The transcriptional coactivators CREB-binding protein and p300 regulate inducible transcription in multiple cellular processes and during the establishment of inflammatory and immune response. Several viruses have been shown to interfere with CREB-binding protein/p300 function, modulating their transcriptional activity. In this study, we report that the viral protein A238L interacts with the amino-terminal region of p300, inhibiting the acetylation and transcriptional activation of NF-ATc2, NF-κB, and c-Jun in stimulated human T cells. We demonstrate that A238L modulates the autoacetylation of p300 without altering its intrinsic histone acetyl transferase activity. Furthermore, we show that the molecular mechanism of the inhibition executed by the viral protein is conducted through blocking protein kinase C (PKC)-p300 interaction and further acetylation in the amino-terminal transactivation domain of the coactivator, and that Ser384, within the CH1 domain, is essential for the full transcriptional activation of the coactivator. Moreover, we show that overexpression of an active form of PKC-θ reverts the A238L-mediated inhibition of the transcriptional activity of p300, showing, for the first time, a PKC-θ-mediated up-regulation of the coactivator. These findings provide new strategies to develop therapies potentially useful in the control of disorders related to p300 deregulation.
The p300 and CREB-binding protein (CBP)3 are pivotal regulators of the inducible transcription of eukaryotic cells (1), and were first identified as proteins interacting with the transcription factor CREB (2) and the adenoviral protein E1A (3). These coactivators act in two different ways. On one hand, they act as a molecular bridge to connect the transcription factors with the transcriptional machinery, including TATA binding protein, transcription factor II B, transcription factor II D, and RNA polymerase II (4). In contrast, p300 and CBP are able to acetylate histones in the chromatin of target promoters, through a histone acetyl transferase (HAT) activity contained in its carboxyl-terminal region (5), thus forcing chromatine to acquire an open structure (6). In addition, recent evidence has demonstrated that HAT activity is extended to nonhistone proteins, including transcription factors (7). CBP/p300 interact with and enhance transactivation of a variety of DNA-binding transcription factors, including p53 (8), E2F family (9), CREB (10), NF-ATc2 (11, 12), NF-κB-p65 (13, 14), or AP-1 family (15, 16), coordinating the transcription of target genes. Therefore, CBP and p300 are involved in regulation of multiple cellular processes, such as proliferation, differentiation, tumor suppression, malignant transformation, and several immunological disorders (1, 17). Interestingly, several reports have demonstrated that certain viral proteins, such as adenovirus E1A (18), SV40 T large Ag (19), E6 and E7 proteins from human papillomavirus (20, 21), or Tax protein from human T cell leukemia virus type I (22) interact with CBP and p300, modulating their transcriptional activity. However, the specific mechanisms through which these viral proteins exert this function remain largely unknown.
CBP/p300 contains two domains presenting transcriptional activity, one in the amino-terminal and the other in the carboxyl-terminal regions, which can act independently and interact simultaneously with the transcriptional machinery and/or with different transcription factors to build the transcriptional activity mediated by these coactivators (23, 24). Transactivation mediated by CBP and p300 is regulated by multiple signaling pathways that promote posttranslational modifications on their transactivation domains (TAD), such as sumoilation, phosphorylation, methylation, and autoacetylation (25, 26, 27). Among them, phosphorylation appears to be one of the most important modifications, because the HAT activity of CBP and p300 is up-regulated by p42 and p44 MAPKs (28), Ca2+/calmodulin-dependent protein kinase IV (29), protein kinase A (30), Akt (31), and the recently demonstrated IκB kinase-α (32). Opposite to this, transcriptional activity of CBP/p300 is negatively controlled by cyclin-E/cdk-2 complex (33) or protein kinase C (PKC)-δ phosphorylation at a conserved residue, Ser89 (34). Although the functional effect of these kinases on p300 transactivation is known, the precise phosphorylation sites and the molecular mechanisms underlying this regulation by phosphorylation remain unclear.
It has been previously reported that the protein A238L from African swine fever virus is a dual inhibitor of the transcription factors NF-ATc2 and NF-κB-p65: the former by blocking activity of the calcineurin phosphatase (35) and the latter by displacement of its DNA-specific recognition sites (36). Moreover, we have previously demonstrated that the viral protein A238L is able to inhibit the transcriptional activation of certain inflammatory mediators through counteracting the transactivation mediated by the transcription factors NF-ATc2, NF-κB-p65, or c-Jun (37, 38).
In this study, we show that the viral protein localizes in the nucleus of Jurkat cells and interacts with the amino-terminal region of p300 and blocks its transactivation, thus inhibiting the acetylation and transcriptional activation of NF-ATc2, NF-κB, and c-Jun, in stimulated T cells. This inhibition is conducted through the inhibition of PKC-p300 interaction in its amino-terminal TAD. We also demonstrate that the phosphorylation of the amino-terminal TAD of p300 by PKC-θ and the subsequent autoacetylation of the coactivator are essential steps in its full transcriptional activation. Moreover, we showed that mutation of Ser384 abrogates the PKC-θ-mediated phosphorylation and autoacetylation of p300, suggesting that this residue is an important PKC-θ target involved in up-regulation of the amino-terminal transactivation activity of p300. We finally demonstrate that overexpression of active PKC-θ recovers the transcriptional activity of the coactivator, showing an essential role for this kinase in p300 transactivation. These data indicate that the molecular mechanism used by A238L to inhibit simultaneously the activation of NF-ATc2, NF-κB-p65, and c-Jun pathways exploits the blockage of the PKC-θ binding to this specific domain of the transcriptional coactivator.
These findings provide new strategies to develop tools potentially useful in the control of several disorders related to p300 deregulation.
Materials and Methods
Cell culture and reagents
Jurkat human leukemia T cell line was obtained from American Type Culture Collection and cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml gentamicin, and nonessential amino acids. Cells were grown at 37°C in 7% CO2 in air saturated with water vapor. Jurkat cells were stimulated by PMA (Sigma-Aldrich) at 15 ng/ml and A23187 calcium ionophore (Ion; Sigma-Aldrich) at 1 μM. Generation of A238L stably expressing Jurkat cells (Jurkat-A238L) and its control cell line (Jurkat-pcDNA) was previously described (39).
