HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Granja, A. G. Articles by Revilla, Y. Search for Related Content PubMed PubMed Citation Articles by Granja, A. G. Articles by Revilla, Y. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Substance via MeSH

A238L Inhibits NF-ATc2, NF-B, and c-Jun Activation through a Novel Mechanism Involving Protein Kinase C--Mediated Up-Regulation of the Amino-Terminal Transactivation Domain of p300¹

Aitor G. Granja^*, Neil D. Perkins and Yolanda Revilla^2,*

^* Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain; and School of Life Sciences, Division of Gene Regulation and Expression, University of Dundee, Dundee, Scotland, United Kingdom

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
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-B, 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- reverts the A238L-mediated inhibition of the transcriptional activity of p300, showing, for the first time, a PKC--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.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
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-B-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 IB kinase- (32). Opposite to this, transcriptional activity of CBP/p300 is negatively controlled by cyclin-E/cdk-2 complex (33) or protein kinase C (PKC)- 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-B-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-B-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-B, 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- 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--mediated phosphorylation and autoacetylation of p300, suggesting that this residue is an important PKC- target involved in up-regulation of the amino-terminal transactivation activity of p300. We finally demonstrate that overexpression of active PKC- 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-B-p65, and c-Jun pathways exploits the blockage of the PKC- 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

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
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-B-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- (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.

Immunoprecipitation

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-B-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- (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.

Plasmid constructs

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-B-p65 reporter plasmid (pNF3TKLuc) contains a trimer of the NF-B-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-p300HAT, 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- (pEF-PKC- wt and pEF-PKC- 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 x 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- 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- (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 [³²P]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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
p300 overexpression reverts the A238L-mediated inhibition of inducible transcription

Previous data from our laboratory showed that A238L inhibits NF-AT, NF-B, 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-B-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. 1A 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. 1B)-, NF-B-p65 (Fig. 1C)-, or c-Jun (Fig. 1D)-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. 1D). 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 p300HAT on AP-1 transcriptional activity because CBP might overlap the coactivating activity mediated by p300 in that condition.

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Effect of p300 overexpression in the transcriptional activation of NF-ATc2, NF-B, and AP-1. A, Western blot showing the synthesis of A238L viral protein in whole-cell extracts from Jurkat-pcDNA or Jurkat-A238L cells prepared at the indicated times after stimulation with 15 ng/ml PMA plus 1 µM Ion (PMA/Ion). Jurkat-pcDNA () or Jurkat-A238L () cells were transiently transfected with NF-ATc2 (B), NF-B (C), or c-Jun (D) reporter plasmids (250 ng/106 cells), and with pCI-p300 wt or p300 HAT deletion mutant (pCI-p300HAT) expression plasmids (1 µg/106 cells), as described in Materials and Methods. After transfection, cells were cultured in the absence (–) or presence (+) of PMA/Ion, as indicated in the figure, and assayed for luciferase activity. Extracts were normalized to Renilla luciferase activity. Results from triplicate assays are shown in RLU/µg protein (mean ± SD).

A238L prevents the association of NF-AT, NF-B-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-B, 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-B, 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. 2A), NF-B-p65 subunit (Fig. 2B), and c-Jun (Fig. 2C), but not c-Fos (Fig. 2D), 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.

View larger version (52K): [in this window] [in a new window]   FIGURE 2. A238L inhibits the association between p300 and the transcription factors NF-ATc2, NF-B, and c-Jun, and their p300-mediated acetylation. Nuclear extracts from 107 Jurkat-pcDNA and Jurkat-A238L cells treated (+) or not (–) with 15 ng/ml PMA plus 1 µM Ion (PMA/Ion) for 4 h were incubated and immunoprecipitated with 4 µg of rabbit polyclonal specific Abs against the following: NF-ATc2 (A and E), NF-B-p65 (B and F), c-Jun (C and G), or c-Fos (D and H), or preimmune normal IgG as a negative control of coimmunoprecipitation (IgG), as described under Materials and Methods. Immunoprecipitates were separated by SDS-PAGE, electrophoretically transferred to an immobilon membrane, and detected by immunoblotting with the same Abs (pNF-ATc2, p65, c-Jun, or c-Fos) to determine levels of these proteins in the precipitate, or with p300 (p300)-specific Ab to detect the interaction between these transcription factors and p300 (A–D). In parallel, the immunoprecipitates were incubated with an anti-acetylated lysine-specific Ab (Ac.Lys) to detect the levels of acetylated NF-ATc2, NF-B-p65, and c-Jun in the precipitate (E–H). The densitometric analysis shows the ratio between coimmunoprecipitated p300 amount and the precipitated amount of each transcription factor, from three independent experiments (mean ± SD).

