Prokaryotic expression cloning of a novel human tyrosine kinase.
Screening of a human embryonic lung fibroblast cDNA expression library with antiphosphotyrosine antibodies led to isolation of a novel protein kinase. A clone, designated A6, contained a 3-kb cDNA insert with a predicted open reading frame of 350 amino acids. DNA sequence analysis failed to reveal any detectable similarity with previously known genes, and the predicted A6 protein lacked any of the motifs commonly conserved in the catalytic domains of protein kinases. However, the bacterially expressed beta-galactosidase-A6 fusion protein demonstrated both tyrosine and serine phosphorylation in an in vitro kinase assay and phosphorylated exogenous substrates including myelin basic protein specifically on tyrosine residues. The enzyme also displayed biochemical properties analogous to those of other protein tyrosine kinases. The A6 gene was found to be expressed widely at the transcript level in normal tissues and was evolutionarily conserved. Thus, A6 represents a novel tyrosine kinase which is highly divergent from previously described members of this important class of regulatory molecules.
Beeler
J F
JF
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
LaRochelle
W J
WJ
Chedid
M
M
Tronick
S R
SR
Aaronson
S A
SA
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004622:Embryo, Mammalian; D004926:Escherichia coli; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D008168:Lung / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009928:Organ Specificity; D019000:Phosphotyrosine; D014176:Protein Biosynthesis; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases