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Review
. 2012 Dec 18;51(50):9954-65.
doi: 10.1021/bi301441e. Epub 2012 Dec 5.

Redox regulation of epidermal growth factor receptor signaling through cysteine oxidation

Thu H Truong  1 , Kate S Carroll
Affiliations
Review

Redox regulation of epidermal growth factor receptor signaling through cysteine oxidation

Thu H Truong et al. Biochemistry. .

Abstract

Epidermal growth factor receptor (EGFR) exemplifies the family of receptor tyrosine kinases that mediate numerous cellular processes, including growth, proliferation, and differentiation. Moreover, gene amplification and EGFR mutations have been identified in a number of human malignancies, making this receptor an important target for the development of anticancer drugs. In addition to ligand-dependent activation and concomitant tyrosine phosphorylation, EGFR stimulation results in the localized generation of H(2)O(2) by NADPH-dependent oxidases. In turn, H(2)O(2) functions as a secondary messenger to regulate intracellular signaling cascades, largely through the modification of specific cysteine residues within redox-sensitive protein targets, including Cys797 in the EGFR active site. In this review, we highlight recent advances in our understanding of the mechanisms that underlie redox regulation of EGFR signaling and how these discoveries may form the basis for the development of new therapeutic strategies for targeting this and other H(2)O(2)-modulated pathways.