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. 3A 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. 3B), NF-B-p65 (Fig. 3C), and c-Jun (Fig. 3D), 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. 3E), 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. 3F). 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.

View larger version (30K): [in this window] [in a new window]   FIGURE 3. A238L inhibits transactivation mediated by p300 amino-terminal-dependent transcription factors (NF-ATc2, NF-B-p65, and c-Jun), but not others as c-Fos or SP-1. A, Schematic representation of the structural and functional domains of p300. Jurkat-pcDNA and Jurkat-A238L cells were cotransfected with GAL4-luc (50 ng of DNA/106 cells) and the corresponding trans activator (at a final concentration of 150 ng of DNA/106 cells), as follows: B, GAL4-NF-ATc2; C, GAL4-p65; D, GAL4-c-Jun; E, GAL4-c-Fos; and F, GAL4-SP1. Sixteen hours after transfection, the cells were cultured in the absence or presence of 15 ng/ml PMA plus 1 µM Ion (PMA/Ion), as indicated in the figure, during 4 h. In each case, whole-cell extracts were prepared and luciferase activity was assayed. Extracts were normalized to Renilla luciferase activity, as described in Materials and Methods. RLU/µg protein from triplicate transfections (mean ± SD) is shown.

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-B-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. 4A, 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. 4B), or recombinant H₁ histone, to determine histone acetylation activity (Fig. 4C), were developed. The results showed that A238L strongly diminished autoacetylation ability of p300 (Fig. 4B) without altering the acetylation of recombinant H₁ histone (Fig. 4C). 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.

View larger version (29K): [in this window] [in a new window]   FIGURE 4. A238L reduces autoacetylation level of p300 without altering its HAT activity. Nuclear extracts from 107 Jurkat-pcDNA and Jurkat-A238L cells treated (+) or not (–) with PMA/Ion for 4 h were immunoprecipitated with 4 µg of rabbit polyclonal specific Ab against p300, or rabbit preimmune normal IgG as a negative control of immunoprecipitation (IgG). This immunoprecipitated p300 was used in three different assays. A, Immunoprecipitates were analyzed by Western blot with the same p300 Ab to determine levels of these proteins in the precipitate, and with an anti-acetylated lysine-specific Ab (Ac.Lys) to detect acetylated p300, as described in Materials and Methods. The densitometric analysis shows the fold induction level obtained with the ratio between acetylated p300 and total immunoprecipitated p300 proteins, in basal and stimulated conditions, from three independent experiments (mean ± SD). B, Immunoprecipitated p300 was used in an autoacetylation assay, using purified p300 protein as substrate, as described in Materials and Methods. The densitometric analysis shows the fold induction level obtained with the ratio between radiolabeled acetylated p300 and total immunoprecipitated p300 proteins, in basal and stimulated conditions, from three independent experiments (mean ± SD). C, Immunoprecipitated p300 was used in a HAT assay, as described under Materials and Methods, using purified H1 histone as substrate. The densitometric analysis shows the fold induction level obtained with the ratio between radiolabeled acetylated H1 histone and total immunprecipitated p300 proteins, in basal and stimulated conditions, from three independent experiments (mean ± SD).

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. 5A), 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. 5A), 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. 5B). 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. 5B) 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.

View larger version (54K): [in this window] [in a new window]   FIGURE 5. A238L colocalizes and associates with endogenous p300 in the nucleus of stimulated Jurkat T cells. A, The wt Jurkat cells were transiently transfected with empty plasmid pcDNA3.1, or SV5-tagged A238L expression plasmid, pcDNA-A238L-SV5, as described under Materials and Methods. Twenty-four hours after transfection, cells were incubated in the absence (control) or presence of PMA/Ion during 30 or 60 min. Then the cells were labeled with anti-SV5 Ab (green), anti-p300 Ab (red), and DAPI to show nuclear staining (blue), and examined by confocal microscopy. The figure shows images corresponding to one of three independent experiments performed. B, Cytoplasmic and nuclear extracts from 107 wt Jurkat cells transiently transfected with pcDNA3.1 or pcDNA-A238L-SV5, treated (+) or not (–) with PMA/Ion for 4 h, were incubated and immunoprecipitated with 4 µg of rabbit polyclonal specific Ab against p300, or rabbit preimmune normal IgG as a negative control of immunoprecipitation (IgG). Immunoprecipitates were analyzed by Western blot using the same Ab (p300) to determine levels of these proteins in the precipitate, and anti-SV5 (SV5) to detect levels of A238L-SV5 associated with p300. The densitometric analysis shows the ratio between coimmunoprecipitated A238L-SV5 and total p300 present in the precipitate, from three independent experiments (mean ± SD).

