Redox regulation of tyrosine kinase signalling: more than meets the eye

Src family kinases

The Src family kinases (SFK) represent one of the most widely studied families of NRTKs (65). Structurally, SFKs consist of an N-terminal Src homology (SH) 4 domain that contains lipid modification sites, a poorly conserved ‘unique’ domain, an SH3 domain that binds to specific proline-rich sequences, an SH2 domain that can bind to specific tyrosine phosphorylated proteins, and a C-terminal tyrosine kinase domain followed by an unstructured negative regulatory C-terminal peptide tail. In their inactive state, SFKs are held in a ‘clamped’ autoinhibited state via intramolecular binding of a C-terminal phosphotyrosine (pY527; based on chicken c-Src numbering) to their SH2 domains, as well as binding of their SH3 domains to a polyproline sequence within the kinase linker domain (66). Activation of SFKs occurs in a mechanism initiated by dephosphorylation of pY527 (or its homolog) and results in detachment of the SH2/SH3 domains from the back of the kinase domain, thereby inducing ‘unclamping’ and allowing for recognition of these SH2/SH3 domains by target proteins (66). In a concerted fashion, SH2/SH3 domain detachment ‘unclamps’ the kinase domain in an allosteric mechanism leading to unfolding of the activation loop, exposing a conserved A-loop tyrosine (Y416 in Src) for phosphorylation, and inward rotation of the αC-helix. PTP-dependent dephosphorylation of Y416 and phosphorylation of Y527 by, e.g. C-terminal Src kinase (CSK) can reverse this activation and restore the inactive state (67).

In addition to these classical SFK regulating mechanisms, recent discoveries highlight the impact of redox regulation on the inactive/active state equilibrium of these kinases through direct Cys oxidation, although these mechanisms vary between the different family members based on the evolutionary conservation of Cys residues (50) (Fig. 3A). The Src protein contains 9 Cys residues that are distributed across its SH2 and kinase domains (Fig. 3B). None of these cysteines are involved in structural disulphide bonding, and several are contained within the conserved CXXXXXXXMXXCW motif. The first indication that Src is redox regulated came from studies showing that various Cys-SH targeting alkylating agents can abrogate Src activity (68). Later, it was observed that exposure of fibroblasts or immunoprecipitated c-Src to NO-generating molecules could in fact promote Src activation, independent of its Y527 phosphorylation status, and this was found to be associated with redox-dependent formation of putative Src multimers through disulphide formation, although for such disulphide formation was not directly demonstrated (69). Follow up studies by the same group suggested a role for the four clustered C-terminal Cys residues (including the CXXXXXXXMXXCW motif C487 and 498) in Src activation induced by HgCl₂ or P-SNO (49–51). These and subsequent studies suggested that increased cellular NO^• production in, e.g. breast cancers could promote cell invasion and oncogenesis by S-nitrosylation of Src and/or associated disulphide formation (49, 70).

Giannoni et al. highlighted an alternative mechanism of Src redox regulation by cellular ROS in the context of anchorage-dependent growth. They found that Src activation was associated with cysteine oxidation within the Src protein following adhesion-induced activation of 5-lipoxygenase via integrin signalling, and mutation studies implicated C245 within the SH2 domain and C487 within the CXXXXXXXMXXCW motif in such oxidative regulation (45). Subsequent studies by the same authors also implicated these Cys residues in redox-dependent Src activation in increased anoikis resistance via EGFR-dependent mechanisms (71, 72). The authors postulated formation of a putative intramolecular disulphide bond between these residues in such oxidative Src activation (45), although this would seem unlikely from a structural perspective since these Cys are quite distant from each other in the inactive Src structure (e.g.Fig. 2B). However, since Src activation was recently shown to involve the formation of asymmetric dimers (73), is it plausible that intermolecular disulphide bonds between two Src monomers may be involved. Other studies have highlighted a role for C487 in redox-dependent Src activation in angiotensin II-mediated cellular hypertrophy or in IGF-1 signalling during hyperglycaemia, which was linked to cellular H₂O₂ production by the NADPH oxidase isoform NOX4 (40, 48). NOX activation was also associated with Src activation in the context of TGF-β signalling, and mutagenesis studies suggested the involvement of C245, C277, C487 and C498 (46). Although none of these studies actually demonstrated the ability of H₂O₂ to directly enhance Src activity, this was demonstrated more recently in studies with immunoprecipitated Src (74).

Most of the Cys residues highlighted above are conserved among all SFKs, and other SFKs have also been shown to be regulated by ROS. For example, Fyn and Yes represent ubiquitously expressed SFK members that also contain a CXXXXXXXMXXCW motif (Fig. 3), and activation of Fyn in keratinocytes in response to solar UV-light exposure was found to involve oxidation of C488 (homologous to Src C487), which was thought to represent a potential redox sensing mechanism to induce protective cellular apoptosis and prevent melanoma (52). In a model of zebrafish tail fin regeneration, haematopoietic cell-specific Lyn was found to be a critical mediator of neutrophil recruitment to areas of high ROS production at injury sites, which was dependent on oxidation of the kinase CXXXXXXXMXXCW motif residue C466 (homologous to Src C487) (53). Similar cysteine residues were also implicated in HgCl₂-induced activation of Yes (C491/C506; homologous to Src C483/C498) and Lyn (C479; homologous to Src C498) (51) and in redox-dependent activation of the lymphocyte-specific SFK Lck (at residues C464 and C475, homologous to Src C487 and C498, respectively) (36). Finally, Hck was also found to be activated by the oxidant peroxynitrite (ONOO⁻), although no specific Cys residues were implicated (54).

In aggregate, these various studies strongly implicate the importance of CXXXXXXXMXXCW motif cysteines within αH-helix of SFK kinase domains, perhaps in combination with other Cys residues, in redox-mediated activation of several SFKs. However, the mechanisms by which modification of these cysteines control kinase function are still not understood, largely because the relevant cysteine modifications have not been identified. No structural studies exist, to our knowledge, to directly link oxidation of CXXXXXXXMXXCW motif cysteines to SFK activation, and this region is typically thought to be rather inconsequential for kinase activation based on available structural studies (66). Indeed, recent molecular dynamics (MD) simulations of Src C498 oxidation (to its primary oxidation product P-SOH) did not reveal significant structural alterations that would explain increases in kinase function (44). We therefore speculate that redox-dependent modification of these cysteines may involve altered interactions with other associated proteins (such as other SFK monomers), potentially via disulphide-mediated interactions, but this has to date not been addressed directly.

Unique cysteine modifications in redox-dependent activation of Src

Our research group recently demonstrated the importance of the NOX isozyme DUOX1 in airway epithelial wound responses via ATP-dependent stimulation of purinergic P2YR2 receptors and redox-dependent activation of Src (75). Src activation by P2YR2 stimulation was found to involve recruitment of Src to the P2YR2 via its PXXP domain, and also appears to involve recruitment of DUOX1. We attempted to associate Src activation with specific redox-based modifications within the Src protein (P-SOH and P-SSG), which suggested that Src activation corresponded most closely with formation of P-SOH (41). Studies with recombinant Src protein indicated that H₂O₂ was capable of enhancing Src kinase activity, whereas formation of P-SSG by addition of GSSG was ineffective (41). Mass spectrometry analysis using P-SOH-specific probes, to identify the main redox-sensitive cysteines within recombinant Src protein, highlighted prominent sulphenylation of two cysteines that are rather unique within Src compared to other SFK members, namely C185 within the SH2 domain and C277 within the ATP binding region of the kinase domain (Figs 2 and 3). Mutation of either cysteine significantly attenuated H₂O₂-dependent Src activation, both in studies with recombinant proteins in vitro and in a cellular context (44). Moreover, MD simulations demonstrated that introduction of sulphenylated forms (P-SOH) of either C185 or C277 within the autoinhibited Src crystal structure (PDB 2SRC) promoted electrostatic and structural changes that are consistent with Src activation mechanisms (44). Indeed, modelling of C277-SOH indicated that this modification directly influences the A-loop, promoting energetically favourable unfolding and solvent exposure of Y416. Inclusion of C185-SOH altered interactions with the inhibitory pY527, destabilizing its binding affinity to the SH2 domain and enhancing its solvent accessibility and potential susceptibility to dephosphorylation (44). Since both cysteine residues appeared to contribute to ROS-dependent Src activation in a cellular context, we proposed a stepwise redox-based mechanism of Src activation, involving the successive oxidation of both C277 and C185 to P-SOH (44). Subsequent S-glutathionylation of either residue would be expected to lock the Src kinase into its active ‘unclamped’ state until these modifications are reversed by cellular reductases (41).

The most intriguing aspect of our recent studies relates to the fact that it concerns relatively unique cysteines that are not present in most other SFK members, although similar cysteines are found in other PTKs. C277 is conserved in only two other SFK members and its function is largely unknown, and has therefore been targeted by newly developed covalent Src inhibitors (38). Studies with recombinant Src proteins indicated that removal of DTT in the kinase buffer causes loss of kinase activity, and this was attributed to formation of disulphide-linked homodimers via C277 (47), but the significance of such a disulphide-based mechanisms of Src inactivation has to date not been demonstrated in a cellular context. The importance of C185 in the SH2-domain of Src is also poorly understood, and all other SFKs contain a serine in the corresponding position (Fig. 3A), although similar cysteines can be found in other SH2 domains (43). Mutation studies with recombinant Src proteins suggested that the presence of a cysteine in this position may weaken binding affinity to phosphotyrosine-containing peptides that mimic the autoinhibitory pY527-containing domain, and that Src may thus be more readily activated compared to other SFKs (76). A more recent study implicated Src C185 in recognition of substrates through formation of an intermolecular disulphide, as was demonstrated for the Src substrate cortactin (43).

