C114 JBCD3

C114 is a novel IL-11-inducible nuclear dsRNA-binding protein that inhibits

Protein Kinase R*

Zhan Yin‡,§ , Jennifer Haynie‡, §§, Bryan R. G. Williams¶ and Yu-Chung Yang‡, **

From the ‡Department of Pharmacology and Cancer Center, Case Western Reserve

University School of Medicine, Cleveland, OH 44106-4965, and ¶Department of Cancer

Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195

*This work was supported by grants from National Institute of Health (RO1 HL48819

and RO1 CA78433 to Y.C.Y. and RO1 AI34039 and PO1 CA62220 to B.R.G.W.).

§Current address: Department of Cancer Biology, Lerner Research Institute, Cleveland

Clinic Foundation, Cleveland, OH 44195

§§Current address: Department of Animal Sciences, Purdue University, West Lafayette,

IN 47907

**To whom correspondence should be addressed: Department of Pharmacology, Case

Western Reserve University School of Medicine, 2109 Adelbert Road, W353, Cleveland,

OH 44106-4965. Tel. 216-368-6931; Fax: 216-368-3395; E-mail: [email protected].

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 4, 2003 as Manuscript M212969200 by guest on February 3, 2018

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We have identified a cDNA (named C114) that encodes novel transcripts

induced by IL-11 in mouse 3T3 L1 cells. Northern analysis of RNAs from multiple

mouse tissues detects two C114 transcripts of ~1.0 kb and ~ 2.0 kb with the highest

expression in liver, testis, brain and kidney. The C114 cDNA contains an open

reading frame of 187 amino acids with a predicted mass of 21 kDa. Three putative

nuclear localization signals (NLSs) are predicted at amino acids 83-88, 126-131 and

167-178. Using GFP-C114 fusion plasmids, amino acids 126-131 are shown to be

essential for the nuclear localization of C114. An arginine-rich region (amino acids

98-143) spanning the NLSs (amino acids 126-131) exhibits a double-stranded RNA

(dsRNA) binding activity. Competition experiments with different RNA

homopolymers demonstrate that C114 preferentially binds to poly (I:C). Similar to

other dsRNA binding proteins, C114 binds to the dsRNA-activated protein kinase,

PKR, via dsRNA-binding domains (DRBDs) of PKR and the N-terminal region of

the C114 protein. In vitro kinase assays indicate that C114 inhibits PKR activation

via a dsRNA-independent mechanism. Over-expression of C114 protein inhibits the

induction of eIF-2α phosphorylation following poly(I:C) treatment. This is the first

demonstration of a novel PKR modulator induced by a gp130 superfamily cytokine

which may play a role in cytokine mediated biological functions.

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IL-11 is a bone marrow fibroblast-derived cytokine with a variety of in vitro

biological activities within the hematopoietic, lymphopoietic, hepatic, adipose, bone, and

neuronal systems. It was originally isolated from a primate bone marrow-derived stromal

cell line based on its ability to stimulate the proliferation of an IL-6-dependent mouse

plasmacytoma cell line, T1165 (1). Meanwhile, Kawashima et al. isolated a cDNA

encoding a novel adipogenesis inhibitory factor that inhibits adipogenesis of mouse 3T3

L1 cells. The cDNA sequence of this factor is identical to IL-11 (2). Using biological

assays and 125I-IL-11 binding analysis, we previously demonstrated the existence of

functional IL-11 receptors on 3T3 L1 mouse preadipocytes (3). In spite of the

participation of IL-11 in diverse physiological processes, its precise role in cell growth

and differentiation remains unclear.

PKR 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-2α. Phosphorylation of eIF-2α 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 anti-viral 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, IFN-α, IFN-γ, IL-1,

TNF-α and PDGF, through a variety of effectors including NF-κB, IRF-1, ATF-2,

STAT3 and eIF-2α (4, 11). Although PKR has been implicated in different cytokine


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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, TAR RNA-binding protein,

ribosomal protein L18, NF90 and its homologues (12-14). On the other hand, other

cellular proteins such as PACT and RAX function as PKR activators (15). Many of these

proteins also contain well-defined 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.

