The HIV-1 Tat protein is a potent transactivator of viral gene expression and plays an essential role in viral replication, interacting mainly with the transactivation responsive RNA element, located within the long terminal repeat (LTR) of the viral genome. Tat is translated from a multiply spliced mRNA containing two coding exons: the first exon encodes 72 amino acids and is relatively conserved between the related HIV-1, HIV-2 and SIV viruses, whereas the second exon is less well conserved and varies considerably in length from 14 to 31 amino acids.
Five distinct functional domains have been characterized in the Tat protein: N-terminal (amino acids 1-21), cysteine-rich (amino acids 22-37), core (amino acids 38-48), basic (amino acids 49-57) and C-terminal (amino acids 73-86/101). The activation domain of Tat (amino acids 1-48) includes the N-terminal and the conserved cysteine-rich and core regions, whereas the basic domain consists of a stretch of basic amino acids necessary for nuclear localization and binding to TAR RNA. Additional functions have been mapped in the C-terminal domain of Tat encoded by the second exon, which contains an RGD motif typical of extracellular matrix proteins.
It has been demonstrated that Tat protein is actively released by infected cells[2-3], and influences the survival/growth of CD4 T cells[4-8]. However, a crucial, still not completely understood, issue is how extracellular Tat elicits its biological effects on T cell survival/growth. In fact, Tat protein can either be taken up by intact cells, reaching the nucleus quite rapidly, or it can interact with a variety of surface receptors, including integrin receptors [7,10-11] and members of the vascular endothelial growth factor (VEGF) family[12-13]. It has also been shown that the addition of extracellular Tat in culture rapidly activates a variety of signal transduction pathways, [14-16], including extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) [17-18]and c-Jun N-terminal kinase (JNK), which play a major role in T cell activation[17,20].
In this context, the aims of our study were: to characterize the dose-response of JNK and ERK/MAPK activity to extracellular Tat in CD4 T cells; to elucidate the role of the second exon of Tat in activating JNK and ERK/MAPK; and to ascertain whether GST-Tat internalization is required or not to stimulate JNK and ERK/MAPK.
Materials and methods
Jurkat CD4 T lymphoblastoid cell line (American Type Culture Collection, Rockville, Maryland, USA), was maintained in RPMI 1640 (Gibco Laboratories, Grand Island, New York, USA) supplemented with 10% foetal calf serum (FCS; Gibco) at 37°C in 5% CO2. HL3T1 cell line, a HeLa-derived cell clone containing an integrated copy of the HIV-1 long terminal repeat (LTR), was maintained in agr;-MEM supplemented with 10% FCS at 37°C in 5% CO2. In all experiments, Jurkat cells were serum-starved (RPMI with 0.5% FCS) for 24 h before adding GST-Tat protein in the presence of low (0.5%) or high (10%) levels of FCS.
Production and purification of GST-Tat fusion proteins
Plasmids pGST-Tat2E, pGST-Tat1E, pGST-Tat Argmut and pGST-TatΔN were constructed by cloning the coding regions of both exons of HIV-1HXB2 Tat (GST-Tat2E, 86 amino acids), of the first exon of HIV-1HXB2 (GST-Tat1E, 72 amino acids), and of both exons mutated in the basic region (GST-Tat Argmut, arginine residues 49, 52, 53, 55, 56 and 57 to alanine residues) or deleted in the first 21 amino acids (GST-TatΔN) in the pGEXET vector (Pharmacia, Uppsala, Sweden). The fusion proteins were purified following a previously described procedure. As recombinant GST-Tat proteins were produced in Escherichia coli (American Biotechnologies, Cambridge, Massachusetts, USA), the presence of contaminating endotoxin was checked and excluded in all preparations used in this study by a Limulus Amebocyte Lysate test (Whittakers M.A., Walkersville, Massachusetts, USA). Each batch of GST-Tat fusion proteins was tested for transactivating activity by lypofection on HL3T1 cells. After 48 h, HL3T1 cells were lysed and the clarified lysates were assayed for chloramphenicol acetyltransferase (CAT) activity using volumes of extract corresponding to equal amounts of proteins as determined by the Bio-Rad protein assay system (Bio-Rad Laboratories, Richmond, California, USA).
