Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatases

Signaling through receptor tyrosine kinases (RTKs) is a major mechanism for intercellular communication during development and in the adult organism, as well as in disease-associated processes. The phosphorylation status and signaling activity of RTKs is determined not only by the kinase activity of the RTK but also by the activities of protein tyrosine phosphatases (PTPs). This review discusses recently identified PTPs that negatively regulate various RTKs and the role of PTP inhibition in ligand-induced RTK activation. The contributions of PTPs to ligand-independent RTK activation and to RTK inactivation by other classes of receptors are also surveyed. Continued investigation into the involvement of PTPs in RTK regulation is likely to unravel previously unrecognized layers of RTK control and to suggest novel strategies for interference with disease-associated RTK signaling.     

PTPs versus PTKs: the redox side of the coin

The phosphorylation of tyrosine, and to a lesser extent threonine and serine, plays a key role in the regulation of signal transduction during a plethora of eukaryotic cell functions, including cell activation, cell-cycle progression, cytoskeletal rearrangement and cell movement, differentiation, apoptosis and metabolic homeostasis. In vivo, tyrosine phosphorylation is reversible and dynamic; the phosphorylation states are governed by the opposing activities of protein tyrosine kinases (PTKs)2 and protein tyrosine phosphatases (PTPs). Reactive oxygen species (ROS) act as cellular messengers in cellular processes such as mitogenic signal transduction, gene expression, regulation of cell proliferation, senescence and apoptosis. Redox regulated proteins include PTPs and PTKs, although with opposite regulation of enzymatic activity. Transient oxidation of thiols in PTPs leads to their inactivation by the formation of either an intramolecular S-S bridge or a sulfenyl-amide bond. Conversely, oxidation of PTKs leads to their activation, either by direct SH modification or, indirectly, by concomitant inhibition of PTPs that guides to sustained activation of PTKs. This review focuses on the redox regulation of both PTPs and PTKs and the interplay of their specular regulation.     

ReviewPTPs versus PTKs: The redox side of the coin

The phosphorylation of tyrosine, and to a lesser extent threonine and serine, plays a key role in the regulation of signal transduction during a plethora of eukaryotic cell functions, including cell activation, cell-cycle progression, cytoskeletal rearrangement and cell movement, differentiation, apoptosis and metabolic homeostasis. In vivo, tyrosine phosphorylation is reversible and dynamic; the phosphorylation states are governed by the opposing activities of protein tyrosine kinases (PTKs)² and protein tyrosine phosphatases (PTPs). Reactive oxygen species (ROS) act as cellular messengers in cellular processes such as mitogenic signal transduction, gene expression, regulation of cell proliferation, senescence and apoptosis. Redox regulated proteins include PTPs and PTKs, although with opposite regulation of enzymatic activity. Transient oxidation of thiols in PTPs leads to their inactivation by the formation of either an intramolecular S–S bridge or a sulfenyl–amide bond. Conversely, oxidation of PTKs leads to their activation, either by direct SH modification or, indirectly, by concomitant inhibition of PTPs that guides to sustained activation of PTKs. This review focuses on the redox regulation of both PTPs and PTKs and the interplay of their specular regulation.     

Targeting Src homology 2 domain-containing tyrosine phosphatase (SHP-1) into lipid rafts inhibits CD3-induced T cell activation