MATERIALS AND METHODS

Antibodies and Reagents- 3T3 L1, NIH-3T3 and HEK 293 cells were cultured in

Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum

(FBS) and penicillin/streptomycin. Anti-Flag monoclonal M2 antibody was from Sigma

(St. Louis, MI) and anti-V5 monoclonal antibody from Invitrogen (Carlsbad, CA). The

anti-eIF-2α and phosphospecific anti-eIF-2α antibodies were from Biosource

International (Camarillo, CA). Recombinant human IL-11 was kindly provided by

Genetics Institute (Cambridge, MA). Various polynucleotide resins were from Amersham

Pharmacia (Uppsala, Sweden).

PCR-based cDNA subtraction- Total cellular RNA was isolated from starved or

IL-11-stimulated (500 ng/ml) mouse 3T3 L1 cells using TRIzol reagent (Invitrogen), and


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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 [α-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). 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 RACE. Poly(A)+ RNA blot of mouse tissues (Mouse

MTN Blot, Clontech) was also used for northern analysis.

Cellular localization of C114 protein- The entire coding region and various

mutant forms of C114 cDNA were amplified by PCR using primers containing EcoR I or

BamH I 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 DMEM plus 10% FBS for 48 hours

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.


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Polynucleotide binding studies- In vitro transcription/translation reaction of C114

was carried out using the TNT coupled reticulocyte lysate system (Promega) in the

presence of [35S]-methionine (16). Radiolabeled C114 protein was incubated with various

resins (Amersham) which were first washed and resuspended in wash buffer 1 [20 mM

Tris, pH 7.5, 5 mM β-mercaptoethanol, 4 mM Mg(C2H3O2)2⋅4H2O, 200 mM KCl, 0.5%

NP-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 twice with 500 µl of

wash buffer 2 [20 mM Tris, pH 7.5, 5 mM β-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 2 × 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-labeld

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

epitope- To generate a Flag epitope-tagged version of C114 for expression in mammalian


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cells, the coding region of C114 was amplified by PCR from C114/pGEM-T using 5’-

AGGAATTCTATGGCGAGTCCTGCTGCC-3’ and 5’-ACGGATCCTCGCCCCATGA

TGACGAC-3’ as the forward and reverse primer with BamH I and EcoR I restriction

site, respectively. The 600-base pair PCR product was ligated in-frame with a Flag tag

into the pCMV-2 vector.

Co-immunoprecipitation- HEK 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 references 15 & 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 DTT,

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 PVDF 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).

Pull-down assays- 35S-labeled wild-type or mutated forms of PKR were in vitro

translated using TNT T7 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 were incubated

with Flag-tagged C114 protein in immunocomplexes containing Protein A-agarose and

anti-Flag antibody in 500 µl of pull-down buffer (PDB, 20 mM HEPES-KOH, pH7.5,


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100 mM KCl, 1 mM dithiothreitol, 0.5 mM EDTA, 10% glycerol, and 0.5% NP-40) at

4°C for 2 hr. The resin was washed four times with 500 µl of PDB. 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 PVDF membrane followed by autoradiography, and

processed for western blotting with M2 anti-Flag antibody. Five µl of in vitro translated

35S-labeled proteins were also resolved by SDS-PAGE to visualize the input proteins.

Kinase assays- HEK 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 (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 RIPA buffer (50 mM Tris, pH 7.4, 50 mM KCl, 400 mM

NaCl, 1 mM EDTA, 1 mM DTT, 100 U/ml aprotinin, 0.2 mM PMSF, 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 TNEN buffer (20 mM Tris, 100 mM NaCl, 1

mM EDTA, 0.5% NP-40, 0.1 M PMSF, 100 µM vanadate, 1 mM NaF, pH8.0) and twice

with activity buffer (20 mM Tris, pH 7.5, 50 mM KCl, 2 mM MgCl2, 2 mM MnCl2, 200

U/ml aprotinin, 0.1 mM PMSF, 5% glycerol). The immune complex containing PKR was

incubated with activity buffer containing 1µCi of [γ-32P]ATP at room temperature for 30

minutes. One µg/ml of poly (I:C) or 1U/ml of heparin was added as a PKR activator. In

some samples, kinase activities were assayed in the presence of affinity purified wild-


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type or mutant form IV of C114 protein. Labeled proteins were analyzed by SDS-PAGE

and visualized by autoradiography and western blotting.

