Molecular cloning of HEK, the gene encoding a receptor tyrosine kinase expressed by human lymphoid tumor cell lines.
We describe the molecular cloning of a receptor tyrosine kinase from a cell line (LK63) derived from a case of human pre-B-cell leukemia. We have previously shown that a monoclonal antibody (IIIA4) raised against LK63 recognized a glycosylated, cell-surface 135-kDa molecule (HEK), which displayed tyrosine kinase activity in vitro. The HEK protein was purified by using a IIIA4 antibody column and both N-terminal and internal amino acid sequences were obtained. A 51-mer degenerate oligonucleotide based on the internal amino acid sequence was used to screen an LK63-derived lambda gt10 cDNA library under low-stringency hybridization conditions. One clone of 2.5 kilobases (kb) was isolated and characterized and used to rescreen the library under more-stringent hybridization conditions. A 4.5-kb clone containing the entire HEK coding region was isolated and its complete DNA sequence was determined. The 4.5-kb insert was subcloned into the expression vector CDM8 and transfected into COS cells. COS cells transfected with the sense HEK/CDM8 construct stained specifically with the IIIA4 antibody, thereby confirming that the antigen recognized by the IIIA4 antibody and the expressed protein product of the HEK cDNA clone were identical. DNA sequence analysis revealed that HEK is a newly discovered member of the EPH/ELK family of receptor tyrosine kinases. Northern blot analysis of a number of cell lines demonstrated the expression of 5.5- to 6.0-kb HEK transcripts in LK63 and the T-cell lines JM and HSB-2. Southern blot analysis of DNA from LK63 suggested that the HEK gene was neither amplified nor rearranged in the LK63 tumor.
Wicks
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Lions Clinical Cancer Laboratory, Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Australia.
Wilkinson
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Salvaris
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Boyd
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D005796:Genes; D066298:In Vitro Techniques; D008214:Lymphocytes / Q000201:enzymology*; D008969:Molecular Sequence Data; D005810:Multigene Family; D009363:Neoplasm Proteins / Q000235:genetics*; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D036121:Receptor, EphA3; D011956:Receptors, Cell Surface / Q000235:genetics*; D016415:Sequence Alignment; D014162:Transfection
D009363:Neoplasm Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D004247:DNA; D011505:Protein-Tyrosine Kinases; D036121:Receptor, EphA3