Biochimica et Biophysica Acta 1813 (2011) 1836–1844

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Biochimica et Biophysica Acta

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HIV-1 Tat binds to SH3 domains: Cellular and viral outcome of Tat/Grb2 interaction

Slava Rom b, Marco Pacifici a, Giovanni Passiatore c, Susanna Aprea d, Agnieszka Waligorska a,Luis Del Valle a, Francesca Peruzzi a,⁎a LSU Health Sciences Center, School of Medicine, Stanley S. Scott Cancer Center, New Orleans, LA, USAb Temple University School of Medicine, Department of Pathology and Laboratory Medicine, Philadelphia, PA, USAc Department of Neurology and Neuroscience, Weill Cornell Medical College, New York, NY, USAd Novartis Vaccines, Siena, Italy

⁎ Corresponding author at: LSU Health Sciences CenScott Cancer Center, 533 Bolivar St, New Orleans, LA 72113; fax: +1 504 568 6888.

E-mail address: fperuz@lsuhsc.edu (F. Peruzzi).

0167-4889/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.bbamcr.2011.06.012

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 March 2011Received in revised form 22 June 2011Accepted 24 June 2011Available online 1 July 2011

Keywords:HIV-1 TatGrb2Signal transductionViral replicationHIVESH3 domain

The Src-homology 3 (SH3) domain is one of the most frequent protein recognition modules (PRMs), beingrepresented in signal transduction pathways and in several pathologies such as cancer and AIDS. Grb2(growth factor receptor-bound protein 2) is an adaptor protein that contains two SH3 domains and isinvolved in receptor tyrosine kinase (RTK) signal transduction pathways. The HIV-1 transactivator factor Tatis required for viral replication and it has been shown to bind directly or indirectly to several host proteins,deregulating their functions. In this study, we show interaction between the cellular factor Grb2 and the HIV-1trans-activating protein Tat. The binding is mediated by the proline-rich sequence of Tat and the SH3 domainof Grb2. As the adaptor protein Grb2 participates in a wide variety of signaling pathways, we characterized atleast one of the possible downstream effects of the Tat/Grb2 interaction on the well-known IGF-1R/Raf/MAPKcascade. We show that the binding of Tat to Grb2 impairs activation of the Raf/MAPK pathway, whilepotentiating the PKA/Raf inhibitory pathway. The Tat/Grb2 interaction affects also viral function by inhibitingthe Tat-mediated transactivation of HIV-1 LTR and viral replication in infected primary microglia.

ter, Medical School, Stanley S.0112, USA. Tel.: +1 504 568

ll rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Protein–protein interaction mediated by a conserved sequencemotif in one protein and a protein recognition module (PRM) inanother is a common mechanism of assembling protein complexesparticipating in signal transduction pathways. Src homology 3 (SH3)modules are conserved elements of about 60 amino acids present in alarge number of proteins which participate in a variety of biologicalprocesses [1,2]. Although SH3 domains generally recognize a PxxPmotif in their binding partners, individual SH3 domains might possessdistinct specificities for putative ligands. At least part of this specificityis thought to be determined by the amino acids flanking the coresequence [3,4], making the overall three-dimensional structure of thePxxP-bearing protein specific only for certain SH3-carrying partners.

While protein–protein interactions mediated by SH3 domainsparticipate in many cellular processes, one of the best characterizedsignaling pathways is the tyrosine kinase receptor mediated Rasactivation. Growth factor receptor-binding protein-2 (Grb2), consist-ing of three domains, two SH3 and a single SH2 domain, is one of thecentral factors in the Ras signaling cascade. Grb2 can recruit Rasguanine nucleotide exchange factor, Sos, to the activated tyrosine

kinase receptor at the plasma membrane, where Sos activates Ras [5].Activated Ras then leads to activation of MAPK signaling cascade [6].Besides its critical role in normal development [7], Grb2-mediatedmitogenic signals have been implicated in several human tumors(reviewed in [8]).

The transactivating factor Tat is an 86–101 amino acid polypeptideencoded by the HIV-1 virus [9]. Several studies have demonstrated theimportance of HIV-1 Tat in the viral replication cycle and in thepathogenesis of AIDS [10,11]. Tat displays several important biologicalactivities affecting uninfected and infected cells by a paracrine/autocrine mechanism due to secretion of Tat from infected cells andits uptake by uninfected neighboring cells [12–14]. Tat has beenshown to modulate expression and activity of several cellular factorsderegulating a variety of signal transduction pathways [15]. Forinstance the modulation of signaling pathways such as the MAPK orthe PI3-K pathways by Tat is well documented [16–26]. Given theability of Tat to directly interact with host factors, we sought toexamine the amino acid sequence of this viral protein for the presenceof protein–protein recognition elements. Similar to another HIVprotein, Nef [27], Tat also contains proline-rich motives which areputative SH3-binding domains, one at the N-terminal and the other atthe C-terminus of the protein. In an SH3 domain binding screening,we found that Grb2-derived peptides gave the highest probabilityscore of binding to Tat. GST-pull-down assay further revealed apreferred binding of Tat to the C-terminal SH3 domain of Grb2. Theoutcome of Tat/Grb2 interaction was evaluated in terms of cellular

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and viral activities. In respect to cellular function, Tat/Grb2 interactionresulted in the attenuation of the MAPK pathway while potentiatingthe Raf inhibitory pathway. Of interest, in terms of viral functions, wefound that Tat/Grb2 interaction decreased the activity of Tat on theHIV-1 LTR, as determined by reduced levels of p24 in the medium ofHIV-1 infected human microglia.

