Front. Biol. 2011, 6(2): 156169 DOI 10.

1007/s11515-011-1112-z

REVIEW

Kinases and glutathione transferases: selective and sensitive targeting

Yasemin G. ISGOR, Belgin S. ISGOR (

Chemistry Group, Faculty of Engineering, Atilim University, Ankara 06836, Turkey

Higher Education Press and Springer-Verlag Berlin Heidelberg 2011

Abstract Kinases, representing almost 500 proteins in the human genome, are responsible for catalyzing the phosphorylation reaction of amino acid residues at their targets. As the largest family of kinases, the protein tyrosine kinases (PTKs) have roles in controlling the essential cellular activities, and their deregulation is generally related to pathologic conditions. The recent efforts on identifying their signal transducer or mediator role in cellular signaling revealed the interaction of PTKs with numerous enzymes of different classes, such as Ser/Thr kinases (STKs), glutathione transferases (GSTs), and receptor tyrosine kinases (RTKs). In either regulation or enhancing the signaling, PTKs are determined in close interaction with these enzymes, under specic cellular conditions, such as oxidative stress and inammation. In this concept, intensive research on thiol metabolizing enzymes recently showed their involvement in the physiologic functions in cellular signaling besides their well known traditional role in antioxidant defense. The shared signaling components between PTK and GST family enzymes will be discussed in depth in this research review to evaluate the results of recent studies important in drug targeting for therapeutic intervention, such as cell viability, migration, differentiation and proliferation. Keywords glutathione transferase, protein tyrosine kinase, small molecule inhibitors, c-Src, signal transduction, drug targeting

Introduction
Kinases, representing almost 500 proteins in the human genome, are responsible for catalyzing the phosphorylation reaction of amino acid residues at their targets (Manning et al., 2002). Intensive research in the kinome eld have shown that almost 30% of the whole cellular eukaryotic proteins are generally phosphorylated through the serine (Ser, S), threonine (Thr, T) and tyrosine (Tyr, Y) residues, and hence, they are grouped as protein serine/threonine kinases (STKs), protein tyrosine kinases (PTKs), and dual-specicity kinases (DSK). The latter phosphorylate Tyr in addition to Ser/Thr residues (Hunter and Cooper, 1985; Edelman et al., 1987; Lindberg et al., 1992). Of the 518 genes in the kinome, 95 were identied as expressing PTKs where ve of them are classied as tyrosine kinase pseudo-genes. Among these, 58 enzymes are determined as members of receptor tyrosine

Received November 12, 2010; accepted December 13, 2010 Correspondence: Belgin S. ISGOR E-mail: bisgor@atilim.edu.tr

kinase family (RTK) which is composed of 20 subfamilies with respect to kinase domain sequence. The remaining PTKs are classied as non-receptor tyrosine kinases (NRTKs) with 10 subfamilies with homologous kinase domains in each. PTKs may represent approximately 17% of all kinases in humans, but they were detected in most of the cancers as they constitute more than 60% of oncogenes and proto-oncogenes (Waldmann and Levitzki, 2001). Moreover, four decays of research in the eld were shown that most of the human diseases were associated with the deregulation of PTKs. Considering that the phosphorylated protein can adapt a change in the subcellular location, intrinsic biological activity, protein half-life, as well as its binding ability with other proteins, kinase action is a critical way for signal transduction (Cohen, 2000). It is, therefore, not surprising that more than two dozens of tyrosine kinases from different classes are evaluated as potential targets in cancer therapy, as well as in numerous other diseases, such as osteoporosis, arthritis, psoriasis, and stroke (Thomas and Brugge, 1997; Ben-Bassat and Klein, 2000; Rucci et al., 2008; Vosler and Chen, 2009; Cohen and Fleischmann, 2010; Giamas et al., 2010). Among these enzymes, it was discovered that Src

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tyrosine kinase (c-Src) is a central mediator in transduction of extracellular signals through cell membrane to cytoplasm, and most of the time to the nucleus where gene expression may be modied. It is a prototype member of the Src family kinases (SFKs) which is the largest family of NRTK group, and the rst proto-oncogene product detected with intrinsic protein kinase activity. Since its discovery, the intensive research in the eld revealed c-Src involvement in controlling the essential cellular activities through diverse signaling pathways with respect to other kinases. Being ubiquitously expressed in all bodily tissues, the deregulation in its activity and especially its persistent activation, is therefore attributed for the development and progress of numerous diseases including cancer (Alvarez et al., 2006). To date, c-Src is associated with several biological targets including downstream components of RTK and integrins. It is also shown that c-Src directly interacts with RTKs including epidermal growth factor and platelet-derived growth factor receptors (PDGFR), as well as with kinases including focal adhesion kinase (FAK) and mitogen-activated protein kinases (MAPKs) such as c-Jun N-terminal kinase (JNK). Although the mechanism is not claried yet, c-Src and other members of SFKs are also shown to participate in reactive oxygen species (ROS)-mediated signaling (Chiarugi et al., 2003; Giannoni et al., 2005; Kemble and Sun, 2009; Pani et al., 2009). Very recently, another family of enzymes was reported to directly interact with epidermal growth factor receptor (EGFR) and JNK and found closely related to c-Src associated signaling. These enzymes are the glutathione transferases (GSTs) and participate in the detoxication of a wide array of compounds such as carcinogens, drugs, medicinal plant extracts, pesticides, herbicides, and oxidative stress products (Eaton and Bammler, 1999; Hsu et al., 2002; Di Pietro et al., 2010). As part of the phase II detoxication system (Hayes and Pulford, 1995), GSTs are also shown to provide cellular protection against toxic effects of endogenously produced ROS (Gulick and Fahl, 1995). Besides the deregulations in kinase activity, the increased activity of GSTs has been also associated with human cancers. Additionally, the anticancer drug resistance was attributed to the presence of overactive GST isozymes, such as GST-P. Therefore, to develop therapeutics with full biological response, it is critical to evaluate c-Src mediated signaling mechanisms and its possible interaction with drug metabolizing enzymes, such as glutathione transferases. In this context, we currently evaluate the inhibitors that act on GST, SFK and RTK members to identify molecules with multifunctional targeting ability to provide reduced drug resistance with enhanced therapeutic effectiveness (Kilic et al., 2009; Aydn et al., 2010; Dincer et al., 2010; Isgor et al., 2010b; Kilic-Kurt et al., 2010). With the aim of our current efforts in the eld, this article will review the recent advances in our understanding of c-Src and GST involved signaling mechanisms, and their importance in selective and sensitive enzyme targeting to facilitate drug development efforts for therapeutic intervention.

Physiological activation and function of c-Src tyrosine kinase The comprehensive review on the central role of c-Src in many cellular activities was published before (Thomas and Brugge, 1997), and recently it was studied in more detail, to reveal the role of c-Src mediated signaling in pathways related to cancer and inammation (Lu et al., 2003; Ingley, 2008; Rucci et al., 2008; Ricono et al., 2009). Either overactivated or highly expressed, c-Src was found capable of interacting with various proteins as its downstream targets. This interaction was found slightly transforming, and hence, was weakly associated with cancer development. However, the persistent catalytic activity with overexpression was detected with the advanced cancers of different type and highly associated with malignant transformation (Frame, 2002). The functional domains of c-Src (Fig. 1A) involved in catalytic activities affecting cell functionality are: 1) the catalytic SH1 (Src homology-1) domain, the most conserved domain in all tyrosine kinases containing the ATP binding pocket; 2) the SH2 domain: involved in phosphotyrosinemediated proteinprotein interactions and interacting with negative regulatory Tyr530 residue; 3) the SH3 domain, involved in proline-rich sequence mediated proteinprotein interactions; and 4) SH4 domain, containing myristoylation site (Cowan-Jacob et al., 2005). c-Src has COOH-terminal tail (C-tail) containing negative regulatory Tyr530 and NH2-terminal membrane-association domain (N-tail). Both are identied in regulation of subcellular localization and kinase activity (Fig. 1B), however, they are not dened as functional domains. N-tail is the most divergent region among SFKs attributing unique intermolecular interaction property to each member (Cowan-Jacob et al., 2005; Ingley, 2008). Being c-Src is the prototype, SFKs which is the largest family of nonreceptor PTKs, participate in many cellular activities including differentiation, adhesion, migration, cytoskeletal alterations, and proliferation. For that reason, the enzyme is tightly controlled by positive and negative regulations in a cell. c-Src is positively regulated through autophosphorylation of Tyr419, which is also indispensable for kinase activity. The negative regulation, on the other hand, involves Tyr530 residue and it arises from the intramolecular interaction between phosphorylated Tyr530 (pTyr530) on C-tail and SH2 domain resulting in somehow a closed, and therefore, an inactive conformation (Fig. 1C). Dephosphorylation of this residue displaces SH2 domain from C-tail and protein resumes the open and so the active conformation (Fig. 1D). This conguration exposes the catalytic domain, SH1, for ATP binding, and also exposes SH2 and SH3 domains to associate with other signaling molecules (Bjorge et al., 2000; Frame, 2002; Martin, 2004). Upon activation, the enzyme relocation occurs from cell periphery to membrane where it attaches through myristoylated SH4 domain, and, in turn, becomes involved in ligand-activated signaling processes. In addition to a variety of proteins, this process

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generally involves the direct interaction with RTKs, such as EGFR, broblast growth factor (FGFR), hepatocyte growth factor (HGFR), and PDGFR, or the indirect interaction through downstream components of related RTKs (Tice et al., 1999; Ha et al., 2008; Huveneers and Danen, 2009). Structural and functional properties of glutathione transferases GSTs generally participate in the binding, transport, and detoxication of a wide array of endogenous and exogenous compounds such as carcinogens, drugs, medicinal plant extracts, pesticides, herbicides, and oxidative stress products (Igarashi et al., 1986; Manal et al., 2007; Grifth et al., 2010). They are characterized as multi-functional proteins having ubiquitous expression prole with varying subcellular levels, and therefore classied as: 1) the microsomal GSTs which is also known as the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG); 2) the cytosolic or soluble GSTs, and 3) the mitochondrial GSTs. Of those, with 18 members, the cytosolic GSTs constitute the largest group of all three families. Most of the subcellular activities in cytotoxic defense are attributed to the members of cytosolic GSTs, by providing cellular protection against antioxidant stress. Although the presence of GSTs was rst demonstrated

in rat tissues, it is now known that at least seven classes of enzyme subfamilies (isoforms or isozymes) are present in almost all mammalian tissues. These subfamilies are formed with respect to their structural homology, substrate specicity, and immunological cross-reactivity. In this context, the human cytosolic GSTs are classied as GST alpha (A), mu (M), pi (P), sigma (S), theta (T), omega (O) and zeta (Z) (Hayes et al., 2005). Their roles in disease progression are mostly attributed in the development of asthma, cardiovascular diseases and cancer. Among all cytosolic GSTs, GST A, M and P are found as the most multifunctional enzymes acting as GSH transferases, peroxidases, isomerases, and signaling proteins. Independent of tissue distribution and subcellular localization, all isozymes use reduced glutathione (GSH) as an acceptor species, but they differ in the specicity of substrates to be transferred to the sulfhydryl moiety (cysteine thiol) of GSH. Despite the fact that the GSTs are highly conserved among the diverse array of species and throughout the evolution, frequently these enzymes show higher degree of polymorphism and generally they contain several subunits. But in each subunit there is a GSH binding site (G-site) in the N-terminal domain which is also catalytically independent active site, and a hydrophobic substrate (H-site) binding site located in the C-terminal domain (Fig. 2). The majority of functional GSTs are shown

Figure 1 The ribbon diagram for the crystal structure of human c-Src complexed with anticancer drug imatinib (A) is adapted from Protein DataBank Accession No: 1Y57. The linear representation of functional domains of c-Src with opposing regulatory Tyr(Y) residues is shown (B). The inactive (C) and active (D) conformational change is illustrated. The cartoon also shows the membrane attachment through N-myristoylated tail (C and D).

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to form homo- or heterodimers, and the latter results in isoforms with signicantly larger sizes. The homodimer GST isozymes present in human is given in Fig. 3 where the ribbon diagrams for the crystal structures were adapted from Protein DataBank with accession numbers 1PKW for GST A1-1; 11GS for GST P1-1; 1YJ6 for GST M1-1; 2C3N for GST T11; 1YZX for GST K1-1; 1EEM for GST O1-1; 1FW1 for GST Z1-1; 2H8A for MGST-1 (Oakley et al., 1997; Board et al., 2000; Polekhina et al., 2001; Li et al., 2005; Grahn et al., 2006; Holm et al., 2006; Tars et al., 2006; Patskovsky et al., 2009).

the action on xenobiotic metabolism, in addition to signaling mechanisms in tissue specic manner. GST Z isozyme was identied previously in the catabolism of phenylalanine and tyrosine amino acid residues, and also for glyoxalate formation. For xenobiotic-mediated induction of GSTs, different but synchronized mechanisms become active at expression level for distinct members of cytosolic GSTs, especially upon exposure to polycyclic aromatic hydrocarbons (PAH), isothiocyanates, aromatic or phenolic antioxidants, and ROS. In response to various extracellular stimuli, the GST expression is regulated by transcription factors, either at transcriptional or posttranscriptional levels. For GST A isoforms, the xenobiotic response element (XRE) and antioxidant response element (ARE) gene sequences are found functional at transcriptional level upon exposure to xenobiotics. For GST P isoforms, functional AP-1 (activating protein-1) was dened as response elements, and NFB response element was dened mediator for induction upon antioxidant exposure. On the other hand, regulation of GST M and T isoforms at gene expression level is not fully claried yet.

GST and c-Src involved signaling pathways

The efforts in the eld of developing novel druggable targets, eventually, were facilitated through the studies to identify the targets as signaling components involved in the processes for disease development and progress. Moreover, the off-target enzymes, some of which are detoxication or defense components, were also analyzed for possible participation in the process for drug resistance. The results of these analyses were evaluated together to develop multifunctional therapeutics for various diseases. In this perspective, the enzymes containing and utilizing thiols, as part of detoxication system, are shown to have a well established role in cell defense against prooxidant damage through catalyzing reactions to neutralize and conjugate ROS and free radicals. Lately it was shown that these enzymes can also mediate physiological functions by virtue of direct interaction of thiol moiety in their structure with free radicals and other oxidants (Townsend et al., 2008; Singh et al., 2010), and such interaction was also related with kinase mediated signaling (Lo et al., 2004; Okamura et al., 2009a; Okamura et al., 2009b). These reports in the current literature bring the possibility that we may still have primitive understanding of the physiological functions of kinase and detoxication system enzymes on the use of substrates, effectors and regulators under specic conditions. In this perspective, although, only few studies have been published to date, the earlier and recent results point out that possibly these enzymes work in a concerted way to mediate cellular events including protein synthesis and folding, signal transduction, and cell proliferation, through the interaction with down-

Figure 2 The ribbon diagram for crystal structure of the human GST A4 monomer (single subunit) representing secondary (hydrophobic) substrate and GSH binding sites. Adapted from Protein DataBank, accession number: 1GUL.

Among these, GST A is expressed in many tissues and especially used as diagnostics plasma marker to detect liver damage under pathological conditions. GST A mostly exhibits GSH peroxidase activity in lipid metabolism, such as conjugation of fatty acids and isomerism of steroids, important in stress signaling. GST M is highly abundant in skeletal and cardiac muscles and identied in Ca2+ channel regulation important for cardiac and muscle pathology. The more detailed information on GST function in drug resistance and cancer development was provided with the recent review of Di Pietro et al. (2010). GST P is ubiquitously expressed in bodily tissues and identied in carcinogenesis, aging and neurodegeneration; and regulation in kinase signaling. Moreover, it is known to act in antitumor drug resistance mechanisms. GST P and M was also detected in the brain region called black substance (substantia nigra) and detected in human brain tumors (Baez et al., 1997; Smeyne et al., 2007). GST S is known to be involved in prostaglandin synthesis, in addition to xenobiotic conjugation with GSH. GST T is newly identied in a mammalian tissue in an active form and, so far, identied with

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Figure 3 The ribbon diagrams for the crystal structures of human GSTs complexed with GSH and inhibitors. Except for MGST-1, all isoforms shown here are homodimers. The representations were adapted from Protein DataBank with accession numbers: 1PKW (GST A1-1), 11GS (GST P1-1), 1YJ6 (GST M1-1), 2C3N (GST T1-1), 1YZX (GST K1-1), 1EEM (GST O1-1), 1FW1 (GST Z1-1), and 2H8A (MGST-1).

stream proteins in signaling cascades. Here, we provide the information recently published on commonly shared signaling components between glutathione transferases and c-Src as central signaling component, to our understanding of their concerted possible roles in disease progress and in therapeutic targeting, especially with conditions related to various cancers of human. Interaction of c-Src with proteins in signal transduction pathways The signaling complexes associated with RTKs and integrins are shown to employ c-Src as a signaling molecule (Fig. 4) or to interact closely with to develop full biological response (Playford and Schaller, 2004; Huveneers and Danen, 2009), and hence, the robust and persistent RTK signaling was attributed mostly to the physical interaction of c-Src with RTKs. In a study to demonstrate c-Src mediated regulation of phosphatidylinositol 3-kinase (PI3K) signaling, it was shown that the inhibition of PTEN (phosphatase and tensin homolog protein) function by overactive c-Src leads to alterations in signaling through the PI3K/AKT pathway (Lu et al., 2003). Under normal conditions, the activated PI3K catalyzes the conversion of phosphatidylinositol-3,4-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-trisphosphate (PIP3) which, in turn, recruits phosphoinositide-dependent kinase-1 (PDK-1) to the plasma membrane where it phosphorylates and

activates Akt. PTEN is a protein with PIP3-3-phosphatase activity that dephosphorylates PIP3 to PIP2, and hence, downregulating the Akt activity (Planchon et al., 2008). Therefore, its inhibition by overactive c-Src results in a concomitant increase in Akt activity. The activation of c-Src was also involved in PI3K cascade by p70-ribosomal S6 kinase-1 (P70S6K-1) which acts downstream of PDK-1 of PI3K pathway. It was reported that overactive c-Src may provide a necessary signal for the activation of P70S6K-1 as well as for the activation of other S6 kinases (Shah et al., 2002). Not only over activation of c-Src, but also its interaction with other bio-molecules results in changes in signaling processes which depends on the closely related or overlapping downstream protein substrates of both c-Src and other enzymes. Such that, the stable c-Src-focal adhesion kinase (FAK) complex formation was shown to be central to several c-Src mediated alterations including mitogenactivated protein kinase (MAPK) signaling (Playford and Schaller, 2004; Berrier and Yamada, 2007). Furthermore, this complex was also shown to stimulate PI3K/AKT pathway (Carlucci et al., 2008) in addition to c-Src mediated PTEN inhibition. Focal adhesion kinase is a cytosolic PTK localized at the cell membrane where the cytoskeleton interacts with proteins of the extracellular matrix and involved in cellular adhesion which regulates the cell migration and anchorage-dependent differentiation. Soluble growth factor activation is known to recruit FAK-integrin association which leads to tyrosine

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Figure 4 Recently published c-Src mediated signaling pathways. Under different physiological conditions some of the cellular reactions are presented, where c-Src is either signaling or regulatory component. A few phosphorylated proteins upon aberrant activation of c-Src are demonstrated to reect the latest ndings in the eld. The c-Src activation summarized here with blue arrows are due to direct interaction with RTKs, except EGFR which is not shown for simplication. The black arrows correspond to the involvement of external or internal ROS in c-Src regulation. The downstream effects upon c-Src phosphorylation through RTKs or ROS are represented with green arrows. The purple arrows are used to demonstrate c-Src-FAK complex formation and related downstream effects. Here, E-Cad stands for E-cadherin and P for phosphorylation (upon c-Src action). The dashed line (red) stands for the direct inhibition of the target (PTEN), and solid lines correspond to the established interactions such as activation of enzymes, transcription factors (TFs) or initiation of translation, where the mechanism is almost claried to date. Proteins with dark shades represent the enzymes critical for cell physiology for both normal and pathologic conditions.

autophosphorylation of FAK at Tyr397 that forms stable c-SrcFAK complex through Src-SH2 binding and enhancing the kinase activity of c-Src (McLean, et al., 2005; Playford and Schaller 2004). In receptor mediated signaling, c-Src was shown to phosphorylate several sites in FAK which employs the growth factor receptor-bound protein 2 (Grb-2) adapter molecule. Followed by the Grb-2 enrollment, the activation of related signaling pathways occurs, including Ser/Thr kinase signaling, namely mitogen-activated protein kinase (MAPK) pathway. Here, c-Src catalyzed the phosphorylation of Tyr residue on FAK was proposed to create a binding site for a complex, formed between Grb-2 and Ras-guanine exchange factor (Sos, son of sevenless gene product), and nally leading to Grb-2/Sos/Ras/Raf/MEK/MAPK pathway activation (Schlaepfer et al., 1994; Lu et al., 2003).

