Ligand-independent phosphorylation of Y869 (Y845) links mutant EGFR signaling to stat-mediated gene expression.
Activating mutants of EGFR have been identified in a subset of non-small-cell lung cancers. To investigate mutant-driven signaling, we focused on Y869, a residue in the same activation loop where the L858R and L861Q mutations are located. We observed ligand-independent phosphorylation of Y869 in 32D cells EGFR(L858R) and EGFR(L861Q). The EGFR tyrosine kinase inhibitor (TKI) erlotinib inhibited Y869 P-EGFR in intact cells as well as in a cell-free kinase reaction. Expression of kinase domain of EGFR(L858R) and EGFR(L861Q) exhibited auto-phosphorylation of Y869; this was inhibited by EGFR TKIs but not by Src kinase inhibitor. P-Y859 of EGFR-mediated downstream component, STAT5, was also analyzed. Y694 P-STAT5 was eliminated by erlotinib treatment. Analysis of immune-complexes showed constitutive association of mutant EGFRs with STAT5 and Src which was unaffected by erlotinib or PP1. On the other hand, 32D-EGFR(WT) exhibited constitutive STAT5 phosphorylation and association of EGFR with JAK2. In these cells, a JAK2 inhibitor abrogated P-STAT5 whereas mutant EGFRs did not associate with JAK2. Expression of c-myc was regulated by EGFR/STAT5 signaling in cells expressing EGFR(L858R) and EGFR(L861Q). Our results suggest that ligand-independent and Src activity-independent phosphorylation of Y869 in mutant EGFR regulates STAT5 activation and c-myc expression.
Yang
Seungchan
S
Department of Medicine, Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University School of Medicine, Nashville, TN, USA.
Park
Kyungho
K
Turkson
James
J
Arteaga
Carlos L
CL
D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D000069347:Erlotinib Hydrochloride; D016259:Genes, myc; D006801:Humans; D008024:Ligands; D009154:Mutation; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology; D050799:STAT5 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D008024:Ligands; D011799:Quinazolines; D050799:STAT5 Transcription Factor; D000069347:Erlotinib Hydrochloride; D066246:ErbB Receptors; D016392:Proto-Oncogene Proteins pp60(c-src)