Regulation of JNK signaling by GSTp

Victor Adler; Zhimin Yin; Serge Y. Fuchs; Miriam Benezra; Lilliam Rosario; Kenneth D. Tew; Matthew R. Pincus; Mohinder Sardana; Colin J. Henderson; C.Roland Wolf; Roger J. Davis; Ze'ev Ronai

doi:10.1093/emboj/18.5.1321

Regulation of JNK signaling by GSTp

Adler, Victor; Yin, Zhimin; Fuchs, Serge Y.; Benezra, Miriam; Rosario, Lilliam; Tew, Kenneth D.; Pincus, Matthew R.; Sardana, Mohinder; Henderson, Colin J.; Wolf, C.Roland; Davis, Roger J.; Ronai, Ze'ev 1999-03-01 00:00:00 The EMBO Journal Vol.18 No.5 pp.1321–1334, 1999 shock or inﬂammatory cytokines (Galcheva-Gargova et al., Victor Adler, Zhimin Yin, Serge Y.Fuchs, 1994; Kyriakis et al., 1994; Westwick et al., 1994). JNK Miriam Benezra, Lilliam Rosario , 1 2 activation in response to UV irradiation is mediated by Kenneth D.Tew , Matthew R.Pincus , PAK 3 4 upstream signaling components, including cdc42, p21 , Mohinder Sardana , Colin J.Henderson , ASK1, MLK, MEKK1, SEK1/MKK4, MKK7 (Coso et al., 4 5 C.Roland Wolf , Roger J.Davis and 1995; Fanger et al., 1997; Tournier et al., 1997; reviewed Ze’ev Ronai ras by Ip and Davis, 1998) and p21 (Minden et al., 1994; Adler et al., 1995a, 1996), in concert with nuclear DNA The Ruttenberg Cancer Center, Mount Sinai School of Medicine, One Gustave L.Levy Place, Box 1130, New York, NY 10029-6574, lesions (Adler et al., 1995b). Different forms of stress Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, have been shown to mediate JNK activation via various PA, Department of Pathology and Laboratory Medicine, SUNY cellular pathways (Adler et al., 1995c). Activated JNK Health Science Center, Brooklyn, NY, Protein Sequencing Facility, phosphorylates the transcription factors c-Jun, ATF2, p53 Merck Sharp & Dohme, West Point, PA, USA, Imperial Cancer Research Fund, Molecular Pharmacology Unit, Dundee, DD1 9SY, UK and ELK-1 (Kyriakis et al., 1994; Gupta et al., 1995; and Howard Hughes Medical Institute, University of Massachusetts, Whitmarsh et al., 1995; Adler et al., 1997; Fuchs et al., Worcester, MA, USA 1998a). Phosphorylation by JNK has been implicated in Corresponding author stabilization (Fuchs et al., 1997, 1998b) and transcriptional e-mail: [email protected] activity (Kyriakis et al., 1994) of its substrates, which directly contribute to the mammalian stress response Studies of low basal Jun N-terminal kinase (JNK) through changes in the cell cycle, DNA repair or apoptosis activity in non-stressed cells led us to identify a JNK (Xia et al., 1995; Fuchs et al., 1998a; Kasibhatla et al., inhibitor that was puriﬁed and identiﬁed as glutathione 1998). S-transferase Pi (GSTp) and was characterized as Despite the signiﬁcant advances in identifying the a JNK-associated protein. UV irradiation or H O 2 2 components of stress-activated protein kinases, the mech- treatment caused GSTp oligomerization and dissoci- anisms underlying the regulation of JNK before and ation of the GSTp–JNK complex, indicating that immediately after stress are not well understood. In cells it is the monomeric form of GSTp that elicits JNK maintained under normal growth conditions, the basal inhibition. Addition of puriﬁed GSTp to the Jun– activity of JNK is low, although JNK phosphorylation by JNK complex caused a dose-dependent inhibition of upstream kinases occurs in response to growth factors JNK activity. Conversely, immunodepleting GSTp from (Minden et al., 1994) and hence should also be observed protein extracts attenuated JNK inhibition. Further- in cells proliferating under normal growth conditions. more, JNK activity was increased in the presence of While some unstressed cell types have been found to speciﬁc GSTp inhibitors and a GSTp-derived peptide. contain high levels of JNK activity (Dhar et al., 1996), Forced expression of GSTp decreased MKK4 and the reason for this high basal level is unknown. A 2- to JNK phosphorylation which coincided with decreased 4-fold increase in constitutive JNK activity has been JNK activity, increased c-Jun ubiquitination and reported and, while this is lower than the activation upon decreased c-Jun-mediated transcription. Co-transfec- exposure to DNA-damaging agents, it is equivalent to tion of MEKK1 and GSTp restored MKK4 phos- tumor necrosis factor-α (TNF-α) treatment and the degree phorylation but did not affect GSTp inhibition of JNK of activation elicited by multiple exposures to low dose activity, suggesting that the effect of GSTp on JNK is UV-B (Adler et al., 1995d). As a key component in independent of the MEKK1–MKK4 module. Mouse regulating the stability and activity of its substrates (Fuchs embryo ﬁbroblasts from GSTp-null mice exhibited a et al., 1998c; Kyriakis et al., 1994), changes in JNK’s basal high basal level of JNK activity that could be reduced activity could inﬂuence key cellular functions, including by forced expression of GSTp cDNA. In demonstrating growth, apoptosis and transformation. the relationships between GSTp expression and its Neither transcript nor protein levels of JNK are affected association with JNK, our ﬁndings provide new insight by stress. JNK activation is accomplished upon its into the regulation of stress kinases. phosphorylation by upstream kinases in response to stress Keywords: GSTp/JNK/signaling/stress kinase (Coso et al., 1995; Tournier et al., 1997). Dephosphoryla- tion of JNK at a later stage (Cavigelli et al., 1996; Hanada et al., 1998) is thought to constitute the primary mechanism underlying the regulation of JNK activity as a kinase. Introduction Nevertheless, several studies have demonstrated the exist- Jun N-terminal kinases (JNKs) belong to the multi-member ence of JNK inhibitors in normal growing cells. Thus, family of stress kinases that are activated transiently in stress-mediated JNK activation may be independent of its response to UV- or X-irradiation, heat shock, osmotic upstream kinases. Among the cellular components © European Molecular Biology Organization 1321 V.Adler et al. involved in regulating JNK substrates is a ‘δ inhibitor’ 1995b), we refer to the Jun-bound kinase as JNK and to which blocks transactivation of c-Jun by interacting with the overall complex as Jun–JNK. the δ domain (Baichwal et al., 1991). This is an integral When cellular extracts prepared from unstressed cells 27 amino acid sequence (30–57) from the N-terminal were added to the pre-formed Jun–JNK complex before region of c-Jun required for JNK binding to c-Jun, enabling adding [γ- P]ATP, c-Jun phosphorylation was inhibited Jun phosphorylation (Adler et al., 1992) or ubiquitination by 80% (Figure 1A, compare lanes 1 and 2). To elucidate (Treier et al., 1994; Fuchs et al., 1996). Other JNK further the nature of this inhibition, proteins derived from cip/waf1 regulatory proteins include p21 , which has been the non-stressed cells were absorbed by pre-incubation shown to exert greater inhibition of JNK activity in with increasing amounts of Jun–JNK. Subsequently, response to stress (Shim et al., 1996). beads-bound c-Jun–JNK complexes were spun and the Our interest in exploring the nature of JNK’s low basal supernatant, which lacked Jun–JNK-bound proteins, was activity originated in the observation that extracts of non- tested to determine its ability to alter the degree of JNK- stressed cells efﬁciently inhibited c-Jun phosphorylation mediated Jun phosphorylation. Increasing the amounts of when added to a solid-phase kinase reaction. Our further Jun–JNK complexes used to absorb the putative inhibitor studies led to the puriﬁcation, identiﬁcation and character- resulted in a dose-dependent decrease of inhibitory activity (Figure 1A, lanes 3–6). To determine whether this inhibitor ization of glutathione S-tranferase Pi (GSTp) as a JNK activity was heat stable, the protein fraction that contained inhibitor, described in this report. the inhibitor activity was heated to 95°C for 5 min. This GSTs comprise a multigene family, of which GSTp is treatment abolished the fraction’s ability to block Jun the most prevalent and ubiquitous non-hepatic isozyme phosphorylation by JNKs (not shown). (Jakoby, 1978). Among cellular functions attributed to GSTs are ligand binding and xenobiotic detoxiﬁcation UV irradiation abolishes JNK inhibitor activities (Tew, 1994). Reduced glutathione (GSH) binds to the ‘G’ The presence of JNK inhibitory activity in non-stressed site of GSTp (and other GST isozymes) and plays an cells prompted us to determine possible changes to the important role in detoxiﬁcation of reactive oxygen species inhibitory activity after UV irradiation. As shown in Figure (ROS) and the maintenance of the cellular redox state 1B, extracts prepared after UV treatment lacked inhibitory (Sato et al., 1989). activity. The decrease in JNK inhibitor activity depended Among factors implicated in regulating JNK activity on the UV dose. Whereas a 10 J/m dose (a 6 s exposure) are ROS and altered redox potential (Adler et al., 1995c; caused a 40% reduction (as quantiﬁed using a phospho- Gomez-del-Arco et al., 1996; Cui et al., 1997; Wilhelm imager), a 40 J/m dose (a 25 s exposure) completely et al., 1997). ROS have also been associated with regula- abolished the inhibition. In all instances, analysis was tion of other signaling cascades, e.g. certain isozymes of performed using the same amount (20 μg) of protein protein kinase C (PKC; Konisshi et al., 1997) and mitogen- prepared 30 min after administering UV irradiation activated protein kinase (MAPK; Guyton et al., 1996). (Figure 1B). A correlation between JNK activation and The addition of exogenous oxidants or anti-oxidants has the inactivation of its inhibitory activity was also noted been found to inﬂuence the activation of MAPK/JNK in human melanoma cells, which require a 60 J/m dose (Lo et al., 1996; Wilhelm et al., 1997). for JNK activation. In melanoma cells, 40 J/m doses Changes in ROS can also directly inﬂuence transcrip- caused a 50% inhibition, whereas a 60 J/m dose abolished tional activity, as demonstrated for NF-κB (Wang et al., JNK inhibitory activities (not shown). 1995), Ref-1 (Xanthoudakis et al., 1992) and c-Jun (Gomez-del-Arco et al., 1996). Thus, evidence is Puriﬁcation of the JNK inhibitor accumulating that redox status can play an integral role To purify the putative inhibitor, extracts of normally in kinase-mediated stress response pathways. Our growing cells were subjected to the six steps described characterization of GSTp as an endogenous regulator of below. After each step, the presence of the putative JNK activity provides a novel function for a protein inhibitor (tested by measuring inhibition of c-Jun phospho- with previously characterized catalytic and ligand-binding rylation) was monitored in a solid-phase kinase assay properties. using the Jun–JNK (1 μg) complex. Proteins from non- his stressed cells were ﬁrst precipitated in ammonium sulfate (50%). After dialysis against kinase buffer, the non- Results precipitated proteins were concentrated on membranes A JNK inhibitor is present in cellular extracts of with various molecular weight cut-offs. Inhibitor activity normally growing cells was found in the ﬂow-through of both 100 and 30 kDa JNK activities were measured in solid-phase kinase reac- cut-off ﬁlters; it was retained on the 10 kDa membrane, tions using Jun as a substrate. Proteins prepared from indicating that the inhibitor is between 10 and 30 kDa in UV-treated 3T3-4A mouse ﬁbroblast cells exhibited molecular weight (Figure 1C). The 10–30 kDa fractions marked phosphorylation of c-Jun. The kinetics of JNK were loaded on a gel ﬁltration column; several fractions activation in these cells increased within 1–5 min, reaching within the 20–25 kDa range (based on calibration of peak levels within 20 min and declining to basal levels the gel ﬁltration column with known molecular weight within 3–4 h (Adler et al., 1995b). This is a transient standards) retained inhibitory activity (Figure 1D). Active activation that is monitored as a multifold increase of fractions were pooled and fractionated on a MonoQ anion kinase activity relative to the control (untreated) cells. exchange column; a 70 mM NaCl eluate was found to Since the assays are performed under conditions that contain most of the inhibitor activity (Figure 1E). Active enable selective binding of JNK isozymes (Adler et al., MonoQ fractions were then loaded onto a phenyl- 1322 Regulation of JNK signaling by GSTp Fig. 1. (A) UV-mediated JNK activation is inhibited by proteins obtained from cells maintained under normal growth conditions. Phosphorylation of Jun by JNK (lane 1) is inhibited when the pre-formed complex (Jun–JNK; total 1 μg at 1:1 ratio) is incubated with proteins (10 μg) from untreated cells before adding the [γ- P]ATP (lane 2). Pre-incubation of proteins from non-stressed cells with increasing concentrations of Jun–JNK complex resulted in a dose-dependent decrease of inhibitory activity (lanes 3–6 represent increasing amounts of the pre-formed complex 0.25 , 0.5, 1 and 2 μg, respectively). (B) UV irradiation abolishes inhibitor activities. Pre-formed Jun–JNK complex was incubated with whole-cell extract prepared 30 min after sham or UV-C irradiation at the doses indicated prior to addition of [γ- P]ATP. (C) Puriﬁcation of JNK inhibitor on molecular weight cut-off columns. Whole-cell extracts from non-stressed cells were subjected to an ammonium sulfate precipitation and the supernatants were fractionated on the indicated