The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kwon, M.-J.
Right arrow Articles by Chang, C.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kwon, M.-J.
Right arrow Articles by Chang, C.-H.
The Journal of Immunology, 2006, 177: 950-956.
Copyright © 2006 by The American Association of Immunologists

Protein Kinase C{delta} Is Essential to Maintain CIITA Gene Expression in B Cells1

Myung-Ja Kwon*, Jae-Won Soh{dagger} and Cheong-Hee Chang2,*

* Department of Microbiology and Immunology, Indiana University School of Medicine and Walther Oncology Center, Indianapolis, IN 46202; and {dagger} Department of Chemistry, Inha University, Incheon, Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Expression of MHC class II genes requires CIITA. Although the transactivation function of CIITA is well characterized, the signaling events that regulate CIITA expression are less understood. In this study, we report that CIITA expression in B cells depends on protein kinase C{delta} (PKC{delta}). PKC{delta} controls CIITA gene transcription mainly via modulating CREB recruitment to the CIITA promoter without affecting CIITA mRNA stability. Inhibition of PKC{delta} by a pharmacological inhibitor or knocking down of endogenous PKC{delta} expression by small interfering RNA reduced CREB binding to the CIITA promoter. The decrease of CIITA gene expression in the presence of the PKC{delta} inhibitor was prevented by ectopically expressing a constitutively active form of CREB. In addition, histone acetylation of the CIITA promoter is regulated by PKC{delta} since the PKC{delta} inhibitor treatment or PKC{delta} small interfering RNA resulted in decreased histone acetylation. Taken together, our study reveals that PKC{delta} is an important signaling molecule necessary to maintain CIITA and MHC class II expression in B cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Major histocompatibility complex class II genes are constitutively expressed in professional APC such as B lymphocytes and dendritic cells and are inducible by IFN-{gamma} in macrophages or other cell types (1, 2, 3, 4, 5). Studies have shown that protein kinase C (PKC)3 is involved in constitutive as well as inducible expression of MHC class II genes (6, 7, 8, 9). In B cells, activated HLA-DR expression by PMA is dependent on PKC (8). This activation is mediated by the proximal elements of the DR{alpha} promoter (8). IFN-{gamma}-inducible expression of the MHC class II gene is also regulated by PKC (9). Both constitutive and inducible expression of the MHC class II gene require CIITA (10, 11). CIITA is not a DNA-binding protein. Rather, CIITA acts as a scaffolding protein by interacting with itself and other transcription factors to activate the MHC class II promoter (12, 13, 14, 15, 16). Three different isoforms of CIITA have been identified, each transcribed from a separate, independently regulated promoter designated as pI, pIII, and pIV (17). In B cells, pIII is primarily used for the constitutive expression of CIITA. pI and pIV are responsible for CIITA transcription in monocytes/dendritic cells and cells that express CIITA upon IFN-{gamma} treatment, respectively (18).

A recent study reported that PKC{alpha} enhances the ability of IFN regulatory factor 1 to activate the IFN-{gamma}-inducible promoter of the CIITA gene, which is responsible for increased MHC class II expression (9). In contrast, PMA treatment of promyelocytic cells induces them to differentiate into macrophage-like cells and destabilizes CIITA mRNA, resulting in reduced MHC class II expression (19). Therefore, PKC seems to regulate CIITA expression in a cell-type specific manner. However, the underlying mechanisms by which PKC induces MHC class II expression in B cells are not understood.

PKC is a serine/threonine-specific protein kinase and is involved in a number of important biological events, such as cell cycle progression, apoptosis, differentiation, and immune responses (20, 21, 22, 23). At present, 11 different PKC isoforms have been identified and grouped into three subsets based on their ability to respond to Ca2+ and/or diacylglycerols (24, 25). Both the classical PKC isoforms, {alpha}, betaI, betaII, and {gamma}, and the novel PKC isoforms, including {delta}, {epsilon}, {theta}, and {eta} are activated by diacylglycerols or PMA. The classical PKC, but not the novel PKC, respond to a change of the intracellular calcium level. Unlike classical and novel PKC isoforms, atypical PKC isoforms, such as {zeta} and {lambda}{tau}, do not respond to either Ca2+ or PMA.

B cells express several PKC isoforms that have different substrate specificities as well as unique functions (26, 27). In mature B cells, PKCbeta controls NF-{kappa}B activation and cell survival (23). PKC{zeta}-deficient mice show an alteration in the development of secondary lymphoid tissues due to the impairment of signaling through the BCR, which results in inhibition of cell proliferation and survival (28). PKC{delta} is highly expressed in B cells and regulates B cell tolerance (26, 29). Recently, it has shown that PKC{delta} activates CREB in B cells upon BCR ligation (30). CREB belongs to the CREB/activating transcription factor bZip family of transcription factors and binds to a cAMP-responsive element (CRE) as a homodimer or a heterodimer with either CREB/activating transcription factor or AP-1 transcription factors (31). CREB activates numerous genes including the MHC class II and the CIITA gene by directly binding to the promoters (32, 33). For the CIITA gene, several CREs have been identified in the proximal promoter and in the 5'-untranslated region of pIII (33). In addition, CREB binding to CRE on the CIITA promoter is necessary to activate CIITA gene expression (33).

In this study, we investigated the role of PKC{delta} in the regulation of CIITA gene expression in B cells. Our data show that PKC{delta} controls CIITA gene expression by modulating phosphorylation of CREB that affects CREB recruitment to the CIITA promoter. In addition, PKC{delta} is necessary for maximum histone acetylation of the CIITA promoter. Together, these data support a critical role for PKC{delta} in B cell CIITA gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells

Raji, M12, and splenic B cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/ml penicillin/streptomycin. T2 and Priess, provided by Dr. J. Blum (Indiana University, Indianapolis, IN), were grown in IMDM with the same supplements. Mouse splenic B cells were prepared as described previously (34).

