Molecular Cloning of a T Cell-specific Adapter Protein (TSAd) Containing an Src Homology (SH) 2 Domain and Putative SH3 and Phosphotyrosine Binding Sites*

Anne SpurklandDagger §, Jan E. BrinchmannDagger , Gunnar MarkussenDagger , Florence Pedeutour, Else Munthepar , Tor Lea**, Frode VartdalDagger , and Hans-Christian Aasheimpar

From the Dagger  Institute of Transplantation Immunology, National Hospital, 0027 Oslo, Norway,  URA CNRS, Faculté de Medicine, Avenue de Valombrose, Nice, France, the ** Institute of Immunology and Rheumatology, National Hospital, 0027 Oslo, Norway, and the par  Department of Immunology, Radiumhospitalet, 0310 Oslo, Norway

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Adapter proteins link catalytic signaling proteins to cell surface receptors or downstream effector proteins. In this paper, we present the cDNA sequence F2771, isolated from an activated CD8+ T cell cDNA library. The F2771 cDNA encodes a novel putative adapter protein. The predicted amino acid sequence includes an SH2 domain as well as putative SH3 and phosphotyrosine binding interaction motifs, but lacks any known catalytic domains. The expression of the gene is limited to tissues of the immune system and, in particular, activated T cells. The protein expressed by F2771 cDNA in transfected COS cells is localized in the cytoplasm. A polyclonal antiserum raised against an F2771-encoded peptide reacts with a tyrosine-phosphorylated 52-kDa protein expressed in phytohemagglutinin-stimulated peripheral blood mononuclear cells. The gene is localized to chromosome 1q21, a region often found to be aberrant in lymphomas. The T cell-specific expression and the rapid induction of mRNA expression upon receptor binding, as well as the lack of catalytic domains in the presence of protein interaction domains, indicate that the F2771 gene encodes a novel T cell-specific adapter protein (TSAd) involved in the control of T cell activation.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Activation of T cells involves interaction of the T cell antigen receptor (TCR)1 with complexes of peptides and major histocompatibility complex class I or II molecules. Depending on the peptide and the antigen-presenting cell, the initial contact of the TCR with the complex of peptide bound to major histocompatibility complex molecules may lead to activation, anergy, or T cell apoptosis (1). The transduction of signals from the cell membrane to the nucleus is mediated by transient interactions between various signaling proteins, among which are tyrosine kinases, phosphatases, and adapter proteins without catalytic function. The signaling process is complex and still not well understood (2).

Phosphorylation and dephosphorylation events act as triggers for transient interaction between the various signal transducing proteins. Recognition of specific protein sequences containing phosphorylated tyrosines is mediated by particular conserved modular binding domains, such as the Src homology 2 (SH2) domain (3, 4) and the phosphotyrosine binding (PTB) domain (5). Signal transducing proteins may also have binding domains, which recognize structures that do not undergo enzymatic changes during signal transduction, such as the Src homology 3 (SH3) domain. SH3 domains bind to proline-rich sequences (6, 7). This may localize the SH3 domain-containing protein to proline-rich structures like the cytoskeleton. Additionally, SH3 domains of cytoplasmic enzymes may aid in the recruitment of specific substrates for the enzyme (3). Not all signal transducing proteins have catalytic functions. Some proteins, like Slap (8), Crk (9), Shc (10), Nck (11), Lnk (12), and Grb2 (13), function as adapters, linking catalytic signaling proteins to receptors at the cell membrane or to downstream effectors like Ras (14).

We initiated experiments to identify genes that are specifically expressed in activated CD8+ T cells using a subtractive strategy. By using this approach, we have isolated and characterized a novel gene that is strongly expressed in T cells shortly after activation. The gene continues to be expressed in long term cultures of activated T cells. We propose that this protein, due to the presence of an SH2 domain and putative SH3 and PTB binding motifs, is an adapter molecule involved in protein-protein interactions during T cell activation. This novel protein may be termed TSAd, for T cell SH2 domain-containing adapter protein.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Antisera-- A polyclonal rabbit antiserum was raised against a synthetic peptide (AEEVPGEGSLFLQAETRAWF) derived from amino acid (aa) position 59-78 of the deduced F2771 polypeptide by immunizing rabbits with peptide conjugated to keyhole limpet hemocyanin. Anti-TCR mAb T10B9 was obtained as a gift from J. S. Thomson, University of Kentucky Medical Center, Lexington, KY. Biotinylated goat anti-rabbit IgG and biotinylated goat anti-mouse IgG were obtained from Southern Biotechnology Associates (Birmingham, AL), and QIAexpress MRGSHis IgG was obtained from Qiagen (Chatsworth, CA). Anti-p56lck antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). For detection of phosphotyrosine, the monoclonal antibody 4G10 (Upstate Biotechnology, Inc.) was used.