Altogether our results provide evidence for a direct interaction ofTat with Grb2, and possibly other SH3-bearing proteins, suggesting abroad-spectrum mechanism for Tat-mediated deregulation of signal-ing pathways.

2. Materials and methods

2.1. Cell culture, transfection, infection, recombinant proteins andinhibitors

The human Glioblastoma cell line LN229 was maintained inDulbecco's modified Eagle's medium (DMEM) supplemented with10% fetal bovine serum (GIBCO Invitrogen Co., Carlsbad, CA). Primaryhuman fetal microglia cells were isolated and cultured as previouslydescribed [28]. Preparation of HIV-1, strain JRFL, stock and infection ofprimary microglia were performed at a multiplicity of infection (MOI)of 0.1, as described previously [28].

For transient expression experiments, LN229 cells were seeded on60 mm dishes (BD Falcon™, Oakville, Ontario, Canada) at a density of5×105 cells/plate, and the transfection was performed using FuGENE6 Transfection reagent (Roche Diagnostics Corp., Indianapolis, IN)according to the manufacturer's instructions. When required, 24 hafter transfection cells were washed with PBS twice and starved for24 h in DMEMwithout FBS. Stimulationwas done by IGF-1 (40 ng/ml;Invitrogen, Carlsbad, CA) for 0, 2 and 10 min and was stopped bymedium removal and placing on ice.

Recombinant Tat101 was from Immunodiagnostics (Woburn,MA). Inhibitors, LY 294002 and H-89, were purchased from BioMol(BIOMOL Int., Plymouth Meeting, PA).

2.2. Antibodies and Western blots

Monoclonal antibody specific to Grb2 was purchased from BDTransduction Laboratories™ (BD Biosciences, San Jose, CA). Anti-bodies specific to phospho-Akt (Ser473), Akt, phosphorylated formsof Erk1/2 (Thr202/Tyr204), total Erk1/2, phospho-c-Raf (Ser259),total IFG-IR and Gab-1 were purchased from Cell Signaling Inc.(Danvers, MA). Polyclonal antibody specific to total c-Raf protein waspurchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Cells were collected by scraping the plates in the presence of PBS,followed by centrifugation and lysis of the cell pellet in theappropriate volume of RIPA buffer (50 mM Tris pH 7.4, 1% NP-40,0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,1 mM PMSF, 1 mM sodium orthovanadate, phosphatase inhibitorsand protease inhibitor cocktails). Fifteen to fifty micrograms of wholecell lysates was separated on 4–15% SDS-PAGE (BioRad, Hercules, CA).Sample proteins separated by SDS-PAGE were transferred ontonitrocellulose membranes (BioRad) using BioRad semi-dry blottingsystem (BioRad, Hercules, CA).

2.3. Immunohistochemistry

Five samples of formalin-fixed, paraffin-embedded HIV-Encepha-lopathy samples were collected from the NIH funded, HIV Nationaltissue consortium at the Manhattan Brain Bank, Mount Sinai School ofMedicine. All samples were from the cortex and sub-cortical whitematter of the frontal lobe, and histopathologically all contain microglialnodules and perivascular cuffs of inflammatory cells, including giantmultinucleated cells, pathognomonic of HIV-Encephalitis. According to

the clinical data provided by theHIVBrain Bank, allfive patients showedsigns of dementia, ranging from mild to moderate.

For immunohistochemistry, four micron thick sections were cutusing a microtome and placed on charged glass slides. Immunohis-tochemistry was performed using the avidin–biotin peroxidasemethodology, according to the manufacturer's instructions (VectorLaboratories, Burlingame, CA). Our protocol includes deparaffination,rehydration in alcohol up to water, antigen retrieval in citrate bufferpH 6.0 at 98 °C, endogenous peroxidase quenching and blocking innormal horse serum for 2 h at room temperature. Primary antibodiesused included a mouse monoclonal anti-Grb2 (Santa Cruz; 1:100dilution) and a mouse monoclonal anti-Tat (ImmunoDiagnostics, Inc.,Woburn, MA; 1:500 dilution). After rinsing with PBS, secondarybiotinylated antibodies were incubated for 1 h (1:200 dilution),followed by avidin–biotin peroxidase complex incubation (ABC Elitekit, Vector Laboratories, Burlingame, CA) and developed with diamino-benzidine (DAB, Boehringer Ingelheim, Ingelheim, Germany), counter-stained with hematoxylin and coverslipped.