membranes with a cut-off mol. wt of 100, 30 or 10 kDa before being added to pre-formed Jun–JNK complex, followed by addition of [γ- P]ATP (the basal activity in UV-treated cells is shown in lane JNK). Shown is an autoradiograph of the phosphorylated c-Jun. (D) Gel ﬁltration of 3–30 kDa fractions. Fractions 30 kDa were concentrated and loaded (50 μl) onto a Superdex 75 column. From each fraction, 20 μl were added to the pre-formed Jun–JNK complex to measure the ability to inhibit JNK activity (the ﬁrst lane points to JNK activity with UV-treated extract before this inhibition; numbers represent the respective fractions). (E) Fractionation of JNK inhibitor via an anion exchange column. Fractions 18–24 obtained by Superdex 75 separation were pooled and applied to a MonoQ column. The fractions were assayed for inhibitor activities as shown in the inset. (F) Identifying the inhibitor component on the basis of its binding to Jun–JNK complex. Jun or Jun–JNK his his complex was incubated with the phenyl-Sepharose eluate (A) or with a puriﬁed form of GSTp (B). Beads-bound material was washed and separated on SDS–PAGE. Silver staining of bound proteins is shown in the left panel, whereas the immunoblot of a duplicate gel with antibodies to GSTp is shown on the right. The arrow points to the position of GSTp. 1323 V.Adler et al. Table I. Puriﬁcation of JNK inhibitor Puriﬁcation step Protein Total Speciﬁc activity (mg) activity (U) ( Jun–JNK) his Total lysate 80 10 160 0.127 Ammonium sulfate 32 8128 0.254 (50%) Membrane cut-off 6.7 6096 0.91 MonoQ 2.1 5425 2.69 Phenyl-Sepharose 1.4 4820 3.46 Jun–JNK 0.063 4511 70.70 his All puriﬁcation steps were performed using the Jun–JNK complex his (on nickel beads) as a substrate to measure the degree of JNK activity/ inhibition (see Materials and methods for details). Total activity is deﬁned in units:1Uisthe ability to decrease JNK activity by 50%. Speciﬁc activity is calculated as inhibitor activity of 1 μg of protein. Sepharose column in the presence of 0.6 M ammonium sulfate. Using decreased concentrations of ammonium sulfate, the inhibitor activity was found in 0.2 M eluate fractions (not shown). The active fractions from the phenyl-Sepharose column were incubated in a Jun– his JNK column. Analysis of the Jun–JNK-bound material his revealed a single band on silver-stained gels (Figure 1F). Microsequencing analysis of the corresponding band obtained after a large-scale puriﬁcation (Table I) revealed a 12 amino acid peptide (Pro-Pro-Tyr-Thr-Val-Val-Tyr- Phe-Pro-Val-Arg-Gly) that exhibited 100% homology to the human form of GSTp. To conﬁrm this protein’s identity, a puriﬁed form of GSTp was separated parallel to the Jun–JNK-bound material and subjected to Western his blot analysis using polyclonal antibodies to GST. Both migration and immunoreactivity of puriﬁed GSTp were identical to those of the Jun–JNK-bound material his (Figure 1F). GST inhibition of JNK activity does not alter Fig. 2. (A) GSTp as a JNK inhibitor. GSTp was added at the indicated phosphorylation of Jun or JNK concentrations (micrograms) to the pre-formed Jun–JNK complex and To determine whether GSTp affected the degree of JNK the level of Jun phosphorylation was measured by means of autoradio- graphy. (B) GSTp does not affect JNK phosphorylation. To measure or Jun phosphorylation, increasing concentrations of GSTp the effect of GSTp on JNK phosphorylation, JNK was immuno- (puriﬁed form) were added to pre-formed Jun–JNK com- precipitated from UV-treated cells and incubated with GSTp (at the plex which contained the phosphorylated form of JNK microgram concentrations indicated) followed by Western blot with obtained from UV-treated cells. GSTp decreased JNK antibodies to phospho-JNK (upper panel). The lower panel depicts the level of JNK on the same blot after re-probing with antibodies to JNK. phosphorylation of c-Jun in a dose-dependent manner (C) As a positive control, JNK from UV-treated cells was incubated (within a range of 0.05–1 μg) (Figure 2A), but it did not with no protein (lane C), dual speciﬁcity phosphatase (lane PP; decrease the number of phosphate groups on JNK, as Ishibashi et al., 1992) or GSTp (lane GSTp) before carrying out revealed by immunoblots with phospho-JNK antibodies immunoblot analysis with antibodies to phospho-JNK. Quantiﬁcation (Figure 2B). Dual activity protein phosphatase (Ishibashi via densitometer scanning revealed 35% inhibition of JNK phosphorylation by PP, whereas GSTp did not elicit such inhibition. et al., 1992) was used as a positive control in these (D) GSTp does not alter c-Jun phospho groups. The autoradiograph reactions (Figure 2C). depicts the c-Jun phosphorylation level after incubation with the To determine whether GSTp affects the number of inhibitor for the indicated time periods (minutes) before or after phospho groups on c-Jun, cellular extracts from normally phosphorylation by JNK. growing cells were incubated with pre-formed Jun–JNK complex for the indicated periods of time before or after the phosphorylation step with [γ- P]ATP. The extent of identiﬁed GSTp as the associated protein. A marked c-Jun phosphorylation was not altered when cell extracts decrease in this association was found in proteins prepared were added after the phosphorylation reaction (Figure after UV irradiation (Figure 3A). In addition to GSTp, 2D). This observation suggested that GSTp did not reduce isozymes of the GSTα and GSTμ families were also the number of phospho groups on c-Jun. capable of associating with the Jun–JNK complex in vitro (Figure 3A). GSTp exhibited greater JNK inhibitory activ- Effect of different GST isoforms on JNK activity ity than did GSTμ, which was more potent than GSTα Incubation of whole-cell extract prepared from non- (Figure 3B). Bacterially expressed GST (GST-2T) also stressed mouse ﬁbroblasts with the Jun–JNK complex mediated JNK inhibition (Figure 3B). This excludes the his 1324 Regulation of JNK signaling by GSTp possibility that the inhibitor activity was dependent on blocked GSTp inhibition of JNK activity (Figure 3F). any putative GST-associated cellular component. TER-317, structurally similar to TER-117, yet void of GSTp inhibitory properties, failed to affect GSTp inhibi- Dose-dependent inhibition of JNK by GSTp tion of JNK. Similarly, TER-199, an inactive prodrug GSTp inhibition of JNK activities was determined using of TER-117 (Flatgaard et al., 1993), was also without six concentrations of puriﬁed GSTp added to either full- effect in vitro. length or N-terminal forms of c-Jun. The addition of Molecular dynamic calculations on the energy- 5–89 puriﬁed GSTp to JNK complexed with GST–Jun or minimized X-ray crystal structure of GST were performed full-length Jun led to a concentration-dependent inhibition of to identify the most ﬂexible regions of the protein, i.e. his c-Jun phosphorylation (Figure 3C). The greater inhibitory those that were computed to have the highest degree of 5–89 capacity of GSTp observed for GST–Jun - compared ﬂuctuations. These regions are most likely to undergo full-length with Jun -based JNK complexes can be attributed signiﬁcant conformational changes during catalysis or his to the different conformations of the recombinant sub- ligand binding. Two such ﬂexible domains, corresponding 5–89 full-length strates (N-terminal GST–Jun versus Jun ). A respectively to amino acids 36–50 and 194–201, which his 5–89 higher degree of inhibition of the GST–Jun fusion can participate in the GST–Jun–JNK association and protein is not likely to occur as a result of GST–GST inhibition, were tested. interactions, which would require other experimental con- Peptides corresponding to each of these domains were ditions and result in different kinetics. Nevertheless, using synthesized, and their ability to alter GSTp inhibitor 5–89 full-length either GST–Jun or Jun as substrates, the activity was determined in vitro. As shown, the GSTp- his degree of GSTp inhibitory activity appeared to be linear derived peptide from amino acids 194–201 (at the within the range of 25–180 ng of GSTp (per microgram C-terminal domain designated P1) abrogated GSTp inhibi- of pre-formed Jun–JNK complex; Figure 3C). GSTp also tion of JNK activity (Figure 3F). Conversely, neither P2 inhibited JNK activity when added to a soluble form of (aa 36–50; Figure 3F) nor three unrelated, non-GST-based Jun–JNK complex (not shown). peptides of varying lengths (8–24 amino acids; not shown) his were capable of affecting GSTp inhibition of JNK. Treat- Speciﬁcity of GST inhibition ment of cells with TER-199 (the prodrug form of TER- To determine the speciﬁcity of the JNK inhibitor, puriﬁed 117 which is converted to active form in vivo and has GSTp was incubated with substrates for other protein been shown to elicit effective inhibition of GST activity kinases. Incubation with PKA, PKC, casein kinase II in vivo; Flatgaard et al., 1993) led to a 2-fold increase in (CKII) or MAPK revealed 6, 3, 36 and 9% inhibition, basal JNK activity in non-stressed cells. UV irradiation respectively. Under the same conditions, GST elicited also increased (~2.5-fold) JNK activation in TER-199- 86% inhibition of JNK (Figure 3D). In all cases, the treated cells (Figure 3G). That TER-199 also affected activities of the various kinases tested were normalized JNK activities after UV irradiation suggests that the dose (c.p.m./μg protein and an equal ratio between the respect- of UV irradiation was insufﬁcient to mediate complete ive substrate and GSTp) to ensure an equal degree of inactivation of GST as a JNK inhibitor; thus, the presence substrate phosphorylation. of a speciﬁc GSTp inhibitor led to an additive effect on overall JNK activities in these cells. The effect of these GSTp immunodepletion attenuates JNK inhibition GSH peptidomimetic agents is likely to be mediated either We further elucidated the ability of GSTp to inhibit JNK by altered conformation of the GSTp molecule or by a activity by modulating its levels or activity, or both, competition for the binding site, as non-active (bacterially in vitro and in vivo. The in vitro kinase assay in which produced) GST was also capable of eliciting JNK inhibi- proteins from non-stressed cells were added to the pre- tion (Figure 3B). formed Jun–JNK complex revealed a dose-dependent increase in the degree of JNK inhibition (of up to 80%). Changes in ROS affect GST oligomerization and its Immunodepletion of GST from whole-cell extract association with, and inhibition of, JNK decreased the degree of JNK inhibition from 80 to 45% We next assessed whether modulation of the cellular redox (Figure 3E, lane GST-1). Repeated immunodepletion by potential would affect JNK inhibition by GSTp. We GST antibodies further decreased the degree of JNK monitored the GST–JNK complex in vivo by means of inhibition to 20% (Figure 3E, lane GST-2). Control immun- immunoprecipitations followed by immunoblot analysis. odepletion reactions with normal rabbit serum (NRS) and Exposure of mouse ﬁbroblast cells to either UV or H O 2 2 protein A/G beads did not affect the degree of JNK reduced the amount of the JNK–GSTp complex and inhibition (Figure 3E, lanes NRS-1 and NRS-2, increased JNK activity, whereas pre-treating cells with the respectively). free radical scavengers N-acetylcysteine (NAC) or the ethyl-ester of glutathione (eeGSH) prevented JNK dissoci- Speciﬁc GSTp inhibitors and a GSTp-derived ation from GSTp and maintained GSTp inhibitory activity peptide efﬁciently alter JNK inhibition (Figure 4A I–III). These observations indicate that GST– As an independent approach to inhibit GSTp, we used JNK–Jun association is inversely correlated with JNK speciﬁc inhibitors which were shown to inactivate GSTp activity. To elucidate further changes to GST that take enzymatic activity in vitro and in vivo (Flatgaard et al., place upon altered redox conditions, we measured the 1993). Adding TER-117, a speciﬁc in vitro GSTp inhibitor, possible formation of GST–GST dimers/multimers through to a Jun–JNK complex at the same time as GSTp prevented disulﬁde bonds on neighboring cysteines. Monitoring the GSTp inhibition of JNK activity. Similarly, TER-293, migration of GSTp on non-reducing SDS–PAGE revealed another GSH peptidomimetic related to TER-117, also decreased monomer/dimer forms of GSTp in UV- or 1325 V.Adler et al. H O -treated cells (Figure 4A IV). Instead, a high IV, the single arrow in panel a points to the dimer and 2 2 molecular weight band corresponding to a multimer form panel b shows the monomer). H O treatment is known 2 2 of GSTp was detected (Figure 4A IV, upper arrow in to cause the formation of intra- or intersubunit disulﬁde panel a). Pre-treatment with NAC or eeGSH maintained bonds between cysteines at amino acids 47 and 101 of the lower molecular weight forms of GSTp (Figure 4A GST (Shen et al., 1993), resulting in a multimerization 1326 Regulation of JNK signaling by GSTp of GST subunits which no longer accommodates JNK efﬁciently increased MKK4 phosphorylation. Conversely, molecules. forced expression of GSTp reduced the level of MKK4 phosphorylation in a dose-dependent manner (Figure 5BI). Monomer form of GSTp mediates JNK inhibition Under the same conditions, GSTp did not alter phospho- The observation of changes in GSTp from monomer/ rylation of ERK1 (Figure 5BII). Treatment of GSTp- dimer to multimer forms upon exposure to increased ROS transfected cells with TER-199, an inhibitor of GSTp, prompted