DOMINANT-NEGATIVE MUTANTS OF GRB2 INDUCED REVERSAL OF THE TRANSFORMEDPHENOTYPES CAUSED BY THE POINT MUTATION-ACTIVATED RAT HER-2 NEU/

To clarify the role of the Shc-Grb2-Sos trimer in the oncogenic signal ing of the point mutation-activated HER-2/neu receptor tyrosine kinase (named p185), we interfered with the protein-protein interactions in the Shc . Grb2 . Sos complex by introducing Grb2 mutants with deletion s in either amino- (Delta N-Grba) or carboxyl(Delta C-Grb2) terminal S H3 domains into B104-1-1 cells derived from NIH3T3 cells expressing th e point mutation-activated HER-2/neu. We found that the transformed ph enotypes of the B104-1-1 cells were largely reversed by the Delta N-Gr ba. The effect of the Delta C-Grba was much weaker. Biochemical analys is showed that the Delta N-Grba was able to associate She but not p185 or Sos, while the Delta C-Grba bound to Shc, p185, and Sos. The p185- mediated Ras activation was severely inhibited by the Delta N-Grb2 but not the Delta C-Grb2. Taken together, these data demonstrate that int erruption of the interaction between She and the endogenous Grb2 by th e Delta N-Grb2 impairs the oncogenic signaling of the activated p185, indicating that (i) the Delta N-Grb2 functions as a strong dominant-ne gative mutant, and (ii) Shc/Grb2/Sos pathway plays a major role in med iating the oncogenic signal of the activated p185. Unlike the Delta N- Grb2, Delta C-Grb2 appears to be a relatively weak dominant-negative m utant, probably due to its ability to largely fulfill the biological f unctions of the wild-type Grb2.