For double labeling, the first steps of the protocol are similar to theprotocol described above. However, after incubation with the firstprimary antibody, a secondary fluorescein tagged antibody wasincubated for 1 h at room temperature, and after rinsing thoroughly,the second primary antibody was incubated overnight. Finally asecond secondary antibody tagged with rhodamine was incubated for1 h. Sections were coverslipped with aqueous based mounting mediacontaining DAPI and visualized under a fluorescent microscope(Olympus BX61 equipped with a DP72 camera, Center Valley, PA).Deconvolution of selected pictures was performed using the decon-volution software Slide Book 5 (Intelligent Imaging Innovations,Denver, CO).

2.4. Plasmids and GST-fusion protein construction

GST-fusion Grb2 full length and the various truncated constructs(ΔN–SH3, ΔC–SH3 and ΔNC–SH3) were generated by PCR using astemplate the sequence of human Grb2 contained into the pGex-2T–Grb2plasmid (a kindgift of Dr. Robert J. Sheaff). The primerswere: Grb2Forward 5′-ACGGATCCATGGAAGCCATCGCCAAATATGAC, Grb2 Reverse654 Stop 5′-ACCTCGAGTTAGACGTTCCGGTTCACGGGGG, Grb2 Δ1–69Forward 5′-ACGGATCCATGGAAGAAATGCTTAGCAAACAGCGG, andGrb2 Δ128–198 Reverse-stop 5′-ACCTCGAGCCGCAGGAATATCTGCT-GGTTTCTGAGACAGATGT. Those primers were used in various combi-nations to obtain the desired amplified sequences that weresubsequently cloned into pGex-5x1 expression vector (GE HealthcareBio-Sciences Corp., Piscataway, NJ) between BamHI and XhoI sites(labeled in bold). Wild type and deletion mutants of Grb2 were alsosubcloned into pmCherry vector (BD Biosciences Clontech, MountainView, CA) between NotI and EcoRI sites.

The GST–Tat (truncated Tat86, Tat72, Tat50) constructs havebeen previously described [29]. To generate GST–Tat101 wild type fusionprotein the PCR primers were: Forward 5′-ACAAGGTACCATGGAGCCAG-TAGATCCTAGC, and Reverse 5′-ACGCGGCCGCTCAAGCGCTCGGATCT-GTCTCTG.

To generate pEYFP-Tat101 fusion protein, the PCR primers were:Forward 5′-ACAAGGTACCATGGAGCCAGTAGATCCTAGC, and Reverse5′-ACGGATCCTCAAGCGCTCGGATCTGTCTCTG. The PCR product wascloned into pEYFP-C1 plasmid (BD Biosciences Clontech, MountainView, CA) between KpnI and BamHI sites.

2.5. GST-pull-down assays

Expression and purification of GST fusion proteins were performedaccording to the manufacturer's instructions. 300–400 μg of proteinlysates from LN229 cells was incubated with purified GST–Tat fusionproteins (1 μg) bound to glutathione-agarose beads or from Tat-expressing LN229 cell lysates that were incubated with GST–Grb2

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fusion proteins bound to glutathione-agarose beads for 2 h at 4 °C atrotator. The glutathione-agarose resin was centrifuged at 14,000 ×gfor 3 min and washed 3 times with ice-cold RIPA buffer. Precipitatedproteins were suspended in 2× SDS protein-loading buffer, boiled for5 min, and subjected to 4–15% SDS-PAGE; this was followed byelectrotransfer and immunoblot analysis as described above.

2.6. SH3 domain prediction

Prediction of SH3 domain interacting sites was performed usingSH3-Hunter (http://cbm.bio.uniroma2.it/SH3-Hunter/) [30].

2.7. Protein kinase A (PKA) kinase assay

PKA activity was measured using commercially available kit (PKAassay kit, Upstate, Lake Placid, NY), according to the manufacturer'sinstructions. In brief, LN229 cells were transfected, serum starved for24 h and IGF-1 stimulated, as described above. Prior to lysis cells werewashed twice with ice-cold PBS, scraped and lysed with RIPA buffer.For measurement of PKA activity in cellular extracts, 60 μg proteinlysates were incubated with kemptide, ATP, cAMP and 2 μCi of [γ-32P]ATP in the presence of PKC/CaMK inhibitor cocktail for 10 min at30 °C. Samples from each reaction were spotted on P81 phospho-cellulose membrane, and the reaction was stopped by immersion ofthe membrane discs in 0.75% phosphoric acid. Amount of incorporat-ed [γ-32P] into the substrate was counted using a multipurposescintillation counter (Beckman LS6500).

2.8. HIV-1 LTR transactivation assay

LN229 cells were co-transfected with pEYFP-Tat101, pmCherry-Grb2 wild type or truncated mutants, an pHIV LTR–Firefly luciferasereporter plasmid [31] and a Renilla luciferase control pRL–TK plasmid(Promega, Madison, WI) using Fugene 6 (Roche Diagnostics Corp.,Indianapolis, IN). Cells were collected 24 h after transfection andsubjected to luciferase assay, as per the manufacturer's recommen-dations (Promega, Madison, WI). Firefly values were normalized withRenilla as a mean of transfection efficiency.

2.9. Statistics

Results were analyzed by an unpaired, two- or one-sided Student'st-test. p-values≤0.05 were considered statistically significant.