us to determine which of the two prevalent restored the level of MKK4 phosphorylation (Figure 5BI). GSTp forms, monomer or dimer, exerts inhibitory activity The observation that under physiological levels of GSTp on JNK. A gel ﬁltration column was used to separate the expression MKK4 was phosphorylated, although JNK monomer from the dimer form of GSTp (Figure 4B). activity was inhibited by GSTp, suggests that GSTp does A puriﬁed form of either monomer or dimer was added not affect JNK kinase under non-stressed conditions. GSTp to a solid-phase kinase reaction in which pre-formed inhibition of JNK could be attributed to the nature of the full-length Jun –JNK complex served as a substrate. As complex between the two proteins. The ﬁnding that his shown, only the monomeric form of GSTp was capable overexpression of GSTp reduces MKK4 phosphorylation of mediating efﬁcient inhibition of JNK phosphorylation suggests that an excess of GSTp can also affect the of c-Jun (Figure 4B). MKK4–JNK module. Further evaluation of the possible interplay between GSTp expression is inversely correlated with JNK GSTp and upstream JNK kinases was carried out in cells activities that had been transfected with ΔMEKK1, an MKK4 In a complementary approach to study GSTp effects on upstream kinase. Forced expression of ΔMEKK1 led to JNK activity, increasing amounts of GSTp cDNA were constitutive phosphorylation of MKK4, even when GSTp transiently transfected into mouse ﬁbroblasts. As shown was co-transfected. Interestingly, although GSTp was not in Figure 5A, GSTp transfection resulted in a dose- able to reduce MKK4 phosphorylation in the presence of dependent decrease in JNK activation by UV. While ΔMEKK, it efﬁciently blocked JNK-mediated phos- transfection of 0.5 μg of GSTp caused a 20% decrease, phorylation of c-Jun (Figure 5BIII). These ﬁndings point 2 μg led to a 40% and 10 μg to a 50% decrease in JNK to a selective effect of GSTp on JNK. GSTp inhibition of activation by UV irradiation. These observations suggest MKK4 depends on the level of GSTp expression and the that it is possible to alter the degree of JNK activation by signal elicited by the upstream kinase (as shown here for GSTp transfection, albeit within the relatively narrow MEKK1). GSTp inhibition of JNK is due primarily to range of 0.5–2 μg. their association, which is released upon the conversion of GSTp from a monomer to a dimer form. Effect of GSTp on phosphorylation of JNK kinase MKK4/JNKK/SEK1 GSTp expression results in higher ubiquitination To elucidate further the mechanism underlying GSTp of c-Jun inhibition of JNK activity, we monitored possible Since JNK efﬁciently targets the ubiquitination of its non- changes at the level of MKK4 phosphorylation. In vitro phosphorylated associated proteins c-Jun, ATF2 and p53, phosphorylation of JNK by MKK4 was not inhibited by we determined the possible effects of GSTp on ubiquitin- GSTp (not shown). In non-stressed 3T3 ﬁbroblasts, there ation of JNK substrates in this reaction. Under non-stress is a basal level of MKK4 phosphorylation, detected by growth conditions, c-Jun exhibits a short half-life, which MKK4 phospho-antibodies (Figure 5BI). UV irradiation is prolonged upon phosphorylation by JNK (Fuchs et al., Fig. 3. (A) GSTp associates with Jun–JNK in vitro. The pre-formed Jun–JNK complex was incubated with whole-cell extract (10 μg) prepared before (WCE cont) or after (WCE UV) UV irradiation or with puriﬁed forms of GST isozymes (Ciaccio et al., 1991), as indicated. Following extensive washes, complex-bound and non-bound (absorbed on Jun–JNK; sup) material was analyzed on immunoblots with polyclonal antibodies that recognize multiple forms of GST (Ramgamaltha and Tew, 1991). Arrows point to the identiﬁed forms of GSTp. (B) Effect of different GST isozymes on JNK activity. Pre-formed Jun–JNK was incubated with the indicated forms of GST (a, α;m, μ;p, π; 2T, bacterially produced form of his GST) puriﬁed as described in Materials and methods before the addition of [γ- P]ATP. Autoradiography demonstrates the degree of c-Jun phosphorylation in the presence of the various GSTs. Quantiﬁcation of phosphorylation is shown in the graph. (C) Dose-dependent effect of GSTp on JNK kinase activity. The puriﬁed form of GSTp was added at the indicated concentrations (per μg of the relevant Jun substrate) to 5–89 32 GST–Jun –JNK (2 μg/reaction) or c-Jun–JNK (7 μg/reaction) prior to the addition of [γ- P]ATP. The degree of inhibitor activity was calculated his based on the ratio between Jun phosphorylation activities in the absence and in the presence of GSTp. The insets show representative autoradiographs of the respective reactions (– reﬂects the degree of phosphorylation without inhibitor added). Quantiﬁcation of three independent experiments is shown in the graph. (D) Speciﬁcity of GST as a JNK inhibitor. The ability of the puriﬁed form of GSTp to inhibit phosphorylation by Src, CKII, MAPK or PKA was tested. Bead-bound substrates were incubated with the respective kinases in the presence of GSTp before [γ- P]ATP was added to initiate the kinase reaction. After phosphorylation, the beads were washed and phosphorylation was quantiﬁed. The values shown represent average results of three different reactions. (E) Immunodepletion of GSTp increases JNK activity. Jun–JNK complex (containing JNK his puriﬁed from UV-treated cells) was incubated with whole-cell extract either from non-stressed cells (10 μg) or subjected to one or two sequential immunodepletions of GSTp (GST-1 or GST-2, respectively) using antibodies to GSTp before addition of [γ- P]ATP. The control reactions with whole-cell extracts treated under the same conditions with NRS and protein A/G beads are also shown. The inhibitory activity depicted was calculated based on values of c-Jun phosphorylation. The lower panel shows an immunoblot indicating the GSTp level after each of the immunodepletion reactions. (F) GSTp inhibitor increases JNK activities in vitro. Jun–JNK or GST–Jun–JNK was incubated with selective GST his inhibitors in the presence of the puriﬁed form of GSTp, prior to the addition of [γ- P]ATP. The degree of Jun phosphorylation in the presence of each of the inhibitors is shown. Peptides tested in parallel represent two ﬂexible domains from GSTp, corresponding to the N- (P2) and the C-terminal regions (P1). (G) GSTp inhibitor increases JNK activities in vivo. Mouse ﬁbroblasts were treated with TER-199, a speciﬁc inhibitor of GSTp, for 2 h followed by either sham or UV treatment (50 J/m ). Whole-cell extract proteins were prepared after 45 min and assayed for JNK activity by means of c-Jun phosphorylation. The inset shows an autoradiograph of Jun phosphorylation (– without; in the presence of his his TER-199), which was quantiﬁed as shown in the graph. 1327 V.Adler et al. 1996, 1997; Musti et al., 1997). Transfection of GSTp cDNA into 3T3 mouse ﬁbroblasts increased the level of c-Jun ubiquitination in vivo (Figure 5C). Since the level of ubiquitinated Jun is inversely correlated with its degree of phosphorylation (Fuchs et al., 1996; Musti et al., 1997), the increase in ubiquitinated c-Jun is an expected result of the GSTp inhibition of basal JNK activity, which reduces the number of c-Jun molecules that undergo phosphorylation. The noticeable increase in ubiquitinated c-Jun molecules provides an example of the physiological signiﬁcance of JNK inhibition under normal growth con- ditions. JNK activity in cells of GSTp null mice We established embryo ﬁbroblast cells from GSTP1/ (–/–) GSTP1/P2(–/–) P2 mice [MEF ] in order to assess JNK activity in a GSTp-free environment. These cells do not express GSTp as monitored by either RT–PCR (data not shown) or Western blot analysis (Figure 6A). Stress in the form of UV irradiation, sorbitol or anisomycin wt markedly increased JNK activity in both MEF and GSTP1/P2(–/–) MEF cells; of these treatments, UV elicited the greater degree of JNK activation (Figure 6B). Interestingly, a higher basal level of JNK activity was found in the GSTP1/P2(–/–) GSTwt MEF than in the MEF (Figure 6B and C). This high activity could be diminished in a dose-dependent manner upon transfection of GSTp cDNA (Figure 6C). Lesser amounts of GSTp cDNA were required to mediate 80–100% inhibition of UV-mediated JNK activation in GSTP1/P2(–/–) GSTwt the MEF cells, as compared with the MEF cells (not shown). In both types of MEFs, transfection of GSTp caused 80–100% inhibition of UV-mediated JNK activation, whereas in mouse ﬁbroblast 3T3 cells, GSTp inhibition reached only 50%. Such differences may be attributed to the overall amount and form (monomer and dimer) of GSTp and/or the levels of other radical scavenger enzymes expressed in each of the cell lines. To elucidate further the possible mechanism by which GSTp elicits its inhibition of JNK, we monitored levels Fig. 4. (A) Free radical scavengers prevent UV- or H O -mediated of JNK phosphorylation before and after stress using JNK 2 2 GSTP1/P2(–/–) GSTp–JNK complex dissociation and maintain low basal JNK activity. phospho-antibodies. In MEF cells, a higher Immunoprecipitations using antibodies to JNK (clone 333; basal level of JNK phosphorylation was seen (Figure PharMingen) were performed on proteins prepared before (–) and after GSTwt 2 6D I), when compared with the MEF cells. Forced UV irradiation (60 J/m )orH O treatment (10 μM added in 2 2 expression of GSTp in these cells revealed an efﬁcient phosphate-buffered saline (PBS) which was replaced after 5 min with medium for another 30 min) of mouse ﬁbroblasts. Cells were pre- reduction in the number of phospho groups on JNK, prior treated with NAC (10 mM) or eeGSH (1 mM added to the medium) to, as well as after, UV irradiation (Figure 6D II). Forced as indicated. JNK immunoprecipitates (from 3 mg of whole-cell expression of a truncated MEKK1 form (ΔMEKK1), extract) were examined by immunoblot analysis using antibodies to which elicits constitutively high levels of JNK activity, GSTp (I). (II) The same blot probed with JNK antibody (clone 333; PharMingen). (III) The level of JNK activity in immunoprecipitates partially restored the levels of JNK phosphorylation monitored by means of Jun phosphorylation. (IV) A GSTp his (Figure 6D II versus I). These observations are in line immunoblot reﬂecting the changes in the migration of GSTp with the effect of GSTp on phosphorylation of MKK4, (immunoprecipitated using antibodies to GSTp from cells treated as and suggest that GSTp elicits inhibition of JNK in vivo indicated in the ﬁgure) under non-reducing (without β-mercaptoethanol in spite of JNK phosphorylation by upstream kinases. in sample buffer) versus reducing SDS–PAGE. The blot shown in (a) was subjected to a 1 min exposure, whereas the blot in (b) was exposed for 1 h. Migration of puriﬁed GSTp is shown on the right Forced expression of GSTp decreases lane marked GSTp. The positions of the dimer (lower arrow), trimer transactivation of c-Jun in GST null cells (double arrow) and large complex (possible tetramer or higher) seen GSTP1/P2(–/–) MEFs derived from GSTp null mice [MEF ] under non-reducing SDS–PAGE conditions (upper arrow) are indicated. Molecular weight markers are indicated on the left panel. as well as from GSTp wild-type mice were co-transfected (B) The monomer form of GST mediates JNK inhibition. GSTp was with GSTp and Jun promoter (5 jun2 target sequence)- puriﬁed by means of gel ﬁltration (Superdex 75) to dissociate GSTP1/P2(–/–) driven luciferase constructs. MEF cells exhib- monomer from dimer forms of GSTp (a). By adding monomer or ited high basal levels of transcriptional activity mediated dimer GSTp forms to the Jun–JNK complex prior to the addition of his by the Jun2 promoter sequence (Figure 6E, control trans- [γ- P]ATP, the monomer form of GSTp was identiﬁed as the actual inhibitor of JNK activity (b). fection with empty construct, pcDNA3). This activity 1328 Regulation of JNK signaling by GSTp reﬂected the high basal JNK activity found in these cells (Figure 6B and C). Forced expression of GSTp efﬁciently reduced high basal Jun-driven transcriptional activity in GSTP1/P2(–/–) the MEF cells (Figure 6E), implicating the capacity of GST to reduce JNK phosphorylation and activity (Figures 6C and D). As the amino acid residues which are important for GST–GST dimerization and enzymatic activity have been identiﬁed previously (Shen et al., 1993), we mutated GSTp at the respective sites and evaluated their ability to elicit JNK inhibition. When GSTP1/P2(–/–) tested in MEF cells, GSTp whose cysteines were mutated at amino acids 47 and 101 (which are required for GSTp dimerization) and GST whose tyrosine was mutated at amino acid 7 (which abrogates catalytic proton transfer activity) were as potent as wild-type GSTp in inhibiting high basal JNK activity (not shown) and Jun-mediated transactivation (Figure 6E). The lower basal levels of Jun-mediated transactivation found in GSTwt MEF were also reduced by each of these constructs. The capacity of GSTp to reduce transcriptional activity mediated by the Jun promoter could be due to reduced phosphorylation of Jun and/or ATF2 by JNK, increased ubiquitination and degradation of c-Jun, or both. These observations further support the hypothesis that GSTp inhibition of JNK does not require GST enzymatic activity and is mediated by its monomeric form. Forced expression of peptide derived from the GSTp C-terminal domain Fig. 