Proteome Characterization of Human NK-92 Cells Identifies Novel IFN

and chemokines, including IFN-γ and TNF-R/β,1,2 that have antimicrobial effects. ... in continuous culture in MEM Alpha Medium (Gibco, Life. Technol...
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Proteome Characterization of Human NK-92 Cells Identifies Novel IFN-r and IL-15 Target Genes Riitta Rakkola,† Sampsa Matikainen,†,‡ and Tuula A. Nyman*,† Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland and National Public Health Institute, Helsinki, Finland Received August 11, 2004

Natural killer (NK) cells are important components of innate immune defense. NK cells kill virus-infected cells and secrete cytokines that are involved in activation of other immune cells. Macrophage-derived cytokines interferon-R (IFN-R) and interleukin-15 (IL-15) are in turn important activators of NK cells, but the receptors and intracellular pathways that are involved in NK cell functions are still incompletely known. Here we have used expression proteomics to find new IFN-R and IL-15 regulated proteins in human NK-92 cells, which have the characteristics of activated NK cells. Cells were stimulated with cytokines for 20 h, lysed, and soluble proteins were separated by two-dimensional electrophoresis, and differentially expressed protein spots were identified with mass spectrometry and database searches. A total of 57 protein spots were found to be reproducibly differentially expressed between control and cytokine stimulated gel pairs, 26 spots being more than 2-fold upregulated and 3 spots being at least 2-fold downregulated. The rest 28 spots showed minor, less than 2-fold changes in their expression levels after quantification. From the differentially expressed protein spots we identified 47 different proteins, most of which are new IFN-R and IL-15 target proteins. Interestingly, we show that e.g., adenylate kinase 2 is highly upregulated by IFN-R and IL-15 stimulation in NK-92 cells. The expression of selected genes with high expression level differences after cytokine stimulation were further studied at mRNA level. Northern blot analysis showed that the genes studied were induced by IFN-R, IL-15, and IL-2 already at 3 h time point, suggesting that they are primary target genes of these cytokines. Keywords: two-dimensional electrophoresis • mass spectrometry • interleukin-15 • interferon-R • NK cells

1. Introduction Natural killer (NK) cells are important cellular mediators of innate immune defense. They are lymphoid cells that, without previous sensitization, can recognize and kill virus-infected and transformed cells. Activated NK cells also produce cytokines and chemokines, including IFN-γ and TNF-R/β,1,2 that have antimicrobial effects. In addition, NK cell-derived cytokines activate other immune cells. NK cell responses are stimulated by cytokines secreted by macrophages during microbial infection, such as IFN-R/β, IL-12, IL-15, and IL-18.3 IFN-R is the key cytokine in the development of innate immune responses against viruses.4 In synergy with IL-18, IFN-R enhances IFN-γ production in NK and T cells.5-7 IFN-γ in turn enhances antigen presentation by DCs and macrophages. This results in effective immune response and ultimately cytotoxic T cells eliminate virus-infected cells. In addition to IFN-R, IL-15 has important functions in NK and T cells. IL-15 is essential for peripheral T * To whom correspondence should be addressed. Current address: Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, FIN-00250 Helsinki, Finland. Tel: +358 30474 2124. E-mail: [email protected]. † Turku Centre for Biotechnology, University of Turku and Åbo Akademi University. ‡ National Public Health Institute. 10.1021/pr049857b CCC: $30.25

 2005 American Chemical Society

cell maturation and it is required for NK cell maturation in vivo. IL-15 receptor (IL-15RR and IL-2/-15Rβ) or IL-15 knock-out mice do not have NK cells.8,9 Despite the importance of IFN-R and IL-15 to NK cell functions, the target genes of these cytokines are still incompletely known. The development of transcriptomics and proteomics techniques has made it possible to do large-scale gene expression profiling studies at the mRNA and protein level, respectively. Transcriptome studies are done using DNA- or oligonucleotide microarrays, whereas in proteome studies one has to combine multiple techniques for protein separation, identification, and characterization. The key techniques in current proteome studies are two-dimensional electrophoresis (2-DE) for protein separation and mass spectrometry (MS) for protein identification and characterization. 2-DE was introduced already in 1975,10 but it is still the most powerful technique available to separate complex protein mixtures. Important for proteomics has also been the developments in biological mass spectrometry, especially the invention of two soft ionization techniques, matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI), which have made protein identification at the low femtomole-level possible. Even though proteomics experiments are technically more challenging than Journal of Proteome Research 2005, 4, 75-82

