From the
Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4965 and the ||Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195
Received for publication, December 19, 2002 , and in revised form, March 4, 2003.
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ABSTRACT |
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INTRODUCTION |
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Protein kinase R (PKR)1 is a 68-kDa serine/threonine kinase that manifests two distinct kinase activities, one for its own phosphorylation and the other for the phosphorylation of other substrates. Once activated, PKR becomes autophosphorylated and acquires the capacity to phosphorylate its well studied substrate, eIF-2. Phosphorylation of eIF-2
inhibits protein synthesis by impairing the activity of guanine nucleotide exchange factor eIF-2B (4). As a serine/threonine kinase found in a latent state in most cells, it plays an important role in cellular antiviral defense. In addition, PKR has been implicated in other cellular processes including apoptosis (5), cellular transformation (6), differentiation (7, 8), splicing (9), and transcription (10). PKR also functions as a signal transducer in signaling pathways activated by different stimuli, including dsRNA, IL-3, interferon-
, interferon-
, IL-1, tumor necrosis factor, and platelet-derived growth factor, through a variety of effectors including NF-
B, IRF-1, ATF-2, STAT3, and eIF-2
(4, 11). Although PKR has been implicated in different cytokine actions, its involvement in IL-11 signaling has not been reported. Meanwhile, the physiological events and signaling pathways that trigger PKR activation by cellular RNA or proteins are not well established (4). Several PKR modulators have been identified. PKR inhibitors include cellular proteins such as P58, HIV-1 transactivating response (TAR) RNA-binding protein (TRBP), ribosomal protein L18, and NF90 and its homologues (1214). On the other hand, other cellular proteins such as PKR activating protein (PACT) and PKR associate protein X (RAX) function as PKR activators (15). Many of these proteins also contain well defined dsRNA-binding domains (DRBDs).
In this study, we identified a novel IL-11-inducible transcript, C114, in 3T3 L1 cells. C114 is a nuclear protein exhibiting higher expression in the nucleolus. C114 interacts with dsRNA and also binds and inhibits the activity of PKR. We propose that this novel cellular protein acts as a negative regulator of PKR.
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MATERIALS AND METHODS |
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PCR-based cDNA SubtractionTotal cellular RNA was isolated from starved or IL-11-stimulated (500 ng/ml) mouse 3T3 L1 cells using TRIzol reagent (Invitrogen), and poly(A) RNA was purified using oligo(dT) beads (Invitrogen). cDNA synthesis and subtraction were done using PCR-Select cDNA subtraction kit (Clontech) following manufacturer's instructions. mRNA of IL-11-treated 3T3 L1 cells was used as a tester, and mRNA of untreated cells was used as a driver. Subtracted PCR products were cloned into pGEM-T Easy vector (Promega) to obtain a subtraction library. DNA fragments were prepared from individual cDNA clones by PCR and used as [-32P]dCTP-labeled hybridization probes for Northern blot analysis. Total RNA extracted from IL-11-treated or untreated 3T3 L1 cells was prepared as mentioned above. RNA was electrophoresed in a 1.0% agarose gel containing formaldehyde and transferred onto a nylon membrane (Hybond N+, Amersham Biosciences). After a cDNA fragment was confirmed by Northern blot to show a differential expression pattern, the full-length cDNA was cloned from a mouse liver cDNA library (Stratagene) by rapid amplification of cDNA ends. Poly(A)+ RNA blot of mouse tissues (Mouse MTN blot, Clontech) was also used for Northern analysis.
Cellular Localization of C114 ProteinThe entire coding region and various mutant forms of C114 cDNA were amplified by PCR using primers containing a EcoRI or BamHI site and cloned into pEGFP (Clontech) so that the coding sequence continued in-frame with that of the enhanced green fluorescent protein (EGFP). These pC114-EGFP hybrid plasmids were transfected into NIH 3T3 cells by LipofectAMINE transfection reagent (Invitrogen). Cells were grown at 37 °C in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum for 48 h and examined by fluorescence microscopy to detect the distribution of C114-EGFP proteins. Mock pEGFP vector and mitochondrial protein-fused EGFP constructs were also transfected into NIH 3T3 cells.
Polynucleotide Binding StudiesIn vitro transcription/translation reaction of C114 was carried out using the TNT-coupled reticulocyte lysate system (Promega) in the presence of [35S]methionine (16). Radio-labeled C114 protein was incubated with various resins (Amersham Biosciences), which were first washed and resuspended in wash buffer 1 (20 mM Tris, pH 7.5, 5 mM -mercaptoethanol, 4 mM Mg(C2H3O2)2·4H2O, 200 mM KCl, 0.5% Nonidet P-40). Equal aliquots of 35S-labeled C114 protein were added to 40 µl of a 1:1 volume of each resin and allowed to incubate at room temperature for 30 min. The resins were spun down, washed three times with 500 µl of wash buffer 1, and washed twice with 500 µl of wash buffer 2 (20 mM Tris, pH 7.5, 5 mM
-mercaptoethanol, 4 mM Mg(C2H3O2)2·4H2O, 50 mM KCl). The proteins bound to beads after the wash were analyzed by SDS-PAGE followed by autoradiography. For quantitation, bound proteins were eluted by the addition of an equal volume of 2x Laemmli loading buffer, and equal aliquots were subjected to scintillation counting to determine the amount of proteins bound to each resin. For analysis, the percentage of proteins bound to each resin was calculated, taking into consideration the specific activity of each deletion construct, and comparison was made relative to the levels of the wild-type protein bound to each resin. For competitive binding studies, analyses were performed as above in the presence of competitor polynucleotides. Various concentrations of competitors (either poly(I·C) or poly(dI·dC)) ranging from 0.05 to 100 µg were added to the in vitro translated 35S-labeled C114 protein followed by the addition of resins. Analysis of bound proteins was carried out as above.
Construction of Eukaryotic Expression Vector Encoding C114 with a FLAG EpitopeTo generate a FLAG epitope-tagged version of C114 for expression in mammalian cells, the coding region of C114 was amplified by PCR from C114/pGEM-T using 5'-AGGAATTCTATGGCGAGTCCTGCTGCC-3' and 5'-ACGGATCCTCGCCCCATGATGACGAC-3' as the forward and reverse primer with a BamHI and EcoRI restriction site, respectively. The 600-bp PCR product was ligated in-frame with a FLAG tag into the pCMV-2 vector.
Co-immunoprecipitationHEK 293 cells were transfected with FLAG epitope-tagged C114 in pCMV-2, FLAG-tagged PACT, and/or V5-tagged PKR in Vet vector (described in Refs. 15 and 17). Transfected cells were washed twice with phosphate-buffered saline, lysed in 20 mM Tris, pH 7.6, 20% glycerol, 2 mM MgCl2, 1 mM dithiothreitol, 100 mM NaCl containing 1% Triton X-100. Cellular debris was removed by spinning at 14,000 rpm for 10 min. Lysates were incubated with either M2 anti-FLAG (Sigma) or anti-V5 (Invitrogen) and Protein A-agarose (Roche Molecular Biochemicals) overnight at 4 °C. The immunocomplexes were washed with lysis buffer and separated by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes and probed with monoclonal anti-V5 or anti-FLAG (1:5,000) followed by appropriate horseradish peroxidase-conjugated secondary antibody and ECL detection (Amersham Biosciences).