Fig. 1. Tat protein contains two putative SH3 domain-binding sites. A) Amino acid sequence oproteins predicted to bind TAT using SH3 Hunter web server (http://cbm.bio.uniroma2.it/SHin the Tat protein. The second column gives names of domains of interacting proteins. Thereliability (sensitivity and precision) of the prediction.

3. Results

3.1. Tat protein contains two putative SH3 domain-binding sites

Bioinformatic analysis using SH3Hunterweb server (http://cbm.bio.uniroma2.it/SH3-Hunter/) [30] revealed the presence of two putativeSH3 binding domains in the amino acid sequence of HIV-Tat full length.Given an input query protein sequence, the server identifies peptidescontaining poly-proline binding motifs (PxxP) and associates them to alist of SH3 domains, in order to create peptide–domain pairs. As shownin Fig. 1A Tat protein sequence contains two putative SH3 domainbinding sites residing in the N-terminal and C-terminal parts of theprotein between amino acids 3–8 and 81–86, respectively. According tothe SH3 Hunter program Tat is predicted to interact with RNA-bindingprotein Fus1 with a significantly high score of 0.976 and reliability(sensitivity and precision) of 95% and 60%, respectively (Fig. 1B).With aslightly lower score of 0.967, the C-terminus of Tat could interact withgrowth factor receptor-bound protein 2 domain C (Grb2-C), Grb2homolog in Caenorhabditis elegans (Sem5), Endophilin-A1 (End2) andEndophilin-A3 (End3) proteins with sensitivity of 57% and withprecision of 70%. Further, Tat is also predicted to interact with muchlower scores but with higher sensitivitywith Grb2-C, signal transducingadaptor molecule SH3 domain and ITAM motif 1 (STAM-1), and End2.Most of the proteins were predicted to bind poly-proline motif locatedbetween 81 and 86 amino acids of Tat protein. However, two domains,End2 and Grb2-C, showed ability to bind the proline rich motif of Tatlocated in the N-terminus.

3.2. Tat directly interacts with Grb2 protein

Since the SH3 domains of Grb2were recurrent in the list we decidedto test Tat–Grb2 interaction in vitro by GST-pull-down assay. The full-length Tat101 and three deletion mutants were cloned into the pGexplasmids (Fig. 2A). Tat101 and Tat1–86 constructs comprised both SH3putative binding sites; Tat1–50 contained the N-terminal putative SH3binding site, whereas Tat50–72was depleted of both. Fig. 2B shows thatTat101 (full length) as well as Tat1–86 deletionmutant was able to pulldown Grb2 protein from LN229 whole cell lysate. Some degree ofbinding was detected with GST–Tat1–50, while the Tat50–72 mutant,lacking both PxxP motifs, failed to pull down Grb2. Next, we generatedTat mutants (pEYFP-Tat101) in which the proline residues within theSH3 binding domains were substituted with alanines as follows: singleSH3 mutants had prolines 3 and 6 (P3/6A) and 81 and 84 (P81/84A)changed, respectively; the double mutant contained all four prolineresidues mutated to alanines (P3/6/81/84A). LN229 cells were trans-fectedwith thesemutants andwith the control Tatwild-type. Results ofGST-pull-down assays show that both single pEYFP-Tat–SH3 mutants

f Tat showing two possible SH3 binding sites (in bold). B) List of SH3 domain containing3-Hunter/). The first column represents amino acid sequence of peptide and its locationlast three columns in the output define respectively the significance (score) and the

Fig. 2. Tat directly interacts with Grb2. A) Scheme of GST–Tat full-length and various mutants cloned into pGex-5x-1 plasmid for GST-fusion protein expression. B) Western blot todetect Grb2 pulled down using wild type Tat101 or the various mutants. C+ represents whole cell lysate (15 μg). C) GST–Grb2 pull-down of Tat from cells transfected with Tatbearing a single SH3-binding domain mutated (P3/6A and P81/84A, respectively), the double mutation (P3/6/81/84A) or the wild type Tat (WT). D) Scheme of GST–Grb2 mutants.Grb2 full-length and three mutants ΔN, ΔC, and ΔN/C were cloned into pGex-5x-1 plasmid for GST-fusion protein expression. E) Western blot to detect Grb2 interaction with Tat incells transfected with Tat101. F) Immunoblot shows that Grb2 wt and Grb2–SH3-C can efficiently pull down Gab1, while recombinant Tat competes with Gab1 for the binding toGrb2 (Panel G).

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were efficiently pulled down by GST–Grb2 (Fig. 2C, lanes 2 and 3),although less efficiently thanwild-type Tat (lane 5). The double mutantof Tat, inwhich a total of four proline residueswere changed to alanines(P3/6/81/84A), was not pulled down by Grb2 (Fig. 2C, lane 4),confirming the prediction analysis that both putative SH3 bindingdomains of Tat may bind Grb2.

