c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr(845) and Tyr(1101) is associated with modulation of receptor function

Accumulating evidence indicates that interactions between the epidermal gro wth factor receptor (EGFR) and the nonreceptor tyrosine kinase c-Src may co ntribute 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 phospho rylation of the receptor substrates Shc and phospholipase gamma as compared with single overexpressors, These parameters correlated with the ability o f c-Src and EGFR to form an EGF-dependent heterocomplex in vivo, Here we pr ovide evidence that association between c-Src and EGFR can occur directly, as shown by receptor overlay experiments, and that it results in the appear ance of two novel tyrosine phosphorylations on the receptor that are seen b oth in vitro and in vivo following EGF stimulation. Edman degradation analy ses and co-migration of synthetic peptides with EGFR-derived tryptic phosph opeptides identify these sites as Tyr(845) and Tyr(1101). Tyr(1101) lies wi thin the carboxyl-terminal region of the EGFR among sites of receptor autop hosphorylation, while Tyr845 resides in the catalytic domain, in a position analogous to Tyr(416) Of c-Src, Phosphorylation of Tyr(416) 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 Tyr(845). Indeed, EGF-induced phosphorylation of Tyr(845) was increased in MDA468 human breast cancer ce lls engineered to overexpress c-Src as compared with parental MDA 468 cells . Furthermore, transient expression of a Y845F variant EGFR in murine fibro blasts resulted in an ablation of EGF-induced DNA synthesis to nonstimulate d levels. Together, these data support the hypothesis that c-Src-mediated p hosphorylation of EGFR Tyr(845) is involved in regulation of receptor funct ion, as well as in tumor progression.