Mutational analysis of conserved residues in the tyrosine kinase domain of the human trk oncogene.
The human trk oncogene (originally identified in a colon carcinoma) was activated by a genetic rearrangement which resulted in replacement of the extracellular ligand-binding domain of the proto-trk transmembrane receptor by non-muscle tropomyosin sequences. The product of the trk oncogene, a protein of 70 kDa (p70trk), possesses tyrosine-specific protein kinase activity, is autophosphorylated in vitro on tyrosine and is phosphorylated on serine, threonine and tyrosine residues in trk-transformed cells. By site-directed mutagenesis of trk oncogene cDNA, the codon for lysine (367) at the putative ATP-binding site was changed to that for methionine and the codons for tyrosines (503 and 504) at the putative autophosphorylation sites were changed to those for phenylalanine. Replacement of Lys-367 by methionine results in a biologically inactive, kinase-negative mutant. Phe-ala mutants of trk showed drastically reduced ability to induce morphologic transformation, anchorage-independent growth and tumorigenicity in mouse NIH3T3 cells and showed reduced in vitro tyrosine kinase activity when assayed by autophosphorylation and phosphorylation of histone as exogenous substrate. The present study indicates the role of these specific conserved residues in regulating the biochemical and biological properties of p70trk oncoprotein.
Mitra
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Department of Molecular Biology, Research Institute, Cleveland Clinic Foundation, Ohio 44195.
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002471:Cell Transformation, Neoplastic; D003001:Cloning, Molecular; D006801:Humans; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D015513:Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D009857:Oncogenes; D010766:Phosphorylation; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D015513:Oncogene Proteins; C065400:oncogene protein trk; D011505:Protein-Tyrosine Kinases