Preparation and Activation of Lymphocytes-- Peripheral blood mononuclear cells (PBM) were obtained from citrate-anticoagulated blood or buffy coats of healthy blood donors by Isopaque-Ficoll (Nycomed AS, Oslo, Norway) gradient centrifugation. Positive selection of cells from buffy coats or PBM using superparamagnetic Dynabeads (Dynal, Oslo, Norway) coated with mAb specific for CD4, CD8, or CD19 has been described previously (15-17). The cells were lysed directly after isolation, or the beads were detached from the cells by incubation for 1 h at 37 °C with goat anti-Fab antiserum (Detachabead, Dynal) (18). Cell subsets thus isolated contained <2% contaminating cells as assessed by flow cytometry and showed >95% viability as determined by acridine orange/ethidium bromide staining.

CD8+ cells were left overnight in RPMI supplemented with 10% pooled human serum at 37 °C, 95% relative humidity and 5% CO2 before being activated by incubation with anti-TCR mAb T10B9 coated onto Dynabeads and interleukin-2 (10 units/ml). The cells were harvested after 72 h, and 30-100 × 106 cells were frozen as cell pellets at -70 °C for later RNA extraction.

For mRNA expression studies, CD4+ and CD8+ T cells were activated with anti-TCR, tetradecanoyl phorbol acetate, or phytohemagglutinin (PHA) (19). B cells were activated with tetradecanoyl phorbol acetate. Cells were harvested after 3 days, or at different time points as shown under "Results."

Cell Lines-- COS-1 cells (CRL-1650) (American Type Culture Collection (ATCC), Rockville, MD) were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Life Technologies, Inc.). Jurkat (TIB-152), MOLT-3 (CRL-1552), KT-1, RAMOS (CRL-1596), THP-1 (TIB-202), and K-562 (CCL 243) cell lines (ATCC) were cultured in RPMI with 10% fetal calf serum. EBV-transformed B cell lines from healthy donors were established in our laboratory. Absence of mycoplasma infection was ascertained by the use of an ATCC mycoplasma PCR detection kit (ATCC).

cDNA Library-- A cDNA library in the pcDNAI plasmid vector was custom made by Invitrogen (San Diego, CA) from 100 × 106 CD8+ T cells activated 3 days previously with anti-TCR coated Dynabeads as described above. cDNA synthesis was performed with a mixture of oligo(dT) primer and random priming. The cDNAs smaller than 800 bp was removed. The resulting cDNA library contained 2.23 × 106 primary recombinants.

RNA Preparation-- Poly(A)+ RNA was isolated directly from 50-70 × 106 lysed cells using 5 mg of Dynabeads oligo(dT)25 (Dynal) as described previously (20). Seven to 10 µg of mRNA were obtained from 50-70 × 106 cells (lymphoid cell lines).

Total RNA was extracted from fresh or frozen pellets of 50 × 106 cells by the guanidine thiocyanate method (21).

Subtraction Probe-- Preparation of a subtraction probe was performed as described previously in detail (22). Briefly, 10 µg of mRNA was isolated from Jurkat cells (subtractor) using oligo(dT)25 as described above and first strand cDNA was synthesized while mRNA was still bound to the Dynabeads, using a combination of avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim) followed by rTth heat-stable polymerase (Perkin Elmer) (22). This leads to immobilized first strand cDNA (subtractor). The subtraction was performed by hybridizing 1 µg of mRNA isolated from anti-TCR activated CD8+ T cells (see above), to the immobilized first strand cDNA subtractor beads. Three rounds of hybridization was performed to ensure optimal subtraction. mRNA hybridizing to the subtractor beads were removed between each round of hybridization. After the last hybridization, the hybridization solution contained the specific target mRNA. This was isolated using oligo(dT)25, and specific target cDNA was synthesized on the beads.