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research articles studies at the mRNA level, it is obvious that proteome studies are needed to complement transcriptomics results to get a more complete picture of a given biological system. The effects of IL-15 in lymphocytes have been studied using microarrays.11,12 Similarly, IFN-R target genes have been identified using oligonucleotide microarrays,13 and a database of these results is available (http://www.lerner.ccf.org/labs/williams/). We have previously used proteomics to identify IFN-R regulated proteins in T cells,14,15 but to our knowledge no studies utilizing cytokine-stimulated NK cells and proteomics tools have been published. In this paper we have used 2-DE combined with MS to identify IFN-R and IL-15 target proteins in human NK-92 cells which have been widely used to study functional characteristics of activated NK cells.(6,7,16-20) With this approach we identified 47 different proteins, most of which are new IFN-R and IL-15 target genes.

2. Materials and Methods NK-92 Cell Line. Human NK-92 cell line21 was maintained in continuous culture in MEM Alpha Medium (Gibco, Life Technologies) supplemented with 12% horse serum (Gibco), 12% FCS (Integro, Zaandam, The Netherlands), 0.2 mM iinositol, 20 mM folic acid, 40 mM β-mercaptoethanol, 2 mM L-glutamine, antibiotics and 100 IU/mL rIL-2 (R&D Systems, Abingdon, United Kingdom). Cytokines. Human leukocyte IFN-R was provided by Dr. Hannele To¨lo¨ (Finnish Red Cross Blood Transfusion Service) and was used at 100 IU/mL. Human rIL-2 and rIL-15 was purchased from R&D Systems and were used at 100 IU/mL and 5 ng/mL, respectively. Stimulation and Labeling of NK-92 Cells. Before cytokine stimulation and metabolic labeling, NK-92 cells were preincubated in a methionine and cysteine-free RPMI medium (Gibco) for 30 min. After this, Redivue Pro-mix L-[35S] in vitro cell labeling mix (Amersham Biosciences, Buckinghamshire, England) and cytokines were added to cell culture medium after which the cells were cultured for 20 h. 2-DE. Protein separation by 2-DE was performed as previously described.22 Briefly, cells were lysed and soluble proteins were absorbed into the 18 cm 3-10 NL IPG-strips (Amersham Biosciences) for 24 h at RT. Isoelectric focusing to a total of 40 kVh was done at +20 °C, and focused strips were equilibrated for 25 min at RT. The second dimension was vertical 12% SDSPAGE with gel thickness of 1 mm. Proteins were detected with silver staining and autoradiography. Autoradiography images were used for the comparison of protein expression levels. Gel comparison was done both manually and with the PDQuest program (version 7.0.1, BioRad, Hercules, CA), and all the gel images were normalized before the comparison. Protein Identification. Differentially expressed proteins were identified with peptide mass fingerprinting (PMF) as previously described.22 Briefly, the gel spot was cut into pieces, which were washed twice with 0.2 M NH4HCO3/ACN (1:1), and dehydrated with ACN. The proteins were reduced with 20 mM dithiothreitol (Sigma), followed by alkylation with 55 mM iodoacetamide (Sigma). The proteins were in-gel digested with trypsin (Sequencing Grade Modified Trypsin, Promega Corporation, Madison, WI) at +37 °C overnight, and the obtained peptides were purified and concentrated using reverse phase nanocolumns (Poros Oligo R3, PerSeptive Biosystems, Framingham, MA). Peptides were directly eluted into the sample plate with saturated R-cyano-4-hydroxycinnamic acid (HCCA, Aldrich Chemical Co. Ltd, Dorset, U.K.) in 0.1% TFA, 60% ACN. Peptide 76