Partial purification of Flag-C114 proteins- HEK 293 cells were transfected with

plasmids expressing Flag-tagged wild-type or mutant form IV of C114 protein (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 TBS buffer (50 mM Tris, pH7.4, 0.15mM NaCl). Bound Flag-tagged fusion protein

was eluted with six 1-ml aliquots of Flag peptide (Sigma) at 0.1 mg/ml in TBS, washed

and concentrated by centrifugation in Centricon 10 (Amicon) and purified Flag-tagged

fusion proteins were rinsed and dissolved in kinase activity buffer.

RESULTS

Cloning of mouse C114- C114 was cloned using PCR-based cDNA subtraction

strategy from 3T3 L1 cells treated with human IL-11 (500 ng/ml) for 30 minutes.

Northern blot analysis confirmed induction of C114 expression in IL-11 treated 3T3 L1

cells as early as 30 min and persisting for 24 hours (Fig. 1A). Sequencing of C114

revealed it is identical to several expressed sequence tags (ESTs) deposited in GenBank.

To obtain the full-length cDNA sequence for C114, PCR-based RACE was performed

using a mouse liver cDNA library based on the overlapping sequences of several mouse

ESTs. The full-length cDNA contains an open reading frame (ORF) of 561 base pairs

which encodes a 187-amino acid polypeptide with a predicted molecular mass of 21 kDa.

The deduced protein sequence contains 3 putative nuclear localization signals (NLSs), a

basic amino acid rich region as well as a highly acidic amino acid tail at its C terminus


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(Fig. 1B). An identical full-length sequence has since been submitted by others (GenBank

Accession No. AK010961). C114 also shares a very high sequence homology with a

recently submitted human hypothetical protein FLJ13902 (Accession No. AK023964).

The striking similarity (91% identity at the amino acid level) suggests that FLJ13902 is

likely a human counterpart of mouse C114. Sequence alignment with the human genome

database revealed that human FLJ13902 gene consists of four exons and is localized at

human chromosome 7q22.1. C114 protein also has significant homology with two other

proteins with unknown functions from C. elegans and yeast (Genbank Accession Nos:

AE003808 and P41880).

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 kb 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 protein- To 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-c). In contrast, the green

fluorescent signal distributed throughout the cytoplasm and nucleus of cells transfected

with pEGFP plasmid (Fig. 2A-a); whereas the signal was detected in mitochondria when

transfected with pEGFP-mitochondrial protein plasmid (Fig. 2A-b).


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There are three clusters of Arg/Lys-rich sequences present in the basic region of

C114 (aa83RHLRRRaa88, aa126KRRKKRaa131 and aa133KLKEKKaa138). 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 hours after transfection of NIH 3T3 cells. The pattern of

various EGFP-∆C114 proteins indicated the last two clusters of the Arg/Lys-rich

sequences (aa126KRRKKRQKLKEKKaa138) are the major NLSs for the C114 protein

(Fig. 2B).

Nucleotide binding analysis of the C114 protein- Since 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 Figure 3A and 3B, 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 (aa1-aa56, mutant fragment III) and the C-terminal peptide

(aa143-aa187, mutant C114 fragment VI) showed only 15% and 25% poly (I:C) binding

capacity compared with wild-type C114 protein, respectively. On the other hand, the

middle region (aa51-aa143,) 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 PKR- Different dsRNA-

binding proteins, including the viral proteins 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 to Flag-PACT (17%), a well characterized

PKR interacting activator (Fig. 4B). The co-immunoprecipitation results also demonstrate

the N-terminal 50 amino acids of C114 are responsible for its association with PKR in

cells (Fig. 4A, lane 4; 4B, 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-b (lanes 1, 2 and 3), wild-type PKR and two deletion mutants containing two dsRNA

binding domains (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 activity- Since several RNA-binding proteins including

cellular proteins such as PACT and NF90 (14-16), 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 (Lane 3, 6, Fig. 5A), However, similar levels of PKR

autophosphorylation were observed with immunoprecipitated V5-PKR from V5-PKR

expression alone or mutant form IV of C114 co-expressing cells (Lane 2, 4, 5, 7, Fig.

