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

Oncogene. 1993 Oct;8(10):2857-62.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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*

Substances

  • Amyloid
  • Membrane Proteins
  • Neoplasm Proteins
  • Receptors, Cell Surface
  • amyloid fibril protein EPS
  • Protein-Tyrosine Kinases
  • Receptor, EphA2
  • Receptor, EphA3
  • Receptor, EphB3