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Dominant negative Tax double mutants as molecular inhibitors for w.t. Tax gene functions
Leukemia Research 33 (2009) 974–979
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Dominant negative Tax double mutants as molecular inhibitors for w.t. Tax gene functions Mahmoud Egbaria a , Yulia Tabakin-Fix b , Mahmoud Huleihel a,∗ a b
Department of Virology and Developmental Genetics, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel Department of Microbiology and Immunology and Cancer Research Center, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel
a r t i c l e
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Article history: Received 19 July 2008 Received in revised form 20 September 2008 Accepted 25 October 2008 Available online 2 December 2008 Keywords: HTLV-1 Tax Cytoplasm Tax mutants Double Tax mutants NF-B CBP
a b s t r a c t Tax plays a key role in HTLV-1 pathogenicity, partly due to its capacity of constitutive NF-B activation. 58Tax does not translocate to the nucleus and traps w.t. Tax molecules in the cytoplasm but still retains the cytoplasmic NF-B activation ability. Therefore, it enhances the w.t. Tax activation of NF-B when co-expressed at lower level than w.t. Tax. However, the double mutants 58Tax (M22) and 58Tax (148) are defective also in the cytoplasmic NF-B activation. They were found as capable of blocking the w.t. Tax-induced NF-B-dependent activation even when expressed at low levels. These double mutants may, therefore, be used as powerful tools for blocking w.t. Tax functions. © 2008 Elsevier Ltd. All rights reserved.
1. Introduction Human T-cell leukemia virus type 1 (HTLV-1) is etiologically associated with adult T-cell leukemia (ATL), tropical spastic paraparesis or HTLV-1 associated myelopathy (TSP/HAM) and certain other clinical disorders [1,2]. The viral Tax protein is widely regarded as an oncoprotein playing a key role in ATL and TSP genesis due to its pleiotropic effects on many cellular processes [1,2]. Tax modulates the expression of numerous genes through the CREB/ATF, SRF and NF-B pathways. Particularly important is Tax ability to activate the NF-B factors. The NF-B family includes the c-Rel proto-oncogene and its related transcription factors, Rel A (p65), Rel B, NF-B1 (p50) and NF-B2 (p52). These factors share a 300-bp N-terminal Rel homology domain (RHD) containing a binding site for a set of related DNA target sequences called B elements, a dimerization region and a nuclear localization signal (NLS) [1,3]. These factors form various combinations of homo- and hetero-dimers, of which p65/p65 and p65/p50 are the most abundant dimers involved in NF-B-mediated gene activation [4]. They affect the expression
∗ Corresponding author at: Ben Gurion University of the Negev, Faculty of Health Sciences, Department of Virology and Developmental Genetics, POB 653, Beer Sheva 84105, Israel. Tel.: +972 8 6479867; fax: +972 8 6479867. E-mail address: [email protected] (M. Huleihel). 0145-2126/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2008.10.023
of numerous cellular and viral genes [5,6] and are involved in the regulation of diverse biological processes, such as embryonic development, immune and inflammatory responses, cell growth, apoptosis, stress responses and oncogenesis [7], as well as the function of several pathogenic viruses [5]. Normally, in most cells the NF-B dimmers are held in the cytoplasm at inactive form bound to inhibitory proteins of the IB (inhibitors of NF-B) family. These proteins contain ankyrin repeats, through which they bind to the NF-B RHD and prevent their nuclear translocation by masking their NLS [8]. Functional activation of the sequestered NF-Bs is triggered by a wide variety of external stimuli [9] and proceeds in two phases. The first phase includes proximal events that lead to IB degradation which releases the sequestered NFB factors to translocate into the nucleus. This initial phase is triggered by external stimuli that induce various specific signaling cascades, each of which may include different downstream signaling kinases [10], but most of them converge, eventually, at activation of a large multi-subunits IB kinase (IKK) complex by various specific protein kinases [11]. The IKK complex consists of two catalytic subunits, IKK␣ and IKK [12] and a regulatory subunit called IKK␥ or NF-B essential modulator (NEMO). IKK␥/NEMO serves as a scaffold for the IKK complex assembly [13] and for the upstream kinases targeted to this complex. By association with IKK␥/NEMO these kinases activate the catalytic subunits IKK␣ and IKK by phosphorylating them at a specific serine residues [3,14]. The activated IKK␣ and IKK phosphorylate specific serine residues
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of the sequestering IBs (Ser32 and Ser36 in IB␣, and Ser19 and Ser23 in IB), thus rendering them accessible for ubiquitination and subsequent proteosomal degradation which liberates the sequestered NF-Bs to translocate to the nucleus [15]. However, the mere nuclear translocation of the NF-B factors is insufficient for inducing NF-B-dependent gene expression. For this purpose, this nuclear translocation needs to be complemented by the second phase of the NF-B activation, which involves binding of the nuclear p65 factor to the CBP/p300 and P/CAF co-activators that are essential for the transcriptional competence of the p65:p65 and p65:p50 dimmers [16]. These co-activators exert their effect through histone acetylation, which induces chromatin conformational modification at the site of the target promoter and facilitates, thereby, the interaction of the enhancer-bound transcriptional activators with the TATAA box-associated basal transcriptional factors [17]. The binding to these co-activators requires phosphorylation of p65 at specific serine residues, which is carried out by PKA or certain other signal-activated serine kinases [18]. In contrast to the transient activation of the NF-B factors by the external signals, Tax renders them constitutively active in HTLV-1 infected cells. Previous studies have suggested that Tax may release NF-B from IB by the following alternative cytoplasmic interactions: (a) Tax is capable of associating with the adaptor IKK␥/NEMO subunit. Tax can also bind to and activate the enzymatic function of the mitogen-activated protein kinase/extracellular signal-regulated kinase 1 (MEKK1) and the NF-B-inducing kinase (NIK) [3,19], which normally mediate the activation of IKK by certain external signals [10]. Thus, Tax can link these activated kinases to IKK␥/NEMO and recruit them to phosphorylate the IKK␣ and IKK subunits independently of external signals [3,19], (b) Tax can bind directly to IKK␣ and IKK and activate their kinase activity independently of their phosphorylation by upstream kinases [20] and (c) Tax can bind directly to the IBs and induce their proteosomal degradation independently of their phosphorylation by IKK [21]. In addition, Tax can link the free p65 to the CBP/p300 co-activators by bridging them together, thus enhancing, in this manner, the transcriptional competence of the