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The Journal of Immunology, 2003, 171: 3636-3644.
Copyright © 2003 by The American Association of Immunologists

Protein Kinase A Regulatory Subunit Type II{beta} Directly Interacts with and Suppresses CREB Transcriptional Activity in Activated T Cells1

Michael R. Elliott*, Mate Tolnay{ddagger}, George C. Tsokos{ddagger} and Gary M. Kammer2,*,{dagger}

* Department of Microbiology and Immunology and {dagger} Section on Rheumatology and Clinical Immunology, Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157; and {ddagger} Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Levels of the type II{beta} regulatory subunit (RII{beta}) of protein kinase A are abnormally high in the nuclei of T cells of some subjects with the autoimmune disorder systemic lupus erythematosus (SLE). However, the role of nuclear RII{beta} in the regulation of T cell function is unknown. Based on previous studies demonstrating that nuclear protein kinase A-RII subunits can modify cAMP response element (CRE)-dependent transcription, we tested the hypothesis that nuclear RII{beta} can alter CRE-directed gene expression in T cells through interaction with the nuclear transcription factor CRE-binding protein CREB. To test this hypothesis, we used the RII{beta}-deficient S49 and the Jurkat T cell lines. In both cell lines, transient transfection of RII{beta} resulted in nuclear localization of a portion of the ectopically expressed RII{beta}. In vitro and in vivo analyses revealed a novel, specific interaction between RII{beta} and CREB that mapped to the N-terminal 135 aa of RII{beta}. In functional studies, RII{beta} inhibited the transcriptional activity of a GAL4-CREB fusion protein by 67% in Jurkat T cells following activation with anti-CD3 and anti-CD28 mAbs. Importantly, deletion of the CREB-binding region of RII{beta} completely abrogated inhibition. Additionally, RII{beta} suppressed CRE-directed reporter gene expression and substantially reduced induction of promoter activity and endogenous protein levels of the CREB-dependent gene, c-fos, in activated T cells. We conclude that nuclear RII{beta} can act as a repressor of CREB transcriptional activity in T cells, providing a potential functional significance for aberrant levels of nuclear RII{beta} in systemic lupus erythematosus T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE)3 is an idiopathic autoimmune disease distinguished by abnormal T lymphocyte effector functions that contribute to impaired cellular immunity. In a proportion of subjects with SLE, a deficiency of T cell protein kinase A (PKA) activity has been identified (1). This deficiency is a component of an overall signaling disorder that may contribute to altered T cell helper and cytotoxic functions (2). Interestingly, the mechanisms underlying deficient activity of each PKA isozyme in SLE differ. Reduced type I PKA (PKA-I) activity is estimated to occur in the T cells of ~80% of SLE subjects (3) and is the result of reduced amounts of type I regulatory (RI) isoforms (4). Deficient PKA-II activity, which is estimated to affect the T cells of ~37% of SLE subjects, is associated with a significant increase in the amount of the {beta} isoform of RII (RII{beta}) in the nucleus compared with controls (5). By contrast, RII{alpha} is not found in the nucleus of normal and SLE T cells (5). Although accumulation of nuclear RII{beta} causes a relative deficiency of PKA-II activity in the cytosol, it is unclear how nuclear RII{beta} affects events in the nucleus such as gene transcription.

PKA is a serine/threonine kinase comprised of two isozymes, PKA-I and PKA-II (6, 7, 8). In its holoenzyme form, each isozyme exists as a tetramer of two R and two C subunits. There are four unique R subunit isoforms (RI{alpha}, RI{beta}, RII{alpha}, and RII{beta}) and three C subunit isoforms (C{alpha}, C{beta}, and C{gamma}), each products of distinct genes (6). PKA isozymes are activated by the binding of cAMP to the A and B binding sites located in the carboxyl two-thirds of the R subunits. Occupancy of R subunits by cAMP results in rapid dissociation of the holoenzyme, allowing the catalytically active C subunit to phosphorylate substrates (6, 7, 8). In human T cells, the PKA-I isozyme is localized predominantly in the plasma membrane (9), whereas the PKA-II isozyme is targeted to specific cytosolic and nuclear structures through interactions with A kinase-anchoring proteins (AKAPs) (10, 11).

Although the PKA-II isozyme is chiefly cytosolic, RII{alpha} and RII{beta} subunits can enter the nucleus as a component of normal cellular processes or in response to specific stimuli (12, 13, 14, 15). Also, a number of transformed cell lines have been shown to support nuclear localization of ectopically expressed RII{beta} (13, 16). Both RII{alpha} and RII{beta} subunits contain four positively charged amino acids (KKRK) at their carboxyl termini that are homologous to the SV40 large T Ag nuclear localization sequence (17). Mutation of this sequence blocks nuclear accumulation of RII{beta} in NIH-3T3 cells (16). In addition, mutation of the autophosphorylation site at Ser114 prevents RII{beta} from transiting to the nucleus (16). However, the molecular processes that direct RII{beta} nuclear translocation are as yet undefined.

Although the function of nuclear RII{beta} remains largely unexplored, there is evidence that it is able to modify cAMP response element (CRE)-dependent gene transcription (13, 18). In one report, RII{beta} was shown to bind directly to a consensus CRE in vitro, forming a higher order complex with the CRE-binding protein (CREB) (18). In this study, overexpression of RII{beta} in NIH-3T3 cells induced activation of a reporter gene under control of the somatostatin CRE. Moreover, when fused to the GAL4 DNA-binding domain, RII{beta} was able to directly stimulate reporter gene transcription (18). However, this study did not examine the possibility that RII{beta} and CREB may be interacting directly and that the observed transactivation by RII{beta} may be mediated via interaction with CREB.