To study the mechanism by which protein tyrosine phosphatases (PTPs) regulate CD3-induced tyrosine phosphorylation, we investigated the distribution of PTPs in subdomains of plasma membrane. We report here that the bulk PTP activity associated with T cell membrane is present outside the lipid rafts, as determined by sucrose density gradient sedimentation. In Jurkat T cells, approximately 5--10% of Src homology 2 domain-containing tyrosine phosphatase (SHP-1) is constitutively associated with plasma membrane, and nearly 50% of SHP-2 is translocated to plasma membrane after vanadate treatment. Similar to transmembrane PTP, CD45, the membrane-associated populations of SHP-1 and SHP-2 are essentially excluded from lipid rafts, where other signaling molecules such as Lck, linker for activation of T cells, and CD3 zeta are enriched. We further demonstrated that CD3-induced tyrosine phosphorylation of these substrates is largely restricted to lipid rafts, unless PTPs are inhibited. It suggests that a restricted partition of PTPs among membrane subdomains may regulate protein tyrosine phosphorylation in T cell membrane. To test this hypothesis, we targeted SHP-1 into lipid rafts by using the N-terminal region of Lck (residues 1--14). The results indicate that the expression of Lck/SHP-1 chimera inside lipid rafts profoundly inhibits CD3-induced tyrosine phosphorylation of CD3 zeta/epsilon, IL-2 generation, and nuclear mobilization of NF-AT. Collectively, these results suggest that the exclusion of PTPs from lipid rafts may be a mechanism that potentiates TCR/CD3 activation.     

Phosphorylation-dependent regulation of T-cell activation by PAG/Cbp,a lipid raft-associated transmembrane adaptor

PAG/Cbp (hereafter named PAG) is a transmembrane adaptor molecule found in lipid rafts. In resting human T cells, PAG is tyrosine phosphorylated and associated with Csk, an inhibitor of Src-related protein tyrosine kinases. These modifications are rapidly lost in response to T-cell receptor (TCR) stimulation. Overexpression of PAG was reported to inhibit TCR-mediated responses in Jurkat T cells. Herein, we have examined the physiological relevance and the mechanism of PAG-mediated inhibition in T cells. Our studies showed that PAG tyrosine phosphorylation and association with Csk are suppressed in response to activation of normal mouse T cells. By expressing wild-type and phosphorylation-defective (dominant-negative) PAG polypeptides in these cells, we found that the inhibitory effect of PAG is dependent on its capacity to be tyrosine phosphorylated and to associate with Csk. PAG-mediated inhibition was accompanied by a repression of proximal TCR signaling and was rescued by expression of a constitutively activated Src-related kinase, implying that it is due to an inactivation of Src kinases by PAG-associated Csk. We also attempted to identify the protein tyrosine phosphatases (PTPs) responsible for dephosphorylating PAG in T cells. Through cell fractionation studies and analyses of genetically modified mice, we established that PTPs such as PEP and SHP-1 are unlikely to be involved in the dephosphorylation of PAG in T cells. However, the transmembrane PTP CD45 seems to play an important role in this process. Taken together, these data provide firm evidence that PAG is a bona fide negative regulator of T-cell activation as a result of its capacity to recruit Csk. They also suggest that the inhibitory function of PAG in T cells is suppressed by CD45. Lastly, they support the idea that dephosphorylation of proteins on tyrosine residues is critical for the initiation of T-cell activation.     

Redox regulation of PTEN and protein tyrosine phosphatases in H(2)O(2) mediated cell signaling

Protein tyrosine phosphatase (PTP) is a family of enzymes important for regulating cellular phosphorylation state. The oxidation and consequent inactivation of several PTPs by H₂O₂ are well demonstrated. It is also shown that recovery of enzymatic activity depends on the availability of cellular reductants. Among these redox-regulated PTPs, PTEN, Cdc25 and low molecular weight PTP are known to form a disulfide bond between two cysteines, one in the active site and the other nearby, during oxidation by H₂O₂. The disulfide bond likely confers efficiency in the redox regulation of the PTPs and protects cysteine-sulfenic acid of PTPs from further oxidation. In this review, through a comparative analysis of the oxidation process of Yap1 and PTPs, we propose the mechanism of disulfide bond formation in the PTPs.     

