Hydrogen Peroxide: A Key Messenger That Modulates Protein Phosphorylation Through Cysteine Oxidation

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Science's STKE  10 Oct 2000:
Vol. 2000, Issue 53, pp. pe1
DOI: 10.1126/stke.2000.53.pe1


Ligand-receptor interactions can generate the production of hydrogen peroxide (H2O2) in cells, the implications of which are becoming appreciated. Fluctuations in H2O2 levels can affect the intracellular activity of key signaling components including protein kinases and protein phosphatases. Rhee et al. discuss recent findings on the role of H2O2 in signal transduction. Specifically, H2O2 appears to oxidize active site cysteines in phosphatases, thereby inactivating them. H2O2 also can activate protein kinases; however, although the mechanism of activation for some kinases appears to be similar to that of phosphatase inactivation (cysteine oxidation), it is unclear how H2O2 promotes increased activation of other kinases. Thus, the higher levels of intracellular phosphoproteins observed in cells most likely occur because of the concomitant inhibition of protein phosphatases and activation of protein kinases.

Substantial evidence suggests that the transient production of hydrogen peroxide (H2O2) is an important signaling event triggered by the activation of many cell surface receptors. Such receptor-mediated production of H2O2 has been studied mostly in phagocytic leukocytes. In these cells, the one-electron reduction of molecular oxygen by a multicomponent reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system generates superoxide (O2·-), which is spontaneously or enzymatically converted to H2O2. However, since the initial observation that insulin induces the production of H2O2 in adipose cells (1), the intracellular generation of this molecule has been detected in various nonphagocytic cells in association with receptor stimulation.

Ligands that elicit an H2O2 response include peptide growth factors such as platelet-derived growth factor (PDGF) (2), epidermal growth factor (EGF) (3), basic fibroblast growth factor (bFGF) (4), insulin (1), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (5); cytokines such as transforming growth factor (TGF)-β1 (6, 7), interleukin-1 (5, 8), interleukin-3 (5), interferon-γ (8), and tumor necrosis factor-α (TNF-α) (4, 8); the T cell antigen receptor (9); and agonists of heterotrimeric guanosine 5°-triphosphate (GTP)-binding protein (G protein)-coupled receptors (GPCRs) such as angiotensin II (10-12), thrombin (13, 14), thyrotropin (8), parathyroid hormone (8, 15), lysophosphatidic acid (LPA) (16, 17), sphingosine 1-phosphate (18), serotonin (19, 20), endothelin (21), acetylcholine (22), platelet-activating factor (23), and bradykinin (24).

Although it remained unclear for many years whether nonphagocytic cells contain an NADPH oxidase system similar to that of phagocytic cells, several types of NADPH oxidase were recently identified in nonphagocytes (25, 26), and some of these enzyme systems were shown to be responsible for H2O2 production induced by PDGF or angiotensin II. Whether these NADPH oxidases are also coupled to receptors for the wide variety of other ligands that stimulate H2O2 production remains to be determined.

Activation of phosphatidylinositol 3-kinase (PI3K) was recently shown to be necessary and sufficient for PDGF-induced production of H2O2 (27). Phosphatidylinositol trisphosphate (PIP3), a product of PI3K, is thought to recruit guanine nucleotide exchange factors for the small GTP-binding protein Rac1 to the cell membrane; this effect is mediated by interaction of the pleckstrin homology domains of these proteins with PIP3 and results in the activation of Rac1. Activated Rac1 in turn stimulates NADPH oxidase.

Ligation of a variety of cell surface receptors also induces protein phosphorylation. Receptors for peptide growth factors such as PDGF, EGF, bFGF, GM-CSF, and insulin are protein tyrosine kinases (PTKs) and undergo autophosphorylation in response to ligand binding (28). Autophosphorylation of most receptor tyrosine kinases (RTKs) triggers the activation of mitogen-activated protein kinases (MAPKs) [through the activation of Ras, MAPK kinase kinases (MAPKKKs), and MAPK kinases (MAPKKs)], protein kinase C (PKC) (through activation of phospholipase C), and protein kinase B (PKB) (through activation of PI3K and phosphoinositide-dependent kinases) (28). The receptors for members of the TGF-β family of ligands are serine-threonine kinases, and they mediate the pleiotropic effects of their ligands by phosphorylating downstream effectors known as Smad proteins (28). The T cell antigen receptor and receptors for interleukins and interferons comprise multiple protein subunits, none of which possesses intrinsic kinase activity; however, on binding of ligand, the receptor complexes recruit and activate nonreceptor PTKs, resulting in activation of MAPK, PKC, and PKB signaling cascades (29, 30). The receptors for the TNF family of proteins also respond to ligand binding by activating MAPKs (31). Finally, GPCRs induce activation of adenylyl cyclase, phospholipase C, and PI3K, which in turn results in the activation of cyclic adenosine 3°,5°-monophosphate (cAMP)-dependent protein kinase, PKC, and PKB, respectively. Furthermore, ligation of most GPCRs results in the stimulation of PTKs and MAPKs (32).