Pull-down Assays35S-labeled wild-type or mutated forms of PKR were in vitro translated using TNT-coupled reticulocyte system from Promega (18). FLAG epitope-tagged C114 protein was immunoprecipitated from transfected HEK 293 cell lysates with the M2 anti-FLAG (Sigma) and Protein A-agarose (Roche Molecular Biochemicals) as described above. Twenty µl of the in vitro translated 35S-labeled proteins was incubated with FLAG-tagged C114 protein in immunocomplexes containing Protein A-agarose and anti-FLAG antibody in 500 µl of pull-down buffer (20 mM HEPES-KOH, pH 7.5, 100 mM KCl, 1 mM dithiothreitol, 0.5 mM EDTA, 10% glycerol, and 0.5% Nonidet P-40) at 4 °C for 2 h. The resin was washed four times with 500 µl of pull-down buffer. Proteins were eluted with 40 µl of Laemmli sample buffer, boiled for 5 min, and resolved by SDS-PAGE. The proteins were transblotted to a polyvinylidene difluoride membrane followed by autoradiography and processed for Western blotting with M2 anti-FLAG antibody. Five µl of in vitro translated 35S-labeled proteins was also resolved by SDS-PAGE to visualize the input proteins.
Kinase AssaysHEK 293 cells were transfected with V5-tagged PKR constructs. In some samples, PKR-expressing plasmid was co-transfected with various forms of FLAG-tagged C114 plasmids (see Fig. 2B). Twenty-four hours after transfection, cells were washed with phosphate-buffered saline, pelleted, and lysed in an equal volume of lysis buffer. After centrifugation at 14,000 rpm for 10 min, the supernatants were collected. PKR was immunoprecipitated from aliquots containing 100 µg of total protein using an anti-V5 monoclonal antibody in radio-immune precipitation buffer (50 mM Tris, pH 7.4, 50 mM KCl, 400 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 100 units/ml aprotinin, 0.2 mM phenylmethylsulfonyl fluoride, 20% glycerol, 1% Triton X-100) at 4 °C overnight. Protein A-agarose was added for an additional hour followed by washing four times with 500 µl of TNEN buffer (20 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 0.1 M phenylmethylsulfonyl fluoride, 100 µM vanadate, 1 mM NaF, pH 8.0) and twice with activity buffer (20 mM Tris, pH 7.5, 50 mM KCl, 2 mM MgCl2, 2 mM MnCl2, 200 units/ml aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, 5% glycerol). The immune complex containing PKR was incubated with activity buffer containing 1 µCi of [-32P]ATP at room temperature for 30 min. One µg/ml poly(I·C) or 1 unit/ml heparin was added as a PKR activator. In some samples, kinase activities were assayed in the presence of the affinity-purified wild-type or mutant form IV of C114 protein. Labeled proteins were analyzed by SDS-PAGE and visualized by autoradiography and Western blotting.
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Partial Purification of FLAG-C114 ProteinsHEK 293 cells were transfected with plasmids expressing FLAG-tagged wild-type or the mutant form IV of C114 protein (see Fig. 2B). Cells expressing FLAG-tagged fusion proteins were harvested and lysed as described above. The clear lysate was then applied to the anti-FLAG M2 (Sigma) and Protein A-agarose for overnight incubation at 4 °C. The agarose beads were then washed three times with Tris-buffered saline buffer (50 mM Tris, pH 7.4, 0.15 mM NaCl). Bound FLAG-tagged fusion protein was eluted with six 1-ml aliquots of FLAG peptide (Sigma) at 0.1 mg/ml in Tris-buffered saline, washed, and concentrated by centrifugation in Centricon 10 (Amicon), and purified FLAG-tagged fusion proteins were rinsed and dissolved in kinase activity buffer.
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RESULTS |
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Northern blot analysis of mRNA prepared from mouse tissues showed that C114 mRNA is widely expressed in mouse tissues with higher abundance in liver, kidney, brain, and heart and low expression in lung and skeletal muscle (Fig. 1C). Two major C114 transcripts of 1.0 and 2.0 kb were detected in most tissues. The length of the cloned cDNA corresponds to the size of the shorter transcript.