respective nuclear NF-B dimers independently of phosphorylation of their p65 subunit [22]. It is currently thought that the release of the NF-B from IB by Tax occurs in the cytoplasm whereas Taxmediated liking of p65 to the transcriptional co-activators occurs in the nucleus [1]. However, we have recently provided evidence that Tax-mediated linking of p65 to the co-activators occurs, first, in the cytoplasm and then the entire complex of Tax, NF-B dimer and the co-activators translocates together to the nucleus, where it exerts high capacity of transcriptional activation [23]. Since Tax is regarded to be responsible for HTLV-1 pathogenicity, blocking its activities might be an attractive therapeutic approach against this virus. A cytoplasmic mutant 58Tax, which lacks the N-terminal 58 amino acids responsible for Tax nuclear translocation, can dimerize with w.t. Tax and trap it in the cytoplasm and thus blocking its nuclear activities [24]. However, these Tax mutants can still exert the cytoplasmic NF-B-activation [25]. Since, as noted above, NF-B activation plays an important role in the oncogenic potential of Tax, this mutant cannot be used safely as an anti-HTLV-1 therapeutic agent unless its NF-B activating capacity is mutagenically impaired. The mutants TaxM22 and TaxM148 are defective in NF-B activation domain and thus are unable to release NF-B factors from their cytoplasmic IBs [17]. Therefore, these factors cannot enter the nucleus and induce gene expression. However, these mutants can still effectively display other nuclear functions of Tax. Therefore, we sought to design Tax mutant which will be able to abrogate all Tax functions without having undesired side effects. Keeping with this line, we developed two double Tax mutants, 58Tax (M22) and 58Tax (M148) which cannot translocate to
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the nucleus and are unable to activate NF-B, but can still heterodimmerize with w.t. Tax and trap it in the cytoplasm. Since CD4 helper T-cells are the main target for HTLV-1 leukemogenicity in human infections [26], we chose to test these mutants in Jurkat cells which retained the original nature of helper T cells with regard to the inducibility of both IL-2 and IL-2Ra in response to extracellular stimuli [27]. We have found that these mutants strongly hamper the activation of CREB/ATF- , SRF- and NF-B-dependent gene expression by w.t. Tax. Our results suggest that these double mutants can block most of Tax activities and may potentially be useful for preventing ATL and curing TSP/HAM. 2. Materials and methods 2.1. Cells In this study we used the following cells: MT2, which are an HTLV-1 infected human T-cells, uninfected human T-cell lines Jurkat and JPX-9 cells [28] which are a Jurkat clone stably transfected with a plasmid expressing w.t. Tax via the inducible promoter of the metalothionine gene that is activated by heavy-metal ions like cadmium (Cd2+ ). JPX-9m cells are a subclone of JPX-9 containing a mutant Tax carrying an in-frame insertion of a single arginine residue at position 63 [29]. This Tax mutant shares the same 40 kDa electrophoretic mobility as the w.t. Tax but is unable to induce gene expression through CREB/ATF, NF-B and SRF pathways [30]. The cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum, glutamine and antibiotics. 2.2. Plasmids and transient transfection assay A reporter plasmid expressing luciferase through a minimal promoter linked to three copies of the consensus NF-B responsive element (pNF-B-Luc, Clontech Labs, Palo Alto, CA) was used to measure the NF-B transcriptional competence. The reporter plasmids expressing -gal through SV40 and hCMV promoters and luciferase through HTLV-1-LTRwere employed in our experiments. The plasmids expressing the HTLV-1 wild type (w.t.) Tax and its mutants, TaxM22 [31] and TaxM148 [22], through the hCMV promoter, were provided by Francoise Bex (Laboratorie de Microbiologie, Universite Libre de Bruxelles, B-1070 Brussels, Belgium) and Tax M29 which is expressed through SV40 promoter was provided by Jean-Michel Mesnard (Laboratoire Infections Retrovirales et Signalisation Cellulaire, Institute de Biologie, Montpellier, France). The mutant Tax M29 is impaired in Tax self-association [32]. The plasmid expressing the truncated 58Tax protein has been described elsewhere [33,34]. In addition, by inserting the M22, the M148 or the M29 mutation into 58Tax, with in vitro site-directed mutagenesis system (Promega kit) and the appropriate primers we generated the double mutants Tax58 (M22), Tax58 (M148) and Tax58 (M29) as instructed by the manufacturer. The indicated quantities of the various plasmids were transfected by electroporation into 107 cells and the total DNA was completed in each transfection mixture to 40 g with an empty plasmid as previously described [35]. The pRL-null vector expressing renilla (pRL-renilla, Promega Corporation, Madison, WI) was included (0.1 g) in each transfection assay for assessing the relative transfection efficiency. The enzymatic activities were assayed in the cell extracts 24 h after transfection and the luciferase activity was normalized according to that of renilla and expressed as fold of the relevant control of each experiment. 2.3. Western blot analysis of the expression and subcellular distribution of the Tax variants Plasmid expressing the indicated Tax mutant was transfected into MT2 cells. To assess the expressions efficiency of these plasmids, the level of their encoded Tax protein was measured in whole cell extracts (50 g protein) at 24 h after transfection by Western blot analysis as described elsewhere [35], using Tax specific monoclonal antibodies recognizing a C-terminal epitope (amino acids KHFRETEV), obtained from AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (Cat. No. 168b17-46-70). To follow the subcellular distribution of these Tax variants, cytoplasmic and nuclear fractions were prepared from the transfected cells and aliquots of 50 g protein were analyzed for the level of the corresponding Tax by Western blot with the same anti-Tax monoclonal antibody. To follow the effect of the Tax mutants on p65 and w.t. Tax nuclear translocation similar aliquots of the nuclear fractions were subjected to Western blot analysis with anti-p65 and anti-Tax antibodies. Equal loading of the different samples of whole cell extracts and the cytoplasmic fractions was assessed by stripping the blot from the first detecting antibodies and re-analyzing it with anti actin antibody. The loading of the nuclear extract samples was assessed with anti-lamin B antibody. Variation in transfection efficiency was assessed by including 10 ng of pRL-Renilla plasmid in each transfection and measuring the enzymatic activity in extract of the transfected cells.
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2.4. Immuno-co-precipitation analysis Aliquots of 150 g protein of the indicated sub-cellular fractions were immunoprecipitated with the specified antibody and the immuno-precipitates were assayed by Western blotting for the indicated co-precipitating proteins with the respective antibody as previously described [34].