Overall, it follows that redox mechanisms of SFK regulation are exceedingly complex and may involve intrinsic mechanisms that induce structural alterations that alter enzyme function, but may also involve redox modifications that impact on interactions with substrates (43) or potentially affect asymmetric Src dimerization as a critical mechanism in promoting autophosphorylation and Src activation (73). Some SFKs, but not Src itself, also contain additional Cys residues within their N-terminal SH4 domains that are modified by S-acylation (palmitoylation) to regulate SFK trafficking and membrane targeting (77). It is not known to date whether these cysteines are also subject to redox regulation.

Other NRTKs

Redox-based mechanisms have also been implicated in regulation of other NRTKs, such as the Janus Kinases (JAK), which are predominantly involved in cytokine signalling and activation of STAT family of transcription factors (78). JAK proteins also contain a CXXXXXXXMXXCW motif (including C1094 and C1105) but, in contrast to SFKs, are generally found to be inactivated by oxidative mechanisms through oxidation of unique cysteines (79). Four distinct Cys residues (C866, C917, C1094 and C1105) in JAK2 were found to be independently responsible for redox-dependent suppression of kinase activity, with C866 and C917 postulated to form a disulphide bond based on their spatial proximity (55). The PTK CSK, which negatively regulates Src by mediating Y527 phosphorylation (80), is redox regulated by formation of an intramolecular disulphide bond within its SH2 domain (between its C122 and C164 residues), and this inhibits kinase activity compared to the fully reduced enzyme (56). Other uniquely regulated non-receptor kinases include the spleen tyrosine kinase (SYK) and its related family members, which are predominantly involved in T-cell activation and adaptive immune signalling (81). SYK, as well as its related family member ZAP-70, were found to be susceptible to Cys-targeted covalent inhibitors as well as H₂O₂, which target Cys residues within their dual SH2 domain motif and thereby prevent these kinases from binding substrate peptides (57). These examples indicate that cysteine oxidation can regulate tyrosine kinase signalling by multiple distinct processes, not only by directly affecting kinase activity but also by regulating kinase signalling more indirectly, e.g. by interfering with phosphopeptide substrate binding.

Epidermal growth factor receptor

RTKs comprise several families of high-affinity cell surface receptors for various growth factors and hormones (82). Among these, the epidermal growth factor receptor (EGFR), a member of the ErbB family, has been extensively studied because of its broad roles in various cellular processes (e.g. proliferation, migration, cell survival, immune signalling) and the prevalence of diverse activating mutations within this protein in various cancers (83). Similar to other RTKs, EGFR is a transmembrane receptor expressed on the cell surface that is activated by one of its seven cognate ligands (e.g. EGF) through homodimerization or heterodimerization with other ErbB family members. Dimerization of the extracellular ectodomain leads to kinase activation through the formation of an asymmetric kinase dimer complex comprising an activator and receiver kinase pair (Fig. 4) followed by EGFR tail (auto)phosphorylation on several tyrosine residues (84). EGFR can also be activated by transactivation processes, in which other kinases (such as SFKs) phosphorylate unique tyrosines within the EGFR kinase domain to enhance kinase function (75). The full-length EGFR protein contains 60 Cys residues, most of which are present in the extracellular domain and are involved in structurally important disulphide bonds (85). However, the intracellular kinase domain also contains six cysteine residues (and some others are also present in the C-terminal phosphorylation tail), including cysteines contained within the previously mentioned CXXXXXXXMXXCW motif within the kinase αH-helix. Since this αH-helix plays a critical role in the activation of EGFR through direct coil–coil interaction with the αC-helix of the receiver kinase in the asymmetric dimer (Fig. 4), it is conceivable that redox-modification of these cysteines could affect such dimer interactions. However, in contrast to studies with SFKs discussed above, no studies to date have addressed the specific function of these CXXXXXXXMXXCW motif cysteines within EGFR.

It has been recognized for over 20 years that EGFR activation can promote production of cellular ROS (e.g. by NOX activation) and that this promotes EGFR-dependent signalling (86, 87). While this was largely attributed to redox-dependent inactivation of PTPs (as described earlier), recent pioneering studies by Kate Carroll et al. have highlighted the ability of ROS to directly regulate EGFR. They illustrated that H₂O₂ can directly activate EGFR by oxidation of a conserved cysteine (C797) within the ATP binding region (59, 60). Among the six cysteines within the EGFR kinase domain, C797 is the most solvent accessible and redox-sensitive, due to its relatively low pK_a (60). Indeed, studies with recombinant EGFR kinase domains showed that, although C797 is not critical for intrinsic kinase activity, the ability of H₂O₂ to enhance kinase activity relies fully on the presence of C797 (59, 60). Redox-dependent EGFR activation by ligand stimulation was found to involve activation of the NOX isoform NOX2, which is recruited to the EGFR signalling complex following EGF stimulation (41, 59). Our research group recently highlighted activation of EGFR in airway epithelial cells in the context of ATP-dependent wound signalling, and this was found to involve activation of a different NOX isoform, DUOX1, which promotes EGFR transactivation by initial redox-dependent activation of Src associated with cysteine oxidation within Src as well as EGFR (41, 75). DUOX1-dependent EGFR activation was also demonstrated to contribute to innate airway responses to allergens and to features of allergic asthma (88, 89). Evaluation of distinct cysteine modifications indicated that H₂O₂-induced EGFR activation is mediated specifically by formation of P-SOH, whereas other modifications (P-SSG, P-SO2/3H) did not appear to alter intrinsic kinase activity (41, 90). MD simulations indicated that formation of C797-SOH can alter local electrostatic interactions due to hydrogen bonding between C797-SOH and a critical R841 residue within the ATP binding region, thereby potentially aiding in coordinating ATP binding (60). Interestingly, C797 within the EGFR kinase domain is also the target for covalent EGFR TKI inhibitors (91, 92) and for inhibition by other thiol-reactive electrophiles such as acrolein (61). Hence, oxidation of EGFR C797 would be expected to negatively impact on efficacy of irreversible kinase inhibitors, as was indeed observed in several studies (60, 90).

The importance of cysteine oxidation in EGFR has been implicated in EGFR-dependent functions in normal epithelial biology (41, 75), but may be dysregulated in the context of lung cancer, especially those involving oncogenic EGFR mutants. For example, the oncogenic L858R/T790M double mutant, which inserts an Arg residue onto the A-loop opposite the active site in addition to the Met gatekeeper mutation, was found to display increased sulphenylation over a higher range of EGF concentrations in comparison to other oncogenic cell lines (60). Alternatively, redox events may also regulate subcellular EGFR trafficking as was illustrated by increased caveolin-dependent perinuclear trafficking in cancer cells under conditions of oxidative stress (93). More recent findings indicated that the absence of DUOX1 in lung cancers can promote EGFR internalization and nuclear localization, and this was associated with altered redox modification(s) within EGFR (42). In this regard, it is important to consider that EGFR is also regulated by palmitoylation on several cysteine residues, including C797, with apparent consequences for EGFR membrane interactions and dimerization, and such palmitoylation has been implicated in development of tumour resistance against EGFR kinase inhibitors (94). In addition to C797, cysteines within the C-terminal tail of EGFR (C1025 and C1122), have also been identified as sites for palmitoylation, regulating receptor turnover and recycling (62). Inhibition of EGFR palmitoylation by inhibiting the palmitoyltransferase DHHC20 was found to result in sustained EGFR activation, and to sensitise cancer cells to EGFR TKI inhibitors (62). Redox modification of C797 or these C-terminal cysteines might therefore indirectly affect EGFR function by interfering with EGFR palmitoylation. Intriguingly, recent studies highlighted development of tumour resistance against covalent EGFR inhibitors that target C797, which was found to involve acquired mutation of this Cys to a Ser (95), which would also suggest that EGFR signalling is in these cases refractory to redox regulation or palmitoylation at this cysteine.

Other RTKs

Other RTKs may be similarly redox regulated based on the presence of conserved cysteines. For example, other ErbB family members such as ErbB2/HER2 or ErbB4/HER4 contain homologous cysteines to C797 in EGFR (35, 39), as do some other RTKs (96), but the importance of redox regulation of these cysteines has not yet been addressed. The insulin-like growth factor 1 receptor (IGF-1R) was recently found to be subject to S-nitrosylation, which was reasoned to be disrupting IGF-1R activation and downstream signaling (63). Fibroblast growth factor receptors (FGFR) contain a cysteine within their ATP binding regions that is homologous to C277 in Src, and studies with recombinant FGFR1 protein showed loss of kinase activity under oxidizing conditions (in the absence of DTT) corresponding to formation of disulphide-based homodimers similar to Src (47), although the relevance for potential redox regulation in a cellular context is unclear. Exposure of vascular endothelial cells to H₂O₂ was found to be capable of promoting intramolecular disulphide bonds within the VEGF receptor, between C1199 and C1206 within its cytoplasmic domain, which resulted in its activation (64). Finally, disulphide-linked dimers were also implicated in the activation of the RET kinase due to UV-mediated oxidation, which was observed to involve C376 within its kinase domain (37).