C-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr⁸⁴⁵ and Tyr¹¹⁰¹ is associated with modulation of receptor function

Accumulating evidence indicates that interactions between the epidermal growth factor receptor (EGFR) and the nonreceptor tyrosine kinase c-Src may contribute to an aggressive phenotype in multiple human tumors. Previous work from our laboratory demonstrated that murine fibroblasts which overexpress both these tyrosine kinases display synergistic increases in DNA synthesis, soft agar growth, and tumor formation in nude mice, and increased phosphorylation of the receptor substrates Shc and phospholipase γ as compared with single overexpressors. These parameters correlated with the ability of c-Src and EGFR to form an EGF-dependent heterocomplex in vivo. Here we provide evidence that association between c-Src and EGFR can occur directly, as shown by receptor overlay experiments, and that it results in the appearance of two novel tyrosine phosphorylations on the receptor that are seen both in vitro and in vivo following EGF stimulation. Edman degradation analyses and co-migration of synthetic peptides with EGFR- derived tryptic phosphopeptides identify these sites as Tyr⁸⁴⁵ and Tyr¹¹⁰¹. Tyr¹¹⁰¹ lies within the carboxyl-terminal region of the EGFR among sites of receptor autophosphorylation, while Tyr⁸⁴⁵ resides in the catalytic domain, in a position analogous to Tyr⁴¹⁶ of c-Src. Phosphorylation of Tyr⁴¹⁶ and homologous residues in other tyrosine kinase receptors has been shown to be required for or to increase catalytic activity, suggesting that c-Src can-influence EGFR activity by mediating phosphorylation of Tyr⁸⁴⁵. Indeed, EGF-induced phosphorylation of Tyr⁸⁴⁵ was increased in MDA468 human breast cancer cells engineered to overexpress c-Src as compared with parental MDA 468 cells. Furthermore, transient expression of a Y845F variant EGFR in murine fibroblasts resulted in an ablation of EGF-induced DNA synthesis to nonstimulated levels. Together, these data support the hypothesis that c-Src-mediated phosphorylation of EGFR Tyr⁸⁴⁵ is involved in regulation of receptor function, as well as in tumor progression.