Amplification of the specific target mRNA was performed essentially as described (22, 23). Poly(A) tails were added to the cDNA by incubating the beads with terminal transferase (Life Technologies, Inc.) and dATP for 15 min at 37 °C. The reaction was stopped by adding 2 µl of 0.5 M EDTA. After washing the beads in TE, 50 µl of a PCR amplification mixture (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 100 mg/ml bovine serum albumin, 0.05% Triton X-100, 2.5 mM MgCl2, 0.2 mM dNTPs, 2.5 units/100 µl Taq polymerase; Perkin Elmer) containing 20 pmol of the T-primer (5'-dTTGCATTGACGTCGACTATCCAGGTTTTTTTTTTTTTTT-3') was added. The T-primer was extended by incubation for 15 min at 30 °C, then 15 min at 40 °C, and 15 min at 72 °C. After 2 min at 94 °C, 15 PCR cycles (94 °C, 1 min, 72 °C 5 min) were performed, followed by a 10-min extension at 72 °C. Reamplification of 5 µl of this reaction was performed for an additional 20 cycles as above in 50 µl of the same PCR amplification mixture containing 50 pmol of L-primer: 5'-dTTGCATTGACGTCGACTATCCAGG-3'. This PCR product was purified using Geneclean kit (Bio 101, Vista, CA), and one-fifth of it was radiolabeled and used as a probe for library screening.

Library Screening-- The library was spread on LB agar containing 12.5 µg/ml ampicillin and 7.5 µg/ml tetracycline. Colony lift using Optitran BA-S 85 reinforced cellulose nitrate membrane (Schleicher & Schuell, Dassel) was performed essentially as described (24). Hybridization was performed in 50% deionized formamide, 0.5% SDS, 5 × SSPE, 5 × Denhardt's solution, and 0.1 mg/ml denatured and sonicated salmon sperm DNA (Sigma) at 42 °C. The PCR-amplified subtraction probe was labeled by random priming using [alpha -32P]dCTP (Amersham, UK). After overnight hybridization, a stringency wash was performed in 0.1 × SSC, 0.1% SDS at 55 °C. Positive colonies were rescreened twice with the same subtraction probe, and positive inserts were partially sequenced (see below). The F2771 clone was one of 15 clones isolated from 5 × 104 colonies. Several full-length F2771 clones were isolated from the same cDNA library as described above using the originally isolated F2771 cDNA as a probe.

Sequencing-- Three near full-length cDNA clones (F2771-19, F2771-21, and F2771-45) as well as the original F2771 cDNA clone were sequenced in both directions using restriction fragment subcloning and oligonucletide primers specific for the F2771 sequence already obtained. Several shorter cDNAs including a 3.5-kilobase pair primary transcript of F2771 (F2771-4.2) were partially sequenced. Sequencing was performed using Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham) following the manufacturer's instruction.

Northern Blot Analysis-- Samples (10 µg) of total RNA were size-fractionated on 1% agarose gel/formaldehyde denaturing gels, transferred to nylon membranes (Hybond-N nylon, 0.45 µm; Amersham) and cross-linked to the membranes by UV light. The membranes were hybridized and washed as described above, using radiolabeled F2771 insert as a probe. All blots were exposed to XAR-5 film at -70 °C using intensifying screens or PhosphorImager.

Chromosomal Localization-- Chromosomal localization of the F2771 gene was performed by PCR amplification using F2771-specific primers and DNA from a monochromosomal somatic cell hybrid DNA panel obtained as a generous gift from the UK HGMP Resource Center, Cambridge, UK.

Chromosomal localization was also performed using FISH on human metaphases as described previously (25). Briefly, 50 ng of the biotinylated (Bionick, Life Technologies, Inc., France) F2771 probe were denaturated 5 min at 70 °C and reannealed with 10 µg of Cot-1 DNA (Life Technologies, Inc.) 30 min at 37 °C, then incubated overnight on metaphase spreads. Revelation of the signal was performed using three layers of avidin-fluorescein. Prodidium iodide (as a counterstain) and 4,5-diamino-2-phenyleindole (for chromosome identification) were added to antifade mounting medium. Slides were observed using a Zeiss Axiophot microscope, and metaphases were analyzed using a high performance cooled CCD camera C4880 (Hamamatsu) interfaced to a 486DX33 PC with a Matrox 640 card (Alcatel software package).

Expression of His-tagged F2771 in COS Cells-- The F2771-19, F2771-21, and F2771-45 inserts representing three different splice variants of the F2771 cDNAs were cloned into pcDNAI-His2, a modified pcDNAI vector encoding a MRGS-His6 tag 3' of the XhoI site in the polylinker of pcDNAI generated by us. The inserts were PCR-amplified using a modified F2771-specific primer introducing an XhoI site at the stop codon at position 1252 and cloned into the XhoI and HindIII site of pcDNAI-His2. The presence of the correct sequence was verified by sequencing.

