PCR mediated detection of a new human receptor-tyrosine-kinase, HEK 2

• Published in: Oncogene Vol. 8; no. 10; p. 2857
• Main Authors: Böhme, B, Holtrich, U, Wolf, G, Luzius, H, Grzeschik, K H, Strebhardt, K, Rübsamen-Waigmann, H
• Format: Journal Article
• Language: English
• Published: England 01.10.1993
• Subjects: Amino Acid Sequence; Amyloid - chemistry; Amyloid - genetics; Animals; Base Sequence; Blotting, Southern; Cell Line; Conserved Sequence; Humans; Membrane Proteins - chemistry; Membrane Proteins - genetics; Molecular Sequence Data; Neoplasm Proteins - chemistry; Neoplasm Proteins - genetics; Open Reading Frames; Polymerase Chain Reaction; Protein-Tyrosine Kinases - chemistry; Protein-Tyrosine Kinases - genetics; Protein-Tyrosine Kinases - isolation & purification; Rats; Receptor, EphA2; Receptor, EphA3; Receptor, EphB3; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - isolation & purification
• ISSN: 0950-9232

We have previously amplified cDNA subfragments of protein-tyrosine-kinases (PTKs) by using the polymerase chain reaction (PCR) and specific sets of oligonucleotide primers derived from nucleotide sequences of their kinase domain. In this study we have used a more directed approach to identify new members of the EPH/elk-family by PCR of human embryonic cDNA: we utilized oligonucleotide primers specifically designed to a highly conserved N-terminal motif and the kinase region of EPH/elk-PTKs in RNA-PCRs. The 5' and 3' elongation of the primary PCR product was achieved by the RACE (rapid amplification of cDNA ends)-technique. Sequence analysis of 3.8 kb of overlapping PCR products allowed to identify a novel receptor-PTK, HEK 2 (human embryo kinase 2), as an additional member of this family, without the need to screen a cDNA library. This approach should be useful for the rapid isolation of other PTK-genes as well. Analysis of genomic DNA placed HEK 2 on chromosome 3. Northern blot analysis demonstrated the expression of a 4.6 kb HEK 2-mRNA in lung, brain, pancreas, liver, placenta, kidney, skeletal muscle, heart and several human cells. In a protein kinase assay with HEK 2-specific immunoprecipitates from the human epidermoid carcinoma cell line A431, a protein of 130 kDa was found phosphorylated.