CREB is a 43-kDa transcription factor found exclusively in the nucleus. CREB binds to the CRE typically as a homodimer, but is transcriptionally inactive until phosphorylated at Ser133 by any one of several protein kinases, including PKA, Ca2+/calmodulin-dependent protein kinase IV, and p38 mitogen-activated protein kinase (reviewed in Refs. 19, 20). Phosphorylation of Ser133 promotes recruitment of the coactivator CREB-binding protein (CBP) or its paralog p300. CBP/p300 mediates gene transcription via its association with the basal transcription machinery of the RNA polymerase II holoenzyme complex (21) and its intrinsic histone acetyltransferase activity (22). However, Ser133 phosphorylation is necessary but not sufficient to mediate CREB transactivation. Indeed, a number of signaling cascades lead to Ser133 phosphorylation without inducing CREB-dependent transcription (19). Although the mechanism of signal discrimination by CREB is unknown, it has been proposed that one or more cofactors may bind CREB and prevent activation in the absence of an appropriate stimulus (19).

The identification of the constitutive RII{beta} subunit in the nucleus of SLE T cells raised the possibility that the subunit could mediate a regulatory effect on gene expression by either binding directly to DNA or by interaction with a transcription factor. In this study, we investigated the hypothesis that the RII{beta} -subunit interacts with CREB in the nucleus of T cells and that this interaction modifies CREB-mediated gene transcription. We show that RII{beta} binds CREB in vitro and in the nucleus of T cells via its amino-terminal region. The binding of RII{beta} to CREB leads to inhibition of CREB transcriptional activity and CRE-dependent transcription in T cells stimulated via the TCR-CD3 complex and CD28 coreceptor. These results suggest that the nuclear RII{beta} subunit functions as a repressor of CREB-dependent transcription in T cells and implicates nuclear RII{beta} as a potential negative regulator of T cell activation. Because nuclear RII{beta} levels are constitutively high in the T cells of some SLE subjects, binding of RII{beta} to CREB could contribute to the T cell effector dysfunctions seen in these individuals by modifying the physiologic expression of genes regulated by CREB.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and Abs

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. Anti-RII{beta}, anti-CD3 (clone UCHT1), and anti-CD28 (clone 28.2) mAbs were purchased from BD Transduction Laboratories (Lexington, KY). Anti-phospho-Ser133 CREB (pCREB), anti-CREB, and anti-c-Fos rabbit polyclonal Abs were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-CREB mAb was purchased from Zymed Laboratories (South San Francisco, CA). HRP-conjugated secondary Abs were purchased from Cell Signaling Technologies (Beverly, MA). Anti-FLAG-HRP and anti-{beta}-actin Abs were purchased from Sigma-Aldrich.

Plasmids

OT-RII{beta}, OT-RII{alpha}, and OT-RI{alpha} were generously provided by Dr. Y. S. Cho-Chung (National Institutes of Health, Bethesda, MD). CMV-RII{beta} was generated by subcloning the full-length RII{beta} cDNA sequence from OT-RII{beta} into the EcoRI site of CMV-Bam (23) using standard subcloning procedures (24).

DNA fragments for FLAG fusion constructs were generated by PCR from plasmid templates using PfuTurbo DNA polymerase (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. The primers used for PCR were engineered to allow KpnI and XbaI restriction sites to be incorporated into the 5' and 3' ends of the amplified fragments, respectively. The 3' primers were designed to allow fusion of the C terminus of the target coding sequence in-frame with the 3xFLAG epitope. PCR products were purified using a QIAquick PCR Purification kit (Qiagen, Valencia, CA) according to manufacturer’s instructions and were subsequently digested with KpnI and XbaI (New England Biolabs, Beverly, MA). Fragments were then purified by agarose gel electrophoresis using the QIAquick Gel Purification kit (Qiagen) according to the manufacturer’s instructions and were subsequently ligated into the KpnI to XbaI sites of the CMV-3xFLAG-14 vector (Sigma-Aldrich). GST fusion constructs were generated in a similar fashion, except EcoRI restriction sites were engineered into the 5' and 3' ends of the PCR-amplified coding sequences and subsequently ligated into the EcoRI site of pGEX-3X (Amersham Pharmacia Biotech, Piscataway, NJ). The 5' primers were engineered to permit fusion of the N terminus of the target coding sequence in-frame with the GST moiety. The amino acids encoded by each RII{beta} deletion construct are indicated in Fig. 4B. The fidelity of all PCR-generated constructs was verified by automated DNA sequencing.



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  		FIGURE 4. Mapping of the CREB-binding domain of RII{beta} subunit. A, Schematic of the major functional domains of the RII{beta} subunit. Amino acids that border the described domains are indicated at the bottom of the illustration. B, Schematic of the RII{beta} deletion mutant constructs used in this study. All RII{beta} coding sequences were fused at the N terminus to GST. The amino acids of RII{beta} present in each construct are denoted in superscript. C, Equal amounts of S49 WCE were incubated with 10 µg of GST protein indicated above the figure. Following GST pull-down, CREB association was detected by SDS-PAGE and immunoblotting using anti-CREB. Input is 10% of the total S49 WCE used in each sample.

 
The GAL4-CREB plasmid was generously provided by Dr. M. Montminy (Salk Institute for Biological Studies, La Jolla, CA). The His6-CREB plasmid was generously provided by Dr. C. McMurray (Mayo Clinic and Foundation, Rochester, MN). 4xCRE-Luc and FR-luciferase reporters were purchased from Stratagene. The TK-RL plasmid was purchased from Promega (Madison, WI). The -360c-fos-luciferase plasmid containing the luciferase reporter gene under control of the promoter region from -360 to + 16 upstream of the transcription start site of the human c-fos gene was generously provided by Dr. G. Stark (Cleveland Clinic Foundation, Cleveland, OH) (25).

Recombinant proteins

GST-RII{beta} and GST-RII{beta} deletion mutant plasmids were transformed into BL21(DE3) Rosetta Escherichia coli (Novagen, Madison, WI), grown until culture OD550 = 0.6–0.8, and induced with 750 µM isopropyl {beta}-D-1-thiogalactopyranoside for 4 h at 37°C. Cells were lysed in BugBuster reagent (Novagen) and affinity purified using GST-Bind Resin (Novagen) according to the manufacturer’s instructions. Purified proteins were dialyzed into 20 mM HEPES (pH 7.0), 100 mM KCl, 1 mM DTT, and 20% glycerol. 6x histidine-tagged CREB protein was synthesized and purified as previously described (26). GST-RII{alpha} protein was generously provided by Dr. K. Taskén (University of Oslo, Oslo, Norway).