Proteolytic processing of the protein tyrosine phosphatase α extracellular domain is mediated by ADAM17/TACE

The receptor protein tyrosine phosphatase alpha (PTPα) is involved in the regulation of tyrosine kinases like the Src kinase and the insulin receptor. As with other PTPs, its function is determined by alternative splicing, dimerisation, phosphorylation and proteolytical processing. PTPα is cleaved by calpain in its intracellular domain, which decreases its potential to dephosphorylate Src kinase. Here, we demonstrate that PTPα is also processed in the extracellular domain. Extracellular processing was exclusively found for a splice variant containing an extra nine amino acid insert three residues amino-terminal from the transmembrane domain. Processing was sensitive to the metalloprotease-inhibitor Batimastat, and CHO-M2 cells lacking a disintegrin and metalloproteinase 17 (ADAM17; tumor-necrosis-factor α converting enzyme) activity were not able to cleave PTPα. After transient overexpression of ADAM17 and PTPα in these cells, processing was restored, proving that ADAM17 is involved in this process. Further characterization of the consequences of processing revealed that dephosphorylation of the insulin receptor or activation of Src was not affected but focus formation was reduced. We conclude that extracellular proteolytic processing is a novel mechanism for PTPα regulation.     

An antibody-based method for monitoring in vivo oxidation of protein tyrosine phosphatases

Regulation of protein tyrosine phosphatases (PTPs) through reversible oxidation of the active site cysteine is emerging as a general, yet poorly characterized, mechanism for control of the activity of this important group of enzymes. This regulatory mechanism was initially described after in vitro treatment of PTPs with oxidizing agents. However, accumulating evidence has substantiated the notion that this mechanism is also operating in vivo, e.g., in association with the transient increase in H(2)O(2) production which occurs after activation of receptor tyrosine kinases. A novel generic antibody-based method for monitoring of PTP oxidation is described. The sensitivity of this strategy has been validated by the demonstration of oxidation of endogenously expressed PTPs after stimulation of cells with growth factors. The method was also instrumental in providing the first evidence for intrinsic differences between PTP domains with regard to sensitivity to oxidation.     

Redox regulation of protein tyrosine phosphatase activity by hydroxyl radical

Substantial evidence suggests that transient production of reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) is an important signaling event triggered by the activation of various cell surface receptors. Major targets of H₂O₂ include protein tyrosine phosphatases (PTPs). Oxidation of the active site Cys by H₂O₂ abrogates PTP catalytic activity, thereby potentially furnishing a mechanism to ensure optimal tyrosine phosphorylation in response to a variety of physiological stimuli. Unfortunately, H₂O₂ is poorly reactive in chemical terms and the second order rate constants for the H₂O₂-mediated PTP inactivation are ~ 10 M^− 1 s^− 1, which is too slow to be compatible with the transient signaling events occurring at the physiological concentrations of H₂O₂. We find that hydroxyl radical is produced from H₂O₂ solutions in the absence of metal chelating agent by the Fenton reaction. We show that the hydroxyl radical is capable of inactivating the PTPs and the inactivation is active site directed, through oxidation of the catalytic Cys to sulfenic acid, which can be reduced by low molecular weight thiols. We also show that hydroxyl radical is a kinetically more efficient oxidant than H₂O₂ for inactivating the PTPs. The second-order rate constants for the hydroxyl radical-mediated PTP inactivation are at least 2–3 orders of magnitude higher than those mediated by H₂O₂ under the same conditions. Thus, hydroxyl radical generated in vivo may serve as a more physiologically relevant oxidizing agent for PTP inactivation. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.     

Oxidative inhibition of receptor-type protein-tyrosine phosphatase kappa by ultraviolet irradiation activates epidermal growth factor receptor in human keratinocytes

Ultraviolet (UV) irradiation rapidly increases tyrosine phosphorylation (i.e. activates) of epidermal growth factor receptors (EGFR) in human skin. EGFR-dependent signaling pathways drive increased expression of matrix metalloproteinases, whose actions fragment collagen and elastin fibers, the primary structural protein components in skin connective tissue. Connective tissue fragmentation, which results from chronic exposure to solar UV irradiation, is a major determinant of premature skin aging (photoaging). UV irradiation generates reactive oxygen species, which readily react with conserved cysteine residues in the active site of protein-tyrosine phosphatases (PTP). We report here that EGFR activation by UV irradiation results from oxidative inhibition of receptor type PTP-kappa (RPTP-kappa). RPTP-kappa directly counters intrinsic EGFR tyrosine kinase activity, thereby maintaining EGFR in an inactive state. Reversible, oxidative inactivation of RPTP-kappa activity by UV irradiation shifts the kinase-phosphatase balance in favor of EGFR activation. These data delineate a novel mechanism of EGFR regulation and identify RPTP-kappa as a key molecular target for antioxidant protection against skin aging.     