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. 6A); the amino-terminal half of p300 (named GAL4-p300(1–1301) and represented in Fig. 6B); the carboxyl-terminal half of p300 (named GAL4-p300 (1239–2414) and represented in Fig. 6C); the amino-terminal transactivation region, containing the CH1 and KIX regulatory domains (named GAL4-p300 (192–703) and represented in Fig. 6D). 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. 6A). As expected, the viral protein interacted with the mutant construct encoding the amino-terminal half of p300 (Fig. 6B), but not with the construction encoding the carboxyl-terminal region (Fig. 6C). 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. 6D).

View larger version (41K): [in this window] [in a new window]   FIGURE 6. A238L interacts with the amino-terminal TAD of p300 and blocks its transcriptional activity. The wt Jurkat T cells were transiently transfected with empty plasmid pcDNA3.1 (pcDNA) or SV5-tagged A238L expression plasmid (A238L), as described under Materials and Methods. In parallel, these cells were cotransfected with the reporter plasmid GAL4-luc, and with different mutant constructs of p300 tagged to GAL4, as follows: A, p300 full length (GAL4-p300 FL); B, mutant p300 construct encoding aa from 1 to 1301 (GAL4-p300 1–1301); C, mutant p300 construct encoding aa from 1239 to 2414 (GAL4-p300 1239-2414); or D, mutant p300 construct encoding aa from 192 to 703 (GAL4-p300 192–703). Twenty-four hours after transfection, cells were incubated in the absence (–) or presence (+) of PMA/Ion for 4 h, and nuclear extracts were prepared and immunoprecipitated with 4 µg of mouse specific mAb against SV5, or mouse preimmune normal IgG as a negative control of immunoprecipitation (IgG). Immunoprecipitates were analyzed by Western blot using the same Ab (SV5) to determine levels of these proteins in the precipitate, and anti-GAL4 (GAL4) to detect levels of A238L-SV5 associated with the corresponding mutant form of p300. The figure shows one immunoprecipitation from three assays conducted independently. In parallel, in each case, whole-cell extracts were prepared and luciferase activity was assayed. Extracts were normalized to Renilla luciferase activity. RLU/µg protein from triplicate transfections (mean ± SD) is shown.

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-B, 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. 7A). The viral protein also displaced PKC from the constructs encoding the amino-terminal region, GAL4-p300 (1–1301) (Fig. 7B), or the CH1/KIX domains, GAL4-p300 (192–703) (Fig. 7C), 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. 7D), which indicates that PKC binding to this region is not affected by the viral protein.

View larger version (52K): [in this window] [in a new window]   FIGURE 7. The amino-terminal TAD of p300 associates with PKC and the viral protein A238L inhibits this interaction. Jurkat-pcDNA and Jurkat-A238L were transfected with different mutant constructs of p300 tagged to GAL4 and described above: A, GAL4-p300 FL; B, GAL4-p300 1–1301; C, GAL4-p300 192–703; and D, GAL4-p300 1239–2414, as described under Materials and Methods. Twenty-four hours after transfection, cells were incubated in the absence (–) or presence (+) of PMA/Ion for 4 h, and nuclear extracts were prepared and immunoprecipitated with 4 µg of rabbit polyclonal specific Ab against PKC, or rabbit preimmune normal IgG as a negative control of immunoprecipitation (IgG). Immunoprecipitates were analyzed by Western blot with the same Ab (PKC) to determine levels of these proteins in the precipitate, and with an anti-GAL4 (GAL4) to detect levels of PKC associated with the corresponding mutant form of p300. The densitometric analysis shows the ratio between coimmunoprecipitated PKC and total p300 present in the precipitate, from three independent experiments (mean ± SD). E, Immunoprecipitated PKC was analyzed by Western blot with an anti-SV5 Ab to detect levels of A238L-SV5 associated with PKC. The densitometric analysis shows the ratio between coimmunoprecipitated A238L and total PKC present in the precipitate, from three independent experiments (mean ± SD). F, In parallel, the immunoprecipitated PKC was used in an in vitro kinase assay, using purified MBP as the substrate. Proteins were separated by SDS-12% PAGE and developed by autoradiography. Densitometric analysis shows the ratio between phosphorylated [32P]MBP and immunoprecipitated PKC, from three independent experiments (mean ± SD).

PKC- 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. 8A). 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.

