Isolation of temperature-sensitive tyrosine kinase mutants of v-abl oncogene by screening with antibodies for phosphotyrosine.
Temperature-sensitive protein-tyrosine kinase (EC 2.7.1.112) mutants of the oncogene v-abl have been obtained by a direct screening of kinase mutants in bacteria. The v-abl oncogene was expressed in Escherichia coli as a trpE/v-abl fusion protein from the trp promoter. The expression plasmid was mutagenized in vitro and then transfected into E. coli. Bacteria that produced defective tyrosine kinases were distinguished from those producing wild-type v-abl kinases by hybridization with antibodies specific for phosphotyrosine. Two independent mutations that generated temperature-sensitive tyrosine kinases were found to be located in a 12-amino acid region in the tyrosine kinase domain of the v-abl-encoded protein. These mutant v-abl oncogenes displayed temperature-sensitive transforming activity when expressed in NIH 3T3 cells. Cells transformed by these temperature-sensitive tyrosine kinase mutants could be shifted between the transformed and untransformed states by changing their growth temperature. These results confirmed the crucial role of tyrosine kinase activity in the v-abl-mediated oncogenesis.
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D051379:Mice; D009154:Mutation; D009857:Oncogenes; D019000:Phosphotyrosine; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D013696:Temperature; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D000906:Antibodies; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D004262:DNA Restriction Enzymes