Cloning and Tyrosine Phosphorylation of a Novel Invertebrate Immunocyte Protein Containing Immunoreceptor Tyrosine-based Activation Motifs*

Immunoreceptor tyrosine-based activation motif (ITAM) plays an important role in signal transduction through mammalian T-cell and B-cell antigen receptors and Fc receptors. The ITAM has been found only in vertebrate immunocytes. Ascidians are intriguing invertebrates from the viewpoint of the evolution of immune systems because they are considered to be ancestors of the vertebrates. We have previously shown that the monoclonal antibody A74 inhibits cellular defense reactions of the ascidian. In the present studies, we found that the A74 antigen protein has two ITAMs and several motifs that are proposed to function in signal transduction. The A74 protein is tyrosine-phosphorylated and associated with other proteins in the initial stages of cellular defense reactions. The ITAMs of the A74 protein are tyrosine-phosphorylated by a c-Src kinase in vitro. The A74 protein provides a key to the understanding of the origin of vertebrate immune systems.

The understanding of innate immunity of protochordates, which occupy a phylogenetic position between vertebrates and true invertebrates and therefore are considered to be ancestors of the vertebrates, provides new insights into the origin and the evolution of acquired immunity of vertebrates. Hemocytes (i.e. immunocytes present in hemolymph) play important roles in host defense of invertebrates (1). In the ascidian Halocynthia roretzi, the hemocytes undergo several cellular defense reactions including phagocytosis and hemocyte aggregation (2).
Monoclonal antibodies that inhibit the cellular defense reactions in H. roretzi have been utilized to define hemocyte membrane-bound molecules that are involved in these reactions (2). The monoclonal antibody A74 inhibits phagocytosis of foreign substances by hemocytes and also aggregation of hemocytes (2,3); the former reaction is triggered by hemocyte-foreign substance interaction (3), whereas the latter is triggered by hemocyte-hemocyte interaction (4). We purified the A74 antigen protein from H. roretzi hemocytes and found that it is a novel membrane glycoprotein with a molecular mass of 160 kDa (3); the molecular mass of its protein portion is approximately 90 kDa.
In this paper we report the cloning of the A74 protein. We found that the A74 protein has two (one typical and one nontypical) ITAMs, 1 which have been reported to play important roles in signal transduction through mammalian TCR, BCR, and FcRs (5,6). We also demonstrated that the ITAMs of the A74 protein are tyrosine-phosphorylated by a c-Src kinase. To our knowledge, this is the first finding concerning ITAM in invertebrates.

EXPERIMENTAL PROCEDURES
cDNA Cloning-The N-terminal amino acid sequence (3) of ascidian A74 protein was used to design degenerate oligonucleotide primers for PCR (5Ј- The primers at concentrations of 10 M were mixed in PCR to amplify the H. roretzi hemocyte cDNA library. PCR was done in 10 mM Tris-HCl, pH 9.5, containing 1.5 mM MgCl 2 , 50 mM KCl, 0.1% Triton X-100, 4 mM deoxynucleotides, and 25 units/ml Taq DNA polymerase (Toyobo). After denaturation at 94°C for 5 min, 30 cycles were run with annealing at 42°C for 2 min, elongation at 72°C for 3 min, and denaturation at 94°C for 1 min. A DNA band migrating at about 50 base pairs was isolated, cloned into a pGEM-T vector (Promega), and transformed into JM109 cells. The A74 cDNA clones were picked from 1 ϫ 10 6 clones of the gt11 cDNA library by phage plaque hybridization (7) using the subcloned N-terminal 51-base pair DNA fragment as a probe. Seventeen independent positive clones were obtained and had inserts of the cDNA of 1.1-3.4 kilobases. The longest insert clone containing the N-terminal amino acid sequence and the 3Ј-end poly(A) ϩ tail was subcloned in the EcoRI site of the pBluescript SK ϩ plasmid DNA. The clone was sequenced on both strands by deletion methods. The nucleotide sequence of the A74 insert cDNA fragment was determined by a Taq dye primer cycle sequencing kit (Applied Biosystems) using an ABI 373A DNA sequencing apparatus (Applied Biosystems).
Immunoprecipitation-Aggregated hemocytes (1 ϫ 10 8 cells) of H. roretzi (3) were washed with filtered artificial seawater and frozen in liquid N 2 . The frozen hemocytes were lysed by homogenization and stirring at 4°C for 30 min in 3 ml of lysis buffer (50 mM Tris-HCl, pH 7.6, containing 0.1% Lubrol PX, 0.15 M NaCl, 0.02% NaN 3 , 10% glycerol, protease inhibitors (10 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 0.2 mM diisopropylfluorophosphate, and 0.2 mM leupeptin), and phosphatase inhibitors (2 mM Na 3 VO 4 and 50 mM NaF)). The lysate was clarified by centrifugation at 12,000 ϫ g for 10 min, and the resulting supernatant was subjected to immunoprecipitation by treatment with * This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan. 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) AB007512.