3. Results 3.1. Effect of the double mutants 58Tax (M22) and 58Tax (M148) on the subcellular distribution of w.t. Tax, p65 and CBP proteins The effect of the Tax double mutants on the subcellular distribution of p65, Tax and CBP proteins was determined by a Western blot analysis of the cytoplasmic and the nuclear fractions of Jurkat cells transiently transfected with w.t. Tax alone or together with increasing amounts of the tested Tax mutants. As a negative control, Jurkat cells were transfected also with Tax58(M29) mutant which is unable to dimerize with other Tax molecule. The transfection efficiency was estimated by co-transfection of these cells with an equal amount of a plasmid expressing the green fluorescent protein (GEF) together with the tested Tax mutants to be 52 ± 7%. The results presented in Fig. 1 show that the double mutant 58Tax (M22) significantly inhibits the nuclear translocation of Tax, p65 and CBP proteins when similar amounts of the mutant and the w.t. Tax (3 g) were co-transfected. However, when high excess of the mutant (12 g) is co-transfected with the w.t. Tax (3 g), it completely inhibits the nuclear translocation of these proteins. Similar results were obtained with the other double mutant 58Tax (M148) (not shown). The 58Tax mutant, on the other hand, can effectively prevent the nuclear translocation of these proteins (Tax, p65 and CBP proteins) only when it is transfected at high excess over the w.t. Tax (about 4-folds higher). Whereas, it confers only about 30% inhibition when transfected at a dose similar to that of the w.t. Tax. It is likely that since the double mutants are not engaged in NF-B activation, higher portion of their protein is free to dimerize with w.t. Tax and block more efficiently its nuclear translocation. The negative control Tax58 (M29), as expected, could not prevent the nuclear translocation of these proteins (Fig. 1). To determine more directly the ability of the Tax mutants to hetero-dimerize with the w.t. Tax and probably with p65 and CBP
Fig. 1. Western blot analysis of the cytoplasmic and nuclear fractions of Jurkat cells transfected Tax mutants. The indicated combinations of w.t. Tax and its cytoplasmic mutants were transfected into Jurkat cells. At 24 h after transfection, aliquots of cytoplasmic and nuclear fractions (100 g protein) were analyzed by Western blotting anti-Tax, anti-p65 and anti-CBP monoclonal antibodies. The presented results are from three repetitive experiments.
Fig. 2. Immuno co-precipitation analysis of the cytoplasmic and nuclear fractions of Jurkat cells transfected with w.t. Tax and Tax mutants. To demonstrate heterodimerization of w.t. Tax with the cytoplasmic Tax mutants, the indicated doses of plasmids expressing w.t. Tax-flag and the specified Tax mutants were transfected into Jurkat cells. At 24 h after transfection, aliquots of cytoplasmic and nuclear fractions (150 g protein) were immuno-precipitated with rabbit anti-flag monoclonal antibody and the imunnoprecipitates were assayed by Western blotting for the indicated co-precipitating proteins with mouse anti-Tax, anti-p65 and anti-CBP monoclonal antibodies. The presented results are from three repetitive experiments.
proteins, 3 g of Tax-flag plasmid was transfected into Jurkat cells together with increasing amounts of each of the Tax mutants. At 24 h later, 150 g aliquots of the cytoplasmic and nuclear fractions of the transfected cells were immuno-precipitated with anti-flag or anti-Tax monoclonal antibodies. Fig. 2 shows that the amount of immuno-precipitated Tax, p65 and CBP proteins were much higher in the nucleus than in the cytoplasm of the cells transfected with the w.t. Tax-flag alone. However, when the cells were co-transfected with 58Tax, only high dose (12 g) of this mutant was able to effectively trap these proteins (w.t. Tax, p65 and CBP) in the cytoplasm, while at lower doses (3–6 g) it displayed only partial cytoplasmic retention of them. On the other hand, the double mutant 58Tax (M22) manifested a potent cytoplasmic trapping of these proteins (w.t. Tax, p65 and CBP) even at as low as 3 g dose. Similar results were obtained with the other double mutant 58Tax (M148) (data not shown). These results support the possibility of a p65-CBP-Tax complex formation in the cytoplasm which is trapped there by the Tax mutants. 3.2. Effect of the double Tax mutants on w.t. Tax activation of various promoters Plasmids expressing reporter genes via different NF-B- or CREB-responsive promoters were transfected into Jurkat cells with increasing amounts of the indicated Tax mutants in the absence or presence of 3 g of w.t. Tax. The results presented in Fig. 3 demonstrate that when transfected separately, the w.t. Tax strongly activates all the tested NF-B-responsive promoters (CMV, SV40 and NF-B) and the CREB/ATF-dependent HTLV-1-LTR whereas, none of the Tax mutants has any effect on these promoters. On the other hand, when increasing amounts of these Tax mutants were cotransfected with the w.t. Tax, at low doses (1–3 g), 58Tax further enhances the activation of the examined promoters by the w.t. Tax, while the double mutants 58Tax (M22) and 58Tax (M148) significantly reduce this Tax mediated activation. Only at higher doses 58Tax can effectively reduce the stimulation of these promoters by the w.t Tax. From these data it is evident that the stimulatory effect displayed by 58Tax at low doses was omitted from the double mutants due to inactivation of their NF-B induction capacity. We hypothesize also that the competition between w.t. Tax and 58Tax on the targets involved in the release of NF-B liberates a higher fraction of the w.t. Tax protein to transport into the nucleus and to be engaged with the promoter’s activation. In contrast,
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Fig. 3. Effect of the various Tax mutants on w.t. Tax activation of different promoters. 1 g of reporter genes controlled by the following promoters: (A) NF-B, (B) SV40, (C) CMV and (D) HTLV-1-LTR and 3 g of w.t. Tax were co-transfected into Jurkat cells alone or together with increasing doses of the various indicated mutants. In addition, the above reporter genes were also co-transfected only with increasing doses of the mutants (without the w.t. Tax). The activities of the various reporters were presented as a percent of their activity in cells transfected only with the reporter gene and the w.t. Tax (without the Tax mutants) as a positive control. A plasmid expressing the Renilla gene was used as a transfection efficiency control. The presented results are an average of three repetitive experiments ± S.D.