5A). Comparable results were observed, when kinase assays were performed using V5-

PKR mixed with partially immuno-affinity purified Flag-C114 or Flag-tagged mutant

form IV of C114 protein in vitro (as described in Materials and Methods, data not

shown). Although the IV mutant form of C114 retained 98% of the poly(I:C) binding


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ability compared with wild-type C114 (Fig. 3D), it lost its binding ability to PKR (Lane

4, Fig. 4A) and the effect on PKR activation. When PKR mixed with IV deletion mutant

of C114, similar levels of PKR phosphorylation could be achieved by the PKR activators

in the same assay (lanes 4 and 7, Fig. 5A). 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 (Lane 6, Fig. 5A). To further test the

negative regulatory role of C114 on PKR function in cells, we examined the levels of

phosphorylation of endogenous eIF-2α in cells following poly(I:C) treatment of cells for

two hours. Overexpression of C114 in HEK 293 or HT 1080 cells resulted in lower levels

of poly(I:C) stimulated eIF-2α phosphorylation (Fig. 5B). In contrast, overexpressed

C114 IV mutant did not complex with PKR (Fig. 4A, 4B), and had no effect on eIF-2α

phosphorylation (Fig. 5C, lane 6). Taken together, these results suggest that C114 inhibits

cellular PKR function through the interaction between C114 and PKR protein.

DISCUSSION

In this paper, we have identified C114 as a novel 21 kDa dsRNA-binding protein

that interacts with PKR and inhibits PKR activity by a dsRNA-independent mechanism.

C114 was isolated through a differential screening for IL-11 inducible genes in a mouse

preadipocyte cell line, 3T3 L1. IL-11 is a multifunctional cytokine in the bone marrow

microenvironment (1, 21). It shares a common signal transducer, gp130, with other IL-6-

type cytokines, such as IL-6, leukaemia inhibitory factor (LIF), oncostatin M (OSM),

ciliary neurotrophic factor (CNTF) and cardiotrophin-1 (CT-1). These cytokines play

important roles in the regulation of complex cellular processes such as gene activation,


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proliferation and differentiation (22). The main components of their signaling pathways

are Janus kinases, Jak1, Jak2 and Tyk2 and the signal transducers and activators of

transcription STAT1 and STAT3 (23). However, mechanisms underlying IL-11 in growth

and differentiation signaling have not been elucidated. Accumulated evidence suggest the

requirement of PKR in response to a variety of biological stimuli, including cytokines

(4). Recently, PKR has been shown to interact with STAT3 activated by PDGF to induce

c-fos expression (11). However, the direct signaling link between IL-6 family cytokines

and PKR function has never been established.

PKR is critical in IFN-mediated anti-viral defense, apoptosis, signal transduction,

cell growth, but also implicated in the onset of differentiation (4, 7-8). Analysis of

phosphoproteins has demonstrated increased phosphorylation of PKR and eIF-2α during

3T3 F442A adipogenesis. Consistently, inhibition of adipocyte differentiation by cat

serum correlated with reduced PKR activation, diminished eIF-2α 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). While insulin induces a

decrease in eIF-2α phosphorylation in chondrocytes (24), IL-11 significantly reduces

LPS-induced binding activity of NF-κB (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


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adipocyte differentiation and potential crosstalk 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

approximately 5:1. Following interferon treatment of Daudi cells for 16 hr, 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 predominatly 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


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fraction when co-expression 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, insulin as well as poly(I:C) treatment, but not interferon-γ,

TGF-β1, 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.

Acknowledgments−−We thank Dr. Ganes C. Sen for kindly providing Flag-

tagged PACT plasmid.

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FIGURE LEGENDS

FIG. 1. Molecular cloning of C114. (A) Time course of C114 mRNA expression

in 3T3 L1 cells after IL-11 treatment. Total RNAs were prepared from 3T3 L1 cells at

various times after treatment with IL-11, and 10 µg of total RNA was loaded. The same

filter was stripped and then hybridized with β-actin as a loading control. (B) Nucleotide

and deduced amino acid sequences of C114 cDNA. Nucleotides sequence is presented in

5’ to 3’ orientation and amino acid sequence is displayed using the single-letter code.

Putative nuclear localization signals are bold and C-terminal acidic region is underlined.

The sequence has been deposited to GenBank by the RIKEN Genome Exploration Group

(Accession No. AK010359). (C) Northern blot analysis of C114 mRNA expression in

mouse tissues. A mouse multiple tissue poly(A)+ RNA blot was probed with 32P-labeled

random primed C114 cDNA. Arrows indicate the two molecular weight markers on the

blot.

FIG. 2. Cellular localization of the C114 protein. (A) pEGFP vector (a),

mitocondrial protein fused GFP construct (b), and C114 fused GFP construct (c) were

transfected into NIH 3T3. Living cells were observed by fluorescence microscopy at 48

hr post-transfection. (B) Structures of wild-type and various deletion mutants of C114 are

shown with the cellular localization observed. Figures in the bottom panel demonstrate

C114 localization in NIH 3T3 cells transfected with pEGFP vector and various GFP-

C114 constructs. N: nuclear localization with high accumulation in nucleolus. C:

cytoplasmic localization.

FIG. 3. Nucleotide binding analysis of the C114 protein. (A) C114 binding to

the poly(I:C) resin. In vitro transcribed/translated 35S-labeled luciferase or C114 protein


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(5µl each) and proteins bound to polynucleotide resins after extensive washing (45 µl

each) were analyzed by SDS-PAGE and autoradiography as indicated. (B)

Polynucleotide binding analysis. C114 was in vitro translated in rabbit reticulocyte

lysates and the 35S-labeled translation products were incubated with excess amounts of

various polynucleotide resins. Bound proteins were eluted and the amount of bound

proteins was determined relative to the luciferase binding. (C) Competitive binding

studies. In vitro translated wild-type C114 was incubated with various concentrations of

competitor nucleic acid [either poly(I:C) or poly(dI:dC), as indicated] followed by

incubation with dsRNA-agarose. Bound proteins were eluted and relative amount

analyzed with wild-type C114 in the absence of any competitors was determined. (D)

Regions of C114 for its poly(I:C) binding. In vitro translated products of various deletion

mutants (as shown in Fig. 2B) were incubated with poly(I:C) resin. Bound proteins were

eluted and amount of bound proteins was determined relative to the wild-type binding.

FIG. 4. C114 interacts with PKR. (A) PKR co-immunoprecipitation assays with

deletion mutants of C114 (Fig. 2B). (B) Relative amount of Flag-C114 and Flag-PACT

associated with PKR. HEK 293 cells were transfected in 10 cm culture dishes with 10 µg

of Flag-C114, Flag-PACT and/or V5-PKR construct DNAs. At 48 hr post-transfection,

cells were harvested and cell extracts prepared. Total cell extracts were used to

immunoprecipitated with anti-Flag mAb, and analyzed by western blot analysis with the

anti-V5 and anti-Flag antibodies. (C) Panel a: structure of wild-type and deletion mutants

of PKR (18); panel b: C114 pull-down assays with deletion mutants of PKR. The

immunoprecipitated Flag-tagged C114 protein was incubated with untagged PKR or its

mutant protein synthesized by in vitro translation using T7 TNT system.


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FIG. 5. Association of C114 and PKR inhibits PKR activation. (A) Inhibition

of PKR activation by C114. In vitro kinase assays of V5-PKR coimmunoprecipitated

with Flag-C114 or mutant form IV of C114 (indicated in Fig. 2B) from transfected HEK

293 cells were performed in the presence or absence of poly(I:C) (1µg/ml) or heparin

(1U/ml). PKR autophosphorylation was detected after proteins were separated by SDS-

PAGE. Equal loading of the input proteins was determined by western blot analysis with

anti-V5. Flag-C114 and Flag-tagged mutant form IV of C114 protein were detected by

western blot with anti-Flag mAb. (B) Inhibition of eIF-2α phosphorylation by C114.

HEK 293 and HT1080 cells were transfected with plasmid expressing Flag-tagged C114

protein 46 hours after transfection, cells were transfected with poly(I:C) (0.6µg/ml) for

different times. (C) Inhibition of eIF-2α phosphorylation by C114 through its PKR

interacting region. HEK 293 cells were transfected with plasmids expressing Flag-tagged

C114 or mutant IV protein 46 hours after transfection, cells were transfected with

poly(I:C) (0.6µg/ml) for 90 minutes. Cell extracts were prepared at time point indicated

and subjected to western blot analysis with monoclonal antibodies to native and

phosphylated forms of eIF-2α. Bar charts are relative activity measurements indicating a

relative quantification for PKR autophosphorylation (Fig. 5A) or eIF-2α phosphorylation

(Fig. 5B, 5C).


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actin

C114

IL-11 treatment 0 0.5 1 6 24 (hr)

1A

1.4kb

Testis

Kid

ney

Skeletal m

uscle

Liver

Lu

ng

Sp

leen

Brain

Heart

2.4kb

1C

1: TAAAGGAGTAGGAGAGCGGCTATGGCGAGTCCTGCTGCCGCTTCTGTGAGA1: M A S P A A A S V R

52: CCGCCCAGGCCCAAGAAAGAGCCGCAGACGCTGGTCATCCCCAAGAATGCG11: P P R P K K E P Q T L V I P K N A

103: GCCGAGGAGCAGAAGCTCAAGCTGGAGCGGCTCATGAAGAACCCGGACAAA28: A E E Q K L K L E R L M K N P D K

154: GCAGTTCCAATTCCAGAGAAAATGAATGAATGGGCTCCTCGAGCGCCTCCA45: A V P I P E K M N E W A P R A P P

205: GAATTTGTCCGAGATGTCATGGGTTCCAGTGCTGGGGCTGGCAGTGGAGAG62: E F V R D V M G S S A G A G S G E

256: TTCCACGTGTATAGGCACCTACGGCGGAGAGAGTACCAGCGGCAGGACTAC79: F H V Y R H L R R R E Y Q R Q D Y

307: ATGGATGCCATGGCTGAGAAGCAAAAACTGGATGCAGAGTTTCAGAAGAGA97: M D A M A E K Q K L D A E F Q K R

358: CTAGAAAAGAATAAAATTGCTGCAGAGGAGCAGACTGCAAAGCGCCGGAAA113: L E K N K I A A E E Q T A K R R K

409: AAGCGCCAGAAGTTAAAAGAGAAGAAGTTACTGGCAAAGAAGATGAAACTT130: K R Q K L K E K K L L A K K M K L

460: GAACAGAAGAAACAGAAGGAAGAACCCAGTCAGTGCCAGGAACAGCATGCC147: E Q K K Q K E E P S Q C Q E Q H A

511: AGCAGCTCTGACGAGGCATCTGAAACAGAGGAGGAGGAAGAGGAGCCCAGC164: S S S D E A S E T E E E E E E P S

562: GTCGTCATCATGGGGCGATGAGGAGTTTCAGCAGTCGTGAGCCCAGCTCAT182: V V I M G R *

613: TCTGTGACCAGCCTCAGAAGAGAAATGGCTGTGTGTGACCAGCTGCTAGAC

664: TGGCTGTGTTGGGAGAAGGCAGGCACCTCCTGCAGCCCTGCCTGCCTGGGG

715: AGCAGAGGGAGCAGACCTTAGACATTTGCAGATTGCATTCTGTATCCTGCC

766: CTGCTCTTGCTGAAGGGGAGTGAGTGCTTCACCTTTCCCGGGTGCCGTTCC

817: CTTCTGCTTGAACTGCAGTGAGCTCTGCGCTGGTTTCTGTGAAGGGAATAT

868: TTATTTAGTAAAGAACAGAAAACCTGAAAA

1B


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2A

pEGFP-C114pEGFP-

Mitochondrialprotein

protein

pEGFP

a b c


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KRRKKRQKLKEKK

con

V

C114 I II III IV

VII VIII IX XVI

C114 Constructs

83-88 126-131 133-138 167-178 187C114 N

170I N

103II C+N

56III C+N

51IV N

98V N

143VI C+N

126 147VII N

VIII N

140 IX C+N

98 138X N

2B

Putative NLSAcidic region


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3B

3C

0

0.2

0.4

0.6

0.8

1

1.2

blank 50 ug/mlpolyI:C