View larger version (54K): [in this window] [in a new window]   FIGURE 8. Ser384 is phosphorylated by PKC-, representing an essential step in full transcriptional activation of p300. A, The wt Jurkat T cells were transiently cotransfected with the reporter plasmid GAL4-luc, and with the corresponding GAL4-p300 construct: GAL4-p300 (FL) wt and Ser384 to alanine mutant (S384A), and GAL4-p300 (192–703) wt and Ser384 to alanine mutant (S384A), as described under Materials and Methods. Twenty-four hours after transfection, cells were incubated in the absence (Control) or presence of PMA/Ion, and whole-cell extracts were prepared and luciferase activity was assayed. Extracts were normalized to Renilla luciferase activity. RLU/µg protein from triplicate transfections (mean ± SD) is shown. B, The wt Jurkat T cells were transiently transfected with the expression plasmids GAL4-p300 (192–703) wt and Ser384 to alanine mutant (S384A), as described in Materials and Methods. The GAL4-p300 proteins were immunoprecipitated by using a specific Ab to GAL4. C, PKC- was immunoprecipitated from wt Jurkat whole-cell extracts. Immunoprecipitated GAL4-p300 (192–703) wt and Ser384 to alanine mutant (S384A) were used as substrate in a PKC- in vitro kinase assay. Proteins were separated by SDS-8% PAGE and developed by autoradiography. Densitometric analysis shows the ratio between phosphorylated [32P]p300 and immunoprecipitated PKC-, from three independent experiments (mean ± SD). D, Effect of PKC- overexpression in the transcriptional activation of p300. Jurkat-pcDNA () or Jurkat-A238L () cells were transiently transfected with pEFneo empty vector, pEF-PKC- wt, or constitutively active mutant of PKC- (pEF-PKC- A/E) expression plasmids (1 µg/106 cells), and with GAL4-p300 full-length (FL) or amino-terminal construct (192–703), together with the reporter plasmid GAL4-luc (250 ng/106 cells each), as described in Materials and Methods. After transfection, cells were cultured in the absence (–) or presence (+) of PMA/Ion, as indicated in the figure, and assayed for luciferase activity. Extracts were normalized to Renilla luciferase activity. RLU/µg protein from triplicate transfections (mean ± SD). E, Jurkat-pcDNA and Jurkat-A238L cells were transiently transfected with GAL4-p300 (192–703) wt, as described in Materials and Methods. Twenty-four hours later, the cells were cultured in the absence (–) or presence (+) of PMA/Ion during 2 h, and whole-cell extracts were prepared and immunoprecipitated with 4 µg of rabbit polyclonal specific Ab against PKC-, or rabbit preimmune normal IgG as a negative control of immunoprecipitation (IgG). Immunoprecipitates were analyzed by Western blot with the same Ab (PKC-) to determine levels of the kinase in the precipitate, and with an anti-GAL4 (GAL4) to detect levels of PKC associated with GAL4-p300 (192–703). The densitometric analysis shows the ratio between coimmunoprecipitated PKC and total p300 present in the precipitate, from three independent experiments (mean ± SD).

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.

View larger version (41K): [in this window] [in a new window]   FIGURE 9. The wt Jurkat T cells were transiently transfected with the expression plasmids GAL4-p300 full length (FL) and amino-terminal (192–703), wt and Ser384 to alanine mutant (S384A), as described in Materials and Methods. Nuclear extracts from 107 transfected Jurkat and Jurkat cells treated (+) or not (–) with PMA/Ion for 4 h were immunoprecipitated with 4 µg of rabbit polyclonal specific Ab against GAL4, or rabbit preimmune normal IgG as a negative control of immunoprecipitation (IgG). These immunoprecipitated GAL4-p300 constructs were analyzed by Western blot with the same GAL4 Ab to determine levels of these proteins in the precipitate, and with an anti-acetylated lysine-specific Ab (Ac.Lys) to detect acetylated p300, as described in Materials and Methods. The densitometric analysis shows the fold induction level obtained with the ratio between acetylated GAL4-p300 and the total immunoprecipitated p300 proteins, in basal and stimulated conditions, from three independent experiments (mean ± SD).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

We have previously described that the protein A238L, encoded by African swine fever virus, inhibits the transcriptional activation of cyclooxygenase-2, TNF-, and inducible NO synthase promoters through a mechanism involving the transactivation of NF-ATc2, NF-B-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-B-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-B-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- 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- activity results in activation of the signal transduction pathways of NF-ATc2, NF-B-p65, and AP-1 (66), but the role played by PKC- 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- nor PKC- was able to phosphorylate the GAL4-p300 (192–703) wt amino terminus of p300, whereas PKC- phosphorylated this fusion protein, revealing for the first time the importance of PKC- in the phosphorylation of this regulatory domain of p300. This fact was corroborated when a constitutively active mutant of PKC- (pEF-PKC- A/E) fully recovered the inhibition induced by A238L, thus enhancing the relevance of PKC- 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- in T cells.