‡ To whom correspondence should be addressed: Dept. of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan. Tel.: 81-11-706-3754; Fax: 81-11-706-4900; E-mail: yoko@ pharm.hokudai.ac.jp. 50 l (wet volume) of A74 antibody-immobilized Sepharose (3) at 4°C for 3 h and subsequently by centrifugation (50 ϫ g, 1 min). The resulting immunoprecipitates were washed three times with the lysis buffer containing 0.5 M NaCl, treated with 0.1 ml of the sample buffer for SDS-PAGE for 5 min at 95°C, and centrifuged. The resulting supernatant was boiled for 5 min after the addition of 5% 2-mercaptoethanol and was then subjected to SDS-PAGE and Western blotting with antiphosphotyrosine antibody PY54 (Affiniti) or PY20 (Leinco Technologies). Mouse IgG-Sepharose was used as a control.
Expression of GST-ITAM Fusion Proteins-DNA fragments corresponding to the N-terminal nontypical ITAM (ITAM (N), amino acids 299 -317), the C-terminal typical ITAM (ITAM (C), amino acids 356 -375), and the two-repeated ITAMs (ITAM (N, C), amino acids 299 -375) were amplified by PCR from the A74 cDNA using the following combinations of forward and reverse primers, at the ends of which EcoRI and XhoI sites, respectively, were included; two forward primers (a, 5Ј-GAATTCAATACAAACTATACGAAC-3Ј, and b, 5Ј-GAATTCCTTTATT-TCCCCCTCAAG-3Ј) and two reverse primers (c, 5Ј-CTCGAGAACT-TCGTTTTTCTCACT-3Ј, and d, 5Ј-CTCGAGCGCCGCTGTGATTGT-3Ј) were designed, and the combinations of primers for ITAM (N, C), ITAM (N), and ITAM (C) were (a ϩ d), (a ϩ c), and (b ϩ d), respectively. The amplified DNA fragments were isolated, digested with EcoRI and XhoI (Toyobo), and cloned into the pGEX-4T-1 expression vector (Pharmacia Biotech Inc.). The respective cloned sequences were confirmed by DNA sequencing. Three GST fusion protein expression vectors were used to transform Escherichia coli DH5␣ to ampicillin resistance. The expressed fusion proteins were purified using glutathione-agarose beads (Sigma) according to the instruction manual.
Tyrosine Phosphorylation of GST-ITAM Fusion Proteins-The purified GST-ITAM fusion proteins were tyrosine-phosphorylated with recombinant human c-Src kinase (8). Three GST-ITAM fusion proteins bound to glutathione-agarose beads were washed and suspended in phosphorylation buffer (20 mM Tris-HCl, pH 7.5, containing 10 mM MgCl 2 , 1 mM dithiothreitol, 0.1% Triton X-100, and 0.5 mM Na 3 VO 4 ). GST was used as a control. Then 50 units/ml c-Src kinase (Upstate Biotechnology, Inc.) and 5 mM ATP were added to start the reaction, and the solutions were incubated at 30°C for 24 h with gentle shaking. The beads were then collected and washed with the phosphorylation buffer and also with 20 mM Tris-HCl, pH 7.5, containing 2 mM MgCl 2 and 1 mM dithiothreitol. The fusion proteins were eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 9.6, and were subjected to SDS-PAGE, followed by Western blotting with anti-phosphotyrosine antibody PY20 and also by protein staining with Coomassie Brilliant Blue R250.

RESULTS AND DISCUSSION
Cloning and Northern Blot Analysis-In a previous study (3), we purified the A74 protein from ascidian hemocytes and determined its N-terminal peptide sequence. The N-terminal sequence was used to design degenerate oligonucleotide primers for PCR of the H. roretzi hemocyte gt11 cDNA library. The deduced amino acid sequences of the PCR products were found to include the N-terminal peptide sequence (3) of the A74 protein. The N-terminal 51-base pair PCR product was used as a probe for screening the hemocyte gt11 cDNA library to isolate the A74 cDNA clone. On Northern blot analysis of the A74 mRNA using 32 P-labeled A74 cDNA as a probe, a single transcript of approximately 3.4 kilobases was detected only in hemocytes of H. roretzi, whereas the expression was low or zero in other tissues (Fig. 1).
