Activation of the proto-oncogene p60c-src by point mutations in the SH2 domain.
To investigate the importance of a conserved region spanning residues 137 to 241 in the noncatalytic domain of p60c-src (SH2 region), we used oligonucleotide-directed mutagenesis to change residues that are highly conserved in this region. Chicken embryo fibroblasts infected with a p60c-src variant containing arginine instead of tryptophan at residue 148 (W148R) appeared more rounded than cells overexpressing a normal c-src gene, and they formed colonies in soft agar. p60c-src variants containing serine instead of arginine at residue 155 (R155S) or isoleucine instead of glycine at residue 170 (G170I) also appeared transformed and were anchorage independent, but to a lesser extent than W148R. Mutation of residue 201 from histidine to leucine (H201L) had no observable effect. The in vitro kinase activity of cells infected with W148R or G170I was elevated twofold. Expression of p60W148R (or, to a lesser extent, of p60G170I) increased the number of proteins phosphorylated on tyrosine in infected cells. All of the mutants were phosphorylated in vivo on Tyr-527, instead of Tyr-416 as observed for p60v-src. Immunoprecipitated p60W148R and p60G170I were found to be associated with a phosphatidylinositol kinase activity, a factor which appears to be necessary for transformation by tyrosine-specific protein kinases. These results show that a single point mutation in the SH2 region of the cellular src gene can activate its transforming potential. This type of activation is in a new category of alterations at the amino terminus that activate but do not cause a shift in phosphorylation at the carboxy terminus.
O'Brien
M C
MC
Rockefeller University, New York, New York 10021-6399.
Fukui
Y
Y
Hanafusa
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D002642:Chick Embryo; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics*; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011519:Proto-Oncogenes; D012190:Retroviridae / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D015345:Oligonucleotide Probes; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src)