JOURNAL OF VIROLOGY, Apr. 1996, p. 2525–2532 0022-538X/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 70, No. 4
Human T-Cell Lymphotropic Virus Type 1 Tax1 Activation of NF-kB: Involvement of the Protein Kinase C Pathway PAUL F. LINDHOLM,† MEHRNAZ TAMAMI, JAMES MAKOWSKI,†
AND
JOHN N. BRADY*
Laboratory of Molecular Virology, National Cancer Institute, Bethesda, Maryland 20892-5005 Received 16 June 1995/Accepted 15 January 1996
Human T-cell lymphotropic virus type 1 Tax1 induces the activation and nuclear localization of the cellular transcription factor, NF-kB. Treatment of cells with calphostin C, a protein kinase C (PKC) inhibitor, blocked induction of NF-kB DNA binding activity in human T-cell lymphotropic virus type 1-transformed C81 cells and Tax1-stimulated murine pre-B cells, suggesting that PKC was an important intermediate in the NF-kB induction pathway. We further demonstrate that Tax1 associates with, and activates, PKC. PKC was coimmunoprecipitated with anti-Tax1 sera from Tax1-expressing MT4 extracts and Jurkat extracts in the presence of exogenous Tax1 protein. In addition, the glutathione-S-transferase-Tax1 protein bound specifically to the alpha, delta, and eta PKC isoenzymes synthesized in rabbit reticulocyte lysates. The addition of Tax1 to in vitro kinase reaction mixtures leads to the phosphorylation of Tax1 and an 18-fold increase in the autophosphorylation of PKC. Transfection of Jurkat cells with wild-type Tax1 stimulated membrane translocation of PKC. In contrast, Tax1 mutant M22, which fails to stimulate NF-kB-dependent transcription, failed to stimulate membrane translocation of PKC. Tax1 did not directly increase PKC phosphorylation of IkBa. Our results are consistent with a model in which Tax1 interacts with PKC and stimulates membrane translocation and triggering of the PKC pathway. Subsequent steps in the PKC cascade likely stimulate phosphorylation of IkBa. Human T-cell lymphotropic virus type 1 (HTLV-1) infection is associated with adult T-cell leukemia and tropical spastic paraparesis/HTLV-1-associated myelopathy. HTLV-1 Tax1, a viral regulatory protein, is essential for virus replication and cellular transformation. Tax1 positively regulates transcription of the HTLV-1 long terminal repeat as well as other viral and cellular genes including interleukin-2R alpha (IL-2Ra), IL-2, IL-6, granulocyte macrophage colony-stimulating factor, c-myc, c-fos, lymphotoxin (tumor necrosis factor beta), Krox20,24, and the human immunodeficiency virus type 1 long terminal repeat (2, 7, 10, 17, 19, 24, 25, 32, 41, 42, 47, 50, 53, 56, 59, 63a, 69, 74, 76, 77, 80). Tax1 activates transcription of these promoters by modulating the DNA binding activity of transcription factors which include CREB, ATF, SRF, and NF-kB (10, 17, 19, 21, 32, 58, 59, 68, 69). Direct interaction of Tax1 with each of these transcription factors has been reported (7, 15, 22, 47, 78). In vivo studies further suggest that protein kinases, similar to their involvement in human immunodeficiency virus and bovine leukemia virus transcription (1, 14, 31, 33, 36, 75), are important in Tax1 transactivation of the HTLV-1 long terminal repeat (27, 59, 75). NF-kB is sequestered in the cytoplasm by IkB inhibitor proteins which contain ankyrin repeats and putative phosphorylation sites for protein kinase C (PKC) and phosphoinositol-3 kinase (23, 26). Tax1, phorbol esters, tumor necrosis factor alpha, lipopolysaccharide, and IL-1 stimulate NF-kB in vivo and have been shown to induce, in a sequential manner, IkB release from NF-kB complexes, IkB degradation, and synthesis of IkB mRNA and protein in vivo (3, 6, 11, 16, 61, 65, 66, 70, 72, 73). IkB release and degradation are temporally related to the appearance of phosphorylated IkB (6, 16). The kinases
involved in IkB phosphorylation and inactivation have not been unambiguously identified, but several studies have demonstrated that IkB activity could be modified in vitro by purified protein kinases including PKC (23, 45). We have previously shown Tax1 protein in the extracellular media of HTLV-1 Tax1-expressing transformed cell lines (43). Further, purified Tax1 protein can be taken up by uninfected cells. Cellular Tax1 uptake was followed by a rapid and transient increase in NF-kB DNA binding, which did not require de novo protein or mRNA synthesis (43, 44). In this study, we demonstrate that in vivo NF-kB induction by extracellular Tax1 or tetradecanoyl phorbol acetate (TPA) can be blocked by the PKC inhibitor calphostin C. The PKC inhibitor also decreased the constitutive activation of NF-kB in HTLV-1-transformed cells. This study further describes a novel physical interaction between Tax1 and PKC. The interaction of Tax1 and PKC is shown to stimulate PKC autophosphorylation in the absence of the cofactors diolein and phosphatidylserine. Jurkat T lymphocytes transfected with Tax1-expressing plasmids, which stimulate NF-kB, lead to PKC translocation to the particulate cellular membrane fraction, suggesting that Tax1 activates PKC in vivo. MATERIALS AND METHODS Cells. 70Z/3 cells are mouse pre-B lymphocytes (ATCC T1B 158; originator, P. Kincade, Sloan-Kettering Institute, Rye, N.Y.) derived from a methyl nitrosourea-induced tumor in a (C57BL/6 3 DBA/2)F1 mouse. Cells were cultured according to American Type Culture Collection specifications. MT4 and C81-66 cells are HTLV-1-transformed human cord blood lymphocytes derived by cocultivation with HTLV-1-infected cells (64). Jurkat E6.1 cells were obtained from the National Institutes of Health AIDS repository. Construction of plasmids. The tax gene was directionally subcloned into pGEX-2T at BamHI and EcoRI restriction sites from pCRII-tax. pCRII-tax was created by PCR amplification of the tax gene from pHTLV-1-tax with an upstream primer, 59-CCAAGCTGGATCCATGGCCCACTTCCCAGGG-39, containing a BamHI site and a second, downstream primer, 59-GGGAGACGTCA GAGCCTTAGTCTGGGCCCTG-39, containing an EcoRI site. The orientation of the tax gene was confirmed by restriction of pGEX-2T-tax with TthIII 1. The NF-kB p65 expression vector pSKp65 was kindly provided by G. Nolan and D. Baltimore. The full-length clones for bovine PKC alpha (PKC-a), mouse PKC delta (PKC-d), and PKC eta (PKC-h) in PVL1393 were kindly provided by M. G.