5. (A) Transfection of GSTp into mouse ﬁbroblasts reduces JNK activities. The ﬁgure shows the extent of JNK activation by UV in cells transfected with GSTp cDNA as compared with mock-transfected controls (100%). GSTp cDNA was co-transfected with the β-gal (0.5 μg) construct into 3T3 cells (via lipofection; lipofectamine, Gibco- BRL). Control empty vector was added to reach a constant amount of transfected DNA (15 μg). At 48 h post-transfection, cells were subjected to UV treatment (50 J/m ) and whole-cell extract proteins were prepared 45 min later. The inset depicts an immunoblot performed on the same protein extracts, revealing the respective increase in expression of GSTp (– reﬂects mock-transfected, lanes 2–4 represent 0.5, 2 and 10 μg of GSTp cDNA, respectively). Whole-cell extracts (normalized to transfection efﬁciency based on β-galactosidase values) were used for a kinase reaction using the c-Jun substrate (2 μg). his (B) (I) Forced expression of GSTp reduces MKK4 phosphorylation. Left panel: mouse 3T3 ﬁbroblasts analyzed for MKK4 phosphorylation using MKK4 phospho-antibodies. Forced expression of GSTp was achieved via lipofection of GSTp cDNA at the indicated concentrations (micrograms). TER-199 (at the micromolar concentration indicated in the ﬁgure) was added 22 h after transfection (2 h before protein preparations). The lower panel depicts the overall expression level of MKK4 (using non-phospho-antibodies). Right panel: level of MKK4 phosphorylation upon GSTp expression (0.5 μg) or UV irradiation (30 J/m ). PC represents positive control of phosphorylated MKK4. (III) Forced expression of GSTp does not affect ERK1 phosphorylation. Proteins prepared as indicated in (I) were subjected to immunoblot analysis using ERK1 phospho-antibodies. (III) Forced expression of ΔMEKK1 blocks GSTp effect on MKK4 but not inhibition of JNK phosphorylation of c-Jun. Mouse 3T3 ﬁbroblasts were co-transfected with ΔMEKK1 cDNA (1 μg) and empty vector (pcDNA3 to a total of 2.5 μg) or GSTp cDNA at the indicated concentrations. Proteins prepared 24 h after transfection were subjected to JNK immunokinase reaction (upper panel) using Jun as a substrate, or to immunoblot his analysis using antibodies to the phosphorylated form of MKK4 (middle panel). Analysis using non-phospho-MKK4 antibodies is shown in the lower panel. (C) Ubiquitination of c-Jun increases upon GSTp expression. Mouse 3T3 cells were co-transfected with Jun, GSTp and his Ub-HA as indicated in the ﬁgure. Jun was puriﬁed on Ni beads as his described (Terier et al., 1994) and the degree of ubiquitinated Jun was assessed by immunoblots with antibodies to hemagglutinin (HA). The area reﬂecting the polyubiquitination is marked on the right panel. The expression of c-Jun is shown on the lower panel. 1329 V.Adler et al. (which blocked GSTp inhibition in vitro) increased including c-Jun, ATF2 and p53 (Fuchs et al., 1996, 1998c; basal JNK kinase and c-Jun transcriptional activities, Musti et al., 1997), and to play a key role in maintenance respectively (Z.Yin, V.Adler and Z.Ronai, unpublished of controlled cell growth. Indeed, forced expression of observations). By monitoring c-Jun transcriptional activit- GSTp in 3T3 ﬁbroblasts increased the degree of Jun ies, these experiments point to the biological signiﬁcance ubiquitination and decreased Jun-mediated transactivation. of GSTp as an inhibitor of JNK. GSTp inhibition of JNK is found primarily in normally growing non-stressed cells. Stress, as shown in the case of UV irradiation, decreases this inhibition in a dose- Discussion dependent manner. Important to our understanding of In the present study, we identify and characterize GSTp GSTp’s ability to elicit association with JNK and inhibition as a JNK inhibitor. Our data point to an additional cellular of this kinase is the ﬁnding that the inhibitory activities mechanism that is involved in the regulation of JNK are conﬁned to the monomeric form of GSTp. When ﬁrst activity before and after stress. In non-stressed cells, the identiﬁed, the Jun–JNK-associated protein had a mol. wt basal levels of JNK phosphorylation are low, in spite of of 23 kDa, the characteristic size for the monomeric form constitutive stimulation by growth factors and endogen- of GSTp. Immunoprecipitation reactions always identiﬁed ously formed ROS. Maintaining a low basal JNK activity the monomeric form of GSTp as the Jun–JNK-associated is believed to affect the half-life of JNK substrates, protein. Similarly, the ability of the monomer (but not the dimer) to elicit JNK inhibitory activity in vitro supports the role of the monomer GSTp in JNK inhibition in vivo. UV irradiation reduces GSTp–JNK association, probably as a result of the formation of GST–GST dimers and multimers; because of disulﬁde bond-induced steric con- straints, dimers/multimers cannot accommodate the Jun– JNK complex. ROS scavengers, such as NAC or eeGSH, inhibit the formation of GSTp multimers, prevent GSTp dissociation from Jun–JNK and maintain the low basal activity of JNK as a kinase. The switch from a monomer to a dimer/multimer form is likely to provide the underlying mechanism for GSTp’s ability to sense and transmit changes in redox potential as a regulator of JNK signaling. Thus, based on its cellular conformation, GSTp dictates the association and inhibition of JNK. Importantly, while our studies demonstrate the effect of ROS elicited by UV and H O on GSTp dimerization, certain type of stress 2 2 Fig. 6. (A) Expression of GST in GSTp null mice. Expression of GST GSTP1/P2(–/–) GSTwt in MEF (lanes A) and MEF (lanes B) was determined using speciﬁc antibodies to GSTP1-1 (Henderson et al., 1998). (B) Basal and induced JNK activity in GSTp null cells. Proteins prepared (–/–) from either GSTP1/P2 (null) or GST wt MEF (MEF wt) before (C) or after exposure to UV irradiation (UV; 40 J/m ), sorbitol (S; 0.6 M) or anisomycin (A; 10 μg/ml) were subjected to a solid-phase kinase reaction using c-Jun as substrate. Levels of phosphorylated Jun are shown. (C) Basal JNK activity in MEF cells of GSTp null mice can be reduced by forced expression of GSTp. JNK activity was monitored in MEF cells derived from wild- –/– type (MEF wt) or GST null (GST P1/P2 ) mice transfected via lipofection with either empty vector (4 μg; ﬁrst lane on left) or GSTp cDNA (0.5, 2 or 4 μg). JNK activity was determined 24 h after transfection into each of the MEF lines. (D) JNK phosphorylation is affected by GST expression. JNK phosphorylation was monitored in GSTwt GSTP1/P2(–/–) MEF and MEF cells before (C) and after UV irradiation (UV) as well as after transfection with the constitutively active form of MEKK1 (ΔMEKK1). The effect of the empty vector (I) or GSTp cDNA (II–IV) on overall JNK phosphorylation (I and II) is shown. The levels of JNK (III) and GSTp expression (IV) are also shown. JNK phosphorylation was determined 30 min after mock or UV irradiation (which was administered 24 h after transfection) using antibodies to phosphorylated residues 183 and 185 on JNK (P-JNK). The control reactions using antibodies that recognize non- phosphorylated forms are shown in (III). (E) Transactivation of the (–/–) Jun-driven luciferase construct in GSTP1/P2 cells. Forced expression of GSTp or the indicated mutant forms in MEFs of (–/–) GSTpwt or MEFs of GSTP1/P2 mice was used to determine the effect on Jun transcriptional activities. Each of the GSTp constructs was co-transfected with the Jun2-luciferase vector (consisting of ﬁve repeats of the Jun2 sequence TGACATCA). The amount of luciferase activity was quantitated 24 h after transfection. Values shown were normalized with respect to transfection efﬁciency. 1330 Regulation of JNK signaling by GSTp Fig. 7. Model of GST inhibition of JNK signaling. Based on our ﬁndings, the following model is proposed: under non-stressed conditions, GSTp can be free or part of a complex with Jun–JNK. Upon stress, in which ROS are formed, GSTp forms dimers and larger aggregates which cannot accommodate Jun–JNK, thus enabling JNK phosphorylation of c-Jun, which as a result is a stable and active transcription factor. (i.e. certain cytokines) are not expected to cause the same as forced expression of GSTp did not affect ERK1/2 degree of ROS, and yet are strong inducers of JNK. The phosphorylation. Our data also imply that MEKK1 is not latter suggests that dissociation of GSTp from JNK may a target for GSTp inhibition; other MKK4 upstream also occur due to changes other than disulﬁde bond-based kinase(s), which include ASK1, TAKs, MLKs and GCKs, dimerization. The effect of post-translational modiﬁcations are currently being examined. Of interest is the observation of GSTp on its association with JNK is being investigated. that UV doses (10 J/m ) which decrease GST inhibitory The ﬁnding that GSTp transfection reduces basal and activities are insufﬁcient for JNK activation, which UV-inducible levels of JNK phosphorylation in vivo pro- requires doses 20 J/m . This suggests that conversion vides further insight into the mechanism by which GSTp of the monomer to the dimer may precede JNK ability to elicits inhibition of JNK signaling. It is possible to rescue elicit kinase activity MKK4/7, and is in agreement with GSTp-mediated inhibition of JNK phosphorylation by the ﬁnding that GSTp efﬁciently reduces JNK phospho- transfecting a constitutively active form of MEKK1. rylation of c-Jun in vivo, even when its upstream kinase Independently, under physiological conditions, GSTp MKK4 is active. inhibition of JNK takes place in spite of activated MKK4, That speciﬁc inhibitors of GSTp efﬁciently decrease possibly through direct inhibition of JNK-mediated Jun GSTp inhibition of JNK both in vitro (TER-117, TER-287) phosphorylation, supporting our original in vitro observa- and in vivo (TER-199) suggests that their binding to GSTp tion where GSTp inhibited c-Jun phosphorylation by an is within the domain which is also required for association activated form of JNK. The ability of GSTp to block JNK with Jun–JNK or, alternatively, that they alter the con- activity in spite of MKK4 and JNK phosphorylation formation of GSTp, affecting its association with JNK. by MEKK1 suggests that the nature of the GST–JNK Our results suggest that GSTp does not require transferase association disables JNK activity as a kinase. That endo- activity to mediate inhibition of JNK, in as much as genous GSTp does not affect the MKK4 phosphorylation bacterially produced GST, as well as GSTp mutated on level in non-stressed cells provides further evidence for Tyr7, which is essential for its catalytic activity, efﬁciently selective inhibition of the Jun–JNK module by physio- inhibited JNK enzyme and Jun transcriptional activities, logical levels of GSTp. That high expression of GSTp respectively. also reduces MKK4 phosphorylation could be attributed Important conﬁrmation of the ﬁnding that GSTp is a to possible inclusion of MKK4 with the GST–JNK module. regulator of JNK kinase activity comes from the use of (–/–) Our data suggest that the interplay between upstream cells from GSTP1/P2 -null mice (Henderson et al., (–/–) MKK4 signaling and the amount of monomer GSTp may 1998). GSTP1/P2 -derived MEFs revealed a noticeably determine the effect of GSTp on MKK4. Interplay between higher basal level of JNK activity, which was also reﬂected MEKK1–MKK4 and MKK4–JNK modules (Xia et al., in JNK’s phosphorylation and Jun-driven transcriptional GSTP1/P2(–/–) 1998) and their existence in complex with scaffold protein, activities. Transfection of GSTp into MEF cells which determines the speciﬁcity of these signaling cas- caused a decrease in high basal JNK phosphorylation, cades (Whitmarsh et al., 1998), may explain the nature kinase activity and Jun-mediated transactivation, further of changes elicited with different expression levels of supporting the role of GSTp as an inhibitor of JNK GSTp. The speciﬁcity of GSTp-mediated JNK inhibition signaling. was demonstrated via comparison of different protein It is well documented that GSTp levels vary between kinases in a solid-phase reaction, as well as in vivo, different cell types (Tew, 1994). This, together with 1331 V.Adler et al. previously (Ramgamathan and Tew, 1991; Henderson et al., 1998). variable cellular compartmentalization and the expression Antibodies to the phosphorylated form of MKK4, ERK1/2 were obtained of other detoxiﬁcation enzymes, will contribute to vari- from New England Biolabs. ability of GSTp inhibition of JNK activity. Indeed, Immunoprecipitations were carried out using 1 mg of protein extracts comparison of 3T3 ﬁbroblasts and MEFs revealed that and 500 ng of the antibodies, for 16 h at 4°C. Protein A/G beads (Gibco- BRL) were added (15 μl) for 30 min at room temperature before while GSTp transfection causes 100% inhibition in MEFs, washes were carried out in PBS supplemented with Tween-100 (0.5%). it is limited to 50% in the 3T3 ﬁbroblasts. Immunoblot analysis was performed using 50 μg of whole-cell extract Overexpression of GSTp has been associated with that had been separated on SDS–PAGE (10%) followed by electrotransfer transformation to malignancy (Sato, 1989) and acquired to PVDF membrane. Ponceau staining was carried out to conﬁrm equal resistance to electrophilic anticancer drugs (Nakagawa loading, followed by blocking (5% non-fat milk) and reaction with the appropriate antibodies (diluted 1:3000) for 16 h at 4°C. Reactions et al., 1990; Tew, 1994). The