The Nef protein of HIV-1 is a key promoter of disease progression, owing to its dramatic yet ill-defined impact on viral replication. Previously, we have shown that Nef enhances embryonic ectodermal development Tat-mediated transcription in a manner depending on Lck and the cytoplasmic sequestration of the transcriptional repressor embryonic ectodermal development. In this study, we report that Lck is activated by Nef and targets protein kinase Cθ downstream, leading to the translocation of the kinase into membrane microdomains. Although microdomain-localized protein kinase Cθ is thought to induce the transcription factor NFκB, we unexpectedly failed to correlate Nef-induced signaling events with enhanced NFκB activity. Instead, we observed an increase in ERK MAPK activity. We conclude that Nef-mediated signaling cooperates with Nef-induced derepression and supports HIV transcription through an ERK MAPK-dependent, but NFκB-independent, pathway.

The Nef protein of human and simian immunodeficiency viruses is an important pathogenicity factor that is required for the maintenance of high viral loads and the progression to AIDS (1, 2, 3). The underlying molecular mechanisms remain ill-defined. Several Nef in vitro functions including the down-regulation of cell surface receptors (4, 5, 6, 7, 8), the enhancement of virion infectivity (9, 10), and the modulation of cellular signal transduction to lower the threshold for T cell activation (11, 12) have been identified. Their relative contribution to and overall impact on viral replication, however, remains unknown. Given the tremendous effect of Nef on viral load it seemed likely that Nef directly targets HIV transcription.

In fact, recently we demonstrated that Nef enhances HIV transcription through a derepression mechanism. Derepression was found to be caused by Nef-mediated nuclear exclusion of embryonic ectodermal development (EED), a transcriptional repressor of the family of Polycomb group proteins. For unknown reasons, Nef-mediated induction of HIV transcription also strictly depended on the presence and activity of the lymphocyte-specific tyrosine kinase Lck (13) suggestive of a requirement for positive signaling events in addition to derepression.

In the cytoplasm, EED associates with Nef and organizes the formation of a previously identified Nef-associated kinase complex (NAKC)5 containing Lck and a serine/threonine kinase of the novel protein kinase C (PKC) family (14, 15). A highly conserved α helix within the N terminus of Nef serves as the EED/NAKC interaction site. Although deletion of this region abolished Nef-induced enhancement of viral replication, Nef-mediated CD4 down-regulation was preserved (14). Continuing studies suggested that association of nPKC with Nef stimulates PKC kinase activity resulting in PKC-induced phosphorylation of Nef. This again led to perinuclear targeting of Nef and increased HIV transcription/replication (15).

Both the mechanism of PKC activation and the consequences of the Nef/Lck interaction have yet to be delineated. Based on the finding that Lck kinase activity is required for Nef-induced HIV transcription, we asked whether Lck is activated upon binding to Nef/NAKC assembly and whether it is involved in the activation of nPKC.

In this study, we demonstrated that Lck kinase activity is up-regulated upon association with Nef, leading to tyrosine phosphorylation of PKCθ and its subsequent translocation into membrane microdomains. Contrary to expectations, these events did not enhance NFκB activity but instead correlated with increased ERK MAPK activity. In summary, these results suggest that Nef promotes HIV transcription through a mechanism involving promoter derepression and synchronized ERK MAPK-dependent signaling events.

All cell lines were cultured at 37°C and 5% CO2. The human leukemia T cell line Jurkat (E6-1) and its Lck-deficient (J.CaM1.6) and IKKγ-deficient (JM 4.5.2) variants were maintained in RPMI 1640 supplemented with 10% FCS. They were transfected by electroporation or with polycationic liposomes using Metafectene Pro (Biontex) according to the manufacturer’s instructions. The Jurkat NefmER cell line was established in our laboratory as described previously (13). It is a derivate of the commercially available Jurkat Tet On cell line (Clontech), which stably expresses a tetracycline-responsive transcriptional activator consisting of the reverse Tet repressor fused to the C-terminal activation domain of the herpes simplex VP16 protein. Stable transfection of a chimeric transgene consisting of Nef and the tamoxifen-sensitive version of the estrogen receptor (mER for mutant estrogen receptor) under the control of the Tet-responsive PhCMV*-1 promoter resulted in the generation of a double-inducible cell line in which Nef expression and function are stimulated by the addition of 1 μg/ml doxycycline and 100 nM 4-hydroxytamoxifen (Sigma-Aldrich), respectively. The Jurkat NefmER cell line was maintained in RPMI 1640 supplemented with 10% FCS, 0.2 μg/ml puromycin, and 100 μg/ml G418. The human embryonic kidney cell line 293T was cultured in DMEM supplemented in 10% FCS. These cells were transfected by calcium phosphate according to the standard protocol or with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

The monoclonal anti-CD8 Ab OKT-8 was obtained and used as described previously (16). The mAbs anti-c-myc (9E10) and anti-AU1 (HISS Diagnostics), anti-β-actin (Sigma-Aldrich), anti-Lck (3A5) and anti-p65 (F-6, Santa Cruz Biotechnology), anti-phosphotyrosine (4G10, Upstate Biotechnology), anti-PKCθ (27), anti-IKKγ (C73-764), and anti-IKKα (B78-1, BD Biosciences), anti-Transferrin Receptor (H68.4, Zymed Laboratories) as well as the polyclonal Abs anti-p-Src (Y418) (Biosource), anti-ERK1/2 and anti-phospo-ERK1/2 (Cell Signaling Technology), and the HRP-conjugated secondary Abs (Promega) were all used according to the manufacturer’s instructions.

