Cloning and Characterization of HTK a Novel Transmembrane Tyrosine Kinase of the EPH Subfamily*

Using a polymerase chain reaction based strategy, we identified a novel transmembrane tyrosine kinase in CD34+ human bone marrow cells and a human hepato- cellular carcinoma cell line, Hep3B. This protein, hepatoma transmembrane kinase or Htk, shares amino acid similarity with the EPH subfamily of tyrosine kinases. The HTK gene is located on human chromosome 7. The predicted 987-amino acid sequence of Htk includes a transmembrane region and signal sequence. In the predicted extracellular domain, a cysteine-rich region and tandem fibronectin type 111 repeats are present while a single uninterrupted catalytic domain is present in the intracellular domain. These features are consistent with other members of the Eph subfamily. Antibodies raised against Htk extracellular domain immunoprecipitated a 120-kDa protein from either in vitro translated HTK or Hep3B cells which localized primarily to the Hep3B membrane subcellular fraction. Purified in vitro translated Htk was enzymatically active and autophospho- rylated on tyrosine in kinase assays. Furthermore, antibodies against Htk ECD were agonistic, inducing Htk tyrosine phosphorylation in transfected MH3T3 cells. Northern blot analysis demonstrated a single HTK tran- script abundantly

Using a polymerase chain reaction based strategy, we identified a novel transmembrane tyrosine kinase in CD34+ human bone marrow cells and a human hepatocellular carcinoma cell line, Hep3B. This protein, hepatoma transmembrane kinase or Htk, shares amino acid similarity with the EPH subfamily of tyrosine kinases. The HTK gene is located on human chromosome 7. The predicted 987-amino acid sequence of Htk includes a transmembrane region and signal sequence. In the predicted extracellular domain, a cysteine-rich region and tandem fibronectin type 111 repeats are present while a single uninterrupted catalytic domain is present in the intracellular domain. These features are consistent with other members of the Eph subfamily. Antibodies raised against Htk extracellular domain immunoprecipitated a 120-kDa protein from either in vitro translated HTK or Hep3B cells which localized primarily to the Hep3B membrane subcellular fraction. Purified in vitro translated Htk was enzymatically active and autophosphorylated on tyrosine in kinase assays. Furthermore, antibodies against Htk ECD were agonistic, inducing Htk tyrosine phosphorylation in transfected MH3T3 cells. Northern blot analysis demonstrated a single HTK transcript abundantly present in placenta and in a range of primary tissues and malignant cell lines. HTK appears to be expressed in fetal but not adult brain and in primitive and myeloid but not lymphoid hematopoietic cells. The novel transmembrane protein, Htk, may function as a receptor with an expression pattern suggesting a role in events mediating differentiation and development.
Tyrosine kinases mediate critical events in regulating the proliferation and differentiation of eukaryotic cells. The subset of transmembrane tyrosine kinases often serve as receptors for growth factors and have been implicated in developmental processes as well as in malignant transformation (1-4). Among the transmembrane tyrosine kinases there are several subfamilies which may be distinguished according to specific features of their extracellular and/or intracellular domains (3, 4). The presence of a cysteine-rich extracellular region is characteristic of subfamilies typified by the EGF' receptor, the in-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: EGF, epidermal growth factor; PCR, polymerase chain reaction; bp, base pair(s); PAGE, polyacrylamide gel sulin receptor or, the orphan receptor, Eph, respectively. The EGF receptor subfamily typically has two or three such cysteine-rich domains while the insulin receptor subfamily has a single short cysteine-rich region on one of the subunits generated from a proteolytically cleaved precursor protein. EPH a n d related genes typically encode proteins with a single cysteinerich domain and two fibronectin type I11 repeats in the extracellular region on a single, non-cleaved protein (5-14). In addition this subfamily usually encodes a single kinase domain which, unlike the platelet-derived growth factor receptor or kit subfamily, is not interrupted by a kinase insert domain. The gene reported here appears to encode a protein with features typical of the Eph subfamily. Patterns of expression of certain members of this subfamily suggest functional importance in mediating developmental events, particularly in the nervous system (7, 10, 11, 15-18). Additionally, overexpression of the EPH gene can induce tumorigenicity (19).
