EGFR S1166 Phosphorylation Induced by a Combination of EGF and Gefitinib Has a Potentially Negative Impact on Lung Cancer Cell Growth

Phosphorylation of protein plays a key role in the regulation
of cellular signal transduction and gene expression. In recent years,
targeted mass spectrometry facilitates functional phosphoproteomics
by allowing specific protein modifications of target proteins in complex
samples to be characterized. In this study, we employed multiple reaction
monitoring (MRM) to examine the influence of gefitinib (also known
as Iressa) on the phosphorylation sites of EGFR protein before and
after EGF treatment. By coupling MRM to MS/MS, 5 phosphotyrosine (Y1110,
Y1172, Y1197, Y1069, and Y1092) and 1 S/T (T693) sites were identified
on EGFR. Y1197 and T693 were constitutively phosphorylated. All phosphorylation
sites were sensitive to gefitinib treatment except T693. Interestingly,
gefitinib treatment induced phosphorylation of S1166 only in the presence
of EGF. We further showed that lung cancer cells overexpressing phosphomimic
S1166D EGFR mutant possessed significantly lower growth and proliferation
property compared to wildtype EGFR-expressing cells. While the function
and mode of regulation of S1166 remain unclear, our data supports
the notion that S1166 represents a regulatory site that exerts a negative
regulation on growth and proliferation of cancer cells. The data presented
has implication in our understanding of dynamic drug (gefitinib)–target
(EGFR) interaction and in improving the efficacy of target-directed
therapeutics.