10.1128/MCB.20.7.2619-2627.2000.

2000, 20(7):2619. DOI:Mol. Cell. Biol. B. Matija Peterlin

andGeyer, Bing Jiang, Wen Luo, Arie Abo, Arthur S. Alberts Oliver T. Fackler, Xiaobin Lu, Jeffrey A. Frost, Matthias in Cellular Activation by Nefp21-Activated Kinase 1 Plays a Critical Role

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MOLECULAR AND CELLULAR BIOLOGY,0270-7306/00/$04.0010

Apr. 2000, p. 2619–2627 Vol. 20, No. 7

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

p21-Activated Kinase 1 Plays a Critical Role inCellular Activation by Nef

OLIVER T. FACKLER,1 XIAOBIN LU,2 JEFFREY A. FROST,3 MATTHIAS GEYER,1

BING JIANG,1 WEN LUO,1 ARIE ABO,2 ARTHUR S. ALBERTS,4 AND B. MATIJA PETERLIN1*

Howard Hughes Medical Institute, Departments of Medicine, Microbiology, and Immunology,1 and Cancer ResearchInstitute,4 University of California at San Francisco, San Francisco, California 94143-0703; Onyx Pharmaceuticals,

Richmond, California 948062; and University of Texas Southwestern Medical Center, Dallas, Texas 75235-90413

Received 2 September 1999/Returned for modification 27 October 1999/Accepted 14 January 2000

The activation of Nef-associated kinase (NAK) by Nef from human and simian immunodeficiency viruses iscritical for efficient viral replication and pathogenesis. This induction occurs via the guanine nucleotideexchange factor Vav and the small GTPases Rac1 and Cdc42. In this study, we identified NAK as p21-activatedkinase 1 (PAK1). PAK1 bound to Nef in vitro and in vivo. Moreover, the induction of cytoskeletal rearrange-ments such as the formation of trichopodia, the activation of Jun N-terminal kinase, and the increase of viralproduction were blocked by an inhibitory peptide that targets the kinase activity of PAK1 (PAK1 83-149). Theseresults identify NAK as PAK1 and emphasize the central role its kinase activity plays in cytoskeletal rear-rangements and cellular signaling by Nef.

Nef is a 27- to 35-kDa myristylated accessory protein whichis unique to primate lentiviruses human immunodeficiency vi-rus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus(SIV). Although dispensable for viral replication in cell lines,Nef is critical for maintaining high levels of viremia and for theprogression to AIDS in the infected host. Infections of rhesusmacaques with SIV containing large deletions in the nef genedid not lead to disease. In contrast, rapid reversions of dele-terious point mutations and small deletions in the nef generestored the full pathogenic potential of the virus (19, 20, 40).The importance of Nef was also demonstrated for HIV-1 in theSCID/hu mouse (17) and in humans who were infected withviral strains carrying deletions in the nef gene (10, 22).

Whereas the importance of Nef for the pathogenesis of HIVand SIV is undisputed, its exact mechanism of action remainselusive. At least three different functions have been attributedto Nef. First, Nef directs CD4 and major histocompatibilitycomplex class I determinants to endosomal compartments andtheir degradation (14, 25, 27, 31, 42). Second, Nef increasesviral infectivity at a postentry step in the viral replicative cycle(2, 7, 41). Third, depending on its intracellular localization, Nefactivates or inhibits cellular signaling pathways (4). For theselatter effects, Nef interacts with a number of cellular tyrosineand serine-threonine kinases. Members of the Src kinase fam-ily can bind to the N terminus or the proline-rich motif in Nef(5, 24, 37). Additionally, the guanine nucleotide exchange fac-tor (GEF) Vav binds to this motif in Nef (12). This interactionleads to the activation of the GEF activity of Vav, which in-duces cytoskeletal rearrangements and downstream effectorfunctions. Vav recruits the small GTPases Rac1 and Cdc42,which are required for the activity of Nef-associated kinase(NAK) (8, 16, 26). The association with and activation of NAKby Nef requires the conserved proline-rich motif and diargin-ine residues in the core domain of Nef (28, 39, 45). Whenrhesus macaques were infected with SIV bearing mutations in

either motif, monkeys failed to develop AIDS until reversionsof prolines or arginines were observed (20, 40).