In most experiments, increasing concentrations of soluble GST-Tat proteins were added in culture to 24 h serum-starved Jurkat cells. In some cases, 48-well flat-bottomed polystyrene plates (Costar, Cambridge, Massachusetts, USA), were coated overnight at 4°C either with 1μM of GST-Tat fusion proteins or GST alone, as described previously[7,23]. Plates were then rinsed with AIM-V serum-free medium (Gibco BRL, Grand Island, New York, USA) to remove non-adherent proteins and medium was immediately added to the plates after the final wash. The amount of Tat coated on each well was estimated by an enzyme-linked immunosorbent assay by using anti-Tat polyclonal IgG (Intracell, Cambridge, MA, USA) followed by horseraddish peroxidase-conjugated goat anti-rabbit IgG (Dako, Copenhagen, Denmark), as described previously. Tat-coated plates were examined before and after 80 min of culture with Jurkat cells.
JNK and ERK/MAPK assays
In most cases, the JNK and ERK/MAPK assays were performed on lysates obtained from Jurkat cells pre-treated with various concentrations of soluble or immobilized Tat protein for up to 120 min. Jurkat cells (3-10×106) were lysed in 50μl of a lysis buffer containing 1μg/ml leupeptin, 1μg/ml aprotinin, 2mM Na3VO4, 10mM NaF. Nuclei were removed by centrifugation and the cytoplasmic extract was combined with 450μl of kinase buffer (20mM HEPES pH 7.4, 2mM EGTA, 50mM β-glycerophosphate, 1% Triton X-100, 10% glycerol, 1mM dithiothreitol, 2mM phenylmethanesulfonyl fluoride, 1μg/ml leupeptin, 1μg/ml aprotinin, 2mM Na3VO4, 10mM NaF). Kinases were immunoprecipitated with goat polyclonal anti-JNK1 IgG (0.5μg, C-17; Santa Cruz Biotechnology, Santa Cruz, California, USA) or with goat polyclonal anti-ERK1 (MAPK p44, 0.5μg, C-16; Santa Cruz) plus goat polyclonal anti-ERK2 (MAPK p42, 0.5μg, C-14; Santa Cruz) IgG plus 20μl protein A-Sepharose beads for 16 h. Beads were washed twice each with kinase buffer, high salt buffer (100mM Tris-HCl pH 7.6, 500mM LiCl, 0.1% Triton X-100, 1mM dithiothreitol) and assay buffer (20mM MOPS pH 7.2, 2mM EGTA, 10mM MgCl2, 0.1% Triton X-100, 1mM dithiothreitol). After the last wash, beads were left as a suspension in an equal volume of assay buffer. Kinase reactions were carried out at 30°C for 20 min after the addition of 20μl of substrate (1-3μg GST-c-Jun, and ERK-specific myelin basic protein peptide, for JNK1 and ERK1/2, respectively, both from Santa Cruz) and 15μl ATP mix (50mM MgCl2, 200μM ATP, 5-10μCi [γ-32P]ATP. Reactions were stopped by adding 25μl of 6× Laemmli buffer and boiling for 5 min. Samples were fractionated on SDS-polyacrylamide gels, electroblotted onto nitrocellulose membrane and autoradiographed or subjected to Western blot analysis using the same anti-JNK1 goat polyclonal IgG as that used for immunoprecipitation. In some experiments, JNK1 immunoprecipitated from untreated Jurkat cells were supplemented with GST-Tat 2E for 80 min at room temperature before performing the kinase assay.