It is well known that the angiogenesis and vascular permeability is directly mediated by vascular endothelial growth factor (VEGF) and this factor is responsible for proliferative signaling. Moreover, the endothelial cells are known to be contact-inhibited in their growth and poorly respond to proliferation signals of VEGF. This occurs upon binding of vascular endothelial cadherin (E-cadherin, CD144), a calcium-dependent cell-cell adhesion glycoprotein, to VEGFR and inhibiting its signaling activity (Ha et al., 2008). Lately, in vascular endothelial cells, c-Src and its negative regulator C-terminal Src kinase (CSK) shown to be associated with E-cadherin, and explained by the requirement of c-Src for E-cadherin ligation (McLachlan et al., 2007). Although the contribution of c-Src and SFKs to this mechanism is known for years, c-Src mediated phosphorylation of E-

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cadherin on Tyr685, the direct involvement of c-Src in the mechanism by regulating its adhesive activity, was recently reported (Wallez et al., 2006; Wallez et al., 2007). Furthermore, it was found that c-Src is also necessary for E-cadherin ligation to activate PI3-kinase signaling which could restore the functional defects that occur when c-Src signaling is blocked (McLachlan et al., 2007). Also it was shown that VEGF-mediated activation of Akt and endothelial nitric-oxide synthase was reduced upon the inhibition of SH2 domain-containing tyrosine phosphatase (SHP2), c-Src, and E-cadherin (Ha et al., 2008). EGFR tyrosine kinase has been shown to stimulate invasion and metastasis of carcinoma cells through b5 integrin activation and c-Src-mediated phosphorylation of Crk-associated substrate (CAS), thereby leading to activation of Rap1, a small GTPase able to modulate cell adhesion (Ricono et al., 2009). The mechanism was presumed that EGFR provides docking sites to activate intracellular signaling cascades upon homodimerization or heterodimerization, and then, the phosphorylation of receptor leads the conformational change in protein, in three-dimensional space (Ingley, 2008). In erythropoietin receptor (EPOR)-mediated signaling (Okutani et al., 2001), c-Src was shown to directly phosphorylate the Tyr694 residue at the activation site of signal transducers and activators of transcription-5A (STAT5A). STAT proteins, STAT5A and STAT5B, are the transcription factors activated by phosphorylation on tyrosine residues after cytokine stimulation. The active forms of STATs are shown to interact with the regulatory subunit of the PI3K, and hence, activate the related signaling cascades in cell growth and survival. In vitro binding experiments were also demonstrated the PI3K-EPOR interaction through the binding of alpha SH2 domains of PI3K regulatory unit p85 to EPOR-Tyr residue after phosphorylated by c-Src. Accordingly, c-Src was shown to transduce the EPO-induced erythroid differentiation signals by regulating PI3K activity. Grb-2-associated binder-2 (Gab-2) was also found as an essential component for activated-STAT induced cell proliferation, where the required activation of ERK1/2 and Akt was observed only after binding of Gab-2 to PI3K. Conversely, inhibition of this interaction was reported to prevent STAT-induced cell proliferation, as well as ERK1/2 and Akt phosphorylation (Nyga et al., 2005; Harir et al., 2007). In integrin mediated signaling, it was recently shown that upon binding to its ligand, the cytoplasmic tail of integrin interact with SH3 domain of c-Src. The outcome is the subsequent activation of Rac protein as well as the subsequent components in Ras/Raf/MEK pathway (Huveneers and Danen, 2009). FAK-c-Src complex also reported to phosphorylate the components in actin cytoskeleton reorganization and migration, such as CAS, paxillin, and p190RhoGAP (Playford and Schaller, 2004). Beyond the role in signaling through interaction with numerous proteins, previous studies also indicated that SFKs

participate in ROS mediated signaling in a way that they either activated or inactivated by H2O2 or any other ROS present, however the mechanism is not fully understood yet (Abe et al., 1997; Jope et al., 2000; Chiarugi et al., 2003; Ingley, 2008). One of the reports on this context was shown that the oxidative stress-mediated activation of c-Src is essential for the mitogen-activated protein kinase 1 (BMK1) activation that is mediated through H2O2 participation (Abe et al., 1997). Later on, peroxynitrite, another ROS source was shown to affect the signaling pathways through MAPKs, such as ERK1/2 and p38 in neuroendocrine cells (Jope et al., 2000). In this study, the activation of p38 upon peroxynitrite exposure was found independent of the EGF receptor recruitment, but directly related with the participation of the cell type specic mechanisms involving the activation of calcium/calmodulin-dependent kinase II (CDK2) and SFKs, where the nerve growth factor (NGF) is the regulatory component. It was also shown that the oxidative stress induced protein kinase D (PKD) activation through protein kinase C (PKC) dependent signaling is partially controlled by SFKs, where PKD activation was reduced by SFK inhibitors (Waldron et al., 2004). In vivo studies on oxidative stressinduced SFK activation revealed that the activation or inactivation of SFKs (Khadaroo et al., 2004) is related with the level of ROS present. Dose dependent regulation of SFKs or other PTK activity were, consequently, proposed as a protective mechanism of cells against the inammatory activation (Kemble and Sun, 2009). Beyond these, the studies indicated the persistent activation of c-Src upon overexpression of receptor protein tyrosine phosphatase- (RPTP), demonstrating the critical role of RPTP for ROS-signaling besides its positive regulatory role on SFKs (Hao et al., 2006). Studies that present the redox regulator function of ROS in intracellular kinase mediated signaling was explained as alterations in kinase activity through the oxidation of cysteine residue (Cys277) that is conserved in enzymes catalytic domain. The disulde dimer formation between oxidized cSrc and CSK was reported only with SFK members Src, Yes, and Fgr containing this conserved Cys residue, and the resulting overactivation of SFKs was attributed to CSK mediated regulation upon Cys oxidation (Giannoni et al., 2005; Kemble and Sun, 2009), as a result of the reduced or oxidized status of the cellular environment (Chiarugi et al., 2003; Giannoni et al., 2005; Ingley, 2008; Kemble and Sun, 2009). Interaction of GST with proteins in signal transduction pathways Glutathione transferases were known for years as part of the defense system against toxic chemicals with internal or external sources, and associated not only with cell detoxication and survival but also cancer development or prevention (Hayes and Pulford, 1995; Eaton and Bammler, 1999). In addition to essential role of GSTs in detoxifying system,