The resulting constructs were transfected into COS-1 cells by the DEAE Dextran method (26). The cells were transferred to Chamber slide culture chambers (Nunc, Napierville, IL) the day after transfection. Three or four days later, the cells were fixed in 100% acetone and incubated in a 1/200 dilution of QIAexpress MRGS·His antibody at room temperature for 30 min. Visualization was done using the alkalic phosphatase anti-alkalic phosphatase technique (DAKO, Glostrup, Denmark). Cellular localization of His-labeled protein was assessed by microscopy.

Western Blot of Cell Lysates-- Cell lysates were prepared by lysis of cells in phosphate-buffered saline supplemented with 10 mM Na2+ orthovanadate, 50 mM iodoacetamide, 1% Nonidet P-40, and 1% phenylmethylsulfonyl fluoride. Protein concentration was assessed by colorimetric detection using Bio-Rad DC protein assay. Five µg of each lysate was run on a precast 16% Tris-glycine polyacrylamide gel (Novex, San Diego, CA). Alternatively, immunoprecipitation of proteins from the cell lysates was performed using the indicated antibodies and Sepharose A according to standard methods (19). Electrophoresis and blotting of the gel onto nitrocellose was performed according to standard methods (26). The blots were incubated with the appropriate antibodies, and signals were developed using ECL chemiluminescence detection kit (Amersham, UK).

Sequence Analysis-- Data base searches were performed using the Blast (National Center for Biotechnology Information) and Fasta and Tfasta programs from the GCG program package (27). Search for protein motifs was also performed using the compilation of protein sequence analysis programs at the UK HGMP Resource Center, Cambridge, UK.2 The alignment of multiple SH2 domains was performed using the pfscan program.3

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Isolation of F2771, a Novel Human cDNA Clone Encoding a Polypeptide Containing an SH2 Domain-- Using a subtractive strategy, we isolated a novel cDNA clone, F2771, derived from anti-TCR activated human CD8+ T cells. Initial Northern blot analysis showed that the F2771 cDNA hybridized to a single band of approximately 1700 bp, expressed in activated CD8+ cells and not in the Jurkat cell line used as subtractor, thus confirming the efficiency of the subtraction (data not shown).

The entire F2771 cDNA sequence was found to consist of 1592 bp, excluding the nucleotide polyadenylate stretch (Fig. 1A). A common AAUAAA polyadenylation signal is localized at position 1556. The F2771 sequence has an open reading frame from position 2 to position 1254. Attempts to identify a transcription start site upstream of the obtained F2771 sequence, either by primer extension analysis of RNA or PCR amplification of the 5' F2771 ligated to the pcDNAI vector, failed to reveal a more 5' start site (data not shown). Thus, the first putative initiation codon is at nucleotide 87, within a sequence (GCTCTCATGG) that shows moderately good similarity to the Kozak translation initiation consensus sequence (XCXGCCATGG; Ref. 28). This putative initiation codon yields a predicted polypeptide chain of 389 aa with a predicted molecular mass of 43 kDa. Two other F2771 hybridizing cDNAs representing two different alternate splice variants of the F2771 gene were isolated. One of these cDNAs may employ a different start site at nucleotide 171 (ACAGACATGA) yielding a polypeptide of 361 aa. The other cDNA includes a 30-bp insertion at position 399, yielding a predicted polypeptide of 399 aa. The F2771 sequence showed only low overall homology to any genes or gene families. However, several expressed sequence tags (29) with high degree of similarity to F2771 were identified in the EST (expressed sequence tag) Data Bank.


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Fig. 1.   F2771 nucleotide and deduced amino acid sequences. A, the translation of the F2771 sequence is shown below the nucleotide sequence. Nucleotide and amino acid positions are indicated on the left of each lane. The ATG171 representing an alternate start site and the putative poly(A) addition signal AATAAA at position 1554 are underlined. The SH2 domain is underlined, and proline-rich sequences are stippled. An arrow (up-arrow  ) indicates the position of a 10-aa insertion (RVRPPLSVTH) found in one cDNA clone (F2771-45). Prolines and arginines separated by two amino acids are marked in bold. Tyrosine residues residing within an NPXY motif is double underlined. Filled circles (bullet ) indicate six putative Ser/Thr phosphorylation sites. An N-glycosylation site is indicated with open diamonds (diamond ). The cDNA sequence is available through GenBank (accession no. AJ000553). B, alignment of the SH2 domain of F2771 (aa 94-186) with the SH2 domains of other proteins. The sequences of the SH2 domain proteins are taken from the SwissProt data base. Boxes are drawn by the PrettyPlot program in the extended GCG program package (27). C, schematic presentation of the F2771 protein.