Cell culture and transfection

The BALB/c-derived S49 murine thymoma cell line was obtained from American Type Culture Collection (ATCC TIB-28 Manassas, VA) and maintained at a density of 2 x 105 to 0.2–1.0 x 106 cells/ml in DMEM supplemented with 10% heat-inactivated horse serum (HyClone, Logan, UT), 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 mM HEPES (pH 7.4). The E6-1 clone of Jurkat cells was maintained at a density of 0.5–2 x 106 cells/ml in RPMI 1640 supplemented with 10% heat-inactivated FCS (HyClone), 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 mM HEPES (pH 7.4).

Cells were seeded at 5 x 105/ml in fresh medium 24 h before transfection. Transient transfections were performed by electroporation using the Gene Pulser II (Bio-Rad, Hercules, CA). For S49 cells, 2 x 107 cells were collected and washed once in DMEM. The amount of plasmid DNA indicated in each figure was added to cells in a total volume of 0.4 ml of DMEM. Electroporation of cells was performed in 0.4-cm cuvettes at 300 V and 825 µF. Jurkat cells were transfected similarly, except 1 x 107 cells per transfection were mixed with the amount of DNA indicated in each figure in a total volume of 0.4 ml of RPMI 1640 medium and electroporated at 300 V and 975 µF. Immediately following electroporation, cells were transferred to 10 ml of prewarmed complete medium and incubated at 37°C/5% CO2 for 20–48 h before use in experiments. Transfection efficiency was determined by flow cytometric quantitation of enhanced green fluorescent protein (EGFP)-positive cells 24 h after being transfected with 25 µg of EGFP-C1 plasmid (Clontech Laboratories, Palo Alto, CA). Percentage of transfected cells was routinely 30 and 50% for S49 and Jurkat, respectively (data not shown).

Jurkat T cells were stably transfected with FLAG or RII{beta}-FLAG plasmids by electroporation with 30 µg of plasmid DNA. Forty-eight hours posttransfection, cells were cultured in 1 mg/ml geneticin (Invitrogen, San Diego, CA) for 2–3 wk. Cells were subcloned by limiting dilution in medium containing 0.5 mg/ml geneticin, and RII{beta}-FLAG subclones were screened for expression by immunoblotting with anti-FLAG. FLAG subclones were obtained by picking geneticin-resistant colonies.

Cell fractionation

For isolation of nuclear and cytoplasmic fractions, 1 x 107 cells were washed twice in PBS and resuspended in 400 µl of buffer A (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5 mM DTT, and 1x protease inhibitor mixture I; Calbiochem, La Jolla, CA) and incubated on ice for 15 min. After the addition of 20 µl of 10% Nonidet P-40, cells were vortexed for 10 s and incubated on ice for 15 min. Nuclei were pelleted at 4000 x g for 30 s at 4°C. The supernatant containing the cytoplasmic fraction was withdrawn and stored at -70°C. The nuclear pellet was then washed once in buffer A to remove any residual cytoplasmic fraction. Nuclear proteins were then extracted from the pellet in 50 µl of buffer C (20 mM HEPES (pH 7.9), 400 mM NaCl, 0.5 µM EDTA, 1 mM EGTA, 1 mM DTT, and 1x protease inhibitor mixture I) for 1 h at 4°C on a rocking platform, followed by centrifugation at 16,000 x g for 10 min at 4°C. The supernatant containing the nuclear extract was used immediately or stored at -70°C.

GST pull-down and immunoprecipitations

Whole cell extracts (WCE) were prepared by lysing 2 x 107 cells in 1 ml of IPC150 buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA, 1 mM DTT, and 1x protease inhibitor mixture I) on ice for 30 min followed by centrifugation at 16,000 x g for 20 min at 4°C. For GST pull-downs using WCE, 500-1000 µg of cleared WCE was mixed with 10 µg of GST protein and rocked gently overnight at 4°C. For GST pull-downs using recombinant His6-CREB, 100 ng of GST protein was incubated with 100 ng of purified His-CREB protein in 50 mM KCl, 20 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM DTT, and 350 µg/ml BSA for 2 h at 4°C. GST proteins were precipitated from binding reactions using a 25-µl bed volume of GST-Bind agarose beads (Novagen) for 2 h at 4°C, followed by three 1-ml washes in 1x Bind/Wash buffer (Novagen). Wash buffer for binding reactions using recombinant proteins also included 1% sodium deoxycholic acid, 0.5% Triton X-100, and 0.1% SDS. Washed beads were boiled for 10 min in SDS sample buffer, and the supernatant was subjected to SDS-PAGE and immunoblotting.

For CREB immunoprecipitation, cytoplasmic and nuclear extracts were prepared from 1 x 107 cells and precleared with 10 µg of normal rabbit IgG (Upstate Biotechnology) followed by incubation with 10 µg of anti-CREB (Upstate Biotechnology) overnight at 4°C. Immune complexes were precipitated with a 25-µl bed volume of protein A-agarose beads (Sigma-Aldrich) for 2 h at 4°C, followed by three 1-ml washes in IPC150. Beads were boiled in SDS sample buffer and subjected to SDS-PAGE and immunoblotting.

T cell activation and luciferase assays

Wells of a 24-well plate were coated with 400 µl of PBS containing 10 µg/ml anti-CD3 and incubated overnight at 4°C. Twenty to 24 h posttransfection, 2.5–5 x 106 Jurkat cells were collected, plated in anti-CD3-coated wells, and treated with a final concentration of 3 µM forskolin and 2 µg/ml anti-CD28 Ab. The plate was then centrifuged at 250 x g for 5 min to settle the cells on the bottom of the plate. Cells were incubated at 37°C/5% CO2 for 6 h before luciferase activity determination.

Luciferase activity was quantified from cell extracts using the dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions. Briefly, following treatment, cells were pelleted by centrifugation and washed once in PBS, pellets were lysed in 200 µl of 1x passive lysis buffer, and incubated for 10 min at room temperature. Lysates were cleared by centrifugation at 16,000 x g for 5 min at room temperature. Twenty microliters of the cleared lysate was used in the dual-luciferase reporter assay. Samples were normalized for transfection efficiency by inclusion of 2 µg of TK-RL control plasmid in each transfection and measurement of Renilla luciferase activity.

