of April 12, 2018.This information is current as

SignalingγReceptor and Regulates IFN-γAnnexin V Associates with the IFN-

Moeenrezakhanlou and Neil E. ReinerCarlos Leon, Devki Nandan, Martin Lopez, Alireza

http://www.jimmunol.org/content/176/10/5934doi: 10.4049/jimmunol.176.10.5934

2006; 176:5934-5942; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/176/10/5934.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2006 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Annexin V Associates with the IFN-� Receptor and RegulatesIFN-� Signaling1

Carlos Leon,2*† Devki Nandan,2* Martin Lopez,* Alireza Moeenrezakhanlou,* andNeil E. Reiner3*†

Many of the biological activities of IFN-� are mediated through the IFN-�R3-linked Jak-Stat1� pathway. However, regulation ofIFN-� signaling is not fully understood, and not all responses to IFN-� are Stat1� dependent. To identify novel elements involvedin IFN-� cell regulation, the cytoplasmic domain of the R2 subunit of the human IFN-�R was used as bait in a yeast two-hybridscreen of a human monocyte cDNA library. This identified annexin A5 (AxV) as a putative IFN-�R binding protein. The inter-action was confirmed in pull-down experiments in which a GST-R2 cytoplasmic domain fusion protein was incubated withmacrophage lysates. Furthermore, immunoprecipitation using anti-IFN-�R2 Abs showed that AxV interacted with IFN-�R2 toform a stable complex following incubation of cells with IFN-�. In 293T cells with reduced expression of AxV, brought about bysmall interfering RNA targeting, activation of Jak2 and Stat1� in response to IFN-� was enhanced. Inhibition of cell proliferation,a hallmark of the IFN-� response, also was potentiated in HeLa cells treated with small interfering RNA directed at AxV. Takentogether, these results suggest that through an inducible association with the R2 subunit of the IFN-�R, AxV modulates cellularresponses to IFN-� by modulating signaling through the Jak-Stat1 pathway. The Journal of Immunology, 2006, 176: 5934–5942.

I FN-� has pleiotropic immunomodulatory and proinflamma-tory activities and is pivotally involved in host defenseagainst infection and malignancy (1, 2). The current para-

digm of IFN-� signaling was assembled during the 1990s (3–5).The IFN-�R is a heterotetramer comprised of two �-chains (R1)4

and two �-chains (R2) that are constitutively associated with Jak1and Jak2, respectively. Upon ligand binding to IFN-�R1, activa-tion of Jak1 and Jak2 ensues leading to phosphorylation of ty-rosine440 on IFN-�R1, thereby generating a docking site for thetranscription factor Stat1�. Once at the activated receptor com-plex, Stat1� monomers homodimerize and are phosphorylated ontyrosine701. The phosphorylated homodimer disassociates from thereceptor and translocates to the nucleus where it binds to IFN-�activated sequences (GAS) elements in IFN-�-regulated genes (6).Stat1� also undergoes phosphorylation on serine727 in the cytosol(7), before moving to the nucleus, an event that significantly poten-tiates its ability to activate gene transcription (8). The importance ofthis phosphorylation event is illustrated by the finding that the trans-activation potential of a S727A mutant of Stat1� was reduced by80% (9).

Multiple mechanisms have been shown to negatively regulatethe activity of Stat1�, including dephosphorylation by dual spec-ificity phosphatases (10), arginine methylation (11), ubiquitinationand consequent degradation by the proteasome (12), and bindingof PIASy, a protein inhibitor of the activated Stat1�, which re-presses Stat1�-mediated gene activation (13). The Jak-Stat1� sig-naling pathway also is regulated by contraints on activation of Jak1and Jak2 imposed by their association with tyrosine phosphatases,such as SHP-1 (14, 15) and SHP-2 (16, 17). In addition, the IFN-�-inducible tyrosine phosphorylation of Jak2 on residue ty-rosine1007, leads to the recruitment of suppressor of cytokine sig-naling 1 and polyubiquitination of Jak2, resulting in its rapiddegradation through the proteasome (18). Signaling through theJak-Stat1� pathway also has been shown to be negatively regu-lated via the association of Jak2 with the human DnaJ protein,hTid-1 (19).

Although many biologic responses to IFN-� are regulated byStat1�, recent evidence indicates that IFN-� signaling uses ele-ments other than or in addition to Stat1�. For example, microarraydata generated using cells derived from Stat1� null mice hasshown that there are at least 200 genes regulated by IFN-� in theabsence of this transcriptional activator (20). In this regard, a di-verse group of signaling elements distinct from the Jak-Stat1�pathway have been implicated in regulating IFN-� signaling (re-viewed in Refs. 21 and 22), and it is likely that others are yet to becharacterized.

To identify novel components involved in IFN-� signaling, wedesigned a yeast two-hybrid screen using the intracellular domainof R2 subunit of the IFN-�R as bait to screen a monocyte cDNAlibrary. This approach resulted in the identification of annexin V(AxV) as an IFN-�R2 interacting protein. To address the role ofAxV in IFN-� signaling, small interfering RNA (siRNA) was usedto silence the AxV gene. Cells with reduced AxV expressionshowed increased responses to IFN-� for activation of Jak2 andStat1�, and IFN-�-induced growth arrest also was potentiated un-der these conditions. These findings are consistent with a role forAxV in modulating signaling through the Jak-Stat1� pathway.

*Department of Medicine, Division of Infectious Diseases, and †Department of Mi-crobiology and Immunology, University of British Columbia Faculties of Medicineand Science, and Vancouver Coastal Health Research Institute, Vancouver, BritishColumbia, Canada

Received for publication September 7, 2005. Accepted for publication February27, 2006.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Canadian Institutes of Health Research OperatingGrants MOP-8633 (to N.E.R.) and FRN-38005 (to D.N.).2 C.L. and D.N. contributed equally to this work.3 Address correspondence and reprint requests to Dr. Neil E. Reiner, Division ofInfectious Diseases, University of British Columbia, Room 452D, 2733 HeatherStreet, Vancouver, BC, Canada, V5Z 3J5. E-mail address: ethan@interchange.ubc.ca.4 Abbreviations used in this paper: R1, �-chain of IFN-�R; R2, �-chain of IFN-�R;GAS, �-activated sequence; AxV, annexin V; GAD, Gal-4 activator domain; GBD,Gal-4 binding domain; SH2, Src homology 2; siRNA, small interfering RNA; shRNA,short hairpin RNA; VEGFR, vascular endothelial growth factor receptor.

