration of p95-C2 at largerecycling vesicles observed in cells transfected with pFlag-P95-C2 only (Fig. 9A), p95-C2 was found redistributed tothe cell periphery in cotransfected cells (Fig. 9B). Thecotransfected cells showed the appearance of large

lamellipodia (Fig. 9B,C,E) when compared with cellsexpressing p95-C2 alone, or with cells coexpressing p95-C2and an inactive form of the GTPase (Fig. 9D,F). P95-C2 wasoften found concentrated with V12-Rac at the edge oflamellipodia (Fig. 9C,E). Interestingly, N17Rac colocalizedwith p95-C2 at the large intracellular vesicles (Fig. 9D,F).Similar findings were obtained by coexpressing p95-C3 andV12Rac. In contrast to the concentration of the p95-C3 proteinat large intracellular vesicles (Fig. 6A), in the cotransfectedcells, which had evident, large lamellipodia, the localization ofp95-C3 was often diffuse, with some concentration atperipheral areas together with V12Rac (Fig. 9G,H). Fewerlarge vesicles were still evident only in some cotransfectedcells (Fig. 9I). In these cases, colocalization at the largevesicles of p95-C3 and V12Rac was obvious. Interestingly,paxillin also co-distributed with p95-C2 and p95-C3 at the cellperiphery in cells co-expressing V12Rac (Fig. 9L-O).Similarly, both PAK (Fig. 9R,S) and PIX (Fig. 9T,U) co-distributed with p95-C3 and p95-C2 at the cell edge in cellsexpressing V12Rac. Under these conditions, Rab11 showed adiffuse punctuate pattern (Fig. 9P,Q).

A similar colocalization with V12Rac at the edge of

JOURNAL OF CELL SCIENCE 114 (24)

Fig. 9.Relocalization of p95-C2, p95-C3,paxillin, PIX, and PAK to the cell peripheryby activated Rac. Fibroblasts weretransfected or cotransfected with thefollowing plasmids: pFlag-p95-C2 (A);pFlag-p95-C2 and pFlag-V12-Rac1B(B,C,E,L,M,P,Q); pFlag-p95-C2 and pFlag-N17-Rac1B (D,F); pFlag-p95-C3 and pFlag-V12-Rac1B (G-I,N,O); pFlag-p95-N4 andpFlag-V12-Rac1B (J,K); pFlag-p95-C3,pCMV6m/Pak1, and pFlag-V12-Rac1B(R,S); pFlag-p95-C3, pXJ40-HA-βPIX, andpFlag-V12-Rac1B (T,U). Cells were fixedand processed for indirectimmunofluorescence one day aftertransfection. The same field is shown in(C,E), (D,F), (G,H), (J,K), (L,M), (N,O),(P,Q), (R,S), and (T,U). Bars, 10 µm. V12-Rac induces the localization of both p95-C2(B,C,E) and p95-C3 (G,H) at the cellperiphery, together with paxillin (L-O), Pak(R, S), and Pix (T,U). V12-Rac does notinfluence the localization of p95-N4 (J,K).

4517Regulation of p95-APP1 localization

cotransfected cells was also observed with full length p95-APP1 and with p95-N5 (not shown). by contrast, V12Rac wasunable to induce the redistribution at the cell periphery of theN-terminal p95-N4 polypeptide, which lacks the PIX-bindingdomain (Fig. 9J,K), thus implicating the PIX/PAK complexin the recruitment of p95-APP1 by V12Rac. Moreover, thecoexpression of p95-N4 did not affect V12Rac-inducedruffling or lamellipodia formation. These data indicate that thep95-APP1/PIX/PAK complex may undergo redistribution atsites of Rac activation.

To test whether the activation of endogenous Rac is ableto induce the recruitment of ARF-GAP-deficient p95polypeptides at the cell periphery, we have looked at thedistribution of p95-C2 in COS7 cells treated with PDGF. Intransfected cells we found that p95-C2 localized at largevesicles (Fig. 10A). In cells treated with PDGF (Fig. 10B-E),as well as in a fraction of untreated cells, in which ruffles werestill evident (not shown), p95-C2 co-localized with actin atruffles and lamellipodia. This result indicates that p95-C2 maylocalize to sites of endogenous Rac activation.

