Differential Requirement for p56lck in HIV-tat Versus TNF-Induced Cellular Responses: Effects on NF-κB, Activator Protein-1, c-Jun N-Terminal Kinase, and Apoptosis

Sunil K. Manna and Bharat B. Aggarwal
J Immunol May 15, 2000, 164 (10) 5156-5166; DOI: https://doi.org/10.4049/jimmunol.164.10.5156

Abstract

HIV-tat protein, like TNF, activates a wide variety of cellular responses, including NF-κB, AP-1, c-Jun N-terminal kinase (JNK), and apoptosis. Whether HIV-tat transduces these signals through the same mechanism as TNF is not known. In the present study we investigated the role of the T cell-specific tyrosine kinase p56lck in HIV-tat and TNF-mediated cellular responses by comparing the responses of Jurkat T cells with JCaM1 cells, an isogeneic lck-deficient T cell line. Treatment with HIV-tat protein activated NF-κB, degraded IκBα, and induced NF-κB-dependent reporter gene expression in a time-dependent manner in Jurkat cells but not in JCaM1 cells, suggesting the critical role of p56lck kinase. These effects were specific to HIV-tat, as activation of NF-κB by PMA, LPS, H2O2, and TNF was minimally affected. p56lck was also found to be required for HIV-tat-induced but not TNF-induced AP-1 activation. Similarly, HIV-tat activated the protein kinases JNK and mitogen-activated protein kinase kinase in Jurkat cells but not in JCaM1 cells. HIV-tat also induced cytotoxicity, activated caspases, and reactive oxygen intermediates in Jurkat cells, but not in JCaM1 cells. HIV-tat activated p56lck activity in Jurkat cells. Moreover, the reconstitution of JCaM1 cells with p56lck tyrosine kinase reversed the HIV-tat-induced NF-κB activation and cytotoxicity. Overall, our results demonstrate that p56lck plays a critical role in the activation of NF-κB, AP-1, JNK, and apoptosis by HIV-tat protein but has minimal or no role in activation of these responses by TNF.

Human immunodeficiency virus-1 tat (HIV-tat) protein is a virally encoded transactivating protein of 76 aa that plays a critical role in viral replication and is conserved in genomes of all primate lentiviruses (1, 2). Although the primary role of HIV-tat is in regulating productive and processive transcription from the HIV-1 long terminal repeat, it also displays activities consistent with that of a secreted cytokine, TNF. It induces the expression of various cytokines including IL-6, TNF-β, IL-2, and TGF-β; and cytokine receptors including IL-2R and IL-4R (3, 4, 5, 6, 7, 8). HIV-tat stimulates the growth of some cells (8, 9, 10) and induces apoptosis in others (11, 12, 13). Like TNF, HIV-tat induces the expression of various adhesion molecules (14) and metalloproteinases (15).

The molecular mechanisms by which HIV-tat signals for this wide array of biological functions remain unknown. Some, however, could follow by alterations in the activation pathways of protein kinases and specific transcription factors. For example, HIV-tat activates the transcription factors NF-κB (16, 17) and AP-1 (18) and associated kinases of the mitogen-activated protein (MAP)3 kinase (MAPK) family, including c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and MAPK kinase (MAPKK) (18).

Several lines of evidence suggest that the T cell-specific tyrosine kinase-p56lck may play a critical role in HIV-tat signaling. p56lck is a cytoplasmic tyrosine kinase, has a molecular size of 56 kDa, is a member of the src family that is expressed highly in T cells, and binds to the cytoplasmic domain of CD4 receptor (19). It is required for T cell signaling in the human Jurkat T cell leukemia line (20). Several reports indicate that p56lck may play a role in HIV-1 infection. For instance, the HIV-1 derived protein Nef-1 has been shown to bind to p56lck (21). Defective expression of p56lck was detected in infants with severe combined immunodeficiency (22). This protein tyrosine kinase is required for triggering NF-κB activation upon interaction of HIV-1 envelope glycoprotein gp120 with cell surface CD4 (23). The suppression of T cell Ag response by the HIV gp120 was, however, found to be p56lck-independent (24). The precise role p56lck plays in the HIV-tat induced signaling is not yet known, however. To explore this question, we used JCaM1 cell line, a genetic variant of Jurkat deficient in p56lck protein due to the deletion of exon 7 in p56lck mRNA (25). We compared the cellular responses induced by HIV-tat with TNF in this cell line. We also used JCaM1 cells that had been reconstituted by transfection with the p56lck gene (20). The studies indicated that HIV-tat can activate p56lck and it is required for HIV-tat-induced activation of NF-κB, AP-1, JNK, MAPKK, and apoptosis. Although several cellular responses of HIV-tat mimicked those of TNF, p56lck was found to be not essential for TNF-induced cellular responses.

Materials and Methods

Materials

HIV-tat protein was obtained from AIDS Research and Reference Reagent Program (National Institute of Allergy and Infectious Diseases, Rockville, MD). Anti-JNK1 Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The phospho-specific anti-p44/42 MAPK (Thr202/Tyr204) Ab was obtained from New England Biolabs (Beverly, MA). Antibiotics-antimycotics (penicillin, streptomycin, and amphotericin B), RPMI 1640 medium, and FBS were obtained from Life Technologies (Grand Island, NY). Glycine, PMA, LPS, ceramide, NaCl, calpain inhibitor I, N-acetylleucylleucylnorleucinal (ALLN), and BSA were obtained from Sigma (St. Louis, MO). Bacteria-derived recombinant human TNF, purified to homogeneity with a specific activity of 5 × 107 U/mg, was kindly provided by Genentech (South San Francisco, CA). Ab against IκBα and double-stranded oligonucleotide having the AP-1 consensus sequence were obtained from Santa Cruz Biotechnology. Poly(ADP-ribose) polymerase (PARP) Ab was purchased from PharMingen (San Diego CA). Phospho-IκBα (Ser32) Ab was purchased from New England Biolabs.