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masses were measured with MALDI-TOF MS (Voyager DE PRO, PerSeptive Biosystems, Houston, Texas, USA) in positive ion reflector mode. Spectra were internally calibrated using autoproteolytic trypsin fragments, or with standard peptides (Cal Mix 2, Sequazyme peptide mass standard kit, PE Biosystems, Framingham, MA). Database searches were done with Mascot (http://www.matrixscience.com) program against NCBI and SWISS-PROT databases. In cases where PMF did not result in positive identification, the peptides after in-gel digestion were analyzed by nanoLCMS and -MS/MS. For this, the peptides were extracted twice with 150 µL of 5% formic acid/50% acetonitrile, the extracts were pooled, and dried in a vacuum centrifuge. The resulting peptides were analyzed by automated nanoscale capillary LCMS/MS using an Ultimate capillary LC system, Famos autosampler, and Switchos II (LC Packings, The Netherlands) coupled to a quadrupole time-of-flight mass spectrometer (QStar Pulsar, ABI/MDS-SCIEX, Toronto, Canada). The peptides were dissolved in 15 µL 2% formic acid, and desalting and concentration were done with a 300 µm × 5 mm PepMap -precolumn (LC Packings, The Netherlands). Reversed-phase separations were carried out using a 75 µm × 15 cm Pepmapcolumn at a flow rate of 200 nl/min. Solvent A was 0.1% formic acid/5% ACN and solvent B was 0.1% formic acid/95% ACN. Peptide separation was done with a linear gradient of 5-60% solvent B in 30 min. Database searches were done with program Mascot (www. matrixscience.com). RNA Isolation and Northern Blot Analysis. NK-92 cells were treated with cytokines for 3, 6, 12, and 24 h. Total cellular RNA was isolated by Rneasy kit (Qiagen, Valencia, CA) according to manufacturers instructions. Samples containing equal amounts of RNA (10 µg) were size fractionated on 1% formaldehydeagarose gel, transferred to a nylon membrane (Hybond; Amersham, Buckhingshamhire, U.K.) and hybridized with different probes. Probes for adenylate kinase 2 (AK2), gamma-actin, glucose-regulated protein 58 (GRP58/ER-60/ ERp57), heat shock 70 kDa protein (MTHSP75/PBP74/GRP75/mortalin), and leucine aminopeptidase (LAP) were RT-PCR-cloned from total RNA obtained from IL-15-induced NK-92 cells. The PCR was performed with 5′ to 3′ primers listed in Table 1. The PCR products were subcloned into BamHI site of pGEM3-Zf(+) vector (Promega, Madison, WI) and sequenced. Interferon stimulated gene 15 (ISG15) probe was amplified from ISG15 cDNA with following oligonucleotides TGACGGTGAAGATGC (sense), and TCGAAGGTCAGCCAG (antisense). Ethidium bromide staining of ribosomal RNA bands was used to ensure equal RNA loading. The probes were labeled with [R-32P] dATP (3000 Ci/mmol, Amersham) using a random primed DNA labeling kit (Boehringer Mannheim, Mannheim, Germany). The membranes were hybridized in Ultrahyb buffer (Ambion, TX) overnight after which they washed twice at room temperature and once at +60 °C in 1 X SSC/0.1% SDS for 30 min and exposed to Kodak AR X-omat films at -70 °C using intensifying screens.