Grb2protein carries two SH3domains, SH3-1 (N-terminal) and SH3-2 (C-terminal), separated by one Src homology 2 (SH2) domainpositioned in the middle of the protein. We cloned three GST-taggedtruncated fragments of Grb2 consisting of either SH3-1 (ΔC) or SH3-2(ΔN), or depleted of both SH3 domains (ΔN/C) aswell as the full length(WT) (Fig. 2D). The four Grb2 expressing constructs were examined fortheir ability to pull down Tat from Tat-expressing cells (Fig. 2E). Resultsconfirmed that Grb2 full length directly interactedwith Tat, and, amongthe mutants, the C-terminal SH3 domain of Grb2 (ΔN) pulled down Tatmore efficiently than theN-terminal domain (ΔC) or themutant lackingboth SH3 domains (ΔN/C).

The Grb2-associated binder 1 (Gab1) protein has been reported tobind with high affinity to the C-terminal SH3 domain of Grb2 [32]. Thespecificity of such Grb2/Gab1 interaction was confirmed in our systemby GST pull-down experiments showing that Gab1 can be efficientlypulleddownbyGrb2–SH3-Cbut notGrb2–SH3-NorGrb2–SH2(Fig. 2F).We then performed a competition assay in which Gab1 was pulleddown by GST–Grb2–SH3-C in the absence or presence of recombinantTat (Fig. 2G).Asexpected, Grb2–SH3-CpulleddownGab1 in the absenceof Tat; however, addition of 1 μg of recombinant Tat efficientlycompeted with Gab1 for binding to the C-terminal domain of Grb2.

Taken together these results suggest that the interaction betweenTat and Grb2 is mediated by the PxxP motifs of Tat and the C-terminalSH3 domain of Grb2.

The Tat–Grb2 interactionwas additionally evaluated in cases of HIV-Encephalitis. Fig. 3 shows representative images of HIVE brain tissuesamples immunolabeled with Grb2 (Fig. 3A) and Tat (Fig. 3B and C). Ofnote, Tat-positive cells, mainly reactive astrocytes, are present only inareas of inflammation (Panel B), while areas within the same brainsample which do not show signs of inflammation are also negative forTat expressing cells (Panel C). Immunofluorescence performed on a

consecutive section demonstrates colocalization between Tat and Grb2in the cytoplasm of reactive astrocytes (Fig. 3E, D, and F); whileimmunohistochemistry for p24 (Panel G) shows areas of viralreplication. Immunofluorescence analysis further confirmed that p24-positive cells are also positive for Tat (Fig. 3H–J).

Immunofluorescence analysis of Tat/Grb2 interaction was addi-tionally performed on LN229 cell cultures. Panels A through F of Fig. 4show representative images of Grb2 immunolabeling (in red) in cellstransfected with the control pEYFP or YFP-Tat plasmids (in green),respectively. The colocalization of Tat with Grb2 is enhanced in thesecells due to the subcellular distribution of Tat, which takes Grb2 to thenuclear compartment.

3.3. Impairment of ERK1/2 activation by Tat

Next, we asked whether the direct binding of Tat with Grb2 wasfunctional. To this end we have utilized the LN229 Glioblastoma cellline and the IGF-1R signaling system. We first examined the phos-phorylation status of ERK1/2 in Tat-expressing Glioblastoma cellsupon IGF-1 stimulation. LN229 cells were transfected with pEYFP-Tator pEYFP, as a control, serum-starved for 24 h and then stimulatedwithIGF-1 for 2 and 10 min (Fig. 5). Results fromWestern blot analysis showbasal levels of phospho-ERKs at time 0. IGF-1 stimulation in pEYFP-transfected cells resulted in increased activation of ERKs after 2 or10 min of stimulation; whereas less phosphorylation of ERK1/2 wasobserved in Tat-transfected cells at the same time points. Reducedphosphorylation of ERKs might be due, at least in part, to the Tat-mediated nuclear translocation of Grb2, as we observed by immuno-fluorescence (Fig. 4). No differencewas observed in respect to phospho-Akt levels (data not shown). Expression of Tat in LN229 did not affectlevels of expression of IGF-IR (Fig. 5, lower panel).

3.4. Tat-mediated increased phosphorylation of c-Raf S259 inhibitorysite involves the binding of Tat with Grb2

To get inside the functional implication of Grb2–Tat interaction,we analyzed molecules in the Raf–MEK–MAPK pathway. No changes

Fig. 3. Detection of Grb2 and HIV-1 Tat in HIV-Encephalitis. Immunohistochemistry analysis shows the presence of Grb2 in the cytoplasm of reactive astrocytes in areas of neuro-inflammation (Panel A). The HIV-1 transactivator protein Tat is detected in endothelial cells, perivascular macrophages and robustly in the cytoplasm of reactive astrocytes in areasof encephalitis (Panel B). In contrast, non-affected areas of the brain within the same section show no expression of Tat (Panel C). Double labeling corroborates the modest presenceof Grb2 (Panel D, rhodamine), and abundant expression of Tat (Panel E, fluorescein), in the cytoplasm of astrocytes, where the two proteins co-localize (Panel F), as demonstrated bydeconvolution imaging (insets). G) Immunohistochemistry analysis to detect p24 in HIVE. H, I, J) Immunolabeling shows the presence of Tat in reactive astrocytes also positive forp24. Original magnification for all panels is 600× and 1000× in the insets.