Reagents and Abs

PMA, Lactacystin, actinomycin D, Ro-31-8425, GO-6976, and Rottlerin were obtained from Calbiochem. MTT and chloroquine were purchased from Sigma-Aldrich. Human PKC{delta} (sc-36253) and control small interfering (si) RNA (sc-37007) were purchased from Santa Cruz Biotechnology. Abs against beta-actin and ubiquitin were obtained from Sigma-Aldrich and Santa Cruz Biotechnology, respectively. Anti-RFX5 and anti-phospho-CREB and anti-CREB were purchased from Rockland and Cell Signaling Technology, respectively. Anti-CIITA and anti-human MHC class II Ab were described elsewhere (34). Abs against CREB and acetylated histone H4, and CIITA (sc-13556X) for chromatin immunoprecipitation (ChIP) were purchased from Upstate Biotechnology and Santa Cruz Biotechnology, respectively.

MTT assay

Cell viability was determined using the colorimetric assay with MTT. The MTT assay measures cell survival based on mitochondrial conversion of MTT from a soluble tetrazolium salt into an insoluble colored formazan precipitate, which is dissolved in DMSO and quantitated by spectrophotometry. Cells (5 x 104) were seeded in 48-well plates in the presence of 5 µM Rottlerin or in an equal volume of DMSO for the indicated time. MTT (1 mg/ml final concentration) was added to the cells at the end of the experiments followed by incubation for 1 h at 37°C. DMSO was added to lyse the cells before measuring absorbance at 590 nm.

Transient transfection

Wild-type (WT) and a dominant negative (DN) mutant (K376R) of PKC{delta}, MHC class II E{alpha}-driven promoter, were reported previously (35). The CIITA promoter-driven luciferase construct was obtained from Dr. J. P.-Y. Ting (University of North Carolina, Chapel Hill, NC) (39). The constitutively active (CA-CREB) and the repressive form of CREB (R-CREB) that were described previously (40, 41) were obtained from Dr. R. P. S. Kwok (University of Michigan, Ann Arbor, MI). Raji cells (5 x 106) were transfected with 15 µg of the reporter plasmid using electroporation. Twenty-four hours after transfection, cells were treated with Rottlerin or DMSO for 6 h. In some experiments, the reporter was cotransfected with WT, DN PKC{delta}, CA-CREB, or R-CREB (15 µg) expression vector for 48 h. The total DNA amount was adjusted by adding empty vector DNA. PKC{delta} or control siRNA along with the GFP expression vector was introduced using Nucleofection with the reagents and the protocol provided by the manufacturer (Amaxa). GFP-positive cells were sorted by flow cytometry. Cell lysates were prepared and used for luciferase assay as described previously(36), immunoblot, or ChIP assay. Luciferase activity in each group was normalized by protein concentration. The results were expressed as relative luciferase activity. All experiments were performed in duplicate and repeated several times with similar results.

Quantitative real-time RT-PCR (qRT-PCR) and RT-PCR

Total RNA was prepared using TRIzol (Invitrogen Life Technologies). cDNA preparation and qRT-PCR were performed as described elsewhere (42). Primers and concentrations used for qRT-PCR were: human CIITA sense (900 nM), 5'-CTGAAGGATGTGGAAGACCTGGGAAAGC-3'; antisense (900 nM), 5'-GTCCCCGATCTTGTTCTCACTC-3'; human actin sense (300 nM), 5'-CCTTCCTGGGCATGGACTCCT-3'; and human actin antisense (300 nM), 5'-GGAGCA-ATGAATCTTGATCTT-3'.

RT-PCR was performed as described previously (43). The primers for the mouse CIITA gene were: sense, 5'-CTCAGCCTTAGGAGGGACTTG-3'; and antisense, 5'-GACCTGGATCGTCTCGTGCAG-3'. Primers for mouse hypoxanthine phosphoribosyltransferase were: sense, 5'-AGCTGTGGACAAAGCCAACT-3'; and antisense, 5'-TTGGGCTCTCTCAGTTCCAC-3'.

Immunoblotting

Total cell lysates were used for Western blot analysis as previously described (34). To assess CIITA expression, 30 µg of proteins from cell lines or 100 µg of splenic B cell lysates was subjected to SDS-PAGE on 6% slab gels. The same membrane was used to detect CIITA followed by RFX5. To examine MHC class II or actin, 10% slab gels were used. Relative expression levels of each band were calculated using an optical unit obtained by a densitometric analysis (FluorChem 8900 Imaging System; Alpha Innotech). To detect the CREB signal, 15 or 40 µg of protein lysates was used for SDS-PAGE. Phosphorylation of CREB was detected with the anti-pCREB Ab. The same membrane was used to detect total CREB. PKC{delta} protein level was assessed with 30-µg protein lysates. The amount of ubiquitin was determined with the Ab recognizing ubiquitin using 15 µg of protein lysates.

ChIP

ChIP assay was performed using the Upstate Biotechnology protocol (<http://www.upstate.com/misc/protocols.q.prot.e.chips/>). Approximately 1 x 107 cells were used for each chromatin preparation. The chromatin sample from cells cross-linked with formaldehyde and sonicated was precleared with salmon sperm DNA and protein A-Sepharose. Two percent of the sample was used for input. One-seventh of the samples (1.5 x 106 cells) was used for immunoprecipitation with the Ab recognizing CREB, acetylated histone H4, or CIITA. Immune complexes were collected with protein A-Sepharose beads and eluted. After reverse cross-linking and digestion of proteins with proteinase K, the DNA was purified by phenol/chloroform extraction. PCR was performed using primers designed to amplify the 184-bp fragments which contain CRE of the CIITA promoter pIII (33) or the 90-bp fragment which contains the WXY region of the DR{alpha} promoter (32).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Inhibition of PKC{delta} down-regulates the level of CIITA protein

To investigate whether PKC has an effect on CIITA expression, we treated Raji cells with PMA to activate PKC. We observed that the CIITA protein level was enhanced by PMA treatment, while RFX5, another transcription factor for MHC class II, was not affected (Fig. 1A, first lane of each group). CIITA protein is often detected as a doublet due to phosphorylation (36, 37, 38) and PMA increased the expression of both species of CIITA protein. PMA is known to activate several PKC isoforms (24, 25); therefore, we next examined which PKC isoforms are responsible for the change in the CIITA protein level. We treated Raji cells with pharmacological PKC blockers with different specificities: Ro-31-8425 for PKC{alpha}, beta, {gamma} and {epsilon}; GO-6976 for PKC{alpha} and beta; and Rottlerin for PKC{delta}. As shown in Fig. 1A, Rottlerin, but not the other inhibitors, severely diminished the CIITA protein level, regardless of PMA treatment, indicating the involvement of PKC{delta}. Despite a lower level of CIITA, the amount of MHC class II protein remained unchanged, possibly due to a longer half-life of MHC class II protein (Fig. 1A, third panel). RFX5 levels were comparable among all conditions (Fig. 1A, second panel).