the double mutants do not alter the fraction of the w.t. Tax protein engaged with the NF-B activation on one hand and on the other hand higher fraction of their own protein is free to dimerize with w.t. Tax and therefore even at low doses they can effectively reduce its activatory effect on these promoters. Taken together, these data indicate that the double mutants 58Tax (M148) and 58Tax (M22) are able to inhibit the activation of both the NF-Band CREB-responsive promoters by the w.t. Tax much more effectively than 58Tax. Moreover, they are safer for use as a potential anti-HTLV-1 therapy since they are free of the undesired stimulatory effect of their low doses on the NF-B activation by the w.t. Tax. Based on the above results, the double mutant Tax can be considered as a potent inhibitor of all w.t. Tax activities. Yet, in all these experiments both w.t. Tax and the examined mutants were transiently transfected into the target cells. In the next step we examined the effect of these mutants on permanently expressed w.t. Tax. For this purpose, each of the examined mutants (the single mutants or the double) were transfected together with the above mentioned reporter plasmids into the HTLV-1-infected MT-2 cells or into JPX-9 cells which are Jurkat subclone containing integrated plasmid expressing w.t. Tax via an inducible promoter of the metalothionine gene which is activated by heavy metals like Cd2+ [28]. Fig. 4 presents the results of the NFB-Luc reporter. Similar results were obtained also with the other reporters (data not shown). These results demonstrate that 58Tax slightly stimulated the activity of the NFB-dependent promoter (Fig. 4) as well as of the other promoters (data not shown) induced by the internal cellular w.t. Tax, while higher amounts (8–12 g) of this mutant reduced the activity of theses promoters. Unlike 58Tax, 3 g of each of the two double mutants significantly reduced the NFB promoter activity in both of these cells (Fig. 4).
4. Discussion Transcription factors of the NF-B family regulate the expression of many cellular and viral genes [5,6] and consequently affect diverse biological processes, including oncogenesis [7]. Therefore, a substantial part of the oncogenic potential of HTLV-1 Tax protein is ascribed to its capacity of inducing a constitutive activation of the transcriptional competence of these factors [36]. The current view is that Tax affects NF-B through cytoplasmic and nuclear functions. In the cytoplasm, Tax acts to release NF-Bs from their IB proteins and facilitate their transport to the nucleus. Then, in the nucleus Tax interacts with the p65 member of the NF-B family and links it with the CBP/p300 and P/CAF co-activators, which are essential for the transcriptional activity of p65-containing NFB dimers. This and other Tax nuclear functions which activate a wide range of cellular genes involved in central biological pathways contributes to Tax oncogenic potential [1]. Trapping of the w.t. Tax in the cytoplasm by 58Tax mutant can avoid its nuclear activities. However, 58Tax is still capable of executing the release of NF-Bs from IBs in the cytoplasm and enables thereby their transport to the nucleus. Consequently this mutant retains a substantial oncogenic potential of its own [37]. This property should disqualify 58Tax for serving as a possible means for clinical anti-HTLV-1 gene therapy, unless its NF-B activating capacity is eliminated. On this ground, we have developed two double Tax mutants, Tax58(M22) and Tax58(M148) which cannot translocate to the nucleus and unable to activate NF-B, but can still heterodimerize with w.t. Tax and trap it in the cytoplasm. We found that these double mutants were indeed incapable of nuclear translocation, but could still dimerize with w.t. Tax and trap it in the cytoplasm (Figs. 1 and 2). However, unlike 58Tax, these double mutants were also incapable of releasing the NF-B factors form
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w.t. Tax is involved in forming this ternary complex in the absence of its cytoplasmic mutants, the entire complex moves rapidly to the nucleus, where it binds to and activates promoters of NF-Bdependent genes. Therefore, we and others could not detect so far this ternary complex in the cytoplasm of HTLV-1 infected or w.t. Tax-expressing cells. This notion is particularly important for developing therapeutic drugs aimed to block Tax-mediated linkage of p65 to CBP/p300/PCAF co-activators. Such drugs should be designed to accumulate and efficiently act within the cytoplasm because action of such drugs in the nucleus will supposedly be ineffective. Conflict of interest All authors confirm that they have not any financial and personal relationships with other people or organisations that could inappropriately influence (bias) their work. Acknowledgment This study on HTLV-1 is supported by grants from the Israel Science Foundation (ISF). References
Fig. 4. Effect of the double mutants on NF-B-dependent promoter activity in MT2 and JPX-9 cells. MT-2 (A) or Cd2+ JPX-9m (B) cells were transfected with a plasmid expressing the NF-B-dependent promoter (1 g) alone or together with the indicated increasing amounts of various mutants. The activity of the reporter was presented as a percent of its activity in cells transfected only with the reporter gene and the w.t. Tax (without the Tax mutants) as a positive control. Plasmid expressing the Renilla gene was used as a transfection effeciency control. The presented results are an average of three repeated experiments ± S.E.
their cytoplasmic inhibitors. Our results showed also that these double mutants abolished the w.t. Tax cytoplasmic and nuclear activities at 3–4-fold lower doses than 58Tax. Furthermore, we noted that low doses of 58Tax rather stimulated the w.t. Tax mediated NF-B-dependent reporter activation. It is reasonable to postulate that these low doses are not sufficient for efficient blocking the nuclear translocation of the w.t. Tax but can still dissociate NF-B from IBs, so that under these conditions they increase the amount of the nuclear NF-B factors that can be transcriptionally activated by the nuclear w.t. Tax. In contrast, however, no such stimulation was induced by similar low doses of the double mutants, most likely because they could not provide free NF-B for transcriptional activation by the w.t. Tax. Moreover, as can be seen from Fig. 3 the w.t. Tax induced activation of different NF-B or CREBresponsive promoters was reduced even by these low doses of the double mutants. It is likely that since these double mutants are not engaged in NF-B activation, higher portion of their protein is free to dimerize with w.t. Tax and block more efficiently its nuclear translocation. Notably, we found that the inhibitory levels of 58Tax and the double mutants were able to bind the w.t. Tax, p65 (RelA) and CBP factors and trap the Tax-p65 (RelA)–CBP ternary complex in the cytoplasm (Fig. 2). Thus in contrary to the above mentioned current view on the separate cytoplasmic and nuclear Tax functions in NF-B activation, this finding suggests that Tax links the p65 containing NF-B dimmers already in the cytoplasm. However, when
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[30] Ng PW, Iha H, Iwanaga Y, Bittner M, Chen Y, Jiang Y, et al. Genome-wide expression changes induced by HTLV-1 Tax: evidence for MLK-3 mixed lineage kinase involvement in Tax-mediated NF-B activation. Oncogene 2001;20:4484–96. [31] Smith MR, Green WC. Identification of HTLV-I tax transactivator mutants exhibiting novel transcriptional phenotype. Genes Dev 1990;4:1875– 85. [32] Basbous J, Bazrbachi A, Granier C, Devaux C, Mesnard JM. The central region oh human T-cell leukemia virus type 1 Tax protein contains distinct domains involved in subunit dimerization. J Virol 2003:13028–35. [33] Gitlin SD, Lindholm PF, Marriott SJ, Brady JN. Transdominant human T-cell lymphotropic virus type I tax1 mutant that fails to localize to the nucleus. J Virol 1991;65:2612–21. [34] Torgeman A, Mor-Vaknin N, Zelin E, Ben-Aroya Z, Lochelt M, Flugel RM, et al. Sp1–p53 heterocomplex mediates activation of HTLV-I long terminal repeat by 12-O-tetradecanoylphorbol-13-acetate that is antagonized by protein kinase C. Virology 2001;281:10–20. [35] Mor-Vaknin N, Torgeman A, Galron D, Lochelt M, Flugel RM, Aboud M. The long terminal repeats of human immunodeficiency virus type-1 and human T-cell leukemia virus type-I are activated by 12-O-tetradecanoylphorbol-13-acetate through different pathways. Virology 1997;232:337–44. [36] Peloponese JMJ, Jeang KT. Role for Akt/protein kinase B and activator protein-1 in cellular proliferation induced by the human T-cell leukemia virus type 1 tax oncoprotein. J Biol Chem 2006:8927–38. [37] Nicot C, Tie F, Giam CZ. Cytoplasmic forms of human T-cell leukemia virus type 1 Tax induce NF-B activation. J Virol 1998;72:6777–84.
