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Analysis of protein-protein interactions involved in the activation of the Shc/Grb-2 pathway by the ErbB-2 kinase
A Ricci1, L Lanfrancone, R Chiari
1Laboratory of Immunology, Regina Elena Cancer Institute, Roma, Italy.
Abstract:
In murine fibroblasts activation of the Shc/Grb-2 pathway by the ErbB-2 kinase involves tyrosine phosphorylation of Shc products and the formation of Shc/ErbB-2, Shc/Grb-2 and Grb-2/ErbB-2 complexes. Tyr 1139 of ErbB-2 bound to the Grb-2 SH2 domain in vitro as well as in intact cells. Tyr 1221 and 1248 are binding sites of gp185ErbB-2 for Shc SH2 domain in vitro whereas Tyr 1196 and 1248 are major binding sites of ErbB-2 for Shc PTB domain. Inhibition of Shc/ErbB-2 complex formation in intact cells was obtained by simultaneous mutational inactivation of Shc SH2 and Shc PTB binding sites of gp185ErbB-2. Shc/ErbB-2 complexes are formed upon activation of the ErbB-2 kinase and tyrosine phosphorylation of Shc proteins; they are located in both cytosol and cellular membranes. ErbB-2 activation induces also translocation of Grb-2 from cytosol to membranes. This network of protein-protein interactions may reflect the ability of the Shc/Grb-2 pathway to act as a molecular switch controlling different cellular functions regulated by RTK activation. In fact the Ras GDP exchanger mSOS was recruited in Grb-2/ErbB-2 complexes; furthermore besides mSOS, other polypeptides present in either cytosolic or membrane preparations were able to complex in vitro with Grb-2 SH3 domains.
Insights
The Shc/Grb-2 pathway activation by ErbB-2 kinase involves specific tyrosine phosphorylation events and complex formation. This interaction network acts as a molecular switch, controlling cellular functions downstream of receptor tyrosine kinase (RTK) activation.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Biochemistry
Background:
- The ErbB-2 receptor tyrosine kinase (RTK) plays a crucial role in cell growth and differentiation.
- Activation of the Shc/Grb-2 signaling pathway is a key event downstream of RTK activation.
- Understanding the precise molecular interactions within this pathway is essential for deciphering cellular regulation.
Purpose of the Study:
- To elucidate the specific molecular interactions involved in the activation of the Shc/Grb-2 pathway by ErbB-2.
- To identify the binding sites and complex formation dynamics between ErbB-2, Shc, and Grb-2.
- To investigate the functional consequences of these interactions on cellular signaling.
Main Methods:
- Murine fibroblast cell culture
- Tyrosine phosphorylation analysis
- Co-immunoprecipitation assays
- Site-directed mutagenesis
- In vitro binding assays
Main Results:
- ErbB-2 kinase activation leads to tyrosine phosphorylation of Shc proteins and the formation of Shc/ErbB-2, Shc/Grb-2, and Grb-2/ErbB-2 complexes.
- Specific tyrosine residues on ErbB-2 (Tyr 1139, 1221, 1248) and Shc (Tyr 1196) were identified as critical binding sites for Grb-2 and Shc domains, respectively.
- Inhibition of Shc/ErbB-2 complex formation by mutating these binding sites blocked pathway activation.
- ErbB-2 activation induced translocation of Grb-2 from the cytosol to cellular membranes.
- The Ras GDP exchanger mSOS was recruited to Grb-2/ErbB-2 complexes, indicating downstream signaling activation.
Conclusions:
- The Shc/Grb-2 pathway acts as a molecular switch regulated by ErbB-2 activation through specific protein-protein interactions.
- These interactions facilitate the recruitment of downstream signaling molecules like mSOS, controlling cellular functions.
- The localization of these complexes in both cytosol and membranes highlights the dynamic nature of RTK signaling.