* Corresponding author. Mailing address: Laboratory of Molecular Virology, National Cancer Institute, Building 41, Room B403, Bethesda, MD 20892. Phone: (301) 496-6201. Fax: (301) 496-4951. Electronic mail address:
[email protected]. † Present address: Department of Pathology, Medical College of Wisconsin, Milwaukee, WI 53226. 2525
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Kazanietz and P. M. Blumberg. The full-length 2.1-kb PKC-a was excised by partial digestion with BamHI and EcoRI. The partial PKC-a fragment, 1.7 kb in length, was excised by digestion with BamHI and EcoRI. The full-length 2.3-kb PKC-d cDNA was obtained by restriction of pVL1393 PKC-d with BamHI and EcoRI. The PKC cDNAs were directionally cloned into pSP 72 vector (Promega) at BamHI and EcoRI restriction sites. The full-length 2.1-kb PKC-h fragment was obtained from pVL1393 PKC-h by restriction with EcoRI and subcloned into psp 72. Protein expression and purification. Tax1 protein was expressed in Escherichia coli and purified as previously described (44). Control extracts prepared from E. coli (HB101 strain) were prepared by the same methods as the Tax1 protein extract of E. coli (44). The purity of the Tax1 and HB101 extracts was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining as previously reported (43, 44). Glutathione-S-transferase (GST)Tax1 protein or GST proteins were generated in E. coli by growth of the transformed HB101 strain in LB with 100 mg of ampicillin per ml. The cultures were grown to an optical density of 0.6 and then induced by the addition of 0.5 mM IPTG (isopropyl-b-D-thiogalactopyranoside) for 3 h. The GST fusion proteins were obtained by sonication of the bacterial pellet in buffer containing 50 mM Tris, (pH 7.5), 250 mM NaCl, 0.1% Nonidet P-40 (NP-40), 1 mg of leupeptin per ml, 1 mg of aprotinin per ml, and 100 mg of phenylmethylsulfonyl fluoride (PMSF) per ml. The extract was centrifuged was centrifuged at 100,000 3 g for 30 min, and the supernatant was collected and reacted with glutathione-Sepharose beads (pH 7.5) at 48C. The GST proteins were eluted by the addition of 20 mM glutathione (pH 7.5) for 10 min and then dialyzed in buffer D. The GST proteins were analyzed by Coomassie blue staining to quantitate recovery of the 27-kDa GST and 67-kDa GST-Tax1 proteins. The [35S]methionine-labelled PKC isoenzymes or NF-kB p65 was in vitro transcribed and translated in rabbit reticulocyte lysates by using the TNT system (Promega). The protein production and yield were confirmed by SDS-PAGE and autoradiography. Assay of NF-kB induction. Murine 70Z/3 pre-B cells were plated at 106 cells per ml in 15 ml of RPMI medium and cultured as described above. The cells were cultured overnight prior to stimulation with 25 nM purified Tax1 protein, an equal volume of bacterial extract, or 50 ng of TPA per ml. For some experiments, the cells were stimulated in the presence of the selective protein kinase inhibitors herbimycin A, H-8, or calphostin C (37). Cellular extracts were prepared by the method of Osborn et al. (55). For gel shift assays, the murine immunoglobulin kappa (Igk)-enhancer NF-kB oligonucleotides 59-GATCCAGAGGGGACTTT CCGAGAG-39 were Klenow labelled with [32P]dGTP (Amersham), desalted with G-25 spin columns (Boehringer Mannheim), ethanol precipitated, and resuspended in 10 mM Tris (pH 7.5)–1 mM EDTA. The gel shift reactions were carried out in a volume of 20 ml in gel shift reaction buffer (10 mM Tris HCl [pH 7.5], 40 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol) with 6 mg of nuclear or cytoplasmic extract, 0.5 to 2 ng (;50,000 cpm) of labelled NF-kB oligonucleotide, and 3 mg of poly(dI-dC/dI-dC) (Pharmacia) at room temperature for 20 min. Some reaction mixtures were incubated with a 100-fold excess of unlabelled mutant or wild-type Igk NF-kB oligonucleotides to identify the specific NF-kB gel shift complex. Gel shift reactions were carried out in parallel with an octamer probe as previously described (67). Protein kinase and Tax1 binding assays. The protein kinase reaction (mixture volume, 40-ml) were carried out in buffer containing 30 mM Tris (pH 7.5), 100 mM KCl, 6 mM MgCl2, and 500 mM CaCl2 (unless otherwise specified). Phosphatidylserine (10 mg/ml), diolein (1 mg/ml) (except as