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

J Biol Chem. 1995 Dec 22;270(51):30717-24. doi: 10.1074/jbc.270.51.30717.

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- (delta N-Grb2) or carboxyl-(delta C-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 delta N-Grb2. The effect of the delta C-Grb2 was much weaker. Biochemical analysis showed that the delta N-Grb2 was able to associate Shc but not p185 or Sos, while the delta C-Grb2 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 interruption of the interaction between Shc and the endogenous Grb2 by the delta N-Grb2 impairs the oncogenic signaling of the activated p185, indicating that (i) the delta N-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 delta N-Grb2, delta C-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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells
  • Adaptor Proteins, Signal Transducing*
  • Animals
  • ErbB Receptors / metabolism
  • GRB2 Adaptor Protein
  • Mice
  • Mutagenesis
  • Phenotype
  • Point Mutation*
  • Proteins / metabolism*
  • Rats
  • Receptor, ErbB-2 / biosynthesis
  • Receptor, ErbB-2 / metabolism*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / metabolism
  • Sequence Deletion
  • Transfection
  • src Homology Domains

Substances

  • Adaptor Proteins, Signal Transducing
  • GRB2 Adaptor Protein
  • Grb2 protein, mouse
  • Grb2 protein, rat
  • Proteins
  • Recombinant Proteins
  • ErbB Receptors
  • Receptor, ErbB-2