Development of a modified in-gel assay to identify protein tyrosine phosphatases that are oxidized and inactivated in vivo

Protein tyrosine phosphatases (PTPs) comprise a superfamily of enzymes that control a diverse array of signal transduction pathways. However, the function and regulation of many of these enzymes remain undefined. Previous studies have shown that the optimal tyrosine phosphorylation response to various exogenous stimuli requires the production of reactive oxygen species (ROS). It has been proposed that ROS might transiently inactivate inhibitory PTPs, thus facilitating tyrosine phosphorylation-dependent signaling. Interestingly, the unique chemistry of the invariant, active site Cys residue located in the signature motif renders it highly susceptible to oxidation, leading to the inactivation of PTPs. We have developed a novel strategy to identify those PTPs that are oxidized and therefore, inactivated in response to extracellular stimuli. Iodoacetic acid (IAA) was used to alkylate selectively the thiolate anion of the active site Cys in the reduced PTPs. In contrast, any PTPs in which the active site Cys had been oxidized in response to the stimulus were resistant to alkylation. Following this key step to differentiate between the two pools of PTPs, the oxidized phosphatases were reduced back to the active state during the process of a standard in-gel PTP activity assay. This novel technique revealed, for the first time, that multiple cellular PTPs were indeed oxidized and inactivated in response to exogenous hydrogen peroxide. We have used this technique extensively to show that the ligand-stimulated production of intracellular hydrogen peroxide reversibly regulates the activity of specific PTPs in vivo. By defining the precise PTP targets of intracellular oxidants, the mechanistic details of signal transduction can be delineated. Due to the potential use of this method in finding the molecular targets of intracellular oxidants in diverse signaling pathways, we describe here the theoretical background and the detailed protocols of the modified in-gel PTP assay.     

Global analysis of protein tyrosine phosphatase activity with ultra-sensitive fluorescent probes

Protein tyrosine phosphatases (PTPs) consist of a large family of enzymes known to play important roles in controlling virtually all aspects of cellular processes. However, assigning functional significance of PTPs in normal physiology and in diseases remains a major challenge in cell signaling. Since the function of a PTP is directly associated with its intrinsic activity, which is subject to post-translational regulation, new tools are needed to monitor the dynamic activities of PTPs, rather than mere abundance, on a global scale within the physiologically relevant environment of cells. To meet this objective, we report the synthesis and characterization of two rhodamine-conjugated probes that covalently label the active site of the PTPs in an activity-dependent manner, thus providing a direct readout of PTP activity and superior sensitivity, robustness, and quantifiability to previously reported biotinylated probes. We present evidence that the fluorescent probes can be used to identify new PTP markers and targets for potential diagnosis and treatment of human diseases. We also show that the fluorescent probes are capable of monitoring H(2)O(2)-mediated PTP inactivation, which should facilitate the study of regulated H(2)O(2) production as a new tier of control over tyrosine phosphorylation-dependent signal transduction. The ability to profile the entire PTP family on the basis of changes in their activity is expected to yield new functional insights into pathways regulated by PTPs and contribute to the discovery of PTPs as novel therapeutic targets.     

A one-step method to identify MAP kinase residues involved in inactivation by tyrosine- and dual-specificity protein phosphatases

MAP kinases (MAPKs) are enzymes directly involved in the control of cellular homeostasis in response to external cues, from differentiation and developmental processes to cell transformation. The activation status of MAPKs, both in magnitude and in duration, reflects the balance of phosphorylation at their Thr and Tyr regulatory residues by specific MAPK kinases and their dephosphorylation by inactivating protein serine/threonine phosphatases (PPs) and protein tyrosine phosphatases (PTPs). The dephosphorylation of MAPKs by PTPs relies on molecular docking between the two enzymes at specific interaction sites. Here we outline a one-step method to identify ERK1/2 and p38α mutations that prevent binding and inactivation by PTPs (tyrosine- or dual-specificity phosphatases) based on the use of anti-pTyr antibodies and cell lysis buffers lacking or containing the broad PTP inhibitor sodium orthovanadate (Na₃VO₄).     