3. Results During viral infection, macrophages simultaneously secrete IFN-R and IL-15, which activate NK cells. In the present study we have used expression proteomics to find new IFN-R and IL-15 regulated proteins in human NK-92 cells. These cells have the characteristics of activated NK cells, and therefore they are a useful model to study NK cell biology. The cells were

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Figure 1. Representative 2-DE protein patterns from control and IFN-R and IL-15 stimulated human NK-92 cells. Control and cytokine stimulated NK-92 cells were lysed, and soluble proteins were separated by 2-DE. Proteins in the resulting gels were detected by autoradiography (A) and silver-staining (B). Autoradiography images were used for protein quantification, and the differentially expressed protein spots are marked to the images.

stimulated with IFN-R and IL-15 and labeled with 35S-Met/Cys for 20 h. After that, the cells were harvested and lysed, and soluble proteins were separated by 2-DE. Proteins in the resulting gels were detected by silver-staining and autoradiography. To find up- and downregulated protein spots by IFN-R and IL-15 the autoradiography patterns obtained from control and cytokine stimulated NK-92 cells were first compared manually and then with the program PDQuest. A total of 57 protein spots were found to be differentially expressed in at least two independent experiments. The differences in expression levels were quantified from the autoradiography patterns using software PDQuest. From the 57 differentially expressed spots found from the 2-DE gels by manual inspection and PDQuest analysis, 26 spots were at least 2-fold upregulated by IFN-R and IL-15 treatment, 2 spots were present only in control gels, 1 spot was almost 3-fold downregulated, and 28 spots showed minor, less than 2-fold differences in expression levels. A representative silver-stained and autoradiography 2-DE gel images from control and cytokine stimulated cells are shown in Figure 1, and the differentially expressed protein spots are marked to the images.

Table 1. RT-PCR Primers

For identification, the differentially expressed protein spots were cut out from the gels, in-gel digested, and the resulting peptides were analyzed by MS and database searches. From the 57 spots, we identified proteins from 47 spots. In the remaining 10 spots the protein amount was too low for identification, even when spots were pooled from several 2-DE gels before in-gel digestion.The identification results together Journal of Proteome Research • Vol. 4, No. 1, 2005 77

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Table 2. Differentially Expressed Proteins from IFN-R and IL-15 Stimulated Human NK-92 Cellsa

a na: no corresponding access number found. nm: could not be measured because there were no comparable spot in cytokine stimulated gel *In spots 6-a, 13, 15-c, 17-a, 24-b, and 28-c, two proteins were identified from one spot, and the fold difference implies to the corresponding spot. The protein spots indicated in Figure 1 were quantitated from normalized autoradiography images using the software PDQuest, and identified using mass spectrometry and database searches. All of the identification results were significant (p < 0.05) according to database search program Mascot. After identification the proteins were classified into different functional classes according to their known function.

with the expression level differences obtained with PDQuest analysis are shown in Table 2. The identified proteins were also classified into different classes according to their known cellular functions (Table 2). Two different proteins were identified from two spots that were more than 2-fold upregulated by IFN-R and IL-15 treatment: spot 13 contained both leucine aminopeptidase and glucose-6-phosphate dehydrogenase and spot 28-c PESTcontaining nuclear protein and GTP-binding protein SAR1a. Also four spots showing less than 2-fold upregulation were found to contain two diffent proteins, namely spot 6-a cleavage stimulation factor subunit 2, 64 kDa isoform and far upstream element binding protein 2, spot 15-c glutathione synthetase and similar to tubulin alpha 2 -protein, spot 17-a ErbB3 binding protein EBP1 and proliferation-associated protein 2G4, and 78

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spot 24-b breast carcinoma amplified sequence 2 -protein and pyruvate kinase. From these proteins leucine aminopeptidase is known to be upregulated by IFN-R, and it is likely that it has contributed to the observed upregulation also in this study. However, without further studies it is not possible to say whether only one of the proteins or both proteins in the spot have contributed to the observed differences in expression levels. GTP-binding protein SAR1a (spots 28-a, b, and c) was identified from three different spots. From the three spots containing SAR1a, two spots (28-a and 28-c) were upregulated by IL-15 and IFN-R stimulation, and spot 28-b was present only in control gels. However, spot 28-c also contained PESTcontaining nuclear protein, which might have also contributed to the upregulation of the spot. Four proteins were identified

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Figure 2. IFN-R induces AK2, GRP58, ISG15, LAP, and MTHSP75 mRNA syntheses in NK-92 cells. NK-92 cells were stimulated with IFN-R for 3, 6, 12, and 24 h. Total cellular RNA was isolated and Northern blot analyses with AK2, GRP58, ISG15, LAP, and MTHSP75 probes were performed. Ethidium bromide staining of rRNA bands was used to confirm equal RNA loading.

from two different spots, namely cleavage stimulation factor subunit 2 (spots 3 and 4-b), far upstream element binding protein 1 (spots 7-a and -b), IFP-53 (spots 12 and 20-a), and pyruvate kinase (spots 21 and 24-b). Both spots containing far upstream element binding protein 1 and IFP-53 showed higher expression in IFN-R and IL-15 stimulated NK-92 cells than in unstimulated cells suggesting that the total expression of these proteins is higher after cytokine treatment. With CSTF2, spot 3 was slightly upregulated and spot 4-b downregulated after cytokine treatment. In addition to these two spots, we identified another isoform of CSTF2, namely cleavage stimulation factor subunit 2, 64 kDa isoform (spot 6-a) to be upregulated by IFN-R and IL-15 treatment. Also, from T-complex protein 1, which is composed of at least eight subunits, we identified three subunits (spots 2-b, 9 and 10) to be upregulated by IFN-R and IL-15 stimulation. We also identified different heterogeneous nuclear ribonucleoproteins from many spots (6-b, 22, 30-b, and 33-b), which are mainly involved in transcription and RNA processing. Pyruvate kinase exists in the database as multiple isoforms with almost the same sequences, and it was not possible to determine the exact isoform(s) present in our 2-DE gels based on PMF data obtained as we could not find any peptide(s) matching to only single isoform. From the two spots containing pyruvate kinase, spots 21 and 24-b, the former was 2.8-fold downregulated whereas the latter was 1.5-fold upregulated by IFN-R and IL-15 treatment. However, spot 24-b also contained another protein, namely breast carcinoma amplified sequence 2 which might affect to the upregulation in the spot. The

theoretical MW of pyruvate kinase is 58.5 kDa, whereas the theoretical MW of breast carcinoma amplified sequence 2 is 26.2 kDa, and the spot location in the 2-DE gels corresponds better to the latter MW. When looking at the PMF data obtained from the two pyruvate kinase spots, only peptides from the N-terminal side were found, covering amino acids 33-294 in spot 21 and amino acids 33-224 in spot 24-b suggesting that pyruvate kinase is present in the gels as a N-terminal fragment. Several studies have shown that protein and mRNA levels do not always correlate well.23-26 Thereby selected IFN-R and IL-15 regulated proteins in NK-92 cells were further studied at the mRNA level using Northern blot analysis. The cells were stimulated with IFN-R, IL-2, or IL-15 for 3, 6, 12, or 24 h, total cellular RNA was isolated, and Northern blot analyses were performed. IL-2 was included to our studies since it has been shown that IL-2 and IL-15 induce a highly similar set of genes in lymphocytes.11 Only proteins with high expression level differences after IFN-R and IL-15 treatment and/or with significant biological importance were chosen. The genes studied were following: adenylate kinase 2 (AK2), gamma-actin, glucose-regulated protein 58 (GRP58/ER-60/ ERp57), heat shock 70 kDa protein (MTHSP75/PBP74/GRP75/mortalin), interferon stimulated gene 15 (ISG15), and leucine aminopeptidase (LAP). IFN-R clearly activated AK2, ISG15, and LAP mRNA expression at all timepoints studied in NK-92 cells (Figure 2). IFN-R treatment also upregulated GRP58 mRNA expression, although to a lesser degree than with AK2, ISG15, and LAP (Figure 2). In contrast, MTHSP75 mRNA expression was only slightly induced by IFN-R treatment (Figure 2). Both IL-2 and IL-15 induced clear AK2, LAP, ISG15, and MTHSP75 mRNA expression in NK92 cells (Figure 3). IL-2 and IL-15 stimulation of NK-92 cells had a modest enhancing effect on GRP58 mRNA expression (Figure 3). In contrast to other genes studied, cytokine treatment had no effect on gamma-actin mRNA expression in NK92 cells (data not shown).

