Nitrotyrosine mimics phosphotyrosine binding to the SH2 domain of the src family tyrosine kinase lyn

The nitration of tyrosine residues in protein occurs through the action of reactive oxygen and nitrogen species and is considered a marker of oxidative stress under pathological conditions. The most active nitrating species so far identified is peroxynitrite, the product of the reaction between nitric oxide and superoxide anion. Previously, we have reported that in erythrocytes peroxynitrite irreversibly upregulates lyn, a tyrosine kinase of the src family. In this study we investigated the possible role of tyrosine nitration in the mechanism of lyn activation. We found that tyrosine containing peptides modelled either on the C-terminal tail of src kinases or corresponding to the first 15 amino acids of human erythrocyte band 3 were able to activate lyn when the tyrosine was substituted with 3-nitrotyrosine. The activity of nitrated peptides was shared with phosphorylated but not with unphosphorylated, chlorinated or scrambled peptides. Recombinant lyn src homology 2 (SH2) domain blocked the capacity of the band 3-derived nitrotyrosine peptide to activate lyn and we demonstrated that this peptide specifically binds the SH2 domain of lyn. We propose that nitropeptides may activate src kinases through the displacement of the phosphotyrosine in the tail from its binding site in the SH2 domain. These observations suggest a new mechanism of peroxynitrite-mediated signalling that may be correlated with the upregulation of tyrosine phosphorylation observed in several pathological conditions.     

Activation of src tyrosine kinases by peroxynitrite.

In this study, we demonstrate that the phosphorylation activity of five tyrosine kinases of the src family from both human erythrocytes (lyn, hck and c-fgr) and bovine synaptosomes (lyn and fyn) was stimulated by treatment with 30-250 microM peroxynitrite. This effect was not observed with syk, a non-src family tyrosine kinase. Treatment of kinase immunoprecipitates with 0.01-10 microM peroxynitrite showed that the interaction of these enzymes with the oxidant also activated the src kinases. Higher concentrations of peroxynitrite inhibited the activity of all kinases, indicating enzyme inactivation. The addition of bicarbonate (1.3 mM CO2) did not modify the upregulation of src kinases but significantly protected the kinases against peroxynitrite-mediated inhibition. Upregulation of src kinase activity by 1 microM peroxynitrite was 3.5-5-fold in erythrocytes and 1.2-2-fold in synaptosomes, but this could be the result, at least in part, of the higher basal level of src kinase activity in synaptosomes. Our results indicate that peroxynitrite can upregulate the tyrosine phosphorylation signal through the activation of src kinases.     

Peroxynitrite modulates the activation of p38 and extracellular regulated kinases in PC12 cells

Although peroxynitrite appears to contribute to neuronal dysfunction in several neurodegenerative disorders, little is known about how peroxynitrite affects cellular signaling processes. This study investigated if peroxynitrite affects the mitogen-activated protein kinases, extracellular-regulated kinases 1 and 2 (ERK1/2) and p38. Exposure of PC12 cells to 500 microM peroxynitrite activated ERK1/2 and p38 within 5 min and this was followed by gradual decreases in activation over the next 25 min. Activation of ERK1/2 by peroxynitrite was mediated by activation of the epidermal growth factor (EGF) receptor in a calcium/calmodulin-dependent kinase II- and src family tyrosine kinase-dependent manner, as it was blocked by the selective EGF receptor inhibitor AG1478, by KN62, an inhibitor of calcium/calmodulin-dependent kinase II, and by PP1, a src family tyrosine kinase inhibitor. Activation of p38 by peroxynitrite was independent of the EGF receptor, required activation of calcium/calmodulin-dependent kinase II and src family tyrosine kinases, and was modulated by nerve growth factor (NGF) in a time-dependent manner. Pretreatment with NGF (2 h) attenuated, whereas cotreatment with NGF potentiated, peroxynitrite-induced activation of p38. Thus, peroxynitrite activates ERK1/2 and p38, activation of EGF receptors, calcium/calmodulin-dependent kinase II, and src family tyrosine kinases participate in these signaling responses to peroxynitrite, and peroxynitrite- and NGF-induced signaling activities converge on p38.     

