PCR mediated detection of a new human receptor-tyrosine-kinase, HEK 2.
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.
Böhme
B
B
Chemotherapeutisches Forschungsinstitut, Georg-Speyer-Haus, Frankfurt, Germany.
Holtrich
U
U
Wolf
G
G
Luzius
H
H
Grzeschik
K H
KH
Strebhardt
K
K
Rübsamen-Waigmann
H
H
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D017124:Conserved Sequence; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D051381:Rats; D036104:Receptor, EphA2; D036121:Receptor, EphA3; D036223:Receptor, EphB3; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000302:isolation & purification*
D000682:Amyloid; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011956:Receptors, Cell Surface; C046640:amyloid fibril protein EPS; D011505:Protein-Tyrosine Kinases; D036104:Receptor, EphA2; D036121:Receptor, EphA3; D036223:Receptor, EphB3