The CD8Nef (HIV-1SF2) chimeras used for 293T cell transfection were generated by a two-step PCR procedure and cloned into the pRcCMV expression vector as described previously (14). For T cell transfection, pEF expression plasmids encoding various CD8Nef constructs (wild-type, Δ12–39, Δ12–39.EDAA), Lck, kinase-deficient Lck (Lck.K273R), Hck, and various PKCθ mutants (wild-type, kinase-deficient K409R, and constitutive active A148E) were used.

Jurkat NefmER cells (1 × 108 per sample) were stimulated as indicated and lysed in 1 ml of ice-cold MBS (25 mM MES (pH 6.5), 150 mM NaCl (pH 6.5), 0.5% Triton X-100, 2 mM Na3VO4, and protease inhibitors). Lysates were homogenized by repeated passage through a 21-gauge needle, mixed with 1 ml of 85% sucrose in MBS and carefully overlaid with 6 ml of 35% sucrose and 3 ml of 5% sucrose in MBS. Following ultracentrifugation at (200,000 × g, 4°C, 16 h), 11 fractions of 1 ml were collected from the top of the gradient and analyzed by Western blotting.

DRM flotation assays of 293T cell lysates were performed as described previously (17). In brief, 293T cells were transfected with Lipofectamine 2000 (Invitrogen). Twenty-four hours posttransfection, cells were collected and lysed in ice-cold TXNE (1% Triton X-100, 50 mM Tris-HCL (pH 7.4), 150 mM NaCl, 5 mM EDTA, and protease inhibitors). Homogenization was conducted by pipetting up and down 50 times. The lysates were then adjusted to 40% Optiprep and overlaid with 2.5 ml of 28% Optiprep and 0,6 ml of TXNE. After ultracentrifugation (SW60Ti rotor, 35,000 rpm, 4°C, 3 h) eight fractions of 500 μl were collected from the top of the gradient and analyzed by Western blotting.

The purity of detergent-resistant and soluble fractions was confirmed by Western blotting with peroxidase-conjugated cholera toxin B and α-Transferrin Receptor, respectively.

Using Metafectene Pro (Biontex) various T cell lines were transfected with the reporter plasmid pLTR luc (firefly luciferase under the control of the HIV promoter), an internal control reporter pGL.4.70 (promoterless renilla luciferase plasmid), suboptimal levels of Tat, and different combinations of wild-type or mutant Lck, PKC and Nef expression plasmids. Sixteen hours post transfection, cell lysates were prepared and assayed for luciferase activity using the Dual Luciferase Assay System from Promega. Relative luciferase activities were calculated by normalizing firefly luciferase activities to renilla luciferase activities. The experiments were performed in triplicate at least three independent times. Mean values were calculated and plotted as fold induction over background (pLTR luc alone) with error bars indicating the SEM.

Transfected or stimulated cells were lysed in 1 ml of lysis buffer (0.5% Nonidet P-40, 2 mM EDTA, 137 mM NaCl, 50 mM Tris, 10% glycerol (pH 8.0), freshly supplemented with 2 mM PMSF, 20 mM sodium fluoride, and 2 mM sodium orthovanadate) for 1 h at 4°C. Following a 10-min centrifugation step at 13,000 rpm and 4°C to pellet unsolubilized cell debris, supernatants were collected and incubated with 30 μl of protein A-Sepharose beads (Amersham Biosciences) and 1 μg of specific Ab for an additional hour at 4°C. The bead-bound immunocomplexes were then washed three times with high salt wash buffer (0.5% Nonidet P-40, 450 mM NaCl, 2 mM EDTA, 50 mM Tris, 10% glycerol (pH 8.0)), resuspended in Laemmli sample buffer and separated by SDS-PAGE. The separated proteins were transferred to nitrocellulose and Western blotting was performed according to standard procedures using ECL substrate (Pierce).

For in vitro kinase assay, protein A-Sepharose-bound immunocomplexes were subject to three washes with high salt buffer as described above and one additional wash with kinase activation buffer (KAB: 1% Triton-X, 137 mM NaCl, 50 mM Tris, 5 mM MgCl2, (pH 8.0)). Subsequently, they were resuspended in 50 μl of KAB containing 10 μCi 32P γdATP and incubated for 10 min at room temperature. The kinase reaction was stopped through the addition of 1 ml of high salt buffer. The immuncomplexes were pelleted, washed three times, and resuspended in Laemmli sample buffer. The degree of phosphorylation was quantified by autoradiography following protein separation by SDS-PAGE.

To assess IκB phosphorylation, recombinant GST-IκB was added before the in vitro kinase reaction. To stop the reaction, Laemmli sample buffer was added directly to the sample without previous wash steps.

Jurkat cells (1 × 107 per sample) were stimulated as indicated and subsequently resuspended in 300 μl of hypotonic lysis buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.4% NP40, 1 mM DTT, freshly supplemented with 2 mM PMSF). The samples were homogenized by pipetting up and down several times and incubated on ice for 5 min. Subsequently, the nuclei were pelleted by centrifugation. The postnuclear supernatant was transferred to a fresh tube while the pelleted nuclei were washed with 500 μl of hypotonic buffer in the absence of detergent and then lysed through repeated vortexing in 50 μl of nuclear extraction buffer (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, freshly supplemented with 2 mM PMSF). Both the nuclear extract and the postnuclear supernatant were centrifuged again for 10 min at 13,000 rpm. Protein concentration of the supernatants was assessed and equal amounts of protein were loaded on a SDS gel.