Immunocytochemical detection of phospho-Ser63 c-Jun
Jurkat T cells (2×105) were supplemented with 0.1μM GST-Tat2E or 200μM As3+ for the times indicated. Cells were spun on to coverslips and fixed 5 min in cold acetone. After washing twice with phosphate-buffered saline (PBS), and blocking in PBS-1% bovine serum albumin (Sigma Chemicals, St Louis, Missouri, USA), the cells were treated for 60 min at 37°C with a 1:100 dilution of a mouse monoclonal antibody recognizing c-Jun phosphorylated on Ser63 (PhosphoPlus c-Jun Ab Kit, Biolabs, Beverly, MA, USA), followed by second layer labelling using the ABC kit (Dako). Negative controls consisted of: (i) cells untreated with either c-Jun Ser63 primary antibody or ABC reagent (background endogenous peroxidase); (ii) cells treated only with ABC reagent (background ABC reagent). The presence and distribution of c-Jun protein phosphorylated on Ser63 was investigated by optical microscopy and printed on an Ilford (Mobberley, Cheshire, UK) 100 Delta black and white film.
Western blotting analysis of c-Jun protein
Samples of 2×106 viable Jurkat cells were pre-treated or not with 100μM cycloheximide for 60 min at 37°C and then seeded in RPMI with 10% FCS and with or without 0.1μM GST-Tat2E for a further 120 min. Cell homogenates containing approximately 100μg protein, were separated on 10% acrylamide gels and blotted onto nitrocellulose filters. Blotted filters were blocked for 30 min in a 3% suspension of dried skimmed milk in PBS, and incubated overnight at 4°C with 1:500 dilution of rabbit polyclonal anti-c-Jun IgG (H-79; Santa Cruz). Filters were washed and incubated further 1 h at room temperature with 1:1500 dilution of peroxidase-conjugated goat anti-rabbit IgG (Sigma, St Louis, MO, USA) in 1% bovine serum albumin. Specific reactions were revealed with the ECL Western blotting detection reagent (Amersham Corp., Arlington Heights, Illinois, USA).
The results are expressed as mean±SD of at least three separate experiments performed in duplicate. Two tailed Student‚s t test for unpaired data was used for statistical comparison.
GST-Tat2E activates JNK in a dose-dependent manner
A series of proteins expressed in bacteria as GST fusion proteins were used in this study (Fig. 1a): full-length GST-Tat2E, one-exon GST-Tat1E, which lacks the amino acids encoded by the second exon of the tat gene, full length Tat mutated in the basic domain (GST-Tat Argmut), and GST-TatΔN, which has a deletion of the N-terminal 21 amino acids. The biological activity of GST-Tat fusion proteins was monitored in a transactivation assay performed on HL3T1 cells, stably transfected with an HIV-1 LTR-CAT construct (Fig. 1b). When lipofected into HL3T1 at the concentration of 100nM, GST-Tat2E and GST-Tat1E, but not GST-Tat Argmut or GST-TatΔN, potently stimulated CAT activity.
To determine precisely the time-course and dose-dependence effect on JNK, Jurkat cells were treated for up 80 min with increasing concentrations (0.1-100nM) of full-length GST-Tat2E in RPMI medium containing either 10% FCS or 0.5% FCS. The addition in culture of soluble GST-Tat2E stimulated JNK activity progressively from 20 min (P<0.01) onwards (Fig. 2a). The effect of GST-Tat2E was strongest under serum-starvation conditions (RPMI with 0.5% FCS), when a clear-cut, dose-dependent stimulation of JNK was noted with maximal activation at the highest concentration (0.1μM) of GST-Tat2E used (Fig. 2b). On the other hand, none of the other GST-Tat fusion proteins used in this study stimulated JNK activity over the background level observed in the presence of GST alone (Fig. 2c).
To ascertain further whether the activation of JNK required Tat internalization and protein-protein interaction with JNK or whether it was mediated by specific interactions with receptors present on the cell surface of Jurkat cells, two distinct approaches were used. In the first approach Jurkat cells were seeded on to plates pre-coated overnight with immobilized GST-Tat2E, GST-Tat1E and GST for up to 80 min. As shown in Fig. 3a, also when immobilized on plastic, GST-Tat 2E but not GST-Tat1E or GST efficiently stimulated JNK activity. To be sure that the Tat-mediated activation of JNK was truly due to extracellular Tat, the amount of GST-Tat immobilized on plastic was evaluated before and after seeding Jurkat cells on Tat-coated plates for 80 min by enzyme-linked immunosorbent assay. In three separate experiments, the enzyme immune adsorbance optical density values were similar before (0.124±0.32) and after (0.118±0.23) performing the cell cultures. These values corresponded to a concentration of GST-Tat2E protein attached to the plastic of approximately 2% (0.1μM) of that added to the plate initially (5μM).