The predicted polypeptide of F2771 contains a putative SH2 domain, which is located at aa positions 95-186 (Fig. 1, A and B) (30, 31). In the C-terminal part of the F2771 sequence (aa 239-389), the motif RPKPXXP is repeated twice, and there are also several other repeats of ((R/P)XX)n. These motifs may represent binding sites for SH3 domains (32). Two of the 10 tyrosines of the F2771 sequence are found within an NPXY motif, i.e. Tyr-216 and Tyr-280 (Fig. 1A). When phosphorylated on tyrosine, NPXY motifs may represent binding sites for PTB domains (33). A schematic representation of the F2771 cDNA is given in Fig. 1C.

The F2771 Gene Is Localized to Chromosome 1q21-- By using a monochromosomal cell hybrid DNA panel and F2771-specific primers, we localized the F2771 gene to chromosome 1 (data not shown). These data were further confirmed by FISH analysis. Fluorescent signals were observed in the 1q21 position (Fig. 2) on 20 human metaphases from two unrelated healthy donors (one male and one female), respectively.


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Fig. 2.   Chromosomal localization of the F2771 gene: chromosomal localization performed by FISH. The idiogram and the chromosome 1 counterstained with 4,5-diamino-2-phenyleindole (a) and propidium iodide (b), respectively, illustrate the chromosomal localization at 1q21. Arrowheads indicate chromosomes 1 with a fluorescent signal at 1q21 band on a partial normal metaphase countertained with propidium iodide (c).

The F2771 Gene Is Abundantly Expressed in Lymphoid Tissue-- The initial Northern blot analysis demonstrated that the F2771 cDNA hybridized to a single band of approximately 1700 bp, expressed in activated CD8+ cells but not in the T leukemic cell lines Jurkat. When examining the tissue distribution, the F2771 gene was found to be expressed in peripheral blood leukocytes (PBL), thymus, and spleen (Fig. 3A). Furthermore, we found that the F2771 gene was expressed in tetradecanoyl phorbol acetate-activated CD4+ and CD8+ cells, but not in resting CD4+ and CD8+ T cells, nor in B cells (Fig. 3B). The resting cells were obtained by positive selection from buffy coats using anti-CD4, anti-CD8, or anti-CD19 Dynabeads, respectively, and directly lysed after a 30-min incubation at 4 °C with the beads. Several lymphoid cell lines (the KT-1 and MOLT-3, the B lymphoma cell line Ramos, the erythroleukemoid cell line K-562, and the myelocyte cell line THP-1), as well as the monkey fibroblast cell line COS-1, did not express F2771 mRNA as demonstrated by Northern blot analysis (data not shown).


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Fig. 3.   Expression of F2771 in different human tissues. Panel A represents commercial tissue blots (multiple human tissue blot 1 and 2, CLONTECH). Each lane shows hybridization to 2 µg of mRNA of the indicated tissue. Panel B represents a Northern blot of 1 µg of mRNA/lane from positively selected B cells, CD4+ cells, and CD8+ cells using Dynabeads oligo(dT)25.

When probing 105 cDNA clones from the cDNA library of activated CD8+ T cells using the initially isolated F2771 cDNA as a probe, we isolated 16 different F2771 clones. Thus, we were able to estimate the frequency of F2771 expression to be higher than 1/10,000 transcripts.

The Expression of the F2771 Gene Is Rapidly Induced after Activation of T Cells-- Exposure of freshly isolated PBM to the plastic of culture bottles activate monocytes and thereby possibly T cells (34). We observed that after a 2-h incubation of PBM on plastic at 37 °C to remove adherent cells, the F2771 gene was already induced to maximal mRNA expression in the nonadherent PBM fraction (Fig. 4A). Furthermore, we found that positive selection of CD4+ cells by cross-linking of the CD4+ receptor using immunomagnetic beads (15) induced the expression of the F2771 mRNA within 2 h when incubated in RPMI plus 10% human serum at 37 °C (Fig. 4B). Similar data were obtained by cross-linking of the CD8 receptor or the TCR (data not shown). The increased levels of F2771 mRNA were sustained for at least 24 h in unstimulated culture of positively selected T cells after the removal of the immunomagnetic beads using anti-Fab (Fig. 4C, lanes labeled 0 h CD4-cells and 0 h CD8-cells). In CD4+ but not in CD8+ cells, the F2771 expression in these T cells could be further increased by cross-linking of TCR (Fig. 4C). Finally, we found that the F2771 gene continues to be expressed throughout long term cultures of activated CD8+ T cells (Fig. 4C, right panel).