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between Tat-transfected and control cells were observed when westudied the phosphorylation status of c-Raf on the activation sitesY340/Y341 (data not shown). We then determined the levels of c-Rafphosphorylation on Serine 259 (S259) inhibitory site in LN229 cellstransfected with pEYFP, pEYFP-Tat, pEYFP-Tat and pmCherry–Grb2 orpmCherry–Grb2 only. Cells, transfected and controls, were serum-starved for 24 h prior to stimulation with IGF-1 for 2 and 10 min(Fig. 6A). No phosphorylation was observed in control (pEYFP-

Fig. 4. Co-localization of Grb2 and Tat in LN229 cells. Representative images show Tat and GNote the diffuse cytoplasmic immunolabeling of Grb2 in control pEYFP transfected cells (Panmagnification is 100×.

transfected cells) at time zero, and addition of IGF-1 resulted in higherlevels of phosphorylated c-Raf, confirming previous studies [33]. Tatexpressing cells showed high level of phosphorylation of c-Raf (S259)without IGF-1 stimulation and treatment with IGF-1 did not furtherincrease phospho-c-Raf levels (Fig. 6A, lanes 4–6). If Tat/Grb2interaction is involved in increased levels of phospho-c-Raf, over-expression of Grb2 might restore IGF-1-dependent phosphorylationof c-Raf on S259. PmCherry–Grb2 vector was transfected alone or

rb2 co-localization in LN229 cells transfected with pEYFP and pEYFP-Tat, respectively.els B and C), and its nuclear translocation when Tat is present (Panels E and F). Original

Fig. 5. Inhibitory effect of Tat in IGF-IR-mediated ERK1/2 phosphorylation. Represen-tative Western blot showing activation of MAP kinases Erk1/2 by IGF-1 (40 ng/ml) inpEYFP or pEYFP-Tat expressing cells at the time points of 0, 2 and 10 min (upper panel).Total ERKs indicate equal loading (middle panel). Expression of IGF-IR is not affectedby Tat (lower panel).

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together with pEYFP-Tat. Fig. 6A (lanes 7–9) shows that over-expression of Grb2 was able to reduce phosphorylation of c-Raf(S259) in Tat-expressing cells. In general, overexpression of Grb2alone resulted in a slightly higher level of phospho-c-Raf S259 at time0, but, upon induction with IGF-1, the cells behaved essentially as thecontrols (Fig. 6A, lanes 10–12). Next, we determinedwhich SH3 site ofGrb2 is more important in preventing the Tat-mediated increase ofphospho-c-Raf S259. LN229 cells were transfected with Tat togetherwith Grb2 full-length and the three mutants, ΔN, ΔC, and ΔN/C.Serum-starvation and stimulation were done as described in Panel A.Expression of Grb2 WT inhibited phosphorylation of c-Raf S259 uponIGF-1 stimulation (Fig. 6B, lanes 1–3). Expression of Grb2–ΔC orGrb2–ΔN/C had little effect on phosphorylation of c-Raf (Fig. 6B, lanes7–12). Conversely, the C-terminal SH3 domain of Grb2 was able toinhibit phosphorylation of S259 inhibitory site of c-Raf in LN229 Tat-transfected cells even better than full-length Grb2 protein (Fig. 6B,lanes 4–6). As a result of inhibition of phosphorylation of c-Raf S259caused by WT or ΔN Grb2 expression, ERK activation was restored inthose samples (data not shown).

3.5. Protein kinase A (PKA) phosphorylates c-Raf (S259)

Previous studies have shown that c-Raf could be phosphorylatedon its inhibitory site S259 by two kinases protein kinase A (PKA) orAKT/protein kinase B (PKB) [34–36]. To investigate which of thesetwo kinases was involved in the phosphorylation of c-Raf (S259) in

Fig. 6. Phosphorylation of S259 inhibitory site of c-Raf is increased in Tat-transfectedGlioblastoma LN229 cells. A) Western blot analysis to detect phosphorylation levels ofc-Raf S259. Fold ratio of time 0 (lane 1) accepted as 1. B) Phosphorylation levels of c-RafS259 and Erk1/2 in Tat overexpressing LN229 cells transfected together with Grb2 full-length and three mutants ΔN, ΔC, and ΔN/C were detected by Western blot. Total c-Rafindicates equal loading. Experiments were repeated at least 3 times; representativeWestern blot is shown in the figure.

our system we used specific compounds to inhibit PKA or PI3-K.LN229 cells were transfected with pEYFP (lanes 1–4) or pEYFP-Tat(lanes 5–8), serum-starved for 24 h, pretreated with specific in-hibitors (50 μM of LY294002, and 25 μM of H89, lanes 3 and 7, andlanes 4 and 8, respectively) for 30 min and stimulated with IGF-1 forthe indicated times (Fig. 7A). Results illustrate inhibition of c-Raf S259phosphorylation only in control cells treated with H89, a specificinhibitor of PKA. PI3-K specific inhibitor LY294002 did not have anynoticeable consequence on S259 phosphorylation. Of interest, in cellsexpressing Tat, both compounds had an inhibitory effect on Rafphosphorylation.