ﬁnding that GSTp is a were visualized using enhanced chemiluminesence (ECL) reagents modulator of JNK inhibitor and the relationship between (Amersham). Analysis of GSTp migration was performed as indicated expression of this protein and JNK inhibition suggests (Shen et al., 1993), using material that had been immunoprecipitated that cancer cells prone to overexpress GSTp may exhibit (0.3 μg of GSTp antibodies and 1 mg of proteins) from cells. Where indicated, separation under non-reducing conditions was performed in high intrinsic JNK inhibitory activity. In this model, tumor SDS–PAGE without including the reducing agent β-mercaptoethanol in cells overexpressing GSTp may escape apoptosis, which sample buffer. has been implicated as one of the end-points of JNK activity (Xia et al., 1995; Kasibhatla et al., 1998). Peptides and inhibitor The emerging model suggests that through its associ- Selective inhibitors of GST, including TER-117, TER-199, TER-291 and TER-317, were synthesized, puriﬁed to 93% purity, and kindly ation with the Jun–JNK complex under non-stressed condi- provided by Telik Inc. (San Francisco, CA). Peptides corresponding to tions, GSTp inhibits JNK phosphorylation and activity ﬂexible domains on GST [amino acids 194–201, sequence SSPEHVNR (Figure 7). Changes in the level of ROS elicited by UV (P1), and amino acids 36–50, sequence TIDTWMQGLLKPTCL (P2)] irradiation or H O treatments decrease the amount of were synthesized (Peptide Technologies Corp., Gaithersburg, MD) and 2 2 puriﬁed by HPLC to 98.5%. monomeric GSTp, resulting in a reduction of the GST– JNK complex, thereby enabling JNK phosphorylation UV irradiation and H O treatment 2 2 (Figure 7). The fact that transcriptionally active p53 and Cells in the logarithmic growth phase were exposed to UV-C (60 J/m , c-Jun directly affect transcription of radical scavenging which requires a 15 s exposure in PBS with the lids off), followed by enzymes including GST (Polyak et al., 1997; Komarova addition of medium and incubation for 45 min. When indicated, NAC (10 mM) was added to the cells 1 h prior to UV irradiation. For H O et al., 1998) points to the possible existence of an 2 2 treatment, medium from the culture dish was mixed with freshly diluted autoregulatory loop for GSTp regulation of JNK. Under H O (10 μM) and immediately applied to the ﬁbroblasts (10 cells). 2 2 such autoregulation, stress-mediated GSTp dimerization/ When indicated, H O was added in the presence of 1 mM eeGSH 2 2 multimerization could enable JNK activation, yielding (Sigma). transcriptionally active p53/Jun and, in turn, newly synthe- Protein kinase assays sized GSTp that re-forms a complex with JNK to limit Protein kinase assays were carried out using a fusion protein, GST–Jun the degree and duration of JNK kinase activity. Our studies (amino acids 5–89; Adler et al., 1992) or c-Jun (full-length; Treier his support this model, which provides a possible underlying et al., 1994) as a substrate. JNK2 was puriﬁed from UV-treated 3T3-4A mechanism for the regulation of stress kinases by altered cells as previously described (Adler et al., 1995a). The purity of bacterially produced c-Jun and of 3T3-4A-derived JNK was conﬁrmed redox potential (Adler et al., 1996; Gomez del Arco, by silver-stained SDS–PAGE. Solid-phase JNK assays were carried out 1996; Kuo et al., 1996; Wilhelm et al., 1997; reviewed as previously described (Adler et al., 1995b, 1996). Brieﬂy, the GST– by Finkel, 1998). Thus, as a guardian of JNK activities 5–89 full-length Jun or Jun fusion proteins (0.5 μg/assay) were bound to his in normally growing cells, GSTp may serve as a sensor glutathione–Sepharose or nickel beads, before incubation with the puriﬁed form of JNK (0.5 μg/assay) in the presence of kinase buffer of intracellular changes in redox potential that are elicited [20 mM HEPES, pH 7.6, 1 mM EGTA, 1 mM dithiothreitol (DDT), by various forms of stress. 2 mM MgCl , 2 mM MnCl , 5 mM NaF, 1 mM NaVO , 50 mM NaCl] 2 2 3 at room temperature for 15 min. The beads were pelleted and washed extensively with PBST [150 mM NaCl, 16 mM sodium phosphate, Materials and methods pH 7.5, 1% Triton X-100, 2 mM EDTA, 0.1% β-mercaptoethanol, 0.2 mM phenylmethylsulfonyl ﬂuoride (PMSF) and 5 mM benzamidine], Cells and protein preparation 32 before they were incubated with [γ- P]ATP (50 c.p.m./fmol; Amersham) The mouse ﬁbroblast cell line 3T3-4A (Adler et al., 1995a), kindly in the presence of kinase buffer. Following extensive washing, the provided by Dr Claudio Basilico, was maintained in Dulbecco’s modiﬁed phosphorylated Jun was boiled in SDS sample buffer and the eluted Eagle’s medium (DMEM) supplemented with 10% calf serum and proteins were run on a 15% SDS–polyacrylamide gel. The gel was antibiotics (Gibco-BRL). Cells were grown at 37°C with 5% CO . MEFs dried, and phosphorylation of the Jun substrate was determined by (–/–) from GSTP1/P2 cells and wild-type control animals were prepared autoradiography, followed by quantiﬁcation with a phosphoimager using standard protocols. MEFs were maintained in DMEM supple- (Bio-Rad). mented with 10% fetal bovine serum (FBS) for no longer than 2 weeks. To assay for the presence of the JNK inhibitor, we used Jun–JNK as Proteins were prepared from cells as previously described (Adler et al., a pre-formed complex. GST–Jun or Jun (0.5 μg) was ﬁrst pre-incubated his 1995a). In all cases, buffers contained a protease ‘cocktail’ (1 μg/ml of with the puriﬁed form of active JNK (0.5 μg), thus forming a Jun–JNK pepstatin, leupeptin and aprotinin) and phosphatase inhibitors (1 mM complex which subsequently was incubated with protein extracts (for sodium orthovanadate and 5 mM sodium ﬂuoride). 30 min) before addition of [γ- P]ATP (for 10 min) at room temperature. Important for the formation of these pre-formed complexes is the ratio Chemicals between Jun and JNK molecules (1:1 molar ratio) and the saturation of H O , eeGSH, GST and NAC were purchased fom Sigma. 2 2 the nickel or glutathione beads with EDTA or glutathione to prevent non-speciﬁc binding of cellular proteins. In all cases, the buffers used Antibodies, immunoprecipitations and immunoblots contained a cocktail of protease and phosphatase inhibitors (Adler et al., Antibodies to c-Jun, PKA and ERK1 were purchased from Santa Cruz. 1995c, 1997). Antibodies to JNK were obtained from PharMingen. Antibodies to CKII Other kinase assays were performed with peptides that are known to were purchased from Upstate Biotechnology and antibodies to phospho- serve as speciﬁc substrates for Src tyrosine kinases, MAPKs and for JNK were purchased from Promega. Antibodies to GSTs were as decribed PKA, all of which were purchased from Santa Cruz Biotech. Co. (Santa 1332 Regulation of JNK signaling by GSTp Cruz, CA). Histone H1 (Sigma) was used as a substrate for CKII. In Adler,V., Pincus,M.R., Brandt-Rauf,P. and Ronai,Z. (1995a) Complexes ras all cases, the substrates were covalently bound to beads using the of p21 with jun N-terminal kinase and Jun proteins. Proc. Natl immunolink kit (Pierce) according to the manufacturer’s recommenda- Acad. Sci. USA, 92, 10585–10589. tions. In these assays, 20 μl of bead-coupled peptides (1 μg) were Adler,V., Fuchs,S.Y., Kim,J., Kraft,A., King,M.P., Pelling,J. and Ronai,Z. incubated with the source of kinase in the presence or absence of the (1995b) Jun-NH -terminal kinase activation mediated by UV-induced inhibitor fraction, followed by extensive washing and quantiﬁcation. DNA lesions in melanoma and ﬁbroblast cells. Cell Growth Differ., 6, 1437–1446. Puriﬁcation of JNK inhibitor Adler,V., Shaffer,A., Kim,J., Dolan,L. and Ronai,Z. (1995c) UV- Protein extracts prepared from 3T3-4A mouse ﬁbroblast cells (4 mg/ml) irradiation and heat shock mediate JNK activation via alternate were precipitated with ammonium sulfate (50%). The precipitated pathways. J. Biol. Chem., 270, 26071–26077. material was dialyzed against kinase buffer and subjected to fractionation Adler,V., Polotskaya,A., Kim,J., Dolan,L., Davis,R., Pincus,M. and on small concentrators with cut-off membranes of 100 and 30 kDa Ronai,Z. (1995d) Dose rate and mode of exposure are key factors in (Millipore, Bedford, MA). Material that passed through the 100 kDa JNK activation by UV-irradiation. Carcinogenesis, 17, 2073–2076. membrane was placed on a 30 kDa column and the ﬁltrate (30 kDa) Adler,V., Pincus,M.R., Polotskaya,A., Montano,X., Friedman,F. and was concentrated further to a volume of 50 μl using a 10 kDa membrane Ronai,Z. (1996) Activation of c-Jun NH -kinase by UV irradiation is (Amicon). Concentrated material that was 10–30 kDa in size was ras dependent on p21 . J. Biol. Chem., 271, 23304–23309. separated on a Superdex 75 column (SMART system; Pharmacia) pre- Adler,V. et al. (1997) Conformation-dependent phosphorylation of p53. calibrated with respect to the position of the expected molecular weight Proc. Natl Acad. Sci. USA, 94, 1686–1691. using a combination of protein standards. Using kinase buffer and a Baichwal,V.R., Park,A. and Tjian,R. (1991) Control of c-Jun activity by ﬂow rate of 40 μl/min, fractions (200 μl) were collected and tested for interaction of a cell speciﬁc inhibitor with regulatory domain delta, inhibitor activity. In these assays, 20 μl fractions were added to pre- differences between c-jun and v-jun. Nature, 352, 165. formed Jun–JNK complex before addition of [γ- P]ATP in the his Cavigelli,M., Li,W.W., Lin,A., Su,B., Yoshioka,K. and Karin,M. (1996) presence of kinase buffer and a cocktail of protease and phosphatase The tumor promoter arsenite stimulates AP1 activity by inhibiting a inhibitors. Active fractions were pooled and applied to a MonoQ column JNK phosphatase. EMBO J., 15, 6269–6279. with a gradient of 20–500 mM NaCl in kinase buffer at a ﬂow rate of Ciaccio,P.J., Tew,K.D. and La Creta,F.P. (1991) Enzymatic conjugation 75 μl/min. MonoQ fractions were tested for inhibitory activity, and of chlorambucil with glutathione by human glutathione S-transferases positive fractions were pooled and loaded on a phenyl-Sepharose column and inhibition by ethacrynic acid. Biochem. Pharmacol., 42, 1504– in a buffer consisting of 20 mM K HPO pH 7.5, 10% glycerol and 2 4 0.6 M ammonium sulfate. Using a gradient elution scheme, the inhibitory Coso,O.A, Chiariello,M., Yu,J.C., Teramoto,H., Crespo,P., Xu,N., Miki,T. component was found in the 0.2 M ammonium sulfate fraction. To adjust and Gutkind,J.S. (1995) The small GTP-binding proteins Rac1 and for kinase buffer and to concentrate the fraction that contained inhibitory Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell, activity, the phenyl-Sepharose eluate was concentrated ona3kDa 81, 1137–1146. column and applied to a pre-formed Jun–JNK complex. This fraction his Cui,X.L. and Douglas,J.G. (1997) Arachidonic acid activates c-jun inhibited JNK activity (Table I). Material that was bound to the Jun– his N-terminal kinase through NADPH oxidase in rabbit proximal tubular JNK complex revealed a single protein on silver-stained SDS–PAGE. epithelial cells. Proc. Natl Acad. Sci. USA, 94, 3771–3776. Dhar,V., Adler,V., Lehman,A. and Ronai,Z. (1996) Impaired jun-NH - Microsequencing A large-scale preparation of the JNK inhibitor (Table I) was subjected terminal kinase activation by ultraviolet irradiation in ﬁbroblasts of to multiple separation steps as described above, followed by SDS–PAGE patients with Cockayne syndrome complementation group B. Cell and blotting onto a PVDF membrane. The purity of the single protein Growth Differ., 7, 841–846. identiﬁed by Ponceau staining on the PVDF membrane was conﬁrmed Fanger,G.R., Gerwins,P., Widmann,C., Jarpe,M.B., Johnson,G.L. (1997) in parallel by silver staining of the same material. The band identiﬁed MEKKs, GCKs, MLKs, PAKs, TAKs and tpls: upstream regulators on the PVDF membrane was excised and subjected to N-terminal of the c-Jun amino terminal kinases? Curr. Opin. Genet. Dev., 7, 67–74. analysis on an ABI protein sequencer Model 494 equipped with a Model Finkel,T. (1998) Oxygen radicals and signaling. Curr. Opin. Cell Biol., 140C phenylthiohydantoin microanalyzer. The sequence Pro-Pro-Tyr- 10, 248–253. Thr-Val-Val-Tyr-Phe-Pro-Val-Arg-Gly, which was obtained at the Flartaard,J.E., Bauer,K.E. and Kauvar,L.M. (1993) Isozyme speciﬁcity 10 pmol level, has 100% homology to the human GST P1-1. of novel glutathione S-transferase inhibitors. Cancer Chemother. Pharmacol., 33, 63–70. Puriﬁcation of GSTp Fuchs,S.Y. Dolan L.R., Davis,R.J. and Ronai,Z. (1996) JNK targets GSTp was puriﬁed from human placenta. GSTα and GSTμ were puriﬁed the ubiquitination of c-Jun in a phosphorylation-dependent manner. from human liver as described (Ciaccio et al., 1991), followed by Oncogene, 13, 1531–1535. Superdex 75 gel ﬁltration (SMART). Purity was conﬁrmed by silver- Fuchs,S.Y., Xie,B., Adler,V., Fried,V.A., Davis,R.J. and Ronai,Z. (1997) stained SDS–PAGE. c-Jun NH -terminal kinases target the ubiquitination of their associated transcription factors. J. Biol. Chem., 272, 32163–32168. Transcription and ubiquitination assays Fuchs,S.Y., Adler,V., Pincus,M.R. and Ronai,Z. 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Control of c-Jun activity by interaction of a cell-specific inhibitor with regulatory domain δ: Differences between v- and c-Jun
Cell, 63
A. Whitmarsh, J. Cavanagh, C. Tournier, Jun Yasuda, R. Davis (1998)
A mammalian scaffold complex that selectively mediates MAP kinase activation.