Cell lines

The cell lines Jurkat (human T cells), and JCaM1 (p56lck-deficient) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). JCaM1 cells transfected with the p56lck gene were kindly supplied by Dr. Arthur Weiss (University of California, San Francisco, CA). The characterization of these cells has been previously reported (20). All cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1× antibiotics-antimycotics.

NF-κB activation assay

To assay NF-κB activation, we prepared nuclear extracts and performed EMSA as described (26).

AP-1 activation assay

The activation of AP-1 was determined as described (26).

Western blot for IκBα

To assay IκBα, postnuclear (cytoplasmic) extracts were prepared (26) from treated cells and resolved on 10% SDS-polyacrylamide gels. After electrophoresis, the proteins were electrotransferred to nitrocellulose filters, probed with rabbit polyclonal Abs against either phospho-IκBα or IκBα, and detected by enhanced chemiluminescence (ECL, Amersham-Pharmacia Biotechnology, Arlington Heights, IL).

c-Jun kinase assay

The c-Jun kinase assay was performed by a modified method as described earlier (26). Briefly, after treatment of cells (3 × 106/ml) with TNF for 10 min, cell extracts were prepared by lysing cells in buffer containing 20 mM HEPES (pH 7.4), 2 mM EDTA, 250 mM NaCl, 1% Nonidet P-40, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mM PMSF, 0.5 μg/ml benzamidine, and 1 mM DTT. Cell extracts (150–250 μg/sample) were immunoprecipitated with 0.03 μg anti-JNK Ab for 60 min at 4°C. Immune complexes were collected by incubation with protein A/G Sepharose beads for 45 min at 4°C. The beads were washed with lysis buffer (4 × 400 μl) and kinase buffer (2 × 400 μl: 20 mM HEPES (pH 7.4), 1 mM DTT, and 25 mM NaCl). Kinase assays were performed for 15 min at 30°C with GST-Jun1–79 as a substrate (2 μg/sample) in 20 mM HEPES (pH 7.4), 10 mM MgCl2, 1 μM DTT, and 10 μCi [γ-32P]ATP. Reactions were stopped with the addition of 15 μl of 2× SDS sample buffer, boiled for 5 min, and subjected to SDS-PAGE (9%). GST-Jun1–79 was visualized by staining with Coomassie blue, and the dried gel was analyzed by a PhosphorImager (Molecular Dynamics, San Jose, CA).

MAPKK assay

Cells were treated with TNF or with different concentrations of HIV-tat protein for 30 min at 37°C. The cells were washed with PBS and extracted with lysis buffer containing 20 mM HEPES (pH 7.4), 2 mM EDTA, 250 mM NaCl, 0.1% Nonidet P-40, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mM PMSF, 0.5 μg/ml benzamidine, 1 mM DTT, and 1 mM sodium orthovanadate. A 50-μg aliquot of protein was resolved on each lane on 10% SDS-PAGE, electrotransferred onto nitrocellulose membrane, and probed with the phospho-specific anti-p44/42 MAPK (Thr202/Tyr204) Ab (New England Biolabs) raised in rabbits (1:3000 dilution). The membrane was then incubated with peroxidase-conjugated anti-rabbit IgG (1:3000 dilution), and bands were detected by ECL (Amersham).

NF-κB-dependent reporter gene transcription

HIV-tat-induced NF-κB-dependent reporter gene transcription was measured as previously described (27). Briefly, cells (5 × 106) in 5 ml medium were plated in 6-well plates and then transfected with plasmid DNA (2.5 μg) for NF-κB promoter DNA that had been linked to heat-stable secretory alkaline phosphatase (SEAP) by the calcium phosphate method. After 10 h at 37°C, cells were washed, reincubated in fresh medium for 10 h, and then aliquoted 1 × 106 cells in each well and treated with different concentrations of either TNF or HIV-tat. Twenty four hours later, cell culture-conditioned medium was harvested and analyzed (25 μl) for alkaline phosphatase activity essentially as described by the protocol of Clontech (Palo Alto, CA). The activity of SEAP was assayed on a 96-well fluorescent plate reader (Fluoroscan II, Lab Systems, Chicago, IL) with excitation set at 360 nm and emission at 460 nm. This reporter system was specific because TNF-induced NF-κB SEAP activity was inhibited by overexpression of IκBα mutants lacking either Ser32 or Ser36 (27). To determine the transfection efficiency, cells were cotransfected with plasmid containing β-galactosidase gene.

Cytotoxicity assay

The HIV-tat-induced cytotoxicity was measured by the modified tetrazolium salt MTT assay (26). Briefly, cells (10,000 cells/well) were incubated in the presence or absence of the indicated test sample in a final volume of 0.1 ml for 24 h at 37°C. Thereafter, 0.025 ml of MTT solution (5 mg/ml in PBS) was added to each well. After a 2-h incubation at 37°C, 0.1 ml of the extraction buffer (20% SDS, 50% dimethyl formamide) was added. After an overnight incubation at 37°C, the OD at 590 nm were measured using a 96-well multiscanner autoreader (model MR 5000, Dynatech Laboratories, Chantilly, VA), with the extraction buffer as a blank.

