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Src phosphorylation of the epidermal growth factor receptor at novel sites mediates receptor interaction with Src and
D R Stover1, M Becker, J Liebetanz
1Research Department, Ciba Geigy Limited, Basel, Switzerland.
Abstract:
Following ligand binding, the epidermal growth factor receptor (EGF-R) autophosphorylates itself on tyrosine residues located in its carboxyl terminus; in vitro, three sites are highly phosphorylated, while two other sites are phosphorylated to lesser extents. In the presence of the Src protein-tyrosine kinase, in vitro phosphorylation of the minor autophosphorylation sites was increased, and four additional residues were phosphorylated. Following EGF stimulation, two (Tyr-891 and Tyr-920) were found to be phosphorylated in a colorectal cell line (DLD-1) and in a breast tumor cell line (MCF7). The remaining in vitro sites were not found to be highly phosphorylated in vivo. The sequences surrounding Tyr-891 and Tyr-920 match the reported consensus binding sequences for the SH2 domains of Src and the regulatory domain of phosphatidylinositol 3-kinase (p85 alpha), respectively. In vitro, both of these proteins were found to bind to Src-phosphorylated EGF-R with approximately 100-fold greater affinity than to autophosphorylated EGF-R, demonstrating that Src creates new sites for SH2 binding. Furthermore, Csk-inactivated Src was activated by interaction with Src-phosphorylated EGF-R but not by autophosphorylated EGF-R. Upon EGF treatment of MCF7 or three colorectal carcinoma cell lines (WiDr, DLD-1, and LS174T), the EGF-R coimmunoprecipitated with both p85 alpha and Src. Evidence is also presented that suggests that an EGF-R-related protein, ErbB2, may be involved in similar Src-mediated interactions. These data demonstrate that EGF-R is phosphorylated in vivo at non-autophosphorylation sites and that these novel sites can act as docking sites for Src, P85 alpha, and potentially other SH2-containing proteins. In addition, the data suggest a tyrosine phosphatase-independent mechanism for the elevation of Src activity in cells exposed to growth factors. Overexpression of Src, EGF-R, and/or ErbB2 in breast and colorectal tumor cells suggests the potential that such interactions may contribute to the transformed phenotype of these carcinomas.
Insights
Epidermal growth factor receptor (EGF-R) phosphorylation by Src creates new binding sites for Src and phosphatidylinositol 3-kinase (p85 alpha). These interactions may drive cancer cell growth and suggest a novel mechanism for elevated Src activity in tumors.
Area of Science:
- Cellular signaling and cancer biology
- Protein phosphorylation and kinase interactions
Background:
- Epidermal growth factor receptor (EGF-R) autophosphorylation is crucial for cellular signaling.
- The role of Src protein-tyrosine kinase in modifying EGF-R phosphorylation sites was previously unclear.
Purpose of the Study:
- To investigate novel in vivo phosphorylation sites on EGF-R beyond autophosphorylation.
- To determine if Src-mediated phosphorylation creates new binding sites for SH2-containing proteins.
- To explore the functional consequences of these interactions in cancer cells.
Main Methods:
- Phosphorylation analysis of EGF-R in cancer cell lines (DLD-1, MCF7) after EGF stimulation.
- In vitro kinase assays with EGF-R, Src, and identified SH2-domain proteins (Src, p85 alpha).
- Coimmunoprecipitation assays to detect protein-protein interactions in treated cells.
Main Results:
- EGF-R is phosphorylated in vivo at Tyr-891 and Tyr-920, sites not heavily phosphorylated by autophosphorylation alone.
- Src-mediated phosphorylation significantly enhances binding affinity for Src and phosphatidylinositol 3-kinase (p85 alpha) via their SH2 domains.
- EGF-R coimmunoprecipitated with Src and p85 alpha in EGF-stimulated cancer cells, indicating complex formation.
Conclusions:
- Src creates novel docking sites on EGF-R for SH2-containing proteins, impacting cellular signaling.
- These interactions may contribute to the transformed phenotype of breast and colorectal carcinomas.
- A tyrosine phosphatase-independent mechanism for Src activation by growth factors is suggested.