NAK was first described as two phosphoproteins with ap-parent molecular masses of 62 (p62) and 72 kDa (p72) (38).p62 is a cellular serine-threonine kinase that is recognized byantibodies against N- and C-terminal epitopes of p21-activatedkinase 1 (PAK1), and p72 is most likely the substrate of NAK(26, 30, 40). To date, four different members of the PAK family(PAK1 to PAK4) have been identified. They contain similarN-terminal regulatory domains composed of three proline-richmotifs, a GTPase binding site (Cdc42-Rac1 interactive binding[CRIB] domain) and a stretch of acidic amino acids (aa) (seeFig. 2A). Their C termini contain large catalytic domains (forreviews, see references 23 and 43). Whereas PAK1 and PAK2play identical roles in cytoskeletal rearrangements and cellularsignaling such as the activation of Raf-1, PAK2 is also involvedin apoptotic cell death (13, 21, 35). PAK3 is expressed exclu-sively in the brain, and PAK4 is localized to the Golgi appa-ratus (1, 21). Although previous studies documented the im-munological relationship between NAK and the PAK family(26, 30, 40), they could not demonstrate a direct physical andfunctional interaction between Nef and a specific PAK isoformand thus failed to identify NAK.

In this study, we sought to identify the specific PAK thatprovides the kinase activity of NAK. In vivo and in vitro studiesrevealed that NAK can be PAK1. Furthermore, an inhibitoryfragment that was reported to be specific for PAK1 blocked theactivation of NAK, the formation of trichopodia, and the in-duction of Jun N-terminal kinase (JNK) and increased viralproduction in a manner that depended on Nef. After submis-sion of this work, another group using a different allele of Nefsuggested that NAK could also be PAK2 (34). Taken together,these studies identify NAK as two members of the PAK familythat have interchangeable effects on the actin cytoskeleton andcellular signaling cascades.

MATERIALS AND METHODS

Plasmids. Plasmids encoding Vav, CD8-Nef fusion proteins, and Nef.GFP andNefPP-AA.GFP were described earlier (12, 26). NefRR-LL.GFP was generated bysubcloning the HIV-1SF2nef with the RR-LL mutation into the green fluorescentprotein (GFP) vector pEGFP-N1 (Clontech, Palo Alto, Calif.). Nucleotide se-quences of novel constructs were confirmed by DNA sequencing. Proviral con-

* Corresponding author. Mailing address: UCSF Box 0703, 3rd andParnassus Ave., San Francisco, CA 94143-0703. Phone: (415) 502-1902. Fax: (415) 502-1901. E-mail: matija@itsa.ucsf.edu.

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structs of HIV-1SF2 and HIV-1SF2DNef were gifts from Cecilia Cheng-Mayer(Aaron Diamond AIDS Research Center, New York, N.Y.). The expressionplasmid for glutathione S-transferase (GST)–PAK1 was generated by subcloninga fragment encoding aa 1 to 436 of PAK1 into the pGEX-2TK vector. Theeukaryotic expression plasmid for PAK1 83-149 was described earlier (13). Theexpression plasmid for myc-tagged PAK1 was a generous gift from Art Weiss(University of California, San Francisco).

Cell lines, transfection, and microinjection. Jurkat and COS cells were culti-vated as described earlier (26). Transfections were performed by electroporationfor Jurkat cells and by lipofection using Lipofectamine (GIBCO, Grand Island,N.Y.) for COS cells following standard protocols. For microinjection experi-ments, NIH 3T3 fibroblasts were cultured in 10% fetal calf serum (FCS) inDulbecco modified Eagle medium and were plated onto glass coverslips aspreviously described (3). Following incubation in low-serum media containing0.1% FCS for 36 h prior to injection, cells were microinjected with an Eppendorf5171/5246 microinjection apparatus by using pulled borosilicate glass capillaries.Plasmids (10 mg/ml unless indicated otherwise) were diluted 1:1 in Dulbecco’sphosphate-buffered saline with deionized water.

Immunoprecipitation. Transfected COS cells were washed with phosphate-buffer saline (PBS) twice and were lysed in 1 ml of kinase extraction buffer(KEB) containing 1% NP-40, 10 mM Tris (pH 7.8), 150 mM NaCl, 2 mM EDTA,and protease and phosphatase inhibitors at 4°C for 20 min. The cell lysates werecentrifuged at 12,000 3 g for 10 min at 4°C, and the supernatants were incubatedwith 1 ml of anti-N20 (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) oranti-CD8 antibody, respectively, at 4°C for 1 h. The samples were then mixedwith 40 ml of protein A-conjugated agarose beads for 2 h, and the immunopre-cipitations were washed four times and resuspended in sodium dodecyl sulfatesample buffer. Western blot analysis of the samples was performed followingstandard procedures by using the ECL kit (Amersham, Arlington Heights, Ill.).

Purification of the Nef-NAK complex. Nef-NAK complexes were isolated fromJurkat cells transiently expressing the hybrid CD8-Nef protein. Cells (107 per ml)were lysed in KEB and were loaded onto a MonoQ column (Pharmacia, Pisacat-away, N.J.) previously equilibrated with KEB lacking salt. Proteins were elutedfrom the column in 1-ml fractions by washing with 30 ml of KEB with increasingsalt concentrations (0 to 0.5 M NaCl). Individual fractions were then assayed forprotein content by Western blotting and were subjected to in vitro kinase analysisfollowing immunoprecipitation with the anti-CD8 antibody.

Immunofluorescence and digital imaging. Three hours after the microinjec-tion with expression plasmids coding for hybrid Nef.GFP proteins or GFP alone,cells were fixed with 3.7% (vol/vol) formaldehyde-PBS for 10 min, were perme-abilized with 0.3% (vol/vol) Triton X-100, and were incubated with tetrameth-ylrhodamine isocyanate (TRITC)-phalloidin in PBS for 1 h in a humidifiedatmosphere at ambient room temperature. c-Jun serine-63 phosphorylation (ex-pressed from EF-NLex.Jun [33]) was monitored by indirect immunofluorescenceby using phosphospecific antisera (New England Biolabs, Beverly, Mass.) andTexas red donkey anti-rabbit antisera (Jackson ImmunoResearch Laboratories,West Grove, Pa.) as previously described (3). Fluorescence was monitored witha Leica DMXRA microscope using 1003 or 403 magnification oil-immersionobjectives (numerical aperture, 1.7). Images were captured with fixed exposuretimes with a Diagnostics CCD camera and were processed identically with AdobePhotoshop as PICT files, and figures were assembled with ClarisDraw.

Antibodies. The polyclonal rabbit sera against PAK1, hemagglutinin (HA),and Myc were purchased from Santa Cruz Biotechnology, Inc. The monoclonalantibody (MAb) against Nef from HIV was a generous gift from Earl Sawai,University of California, Davis. The MAb against human CD8 was from ArtWeiss, University of California, San Francisco.

In vitro kinase assay. In vitro kinase reactions were performed as describedearlier (26). Briefly, 5 3 106 transfected cells were lysed in 1 ml of KEB, andcleared supernatants were immunoprecipitated with 1 ml of anti-CD8 MAb.After extensive washing, the immunoprecipitated complex was resuspended in100 ml of kinase assay buffer containing 10 mCi of [g-32P]ATP per ml. The kinasereaction was conducted at room temperature for 5 min, and the samples werewashed and subjected to autoradiography.

GST fusions and in vitro binding studies. The GST-PAK1 fusion protein wasexpressed in Escherichia coli and was purified on glutathione-Sepharose beads.Equal amounts of GST and GST fusion protein immobilized on the beads wereincubated with purified HIV-1SF2 Nef or mutant HIV-1SF2NefRR-LL proteinexpressed from baculovirus in insect cells. No contaminating proteins were de-tected in these preparations of Nef. The binding reaction was performed at 4°Cfor 2 h in KEB. The beads were then washed three times with KEB containing1 M NaCl, and bound proteins were separated by sodium dodecyl sulfate–12%polyacrylamide gel electrophoresis and were analyzed by Western blotting.

Virus production assay. Jurkat cells (5 3 106) were electroporated understandard conditions with 5 mg of proviral DNA and 15 mg of VavDDH, Sek-AL,PAK1 83-149, or empty plasmid vector, and were subsequently cultured in 5 mlof medium. At 48 h posttransfection, cell culture supernatants were harvestedand filtered through a 45-mm-pore-size filter (Millipore, Bedford, Mass.) andwere stored at 270°C. Virus production was quantified by p24Gag enzyme-linkedimmunosorbent assay (NEN Life Science Products, Boston, Mass.). To distin-guish between intracellular and extracellular p24Gag, pellets from 1 ml of cellsuspension were washed in PBS and lysed in 1 ml of KEB, and p24 concentra-

tions in cell supernatant and cell lysate were determined by p24Gag enzyme-linked immunosorbent assay.