Peroxynitrite-dependent activation of src tyrosine kinases lyn and hck in erythrocytes is under mechanistically different pathways of redox control

Peroxynitrite, the product of superoxide and nitric oxide radicals, is considered one of the major oxidants formed in vivo under intense oxidative stress. We have previously reported the upregulation by peroxynitrite of src kinase activity in red blood cells. In this study, we investigated the mechanisms of peroxynitrite action and we demonstrate that two src kinases (lyn and hck) are activated through different pathways involving cysteine-dependent or -independent oxidations. Activation of hck by peroxynitrite or by hydrogen peroxide could be explained by reversible SH redox changes, whereas lyn was unaffected by hydrogen peroxide and its direct activation by peroxynitrite occurred through a still unknown modification(s) not reverted by SH reduction or inhibited by SH alkylation. Moreover, lyn could be activated also downstream by peroxynitrite-activated hck. The cross talk between lyn and hck was selective, since activated hck did not activate the non-src kinase syk. This study illustrates the complexity of redox-dependent src regulation and suggests that one reason for src heterogeneity may be a peculiar difference in their sensitivity to physiological oxidants. Irrespectively of the activation pathway, the final effect of peroxynitrite is the amplification of tyrosine-dependent signaling, a finding of general interest in nitric oxide-related pathophysiology.     

Peroxynitrite Induces Tyrosine Nitration and Modulates Tyrosine Phosphorylation of Synaptic Proteins

Peroxynitrite, the product of the radical-radical reaction between nitric oxide and superoxide anion, is a potent oxidant involved in tissue damage in neurodegenerative disorders. We investigated the modifications induced by peroxynitrite in tyrosine residues of proteins from synaptosomes. Peroxynitrite treatment (> or =50 microM) induced tyrosine nitration and increased tyrosine phosphorylation. Synaptophysin was identified as one of the major nitrated proteins and pp60src kinase as one of the major phosphorylated substrates. Further fractionation of synaptosomes revealed nitrated synaptophysin in the synaptic vesicles, whereas phosphorylated pp60src was enriched in the postsynaptic density fraction. Tyrosine phosphorylation was increased by treatment with 50-500 microM peroxynitrite and decreased by higher concentrations, suggesting a possible activation/inactivation of kinases. Immunocomplex kinase assay proved that peroxynitrite treatment of synaptosomes modulated the pp60src autophosphorylation activity. The addition of bicarbonate (CO2 1.3 mM) produced a moderate enhancing effect on some nitrated proteins but significantly protected the activity of pp60src against peroxynitrite-mediated inhibition so that at 1 mM peroxynitrite, the kinase was still more active than in untreated synaptosomes. The phosphotyrosine phosphatase activity of synaptosomes was inhibited by peroxynitrite (> or =50 microM) but significantly protected by CO2. Thus, the increase of phosphorylation cannot be attributed to peroxynitrite-mediated inhibition of phosphatases. We suggest that peroxynitrite may regulate the posttranslational modification of tyrosine residues in pre- and postsynaptic proteins. Identification of the major protein targets gives insight into the pathways possibly involved in neuronal degeneration associated with peroxynitrite overproduction.     

Differential effects of quercetin and resveratrol on Band 3 tyrosine phosphorylation signalling of red blood cells

The protective effects of eating fruits and vegetables in the prevention of several degenerative pathologies have been attributed at least in part to the antioxidant and anti-inflammatory properties of polyphenols. In this study, we investigated the effects of two polyphenols, quercetin and resveratrol, on red blood cell Band 3 tyrosine phosphorylation signalling activated by peroxynitrite. Peroxynitrite is a physiological oxidant scavenged largely by the erythrocyte and formed by the reaction between nitrogen monoxide and superoxide anion. Quercetin and its structurally analogous (+)-catechin inhibited the peroxynitrite-dependent upregulation of Band 3 tyrosine phosphorylation. Quercetin was found to downregulate the activity of syk, which is upstream in the Band 3 tyrosine phosphorylation cascade, and partially prevented peroxynitrite-mediated phosphotyrosine phosphatase inhibition. Resveratrol and hydroxytyrosol, unexpectedly, amplified peroxynitrite-dependent upregulation of Band 3 tyrosine phosphorylation through the activation of lyn, a kinase of the src family. The present results clearly indicate that polyphenols may activate cell transduction pathways in different and sometimes opposite ways.     

Differential association of cellular proteins with family protein-tyrosine kinases

We have sought to identify candidate substrates for src family protein-tyrosine kinases potentially important for transformation. Transfected NIH/3T3 cells, each overexpressing a normal or activated version of the fyn, fgr, or src translational product, were examined using antibody to phosphotyrosine as a probe. Expression of each cDNA induced similar but distinct patterns of tyrosine phosphorylated cellular proteins, with the extent of phosphorylation being greatest in cells expressing an activated kinase. A 70-kDa tyrosine-phosphorylated protein was found to associate with the activated fyn gene product. A protein designated p130, tyrosine phosphorylated in vitro, and in vivo, was found to physically associate with the activated product of each src family gene examined. Physical interaction of three different highly transforming tyrosine kinases with a common cellular protein suggests that p130 may play an important role in transformation induced by src family kinases.     

