Real-time imaging elucidates the role of H2O2 in regulating kinetics of epidermal growth factor-induced and Src-mediated tyrosine phosphorylation signaling

Abstract

Reversible oxidation is emerging as an important regulatory mechanism in protein tyrosine phosphor-ylation. Generation of hydrogen peroxide (H 2O2), upon growth factor stimulation, is hypothesized to inhibit activity of protein tyrosine phosphatases (PTPs). This ensures that protein tyrosine kinases can elevate the steady-state level of protein tyrosine phosphorylation, which then allows propagation of the tyrosine phosphorylation signal. However, the effects of H2O2 on the kinetics of tyrosine phosphorylation signaling remain poorly understood, especially in living cells. Therefore, we used a genetically encoded Src kinase-specific biosensor based on fluorescence resonance energy transfer (FRET) to image the kinetics of the Src-mediated tyrosine phosphorylation signaling (Src signaling) induced by epidermal growth factor (EGF). We examined the kinetics under increased and decreased H2O2 levels. Through a straightforward, quantitative analysis method which characterized the signaling kinetics, we demonstrated that H2O2 modulated the amplitude and duration of the signal by inhibiting PTPs' activity. Our evidence also suggested the effect of H2O2 on Src activation is mediated by H2O2-dependent inhibition of PTPs. Furthermore, we provide evidence showing global elevation of intracellular H2O2 level attenuates EGF-induced Src signaling. © 2012 Society of Photo-Optical Instrumentation Engineers (SPIE).