indicated), and ATP (10 mM) were added to the reaction mixtures (35). The buffer was freshly sonicated with 10 brief bursts prior to the addition of the remaining reactants. For reaction mixtures in which the calcium concentrations were varied, the buffer system of Fabiato and Fabiato was employed (13). Tax1 or control extract (0.5 to 1.0 mg) and [g-32P]ATP (0.2 mM final concentration, 8.9 3 107 cpm; Amersham) were included. Purified rat brain PKC (0.1 to 0.2 U, 30 to 60 ng; Calbiochem) was added simultaneously to each reaction mixture for 20 min at 258C. The reaction mixtures were subsequently placed on ice and either immunoprecipitated or precipitated with 100% acetone at 2208C as indicated. The acetone precipitation reaction mixtures were held at 2708C for 1 h and then washed three times with 80% acetone at 2208C prior to SDS-PAGE and autoradiography. For coimmunoprecipitation experiments, 450 ml of buffer containing 50 mM Tris (pH 7.5), 100 mM NaCl, and 1 mM EDTA (TNE) with 200 mM Na3VO4 and 0.5% NP-40 at 48C was added to the above-described reaction mixtures and then 1 to 2 mg of anti-Tax1 or preimmune (IgG2a) (PharMingen) antibodies was added for 45 min at 48C with gentle mixing. The reaction mixtures were incubated with 100 ml of 50% protein A-Sepharose beads for 1 h at 48C with gentle mixing and washed five times with TNE containing 200 mM Na3VO4 and 0.5% NP-40 at 48C. The immunoprecipitates were eluted with SDS-PAGE denaturation buffer prior to analysis by SDS-PAGE and autoradiography. For coimmunoprecipitation of Tax1 and PKC from cellular extracts, 32P-labelled PKC was equilibrated with cellular cytoplasmic extracts at 48C for 20 min prior to the addition of Tax1 and/or anti-Tax1 antibodies. Reimmunoprecipitation was done with the addition of 50 ml of sample buffer containing 2% SDS, 100 mM dithiothreitol, and 50 mM Tris (pH 7.0). The samples were heated for 5 min at 958C, and then 1 ml of TNE buffer containing 0.5% NP-40 and 200 mM Na3VO4 was added. The sample was gently centrifuged, and the supernatant was removed. The reimmunoprecipitation was then performed as described above with anti-PKC antibodies. For the GST pull-down assays, the [35S]methionine-labelled PKC isoenzymes or NF-kB
J. VIROL. was reacted with approximately 0.5 mg of either GST or GST-Tax1 proteins in TNE buffer with 0.5% NP-40 for 45 min at 48C. Glutathione-Sepharose beads (50 ml of a 50% suspension) were added for an additional 45 min at 48C with gentle mixing. The reaction mixtures were washed five times with the same buffer and then analyzed by SDS-PAGE and autoradiography. The amount of radioactivity in the gels was quantitated by scintillation counting and PhosphorImage analysis. PKC translocation assay. Jurkat T-cells (107) were washed in RPMI 1640 medium without fetal calf serum and electroporated under conditions of 107 cells per 0.25 ml of RPMI 1640 at 250 V and 800 mF with pcTax M47 or control vector DNA. The transfected cells were placed in 5 ml of RPMI 1640 with 0.5% fetal calf serum and harvested at 18, 24, and 48 h. Plates of nontransfected cells (107 cells per 5 ml) were prepared for negative control and TPA-stimulated cultures. Positive controls for PKC membrane translocation were obtained by treating the cells with 50 ng of TPA for 1 h. The cells were harvested and washed three times in phosphate-buffered saline (PBS) without calcium or magnesium, and extracts were prepared as described previously (46, 71). Briefly, the cells were resuspended in 500 ml of buffer A (20 mM Tris HCl [pH 7.5], 0.5 mM ethylene glycol-bis(b-aminoethyl ether)-N,N,N9,N9-tetraacetic acid [EGTA], 2 mM EDTA, 2 mM PMSF, 10 mg of leupeptin per ml, 10 mg of aprotinin per ml), sonicated by seven brief bursts with a Branson sonifier with a microtip setting of 1, and centrifuged at 100,000 3 g for 1 h at 48C. The supernatant and pellet, corresponding to the soluble cytoplasmic fraction and insoluble particulate membrane fraction, respectively, were adjusted to 1% SDS and 2% b-mercaptoethanol. The extracts were analyzed by Western blot (immunoblot) analysis with rabbit anti-PKC (1:2,000; Upstate Biotechnology, Inc.). The image was developed with Amersham enhanced chemiluminescence Western blotting reagents and exposed to Kodak XAR autoradiography film.