SDS-PAGE and immunoblotting

Immunoblotting was performed as previously described (27). Briefly, proteins separated by 10% SDS-PAGE were transferred to Immobilon-P (Millipore, Bedford MA) for 1 h at 100 V. All primary Ab incubations were conducted overnight at 4°C, followed by incubation with appropriate secondary HRP-conjugated Abs for 2 h at room temperature. Immunoblots were developed using Western Lightning ECL reagent (PerkinElmer Life Sciences, Boston, MA).

Levels of CREB and pCREB were determined by lysing equivalent numbers of cells in SDS sample buffer and sonication for a total of 10 pulses at 1 s/pulse (Sonifier 250; Branson Products, Poway, CA). After boiling lysates for 10 min, an equal volume of lysate from each sample was analyzed by 10% SDS-PAGE and immunoblotting with anti-pCREB. Blots were stripped (200 mM glycine, pH 2.0) and reprobed with anti-CREB.

For determination of c-Fos protein levels, cells were washed once in PBS and cultured overnight in serum-free medium. Equivalent numbers of cells were stimulated for 3 h as indicated in the legend for Fig. 8. Cells were then washed in PBS and lysed in 400 µl of SDS sample buffer, sonicated for ten 1-s pulses, and boiled for 5 min. Equivalent volumes of lysate were analyzed by SDS-PAGE and immunoblotting with anti-c-Fos.



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  		FIGURE 8. RII{beta} inhibits c-Fos protein induction. Jurkat cells stably transfected with FLAG (lanes 1–3) or RII{beta}-FLAG (lanes 4–6) were stimulated for 3 h with anti-CD3, anti-CD28, and 3 µM forskolin (lanes 2 and 5), 50 ng/ml PMA, 1 µM ionomycin, anti-CD28, and 3 µM forskolin (lanes 3 and 6), or vehicle (lanes 1 and 4). Equivalent amounts of lysate were analyzed by SDS-PAGE and immunoblotting with anti-c-Fos, anti-FLAG, and anti-{beta}-actin.

 
Confocal immunofluorescence microscopy

Compartmentalization of the RII{beta} subunit in S49 cells was assessed by laser-scanning confocal immunofluorescence microscopy using methods previously described (5).

Statistical analysis

Statistical significance (p <= 0.05) was calculated using the paired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ectopically expressed RII{beta} localizes to the nucleus of T cells

To study the nuclear RII{beta} subunit in T cells, we used the PKA-II deficient, BALB/c-derived S49 and human Jurkat T cell lines. S49 cells express negligible levels of RII{beta} protein compared with BALB/c splenic T cells (Fig. 1A). Transient transfection of an RII{beta} expression plasmid restored RII{beta} expression (Fig. 1A). Immunoblot analysis and confocal immunofluorescence microscopy of RII{beta}-transfected S49 cells demonstrated that a substantial proportion (~30%) of the ectopically expressed RII{beta} protein localized to the nucleus, whereas the remainder was cytosolic (Fig. 1, B and C). We also examined RII{beta} localization in Jurkat cells, which express all four R subunit isoforms at levels similar to those seen in primary human T cells (data not shown). Overexpression of RII{beta} by transient transfection led to accumulation of RII{beta} in the nucleus of Jurkat cells similar to that seen in S49 cells (data not shown). The ability to assay cellular functions in the absence or presence of RII{beta} as well as the nuclear accumulation of RII{beta} in transfected S49 and Jurkat cells made these cell types suitable for examining the role of nuclear RII{beta} in T cells.



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  		FIGURE 1. Ectopically expressed RII{beta} localizes to the nucleus of RII{beta}-deficient S49 T cells. A, S49 cells were transfected with no plasmid DNA (mock), 20 µg of empty CMV-Bam vector (vector), or 20 µg of CMV-RII{beta} (RII{beta}). Twenty-four hours posttransfection, 20 µg of WCE was separated by SDS-PAGE and analyzed by immunoblotting with anti-RII{beta}. Twenty micrograms of WCE from CD3+ cells purified from BALB/c splenocytes (T cell) was included to show wild-type RII{beta} expression. B, S49 cells were transfected with 20 µg of RII{beta}-FLAG. Twenty-four hours posttransfection, nuclear (Nuc.) and cytoplasmic (Cyto.) fractions were prepared and 10 µg of each was analyzed by immunoblotting using anti-FLAG (RII{beta}). The blot was reprobed with Abs to CREB and {beta}-actin to determine the quality of the cytoplasmic and nuclear fractions, respectively. C, S49 cells were transfected with 20 µg of RII{beta}-FLAG. Twenty-four hours posttransfection, cells were immunostained with anti-FLAG (RII{beta}, green) and treated with propidium iodide (PI, red) to visualize the nucleus. Photomicrographs are a 1-µm-thick planar view through the center of the cells obtained by confocal immunofluorescence microscopy. Of the three cells shown, only the middle cell is transfected. The lower right photomicrograph is an overlay of propidium iodide and RII{beta} staining (PI + RII{beta}). The lower left photomicrograph shows the same field under differential interference contrast (DIC) microscopy.

 
RII{beta} binds CREB in vitro and in vivo

Previous analyses of nuclear lysates from nonhemopoietic cells using gel shift assays suggested that the RII{beta} subunit can bind a consensus CRE in vitro, forming a higher order complex with CRE-binding protein (CREB) (18). Because our analyses revealed that RII{beta} did not bind to a c-fos-specific CRE oligonucleotide by EMSA (data not shown), we considered the possibility that RII{beta} may be interacting directly with CREB. As an initial approach, we determined whether RII{beta} could bind CREB in pull-down assays using bacterially expressed, purified GST-RII{beta} incubated with cell extracts from several T cell lines. GST-RII{beta} pulled down CREB from Jurkat and S49 cells whereas GST alone did not bind CREB (Fig. 2A). GST-RII{beta} also specifically pulled down CREB from WCE of primary human T cells and the Raji B cell line (data not shown). To determine whether the interaction between RII{beta} and CREB is direct or dependent on other factors, binding assays were performed using bacterially expressed, purified histidine-tagged CREB and GST-RII{beta} or GST-RII{alpha}. GST-RII{beta}, but not GST-RII{alpha}, precipitated CREB in the absence of other eukaryotic factors (Fig. 2B). These results demonstrate that RII{beta} can interact directly with CREB; however, we cannot exclude the possibility that optimal interaction between RII{beta} and CREB is dependent on the formation of a multimeric complex with other cellular cofactors.