100 ug/mlpolyI:C

50 ug/mlpolydI:dC

100 ug/mlpolydI:dC

nucleotide competitor

0

10

2030

40

50

6070

80

90

polyA polyU polyI:C

nucleotide resin

luciferase

C114 protein

3D

3A84.0 kDa

52.2 kDa

36.3 kDa

30.2 kDa

21.9 kDa

Luc C114 Luc C114 C114 C114I:C I:C A U

1 2 3 4 5 6

Rel

ativ

e B

ind

ing

Rel

ativ

e B

ind

ing

Rel

ativ

e B

ind

ing

0

0.2

0.4

0.6

0.8

1

1.2

full-length

I II III IV V VI aa51-aa143

C114 construct


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4AV5-PKR + + + +Flag-C114 WT II III IV

IP: anti-FlagWB: anti-V5

IP: anti-FlagWB: anti-Flag

Total cell lysate WB: anti-V5

1 2 3 4

1--++-+-

WB:anti-V5

WB:anti-Flag

Flag-C114Flag-C114 IVFlag-vectorV5-PKRFlag-PACTIP:anti-FlagIP:anti-V5

2--++--+

3+--+-+-

4+--+--+

5-+-+-+-

6-+-+--+

7---+++-

8---++-+

4B

Relative amount of Flag-tagged protein detected with anti-Flag Ab

0 0

1

0.145

0.98

0

1.31

0.23

00.20.40.60.8

11.21.4

1 2 3 4 5 6 7 8

Relative amount of PKR detected with anti-V5 Ab

0

1

0.21

0.98

0

0.88

0.14

0.68

0

0.2

0.4

0.6

0.8

1

1.2

1 2 3 4 5 6 7 8


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4C DRBD1 DRBD2 Kinase Domain

WT

DRBD

7M

KD

a

WT DRBD 7M KD Luc Con

Input protein

Pull-down

WB: anti-Flag

b

1 2 3 4 5 6

PKR constructs


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V5-PKR Flag-C114Flag-C114 IV Flag-vector I:C(1µg/ml)Heparin(1U/ml)

WB: anti-Flag

WB: anti-V5

PKR auto-phosphorylation

5A 1

+--+--

2

+--++-

3

++--+-

4

+-+-+-

5

+--+-+

6

++---+

7

+-+--+

1

5.2

2.1

5.85.4

2.2

3.9

0

1

2

3

4

5

6

7

1 2 3 4 5 6 7

Rel

ativ

e A

ctiv

ity


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5B Flag-C114 - - + - + - + Flag-vector + + - + - + -I:C(0.6µg/ml) 0 60 60 90 90 120 120 (min)

HT1080 cells

Flag-C114 - - + - + - + - + Flag-vector + + - + - + - + -I:C (0.6µg/ml) 0 30 30 60 60 90 90 120 120 (min)

293 cells

1

5.12

2.47

5.6

2.74

4.48

1.8

0

1

2

3

4

5

6

1 2 3 4 5 6 7

Rel

ativ

e A

ctiv

ity

1

1.8

1.1

3.4

1.5

3.6

1.5

10.7

0

0.5

1

1.5

2

2.5

3

3.5

4

1 2 3 4 5 6 7 8 9

Rel

ativ

e A

ctiv

ity

1 2 3 4 5 6 7

1 2 3 4 5 6 7 8 9

WB: anti-EIF-2α-P

WB: anti-EIF-2α

WB: anti-Flag

WB: anti-EIF-2α-P

WB: anti-EIF-2α

WB: anti-Flag


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WB: anti-EIF-2α-P

WB: anti-EIF-2α

WB: anti-Flag

Flag-C114Flag-C114 IV

Flag-vector

I:C (0.6µg/ml)

1--+

-

2+--

-

3-+-

-

4--+

+

5+--

+

6-+-

+

1 0.97 0.95

2.33

1.21

1.78

0

0.5

1

1.5

2

2.5

1 2 3 4 5 6

5C

Rel

ativ

e A

ctiv

ity by guest on February 3, 2018

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Zhan Yin, Jennifer Haynie, Bryan R.G. Williams and Yu-Chung Yangkinase R

C114 is a novel IL-11-inducible nuclear dsRNA-binding protein that inhibits protein

published online April 4, 2003J. Biol. Chem.

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C114 JBCD3