RESULTS Tax1 induction of NF-kB DNA binding is blocked by calphostin C. Protein kinase inhibitors were added to 70Z/3 preB-lymphocyte cell cultures during stimulation to determine the role of specific protein kinases in the activation of NF-kB. Calphostin C (Calbiochem) is a selective inhibitor of PKC; 1 mM calphostin C inhibits PKC but does not interfere with cyclic nucleotide-dependent protein kinases (50% inhibitory concentration of .25 mM). H-8{N-[2-(methylamino)-ethyl]-5isoquinolinesulfonamide HCl} is a selective inhibitor of cyclic AMP-dependent kinases (Ki of 1.2 mM) and cyclic GMP-dependent protein kinase (Ki of 0.48 mM). We used H-8 at 5 mM, which inhibits cyclic nucleotide-dependent kinases but not PKC (Ki of 15 mM). Herbimycin A (Gibco BRL) at 12 mM is a potent inhibitor of tyrosine kinases (65). The kinase inhibitors were added to the cells for 1 h prior to the addition of the Tax1 protein or TPA stimulus. Because the action of calphostin C requires light, this inhibitor was used in the presence of light (12). Consistent with previous results, Tax1 and TPA induced NF-kB binding to the Igk-enhancer NF-kB DNA probe (Fig. 1A and B, lanes 3). NF-kB binding was specific, as determined by gel shift competition assays (Fig. 1A and B, lanes 1 to 3). While 5 mM H-8 did not block Tax1 or TPA induction of NF-kB DNA binding (Fig. 1A, lane 4, and B, lane 5), 1 mM calphostin C blocked both Tax1 and phorbol ester (TPA) induction of NF-kB (Fig. 1A, lane 5, and B, lane 6). In addition, the tyrosine kinase inhibitor herbimycin A (12 mM) did not block NF-kB induction by either Tax1 or TPA (Fig. 1B, lane 4) (data not shown). Gel shift assays were also performed with the same extracts and an octamer binding DNA probe. No significant changes were observed in the levels of octamer binding during cell stimulation or in the presence of the protein kinase inhibitors (Fig. 1A and B). The specific octamer binding complexes were identified by competition with a 100-fold excess of wild-type, but not mutant, competitor oligonucleotides (Fig. 1A, lanes 7 to 9, and B, lanes 8 to 10). These data indicate that the PKC inhibitor calphostin C specifically inhibited Tax1 or TPA-induced NF-kB DNA binding. HTLV-1-transformed, Tax1-expressing C81-66 lymphocytes show constitutive expression of nuclear NF-kB DNA binding activity (Fig. 1C, lane 3). Treatment of C81-66 cell cultures
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FIG. 1. Assay of NF-kB DNA binding activity in cells treated with protein kinase inhibitors. (A) 70Z/3 cellular extracts were analyzed for gel shift activity with the Igk light chain NF-kB probe (lanes 1 to 6) or an octamer binding probe (lanes 7 to 12) (74). The cells were stimulated with 25 nM Tax1 (lanes 1 to 5 and 7 to 11) in the presence of the inhibitor H-8 (5 mM) (lanes 4 and 10) or calphostin C (1 mM) (lanes 5 and 11). Unstimulated cell extracts were utilized as controls (lanes 6 and 12). (B) 70Z/3 cellular extracts were prepared from cells stimulated with 50 ng of TPA per ml and in the presence of 5 mM H-8 (lanes 5 and 12), 500 ng of herbimycin A per ml (lanes 4 and 11), or 1 mM calphostin C (lanes 6 and 13). The extracts were tested for gel shift activity with the Igk light chain NF-kB probe (lanes 1 to 7) and the octamer probe (lanes 8 to 14). A 100-fold excess of wild-type (W.T.) and mutant (Mut.) competitor oligonucleotides were included in the indicated reaction mixtures to establish the specificity of the gel shift reactions. (C) HTLV-1 Tax1-expressing C81-66 cells were untreated (lanes 1 to 3) or treated with 0.1 (lane 4), 0.5 (lane 5), or 1 mM (lane 6) calphostin C or 50 ,250 (lane 8), or 500 (lane 9) ng of herbimycin A per ml. A 100-fold excess of wild-type (lane 1) or mutant (lane 2) competitor oligonucleotides were added to reaction mixtures to identify specific NF-kB complexes.
with 0.5 or 1.0 mM calphostin C for 3 h significantly reduced NF-kB DNA binding activity (Fig. 1C, lanes 3 to 6). In contrast, herbimycin A treatment (50 to 500 ng/ml) of C81-66 cells did not reduce NF-kB DNA binding activity (Fig. 1C, lanes 7 to 9). These results suggest that PKC plays an important role in the Tax1-mediated NF-kB induction pathway in transformed cells. Tax1 protein binds to PKC in cellular extracts. Immunoprecipitation experiments were performed with [35S]methioninelabelled HTLV-1-transformed Tax1-expressing MT4 or Jurkat cytoplasmic extracts to analyze potential interactions of Tax1 with PKC (Fig. 2A). The [35S]methionine-labelled cytoplasmic extracts were immunoprecipitated with anti-Tax1 or control monoclonal antibodies (Fig. 2A, lanes 1 and 2). Tax1 was specifically immunoprecipitated with the anti-Tax1 serum (Fig. 2A, lane 1). Several other protein bands, with sizes of 46 to 130 kDa, were present in the immunoprecipitate, but their speci-
ficities were difficult to interpret because of background bands in the control immunoprecipitation reactions. To specifically determine if PKC was coprecipitated along with Tax1, each immunoprecipitation reaction mixture was washed twice with 20 volumes of immunoprecipitation buffer and denatured with 1 volume of buffer containing 50 mM Tris-HCl (pH 7.0), 100 mM dithiothreitol, and 2% SDS. 35S-labelled proteins were then reprecipitated with anti-PKC or control preimmune sera in immunoprecipitation buffer containing 0.5% NP-40 and 0.1% SDS. The reaction mixtures were washed four times and analyzed by SDS-PAGE and autoradiography for 35S-labelled PKC. Reimmunoprecipitation of the eluted anti-Tax1 immunoprecipitate complexes with anti-PKC antisera confirmed the presence of PKC in the immunoprecipitate from the MT4 extracts (Fig. 2B, lane 5). In parallel immunoprecipitation reactions, 35S-labelled PKC was not immunoprecipitated in the absence of Tax1 protein in Jurkat extracts (Fig. 2B, lanes 1 to 4) or in the presence of preimmune antibodies (Fig. 2B, lanes 2, 4, 6, and 8). To further demonstrate the interaction of Tax1 and PKC in cell extracts, exogenous 32P-labelled PKC was incubated with Tax1-containing C81 extracts or control Jurkat extracts and then subjected to immunoprecipitation analysis with anti-Tax1 sera. PKC (60 ng) was autophosphorylated with 20 mCi of [g-32P]ATP and incubated with 400 mg of C81 or Jurkat cel-
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FIG. 2. Assay of PKC binding to Tax1 in cellular extracts. (A) 35S-labelled MT4 and Jurkat cellular extracts were immunoprecipitated with anti-Tax1 antibodies (lanes 1 and 3) or preimmune IgG2a (lanes 2 and 4). (B) The immunoprecipitate pellets from reaction mixtures parallel to those for which the results are shown in panel A were denatured and reimmunoprecipitated with anti-PKC antibodies (lanes 1, 3, 5, and 7) or preimmune antibodies (lanes 2, 4, 6, and 8). (C) 32P-labelled PKC protein was immunoprecipitated in the Tax1-containing C81 cytoplasmic extract (lane 1) but not in the control non-Tax1-expressing Jurkat cytoplasmic extract (lane 2). Reimmunoprecipitation with anti-PKC (lane 5) but not preimmune antibodies (lane 9) confirmed the identity of the PKC from the anti-Tax1 immunoprecipitation. (D) In vitro-transcribed and translated PKC-a (lanes 1 to 4), PKC-d (lanes 5 and 6), or PKC-h (lanes 7 and 8) was analyzed for binding to GST (lanes 1, 3, 5, and 7) or GST-Tax1 (lanes 2, 4, 6, and 8) by the GST interaction assay followed by SDS-PAGE and autoradiography. Molecular masses (in kilodaltons) are indicated on the left.
lular extracts. A Tax1-PKC complex was precipitated with antiTax1 sera in the C81 but not the control Jurkat cell extracts (Fig. 2C, lanes 1 and 2). The amount of 32P-labelled PKC detected in the C81 extract was 20-fold greater than that observed in the control Jurkat cells. Reimmunoprecipitation of the eluted anti-Tax1 immunoprecipitate complexes with antiPKC antisera confirmed the identity of PKC in the immunoprecipitates from C81 cytoplasmic extracts (Fig. 2C, lanes 3 and 4). The lower-molecular-weight protein band migrating at approximately 42 kDa is likely a proteolytic fragment of PKC since it was not present in the original immunoprecipitation. PKC was not reimmunoprecipitated in the presence of preimmune antisera (Fig. 2C, lane 5). To verify the interaction of Tax1 and PKC, we performed bidirectional coimmunoprecipitation of Tax and PKC following an in vitro kinase reaction. PKC was detected in immunoprecipitates with Tax1 by using anti-Tax1 sera but not preim-
mune sera. Similarly, Tax1 was detected in immunoprecipitates with PKC by using anti-PKC sera but not preimmune sera (data not shown). Tax1 protein binds to in vitro-translated PKC. GST binding assays were performed to analyze Tax1 protein binding to [35S]methionine-labelled PKC isoenzymes translated in vitro in rabbit reticulocyte lysates. The GST-Tax1 protein used in these assays is transcriptionally active following electroporation into Jurkat T lymphocytes (data not shown). Glutathione-Sepharose beads containing equal quantities of GST or the GSTTax1 fusion protein were incubated with PKC-a, PKC-d, or PKC-h. PKC-a and PKC-h were specifically precipitated in the presence of GST-Tax1 but not the control GST protein (Fig. 2D, lanes 1 to 4, 7 and 8). The specific interaction of GST-Tax1 with PKC-d was not unambiguous, because of the increased level of binding to the GST control protein (Fig. 2D, lanes 5 and 6). The specificity of the Tax1-PKC interaction was further
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FIG. 4. PKC translocation assay. (A) Jurkat cells were untreated (2TPA) or treated with 50 ng of TPA per ml for 1 h (1TPA). The cells were harvested, washed three times in PBS without calcium or magnesium, resuspended in 500 ml of buffer A (20 mM Tris HCl [pH 7.5], 0.5 mM EGTA, 2 mM EDTA, 2 mM PMSF, 10 mg of leupeptin per ml, 10 mg of aprotinin per ml), and centrifuged at 100,000 3 g for 1 h at 48C. The supernatant corresponding to the soluble cytoplasmic fraction was adjusted to 1% SDS and 2% b-mercaptoethanol. Similarly, the pellet corresponding to the particulate membrane fraction was resuspended in 1% SDS–2% b-mercaptoethanol. The extracts were analyzed by SDS– 10% PAGE and Western blot. The blots were probed with rabbit anti-PKC (1:2,000). The image was developed with Amersham enhanced chemiluminescence Western blotting reagents and exposed to Kodak XAR autoradiography film. (B) Analysis of PKC translocation following transfection of Jurkat cells with NF-kB-activating pcTax M47 versus non-NF-kB-activating mutant pcTax M22. Cytoplasmic and particulate membrane fractions were isolated as described in Materials and Methods and analyzed by Western blot with anti-PKC antisera. The Western blot of membrane-associated PKC is shown above the graph. Quantitation of the bands was done with a Molecular Dynamics densitometer.