Western blot analysis
Cytosolic and nuclear extracts from Jurkat wild-type (wt), Jurkat-pcDNA, and Jurkat-A238L cells unstimulated or stimulated with PMA/Ion were prepared, as previously described (39). Briefly, cells were harvested by centrifugation, washed twice with PBS, and resuspended in 500 μl of buffer A (10 mM HEPES (pH 7.6), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.75 mM spermidine, 0.15 mM spermine, 1 mM DTT, 0.5 mM PMSF, 10 mM Na2MoO4, and 2 μg/ml each of inhibitors leupeptin, aprotinin, and pepstatin A). After 15 min at 4°C, 5 μl of a 10% Nonidet P-40 solution was added. Samples were vortexed for 10 s and centrifuged for 20 min at 3000 rpm and 4°C. The supernatants were used as cytosolic extracts. To avoid cytosolic contamination, nuclei were washed twice with 200 μl of buffer A. For nuclear protein extraction, 50 μl of buffer C (20 mM HEPES (pH 7.6), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 10 mM Na2MoO4, and 2 μg/ml each of inhibitors leupeptin, aprotinin, and pepstatin A) was added, and nuclear pellets were incubated for 30 min at 4°C with gentle agitation. Samples were centrifuged for 10 min at 14,000 rpm and 4°C, and supernatants were used as nuclear extracts. Whole-cell protein extracts from wt Jurkat cells were prepared, as previously described (37), using radioimmunoprecipitation assay (RIPA) buffer, containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, and 0.25% Na-deoxycholate, and supplemented with protease inhibitor mixture tablets (Roche). In any case, protein concentration was determined by the bicinchoninic acid (BCA) spectrophotometric method (Pierce). Cell lysates were fractionated by SDS-PAGE and electrophoretically transferred to an Immobilon extra membrane (Amersham), and the separated proteins reacted with specific primary Abs. The Abs used were as follows: NF-ATc2 (sc-7296; Santa Cruz Biotechnology), NF-κB-p65 (sc-109; Santa Cruz Biotechnology), c-Jun (sc-45; Santa Cruz Biotechnology), c-Fos (sc-52; Santa Cruz Biotechnology), p300 (sc-584; Santa Cruz Biotechnology), acetylated lysine (Ac-K-103; Cell Signaling Technology), GAL4 (sc-577; Santa Cruz Biotechnology), PKC (sc-10800; Santa Cruz Biotechnology), PKC-θ (sc-212; Santa Cruz Biotechnology), V5-TAG (MCA1360; Serotec), β-actin (AC-15; Sigma-Aldrich), and A238L polyclonal antiserum, which was previously described (39). Membranes were exposed to HRP-conjugated secondary Abs (Amersham Biosciences), followed by chemiluminescence (ECL; Amersham Biosciences) detection by autoradiography. Densitometric analysis was performed by using TINA 2.0 software.
Whole-cell extracts and nuclear extracts were prepared from 80–90% confluent Jurkat cells treated with or without PMA/Ion for 4 h, and their protein concentrations were determined, as described above. The extracts were incubated with specific Abs, as follows: NF-ATc2 (sc-7296; Santa Cruz Biotechnology), NF-κB-p65 (sc-109; Santa Cruz Biotechnology), c-Jun (sc-45; Santa Cruz Biotechnology), c-Fos (sc-52; Santa Cruz Biotechnology), p300 (sc-584; Santa Cruz Biotechnology), PKC (sc-10800; Santa Cruz Biotechnology), PKC-θ (sc-212; Santa Cruz Biotechnology), V5-TAG (MCA1360; Serotec), or a rabbit or mouse preimmune normal IgG as a negative control, at a final concentration of 4 μg/ml. The samples were incubated at 4°C overnight. Protein A/G-Sepharose beads (Sigma-Aldrich) were added, incubated for 3 h at 4°C, and centrifuged. The beads were washed three times with wash buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40). The immunoprecipitates were mixed with SDS loading buffer and analyzed by 4–15% SDS-PAGE, followed by Western blotting.
The pcDNA-A238L expression plasmid was generated, as described (39). The NF-AT-luc, containing three tandem copies of the distal NF-ATc2/AP-1 (position −286 to −257) binding site of the IL-2 promoter (40), was a gift from G. Crabtree (Department of Pathology and Developmental Biology, Howard Hughes Medical Institute, Stanford University Medical School, Stanford, CA). The NF-κB-p65 reporter plasmid (pNF3TKLuc) contains a trimer of the NF-κB-p65-binding motif of the H-2k gene upstream of the thymidine kinase minimal promoter and the luciferase reporter gene (41). AP-1-Luc plasmid includes the AP-1-responsive (−73 to +63 bp) region of the human collagenase promoter fused to the luciferase gene (42). The pGAL4-hNF-ATc2 construct containing the first 1–451 aa of human NF-ATc2 fused to the DNA-binding domain (DBD) of yeast GAL4 transcription factor was originated, as described previously (43). The GAL4-p65 construct has the yeast GAL4 DBD fused to the carboxyl-terminal TAD of p65, and was generated as described (44). The GAL4-c-Jun plasmid expressing the first 166 aa of the human c-Jun fused to the DBD of the yeast GAL4 transcription factor was generated, as described (45). GAL4-c-Fos was a gift from M. Fresno (Centro de Biología Molecular Severo Ochoa, Madrid, Spain). GAL4-Sp1 was supplied by S. Roberts (Division of Gene Expression, Department of Biochemistry, University of Dundee, Dundee, Scotland, U.K.). The GAL4-luciferase construct (pGAL4-Luc) contains five GAL4 DNA consensus binding sites derived from the yeast GAL4 gene fused to luciferase reporter gene (46). The GAL4-p300 full-length construct and the mutants (192–703, 1–1301, and 1239–2414) were generated, as described (47). The p300 wt expression plasmid pCI-p300 and its HAT deletion mutant, pCI-p300ΔHAT, were a gift from J. Boyes (Institute of Cancer Research, London, U.K.) and generated, as described (48). The pCDNA3-A238L-SV5 was a gift from L. Dixon (Institute for Animal Health, Woking, Surrey, U.K.) and generated, as described (35). The expression plasmids for wt and constitutively active mutant forms of PKC-θ (pEF-PKC-θ wt and pEF-PKC-θ A/E, respectively) were a gift from M. Villalba (Institut de Génétique Moléculaire de Montpellier, Centre National de la Recherche Scientifique-Unité Mixte de Recherche, Montpellier, France), and generated, as described (49). The pEFneo empty plasmid was a gift from M. Fresno (Centro de Biología Molecular Severo Ochoa, Madrid, Spain). GAL4-p300(FL)Ser384Ala and GAL4-p300(192–703)Ser384Ala were generated by using QuickChange Site-Directed Mutagenesis Kit (Stratagene) following manufacturer’s instructions. Oligonucleotides used for mutagenesis were as follows: p300S384A, forward (5′-CCACATGACACACTGCCAG-GCAGGCAAGTCTGCCAAGTGGC-3′) and reverse (5′-GCCACTTGGCAGACTTG-CCTGCCTGGCAGTGTGTCATGAGG-3′). Serine to alanine substitution is underlined. The plasmid pRL-tk-luc (Promega) was used in each case to evaluate transfection efficiency.