4. Discussion NK cells are important components of innate immune defense. They recognize and kill virus-infected and transformed cells. NK cells are activated by macrophage-derived cytokines which enhance their cytotoxicity and IFN-γ production. During virus infection IFN-R and IL-15 are the most important activators of NK cells.3 NK cell-derived IFN-γ in turn activates macrophages and promotes adaptive Th1 immunity, which is involved in the eradication of microbial pathogens.27,28 In this report we have used expression proteomics to find and identify novel IFN-R and IL-15 regulated proteins in NK-92 cells. We have previously used this methodology, 2-DE for protein separation and quantification, and MS for protein identification to characterize human T cells after various treatments.14,15,29 In this study, 57 protein spots were found in the comparison between control and cytokine stimulated gel pairs to be differentially expressed. Twenty-six spots were more than 2-fold up-regulated and 3 spots were at least 2-fold down-regulated by IFN-R and IL-15 in NK-92 cells. From the 57 differentially expressed spots we were able to identify proteins from 47 spots, whereas the remaining 10 spots contained too little material for identification. From the separation methods available in proteome studies, 2-DE is the method of choice for very complex proteins mixtures. However, when trying to separate proteins from total cell lysate using only one, wide-range pI-gradient in the first Journal of Proteome Research • Vol. 4, No. 1, 2005 79

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both IFN-R and IL-15 in NK-92 cells. However, further studies are required to clarify the role of adenylate kinase in NK cell biology. Several reports have demonstrated that protein and mRNA expression levels do not always correlate with each other.23-26 In this report, we found out that γ-actin protein expression is upregulated by IFN-R and IL-15 in NK-92 cells (Figure 1) despite that these cytokines had no effect on γ-actin mRNA expression (data not shown). Therefore, our results suggest that γ-actin expression is regulated at postranscriptional level. It is possible that cytokine treatment of NK cells enhances γ-actin protein half-life which could lead to enhanced accumulation of γ-actin protein. In accordance with our study it has been shown that IL-15 upregulates cytoskeletal actin (β and γ) expression in human neutrophils.34 Actin is a cytoskeletal protein and one of the most highly conserved proteins in evolution.35 This is likely due to its major role in basic functions in cell motility, phagocytosis, cell permeability, movement of organelles, anchorage of many surface receptors, signal transduction, cell-cell interactions and mRNA localization.36 Therefore, it is possible that cytokine-induced upregulation of γ-actin protein expression contributes to NK cell responses.

Figure 3. IL-2 and IL-15 induce AK2, GRP58, ISG15, LAP, and MTHSP75 mRNA syntheses in NK-92 cells. NK-92 cells were stimulated with IL-2 or IL-15 for 3, 6, 12 or 24 h. Total cellular RNA was isolated and Northern blot analyses with AK2, GRP58, ISG15, LAP, and MTHSP75 probes were performed. Ethidium bromide staining of rRNA bands was used to confirm equal RNA loading.

IFN-R and IL-15 upregulated both GRP58 mRNA and protein expression in NK-92 cells. GRP58 is a protein chaperone with thiol-dependent protein disulfide isomerase activitiy.37-41 It is involved in chaperoning of many proteins such as MHC class I42-45 and CD1d46 which are molecules involved in antigen presentation.47 In addition to chaperone function, GRP58 has been shown to involved in regulation of cell signaling. Intracellular signaling of many cytokines is transduced by family of Janus kinases (JAK) and Signal Transducers and Activators of Transcription (STAT) proteins. GRP58 has been shown to regulate STAT3 signaling by sequestration of activated STAT3. Both IFN-R and IL-15 can activate STAT3.17,48,49 Therefore, it is likely that IFN-R and IL-15 induced GRP58 protein negatively regulates cytokine-activated STAT3 response in NK cells.

