Dominant-negative mutants of Grb2 induced reversal of the transformed phenotypes caused by the point mutation-activated rat HER-2/Neu.
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.
Department of Tumor Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
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Xie
A M
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Pendergast
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Hung
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D066246:ErbB Receptors / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D051379:Mice; D016296:Mutagenesis; D010641:Phenotype; D017354:Point Mutation; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D066246:ErbB Receptors; D051380:GRB2 Adaptor Protein; D011506:Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; C495949:Grb2 protein, mouse; C495950:Grb2 protein, rat