Src-dependent phosphorylation of the EGF receptor Tyr-845 mediates Stat-p21waf1 pathway in A431 cells

Background: Cell surface receptor for the epidermal growth factor (EGFR) and cytoplasmic tyrosine kinase c-Src co-operate in several cellular functions such as proliferation and apoptosis. Our previous studies have shown that ectopic expression of the adaptor protein p52(shc) or p66(shc), but not p46(shc), and EGF stimulation lead to the activation of c-Src that is accompanied by phosphorylation of signal transducers and activators of transcription (Stat) in A431 cells. Results: Here, we show that by using A431 cells as a model system, expression of p52(shc), or cell stimulation with EGF or H2O2 leads to phosphorylation of EGFR on Tyr 845 that is located to the activation segment of the catalytic domain. The phosphorylation of Tyr 845 can be inhibited by PP2, but not by AG1478, and is associated with Src activation and Stat 3/5 phosphorylation, but not with MAP (mitogen-activated protein) kinase phosphorylation. Phosphorylation of Stat 3/5 in response to p52(shc) expression, EGF or H2O2 could also be inhibited by introduction into cells of phospho-Tyr 845-specific antibody or by expression of dominant-negative version of c-Src. Co-incubation of purified c-Src and EGFR results in phosphorylation of Tyr 845 in vitro, indicating that c-Src can directly phosphorylate EGFR on Tyr 845. Conclusion: These results indicate that multiple signals for c-Src activation can promote Stat 3/5 phosphorylations through Src-dependent phosphorylation of EGFR on Tyr 845.