The Journal of Immunology

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Materials and MethodsReagents and chemicals

Anti-phosphotyrosine mAb 4G10 and rabbit polyclonal sera to Jak2 wereobtained from Upstate Biotechnology; anti-IFN-�R2 was from PBL Bio-chemical Laboratories. Abs to Gal binding domain, GST, AxV, AxII, abl,Stat1�, actin, and IFN-�R1 were purchased from Santa Cruz Biotechnol-ogy. Ab to phosphoJak2 was from BioSource International. The rhIFN-�was a gift from Genentech. RPMI 1640, DMEM, HBSS, PBS, and proteaseinhibitors PMSF, pepstatin A, aprotinin, leupeptin, and Ab to phospho-serine were obtained from Sigma-Aldrich. BCECF-AM and BAPTA-AMwere from Molecular Probes.

Cell culture

THP-1 and human embryonic kidney 293T cells were obtained from theAmerican Type Culture Collection. THP-1 cells were cultured in RPMI1640 supplemented with 10% FCS (HyClone), 100 �g/ml penicillin, 100�g/ml streptomycin, and 10 mM HEPES. The 293T cells were grown inDMEM supplemented with 10% FCS, 0.1 mM minimal nonessential aminoacids, 10 mM L-glutamine, 20 mM HEPES, and 100 �g/ml each of peni-cillin and streptomycin.

Electrophoresis and immunoblotting

Protein samples were separated by SDS-PAGE, transferred to nitrocellu-lose membranes (Bio-Rad), and blocked with either 3% BSA (Sigma-Aldrich) or 5% milk in 1� TBS with 0.1% Tween 20 for 1–2 h and thenprobed with the indicated primary Ab diluted in blocking solution for 2 hor as otherwise specified. Membranes were washed three times, incubatedwith the appropriate secondary Ab coupled to peroxidase, and proteinswere detected by ECL (Amersham Biosciences).

Cloning of the intracellular domain of the R2 subunit of theIFN-�R as bait for yeast two-hybrid screening

The intracellular domain of the IFN-�R2 subunit (residues 265–334) wascloned by PCR into the EcoRI and BglII restriction sites of the pYTH9shuttle vector (provided by GlaxoSmithKline) as a fusion with Gal-4 bind-ing domain (GBD-R2). The cytoplasmic domain of the IFN-�R1 subunit(residues 262–489) was cloned in an identical fashion (GBD-R1) and usedas a control for specificity. Both constructs were checked by DNA se-quencing to confirm the correct frame and the absence of any PCR errors.The constructs and the vector alone, which also contained a Trp gene, werelinearized with XbaI and used to stably transform yeast strain SDY191(SDY191/R1, SDY191/R2, and SDY191/GBD) (23). The Saccharomycescerevisiae strain SDY191 is auxotrophic for the selectable markers tryp-tophan, leucine, and histidine and encodes Escherichia coli LacZ and yeastHIS3 genes as reporters under the control of the Gal-4 transcription factor.Integration of the construct into the yeast strain was assessed by PCR ofyeast genomic DNA, and expression of the fusion protein was determinedby Western blot using anti-GBD Ab (data not shown).

Construction and screening of a cDNA library for yeasttwo-hybrid screening

An expression library in the HybriZap vector (Stratagene) was constructedaccording to the manufacturer’s specifications using mRNA extracted fromhuman monocytes treated with IFN-� (provided by Dr. A. Mui, Universityof British Columbia). The quality of the library was assessed by the numberof clones, the sizes and percentage of inserts, and the presence of IFN-�-inducible genes as determined by PCR (data not shown). Screening of thelibrary used 1 million clones and was conducted in the presence of 15 mM3-aminotriazole to prevent leakage of histidine expression. Candidate in-teracting clones were examined further by for �-galactosidase activity.Double-positive clones were amplified for plasmid extraction, and the latterwere used to transform bacteria in selective medium. Plasmids isolatedfrom these bacteria were used to retransform yeast cells recombinant forthe bait construct to confirm the interaction. Negative controls consisted oftransforming recombinant yeast strain SDY191/R2 with the Gal-4 activatordomain (GAD) alone and by transformation of yeast recombinant for theGBD alone with the putative interacting clone. Plasmids with the correctphenotype were sent for DNA sequencing (NAPS Unit, University of Brit-ish Columbia).

Cloning of the cytoplasmic domain of IFN-�R2 as a GST fusionprotein and GST pull-down experiments

The cytoplasmic domain of IFN-�R2 (residues 269–334) was cloned intopGEX4T using the EcoRI and XhoI restriction sites. As a specificity con-

trol, the cytoplasmic domain of IFN-�R1 (residues 267 to 489) also wascloned in the same vector using the BamHI and SalI restriction sites.Clones containing inserts were sequenced, and those in frame with GSTand without mutations were grown at 37°C in 2� YTA medium with 100�g/ml ampicillin up to an OD of 0.6. Isopropyl �-D-thiogalactoside wasadded to a final concentration of 0.1 mM, and bacteria were grown at 28°Cfor 1.5 h, after which they were pelleted and disrupted in PBS with 1 mMEDTA by sonication. Sonicates were incubated with 0.1% Triton X-100and 0.1 mM PMSF on ice for 5 min and cleared by centrifugation at a speedof 10,000 � g at 4oC for 15 min. Fusion protein was purified using glu-tathione beads as suggested by the manufacturer and analyzed by SDS-PAGE and immunoblotting with anti-GST Ab. For GST pull-downs, pu-rified fusion protein bound to glutathione beads was incubated with wholecell lysates from THP-1 cells at 4oC for 2 h. Beads were then washed withPBS/0.5% Triton X-100 three times, and bound material was released byboiling coated beads in 2� SDS-Laemmli buffer. Eluted samples wereanalyzed by SDS-PAGE and immunoblotting with an anti-AxV Ab.

