1 research outputs found
Temporal Resolution of Autophosphorylation for Normal and Oncogenic Forms of EGFR and Differential Effects of Gefitinib
Epidermal growth factor receptor (EGFR) is a member of
the ErbB family of receptor tyrosine kinases (RTK). EGFR overexpression
or mutation in many different forms of cancers has highlighted its
role as an important therapeutic target. Gefitinib, the first small
molecule inhibitor of EGFR kinase function to be approved for the
treatment of nonsmall cell lung cancer (NSCLC) by the FDA, demonstrates
clinical activity primarily in patients with tumors that harbor somatic
kinase domain mutations in EGFR. Here, we compare wild-type EGFR autophosphorylation
kinetics to the L834R (also called L858R) EGFR form, one of the most
common mutations in lung cancer patients. Using rapid chemical quench,
time-resolved electrospray mass spectrometry (ESI-MS), and Western
blot analyses, we examined the order of autophosphorylation in wild-type
(WT) and L834R EGFR and the effect of gefitinib (Iressa) on the phosphorylation
of individual tyrosines. These studies establish that there is a temporal
order of autophosphorylation of key tyrosines involved in downstream
signaling for WT EGFR and a loss of order for the oncogenic L834R
mutant. These studies also reveal unique signature patterns of drug
sensitivity for inhibition of tyrosine autophosphorylation by gefitinib:
distinct for WT and oncogenic L834R mutant forms of EGFR. Fluorescence
studies show that for WT EGFR the binding affinity for gefitinib is
weaker for the phosphorylated protein while for the oncogenic mutant,
L834R EGFR, the binding affinity of gefitinib is substantially enhanced
and likely contributes to the efficacy observed clinically. This mechanistic
information is important in understanding the molecular details underpinning
clinical observations as well as to aid in the design of more potent
and selective EGFR inhibitors