FIG. 3. Analysis of Tax1 effect on PKC autophosphorylation in vitro. (A) PKC was incubated with Tax1 (lanes 1 to 4) or control HB101 extract (lanes 5 to 8) for 20 min at 258C prior to precipitation with 4 volumes of acetone at 2808C. The precipitates were washed three times with 80% acetone at 2208C and analyzed by SDS-PAGE. Molecular masses (in kilodaltons) are indicated on the left. PS, phosphatidylserine. (B) Time course analysis of PKC autophosphorylation. PKC was incubated in the presence of [g-32P]ATP and purified Tax1 protein or control extract. The reaction mixtures were incubated for the indicated times at 258C, acetone precipitated, washed three times with 80% acetone at 2208C, and analyzed by SDS-PAGE. The radioactivity of the 32P-labelled PKC (in counts per minute) was determined by scintillation counting for and reaction mixtures with (1) and without (2; control) the addition of Tax1.
demonstrated in control experiments in which Tax1 failed to interact with in vitro-translated TBP, ATF1, ATF2, TFIIA-b, and TFIIA-g (data not shown). The interaction of GST-Tax1 with in vitro-translated PKC, in the absence of added ATP, suggests that posttranslational modification of PKC was not required for its interaction with Tax1. PKC autophosphorylation is increased by Tax1 protein in vitro. To analyze the effect of Tax1 protein on the overall enzymatic activity of PKC, recombinant purified Tax1 or control extracts, prepared exactly as was the Tax1 preparation, were added to PKC in the presence of [g-32P]ATP and PKC buffer. The 32P-labelled products were acetone precipitated and analyzed by SDS-PAGE and autoradiography. Subsequently, 32P-labelled PKC bands were quantitated. In reaction mixtures containing the Tax1 protein (Fig. 3A), a 10-foldgreater level of 32P-labelled PKC autophosphorylation was ob-
served. Interestingly, autophosphorylation of PKC in the presence of Tax1 protein occurred independent of the presence of the lipid cofactors (Fig. 3A, lanes 1 to 4). In a separate series of experiments, the autophosphorylation of PKC was evaluated in the presence of Tax1, carbonic anhydrase (with a similar molecular weight and isoelectric point), or human immunodeficiency virus type 1 Tat. In these experiments, increased autophosphorylation of PKC was observed only in the presence of Tax1. PKC did not show increased autophosphorylation in the presence of carbonic anhydrase or Tat. In a time course analysis, PKC showed a rapid increase in autophosphorylation within 30 s in the presence of Tax1 protein (Fig. 3B). Quantitatively, a 30-fold increase in PKC autophosphorylation was observed during this initial phase of the reaction. The level of autophosphorylated PKC increased gradually over the next 10 min of the reaction. Tax1 protein expression increases PKC membrane translocation in Jurkat cells. We next performed experiments to determine whether Tax1 or Tax1 mutants would cause PKC activation in vivo. Membrane translocation of PKC has been demonstrated during stimulation of cells with several agents including the phorbol ester TPA (46, 49, 70). We examined membrane translocation of protein kinase C by Western blot analysis of soluble and particulate fractions of cells as described previously (46). Our studies demonstrated a shift of PKC localization from soluble, cytoplasmic fraction to the insoluble membrane fraction during stimulation with TPA (Fig. 4A). Similarly, an increased percentage of PKC was detected in the insoluble membrane fractions following transfection of cells with Tax1 capable of inducing NF-kB. Transfection with the Tax1 mutant M47, which has been shown to transactivate the human immunodeficiency virus promoter and causes NF-kB activation, increased PKC translocation to the insoluble membrane fraction (Fig. 4B). Similar results were obtained with wild-
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FIG. 5. Effect of Tax1 on PKC phosphorylation of IkBa. Bacterially expressed IkBa was incubated in the presence of PKC or Tax1 and PKC for 20 min at 258C. Subsequently, the reaction mixtures were immunoprecipitated with preimmune or anti-IkBa antisera and analyzed by SDS-PAGE and autoradiography. The 38-kDa 32P-IkBa was detected only in the lanes immunoprecipitated with the anti-IkBa antibody. The presence of IkBa, PKC, and Tax1 in the incubation mixture is indicated above each lane. Immunoprecipitations were done with preimmune (lanes 2, 4, 6, 8, 10, and 12) or anti-IkBa (lanes 1, 3, 5, 7, 9, and 11) sera. The 38-kDa (kd) IkBa fragment is indicated by the arrow.