Selective regulation of hydrogen peroxide signaling by receptor tyrosine phosphatase-alpha

Reactive oxygen species (ROS) are constantly produced in the human body and are involved in the pathogenesis of aging, cardiovascular diseases, and cancer. Emerging evidence indicates that oxidation and inhibition of protein tyrosine phosphatases (PTPs) are critical for ROS signal transduction. However, the role of individual PTPs in ROS signaling remains unclear. Here, we demonstrated that the receptor-like PTP alpha (RPTP alpha) was an effector of H2O2, the most stable form of ROS. H2O2 at nontoxic concentration rapidly induced the association of RPTP alpha with Src family kinases, platelet-derived growth factor receptor-beta, and protein kinase D in various cultured cells, although it markedly suppressed RPTP alpha phosphorylation on Tyr-789. We further identified that RPTP alpha selectively regulated the signal transduction pathways induced by H2O2. Particularly, RPTP alpha was required for the activation of protein kinase D and for the modulation of p130Cas tyrosine phosphorylation in response to H2O2. In contrast, the H2O2-induced inactivation of Src family kinases and suppression of paxillin phosphorylation on Tyr-118 were both largely independent of RPTP alpha. Our findings indicate that H2O2 signaling pathways are selectively regulated by RPTP alpha in cells, which may provide new insights into the functional regulation of ROS signal transduction by PTPs.     

Receptor-stimulated oxidation of SHP-2 promotes T-cell adhesion through SLP-76-ADAP

Receptor-stimulated generation of intracellular reactive oxygen species (ROS) modulates signal transduction, although the mechanism(s) is unclear. One potential basis is the reversible oxidation of the active site cysteine of protein tyrosine phosphatases (PTPs). Here, we show that activation of the antigen receptor of T cells (TCR), which induces production of ROS, induces transient inactivation of the SH2 domain-containing PTP, SHP-2, but not the homologous SHP-1. SHP-2 is recruited to the LAT-Gads-SLP-76 complex and directly regulates the phosphorylation of key signaling proteins Vav1 and ADAP. Furthermore, the association of ADAP with the adapter SLP-76 is regulated by SHP-2 in a redox-dependent manner. The data indicate that TCR-mediated ROS generation leads to SHP-2 oxidation, which promotes T-cell adhesion through effects on an SLP-76-dependent signaling pathway to integrin activation.     

Inhibition of protein tyrosine phosphatases by amino acid, peptide, and protein hydroperoxides: potential modulation of cell signaling by protein oxidation products

Reaction of radicals in the presence of O2, or singlet oxygen, with some amino acids, peptides, and proteins yields hydroperoxides. These species are key intermediates in chain reactions and protein damage. They can be detected in cells and are poorly removed by enzymatic defenses. Previously we have shown that peptide and protein hydroperoxides react rapidly with thiols, with this resulting in inactivation of some thiol-dependent enzymes. In light of these data, we hypothesized that inactivation of protein tyrosine phosphatases (PTPs), by hydroperoxides present on oxidized proteins, may contribute to cellular and tissue dysfunction by modulation of phosphorylation-dependent cell signaling. We show here that PTPs in cell lysates, and purified PTP-1B, are inactivated by amino acid, peptide, and protein hydroperoxides in a concentration- and structure-dependent manner. Protein hydroperoxides are particularly effective, with inhibition occurring with greater efficacy than with H2O2. Inactivation involves reaction of the hydroperoxide with the conserved active-site Cys residue of the PTPs, as evidenced by hydroperoxide consumption measurements and a diminution of this effect on blocking the Cys residue. This inhibition of PTPs, by oxidized proteins containing hydroperoxide groups, may contribute to cellular dysfunction and altered redox signaling in systems subject to oxidative stress.     

