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Review TRENDS in Biochemical Sciences Vol.28 No.9 September 2003 509 Redox regulation of protein tyrosine phosphatases during receptor tyrosine kinase signal transduction Paola Chiarugi and Paolo Cirri Dipartimento di Scienze Biochimiche, Università di Firenze, viale Morgagni 50, 50134 Firenze, Italy In addition to protein phosphorylation, redox-dependent post-translational modification of proteins is emerging as a key signaling system that has been conserved throughout evolution and that influences many aspects of cellular homeostasis. Both systems exemplify dynamic regulation of protein function by reversible modification, which, in turn, regulates many cellular processes such as cell proliferation, differentiation and apoptosis. In this article we focus on the interplay between phosphorylation- and redox-dependent signaling at the level of phosphotyrosine phosphatase-mediated regulation of receptor tyrosine kinases (RTKs). We propose that signal transduction by oxygen species through reversible phosphotyrosine phosphatase inhibition, represents a widespread and conserved component of the biochemical machinery that is triggered by RTKs. Activation of receptor tyrosine kinases (RTKs) is implicated in cell proliferation, motility, actin reorganization and chemotaxis. Tight regulation of RTK signaling is therefore crucial for eliciting an appropriate type and level of response to external stimuli. It has been proposed that protein tyrosine phosphatases (PTPs) negatively regulate RTK activity and downstream signaling [1]. Recent work on PTPs has demonstrated that these enzymes undergo reversible inhibition through oxidation of active-site cysteine residues [2]. Reactive oxygen species in receptor-mediated signal transduction During the past decade, reactive oxygen species (ROS), 2 namely H2O2, Oz2 2 and OH , have been identified as important mediators of cell growth, differentiation and apoptosis (Fig. 1). Mitochondria are the predominant source of ROS in all cell types, 1– 5% of electrons from the respiratory chain being typically diverted to the formation of Oz2 2 by ubiquinone-dependent reduction [3] (Fig. 1a). Recent evidence supports the hypothesis that mitochondrial ROS production is not only related to apoptotic cell death [4] but, in many conditions, assumes an essential signaling significance, for example, during tumor necrosis factor-a (TNFa) stimulation [5], hypoxia [6] and integrin signaling [7]. Corresponding author: Paolo Chiarugi ([email protected]). In addition to mitochondria, another cellular source of ROS is NADPH oxidase. This is a multi-protein complex (originally characterized in leukocytes) that is formed by membrane (gp91phox and p22phox) and cytosolic (Rac, p67phox, p47phox and p40phox) proteins [8]. Membrane oxidases that are similar to the phagocytic NADPHoxidase complex are expressed almost ubiquitously in non-phagocytic cell types [9]. NADPH oxidase catalyzes the one-electron reduction of O2 to Oz2 2 , which spontaneously or enzymatically dismutes to H2O2 (Fig. 1b). Several lines of evidence demonstrate that NADPH oxidase is specifically involved in the generation of ROS by soluble growth factors such as transforming growth factor-b1 [10,11], interleukin-1 [12], TNFa [13], insulin [14], platelet-derived growth factor (PDGF) [15], epidermal growth factor (EGF) [16], angiotensin II [17], thrombin and lysophosphatidic acid [18]. Growth-factor stimulation can induce intracellular ROS production via the activation of the phosphatidylinositol 3-kinase (PI3K) pathway, resulting in the activation of the small GTPase Rac1, which, in turn, stimulates NADPH-oxidase activity [19]. These findings suggest that ROS formation plays a role in mitogenic signaling elicited by cytokines and growth factors. Finally, growth-factor-stimulated ROS production can also be generated via a phospholipase A2-dependent mechanism. Indeed, growth factors activate the phospholipase A2-dependent lipoxygenase in a Rac1-dependent manner [20], which generates Oz2 2 and H2O2 as by-products. Therefore, it seems that the small GTPase Rac1 is central in the regulation of ROS signaling. This assumption is further strengthened by the recent observation that Rac 1, in addition to its