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Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Dominant negative Tax double mutants as molecular inhibitors for w.t. Tax gene functions Mahmoud Egbaria a , Yulia Tabakin-Fix b , Mahmoud Huleihel a,∗ a b
Department of Virology and Developmental Genetics, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel Department of Microbiology and Immunology and Cancer Research Center, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel
a r t i c l e
i n f o
Article history: Received 19 July 2008 Received in revised form 20 September 2008 Accepted 25 October 2008 Available online 2 December 2008 Keywords: HTLV-1 Tax Cytoplasm Tax mutants Double Tax mutants NF-B CBP
a b s t r a c t Tax plays a key role in HTLV-1 pathogenicity, partly due to its capacity of constitutive NF-B activation. 58Tax does not translocate to the nucleus and traps w.t. Tax molecules in the cytoplasm but still retains the cytoplasmic NF-B activation ability. Therefore, it enhances the w.t. Tax activation of NF-B when co-expressed at lower level than w.t. Tax. However, the double mutants 58Tax (M22) and 58Tax (148) are defective also in the cytoplasmic NF-B activation. They were found as capable of blocking the w.t. Tax-induced NF-B-dependent activation even when expressed at low levels. These double mutants may, therefore, be used as powerful tools for blocking w.t. Tax functions. © 2008 Elsevier Ltd. All rights reserved.
1. Introduction Human T-cell leukemia virus type 1 (HTLV-1) is etiologically associated with adult T-cell leukemia (ATL), tropical spastic paraparesis or HTLV-1 associated myelopathy (TSP/HAM) and certain other clinical disorders [1,2]. The viral Tax protein is widely regarded as an oncoprotein playing a key role in ATL and TSP genesis due to its pleiotropic effects on many cellular processes [1,2]. Tax modulates the expression of numerous genes through the CREB/ATF, SRF and NF-B pathways. Particularly important is Tax ability to activate the NF-B factors. The NF-B family includes the c-Rel proto-oncogene and its related transcription factors, Rel A (p65), Rel B, NF-B1 (p50) and NF-B2 (p52). These factors share a 300-bp N-terminal Rel homology domain (RHD) containing a binding site for a set of related DNA target sequences called B elements, a dimerization region and a nuclear localization signal (NLS) [1,3]. These factors form various combinations of homo- and hetero-dimers, of which p65/p65 and p65/p50 are the most abundant dimers involved in NF-B-mediated gene activation [4]. They affect the expression
∗ Corresponding author at: Ben Gurion University of the Negev, Faculty of Health Sciences, Department of Virology and Developmental Genetics, POB 653, Beer Sheva 84105, Israel. Tel.: +972 8 6479867; fax: +972 8 6479867. E-mail address: [email protected] (M. Huleihel). 0145-2126/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2008.10.023
of numerous cellular and viral genes [5,6] and are involved in the regulation of diverse biological processes, such as embryonic development, immune and inflammatory responses, cell growth, apoptosis, stress responses and oncogenesis [7], as well as the function of several pathogenic viruses [5]. Normally, in most cells the NF-B dimmers are held in the cytoplasm at inactive form bound to inhibitory proteins of the IB (inhibitors of NF-B) family. These proteins contain ankyrin repeats, through which they bind to the NF-B RHD and prevent their nuclear translocation by masking their NLS [8]. Functional activation of the sequestered NF-Bs is triggered by a wide variety of external stimuli [9] and proceeds in two phases. The first phase includes proximal events that lead to IB degradation which releases the sequestered NFB factors to translocate into the nucleus. This initial phase is triggered by external stimuli that induce various specific signaling cascades, each of which may include different downstream signaling kinases [10], but most of them converge, eventually, at activation of a large multi-subunits IB kinase (IKK) complex by various specific protein kinases [11]. The IKK complex consists of two catalytic subunits, IKK␣ and IKK [12] and a regulatory subunit called IKK␥ or NF-B essential modulator (NEMO). IKK␥/NEMO serves as a scaffold for the IKK complex assembly [13] and for the upstream kinases targeted to this complex. By association with IKK␥/NEMO these kinases activate the catalytic subunits IKK␣ and IKK by phosphorylating them at a specific serine residues [3,14]. The activated IKK␣ and IKK phosphorylate specific serine residues
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of the sequestering IBs (Ser32 and Ser36 in IB␣, and Ser19 and Ser23 in IB), thus rendering them accessible for ubiquitination and subsequent proteosomal degradation which liberates the sequestered NF-Bs to translocate to the nucleus [15]. However, the mere nuclear translocation of the NF-B factors is insufficient for inducing NF-B-dependent gene expression. For this purpose, this nuclear translocation needs to be complemented by the second phase of the NF-B activation, which involves binding of the nuclear p65 factor to the CBP/p300 and P/CAF co-activators that are essential for the transcriptional competence of the p65:p65 and p65:p50 dimmers [16]. These co-activators exert their effect through histone acetylation, which induces chromatin conformational modification at the site of the target promoter and facilitates, thereby, the interaction of the enhancer-bound transcriptional activators with the TATAA box-associated basal transcriptional factors [17]. The binding to these co-activators requires phosphorylation of p65 at specific serine residues, which is carried out by PKA or certain other signal-activated serine kinases [18]. In contrast to the transient activation of the NF-B factors by the external signals, Tax renders them constitutively active in HTLV-1 infected cells. Previous studies have suggested that Tax may release NF-B from IB by the following alternative cytoplasmic interactions: (a) Tax is capable of associating with the adaptor IKK␥/NEMO subunit. Tax can also bind to and activate the enzymatic function of the mitogen-activated protein kinase/extracellular signal-regulated kinase 1 (MEKK1) and the NF-B-inducing kinase (NIK) [3,19], which normally mediate the activation of IKK by certain external signals [10]. Thus, Tax can link these activated kinases to IKK␥/NEMO and recruit them to phosphorylate the IKK␣ and IKK subunits independently of external signals [3,19], (b) Tax can bind directly to IKK␣ and IKK and activate their kinase activity independently of their phosphorylation by upstream kinases [20] and (c) Tax can bind directly to the IBs and induce their proteosomal degradation independently of their phosphorylation by IKK [21]. In addition, Tax can link the free p65 to the CBP/p300 co-activators