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novel roles in stress and growth factor-induced signaling, cell proliferation, immune response, differentiation, cellular transformation, and apoptosis, were recently attributed to GSTs under different physiological conditions (Fig. 5). The detailed mechanisms of these pathways were recently explained to some extent, and most of these are still to be claried. The GST participation in signaling, is generally studied with the GST P isozyme since it is highly expressed in aggressive tumors of human, and its over activity and expression are associated with the poor prognosis of the disease, that is the reduced survival rate. In addition to this, its over activity was associated with drug resistance against several anticancer agents. GST gene expression, in response to ROS, has been shown to be regulated by virtue of activating the transcription factors at transcriptional or posttranscriptional levels that occurs in an organized manner. Furthermore, under oxidative stress, all members of cytosolic GSTs, namely GST A, M, P, S, T, O and

Z, and in particular the GST A, have been conrmed to be positively regulated by TNF, IL6, and EGF via survival signaling pathways for liver tissue regeneration (Desmots et al., 2002). The mechanism of ROS-mediated response resulting in the direct alterations has been claried as the changes in activity and expression levels of several kinases and transcription factors, through three decades of research. On the contrary, the indirect alterations was only dened within the last decade and reported to occur through the cysteine rich proteins, represented by thioredoxin and GSTs, due to their sensitivity for oxidative stress (Adler et al., 1999b). The increase in the endogenously produced ROS, and in most cases the exposure to exogenous genotoxic agents including irradiation, inammatory cytokines and chemical carcinogens induction, are known to cause the altered redox potential affecting the stress-activated signaling cascades. In this respect, ROS and the consequent alterations in redox

Figure 5 Recent advancements in GST involved signaling pathways. Under different physiological conditions, GST isozymes appear to function in regulation or in signaling as substrates for various kinases. The positive effect of RTKs on GST expression is shown but detail not given. The protein components interacting with GSTs or inducing their expression are shown with dark gray shades. The amino acid residues important in GST activation upon STK or PTK catalysis are given in light gray shades. GST stands for all soluble isozymes unless any symbol for specic isozyme was used with. In the gure, P corresponds to phosphorylation (after kinase action), and S (on the outside of shapes representing GSTs) stands for the thiol group of GST required for dimerization. It should be noted that the dimerization of GSTs, transferred from nucleus to cytosol, is not shown to simplify the presentation.

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potential may be granted for the primary intracellular changes in protein kinase regulations by virtue of connecting external stimuli with signal transduction in stress response. In the presence of different types of thiol-compounds, the change in thiol-utilizing enzyme activities with respect to alterated cellular redox potential were also shown the role of thiolcompound utilization under oxidative stress. In this regard, in vitro GSH and ROS exposure models shown that ROS can directly react with all sulfhydryl containing molecules where the released protons are important in signal transduction (Nordberg and Arnr, 2001). Under oxidative stress, GST P was dened as a substrate for two Ser/Thr protein kinases, cAMP-dependent protein kinase A (PKA) and PKC, where Ser42 and Ser184 residues of GST P protein was shown as the putative phosphorylation sites for both PKA and PKC (Lo et al., 2004). The result was twofold increase in catalytic efciency of the GST P after phosphorylation. Very recently, it was revealed that treatment with cisplatin, one of the conventional anticancer drugs, PKC activation and associated phosphorylation of GST P increases glutathionyl-platinum formation, decreases the formation DNA interstrand cross-link and increased cisplatin resistance of tumor (Singh et al., 2010). In human glioma and breast cancer cells, GST P was dened as downstream substrate for EGFR, where Tyr7 and Tyr198 residues were dened as the major EGFR-specic phosphorylation sites in the GST P1 protein. The outcome was signicant increase in GST P enzymatic activity with respect to unphosphorylated protein, and hence, the decrease in cisplatin sensitivity. Moreover, it was also reported that a clinically active EGFR inhibitor, Lapatinib, was signicantly reversed the EGF-induced cisplatin resistance revealing the elevated GST P1 and activated EGFR in tumors and their novel role in drug resistance (Okamura et al., 2009a; Okamura et al., 2009b). Very recently, the agents with antioxidant capacity were suggested to induce GST expression and concomitantly activate the PI3K-Akt/ERK-RSK1-mTOR pathway which, in turn, leads the activation of transcription factors required for essential processes in supporting cell viability (Kim and Lee, 2007). On the other hand, it was reported that GST binds and inhibits the basal activity of JNK in non-stressed cells, however can be released after induction of oxidative stress by UV irradiation or hydrogen peroxide exposure (Adler et al., 1999a). Under normal physiologic conditions, the phosphorylation and activation of JNK is mediated through serine/ threonine-specic protein kinases MEKK1 (MAP kinase kinase kinase) and MKK4 (mitogen-activated protein kinase kinase 4). However, it was reported that overexpression of GST P results in decreased MKK4 and JNK phosphorylation. These changes subsequently cause the reduction in JNK activity accompanied by increased c-Jun ubiquitination that leads JNK degradation, and hence, decreased c-Jun-mediated transcription. On the other hand GST P-JNK interaction was found independent of MEKK1/MKK4 pathway, and may

