Molecular cloning of a T cell-specific adapter protein (TSAd) containing an Src homology (SH) 2 domain and putative SH3 and phosphotyrosine binding sites.

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.

described previously (15)(16)(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 Ͻ2% contaminating cells as assessed by flow cytometry and showed Ͼ95% viability as determined by acridine orange/ethidium bromide staining.
CD8ϩ cells were left overnight in RPMI supplemented with 10% pooled human serum at 37°C, 95% relative humidity and 5% CO 2 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 ϫ 10 6 cells were frozen as cell pellets at Ϫ70°C for later RNA extraction.
cDNA Library-A cDNA library in the pcDNAI plasmid vector was custom made by Invitrogen (San Diego, CA) from 100 ϫ 10 6 CD8ϩ 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 ϫ 10 6 primary recombinants.
Total RNA was extracted from fresh or frozen pellets of 50 ϫ 10 6 cells by the guanidine thiocyanate method (21).
Subtraction Probe-Preparation of a subtraction probe was performed as described previously in detail (22). Briefly, 10 g 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 g of mRNA isolated from anti-TCR activated CD8ϩ 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 l of 0.5 M EDTA. After washing the beads in TE, 50 l 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 MgCl 2 , 0.2 mM dNTPs, 2.5 units/100 l Taq polymerase; Perkin Elmer) containing 20 pmol of the T-primer (5Ј-dTTGCATTGACGTCGACTATCCAGGTTTTTTTTTTTTT-TT-3Ј) 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 l of this reaction was performed for an additional 20 cycles as above in 50 l of the same PCR amplification mixture containing 50 pmol of L-primer: 5Ј-dTTGCATTGACGTCGACTATCCAGG-3Ј. 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 g/ml ampicillin and 7.5 g/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 ϫ SSPE, 5 ϫ 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 [␣-32 P]dCTP (Amersham, UK). After overnight hybridization, a stringency wash was performed in 0.1 ϫ 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 ϫ 10 4 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 g) of total RNA were sizefractionated on 1% agarose gel/formaldehyde denaturing gels, transferred to nylon membranes (Hybond-N nylon, 0.45 m; 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 Ϫ70°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 g 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-His 6 tag 3Ј 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 MRGS⅐ 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 Na 2ϩ 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 g 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 pro- 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).
tein 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
Isolation of F2771, a Novel Human cDNA Clone Encoding a Polypeptide Containing an SH2 Domain-Using a subtractive T Cell-specific Adapter Protein 4542 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 5Ј F2771 ligated to the pcDNAI vector, failed to reveal a more 5Ј 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 (XCXGC-CATGG; 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.
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.
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 CD8ϩ 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 CD4ϩ and CD8ϩ cells, but not in resting CD4ϩ and CD8ϩ 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).
When probing 10 5 cDNA clones from the cDNA library of activated CD8ϩ 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 CD4ϩ cells by cross-linking of the CD4ϩ 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 CD4ϩ but not in CD8ϩ 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 CD8ϩ T cells (Fig. 4C, right panel). 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 Cterminal histidine tag (MRGS-His 6 ). Microscopy of COS-1 cells transiently transfected with these constructs, visualized using an anti-His-specific antibody and the alkalic phosphatase antialkalic 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.
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 CD4ϩ and CD8ϩ 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 posi-tively selected CD4ϩ and CD8ϩ T cells treated in a similar way (Fig. 4C).
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).
Attempts to coprecipitate the p56 lck protein from lysates of PHA blasts, using the anti-F2771 antiserum or vice versa using an antiserum against the p56 lck protein, failed to reveal an association between the two proteins ( Fig. 7A and data not shown). DISCUSSION 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 3 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 ϩ3 or Ϫ3 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 p56 lck and the p59 fyn 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 p59 fyn 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 p56 lck tyrosine kinase, since p56 lck is associated with these coreceptors (47). However, we were not able by coimmunoprecipitation studies to demonstrate an association between the p56 lck 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-p56 lck -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)."