Previously, we demonstrated that Nef enhances Tat-dependent HIV transcription in an Lck-dependent fashion (13). To further investigate the cooperation between Nef and Lck, we applied a previously established HIV transcription reporter assay (13). The Lck-deficient Jurkat variant J.CaM1.6 was cotransfected with a HIV LTR luciferase reporter construct, suboptimal levels of Tat, wild-type, or kinase-deficient Lck (Lck.KR), and wild-type or N-terminally mutated Nef fused to the extracellular domain of CD8 (CD8Nef; CD8NefΔ12–39). Previous studies indicated that Nef function requires membrane localization (18). Because Nef is rapidly internalized following membrane targeting, CD8-tagging serves to stabilize Nef at the membrane thus generally leading to more pronounced effects (16). Analysis of cell-associated luciferase activities confirmed that CD8Nef and Lck cooperatively induce the HIV promoter. By contrast, Lck or CD8Nef alone only marginally augmented Tat-dependent HIV promoter activity (Fig. 1). Importantly, the observed CD8Nef/Lck cooperation critically depended on two conditions; first, the presence of an N-terminal α helix in Nef that serves as the binding site for NAKC (13, 14, 15), as CD8NefΔ12–39 failed to enhance but rather down-modulated Lck-mediated HIV transcription, and second, Lck kinase activity, as wild-type CD8Nef failed to cooperate with kinase-deficient Lck. For comparison, we also evaluated the ability of untagged Nef to cooperate with Lck in the induction of HIV transcription. Again, we observed a decrease in Nef/Lck-induced LTR promoter activity with the Nef construct lacking the previously described N-terminal α helix. Taken together, these results suggested that Nef-induced HIV transcription requires the recruitment of functional Lck to the N terminus of Nef.

FIGURE 1.

Nef-mediated increase of HIV transcription requires the Nef N terminus and Lck kinase activity. Lck-deficient JCaM.1 T lymphocytes were transiently transfected with a pLTR luciferase reporter construct (0.3 μg), a control reporter plasmid (pGL4.70; 0.3 μg), suboptimal levels of Tat (0.05 μg), wild-type Lck or kinase-deficient Lck.KR (2 μg) and either untagged or CD8-tagged wild-type or N-terminally mutated Nef (Δ12–39; 3.5 μg). Sixteen hours posttransfection, cell lysates were prepared and luciferase activities assessed. Relative luciferase activities were calculated by normalizing firefly luciferase (pLTR luc) to Renilla luciferase (pGL4.70) activities. Shown is fold increase, calculated as the relative luciferase activity of the sample divided by the relative luciferase activity of the control (i.e., cells transfected with pLTR luc alone).

FIGURE 1.

Nef-mediated increase of HIV transcription requires the Nef N terminus and Lck kinase activity. Lck-deficient JCaM.1 T lymphocytes were transiently transfected with a pLTR luciferase reporter construct (0.3 μg), a control reporter plasmid (pGL4.70; 0.3 μg), suboptimal levels of Tat (0.05 μg), wild-type Lck or kinase-deficient Lck.KR (2 μg) and either untagged or CD8-tagged wild-type or N-terminally mutated Nef (Δ12–39; 3.5 μg). Sixteen hours posttransfection, cell lysates were prepared and luciferase activities assessed. Relative luciferase activities were calculated by normalizing firefly luciferase (pLTR luc) to Renilla luciferase (pGL4.70) activities. Shown is fold increase, calculated as the relative luciferase activity of the sample divided by the relative luciferase activity of the control (i.e., cells transfected with pLTR luc alone).

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Because Lck functionality appears to be critical for Nef-mediated induction of HIV transcription, we asked whether Lck is activated as a consequence of its association with Nef/NAKC. To assess this question, 293T cells were transiently transfected with expression plasmids for Lck or the macrophage-specific Src kinase Hck and two Nef mutants fused to the extracellular domain of CD8. The 1–49 Nef mutant only contains the first 49 aa of Nef, the region containing the NAKC binding domain, whereas the full-length Δ12–39 Nef mutant lacks this very site. Of note, both Hck (19) and Lck (20, 21) were previously reported to associate with a PXXP-motif at position 73–76 in the Nef core domain. However, only Hck but not Lck kinase activity is induced as a consequence thereof. Following immunoprecipitation of the various CD8Nef constructs, the precipitates were analyzed for Lck/Hck binding by Western blotting (Fig. 2,A) or kinase activity by in vitro kinase assays (Fig. 2,B). In contrast to Hck, which required the PXXP-motif for binding, Lck associated with the Nef N terminus as well as the core domain (Fig. 2,A). In agreement with published data, binding of Hck to the core domain of Nef activated its kinase activity (Fig. 2,B, lower panel). Conversely, interaction with the Nef N terminus but not the core domain potently stimulated Lck kinase activity (Fig. 2 B, upper panel).

FIGURE 2.

Nef stimulates Lck kinase activity. 293T cells were transiently transfected with myc-tagged Lck or Hck and various CD8-tagged Nef constructs as indicated. One day posttransfection, cell lysates were prepared and immunoprecipitated with anti-CD8 Ab (A and B) or anti-myc Ab (C). The immunoprecipitates were then analyzed for Lck/Hck interaction by Western blotting with anti-myc Ab (A) or subject to in vitro kinase assay to assess the activity of the precipitated kinases (B and C). Fold induction of Lck activity compared with basal levels was calculated following quantification by densitometric analysis using the Kodak Image Station 2000R (C). Equal expression of Lck/Hck was confirmed.

