SH2 mutants of c-src that are host dependent for transformation are trans-dominant inhibitors of mouse cell...

The c-src gene encodes a membrane-associated protein-tyrosine kinase, pp60c-src, whose substrates and regulators have not been identified. In an effort to obtain mutants that might assist the search for proteins that interact with pp60c-src, we have generated by site-directed mutagenesis two alleles of chicken c-src that are host dependent for transformation and inhibit transformation of mouse cells by activated c-src in a trans-dominant manner. These alleles, named M6 and M9, encode nonconservative changes within the highly conserved FLVRES sequence in the src homology-2 (SH2) region of pp60c-src, as well as an activating change, Y527F, near the carboxyl terminus. M6 and M9 transform chicken embryo fibroblasts (CEF) more efficiently than the parental allele (Y527F c-src), but fail to transform mouse NIH-3T3 cells. The product of M6-src is less stable than activated pp60c-src in NIH-3T3 cells and shows decreased protein tyrosine kinase activity on all tested substrates; the product of M9-src, however, is stable, has a novel pattern of substrate preference for tyrosine phosphorylation in vitro, and induces a pattern of phosphotyrosine-containing proteins in mouse cells that is similar to that induced by Y527F c-src, even though it fails to transform these cells. Both M6 and M9 inhibit transformation of NIH-3T3 cells by activated c-src in a dose-dependent manner, as assayed by resistance of M6- or M9-expressing cells to transformation by Y527F c-src or by morphological reversion of cells previously transformed by activated c-src following introduction of M6 or M9. When reversion occurs, the concentration of protein encoded by the active allele declines, without change in level of src mRNA or rate of src protein synthesis, implying that the products of M6 and M9, when present at adequate levels, can destabilize transformation-competent src protein. These alleles offer new opportunities for interfering with the actions of src-related genes and for isolating cellular factors required for the functions of those genes.