Platelet-derived growth factor receptor sequences important for binding of src family tyrosine kinases.

We previously demonstrated that src family tyrosine kinases associate with activated platelet-derived growth factor receptors. We have now investigated the requirement for tyrosine phosphorylation sites in the human platelet-derived growth factor receptor for this binding. Tyrosine 857, but not tyrosine 751, is required for efficient association. Furthermore, even though src family tyrosine kinases associate with receptors lacking tyrosine 751, they are not activated, implying that association does not invariably lead to activation.     

CSK: a protein-tyrosine kinase involved in regulation of src family kinases.

The functions of src family protein-tyrosine kinases are thought to be regulated negatively by the phosphorylation of highly conserved tyrosine residues close to their carboxyl termini. Recently we have purified and cloned a protein-tyrosine kinase (designated as CSK) that can specifically phosphorylate the negative regulatory site of p60c-src. To elucidate the relationship between CSK and other types of src family kinases, we investigated the tissue distribution of CSK and examined whether CSK could phosphorylate the negative regulatory sites of src family kinases other than p60c-src. Western blot analysis indicated that CSK was enriched at the highest level in lymphoid tissues in which the expression of p60c-src is considerably lower than those of other types of src family kinases. CSK phosphorylated p56lyn and p59fyn, which are known to be expressed in lymphoid tissues at a relatively high level. The putative regulatory site, tyrosine 508, was found to be essential for phosphorylation in p56lyn, and the kinase activities of these src family kinases were repressed by phosphorylation with CSK. These findings raise the possibility that CSK might act as a universal regulator for src family kinases.     

A Reactive Oxygen Species-Mediated,Self-Perpetuating Loop Persistently Activates Platelet-Derived Growth Factor Receptor α

The platelet-derived growth factor (PDGF) receptors (PDGFRs) are central to a spectrum of human diseases. When PDGFRs are activated by PDGF, reactive oxygen species (ROS) and Src family kinases (SFKs) act downstream of PDGFRs to enhance PDGF-mediated tyrosine phosphorylation of various signaling intermediates. In contrast to these firmly established principles of signal transduction, much less is known regarding the recently appreciated ability of ROS and SFKs to indirectly and chronically activate monomeric PDGF receptor α (PDGFRα) in the setting of a blinding condition called proliferative vitreoretinopathy (PVR). In this context, we made a series of discoveries that substantially expands our appreciation of epigenetic-based mechanisms to chronically activate PDGFRα. Vitreous, which contains growth factors outside the PDGF family but little or no PDGFs, promoted formation of a unique SFK-PDGFRα complex that was dependent on SFK-mediated phosphorylation of PDGFRα and activated the receptor''s kinase activity. While vitreous engaged a total of five receptor tyrosine kinases, PDGFRα was the only one that was activated persistently (at least 16 h). Prolonged activation of PDGFRα involved mTOR-mediated inhibition of autophagy and accumulation of mitochondrial ROS. These findings reveal that growth factor-containing biological fluids, such as vitreous, are able to tirelessly activate PDGFRα by engaging a ROS-mediated, self-perpetuating loop.     

Peroxynitrite reactivity with amino acids and proteins

Summary. Peroxynitrite, the product of the fast reaction between nitric oxide (NO) and superoxide O₂ ^– radicals, is an oxidizing and nitrating agent which is able to traverse biological membranes. The reaction of peroxynitrite with proteins occurs through three possible pathways. First, peroxynitrite reacts directly with cysteine, methionine and tryptophan residues. Second, peroxynitrite reacts fast with transition metal centers and selenium-containing amino acids. Third, secondary free radicals arising from peroxynitrite homolysis such as hydroxyl and nitrogen dioxide, and the carbonate radical formed in the presence of carbon dioxide, react with protein moieties too. Nitration of tyrosine residues is being recognized as a marker of the contribution of nitric oxide to oxidative damage. Peroxynitrite-dependent tyrosine nitration is likely to occur through the initial reaction of peroxynitrite with carbon dioxide or metal centers leading to secondary nitrating species. The preferential protein targets of peroxynitrite and the role of proteins in peroxynitrite detoxifying pathways are discussed.     