FIGURE 2.

Nef stimulates Lck kinase activity. 293T cells were transiently transfected with myc-tagged Lck or Hck and various CD8-tagged Nef constructs as indicated. One day posttransfection, cell lysates were prepared and immunoprecipitated with anti-CD8 Ab (A and B) or anti-myc Ab (C). The immunoprecipitates were then analyzed for Lck/Hck interaction by Western blotting with anti-myc Ab (A) or subject to in vitro kinase assay to assess the activity of the precipitated kinases (B and C). Fold induction of Lck activity compared with basal levels was calculated following quantification by densitometric analysis using the Kodak Image Station 2000R (C). Equal expression of Lck/Hck was confirmed.

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For confirmation, we repeated the experiment in a reverse orientation, immunoprecipitating Lck after cotransfection of different CD8Nef constructs. In general, CD8Nef constructs containing the Nef N terminus (CD8Nef, CD8Nef.1–49 and CD8Nef.EDAA, an internalization-defective Nef mutant) increased Lck kinase activity, whereas constructs lacking the N-terminal α helix (CD8NefΔ12–39 and CD8NefΔ12–39.EDAA) failed to significantly enhance Lck kinase activity. The rather weak effect of wild-type CD8Nef in comparison to CD8Nef.1–49 and CD8Nef.EDAA may be explained by its enhanced internalization, which occurs despite the presence of the CD8 tag. Accordingly, due to the absence of the C-terminal ED internalization motif (22), CD8Nef.1–49 and CD8Nef.EDAA were likely more effective in stimulating Lck kinase activity. Although the results in Fig. 2,C are less striking than in Fig. 2 B, they confirm that the increase in Nef-associated Lck kinase activity is mirrored by an increase in total cellular Lck kinase activity.

To confirm these findings in the more physiological T cell environment, we used a previously described Nef-inducible Jurkat T cell line (13). This cell line controls Nef expression on the transcriptional and protein level. Induction with doxycycline induces the expression of an inactive NefmER (mER for mutant estrogen receptor) fusion protein, which is rendered functional within minutes of tamoxifen addition to the culture medium. Using this cell line, we assessed the kinetics of endogenous Lck kinase activation following induction of Nef expression/function and immunoprecipitation of Lck. We observed a transient increase in Lck kinase activity, which peaked at 10 min of Nef expression (data not shown). After 60 min, kinase activity returned to background levels most likely due to rapid internalization/inactivation of Nef (16). In addition, overall protein tyrosine phosphorylation levels in the cell extracts increased correlatively with Lck kinase activity, which may suggest phosphorylation of multiple downstream targets by active Lck. Taken together, the results clearly demonstrated that binding of Lck to the Nef N terminus stimulates Lck kinase activity.

Next, we asked which downstream factors are targeted by Nef-activated Lck. Recently, we identified the serine/threonine kinase contained in NAKC as a member of the novel PKC family (15). We found that PKC is activated upon binding to Nef, but the mechanism of its activation remained unresolved. In 2000, Liu et al. suggested that Lck regulates PKCθ function during T cell activation via Lck-mediated tyrosine phosphorylation (23). We explored the possibility that the NAKC effect on HIV transcription is due to Lck-mediated activation of PKCθ. Although we were previously unable to exclude PKCδ as a possible Nef interaction partner (15), we here concentrate on PKCθ, as this family member appears to be the major NAKC component in T cells.

We applied the above-described reporter assays and found that coexpression of wild-type Lck and PKCθ in Lck-deficient J.CaM1.6 cells enhanced LTR promoter activity ∼20-fold even in the absence of Nef (Fig. 3,A). The observation that transcription was decreased in the presence of either kinase-deficient Lck (Lck.KR) or kinase-deficient PKCθ (PKC.KR) supported the involvement of both kinases in HIV transcriptional regulation. Importantly, expression of constitutive active PKCθ (PKC.AE) along with kinase-deficient Lck not only rescued but greatly enhanced HIV promoter activity, an effect that was partially replicated by cotransfection of wild-type but not N-terminally mutated Nef along with wild-type PKCθ and Lck. Taking into account the findings of Liu et al. that Lck regulates PKCθ function during T cell activation and our previous observation that wild-type but not N-terminally mutated Nef activates Lck, these results suggested that the Nef effect on transcription may be due to Nef-activated Lck targeting PKCθ downstream. To further investigate this hypothesis, we cotransfected various Lck, PKC, and Nef constructs into 293T cells and immunoprecipitated either Lck or PKCθ. Subsequent Western blotting with α-PKC or α-phosphotyrosine Ab 4G10, respectively, confirmed the findings of Liu et al. that Lck and PKCθ interact resulting in PKCθ tyrosine phosphorylation (Fig. 3,B, lane 2). Importantly, however, this interaction was more pronounced in the presence of Nef leading to an enhancement of PKCθ tyrosine phosphorylation (Fig. 3,B, lane 3), which is likely due to both increased Lck binding and Nef-mediated enhancement of Lck activity. The latter was demonstrated in Fig. 2 and is here evident in the increase in Lck tyrosine phosphorylation in the presence of wild-type but not N-terminally mutated Nef. Accordingly, Δ12–39 Nef promoted PKCθ tyrosine phosphorylation to a significantly lesser extent than wild-type Nef (Fig. 3,B, lane 5). Of note, while PKCθ tyrosine phosphorylation critically depended on cotransfection of functional Lck also the Lck/PKCθ interaction appeared to be regulated by Lck kinase activity (Fig. 3 B, lane 4) suggesting that PKCθ phoshorylation may strengthen its interaction with Lck.

FIGURE 3.

PKCθ is a downstream effector of Lck and cooperatively promotes HIV transcription. A, J.CaM1.6 T lymphocytes were transfected with a pLTR luciferase reporter construct, a control reporter plasmid (pGL4.70), suboptimal levels of Tat and different Lck (Lck, Lck.KR), PKCθ (PKC, PKC.AE), and Nef (CD8Nef, CD8NefΔ12–39) constructs as indicated. Sixteen hours posttransfection, firefly luciferase activity (pLTR luc) in the cell lysates was assessed, normalized to control renilla luciferase activity (pGL4.70) and presented as fold induction over the control (pLTR luc alone). The pGL4.70 reporter was not significantly affected by expression of Nef or other signaling molecules (see Supplemental Fig. 1). B, Cell lysates of 293T cells expressing PKCθ along with either wild-type or kinase-deficient Lck (Lck.KR) and wild-type or N-terminally mutated Nef (NefΔ12–39) were immunoprecipitated with anti-Lck and Western blotted with anti-PKCθ to analyze the interaction of PKCθ with Lck or immunoprecipitated with anti-PKCθ and Western blotted with anti-phosphotyrosine Ab 4G10 to assess PKCθ as well as associated Lck tyrosine phosphorylation. Equal expression levels of Lck and PKCθ were confirmed by Western blotting with anti-Lck and anti-PKCθ, respectively. C, Jurkat T cells were electroporated with pSuper plasmid expressing shRNA targeting PKCθ or empty vector as control. On day 2 and 3, the cells were again transfected with pLTR luc, suboptimal levels of Tat, Lck, and Nef. The following day cell-associated luciferase activities were assessed and analyzed as previously described. Knockdown efficiency was monitored by Western blotting of the whole cell lysates with anti-PKCθ on day 2, 3, and 4 postelectroporation.

FIGURE 3.

PKCθ is a downstream effector of Lck and cooperatively promotes HIV transcription. A, J.CaM1.6 T lymphocytes were transfected with a pLTR luciferase reporter construct, a control reporter plasmid (pGL4.70), suboptimal levels of Tat and different Lck (Lck, Lck.KR), PKCθ (PKC, PKC.AE), and Nef (CD8Nef, CD8NefΔ12–39) constructs as indicated. Sixteen hours posttransfection, firefly luciferase activity (pLTR luc) in the cell lysates was assessed, normalized to control renilla luciferase activity (pGL4.70) and presented as fold induction over the control (pLTR luc alone). The pGL4.70 reporter was not significantly affected by expression of Nef or other signaling molecules (see Supplemental Fig. 1). B, Cell lysates of 293T cells expressing PKCθ along with either wild-type or kinase-deficient Lck (Lck.KR) and wild-type or N-terminally mutated Nef (NefΔ12–39) were immunoprecipitated with anti-Lck and Western blotted with anti-PKCθ to analyze the interaction of PKCθ with Lck or immunoprecipitated with anti-PKCθ and Western blotted with anti-phosphotyrosine Ab 4G10 to assess PKCθ as well as associated Lck tyrosine phosphorylation. Equal expression levels of Lck and PKCθ were confirmed by Western blotting with anti-Lck and anti-PKCθ, respectively. C, Jurkat T cells were electroporated with pSuper plasmid expressing shRNA targeting PKCθ or empty vector as control. On day 2 and 3, the cells were again transfected with pLTR luc, suboptimal levels of Tat, Lck, and Nef. The following day cell-associated luciferase activities were assessed and analyzed as previously described. Knockdown efficiency was monitored by Western blotting of the whole cell lysates with anti-PKCθ on day 2, 3, and 4 postelectroporation.

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To substantiate the argument that PKCθ is involved in Nef-mediated HIV transcription, we knocked down endogenous PKCθ expression in Jurkat T cells by RNA interference and measured the induction of HIV transcription by Lck and Nef (Fig. 3 C). On day 3 of shPKCθ expression, Nef/Lck-mediated HIV transcription was decreased to 57% and on day 4 of shPKCθ expression to 27%. These figures correlated well with PKCθ knockdown efficiency, which was 49% on day 3 and 74% on day 4.

In summary, the results established that PKCθ is a downstream target of Nef-activated Lck, regulated through Lck-mediated phosphorylation, and is part of a signaling cascade initiated by Nef to promote HIV transcription.

Recruitment of PKCθ to membrane microdomains at the immunological synapse represents a hallmark of T cell activation (24). Because Nef has been shown to localize to glycolipid-enriched microdomains and has been proposed to activate or at least prime T cells for activation (12, 16, 25), we asked whether Nef induces the recruitment of PKCθ into membrane microdomains as part of its activating effect on T cells.

We used a flotation assay to isolate DRMs after cell lysis in cold detergent and analyzed the DRM localization of transfected PKCθ in 293T cells upon coexpression of Nef or phorbol ester (PMA) treatment as positive control. Similar to PMA stimulation, Nef expression induced the enrichment of PKCθ in DRMs floating at the top of the gradient (Fig. 4,A; fraction 2). In parallel, we investigated the DRM localization of endogenous PKCθ in the Nef-inducible Jurkat T cell line following induction of Nef expression or stimulation with PMA. Again, we observed PKCθ but not PKCδ (data not shown) redistribution to DRMs within 15 min of Nef expression (Fig. 4 B).

FIGURE 4.

Nef expression recruits PKCθ into membrane microdomains. 293T cells overexpressing PKCθ (A) or Jurkat NefmER T lymphocytes (B) were stimulated with phorbol ester (PMA) for 15 min before cell lysis and subsequent discontinuous density ultracentrifugation and compared with 293T cells coexpressing PKCθ and Nef.GFP (A) or Jurkat NefmER cells induced for Nef expression by overnight incubation with doxycyline and 15 min stimulation with tamoxifen (B), respectively. Detergent-resistent membrane (DRM) and soluble (S) fractions were collected and analyzed for expression of PKCθ and Nef (A) or PKCθ, Nef, Lck and IKKα (B). Western blotting with peroxidase-conjugated cholera toxin B (CTxB) and anti-Transferrin receptor Ab served to identify detergent-resistant and soluble fractions.

FIGURE 4.

Nef expression recruits PKCθ into membrane microdomains. 293T cells overexpressing PKCθ (A) or Jurkat NefmER T lymphocytes (B) were stimulated with phorbol ester (PMA) for 15 min before cell lysis and subsequent discontinuous density ultracentrifugation and compared with 293T cells coexpressing PKCθ and Nef.GFP (A) or Jurkat NefmER cells induced for Nef expression by overnight incubation with doxycyline and 15 min stimulation with tamoxifen (B), respectively. Detergent-resistent membrane (DRM) and soluble (S) fractions were collected and analyzed for expression of PKCθ and Nef (A) or PKCθ, Nef, Lck and IKKα (B). Western blotting with peroxidase-conjugated cholera toxin B (CTxB) and anti-Transferrin receptor Ab served to identify detergent-resistant and soluble fractions.

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To determine the relevance of microdomain recruitment of PKCθ for HIV transcription, we monitored the Nef/Lck-mediated induction of HIV transcription in cells treated with the cholesterol depleting chemical compound methyl-β-cyclodextrin (MβCD), which inhibits microdomain formation. To our surprise, HIV transcription was maximally enhanced in the presence of 10 mM MβCD for 1 or 2 h before cell lysis (see Supplemental Fig. 2).6 When we assessed the effects of MβCD treatment biochemically, we found that both Lck phosphorylation and overall tyrosine phosphorylation markedly increased within 15 min and ERK phosphorylation within 30 min of MβCD treatment (see Supplemental Fig. 2). These findings are in line with previous results by Kabouridis et al. (26), who demonstrated that MβCD treatment induces signaling events similar to T cell activation. Cholesterol depletion may thus lead to aberrant signaling events possibly due to abolishing the compartimentalization of signaling molecules within the plasma membrane. However, a clear interpretation of these results is difficult, as MβCD is highly toxic leading to significant cell death as early as 30 min after addition to the culture medium.

In summary, the results demonstrated that similar to T cell activation events Nef expression induces the translocation of PKCθ into membrane microdomains.

PKCθ is a known and important signaling intermediate that connects T cell activation signals with the transcription factor NFκB and recruitment of PKCθ to membrane microdomains is believed to serve as a prerequisite to this effector function (27). TCR-induced NFκB activation has been demonstrated to require the CARMA/Bcl10/MALT1 (CBM) complex, which mediates the activation of the IKK signalosome through a complex mechanism involving the ubiquitin ligase TRAF6 and the TGF-activated kinase 1 complex (28). In T cells, CBM assembly was suggested to depend on PKCθ-mediated phosphorylation of membrane-associated CARMA, which triggers a conformational change in CARMA resulting in the recruitment of the other CBM subunits Bcl10 and MALT1 (29, 30). In addition, PKCθ has been proposed to directly recruit the IKK complex to membrane microdomains (27, 31). Therefore, we asked whether Nef-induced PKCθ microdomain translocation activates NFκB. We first immunoprecipitated IKKγ-associating IKK complexes from cells induced for Nef expression and assessed phosphorylation of recombinant GST-IκB protein in vitro. Unlike PMA stimulation, induction of Nef expression did not result in a detectable increase in IκB phosphorylation (Fig. 5,A). In line with these results, Nef expression failed to induce the nuclear translocation of the RelA/p65 NFκB subunit (Fig. 5,A). In addition, Nef still activated HIV transcription in the mutant Jurkat T cell line JM 4.5.2 that lacks IKKγ expression (Fig. 5 B).

FIGURE 5.

Nef does not activate NFκB. A, Jurkat NefmER cells were either stimulated with PMA for 15 min or induced for Nef expression for increasing periods of time as indicated. Cell lysates were prepared, immunoprecipitated with α-IKKγ Ab and subject to in vitro kinase assay in the presence of recombinant GST-IκB as phosphorylation substrate. Following SDS-PAGE and transfer to nitrocellulose, the degree of GST-IκB phosphorylation was assessed by autoradiography. In parallel, nuclear extracts were prepared and assayed for p65 expression by Western blotting. Nef expression was confirmed and served as a loading control. B, IKKγ-deficient and wild-type Jurkat cells were transfected with pLTR luc, suboptimal levels of Tat and Nef. Sixteen hours posttransfection, lysates were prepared and luciferase activities measured. Shown is fold increase, calculated as the relative luciferase activity divided by the relative luciferase of the control (i.e., cells transfected with pLTR luc alone).