Because H2O2 is a small, diffusible, and ubiquitous molecule that can be synthesized and destroyed rapidly in response to external stimuli, it meets all of the important criteria for an intracellular messenger. Recent evidence has implicated H2O2 as an intracellular messenger that modulates the extent of protein phosphorylation on either serine-threonine or tyrosine residues (2, 3, 31, 33). The importance of H2O2 production in growth factor signaling has been demonstrated by blocking its accumulation: Exogenously introduced catalase completely inhibited PDGF-induced tyrosine phosphorylation as well as MAPK activation in rat vascular smooth muscle cells (2). Similar results were obtained with catalase-treated A431 cells stimulated with EGF (3). The link between ligand-induced H2O2 generation and protein phosphorylation was further strengthened by the observation that exogenous H2O2 mimics growth factors by inducing protein tyrosine phosphorylation and MAPK activation (1, 34, 35). Because the extent of protein phosphorylation in a cell reflects the balance between the opposing actions of protein kinases and phosphatases, either activation of kinases or inhibition of phosphatases would be expected to shift the equilibrium toward phosphorylation.

Unlike cAMP and cyclic guanosine 3°,5°-monophosphate, H2O2 is too simple structurally to be recognized specifically by a protein. It is thus unlikely that the modulation of protein phosphorylation by H2O2 is mediated by the reversible binding of this molecule to protein kinases or phosphatases. On the other hand, H2O2 is a mild oxidant that can oxidize cysteine residues in proteins to cysteine sulfenic acid or disulfide, both of which are readily reduced back to cysteine by various cellular reductants. Because the pKa (where Ka is the acid constant) of the sulfhydryl group of most cysteine residues (Cys-SH) is ~8.5 and because this group is less readily oxidized by H2O2 than is the cysteine thiolate anion (Cys-S-), few proteins might be expected to possess a Cys-SH that is vulnerable to oxidation by H2O2 in cells. However, certain protein cysteine residues do exist as thiolate anions at neutral pH as a result of the lowering of their pKa values by charge interactions between the negatively charged thiolate and nearby positively charged amino acid residues. Proteins with low-pKa cysteine residues include protein tyrosine phosphatases (PTPs). All PTPs contain an essential cysteine residue (pKa 4.7 to 5.4) in the signature active site motif, His-Cys-X-X-Gly-X-X-Arg-Ser/Thr (where X is any amino acid), that exists as a thiolate anion at neutral pH (36). This thiolate anion contributes to the formation of a thiol-phosphate intermediate in the catalytic mechanism of PTPs. The active-site cysteine is the target of specific oxidation by various oxidants, including H2O2, and this modification can be reversed by incubation with thiol compounds such as dithiothreitol and reduced glutathione (37-39). These observations suggest that PTPs might undergo H2O2-dependent inactivation in cells, resulting in a shift in the equilibrium with PTKs toward protein phosphorylation.