by bridging them together, thus enhancing, in this manner, the transcriptional competence of the respective nuclear NF-B dimers independently of phosphorylation of their p65 subunit [22]. It is currently thought that the release of the NF-B from IB by Tax occurs in the cytoplasm whereas Taxmediated liking of p65 to the transcriptional co-activators occurs in the nucleus [1]. However, we have recently provided evidence that Tax-mediated linking of p65 to the co-activators occurs, first, in the cytoplasm and then the entire complex of Tax, NF-B dimer and the co-activators translocates together to the nucleus, where it exerts high capacity of transcriptional activation [23]. Since Tax is regarded to be responsible for HTLV-1 pathogenicity, blocking its activities might be an attractive therapeutic approach against this virus. A cytoplasmic mutant 58Tax, which lacks the N-terminal 58 amino acids responsible for Tax nuclear translocation, can dimerize with w.t. Tax and trap it in the cytoplasm and thus blocking its nuclear activities [24]. However, these Tax mutants can still exert the cytoplasmic NF-B-activation [25]. Since, as noted above, NF-B activation plays an important role in the oncogenic potential of Tax, this mutant cannot be used safely as an anti-HTLV-1 therapeutic agent unless its NF-B activating capacity is mutagenically impaired. The mutants TaxM22 and TaxM148 are defective in NF-B activation domain and thus are unable to release NF-B factors from their cytoplasmic IBs [17]. Therefore, these factors cannot enter the nucleus and induce gene expression. However, these mutants can still effectively display other nuclear functions of Tax. Therefore, we sought to design Tax mutant which will be able to abrogate all Tax functions without having undesired side effects. Keeping with this line, we developed two double Tax mutants, 58Tax (M22) and 58Tax (M148) which cannot translocate to
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the nucleus and are unable to activate NF-B, but can still heterodimmerize with w.t. Tax and trap it in the cytoplasm. Since CD4 helper T-cells are the main target for HTLV-1 leukemogenicity in human infections [26], we chose to test these mutants in Jurkat cells which retained the original nature of helper T cells with regard to the inducibility of both IL-2 and IL-2Ra in response to extracellular stimuli [27]. We have found that these mutants strongly hamper the activation of CREB/ATF- , SRF- and NF-B-dependent gene expression by w.t. Tax. Our results suggest that these double mutants can block most of Tax activities and may potentially be useful for preventing ATL and curing TSP/HAM. 2. Materials and methods 2.1. Cells In this study we used the following cells: MT2, which are an HTLV-1 infected human T-cells, uninfected human T-cell lines Jurkat and JPX-9 cells [28] which are a Jurkat clone stably transfected with a plasmid expressing w.t. Tax via the inducible promoter of the metalothionine gene that is activated by heavy-metal ions like cadmium (Cd2+ ). JPX-9m cells are a subclone of JPX-9 containing a mutant Tax carrying an in-frame insertion of a single arginine residue at position 63 [29]. This Tax mutant shares the same 40 kDa electrophoretic mobility as the w.t. Tax but is unable to induce gene expression through CREB/ATF, NF-B and SRF pathways [30]. The cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum, glutamine and antibiotics. 2.2. Plasmids and transient transfection assay A reporter plasmid expressing luciferase through a minimal promoter linked to three copies of the consensus NF-B responsive element (pNF-B-Luc, Clontech Labs, Palo Alto, CA) was used to measure the NF-B transcriptional competence. The reporter plasmids expressing -gal through SV40 and hCMV promoters and luciferase through HTLV-1-LTRwere employed in our experiments. The plasmids expressing the HTLV-1 wild type (w.t.) Tax and its mutants, TaxM22 [31] and TaxM148 [22], through the hCMV promoter, were provided by Francoise Bex (Laboratorie de Microbiologie, Universite Libre de Bruxelles, B-1070 Brussels, Belgium) and Tax M29 which is expressed through SV40 promoter was provided by Jean-Michel Mesnard (Laboratoire Infections Retrovirales et Signalisation Cellulaire, Institute de Biologie, Montpellier, France). The mutant Tax M29 is impaired in Tax self-association [32]. The plasmid expressing the truncated 58Tax protein has been described elsewhere [33,34]. In addition, by inserting the M22, the M148 or the M29 mutation into 58Tax, with in vitro site-directed mutagenesis system (Promega kit) and the appropriate primers we generated the double mutants Tax58 (M22), Tax58 (M148) and Tax58 (M29) as instructed by the manufacturer. The indicated quantities of the various plasmids were transfected by electroporation into 107 cells and the total DNA was completed in each transfection mixture to 40 g with an empty plasmid as previously described [35]. The pRL-null vector expressing renilla (pRL-renilla, Promega Corporation, Madison, WI) was included (0.1 g) in each transfection assay for assessing the relative transfection efficiency. The enzymatic activities were assayed in the cell extracts 24 h after transfection and the luciferase activity was normalized according to that of renilla and expressed as fold of the relevant control of each experiment. 2.3. Western blot analysis of the expression and subcellular distribution of the Tax variants Plasmid expressing the indicated Tax mutant was transfected into MT2 cells. To assess the expressions efficiency of these plasmids, the level of their encoded Tax protein was measured in whole cell extracts (50 g protein) at 24 h after transfection by Western blot analysis as described elsewhere [35], using Tax specific monoclonal antibodies recognizing a C-terminal epitope (amino acids KHFRETEV), obtained from AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (Cat. No. 168b17-46-70). To follow the subcellular distribution of these Tax variants, cytoplasmic and nuclear fractions were prepared from the transfected cells and aliquots of 50 g protein were analyzed for the level of the corresponding Tax by Western blot with the same anti-Tax monoclonal antibody. To follow the effect of the Tax mutants on p65 and w.t. Tax nuclear translocation similar aliquots of the nuclear fractions were subjected to Western blot analysis with anti-p65 and anti-Tax antibodies. Equal loading of the different samples of whole cell extracts and the cytoplasmic fractions was assessed by stripping the blot from the first detecting antibodies and re-analyzing it with anti actin antibody. The loading of the nuclear extract samples was assessed with anti-lamin B antibody. Variation in transfection efficiency was assessed by including 10 ng of pRL-Renilla plasmid in each transfection and measuring the enzymatic activity in extract of the transfected cells.