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  		FIGURE 2. RII{beta} binds CREB in vitro. A, Equivalent amounts of GST or GST-RII{beta} were used to pull-down proteins from lysates of Jurkat and S49 T cells. Resulting precipitates were separated by SDS-PAGE and immunoblotted with anti-CREB. Input lanes show 10% of input lysate used in each pull-down. B, One hundred nanograms of purified His6-CREB protein was incubated with equivalent amounts of GST, GST-RII{alpha}, or GST-RII{beta} recombinant protein and analyzed for interaction by GST pull-down followed by SDS-PAGE and immunoblotting using anti-CREB. Input lanes contain 10% of CREB used in each reaction.

 
Although we could show that RII{beta} interacts with CREB in vitro and in pull-down assays using T cell extracts, it was important to determine whether CREB and RII{beta} interact in vivo. To investigate this question, coimmunoprecipitation experiments were performed using S49 and Jurkat cells transfected with a 3xFLAG epitope-tagged RII{beta} expression plasmid (RII{beta}-FLAG). RII{beta}-FLAG, but not RI{alpha}-FLAG, coimmunoprecipitated with endogenous CREB in S49 cells (Fig. 3A). This interaction took place in the nucleus, as RII{beta}-FLAG coimmunoprecipitated with CREB from the nuclear fraction of S49 cells but not from the cytosolic fraction (Fig. 3B). We also observed coimmunoprecipitation of endogenous CREB and RII{beta}-FLAG in Jurkat T cells (Fig. 3C). These results demonstrate that RII{beta} interacts specifically with CREB in the nucleus of Jurkat and S49 T cells.



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  		FIGURE 3. RII{beta} interacts with CREB in the nucleus of T cells. A, S49 cells were transfected with 20 µg of RI{alpha}-FLAG (lane 1) or RII{beta}-FLAG (lane 2) expression plasmids. Forty hours posttransfection, WCE were prepared and immunoprecipitated (IP) with anti-CREB. Resulting complexes were examined by SDS-PAGE and immunoblotting with anti-FLAG-HRP Ab. Upper blot shows results of immunoprecipitation. Lower blot shows 10% of input WCE from each immunoprecipitate. B, S49 cells were transfected with RII{beta}-FLAG as in A. Nuclear (Nuc.) and cytoplasmic (Cyto.) extracts were prepared as described in Materials and Methods. Eighty micrograms of nuclear extract or 200 µg of cytoplasmic extract was immunoprecipitated with anti-CREB. Immunoprecipitates were analyzed by SDS-PAGE, followed by immunoblotting with anti-FLAG-HRP (upper blot). Lower blot shows 10% of the input from each fraction analyzed by SDS-PAGE and immunoblotting with anti-FLAG-HRP. C, Jurkat T cells were transfected with 20 µg of RII{beta}-FLAG plasmid. Forty hours after transfection, WCE were prepared and IP with anti-CREB ({alpha}-CREB) or normal rabbit IgG (rIgG). Resulting complexes were examined by SDS-PAGE and immunoblotting with anti-FLAG-HRP Ab. Input indicates 10% of WCE used in immunoprecipitation (IP).

 
CREB-binding region of RII{beta} maps to the N terminus

To identify the region of RII{beta} that mediates binding to CREB, a series of RII{beta} deletion mutant-GST fusion proteins were tested for their ability to bind CREB from S49 extracts. The domains of RII{beta} that contain the sites for cAMP binding, C subunit interaction, and AKAP binding are shown schematically in Fig. 4A. The positions of the truncations were designed so that previously characterized domains of RII{beta} would remain intact, thus allowing us to assess the functionality of the truncated proteins (Fig. 4B). The domains for AKAP interaction and C subunit binding were determined to be functional in the purified deletion mutants by GST pull-down of Jurkat extracts and immunoblotting for AKAP79 and C subunit (data not shown). The RII{beta} dimerization domain was assumed to be functional as well because AKAP binding requires RII dimerization (28).

Full-length RII{beta} and a fragment containing aa 1–135 of RII{beta} (RII{beta}1–135) precipitated CREB comparably (Fig. 4C, lanes 3 and 5). Accordingly, deletion of residues 1–134 (RII{beta}135–418) completely abrogated CREB binding, indicating that aa 1–134 alone are sufficient to mediate CREB interaction (Fig. 4C, lane 7). Interestingly, deletion of the first 49 residues of RII{beta} (RII{beta}50–418) caused a substantial decrease in binding over that seen with RII{beta}1–135 (Fig. 4C, compare lane 6 to 5), while RII{beta}1–50 failed to bind CREB completely (Fig. 4C, lane 4). Importantly, the RII{beta}50–418 protein was able to bind the C subunit at similar levels as wild-type RII{beta}, indicating that the decrease in CREB binding by RII{beta}50–418 was not caused by protein misfolding (data not shown). From these data we conclude that aa 50–134 of RII{beta} are absolutely required for binding to CREB and that RII{beta} dimerization and AKAP-binding domains at residues 1–50 alone are not sufficient to mediate CREB interaction. However, the reduced binding of RII{beta}50–418 compared with RII{beta}1–135 suggests that residues 1–50 may be critical for optimal CREB interaction.

RII{beta} inhibits CREB transcriptional activity following T cell activation

Transcriptional activation by CREB is required for an efficient T cell response (29). Activation of T cells through the TCR-CD3 complex induces CREB phosphorylation at Ser133 through multiple kinase pathways. However, Ser133 phosphorylation is necessary but not sufficient to cause recruitment of CBP and subsequent CRE-dependent gene transcription. In Jurkat T cells, for example, it has been shown that a suboptimal dose of the cAMP-elevating agent, forskolin, is required to induce CREB transcriptional activity (30).

To quantify the transcriptional activity of CREB in response to TCR activation, Jurkat T cells were transiently transfected with a GAL4-CREB chimeric expression construct encoding full-length CREB fused to the GAL4 DNA-binding domain along with the FR-Luc reporter plasmid containing the luciferase gene downstream of a 5xGAL4 response element. As expected, treatment for 6 h with anti-CD3 or anti-CD28 singly or together failed to activate CREB (Fig. 5A). However, the combination of 3 µM forskolin and anti-CD28 or anti-CD3 increased luciferase activity by 8- and 20-fold over vehicle-treated cells, respectively, while this dose of forskolin alone produced a <4-fold increase in activity (Fig. 5A). When cells were treated with anti-CD3, anti-CD28, and forskolin together, the additive effect yielded a 24-fold increase in luciferase activity over control-treated cells (Fig. 5A). These data verify that sufficient levels of cAMP are required for CREB activation with anti-CD3 in Jurkat cells and also demonstrate that cAMP is required for early activation of CREB in response to signaling via the CD28 coreceptor.



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  		FIGURE 5. RII{beta} inhibits GAL4-CREB activity stimulated in activated T cells. A, Jurkat cells were transfected with 10 µg of GAL4-CREB and 10 µg of FR-luciferase reporter plasmid. Twenty-four hours posttransfection, cells were stimulated with 3 µM forskolin (Fsk), anti-CD3, and anti-CD28 alone or in combination, as indicated, for 6 h and luciferase activity was determined. B, Jurkat cells were transfected with 10 µg of GAL4-CREB expression plasmid, 10 µg of FR-luciferase reporter plasmid, and 7.5 µg of either FLAG, RII{beta}-FLAG (RII{beta}), or RII{beta}135–418-FLAG (RII{beta}135–418) expression plasmids. Cells were stimulated for 6 h with 3 µM forskolin, anti-CD3, and anti-CD28 (Stim.) or left untreated (Con.) and luciferase activity was determined. GAL4-CREB-driven luciferase activity was normalized to Renilla luciferase activity for each sample. Results shown are the average of two (A) or three (B) experiments ± SD. C, Relative expression of FLAG-tagged proteins from transfected cells in B determined by SDS-PAGE of 20 µg of WCE from stimulated cells in B followed by immunoblotting with anti-FLAG-HRP (upper panel). Blot from upper panel was reprobed with anti-{beta}-actin to control for protein loading (lower panel). Relative molecular weights in thousands are shown to the left of the panel. One of three representative experiments is shown.

 
Because we were interested in understanding the effect of the RII{beta} interaction on CREB activity in the context of an immunological response, we assessed the ability of GAL4-CREB to activate transcription of the luciferase reporter gene in the presence or absence of RII{beta} following T cell activation. Jurkat cells were transiently cotransfected with the GAL4-CREB fusion construct plus a FLAG expression vector encoding either full-length RII{beta}, an RII{beta} deletion mutant missing aa 1–134 (RII{beta}135–418), or the empty vector (FLAG). The RII{beta}135–418 construct was used to assess the role of the CREB-binding domain of RII{beta} on CREB transcriptional activity. Overall, the addition of the FLAG expression constructs in Fig. 5B resulted in a decrease in total GAL4-CREB activity stimulated by anti-CD3 and anti-CD28 compared with the results in Fig. 5A. Still, a 12-fold increase in CREB activity was observed in anti-CD3/anti-CD28/forskolin-treated cells compared with control-treated cells (Fig. 5B). Transfection of the full-length RII{beta} expression plasmid caused a 67% decrease (p = 0.0002) in luciferase activity following stimulation (Fig. 5B). By comparison, the RII{beta}135–418 deletion mutant inhibited GAL4-CREB activity by 14% (p = 0.003; Fig. 5B). Therefore, the N-terminal 134 aa of RII{beta}, which alone can bind CREB, are required for RII{beta} to inhibit CREB transcriptional activity substantially.

RII{beta} expression does not inhibit CREB Ser133 phosphorylation

The transcriptional activity of CREB is dependent on Ser133 phosphorylation. Thus, we considered the possibility that the inhibition of CREB activity by RII{beta} seen in Fig. 5B may be due to a decreased level of Ser133 CREB phosphorylation. To address this possibility, Jurkat cells were transfected with either the empty FLAG, RII{beta}-FLAG, or RII{beta}135–418 plasmids and stimulated as in Fig. 5B. The extent of CREB phosphorylation at Ser133 (pCREB) was determined by immunoblotting using a phospho-Ser133-specific CREB Ab. pCREB and total CREB levels were quantified by densitometry, and the relative level of pCREB was expressed as the ratio of pCREB:CREB for each sample. Stimulation resulted in an increase in pCREB of ~4-fold in FLAG-transfected cells and was not decreased by RII{beta} (Fig. 6, A and B). A modest increase in pCREB levels was observed in the RII{beta}135–418-transfected cells over the FLAG-transfected cells. It is important to note that the efficiency of transfection was determined to be 45–50% as judged by flow cytometry of Jurkat cells transfected with EGFP (data not shown). Thus, a substantial change in pCREB levels caused by the ectopically expressed proteins would be detectable in these experiments. Taken together, these experiments demonstrate that RII{beta} overexpression does not inhibit CREB phosphorylation under the same conditions that promote activation of GAL4-CREB. Thus, the inhibition of CREB activity by RII{beta} shown in Fig. 5B does not appear to be associated with a decrease in CREB Ser133 phosphorylation.



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  		FIGURE 6. RII{beta} does not inhibit Ser133 CREB phosphorylation in Jurkat T cells. A, Jurkat cells were transfected with 7.5 µg of FLAG (lanes 1 and 4), RII{beta}-FLAG (lanes 2 and 5), or RII{beta}135–418-FLAG (lanes 3 and 6) expression plasmid. Twenty-four hours posttransfection, cells were left untreated (Con.) or stimulated with 3 µM foskolin, anti-CD3, and anti-CD28 for 6 h (Stim.). Following treatment, equivalent numbers of cells from each sample were lysed for pCREB analysis, as described in Materials and Methods. Equal volumes of lysate from each sample were analyzed by SDS-PAGE followed by immunoblotting for total CREB (lower panel) and phospho-Ser133-CREB (pCREB, upper panel). B, The level of pCREB for each sample in A was determined by densitometry using the Alpha Innotec Imaging System. Relative levels of pCREB are expressed as the ratio of pCREB:CREB for each sample in arbitrary densitometric units (ADU). One of three representative experiments is shown.