selectively prevent the oncogenic ras-p21-induced cell transformation. The binding ability of GST to JNK was also shown for GST A and M with in vivo studies (Villafania et al., 2000). Tissue specicity is an important factor to consider for expression and regulation of drug metabolizing enzymes including GSTs which are highly abundant in the liver of various species due to its role in detoxication system especially in human, sheep and monkey, and recently their active presence in bovine liver was also shown (Isgor et al., 2010a). Accumulating body of evidences revealed the involvement of GSTs in the regulation of hormone- and growth factor-mediated cellular signaling pathways, particularly on the hepatic drug metabolizing enzyme expression, under both physiologic and pathological conditions. In this context, the increase in GST A protein expression in the presence of increased insulin was reported, whereas the determined levels of GST A and P were reduced signicantly with glucagon. On the contrary, no effect of insulin or glucagon treatment on GST M was observed in vitro (Kim et al., 2003). Later, it was shown that the levels of GST A1 and A3 were increased in the presence of insulin in a dose dependent manner. This activation was prevented with the presence of PI3K, mTOR and p70S6K inhibitors, as well as the dominant negative or kinase inactive Akt. It was also shown that the insulin-mediated increase in GST A protein levels in hepatocytes is independent of MEK1 inhibition that lead to ERK1/2 pathway blockage, in addition to p38 MAPK, HSP27 and PKC inhibition in hepatocytes (Kim et al., 2006). Consequently, PI3K/Akt/p70S6K signaling was determined functioning in the insulin-mediated upregulation of the antioxidant defense system, and potentially enhances the hepatocytes susceptibility to xenobiotic-or oxidative stressmediated damage (Kim and Novak, 2007). In terms of GST catalytic activity, the positive effect of prolactin was shown on a study tailored to identify the drug resistance against cisplatin (LaPensee et al., 2009). Here, the prolactin, a survival factor in malignant and non-malignant cells, was shown to induce GST M expression, and therefore reduces the effectiveness of cisplatin. GST M overexpression was also shown to be a negative regulator of apoptosis-related signaling cascades including the chemotherapy through glucocorticoid drug dexamethasone. The observed dexamethasone resistance was due to the suppression of Bim, a proapoptotic protein and family member of B cell lymphoma2 (Bcl-2), by dual mechanism where the suppression of dexamethasone-induced p38-MAPK and upregulation of nuclear factor kappa B (NFB) activity occur concomitantly (Hosono et al., 2010). In GST P decient animal model, cytokine induced myeloproliferation was detected with downregulation of the SHP-1 (Src homology 2 domaincontaining tyrosine phosphatase-1) and SHP-2, the negative regulator of JAK/STAT signaling in hematopoietic cells, and associated with increased activation of JNK and STATs (Gate et al., 2004). The GST P involved JNK and STAT activation

Yasemin G. ISGOR and Belgin S. ISGOR

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was further veried with the use of a glutathione disulde mimetic (NOV-002), a GST inhibitor approved for clinical studies. Upon exposure to NOV-002, the reduction of cellular thio-protein levels was determined with leukemia cells accompanied with the increase of S-glutathionylation of cell proteins, such as actin. Additionally, the involvement of GST P in cellular signaling was shown with the detection of p38 MAPK, JNK and ERK activation with dose dependent phosphorylation of Akt, JAK2, and STAT5. Clinical studies with NOV-002 were shown the improved tolerance to chemotherapy with increase in survival rate when administered in combination with standard chemotherapeutics in advanced non-small cell lung cancer patients (Townsend et al., 2002; Townsend et al., 2008; Uys et al., 2010). Besides the advancements of cancers in relation to the oxidative stress induced GST activation, recently it was reported that normal keratinocytes may represent a vitiligo-like cytokine pattern after exposure to 4-hydroxy-2-nonenal (HNE), another ROS source. The exposure to HNE was shown to induce overproduction of H2O2, followed by transient inhibition of Erk1/2 and Akt phosphorylation, and these were accompanied by upregulation of catalase and GST M genes (Kostyuk and Potapovich, 2009; Kostyuk et al., 2010). The physiological implications of GST regulation in response to pathophysiologic conditions may require further evaluation to clarify the involvement with signaling components with respect to organ, sex and specie specic manner.

Concluding remarks
In the eld of drug discovery and development, it is indispensable to dene the targets, which are usually functional proteins, responsible either in the occurrence or persistence of the pathophysiologic conditions. The early studies have shown that these targets are also the natural components of normal physiologic functioning and they only become pathologic after aberrant activity of other critical components in the cellular physiology. In the following years, the attempts have been focused on identifying the druggable targets through acellular and cellular assay platforms, as well as animal studies with molecular biology tools. Since the rst kinase mechanism identied 60 years ago, these studies revealed the most intriguing results demonstrating that the components of protein phosphorylation networking is essential for a balance between normal and pathologic conditions. Since then, the accumulating evidence revealed that 60% of oncogenes and proto-oncogenes are PTKs. These ndings were appreciated by pharmaceutical industry; more than two dozens of tyrosine kinases from different classes were evaluated as potential druggable targets. Over the past six years, seven new kinase inhibitors have been approved for clinical studies and generally they are NRTK inhibitors with dual specicity or with some RTK afnity. Among those, the most difcult target appears to be c-Src due to its central role in signaling and its structural homology to other SFKs. Since