role in NADPH and lipoxygenase activation, is also involved in mitochondrial oxidant production [7]. PTPs as intracellular targets of ROS Recent evidence has claimed a role for H2O2 as an intracellular messenger that regulates protein phosphorylation of tyrosine residues [15,16]. Unlike other known second messengers, H2O2 is not directly recognized by a sensor protein, instead it modulates intracellular protein phosphorylation by the reversible modification of downstream effectors. H2O2 is a mild oxidant and might http://tibs.trends.com 0968-0004/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0968-0004(03)00174-9 Review 510 (a) TRENDS in Biochemical Sciences (b) TNFα Ceramide Hypoxia p53 Apoptosis Integrin signaling Serum deprivation Oncogenic Ras I II Vol.28 No.9 September 2003 Growth factors Cytokines Hormones Immunological stimuli Oncogenic Ras Hypoxia/reoxygenation Cytochrome c III IV UQ/UQ-H2 O2• – Dismutation H2O2 NADPH oxidase Lipoxygenase Rac NADPH 2AA + O2 + NADP+ + H+ 2O2• – 2HPETE 2O2 Dismutation H2O2 Ti BS Fig. 1. Biological stimuli associated with the generation of reactive oxygen species (ROS). The main sources of intracellular ROS are illustrated and the specific stimuli that elicit the response are listed [57]. (a) The mitochondrial generation of ROS represents a relevant by-product of electron flow through the respiratory chain (complexes 1 –4 are indicated in blue). Oxygen is converted to Oz2 2 by ubiquinone (UQ)-mediated one-electron reduction, which is then converted to H2O2 by spontaneous or enzymaticdismutation that is mediated by superoxide dismutase. Mitochondrial ROS production is itself modulated by a variety of stimuli (listed), and, at least for integrin signaling, is reported to be Rac dependent [7]. (b) Upon activation, both NADPH oxidase and lypoxygenase (or cycloxygenase) are translocated to the membrane and, therefore, the small GTPase Rac is activated. Both oxidases produced Oz2 2 , which is then converted to H2O2 by spontaneous or enzymatic-dismutation mediated by superoxide dismutase. Abbreviations: AA, arachidonic acid; HPETE, hydroperoxyeicosatetraenoaic acid. oxidize cysteine residues in proteins to cysteine sulfenic acid or disulfide, both of which are readily re-reduced to cysteine by various cellular reductants. Proteins with low-pKa cysteine residues, which are vulnerable to oxidation by H2O2, include transcription factors such as the nuclear factor k-B [21], activator-protein 1 [22], hypoxiainducible factor [23], p53 [24], the p21Ras family of protooncogenes [25] and, notably, PTPs. All PTPs contain an essential cysteine residue in the signature active-site motif Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Arg that exists as a thiolate anion at neutral pH [26]. This thiolate anion contributes to the formation of a thiol-phosphate intermediate in the catalytic mechanism of PTPs. Oxidation of the active-site cysteine of PTPs to the cysteine sulfenic derivative by various oxidant agents, including H2O2, leads to enzymatic inactivation, however, this modification can be reversed by incubation with thiol compounds [27– 29]. These observations suggest that oxidation of the cysteine residue of PTPs might occur in vivo in response to ROS or to an increase in redox potential. Recent work has provided insight into how PTPs might transduce oxidative stress conditions. A reversible oxidation was first demonstrated for PTP1B during EGF [29] and insulin [14] signaling, and then for low molecular weight-PTP (LMW-PTP) during PDGF stimulation [30]. Both PTP1B and LMW-PTP fully rescue their phosphatase activity due to re-reduction 30 min after receptor activation [31,32]. Reversible oxidation of Src-homology 2 domain (SHP-2) PTP following PDGF stimulation has also been reported [33]. In addition, Rhee and coworkers have shown intramolecular disulfidebridge-dependent in vitro redox regulation of lipid phosphatase PTEN (phosphatase and tensin homolog) [34]. Savintsky and colleagues have provided evidence for Cdc25C phosphatase degradation that is triggered by H2O2-induced disulfide-bond formation between the activesite cysteine and another invariant cysteine residue [35]. http://tibs.trends.com Finally, as detected by fluorescence resonance energy transfer, H2O2-dependent Cys723 oxidation stabilizes dimers of transmembrane receptor PTPa (RPTPa), resulting in phosphatase inactivation [36]. RTK activation via redox regulation of PTPs It is well established that ligand binding to RTKs increases their intrinsic tyrosine kinase activity [37]. The tyrosine phosphorylation level of an RTK is given by the ratio between its intrinsic tyrosine kinase activity and the coordinated activity of PTPs (see Table 1 for an outlook of PTPs identified as negative regulators of RTKs). Growing evidence suggests that inhibition of PTP activity, as well as reduced sensitivity of RTKs to dephosphorylation [38], occurs after RTK dimerization and, thereby, also contributes to the ligand-induced increase in RTK tyrosine phosphorylation [1]. Here, we focus on the emerging hypothesis that the transient negative regulation of PTPs – due to oxidants produced in response to RTK-ligand stimulation – represents a strategy that has been adopted by cells to promote RTK signaling by avoiding its prompt inactivation by PTPs. The functional relevance of ROS-mediated PTP inhibition in growth factor signaling has been demonstrated by blocking their accumulation. First, Sundaresan provided the initial piece of evidence by demonstrating that the overexpression of catalase in vascular smooth muscle cells blocked PDGF receptor (PDGF-R)-induced tyrosine phosphorylation of extracellular signal-regulated kinase (ERK), as well as PDGFinduced DNA synthesis and migration [15]. Second, interference with H2O2 production through catalase loading of A431 cells dramatically reduced tyrosine phosphorylation of EGF receptor (EGF-R) [16]. Third, catalase pretreatment abolished both the insulin-stimulated production of ROS and the inhibition of PTP1B, and was associated with reduced tyrosine phosphorylation of insulin receptor [14]. Review TRENDS in Biochemical Sciences 511 Vol.28 No.9 September 2003 Table 1. Protein tyrosine phosphatases (PTPs) identified as negative regulators of receptor tyrosine kinases (RTKs): the investigation method and the documented redox regulation for every RTK/PTP couple was indicateda PTPs RTKs Investigation method Redox regulation Refs LMW-PTP PDGF-R Expression of dominant negative mutant/ co-immunoprecipitation Two hybrid system Expression of dominant negative mutant/ co-immunoprecipitation Mouse gene knockout Expression of dominant negative mutant Overexpression Overexpression Expression of dominant negative mutant Overexpression/co-immunoprecipitation Mice with natural gene mutation Mice with natural gene mutation Yes [30,58] Not yet Not yet [59] [60] Yes Yes Not yet Not yet Not yet Not yet Not yet Not yet In vitro Yes Not yet [14,61] [16,62] [63] [42] [64] [65] [66] [67] [27] [68] [69] Not yet Not yet Not yet Not yet In vitro Not yet Reported for exogenous oxidative stress Not yet Not yet [70] [71] [71] [72] [28] [73] [36] [74] [73] VEGF-R2 Insulin-R PTP1B DEP-1 SHP-1 SHP-2 T cell PTP LAR RPTPa RPTPs RPTPe Insulin-R EGF-R IGF1-R PDGF-R PDGF-R EGF-R CSF-1 Ros – PDGF-R EGF-R EGF-R HGF-R Insulin-R – Insulin-R – EGF-R Insulin-R In vitro dephosphorylation, association Expression of dominant negative mutant/ co-immunoprecipitation Expression of dominant negative mutant Antisense oligonucleotides Antisense oligonucleotides Antisense oligonucleotides Overexpression Antisense oligonucleotides/overexpression Overexpression a Abbreviations: CSF-1, colony-stimulating factor-1; DEP-1, density enhanced phosphatase-1; EGF, epidermal growth factor; EGF-R, EGF receptor; HGF-R, hepatocyte growth factor receptor; IGF-R, insulin-like growth factor receptor; insulin-R, insulin receptor; LAR, leukocyte-associated receptor; LMW-PTP, low molecular weight protein tyrosine phosphatase; PDGF-R, platelet-derived growth factor receptor; PTP, protein tyrosine phosphatase; RPTP, receptor-PTP; SHP, Src homology phosphatase; VEGF-R2, vascular endothelial growth factor receptor-2. Finally, blocking ROS production in PDGF-stimulated cells, either achieved by catalase pretreatment or inhibition of the NADPH oxidase by diphenyl iodide, led to the reduction of PDGF-R tyrosine phosphorylation [39]. In addition, during PDGF stimulation the kinetics of ROS production, PTP redox inhibition and receptor phosphorylation displayed an excellent alignment, suggesting a strict temporal correlation among these events [39]. Hence, it is likely that the redox inhibition of PTPs has an important role in RTK signaling, and that the rescue (via re-reduction) of the PTP catalytic activity after oxidation is followed by a dephosphorylation of activated receptor, thereby terminating the signal elicited from the receptor. Thus, the ROS produced after RTK engagement might be considered as intracellular second messengers that are involved in the signal-transduction machinery of soluble hormones. These second messengers form a feedback loop that, through the inhibition of PTPs, upregulates RTK tyrosine phosphorylation and, ultimately, leads to their own activation (Fig. 2). Although many PTPs can interact with more than one RTK and vice versa, PTP modulation of RTKs can offer different levels of specificity. We can speculate that celland tissue-specific expression of PTPs and RTKs provide the first level of selectivity. A possible second level of selectivity is provided by substrate specificity through siteselective dephosphorylation of RTK-specific tyrosine(s) by PTPs. Indeed, RTK signaling can be affected by general dephosphorylation, including dephosphorylation of regulatory phosphotyrosines, or by dephosphorylation of binding sites for downstream signaling molecules. The effects might be very different: dephosphorylation of the RTK regulatory http://tibs.trends.com RTK ligand NADPH oxidase P P Rac P RTK P P ROS P SH PTP Downstream signaling Reduced/ active PTP SOH Oxidated/ inactive Reducing agents Ti BS Fig. 2. Reactive oxygen species (ROS) as modulators of receptor tyrosine kinase (RTK) tyrosine phosphorylation. Ligand-induced activation of RTKs leads to the phosphatidyl inositol 3-kinase (PI3K)-mediated activation of Rac, which, in turn, switches on the production of ROS from NADPH oxidase. Among intracellular ROS targets are protein tyrosine phosphatases (PTPs), which are catalytically inactivated by oxidation, hence allowing the sustained RTK phosphorylation or activation required for cell-proliferation induction. The subsequent decrease of intracellular ROS concentration is accompanied by the recovery of PTP enzymatic activity (with a re-reduction mechanism that is not well defined). Full re-activated PTPs leads to RTK dephosphorylation and to the termination of the signal. 512 Review TRENDS in Biochemical Sciences tyrosines leads to subsequent termination of the signal, whereas site-specific dephosphorylation modulates single signal-transduction pathways. Dephosphorylation of the kinase-activation residues has been reported for LMW-PTP on PDGF-R Tyr857 [40] and for PTP1B on the insulin receptor [41]. By contrast, site-specific dephosphorylation of specific RTK tyrosines has been reported for densityenhanced phosphatase-1 (DEP-1), which displays siteselectivity dephosphorylation of PDGF-R, preferring Tyr763, Tyr771 and Tyr778 [42], and for SHP2, which reveals preferential dephosphorylation of Tyr771, Tyr751 and Tyr750 of PDGF-R [43]. In this scenario, we hypothesize that a third level of selectivity on PTP modulation of RTK signaling could come from different sensivity of PTPs to ROS-mediated inactivation, owing to possible differences in pKa values of target cysteine residues, and/or differences in the ability to rescue the enzymatic activity after re-reduction. Although great differences have been reported in the pKa values of catalytic cysteine residues among PTPs [44], the real differential vulnerability of PTPs to oxidation by H2O2 remains to be addressed. This discriminating regulation of PTPs, together with other post-translational modifications affecting PTP enzymatic activity, might cause specific responses in RTK signal transduction. Intriguing findings suggest that redox regulation of PTPs might be of pathophysiological significance. For example, recent reports have shown reduced basalinduced and growth-factor-induced ROS production in conditions of cell-growth inhibition [45,46]. Thus, antiproliferative conditions might shift the redox equilibrium of PTPs towards the reduced state, which would favor PTP activity and ensure silencing of RTK-dependent mitogenic signaling. Conversely, the constitutive high levels of H2O2 that are found in tumor cells [47,48] might cause