Figure 1
View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 1. Reduction of CIITA protein in B cells by a PKC{delta} inhibitor. A, The effect of PKC on CIITA protein. Raji cells were treated with PKC inhibitors (Ro-31-8425 (RO, 500 nM), GO-6976 (GO, 200 nM), Rottlerin (RT, 5 µM), or DMSO (DM)) for 30 min followed by PMA (20 nM) treatment for 6 h. Cells were lysed and immunoblot was performed using the Ab as indicated. To assess CIITA protein levels, 30 µg of total cell lysates was loaded on a 6% gel. The same membrane was used to detect CIITA and then RFX5. To examine MHC class II and actin expression, a 10% slab gel was used. B, The dose effect of Rottlerin. Raji cells were treated for 6 h at a varying dose of Rottlerin as indicated. Western blot was performed the same way as in A. C, Raji cells were treated with 5 µM Rottlerin for different lengths of time as indicated. Western blot was performed the same way as in A. D, MTT assay was performed as described in Materials and Methods. The MTT values of Raji cells without Rottlerin were set at 100%. E, Human B cell lines (T2 and Priess) and F, the mouse B cell line M12 and B220-positive splenic cells were treated with Rottlerin (5 µM) for 6 h. For splenic B cells, 100 µg of cell lysates was used. Relative expression levels shown were calculated using optical units obtained by the densitometric analysis. Values were normalized to time 0 or DMSO-treated cells. Data are representative of at least three independent experiments.

 
Having observed that Rottlerin down-regulates CIITA, we further investigated the effect of the dose of the inhibitor and the kinetics. CIITA protein level was decreased in a dose- and time-dependent manner upon Rottlerin treatment (Fig. 1, B and C). Again, RFX5, MHC class II, and actin levels remained equivalent among all conditions tested. The decrease in CIITA expression was not due to cytotoxicity of Rottlerin since cell viability was not significantly altered by the Rottlerin treatment (Fig. 1D)

Next, we tested whether the reduction of CIITA protein levels by Rottlerin could be observed in other B cells. To address this, we examined two additional human B cell lines (T2 and Priess), a mouse B cell line (M12), and splenic B220+ B cells. All B cells responded similarly to Rottlerin by showing decreased CIITA protein in the presence of Rottlerin (Fig. 1, E and F). Taken together, the data suggest that PKC{delta} seems to be important for CIITA protein expression in B cells.

Decreased CIITA protein by the PKC{delta} inhibitor was not due to enhanced protein degradation

It is reported that the half-life of CIITA protein is ~30 min and that CIITA is degraded by the proteosome-mediated pathway (44). Therefore, it is possible that the effects of PMA and Rottlerin that we have observed might have been due to an alteration in the stability of CIITA protein. To test this, we compared the level of CIITA protein in Raji cells that were cultured with or without the proteosome inhibitor lactacystin for 30 min before a 6-h culture in the presence or absence of Rottlerin. As shown in Fig. 2A, CIITA protein levels were comparable among cells treated in the described conditions. Lactacystin treatment resulted in an accumulation of ubiquitinated proteins, indicating that lactacystin prevented the degradation of ubiquitinated proteins by proteosomes (Fig. 2A, bottom panel). Neither MG132 nor ALLN, other proteosome inhibitors, prevented the effects of PKC{delta} inhibition (data not shown). We next tested a lysosome inhibitor, chloroquine, because lysosomes are also involved in protein degradation (45). Similar to proteosome inhibitor-treated cells, chloroquine did not prevent the reduction of CIITA protein by Rottlerin (Fig. 2B). Therefore, the reduced CIITA protein level resulting from treatment with Rottlerin is unlikely due to enhanced degradation of CIITA protein.


Figure 2
View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 2. CIITA protein levels in the presence of a proteosome or a lysosome inhibitor. The effect of a proteosome inhibitor (A) or a lysosome inhibitor (B) on CIITA protein. Raji cells were pretreated with lactacystin (10 µM; A) or chloroquine (100 µM; B) for 30 min followed by Rottlerin treatment for 6 h. DMSO-treated cells were used as a control. Western blot was performed as in Fig. 1, except that 15 µg of lysates was used to assess ubiquitination.

 
PKC{delta} inhibitor down-regulates the CIITA mRNA level

We next investigated whether the reduction of CIITA protein by Rottlerin results from decreased CIITA mRNA. RNA was prepared from Raji cells that were treated with the PKC inhibitors described above and analyzed by qRT-PCR. The amount of CIITA but not MHC class II mRNA was significantly reduced in Raji cells treated with Rottlerin but not with the other PKC inhibitors (Fig. 3, A–C). Consistent with the protein data, other B cells, including splenic B cells, showed a significant decrease in CIITA mRNA levels upon Rottlerin treatment (Fig. 3, D and E). Thus, the changes in CIITA mRNA following Rottlerin treatment corresponded to the protein level, suggesting that PKC{delta} regulates CIITA expression at the mRNA level.


Figure 3
View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 3. PKC{delta} inhibitor down-regulates CIITA mRNA levels. Raji cells were treated as in Fig. 1, and RNA was prepared and subjected to qRT-PCR to measure the amount of CIITA (A) or DR{alpha} (B) mRNA. C, The decrease of CIITA mRNA after Rottlerin treatment. Raji cells were treated with 5 µM Rottlerin and harvested at the indicated time. RNA was prepared and analyzed as in A. D and E, CIITA mRNA levels in T2 and Priess (D), and M12 and splenic B cells (E) with or without Rottlerin (5 µM) treatment for 6 h. RNA was prepared and subjected to qRT-PCR (D) or RT-PCR (E). All qRT-PCR data were normalized against mRNA levels of the actin gene. The data of qRT-PCR represent the mean and SE of three independent experiments.

 
PKC{delta} regulates CIITA promoter activity

The decrease in CIITA mRNA could be due to a change in mRNA stability or promoter activity. To distinguish these possibilities, we first compared CIITA mRNA stability by measuring the half-life of CIITA mRNA. Raji cells were pretreated with Rottlerin or DMSO for 1 h and then actinomycin D was added to stop new RNA synthesis. Total RNA was prepared and the amount of CIITA mRNA was measured using qRT-PCR. As shown in Fig. 4A, we did not observe a significant difference in CIITA mRNA stability in the presence or absence of Rottlerin.


Figure 4
View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 4. PKC{delta} regulates CIITA promoter activity. A, The effect of Rottlerin on CIITA mRNA stability. Raji cells were treated with actinomycin D (5 µg/ml) at the indicated time after a 1-h pretreatment of Rottlerin or DMSO. Cells were harvested at the indicated time points and CIITA mRNA levels were measured by qRT-PCR as in Fig. 3. B and C, The effect of Rottlerin on CIITA (B) and MHC class II (C) promoter activity. Raji cells were transiently transfected with the luciferase reporter. Next day, cells were treated with Rottlerin for 6 h. Cell lysates were prepared and used to measure luciferase activity. D and E, The effect of PKC{delta} mutant on CIITA (D) and MHC class II (E) promoter activity. Transient transfections of Raji cells were performed with the luciferase reporter along with the expression vector encoding WT or DN mutant PKC{delta}. Cell lysates were prepared to assess the luciferase activity. Luciferase activity in each group was normalized by protein concentration. The results were expressed as relative luciferase activity (RLA) compared with the value obtained from DMSO-treated or empty vector-transfected cells. All experiments were performed in duplicate and repeated several times with similar results.

 
We next tested whether Rottlerin affects the promoter activity of CIITA. To do this, we transfected Raji cells with the CIITA promoter pIII-driven luciferase reporter followed by Rottlerin treatment. When we compared luciferase activity, we observed a decrease in the activity of the CIITA promoter in Rottlerin vs control-treated cells (Fig. 4B). Since CIITA is a critical transcription factor for MHC class II gene expression, we expected that the decrease in CIITA caused by Rottlerin would also down-regulate MHC class II gene expression. Indeed, luciferase activity driven by the MHC class II promoter was significantly diminished by Rottlerin treatment (Fig. 4C).

To further confirm whether the Rottlerin effect was mediated by PKC{delta}, we cotransfected the expression vector encoding WT or a DN PKC{delta} mutant along with the luciferase reporters. Consistent with the effect of Rottlerin, cells cotransfected with DN but not WT PKC{delta} showed a decrease in both CIITA and MHC class II promoter-driven luciferase activity (Fig. 4, D and E). Together, the data suggest that PKC{delta} regulates CIITA gene expression, which in turn controls the MHC class II gene in B cells.

Regulation of CIITA expression by PKC{delta} is mediated by CREB

CREB binds to the CIITA promoter and is necessary for CIITA gene expression (33). In addition, a recent study demonstrated that PKC{delta} phosphorylates CREB (30). Therefore, we wondered whether the PKC{delta} effect on CIITA gene expression is mediated via CREB phosphorylation. To examine this possibility, we first assessed the phosphorylation status of CREB. Phosphorylated CREB (pCREB) was present in Raji cells but the level was decreased by Rottlerin treatment either with or without PMA (Fig. 5A). Therefore, CREB is constitutively phosphorylated in B cells and phosphorylation is regulated by PKC{delta} signaling. Since pCREB is known to bind DNA and activates transcription, we tested whether CREB recruitment to the CIITA promoter is altered in the presence of Rottlerin using a ChIP assay. As we expected, CREB bound to the CIITA promoter constitutively and its binding was attenuated in the presence of Rottlerin (Fig. 5B). CREB also binds the histone acetyltransferase (HAT) CREB binding protein (CBP), resulting in enhanced histone acetylation (40). Therefore, we compared the amount of histone acetylation of the CIITA promoter. Consistent with the data showing CREB binding on the CIITA promoter, histone acetylation was greatly diminished in cells treated with Rottlerin (Fig. 5B, middle panel).


Figure 5
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 5. PKC{delta} regulates CIITA expression by modulating CREB. A, PKC{delta} regulates CREB phosphorylation. CREB phosphorylation was assessed in Raji cells under the indicated conditions. Raji cells were treated with Rottlerin (5 µM) for 1 h and then with or without PMA (20 nM) for 30 min. Total lysates (40 and 15 µg, left and right panel, respectively) were used to compare pCREB followed by total amounts of CREB. B, The effect of CREB binding and histone acetylation of the CIITA promoter. The same Raji cells used in A were subjected to the ChIP assay as described in Materials and Methods. The Abs used for immunoprecipitation were indicated. Semiquantitative PCR was performed using the samples that were serially diluted by 2-fold. Input shows 2% of total cell lysates. C, The effects of CREB mutants on CIITA promoter activity. Raji cells were transfected with CA-CREB, a repressor mutant of CREB (R-CREB), or an empty vector (Vector) in combination with the CIITA promoter-driven luciferase reporter. Rottlerin was added during the last 6 h before lysis. Cell lysates were prepared to assess luciferase activity. Luciferase activity in each group was normalized by protein concentration. The results were expressed as relative luciferase activity against that of empty vector-transfected cells. All experiments were performed in duplicate and repeated several times with similar results. Data are representative of three independent experiments.

 
If the decrease of CIITA promoter activity is due to the inefficient recruitment of CREB to the promoter, ectopic expression of a constitutively active form of CREB should restore the promoter activity. To test this, we cotransfected a constitutively active (CA-CREB) or a mutant form of CREB (R-CREB) along with the CIITA promoter-driven luciferase reporter. CA-CREB was made by replacing six residues (RRPSYR, which includes S133) from the KID domain of CREB with six residues (DIEDML) from the sterol response element-binding protein, a transcriptional factor that binds CBP in the absence of phosphorylation (40). As shown in Fig. 5C, CA-CREB but not R-CREB induced the CIITA promoter activity significantly. More importantly, CIITA promoter activity remained high in the presence of Rottlerin when CA-CREB was expressed. Therefore, PKC{delta} appears to regulate CIITA gene expression by modulating CREB phosphorylation and influencing CREB binding to the CIITA promoter.

Reduction of endogenous PKC{delta} protein down-regulates CIITA

So far, we have shown that CIITA gene expression is regulated by PKC signaling by treating cells with a pharmacological inhibitor or expressing a dominant mutant form of PKC{delta}. To test whether the reduction of endogenous PKC{delta} would result in a negative effect on CIITA expression, we used a knock-down approach using siRNA. Raji cells were transfected with control or PKC{delta} siRNA along with an expression vector encoding GFP. GFP-positive cells were then sorted to enrich siRNA-transfected cells and cell lysates were prepared to compare CIITA expression. As expected, PKC{delta} siRNA-, not control siRNA-, transfected cells showed a significant decrease in the PKC{delta} level (Fig. 6A). Treatment with PKC{delta} siRNA not only decreased CIITA protein levels, but also MHC class II expression, which differs from results using Rottlerin. Again, there was no difference in the levels of RFX5 between control and siRNA-transfected cells (Fig. 6A). When we assessed CREB recruitment and histone acetylation of the CIITA promoter, both were diminished in PKC{delta} siRNA-transfected cells (Fig. 6B). As a consequence, CIITA binding to the MHC class II promoter was also reduced (Fig. 6C). Therefore, PKC{delta} plays an important role in maintaining CIITA gene expression and consequently regulating MHC class II gene expression in B cells.


Figure 6
View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 6. Reduction of endogenous PKC{delta} down-regulates CIITA expression. A, Control or PKC{delta} siRNA with the GFP-expressing vector was transfected to Raji cells. Next day, GFP-positive cells were sorted using flow cytometry and cell lysates were prepared for Western blot analysis as in Fig. 1A. The Abs used for Western blot are shown. B, CREB recruitment and histone acetylation. The same cells prepared in A were used for the ChIP assay with the indicated Abs to measure their binding on the CIITA promoter. C, CIITA binding on the DR{alpha} promoter. The ChIP experiment was performed as in A using the anti-CIITA Ab. Semiquantitative PCR was performed with the samples serially diluted by 2-fold. Input shows 2% of total cell lysates.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Proper CIITA expression is critical for Ag presentation by MHC class II in APC (1). In this study, we show that constitutive expression of the CIITA gene in B cells is maintained by PKC{delta}. PKC{delta} regulates CIITA gene expression through modulating CREB recruitment to the CIITA promoter. In addition, histone acetylation of the CIITA promoter is regulated by PKC{delta}. The basal activity of PKC{delta} seems to be important for CIITA gene expression, since the CIITA protein level was decreased in PKC{delta} siRNA-transfected cells, in the absence of an external stimulus. Consistent with this observation, pCREB in B cells appears to be sufficient to maintain CIITA gene expression. Moreover, constitutive phosphorylation of CREB and CREB binding to the CIITA promoter is at least partly PKC{delta} dependent as demonstrated by reduced pCREB levels and promoter occupancy following Rottlerin treatment.

CREB also binds to the X2 box of the MHC class II promoter and activates transcription (32). PKC is known to up-regulate MHC class II expression (8) and, therefore, the positive effect of PKC on MHC class II gene expression may be mediated by direct activation of the MHC class II promoter by CREB, through the regulation of CIITA gene expression, or by both. However, we did not observe a difference in MHC class II protein or mRNA in cells treated with Rottlerin for 6 h. This is most likely due to longer half-lives of MHC class II mRNA and protein compared with those of CIITA. Consistent with this notion, cells expressing less endogenous PKC{delta} 24 h following PKC{delta} siRNA transfection expressed a low amount of MHC class II protein. We also showed that the RFX5 level was not changed in the presence of the PKC{delta} inhibitor or PKC{delta} siRNA-transfected cells. Therefore, CIITA is susceptible to inhibition of PKC{delta} signaling which results in down-regulation of MHC class II expression.

We also observed diminished histone acetylation on the CIITA promoter in the presence of the PKC{delta} inhibitor as well as in cells transfected with PKC{delta} siRNA. Histone acetylation is mediated by a family of histone acetyltransferases (HAT) and therefore modulating the enzymatic activity of HAT would affect gene expression. There are at least two possible explanations for the reduced histone acetylation at the CIITA promoter. First, PKC{delta} can promote HAT activity directly. However, a study reported that PKC{delta} inhibits intrinsic HAT activity of p300 in HeLa cells (46). This report contradicts our finding that the inhibition of PKC{delta} decreases histone acetylation of the CIITA promoter. Although we cannot rule out that PKC{delta} modulates other HAT members, the decrease in histone acetylation of the CIITA promoter may not be a direct consequence of the PKC{delta} effect on HAT. Alternatively and more likely, the recruitment of CREB-interacting HAT such as CBP to the CIITA promoter could be diminished if less CREB is available on the promoter. CREB is phosphorylated by PKA as well as PKC and pCREB binds to the target promoter activating transcription (47, 48). It remains to be determined whether PKC{delta}-mediated phosphorylation of CREB is necessary for CREB to interact with CBP and to maintain CIITA gene expression in B cells.

We showed that PKC{delta} inhibition did not alter CIITA mRNA stability in Raji B cells. However, it has been shown that PMA reduces CIITA mRNA levels in myelomonocytic cells due to decreased mRNA stability (19), suggesting that the effect of the PKC on CIITA expression is cell type specific. This is not surprising since CIITA expression is cell-type dependent, and each cell type might operate through a distinct mechanism to regulate MHC class II expression (17). Moreover, the CIITA locus has three promoters and each promoter is transcribed in a cell-type dependent manner (19). The regulatory elements of each promoter are different and thus their response to PKC would be distinct as well. In this regard, PKC{alpha} is reported to regulate the IFN-{gamma}-inducible promoter of the CIITA gene in macrophage cell lines (9), though we observed no significant difference in CIITA expression by a PKC{alpha} inhibitor in B cells (Figs. 1A and 3A). Because a CREB binding site is not found within the proximal region of the IFN-{gamma}-inducible promoter of the CIITA gene (18), it is likely that PKC{alpha} and PKC{delta} utilize different promoter elements to regulate CIITA gene transcription. Indeed, PKC{alpha}-mediated regulation of CIITA expression depends on IFN regulatory factor 1 (9), while, as we demonstrated in this study, PKC{delta} modulates CREB.

Mice deficient in PKC{delta} show a normal development of lymphocytes but enlarged spleens and lymph nodes (29). This is attributed to a higher B cell proliferation rate in PKC{delta}–/– compared with the control mice, suggesting that PKC{delta} is involved in negative regulation of B cell proliferation (49). PKC{delta}–/– mice also exhibit autoreactive anti-DNA and anti-nuclear Abs in the serum (29). Therefore, PKC{delta} may be essential for inducing tolerance of B cell-mediated immune responses. However, it was not reported whether MHC class II expression is altered in PKC{delta}–/– B cells. If there is no difference in MHC class II expression in PKC{delta}–/– B cells, it could be due to the functional redundancy among PKC isoforms with respect to MHC class II gene expression. Further investigation is warranted to delineate signaling pathways responsible for maintaining or inducing CIITA gene expression, which ultimately controls the Ag presentation function of B cells.


   Acknowledgments
 
We thank Dr. Janice Blum for providing T2 and Priess cells, Dr. Roland Kwok for CREB expression vectors, and Brian McCarthy and Randy Wireman for technical support. We also thank Kevin Nickerson and Drs. Mark Kaplan, Wei Li, Yongxue Yao, and Elim Shao for their critical reading of this manuscript and helpful discussion.


   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 partly supported by National Institutes of Health Grant AI53556 (to C.-H.C.) and the Indiana Genomics Initiative (INGEN) of Indiana University. INGEN is supported in part by a Lily Endowment. Back

2 Address correspondence and reprint requests to Dr. Cheong-Hee Chang, Department of Microbiology and Immunology, Walther Oncology Center, R2-302, 950 West Walnut Street, Indiana University School of Medicine, Indianapolis, IN 46202-5188. E-mail address: chechang{at}iupui.edu Back

3 Abbreviations used in this paper: PKC, protein kinase C; qRT-PCR, quantitative real-time RT-PCR; HAT, histone acetyltransferase; WT, wild type; DN, dominant negative; CA-CREB, constitutively active CREB; R-CREB, repressive form of CREB; CRE, cAMP-responsive element; si, small interfering; ChIP, chromatin immunoprecipitation; pCREB, phosphorylated CREB; CBP, CREB binding protein. Back

Received for publication December 30, 2005. Accepted for publication April 25, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Mach, B., V. Steimle, E. Martinez-Soria, W. Reith. 1996. Regulation of MHC class II genes: lessons from a disease. Annu. Rev. Immunol. 14: 301-331. [Medline]
  2. Inaba, K., S. Turley, T. Iyoda, F. Yamaide, S. Shimovama, C. Reise Sours, R. N. Germain, I. Mellman, R. M. Steinman. 2000. The formation of immunogenic major histocompatibility complex class II-peptide ligands in lysosomal compartments of dendritic cells is regulated by inflammatory stimuli. J. Exp. Med. 191: 927-936. [Abstract/Free Full Text]
  3. Steinman, R. M., K. Inaba, S. Turley, P. Pierre, I. Mellman. 1999. Antigen capture, processing, and presentation by dendritic cells: recent cell biological studies. Hum. Immunol. 60: 562-567. [Medline]
  4. O’Keefe, G. M., V. T. Nguyen, L. L. Ping Tang, E. N. Benveniste. 2001. IFN-{gamma} regulation of class II transactivator promoter IV in macrophages and microglia: involvement of the suppressors of cytokine signaling-1 protein. J. Immunol. 166: 2260-2269. [Abstract/Free Full Text]
  5. Collins, T., A. J. Korman, C. T. Wake, J. M. Boss, D. J. Kappes, W. Fiers, K. A. Ault, M. A. Gimbrone, Jr, J. L. Strominger, J. S. Pober. 1984. Immune interferon activates multiple class II major histocompatibility complex genes and the associated invariant chain gene in human endothelial cells and dermal fibroblasts. Proc. Natl. Acad. Sci. USA 81: 4917-4921. [Abstract/Free Full Text]
  6. Lee, Y. J., R. B. Panek, M. Huston, E. N. Benveniste. 1995. Role of protein kinase C and tyrosine kinase activity in IFN-{gamma}-induced expression of the class II MHC gene. Am. J. Physiol. 268: C127-C137. [Medline]
  7. Brown, D., M. Faris, M. Hilburger, B. S. Zwilling. 1994. The induction of persistence of I-A expression by macrophages from Bcgr mice occurs via a protein kinase C-dependent pathway. J. Immunol. 152: 1323-1331. [Abstract]
  8. Setterblad, N., I. Onyango, U. Pihlgren, L. Rask, G. Andersson. 1998. The role of protein kinase C signaling in activated DRA transcription. J. Immunol. 161: 4819-4824. [Abstract/Free Full Text]
  9. Giroux, M., M. Schmidt, A. Descoteaux. 2003. IFN-{gamma}-induced MHC class II expression: transactivation of class II transactivator promoter IV by IFN regulatory factor-1 is regulated by protein kinase C-{alpha}. J. Immunol. 171: 4187-4194. [Abstract/Free Full Text]
  10. Reith, W., B. Mach. 2001. The bare lymphocyte syndrome and the regulation of MHC expression. Annu. Rev. Immunol. 19: 331-373. [Medline]
  11. Chang, C.-H., S. Guerder, S. C. Hong, W. van Ewijk, R. A. Flavell. 1996. Mice lacking the MHC class II transactivator (CIITA) show tissue-specific impairment of MHC class II expression. Immunity 4: 167-178. [Medline]
  12. Harton, J. A., J. P. Ting. 2000. Class II transactivator: mastering the art of major histocompatibility complex expression. Mol. Cell. Biol. 20: 6185-6194. [Free Full Text]
  13. Masternak, K., A. Muhlethaler-Mottet, J. Villard, M. Zufferey, V. Steimle, W. Reith. 2000. CIITA is a transcriptional coactivator that is recruited to MHC class II promoters by multiple synergistic interactions with an enhanceosome complex. Genes Dev. 14: 1156-1166. [Abstract/Free Full Text]
  14. Sisk, T. J., S. Roys, C.-H. Chang. 2001. Self-association of CIITA and its transactivation potential. Mol. Cell. Biol. 21: 4919-4928. [Abstract/Free Full Text]
  15. Scholl, T., S. K. Mahanta, J. L. Strominger. 1997. Specific complex formation between the type II bare lymphocyte syndrome-associated transactivators CIITA and RFX5. Proc. Natl. Acad. Sci. USA 94: 6330-6334. [Abstract/Free Full Text]
  16. Steimle, V., L. A. Otten, M. Zufferey, B. Mach. 1993. Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome). Cell 75: 135-146. [Medline]
  17. LeibundGut-Landmann, S., J. M. Waldburger, M. Krawczyk, L. A. Otten, T. Suter, A. Fontana, H. Acha-Orbea, W. Reith. 2004. Mini-review: specificity and expression of CIITA, the master regulator of MHC class II genes. Eur. J. Immunol. 34: 1513-1525. [Medline]
  18. Muhlethaler-Mottet, A., L. A. Otten, V. Steimle, B. Mac. 1997. Expression of MHC class II molecules in different cellular and functional compartments is controlled by differential usage of multiple promoters of the transactivator CIITA. EMBO J. 16: 2851-2860. [Medline]
  19. De Lerma Barbaro, A., F. A. Procopio, L. Mortara, G. Tosi, R. S. Accolla. 2005. The MHC class II transactivator (CIITA) mRNA stability is critical for the HLA class II gene expression in myelomonocytic cells. Eur. J. Immunol. 35: 603-611. [Medline]
  20. Grumont, R., P. Lock, M. Mollinari, F. M. Shannon, A. Moore, S. Gerondakis. 2004. The mitogen-induced increase in T cell size involves PKC and NFAT activation of Rel/NF-{kappa}B-dependent c-myc expression. Immunity 21: 19-30. [Medline]
  21. Hinshaw, J. A., C. M. Mueller, D. W. Scott, M. S. Williams. 2003. B cell receptor signaling mediates immediate protection from Fas-induced apoptosis upstream of caspase activation through an atypical protein kinase C isozyme and de novo protein synthesis. Eur. J. Immunol. 33: 2490-2500. [Medline]
  22. Mancini, A., A. Koch, A. D. Whetton, T. Tamura. 2004. The M-CSF receptor substrate and interacting protein FMIP is governed in its subcellular localization by protein kinase C-mediated phosphorylation, and thereby potentiates M-CSF-mediated differentiation. Oncogene 23: 6581-6589. [Medline]
  23. Saijo, K., I. Mecklenbrauker, C. Schmedt, A. Tarakhovsky. 2003. B cell immunity regulated by the protein kinase C family. Ann. NY Acad. Sci. 987: 125-134. [Medline]
  24. Nishizuka, Y.. 1992. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258: 607-614. [Abstract/Free Full Text]
  25. Nishizuka, Y.. 1995. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J. 9: 484-496. [Abstract]
  26. Mischak, H., W. Kolch, J. Goodnight, W. F. Davidson, U. Rapp, S. Rose-John, J. F. Mushinski. 1991. Expression of protein kinase C genes in hemopoietic cells is cell-type- and B cell-differentiation stage specific. J. Immunol. 147: 3981-3987. [Abstract]
  27. Sidorenko, S. P., C. L. Law, S. J. Klaus, K. A. Chandran, M. Takata, T. Kurosaki, E. A. Clark. 1996. Protein kinase C µ (PKC µ) associates with the B cell antigen receptor complex and regulates lymphocyte signaling. Immunity 5: 353-363. [Medline]