type Tax1. In contrast, transfection with the Tax1 mutant M22, which is defective for NF-kB activation, did not cause PKC translocation to the particulate membrane fraction. Our studies suggest that Tax1 and a Tax1 mutant which activates NF-kB are able to activate PKC, while the Tax1 mutant M22, which does not activate NF-kB, also does not cause PKC membrane localization or activation. Tax1 does not stimulate PKC phosphorylation of IkB in vitro. In view of the role of IkB in NF-kB regulation and the known effect of IkB phosphorylation in its dissociation from the NF-kB complex, it was of interest to determine if the presence of Tax1 would increase PKC phosphorylation of IkBa. The bacterially expressed 38-kDa IkBa (MAD3) was incubated with purified PKC in the presence or absence of Tax1 protein. Subsequently, the reaction mixture was immunoprecipitated with rabbit anti-IkBa or preimmune sera, and the immunoprecipitates were analyzed by SDS-PAGE and autoradiography. Incubation of IkBa with PKC, consistent with previous reports, results in the phosphorylation of IkBa, which was specifically immunoprecipitated with anti-IkBa antisera (Fig. 5, lanes 3 and 4). The addition of Tax1 protein to the reaction mixture did not increase the level of IkBa phosphorylation (Fig. 5, lanes 7 and 8). These results suggest that while Tax1 increases the PKC activity, it does not preferentially stimulate phosphorylation of IkB in the presence of multiple substrates. In a separate series of experiments, we also tested the ability of Tax1 and PKC to regulate the activity of IkBa. Consistent with the results of the phosphorylation analysis, Tax1 did not directly decrease the inhibitory effect of IkBa on NF-kB binding activity (data not shown). Because Tax1 did not affect direct PKC phosphorylation of the IkBa substrate in vitro, we hypothesize that additional intermediate steps are required for Tax1-induced modification of IkBa in vivo. It is important to emphasize that these results do not contradict previous reports that Tax1 activates NF-kB through phosphorylation or inactivation of IkB but suggests that the inactivation of IkB is not a direct function of Tax1. DISCUSSION In this study we provide evidence that PKC is an important intermediate in the NF-kB induction pathway. Treatment of cells with calphostin C, a selective PKC inhibitor, inhibits Tax1 induction of NF-kB. We further demonstrate that Tax1 protein physically interacts with PKC through immunoprecipitation and protein-protein binding assays of PKC in cellular extracts. Interestingly, coimmunoprecipitation of Tax1 with PKC was optimal in the presence of calcium and reducing conditions (data not shown). It has been reported that PKC binds to
J. VIROL.
nuclear proteins (8) and receptors for activated C kinase (49). The latter proteins bind to PKC in a phospholipid-, calcium-, and diacylglycerol-dependent manner. In this respect, Tax1 interaction with PKC is unique because it does not require PKC phosphorylation or activation. The primary amino acid sequence involved in PKC interaction with the activated C kinase receptor proteins was not detected in Tax1. In addition to a physical interaction between Tax1 and PKC, a significant increase in PKC autophosphorylation was observed in the presence of added Tax1 protein. PKC autophosphorylation may have important biologic effects since autophosphorylation of PKC has been reported to regulate phorbol ester-mediated down-regulation, calcium sensitivity, histone phosphorylation, and activation of PKC (38, 51, 54, 79). PKC autophosphorylation has been observed in the amino terminus, hinge region, and carboxy-terminal sites (20). Although PKC phosphorylation near the catalytic site and autophosphorylation at the carboxy-terminal Thr-641 are important for the generation of the mature catalytically active enzyme (18), the function of other autophosphorylation sites remains to be established. The function of Tax1-induced PKC autophosphorylation and membrane translocation is under investigation. NF-kB transcription factors are normally retained in the cytoplasm by inhibitor proteins such as IkB, p100, and p105. In vivo induction of NF-kB by a variety of stimuli, including phorbol esters, tumor necrosis factor alpha, lipopolysaccharide, and IL-1, are associated with phosphorylation and release of IkB from NF-kB complexes followed by rapid proteolytic degradation. Active NF-kB then stimulates synthesis of IkB mRNA and protein (6, 11, 16, 61, 73). Phosphorylation of the 38-kDa IkB inhibitor may be regulated in vitro by several protein kinases including PKC (23, 45). Tax1 has been reported to induce NF-kB by several mechanisms, including regulation of posttranslational modifications, direct interactions with NFkB, and transcriptional control (28, 29, 34, 39). To test the hypothesis that Tax1 stimulated direct PKC phosphorylation of IkBa, purified IkBa was incubated in the presence of Tax1 and PKC. No stimulation of IkBa phosphorylation was observed. While a negative result is somewhat difficult to interpret, the most straightforward interpretation of these results suggest that Tax1 does not regulate direct phosphorylation of IkB through PKC. Two independent laboratories have found that IkB is constitutively phosphorylated in HTLV-1-transformed cells (40, 73). In a permanently transfected Jurkat cell line in which Tax1 expression was dependent on the addition of heavy metals, early induction of NF-kB binding was associated with degradation of the IkB inhibitor (34). If PKC is directly involved in modification of IkB, additional cofactors besides Tax1 are likely required. Consistent with this hypothesis, it has been previously reported that NF-kB activation by Tax1 is an indirect process that