It has been previously demonstrated that nuclear IB kinase--dependent CBP phosphorylation causes dissociation between p53 and CBP and increases NF-B-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- 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- 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- up-regulates p300 amino-terminal TAD in T cells, thus enhancing the transactivation mediated by NF-ATc2, NF-B-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.

	Acknowledgments

 
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.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

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

¹ This work was supported by 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.

² Address correspondence and reprint requests to Dr. Yolanda Revilla, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Madrid 28049, Spain. E-mail address: yrevilla{at}cbm.uam.es

³ 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.

Received for publication September 12, 2007. Accepted for publication December 7, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Goodman, R. H., S. Smolik. 2000. CBP/p300 in cell growth, transformation, and development. Genes Dev. 14: 1553-1577. [Free Full Text]
2. Chrivia, J. C., R. P. Kwok, N. Lamb, M. Hagiwara, M. R. Montminy, R. H. Goodman. 1993. Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365: 855-859. [Medline]
3. Eckner, R., M. E. Ewen, D. Newsome, M. Gerdes, J. A. DeCaprio, J. B. Lawrence, D. M. Livingston. 1994. Molecular cloning and functional analysis of the adenovirus E1A-associated 300-kD protein (p300) reveals a protein with properties of a transcriptional adaptor. Genes Dev. 8: 869-884. [Abstract/Free Full Text]
4. Chan, H. M., N. B. La Thangue. 2001. p300/CBP proteins: HATs for transcriptional bridges and scaffolds. J. Cell Sci. 114: 2363-2373. [Abstract/Free Full Text]
5. Ogryzko, V. V., R. L. Schiltz, V. Russanova, B. H. Howard, Y. Nakatani. 1996. The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87: 953-959. [Medline]
6. Brownell, J. E., C. D. Allis. 1996. Special HATs for special occasions: linking histone acetylation to chromatin assembly and gene activation. Curr. Opin. Genet. Dev. 6: 176-184. [Medline]
7. Soutoglou, E., N. Katrakili, I. Talianidis. 2000. Acetylation regulates transcription factor activity at multiple levels. Mol. Cell 5: 745-751. [Medline]
8. Gu, W., R. G. Roeder. 1997. Activation of p53 sequence-specific DNA binding by acetylation of the p53 carboxyl-terminal domain. Cell 90: 595-606. [Medline]
9. Martinez-Balbas, M. A., U. M. Bauer, S. J. Nielsen, A. Brehm, T. Kouzarides. 2000. Regulation of E2F1 activity by acetylation. EMBO J. 19: 662-671. [Medline]
10. Arany, Z., W. R. Sellers, D. M. Livingston, R. Eckner. 1994. E1A-associated p300 and CREB-associated CBP belong to a conserved family of coactivators. Cell 77: 799-800. [Medline]
11. Garcia-Rodriguez, C., A. Rao. 1998. Nuclear factor of activated T cells (NFAT)-dependent transactivation regulated by the coactivators p300/CREB-binding protein (CBP). J. Exp. Med. 187: 2031-2036. [Abstract/Free Full Text]
12. Avots, A., M. Buttmann, S. Chuvpilo, C. Escher, U. Smola, A. J. Bannister, U. R. Rapp, T. Kouzarides, E. Serfling. 1999. CBP/p300 integrates Raf/Rac-signaling pathways in the transcriptional induction of NF-ATc during T cell activation. Immunity 10: 515-524. [Medline]
13. Gerritsen, M. E., A. J. Williams, A. S. Neish, S. Moore, Y. Shi, T. Collins. 1997. CREB-binding protein/p300 are transcriptional coactivators of p65. Proc. Natl. Acad. Sci. USA 94: 2927-2932. [Abstract/Free Full Text]
14. Zhong, H., R. E. Voll, S. Ghosh. 1998. Phosphorylation of NF-B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol. Cell 1: 661-671. [Medline]
15. Lee, J. S., R. H. See, T. Deng, Y. Shi. 1996. Adenovirus E1A down-regulates cJun- and JunB-mediated transcription by targeting their coactivator p300. Mol. Cell. Biol. 16: 4312-4326. [Abstract]