  28. Martin, P., A. Duran, S. Minguet, M. L. Gaspar, M. T. Diaz-Meco, P. Rennert, M. Leitges, J. Moscat. 2002. Role of {zeta} PKC in B-cell signaling and function. EMBO J. 21: 4049-4057. [Medline]
  29. Mecklenbrauker, I., K. Saijo, N. Y. Zheng, M. Leitges, A. Tarakhovsky. 2002. Protein kinase C{delta} controls self-antigen-induced B-cell tolerance. Nature 416: 860-865. [Medline]
  30. Blois, J. T., J. M. Mataraza, I. Mecklenbrauker, A. Tarakhovsky, T. C. Chiles. 2004. Cell receptor-induced cAMP-response element-binding protein activation in B lymphocytes requires novel protein kinase C{delta}. J. Biol. Chem. 279: 30123-30132. [Abstract/Free Full Text]
  31. Yamamoto, K. K., G. A. Gonzalez, W. H. Biggs, III, M. R. Montminy. 1998. Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature 334: 494-498.
  32. Moreno, C. S., G. W. Beresford, P. Louis-Plence, A. C. Morris, J. M. Boss. 1999. CREB regulates MHC class II expression in a CIITA-dependent manner. Immunity 10: 143-151. [Medline]
  33. Van der Stoep, N., E. Quinten, P. J. van den Elsen. 2002. Transcriptional regulation of the MHC class II trans-activator (CIITA) promoter III: identification of a novel regulatory region in the 5'-untranslated region and an important role for cAMP-responsive element binding protein 1 and activating transcription factor-1 in CIITA-promoter III transcriptional activation in B lymphocytes. J. Immunol. 169: 5061-5071. [Abstract/Free Full Text]
  34. Yee, C. S., Y. Yao, Q. Xu, B. McCarthy, D. Sun-Lin, M. Tone, H. Waldmann, C.-H. Chang. 2005. Enhanced production of IL-10 by dendritic cells deficient in CIITA. J. Immunol. 174: 1222-1229. [Abstract/Free Full Text]
  35. Soh, J. S., I. B. Weinstein. 2003. Roles of specific isoforms of protein kinase C in the transcriptional control of cyclin D1 and related genes. J. Biol. Chem. 278: 34709-34716. [Abstract/Free Full Text]
  36. Sisk, T. J., K. Nickerson, R. P. Kwok, C.-H. Chang. 2003. Phosphorylation of class II transactivator regulates its interaction ability and transactivation function. Int. Immunol. 15: 1195-1205. [Abstract/Free Full Text]
  37. Tosi, G., N. Jabrane-Ferrat, B. M. Peterlin. 2002. Phosphorylation of CIITA directs its oligomerization, accumulation and increased activity on MHCII promoters. EMBO J. 21: 5467-5476. [Medline]
  38. Greer, S. F., J. A. Harton, M. W. Linhoff, C. A. Janczak, J. P. Ting, D. E. Cressman. 2004. Serine residues 286, 288, and 293 within the CIITA: a mechanism for down-regulating CIITA activity through phosphorylation. J. Immunol. 173: 376-383. [Abstract/Free Full Text]
  39. Piskurich, J. F., Y. Wang, M. W. Linhoff, L. C. White, J. P. Ting. 1998. Identification of distinct regions of 5' flanking DNA that mediate constitutive, IFN-{gamma}, STAT1, and TGF-beta-regulated expression of the class II transactivator gene. J. Immunol. 160: 233-240. [Abstract/Free Full Text]
  40. Cardinaux, J. R., J. C. Notis, Q. Zhang, N. Vo, J. C. Craig, D. M. Fass, R. G. Grennan, R. H. Goodman. 2000. Recruitment of CREB binding protein is sufficient for CREB-mediated gene activation. Mol. Cell. Biol. 20: 1546-1552. [Abstract/Free Full Text]
  41. Laurance, M. E., R. P. Kwok, M. S. Huang, J. P. Richards, J. R. Lundblad, R. H. Goodman. 1997. Differential activation of viral and cellular promoters by human T-cell lymphotropic virus-1 tax and cAMP-responsive element modulator isoforms. J. Biol. Chem. 272: 2646-2651. [Abstract/Free Full Text]
  42. Gourley, T. S., D. R. Patel, K. Nickerson, S.-C. Hong, C.-H. Chang. 2002. Aberrant expression of Fas ligand in mice deficient for the MHC class II transactivator. J. Immunol. 168: 4414-4419. [Abstract/Free Full Text]
  43. Chang, C.-H., J. D. Fontes, M. Peterlin, R. A. Flavell. 1994. Class II transactivator (CIITA) is sufficient for the inducible expression of major histocompatibility complex class II genes. J. Exp. Med. 180: 1367-1374. [Abstract/Free Full Text]
  44. Schnappauf, F., S. B. Hake, M. M. Camacho Carvajal, S. Bontron, B. Lisowska-Grospierre, V. Steimle. 2003. N-terminal destruction signals lead to rapid degradation of the major histocompatibility complex class II transactivator CIITA. Eur. J. Immunol. 33: 2337-2347. [Medline]
  45. Tjelle, T. E., A. Brech, L. K. Juvet, G. Griffiths, T. Berg. 1996. Isolation and characterization of early endosomes, late endosomes and terminal lysosomes: their role in protein degradation. J. Cell Sci. 109: 2905-2914. [Abstract]
  46. Yuan, L. W., J. W. Soh, I. B. Weinstein. 2002. Inhibition of histone acetyltransferase function of p300 by PKC{delta}. Biochim. Biophys. Acta 1592: 205-211. [Medline]
  47. Xie, H., T. L. Rothstein. 1995. Protein kinase C mediates activation of nuclear cAMP response element-binding protein (CREB) in B lymphocytes stimulated through surface Ig. J. Immunol. 154: 1717-1723. [Abstract]
  48. Mayr, B. M., G. Canettieri, M. R. Montminy. 2001. Distinct effects of cAMP and mitogenic signals on CREB-binding protein recruitment impart specificity to target gene activation via CREB. Proc. Natl. Acad. Sci. USA 98: 10936-10941. [Abstract/Free Full Text]
  49. Miyamoto, A., K. Nakayama, H. Imaki, S. Hirose, Y. Jiang, M. Abe, T. Tsukiyama, H. Nagahama, S. Ohno, S. Hatakeyama, K. I. Nakayama. 2002. Increased proliferation of B cells and auto-immunity in mice lacking protein kinase C{delta}. Nature 416: 865-869. [Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
S. W. Wong, M.-J. Kwon, A. M. K. Choi, H.-P. Kim, K. Nakahira, and D. H. Hwang
Fatty Acids Modulate Toll-like Receptor 4 Activation through Regulation of Receptor Dimerization and Recruitment into Lipid Rafts in a Reactive Oxygen Species-dependent Manner
J. Biol. Chem., October 2, 2009; 284(40): 27384 - 27392.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. Sebastian, M. Serra, A. Yeramian, N. Serrat, J. Lloberas, and A. Celada
Deacetylase Activity Is Required for STAT5-Dependent GM-CSF Functional Activity in Macrophages and Differentiation to Dendritic Cells
J. Immunol., May 1, 2008; 180(9): 5898 - 5906.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. N. Voong, A. R. Slater, S. Kratovac, and D. E. Cressman
Mitogen-activated Protein Kinase ERK1/2 Regulates the Class II Transactivator
J. Biol. Chem., April 4, 2008; 283(14): 9031 - 9039.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kwon, M.-J.
Right arrow Articles by Chang, C.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kwon, M.-J.
Right arrow Articles by Chang, C.-H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS