Calcineurin/nuclear factors of activated T cells (NFAT)-activating and immunoreceptor tyrosine-based activation motif (ITAM)-containing protein (CNAIP), a novel ITAM-containing protein that activates the calcineurin/NFAT-signaling pathway.

We report in this study the identification and characterization of a novel protein that we designated as calcineurin/NFAT-activating and immunoreceptor tyrosine-based activation motif (ITAM)-containing protein (CNAIP). The predicted 270-amino acid sequence contains an N-terminal signal peptide, an immunoglobin domain in the extracellular region, a transmembrane domain and an ITAM in the cytoplasmic tail. Quantitative reverse transcription-PCR showed that CNAIP was preferentially expressed in neutrophils, monocytes, mast cells, and other immune-related cells. Co-transfection of CNAIP expression constructs with luciferase reporter plasmids in HMC-1 cells resulted in activation of interleukin-13 and tumor necrosis factor-alpha promoters, which was mediated through the calcineurin/NFAT-signaling pathway. Mutation of either or both tyrosines in the ITAM abolished transcriptional activation induced by CNAIP, indicating that the ITAM is indispensable for CNAIP function in activating cytokine gene promoters. Thus, it is concluded that CNAIP is a novel ITAM-containing protein that activates the calcineurin/NFAT-signaling pathway and the downstream cytokine gene promoters.

It is well recognized that the first level regulation of activation or inhibition of an immune response occurs at the cell surface receptor site (1). The signals sensed by the receptors are relayed through their cytoplasmic signaling modules or adaptor molecules to regulate various cellular activities. The immunoreceptor tyrosine-based activation motif (ITAM) 1 is one of such signal modules present in cytoplasmic tails of many antigen and Fc receptors such as T cell receptor (TCR), B cell receptor (BCR), Fc⑀RI␤, and Fc⑀RI␥. The consensus of ITAM (YXX(L/I)X 6 -8 YXX(L/I)) has been deduced from sequence analysis of existing ITAM-containing receptors (2). In T cells, following the antigen binding to TCR ␣ and ␤ chain, the two tyrosine residues in the ITAM are phosphorylated by Src-family protein tyrosine kinase, Lck or Fyn (1). The phosphotyrosines, in turn, participate in and physically interact with the signal-amplifying kinase, Syk/ZAP-70, as well as other SH2 domain-containing proteins, which leads to the activation of downstream signaling pathways (1). Analogous events occur in B cells, basophils, mast cells, and other immune cells (3).
During a comprehensive search for novel activating receptors expressed on cell surface using the bioinformatics approach, we identified a human gene that encodes a novel ITAMcontaining protein. Expression of this protein in HMC-1 cells activated transcription of IL-13 and TNF-␣ promoters, which was mediated through the calcineurin/NFAT-signaling pathway. Therefore, the newly identified protein was named calcineurin/NFAT activating and ITAM-containing protein, or CNAIP. We report here its identification, gene structure and stimulatory role for NFAT.

EXPERIMENTAL PROCEDURES
Identification and in Silico Characterization of CNAIP-The protein data base compiled by International Protein Index (IPI) (www.ensembl. org/IPI/) containing ϳ65,000 protein sequences (as of March 2002) was used as data source in this study. A hidden Markov model (HMM)-based method was employed for Ig-domain search against IPI data base. The HMM, which was built from an alignment of 113 confident Ig domains and calibrated using the program HMMER, was obtained from the Pfam (version 6.6) data base (25). To identify ITAM-containing proteins, a PROSITE-formatted motif profile was first constructed based on the common features of ITAM motif, and the software "seedtop" (NCBI) was used to perform the search. Large-scale transmembrane region prediction for all of the IPI proteins was carried out by using software TMHMM version 2.0 (26) (www.cbs.dtu.dk/services/TMHMM/). The three sets of genes gen-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AY247409.
§ To whom correspondence may be addressed. Tel.: 713-578-4173; Fax: 713-578-5001; E-mail: byao@tanox.com. 1 The abbreviations used are: ITAM, immunoreceptor tyrosine-based activation motif; AP-1, activator protein 1; BCR, B cell receptor; Ig, immunoglobulin; HMM, hidden Markov model; NFAT, nuclear factors of activated T cells; CNAIP, calcineurin and NFAT-activating and ITAM-containing protein; TCR, T cell receptor; IL, interleukin; TNF-␣, tumor necrosis factor-␣; RT, reverse transcription; luc, luciferase; FACS, fluorescence-activated cell sorter; TREM, triggering receptor expressed on myeloid cells molecule; contig, group of overlapping clones; C t , threshold cycle. erated above were then overlapped to identify molecules with all of the common features by a relational data base system. In silico cloning was carried out as follows: the starting sequence was blasted against the human database of expressed sequence tag (NBCI) data base, the resulting hits were assembled as a contig, which was then reblasted against database of expressed sequence tag. This process was proceeded in a cyclic fashion until no more new expressed sequence tag hits could be found. Protein signal peptide prediction was performed by using both SignalP (27) and SOSUIsignal (28). Potential glycosylation sites were identified by PROSITE (29). Protein fold recognition was performed by using 3D-PSSM (30,36).
Cell Culture-Human HMC-1 and 293T cells were routinely cultured in Iscove's modified Dulbecco's medium and Dulbecco's modified Eagle's medium, respectively, supplemented with 10% fetal calf serum (Invitrogen), 1 g/ml penicillin and streptomycin. Cyclosporin A (Calbiochem, San Diego, CA) was added to the culture medium at the indicated concentration.
The cDNA Cloning and Expression-The cDNA encompassing CNAIP coding region was amplified by PCR and cloned into pcDNA3.1D/V5-His-TOPO (Invitrogen) by using two oligonucleotide primers derived from the predicted cDNA sequence. The cDNA sequence was verified by sequencing the entire insert, and the construct was termed CNAIP-V5C. An alternative CNAIP expression plasmid termed CNAIP-V5N was constructed by subcloning a PCR fragment into pSecTag/FRT/V5-His-TOPO vector (Invitrogen), which contains the truncated CNAIP coding region from the 43rd amino acid to the stop codon and a V5 tag fused at the N-terminal truncation site. Mutagenesis of tyrosine residues in the ITAM was generated on CNAIP-V5N backbone by PCR SOEing as described (31), and verified by sequencing. A full-length construct for expression of wild type CNAIP was generated by cloning the entire coding region sequence into pcDNA3.1. The cDNA sequence was verified by sequencing the entire insert on both strands.
Transfection and Western Blotting-For transient transfection, six to eight g of protein expression plasmids were transfected into 293T or HMC-1 cells in a 50-mm dish with LipofectAMINE 2000 (Invitrogen) according to manufacturer's instructions. The transfected cells were cultured for 40 -48 h and lysed in either 1ϫ sample buffer directly or protein extraction buffer (20 mM Tris-HCl, 1 mM EDTA, 70 mM KCl, pH 7.6) through freeze-thaw cycles. The membrane protein was separated from the soluble proteins by high-speed centrifugation. The protein extracts produced by cell lysis were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane, which was blocked with 5% dry milk in TBST (10 mM Tris, pH 7.8, 150 mM NaCl, with 0.05% Tween), then incubated with horseradish peroxidase-labeled anti-V5 monoclonal antibody (Invitrogen), and the signal was developed by enhanced chemiluminescence (ECL) assay kit (Amersham Biosciences).
First-strand cDNA Synthesis-Total RNA samples of various human tissues were obtained from Clontech, and total RNAs from cultured cells were purified by using the RNeasy mini kit (Qiagen, Valencia, CA). First-strand cDNA was synthesized using the total RNA and Super-Script II reagent (Invitrogen). Briefly, the reaction was set in a volume of 19 l with 1.5 g of total RNA, 2 pmol of each primer, 4 l of 5ϫ first-strand buffer (10 mM dithiothreitol and 0.2 mM dNTP). The mixture was heated to 70°C for 10 min and then cooled to 42°C when 1 l (200 units) SuperScript II was added. The mixture was then incubated for 1 h at 42°C and then heated to 70°C for 15 min. RT-PCR was performed as duplex RT-PCR using ␤-actin as a standard.
Real-time Quantitative PCR-Oligonucleotide primers were designed using Primer Express 2.0 (Applied Biosystems, Inc., Foster City, CA) and were synthesized and used in RT-PCR reactions to monitor the real-time expression of CNAIP. RNA samples were isolated from the following tissues and cells: brain, heart, kidney, liver, lung, spleen, monocytes, Daudi (a Burkitt's lymphoma cell line), HPB-ALL (a T cell leukemia cell line), THP-1 (acute monocytic leukemia; lymphocytes), Jurkat (a T cell leukemia cell line), HMC-1 (a mast cell line), human vascular endothelial cells (primary human vascular endothelial cells), neutrophils, peripheral blood mononuclear cells, and four different batches of in vitro cultured cord-blood-derived mast cell samples. Realtime quantitative PCR was performed with the ABI Prism 7900 (Applied Biosystems, Inc.) sequence detection system, using Taqman reagents, according to the manufacture's instructions. Equal amounts of each of the RNAs from the tissues and cell lines indicated above were used as PCR templates in reactions to obtain the threshold cycle (C t ), and the C t was normalized using the known C t from 18 S RNAs to obtain ⌬C t . To compare relative levels of gene expression of CNAIP in different tissues and cell lines, ⌬⌬C t values were calculated by using the lowest expression level as the base, which were then converted to real fold expression difference values.
Luciferase Reporter Assay-Luciferase reporter plasmids, NFAT-luc, NF-B-luc, and AP-1-luc were purchased from Clontech. The luciferase reporter plasmids, IL-13-luc, TNF-␣-luc, and Fc⑀RI␣-luc, were constructed by inserting 1.7 kb, 2.4 kb and 1.5 kb promoter sequences derived from IL-13, TNF-␣, and Fc⑀RI␣ genes into pTA-Luc vector (Clontech), respectively. All of the promoter sequences used were starting from a putative transcription start site extending to the 5Ј upstream region. HMC-1 cells were seeded onto a 24-well culture plate at the density of 0.2 million cells per milliliter of medium. Three plasmids, a firefly luciferase reporter with a defined promoter, CNAIP expression plasmid, and Renilla luciferase control plasmid, pRL-SV40 (Promega, Inc., Madison, WI), were co-transfected into HMC-1 cells. Cells were harvested 40 -46 h after transfection and lysed in lysis buffer. Both firefly and Renila luciferase activities were assayed with the dual luciferase assay kit (Promega). The fluorescent light emission was determined by TD-20 luminometer (Turner Design, Sunnylvale, CA).
Immunostaining-The transfected 293T cells were washed and preincubated at 4°C for 20 min in the enzyme-free cell dissociation buffer containing 1% bovine serum albumin (Invitrogen). Cells were then incubated with fluorescein isothiocyanate-conjugated anti-V5 monoclonal antibody (10 g/ml) (Invitrogen) in the same buffer for 30 min. After three washes, cells were fixed in 1 ϫ phosphate-buffered saline with 1% paraformaldehyde. Alternatively, the cells were first fixed in methanol for 5 min at room temperature and rinsed three times before the immunostaining. The samples were analyzed by FACScan (BD Biosciences) or fluorescence microscopy.

Identification and Molecular
Cloning of CNAIP-Non-redundant human protein data base IPI was searched for novel molecules containing 1) Ig domain, 2) ITAM, and 3) transmembrane region. Those are common features shared by many signal-activating receptors mediating immune system functions, including components of TCR, BCR, Fc⑀RI, and several other recently identified activating receptors (2,3,(32)(33)(34)(35). A hypothetical protein sequence labeled as "IPI00086590" was identified that satisfied all three criteria. In silico cDNA-cloning procedure was used to derive its full-length cDNA sequence.
To verify this cDNA, we designed two oligo primers encompassing the putative starting methionine codon and stop codon, and amplified the cDNA from human monocytes by RT-PCR. Two cDNA clones were isolated, sequenced, and found to be identical to the in silico cloning-derived coding region sequence (Fig. 1A). The analysis of immediate 5Ј flanking sequence to the coding region revealed a perfect Kozak motif, and several inframe stop codons preceding the predicted initiation methionine. Furthermore, this cDNA contains a putative signal peptide starting from the initiation methionine with a predicted cleavage site between amino acids 42 and 43. Based on the structural features and functionality that we elucidated, we designated this protein calcineurin/NFAT-activating and ITAM-containing protein, or CNAIP.
CNAIP encodes a polypeptide with 270 amino acid residues and a calculated molecular mass of ϳ30 kDa. It is predicted to be a type I transmembrane protein, which contains a putative signal peptide at the N-terminal (amino acids 1-42), an Igdomain (amino acids 50 -150) in the extracellular region, a transmembrane domain (amino acids 164 -186), and an ITAM (amino acids 220 -235) in the cytoplasmic region (Fig. 1B). One potential N-glycosylation site was found in the extracellular region (amino acids 107-110). The Ig-domain most likely adopts a V-type fold based on 3D-PSSM fold recognition algorithm (30,36). CNAIP has been mapped to chromosome 22q13.2 by sequence similarity search. Alignment of cDNA with genomic sequence showed that the coding region of CNAIP comprises six exons (Fig. 1C). Sequence similarity search against various public data bases showed that CNAIP does not share statistically significant similarity with any known proteins.
Tissue Distribution of CNAIP-The CNAIP mRNA expres-sion levels in a number of human cells and tissues were assessed using real time quantitative RT-PCR. The results showed that CNAIP was highly expressed in neutrophils, primary monocytes and monocytic cell lines (THP-1), lymphocytes, and in vitro cultured mast cells derived from cord blood (Fig. 2). The expression in spleen and lung was also evident. In contrast, the CNAIP expression in all other tissues and cells (brain, heart, kidney, liver, Daudi, HPB-ALL, Jurkat, HMC-1, and HUVAC) was low, suggesting that the primary role of CNAIP may be restricted to the immune system. Interestingly, the CNAIP expression level in more mature mast cells (cultured for 7 weeks and 86% tryptase-stained positive) is about 3-fold higher than that in the earlier stage of mast cells (cultured for 4 weeks and 17% tryptase-stained positive). Expression and Subcellular Localization of CNAIP Protein-To characterize the CNAIP gene product, we made two expression constructs, one with a V5 tag fused in frame to the C terminus of CNAIP coding region (CNAIP-V5C) and the other with the native signal peptide replaced by a heterologous signal peptide in the vector, which is immediately followed by a V5 tag fused to the N-terminal region of the truncated CNAIP (CNAIP-V5N). Transient transfection of CNAIP-V5C into 293T cells showed two protein bands of ϳ33 and 36 kDa in Western blot, which are absent in cells transfected with the empty vector (data not shown). In protein fractions prepared by freeze-thaw cycle and high-speed centrifugation, the 33 and 36 kDa proteins were only detected in the insoluble membrane fraction, not in the soluble cytosol fraction (Fig. 3), indicating that CNAIP is a membrane-associated protein.
Immunofluorescence staining was then performed to further determine the subcellular localization and the orientation of CNAIP in the membrane. FACS analysis of 293T cells transfected with CNAIP-V5N or CNAIP-V5C showed that N terminus-tagged CNAIP was detected only in the living cells transfected with CNAIP-V5N (Fig. 4) but not in the living cells transfected with CNAIP-V5C (data not shown). However, fixation of the cells transfected with the two expression constructs resulted in unambiguous detection of both the N terminus-and C terminus-tagged protein by FACS and fluorescent microscopy (data not shown). These results indicated that CNAIP is a transmembrane protein with the N terminus exposed to the outside of the cellular membrane. CNAIP Activates the Calcineurin/NFAT-signaling Pathway-The co-existence of Ig domain and ITAM motif in CNAIP, along with the preferential expression in immune cells, strongly suggest that CNAIP may function as an activating receptor in immune system. To test if CNAIP can activate the transcription of cytokine genes, we co-transfected CNAIP-V5N with luciferase reporter constructs that were linked to IL-13, TNF-␣, or Fc⑀RI␣ promoter in HMC-1 cells. The luciferase reporter assays showed that CNAIP increased IL-13 and TNF-␣ promoter activities by ϳ14and ϳ5-fold, respectively, as compared with the expression vector control. In contrast, the Fc⑀RI␣ promoter-linked luciferase activity was not changed (Fig. 5A).
It was well documented that NFAT is involved in transcriptional activation of cytokine genes, such as IL-13 (18) and TNF-␣ (20), and that ITAM-containing receptors, upon ligand binding, can lead to the activation of several transcription factors including NFAT (37), NF-B (38), and AP-1 (39). To test whether NFAT, NF-B, and/or AP-1 are potential downstream targets of the CNAIP-signaling pathway, we assessed the NFAT, NF-B, or AP-1 luciferase reporter activity by transient transfection with CNAIP-V5N in HMC-1 cells. Expression of CNAIP elevated NFAT luciferase reporter activity by ϳ19-fold as compared with the transfection with the vector only, whereas the NF-B or AP-1 luciferase reporter activities showed no significant changes (Fig. 5A). To confirm that fulllength CNAIP with its native signal peptide can also activate NFAT, we co-expressed a full-length construct of CNAIP with an NFAT luciferase reporter. Expression of full-length CNAIP also elevated NFAT luciferase reporter by ϳ9-fold as compared with the transfection with a control vector (Fig. 5C). These data were reproducible in three separate experiments.
Because calcium flux and calcineurin activation are signaling events upstream of NFAT activation (12), we tested whether the CNAIP/calcineurin/NFAT-signaling pathway could be blocked by cyclosporin A, a immune-suppressive agent which specifically inhibits calcineurin activation. Addition of 1 M of cyclosporin A to the culture inhibited CNAIP-mediated NFAT activation by ϳ90% (Fig. 6), indicating that CNAIP can activate calcineurin/ NFAT-mediated signaling cascade and therefore activate transcription of NFAT-regulated cytokine genes.
Calcineurin/NFAT Activation by CNAIP Is Mediated by ITAM Motif-Sequence analysis of CNAIP revealed a good match of ITAM in the cytoplasmic tail (Fig. 1B). To determine whether the putative ITAM motif mediates the signal transduction, we generated three CNAIP mutants (Y220A, Y231A, and Y220A/Y231A) by replacing the two tyrosines (Tyr-220 and Tyr-231) in the ITAM to alanine individually or in combination. Transfection of either of these mutants into HMC-1 cells failed to activate IL-13, TNF-␣, or NFAT luciferase reporter activity (Fig. 5). The relative luciferase reporter activity in these cells was comparable with that of cells transfected with vector control (Fig. 5A). Western blot analysis showed that mutant proteins were expressed at similar level as that of wild type CNAIP (Fig. 5B), indicating that mutations do not affect the expression and stability of CNAIP in these cells. These data indicated that activation of the calcineurin/NFAT-signaling pathway by CNAIP is mediated by ITAM, and both tyrosine residues are required for the ITAM-mediated function in CNAIP.