Rapid and irreversible inactivation of protein tyrosine phosphatases PTP1B, CD45, and LAR by peroxynitrite.

Protein tyrosine phosphatases (PTPs) contain an essential thiol in the active site which may be susceptible to attack by nitric oxide-derived biological oxidants. We assessed the effects of peroxynitrite, nitric oxide, and S-nitrosoglutathione on the activity of three human tyrosine phosphatases in vitro. The receptor-like T-cell tyrosine phosphatase (CD45), the non-receptor-like tyrosine phosphatase PTP1B, and leukocyte-antigen-related (LAR) phosphatase were all irreversibly inactivated by peroxynitrite in less than 1 s with IC(50) values of 相似文献   

Redox regulation of MAP kinase phosphatase 3

MAP kinase phosphatase 3 (MKP3) is a protein tyrosine phosphatase (PTP) for which in vivo evidence suggests that regulation can occur by oxidation and/or reduction of the active site cysteine. Using kinetics and mass spectrometry, we have probed the biochemical details of oxidation of the active site cysteine in MKP3, with particular focus on the mechanism of protection from irreversible inactivation to the sulfinic or sulfonic acid species. Like other PTPs, MKP3 was found to be rapidly and reversibly inactivated by mild treatment with hydrogen peroxide. We demonstrate that unlike the case for some PTPs, the sulfenic acid of the active site cysteine in MKP3 is not stabilized in the active site but instead is rapidly trapped in a re-reducible form. Unlike the case for other PTPs, the sulfenic acid in MKP3 does not form a sulfenyl-amide species with its neighboring residue or a disulfide with a single proximate cysteine. Instead, multiple cysteines distributed in both the N-terminal substrate-binding domain (Cys147 in particular) and the C-terminal catalytic domain (Cys218) are capable of rapidly and efficiently trapping the sulfenic acid as a disulfide. Our results extend the diversity of mechanisms utilized by PTPs to prevent irreversible oxidation of their active sites and expand the role of the N-terminal substrate recognition domain in MKP3 to include redox regulation.     

LAR protein tyrosine phosphatase receptor associates with TrkB and modulates neurotrophic signaling pathways

The identities of receptor protein tyrosine phosphatases (PTPs) that associate with Trk protein tyrosine kinase (PTK) receptors and modulate neurotrophic signaling are unknown. The leukocyte common antigen-related (LAR) receptor PTP is present in neurons expressing TrkB, and like TrkB is associated with caveolae and regulates survival and neurite outgrowth. We tested the hypothesis that LAR associates with TrkB and regulates neurotrophic signaling in embryonic hippocampal neurons. Coimmunoprecipitation and coimmunostaining demonstrated LAR interaction with TrkB that is increased by BDNF exposure. BDNF neurotrophic activity was reduced in LAR-/- and LAR siRNA-treated LAR+/+ neurons and was augmented in LAR-transfected neurons. In LAR-/- neurons, BDNF-induced activation of TrkB, Shc, AKT, ERK, and CREB was significantly decreased; while in LAR-transfected neurons, BDNF-induced CREB activation was augmented. Similarly, LAR+/+ neurons treated with LAR siRNA demonstrated decreased activation of Trk and AKT. LAR is known to activate the Src PTK by dephosphorylation of its negative regulatory domain and Src transactivates Trk. In LAR-/- neurons, or neurons treated with LAR siRNA, phosphorylation of the Src regulatory domain was increased (indicating Src inactivation), consistent with a role for Src in mediating LAR's ability to up-regulate neurotrophic signaling. Interactions between LAR, TrkB, and Src were further confirmed by the findings that Src coimmunoprecipitated with LAR, that the Src inhibitor PP2 blocked the ability of LAR to augment TrkB signaling, and that siRNA-induced depletion of Src decreased LAR interaction with TrkB. These studies demonstrate that receptor PTPs can associate with Trk complexes and promote neurotrophic signaling and point to receptor PTP-based strategies as a novel approach for modulating neurotrophin function.