Activation of pp60c-src transforming potential by mutations altering the structure of an amino terminal domain...

The overexpression of the c-src gene product, pp60c-src, in avian and rodent embryo cells is not sufficient to induce cellular transformation. In this study we report that structural alterations within an amino terminal domain of pp60c-src, the exon 3 domain (residues 84-115) activate the oncogenic potential of the c-src gene product. Site-directed mutagenesis of the c-src gene was used to generate c-src variants encoding pp60c-src proteins with the following amino acid alterations: tyr 90 to phe (pm90F); tyr 92 to phe (pm92F); arg 95 to either trp, lys, glu or gln (pm95W, 95K, 95E and 95Q, respectively), and deletion of residues 92-95 (dl92). C-src variants encoding proteins with the alteration of arg 95 to trp, glu, or lys, or containing the deletion of residues 92-95, induced alterations in cell morphology and promoted growth in soft agar as well as changes in glucose transport and in vivo tyrosine phosphorylation of cellular proteins (including calpactin I heavy chain, p36). Analysis of in vivo phosphorylation of the transforming variant src proteins revealed little detectable alteration in the phosphorylation of tyr 527, a putative site of tyrosine kinase regulation. Our results suggest that structural alterations within a domain distal to the catalytic (kinase) domain activate pp60c-src kinase activity and, concomitantly, oncogenic potential. Furthermore, we suggest that the exon 3 domain of pp60c-src may contribute to the regulation and/or substrate specificity of the c-src protein.