DISCUSSION
In the present report, we describe the identification of a novel ITAM-containing protein, CNAIP, that activates NFAT, IL-13, and TNF-␣ promoter activity. Our data suggest the CNAIP may function as an activating receptor. ITAM-containing receptors are a divergent group of cell surface membrane proteins that are expressed in immune-related cells and regulate cell growth, maturation, apoptosis, and cell activation. Members of this family include Ig␣, Ig␤, TCR-, TCR-, CD3␥, CD3␦, CD3⑀, Fc⑀RI␤, and Fc⑀RI␥. Some of them contain Ig or Ig-like domains in the extracellular region, which are involved in protein-protein interaction. Similar to those receptors, CNAIP contains an Ig domain in the extracellular region. Some ITAM-containing receptors transduce signal within a multisubunit of immune receptor complexes such as TCR and BCR. It is not clear at present whether CNAIP is a component of an immune receptor complex or functions as a single unit. Interestingly, overexpression of CNAIP can activate downstream effectors without ligand binding or antibody cross-linking. The mechanisms by which CNAIP activates the signaling pathway remain to be determined. However, it is possible that overexpression of CNAIP proteins in the cells results in the aggregation or clustering of the receptors on cell surface, which leads to the recruitment of downstream effector to the receptor molecules that subsequently lead to its activation.
The ITAM consensus sequence (YxxL/Ix 6 -8 YxxL/I) contains two appropriately spaced tyrosine residues (33). Following receptor engagement, phosphorylation of the two tyrosines by Src family kinases creates temporary anchors for SH2-containing signaling proteins that recruit to the receptor site (3). In our studies, mutation of either of the two tyrosine residues in the ITAM of CNAIP abolished the activating activity mediated by CNAIP. Thus, like other ITAM-containing activating receptors (40), the phosphorylation of the two tyrosine residues is critical for its activity. It was well documented that in mast cells the tyrosines in the ITAM of Fc⑀RI ␤ and ␥ subunits are rapidly phosphorylated upon IgE cross-linking. The phosphotyrosines are then engaged in recruiting and activating Src-related protein receptor kinases, Lyn and Fyn, and signal amplifying kinase, Syk (33). The reminiscent functionality of ITAMs found in other immunoreceptor complexes was established through extensive experimental research, such as those in TCR and BCR (1). It is conceivable that CNAIP may transduce its activating signal through the ITAM in the manner similar to that of the other ITAM-containing proteins. However, the divergence and multiplicity of ITAM domains might determine which Src-related receptor kinase(s) and SH2-containing protein would be recruited (3). Because the ITAM sequence of CNAIP displays no close similarity to any existing ITAMs except for the four conserved positions (Fig. 1B), it is not known yet what kinase(s) or SH2-containing protein(s) are recruited to the ITAM of CNAIP.
The gene structure and topology of CNAIP resemble other activating molecules such as NKp44 and TREMs (triggering receptor expressed on myeloid cells molecules). All those molecules are type I transmembrane protein with a single Ig domain in the extracellular region, and transduce signals via ITAM motif, which is contained in either the cytoplasmic region of the molecule or its associated adaptor molecule DAP12. Moreover, both TREMs and CNAIP are abundantly expressed in neutrophils and monocytes. However, sequence similarity between CNAIP and TREM and NKp44 is very low (Ͻ10%) and statistically insignificant. TREM-1 was recently characterized and found to function as an activating receptor (41). It is expressed at high levels on neutrophils and monocytes that infiltrate human tissues infected with bacteria and plays a critical role in acute inflammatory responses to bacteria (41). It is tempting to speculate that CNAIP may play similar roles in acute inflammation, which are characterized by an exudate of neutrophils and monocytes (42). Further studies are aimed to identify the functional role of CNAIP in the immune responses.