 
RII{beta} inhibits CRE-directed transcription following T cell activation

Because CREB activates transcription of genes containing CRE cis-acting sequences, we examined the effect of RII{beta} on the transcription of a luciferase reporter gene under control of a 4x consensus CRE promoter (4xCRE-Luc). As with GAL4-CREB, a suboptimal dose of forskolin was required to induce CRE-dependent transcription in response to treatment with anti-CD3 and anti-CD28 for 6 h (data not shown and Fig. 7A). Compared with the empty vector control, cotransfection of RII{beta} inhibited 4xCRE-directed luciferase activity by 66% (Fig. 7A). By contrast, the CREB-binding mutant RII{beta}135–418 failed to inhibit CRE promoter activity (Fig. 7A). These data demonstrate that the suppression of CREB transcriptional activity by RII{beta} leads to a loss of transcriptional activity from a functional CRE and that this inhibition is dependent on the CREB-binding domain of RII{beta}.



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  		FIGURE 7. RII{beta} inhibits activity of promoters containing functional CREs. A, Jurkat T cells transfected with 10 µg of 4xCRE-luciferase reporter plasmid and 7.5 µg of FLAG, RII{beta}-FLAG, or RII{beta}135–418-FLAG expression plasmid were left untreated (control (Con.)) or stimulated (Stim.) with 3 µM forskolin, anti-CD3, and anti-CD28 for 6 h. B, Jurkat cells were transiently transfected with 10 µg of -360c-fos-luciferase reporter plasmid and 10 µg of FLAG, RII{beta}-FLAG, or RII{beta}135–418-FLAG expression plasmid. Cells were left untreated or stimulated as in A. 4xCRE- and -360c-fos-driven luciferase activity were normalized to Renilla luciferase activity in each sample. Results shown are the average of three experiments ± SD.

 
Inhibition of c-fos expression by RII{beta}

CREB regulates transcription of the protooncogene c-fos via a CRE located at the -60 position relative to the transcription initiation start site (31). In T cells, CREB activity is critical for the induction of c-fos mRNA following activation (29). To determine whether the inhibition of CREB by RII{beta} would impair c-fos expression, we examined the effect of RII{beta} on induction of c-fos promoter activity and endogenous c-Fos expression. In transient transfection assays, the basal c-fos promoter (-360 to + 16) was inhibited 37% by RII{beta} (p = 0.02), but not the deletion mutant RII{beta}135–418, following activation via the CD3 and CD28 receptors (Fig. 7B). To determine what effect RII{beta} has on the level of endogenous c-Fos protein following T cell activation, Jurkat T cells were stably transfected with the empty FLAG vector or RII{beta}-FLAG. In total, we examined three independently derived FLAG and RII{beta}-FLAG stable lines and found the levels of c-Fos following T cell activation were reduced by 50–90% in the RII{beta}-FLAG compared with the FLAG control lines (Fig. 8 and data not shown). These results indicate that the inhibition of CREB by RII{beta} leads to a decrease in the promoter activity and endogenous expression of a CREB-dependent gene.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our rationale for this study was 2-fold: first, several lines of evidence point to nuclear RII subunits as key regulators of CRE-dependent transcription (13, 18). Second, the significance of increased nuclear RII{beta} in SLE T cells remains unclear (5). We considered the possibility that such aberrant compartmentalization could modify the expression of genes that possess a CRE in their cis-acting regions. In this study, we describe a novel interaction between a PKA R subunit and a mammalian transcription factor. We show that ectopically expressed RII{beta} localizes to the nucleus of Jurkat and RII{beta}-deficient S49 T cells where it binds CREB in the nucleus. In vitro, recombinant RII{beta} binds directly to recombinant CREB in the absence of other cellular factors, and this interaction occurs via the N-terminal 135 aa of RII{beta}. The binding of CREB by RII{beta} subsequently inhibits both GAL4-CREB- and CRE-dependent gene expression stimulated by signaling through the CD3 and CD28 surface receptors of the Jurkat T cell.

Of the three PKA regulatory subunits that we examined (RI{alpha}, RII{alpha}, and RII{beta}), only RII{beta} was able to bind CREB. This observation suggests that the RII{beta} protein contains a unique structural element(s) that mediates CREB interaction. RII{alpha} and RII{beta} are >70% identical between residues 1–40 and 100–418 (404 of RII{alpha}). By contrast, the proteins share only 18% identity between positions 40 and 100. Results from our binding studies with RII{beta} deletion mutants indicate that residues 50–135 are absolutely required for CREB binding and that 1–50, while not able to bind CREB alone, are necessary for optimal RII{beta}-CREB interaction. Considering these data, we conclude that the region of RII{beta} most likely to mediate direct interaction with CREB is contained within aa 50–135. Because residues 1–50 of RII{beta} contain the homodimerization domain, it is conceivable that dimerization may be required for the formation of a stable RII{beta}-CREB complex.

The amino-terminal one-third of the R subunits mediates all known R subunit-protein interactions, including interactions with AKAPs and the PKA C subunit. We investigated a role for the C subunit in the formation of an RII{beta}-CREB complex and found that the C subunit did not coimmunoprecipitate with CREB (data not shown). Moreover, purified RII{beta} was able to bind CREB in vitro in the absence of the C subunit. Taken together, these data support the idea that RII{beta} can bind CREB without forming a holoenzyme. We also did not detect the only known nuclear AKAP, AKAP95, in an anti-CREB immune complex from S49 cells (data not shown). Thus, neither the C subunit nor AKAP95 appear to be components of the RII{beta}-CREB complex. However, it is conceivable that the RII{beta}-CREB complex could interact with an unidentified nuclear AKAP. Finally, CREB itself does not appear to fit the criteria of an AKAP because 1) the PKA holoenzyme is not present and 2) the AKAP-binding domain of RII{beta} does not bind CREB (10).