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Fig. 4.   Kinetics of the expression of the F2771 gene. Panel A shows induction of F2771 mRNA expression in the nonadherent fraction of PBM incubated in unstimulated culture in a plastic bottle for 2 h at 37 °C (labeled 2 h PBM). Freshly isolated PBM (0 h PBM) and PBM stimulated with PHA and interleukin-2 for 6 days are included as controls. Panel B represent CD4+ T cells positively selected with immunomagnetic beads covered with anti-CD4. Positively selected cells were incubated with beads present at 37 °C for the indicated time points, whereupon RNA was extracted. Panel C represents F2771 expression in positively selected CD4+ or CD8+, detached from the beads and incubated for 24 h at 37 °C (lanes labeled 0 h CD4-cells or 0 h CD8-cells). These cells were then activated with anti-TCR-coated immunomagnetic beads, and RNA was extracted at the indicated time points. Right part of panel C shows expression of F2771 gene in long term cultures of PBM or CD8+ T cells activated with PHA or anti-CD3 cross-linking. Number of days in culture is indicated above each lane.

F2771 Protein Is Localized to the Cytosol-- By the use of "analysis and prediction of protein sorting signals" (PSORT; Ref. 35), the F2771 protein was found to lack a hydrophobic signal sequence, transmembrane regions and nuclear localization signals. This indicates that the F2771 protein is an intracytosolic protein.

To verify this prediction, three cDNA clones (F2771-19, F2771-21, and F2771-45) encoding F2771 polypeptides of 361, 389, and 399 aa, respectively, were modified to encode a C-terminal histidine tag (MRGS-His6). Microscopy of COS-1 cells transiently transfected with these constructs, visualized using an anti-His-specific antibody and the alkalic phosphatase anti-alkalic phosphatase technique, showed staining of the cytoplasm and not the nuclei of positive cells (Fig. 5). This observation supports the prediction that the F2771 protein is an intracytosolic protein.


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Fig. 5.   Cellular localization of F2771. His tag-labeled F2771 expressed in COS-1 cells was visualized by using an anti-His antibody and the indirect alkalic phosphatase anti-alkalic phosphatase technique. Panels a-c show cells transfected with three different histidine-tagged splice variants of the F2771 gene (encoding polypeptides of 389, 399, or 360 aa, respectively). Panel d shows COS-1 cells transfected with the pcDNAI-His2 vector.

An F2771-specific Antiserum Identifies a Tyrosine-phosphorylated 52-kDa Protein in Cell Lysates from PHA-stimulated PBM-- A polyclonal rabbit antiserum was raised against a peptide derived from aa 56 to aa 78 in the predicted F2771 polypeptide sequence. In Western blots of cell lysates, this antiserum detected one protein band at 52 kDa expressed in PHA blasts and freshly isolated PBM, but not in Jurkat, KT-1, MOLT-3, or K562 leukemic cell lines, or in EBV-transformed B cells (Fig. 6A). The 52-kDa protein could be detected by Western blot in freshly isolated CD4+ and CD8+ T cells (Fig. 6B). Protein expression was increased in cells left overnight in unstimulated culture (cells labeled 0 h CD4-cells and 0 h-CD8 cells). A further increase in protein expression was observed in these T cells after 1 day of PHA stimulation (Fig. 6B). This corresponds to the observed changes in F2771 mRNA expression in positively selected CD4+ and CD8+ T cells treated in a similar way (Fig. 4C).


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Fig. 6.   Expression of the F2771 protein in lymphoid cells. A, Western blot of cell lysates derived from PHA blasts, the T leukemic cell lines Jurkat, KT1, MOLT-3, EBV-transformed B cells, the erythroleukemic cell line K562, and freshly isolated PBM. The blot was stained with a polyclonal rabbit antiserum specific for a F2771 peptide. Position of 52 kDa band is indicated with an arrow. B, Western blot of lysates from positively selected CD4+ and CD8+ T cells lysed directly after isolation, lysed after 1 day in unstimulated culture (labeled 0 h CD4-cells and 0 h CD8-cells), and after 1 day of PHA stimulation. The blot was stained with a polyclonal rabbit antiserum specific for a F2771 peptide.

The F2771 antiserum could immunoprecipitate a 52-kDa protein from lysates of PHA blasts (Fig. 7A). F2771 immunoprecipitates from lysates of PBM stimulated with PHA for 1 and 2 days reacted with an anti-phosphotyrosine antibody. However, phosphorylation of the immunoprecipitated protein was also observed in lysates from freshly isolated PBM (Fig. 7B).


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Fig. 7.   Immunoprecipitation and tyrosine phosphorylation of the F2771 protein. Panel A shows immunoprecipitates from PBM stimulated with PHA for 6 days, using anti-p56lck antibody and anti-F2771 antibody. Staining with F2771 antiserum is shown in the upper part, whereas staining with p56lck antiserum is shown in the middle part and staining with anti-phosphotyrosine is shown in the lower part. Positions of 56 kDa and 52 kDa in the lower part are indicated with arrows. Panel B shows F2771 immunoprecipitates from lysates of PBM and PBM stimulated with PHA for 1 and 2 days of staining with anti-phosphotyrosine antibody.

Attempts to coprecipitate the p56lck protein from lysates of PHA blasts, using the anti-F2771 antiserum or vice versa using an antiserum against the p56lck protein, failed to reveal an association between the two proteins (Fig. 7A and data not shown).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

We have identified and characterized a novel human cDNA (F2771) encoding a putative adapter protein with T cell-specific expression. The F2771 cDNA encodes a protein with predicted molecular mass of 43 kDa, including an SH2 domain, as well as putative SH3 and PTB interaction motifs. The polypeptide sequence contains no homology to known catalytic domains.

The presence of an SH2 domain in the F2771 sequence indicates that the F2771 protein is involved in phosphotyrosine-dependent protein interactions. SH2 domains are conserved regions of approximately 100 aa, found in many cytoplasmic signaling molecules (3, 31). Tyrosine phosphorylation acts as a switch to induce binding of SH2 domains, thereby mediating the formation of heterodimeric protein complexes. The F2771 aa 117-120 encodes YLVR instead of the highly conserved FLVR sequence found in most SH2 domains. Mutation studies where the phenylalanine of this motif was replaced by tyrosine (Phe right-arrow Tyr) in the SH2 domain of the c-Src oncoprotein showed that this substitution has no major effect on the transforming activity of the Src protein (36). The F2771 SH2 domain, like all SH2 domains described so far, contains at position 120 an arginine that is crucial for phosphotyrosine binding (37). Thus, the putative SH2 domain region in the F2771 sequence probably forms a functional phosphotyrosine binding domain. However, we have isolated one variant cDNA, where a 10-aa insertion is included in the SH2 domain at aa position 102 (Fig. 1A). This insertion may distort the structure of the SH2 domain, thus rendering the domain dysfunctional.

The deduced F2771 protein displays other sequences that may mediate protein-protein interactions. Many signaling proteins, with or without catalytic function, contain SH3 domains (2), which are conserved regions of 60-85 aa residues involved in cell polarization and in subcellular localization of proteins (38). The consensus SH3 binding motif is PXXP, with different SH3 domains having different preferences for particular sequence motifs (3, 32). Arginine at position +3 or -3 from the PXXP motif also seems to be part of the SH3 binding motif (32). The C-terminal proline-rich F2771 sequence contains, in addition to eight PXXP motifs, seven RXXP or PXXR motifs (Fig. 1A). Furthermore, the motif RPKPXXP is found twice in the F2771 sequence. Thus, it is highly probable the F2771 protein contains binding sites for SH3 domains.

Two NPXY motifs are found in the F2771 sequence. Phosporylated NPXY have been found to be bound by the PTB domains of Shc and insulin receptor substrate-1 (33). We observed that the F2771 protein is tyrosine-phosphorylated in vivo. If the tyrosines of the NPXY motifs of the F2771 sequence are phosphorylated, these motifs could interact with PTB domain-containing proteins.