Immunoblot analysis of PARP degradation

TNF-induced apoptosis was examined by proteolytic cleavage of PARP (26). Briefly, Jurkat and JCaM1 cells (2 × 106/ml) were activated with different concentrations of TNF or HIV-tat for 24 h, and then cell extracts were prepared by incubating the cells for 30 min on ice in 0.05 ml buffer containing 20 mM HEPES (pH 7.4), 2 mM EDTA, 250 mM NaCl, 0.1% Nonidet P-40, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mM PMSF, 0.5 μg/ml benzamidine, and 1 mM DTT for 30 min. The lysate was centrifuged, and the supernatant was collected. Cell extract protein (50 μg) was resolved on 7.5% SDS-PAGE, electrotransferred onto a nitrocellulose membrane, blotted with mouse anti-PARP Ab, and then detected by ECL (Amersham). Apoptosis was represented by the cleavage of 116-kDa PARP into a 85-kDa peptide product.

Measurement of reactive oxygen intermediates (ROI)

The production of ROI after treatment of cells with HIV-tat was determined by flow cytometry as described (28) by using the dye dihydrorhodamine (DHR123). R123 fluorescence intensity resulting from DHR123 oxidation was measured by a FACScan flow cytometer (Becton Dickinson) with excitation at 488 nm and detection between 515 and 550 nm. Data analysis was performed using LYSYSII software (Becton Dickinson).

p56lck kinase assay

The p56lck immuncomplex kinase assay was performed by a modified method (20). Briefly, after treatment of cells (5 × 106/ml) with either TNF or HIV-tat for 15 min, cell extracts were prepared by lysing cells in buffer containing 20 mM HEPES (pH 7.4), 2 mM EDTA, 250 mM NaCl, 1% Nonidet P-40, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mM PMSF, 0.5 μg/ml benzamidine, and 1 mM DTT. Cell extracts (800 μg/sample) were immunoprecipitated with 0.5 μg anti-p56lck Ab for 12 h at 4°C. Immune complexes were collected by incubation with protein A/G Sepharose beads for 1 h at 4°C. The beads were washed with lysis buffer (4 × 400 μl) and kinase buffer (2 × 400 μl: 20 mM HEPES (pH 7.4), 1 mM DTT, and 25 mM NaCl). Kinase assays were performed for 30 min at 37°C in 20 mM HEPES (pH 7.4), 10 mM MgCl2, 1 mM DTT, and 10 μCi [γ-32P] ATP. Reactions were stopped with the addition of 15 μl of 2× SDS sample buffer, boiled for 5 min, and subjected to SDS-PAGE (9%). p56lck autophosphorylation band was analyzed by a PhosphorImager (Molecular Dynamics).

Results

To study the role of p56lck in HIV-tat signaling, we used JCaM1 cells, which are known to be p56lck-deficient. Jurkat cells were used as p56lck-positive control. Most HIV-tat-induced cellular responses are similar to those of TNF. To determine how HIV-tat signaling differs from that of TNF, the two agents were compared throughout this study. The concentrations of HIV-tat used in our studies are comparable with that used previously (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) and are physiologically acheivable (10).

NF-κB activation

Although HIV-tat activates NF-κB (16, 17), whether activation requires p56lck is not known. Jurkat and JCaM1 cells were treated with various concentrations of either TNF or HIV-tat for 30 min, and nuclear extracts were prepared and examined for NF-κB activation by EMSA (Fig. 1⇓A). TNF activated NF-κB in both Jurkat and JCaM1 cells in a dose-dependent manner, with optimum activation at around 100 pM (upper panels). However, the overall activation of NF-κB was slightly lower in JCaM1 cells than in Jurkat cells (5.6 vs 4.4). HIV-tat activated NF-κB in a dose-dependent manner in Jurkat cells, but no activation was found in JCaM1 cells (lower panels). These results suggest that p56lck kinase is required for HIV-tat-induced but is less essential for TNF-induced activation. To determine whether this effect is time-dependent, Jurkat and JCaM1 cells were treated with either TNF (100 pM) or HIV-tat (50 ng/ml) for different times and then examined for NF-κB activation (Fig. 1⇓B). TNF activated NF-κB in both cell types with similar kinetics. However, the overall activation of NF-κB was slightly lower in JCaM1 cells than in Jurkat cells (5.9 vs 3.3). HIV-tat activated NF-κB in a time-dependent manner in Jurkat cells but did not significantly activate NF-κB in p56lck-deficient JCaM1 cells, again suggesting that lack of p56lck has more dramatic effect on HIV-tat-induced NF-κB activation than that on TNF.

FIGURE 1.
FIGURE 1.

Effect of TNF and HIV-tat on NF-κB activation. A, Jurkat and JCaM1 cells (2 × 106/ml) were stimulated with different concentrations of TNF or HIV-tat for 30 min. After these treatments, nuclear extracts were prepared and then assayed for NF-κB as described in Materials and Methods. Fold NF-κB activation was calculated based on levels in untreated Jurkat cells as one. B, Cells were incubated at 37°C with 0.1 nM TNF and 50 ng/ml HIV-tat for the indicated times. After these treatments nuclear extracts were prepared and then assayed for NF-κB. Fold NF-κB activation was calculated based on levels in untreated Jurkat cells as one. C, H2O2, okadaic acid, TNF, and HIV-tat on NF-κB activation. Jurkat and JCaM1 cells were stimulated with PMA (25 ng/ml), SA-LPS (1 μg/ml), H2O2 (250 μM), okadaic acid (500 nM), TNF (0.1 nM), and HIV-tat (50 ng/ml) for 30 min at 37°C. After these treatments nuclear extracts were prepared and then assayed for NF-κB. D, Supershift and specificity of the NF-κB. Nuclear extracts were prepared from untreated or HIV-tat-treated (50 ng/ml) Jurkat cells (2 × 106/ml), incubated for 15 min with different Abs and unlabeled NF-κB probe, and then assayed for NF-κB as described in Materials and Methods.

NF-κB activation induced by PMA, TNF, LPS, and H2O2

NF-κB is activated by a wide variety of stimuli (29), some of whose pathways differ (30). Thus we sought to examine whether p56lck is required for NF-κB activation induced by PMA, LPS, and H2O2. Jurkat and JCaM1 cells were stimulated with PMA (25 ng/ml), serum-activated (SA)-LPS (1 μg/ml), H2O2 (250 μM), TNF (0.1 nM), and HIV-tat (50 ng/ml) for 30 min at 37°C. After these treatments, nuclear extracts were prepared and then assayed for NF-κB by EMSA (Fig. 1⇑C). PMA, LPS, TNF, and H2O2 activated NF-κB in both cell types, but again HIV-tat activated the transcription factor only in Jurkat cells. These results suggest that the mechanism of activation of NF-κB by HIV-tat differs from that of other inducers.

Components of HIV-tat-induced NF-κB and specificity

Activated NF-κB typically consists of p50 and p65 homodimers or heterodimers (29). To determine the composition of the HIV-tat-induced NF-κB complex, we prepared nuclear extracts from untreated or HIV-tat-treated (50 ng/ml) Jurkat cells (2 × 106/ml), incubated them for 15 min with different Abs or unlabeled NF-κB probe, and then assayed them for NF-κB by EMSA (Fig. 1⇑D). Both anti-p50 and anti-p65 Abs supershifted the NF-κB complex, whereas irrelevant anti-cyclin D1, anti-c-Rel or preimmune serum had no effect on the complex. The NF-κB band disappeared by competition with wild-type oligonucleotides but not with mutant oligonucleotides.

IκBα degradation and phosphorylation

NF-κB activation by most inducers requires IκBα degradation (29). Previously it has been shown that NF-κB activation induced by UV, pervanadate (PV), or reoxygenation does not coincide with IκBα degradation (31). Whether p56lck is required for HIV-tat-induced IκBα degradation was also examined (Fig. 2⇓A). TNF-induced IκBα-degradation reached maximum at 15 min in both Jurkat and JCaM1 cells. HIV-tat-induced IκBα degradation occurred as early as 5 min and reached maximum at 15 min in Jurkat cells (upper panel). No HIV-tat-induced IκBα degradation was observed in p56lck-deficient JCaM1 cells (lower panel).

FIGURE 2.
FIGURE 2.

Effect of TNF or HIV-tat on phosphorylation and degradation of IκBα. A, Both Jurkat and JCaM1 cells were stimulated with either 0.1 nM TNF or 50 ng/ml HIV-tat for different times at 37°C and then assayed for IκBα in cytosolic fractions. Western blot for β-actin shows equal loading of the samples in each case. B, Cells were incubated with ALLN (100 μg/ml) for 1 h before treating with TNF (0.1 nM) or HIV-tat for 15 min, and then the Western blot analysis was done by using Abs against phosphorylated IκBα. The same blot was stripped and reprobed with nonphosphorylated IκBα.

HIV-tat did not induce IκBα phosphorylation in JCaM1 cells

It has been previously shown that PV does not induce IκBα phosphorylation in Lck-deficient JCaM1 cells (31). Here we examined what affect p56lck had on HIV-tat-induced IκBα phosphorylation. To stabilize the phosphorylated form of IκBα, cells were treated with the proteosome inhibitor ALLN (32). To detect the phosphorylated form of IκBα on the Western blot, we used Abs specific to the Ser32 phosphorylated form of IκBα. As shown in Fig. 2⇑B, TNF induced the phosphorylation of IκBα in both Jurkat and JCaM1 cells. HIV-tat induced IκBα phosphorylation in Jurkat cells but not in JCaM1 cells, suggesting that p56lck is required for HIV-tat-induced IκBα phosphorylation. Because p56lck is a protein tyrosine kinase and the IκBα phosphorylation detected is on serine, p56lck must regulate an IκB kinase that phosphorylates IκBα directly. However, we and others have shown that PV induces phosphorylation of IκBα at Tyr42 (31, 33).

NF-κB-dependent reporter gene expression

NF-κB binding to the DNA and IκBα degradation is not sufficient to suggest that p56lck is required for NF-κB-dependent reporter gene expression (34). Therefore, the effect of p56lck on HIV-tat-induced reporter gene expression was examined. As shown in Fig. 3⇓, TNF induced reporter gene expression in both Jurkat and JCaM1 cells in a dose-dependent manner, with almost 4-fold induction occurring at 1 nM. The transfection of cells with plasmid containing dominant negative form of IκBα abolished the TNF-induced NF-κB reporter activity, suggesting that gene induction is NF-κB-dependent. HIV-tat also induced expression in a dose-dependent manner in Jurkat cells but not in JCaM1 cells, suggesting that p56lck was also required for HIV-tat-induced NF-κB mediated reporter gene expression. When examined for the potential difference for the transfection efficiency by β-galactosidase assay, no difference was found between Jurkat and JCaM1 cells.