Transfection and luciferase assays
Generation of A238L stably expressing Jurkat cells was done as described previously (39). Jurkat cells were transfected with 250 ng of specific reporter plasmids or 1 μg of expression plasmids per 106 cells using the LipofectAMINE Plus Reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions, and mixing in Opti-MEM (Invitrogen Life Technologies) in a six-well plate. In cotransfection assays, the indicated doses of the corresponding expression plasmid/106 cells were added. The cells were incubated at 37°C during 4 h, washed, incubated in serum-free medium for 24 h, and treated with or without PMA/Ion. As a transfection control for luciferase assays, the Renilla luciferase control plasmid pRL-TK-luc (Promega) was cotransfected in all of the experiments. At the indicated poststimulation times, cells were lysed with 200 μl of Cell Culture Lysis Reagent (Promega) and microcentrifuged at full speed for 5 min at 4°C, and 20 μl of each supernatant was used to determine Firefly and Renilla luciferase activity in a Monolight 2010 luminometer (Analytical Luminescence Laboratory) using Dual Luciferase Assay System (Promega). Transfections were normalized to Renilla luciferase activity, and results were expressed as the relative luminescence units after normalization of protein concentration determined by the BCA method, as indicated in the figure legends. Transfection experiments were performed in triplicate, and the data were presented as the mean of the relative luminescence units (RLU) (mean ± SD).
Immunofluorescence and confocal microscopy
Jurkat cells were transfected with pcDNA 3.1 or pcDNA-A238L(SV5) (at a final concentration of 1 μg/106 cells), as described above. Then the cells were grown on poly(l-lysine) (Sigma-Aldrich)-treated coverslips to 2 × 105 cells/cm2. Twenty-four hours posttransfection, the cells were cultured in the absence (control) or presence of PMA/Ion during 30 or 60 min. The cultures were rinsed three times with PBS and fixed with cold 99.8% methanol (Merck) for 15 min at −20°C, before rehydrating twice with PBS and blocking with 1% BSA in PBS for 10 min at room temperature. The cells were incubated during 2 h with the specific Ab against p300 (sc-584; Santa Cruz Biotechnology) or SV5-tag (MCA1360; Serotec), rinsed extensively with PBS, and then incubated with the specific secondary Abs (Alexa; Molecular Probes) for 1 h at room temperature in the dark. Then, to show nuclear staining, the cells were labeled with 4′,6′-diamidino-2-phenylindole (DAPI; Molecular Probes). Finally, the cells were rinsed successively with PBS, distilled water, and ethanol, and mounted with a drop of Mowiol on a microslide. Visualization of stained cultures was performed under a fluorescence Axioskop2 plus (Zeiss) microscope coupled to a color charge-coupled device camera or to Confocal Microradiance (Bio-Rad) equipment. Images were digitalized, processed, and organized with Metamorph, Lasersharp2000 v.4, Adobe Photoshop 7.0, Adobe Illustrator 10, and Microsoft PowerPoint SP-2 software.
Solid-phase in vitro phosphorylation kinase assay
We used 2 μg of myosin-binding protein (MBP) (sc-4113; Santa Cruz Biotechnology) or immunoprecipitated GAL4-p300 (192–703)wt and GAL4-p300(192–703)S384A as the substrate for in vitro phosphorylation, in which immunoprecipitated pan-PKC or PKC-θ from Jurkat cells was assayed. Whole-cell extracts from 107 Jurkat cells cultured in the absence or presence of PMA/Ion during 30 min were prepared. The cells were lysed in RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1% Nonidet P-40, and supplemented with phosphatase inhibitors (1 mM NaVO3, 10 mM NaF, and 10 mM Na2MoO4) and protease inhibitors (0.5 mM PMSF, 1 μg of pepstatin, 2 μg of leupeptin, and 2 μg of aprotinin per ml).
Cleared extracts were incubated overnight with 4 μg of specific Ab against PKC (sc-10800; Santa Cruz Biotechnology) or PKC-θ (sc-212; Santa Cruz Biotechnology) to immunoprecipitate them. Precipitates were finally resuspended in kinase buffer containing 20 mM HEPES (pH 7.6), 20 mM MgCl2, 20 mM β-glycerophosphate, 20 μM ATP, and 1 μCi of [γ32P]ATP (sp. act., 3000 Ci/mol) supplemented with phosphatase inhibitors and mixed with the corresponding substrate. After 30 min at 30°C, the kinase reaction was terminated by washing with TNT buffer containing 20 mM Trizma base (pH 7.5), 200 mM NaCl, and 1% Triton X-100, and supplemented with protease inhibitor mixture tablets (Roche). Phosphorylated proteins were separated in a SDS-12% PAGE, dried, and developed by autoradiography.
Immunoprecipitation HAT assay
Jurkat cells were resuspended in RIPA lysis buffer, containing 50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.5% Nonidet P-40, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 100 μM PMSF, and 2 mM DTT. The lysis mixture was incubated on ice for 30 min to extract the whole cell proteins and centrifuged at 12,000 rpm at 4°C for 10 min. The supernatant protein concentration was determined by BCA method, as described above. For immunoprecipitation, the protein concentration was adjusted to 1 μg/μl in 500 μl. Protein extracts were incubated with a specific Ab against p300 (sc-584; Santa Cruz Biotechnology) or a rabbit preimmune normal IgG as a negative control, at a final concentration of 4 μg/ml. The samples were incubated at 4°C overnight. Protein A/G-Sepharose beads (Sigma-Aldrich) were added, incubated for 3 h at 4°C, and centrifuged. The beads were washed three times with RIPA lysis buffer. The beads were washed with HAT buffer (1 mM PMSF, 50 mM Tris-HCl (pH 8.0), 10% glycerol, 10 mM butyric acid, 0.2 mM EDTA, and 1 mM DTT), and a standard HAT assay was performed containing the immunoprecipitated p300 with the corresponding substrate (either 200 ng of purified p300 (Active Motif) or 2 μg of purified H1 histone (Sigma-Aldrich)). The mixture was incubated at 30°C for 1 h with 90 pmol of [14C]acetyl-CoA (Amersham Biosciences). Reaction was subjected to SDS-PAGE and viewed following autoradiography of the gel.
p300 overexpression reverts the A238L-mediated inhibition of inducible transcription
Previous data from our laboratory showed that A238L inhibits NF-AT, NF-κB, and c-Jun separately (37), but the molecular mechanism used by the viral protein to exert its function remains largely unknown. We hypothesize that the key of the inhibitory role of A238L relies on p300 regulation, one element common to NF-ATc1, NF-κB-p65, and c-Jun activation pathways. To test this hypothesis, we have generated Jurkat cells that stably express the A238L gene by transfection with pcDNA-A238L, followed by selection using G418, as described under Materials and Methods. Fig. 1,A shows the expression of specific protein levels for A238L in Jurkat cells transfected with pcDNA-A238L and not in the cells transfected with the empty pcDNA plasmid. Next, Jurkat-A238L or control cells Jurkat-pcDNA were transfected with different luciferase reporter constructs under the control of NF-ATc2 (Fig. 1,B)-, NF-κB-p65 (Fig. 1,C)-, or c-Jun (Fig. 1,D)-specific response sequences. The results showed that, after PMA/Ion stimulation, A238L simultaneously inhibited the activation of the reporter constructs assayed, and that effect was reverted by overexpression of wt p300, but not by p300 HAT-defective mutant. This result indicates that the inhibitory effect of A238L involves the transactivation mediated by the acetyl transferase activity of the coactivator. In contrast, it is noteworthy that the ectopic expression of the p300 HAT-defective mutant did not have a negative effect in Jurkat-pcDNA transfected with the luciferase-driven AP-1 contruct (Fig. 1 D). It has been previously reported that not only p300, but also CBP possesses the ability of activating AP-1 transcriptional factor (16), particularly to c-Jun family member (50). This could explain the absence of a negative effect of p300ΔHAT on AP-1 transcriptional activity because CBP might overlap the coactivating activity mediated by p300 in that condition.
A238L prevents the association of NF-AT, NF-κB-p65, and c-Jun with p300 and the subsequent acetylation mediated by the coactivator
Because A238L was shown to inhibit the transcriptional activity of NF-ATc2, NF-κB, and c-Jun simultaneously (37), and taken in account that the recruitment of p300 is an essential step in the activation pathway of these transcription factors (11, 15, 33), we have investigated whether the above mentioned factors were able to associate with p300 under stimulation conditions in the presence of the viral protein. To achieve this, we prepared nuclear extracts from resting and PMA/Ion-stimulated Jurkat-pcDNA and Jurkat-A238L cells, and the interaction between NF-ATc2, NF-κB, and c-Jun with the coactivator p300 was assessed by immunoprecipitation with specific Abs for the corresponding transcription factors or a control serum. The presence of p300 in the immunoprecipitates was analyzed by Western blot. The results, shown in Fig. 2, indicate that p300 complexes were increased by PMA/Ion, but, more important, the presence of A238L inhibited the interaction of p300 with NF-ATc2 (Fig. 2,A), NF-κB-p65 subunit (Fig. 2,B), and c-Jun (Fig. 2,C), but not c-Fos (Fig. 2 D), a transcriptional factor dependent on carboxyl-terminal TAD activity of p300. A control preimmune IgG did not precipitate a p300-containing complex. The densitometric analysis shows that A238L diminished the binding of p300 in the nucleus both at basal and PMA/Ion-stimulated cellular states, suggesting that the transcriptional inhibition by the viral protein is dependent on its ability to compete with the transcription factors for binding to p300 coactivator, although a direct interaction between A238L and p300 has not yet been demonstrated.
To explore the link of the A238L-mediated inhibition on the transcription factors-p300 interaction with a direct functional consequence, we analyze the acetylation level of the transcriptional factors involved because acetylation of specific residues by CBP and p300 is required to enhance their transcriptional activity (17). To accomplish this objective, we prepared nuclear extracts from resting and PMA/Ion-treated Jurkat-pcDNA and Jurkat-A238L cells, which were used to immunoprecipitate proteins with specific Abs against NF-ATc2, NF-κB-p65, c-Jun, and c-Fos, respectively. Next, the acetylation in the immunoprecipitate was detected by Western blot by using an anti-acetyl lysine Ab, as described under Materials and Methods. As shown in the figure, acetylation of NF-ATc2 (Fig. 2,E), p65 (Fig. 2,F), and c-Jun (Fig. 2,G), but not c-Fos (Fig. 2 H), was clearly induced after PMA/Ion treatment. Interestingly, the expression of the viral protein resulted in strong reduction of the acetylated form of NF-ATc2, NF-κB-p65, and c-Jun transcription factors, which interact with the amino-terminal region of p300, whereas the acetylation level of c-Fos, a transcription factor that interacts with the carboxyl-terminal domain of the coactivator, was unaltered under stimulation conditions. These results indicate that one of the functional consequences of the absence of interaction between NF-ATc2, NF-κB-p65, and c-Jun with the coactivator p300, induced by the viral protein A238L, is a strong decrease in their acetylation levels.