16. Benkoussa, M., C. Brand, M. H. Delmotte, P. Formstecher, P. Lefebvre. 2002. Retinoic acid receptors inhibit AP1 activation by regulating extracellular signal-regulated kinase and CBP recruitment to an AP1-responsive promoter. Mol. Cell. Biol. 22: 4522-4534. [Abstract/Free Full Text]
17. Vo, N., R. H. Goodman. 2001. CREB-binding protein and p300 in transcriptional regulation. J. Biol. Chem. 276: 13505-13508. [Free Full Text]
18. Lundblad, J. R., R. P. Kwok, M. E. Laurance, M. L. Harter, R. H. Goodman. 1995. Adenoviral E1A-associated protein p300 as a functional homologue of the transcriptional co-activator CBP. Nature 374: 85-88. [Medline]
19. Eckner, R., J. W. Ludlow, N. L. Lill, E. Oldread, Z. Arany, N. Modjtahedi, J. A. DeCaprio, D. M. Livingston, J. A. Morgan. 1996. Association of p300 and CBP with simian virus 40 large T antigen. Mol. Cell. Biol. 16: 3454-3464. [Abstract]
20. Bernat, A., N. Avvakumov, J. S. Mymryk, L. Banks. 2003. Interaction between the HPV E7 oncoprotein and the transcriptional coactivator p300. Oncogene 22: 7871-7881. [Medline]
21. Patel, D., S. M. Huang, L. A. Baglia, D. J. McCance. 1999. The E6 protein of human papillomavirus type 16 binds to and inhibits co-activation by CBP and p300. EMBO J. 18: 5061-5072. [Medline]
22. Kwok, R. P., M. E. Laurance, J. R. Lundblad, P. S. Goldman, H. Shih, L. M. Connor, S. J. Marriott, R. H. Goodman. 1996. Control of cAMP-regulated enhancers by the viral transactivator Tax through CREB and the co-activator CBP. Nature 380: 642-646. [Medline]
23. Swope, D. L., C. L. Mueller, J. C. Chrivia. 1996. CREB-binding protein activates transcription through multiple domains. J. Biol. Chem. 271: 28138-28145. [Abstract/Free Full Text]
24. Stiehl, D. P., D. M. Fath, D. Liang, Y. Jiang, N. Sang. 2007. Histone deacetylase inhibitors synergize p300 autoacetylation that regulates its transactivation activity and complex formation. Cancer Res. 67: 2256-2264. [Abstract/Free Full Text]
25. Chevillard-Briet, M., D. Trouche, L. Vandel. 2002. Control of CBP co-activating activity by arginine methylation. EMBO J. 21: 5457-5466. [Medline]
26. Girdwood, D., D. Bumpass, O. A. Vaughan, A. Thain, L. A. Anderson, A. W. Snowden, E. Garcia-Wilson, N. D. Perkins, R. T. Hay. 2003. P300 transcriptional repression is mediated by SUMO modification. Mol. Cell 11: 1043-1054. [Medline]
27. Thompson, P. R., D. Wang, L. Wang, M. Fulco, N. Pediconi, D. Zhang, W. An, Q. Ge, R. G. Roeder, J. Wong, et al 2004. Regulation of the p300 HAT domain via a novel activation loop. Nat. Struct. Mol. Biol. 11: 308-315. [Medline]
28. Liu, Y. Z., N. S. Thomas, D. S. Latchman. 1999. CBP associates with the p42/p44 MAPK enzymes and is phosphorylated following NGF treatment. NeuroReport 10: 1239-1243. [Medline]
29. Chawla, S., G. E. Hardingham, D. R. Quinn, H. Bading. 1998. CBP: a signal-regulated transcriptional coactivator controlled by nuclear calcium and CaM kinase IV. Science 281: 1505-1509. [Abstract/Free Full Text]
30. Brouillard, F., C. E. Cremisi. 2003. Concomitant increase of histone acetyltransferase activity and degradation of p300 during retinoic acid-induced differentiation of F9 cells. J. Biol. Chem. 278: 39509-39516. [Abstract/Free Full Text]
31. Liu, Y., C. E. Denlinger, B. K. Rundall, P. W. Smith, D. R. Jones. 2006. Suberoylanilide hydroxamic acid induces Akt-mediated phosphorylation of p300, which promotes acetylation and transcriptional activation of RelA/p65. J. Biol. Chem. 281: 31359-31368. [Abstract/Free Full Text]
32. Huang, W. C., T. K. Ju, M. C. Hung, C. C. Chen. 2007. Phosphorylation of CBP by IKK promotes cell growth by switching the binding preference of CBP from p53 to NF-B. Mol. Cell 26: 75-87. [Medline]
33. Perkins, N. D., L. K. Felzien, J. C. Betts, K. Leung, D. H. Beach, G. J. Nabel. 1997. Regulation of NF-B by cyclin-dependent kinases associated with the p300 coactivator. Science 275: 523-527. [Abstract/Free Full Text]