DISCUSSION

We have identified the Rab11-positive recycling compartmentas the endocytic compartment where p95-derived polypeptideslocalize upon expression in fibroblasts. Accumulation of p95-APP1 and of other components of the p95 complex in thiscompartment depends on the deletion or mutation of the ARF-GAP domain of p95-APP1. We have also demonstrated thatRac activation induces the localization of the p95 complex atthe periphery of the cell. Together, our results suggest that p95-APP1 is implicated in the recycling of endocytosed membranesto the cell surface, at sites of Rac-mediated protrusive activity,

and implicate the ARF-GAP domain of p95-APP1, paxillin,and Rac activation in the regulation of the subcellularlocalization of the p95-complex.

The C-terminal p95-C fragment of p95-APP1 induces theformation of protrusions, while this effect is not observed inp95-C2-expressing cells (Di Cesare et al., 2000). While p95-Cis evidently localized at sites of protrusion, p95-C2 ispredominantly concentrated at large intracellular vesicles.These striking differences originate from the presence (in p95-C2) or absence in (p95-C) of the SHD domain. As for GIT1(Zhao et al., 2000), we found that the SHD domain is requiredfor the interaction of p95-APP1 with PIX, a putativeexchanging factor for Rac and Cdc42 (Oh et al., 1997;Bagrodia et al., 1998; Manser et al., 1998). Our findingssupport a role of PIX in the localization of the p95-complex toRab11-positive large vesicles. Rab11 is a small GTP-bindingprotein required for transferrin recycling through thepericentriolar recycling endosomes (Ullrich et al., 1996; Renet al., 1998), and represents a functional marker for thiscompartment. The large Rab11-positive vesicles seem to formspecifically as a consequence of PIX-mediated accumulationof p95-APP1 in the endosomal compartment. The specificityis supported by the observation that only markers for theendosomal recycling compartment accumulate at thesevesicles, while earlier and later endosomal compartments arenot affected. The deletion (p95-C3) or mutation (p95-K39) ofthe ARF-GAP domain also induce accumulation to Rab11-

Fig. 10.Localization of p95-C2 at the cell periphery. COS7 cellstransfected for 20 hours with pFlag-p95-C2 were incubated for 3hours without serum, before incubation for 10 minutes with mediumcontaining 50 ng/ml of PDGF (B-E). After fixation, cells wereimmunostained for p95-C2 (A,B,D), and incubated withfluorescently-labelled phalloidin (C,E). Same fields are shown inB,C, and in D,E. Bar, 10µm.

PM

GTP-Rac

p95-APP1

paxillin

PIX

PAKR.E.

GAP

Ank

(Rab11)

Arf

Fig. 11.Model for the role of the p95 complex in membranerecycling from the endocytic compartment. P95-APP1 interacts withPIX which in turn interacts with PAK. Moreover, the C-terminalportion of p95-APP1 can interact with paxillin, and induce paxillinrelocalization away from focal adhesions. Both PIX and the ankyrinrepeats of p95-APP1 may induce recruitment of the p95 complex tothe endosomal compartment. In particular, PIX is required for therecruitment to Rab11-positive recycling endosomes (R.E.).Internalized membrane can normally be recycled back to the plasmamembrane (PM) from the Rab11-positive recycling compartment.According to this model, the lack of a functional ARF-GAP domainmay interfere with membrane recycling by preventing ARF-mediatedvesicle budding from the recycling compartment, thus inducing anabnormal accumulation of large Rab11-positive vesicles. By contrast,activation of Rac at the cell surface may induce translocation of thep95 complex, and possibly of recycling vesicles, to the cell surfacevia the interaction with the Rac effector PAK. This would result inthe transport to the cell periphery of new focal adhesion components(paxillin) and of part of the machinery required for Rac-mediatedactin assembly (PAK, PIX). See text for more details.

4518

positive structures, showing that the alteration of the ARF-GAP domain of p95-APP1 is responsible for the formation ofthe abnormal recycling compartment. In vitro ARF-GAPactivity of the highly homologous GIT1 on ARF6 has beenrecently demonstrated (Vitale et al., 2000). The stronghomology between avian p95-APP1 and mammalian GIT1,and the colocalization of the N-terminal portion of p95-APP1with N27-ARF6 in the endocytic compartment suggest that thisprotein is a candidate ARF6 regulator in vivo. The fact that wehave not been able to detect any ARF-GAP activity of p95-APP1 on ARF6 in vitro indicates that the ARF-GAP activityof p95-APP1 may be finely regulated in the cell. The conservedarginine corresponding to arginine 39 of p95-APP1dramatically reduces the ARF-GAP activity of a number ofARF-GAPs in vitro (Mandiyan et al., 1999; Randazzo et al.,2000; Szafer et al., 2000; Jackson et al., 2000). We would liketo speculate that like ARF1 in the Golgi (Roth, 1999), ARF6regulates vesicle formation during recycling betweenendosomes and the plasma membrane. According to our model(Fig. 11), the effects of the ARF-GAP mutants on the Rab11-recycling compartment is due to the inability of the mutatedp95-APP1 to induce GTP hydrolysis, necessary for ARF-mediated vesicle budding from recycling endosomes. Theoverexpressed ARF-GAP mutants, by interacting via PIX withthe endosomes, would compete with the endogenous p95-APP1, and act as dominant negative forms, inhibiting p95-APP1 function in vesicle formation. The abnormal recyclingcompartment would be generated by the accumulation ofinternalized membranes, not compensated by membranerecycling in cells overexpressing the ARF-GAP mutants.

The p95-C construct missing the PIX-binding domaininduces depletion of paxillin from focal adhesions, and itsaccumulation at the cell periphery, where it enhancesprotrusive activity. Polypeptides with further deletion of thepaxillin binding region (p95-C5) do not influence theprotrusive activity, nor paxillin distribution. By contrast,constructs including the PIX-binding region and the paxillinbinding region induce a striking relocalization of paxillin, PIXand PAK1 to the Rab11-positive large vesicles. These datasupport a model according to which, paxillin, once removedfrom focal adhesions, travels with the p95-complex, via therecycling compartment, to the cell front, where it participatesin the formation of new focal complexes (Fig. 11).

The absence of the PIX-binding region from the N-terminalp95-N4 construct leads to accumulation of this protein in adistinct population of endocytic vesicles. In contrast to thelarge Rab11-positive vesicles, the small p95-N4-positivevesicles are only weakly overlapping with the population ofvesicles stained with anti-Rab11 antibodies. This localizationof p95-N4 is mediated by the first ankyrin repeat (Di Cesare etal., 2000), and is independent of the SHD-mediatedlocalization of p95 to the large vesicles. We have previouslyshown that p95-N4 and N27-ARF6 colocalize in these vesicles.Further work will be required to understand whether the twoidentified structures correspond to two altered, functionallydistinct endosomal subcompartments. This hypothesis issupported by the finding that PIX and the transferrin receptorare excluded from the small p95-N4-positive vesicles, whileboth are present into the large Rab11-positive vesicles. Thetransferrin receptor cycles between the plasma membrane andthe recycling endosomes, and can be induced to accumulate

intracellularly by ligand internalization (Marsh et al., 1995). Innon-transfected CEFs, a clear surface distribution of thetransferrin receptor is observed. The concentration of thereceptor in the large Rab11-positive vesicles in cellstransfected with p95-C2 or p95-C3 indicates that recycling tothe cell surface is altered in these cells, leading to accumulationinto an abnormally enlarged recycling compartment.