Given the importance of the transmembrane tyrosine kinases in the regulation of a spectrum of cellular events, we undertook a strategy to identify novel members of the tyrosine kinase gene family focusing on sequences encoding transmembrane proteins. Reported here is a novel member of the EPH subfamily which has tissue restricted expression and functional characteristics of a tyrosine kinase.
Northern Blotting-Poly(A)-selected RNA was electrophoresed through a 1.2% agarose, 2.2 M formaldehyde gel and transferred to a nylon filter. Prepared or commercially obtained filters were hybridized in 50% formamide at 42 "C to 32P-labeled HTK, glyceraldehyde-3-phosphate dehydrogenase, or actin (data not shown) cDNA inserts and washed under stringent conditions (final wash 0.1 x SSC, 0.2% SDS at 65 "C). SSC is 0.15 M NaC1, 0.015 M Na,.citrate, pH 7.6.
Antibodies-An HTK extracellular domain-human IgG, PC fusion gene was constructed and fusion protein produced as previously described (23). Rabbit polyclonal antiserum was raised using the fusion protein as immunogen. Htk specificity of the immunized rabbit serum was assessed by flow cytometric analysis of NIH3T3 cells transfected with full-length HTK or vector alone using a 1:200 dilution of preimmune serum or anti-Htk-IgG Fc serum. Significant peak shifts were observed in several HTK expressing clones as compared to either preimmune serum or vector alone transfectant controls.
In Vitro Danscription and Danslation-Transcription was performed on 2 pmol of linearized HTK or FLAG-HTK containing plasmid at 37 "C for 1 h in a 50-p1 volume containing 10 nm dithiothreitol, 2.5 pg of bovine serum albumin, 0.25 m M each dNTP, 0.5 M m'GRNA cap (New England Biolabs, Beverly, MA), 2.5 units of RNasin (Promega, Madison, WI), 3 units of T3 RNA polymerase (Pharmacia, Piscataway, NJ). 1 pg of DNase (New England Biolabs) was added for 15 min at 37 "C prior to phenollchloroform extraction and ethanol precipitation. Translation was performed using the Promega rabbit reticulocyte lysate kit according to the manufacturer's specifications with or without [35Slmethionine (350 pCi) labeling. Sample buffer containing SDS and P-mercaptoethanol was added before boiling and 10% SDS-PAGE.
HTK Expression in NIH3T3 Cells-A 4038-bp ClaI-XbaI cDNA fragment containing 32 bp of linker sequence, 37 bp of pBluescript (Stratagene) polylinker, and the entire 3969-bp HTK cDNA was subcloned into the expression vector pRIS (Genentech, Inc.) under the control of the Rous sarcoma virus long terminal repeat promoter. NIH3T3 cells maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum were co-transfected with pRIS-HTK and pNeo (an SV40 based vector containing the neomycin resistance marker) by the calcium phosphate method as described (24). Neomycinresistant colonies were selected 48 h after transfection with Geneticin (Life Technologies Inc.) at 400 pg/ml. Fourteen days later individual resistant colonies were isolated, expanded, and analyzed by flow cytometry for Htk expression using rabbit polyclonal antiserum.
Cell 150 m M NaC1) blocked by incubating in TBST (TBS with 0.05% Tween 20) containing 5% dry milk (Carnation) for 2-5 h. Filters were washed four times for 5 min each in TBST and incubated overnight with antiphosphotyrosine antibody (1:5000 dilution in TBST). Filters were washed four times for 5 min each in TBST and incubated for 2 h with the horseradish peroxidase-labeled anti-mouse secondary antibody (Amersham) at a 1:3000 dilution in TBST. After washing four times, the enhanced chemiluminescence system (ECL, Amersham) was used for detection for 10 s to 2 min.
Antibody-induced Phosphorylation Assay-Cells were plated at a density of 5 x lo5 cells/well in a 6-well plate and, after 24 h, were serum starved for 1 h prior to adding preimmune or immune serum at a 1:50 dilution for 30 min. Cells were then washed in phosphate-buffered saline and lysed in either 2 x sample buffer or Nonidet P-40 lysis buffer as described above. Either crude lysates or immunoprecipitated cell lysates were then separated ria 612% gradient SDS-PAGE and analyzed by anti-phosphotyrosine immunoblot as described above.

