GROWTH-INHIBITION BY DOMINANT-NEGATIVE MUTATIONS OF THE NEU-ENCODED ONCOPROTEIN

In the present study, kinase-deficient mutants of the neo gene were co nstructed in order to generate dominant-negative receptor molecules, w hich should abolish phosphorylation of receptor complexes, One constru ct carried a mutation of the putative ATE-binding site (K758M), while the other mutant was generated by deletion of the kinase domain (ID400 ), Neither receptor showed phosphorylation by in vitro kinase assay. W hen NIH3T3 fibroblasts were co transfected by the oncogenic neu gene a nd one of either construct, the transforming effect could be partially reversed, Therefore, kinase-negative mutations of the neo-encoded rec eptor seemed to have a dominant-negative effect on the action of the a ctivated protein, To test this hypothesis, rat neurinoma cell lines co ntaining oncogenic neu genes were transfected with the constructs. Exp ression of the kinase-defective mutants and reduced phosphorylation co uld be detected in different clones derived from single transfected ce lls, Striking growth inhibition and reduction of colony formation in s oft agar were observed in these cell lines when compared with untransf ected cells, Thus, kinase-deficient mutants exert a dominant-negative effect on phosphorylation of receptor complexes, resulting in a revers ion of the transformed phenotype. (C) 1997 Wiley-Liss, Inc.