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.
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Bobby Fachrizal
BF
Assiddiq
Kah Yap
KY
Tan
Weiyi
W
Toy
Siew Pang
SP
Chan
Poh Kuan
PK
Chong
Yoon Pin
YP
Lim
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D004815:Epidermal Growth Factor / Q000502:physiology*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000077156:Gefitinib; D006801:Humans; D008175:Lung Neoplasms; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011799:Quinazolines / Q000494:pharmacology*; D012694:Serine / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000378:metabolism
D000970:Antineoplastic Agents; D010446:Peptide Fragments; D011799:Quinazolines; D014443:Tyrosine; D012694:Serine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D000077156:Gefitinib