FIGURE 3.
FIGURE 3.

Effect of TNF or HIV-tat on NF-κB-dependent reporter gene expression. Both Jurkat and JCaM1 cells were transiently transfected with NF-κB-SEAP reporter gene for 10 h, washed three times, incubated for 10 h, and then exposed to different concentrations of TNF or HIV-tat for 24 h; the supernatants were then assayed for SEAP activity as described in Materials and Methods. Results are expressed as fold activity over the nontransfected control.

AP-1 activation

Most agents that activate NF-κB also activate AP-1. Our laboratory has recently shown that HIV-tat can activate AP-1 (18), but whether activation requires p56lck is not known. To determine the role of Lck in AP-1 activation, Jurkat and JCaM1 cells were treated with various concentrations of either TNF or HIV-tat for 30 min, and the nuclear extracts were prepared and examined for AP-1 activation by EMSA (Fig. 4⇓A). TNF activated AP-1 in both Jurkat and JCaM1 cells in a dose-dependent manner, with optimum activation at around 100 pM (upper panels). HIV-tat activated AP-1 in a dose-dependent manner in Jurkat cells, but no activation was found in JCaM1 cells (lower panels). These results suggest that p56lck kinase is also not required for TNF-induced AP-1 activation but is required for HIV-tat-induced activation. To determine whether this effect is time-dependent, we treated Jurkat and JCaM1 cells with either TNF (100 pM) or HIV-tat (50 ng/ml) for different times and then examined for AP-1 activation (Fig. 4⇓B). TNF activated AP-1 in both cell types with similar kinetics. HIV-tat activated AP-1 in Jurkat cells in a time-dependent manner, but again no significant activation was observed in p56lck-deficient JCaM1 cells.

FIGURE 4.
FIGURE 4.

Effect of TNF and HIV-tat on AP-1 activation. A, Jurkat and JCaM1 cells (2 × 106/ml) were stimulated with different concentrations of TNF and HIV-tat for 30 min. After these treatments, nuclear extracts were prepared and then assayed for AP-1 as described in Materials and Methods. Fold NF-κB activation was calculated based on levels in untreated Jurkat cells as one. B, Cells were incubated at 37°C with 0.1 nM TNF and 50 ng/ml HIV-tat for the indicated times. After these treatments nuclear extracts were prepared and then assayed for AP-1. Fold NF-κB activation was calculated based on levels in untreated Jurkat cells as one. C, Effect of PMA, LPS, H2O2, okadaic acid, TNF, and HIV-tat on AP-1 activation. Jurkat and JCaM1 cells were stimulated with PMA (25 ng/ml), SA-LPS (1 μg/ml), H2O2 (250 μM), okadaic acid (500 nM), TNF (0.1 nM), and HIV-tat (50 ng/ml) for 30 min at 37°C. After these treatments nuclear extracts were prepared and then assayed for AP-1. Fold NF-κB activation was calculated based on levels in untreated Jurkat cells as one. D, Supershift and specificity of the AP-1. Nuclear extracts were prepared from untreated or HIV-tat-treated (50 ng/ml) Jurkat cells (2 × 106/ml), incubated for 15 min with different Abs and unlabeled AP-1 probe, and then assayed for AP-1 as described in Materials and Methods. PIS, preimmune serum.

Like NF-κB, AP-1 can be activated by a wide variety of stimuli (35). To examine whether p56lck is required for AP-1 activation induced by PMA, LPS, and H2O2, Jurkat and JCaM1 cells were stimulated with PMA (25 ng/ml), SA-LPS (1 μg/ml), H2O2 (250 μM), TNF (0.1 nM), or HIV-tat (50 ng/ml) for 30 min at 37°C. After these treatments nuclear extracts were prepared and then assayed for AP-1 by EMSA (Fig. 4⇑C). PMA, LPS, TNF, and H2O2 activated AP-1 in both cell types, but HIV-tat activated it only in Jurkat cells. These results suggest that the mechanism of activation of AP-1 by HIV-tat differs from that of other inducers.

To determine the composition of HIV-tat-induced AP-1 complex, nuclear extracts were prepared from untreated or HIV-tat treated (50 ng/ml) Jurkat cells (2 × 106/ml), incubated for 15 min with different Abs and unlabeled NF-κB probe, and then assayed for AP-1 by EMSA (Fig. 4⇑D). Both anti-c-fos and anti-c-jun Abs supershifted the AP-1 complex, whereas irrelevant anti-cyclin D1, anti-c-Rel, or preimmune serum had no effect on the complex. The AP-1 band disappeared by competition with wild-type oligonucleotides.