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Figures

Figure 1
Figure 1
Timeline outlining key events and discoveries relating to redox regulation of EGFR signaling through cysteine oxidation.
Figure 2
Figure 2
Oxidative modification of cysteine residues by hydrogen peroxide (H2O2). (A) The initial reaction product of a thiolate with H2O2 yields sulfenic acid (RSOH). This modification, also known as sulfenylation, is reversible and can be directly reduced back to the thiol form or indirectly through disulfide bond formation. (B) Sulfenic acids can be stabilized by the protein microenvironment and/or undergo subsequent modification. For example, they can condense with a second cysteine in the same or different protein to generate a disulfide bond. Alternatively, reaction with the low molecular weight thiol glutathione (GSH, red circle) affords a mixed disulfide through a process known as S-glutathionylation. In a few proteins, such as PTP1B, nucleophilic attack of a backbone amide on RSOH results in sulfenamide formation. Sulfenyl groups can also oxidize further to the sulfinic (RSO2H) and/or sulfonic (RSO3H) acid form under conditions of high oxidative stress.
Figure 3
Figure 3
EGFR signaling pathways and general mechanisms for thiol-based redox modulation of signaling proteins. (A) EGF binding to EGFR induces receptor dimerization, followed by autophosphorylation of tyrosine (Tyr) residues (red circles) within its cytoplasmic domain. In turn, these phosphorylated Tyr residues serve as docking sites for associating proteins to activate a number of downstream signaling cascades. Two such pathways, Ras/ERK and PI3K/AKT, are shown here for simplicity. The EGF-EGFR interaction also triggers the assembly and activation of NADPH oxidase (Nox) complexes, followed by subsequent production of H2O2 through spontaneous dismutation or action of SOD. Once formed, endogenous H2O2 may pass through specific aquaporin (AQP) channels and/or diffuse across the membrane to reach the intracellular cytosol. Transient increases in H2O2 leads to the oxidation of local redox targets. (B) Model for redox-dependent signal transduction. Protein tyrosine kinases (PTKs) catalyze the transfer of γ-phosphoryl groups from ATP to tyrosine hydroxyls of proteins, whereas protein tyrosine phosphatases (PTPs) remove phosphate groups from phosphorylated tyrosine residues. PTPs function in a coordinated manner with PTKs to control signaling pathways to regulate a diverse array of cellular processes. (C) Regulatory cysteines in protein kinases can undergo oxidation/reduction to modulate their function. Depending on the kinase, redox modifications can stimulate or inhibit function. (D) Oxidation of the conserved active site cysteine residue in PTPs inactivates these enzymes, and can be restored by reducing the oxidized residue to its thiol form. SOx: oxidized cysteine.
Figure 4
Figure 4
Redox regulation of peroxiredoxins (Prxs) during EGFR signaling. Receptor activation results in localized phosphorylation and inactivation of peroxiredoxin I (PrxI) by PTKs, such as the redox-regulated cytoplasmic Src (c-Src). Deactivation of PrxI diminishes the redox-buffering capacity adjacent to the cell membrane, allowing for a transient and localized increase in H2O2 levels for signal transduction. Additionally, elevated H2O2 concentrations can inactivate Prx2 by oxidation of its catalytic cysteine to sulfinic acid.
Figure 5
Figure 5
General strategy for detecting protein cysteine sulfenylation (RSOH) in cells. (A) Chemoselective reaction between sulfenic acid and 5,5-dimethyl-1,3-cyclohexanedione (dimedone, 1). (B) Azide and alkyne-functionalized small-molecule probes for trapping and tagging protein sulfenic acids include DAz-2 (2) and DYn-2 (3). (C) Detection of protein sulfenic acids in living cells. Target cells are incubated with cell-permeable probes to trap and tag protein sulfenic acids in situ. In subsequent steps, lysates are prepared and tagged proteins are further elaborated by attachment of biotin or fluorescence labels via click chemistry and enables detection by Western blot or in-gel fluorescence. Alternatively, biotinylated proteins may be enriched for proteomic analysis.
Figure 6
Figure 6
Model for H2O2-dependent regulation of EGFR tyrosine kinase activity. (A) Binding of EGF induces production of H2O2 through Nox2. Nox-derived H2O2 directly modifies EGFR cysteine (Cys797) to sulfenic acid in the active site, which enhances its tyrosine kinase activity. Endogenous H2O2 can also oxidize and deactivate localized PTPs, leading to a net increase in EGFR phosphorylation. (B) Crystal structure of the EGFR kinase domain (PDB 3GT8) bound to AMP-PNP, a hydrolysis resistant ATP analog, and Mg2+. Dashed yellow lines and accompanying numbers indicate the distance (Å) between the γ-sulfur atom of Cys797 and key substrate functional groups. Note also that Cys797 can adopt different rotamers and sulfenylation of this residue may enhance its ability to participate in electrostatic and hydrogen-bonding interactions with its substrate. (C) Abbreviated sequence alignment of EGFR and nine other kinases that harbor a cysteine at the structural position that corresponds to Cys797 (adapted from ref. 112).
Figure 7
Figure 7
Covalent cysteine-based protein targeting strategies. (A) Conventional covalent inhibitors of kinases inactivate their target through covalent attachment to the cysteine thiol functional group. However, the electrophilic center (e.g., acrylamide, haloacetamide, vinyl sulfonamide) that reacts with the cysteine can exhibit nonspecific reactivity toward other cellular thiols, including glutathione present at millimolar concentrations inside mammalian cells. The electrophile may also react with other nucleophilic functionalities present in biological systems (amino and imidazole groups of amino acids, various reactive sites in nucleic acid bases, water). (B) Orthogonal strategy as one potential mechanism to address issues associated with employing an electrophilic functional group to target one cysteine among a sea of biological nucleophiles. According to this approach, active site-directed small-molecule inhibitors containing a reactive nucleophilic substituent form a covalent bond with a sulfenic acid-modified cysteine side chain. Such modifications form transiently in specific proteins during H2O2-mediated signal transduction in normal cells, but form constitutively in diseases associated with chronically elevated levels of H2O2, including cancer. In the sulfenic acid oxidation state, the electron-deficient sulfur exhibits enhanced electrophilic character that can be selectively targeted by certain nucleophilic compounds. Because sulfenic acid is a unique chemical moiety in biochemistry, this strategy might decrease the potential for off-target activity while retaining the advantages gained by covalent targeting.
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