the discovery of c-Src three decades ago, it still receives great attention as universal therapeutic target today, even though there is no selective therapeutics modifying c-Src activity reached the clinic yet. Although the expression levels and abundance of SFK members, other than c-Src, may vary depending on tissue type and this property makes it feasible to target them, c-Src is expressed in every tissue with reasonable amount and activity. This later information brings the difculty to modify its function without affecting other kinases with structural similarity. For solving the problems arisen due to the reduced effectiveness of drugs targeting multiple kinases, combined therapy is generally the method chosen. With conventional chemotherapy, the problem appears to be the reduced effectiveness of the therapeutics upon prolonged exposure, if not given at high doses. The systemic toxicity issues preventing the use of high dose applications with the effect of detoxifying system enzymes were addressed for a long time, however, the combined therapy only found successful for only a small portion of the cancer types. It should be noted that targeted delivery approach, a method to deliver drugs either encapsulated in a biocompatible nanomaterial or in liposomes to target tissue and organs, has been developed to improve drug effectiveness and reduce systemic toxicity. However, in clinical studies, such approach does not seem to solve the failure of drugs arisen from their unappreciated ability to interact with offtargets in signaling pathways. Current approaches with c-Src inhibitor studies appear to be promising, even though the selectivity and effectiveness are still the problem to be solved. In this sense, the latest ndings with c-Src function upon oxidative stress revealed its novel role in ROS mediated signaling which seems important to understand its behavior under the conditions that GSTs are highly active to restore the cellular redox balance. It was reported that c-Src is activated at oxidative conditions where the Cys residues in the conserved domain is oxidation vulnerable. The outcome of this process is disulde dimerization between c-Src and CSK depending on the level of ROS present (Kemble and Sun, 2009; Sun and Kemble, 2009). In the same manner, cellular oxidation levels can be regulated by GSTs, known to construct disulde bridges between several subunits of the same isoforms and catalyze reactions through glutathione transfer by thiol linkage. This enzyme, very recently, was also identied as substrates of various kinases such as PKA, PKC and EGFR. These kinases, so far identied as GST positive regulators under different conditions, are either in direct interaction with c-Src or part of c-Src mediated signaling events (Adler et al., 1999a; Allan et al., 2001; Okutani et al., 2001; Townsend et al., 2002; Adler and Pincus, 2004; Rodriguez et al., 2005; McIlwain et al., 2006; Townsend et al., 2006; Kim and Lee, 2007; Okamura et al., 2009b; Bordeleau et al., 2010; Crout et al., 2010; Hosono et al., 2010; Scodelaro Bilbao et al., 2010; Singh et al., 2010). Except for disease progression, xenobiotic/drug toxicity, and redox balance, only a little research with GSTs have been performed

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with respect to their roles in signaling pathway. In this context, even with the well documented role of GST A and P isozymes, only recently the GSTs have been attributed as kinase substrates under specic conditions. In this respect, it can be assumed that the limitations of currently available methods may prevent our understanding of kinase contributions to signaling for enzymes as kinase targets but difcult to identify. This can be true for determining the activity GSTs, due to absence of non-physiological or overlapping substrates to detect activity levels of some isozymes such as GST S, M and T. Although the presence of GSTs can be determined to some extent, the complexity to identify the activity levels of GST family of enzymes in various tissues is, therefore, still challenging. On the contrary, the studies with specic isozymes, such as GST P, M and A revealed their involvement in many signaling pathways to some extent, however until recently, their aberrant activity was not attributed to the presence of possible phosphorylation sites at enzymes conserved domains. Interestingly GST P, one of the well studied isoform, was reported as both substrates of STKs (PKA and PKC) and RTK (EGFR). It exhibits the possible involvement with c-Src, the more active kinase compared to EGFR, PKA or PKC under oxidative stress. Moreover the c-Src involved PI3K/AKT, MEKK1/JNK pathways, as well as c-Src-FAK complex related downstream effects, appear to be shared with active GSTs with the mechanism not resolved clearly yet. It can be discussed that we still know less about kinases, their substrates and regulators; however, clarifying functional effects of specic phosphorylation events with well-designed projects, such as those with GSTs, and relating those with cellular physiology may help to improve our understanding of kinases. In terms of GSTs, the recent studies with GST inhibitors resulted in two clinically available formulations: one with stabilized formulation of disodium glutathione disulde (GSSG; oxidized glutathione) and cisplatin, NOV 002, and the other, ezatiostat, is a small-molecule glutathione analog. NOV 002 was reported with potential chemoprotective and immunomodulating activities by inducing phosphorylation of proteins such as ERK and p38 which are critical in the regulation of cell proliferation and apoptosis, whereas, ezatiostat, shown to restore JNK and MAPK pathway activities, induce MAPKmediated cellular proliferation and differentiation, as well as the hematopoietic precursor cell maturation at bone marrow. Despite these successes, there is still less known about the function of other isoforms of GSTs in signaling, since there are no inhibitors of them available for preclinical in-depth analysis or clinical studies yet. In terms of GST P inhibition, it is known to enhance the drug sensitivity for various chemotherapeutics in model systems, and therefore improving the effectiveness of drugs against solid tumors. Therefore, the substantial enthusiasm for advancing c-Src and GST inhibitor development and using in appropriate combinations with other clinically available chemotherapeutics may enhance the therapeutic

value of the drugs by improving selectivity and sensitivity of compounds developed for therapeutic interventions.

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