Science, 281 5383

Publisher: Springer Journals
Copyright: Copyright © European Molecular Biology Organization 1999
ISSN: 0261-4189
eISSN: 1460-2075
DOI: 10.1093/emboj/18.5.1321
Publisher site: See Article on Publisher Site

Abstract

The EMBO Journal Vol.18 No.5 pp.1321–1334, 1999 shock or inﬂammatory cytokines (Galcheva-Gargova et al., Victor Adler, Zhimin Yin, Serge Y.Fuchs, 1994; Kyriakis et al., 1994; Westwick et al., 1994). JNK Miriam Benezra, Lilliam Rosario , 1 2 activation in response to UV irradiation is mediated by Kenneth D.Tew , Matthew R.Pincus , PAK 3 4 upstream signaling components, including cdc42, p21 , Mohinder Sardana , Colin J.Henderson , ASK1, MLK, MEKK1, SEK1/MKK4, MKK7 (Coso et al., 4 5 C.Roland Wolf , Roger J.Davis and 1995; Fanger et al., 1997; Tournier et al., 1997; reviewed Ze’ev Ronai ras by Ip and Davis, 1998) and p21 (Minden et al., 1994; Adler et al., 1995a, 1996), in concert with nuclear DNA The Ruttenberg Cancer Center, Mount Sinai School of Medicine, One Gustave L.Levy Place, Box 1130, New York, NY 10029-6574, lesions (Adler et al., 1995b). Different forms of stress Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, have been shown to mediate JNK activation via various PA, Department of Pathology and Laboratory Medicine, SUNY cellular pathways (Adler et al., 1995c). Activated JNK Health Science Center, Brooklyn, NY, Protein Sequencing Facility, phosphorylates the transcription factors c-Jun, ATF2, p53 Merck Sharp & Dohme, West Point, PA, USA, Imperial Cancer Research Fund, Molecular Pharmacology Unit, Dundee, DD1 9SY, UK and ELK-1 (Kyriakis et al., 1994; Gupta et al., 1995; and Howard Hughes Medical Institute, University of Massachusetts, Whitmarsh et al., 1995; Adler et al., 1997; Fuchs et al., Worcester, MA, USA 1998a). Phosphorylation by JNK has been implicated in Corresponding author stabilization (Fuchs et al., 1997, 1998b) and transcriptional e-mail: [email protected] activity (Kyriakis et al., 1994) of its substrates, which directly contribute to the mammalian stress response Studies of low basal Jun N-terminal kinase (JNK) through changes in the cell cycle, DNA repair or apoptosis activity in non-stressed cells led us to identify a JNK (Xia et al., 1995; Fuchs et al., 1998a; Kasibhatla et al., inhibitor that was puriﬁed and identiﬁed as glutathione 1998). S-transferase Pi (GSTp) and was characterized as Despite the signiﬁcant advances in identifying the a JNK-associated protein. UV irradiation or H O 2 2 components of stress-activated protein kinases, the mech- treatment caused GSTp oligomerization and dissoci- anisms underlying the regulation of JNK before and ation of the GSTp–JNK complex, indicating that immediately after stress are not well understood. In cells it is the monomeric form of GSTp that elicits JNK maintained under normal growth conditions, the basal inhibition. Addition of puriﬁed GSTp to the Jun– activity of JNK is low, although JNK phosphorylation by JNK complex caused a dose-dependent inhibition of upstream kinases occurs in response to growth factors JNK activity. Conversely, immunodepleting GSTp from (Minden et al., 1994) and hence should also be observed protein extracts attenuated JNK inhibition. Further- in cells proliferating under normal growth conditions. more, JNK activity was increased in the presence of While some unstressed cell types have been found to speciﬁc GSTp inhibitors and a GSTp-derived peptide. contain high levels of JNK activity (Dhar et al., 1996), Forced expression of GSTp decreased MKK4 and the reason for this high basal level is unknown. A 2- to JNK phosphorylation which coincided with decreased 4-fold increase in constitutive JNK activity has been JNK activity, increased c-Jun ubiquitination and reported and, while this is lower than the activation upon decreased c-Jun-mediated transcription. Co-transfec- exposure to DNA-damaging agents, it is equivalent to tion of MEKK1 and GSTp restored MKK4 phos- tumor necrosis factor-α (TNF-α) treatment and the degree phorylation but did not affect GSTp inhibition of JNK of activation elicited by multiple exposures to low dose activity, suggesting that the effect of GSTp on JNK is UV-B (Adler et al., 1995d). As a key component in independent of the MEKK1–MKK4 module. Mouse regulating the stability and activity of its substrates (Fuchs embryo ﬁbroblasts from GSTp-null mice exhibited a et al., 1998c; Kyriakis et al., 1994), changes in JNK’s basal high basal level of JNK activity that could be reduced activity could inﬂuence key cellular functions, including by forced expression of GSTp cDNA. In demonstrating growth, apoptosis and transformation. the relationships between GSTp expression and its Neither transcript nor protein levels of JNK are affected association with JNK, our ﬁndings provide new insight by stress. JNK activation is accomplished upon its into the regulation of stress kinases. phosphorylation by upstream kinases in response to stress Keywords: GSTp/JNK/signaling/stress kinase (Coso et al., 1995; Tournier et al., 1997). Dephosphoryla- tion of JNK at a later stage (Cavigelli et al., 1996; Hanada et al., 1998) is thought to constitute the primary mechanism underlying the regulation of JNK activity as a kinase. Introduction Nevertheless, several studies have demonstrated the exist- Jun N-terminal kinases (JNKs) belong to the multi-member ence of JNK inhibitors in normal growing cells. Thus, family of stress kinases that are activated transiently in stress-mediated JNK activation may be independent of its response to UV- or X-irradiation, heat shock, osmotic upstream kinases. Among the cellular components © European Molecular Biology Organization 1321 V.Adler et al. involved in regulating JNK substrates is a ‘δ inhibitor’ 1995b), we refer to the Jun-bound kinase as JNK and to which blocks transactivation of c-Jun by interacting with the overall complex as Jun–JNK. the δ domain (Baichwal et al., 1991). This is an integral When cellular extracts prepared from unstressed cells 27 amino acid sequence (30–57) from the N-terminal were added to the pre-formed Jun–JNK complex before region of c-Jun required for JNK binding to c-Jun, enabling adding [γ- P]ATP, c-Jun phosphorylation was inhibited Jun phosphorylation (Adler et al., 1992) or ubiquitination by 80% (Figure 1A, compare lanes 1 and 2). To elucidate (Treier et al., 1994; Fuchs et al., 1996). Other JNK further the nature of this inhibition, proteins derived from cip/waf1 regulatory proteins include p21 , which has been the non-stressed cells were absorbed by pre-incubation shown to exert greater inhibition of JNK activity in with increasing amounts of Jun–JNK. Subsequently, response to stress (Shim et al., 1996). beads-bound c-Jun–JNK complexes were spun and the Our interest in exploring the nature of JNK’s low basal supernatant, which lacked Jun–JNK-bound proteins, was activity originated in the observation that extracts of non- tested to determine its ability to alter the degree of JNK- stressed cells efﬁciently inhibited c-Jun phosphorylation mediated Jun phosphorylation. Increasing the amounts of when added to a solid-phase kinase reaction. Our further Jun–JNK complexes used to absorb the putative inhibitor studies led to the puriﬁcation, identiﬁcation and character- resulted in a dose-dependent decrease of inhibitory activity (Figure 1A, lanes 3–6). To determine whether this inhibitor ization of glutathione S-tranferase Pi (GSTp) as a JNK activity was heat stable, the protein fraction that contained inhibitor, described in this report. the inhibitor activity was heated to 95°C for 5 min. This GSTs comprise a multigene family, of which GSTp is treatment abolished the fraction’s ability to block Jun the most prevalent and ubiquitous non-hepatic isozyme phosphorylation by JNKs (not shown). (Jakoby, 1978). Among cellular functions attributed to GSTs are ligand binding and xenobiotic detoxiﬁcation UV irradiation abolishes JNK inhibitor activities (Tew, 1994). Reduced glutathione (GSH) binds to the ‘G’ The presence of JNK inhibitory activity in non-stressed site of GSTp (and other GST isozymes) and plays an cells prompted us to determine possible changes to the important role in detoxiﬁcation of reactive oxygen species inhibitory activity after UV irradiation. As shown in Figure (ROS) and the maintenance of the cellular redox state 1B, extracts prepared after UV treatment lacked inhibitory (Sato et al., 1989). activity. The decrease in JNK inhibitor activity depended Among factors implicated in regulating JNK activity on the UV dose. Whereas a 10 J/m dose (a 6 s exposure) are ROS and altered redox potential (Adler et al., 1995c; caused a 40% reduction (as quantiﬁed using a phospho- Gomez-del-Arco et al., 1996; Cui et al., 1997; Wilhelm imager), a 40 J/m dose (a 25 s exposure) completely et al., 1997). ROS have also been associated with regula- abolished the inhibition. In all instances, analysis was tion of other signaling cascades, e.g. certain isozymes of performed using the same amount (20 μg) of protein protein kinase C (PKC; Konisshi et al., 1997) and mitogen- prepared 30 min after administering UV irradiation activated protein kinase (MAPK; Guyton et al., 1996). (Figure 1B). A correlation between JNK activation and The addition of exogenous oxidants or anti-oxidants has the inactivation of its inhibitory activity was also noted been found to inﬂuence the activation of MAPK/JNK in human melanoma cells, which require a 60 J/m dose (Lo et al., 1996; Wilhelm et al., 1997). for JNK activation. In melanoma cells, 40 J/m doses Changes in ROS can also directly inﬂuence transcrip- caused a 50% inhibition, whereas a 60 J/m dose abolished tional activity, as demonstrated for NF-κB (Wang et al., JNK inhibitory activities (not shown). 1995), Ref-1 (Xanthoudakis et al., 1992) and c-Jun (Gomez-del-Arco et al., 1996). Thus, evidence is Puriﬁcation of the JNK inhibitor accumulating that redox status can play an integral role To purify the putative inhibitor, extracts of normally in kinase-mediated stress response pathways. Our growing cells were subjected to the six steps described characterization of GSTp as an endogenous regulator of below. After each step, the presence of the putative JNK activity provides a novel function for a protein inhibitor (tested by measuring inhibition of c-Jun phospho- with previously characterized catalytic and ligand-binding rylation) was monitored in a solid-phase kinase assay properties. using the Jun–JNK (1 μg) complex. Proteins from non- his stressed cells were ﬁrst precipitated in ammonium sulfate (50%). After dialysis against kinase buffer, the non- Results precipitated proteins were concentrated on membranes A JNK inhibitor is present in cellular extracts of with various molecular weight cut-offs. Inhibitor activity normally growing cells was found in the ﬂow-through of both 100 and 30 kDa JNK activities were measured in solid-phase kinase reac- cut-off ﬁlters; it was retained on the 10 kDa membrane, tions using Jun as a substrate. Proteins prepared from indicating that the inhibitor is between 10 and 30 kDa in UV-treated 3T3-4A mouse ﬁbroblast cells exhibited molecular weight (Figure 1C). The 10–30 kDa fractions marked phosphorylation of c-Jun. The kinetics of JNK were loaded on a gel ﬁltration column; several fractions activation in these cells increased within 1–5 min, reaching within the 20–25 kDa range (based on calibration of peak levels within 20 min and declining to basal levels the gel ﬁltration column with known molecular weight within 3–4 h (Adler et al., 1995b). This is a transient standards) retained inhibitory activity (Figure 1D). Active activation that is monitored as a multifold increase of fractions were pooled and fractionated on a MonoQ anion kinase activity relative to the control (untreated) cells. exchange column; a 70 mM NaCl eluate was found to Since the assays are performed under conditions that contain most of the inhibitor activity (Figure 1E). Active enable selective binding of JNK isozymes (Adler et al., MonoQ fractions were then loaded onto a phenyl- 1322 Regulation of JNK signaling by GSTp Fig. 1. (A) UV-mediated JNK activation is inhibited by proteins obtained from cells maintained under normal growth conditions. Phosphorylation of Jun by JNK (lane 1) is inhibited when the pre-formed complex (Jun–JNK; total 1 μg at 1:1 ratio) is incubated with proteins (10 μg) from untreated cells before adding the [γ- P]ATP (lane 2). Pre-incubation of proteins from non-stressed cells with increasing concentrations of Jun–JNK complex resulted in a dose-dependent decrease of inhibitory activity (lanes 3–6 represent increasing amounts of the pre-formed complex 0.25 , 0.5, 1 and 2 μg, respectively). (B) UV irradiation abolishes inhibitor activities. Pre-formed Jun–JNK complex was incubated with whole-cell extract prepared 30 min after sham or UV-C irradiation at the doses indicated prior to addition of [γ- P]ATP. (C) Puriﬁcation of JNK inhibitor on molecular weight cut-off columns. Whole-cell extracts from non-stressed cells were subjected to an ammonium sulfate precipitation and the supernatants were fractionated on the indicated membranes with a cut-off mol. wt of 100, 30 or 10 kDa before being added to pre-formed Jun–JNK complex, followed by addition of [γ- P]ATP (the basal activity in UV-treated cells is shown in lane JNK). Shown is an autoradiograph of the phosphorylated c-Jun. (D) Gel ﬁltration of 3–30 kDa fractions. Fractions 30 kDa were concentrated and loaded (50 μl) onto a Superdex 75 column. From each fraction, 20 μl were added to the pre-formed Jun–JNK complex to measure the ability to inhibit JNK activity (the ﬁrst lane points to JNK activity with UV-treated extract before this inhibition; numbers represent the respective fractions). (E) Fractionation of JNK inhibitor via an anion exchange column. Fractions 18–24 obtained by Superdex 75 separation were pooled and applied to a MonoQ column. The fractions were assayed for inhibitor activities as shown in the inset. (F) Identifying the inhibitor component on the basis of its binding to Jun–JNK complex. Jun or Jun–JNK his his complex was incubated with the phenyl-Sepharose eluate (A) or with a puriﬁed form of GSTp (B). Beads-bound material was washed and separated on SDS–PAGE. Silver staining of bound proteins is shown in the left panel, whereas the immunoblot of a duplicate gel with antibodies to GSTp is shown on the right. The arrow points to the position of GSTp. 1323 V.Adler et al. Table I. Puriﬁcation of JNK inhibitor Puriﬁcation step Protein Total Speciﬁc activity (mg) activity (U) ( Jun–JNK) his Total lysate 80 10 160 