The protein tyrosine kinase p56lck is required for triggering NF-kappaB activation upon interaction of human immunodeficiency virus type 1 envelope glycoprotein gp120 with cell surface CD4

Abstract

We have previously shown that NF-kappaB nuclear translocation can be observed upon human immunodeficiency virus type 1 (HIV-1) binding to cells expressing the wild-type CD4 molecule, but not in cells expressing a truncated form of CD4 that lacks the cytoplasmic domain (M. Benkirane, K.-T. Jeang, and C. Devaux, EMBO J. 13:5559-5569, 1994). This result indicated that the signaling cascade which controls HIV-1-induced NF-kappaB activation requires the integrity of the CD4 cytoplasmic tail and suggested the involvement of a second protein that binds to this portion of the molecule. Here we investigate the putative role of p56(lck) as a possible cellular intermediate in this signal transduction pathway. Using human cervical carcinoma HeLa cells stably expressing CD4, p56(lck), or both molecules, we provide direct evidence that expression of CD4 and p56(lck) is required for HIV-1-induced NF-kappaB translocation. Moreover, the fact that HIV-1 stimulation did not induce nuclear translocation of NF-kappaB in cells expressing a mutant form of CD4 at position 420 (C420A) and the wild-type p56(lck) indicates the requirement for a functional CD4-p56(lck) complex.

FIG. 1

Cell surface expression of CD4 molecules in HeLa cell lines. Cells were incubated with medium alone (white histograms) or 50 μg of anti-CD4 MAb BL4 per ml (black histograms). MAb binding was detected by a fluorescein isothiocyanate-labeled GAM Ig. The fluorescence intensity was recorded in the log mode.

FIG. 2

Detection of p56^lck expression by Western blot analysis. HeLa CD4⁺/p56^lck, HeLa CD4⁺, HeLa p56^lck, HeLa CD4⁺ Cyt⁻, HeLa CD4 (C420S)/p56^lck, HeLa CD4 (S408A)/p56^lck, and HeLa CH4 extracts containing 50 μg of total cellular proteins were electrophoresed in an SDS–10% polyacrylamide gel and blotted to a PVDF membrane. The membrane was incubated with a mixture of anti-p56^lck and antiactin MAbs and then reacted with GAM Ig-peroxidase conjugate. Bound MAbs were revealed by incubation of the membrane with ECL reagent and exposure to Hyperfilm-ECL. Controls consist of lysates from MT2 cells (a human T-cell leukemia virus type 1-transformed CD4⁺ T-cell line which lacks p56^lck expression) and CEM cells (a CD4⁺ T-cell line which expresses p56^lck).

FIG. 3

Analysis of CD4-p56^lck interaction by coimmunoprecipitation. One milligram of total cellular protein was immunoprecipitated with 13B8-2 anti-CD4 MAb. After washing, the immunoprecipitates were electrophoresed in SDS-PAGE (10% polyacrylamide) gels, transferred to PVDF membrane, and hybridized with anti-p56^lck MAbs. MAb staining was revealed by incubation of the membrane with a 1:3,000 dilution of GAM Ig. Immunoprecipitates from MT2 and CEM cellular extracts are shown as controls.

FIG. 4

Effect of iHIV treatment on NF-κB nuclear translocation in different HeLa cell lines analyzed by EMSA. Nuclear extracts prepared from HeLa CD4⁺/p56^lck, HeLa CD4⁺, HeLa p56^lck, HeLa CD4⁺ Cyt⁻, HeLa CD4(S408A)/p56^lck, HeLa CD4(C420S)/p56^lck, and HeLa CH4 cell lines cultured for 4 h in medium alone, medium containing iHIV- 1 (iHIV +) or medium supplemented with 20 ng of PMA per ml (PMA +) were reacted with radiolabeled double-stranded NF-κB oligonucleotide (HIV- 1Lai LTR sequence). The samples were electrophoresed and analyzed by autoradiography.

FIG. 5

Induction of HIV- 1 LTR transactivation by gp120-CD4 interaction. (A) Expression of gp120 on A2.01/gp120 cells (black histogram) was monitored by indirect flow cytometry. Background reactivity of anti-gp120 antibodies with gp120-negative A2.01 parental cells is shown as a control (white histogram). (B) Expression of CD4 on HeLa P4 and HeLa P4p56 (black histogram) was monitored by flow cytometry, as described in the legend to Fig. 1. The background of the probe is shown (white histogram). The CD4⁻ A2.01 cell line was used as a control. (C) Detection of p56^lck expression in HeLa P4p56 by Western blot analysis. The experiment was performed as described in the legend to Fig. 2. (D) Analysis of CD4-p56^lck interaction in HeLa P4p56 cells by coimmunoprecipitation. The experiment was performed as described in the legend of Fig. 3. The CD4⁻ p56^lck-positive A2.01 cell line was used as a control.

FIG. 6

β-Galactosidase activities in HeLa P4 and P4p56 cells. HeLa P4 (A) and HeLa P4p56 (B) cell lines expressing the β-galactosidase reporter gene cloned downstream of the HIV- 1 LTR promoter were cultured in the presence of medium alone (column 1) or medium supplemented with infectious HIV- 1 (column 2) or iHIV- 1 (column 3). In columns 4 and 5, the LTR–β-galactosidase indicator cell lines were cocultured with A2.01 cells or the A2.01/gp120 cells, respectively. After 3 days in culture, adherent cells were harvested and lysed, and β-galactosidase activities were determined by incubation of 150 μl of total cellular extracts with ONPG in an appropriated buffer. (C) HeLa P4p56 cells were treated for 16 h with 250 ng of PTX per ml (column 4) or with control medium (columns 1 to 3) and next cultured for 3 days in the presence of medium alone (column 1), medium supplemented with infectious HIV- 1 (column 2), or iHIV- 1 (columns 3 and 4). β-Galactosidase activities were determined as described above. In order to compare the results obtained from different experiments, all values were normalized according to the β-galactosidase activities obtained following infection of cells by HIV- 1 (100% β-galactosidase activity). Mean absorbances measured with HIV- 1-infected samples were 0.944, 0.472, and 0.452 in panels A, B, and C, respectively.