Tau and src family tyrosine kinases

The interaction between tau and src family non-receptor tyrosine kinases represents a new function for tau. Mediated by the proline-rich region of tau and the SH3 domain of fyn or src, this interaction has the potential to confer novel cellular activities for tau in the growth cone and in the membrane. The subsequent finding that tau is tyrosine phosphorylated has led to the observation that tau in neurofibrillary tangles is tyrosine phosphorylated. Therefore, a role for tyrosine kinases such as fyn in neuropathogenesis is predicted.     

Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation.     

Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1.

src family tyrosine kinases contain two noncatalytic domains termed src homology 3 (SH3) and SH2 domains. Although several other signal transduction molecules also contain tandemly occurring SH3 and SH2 domains, the function of these closely spaced domains is not well understood. To identify the role of the SH3 domains of src family tyrosine kinases, we sought to identify proteins that interacted with this domain. By using the yeast two-hybrid system, we identified p62, a tyrosine-phosphorylated protein that associates with p21ras GTPase-activating protein, as a src family kinase SH3-domain-binding protein. Reconstitution of complexes containing p62 and the src family kinase p59fyn in HeLa cells demonstrated that complex formation resulted in tyrosine phosphorylation of p62 and was mediated by both the SH3 and SH2 domains of p59fyn. The phosphorylation of p62 by p59fyn required an intact SH3 domain, demonstrating that one function of the src family kinase SH3 domains is to bind and present certain substrates to the kinase. As p62 contains at least five SH3-domain-binding motifs and multiple tyrosine phosphorylation sites, p62 may interact with other signalling molecules via SH3 and SH2 domain interactions. Here we show that the SH3 and/or SH2 domains of the signalling proteins Grb2 and phospholipase C gamma-1 can interact with p62 both in vitro and in vivo. Thus, we propose that one function of the tandemly occurring SH3 and SH2 domains of src family kinases is to bind p62, a multifunctional SH3 and SH2 domain adapter protein, linking src family kinases to downstream effector and regulatory molecules.     

Tec/Bmx non-receptor tyrosine kinases are involved in regulation of Rho and serum response factor by Galpha12/13.

A transient transfection system was used to identify regulators and effectors for Tec and Bmx, members of the Tec non-receptor tyrosine kinase family. We found that Tec and Bmx activate serum response factor (SRF), in synergy with constitutively active alpha subunits of the G12 family of GTP-binding proteins, in transiently transfected NIH 3T3 cells. The SRF activation is sensitive to C3, suggesting the involvement of Rho. The kinase and Tec homology (TH) domains of the kinases are required for SRF activation. In addition, kinase-deficient mutants of Bmx are able to inhibit Galpha13- and Galpha12-induced SRF activation, and to suppress thrombin-induced SRF activation in cells lacking Galphaq/11, where thrombin's effect is mediated by G12/13 proteins. Moreover, expression of Galpha12 and Galpha13 stimulates autophosphorylation and transphosphorylation activities of Tec. Thus, the evidence indicates that Tec kinases are involved in Galpha12/13-induced, Rho-mediated activation of SRF. Furthermore, Src, which was previously shown to activate kinase activities of Tec kinases, activates SRF predominantly in Rho-independent pathways in 3T3 cells, as shown by the fact that C3 did not block Src-mediated SRF activation. However, the Rho-dependent pathway becomes significant when Tec is overexpressed.     

Clinical Targeting of Mutated and Wild-Type Protein Tyrosine Kinases in Cancer

Clinical therapies for cancer have evolved from toxic, nontargeted agents to manageable, highly targeted therapies. Protein tyrosine kinases are a family of signaling molecules implicated in nearly every cancer type and are the foundation for the development of modern targeted agents. Recent genomic analyses have identified activating mutations, translocations, and amplifications of tyrosine kinases. Selective targeting of these genetically altered tyrosine kinases has resulted in significant clinical advances, including increased patient survival. This indicates that altered protein tyrosine kinases are the main drivers of many different cancers. However, lost during analyses of genetic lesions are the contributions of activated, wild-type kinases on tumor-dependent pathways. New approaches in phosphoproteomic technologies have identified several wild-type tyrosine kinase activation states, suggesting that non-genetically altered kinases can be essential “nodes” for signal transduction. Here, we summarize the evidence supporting the common mechanisms of protein tyrosine kinase activation in cancer and provide a personal perspective on the kinases BCR-ABL and BTK, as well as nonmutated kinase targets in prostate cancer, through our work. We outline the mechanisms of tyrosine kinase activation in the absence of direct mutation and discuss whether non-genetically altered tyrosine kinases or their associated downstream signaling pathways can be effectively targeted.