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2.4. Immuno-co-precipitation analysis Aliquots of 150 g protein of the indicated sub-cellular fractions were immunoprecipitated with the specified antibody and the immuno-precipitates were assayed by Western blotting for the indicated co-precipitating proteins with the respective antibody as previously described [34].
3. Results 3.1. Effect of the double mutants 58Tax (M22) and 58Tax (M148) on the subcellular distribution of w.t. Tax, p65 and CBP proteins The effect of the Tax double mutants on the subcellular distribution of p65, Tax and CBP proteins was determined by a Western blot analysis of the cytoplasmic and the nuclear fractions of Jurkat cells transiently transfected with w.t. Tax alone or together with increasing amounts of the tested Tax mutants. As a negative control, Jurkat cells were transfected also with Tax58(M29) mutant which is unable to dimerize with other Tax molecule. The transfection efficiency was estimated by co-transfection of these cells with an equal amount of a plasmid expressing the green fluorescent protein (GEF) together with the tested Tax mutants to be 52 ± 7%. The results presented in Fig. 1 show that the double mutant 58Tax (M22) significantly inhibits the nuclear translocation of Tax, p65 and CBP proteins when similar amounts of the mutant and the w.t. Tax (3 g) were co-transfected. However, when high excess of the mutant (12 g) is co-transfected with the w.t. Tax (3 g), it completely inhibits the nuclear translocation of these proteins. Similar results were obtained with the other double mutant 58Tax (M148) (not shown). The 58Tax mutant, on the other hand, can effectively prevent the nuclear translocation of these proteins (Tax, p65 and CBP proteins) only when it is transfected at high excess over the w.t. Tax (about 4-folds higher). Whereas, it confers only about 30% inhibition when transfected at a dose similar to that of the w.t. Tax. It is likely that since the double mutants are not engaged in NF-B activation, higher portion of their protein is free to dimerize with w.t. Tax and block more efficiently its nuclear translocation. The negative control Tax58 (M29), as expected, could not prevent the nuclear translocation of these proteins (Fig. 1). To determine more directly the ability of the Tax mutants to hetero-dimerize with the w.t. Tax and probably with p65 and CBP
Fig. 1. Western blot analysis of the cytoplasmic and nuclear fractions of Jurkat cells transfected Tax mutants. The indicated combinations of w.t. Tax and its cytoplasmic mutants were transfected into Jurkat cells. At 24 h after transfection, aliquots of cytoplasmic and nuclear fractions (100 g protein) were analyzed by Western blotting anti-Tax, anti-p65 and anti-CBP monoclonal antibodies. The presented results are from three repetitive experiments.
Fig. 2. Immuno co-precipitation analysis of the cytoplasmic and nuclear fractions of Jurkat cells transfected with w.t. Tax and Tax mutants. To demonstrate heterodimerization of w.t. Tax with the cytoplasmic Tax mutants, the indicated doses of plasmids expressing w.t. Tax-flag and the specified Tax mutants were transfected into Jurkat cells. At 24 h after transfection, aliquots of cytoplasmic and nuclear fractions (150 g protein) were immuno-precipitated with rabbit anti-flag monoclonal antibody and the imunnoprecipitates were assayed by Western blotting for the indicated co-precipitating proteins with mouse anti-Tax, anti-p65 and anti-CBP monoclonal antibodies. The presented results are from three repetitive experiments.
proteins, 3 g of Tax-flag plasmid was transfected into Jurkat cells together with increasing amounts of each of the Tax mutants. At 24 h later, 150 g aliquots of the cytoplasmic and nuclear fractions of the transfected cells were immuno-precipitated with anti-flag or anti-Tax monoclonal antibodies. Fig. 2 shows that the amount of immuno-precipitated Tax, p65 and CBP proteins were much higher in the nucleus than in the cytoplasm of the cells transfected with the w.t. Tax-flag alone. However, when the cells were co-transfected with 58Tax, only high dose (12 g) of this mutant was able to effectively trap these proteins (w.t. Tax, p65 and CBP) in the cytoplasm, while at lower doses (3–6 g) it displayed only partial cytoplasmic retention of them. On the other hand, the double mutant 58Tax (M22) manifested a potent cytoplasmic trapping of these proteins (w.t. Tax, p65 and CBP) even at as low as 3 g dose. Similar results were obtained with the other double mutant 58Tax (M148) (data not shown). These results support the possibility of a p65-CBP-Tax complex formation in the cytoplasm which is trapped there by the Tax mutants. 3.2. Effect of the double Tax mutants on w.t. Tax activation of various promoters Plasmids expressing reporter genes via different NF-B- or CREB-responsive promoters were transfected into Jurkat cells with increasing amounts of the indicated Tax mutants in the absence or presence of 3 g of w.t. Tax. The results presented in Fig. 3 demonstrate that when transfected separately, the w.t. Tax strongly activates all the tested NF-B-responsive promoters (CMV, SV40 and NF-B) and the CREB/ATF-dependent HTLV-1-LTR whereas, none of the Tax mutants has any effect on these promoters. On the other hand, when increasing amounts of these Tax mutants were cotransfected with the w.t. Tax, at low doses (1–3 g), 58Tax further enhances the activation of the examined promoters by the w.t. Tax, while the double mutants 58Tax (M22) and 58Tax (M148) significantly reduce this Tax mediated activation. Only at higher doses 58Tax can effectively reduce the stimulation of these promoters by the w.t Tax. From these data it is evident that the stimulatory effect displayed by 58Tax at low doses was omitted from the double mutants due to inactivation of their NF-B induction capacity. We hypothesize also that the competition between w.t. Tax and 58Tax on the targets involved in the release of NF-B liberates a higher fraction of the w.t. Tax protein to transport into the nucleus and to be engaged with the promoter’s activation. In contrast,
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Fig. 3. Effect of the various Tax mutants on w.t. Tax activation of different promoters. 1 g of reporter genes controlled by the following promoters: (A) NF-B, (B) SV40, (C) CMV and (D) HTLV-1-LTR and 3 g of w.t. Tax were co-transfected into Jurkat cells alone or together with increasing doses of the various indicated mutants. In addition, the above reporter genes were also co-transfected only with increasing doses of the mutants (without the w.t. Tax). The activities of the various reporters were presented as a percent of their activity in cells transfected only with the reporter gene and the w.t. Tax (without the Tax mutants) as a positive control. A plasmid expressing the Renilla gene was used as a transfection efficiency control. The presented results are an average of three repetitive experiments ± S.D.