Nucleotide and Deduced Amino Acid Sequences of A74 Protein-The A74 cDNA clone consists of 3,390 nucleotides (Fig. 2) and has a poly(A) ϩ tail. A single open reading frame encodes 722 amino acids. The deduced translated protein sequence contains a sequence from residues 20 to 37 that corresponds to the N-terminal amino acid sequence determined from the isolated A74 protein. This result suggests that the mature A74 protein begins with the alanine residue as its N terminus at position 20. The amino acid sequence begins with a typical hydrophobic signal sequence followed by an extracellular region of 275 amino acid residues. In its extracellular region, the A74 protein contains five potential N-linked glycosylation sites (Asn-Xaa-(Thr/Ser)) and five cysteine residues (there is no in-formation concerning disulfide bonds). The putative transmembrane domain is represented by a 26-residue stretch of hydrophobic amino acids followed by a 421-amino acid long cytoplasmic domain.
Domain Organization of A74 Protein-Searches of the Gen-Bank™, EMBL, and DDBJ data bases revealed little significant similarity of the A74 protein to other proteins. The extracellular domain of the A74 protein shows no homology to the known immunoglobulin superfamily or to cell adhesion proteins. In its intracellular domain, however, the A74 protein contains several interesting motifs (Fig. 2), which have been proposed to be involved in tyrosine phosphorylation signaling (5,6,9) and receptor-mediated endocytosis (10,11) in mammals. The first motif is the ITAM (consensus sequence, Tyr-Xaa 2 -(Leu/Ile)-Xaa 6 -8 -Tyr-Xaa 2 -(Leu/Ile)) (5, 6) and is repeated twice near the transmembrane region of the cytoplasmic domain. The first ITAM from Tyr 302 to Lys 317 is a nontypical one, in which the Lys residue occupies the last Leu/Ile of the typical ITAM, whereas the second ITAM from Tyr 357 to Ile 372 is a typical ITAM that is detected in mammalian immunoreceptors (Fig. 3). The ITAM has been reported to function in signal transduction through mammalian TCR, BCR, and FcRs (Fig. 3A), and binding of ligand to the respective receptor triggers phosphorylation of the ITAM at two conserved tyrosine residues by a Src family kinase (5, 6). To our knowledge, this is the first finding concerning ITAM in invertebrate cells including immunocytes.
The second motifs found in the A74 protein are potential SH2/SH3 binding sequences (three SH2 binding sequences (9,12) and one SH3 binding sequence (13)) present in the middle of the cytoplasmic domain (Figs. 2 and 3A). It can be inferred that SH2-containing and SH3-containing proteins bind to these motifs and orchestrate the assembly of signaling complexes downstream of tyrosine kinases. In addition, the A74 protein contains two pairs of trithreonine motifs (14) near the end of cytoplasmic domain (Figs. 2 and 3A) and also eight pairs of dileucine motifs (10,11) in the cytoplasmic domain (Fig. 2).
Tyrosine Phosphorylation of A74 Protein-To address the question as to whether the A74 protein is involved in signal transduction in ascidian cellular defense reactions, we ana-  2. Nucleotide and deduced amino acid sequences of A74 cDNA. The sequence is numbered from the first base at the 5Ј end. The polyadenylation signal, AATAAAA, is underlined. The deduced amino acid sequence matching the N-terminal sequence of purified A74 protein is lyzed tyrosine phosphorylation during hemocyte aggregation in H. roretzi (Fig. 4A). Transient tyrosine phosphorylation of two proteins of 260 and 160 kDa was observed 5-10 min after induction of hemocyte aggregation (4) followed by tyrosine phosphorylation of 90-and 75-kDa proteins, which indicates that there is different timing of tyrosine phosphorylation in the process of hemocyte aggregation. Tyrosine phosphorylation of the A74 protein was detected also during phagocytosis (data not shown). It should be noted that addition of the A74 antibody to the hemocyte suspension triggered tyrosine phosphorylation of the above-mentioned proteins including the A74 protein of 160 kDa (data not shown), which suggests that the A74 antibody has an agonistic activity in a manner similar to that of an anti-CD8 antibody that can activate a tyrosine ki-  4. Tyrosine phosphorylation during ascidian hemocyte aggregation. A, H. roretzi hemocytes collected in a process of hemocyte aggregation were subjected to SDS-PAGE, followed by Western blotting with the anti-phosphotyrosine antibody. The left panel shows time-dependent tyrosine phosphorylation of hemocyte proteins including the A74 protein of 160 kDa, whereas the right panel shows the result on protein staining. B, the immunoprecipitates formed by treatment with the A74 antibody were subjected to SDS-PAGE followed by Western blotting with anti-phosphotyrosine antibody. The A74 protein of 160 kDa was tyrosine-phosphorylated and associated with 90-and 75-kDa proteins, both of which were also tyrosine-phosphorylated.
underlined by a dotted line, the putative N-linked glycosylation sites are indicated by arrowheads, the cysteine residues in a mature extracellular part are indicated by circles, and the putative transmembrane region is underlined by a bold line. The transmembrane region is determined using the TMAP server (EMBL) (18). The Tyr-Xaa 2 -(Leu/Ile) pairs are shown by shaded squares, the potential SH2 binding motifs containing the conserved tyrosine residue are indicated by open squares, the conserved two proline residues in the potential SH3 binding motif are indicated by circles, the trithreonine motifs are underlined, and the dileucine motifs are indicated by asterisks.
nase pathway through a chimeric protein linking the extracellular and transmembrane domains of CD8 to the ITAM-containing cytoplasmic domain of the T cell receptor chain (15).
Association of A74 Protein with Other Proteins-On immunoprecipitation with A74 antibody-immobilized Sepharose from the lysate of aggregated hemocytes followed by Western blotting with anti-phosphotyrosine antibody, at least three proteins of 160, 90, and 75 kDa were co-precipitated and tyrosinephosphorylated (Fig. 4B), indicating that the A74 protein of 160 kDa is tyrosine-phosphorylated and is associated with tyrosinephosphorylated proteins. On SDS-PAGE of the above immunoprecipitates, three protein bands with molecular masses of 105, 75, and 56 kDa were detected by protein staining (data not shown), suggesting that the A74 protein is also associated with nonphosphorylated proteins.
Tyrosine Phosphorylation of GST-ITAM Fusion Proteins-To obtain definitive evidence for the involvement in signal transduction of ITAMs derived from the A74 protein, we expressed three GST fusion proteins carrying the N-terminal nontypical ITAM, the C-terminal typical ITAM, and the twice-repeated ITAMs (Fig. 5A). We demonstrated that each of the two ITAMs present in the A74 protein was tyrosine-phosphorylated by human c-Src kinase in vitro (Fig. 5B). In addition, our preliminary result indicates that a tyrosine-phosphorylated protein of 75 kDa was bound to each of the tyrosine-phosphorylated ITAMs. Taken together, these results lead us to propose that the A74 protein is involved in the initial stage of signal transduction through tyrosine phosphorylation.
Evolutionary Implications-To understand the signal transduction cascade through the A74 protein, we compared the A74 protein-mediated ascidian immune systems with mammalian immune systems, in both of which the ITAM-containing proteins are involved. The mammalian immunocytes use the receptors of oligomeric structures composed of signal recognition and signal-transducing subunits (see Fig. 3A). The receptors catch the respective signals through the former subunits and transduce them through the latter subunits: The ITAM-containing TCR-induced signaling cascade triggers the activation of transcriptional regulators to induce gene expression of cytokines (16,17), and the signal cascade through the ITAM-containing FcR induces phagocytosis (5). In H. roretzi, the A74 antibody inhibits hemocyte aggregation (cell-cell adhesion) and phagocytosis (foreign substance recognition) (3), which suggests that the extracellular domain of A74 protein catches the respective signals in both reactions. In addition, our results provide evidence that the ITAM-containing intracellular domain of A74 protein plays a role in signal transduction. It seems reasonable to suppose that expression of putative immunity genes occurs in the A74 protein-mediated cellular responses in H. roretzi. Thus, in contrast with the mammalian oligomeric immunoreceptors, the A74 protein is a multi-functional single molecule involved in both signal recognition and signal transduction. This implication leads us to hypothesize that a single prototypic immunoreceptor might be separated into two parts, signal recognition and signal-transducing subunits in a process of the evolution. Further investigation of the functions of the respective domains of A74 protein will provide an important key to understanding the origin of vertebrate immune systems.  N)), the C-terminal typical ITAM (ITAM (C)), or the twice-repeated ITAMs (ITAM (N, C)). B, tyrosine phosphorylation of GST-ITAM fusion proteins by human c-Src kinase. Three GST fusion proteins carrying ITAM (N), ITAM (C), and ITAM (N, C), which had been bound to glutathione-agarose beads, were incubated with (ϩ) or without (Ϫ) human c-Src kinase (50 units/ml) in the presence of 5 mM ATP at 30°C for 24 h. After elution from the beads with reduced glutathione, pH 9.6, the fusion proteins were subjected to SDS-PAGE, followed by protein staining (Coomassie Brilliant Blue; CBB) and also by Western blotting with the anti-phosphotyrosine antibody (PY20). GST was used as a control. Three GST-ITAM fusion proteins, but not GST alone, were tyrosine-phosphorylated by the c-Src kinase.