Src Phosphorylation of the Epidermal Growth Factor Receptor at Novel Sites Mediates Receptor Interaction with Src and P85

• Published in: The Journal of biological chemistry 1995-06, Vol.270 (26), p.15591
• Main Authors: David R. Stover, Michael Becker, Janis Liebetanz, Nicholas B. Lydon
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.26.15591

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 (p85α), respectively. In vitro , both of these proteins were found to bind to Src-phosphorylated EGF-R with 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 p85α 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, P85α, 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.