RESULTS

PAK1 is present in the 1-mDa complex that contains Nefand NAK. To identify NAK, we purified Nef complexes fromJurkat cells expressing the hybrid CD8-Nef protein (CN) usingMonoQ gel chromatography followed by immunoprecipitationwith the anti-CD8 antibody. In vitro kinase assays (IVKA)revealed the presence of NAK in fractions 8 to 14 (Fig. 1A).The molecular mass of this complex was estimated to be 1 mDaby size exclusion chromatography (data not shown). In controlexperiments, no kinase activity could be detected in fractionsfrom Jurkat cells expressing the truncated CD8 or the mutanthybrid CD8-NefRR-LL proteins that do not associate with NAK(data not shown; 39). Western blotting revealed the presenceof Nef in the same fractions (Fig. 1A, Western blot and CN).When the membrane was probed with the N20 antibody, onlyone specific band was detected in the fractions that containedNef and NAK (Fig. 1A, Western blot and PAK1). Since theanti-N20 antibody recognizes PAK1 preferentially, the de-tected protein could be PAK1. Reprobing this membrane withother specific antibodies also revealed the presence of Vav inthese fractions (data not shown). Thus, PAK1 is most likelypresent in large complexes containing Nef and NAK in cells.

Nef interacts with PAK1 in cells. Since NAK was identifiedinitially as a kinase activity which coimmunoprecipitates withNef, we wanted to confirm that Nef and PAK1 can interact incells. The hybrid CN and PAK1 were coexpressed in COS cells.Nef was immunoprecipitated with the anti-CD8 antibody, andthese immunoprecipitations were assayed for the presence ofPAK1 by Western blotting with the N20 antibody (Fig. 1B, leftpanel). In these experiments, PAK1 could be detected readilyin anti-CD8 immunoprecipitations from cells expressing thehybrid CN (Fig. 1B, lane 2) but not from cells expressing thetruncated CD8 protein (Fig. 1B, lane 1). To demonstrate thatartificial membrane targeting of Nef via CD8 is not a prereq-uisite for this association between Nef and PAK1, we per-formed reciprocal immunoprecipitations from cells expressingthe hybrid Nef.GFP protein or GFP alone with the anti-N20antibody. Similar to our previous results with the hybrid CN,the hybrid Nef.GFP protein but not GFP alone associated withPAK1 in cells (Fig. 1B, right panel, lanes 3 and 4). We con-clude that Nef interacts with PAK1 in vivo.

Nef binds to PAK1 in vitro. Next, we investigated if PAK1binds to Nef in vitro. Nef was purified from insect cells infectedwith baculovirus and incubated with GST or hybrid GST-PAK1 proteins. As judged by Coomassie blue and silver stain-ing, no contaminating proteins were copurified with Nef frominsect cells (data not shown). The presence of Nef in theseGST pull down assays was analyzed by Western blotting. Aspresented in Fig. 1C, no interaction with Nef was observedwith GST alone (Fig. 1C, lanes 1 and 3). In contrast, the hybridGST-PAK1 protein bound to Nef but not to the mutantNefRR-LL protein (Fig. 1C, lanes 2 and 4) and captured about10% of the input Nef (Fig. 1C, compare lanes 2 and 5). Weconclude that Nef binds to PAK1 and that the RR-LL muta-tion at positions 109 to 110 in Nef abolishes this interaction.

NAK is blocked by an inhibitory fragment from PAK1. Todemonstrate functionally that NAK is PAK1, we examined theability of an inhibitory fragment of PAK1 to block NAK incells. To this end, we assayed NAK in the presence of a peptidecontaining residues 83 to 149 of PAK1 (PAK1 83-149). PAK183-149 has been demonstrated to inhibit the autophosphory-lation of PAK1 by blocking a critical phosphoacceptor site that

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is required for its kinase activity and does not bind to Cdc42nor interfere with the binding of small GTPases to PAK1 (13,47, 48). Moreover, these sequences C terminal to the CRIBdomain in PAK1 are not highly conserved among PAK familymembers (Fig. 2A).

Increasing amounts of the plasmid vector or PAK1 83-149were coexpressed with the hybrid CD8-Nef protein in Jurkatcells, and anti-CD8 immunoprecipitations were then tested forNAK activity. Similar experiments had previously demon-strated that NAK activity depends on the presence of Rac1,