oxidation-dependent PTP inactivation and a subsequent increase in RTK-mediated cell proliferation. These findings might be of pathogenetic relevance to oncogenic transformation and tumor progression. The role of redox regulation of PTPs in the ligand-independent activation of RTKs A large body of experimental evidence suggests that, in addition to endogenous ROS formation, extracellular oxidants affect RTK signaling. Adverse agents such as radiation, exposure to metals, alkylating agents and environmental oxidants have been found to activate RTKs in a ligand-independent manner, which is referred to as RTK trans-activation [49]. Again, oxidation and subsequent inactivation of PTPs seems responsible for RTK trans-activation by extracellular oxidants. For instance, cysteine modification in the PTP catalytic site has been proposed for both UV- and alkylating agentinduced RTK trans-activation [50,51], thus confirming a key role for PTP inhibition in the control of RTK tyrosine phosphorylation, even in the absence of the natural ligand. Moreover, in phagocytes and lymphocytes, NADPH oxidase produces oxidants involved in host defense and inflammation that might result in pro-oxidant conditions for bystander cells [8]. It is an intriguing possibility that the pro-oxidant environment causes redox-regulation of http://tibs.trends.com Vol.28 No.9 September 2003 RTKs (through PTP inhibition) in neighboring cells during inflammation, but it has not been demonstrated. If this interpretation is proven, it could shed a new light on cell signaling during the inflammatory reaction and have important pathophysiological and therapeutic implications. Stimulation of G-protein-coupled receptors (GPCRs) and activation of cell-adhesion receptors can also lead to RTK trans-activation. Although an extracellular pathway has been proposed for EGF-R trans-activation via a metalloprotease-dependent cleavage of heparin-binding EGF [52], the involvement of ROS has been proposed as a causal event for intracellular GPCR-mediated RTK transactivation [2,53,54]. Interestingly, activation of different GPCRs leads to the generation of H2O2, which suggests that subsequent PTP inactivation can contribute to intracellular GPCR-induced trans-activation of RTKs. Indeed, the prevention of H2O2 accumulation by antioxidants blocks RTK stimulation and ERK activation in cells treated with lysophosphatidic acid (LPA), angiotensin II, or serotonin, thereby suggesting a role for PTP redox inhibition in the intracellular RTK trans-activation pathways starting from these GPCRs [53,55,56]. Concluding remarks Mounting evidence indicates PTP redox regulation as pivotal in RTK signaling. Indeed, although growth-factorinduced oxidative PTP inactivation enables correct duration and amplitude of RTK signal transduction, the transient nature of oxidation-dependent PTP inhibition warrants RTK dephosphorylation and signal downregulation. Hence, given the remarkable importance of PTP in cell proliferation and related pathophysiology, further studies are needed to define the complete set of redoxregulated PTPs during RTK signaling, their differential sensivity to oxidants and the ways in which their oxidation is reversed. Acknowledgements We thank G. 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Chem. 270, 23126 – 23131 74 Suarez, P.E. et al. (1999) The transmembrane protein tyrosine phosphatase RPTPs modulates signaling of the epidermal growth factor receptor in A431 cells. Oncogene 18, 4069 – 4079 Could you name the most significant papers published in life sciences this month? Updated daily, Research Update presents short, easy-to-read commentary on the latest hot papers, enabling you to keep abreast with advances across the life sciences. Written by laboratory scientists with a keen understanding of their field, Research Update will clarify the significance and future impact of this research. Our experienced in-house team is under the guidance of a panel of experts from across the life sciences who offer suggestions and advice to ensure that we have high calibre authors and have spotted the ‘hot’ papers. Visit the Research Update daily at http://update.bmn.com and sign up for email alerts to make sure you don’t miss a thing. 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