the double mutants do not alter the fraction of the w.t. Tax protein engaged with the NF-B activation on one hand and on the other hand higher fraction of their own protein is free to dimerize with w.t. Tax and therefore even at low doses they can effectively reduce its activatory effect on these promoters. Taken together, these data indicate that the double mutants 58Tax (M148) and 58Tax (M22) are able to inhibit the activation of both the NF-Band CREB-responsive promoters by the w.t. Tax much more effectively than 58Tax. Moreover, they are safer for use as a potential anti-HTLV-1 therapy since they are free of the undesired stimulatory effect of their low doses on the NF-B activation by the w.t. Tax. Based on the above results, the double mutant Tax can be considered as a potent inhibitor of all w.t. Tax activities. Yet, in all these experiments both w.t. Tax and the examined mutants were transiently transfected into the target cells. In the next step we examined the effect of these mutants on permanently expressed w.t. Tax. For this purpose, each of the examined mutants (the single mutants or the double) were transfected together with the above mentioned reporter plasmids into the HTLV-1-infected MT-2 cells or into JPX-9 cells which are Jurkat subclone containing integrated plasmid expressing w.t. Tax via an inducible promoter of the metalothionine gene which is activated by heavy metals like Cd2+ [28]. Fig. 4 presents the results of the NFB-Luc reporter. Similar results were obtained also with the other reporters (data not shown). These results demonstrate that 58Tax slightly stimulated the activity of the NFB-dependent promoter (Fig. 4) as well as of the other promoters (data not shown) induced by the internal cellular w.t. Tax, while higher amounts (8–12 g) of this mutant reduced the activity of theses promoters. Unlike 58Tax, 3 g of each of the two double mutants significantly reduced the NFB promoter activity in both of these cells (Fig. 4).
4. Discussion Transcription factors of the NF-B family regulate the expression of many cellular and viral genes [5,6] and consequently affect diverse biological processes, including oncogenesis [7]. Therefore, a substantial part of the oncogenic potential of HTLV-1 Tax protein is ascribed to its capacity of inducing a constitutive activation of the transcriptional competence of these factors [36]. The current view is that Tax affects NF-B through cytoplasmic and nuclear functions. In the cytoplasm, Tax acts to release NF-Bs from their IB proteins and facilitate their transport to the nucleus. Then, in the nucleus Tax interacts with the p65 member of the NF-B family and links it with the CBP/p300 and P/CAF co-activators, which are essential for the transcriptional activity of p65-containing NFB dimers. This and other Tax nuclear functions which activate a wide range of cellular genes involved in central biological pathways contributes to Tax oncogenic potential [1]. Trapping of the w.t. Tax in the cytoplasm by 58Tax mutant can avoid its nuclear activities. However, 58Tax is still capable of executing the release of NF-Bs from IBs in the cytoplasm and enables thereby their transport to the nucleus. Consequently this mutant retains a substantial oncogenic potential of its own [37]. This property should disqualify 58Tax for serving as a possible means for clinical anti-HTLV-1 gene therapy, unless its NF-B activating capacity is eliminated. On this ground, we have developed two double Tax mutants, Tax58(M22) and Tax58(M148) which cannot translocate to the nucleus and unable to activate NF-B, but can still heterodimerize with w.t. Tax and trap it in the cytoplasm. We found that these double mutants were indeed incapable of nuclear translocation, but could still dimerize with w.t. Tax and trap it in the cytoplasm (Figs. 1 and 2). However, unlike 58Tax, these double mutants were also incapable of releasing the NF-B factors form
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w.t. Tax is involved in forming this ternary complex in the absence of its cytoplasmic mutants, the entire complex moves rapidly to the nucleus, where it binds to and activates promoters of NF-Bdependent genes. Therefore, we and others could not detect so far this ternary complex in the cytoplasm of HTLV-1 infected or w.t. Tax-expressing cells. This notion is particularly important for developing therapeutic drugs aimed to block Tax-mediated linkage of p65 to CBP/p300/PCAF co-activators. Such drugs should be designed to accumulate and efficiently act within the cytoplasm because action of such drugs in the nucleus will supposedly be ineffective. Conflict of interest All authors confirm that they have not any financial and personal relationships with other people or organisations that could inappropriately influence (bias) their work. Acknowledgment This study on HTLV-1 is supported by grants from the Israel Science Foundation (ISF). References
Fig. 4. Effect of the double mutants on NF-B-dependent promoter activity in MT2 and JPX-9 cells. MT-2 (A) or Cd2+ JPX-9m (B) cells were transfected with a plasmid expressing the NF-B-dependent promoter (1 g) alone or together with the indicated increasing amounts of various mutants. The activity of the reporter was presented as a percent of its activity in cells transfected only with the reporter gene and the w.t. Tax (without the Tax mutants) as a positive control. Plasmid expressing the Renilla gene was used as a transfection effeciency control. The presented results are an average of three repeated experiments ± S.E.
their cytoplasmic inhibitors. Our results showed also that these double mutants abolished the w.t. Tax cytoplasmic and nuclear activities at 3–4-fold lower doses than 58Tax. Furthermore, we noted that low doses of 58Tax rather stimulated the w.t. Tax mediated NF-B-dependent reporter activation. It is reasonable to postulate that these low doses are not sufficient for efficient blocking the nuclear translocation of the w.t. Tax but can still dissociate NF-B from IBs, so that under these conditions they increase the amount of the nuclear NF-B factors that can be transcriptionally activated by the nuclear w.t. Tax. In contrast, however, no such stimulation was induced by similar low doses of the double mutants, most likely because they could not provide free NF-B for transcriptional activation by the w.t. Tax. Moreover, as can be seen from Fig. 3 the w.t. Tax induced activation of different NF-B or CREBresponsive promoters was reduced even by these low doses of the double mutants. It is likely that since these double mutants are not engaged in NF-B activation, higher portion of their protein is free to dimerize with w.t. Tax and block more efficiently its nuclear translocation. Notably, we found that the inhibitory levels of 58Tax and the double mutants were able to bind the w.t. Tax, p65 (RelA) and CBP factors and trap the Tax-p65 (RelA)–CBP ternary complex in the cytoplasm (Fig. 2). Thus in contrary to the above mentioned current view on the separate cytoplasmic and nuclear Tax functions in NF-B activation, this finding suggests that Tax links the p65 containing NF-B dimmers already in the cytoplasm. However, when
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