Coimmunoprecipitation of IFN-�R2 and AxV

Five million THP-1 cells grown in RPMI 1640 complete medium weredifferentiated overnight (12–14 h) using 10 nM PMA in complete medium.After differentiation, cells (70% confluent) were washed with HBSS threetimes and left to rest for 4 h in complete medium. Cells were then eitheruntreated or treated with IFN-� (100 U/ml) for 15 min and then washedwith HBSS and solubilized in digitonin buffer (50 mM Tris (pH 8.0), 150mM NaCl, 0.1% digitonin, 1 mM sodium orthovanadate, 50 mM sodiumfluoride, 1 mM PMSF, 10 �g/ml pepstatin, 10 �g/ml aprotinin, and 10�g/ml leupeptin) for 30 min on ice. Lysates were cleared by centrifugationat 10,000 � g at 4°C for 15 min, and supernatants were incubated withmAbs (both mouse IgG1) specific for either IFN-�R2 or c-abl for 2 h.Protein G (Amersham Biosciences) was then added, and the incubation wascontinued for 45 min after which immune complexes were recovered bycentrifugation at 5,000 � g. Immunoprecipitates were washed in digitoninbuffer three times and solubilized in Laemmli buffer without 2-mercapto-ethanol (nonreducing conditions). After transfer to nitrocellulose, mem-branes were immunoblotted for AxV, IFN-�R2, and c-abl.

Modulation of annexin expression in 293T cells using siRNA

To examine the role of AxV in IFN-� signaling, siRNA constructs weredesigned using a web based platform (�http://katahdin.cshl.org�). The re-gions chosen for targeting were specific to either AxV and or AxII (used asa specificity control) DNA sequences as determined by a BLAST search.Two pairs of partially complementary oligonucleotides (“a” and “b”) wereused to create the pSHAG constructs expressing short hairpin RNA’s(shRNA) targeting AxV.

The following sequences were used to create the pSHAG-AV2.1 con-struct: AV2a, 5�-TCCCCAGATGTATCTCCCTTAATCATGGGAAGCTTGCTATGATTAAGGGGGATACGTCTGGGGATTATTTTTT-3�; andAV2b, 5�-GATCAAAAAATAATCCCCAGACGTATCCCCCTTAATCATAGCAAGCTTCCCATGATTAAGGGAGATACATCTGGGGACG-3�.

The following sequences used to create the pSHAG-AV3.1 construct:AV3a, 5�-CATCAGGGTCTCTGTTAGCCTGAAGGAGGAAGCTTGCTCCTTCGGGCTAGCAGAGGCCCTGGTGCTGTTTTTT-3�; and AV3b,5� -GATCAAAAAACAGCACCAGGGCCTCTGCTAGCCCGAAGGAGCAAGCTTCCTCCTTCAGGCTAACAGAGACCCTGATGCG-3�. Onlythe pSHAG AV2.1 construct effectively reduced cellular levels of AxVprotein, and the pSHAG AV3.1 construct was used as a control for non-specific effects.

A single pair of oligos was designed to generate a shRNA for targetingAxII. The following sequences were used to create the pSHAG AxII con-struct: AxIIa, 5�-TGGGGCACGCTCCGCTCGGTCATGATGCGAAGCTTGGTATTATGACCGAGCGGAGTGTGCCTCACCTTTTTTT-3�; andAxIIb, 5�-GATCAAAAAAAGGTGAGGCACACTCCGCTCGGTCATAATACCAAGCTTCGCATCATGACCGAGCGGAGCGTGCCCCACG-3�.

The oligonucleotide pairs were annealed and cloned into the BseRI/BamHI cloning site of the pSHAG vector that directs the in vivo synthesisof shRNA molecules using a U6 promoter (24). Positive clones were se-lected by the acquisition of both kanamycin resistance and a new HindIIIsite. Clones containing the inserts of interest (pSHAG-AV2.1, pSHAGAV3.1, and pSHAG AxII) were grown, purified using an endotoxin-freeMaxiprep kit (Qiagen), and used to transfect 293T cells. Transfection of thepSHAG constructs was conducted using Lipofectamine 2000 (InvitrogenLife Technologies) according to the manufacturer’s instructions. Cellswere transfected when they reached 95% confluency and were incubatedwith transfection reagent containing plasmid DNA diluted in OptiMEMmedium for 24 h. Medium was then replaced by complete DMEM, andcells were cultured for an additional 48 h to allow for expression of siRNA

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and down-regulation of either AxV or AxII. Transfection efficiency was�95% as determined by cotransfection with a pCMV �-galactosidase ex-pression construct combined with measurement of �-galactosidase activityin situ. The extent to which either AxV or AxII expression was reduced bythe siRNA constructs was assessed by immunoblotting with AxV or AxIIAbs coupled with densitometric analysis using UN-SCAN-IT software(Silk Scientific). Actin was used as a protein loading control.

Western blotting

The 293T cells transfected with either pSHAG (empty vector), pSHAGAV2.1 (referred as AV2.1), or pSHAG AxII plasmids for 24 h were washedwith HBSS and suspended in complete medium for an additional 48 h. Cellswere then either untreated or incubated with IFN-� (50–100 U/ml) for 4 h,followed by washing with HBSS and solubilization in 2� SDS-Laemmli sam-ple buffer by boiling for 5 min. After separation of lysates by SDS-PAGE andtransfer to nitrocellulose membranes, Western blotting was conducted withAbs directed at AxV, AxII, Stat1�, SHP-2, and actin. In addition, Stat1� wasimmunoprecipitated from pSHAG- and pSHAG AV2.1-transfected cells toanalyze the effect of AxV down-regulation on Stat1� tyrosine phosphoryla-tion. IFN-�-induced activation of Jak2 was measured by immunoblotting ofcell lysates using anti-phosphoJak2 pYpY1007/1008, which recognizes the acti-vated form of Jak2 (25).

Growth arrest assays

To examine the influence of AxV levels on IFN-�-mediated growth arrest,HeLa cells transfected with pSHAG, pSHAG AxII, or pSHAG AV2.1 con-structs were seeded in 12-well plates (0.5 � 104 cells per well) and left torest for 24 h. The cells were then treated or not with 100 U IFN-� for 48 h.Cells were washed with HBSS, dislodged using a cell dissociation solution(Sigma-Aldrich), and viable cells were counted in a hematocytometer usingtrypan blue dye. Alternatively, for assessment of cell proliferation based onthymidine incorporation, HeLa cells transfected with either pSHAG orpSHAG AV2.1 constructs were seeded in 6-well plates (1.0 � 104 cells perwell) and left to rest for 24 h. The cells were then treated or not with 100U IFN-� for 16–18 h. [methyl-3H]Thymidine (PerkinElmer) was added toa final concentration of 1 �Ci/ml, and the cells were further incubated for8 h. At the end of metabolic labeling, the cells were washed with PBS andincubated with 5% TCA for 30 min at 4°C. TCA was removed by washingwith PBS, and cells were solubilized in lysis solution (0.5 M NaOH/0.5%SDS), and incorporation of 3H was measured by liquid scintillation count-ing.

For statistical analysis, one-way ANOVA was performed to compareresults between experimental groups, followed by Tukey test for multiplecomparisons. A p value of �0.05 was considered significant. All statisticswere performed using GraphPad Prism software, version 3.0 (GraphPad).

ResultsYeast two-hybrid screening identified AxV as an IFN-�R2interacting protein

Screening of the human monocyte cDNA library in yeast strainSDY191/R2, recombinant for the cytoplasmic domain of IFN-�R2, identified a clone designated D6d with the required pheno-type consisting of the ability to grow in auxotrophic medium com-bined with a positive �-galactosidase assay. The plasmid from thisinteracting clone was amplified and used to re-transformSDY191/R2 and the interaction was confirmed. To control forspecificity of the phenotype, a series of control transfections weredone as outlined in Fig. 1A. Neither growth of yeast nor �-galac-tosidase activity was observed when SDY191/R2 was transformedwith the pACT2 vector expressing only the GAD. Clone D6d alsowas transformed into yeast host SDY191 recombinant for theGBD, and no yeast growth was detected. In addition, whenSDY191 recombinant for the cytoplasmic domain of IFN-�R1 wastransformed with pACT clone D6d, although limited yeast growthwas detected, the �-galactosidase cracking assay was consistentlynegative. Together, these results suggest that the interaction be-tween D6d and the intracellular domain of IFN-�R2 was specific.

The insert in clone D6d was sequenced and was identified asAxV. Fig. 1B shows that the region of AxV contained within thisclone consisted of 826 nucleotides, corresponding to aa 157–319plus the 3� untranslated region in frame with the GAD sequence.

Residues 157–319 encompass domains three and four of AxV,including the C terminus, and these domains are sufficient to forma binding site for calcium and phospholipids.

GST-IFN-�R2 cytoplasmic domain protein specifically interactswith AxV

To seek independent evidence to support the yeast two-hybrid re-sults, the cytoplasmic domains of IFN-�R2 and IFN-�R1 werecloned and expressed as GST fusion proteins. After purificationusing glutathione beads, the purity of the preparations was as-sessed by SDS-PAGE and Coomassie brilliant blue staining (Fig.2A; n � 3) and equivalent amounts of GST, GST-R1 and GST-R2fusion proteins were used in pull-down experiments. When a lysateof IFN-�-treated THP-1 cells was incubated with the fusion pro-teins, AxV bound to the GST-R2 fusion protein (Fig. 2B, lane 2),but not to either GST alone or the GST-R1 fusion (Fig. 2B, lanes1 and 3; n � 3). The absence of AxV binding to either GST orGST-R1 provided additional evidence to support the specificity ofthe interaction of AxV with the cytoplasmic domain of IFN-�R2.

Confirmation of the interaction between IFN-�R2 and AxV invivo by coimmunoprecipitation

Based on the findings from both the yeast two-hybrid screen andthe GST pull-down experiment, we sought evidence to determinewhether AxV and IFN-�R2 associate in vivo. To examine thisquestion, THP-1 cells were either untreated or incubated with

β

FIGURE 1. Specificity of the interaction between the intracellular domainof IFN-�R2 chain and AxV in the yeast two-hybrid system. A, To screen forIFN-�R2 chain interacting proteins, the sequence of human intracellular do-main of IFN-�R2 chain was cloned into GBD vector and recombined into thegenome of yeast strain SDY 191 (GBD-R2). The integration of the constructinto the yeast strain was assessed by PCR of yeast genomic DNA and theexpression of the fusion protein was determined by Western blot using an antiGBD Ab (data not shown). In parallel, the sequence of human intracellulardomain of IFN-�R1 chain also was cloned into GBD and introduced into yeaststrain SDY 191 (GBD-R1). Both of these yeast clones expressing fusion pro-teins were transformed with GAD plasmid containing the sequence of humanAxV. Double transformants were tested for evidence of an interaction usingtwo independent assays (histidine auxotropic growth and the expression of�-galactosidase). B, Schematic representation of a IFN-�R2 interacting clonefrom the monocyte cDNA library. Clone D6d which showed a strong inter-action with R2 was sequenced (826 nucleotides) and identified as AxV. Thisfigure shows the region present in clone D6d, (aa 157–319), including theC-terminal region of AxV and the 3� untranslated region. The AxV sequencepresent in this clone encompassed domains three and four, which are sufficientto form a binding site for calcium and phospholipids. This truncated AxVchain was apparently also sufficient for the interaction with the intracellulardomain of IFN-�R2 receptor.

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IFN-� and then subjected to immunoprecipitation using an Ab di-rected at IFN-�R2. As can be seen in Fig. 3A, AxV coimmuno-precipitated with IFN-�R2, but this was observed only when usinglysates from cells that had been IFN-� treated. When the samemembrane was stripped and reprobed, the results showed that sim-ilar amounts of receptor were brought down from lysates of both

IFN-�-treated and control cells (Fig. 3B, lanes 6 and 7), suggestingthat the interaction is ligand inducible. To examine further thespecificity of the interaction, the membrane containing the proteinsfrom the immunoprecipitation was stripped and reprobed with ananti-AxII Ab (Fig. 3C, lane 1). This Ab only recognized AxII inthe whole cell lysates and not in the immunoprecipitates, indicat-ing that the association between AxV and IFN-�R2 was specific.

Specific down-regulation of AxV expression using siRNA

To examine the role of AxV in IFN-� signaling, siRNA was usedto down-regulate AxV expression in 293T cells. Before undertak-ing these experiments, we verified that 293T cells respond toIFN-� as expected by analyzing ligand-inducible tyrosine phos-phorylation of Stat1�, Jak1, Jak2, and IFN-�R1 (data not shown).Transfection of 293T cells with siRNA construct pSHAG AV2.1brought about a marked reduction in AxV (70–80% reduction), asdetermined by immunoblotting. Specificity of the AV2.1 constructwas determined by analyzing AxII expression that was unaffected(Fig. 4D).

Reduced expression of AxV in 293T cells is associated withenhanced tyrosine phosphorylation of Stat1� in response toIFN-� treatment

To examine whether AxV regulates cell signaling in response toIFN-�, either control (pSHAG) or pSHAG AV2.1 (AV2.1)-trans-fected 293T cells were incubated with IFN-�, and whole cell ly-sates were subjected to immunoprecipitation with an Ab directedat Stat1�. Immune complexes were then separated by SDS-PAGE,transferred to nitrocelullose membranes and probed with anti-phosphotyrosine Ab. As shown in Fig. 5, A and D, the amount oftyrosine phosphorylated Stat1� recovered from pSHAG AV2.1transfected 293T cells after 4 h of IFN-� treatment was increased

A B

GS

T

GST

-R2

GST

-R1

THP

-1 ly

a te

GST-R1

AxV

GST-R2

GST

FIGURE 2. Association of the intracellular domain of IFN-�R2 chain withAxV in vitro. The intracellular domain of R2 was amplified using RT-PCRfrom total RNA of THP-1 cells and cloned into pGEX4T expression vector.IPTG-induced GST-R2 fusion protein was purified using glutathione-agarosebeads. In addition, the intracellular domain of IFN-�R1 chain also was ex-pressed and purified as a GST fusion protein to use as a specificity control. A,Affinity-purified GST-fusion proteins. Lane 1, GST; lane 2, GST-R1; and lane3, GST-R2. B, Interaction of GST-R2 with AxV. PMA differentiated THP-1cell lysates containing a mixture of protease and phosphatase inhibitors wereincubated with GST-fusion proteins (R1 or R2) immobilized on glutathione-Sepharose. After washings, complexes were released by boiling in SDS sam-ple buffer, electrophoresed on 10% SDS-PAGE, and subjected to immuno-blotting with anti-AxV Ab. The data shown are from one of three independentexperiments that yielded similar results.

FIGURE 3. Association of the intracellular domain of IFN-�R2 chain withAxV in vivo. A, PMA differentiated THP-1 cells were treated with IFN-� (100U/ml for 15 min) or left untreated. After stimulation, cells were lysed in dig-itonin containing buffer supplemented with a mixture of protease and phos-phatase inhibitors. Cell lysates were incubated with anti-IFN-�R2 Abs for 2 hat 4°C. Immune complexes were recovered using protein G-Sepharose andreleased into boiling SDS-sample buffer without mercaptoethanol (nonre-duced). Soluble proteins in parallel with an aliquot of total cell lysate wereseparated on 7.5% SDS-PAGE and immunoblotted with AxV Ab. Lane 1,total cell lysate; lanes 2 and 4, immunoprecipitations of lysates from untreatedcells conducted using two different irrelevant Abs; lanes 3 and 5, immuno-precipitations of lysates from IFN-�-treated cells conducted using the sametwo irrelevant Abs; lane 6, immunoprecipitation using anti-IFN-�R2 incubatedwith lysate from control cells; lane 7, anti-IFN-�R2 incubated with lysatesfrom IFN-�-treated cells. The same blot was stripped and reprobed with anti-IFN-�R2 (B) and anti-AxII Abs (C). The data are from one of three indepen-dent experiments that yielded similar results.

FIGURE 4. AxV gene silencing in 293T cells using siRNA. A, TwoAxV-specific nucleotide sequences (21 mers) were selected using a web-based platform for the design of shRNA constructs. Chemically synthe-sized oligonucleotides were cloned into the pSHAG vector that directs thein vivo synthesis of siRNA using a U6 promoter. Plasmids were purifiedfrom positive clones and used for transfecting 293T cells. After 72 h oftransfection, cells were solubilized in boiling SDS-PAGE sample bufferand separated on SDS-PAGE, followed by immunoblotting using anti-AxVAb. B, To verify equal loading, immunoblotting also was performed withan anti-actin Ab. C, Histogram of densitometric analysis of AxV levels incontrol and siRNA down-regulated 293T cells. D, The same blot wasstripped and reprobed with Ab to AxII. Values are mean � SD of fiveindependent experiments that yielded similar results.

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two-fold, compared with that recovered from IFN-�-treated con-trol (pSHAG)-transfected cells (n � 3). These findings suggestedthat AxV may regulate IFN-� signaling pathway by controlling thelevel of tyrosine phosphorylation of Stat1�.

Influence of AxV on activation of Jak2 in response to IFN-�

Because Jak2 has been identified as a kinase involved in the ty-rosine phosphorylation of Stat1�, experiments were done to ex-amine whether activation of Jak2 is influenced by reduced level ofAxV. To investigate this possibility, we used an anti-phosphoJak2Ab that recognizes the activated kinase. As shown in Fig. 6, whencompared with control cells transfected with pSHAG and incu-bated with IFN-�, cells in which AxV was down-regulated showedan approximate twofold increase in Jak2 activation in response toIFN-�. Protein loading between samples was controlled for byimmunoblotting for actin.

AxV regulates growth arrest induced by IFN-�

To identify a phenotype associated with enhanced Jak-Stat activa-tion in cells expressing reduced levels of AxV, the antiproliferativeeffect of IFN-� was examined by two independent methodologies.In the first, the number of viable cells after 48 h of IFN-� treatmentwere counted directly by microscopy. As expected, in bothpSHAG-transfected control cells and pSHAG-AxII-irrelevant con-trol-transfected cells (expressing reduced levels of AxII and con-

trol levels of AxV), IFN-� treatment brought about partial growtharrest (Refs. 26–28 and Fig. 7A). Of interest was the finding thatthe antiproliferative effect of IFN-� was significantly potentiated incells transfected with pSHAG AV2.1, which expressed reducedlevels of AxV (n � 3). It should be pointed out that down-regu-lation of AxV per se, in the absence of IFN-�, did not inducegrowth arrest, compared with untreated control cells (Fig. 7A). Inthe second proliferation assay, we used thymidine incorporationinto DNA to assess the effect of AxV down-regulation on the an-tiproliferative action of IFN-�. As shown in Fig. 7, E and F, basedon thymidine incorporation, AxV down-regulation was again ob-served to bring about enhanced growth arrest mediated by IFN-�,compared with pSHAG-transfected cells treated with IFN-�.

DiscussionGiven its central role in both immune and inflammatory responses,regulation of IFN-� action is a subject of considerable interest.Intuitively, modulation of IFN-� signaling proximally at the levelof the IFN-�R per se would appear to be an ideal locus to exertcontrol. Whereas there is considerable knowledge about negativeregulation of IFN-� signaling at the level of receptor-associatedproteins such as Jak1, Jak2, and Stat1� (29), information aboutregulatory molecules that target receptor subunits directly is lim-ited. Previously, the human DnaJ protein hTid-1 was identified asa Jak2 binding protein in a yeast two-hybrid screen (19). hTid-1also was shown to bind directly to IFN-�R2 in transfected COScells and to form a trimolecular complex with Jak2 and IFN-�R2in COS cells simultaneously transfected with DNA constructs cor-responding to all three proteins (19). Importantly, these associa-tions were found not to require prior incubation of cells withIFN-�. In the present study, AxV was identified as a novel IFN-�R2 binding protein in a yeast two-hybrid screen,. This interactionwas confirmed in GST-IFN-�R2 fusion protein pull-down exper-iments (Fig. 2) and by coimmunoprecipitation of IFN-�R2 and

FIGURE 5. Down-regulation of AxV promotes enhanced tyrosinephosphorylation of Stat1� in response to IFN-�. A, 293T cells were trans-fected with pSHAG or pSHAG AV2.1 plasmids. After 24 h of transfection,cells were washed and rested for another 48 h in complete medium. Cellswere then either untreated or incubated with IFN-� (100 U/ml) for either 15min or 4 h. Cells were then lysed and immunoprecipitated with anti-Stat1�Ab. Immunoprecipitated proteins were separated by SDS-PAGE, trans-ferred to a nitrocellulose, and probed with anti-phosphotyrosine Abs. Thesame membrane was stripped and probed with anti-Stat1� (B) and anti-AxV Abs (C). D, Results of densitometric scanning of the results in A. Thedata are from one of three independent experiments that yielded similarresults.

FIGURE 6. Down-regulation of AxV promotes IFN-�-induced tyrosinephosphorylation of Jak2. A, 293T cells were transfected with pSHAG orpSHAG AV2.1 plasmids. After 24 h of transfection, cells were washed andleft for another 48 h in complete medium and subsequently either untreatedor incubated with IFN-� (100 U/ml) for 4 h. Cells were then lysed directlyin SDS sample buffer and separated on SDS-PAGE, followed by immu-nodetection using anti-phosphoJak2. B, To verify equal loading, immuno-blotting was performed with an anti-actin Ab. C, AxV levels in treatmentgroups by immunoblotting. D, Densitometric scanning of the results in A.The data are from one of three independent experiments that yielded sim-ilar results.

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AxV from IFN-�-treated THP-1 cells (Fig. 3). The finding fromthe coimmunoprecipitation indicated that the interaction was li-gand inducible, and this suggested that AxV may be involved inregulating IFN-� signaling. To the best of our knowledge, this isthe first identification of an inducible association between IFN-�R2 and another cellular protein.

Whereas the biochemical data clearly showed that the interac-tion was ligand inducible (Fig. 3), the yeast two-hybrid screensuggested that the interaction between AxV and R2 was ligandindependent. Although these findings may appear to be contradic-tory, there are other reports in the literature of interactions detectedby yeast two- or three-hybrid screening that were found to be li-gand inducible under native conditions (30–32). There are severalpotential mechanisms to explain this apparent paradox. The ob-servation that a protein-protein interaction is induced by ligand inintact cells may reflect conformational changes, changes in sub-cellular localization, or a posttranslational modification in one orboth proteins. For example, under native conditions, there is thepossibility that a conformational constraint in either one or both ofthe interacting partners may limit the opportunity for an interactionto take place. In the case of AxV and IFN-�R2, IFN-� may inducechanges in the cell leading to removal of a conformational restric-tion, thus promoting an interaction between the binding partners.In contrast, in the yeast two-hybrid system, proteins are importedinto the nucleus where they reside ectopically, and if an interactionbetween bait and prey proteins occurs, a functional transcription

factor is reconstituted, leading to transcription of reporter genes.Ectopic localization may result in a conformational change in oneor both binding partners that favors an interaction. Ectopic expres-sion also may overcome constraints related to subcellular local-ization that exist under native conditions. Notably, the interactionbetween AxV and IFN-�R2 observed in the two-hybrid screeninvolved an AxV clone containing only domains III and IV, in-cluding the C terminus. Because of this, we favor the possibilitythat, under normal conditions, the presence of N-terminal domainsI and II may prevent binding to IFN-�R2 and that this constraint isremoved upon exposure to ligand. However, we cannot excludethe possibility that ligand dependency was related to effects onsubcellular localization or to a posttranslational modification.

Of interest, both in the case of hTid-1 mentioned above (19) andAxV (Figs. 5–7), the evidence suggested that these associationswith IFN-�R2 negatively regulated IFN-� action. The discovery ofthis novel interaction between IFN-�R2 and AxV raises importantquestions about the basis for and biological consequences of theinducible association.

Annexins are a family of 13 structurally related calcium-dependentphospholipid binding proteins (33). They share a conserved 70-aarepeat sequence and have variable N-terminal domains. In general,annexins have been implicated in the maintenance of calcium ho-meostasis, signal transduction, receptor-mediated endocytosis, cellproliferation and growth, ion channel formation, and the secretion ofneurotransmitters and hormones (34, 35). AxV, in particular, has been

FIGURE 7. Reduced levels of AxV potentiate IFN-�growth arrest in HeLa cells. HeLa cells were transfectedwith pSHAG, pSHAG AxII, or pSHAG AV2.1 plas-mids. After 24 h, cells were washed and left for another48 h in complete medium and subsequently were seededin 12-well plates at 0.5 � 105 cells per well. After over-night incubation, cells were either untreated or incu-bated with IFN-� (100 U/ml) for 48 h. Cells were thendislodged and counted in a hematocytometer. A, Thegrowth of the cells in the presence of IFN-� normalizedin respect to the growth of corresponding untreated con-trol cells. The values shown are the mean � SD of re-sults obtained in three independent experiments. For[3H]thymidine incorporation into DNA, control andAxV down-regulated cells were seeded into six-wellplates at 1.0 � 105 cells per well. After overnight incu-bation, cells were either untreated or incubated withIFN-� (100 U/ml) for16–18 h followed by labeling with[3H]thymidine for 8 h, and incorporated [3H]activitywas measured by liquid scintillation counting (E and F).B–D, Results of Western blotting to confirm the specificdown-regulation of AxII (B) or AxV (C). Actin wasused as a loading control. The values shown are themean SD of results obtained in three independentexperiments.

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implicated in regulating cell proliferation (36) and as a protein kinaseC inhibitor (37, 38).

Recently, AxV was shown to bind to and regulate signalingthrough the vascular endothelial growth factor receptor (VEGFR)(39). The finding that AxV bound to the IFN-�R through an as-sociation with the R2 subunit suggests the possibility of a previ-ously unrecognized role for this protein in regulating signal trans-duction in response to diverse agonists. At present, it is not clearwhat accounted for the inducible association of the AxV with R2and whether this was influenced by changes in levels of cytosolicfree calcium or by the phosphorylation status of AxV. Tyrosinephosphorylation of AxV also could potentially facilitate its recruit-ment to IFN-�R2 by stabilizing its interaction with the receptorcomplex through interactions with other signaling components,such as Grb2 or other SH2 adaptor proteins. In this regard, AxVhas been shown to be tyrosine phosphorylated in response to celltreatment with VEGF (39). Nevertheless, when AxV was shown tobe a VEGFR-binding protein, neither the yeast two-hybrid screennor the vitro translation system used to demonstrate this interaction(39) would have allowed for tyrosine phosphorylation of AxV tohave taken place. In fact, in the present study, we were unable tofind any evidence for a change in tyrosine phosphorylation of AxVin response to IFN-� (data not shown). Therefore, this posttrans-lational modification of the protein would not seem to explain itsinteraction with IFN-�R2 in ligand-treated cells.

Another possibility to consider is that the interaction of R2 withAxV could have been influenced by changes in the serine phos-phorylation status of AxV. It has been reported that serine phos-phorylation of AxV promotes its recruitment to the plasma mem-brane (40) and the induction of serine phosphorylation of AxIpromoted its association with the glucocorticoid receptor in re-sponse to ligand (41). However, this would appear unlikely toexplain the inducible association of R2 with AxV, because wewere unable to detect any change in the serine phosphorylationstatus of AxV in IFN-�-treated cells (data not shown).

It is known that cellular responses to IFN-� can be criticallyinfluenced by internalization of R2 (42–46). Because annexinshave been implicated in receptor-mediated endocytosis, it was of

interest to investigate whether down-regulation of AxV inducedcell surface accumulation of R2. This was examined by flow cy-tometry, and the results showed that cell surface expression of R2was equivalent in control and AxV down-regulated cells (data notshown). This finding ruled out the possibility that the observeddifferences in IFN-� signaling were due to the accumulation of R2in the face of reduced levels of AxV.

Given that calcium levels are known to influence the associationof annexins with other proteins (47–49), it also is possible that aconformational change in AxV, the R2 subunit or both, due to anincrease in intracellular free calcium induced by IFN-� (50), couldhave influenced the association of these proteins. However, thispossibility seems unlikely because buffering intracellular free cal-cium with the known calcium chelator BAPTA-AM (51) failed toblock this event (data not shown). We expect that a more detailedunderstanding of the molecular basis for the interaction betweenR2 and AxV will be facilitated by identifying the amino acid res-idues involved in this interaction.