A238L specifically inhibits the transcriptional activity of factors regulated by p300 amino-terminal region CH1/KIX
These results described above suggest that the viral protein might be acting specifically within the amino-terminal TAD of p300. To explore the prospect of a site-directed inhibition of A238L on p300-coactivating activity, we used GAL4 fusion constructs of the transcription factors described above, to analyze their transactivating ability. Fig. 3,A shows a schematic representation of the functional domains of p300, including three cysteine/histidine-rich regions, CH1, KIX, CH2, and CH3, which are able to interact with and coactivate different transcription factors, as follows: a bromodomain, found in mammalian HATs; the cycle repressor domain; and the catalytic HAT domain, in the carboxyl terminus (4). To analyze whether A238L was affecting transactivation mediated by some of these domains, we cotransfected Jurkat-pcDNA and Jurkat-A238L cells with GAL4-luc-reported construct plus with different GAL4-TAD expression plasmids to measure the transactivation mediated by each transcription factor in luciferase reporter assays. Our results showed that amino-terminal region-dependent transcription factors, such as NF-ATc2 (Fig. 3,B), NF-κB-p65 (Fig. 3,C), and c-Jun (Fig. 3,D), were induced after PMA/Ion stimulation in Jurkat-pcDNA control cells, but not in Jurkat-A238L, where their transactivation was strongly inhibited. In contrast, the activity of a carboxyl-terminal region (CH2 and CH3)-dependent transcription factor, such as c-Fos (Fig. 3,E), was not affected by the presence of the viral protein. Moreover, the transcriptional activity of Sp-1, a transcription factor that does not need coactivator function of p300 to enhance its transcriptional activity, was not affected by A238L (Fig. 3 F). Taken together, these data suggest that A238L-mediated regulation of the activity of different transcription factors is conducted through the inhibition of the transactivation activity resident in the amino-terminal region (CH1 and KIX domains) of p300.
A238L reduces autoacetylation level of p300 without altering its HAT activity
It has been demonstrated that CBP and p300 open chromatin by acetylation of the histone tails through their HAT activity, located in the carboxyl-terminal region (6). This HAT activity is extended to transcription factors (7) and CBP/p300 themselves, in an activation loop based in autoacetylation (27). Specifically, it recently has been shown the importance of autoacetylation of the CH1 regulatory domain of p300 in transactivation of inducible transcription factors (51). Because A238L was down-regulating the acetylation of NF-ATc2, NF-κB-p65, and c-Jun, we explore the possibility that the viral protein could interfere with the intrinsic acetyl transferase enzymatic activity of p300. To achieve this, we first analyzed the acetylation level of p300 in nuclear extracts from Jurkat-pcDNA or Jurkat-A238L cultured in the absence or presence of PMA/Ion. Nuclear extracts were immunoprecipitated with an Ab against p300, and the precipitate was analyzed with an Ab that specifically recognizes acetylated lysine. As shown in Fig. 4,A, p300 acetylation level increased after PMA/Ion stimulation, whereas the expression of A238L resulted in a clear reduction of acetylated protein. To further explore the molecular mechanism used by the viral protein, we studied the capacity of p300 to acetylate histones or to autoacetylate itself either in the absence or in the presence of A238L. To achieve this, immunoprecipitation HAT assays using as substrates recombinant p300, to determine autoacetylation activity (Fig. 4,B), or recombinant H1 histone, to determine histone acetylation activity (Fig. 4,C), were developed. The results showed that A238L strongly diminished autoacetylation ability of p300 (Fig. 4,B) without altering the acetylation of recombinant H1 histone (Fig. 4 C). These data demonstrate that A238L does not affect the enzymatic activity of the p300 HAT domain, but reduces the autoacetylation ability of p300, suggesting that the viral protein interferes specifically with the activation of p300 through a region out of the carboxyl-terminal HAT domain, because the enzymatic HAT activity remains intact when recombinant histone was assayed in the presence of A238L.
A238L colocalizes and associates with p300 in the nucleus of stimulated T cells
We have previously demonstrated that A238L shows a nuclear shuttling and colocalizes with overexpressed HA-tagged p300 in the nucleus of stimulated Vero cells (37). In this study, we further analyzed the distribution patterns of localization of A238L and p300, to investigate the possibility of association between these two proteins in the nucleus of stimulated Jurkat T cells. Jurkat cells were transiently transfected with the control plasmid, pcDNA3.1, or the plasmid containing a SV5-tagged A238L gene, pcDNA-A238L-SV5 (Fig. 5,A), and analyzed by confocal microscopy using specific Abs to p300 and SV5 and DAPI staining. The results showed that p300 exhibits a punctuated pattern in the nucleus after PMA/Ion treatment, compatible with inducible nuclear complexes, as previously described (52). Regarding A238L, it localizes preferentially in the cytoplasm of unstimulated T cells and, after treatment with PMA/Ion, was strongly translocated to the nucleus (Fig. 5,A), colocalizing with p300 in these inducible complexes. To extensively assess the possibility of association between p300 and A238L, we next prepared nuclear and cytoplasmic extracts from Jurkat cells transiently transfected with pcDNA3.1 or pcDNA-A238L (SV5), stimulated or not with PMA/Ion. These subcellular extracts were analyzed with specific Abs to p300 and SV5, showing the presence of p300 exclusively in the nuclear fraction, whereas A238L was present preferentially in the cytoplasm of resting A238L-expressing cells, accumulating in the nuclear fraction after stimulation, thus corroborating the results obtained by immunofluorescence (Fig. 5,B). Nuclear and cytoplasmic fractions were further immunoprecipitated with specific Ab to p300 or control serum, and the precipitates were revealed by Western blot with an Ab to SV5. The results (Fig. 5 B) showed that A238L associates with endogenous p300 after PMA/Ion treatment in the nuclear extracts. This result might also indicate that the viral protein is probably altering the acetylation of different transcription factors through direct interference on p300.
A238L interacts with the amino-terminal TAD of p300 and blocks its transcriptional activity
To determine whether the molecular mechanism by which A238L performs its inhibitory function involves the amino-terminal domain of p300 and to further map the specific region modulated by the viral protein, we have used different GAL4-p300 constructs containing the GAL4 DBD fused to the following regions: the full-length sequence of p300 (named GAL4-p300 (FL) and represented in Fig. 6,A); the amino-terminal half of p300 (named GAL4-p300(1–1301) and represented in Fig. 6,B); the carboxyl-terminal half of p300 (named GAL4-p300 (1239–2414) and represented in Fig. 6,C); the amino-terminal transactivation region, containing the CH1 and KIX regulatory domains (named GAL4-p300 (192–703) and represented in Fig. 6,D). Jurkat cells were transiently transfected with pcDNA or pcDNA-A238L-SV5, and cotransfected with the different mutant forms of GAL4-p300, in the absence or presence of PMA/Ion during 2 h. Nuclear extracts were then prepared and incubated with a specific Ab to SV5 tag or control serum. The presence of A238L-SV5 or the corresponding form of GAL4-p300 was next analyzed by Western blot. Our results showed that GAL4-p300 (FL) fusion protein was interacting with A238L, preferentially after PMA/Ion treatment (Fig. 6,A). As expected, the viral protein interacted with the mutant construct encoding the amino-terminal half of p300 (Fig. 6,B), but not with the construction encoding the carboxyl-terminal region (Fig. 6,C). Still more important, we finally found that A238L interacts with the construct encoding the region spanning from aa 192 to 703 of p300, which contains the CH1 and KIX domains (Fig. 6 D).
Independently, we have measured the transcriptional activity of the corresponding mutant forms of p300 in the presence of the viral protein by addition of GAL4-luc reporter plasmid to each of the transfection mixtures containing the different GAL4-p300 constructs containing the GAL4 DBD fused to the specific regions described above. As expected, PMA/Ion treatment of Jurkat T cells increased the transcriptional activity of every construct of p300 (graphics in Fig. 6, A–D). It is noteworthy that all the constructs used showed to be transcriptionally active, suggesting that they are able to recruit the basal transcription complex. In connection to this, and supporting our results, it has been previously described that CBP/p300 contains two different TAD, one in the amino-terminal and the other in the carboxyl-terminal regions, which can act independently and interact simultaneously with the transcriptional machinery and/or with different transcription factors to build the transcriptional activity mediated by these coactivators (53). More important, the viral protein inhibited the transcriptional activity of the GAL4-p300 full-length construct (Fig. 6,A), the transcriptional activity of the amino-terminal half (Fig. 6,B), and the activity mediated by the CH1/KIX region (Fig. 6,D), without altering the transcriptional activity of the carboxyl-terminal half of the coactivator (Fig. 6 C), corroborating the data obtained from the interaction assays shown above. Therefore, taken together, these data demonstrate that the viral protein is directly interfering with the transcriptional activity of the CH1 and KIX domains included in the amino-terminal TAD of p300, thus engaging this region in the inhibitory role of A238L and targeting this regulatory domain in the pathway of activation of proinflammatory molecules modulated by NF-ATc2, NF-κB-p65, and c-Jun.
Ser384 is a novel regulatory residue in CH1 domain of p300 potentially phosphorylated by PKC kinase and blocked by A238L
Because transcriptional activation and repression of both CBP and p300 are regulated by phosphorylation by many different kinases (1), and to establish the molecular mechanism by which the viral protein controls simultaneously the transcriptional activity of NF-ATc2, NF-κB, and c-Jun, most likely through the regulation of CH1 and KIX domains activity, we analyzed the presence of phosphorylable residues in the CH1/KIX domains of p300 by using NetPhosK 1.0 server (54, 55) as a first step to identify residues potentially involved in the function of the viral protein. This approach allowed us to identify a novel residue, the serine in position 384, potentially phosphorylable by PKC, with the highest phosphorylation score (0.866) within the amino-terminal TAD of p300. This finding prompted us to first analyze the importance of this novel residue in the enhancement of the transcriptional activity of p300, focusing in the identification of the specific PKC isotype that phosphorylates Ser384. Next, we planned to determine whether A238L develops the inhibition of p300 transactivation by displacement of the kinase from this regulatory region. To carry this out, we first prepared nuclear extracts from Jurkat-pcDNA and Jurkat-A238L previously transfected with the different GAL4-p300 constructs described above, as follows: GAL4-p300 (FL), GAL4-p300 (1–1301), GAL4-p300 (1239–2414), and GAL4-p300 (192–703), and cultured in the absence or presence of PMA/Ion. These nuclear extracts were immunoprecipitated with an Ab that recognizes PKC family members (pan-PKC) or control serum, and revealed by Western blot using specific Abs to PKCs or GAL4. Our results showed that full-length p300 fused to GAL4 was associated with PKC after PMA/Ion treatment in Jurkat-pcDNA cells. Interestingly, this interaction was displaced by the presence of the viral protein (Fig. 7,A). The viral protein also displaced PKC from the constructs encoding the amino-terminal region, GAL4-p300 (1–1301) (Fig. 7,B), or the CH1/KIX domains, GAL4-p300 (192–703) (Fig. 7,C), thus supporting the hypothesis that A238L specifically inhibits the activation of the amino-terminal TAD of p300 mediated by PKC. When the construct assayed was GAL4-p300 (1239–2414), containing the carboxyl-terminal TAD, A238L did not alter the PKC-p300 interaction (Fig. 7 D), which indicates that PKC binding to this region is not affected by the viral protein.
Moreover, we investigated whether A238L is able to interact with the immunoprecipitated pan-PKCs in whole-cell extracts from Jurkat cells expressing empty pcDNA or SV5-tagged A238L. Fig. 7,E shows that A238L is not present in PKC immunoprecipitates, indicating that the viral protein does not interact with PKC family members. Finally, we analyzed the kinase activity of the immunoprecipitated PKC in a kinase assay using MBP as substrate in cells overexpressing A238L. Fig. 7 F shows that the viral protein did not affect the specific kinase activity observed after PMA/Ion treatment, strongly indicating that the inhibition mediated by the viral protein is conducted specifically by impairing the binding of PKC to the amino-terminal TAD of p300, without altering its enzymatic activity.
PKC-θ overexpression reverts the A238L-mediated inhibition of p300 transcriptional activity through a mechanism involving Ser384 signaling and acetylation of the coactivator
Once demonstrated that the presence of the viral protein blocked the interaction of PKC with the amino terminus of p300, thus inhibiting the transcriptional activity intrinsic to this specific domain, we planned to analyze the relevance of Ser384 in the p300 transactivation. To achieve this, we generated Ser384 to alanine substitution mutants (S384A) on the full-length sequence of p300, GAL4-p300 (FL) S384A construct, and on the amino-terminal region spanning from aa 192 to 703, GAL4-p300 (192–703) S384A construct, which were assayed in transcriptional activity experiments, using the GAL4-luc reporter plasmid. Our results showed that this mutation in full-length p300 is responsible for a dramatic reduction of p300-mediated transactivation, both in basal and stimulated conditions, and that the substitution of Ser384 by alanine in GAL4-p300 (192–703) S384A completely abrogated the transcriptional activity mediated by this region (Fig. 8 A). These data strongly indicate that Ser384 is essential in the amino-terminal transactivation of p300, and directly target this residue in the global regulation of the coactivator.
Previous reports have shown the importance of the PKC family members ζ, ε, and θ in NF-ATc2, NF-κB-p65, and c-Jun activation in T cells (56, 57, 58, 59). Taking in account our data described above, Ser384 seems to be a potential PKC substrate that is moreover probably involved in the regulation performed by A238L. Thus, we next propose to identify the PKC isotype responsible for Ser384 phosphorylation. To carry this out, we first separately overexpressed GAL4-p300 (192–703), wt, and GAL4-p300 (192–703) S384A mutant (Mut) in Jurkat cells. Nuclear extracts were immunoprecipitated with a GAL4-specific Ab, and revealed with a specific Ab against p300, to assess the level of expression of the transfected constructs (Fig. 8,B). The immunoprecipitated extracts were then used as substrates in successive in vitro kinase assays, using PKC-ζ, PKC-ε, and PKC-θ, which were previously obtained from Jurkat and PMA/Ion-stimulated Jurkat cells. The results showed that neither PKC-ζ nor PKC-ε was able to phosphorylate the GAL4-p300 (192–703) wt amino terminus of p300 (data not shown), whereas PKC-θ phosphorylated this fusion protein, revealing the importance of PKC-θ in the phosphorylation of this regulatory domain of p300. Furthermore, a noticeably lower phosphorylation level was observed when GAL4-p300 (192–703) S384A mutant was used as substrate (Fig. 8,C), confirming the relevance of S384 as target of PKC-θ in T cells. To assess that PKC-θ activity was unaltered in our experimental conditions, we show that PKC-θ-mediated MBP phosphorylation was unaffected in an in vitro kinase assay. To further confirm the role of PKC-θ in the A238L-mediated inhibition of the transcriptional activation of p300, Jurkat-pcDNA or Jurkat-A238L cells were transiently transfected with pEFneo empty vector, pEF-PKC-θ wt, or constitutively active mutant of PKC-θ (pEF-PKC-θ A/E) expression plasmids and with GAL4-p300 full-length (FL) or amino-terminal construct (192–703), together with the reporter plasmid GAL4-luc, as described in Materials and Methods. Fig. 8,D shows that overexpression of the constitutively active mutant of PKC-θ (pEF-PKC-θ A/E) fully reverts the inhibition induced by A238L, thus enhancing the relevance of PKC-θ in the mechanism of modulation conducted by the viral protein. In contrast, the expression of PKC-θ wt construct neither reverted the inhibition mediated by the viral protein nor increased the transcriptional activation of none of the constructs used after PMA/Ion stimulation. This result was somehow expected due to the expression of PKC-θ wt was previously shown to be unable to induce Fas ligand promoter activation (49), suggesting that PKC-θ activation is required for its biological function. To demonstrate the link between PKC-θ and the A238L-mediated p300 inhibition, we have immunoprecipitated this kinase from Jurkat-pcDNA and Jurkat-A238L cells, which were previously transfected with GAL4-p300 (192–703) wt. As shown in Fig. 8 E, GAL4-p300 (192–703) was clearly detected in the PKC-θ immunoprecipitates from PMA/Ion-stimulated Jurkat-pcDNA cells, whereas it was nearly absent in the immunoprecipitates from stimulated Jurkat-A238L cells. Taken together, these results strongly suggest that the expression of the viral protein interferes with the association of PKC-θ with amino-terminal region of p300, thus simultaneously controlling NF-κB-p65, NF-ATc2, and c-Jun transactivation, representing a new and sophisticated viral mechanism of blockage of the host inflammatory response.
Finally, and to further assess the functional relevance of S384 in p300 transcriptional activation, we have used Jurkat cells previously transfected with GAL4-p300 (FL) or with GAL4-p300 (192–703), to immunoprecipitate the expressed constructs with an anti-Gal4 Ab or an irrelevant IgG as control. Immunoprecipitates were next analyzed by Western blot by using a specific Ab to acetyl lysine, to investigate the acetylation level of the mutants S384A comparing with the wt expressed constructs. The results, shown in Fig. 9, revealed that the mutation of Ser384 by alanine strongly decreases the acetylation level both of the construct expressing the full-length p300 and the amino-terminal TAD construct, indicating that this residue is probably responsible for the full transcriptional activation of the coactivator.
Multiple signal-activated pathways gather in the transcriptional coactivator proteins CBP and p300, which integrate these signals to coordinate and promote the expression of specific sets of genes in response to diverse physiological processes, such as cell growth, apoptosis, tumor suppression, immune response, or malignant transformation (1, 17).
We have previously described that the protein A238L, encoded by African swine fever virus, inhibits the transcriptional activation of cyclooxygenase-2, TNF-α, and inducible NO synthase promoters through a mechanism involving the transactivation of NF-ATc2, NF-κB-p65, and c-Jun (37, 38, 39). However, the molecular mechanism responsible for the A238L-mediated inhibition remains largely unknown.
Our results showed that the blockage of the transcriptional activity of NF-ATc2, NF-κB-p65, and c-Jun mediated by viral protein in stimulated T cells was reverted by p300 overexpression, thus suggesting that A238L most likely uses a unique mechanism to block simultaneously the activation of several transcription factors. Moreover, we also showed that A238L specifically inhibits the transactivation of transcription factors that require the activity of the amino-terminal TAD of p300. In contrast, the transactivation of carboxyl-terminal TAD-dependent transcription factors, such as c-Fos, or Sp-1, a p300-independent factor, was not affected by the viral protein, indicating that A238L is altering specifically the transcriptional activity of the amino-terminal TAD of p300. We further demonstrate that A238L modulation involves the autoacetylation activity of p300, which is essential in its intrinsic transcriptional activity (60), without altering the HAT activity.
It has been previously shown that certain viruses encode proteins that interact with CBP and/or p300, modulating their activity, such as adenoviral E1A (61), SV40 T large Ag (19), or E6 and E7 proteins from human papillomavirus (20, 21). In fact, p300 was first described as an E1A-interacting protein (3). In this study, we demonstrate that A238L presents a dotted pattern in the nucleus of T cells and colocalizes with endogenous p300 in structures compatible with transcription initiation complexes, as observed by DAPI and immunofluorescence staining (52). We have further assessed the association between A238L and p300, showing that the viral protein is able to interact with the amino terminus of p300, but it does not bind the carboxyl-terminal region of the coactivator.
Most of the viral proteins that regulate p300 inhibit its HAT activity and the activation of CH3-interacting transcription factors, such as p53 or E2F, thus promoting the malignant transformation of the infected cells (19, 21, 62). In contrast, we show in this study that A238L is inhibiting the amino-terminal TAD without altering carboxyl-terminal activity. This is in line with the previously described function of A238L as a suppressor of the innate immune response (37, 38, 39), because, as we have discussed above, the amino-terminal TAD of p300 is essential to enhance the transcriptional activity of immunorelevant factors such as NF-ATc2, NF-κB-p65, or c-Jun (17).
Interestingly, the functional mechanism used by A238L to inhibit p300 activity reminds to the mechanism exploited by Smad nuclear interacting protein 1, a nuclear inhibitor of transcription, that binds to the CH1 domain of p300 regulating multiple transcriptional pathways (63). During evolution, viruses frequently mimic cellular molecules and strategies to evade the host immune response. Future experiments to address the possible structural and functional homology between the viral A238L and the cellular Smad nuclear interacting protein 1 will be developed.
To explore whether the interaction of A238L with CH1 domain of p300 interferes with phosphorylation in this domain, we have first analyzed the putative residues susceptible of undergoing phosphorylation in the amino-terminal region of p300, spanning from aa 192 to 703, which contain the regulatory domains CH1 and KIX. Thus, we have identified a potential PKC site of phosphorylation of p300 at Ser384. We demonstrate that this residue is essential in the activation of the amino-terminal TAD of p300, because mutation of this serine completely abrogated the autoacetylation and the transcriptional activity of the p300 amino terminus. PKC-θ belongs to the novel PKC subfamily, and is mainly expressed in T lymphocytes, playing a critical role in T cell activation by TCR engagement in vivo (64, 65). It has been previously described that, in T cells, PKC-θ activity results in activation of the signal transduction pathways of NF-ATc2, NF-κB-p65, and AP-1 (66), but the role played by PKC-θ in downstream events, such as transactivation or interaction with nuclear coactivators, remains undefined. Therefore, we have analyzed the PKC isotype responsible for Ser384 phosphorylation, showing that neither PKC-ζ nor PKC-ε was able to phosphorylate the GAL4-p300 (192–703) wt amino terminus of p300, whereas PKC-θ phosphorylated this fusion protein, revealing for the first time the importance of PKC-θ in the phosphorylation of this regulatory domain of p300. This fact was corroborated when a constitutively active mutant of PKC-θ (pEF-PKC-θ A/E) fully recovered the inhibition induced by A238L, thus enhancing the relevance of PKC-θ in the modulation mechanism conducted by the viral protein. Finally, we observed a noticeably lower phosphorylation level when GAL4-p300 (192–703) S384A mutant was used as substrate, confirming the relevance of S384 as target of PKC-θ in T cells.
It has been previously demonstrated that nuclear IκB kinase-α-dependent CBP phosphorylation causes dissociation between p53 and CBP and increases NF-κB-p65 transcriptional activity (32). There, the authors suggested that different switches regulated by kinases turn CBP available for specific gene transcription patterns, interfering with others. This prompted us to suggest that A238L might block a PKC-θ switch mediated by phosphorylation of Ser384 on p300 that directs this coactivator to establish a gene transcription pattern specific for inflammatory and immune response, although additional experiments are needed to directly demonstrate the PKC-θ phosphorylation of Ser384 to establish the precise role of A238L in the modulation of CBP/p300 regulation switches. Therefore, we propose a unified mechanism by which PKC-θ up-regulates p300 amino-terminal TAD in T cells, thus enhancing the transactivation mediated by NF-ATc2, NF-κB-p65, and c-Jun, which is efficiently controlled by the viral protein A238L. In addition, Ser384, which is located exactly in the CH1 regulatory domain, within the recently defined lysine-rich region 1 (KR1), a regulatory domain that controls transcriptional enhancement by autoacetylation (24), could link the lack of amino-terminal transcriptional dependent activity observed in the presence of A238L, therefore suggesting that the viral protein possibly blocks the autoacetylation activity of p300 specifically in KR1.
Thus, the modulation conducted by the viral product A238L represents a model to find new pathways and targets for the control of T cell activation in several pathological processes and immunological diseases.
We thank Dr. Manuel Fresno for critical reading of the manuscript and helpful comments. We also thank Maria L. Nogal for excellent technical assistance. The helpful advice of Dr. Angel L. Carrascosa is also very much appreciated.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by grants from Ministerio de Educación y Ciencia (BFU2004-00298/BMC) and by an institutional grant from the Fundación Ramón Areces. A.G.G. was funded by Centro de Investigación en Sanidad Animal.
Abbreviations used in this paper: CBP, CREB-binding protein; BCA, bicinchoninic acid; DAPI, 4′,6′-diamidino-2-phenylindole; DBD, DNA-binding domain; HAT, histone acetyl transferase; Ion, calcium ionophore; MBP, myosin-binding protein; PKC, protein kinase C; RIPA, radioimmunoprecipitation assay; RLU, relative luminescence unit; TAD, transactivation domain; wt, wild type.