On the other hand, when GST-Tat2E, GST-Tat1E or GST were added directly to JNK immunoprecipitates obtained from untreated Jurkat cells, no significant variations of the JNK activity were observed (Fig. 3b). These findings strongly suggest that extracellular GST-Tat 2E activates JNK by recruiting an intracellular signal cascade after specific interaction with cell surface receptors.
Both GST-Tat2E and GST1E activate ERK/MAP
When GST-Tat fusion proteins were tested for their ability to stimulate ERK/MAPK, it was found that both GST-Tat2E and GST-Tat1E stimulated efficiently (P<0.01) the ERK/MAPK activity (Fig. 4a). Of note, the activation of this pathway was maximal at Tat concentrations of 1nM and showed a plateau thereafter. On the other hand, neither GST-Tat Argmutnor GST-TatΔN showed any stimulatory activity (Fig. 4b). Similar results were observed when Jurkat cells were seeded on plates coated with immobilized GST-Tat proteins (data not shown).
Rapamycin selectively blocks the GST-Tat2E-mediated activation of JNK but not of ERK/MAPK
As extracellular Tat activates various intracellular signal transduction pathways very rapidly, we next sought to investigate the effect of different pharmacological inhibitors on the Tat-mediated activation of JNK and ERK/MAPK. Jurkat cells were treated with 100nM wortmannin (a relatively specific inhibitor of phosphatidylinositol 3-kinase), 1μM chelerythrine (a protein kinase C inhibitor), and 10nM rapamycin (an S6-kinase inhibitor) for 30 min, and then supplemented with 0.1 μM GST-Tat2E for additional 80 min.
JNK activity was potently (P<0.01) inhibited by rapamycin, and only marginally (P>0.1) by both wortmannin and chelerythrine (Fig. 5a). On the other hand, none of the pharmacological inhibitors used in this study were able to block the Tat-mediated stimulation of ERK/MAPK (Fig. 5b). A significant (P<0.01) increase of ERK/MAPK activity was observed in cells pre-treated by rapamycin. These findings further support the hypothesis that JNK and ERK/MAPK are recruited by distinct upstream pathways.
GST-Tat2E induces a rapid phosphorylation of c-Jun on Ser63 and de novo synthesis of c-Jun protein
It has been demonstrated that phosphorylation of Ser63 and Ser73 residues in the N-terminal domain of c-Jun protein by JNK stimulates the transcriptional activity of the activating protein 1 (AP1) complex. Thus, an in situ immunocytochemical analysis was performed using a specific monoclonal antibody directed against c-Jun phosphorylated on Ser63 (Fig. 6). GST-Tat2E potently stimulated c-Jun phosphorylation at Ser63 to levels comparable to those observed in the presence of 200μM As3+, used as a positive control.
As c-jun gene expression is positively autoregulated by its product, the AP1 transcription factor, the levels of c-Jun protein were next analysed to ascertain whether the Tat-induced phosphorylation of c-Jun gave rise to a functional AP1. After 120 min treatment with Tat2E, a tenfold increase in the levels of c-Jun protein was noted (Fig. 7), and this increase was abolished completely by cycloheximide, clearly indicating that this phenomenon required de novo protein synthesis.
Several lines of evidence suggest that the cytokine-like activity of Tat protein may be of clinical relevance[3,24-27]. In particular, the contribution of extracellular Tat to the progression of viral infection is highlighted by the ability of neutralizing anti-Tat antibody to reduce the viral load levels in vitro  and in vivo[24-25].
A number of reports have shown that high concentrations of extracellular Tat (0.1-1μM) are able to increase, either directly [8,17,28] or by co-operation with anti-CD3 stimulation[6,7], apoptosis in lymphoid T cells. In this respect, we have previously demonstrated that extracellular Tat triggers simultaneously an anti-apoptotic and a pro-apoptotic signal in Jurkat cells, and that these pathways prevail in the presence of low (1nM or lower) and high (0.1μM or higher) concentrations of extracellular Tat, respectively. In this study, we established that 0.1μM of full-length two-exon Tat could activate JNK potently. The concentrations of GST-Tat2E that induced maximal JNK activation were in the same range as those reported previously to induce CD4 T cell growth inhibition[4-8]. In agreement with Bossy-Wetzel et al., we found also that Tat-mediated JNK activation was enhanced by growth factor deprivation.