0.127 Ammonium sulfate 32 8128 0.254 (50%) Membrane cut-off 6.7 6096 0.91 MonoQ 2.1 5425 2.69 Phenyl-Sepharose 1.4 4820 3.46 Jun–JNK 0.063 4511 70.70 his All puriﬁcation steps were performed using the Jun–JNK complex his (on nickel beads) as a substrate to measure the degree of JNK activity/ inhibition (see Materials and methods for details). Total activity is deﬁned in units:1Uisthe ability to decrease JNK activity by 50%. Speciﬁc activity is calculated as inhibitor activity of 1 μg of protein. Sepharose column in the presence of 0.6 M ammonium sulfate. Using decreased concentrations of ammonium sulfate, the inhibitor activity was found in 0.2 M eluate fractions (not shown). The active fractions from the phenyl-Sepharose column were incubated in a Jun– his JNK column. Analysis of the Jun–JNK-bound material his revealed a single band on silver-stained gels (Figure 1F). Microsequencing analysis of the corresponding band obtained after a large-scale puriﬁcation (Table I) revealed a 12 amino acid peptide (Pro-Pro-Tyr-Thr-Val-Val-Tyr- Phe-Pro-Val-Arg-Gly) that exhibited 100% homology to the human form of GSTp. To conﬁrm this protein’s identity, a puriﬁed form of GSTp was separated parallel to the Jun–JNK-bound material and subjected to Western his blot analysis using polyclonal antibodies to GST. Both migration and immunoreactivity of puriﬁed GSTp were identical to those of the Jun–JNK-bound material his (Figure 1F). GST inhibition of JNK activity does not alter Fig. 2. (A) GSTp as a JNK inhibitor. GSTp was added at the indicated phosphorylation of Jun or JNK concentrations (micrograms) to the pre-formed Jun–JNK complex and To determine whether GSTp affected the degree of JNK the level of Jun phosphorylation was measured by means of autoradio- graphy. (B) GSTp does not affect JNK phosphorylation. To measure or Jun phosphorylation, increasing concentrations of GSTp the effect of GSTp on JNK phosphorylation, JNK was immuno- (puriﬁed form) were added to pre-formed Jun–JNK com- precipitated from UV-treated cells and incubated with GSTp (at the plex which contained the phosphorylated form of JNK microgram concentrations indicated) followed by Western blot with obtained from UV-treated cells. GSTp decreased JNK antibodies to phospho-JNK (upper panel). The lower panel depicts the level of JNK on the same blot after re-probing with antibodies to JNK. phosphorylation of c-Jun in a dose-dependent manner (C) As a positive control, JNK from UV-treated cells was incubated (within a range of 0.05–1 μg) (Figure 2A), but it did not with no protein (lane C), dual speciﬁcity phosphatase (lane PP; decrease the number of phosphate groups on JNK, as Ishibashi et al., 1992) or GSTp (lane GSTp) before carrying out revealed by immunoblots with phospho-JNK antibodies immunoblot analysis with antibodies to phospho-JNK. Quantiﬁcation (Figure 2B). Dual activity protein phosphatase (Ishibashi via densitometer scanning revealed 35% inhibition of JNK phosphorylation by PP, whereas GSTp did not elicit such inhibition. et al., 1992) was used as a positive control in these (D) GSTp does not alter c-Jun phospho groups. The autoradiograph reactions (Figure 2C). depicts the c-Jun phosphorylation level after incubation with the To determine whether GSTp affects the number of inhibitor for the indicated time periods (minutes) before or after phospho groups on c-Jun, cellular extracts from normally phosphorylation by JNK. growing cells were incubated with pre-formed Jun–JNK complex for the indicated periods of time before or after the phosphorylation step with [γ- P]ATP. The extent of identiﬁed GSTp as the associated protein. A marked c-Jun phosphorylation was not altered when cell extracts decrease in this association was found in proteins prepared were added after the phosphorylation reaction (Figure after UV irradiation (Figure 3A). In addition to GSTp, 2D). This observation suggested that GSTp did not reduce isozymes of the GSTα and GSTμ families were also the number of phospho groups on c-Jun. capable of associating with the Jun–JNK complex in vitro (Figure 3A). GSTp exhibited greater JNK inhibitory activ- Effect of different GST isoforms on JNK activity ity than did GSTμ, which was more potent than GSTα Incubation of whole-cell extract prepared from non- (Figure 3B). Bacterially expressed GST (GST-2T) also stressed mouse ﬁbroblasts with the Jun–JNK complex mediated JNK inhibition (Figure 3B). This excludes the his 1324 Regulation of JNK signaling by GSTp possibility that the inhibitor activity was dependent on blocked GSTp inhibition of JNK activity (Figure 3F). any putative GST-associated cellular component. TER-317, structurally similar to TER-117, yet void of GSTp inhibitory properties, failed to affect GSTp inhibi- Dose-dependent inhibition of JNK by GSTp tion of JNK. Similarly, TER-199, an inactive prodrug GSTp inhibition of JNK activities was determined using of TER-117 (Flatgaard et al., 1993), was also without six concentrations of puriﬁed GSTp added to either full- effect in vitro. length or N-terminal forms of c-Jun. The addition of Molecular dynamic calculations on the energy- 5–89 puriﬁed GSTp to JNK complexed with GST–Jun or minimized X-ray crystal structure of GST were performed full-length Jun led to a concentration-dependent inhibition of to identify the most ﬂexible regions of the protein, i.e. his c-Jun phosphorylation (Figure 3C). The greater inhibitory those that were computed to have the highest degree of 5–89 capacity of GSTp observed for GST–Jun - compared ﬂuctuations. These regions are most likely to undergo full-length with Jun -based JNK complexes can be attributed signiﬁcant conformational changes during catalysis or his to the different conformations of the recombinant sub- ligand binding. Two such ﬂexible domains, corresponding 5–89 full-length strates (N-terminal GST–Jun versus Jun ). A respectively to amino acids 36–50 and 194–201, which his 5–89 higher degree of inhibition of the GST–Jun fusion can participate in the GST–Jun–JNK association and protein is not likely to occur as a result of GST–GST inhibition, were tested. interactions, which would require other experimental con- Peptides corresponding to each of these domains were ditions and result in different kinetics. Nevertheless, using synthesized, and their ability to alter GSTp inhibitor 5–89 full-length either GST–Jun or Jun as substrates, the activity was determined in vitro. As shown, the GSTp- his degree of GSTp inhibitory activity appeared to be linear derived peptide from amino acids 194–201 (at the within the range of 25–180 ng of GSTp (per microgram C-terminal domain designated P1) abrogated GSTp inhibi- of pre-formed Jun–JNK complex; Figure 3C). GSTp also tion of JNK activity (Figure 3F). Conversely, neither P2 inhibited JNK activity when added to a soluble form of (aa 36–50; Figure 3F) nor three unrelated, non-GST-based Jun–JNK complex (not shown). peptides of varying lengths (8–24 amino acids; not shown) his were capable of affecting GSTp inhibition of JNK. Treat- Speciﬁcity of GST inhibition ment of cells with TER-199 (the prodrug form of TER- To determine the speciﬁcity of the JNK inhibitor, puriﬁed 117 which is converted to active form in vivo and has GSTp was incubated with substrates for other protein been shown to elicit effective inhibition of GST activity kinases. Incubation with PKA, PKC, casein kinase II in vivo; Flatgaard et al., 1993) led to a 2-fold increase in (CKII) or MAPK revealed 6, 3, 36 and 9% inhibition, basal JNK activity in non-stressed cells. UV irradiation respectively. Under the same conditions, GST elicited also increased (~2.5-fold) JNK activation in TER-199- 86% inhibition of JNK (Figure 3D). In all cases, the treated cells (Figure 3G). That TER-199 also affected activities of the various kinases tested were normalized JNK activities after UV irradiation suggests that the dose (c.p.m./μg protein and an equal ratio between the respect- of UV irradiation was insufﬁcient to mediate complete ive substrate and GSTp) to ensure an equal degree of inactivation of GST as a JNK inhibitor; thus, the presence substrate phosphorylation. of a speciﬁc GSTp inhibitor led to an additive effect on overall JNK activities in these cells. The effect of these GSTp immunodepletion attenuates JNK inhibition GSH peptidomimetic agents is likely to be mediated either We further elucidated the ability of GSTp to inhibit JNK by altered conformation of the GSTp molecule or by a activity by modulating its levels or activity, or both, competition for the binding site, as non-active (bacterially in vitro and in vivo. The in vitro kinase assay in which produced) GST was also capable of eliciting JNK inhibi- proteins from non-stressed cells were added to the pre- tion (Figure 3B). formed Jun–JNK complex revealed a dose-dependent increase in the degree of JNK inhibition (of up to 80%). Changes in ROS affect GST oligomerization and its Immunodepletion of GST from whole-cell extract association with, and inhibition of, JNK decreased the degree of JNK inhibition from 80 to 45% We next assessed whether modulation of the cellular redox (Figure 3E, lane GST-1). Repeated immunodepletion by potential would affect JNK inhibition by GSTp. We GST antibodies further decreased the degree of JNK monitored the GST–JNK complex in vivo by means of inhibition to 20% (Figure 3E, lane GST-2). Control immun- immunoprecipitations followed by immunoblot analysis. odepletion reactions with normal rabbit serum (NRS) and Exposure of mouse ﬁbroblast cells to either UV or H O 2 2 protein A/G beads did not affect the degree of JNK reduced the amount of the JNK–GSTp complex and inhibition (Figure 3E, lanes NRS-1 and NRS-2, increased JNK activity, whereas pre-treating cells with the respectively). free radical scavengers N-acetylcysteine (NAC) or the ethyl-ester of glutathione (eeGSH) prevented JNK dissoci- Speciﬁc GSTp inhibitors and a GSTp-derived ation from GSTp and maintained GSTp inhibitory activity peptide efﬁciently alter JNK inhibition (Figure 4A I–III). These observations indicate that GST– As an independent approach to inhibit GSTp, we used JNK–Jun association is inversely correlated with JNK speciﬁc inhibitors which were shown to inactivate GSTp activity. To elucidate further changes to GST that take enzymatic activity in vitro and in vivo (Flatgaard et al., place upon altered redox conditions, we measured the 1993). Adding TER-117, a speciﬁc in vitro GSTp inhibitor, possible formation of GST–GST dimers/multimers through to a Jun–JNK complex at the same time as GSTp prevented disulﬁde bonds on neighboring cysteines. Monitoring the GSTp inhibition of JNK activity. Similarly, TER-293, migration of GSTp on non-reducing SDS–PAGE revealed another GSH peptidomimetic related to TER-117, also decreased monomer/dimer forms of GSTp in UV- or 1325 V.Adler et al. H O -treated cells (Figure 4A IV). Instead, a high IV, the single arrow in panel a points to the dimer and 2 2 molecular weight band corresponding to a multimer form panel b shows the monomer). H O treatment is known 2 2 of GSTp was detected (Figure 4A IV, upper arrow in to cause the formation of intra- or intersubunit disulﬁde panel a). Pre-treatment with NAC or eeGSH maintained bonds between cysteines at amino acids 47 and 101 of the lower molecular weight forms of GSTp (Figure 4A GST (Shen et al., 1993), resulting in a multimerization 1326 Regulation of JNK signaling by GSTp of GST subunits which no longer accommodates JNK efﬁciently increased MKK4 phosphorylation. Conversely, molecules. forced expression of GSTp reduced the level of MKK4 phosphorylation in a dose-dependent manner (Figure 5BI). Monomer form of GSTp mediates JNK inhibition Under the same conditions, GSTp did not alter phospho- The observation of changes in GSTp from monomer/ rylation of ERK1 (Figure 5BII). Treatment of GSTp- dimer to multimer forms upon exposure to increased ROS transfected cells with TER-199, an inhibitor of GSTp, prompted us to determine which of the two prevalent restored the level of MKK4 phosphorylation (Figure 5BI). GSTp forms, monomer or dimer, exerts inhibitory activity The observation that under physiological levels of GSTp on JNK. A gel ﬁltration column was used to separate the expression MKK4 was phosphorylated, although JNK monomer from the dimer form of GSTp (Figure 4B). activity was inhibited by GSTp, suggests that GSTp does A puriﬁed form of either monomer or dimer was added not affect JNK kinase under non-stressed conditions. GSTp to a solid-phase kinase reaction in which pre-formed inhibition of JNK could be attributed to the nature of the full-length Jun –JNK complex served as a substrate. As complex between the two proteins. The ﬁnding that his shown, only the monomeric form of GSTp was capable overexpression of GSTp reduces MKK4 phosphorylation of mediating efﬁcient inhibition of JNK phosphorylation suggests that an excess of GSTp can also affect the of c-Jun (Figure 4B). MKK4–JNK module. Further evaluation of the possible interplay between GSTp expression is inversely correlated with JNK GSTp and upstream JNK kinases was carried out in cells activities that had been transfected with ΔMEKK1, an MKK4 In a complementary approach to study GSTp effects on upstream kinase. Forced expression of ΔMEKK1 led to JNK activity, increasing amounts of GSTp cDNA were constitutive phosphorylation of MKK4, even when GSTp transiently transfected into mouse ﬁbroblasts. As shown was co-transfected. Interestingly, although GSTp was not in Figure 5A, GSTp transfection resulted in a dose- able to reduce MKK4 phosphorylation in the presence of dependent decrease in JNK activation by UV. While ΔMEKK, it efﬁciently blocked JNK-mediated phos- transfection of 0.5 μg of GSTp caused a 20% decrease, phorylation of c-Jun (Figure 5BIII). These ﬁndings point 2 μg led to a 40% and 10 μg to a 50% decrease in JNK to a selective effect of GSTp on JNK. GSTp inhibition of activation by UV irradiation. These observations suggest MKK4 depends on the level of GSTp expression and the that it is possible to alter the degree of JNK activation by signal elicited by the upstream kinase (as shown here for GSTp transfection, albeit within the relatively narrow MEKK1). GSTp inhibition of JNK is due primarily to range of 0.5–2 μg. their association, which is released upon the conversion of GSTp from a monomer to a dimer form. Effect of GSTp on phosphorylation of JNK kinase MKK4/JNKK/SEK1 GSTp expression results in higher ubiquitination To elucidate further the mechanism underlying GSTp of c-Jun inhibition of JNK activity, we monitored possible Since JNK efﬁciently targets