The ability of intracellularly produced H2O2 to inhibit PTP activity was demonstrated by the observation that the stimulation of A431 cells with EGF selectively reduced the extent of subsequent labeling of the active site cysteine residue of PTP1B by (3H)iodoacetic acid in cell lysates (37). The amount of oxidatively inactivated PTP1B was maximal (30 to 40%) 10 min after exposure of cells to EGF and returned to baseline values by 40 min, suggesting that the oxidation of this phosphatase by H2O2 is reversible in cells. These results, together with the observation that increased levels of PDGF- or EGF-induced protein tyrosine phosphorylation requires H2O2 production, indicate that the activation of an RTK per se by binding of the corresponding growth factor may not be sufficient to increase the steady state level of protein tyrosine phosphorylation in cells. Rather, the concurrent inhibition of PTPs by H2O2 may also be required. This suggests that the extent of tyrosine phosphorylation of PTKs and their substrates would return to basal values after the degradation of H2O2 and the subsequent reactivation of PTPs by electron donors. Experiments with purified PTP1B suggest that the oxidized enzyme is reactivated more effectively by thioredoxin (Trx) than by glutaredoxin or glutathione at the physiological concentrations of these reductants. Thus, Trx might be a physiological electron donor for PTP1B (37), as well as for other protein tyrosine phosphatases.

The proposed roles of H2O2 and Trx in growth factor-induced protein tyrosine phosphorylation are depicted in Fig. 1. This scheme is consistent with the suggestion that the ligand-independent basal activity of RTKs is sufficient to mediate the increase of protein tyrosine phosphorylation observed in cells treated with thiol-alkylating agents or oxidants that induce the inactivation of PTPs (40). Because low-pKa cysteine residues are selectively oxidized by nitric oxide or peroxynitrite, agonists that induce the production of these nitrogen-containing oxidants are also able to increase the extent of protein tyrosine phosphorylation by inhibiting PTPs. Given that purified PTPs are constitutively active and that protein inhibitors or regulatory subunits of these enzymes have not been detected, the regulation of PTPs by cysteine oxidation may represent an important negative control mechanism. In the absence of such a mechanism, the activation of RTKs would result in a futile cycle of phosphorylation and dephosphorylation. The essential role of PI3K in H2O2 production is also indicated (Fig. 1).

Fig. 1.

Scheme depicting the PDGF-induced production of H2O2 and the proposed role of H2O2 as a modulator of protein tyrosine phosphorylation. Y and YP represent tyrosine and tyrosine phosphate, respectively. PIP2, phosphatidylinositol bisphosphate.

Protein serine-threonine phosphatases are regulated by specific regulatory subunits, and the active site residue that forms a phosphorylated intermediate in these enzymes is histidine. However, these phosphatases also may be subject to redox regulation. The family of protein serine-threonine phosphatases is divided into four subfamilies: PP1, PP2A, PP2B, and PP2C. PP2B, also known as calcineurin, contains an Fe(II)-Zn(II) center in its active site and is inactivated by O2·-, probably as a result of oxidation of the dinuclear metal center (41). This O2·--mediated inhibition of PP2B is completely blocked by superoxide dismutase. PP2B is also inactivated by H2O2 in a concentration-dependent manner without a change in the redox state of the dinuclear metal center (42, 43). Two to 3 of the 14 cysteine residues of PP2B are modified during such inactivation, which is reversed by exposure of the enzyme to dithiothreitol or Trx. The inactivation of PP2B by H2O2 has been attributed to the formation of a disulfide bond between Cys228 and Cys256 (43), a pair of cysteine residues that is conserved among members of the PP2B subfamily but not among other subfamilies of protein serine-threonine phosphatases. PP1 and PP2A also contain redox-sensitive cysteine residues (33, 44). Structure-based analysis has identified a potential disulfide oxidoreductase active site, Cys-X-X-Cys, in members of the PP1 subfamily (33). The same sequence is present in the active site of Trx, glutaredoxin, and protein disulfide isomerase and readily forms a disulfide on oxidation. Whether the oxidation of this pair of cysteine residues results in inactivation of PP1 remains to be determined. Inactivation of PP2A has been observed in cells treated with TNF or interleukin-1, both of which induce H2O2 production (44).

Protein kinases are regulated by phosphorylation and through interaction with regulatory proteins. However, both protein serine-threonine kinases and PTKs also appear to be under redox control. Hydrogen peroxide has thus been shown to mediate the activation of the EGF receptor and MAPK in LPA-treated HeLa cells (45), the activation of Janus kinase (JAK) in PDGF-treated Rat-1 cells (46), the activation of PKB in angiotensin II-treated vascular smooth muscle cells (12), and the activation of MAPK in serotonin-treated rat renal mesangial cells (20). The kinase-activating role of H2O2 in these cells was demonstrated by blocking H2O2 production with an NADPH oxidase inhibitor, diphenylene iodonium; by preventing H2O2 accumulation with N-acetylcysteine or catalase; or by the introduction of exogenous H2O2 (12, 20, 45, 46).

Although the mechanism responsible for H2O2-mediated activation remains unknown for most of these protein kinases, cysteine oxidation likely contributes to the activation of certain PTKs. c-Ret is an RTK with a cadherin-like domain in its extracellular region. The production of reactive oxygen species induced by the ultraviolet irradiation of cells expressing c-Ret resulted in the dimerization of a large proportion of c-Ret molecules (47). Dimerization was mediated by the formation of a disulfide between the Cys720 residues of each monomer, and the dimerized receptors were preferentially autophosphorylated, resulting in their activation. This cysteine residue is highly conserved in various nonreceptor PTKs, including Abl, Src, and Lck, suggesting that it might also play a role in the activation of these enzymes. Consistent with this proposal, Cys475 of Lck and Cys498 of Src, the residues equivalent to Cys720 in c-Ret, are crucial for either the catalytic or transforming activity of these kinases.

Some protein kinases are activated by H2O2 as a result of the oxidation of cysteine residues of upstream regulators. One example of such a scenario is the activation of apoptosis signal-regulating kinase 1 [or apoptosis-stimulated kinase 1 (ASK1)] in TNF-treated cells (Fig. 2). ASK1 is a MAPKKK that activates two members of the MAPK family: the stress-activated protein kinases JNK (Jun NH2-terminal kinase) [through activation of stress-activated protein kinase kinase (SEK) 1] and p38 [through activation of mitogen-activated protein kinase kinase kinases (MKK)3 and 6]. The TNF-engaged trimer of TNF receptor 1 (TNFR1) binds a docking protein, known as TNFR1-associated death domain-containing protein (TRADD), through the interaction of death domains in each protein. TRADD, in turn, binds a variety of adapters, including TNFR-associated factor 2 (TRAF2). TRAF2 activates ASK1 through direct interaction. ASK1 also binds with high affinity to the reduced form of Trx (31, 48). Because the binding sites for Trx and TRAF2 on ASK1 overlap, the binding of Trx prevents the association of ASK1 with TRAF2. Oxidation of the Cys-X-X-Cys motif of Trx induces the dissociation of the Trx-ASK1 complex, thereby allowing ASK1 to interact with TRAF2. Interaction with TRAF2 then triggers the oligomerization-dependent activation of ASK1. Thus, H2O2 generated in response to exposure of cells to TNF is expected to promote the dissociation of Trx from ASK1 and the consequent activation of ASK1 by TRAF2 (31). Although the mechanism by which TNF induces the production of H2O2 is unclear, TRAF2 is implicated in this mechanism by the observation that ectopic expression of this protein promotes H2O2 generation (31). The fact that TNF induces the activation of PI3K and Rac suggests that a pathway similar to that triggered by the PDGF receptor also contributes to H2O2 production in TNF-treated cells (49).

Fig. 2.

Regulation of ASK1 by a change in the redox state of Trx in TNF-treated cells. The production of H2O2 in the vicinity of TNFR1 is thought to induce oxidation of Trx. The pathway from TNFR1 to NADPH oxidase has not been well established. DD, death domain.

The oxidation of a specific cysteine residue of Ras also might result in the activation of a downstream protein kinase. The Cys118 residue of Ras is sensitive to oxidizing agents such as H2O2 and nitric oxide (50). Specific oxidation of this residue activates Ras in vitro as a result of an increase in guanosine diphosphate-GTP exchange. The oxidation of H-Ras in various cell lines as a result of the production of nitric oxide induced the activation of downstream protein kinases including extracellular signal-regulated kinases 1 and 2 (isoforms of MAPK), PI3K, and PKB (51, 52). It is likely that the oxidation of Ras by H2O2 will have similar consequences. These observations suggest the existence of a positive feedback loop (consisting of Ras, PI3K, Rac, and NADPH oxidase) in the production of H2O2. In this regard, NIH 3T3 cells that express a constitutively active form of Ras (H-RasVal12) have been shown to produce large amounts of O2·- and H2O2 (34).

