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. 2005 Mar 1;386(Pt 2):215-9.
doi: 10.1042/BJ20041829.

Compartmental oxidation of thiol-disulphide redox couples during epidermal growth factor signalling

Patrick J Halvey  1 , Walter H Watson, Jason M Hansen, Young-Mi Go, Afshin Samali, Dean P Jones
Affiliations

Compartmental oxidation of thiol-disulphide redox couples during epidermal growth factor signalling

Patrick J Halvey et al. Biochem J. .

Abstract

Exogenously added ROS (reactive oxygen species) cause generalized oxidation of cellular components, whereas endogenously generated ROS induced by physiological stimuli activate discrete signal transduction pathways. Compartmentation is an important aspect of such pathways, but little is known about its role in redox signalling. We measured the redox states of cytosolic and nuclear Trx1 (thioredoxin-1) and mitochondrial Trx2 (thioredoxin-2) using redox Western blot methodologies during endogenous ROS production induced by EGF (epidermal growth factor) signalling. The glutathione redox state was measured by HPLC. Results showed that only cytosolic Trx1 undergoes significant oxidation. Thus EGF signalling involves subcellular compartmental oxidation of Trx1 in the absence of a generalized cellular oxidation.

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Figures

Figure 1
Figure 1. Activation of EGFR in HaCaT cells
(A) Western blot of phosphorylated EGFR (top) showing a time-dependent response to EGF (200 ng/ml). (B) DCFH-DA assay for the detection of ROS. An increase in dichlorofluorescein (DCF) fluorescence corresponds to an increase in ROS. Fluorescence measured after 5 min is given in relative fluorescence units. Results are means±S.E.M for at least three separate experiments. Asterisks (*) denote a statistically significant difference (P<0.05) as compared with the control.
Figure 2
Figure 2. Redox states of cytoplasmic and nuclear Trx1 and Trx2 pools
(A, B) Time course of Trx1 oxidation in the cytoplasm (A) and the nucleus (B) following EGF (200 ng/ml) treatment from 0–30 min. Trx1 appears as three distinct bands. The two-disulphide form (top band) is only visible under highly oxidizing conditions. The oxidized band (middle) represents the one-disulphide form and the lower band is the fully reduced form. H2O2 was used as a positive control. (C) Time course of Trx2 redox state following EGF (200 ng/ml) treatment from 0–30 min. Separation of reduced and oxidized bands is based on a mass shift due to AMS alkylation of thiols. tBH (t-butyl hydroperoxide) was used as a positive control.
Figure 3
Figure 3. EGF signalling differentially affects redox pools
Changes in Eh values of (A) cytoplasmic Trx1, (B) nuclear Trx1, (C) Trx2 and (D) GSH in EGF-treated HaCaT cells over 30 min. Values were calculated using the Nernst equation (see the Experimental section). Results are means±S.E.M for at least three separate experiments. Asterisks (*) denote a statistically significant difference (P<0.05) as compared with the control
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