requires activation of a preexistent factor in lymphoid cells (63). Alternatively, phosphorylation of IkB may be regulated by a kinase(s) other than PKC. Our previous studies indicated that Tax1 induction of NF-kB did not require de novo synthesis and that Tax1 did not directly affect the stability of NF-kB/IkB complexes (44). In this regard, it is interesting that extracellular Tax1 stimulation of NF-kB was blocked by calphostin C. Staurosporine or prolonged treatment of cells with phorbol esters, which down-regulate a subset of the PKC isoenzymes (4, 9), did not inhibit Tax1 induction of NF-kB (43). It is possible, therefore, that Tax1 activation of NF-kB requires a specific PKC isoenzyme. Tax1-mediated mobilization of PKC to the membrane likely leads to a cascade of signals which ultimately leads to activation of a serine-threonine kinase, phosphorylation of IkB, and translocation of the active NF-kB to the nucleus of the cell. It is possible that
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ROLE OF PKC IN HTLV-1 Tax1 ACTIVATION OF NF-kB
PKC-induced phosphorylation of Tax1 may be important in further regulation of the NF-kB pathway by Tax1. On the basis of our data, it seems unlikely that Tax1 stimulates PKC to directly phosphorylate IkBa. In addition to participation in virus transcription, Tax1-mediated induction of PKC autophosphorylation or kinase activity may have important effects on the regulation of cell growth and division. PKC has been implicated as one component of the cell growth-regulatory signaling pathway (52, 62). Overexpression of normal PKC or a mutant PKC leads to disordered cell growth and transformation (30, 48, 57). Interestingly, it has recently been reported that PKC stimulation at different stages of the cell cycle mediates phosphorylation of the retinoblastoma protein Rb and activation of cyclin-dependent kinases (81). The p53 cell cycle regulatory protein may also be an important substrate for PKC (5). Along these lines, it would be of interest to analyze the role of the Tax1-PKC interaction in the stabilization of p53 in HTLV-1-transformed cells (60). These studies may provide important insight into the role of Tax1 in transcriptional activation and control of cell growth. REFERENCES 1. Adachi, Y., T. Nosaka, and M. Hatanaka. 1990. Protein kinase inhibitor H-7 blocks accumulation of unspliced mRNA of human T-cell leukemia virus type I (HTLV-I). Biochem. Biophys. Res. Commun. 169:469–475. 2. Alexandre, C., P. Charnay, and B. Verrier. 1991. Transactivation of Krox-20 and Krox-24 promoters by the HTLV-I Tax protein through common regulatory elements. Oncogene 6:1851–1857. 3. Arima, N., J. A. Molitor, M. R. Smith, J. H. Kim, Y. Daitoku, and W. C. Greene. 1991. Human T-cell leukemia virus type I Tax induces expression of the Rel-related family of kB enhancer-binding proteins: evidence for a pretranslational component of regulation. J. Virol. 65:6892–6899. 4. Azzi, A., D. Boscoboinik, and C. Hensey. 1992. The protein kinase C family. Eur. J. Biochem. 208:547–557. 5. Baudier, J., C. Delphin, D. Grunwald, S. Kochbin, and J. J. Lawrence. 1992. Characterization of the tumor suppressor protein p53 as a protein kinase C substrate and a S100b-binding protein. Proc. Natl. Acad. Sci. USA 89:11627– 11631. 6. Beg, A. A., T. S. Finco, P. V. Nantermet, and A. S. Baldwin. 1993. Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of IkBa: a mechanism for NF-kB activation. Mol. Cell. Biol. 13:3301–3310. 7. Beraud, C., G. Lombard-Platet, Y. Michal, and P. Jalinot. 1991. Binding of the HTLV-1 Tax1 transactivator to the inducible 21 bp enhancer is mediated by the cellular factor HEB1. EMBO J. 10:3795–3803. 8. Block, C., S. Freyermuth, D. Beyersmann, and A. N. Malviya. 1992. Role of cadmium in activating nuclear protein kinase C and the enzyme binding to nuclear protein. J. Biol. Chem. 267:19824–19828. 9. Blumberg, P. M. 1991. Complexities of the protein kinase C pathway. Mol. Carcinog. 4:339–344. 10. Brady, J., K.-T. Jeang, J. Duvall, and G. Khoury. 1987. Identification of pX-responsive regulatory sequences within the human T-cell leukemia virus type I long terminal repeat. J. Virol. 61:2175–2181. 11. Brown, K., S. Park, T. Kanno, G. Franzoso, and U. Siebenlist. 1993. Mutual regulation of the transcriptional activator NF-kB and its inhibitor, IkBa. Proc. Natl. Acad. Sci. USA 90:2532–2536. 12. Bruns, R. F., F. D. Miller, R. L. Merriman, J. J. Howbert, W. F. Heath, E. Kobayashi, I. Takahashi, T. Tamaoki, and H. Nakano. 1991. Inhibition of protein kinase C by Calphostin C is light-dependent. Biochem. Biophys. Res. Comm. 176:288–293. 13. Burgess, G. M., J. S. Mckinney, A. Fabiato, B. A. Lesie, and J. W. Putney. 1983. Calcium pools in saponin-permeabilized guinea pig hepatocytes. J. Biol. Chem. 258:15336–15345. 14. Chowdhury, I. H., Y. Koyanagi, S. Kobayashi, Y. Hamamoto, H. Yoshiyama, T. Yoshida, and N. Yamamoto. 1990. The phorbol ester TPA strongly inhibits HIV-1-induced syncytia formation but enhances virus production: possible involvement of protein kinase C pathway. Virology 176:126–132. 15. Connor, L. M., M. N. Oxman, J. N. Brady, and S. J. Marriott. 1993. Twentyone base pair repeat elements influence the ability of a Gal4-Tax fusion protein to transactivate the HTLV-I long terminal repeat. Virology 195:569– 577. 16. Cordle, S., R. Donald, M. A. Read, and J. Hawiger. 1993. Lipopolysaccharide induces phosphorylation of MAD3 and activation of c-Rel and related NF-kB proteins in human monocytic THP-1 cells. J. Biol. Chem. 268:11803– 11810. 17. Cross, S. L., M. B. Feinberg, J. B. Wolf, N. J. Holbrook, F. Wong-Staal, and W. J. Leonard. 1987. Regulation of the human interleukin-2 receptor a chain
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