Next, we determined whether expression of Tat alone or alongwith Grb2 could affect PKA activity. LN229 cells were transfected withpEYFP, pEYFP-Tat, Grb2/pEYFP-Tat and Grb2 alone, serum-starved for24 h, and stimulated with IGF-1 for 0 and 10 min. Activity of PKA inprotein extracts from pEYFP-transfected cells showed slight increaseafter IGF-1 stimulation (Fig. 7B, lanes 1 and 2). Tat-expressing cellsdisplayed a basal level of PKA activity that was about 5-fold higherthan in pEYFP-transfected cells (compare lanes 1 and 3, p≤0.05), andaddition of IGF-1 slightly decreased PKA activity (lane 4). Over-expression of Grb2 in Tat-expressing cells was able to reduce PKAactivity (lanes 5 and 6). When Grb2 was overexpressed alone therewas no statistically significant difference in PKA activity betweenIGF-1 stimulated and non-stimulated cells (lanes 7 and 8).

3.6. Inhibitory action of Grb2 on Tat transactivation and HIV replication

The activity of Tat on the HIV-1 LTRwas evaluated in LN229 cells co-transfectedwith a reporter plasmid containing the viral LTR and variousGrb2 constructs (Fig. 8A). Luciferase activity was determined 24 h post

Fig. 7. Tat-induced PKA activation mediates c-Raf (S259) phosphorylation. A) Westernblot to detect c-Raf S259 phosphorylation and activation of Erk1/2 in LN229 cellstransfected with pEYFP or pEYFP-Tat, pretreated with specific inhibitors (LY29400250 μM, H89 25 μM) for 30 min and stimulated with IGF-1 for the indicated times. B) Thekinase activity of PKA was tested in LN229 cells transfected with pEYFP, pEYFP-Tat,pEYFP-Tat together with Grb2 or Grb2 alone and stimulated with IGF-1 for 0 and10 min. Activity was assayed using commercially available kit. Asterisks indicate valuesstatistically significant (pb0.05). Ns: not statistically significant.

Fig. 8. Suppression of LTR activity and inhibition of HIV-1 replication by overexpressionof Grb2 protein. A) Promoter functional assay showing activation of HIV-LTR by Tat.LN229 cells were co-transfected with pEYFP-Tat, Grb2 wild type or truncated mutants,an HIV LTR–Firefly luciferase reporter plasmid and a Renilla luciferase control pRL-nullplasmid. 24 h later luciferase activity was measured according to the manufacturer'sprotocol. Inhibition of Tat-induced LTR by Grb2 and the indicatedmutants is statisticallysignificant: *p=0.00016, **p=0.00005, #p=0.000316, ##p=0.00907. B) ELISA assayto detect p24 in the supernatant obtained from HIV-infected primary human fetalmicroglia collected at day 1 (D1) andday 3 (D3) post-infectionwithHIV. In theHIV/Grb2sample cells were transfected with Grb2 full-length 1 day prior infection with HIV-1JF-RL. Bar graph shows the average results from three independent experiments.Asterisk indicates pb0.001.

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transfection. Among Tat and the various Grb2 constructs, only Tatactivates the HIV-1 LTR, as expected. In the presence of Grb2 wild typeor themutant containing the C-terminus SH3 domain the activity of Tatwas inhibited about three-fold (70%). The mutant of Grb2 containingthe N-terminus SH3was less efficient in lowering the activity of Tat andluciferase values were reduced about 37%. Even less efficient was theGrb2 mutant lacking both SH3 domains (SH2, 25%).

Finally, we investigated the role of Grb2/Tat interaction on viralreplication. To this end we transfected human primary fetal microgliacells with Grb2 1 day prior to their infection with HIV-1. Levels ofp24 were measured in the supernatant of cultured cells on days 1 and3 post-infection. Fig. 8B shows an approximate 30% decrease in p24in the medium of Grb2-transfected cells compared to control at bothdays 1 and 3.

4. Discussion

The human immunodeficiency virus-1 transactivating factor Tathas been shown to affect cellular functions by interfering with signaltransduction pathways [11,20,23]. This ability of Tat to interact withmany proteins may be due, at least in part, to the presence of specificmotifs in its amino acid sequence. Using SH3 Hunter bioinformaticserver [30] we have identified two putative SH3 binding domainsin the Tat protein sequence and a list of possible interacting SH3domains (Fig. 1) such as those contained in Fus1, Sem5, Grb2-C,

STAM-1, End3 andEnd2proteins. Interestingly, four of themwereeitherGrb2 (Grb2-C), Grb2 C. elegans homolog (Sem5), or Grb2-like proteins(End3 and End2), suggesting some degree of specificity in the SH3recognition sites present in Tat. Sem5 protein is the GRB2 homolog inC. elegans [37,38]. End2 and End3 (full names Endophilin-A2 andEndophilin-A3, respectively) are SH3 domain-containing GRB2-likeproteins implicated in endocytosis [39,40]. STAM-1 is a member of theSTAM (SH3 domain containing signal-transducing adaptor molecule)family of proteins, which are composed of several domain structures,including the VHS (Vps27/Hrs/STAM) domain, ubiquitin-interactingmotif (UIM), Src homology 3 domain, and a coiled-coil (CC) region.Through their CC regions, both STAM-1 and STAM-2 bind Hrs(hepatocyte growth factor-regulated substrate), a protein that islocalized on the cytoplasmic face of the early endosome [41]. Althoughwe investigated thoroughly only the interaction of Tat with Grb2, it istempting to speculate that the interaction of Tat with the abovementioned molecules, and possibly other SH3-bearing proteins, mayresult in deregulation of a wide variety of cellular processes.

