Dominant-negative mutants of Grb2 induced reversal of the transformed phenotypes caused by the point mutation-activated rat HER-2/neu

Abstract

To clarify the role of the Shc-Grb2-Sos trimer in the oncogenic signaling 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 deletions in either amino- (ΔN- Grb2) or carboxyl(δC-Grb2) terminal SH3 domains into B104-1-1 cells derived from NIH3T3 cells expressing the point mutation-activated HER-2/neu. We found that the transformed phenotypes of the B104-1-1 cells were largely reversed by the ΔN-Grb2. The effect of the ΔC-Grb2 was much weaker. Biochemical analysis showed that the AN-Grb2 was able to associate Shc but not p185 or Sos, while the ΔC-Grb2 bound to Shc, p185, and Sos. The p185-mediated Ras activation was severely inhibited by the ΔN-Grb2 but not the ΔC-Grb2. Taken together, these data demonstrate that interruption of the interaction between Shc and the endogenous Grb2 by the ΔN-Grb2 impairs the oncogenic signaling of the activated p185, indicating that (i) the ΔN-Grb2 functions as a strong dominant-negative mutant, and (ii) Shc/Grb2/Sos pathway plays a major role in mediating the oncogenic signal of the activated p185. Unlike the AN-Grb2, ΔC-Grb2 appears to be a relatively weak dominant-negative mutant, probably due to its ability to largely fulfill the biological functions of the wild- type Grb2.