JNK activation

The activation of AP-1 requires the activation of a stress-activated protein kinase, JNK (35). We have previously shown that HIV-tat can activate JNK (18), but whether p56lck is required for this activation is not known. To determine the role of p56lck in JNK activation, Jurkat and JCaM1 cells were treated with various concentrations of either TNF or HIV-tat for 15 min, and the cell extracts were prepared and examined for JNK activation by immune complex kinase assays (Fig. 5⇓). TNF activated JNK in both Jurkat and JCaM1 cells in a dose-dependent manner, with optimum activation at around 1000 pM concentration (upper panels). TNF-induced JNK activation, in comparison, was somewhat depressed in JCaM1 cells as compared with control Jurkat cells (7-fold vs 4-fold). HIV-tat activated JNK in a dose-dependent manner in Jurkat cells, but no activation was found in JCaM1 cells (lower panels). These results suggest that p56lck kinase plays a relatively less important role in TNF-induced JNK activation than it does for HIV-tat-induced activation.

FIGURE 5.
FIGURE 5.

Effect of TNF or HIV-tat on JNK activation. Jurkat and JCaM1 cells were stimulated with different concentrations of either TNF or HIV-tat as indicated for 15 min at 37°C. Then the cells were washed, and pellets were extracted and assayed for JNK activation as described in Materials and Methods. To demonstrate equal loading, 50 μg protein from same extract was analyzed in 9% SDS-PAGE to detect JNK1 by Western blot analysis.

MAPKK activation

The activation of JNK and NF-κB is regulated by an upstream kinase MAPKK, or MAP/ERK kinase (MEK) (35, 36). We have previously shown that HIV-tat can activate MEK (18), but whether p56lck is required for this activation is not known. To determine the role of p56lck in MEK activation, we treated Jurkat and JCaM1 cells with various concentrations of either TNF or HIV-tat for 30 min, prepared the cell extracts, and examined them for MEK activation by Western blot using an Ab that detects the phosphorylated form of MAPK (Fig. 6⇓). TNF activated MEK in both Jurkat and JCaM1 cells in a dose-dependent manner, with optimum activation at around 100 pM concentration (upper panels). HIV-tat activated MEK in a dose-dependent manner in Jurkat cells, but no significant activation was found in JCaM1 cells (lower panels). These results suggest that p56lck kinase plays no significant role in TNF-induced MEK activation, but it does play an important role in HIV-tat-induced activation.

FIGURE 6.
FIGURE 6.

Effect of TNF or HIV-tat on MAPKK activation. Jurkat and JCaM1 cells were stimulated with different concentrations of either TNF or HIV-tat as indicated for 30 min at 37°C. Then the cells were washed, pellets were extracted, and 50 μg protein was analyzed in 10% SDS-PAGE. Western blot was developed against anti-MAPK phosphorylated Ab (New England Biolabs). To show equal loading, the same blot was stripped and reprobed with ERK2 Ab.

Apoptosis induction

Several reports indicate that HIV-tat is cytotoxic to various cell types (11, 12, 13). Whether p56lck is required for the cytotoxic effects of HIV-tat is not known. To determine the role of p56lck in cytotoxicity, Jurkat and JCaM1 cells were treated with various concentrations of either TNF or HIV-tat for 72 h and then examined for cell viability by MTT dye uptake assay (Fig. 7⇓A). TNF induced cytotoxicity in both Jurkat and JCaM1 cells in a dose-dependent manner, the optimum effect occurring around 1 nM concentration (upper panels). HIV-tat-induced cytotoxicity in a dose-dependent manner in Jurkat cells, but no significant cytotoxicity was found in JCaM1 cells (lower panels). These results suggest that p56lck kinase plays no significant role in TNF-induced cytotoxicity but it does play an important role for HIV-tat-induced cytotoxic effects.

FIGURE 7.
FIGURE 7.

Effect of TNF or HIV-tat on cytotoxicity (A) and caspase activation (B). For A, both Jurkat and JCaM1 cells (5 × 103/0.1 ml) were treated with different concentrations of either TNF or HIV-tat for 72 h at 37°C in a CO2 incubator. Relative cell viability was then determined by the MTT method. The results shown are the mean (±SEM) optical density of triplicate assays. For B, cells were incubated with different concentrations of either TNF or HIV-tat for 24 h at 37°C in a CO2 incubator, then the cells were washed, the pellet was extracted, and Western blot performed to detect PARP cleavage.

The cytotoxic effects of TNF are mediated through the activation of a cascade of caspases (37). Caspase-2, -3, -7, and -9 are known to cleave PARP protein. Whether HIV-tat activates these caspases and whether the activation of these caspases requires p56lck are not known. To determine the role of p56lck in caspase activation, we treated Jurkat and JCaM1 cells with various concentrations of either TNF or HIV-tat for 24 h and then prepared the cellular extracts and examined them for PARP cleavage by western blot analysis (Fig. 7⇑B). TNF induced PARP cleavage in both Jurkat and JCaM1 cells in a dose-dependent manner with optimum effect at around 1 nM concentration (upper panels). TNF-induced PARP cleavage was somewhat enhanced in p56lck-deficient JCaM1 cells. HIV-tat induced PARP cleavage in a dose-dependent manner in Jurkat cells, but no significant caspase activation was found in JCaM1 cells (lower panels). These results suggest that p56lck kinase plays an important role in HIV-tat-induced activation of apoptosis, but perhaps plays little role in TNF-induced apoptosis.