Exogenous addition of H2O2 has often been used to mimic the effect of receptor-induced H2O2 production and results in the activation of various protein kinases, including Src family members (Src, Lck, Fyn, and Lyn), Abl, Syk, Zap70, JAK, focal adhesion kinase, PKC, p70 and p90 ribosomal protein S6 kinases, and the receptors for EGF and PDGF, by promoting tyrosine phosphorylation of these enzymes (35, 53-58). However, PTKs that are directly activated by H2O2 and phosphorylate these H2O2-responsive enzymes have not been identified. Studies of Lck activation suggest that the direct target of H2O2 action might not be a PTK; rather, inhibition of PTPs might be responsible for the H2O2-induced activation of Lck. Members of the Src family of PTKs are activated by phosphorylation of a conserved tyrosine residue (Tyr394 in Lck and Tyr416 in Src) in the activation loop of the kinase domain, which evidence suggests is an intermolecular autophosphorylation event. However, expression of a kinase-deficient Lck mutant in fibroblasts that did not contain endogenous Lck or Src resulted in the phosphorylation of Tyr394 of the Lck mutant, to an extent similar to that apparent with wild-type Lck, when the cells were exposed to H2O2 (53). This observation might be explained by the presence in these cells of a non-Src family PTK that catalyzes phosphorylation of the activation loop of Lck. However, given that no such PTK has been identified after an extensive search, it is more likely that Tyr394 of Lck is phosphorylated, albeit slowly, by various nonspecific PTKs. In the absence of a significant opposing dephosphorylation reaction (due to inactivation of PTPs by H2O2), this nonspecific PTK activity might be sufficient to increase the extent of Tyr394 phosphorylation. Several PTPs catalyze the dephosphorylation of Tyr394 of Lck. Similar to Lck, other PTKs, including RTKs, thought to require autophosphorylation for activation might also be activated as a result of H2O2-induced inhibition of PTPs. Thus, the increase in the tyrosine phosphorylation of cellular proteins induced by the inhibition of PTPs by H2O2 or pervanadate is likely mediated initially by basal kinase activities and subsequently by various autophosphorylated kinases.

Exposure of cells to H2O2 induces the tyrosine phosphorylation of various PKC isoforms (α, βI, δ, γ, ϵ, and ζ) (56), which results in their activation in the absence of receptor-mediated stimulation of phospholipase C. The PTK responsible for such phosphorylation of PKCδ has been identified as Abl (54). Treatment of cells with H2O2 thus promoted the association of Abl with PKCδ and the mutual phosphorylation of both proteins. The H2O2-induced activation of Abl, however, likely results from inhibition of PTP activity.

Hydrogen peroxide is also implicated in GPCR signaling. The GPCR agonists LPA, angiotensin-II, thrombin, endothelin, bradykinin, carbachol, serotonin, and isoproterenol induce the tyrosine phosphorylation and consequent activation of RTKs such as the receptors for PDGF, EGF, and insulin-like growth factor as well as trigger the subsequent activation of MAPK signaling pathways (11, 20, 45, 59, 60). The mechanisms that underlie such "transactivation" remain unclear, although Src or the PTK Pyk2, intracellular Ca2+, PKC, and G-protein subunits have been suggested to play a role. Because most of the transactivating GPCR agonists are capable of inducing H2O2 production, inactivation of PTPs by H2O2 likely contributes to the transactivation mechanism. Indeed, the prevention of H2O2 accumulation by antioxidants blocked the stimulation of RTKs and MAPKs in cells treated with LPA, angiotensin II, or serotonin (11, 20, 45).

Even in the absence of receptor stimulation, H2O2 is continuously produced by metabolic reactions in cells. It is also readily converted by the Fe-catalyzed Fenton reaction to hydroxyl radicals, which damage biomolecules indiscriminately (61). To protect themselves from such oxidative damage, all aerobic cells express catalases and peroxidases that eliminate H2O2 (61). The receptor-mediated production of H2O2 is transient and strictly controlled. At least in the case of cells exposed to PDGF, H2O2 production is linked to the generation of another intracellular messenger molecule, PIP3, that is not detectable in unstimulated cells (27). It is likely that the production of and target oxidation by H2O2 are local events restricted to the microdomains of a cell adjacent to stimulated receptors, with H2O2 molecules that diffuse away from such domains being rapidly destroyed. Confirmation of such localized production of and signaling by H2O2 awaits the development of sensitive probes specific for this molecule.


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