FIGURE 5.

Nef does not activate NFκB. A, Jurkat NefmER cells were either stimulated with PMA for 15 min or induced for Nef expression for increasing periods of time as indicated. Cell lysates were prepared, immunoprecipitated with α-IKKγ Ab and subject to in vitro kinase assay in the presence of recombinant GST-IκB as phosphorylation substrate. Following SDS-PAGE and transfer to nitrocellulose, the degree of GST-IκB phosphorylation was assessed by autoradiography. In parallel, nuclear extracts were prepared and assayed for p65 expression by Western blotting. Nef expression was confirmed and served as a loading control. B, IKKγ-deficient and wild-type Jurkat cells were transfected with pLTR luc, suboptimal levels of Tat and Nef. Sixteen hours posttransfection, lysates were prepared and luciferase activities measured. Shown is fold increase, calculated as the relative luciferase activity divided by the relative luciferase of the control (i.e., cells transfected with pLTR luc alone).

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Collectively, these findings suggested that Nef-mediated recruitment of PKCθ into membrane microdomains does not lead to the activation of the transcription factor NFκB. In line with this conclusion, Nef expression failed to translocate IKKα into DRMs (Fig. 4 B), which suggests that PKCθ membrane microdomain translocation is not sufficient to recruit IKK.

Nef’s lack of influence on NFκB led us to the speculation that other T cell signaling pathways are targeted through the translocation of PKCθ into membrane microdomains. Several recent reports indicated a connection between PKCθ and ERK MAP kinase (32, 33). In addition, Schrager et al. (34) demonstrated that Nef enhances TCR-mediated ERK activation. We thus asked whether the Nef/Lck/PKCθ cooperation targets ERK MAPK.

To assess this question, 293T cells were transiently transfected with expression constructs for different Nef, Lck, and PKCθ variants (Fig. 6,A). One day later, whole cell lysates were prepared and assessed for endogenous ERK1/2 phosphorylation. We found that Nef, Lck, and PKCθ cooperatively induce ERK1/2 activation, whereas cotransfection of kinase-deficient Lck or PKCθ abrogated ERK1/2 phosphorylation. However, coexpression of kinase-deficient Lck along with constitutively active PKCθ (PKCθ.AE) rescued ERK1/2 phosphorylation, thus confirming the results from Fig. 3 that PKCθ is downstream of Lck. Importantly, these observations could also be validated in Jurkat T cells (Fig. 6 B), albeit the overall effects were less pronounced, most likely due to poorer transfection efficiencies and the presence of endogenous Lck and PKCθ in these cells.

FIGURE 6.

NAKC assembly stimulates ERK MAPK activity. 293T (A) or Jurkat T (B) cells were transfected with the indicated plasmids. One day post transfection whole cell lysates were prepared and analyzed for phosphorylation of endogenous ERK MAP kinase by Western blotting with anti-phospho-ERK1/2 Ab. Equal protein loading and expression of transfected proteins were both confirmed. C, Jurkat cells were stimulated with anti-CD3 and anti-CD28 in the absence or presence of 1 mM U0126. Cell lysates were prepared and analyzed for ERK phosphorylation by Western blotting with anti-phospho-ERK Ab. In parallel, JCaM1.6 cells were transiently transfected with pLTRluc, suboptimal levels of Tat, Lck, and CD8Nef and either left untreated or treated with 1 mM U0126 for 3 or 4, 5 h or overnight. Luciferase activities in the cell extracts were determined and analyzed as previously described.

FIGURE 6.

NAKC assembly stimulates ERK MAPK activity. 293T (A) or Jurkat T (B) cells were transfected with the indicated plasmids. One day post transfection whole cell lysates were prepared and analyzed for phosphorylation of endogenous ERK MAP kinase by Western blotting with anti-phospho-ERK1/2 Ab. Equal protein loading and expression of transfected proteins were both confirmed. C, Jurkat cells were stimulated with anti-CD3 and anti-CD28 in the absence or presence of 1 mM U0126. Cell lysates were prepared and analyzed for ERK phosphorylation by Western blotting with anti-phospho-ERK Ab. In parallel, JCaM1.6 cells were transiently transfected with pLTRluc, suboptimal levels of Tat, Lck, and CD8Nef and either left untreated or treated with 1 mM U0126 for 3 or 4, 5 h or overnight. Luciferase activities in the cell extracts were determined and analyzed as previously described.

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To confirm the involvement of ERK in Nef/Lck-mediated HIV transcription, we performed luciferase reporter assays in the presence of a pharmacological MEK/ERK inhibitor. Treatment of Jurkat cells with 1 μM U0126 was effective in blocking CD3/CD28-mediated ERK activation. Accordingly, treatment of J.CaM1.6 cells with 1 μM U0126 significantly decreased Lck/Nef-mediated HIV transcription thus corroborating the involvement of ERK in HIV transcriptional regulation (Fig. 6 C).

In summary, these data suggest that the NAKC signalosome inititates a pathway that culminates in the induction of HIV transcription and involves the activation of ERK MAPK.

In our previous studies, we demonstrated that Nef enhances Tat-dependent HIV transcription in an Lck- and PKC-dependent manner (13, 15). Both kinases are members of NAKC, a complex associating with the Nef N terminus (14). However, the function of these kinases within NAKC and the consequences of NAKC assembly are not well-defined. In this study, we demonstrate that formation of NAKC results in the sequential activation of Lck and PKCθ leading to recruitment of PKCθ to membrane microdomains, increased ERK activity, and enhanced HIV transcription.

Lck and PKCθ are both critical for TCR-mediated T cell activation events. In addition, TCR ligation results in Lck-mediated phosphorylation of PKCθ (23). Our studies now implicate this pathway in Nef-induced HIV transcription. First, coexpression of wild-type but not N-terminally mutated Nef along with Lck and PKCθ increases both Lck kinase activity (Fig. 2) and interaction of Lck with PKCθ leading to enhanced tyrosine phosphorylation of PKCθ (Fig. 3,B), increased ERK activity (Fig. 6), and ultimately induction of HIV transcription (Fig. 3,A). Second, while overexpression of kinase-deficient Lck together with wild-type PKCθ or vice versa failed to significantly enhance HIV transcription (Fig. 3,A) and induce ERK activity (Fig. 6), cotransfection of constitutive active PKCθ along with kinase-deficient Lck rescued both HIV transcription (Fig. 3,A) and ERK activity (Fig. 6). Third, Nef/Lck-mediated HIV transcription is inhibited by knockdown of endogenous PKCθ (Fig. 3 C). Taken together, these findings imply that PKCθ is a downstream effector of Lck within the Nef-associated kinase complex and is regulated through Lck-mediated phosphorylation. It is possible that phosphorylation induces a conformational change and/or influences the subcellular localization of PKC, thus facilitating its interaction with other upstream effectors, which may trigger kinase activation.

We have previously demonstrated that association of nPKC with Nef increases its kinase activity and thus suggested that Nef activates PKC (15). We now extend these findings by demonstrating that PKCθ is recruited to membrane microdomains in a Nef-dependent manner. Membrane microdomain recruitment represents a hallmark of PKCθ activation (24). We were therefore surprised to see that Nef-induced PKCθ microdomain localization does not appear to activate the transcription factor NFκB, a well-established target of PKCθ. First, neither IκB phosphorylation nor p65 nuclear translocation was detected in response to Nef expression. Second, IKKγ-deficient cells still supported Nef-mediated HIV transcription. Third, IKKα was not found to be recruited to DRMs upon Nef expression. The latter finding suggests that microdomain localization of PKCθ alone is not sufficient to recruit IKK. Although this contradicts the model of Ghosh and colleagues who proposed that IKK is recruited via PKCθ (31), it is compatible with other reports suggesting that CBM complex formation is crucial for IKK recruitment (35, 36).

Because we observed a clear effect of Nef on HIV transcription, we wondered whether signaling pathways other than the NFκB pathway are targeted. Several lines of evidence pointed to the ERK MAPK pathway (32, 33, 34, 37). In fact, we were able to demonstrate that the Nef/Lck/PKC signalosome activates ERK independent of TCR ligation and that pharmacological inhibition of ERK prevents Nef/Lck-mediated HIV transcription (Fig. 6). Our data are in line with previous reports showing that Nef enhances ERK MAPK activity (34). For reasons that are not entirely clear, these authors found a requirement for TCR costimulation, as Nef expression by itself failed to activate ERK. It is possible that overexpression of Lck and PKCθ compensated for or replaced the TCR signal in the here presented experiments. Conversely, overexpression of Nef alone may lead to early sequestration and/or degradation of the endogenous kinases (16). Thus, coexpression of both kinases may have facilitated a stable and prolonged assembly of NAKC.

ERK is a known activator of transcription factors and histone-modifying enzymes. For example, ERK MAPK targets the transcription factor AP-1 via up-regulation of c-Fos expression. In addition, ERK MAPK was found to negatively regulates LSF occupancy at the HIV promoter (38), which is thought to derepress the HIV promoter. Thus, through a Lck/PKC/ERK-dependent pathway, Nef may influence the chromatin environment and/or negative regulatory elements of the HIV LTR. These findings would be in line with our previous results demonstrating that Nef stimulates HIV transcription through a mechanism involving derepression of the HIV promoter (13). Derepression was found to be linked to the assembly of the Nef-associated kinase complex, which resulted in the nuclear export of the Polycomb group protein EED, a transcriptional repressor that binds to the HIV LTR.

In summary, we propose that the formation of the Nef-associated kinase complex initiates a signaling pathway that cooperates with Tat to induce HIV transcription. By providing a scaffold for the two effector kinases Lck and PKCθ, Nef likely promotes their sequential activation. This then results in enhanced HIV transcription, which may be due to NAKC-mediated ERK activation. However, the precise effect of ERK MAP kinase on HIV transcription awaits further investigation.

We thank Ursula Bommhardt for providing the pGL4.70 Renilla luciferase reporter, Gottfried Baier for the PKC constructs, Arthur Weiss and Kalle Saksela for the Lck and Hck expression plasmids, Melanie Ott for the Tat and HIV LTR luc reporter constructs, Alain Israël for the IKKγ-deficient cell line, and Mark Harris for the anti-Nef serum.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by funds from the Department of Microbiology and Immunology, University of Miami, Miller School of Medicine (grants to A.S.B.) and the Deutsche Forschungsgemeinschaft (Grant TR7013 to O.T.F.).

5

Abbreviations used in this paper: NAKC, Nef-associated kinase complex; EED, embryonic ectodermal development; PKC, protein kinase C; DRM, detergent-resistant membrane; MβCD, methyl-β-cyclodextrin.

6

The online version of this article contains supplementary material.

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