ROI induction

Several reports indicate that ROI is needed for activation of NF-κB, AP-1, JNK, and apoptosis (26, 28). We have shown that ROI are also required for HIV-tat-induced cellular responses (18). Moreover p56lck has been shown to be a redox-sensitive protein kinase (38). Whether p56lck is required for the ligand-induced generation of ROI is not known. To determine the role of p56lck in ROI generation, we treated Jurkat and JCaM1 cells with either TNF (1 nM) or HIV-tat (50 ng/ml) for 3 h and then examined them for ROI generation by FACS analysis using fluorescent dihydrorhodamine dye (Fig. 8⇓). TNF induced ROI generation in both Jurkat and JCaM1 cells, whereas HIV-tat induced ROI in Jurkat cells, but not in JCaM1 cells. These results suggest that p56lck kinase plays no significant role in TNF-induced ROI production but it does play an important role for HIV-tat-induced ROI generation.

FIGURE 8.
FIGURE 8.

Effect of TNF or HIV-tat on ROI generation. Jurkat and JCaM1 cells (5 × 105/ml) were stimulated with either 1 nM TNF or 50 ng/ml HIV-tat for 3 h at 37°C in a CO2 incubator. ROI production was then determined by the flow cytometry method as described in Materials and Methods. The results shown are representative of two independent experiments.

HIV-tat-induced p56lck activation

From the studies indicated above it is clear that p56lck-deficient cells are unable to activate NF-κB, AP-1, JNK, MEK, apoptosis, and ROI induced by HIV-tat. This implies that HIV-tat must mediate its effects through activation of p56lck kinase. We have previously shown that TNF can activate Lck kinase (39). Whether HIV-tat can activate p56lck is, however, not known. To determine the activation of p56lck, we first assayed p56lck protein in Jurkat cells and its absence in JCaM1 cells by Western blot analysis using p56lck Abs. As shown in Fig. 9⇓A, p56lck protein was present in Jurkat cells but not in JCaM1 cells. Then activation of p56lck was examined by treating of cells with different concentration of either TNF or HIV-tat for 15 min and then testing them for autophosphorylation of p56lck (Fig. 9⇓B). TNF induced the autophosphorylation of Lck in Jurkat cells but not in JCaM1 cells in a dose-dependent manner (upper panels). HIV-tat also activated p56lck in Jurkat cells but not in JCaM1 cells (lower panels). These results suggest that p56lck is activated by both TNF and HIV-tat but is required only for HIV-tat-mediated cellular responses.

FIGURE 9.
FIGURE 9.

Level of p56lck expression (A) and effect of TNF or HIV-tat on p56lck autophosphorylation (B). A, Jurkat, JCaM1 and JCaM1/Lck (Lck-reconstituted cell) cells were extracted, and 200 μg protein was analyzed in 9% SDS-PAGE and Western blot was developed against monoclonal anti-Lck and detected by ECL. Western blot for p65 (NF-κB subunit) shows equal loading of the samples in each case. B, Jurkat and JCaM1 cells were stimulated with different concentrations of TNF or HIV-tat for 15 min at 37°C. Then cell extracts were prepared, and 800 μg protein was subjected to immunoprecipitation with anti-Lck Ab. Kinase assay was performed as described in Materials and Methods. Autophosphorylated p56lck was detected by radioactivity. Western blot for p56lck shows equal amount of the protein was immunoprecipitated in each case. Fold induction was calculated based on levels in untreated Jurkat cells as one.

HIV-tat-induced cellular responses can be reversed by transfection of p56lck gene in JCaM1 cells

To further confirm the role of p56lck in HIV-tat signaling, we used JCaM1 cells that had been reconstituted by transfection of the p56lck gene (20). As shown in Fig. 9⇑A, the reconstituted cells expressed p56lck protein and this protein could be activated by TNF and HIV-tat in a dose-dependent manner (Fig. 10⇓A). We further examined these cells for HIV-tat induced NF-κB activation (Fig. 10⇓B) and cytotoxicity (Fig. 10⇓C). The presence of p56lck reversed the HIV-tat-induced NF-κB activation and cytotoxicity in a dose-dependent manner, and it had no significant effect on TNF-induced activation.

FIGURE 10.
FIGURE 10.

Cellular responses are reversed after reconstitution of Lck-deficient JCaM1 cells with Lck. TNF and HIV-tat induces p56lck autophosphorylation (A), NF-κB activation (B) and cytotoxicity (C) in reconstituted JCaM1/Lck cells. A, JCaM1/Lck cells were stimulated with different concentrations of either TNF or HIV-tat for 15 min at 37°C. Then cell extracts were prepared and 800 μg protein was subjected to immunoprecipitation with anti-Lck Ab and then kinase assay was done as described in Materials and Methods. Autophosphorylated p56lck was detected from radioactive band. Western blot for p56lck shows equal amount of the protein was immunoprecipitated in each case. Fold induction was calculated based on levels in untreated Jurkat cells as one. B, JCaM1/Lck cells (2 × 106/ml) were stimulated with different concentrations of TNF and HIV-tat for 30 min. After these treatments, nuclear extracts were prepared and then assayed for NF-κB as described in Materials and Methods. Fold induction was calculated based on levels in untreated Jurkat cells as one. C, JCaM1/Lck cells (5 × 103/0.1 ml) were treated with different concentrations of TNF and HIV-tat for 72 h at 37°C in a CO2 incubator. Relative cell viability was then determined by the MTT method. The results shown are the mean (±SEM) optical density of triplicate assays.

Discussion

Although HIV-tat activates several cellular responses similar to those activated by TNF, whether they are mediated through the same mechanism is not understood. By using p56lck-deficient cells, in this report we demonstrate that lck is required for HIV-tat-induced activation of NF-κB, AP-1, JNK, MEK, and apoptosis, but not for that induced by TNF. The HIV-tat-induced generation of ROI was also defective in p56lck-deficient cells. We found that in control Jurkat cells both TNF and HIV-tat induced p56lck activity, but it was needed only for HIV-tat-induced cellular responses. The transfection of p56lck-deficient cells with p56lck gene reconstituted the HIV-tat-induced cellular responses.