dimension, it is often found that one spot contains multiple proteins. This was true also in this study, as two proteins were identified from one spot in six cases. In addition, we identified one protein from three different spots and four proteins from two different spots. This highlights one of the biggest advantages of 2-DE: it is capable of separating protein isoforms into distinct spots, so that it is possible to determine whether only certain isoform is regulated by a given stimulus. We identified a total of 53 proteins, from which 47 were different proteins. The identified proteins were classified into different functional categories, such as protein destination, transcription and RNA processing, signal transduction, metabolism, intracellular trafficking and cellular organization. To our knowledge most of the proteins identified in this study are novel IFN-R and/or IL-15 regulated proteins. Previous microarray and proteomics 11-15 studies have found only few of the IFN-R and IL-15 target genes which we identified in the present work.

Interestingly, this study showed that MTHSP75 is a IFN-R and IL-15 target gene. MTHSP75 belongs to the heat shock protein 70 (HSP70) family of constitutively expressed proteins.50 In different studies, MTHSP75 has been assigned multiple roles rangingfromstressresponse,51-53 tocellgrowthanddifferentiation54-57 as well as antigen presentation. Intracellular heat shock proteins function as molecular chaperones, which support folding and transport of proteins under physiological conditions and stress. More recently, extracellular HSPs have been shown to be involved in activation of cellular immune responses. HSPs are known to induce inflammatory cytokine response in antigen presenting cells.58 Therefore cytokine-induced production of HSPs by NK cells may be associated with stress response that is followed by activation of antigen presenting cells.

One of the most interesting cytokine-inducible protein was adenylate kinase 2 (Figures 1-3). Adenylate kinase is a ubiquitous enzyme that contributes to the regulation of the cellular adenine and guanine nucleotide pools. Three isozymes, AK1, AK2, and AK3, have so far been characterized which are located in different tissues and in different subcellular sites in cells.30 AK2 is localized in the mitochondrial intermembrane space,31 and it has been proposed to play a role in apoptosis, since it translocates into the cytosol of apoptotic cells concomitantly with cytochrome c.32,33 Therefore, it is of special interest that the mRNA expression of AK2 was found to be up-regulated by

Interferon stimulated gene 15 is one of the most strongly induced genes following IFN-R treatment.13,59 Various cell types release ISG15, which has cytokine-like activity in lymphocytes. It can induce IFN-γ production in T cells and promote proliferation of NK cells.60 ISG15 is also conjugated to intracellular proteins in a process analogous to that for ubiquitin.61 Many proteins involved in cell signaling are conjugated to ISG15. These include Serpin 2a, PLCγ1, ERK1, JAK1 and STAT1.62,63 In accordance with previous studies in other cell types,12,13 ISG15 was found to be upregulated by IFN-R and IL15 stimulation in NK-92 cells.

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Aminopeptidases are enzymes that catalyze the cleavage of amino acids from the amino terminus of protein or peptide substrates. In addition to proteasome system, aminopeptidases can generate MHC-class-I-presented antigenic peptides.64,65 Leucine aminopeptidase is involved in trimming of the N terminus of the precursor peptides.66 Previously it has been shown that LAP gene expression can be activated by IFN-R and IFN-γ.67,15 We show that in addition to IFNs, IL-2 and IL-15 can upregulate LAP expression in NK cells. In conclusion, our data shows that proteome analysis is an efficient method for identifying novel cytokine inducible proteins, and underscores the complementary roles of transcriptomics and proteomics in signal transduction studies. Abbreviations: AK2, adenylate kinase 2; DC, dendritic cell; GRP58, glucose-regulated protein 58; HSP, heat shock protein; IFN, interferon; IL, interleukin; ISG15, interferon stimulated gene 15; JAK, janus kinase; LAP, leucine aminopeptidase; MTHSP75, heat shock 70 kDa protein; NK, natural killer; STAT, signal transducer and activator of transcription.

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