The binding of RII{beta} to CREB inhibited GAL4-CREB- and CRE-directed gene expression. When Jurkat T cells were activated via the TCR-CD3 complex and CD28, RII{beta} inhibited both GAL4-CREB- and CRE-dependent transcriptional activity by about two-thirds. By comparison, the CREB-binding mutant (RII{beta}135–418) failed to inhibit CREB- and CRE-dependent transcription. Thus, the inhibition of CREB activity was dependent on the CREB-binding domain of RII{beta}. We were unable to examine the effect of RII{beta}1–135 on CREB activity in vivo because the nuclear localization sequence (KKRK262–265) would be lost in this construct, preventing nuclear accumulation. Taken together, these results support the concept that RII{beta} acts as a transcriptional repressor of CREB.

The functional significance of RII{beta}-mediated suppression of CREB activity in T cells was shown by examination of the effect of RII{beta} on the CREB-regulated gene c-fos. c-fos is a member of the bZIP family of proteins that dimerize to form the AP-1 transcription factor. We observed significant inhibition of c-fos basal promoter activity by the wild-type, but not the RII{beta} (135 – 418) mutant, indicating that the RII{beta}-CREB interaction was important for this inhibition. Moreover, Jurkat T cells that stably overexpressed wild-type RII{beta} showed a marked inhibition of c-Fos protein expression following T cell activation. These results indicate that the effect of RII{beta} on CREB activity leads to a loss of endogenous expression of a CREB-dependent gene. Future studies are aimed at determining how the suppression of c-Fos by RII{beta} affects gene regulation by AP-1 as well as the effect of RII{beta} on other CREB-regulated genes and on specific effector functions that are important for immune regulation.

Although we observed an inhibition of CREB- and CRE-dependent transcriptional activity in the two T cell lines we examined, overexpression of RII-subunits increases CRE-directed transcription in some nonhemopoietic cell lines (13, 18). At present, we have not tested the capacity of RII{beta} to interact with CREB or to direct CRE-dependent transcription in any nonlymphoid cell lines. However, although these previous studies evaluated the effect of RII{alpha} and RII{beta} on CRE-dependent promoter activity, they did not examine the effect of RII{beta} specifically on CREB activity. Moreover, the means by which RII subunits were shown to modify CRE activity in these studies varied considerably from what we have described. For example, interaction between RII{alpha} and the adenovirus E1A12S protein in the nucleus correlates with the ability of E1A12S to activate a CRE reporter in KB cells (13). The authors hypothesize that this interaction functions to bring the PKA holoenzyme into close proximity with CRE-binding transcription factors, thus enhancing PKA-mediated activation of these factors. Another study demonstrated that RII{beta} binds to the CRE and activates gene transcription directly (18). Because we have observed neither the C subunit in the RII{beta}-CREB complex nor binding of CRE by RII{beta} in T cells, the inhibition of CREB and CRE activity by RII{beta} may reflect a unique role for RII{beta} in regulating gene expression in T cells.

There are several levels at which RII{beta} binding could inhibit CREB function, including phosphorylation, DNA binding, dimerization, and CBP/p300 recruitment. Our results reveal that RII{beta} does not inhibit Ser133 CREB phosphorylation in response to a signal generated from the TCR-CD3 complex and CD28 coreceptor. Moreover, using EMSA, we found that binding of RII{beta} to CREB did not inhibit CREB binding to the human -60 c-fos CRE. Parenthetically, we also did not detect binding of RII{beta} directly to the -60 c-fos CRE. Thus, at least in vitro, RII{beta} does not appear to affect the dimerization and subsequent DNA binding potential of CREB. However, it remains to be determined whether RII{beta} inhibits the binding of CREB to a consensus CRE in T cells. Another potential mechanism by which RII{beta} binding may inhibit CREB transcriptional activation is by preventing binding to CBP/p300. Although we have not yet determined the region of CREB to which RII{beta} binds, other CREB-binding factors down-regulate CREB activity by preventing the interaction of pCREB with CBP/p300 (32). It remains to be determined how RII{beta} binding to CREB affects recruitment of CBP/p300.

Although it has long been known that RII subunits can translocate to the nucleus, their function in that cellular compartment is still not fully understood. We are particularly interested in the role of RII{beta} in the nucleus of T cells because of its possible contribution to T cell dysfunction in SLE. Because our results have demonstrated that RII{beta} binds CREB in the nucleus of T cells and inhibits its transcriptional activity, it will be of interest to learn whether the high levels of nuclear RII{beta} in the T cells of some SLE subjects lead to a down-regulation of CREB activity. Because CREB modifies the expression of genes that regulate T cell homeostasis and effector functions, abnormal nuclear localization of RII{beta} in SLE T cells may alter the expression of these genes and thereby potentially impair T cell functions. However, it is important to note that abnormal levels of nuclear RII{beta} have only been identified in a subset of SLE patients. It remains to be determined whether abnormal levels of nuclear RII{beta} in this subset is associated with specific T cell dysfunctions unique to those individuals. Also, future studies are aimed at determining the specific mechanisms that direct nuclear localization of RII{beta} in this subset of patients.


   Acknowledgments
 
We thank Y. S. Cho-Chung, Cynthia McMurray, Marc Montminy, and Kjetil Taskén for reagents provided. We also thank Steven Mizel and Ryan Shanks for critical review of this manuscript as well as members of the Kammer laboratory for their support and helpful discussions.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI46526 (to G.M.K.), AI42782, and AI49954 (to G.C.T.). M.R.E. was supported by National Research Training Award Grant AI07401. Back

2 Address correspondence and reprint requests to Dr. Gary M. Kammer, Section on Rheumatology and Clinical Immunology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. E-mail address: gmkammer{at}wfubmc.edu Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; PKA, protein kinase A; RI, type I regulatory subunit; RII, type II regulatory subunit; AKAP, A kinase-anchoring protein; CRE, cAMP response element; CBP, CREB-binding protein; EGFP, enhanced green fluorescent protein; WCE, whole cell extract. Back

Received for publication May 1, 2003. Accepted for publication July 29, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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