34. Yuan, L. W., J. E. Gambee. 2000. Phosphorylation of p300 at serine 89 by protein kinase C. J. Biol. Chem. 275: 40946-40951. [Abstract/Free Full Text]
35. Miskin, J. E., C. C. Abrams, L. C. Goatley, L. K. Dixon. 1998. A viral mechanism for inhibition of the cellular phosphatase calcineurin. Science 281: 562-565. [Abstract/Free Full Text]
36. Revilla, Y., M. Callejo, J. M. Rodriguez, E. Culebras, M. L. Nogal, M. L. Salas, E. Vinuela, M. Fresno. 1998. Inhibition of nuclear factor B activation by a virus-encoded IB-like protein. J. Biol. Chem. 273: 5405-5411. [Abstract/Free Full Text]
37. Granja, A. G., M. L. Nogal, C. Hurtado, C. Del Aguila, A. L. Carrascosa, M. L. Salas, M. Fresno, Y. Revilla. 2006. The viral protein A238L inhibits TNF- expression through a CBP/p300 transcriptional coactivators pathway. J. Immunol. 176: 451-462. [Abstract/Free Full Text]
38. Granja, A. G., P. Sabina, M. L. Salas, M. Fresno, Y. Revilla. 2006. Regulation of inducible nitric oxide synthase expression by viral A238L-mediated inhibition of p65/RelA acetylation and p300 transactivation. J. Virol. 80: 10487-10496. [Abstract/Free Full Text]
39. Granja, A. G., M. L. Nogal, C. Hurtado, V. Vila, A. L. Carrascosa, M. L. Salas, M. Fresno, Y. Revilla. 2004. The viral protein A238L inhibits cyclooxygenase-2 expression through a nuclear factor of activated T cell-dependent transactivation pathway. J. Biol. Chem. 279: 53736-53746. [Abstract/Free Full Text]
40. Northrop, J. P., S. N. Ho, L. Chen, D. J. Thomas, L. A. Timmerman, G. P. Nolan, A. Admon, G. R. Crabtree. 1994. NF-AT components define a family of transcription factors targeted in T-cell activation. Nature 369: 497-502. [Medline]
41. Yano, O., J. Kanellopoulos, M. Kieran, O. Le Bail, A. Israel, P. Kourilsky. 1987. Purification of KBF1, a common factor binding to both H-2 and β₂-microglobulin enhancers. EMBO J. 6: 3317-3324. [Medline]
42. Deng, T., M. Karin. 1993. JunB differs from c-Jun in its DNA-binding and dimerization domains, and represses c-Jun by formation of inactive heterodimers. Genes Dev. 7: 479-490. [Abstract/Free Full Text]
43. De Gregorio, R., M. A. Iniguez, M. Fresno, S. Alemany. 2001. Cot kinase induces cyclooxygenase-2 expression in T cells through activation of the nuclear factor of activated T cells. J. Biol. Chem. 276: 27003-27009. [Abstract/Free Full Text]
44. Schmitz, M. L., P. A. Baeuerle. 1991. The p65 subunit is responsible for the strong transcription activating potential of NF-B. EMBO J. 10: 3805-3817. [Medline]
45. Martin, A. G., B. San-Antonio, M. Fresno. 2001. Regulation of nuclear factor B transactivation: implication of phosphatidylinositol 3-kinase and protein kinase C in c-Rel activation by tumor necrosis factor . J. Biol. Chem. 276: 15840-15849. [Abstract/Free Full Text]
46. Minden, A., A. Lin, F. X. Claret, A. Abo, M. Karin. 1995. Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell 81: 1147-1157. [Medline]
47. Snowden, A. W., L. A. Anderson, G. A. Webster, N. D. Perkins. 2000. A novel transcriptional repression domain mediates p21(WAF1/CIP1) induction of p300 transactivation. Mol. Cell. Biol. 20: 2676-2686. [Abstract/Free Full Text]
48. Boyes, J., P. Byfield, Y. Nakatani, V. Ogryzko. 1998. Regulation of activity of the transcription factor GATA-1 by acetylation. Nature 396: 594-598. [Medline]
49. Villalba, M., S. Kasibhatla, L. Genestier, A. Mahboubi, D. R. Green, A. Altman. 1999. Protein kinase C cooperates with calcineurin to induce Fas ligand expression during activation-induced T cell death. J. Immunol. 163: 5813-5819. [Abstract/Free Full Text]
50. Bannister, A. J., T. Oehler, D. Wilhelm, P. Angel, T. Kouzarides. 1995. Stimulation of c-Jun activity by CBP: c-Jun residues Ser63/73 are required for CBP induced stimulation in vivo and CBP binding in vitro. Oncogene 11: 2509-2514. [Medline]