Grb2 is a ubiquitously expressed adaptor protein that has mitogenicproperties and is required for several basic cellular processes [6]. It caninteract with tyrosine phosphorylated substrates, such as tyrosinekinase receptors, via the SH2 domain and with a variety of othersignaling molecules via the two SH3 domains. Grb2 is constitutivelybound to Son of sevenless (Sos), a guanine-nucleotide exchange factorthat promotes GDP–GTP exchange on Ras, whose activation then leadsto the activation ofMAPKpathway [42,43]. Direct interaction of TatwithGrb2 may displace the host protein from its usual binding partners andalter theRas/MAPKpathways. Indeed, our in vitrodata showthat Tat cancompete with Gab1 for binding to the SH3 domain of Grb2, possiblyaltering Gab1 downstream cascade. An additional mechanism bywhichTat may deregulate Grb2 downstream signaling could be mediated, atleast in cell culture, by the subtraction of Grb2 from the cytoplasmicevents. This hypothesis is supported by our data showing nuclearlocalization of Grb2 in Tat-expressing cells (Fig. 4).

Immunohistochemistry analysis of HIVE brain samples revealed amodest presence of Grb2, mainly detected in astrocytes (Fig. 3, PanelsA through F). The pattern of HIV-Tat expression appears very robust inarea of encephalitis, where the viral protein is also detected inendothelial cells, perivascular macrophages and reactive astrocytes. Incontrast, Tat is undetectable in areas of the same brain that are notaffected by inflammation. Colocalization between Tat and Grb2 wasobserved in reactive astrocytes, corroborating the in vitro bindingassays. Of interest to note that, although Grb2 is abundantly expressedin all cell types, immunohistochemistry analysis revealed stronglabeling only in reactive astrocytes. It may indicate the activation/proliferation status of these cells, as previously suggested by C. Russoet al. in their study demonstrating increased Grb2/ShcA/APP interac-tion in reactive astrocytes of Alzheimer's disease (AD) brain samples[44].

Many studies have reported the pleiotropic activity of Tat inderegulating a wide range of signaling pathways by both direct andindirect mechanisms. The presence of SH3-binding domains in thesequence of Tat may account for at least some of the various activi-ties of this viral protein. De la Fuente et al. made an interestingobservation on the ability of Tat to downregulate expression of manycellular genes, particularly on cellular tyrosine kinase receptors (RTK)and their downstream signal transduction members, including theRas–Raf–MEK pathway [45]. Perhaps impairment of the Ras/MEKpathway by Tat/Grb2 interaction may affect transcription of ERK1/2downstream targets. As the cellular signaling pathways are not linearbut cross-talk and overlap, Tat likely affects them at various levels in acomplex network of direct and indirect mechanisms. We have tried toanalyze some of them using a Glioblastoma cell line and the well-known IGF-1R signaling. Following the Ras/Raf/MAPK pathway weshowed that Tat can interact at different levels; it binds to Grb2,subtracting this adaptor molecule to the Ras/MEK pathway, while it

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potentiates the Raf inhibitory pathway by activating PKA. Shifting ofthe substrates along signaling pathways appears to be an interestingfeature of Tat. For instance, we have previously shown the ability ofTat to promote the non-canonical activation of Stat5A in PC12 cellsstimulated with NGF, which results in the inhibition of cellulardifferentiation [46]. Ultimately, since the activity of Tat on cellularprocesses appears to be cell-type specific, the end point of Tat/hostinteraction, including the Tat/SH3–client interaction, could be eitherinhibitory or stimulatory. For example, Tat has been shown tomediateactivation of ERK1/2 in endothelial cells [25] or to inhibit theiractivation in neural cells [47]. On the other hand, the outcome of Tat/host interaction may also affect some aspects of the viral cycle. Weutilized primary human fetal microglia infected with HIV-1 and foundthat over-expression of Grb2 derails the activity of Tat from its naturaltarget, the viral LTR (Fig. 8A), resulting in a lower production of p24 asa measurement of viral replication (Fig. 8B).

In summary, this report provides evidence for Tat/Grb2 directinteraction, which is mediated by the SH3 C-terminal domain of Grb2and polyproline regions of Tat. This interaction is functional andbidirectional: it affects cellular pathways as well as viral replication.Considering the high number of cellular proteins that possess oneor more SH3-binding domains, it is tempting to speculate that the Tat/SH3 interaction might be a general mechanism for Tat to interferewith many signaling pathways, and consequently, many cellularprocesses.

Acknowledgements

Slava Rom, Marco Pacifici, Giovanni Passiatore, Luis Del Valle andFrancesca Peruzzi were previously with the Department of Neurosci-ence and Center for Neurovirology at the Temple University Schoolof Medicine, Philadelphia, PA. This work was supported by NIH grantMH079751 to FP.

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