RESULTS
The method of Wilks (21), which utilizes consensus sequences within related tyrosine kinases (20) to clone fragments of novel tyrosine kinase-related genes, was adapted using the primers described under "Materials and Methods." Following reverse transcriptase PCR amplification of mRNA from the human hepatoma cell line, HepSB, approximately 140 recombinants were characterized by nucleic acid sequencing. Comparative databank analysis revealed four novel sequences of which one, termed HpTK5, was identical in sequence to a frag-1 TCGGCGTCCACCCGCCCAGGGAGAGAGTCAGACCT~CGAGGGCCCCCCAAAC~AGTTCCAGGACGGATCCTACCCGAGTGAGGCGGCGCCATGGAGCTCCG

H E K G A E G P S S V R F L K T S E N R A E L R G L K R G A S Y L 490
500

V Q V R A R S E A G Y G P F G Q E H H S Q T Q L D E S E G W R E Q 520
530

940
950 960 970   The predicted protein sequence includes a transmembrane region (amino acids 538-563) which divides Htk into extracellular (ECD) and intracellular domains. The ECD of 538 amino acids includes a signal peptide of 15 amino acids (38) and a cysteine-rich box containing 20 Cys residues shared among other members of the EPH gene subfamily (Fig. 2) (5-9, 12). In addition, there are two fibronectin type I11 repeats spanning amino acids 321-425 and 435-526. Similar repeats are found in all other EPH-related proteins as well as the tyrosine kinase, AXL, which is not in the EPH subfamily (26). Asn a t positions 208, 340, and 431 are possible sites for N-glycosylation.

T S D P T Y T S S L G G K I P~R W T A P E A .
The putative intracellular domain contains a kinase consensus region from position 613 to 881 (20). This kinase region includes a putative ATP-binding consensus (Gly-X-Gly-X-X-Gly) in subdomain I at positions 622-627 (Gly-Ala-Gly-Glu-Phe-Gly) (20,(27)(28)(29). A Lys a t position 647 (subdomain 11) corresponds to an invariant Lys among tyrosine kinases thought to be critical for the phosphotransfer reaction. Signature regions indicative of substrate specificity suggest that Htk is a tyrosine rather than a serinelthreonine kinase. at positions 783-790 in subdomain VI11 (20,(27)(28)(29). Tyr at positions 601, 619, or 741 are possible substrates for tyrosine kinase activity (30)(31)(32). The 'lJ+Ile-Asp-Pro-Phe-Thr-Tyr-Glu-Asp-Pro sequence (amino acids 5954304) immediately downstream of the membrane spanning domain, has been suggested by others (13) to possibly play a role in Eph subfamily members binding to c-Src based on the similarity of the sequence to a similar, defined functional motif in the platelet-derived growth factor receptor (33). However, according to the analysis of Songyang ct 01. (34), the only phosphorylation site motif with recognition specificity for any of 13 analyzed SH2 domains follows Tyrfiol and is predicted to interact with the SH2 domain of Abl.
The relationship of Htk to two other tyrosine kinase is illustrated in Fig. 2. Hek2 and Elk are the most closely related tyrosine kinases to Htk. They share 79.3 and 76.5% identity within the intracellular domains, respectively, and 45 and 42% identity within the ECD, respectively.
Primers derived from the unique 3"untranslated sequence of HTK were used to identify its human chromosomal localization. The 3'-untranslated region characteristically contains few, if any, intervening sequences ( 3 5 3 6 ) a n d h a s a high degree of diversity among members of gene families (37) making it preferred in this type of analysis. Two independent sets of primers, which were specific for human HTK, were used to amplify DNA from a panel of human-hamster hybrid cell lines. Both sets of primers gave results that were consistent with human chromosome 7 only (single set shown in Fig. 3). Human chromosome 7 also includes the genes for the EGF receptor, hepatocyte growth factor receptor, hepatocyte growth factor, platelet-derived growth factor, and interleukin-6. Karyotypic abnormalities involving this chromosome are common among human leukemias, particularly in aggressive myeloid leukemias that occur following radiation, alkylating agent chemotherapy, or a pre-existing myelodysplastic condition (41 ). Northern blot analysis of human fetal tissues revealed a single transcript of -4 kilobases in heart, lung, liver. and kidney, with a lesser signal detectable in brain (Fig. 4A ). In adult human tissue, no signal was detectable in hrain. while placenta had a particularly intense simal followed by kidney, liver, lung. and pancreas. Skeletal muscle and heart were of lower signal intensity (Fig. 4R ). Cell lines derived from liver. hreast. colon. lung, melanocyte, or cervix bad detectable signal of appropriate size. Message was present in select cell lines of hematopoietic origin. K562 (a primitive myeloid cell with multipotentiaIJ. THP-1 (a monocytoid cell), CMK and SO (both of megakaryocytic origin) were all positive for HTK message, but lymphoid (H9, Jurkat, JH-1, Raji, Ramos) or other select myeloid cells (KG-1 or KMT2) had no detectable transcript hy Northern analysis (Fig. 5 ) .
An Htk ECD-IgG Fc fusion protein was expressed, purified. and used to generate rabbit antiserum which immunoprecipitated a 120-kDa protein from Hep3B cells (Fig. 6C ). The specificity of the antiserum was confirmed hy immunoprecipitation of in vitro translated HTK RNA and HTK transfected NIH3T.7 cells (Figs. 6A and 9). To determine the functional capacity of Htk, in uifro translated Htk was immunoprecipitated, exposed to kinase conditions (Fig.  CiA ), and immunohlotted using a phosphotyrosine-specific monoclonal antibody (Fig. fiR ). These data indicate that Htk is phosphorylated on tyrosine. However, the presence of other bands consistently appearing in the "'Plabeled immunoprecipitation (Fig. 6A. lanr 3 , is representative) suggested that the Htk protein was only partially purified and therefore, it could not be concluded that Htk was enzymatically active.
To overcome this problem, a fusion construct was generated in which an %amino acid epitope (FLAG) was added to the N terminus of Htk. The FLAG-Htk fusion was in vitro translated and immunoprecipitated with a FLAG-specific monoclonal antibody resulting in a single protein of appropriate size (-120 kDa). When subjected to kinase conditions in the presence of ['"PIATP, the Htk-FLAG fusion protein was labeled (Fig. 7 A ) on tyrosine (Fig. 7B) confirming tyrosine autophosphorylation and thereby, the kinase function of Htk.
Cell fractionation of Hep3B cells was performed to confirm the membrane localization of Htk predicted by its amino acid sequence. Fig. 8 demonstrates that Htk segregated predominantly with the membrane fraction, although immunoprecipitated material was evident to a lesser extent in cytosol.
Rabbit antisera to Htk-I@ Fc were tested for their ability to induce Htk phosphorylation in H T K transfected NIH3T3 cells. Htk expressing cells were exposed to antisera and separated by SDS-PAGE either with or without immunoprecipitation. The electrotransferred gel was immunohlotted with anti-phosphotyrosine antibody. Enhanced tyrosine phosphorylation of Htk was observed following exposure to polyclonal antiserum ( Fig.  9) suggesting an agonist-like effect of antibody hinding.
No ligands for the members of the EPif-encoding transmembrane proteins have yet been identified.
A chimeric protein, utilizing the intracellular portion of Elk and EGF receptor ECD, served to induce growth in cells exposed to EGF, suggesting that proliferative signals may be transduced via activation of the tyrosine kinase function f 1.3,. The tyrosine kinase activity of HTK, a novel member of the EPH subfamily, supports the hypothesis that Htk is a signal transducing molecule. Interaction of Htk with an antibody directed against its ECD induces phosphorylation. This provides further support that Htk may serve as a receptor for a ligand that triggers kinase activation. Details of the signaling pathway of Htk may be further explored using antisera as a surrogate ligand.
Differential expression of the i I T K transcript in fetal [vrsus adult brain suggests that Htk may share with other Eph subfamily members, a role in events related to neural development. However, unlike some members of the E P i f subfamily which are exclusively expressed in neurons (401. HTK is widely expressed in other tissues. In particular, HTK is expressed in hematopoietic cells including CD.34+ hematopoietic progenitor cells. The presence of the IfTK message in early hematopoietic cells and cell lines of myeloid lineage, but not in cell lines derived from lymphoid cells, suggests that HTK may have lineage restricted expression. The localization of IfTK on human chromosome 7 is also of interest regarding the hematopoietic system, as karyotypic abnormalities of chromosome 7 are frequently seen in myeloid leukemias (41 ). The expression ofHTK in primary hematopoietic cells and further chromosomal mapping of IfTK in normal and malignant cells are under investigation. Structural analysis of Htk and identification of its ligand will be required to further define its hiological role.