Cellular Localization of the C114 ProteinTo elucidate possible functions of C114, we examined the cellular localization of the C114 protein. The C114 cDNA was fused in-frame into the N or C terminus of EGFP in the pEGFP vector, which expresses a green fluorescent protein to facilitate protein localization in living cells by confocal microscopy. Addition of C114 to pEGFP resulted in a nuclear localization of the C114 fusion protein with enrichment in the nucleolus (Fig. 2A, panel c). In contrast, the green fluorescent signal distributed throughout the cytoplasm and nucleus of cells transfected with pEGFP plasmid (Fig. 2A, panel a), whereas the signal was detected in mitochondria when transfected with pEGFP-mitochondrial protein plasmid (Fig. 2A, panel b).
There are three clusters of Arg/Lys-rich sequences present in the basic region of C114 (83RHLRRR88, 126KRRKKR131, and 133KLKEKK138). To determine the sequences responsible for nuclear localization of C114, wild-type C114 and a series of deletion mutants were expressed as GFP fusion proteins, and confocal fluorescence microscopy was performed 24 h after transfection of NIH 3T3 cells. The pattern of various EGFP-C114 proteins indicated that the last two clusters of the Arg/Lys-rich sequences (126KRRKKRQKLKEKK138) are the major nuclear localization signals for the C114 protein (Fig. 2B).
Nucleotide Binding Analysis of the C114 ProteinSince C114 protein accumulates in nucleolus, and many nucleolar proteins are dsRNA-binding proteins (12, 13), 35S-labeled in vitro translated C114 was tested for its ability to bind various polynucleotide resins. As shown in Fig. 3, A and B, unlike in vitro translated luciferase protein, wild-type C114 bound poly(I·C) but not poly(A) or poly(U) RNA. Competitive binding studies were next performed to compare the binding of wild-type C114 to dsRNA versus dsDNA. For these experiments, in vitro translated C114 was incubated with poly(I·C)-agarose in the presence of either free poly(I·C) or poly(dI·dC) competitors and assayed for the ability to bind to the poly(I·C) resin (Fig. 3C). In the presence of increasing amounts of poly(I·C) RNA, the binding of wild-type C114 to poly(I·C)-agarose decreased 79.5% in the presence of the highest concentration of competitors. In contrast, poly(dI·dC) failed to compete for wild-type C114 binding to the poly(I·C) resin with the highest concentration of competitors. These results suggest that wild-type C114 preferentially binds dsRNA.
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Although C114 clearly binds dsRNA, no known dsRNA-binding motifs were identified in the highly basic amino acid-rich region of C114 by domain search analysis. To determine the regions of C114 that are important for dsRNA-binding, a series of deletion constructs of C114 were assayed for their ability to bind dsRNA (Fig. 3D). The N-terminal 56 amino acids (amino acids 156, mutant fragment III) and the C-terminal peptide (amino acids 143187, mutant C114 fragment VI) showed only 15 and 25% poly(I·C) binding capacity as compared with wild-type C114 protein, respectively. On the other hand, the middle region (amino acids 51143) containing highly basic amino acid residues contains 88% poly(I·C) binding capacity (Fig. 3D). These results suggest that the binding of C114 to dsRNA is mediated by its middle region.
C114 Interacts with the dsRNA-dependent Protein Kinase PKRDifferent dsRNA-binding proteins, including the viral proteins HIV-1 transactivator of transcription (Tat), TRBP, and vaccinia virus E3 protein, and cellular proteins such as PACT and NF90, interact with PKR (13, 18). We therefore tested whether C114 can interact with PKR. Co-immunoprecipitations were performed with overexpressed FLAG-tagged C114 and V5-tagged PKR constructs in HEK 293 cells. When FLAG-tagged C114 was immunoprecipitated with an antibody against FLAG, V5-tagged PKR could be detected when the immunoprecipitates were immumoblotted with anti-V5 antibody (Fig. 4A). In addition, a comparable amount of FLAG-C114 (14%) was found to associate with PKR as compared with FLAG-PACT (17%), a well characterized PKR-interacting activator (Fig. 4B). The co-immunoprecipitation results also demonstrate that the N-terminal 50 amino acids of C114 are responsible for its association with PKR in cells (Fig. 4A, lane 4, and B, lane 6).
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We also mapped the regions of PKR that interact with FLAG-tagged C114 protein. Wild-type PKR or its deletion mutants were individually expressed as [35S]methionine-labeled proteins and incubated with purified FLAG-tagged C114 protein. As shown in Fig. 4C, panel b (lanes 13), wild-type PKR and two deletion mutants containing two DRBDs can interact with the C114 protein. Purified FLAG-C114 associated with Protein A-agarose did not pull down the PKR deletion mutant containing only the kinase domain or in vitro translated luciferase (lanes 4 and 5). These results suggest that C114 can interact with DRBDs in the N terminus of PKR.
C114 Inhibits PKR Kinase ActivitySince several RNA-binding proteins including cellular proteins such as PACT and NF90 (1416) and viral proteins such as TRBP and E3L (19, 20) are known to affect PKR activity, we tested whether C114 can modulate the activity of PKR. Expression constructs of FLAG-tagged C114 or its mutant form IV and V5-tagged PKR were co-transfected into HEK 293 cells. The V5-PKR protein was immunoprecipitated with monoclonal antibody against V5 and co-immunoprecipitated with transfected C114 proteins. When kinase assays were performed, PKR immunoprecipitated from C114-expressing cells was less activated in the presence of poly(I·C) or heparin (Fig. 5A, lanes 3 and 6). However, similar levels of PKR autophosphorylation were observed with immunoprecipitated V5-PKR from V5-PKR expression alone or the mutant form IV of C114 co-expressing cells (Fig. 5A, lanes 2, 4, 5, and 7). Comparable results were observed when kinase assays were performed using V5-PKR mixed with partially immunoaffinity-purified FLAG-C114 or FLAG-tagged mutant form IV of C114 protein in vitro (as described under "Materials and Methods," data not shown). Although the IV mutant form of C114 retained 98% of the poly(I·C) binding ability as compared with wild-type C114 (Fig. 3D), it lost its binding ability to PKR (Fig. 4A, lane 4) and the effect on PKR activation. When PKR mixed with the IV deletion mutant of C114, similar levels of PKR phosphorylation could be achieved by the PKR activators in the same assay (Fig. 5A, lanes 4 and 7). These results suggest that the inhibition of PKR activity is mediated through direct interaction of C114 and PKR and is independent of dsRNA binding. This scenario is supported by the fact that wild-type C114 can also inhibit PKR activation in the presence of heparin (Fig. 5A, lane 6). To further test the negative regulatory role of C114 on PKR function in cells, we examined the levels of phosphorylation of endogenous eIF-2 in cells following poly(I·C) treatment of cells for 2 h. Overexpression of C114 in HEK 293 or HT 1080 cells resulted in lower levels of poly(I·C)-stimulated eIF-2
phosphorylation (Fig. 5B). In contrast, overexpressed C114 IV mutant did not complex with PKR (Fig. 4, A and B) and had no effect on eIF-2
phosphorylation (Fig. 5C, lane 6). Taken together, these results suggest that C114 inhibits cellular PKR function through the interaction between C114 and PKR protein.
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DISCUSSION |
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PKR is critical in interferon-mediated antiviral defense, apoptosis, signal transduction, and cell growth but is also implicated in the onset of differentiation (4, 78). Analysis of phosphoproteins has demonstrated increased phosphorylation of PKR and eIF-2 during 3T3 F442A adipogenesis. Consistently, inhibition of adipocyte differentiation by cat serum correlated with reduced PKR activation, diminished eIF-2
phosphorylation, and elevated growth rate (7, 8), suggesting a role of PKR in adipogenesis. Furthermore, introduction of a kinase-inactive dominant negative PKR mutant into 3T3 L1 prevented proliferation arrest and interfered with adipogenic differentiation (23). In addition to its mitogenic activity on a plasmacytoma cell line, IL-11 was cloned as an adipogenesis inhibitory factor (2). Our observation that C114 is an IL-11-inducible PKR modulator suggests its potential role in adipogenesis. Interestingly, besides IL-11, expression of C114 in 3T3 L1 can also be induced by insulin (data not shown). Although insulin induces a decrease in eIF-2
phosphorylation in chondrocytes (24), IL-11 significantly reduces LPS-induced binding activity of NF-
B (25), a process known to be modulated by PKR (4, 5). Further studies of PKR and C114 interaction may clarify the regulation of PKR in adipocyte differentiation and potential cross-talk between IL-11, insulin signaling pathways, and PKR functions.
PKR has a bipartite structure consisting of N-terminal DRBDs and a C-terminal catalytic domain. Its DRBDs are known to interact with its catalytic domain and act as auto-regulators for PKR activation (26). PACT binds to DRBDs of PKR, which leads to a conformational change facilitating PKR activation (14). C114, like NF90 and many other cellular PKR antagonists, also binds DRBDs of PKR (13). C114 protein inhibits PKR autophosphorylation in a dsRNA-independent manner, suggesting inhibitory effects mediated by its binding to DRBDs of PKR. This interaction may result in a conformational change of PKR, leading to its inability to be activated by polyanionic agents. PKR is mainly localized in the cytoplasm but is also expressed in the nucleolus (27, 28). Under normal condition in Daudi cells, the cytoplasmic to nuclear ratio is 5:1. Following interferon treatment of Daudi cells for 16 h, this ratio could increase to around 17:1 (27). DRBD1 domain in the N terminus of PKR plays an important role for PKR nucleolar localization (27). Moreover, previous findings also demonstrated that cytoplasmic PKR is predominantly in a dimeric form, whereas in the nucleus it is a basic (i.e. unphosphorylated) protein. PKR appears to be actively transported to the nucleus by unknown mechanisms and translocated to the nucleolus by binding to structural RNAs, possibly rRNA (27). In contrast, C114 is predominantly nuclear but has been found in the cytoplasm by fractionation (data not shown). It is highly enriched in nucleolus, determined by its second and third Arg/Lys-rich clusters. Our data also demonstrate that C114 can interact with the DRBDs of PKR protein. Transient transfection experiments showed an increased amount of PKR in the nuclear fraction when coexpressed with C114 in the HEK 293 cells (data not shown), suggesting that C114 might play a role in nuclear import and export of PKR in cells.
In many of the experimental systems, what regulates PKR in the cells has remained an enigma, especially in the cases where synthetic or viral dsRNA is not involved. As a novel cellular inhibitory factor of PKR, expression of C114 in 3T3 L1 cells can be induced by IL-11 and insulin as well as poly(I·C) treatment but not by interferon-, transforming growth factor-
1, IL-4, and IL-9 (data not shown). C114 may modulate intracellular PKR activity under a variety of growth and differentiation conditions and in response to various extracellular stimuli, within and outside the context of virus infection. Further functional studies of this novel PKR modulator should provide important insights into the role of PKR in cell growth and differentiation.
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FOOTNOTES |
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Present address: Dept. of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195.
¶ Present address: Dept. of Animal Sciences, Purdue University, West Lafayette, IN 47907.
** To whom correspondence should be addressed: Dept. of Pharmacology, Case Western Reserve University School of Medicine, 2109 Adelbert Rd., W353, Cleveland, OH 44106-4965. Tel.: 216-368-6931; Fax: 216-368-3395; E-mail: yxy36{at}po.cwru.edu.
1 The abbreviations used are: PKR, protein kinase R; dsRNA, double-stranded RNA; GFP, green fluorescent protein; EGFP, enhanced GFP; DRBD, dsRNA-binding domains; PACT, PKR activating protein; TRBP, HIV-1 transactivating response RNA-binding protein.
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ACKNOWLEDGMENTS |
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REFERENCES |
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