FIG. 1. Nef interacts with PAK1 in vivo and in vitro. (A) Purification of the NAK complex by MonoQ ion exchange chromatography. Nef complexes were isolatedfrom Jurkat cells expressing the hybrid CN using the MonoQ column. Anti-CD8 immunoprecipitations from different fractions were examined for NAK activity (IVKA)and for the presence of Nef and PAK1 (Western blot). (B) Nef from HIV interacts with PAK1 in COS cells. The hybrid CN (left panel), hybrid Nef.GFP (right panel,1 in table), CD8 truncated proteins or GFP alone (2 in table) were coexpressed with PAK1 in COS cells. Cellular lysates were incubated with the anti-N20 (right panel,IP:aPAK1) or the anti-CD8 antibody (left panel, IP:aCD8), and immunoprecipitations were analyzed by Western blotting by using anti-Nef (lanes 3 and 4) or anti-N20(lanes 1 and 2) antibodies, respectively (upper panel, IP). Western blotting of input Nef and PAK1 proteins prior to the immunoprecipitation are presented in thebottom panel (input). (C) Nef binds to PAK1 in vitro. Nef and the mutant NefRR-LL protein were purified from insect cells by using baculovirus, were incubated withthe hybrid GST-PAK1 protein or GST alone, and were passed over glutathione beads. The bound Nef was analyzed by Western blotting by using the anti-Nef antibody(Western blot). Nef proteins (10% of that used in the binding reaction) were loaded on the gel as input control (10% input).

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Cdc42, and Vav (12, 26). When compared to the activity ofNAK from cells transfected with the control plasmid vector(Fig. 2B, lanes 1 and 2), PAK1 83-149 blocked the activity ofNAK in a dose-dependent manner (Fig. 2B, IVKA, lanes 3 and4). Since similar amounts of Nef were present in all immuno-precipitations (Fig. 2B, Western blot), this result confirms theidentity of NAK as PAK1.

Cytoskeletal rearrangements induced by Nef require thekinase activity of PAK1. Previously, we demonstrated that theactivation of Vav by Nef results in cytoskeletal rearrangementsand the activation of the JNK–stress-activated protein kinase(SAPK) cascade (12). Since a mutant Vav protein lacking theGEF activity blocked NAK, we speculated that the down-stream effector functions induced by Nef and Vav are a direct

consequence of the subsequent activation of PAK1. To provethis hypothesis, we investigated the effect of PAK1 83-149 oncytoskeletal rearrangements induced by Nef.

NIH 3T3 cells were injected with plasmid effectors and werestained with TRITC-phalloidin for polymerized actin. Changesin the actin cytoskeleton were observed to be stable for up to14 h. Injected cells were localized via the GFP linked to Nef orexpressed from a coinjected plasmid (12). Whereas the expres-sion of the hybrid Nef.GFP protein alone had no significanteffect on the actin cytoskeleton, the coexpression of Nef andVav led to a dramatic loss of actin stress fibers and the gener-ation of long extentions called trichopodia (Fig. 3, comparepanels 1 and 2 with panels 3 and 4). In sharp contrast, when thekinase activity of PAK1 was inhibited by the coexpression of

FIG. 2. Blocking PAK1 kinase activity inhibits NAK activity. (A) Schematic representation of PAK1 to PAK4 and the inhibitory PAK1 83-149 peptide. Each of thePAK isoforms contains a GTPase binding site (CRIB domain) and a kinase domain. Only PAK4 lacks an acidic domain and a proline-rich binding site for the GEFPIX. The lower panel depicts sequence homologies of all members of the PAK family. Numbers refer to residues in PAK4. (B) The PAK1 83-149 peptide inhibits NAKin Jurkat cells. The hybrid CN was coexpressed with an empty plasmid vector (control vector) or the PAK1 83-149 peptide. Cellular lysates were immunoprecipitatedwith the anti-CD8 antibody followed by an in vitro kinase assay (IVKA). The kinase activity of NAK was revealed by radioautography of gels transferred to membranes.Note that in Jurkat cells, two proteins (p62 and p72) are phosphorylated by NAK. The same blot was also subjected to Western blotting with the anti-Nef antibodyto demonstrate that comparable amounts of the hybrid CN were present in these immunoprecipitations (Western blot).

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PAK1 83-149, Nef and Vav no longer disrupted actin stressfibers and could not form trichopodia. Only the formation ofmembrane ruffles and small lamellipodia was observed (Fig. 3,compare panels 3 and 4 with panels 5 and 6). The coexpressionof Vav with the mutant hybrid NefRR-LL.GFP protein, whichcannot bind and activate PAK1, resulted in the same pheno-type (Fig. 3, panels 7 and 8). Importantly, the microinjection ofPAK1 83-149 alone had no effect (Fig. 3, panels 9 and 10).These results demonstrate that the induction of the kinaseactivity of PAK1 by Nef is critical for the disruption of actinstress fibers and for the formation of trichopodia and thatresidual morphological changes occur independently of thekinase activity of PAK1. These latter effects most likely repre-sent direct effects of activated Rac1 (18, 44). Moreover, cy-toskeletal rearrangements induced by Nef, Vav, and PAK1were more pronounced then the loss of actin stress fibers andthe cytoplasmatic retractions induced by the constitutively ac-tive PAK1 (PAK1 L107F) (Fig. 3, compare panels 3 and 4 withpanels 11 and 12) (13, 48)). As described previously (12), theformation of trichopodia represents a different process fromcytoplasmic retractions induced by the constitutively activePAK1. Highlighting these differences, effects of PAK1 L107Fwere also not sensitive to the coexpression of PAK1 83-149(Fig. 3, panels 13 and 14). Additionally, since the mutant hy-brid NefRR-LL.GFP protein had no effect on PAK1 L107F (Fig.

3, panels 15 and 16), PAK1 must function downstream of Vavin this pathway. This conclusion was confirmed by identicalresults observed with the mutant hybrid NefPP-AA.GFP pro-tein, which cannot activate Vav (data not shown). We concludethat the activation of PAK1 by Nef is critical for the disruptionof actin stress fibers and the formation of trichopodia inducedby Nef and Vav and that membrane ruffles and lamellipodiainduced by Nef independently of the kinase activity of PAK1synergize with this effect.

The kinase activity of PAK1 is required for the activation ofthe JNK-SAPK cascade by Nef. Next, we investigated whetherthe activation of the JNK-SAPK cascade by Vav and Nef is alsomediated by PAK1. To this end, we monitored the phosphor-ylation of the serine at position 63 in Jun in microinjected NIH3T3 cells (3). Vav, the hybrid Nef.GFP or mutant hybridNefRR-LL.GFP proteins, and PAK1 L107F or PAK1 83-149were coexpressed with the N terminus of c-Jun (NLex.JunN[33]). Injected cells were stained with the antibody that recog-nizes only the phosphorylated JunS63. The coexpression of thehybrid Nef.GFP protein and Vav but not the expression of thehybrid Nef.GFP protein alone resulted in the phosphorylationof JunS63 in injected cells (Fig. 4, compare columns 1 and 2).This activation of JNK was similar to that observed with theactivated PAK1 (Fig. 4, column 4). However, no activation ofJNK was observed when the mutant NefRR-LL.GFP protein

FIG. 3. Blocking PAK1 activity prevents cytoskeletal rearrangements induced by Nef and Vav. NIH 3T3 cells were maintained in a low concentration of serum(0.1% FCS-Dulbecco modified Eagle medium) and were microinjected with indicated plasmids (10 mg of each per ml). Cells were fixed for 3 h and were stained withTRITC-phalloidin (right-hand panels) to visualize polymerized actin (see Materials and Methods). Trichopodia refer to extensions bearing minute filopodia (panels3 and 4). Microinjected cells were identified by the expression of GFP (left-hand panels). Proteins expressed from injected plasmids are indicated to the right of panels1 to 16.

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was coexpressed with Vav or when PAK1 83-149 was coex-pressed with Vav and the hybrid Nef.GFP protein (Fig. 4,columns 3 and 5). Moreover, the expression of the PAK183-149 peptide alone did not affect the activity of JNK (Fig. 4,column 7). Similar to its inability to change the actin cytoskel-eton, the expression of the mutant NefPP-AA.GFP protein thatdoes not activate Vav and PAK1 also did not block the acti-vation of JNK by PAK1 L107F (Fig. 4, column 6). Likewise, thePAK1 83-149 peptide did not inhibit the activation of JNK byPAK1 L107F (Fig. 4, column 8). These data suggest that theactivation of PAK1 by Nef induces JNK. We conclude that thekinase activity of PAK1 is required for the formation oftrichopodia and the induction of the JNK-SAPK cascade byNef.

The inhibition of viral replication by PAK1 83-149 dependson Nef. Previously, we reported that the inhibition of NAK bya dominant negative PAK protein (PAK-R) (26) or a mutantVav protein lacking the GEF activity (VavDDH) (12) blockedthe production of HIV-1. Since the coexpression of the PAK183-149 peptide prevented the activation of PAK1 by Nef, weexamined its effects on viral replication. A plasmid containingthe HIV-1SF2 provirus was coexpressed with the empty plasmidvector (Fig. 5, white bars) or with the PAK1 83-149 peptide(Fig. 5, black bars) in Jurkat cells. The production of viralparticles was monitored by measuring levels of p24Gag in the

supernatant of transfected cells (Fig. 5A). As observed with themutant VavDDH protein (Fig. 5, grey bars), the expression ofthe PAK1 83-149 peptide resulted in 80% decreased produc-tion of new virions (Fig. 5A, compare columns 1, 2, and 3).However, neither the mutant VavDDH protein nor the PAK183-149 peptide had a significant effect on the production ofHIV-1SF2 from the provirus that lacked the nef gene (Fig. 5A,compare columns 5, 6, and 7). Since the dominant negativeSek-AL protein (striped bars) had a similar negative effect onviral production (Fig. 5, columns 4 and 8), these effects weremediated by the activation of JNK by Nef. These results dem-onstrate that the inhibition of PAK1 is sufficient to block theeffect of Nef on viral production.

To determine if these effects of Nef reflected increased tran-scription from the viral long terminal repeats, the overall syn-thesis of viral proteins (intracellular p24Gag and p24Gag in thesupernatant) was determined (Fig. 5B). Results mirrored thedata for p24Gag in the supernatant of transfected cells. ThePAK1 83-149 peptide, mutant VavDDH, and Sek-AL proteinsall significantly reduced the overall synthesis of viral proteinsfrom the wild-type HIV-1 but not from the provirus that lackedthe nef gene. We conclude that the major effect of Nef on viralproduction is mediated by increased viral gene expression inJurkat cells.

FIG. 4. Blocking PAK1 activity prevents the activation of the JNK-SAPK cascade by Nef and Vav. NIH 3T3 cells were maintained in a low concentration of serumand were microinjected as described in the legend to Fig. 3 except for the addition of a plasmid coding for the hybrid LexJun protein. Cells were stained with theanti-JunS63P antibody, and stained cells were counted. Expressed proteins are denoted on the bottom of the figure. Whereas the effects of Nef without activation ofPak1 are represented by striped bars, controls for the activity of Nef and Vav as well as Pak1 are shown in black and white, respectively. Standard errors of the meansare presented with error bars. Experiments are representative of three independent injections where more than 50 cells were counted.

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DISCUSSION

In this study, we sought to identify NAK as a specific PAKfamily member, which is activated by Nef to increase thepathogenicity of primate lentiviruses. PAK1 was found in Nefcomplexes and was coimmunoprecipitated with Nef in cells.Furthermore, Nef bound to PAK1 in vitro. The inhibitoryPAK1 83-149 peptide that prevents the autophosphorylation of

PAK1 blocked not only NAK but also the disruption of actinstress fibers, the formation of trichopodia, the induction of theJNK-SAPK cascade, and the production of viruses. We con-clude that PAK1 can provide the kinase activity of NAK, whichis required for the downstream effector functions of Nef.

The identification of NAK as PAK1 is based on structuraland functional criteria. In our immunoprecipitations andWestern blots, the anti-N20 antibody was able to recognizePAK1 with a 100-fold-higher affinity than PAK2. This speci-ficity was confirmed by microsequencing of proteins in com-plexes from Jurkat cells, which were immunoprecipitated withthe anti-N20 antibody, and by the inability of this antibody torecognize overexpressed PAK2, PAK3, or PAK4 in COS cells.Furthermore, we could not detect the association of the hybridCN with any PAK isoform other than PAK1 in cells (data notshown). Additionally, PAK1 bound to Nef in GST pull downassays. Our functional studies also argue for PAK1. Neverthe-less, although the PAK1 83-149 peptide contains sequencesthat are not highly conserved among PAK isoforms, its speci-ficity was examined only with PAK1 and PAK3, where it mostefficiently inhibited PAK1 (13, 48).

Thus, it is possible that PAK1 83-149 also inhibits PAK2. Tothis end, it is of interest that after the submission of our report,another group using a different allele of Nef identified NAK asPAK2 (34). This study was based exclusively on an epitope-specific antibody against PAK2 and presented no functionaldata. However, taken together, these two reports might resolvethe dilemma of NAK. Previous conflicting tryptic peptide mapsof NAK could not differentiate between these two PAK iso-forms. For example, peptide patterns resembling those ofPAK1 and PAK2 were presented for NAK associated with Neffrom SIVmac239 (40) and HIV-1NL4-3, respectively (34). How-ever, for Nef from HIV-1SF2, the peptide pattern of NAK didnot resemble that of PAK2 (26). Since both PAK1 and PAK2have now been identified as NAK by improved binding assaysand the use of epitope-specific antibodies, it is possible thatdifferent alleles of Nef bind preferentially to one or the otherPAK isoform. Alternatively, this specificity could be influencedby levels of expression, subcellular distribution, and context ofthese PAK family members in different cells. However, sincePAK1 and PAK2 are 78% identical, share 91% amino acidhomology, and have interchangeable effects on the actin cy-toskeleton and signaling cascades (23), the choice of eitherpartner serves the need of the virus. Thus, the search for theprecise PAK isoform has instead discovered its pathway andphenotype.

PAK1 and PAK2 (henceforth PAK1) exert profound effectson the actin cytoskeleton and downstream signaling cascades.The extensive disruption of actin stress fibers, cytoplasmic re-traction, and JNK activation require the kinase activity ofPAK1. In sharp contrast, the induction of membrane rufflesand small lamellipodia depends on Rac1 but is kinase inde-pendent (18, 44). The same picture was observed with Nef.The loss of stress fibers and the formation of trichopodia de-pended on Nef, Vav, and PAK1. However, the formation ofmembrane ruffles and small lamellipodia was still observed inthe presence of the PAK1 83-149 peptide or with the mutantNefRR-LL protein, which does not bind to PAK1. In both cases,Nef could still activate Rac1. As discussed previously, trichopo-dia differ from cytoplasmic retractions and most likely repre-sent the local activation of actin reorganization by Nef com-plexes, which are localized in tiny vesicles in these extensions(9, 12). These 1-mDa complexes most likely form in lipid rafts(6, 11, 29, 46) and contain additional signaling intermediates,e.g., Src family kinases that bind to the N terminus of Nef.Indeed, this NAK-C complex, which contains Lck and addi-

FIG. 5. Inhibition of PAK1 activity interferes with viral production. ThePAK1 83-149 peptide as well as mutant VavDDH and Sek-AL proteins inhibitviral production only from proviruses that contain the nef gene. HIV-1SF2 pro-virus or its counterpart, which lacked the nef gene (HIV-1SF2DNef), was coex-pressed in Jurkat cells with the empty plasmid vector (Mock), the mutantVavDDH protein, the dominant negative Sek-AL protein, or the PAK1 83-149peptide. The production of HIV-1 particles was measured after 48 h by quanti-fying levels of p24Gag in the supernatant (A). The overall synthesis of viralproteins was quantified by combining intra- and extracellular levels of p24Gag

(B). Mock transfections were arbitrarily set to 100%. Averages of triplicateexperiments with indicated standard errors of the means are shown.

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tional unknown proteins, is required for the activation of NAK(5).

On the molecular level, both the proline-rich motif anddiarginine residues in Nef are required for the activation ofPAK1 in vitro and for the pathogenesis of SIV in vivo (20, 28,39, 40). Their location allows for the simultaneous binding ofVav and PAK1 to two distinct surfaces on Nef (28). Thisorganization of the Nef complex is in agreement with a studyon the activation of the PAK homologue Ste20 in Saccharo-myces cerevisiae, where Nef bound to Bem1 and Ste20 via theproline-rich motif and the diarginine residues, respectively(32). However, whereas Bem1 stabilized and activated the Nef-Ste20 complex in yeast, Vav does not contribute to the bindingof Nef to PAK1 in mammalian cells (data not shown). Rather,Nef, Rac1, and Cdc42 are localized at the plasma membrane bytheir N-terminal myristyl and C-terminal geranyl moieties, re-spectively. This localization enables the interaction of Nef withthe C-terminal SH3 domain of Vav which induces its GEFactivity for Cdc42 and Rac1. PAK1 is recruited into this com-plex by the diarginine residues in Nef. The activation of Rac1and Cdc42 leads to their subsequent dissociation from Vav andstrongly increases their affinity for PAK1 (13, 36). They bind tothe CRIB domain of PAK1 and activate the kinase. The sub-sequent induction of cytoskeletal rearrangements and activa-tion of the JNK-SAPK cascade then results in increased pro-duction and infectivity of HIV. With the identification of keyplayers in this process, future studies will focus on antiviralstrategies specifically directed against these signaling events.

ACKNOWLEDGMENTS

We thank Michael Armanini for expert secretarial assistance andPierre Chardin, Melanie Cobb, Cecilia Cheng-Mayer, Paul Luciw,Frank McCormick, Pablo Rodriguez-Viciana, Ran Taube, and ArtWeiss for reagents and comments on experiments.

O.T.F. and M.G. kindly acknowledge fellowships from the DeutscheForschungsgemeinschaft and the European Molecular Biology Orga-nisation, respectively. This work was funded by grants from the NIH(1RO1AI38532-01 and GM53032) and the Howard Hughes MedicalInstitute.

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