To examine the role of AxV in regulating IFN-� signaling, wetransfected 293T cells with a pSHAG vector expressing siRNA-targeting AxV sequences. The pSHAG system expressing siRNAhas been shown to efficiently silence both endogenous and exog-enous genes in 293T cells (24). The 293T cells also were selectedfor this purpose both because of the ease with which they aretransfected and because they respond to IFN-� with activation ofthe Jak-Stat1� pathway (Refs. 10 and 52 and Figs. 5 and 6). Aphenotype of hyperresponsiveness to IFN-� in cells with reducedlevels of AxV was initially suggested by antiphosphotyrosine blotsof whole cell lysates. These showed increased tyrosine phosphor-ylation of a 90-kDa protein in IFN-�-treated pSHAG AV2.1-trans-fected cells, compared with pSHAG vector-transfected controlcells (data not shown). The size of this protein suggested that itmight have been Stat1� and prompted investigation of the phos-phorylation status of this IFN-�-regulated transcription factor. Infact, after immunoprecipitation and anti-phosphotyrosine blotting,we found direct evidence for enhanced tyrosine phosphorylation ofStat1� in IFN-�-treated cells with reduced content of AxV (Fig.5). Increased tyrosine phosphorylation of Stat1� was not explained

FIGURE 8. Proposed model for the roleof AxV in regulating IFN-� cell signaling. A,In the absence of ligand, AxV is not associ-ated with the IFN-�R. B, Upon exposure ofcells to IFN-�, AxV is recruited to the R2receptor subunit. The association of AxVwith R2 controls the phosphorylation and ac-tivation states of Jak2 and Stat1�, therebyregulating downstream responses. C, Underconditions where the abundance of AxV islimiting, diminished association of regulatorwith R2 leads to enhanced tyrosine phos-phorylation of Jak2 and Stat1� and to aug-mented cellular responses to IFN-�.

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by increased protein abundance (Fig. 5B) but did correlate withenhanced activation of Jak2 in response to IFN-� (Fig. 6). Of in-terest, enhanced phosphorylation of Stat1� was observed in cellsthat had been exposed to ligand for 4 h, but not in cells treated foronly 15 min. One potential model to explain these findings is thatAxV may recruit a protein tyrosine phosphatase to the vicinity ofthe receptor that acts to modulate the phosphorylation status ofStat1�. Abnormal phosphatase recruitment in cells deficient inAxV could result in sustained and heightened tyrosine phosphor-ylation of Stat1�.

Recent reports indicate that a nonspecific IFN response tosiRNA may occur with a frequency of 30%, although dose-re-lated effects can drive this higher (53–55). Such a response couldhave complicated interpretation of the phenotype that we obtainedin cells with reduced levels of AxV. Three lines of evidence fromour analysis argue strongly against a nonspecific IFN response tosiRNA in our system. First, whereas inhibition of cell growth is ahallmark of the IFN response, introduction of the AxII siRNAcontrol construct into cells did not have an antiproliferative effect(Fig. 7). Moreover, we independently confirmed that this siRNAconstruct was functional leading to reduced expression of the AxIIgene (Fig. 7). Second, in this same experiment, when siRNA con-struct AV2.1 directed at AxV was expressed in HeLa cells in theabsence of IFN-� treatment, no inhibition of cell proliferation wasobserved. Third, induction of IFN responsive genes such as Stat1has been used to monitor nonspecific IFN responses to siRNA(54). However, when we transfected cells with the AV2.1 siRNAconstruct, no induction of Stat1� expression was observed (Fig.5B). Based on these complementary controls, we concluded thatthe impact of silencing the AxV gene was not complicated by anonspecific IFN response.

Taken together, these results suggested that the interaction ofAxV with R2 controls activation levels of Jak2 and Stat1�, andthey are consistent with a model in which AxV normally regulatessignaling through this pathway. One possible mechanism to ex-plain this could involve the association of AxV with R2 interferingwith Jak2 binding because of either overlapping binding sites orsteric hindrance. In this model, reduced levels of AxV would in-crease access of Jak2 to R2 and enhance its activation, and sec-ondarily that of Stat1�, in response to IFN-�. Another possibilityis that AxV may inhibit Jak2 activation either directly or indirectly,as discussed above in the context of Stat1�, by promoting therecruitment of a phosphotyrosine phosphatase to the vicinity of thereceptor.

Although the data available do not allow discrimination betweenthese various alternatives, the model does allow for predictions tobe made. For example, if AxV normally controls the level of Jak2-Stat1 activation, then removing this influence should lead to en-hanced cellular responses to IFN-�. To investigate the impact ofenhanced activation of Jak2 and Stat1� in cells with reduced levelsof AxV, we evaluated IFN-�-induced growth inhibition. Inhibitionof cell proliferation is a hallmark of the IFN response (26–28), andthe antiproliferative response to IFN-� has been shown to be de-pendent on activated Stat1� (26). In the latter study, cells trans-fected with either wild-type Stat1� and/or Stat1� mutants wereexamined. The introduction of wild-type Stat1� enhanced the an-tiproliferative effect of IFN-�, whereas Stat1� mutants reversedIFN-�-induced growth arrest. Consistent with this model, we ob-served that, compared with control cells, the antiproliferative effectof IFN-� was enhanced in cells expressing reduced levels of AxV,but not in those with reduced levels of AxII (Fig. 7). These resultsprovide direct evidence to show modulation of biologic responsesto IFN-� specifically by AxV and suggest that this may be relatedto effects on Jak-Stat1� activation.

In summary, the results of the present study identify a novel rolefor AxV as a ligand-inducible IFN-�R2-binding protein. The pre-dominant impact of this interaction is negative regulation of IFN-�signaling, and this may represent a physiologic mechanism to con-trol signaling through this pathway (Fig. 8). As such, these findingsadd to what is already known about the complex regulation ofIFN-� signaling through other control elements such as SOCS pro-teins (18, 56), SHP-1 and SHP-2 (14, 17, 57–60), hTid-1 (19), andregulated expression of the IFN-�R2 chain itself (61). Finally, al-though the model shown in Fig. 8 suggests that AxV modulatessignaling through the IFN-�-activated Jak-Stat1 pathway, it doesnot preclude the possibility that this interaction also may positionAxV to influence Stat1-independent signaling elements that areactivated downstream of the IFN-�R.

AcknowledgmentsWe thank Dr. Alice Mui (University of British Columbia) for providingcells and monocyte RNA, and Dr. Martin Sims (GlaxoSmithKline) forproviding the pYTH9 shuttle vector and yeast strain SDY191. The tech-nical assistance of Richard Ly and Chrystal Lapinsky is gratefullyacknowledged.

DisclosuresThe authors have no financial conflict of interest.

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