The expression of F2771 at the mRNA level was extensively studied. The gene is not expressed in normal resting or activated B cells, nor in several transformed cell lines and various tissues tested. However, mRNA expression was observed in cells derived from thymus or spleen. While the F2771 gene was expressed at low levels in unstimulated T cells, it was rapidly induced in T cells after triggering through the CD4, CD8, or the CD3 molecules, and also shortly after exposure to adhesion activated monocytes. This rapid induction of mRNA expression could indicate that the gene is involved in the early stages of T cell activation. However, the gene continued to be expressed in long term cultures of activated T cells, indicating a functional role also in the later stages of T cell activation or proliferation.

Proteins involved in signal transmission in T cells may be expressed mainly or exclusively in T cells, but the majority are also expressed in other tissues. The tyrosine kinase ZAP-70 are mainly expressed in T cells (39), whereas the p56lck and the p59fyn kinases are also found in B cells (40, 41) and the central nervous system (42, 43). Several adapter proteins have been reported to have lymphocyte-specific expression. Lnk, containing an SH2 domain and a tyrosine phosphorylation site, is expressed in murine T cells (44). SKAP55 (45) and FYB (46) are adapter proteins that associate with p59fyn and that are mainly expressed in human T cells. Based on the structural features of the F2771 sequence and the pattern of expression, we suggest that the F2771 protein is an adapter protein involved in T cell signaling.

F2771 mRNA expression is rapidly induced upon triggering of the CD4 and CD8 receptors. Thus, a possible candidate with which the F2771 protein might interact is the p56lck tyrosine kinase, since p56lck is associated with these coreceptors (47). However, we were not able by coimmunoprecipitation studies to demonstrate an association between the p56lck protein and the F2771 protein in PHA blasts. This does not exclude the possibility that the F2771 protein could play a role in the CD4-p56lck-dependent signal transduction pathway.

We found by FISH analysis that the F2771 gene is located on chromosome 1q21. Aberrations of chromosome 1 with breakpoint at 1q21-q22 have repeatedly been found in Hodgkin's lymphomas (48). Amplification of 1q21-q22 has been found in soft tissue sarcomas (49). However, the F2771 gene is not found to be amplified in sarcomas.4

We failed to demonstrate expression of the F2771 gene, either at the mRNA or at the protein level, in several lymphoid cells lines derived from different lineages. The lack of expression of the F2771 protein in several T leukemic cell lines, as well as the chromosomal localization of the gene to a region that is often changed in lymphomas, is intriguing, but the significance of this observation is still unclear.

Conclusion-- In conclusion, we have characterized a novel gene, F2771, localized to chromosome 1q21, that encodes a cytosolic protein with no catalytic domains, but with an SH2 domain and several putative SH3 and PTB binding motifs. An antiserum raised against an F2771-derived peptide reacts with a 52-kDa protein expressed in T cells. We have shown that the protein is phosphorylated on tyrosine in vivo. The T cell-specific expression, the sequence structure and the probable cytoplasmic localization suggest that F2771 encode a T cell-specific adapter protein. Thus we propose that the F2771 protein be termed "T cell SH2 domain-containing adapter protein (TSAd)."

    ACKNOWLEDGEMENTS

Arne Deggerdal, Finn Eirik Johansen, Guttorm Haraldsen, and Ludvig M. Sollid have contributed to this work with instructions, ideas, and fruitful discussions. We thank Erik Thorsby and Erlend Smeland for critically reading the manuscript. We acknowledge the technical assistance of Line Bugge Klem, Ingebjørg Knutsen, Inger Johanne Ryen, Ellen Solum Karlstrøm, and Josiane Grosgeorge.

    FOOTNOTES

* This work was supported by Medinnova, Odd Fellow, and The Norwegian Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The 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 GenBankTM/EMBL Data Bank with accession number(s) AJ000553.

§ To whom correspondence should be addressed. Tel.: 47-22-86-85-53; Fax: 47-22-20-36-93.

1 The abbreviations used are: TCR, T cell receptor; SH2, Src homology domain 2; SH3, Src homology domain 3; PTB, phosphotyrosine binding domain; aa, amino acid(s); PBM, peripheral blood mononuclear cells; PCR, polymerase chain reaction; bp, base pair(s); EBV, EpsteinBarr virus; PHA, phytohemagglutinin; FISH, fluorescent in situ hybridization.

2 Protein sequence analysis programs are also available via the World Wide Web (http://expacy.hcuge.ch/www/tools.html).

3 The pfscan program is available via the World Wide Web (http://ulrec3.unil.ch/software/profilescan.html).

4 A. Forus, personal communication.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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