51. Fath, D. M., X. Kong, D. Liang, Z. Lin, A. Chou, Y. Jiang, J. Fang, J. Caro, N. Sang. 2006. Histone deacetylase inhibitors repress the transactivation potential of hypoxia-inducible factors independently of direct acetylation of HIF-. J. Biol. Chem. 281: 13612-13619. [Abstract/Free Full Text]
52. Boisvert, F. M., M. J. Hendzel, D. P. Bazett-Jones. 2000. Promyelocytic leukemia (PML) nuclear bodies are protein structures that do not accumulate RNA. J. Cell Biol. 148: 283-292. [Abstract/Free Full Text]
53. Yuan, W., G. Condorelli, M. Caruso, A. Felsani, A. Giordano. 1996. Human p300 protein is a coactivator for the transcription factor MyoD. J. Biol. Chem. 271: 9009-9013. [Abstract/Free Full Text]
54. Blom, N., S. Gammeltoft, S. Brunak. 1999. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J. Mol. Biol. 294: 1351-1362. [Medline]
55. Schwartz, C., K. Beck, S. Mink, M. Schmolke, B. Budde, D. Wenning, K. H. Klempnauer. 2003. Recruitment of p300 by C/EBPβ triggers phosphorylation of p300 and modulates coactivator activity. EMBO J. 22: 882-892. [Medline]
56. Coudronniere, N., M. Villalba, N. Englund, A. Altman. 2000. NF-B activation induced by T cell receptor/CD28 costimulation is mediated by protein kinase C-. Proc. Natl. Acad. Sci. USA 97: 3394-3399. [Abstract/Free Full Text]
57. San-Antonio, B., M. A. Iniguez, M. Fresno. 2002. Protein kinase C phosphorylates nuclear factor of activated T cells and regulates its transactivating activity. J. Biol. Chem. 277: 27073-27080. [Abstract/Free Full Text]
58. Werlen, G., E. Jacinto, Y. Xia, M. Karin. 1998. Calcineurin preferentially synergizes with PKC- to activate JNK and IL-2 promoter in T lymphocytes. EMBO J. 17: 3101-3111. [Medline]
59. Castrillo, A., D. J. Pennington, F. Otto, P. J. Parker, M. J. Owen, L. Bosca. 2001. Protein kinase C is required for macrophage activation and defense against bacterial infection. J. Exp. Med. 194: 1231-1242. [Abstract/Free Full Text]
60. Santos-Rosa, H., E. Valls, T. Kouzarides, M. Martinez-Balbas. 2003. Mechanisms of P/CAF auto-acetylation. Nucleic Acids Res. 31: 4285-4292. [Abstract/Free Full Text]
61. Felzien, L. K., S. Farrell, J. C. Betts, R. Mosavin, G. J. Nabel. 1999. Specificity of cyclin E-Cdk2, TFIIB, and E1A interactions with a common domain of the p300 coactivator. Mol. Cell. Biol. 19: 4241-4246. [Abstract/Free Full Text]
62. Chakravarti, D., V. Ogryzko, H. Y. Kao, A. Nash, H. Chen, Y. Nakatani, R. M. Evans. 1999. A viral mechanism for inhibition of p300 and PCAF acetyltransferase activity. Cell 96: 393-403. [Medline]
63. Kim, R. H., K. C. Flanders, S. Birkey Reffey, L. A. Anderson, C. S. Duckett, N. D. Perkins, A. B. Roberts. 2001. SNIP1 inhibits NF-B signaling by competing for its binding to the C/H1 domain of CBP/p300 transcriptional co-activators. J. Biol. Chem. 276: 46297-46304. [Abstract/Free Full Text]
64. Pfeifhofer, C., K. Kofler, T. Gruber, N. G. Tabrizi, C. Lutz, K. Maly, M. Leitges, G. Baier. 2003. Protein kinase C affects Ca²⁺ mobilization and NFAT cell activation in primary mouse T cells. J. Exp. Med. 197: 1525-1535. [Abstract/Free Full Text]
65. Sun, Z., C. W. Arendt, W. Ellmeier, E. M. Schaeffer, M. J. Sunshine, L. Gandhi, J. Annes, D. Petrzilka, A. Kupfer, P. L. Schwartzberg, D. R. Littman. 2000. PKC- is required for TCR-induced NF-B activation in mature but not immature T lymphocytes. Nature 404: 402-407. [Medline]
66. Manicassamy, S., S. Gupta, Z. Sun. 2006. Selective function of PKC- in T cells. Cell Mol. Immunol. 3: 263-270. [Medline]

This article has been cited by other articles:

				A. G. Granja, E. G. Sanchez, P. Sabina, M. Fresno, and Y. Revilla African Swine Fever Virus Blocks the Host Cell Antiviral Inflammatory Response through a Direct Inhibition of PKC-{theta}-Mediated p300 Transactivation J. Virol., January 15, 2009; 83(2): 969 - 980. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Granja, A. G. Articles by Revilla, Y. Search for Related Content PubMed PubMed Citation Articles by Granja, A. G. Articles by Revilla, Y. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Substance via MeSH