The accumulation of p95-N4 in the endosomal compartmentmay be explained by the inability of the truncated N-terminalprotein to be relocated to the plasma membrane, and implicatesthe C-terminal portion of p95-APP1 in targeting to the plasmamembrane. One possible mechanism for targeting the p95-complex to the plasma membrane is Rac activation. In fact,overexpression of a constitutively active form of Rac inducesaccumulation of p95-C3 and p95-C2 to the plasma membranetogether with Rac, while activated Rac does not affect thedistribution of p95-N4. Therefore, PIX binding is necessary forRac-mediated localization of the p95 complex to the cellmembrane. Since PIX binds to PAK1, the localization of thep95-complex at the plasma membrane may be mediated by thebinding of PAK1 to Rac (Fig. 11), although we cannot excludethe direct interaction between PIX and Rac. One couldenvisage that the amount of the p95-complex recruited at theplasma membrane would be dependent on the amount of activeRac at the cell surface. Under steady state conditions, theamount of endogenous GTP-Rac would not be sufficient torecruit efficiently the overexpressed p95-C2-complex to thecell surface. The p95-C2-complex would then accumulate atRab11 endosomes via PIX. However, overexpressed V12Racwould be able to recruit a significant amount of the complexto the plasma membrane by interacting with PAK, bycompeting with the PIX-mediated binding of the complex toRab11 endosomes. This idea is supported by the finding ofp95-C2 at sites of endogenous Rac-mediated actin organizationat the cell periphery. The results of the expression of p95-N5and p95-C5 proteins have revealed the importance of the C-terminal paxillin binding domain on protrusive activity andpaxillin relocalization, and argue in favour of a contribution ofpaxillin to the localization of the p95 complex at the cellperiphery. In fact, the lack of the paxillin binding site leavespaxillin predominantly in focal adhesions in cells transfectedwith p95-N5. The p95-N5 with an intact ARF-GAP is affectingthe morphology of the Rab11 compartment, although the p95-N5 protein localizes to vesicles which only partially overlapwith the Rab11 compartment. Our findings imply a complexregulation of the subcellular distribution and trafficking of thep95 complex between endosomes and plasma membrane,which implicate in the process not only a functional ARF-GAPdomain, but also the PIX/PAK complex and the focal adhesionprotein paxillin. The lack of the paxillin binding site couldaffect the distribution of the protein, resulting in its partialassociation to a vesicular compartment that may correspond toa distinct intermediate during the recycling process. Futurework on the complex network of events involved will help tofurther elucidate the proposed model. Interestingly, a numberof ARF-GAPs including GIT2, a member of the same familyof p95-APP1 (Mazaki et al., 2001), and PAPa, a member of thecentaurin family (Kondo et al., 2000), have also beenimplicated in the regulation of paxillin distribution in the cell.

Movement of vesicles to areas of the plasma membraneinvolved in protrusive activity may represent a mechanism to

JOURNAL OF CELL SCIENCE 114 (24)

4519Regulation of p95-APP1 localization

help the forward movement of the front of migrating cells(Bretscher, 1996). The data presented in this study providefurther support to a role of p95-APP1 in coordinatingmembrane traffic from recycling endosomes to sites of Rac-mediated actin reoganization. By using its multi-domainstructure, p95-APP1 may bring together functions related tothe regulation of recycling vesicular traffic, with componentsnecessary to a productive protrusive activity driven by actinpolymerization.

We are grateful to Marino Zerial for the anti-Rab11 antibody, toHarald Stenmark for the anti-EEA1 antibody, to Ed Manser and LouisLim for the pXJ40-HA-βPIX plasmid and the anti-PIX antibody, toVictor Hsu for the anti-ARF6 antibody, and to Gary Bokoch for thepCMV6m/Pak1 plasmid and the anti-Pak1 antibody, and to ChiaraAlbertinazzi for helping with the transfection of COS cells. The mAbLEP100 developed by D. M. Fambrough was obtained by theDevelopmental Studies Hybridoma Bank, The University of Iowa, IA.Special thanks to Ruggero Pardi for critical reading of the manuscript.The financial support of Telethon-Italy (Grant n.1171 to I.d.C.) andof AIRC (Italian Association for Cancer Research) are gratefullyacknowledged. C. Albertinazzi was supported by a fellowship fromFIRC (Italian Federation for Cancer Research). Supported by theUniversity Excellence Center on Physiopathology of CellDifferentiation.

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