This is the first report to indicate that p56lck is required for HIV-tat-mediated activation of NF-κB, AP-1, JNK, MEK, and apoptosis. Three pieces of independent evidence in our studies suggest the role of p56lck in HIV-tat signaling. First, HIV-tat signaling is interrupted in p56lck-deficient cells. Second, HIV-tat can activate p56lck kinase activity in cells where signaling is intact. Third, reconstitution of p56lck-deficient cells with p56lck gene reverses HIV-tat signaling. The activation of NF-κB by HIV-1-derived envelope glycoprotein gp120 has been shown to require p56lck (23). p56lck has also shown to be required for NF-κB activation induced by reoxygenation and by PV (31). Based on p56lck-deficient Jurkat variants, the role of p56lck in PV-induced tyrosine phosphorylation of IκBα and NF-κB activation was suggested (31). Our laboratory (33) and Imbert et al. (31) showed that PV phosphorylation of IκBα at position 42. In the present studies we found that HIV-tat induces serine phosphorylation of IκBα in Jurkat cells but not in p56lck-deficient cells, thus suggesting that p56lck indirectly affects serine phosphorylation of IκBα. Thus p56lck must modulate the function of IκBα kinase IκB kinase, which phosphorylates IκBα (40).

We showed that p56lck is required for HIV-tat-induced activation of JNK and MEK. TNF-induced activation of JNK, and MEK was found to be p56lck-independent. Our results are consistent with a previous report that T-cell Ag receptor-mediated activation of the MAPK pathway requires p56lck kinase (41). Like us, Denny et al. employed p56lck-deficient JCaM1 cells to demonstrate the requirement for p56lck. The activation of JNK by L-selectin was also found to be mediated through p56lck kinase (42).

How p56lck mediates the activation of NF-κB, AP-1, JNK, MEK, and apoptosis by HIV-tat is not clear. The activation of these cellular responses requires the generation of ROI (29, 35, 43, 44). For instance, overexpression of antioxidant enzymes superoxide dismutase and γ-glutamylcysteine synthetase has been shown to suppress the activation of NF-κB, AP-1, JNK, MEK, and apoptosis (26, 45). We found that HIV-tat induces the production of ROI in p56lck-positive Jurkat cells but not in p56lck-deficient cells. Thus it is possible that p56lck-induced ROI generation mediates the activation of NF-κB, AP-1, JNK, MEK, and apoptosis. H2O2 has been shown to activate p56lck enzyme (38), but whether p56lck can mediate ROI generation, has not been reported.

How HIV-tat induces apoptosis in various cell types is not understood (11, 12, 13). We found that HIV-tat induces caspase activation and p56lck is required for HIV-tat-induced apoptosis in Jurkat cells. That p56lck can play a role in apoptosis has been previously reported (46, 47). In agreement with our studies, it was shown recently that p56lck is required for caspase-8 activation and apoptosis in response to ionizing radiation (46). However, another group showed that in nontransformed T lymphocytes the p56lck deficiency induces cell cycle arrest and hypersusceptibitlty to apoptosis (47A ).

Our results demonstrate for the first time that HIV-tat can activate p56lck and the latter plays a major role in HIV-tat signaling. In T cells, p56lck binds to the cytoplasmic domain of CD4 and CD8 receptors. The CD4 is the major receptor for HIV-1 virus. Thus it is not too surprising that HIV-1-derived proteins mediate signaling through activation of p56lck. As indicated above HIV-1-derived gp120 activates NF-κB through p56lck (23). In addition p56lck has been shown to bind directly to an HIV-1-derived protein called Nef (21). This binding leads to the down-regulation of CD4 by HIV-nef (47). HIV-tat is a transcription factor required for HIV-1 replication (48). Although HIV-tat has been used to design therapeutics for AIDS patients (49), our results suggest that inhibitors of p56lck enzyme may also have significant therapeutic potential. Isothiazolones have been shown to inhibit p56lck (50), and thus may prove useful in blocking HIV-1 replication.

Acknowledgments

We thank Dr. N. T. Van for assistance with flow cytometry, and Walter Pagel for critically reading this manuscript.

Footnotes

  • ↵1 This work was supported by a grant from the Clayton Foundation of Research.

  • ↵2 Address correspondence and reprint requests to Dr. Bharat B. Aggarwal, Cytokine Research Section, Department of Bioimmunotherapy, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Room, Box 143, Houston, TX 77030. E-mail address: aggarwal{at}utmdacc.mda.uth.tmc.edu

  • ↵3 Abbreviations used in this paper: MAP, mitogen-activated protein; MAPK, MAP kinase; PV, pervanadate; ALLN, N-acetylleucylleucylnorlucinal; JNK, c-Jun N-terminal kinase; ERK, extracellular response kinase; MEK, MAP/ERK kinase; PARP, poly(ADP-ribose) polymerase; ROI, reactive oxygen intermediates; SEAP, secretory alkaline phosphatase; SA, serum activated.

  • Received December 9, 1999.
  • Accepted March 3, 2000.

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The Journal of Immunology: 164 (10)
The Journal of Immunology
Vol. 164, Issue 10
15 May 2000
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