On the other hand, full-length extracellular Tat maximally activated ERK/MAPK in the same range of concentrations (1nM) previously reported to activate an anti-apoptotic PI-3K/Akt-dependent pathway. In this respect, it has been shown that an unbalanced activation of JNK versus ERK/MAPK with the preponderance of the former pathway leads to T cell anergy. Moreover, GST-Tat1E significantly stimulated ERK/MAPK activity to levels comparable to those induced by GST-Tat2E, indicating that although the C-terminal region of Tat was partially dispensable for ERK/MAPK activation, its presence was absolutely required for JNK activation. These findings are particularly remarkable also in consideration of recent data showing that the presence of the second exon of Tat is essential in mediating T cell apoptosis. Unfortunately, neither TatArgmut nor GST-TatΔN were informative on the domains of Tat protein involved in mediating signal transduction, as both were unable to stimulate JNK and/or ERK/MAPK. These findings only suggest that the structural integrity of correctly folded Tat protein is required to activate JNK.
JNK is known to phosphorylate c-Jun protein on two distinct Ser residues (Ser63 and Ser73), contributing to the activation of AP1[30-32]. This transcription factor is formed by members of the Jun family: Jun-Jun homodimers or Jun heterodimers with other related bZIP proteins, such as members of the Fos and ATF protein families. Although we have not addressed directly the composition of the AP1 complex in Tat-stimulated Jurkat cell extracts, our findings that Tat potently upregulated the levels c-Jun protein strongly suggest that Jun/ATF2 is the predominant AP1 complex in Tat-treated Jurkat cells. In fact, it has been clearly demonstrated that c-Jun expression is positively autoregulated by the Jun/ATF2 heterodimers. It is also noteworthy that Jun/ATF2 mediates the response to genotoxic stress, and, if the stress stimulus is so intense as to induce irreparable damage, its persistent activation may lead to apoptosis.
Experiments performed with pharmacological inhibitors showed that only rapamycin, a relatively specific inhibitor of S6 kinase, significantly inhibited JNK. On the other hand, the same drug was unable to inhibit ERK/MAPK and rapamycin acted synergistically with Tat in activating the ERK/MAPK pathway. Although we did not provide any information on the sequence of intracellular events involved in Tat-mediated JNK activation, these data suggest that different signal transduction pathways probably converge to activate JNK. As Tat protein is formed by multiple domains, which are able to interact with different receptors[3,7,10-13], this may explain why various pathways are simultaneously activated by extracellular Tat, and why the same pharmacological inhibitors differentially modulate the JNK and the ERK/MAPK pathways.
Marcuzzi et al. have reported previously that efficient Tat-mediated LTR-induction in bystander cells requires cell-cell contact. In line with these findings, Verhoef et al.  have demonstrated that Tat-mediated LTR-induction in bystander cells occurs via uptake of Tat in the cell and the nucleus and not via signalling cascades. Interestingly, the same authors have also shown that the second exon of Tat makes a small, but significant contribution to the viral fitness  and although this may be due to a lower efficiency of one-exon Tat versus two-exon-Tat in transactivating HIV-1 LTR in lymphoid cells, it is also possible that activation of JNK by extracellular two-exon Tat contributes to an optimal viral replication.
In conclusion, our data favour the hypothesis that the opposite effects of extracellular Tat on lymphoid T cell survival/growth may depend on the imbalance among: (i) the PI-3K survival signal [14,40] and the ERK/MAPK pathway, both of which are maximally stimulated by nanomolar concentrations of extracellular Tat; and (ii) the JNK pathway, which is maximally activated by micromolar concentrations of Tat.
The authors thank Prof. M. Karin for the plasmid pGEX-c-jun (1-237).
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