the ubiquitination of its non- changes at the level of MKK4 phosphorylation. In vitro phosphorylated associated proteins c-Jun, ATF2 and p53, phosphorylation of JNK by MKK4 was not inhibited by we determined the possible effects of GSTp on ubiquitin- GSTp (not shown). In non-stressed 3T3 ﬁbroblasts, there ation of JNK substrates in this reaction. Under non-stress is a basal level of MKK4 phosphorylation, detected by growth conditions, c-Jun exhibits a short half-life, which MKK4 phospho-antibodies (Figure 5BI). UV irradiation is prolonged upon phosphorylation by JNK (Fuchs et al., Fig. 3. (A) GSTp associates with Jun–JNK in vitro. The pre-formed Jun–JNK complex was incubated with whole-cell extract (10 μg) prepared before (WCE cont) or after (WCE UV) UV irradiation or with puriﬁed forms of GST isozymes (Ciaccio et al., 1991), as indicated. Following extensive washes, complex-bound and non-bound (absorbed on Jun–JNK; sup) material was analyzed on immunoblots with polyclonal antibodies that recognize multiple forms of GST (Ramgamaltha and Tew, 1991). Arrows point to the identiﬁed forms of GSTp. (B) Effect of different GST isozymes on JNK activity. Pre-formed Jun–JNK was incubated with the indicated forms of GST (a, α;m, μ;p, π; 2T, bacterially produced form of his GST) puriﬁed as described in Materials and methods before the addition of [γ- P]ATP. Autoradiography demonstrates the degree of c-Jun phosphorylation in the presence of the various GSTs. Quantiﬁcation of phosphorylation is shown in the graph. (C) Dose-dependent effect of GSTp on JNK kinase activity. The puriﬁed form of GSTp was added at the indicated concentrations (per μg of the relevant Jun substrate) to 5–89 32 GST–Jun –JNK (2 μg/reaction) or c-Jun–JNK (7 μg/reaction) prior to the addition of [γ- P]ATP. The degree of inhibitor activity was calculated his based on the ratio between Jun phosphorylation activities in the absence and in the presence of GSTp. The insets show representative autoradiographs of the respective reactions (– reﬂects the degree of phosphorylation without inhibitor added). Quantiﬁcation of three independent experiments is shown in the graph. (D) Speciﬁcity of GST as a JNK inhibitor. The ability of the puriﬁed form of GSTp to inhibit phosphorylation by Src, CKII, MAPK or PKA was tested. Bead-bound substrates were incubated with the respective kinases in the presence of GSTp before [γ- P]ATP was added to initiate the kinase reaction. After phosphorylation, the beads were washed and phosphorylation was quantiﬁed. The values shown represent average results of three different reactions. (E) Immunodepletion of GSTp increases JNK activity. Jun–JNK complex (containing JNK his puriﬁed from UV-treated cells) was incubated with whole-cell extract either from non-stressed cells (10 μg) or subjected to one or two sequential immunodepletions of GSTp (GST-1 or GST-2, respectively) using antibodies to GSTp before addition of [γ- P]ATP. The control reactions with whole-cell extracts treated under the same conditions with NRS and protein A/G beads are also shown. The inhibitory activity depicted was calculated based on values of c-Jun phosphorylation. The lower panel shows an immunoblot indicating the GSTp level after each of the immunodepletion reactions. (F) GSTp inhibitor increases JNK activities in vitro. Jun–JNK or GST–Jun–JNK was incubated with selective GST his inhibitors in the presence of the puriﬁed form of GSTp, prior to the addition of [γ- P]ATP. The degree of Jun phosphorylation in the presence of each of the inhibitors is shown. Peptides tested in parallel represent two ﬂexible domains from GSTp, corresponding to the N- (P2) and the C-terminal regions (P1). (G) GSTp inhibitor increases JNK activities in vivo. Mouse ﬁbroblasts were treated with TER-199, a speciﬁc inhibitor of GSTp, for 2 h followed by either sham or UV treatment (50 J/m ). Whole-cell extract proteins were prepared after 45 min and assayed for JNK activity by means of c-Jun phosphorylation. The inset shows an autoradiograph of Jun phosphorylation (– without; in the presence of his his TER-199), which was quantiﬁed as shown in the graph. 1327 V.Adler et al. 1996, 1997; Musti et al., 1997). Transfection of GSTp cDNA into 3T3 mouse ﬁbroblasts increased the level of c-Jun ubiquitination in vivo (Figure 5C). Since the level of ubiquitinated Jun is inversely correlated with its degree of phosphorylation (Fuchs et al., 1996; Musti et al., 1997), the increase in ubiquitinated c-Jun is an expected result of the GSTp inhibition of basal JNK activity, which reduces the number of c-Jun molecules that undergo phosphorylation. The noticeable increase in ubiquitinated c-Jun molecules provides an example of the physiological signiﬁcance of JNK inhibition under normal growth con- ditions. JNK activity in cells of GSTp null mice We established embryo ﬁbroblast cells from GSTP1/ (–/–) GSTP1/P2(–/–) P2 mice [MEF ] in order to assess JNK activity in a GSTp-free environment. These cells do not express GSTp as monitored by either RT–PCR (data not shown) or Western blot analysis (Figure 6A). Stress in the form of UV irradiation, sorbitol or anisomycin wt markedly increased JNK activity in both MEF and GSTP1/P2(–/–) MEF cells; of these treatments, UV elicited the greater degree of JNK activation (Figure 6B). Interestingly, a higher basal level of JNK activity was found in the GSTP1/P2(–/–) GSTwt MEF than in the MEF (Figure 6B and C). This high activity could be diminished in a dose-dependent manner upon transfection of GSTp cDNA (Figure 6C). Lesser amounts of GSTp cDNA were required to mediate 80–100% inhibition of UV-mediated JNK activation in GSTP1/P2(–/–) GSTwt the MEF cells, as compared with the MEF cells (not shown). In both types of MEFs, transfection of GSTp caused 80–100% inhibition of UV-mediated JNK activation, whereas in mouse ﬁbroblast 3T3 cells, GSTp inhibition reached only 50%. Such differences may be attributed to the overall amount and form (monomer and dimer) of GSTp and/or the levels of other radical scavenger enzymes expressed in each of the cell lines. To elucidate further the possible mechanism by which GSTp elicits its inhibition of JNK, we monitored levels Fig. 4. (A) Free radical scavengers prevent UV- or H O -mediated of JNK phosphorylation before and after stress using JNK 2 2 GSTP1/P2(–/–) GSTp–JNK complex dissociation and maintain low basal JNK activity. phospho-antibodies. In MEF cells, a higher Immunoprecipitations using antibodies to JNK (clone 333; basal level of JNK phosphorylation was seen (Figure PharMingen) were performed on proteins prepared before (–) and after GSTwt 2 6D I), when compared with the MEF cells. Forced UV irradiation (60 J/m )orH O treatment (10 μM added in 2 2 expression of GSTp in these cells revealed an efﬁcient phosphate-buffered saline (PBS) which was replaced after 5 min with medium for another 30 min) of mouse ﬁbroblasts. Cells were pre- reduction in the number of phospho groups on JNK, prior treated with NAC (10 mM) or eeGSH (1 mM added to the medium) to, as well as after, UV irradiation (Figure 6D II). Forced as indicated. JNK immunoprecipitates (from 3 mg of whole-cell expression of a truncated MEKK1 form (ΔMEKK1), extract) were examined by immunoblot analysis using antibodies to which elicits constitutively high levels of JNK activity, GSTp (I). (II) The same blot probed with JNK antibody (clone 333; PharMingen). (III) The level of JNK activity in immunoprecipitates partially restored the levels of JNK phosphorylation monitored by means of Jun phosphorylation. (IV) A GSTp his (Figure 6D II versus I). These observations are in line immunoblot reﬂecting the changes in the migration of GSTp with the effect of GSTp on phosphorylation of MKK4, (immunoprecipitated using antibodies to GSTp from cells treated as and suggest that GSTp elicits inhibition of JNK in vivo indicated in the ﬁgure) under non-reducing (without β-mercaptoethanol in spite of JNK phosphorylation by upstream kinases. in sample buffer) versus reducing SDS–PAGE. The blot shown in (a) was subjected to a 1 min exposure, whereas the blot in (b) was exposed for 1 h. Migration of puriﬁed GSTp is shown on the right Forced expression of GSTp decreases lane marked GSTp. The positions of the dimer (lower arrow), trimer transactivation of c-Jun in GST null cells (double arrow) and large complex (possible tetramer or higher) seen GSTP1/P2(–/–) MEFs derived from GSTp null mice [MEF ] under non-reducing SDS–PAGE conditions (upper arrow) are indicated. Molecular weight markers are indicated on the left panel. as well as from GSTp wild-type mice were co-transfected (B) The monomer form of GST mediates JNK inhibition. GSTp was with GSTp and Jun promoter (5 jun2 target sequence)- puriﬁed by means of gel ﬁltration (Superdex 75) to dissociate GSTP1/P2(–/–) driven luciferase constructs. MEF cells exhib- monomer from dimer forms of GSTp (a). By adding monomer or ited high basal levels of transcriptional activity mediated dimer GSTp forms to the Jun–JNK complex prior to the addition of his by the Jun2 promoter sequence (Figure 6E, control trans- [γ- P]ATP, the monomer form of GSTp was identiﬁed as the actual inhibitor of JNK activity (b). fection with empty construct, pcDNA3). This activity 1328 Regulation of JNK signaling by GSTp reﬂected the high basal JNK activity found in these cells (Figure 6B and C). Forced expression of GSTp efﬁciently reduced high basal Jun-driven transcriptional activity in GSTP1/P2(–/–) the MEF cells (Figure 6E), implicating the capacity of GST to reduce JNK phosphorylation and activity (Figures 6C and D). As the amino acid residues which are important for GST–GST dimerization and enzymatic activity have been identiﬁed previously (Shen et al., 1993), we mutated GSTp at the respective sites and evaluated their ability to elicit JNK inhibition. When GSTP1/P2(–/–) tested in MEF cells, GSTp whose cysteines were mutated at amino acids 47 and 101 (which are required for GSTp dimerization) and GST whose tyrosine was mutated at amino acid 7 (which abrogates catalytic proton transfer activity) were as potent as wild-type GSTp in inhibiting high basal JNK activity (not shown) and Jun-mediated transactivation (Figure 6E). The lower basal levels of Jun-mediated transactivation found in GSTwt MEF were also reduced by each of these constructs. The capacity of GSTp to reduce transcriptional activity mediated by the Jun promoter could be due to reduced phosphorylation of Jun and/or ATF2 by JNK, increased ubiquitination and degradation of c-Jun, or both. These observations further support the hypothesis that GSTp inhibition of JNK does not require GST enzymatic activity and is mediated by its monomeric form. Forced expression of peptide derived from the GSTp C-terminal domain Fig. 5. (A) Transfection of GSTp into mouse ﬁbroblasts reduces JNK activities. The ﬁgure shows the extent of JNK activation by UV in cells transfected with GSTp cDNA as compared with mock-transfected controls (100%). GSTp cDNA was co-transfected with the β-gal (0.5 μg) construct into 3T3 cells (via lipofection; lipofectamine, Gibco- BRL). Control empty vector was added to reach a constant amount of transfected DNA (15 μg). At 48 h post-transfection, cells were subjected to UV treatment (50 J/m ) and whole-cell extract proteins were prepared 45 min later. The inset depicts an immunoblot performed on the same protein extracts, revealing the respective increase in expression of GSTp (– reﬂects mock-transfected, lanes 2–4 represent 0.5, 2 and 10 μg of GSTp cDNA, respectively). Whole-cell extracts (normalized to transfection efﬁciency based on β-galactosidase values) were used for a kinase reaction using the c-Jun substrate (2 μg). his (B) (I) Forced expression of GSTp reduces MKK4 phosphorylation. Left panel: mouse 3T3 ﬁbroblasts analyzed for MKK4 phosphorylation using MKK4 phospho-antibodies. Forced expression of GSTp was achieved via lipofection of GSTp cDNA at the indicated concentrations (micrograms). TER-199 (at the micromolar concentration indicated in the ﬁgure) was added 22 h after transfection (2 h before protein preparations). The lower panel depicts the overall expression level of MKK4 (using non-phospho-antibodies). Right panel: level of MKK4 phosphorylation upon GSTp expression (0.5 μg) or UV irradiation (30 J/m ). PC represents positive control of phosphorylated MKK4. (III) Forced expression of GSTp does not affect ERK1 phosphorylation. Proteins prepared as indicated in (I) were subjected to immunoblot analysis using ERK1 phospho-antibodies. (III) Forced expression of ΔMEKK1 blocks GSTp effect on MKK4 but not inhibition of JNK phosphorylation of c-Jun. Mouse 3T3 ﬁbroblasts were co-transfected with ΔMEKK1 cDNA (1 μg) and empty vector (pcDNA3 to a total of 2.5 μg) or GSTp cDNA at the indicated concentrations. Proteins prepared 24 h after transfection were subjected to JNK immunokinase reaction (upper panel) using Jun as a substrate, or to immunoblot his analysis using antibodies to the phosphorylated form of MKK4 (middle panel). Analysis using non-phospho-MKK4 antibodies is shown in the lower panel. (C) Ubiquitination of c-Jun increases upon GSTp expression. Mouse 3T3 cells were co-transfected with Jun, GSTp and his Ub-HA as indicated in the ﬁgure. Jun was puriﬁed on Ni beads as his described (Terier et al., 1994) and the degree of ubiquitinated Jun was assessed by immunoblots with antibodies to hemagglutinin (HA). The area reﬂecting the polyubiquitination is marked on the right panel. The expression of c-Jun is shown on the lower panel. 1329 V.Adler et al. (which blocked GSTp inhibition in vitro) increased including c-Jun, ATF2 and p53 (Fuchs et al., 1996, 1998c; basal JNK kinase and c-Jun transcriptional activities, Musti et al., 1997), and to play a key role in maintenance respectively (Z.Yin, V.Adler and Z.Ronai, unpublished of controlled cell growth. Indeed, forced expression of observations). By monitoring c-Jun transcriptional activit- GSTp in 3T3 ﬁbroblasts increased the degree of Jun ies, these experiments point to the biological signiﬁcance ubiquitination and decreased Jun-mediated transactivation. of GSTp as an inhibitor of JNK. GSTp inhibition of JNK is found primarily in normally growing non-stressed cells. Stress, as shown in the case of UV irradiation, decreases this inhibition in a dose- Discussion dependent manner. Important to our understanding of In the present study, we identify and characterize GSTp GSTp’s ability to elicit association with JNK and inhibition as a JNK inhibitor. Our data point to an additional cellular of this kinase is the ﬁnding that the inhibitory activities mechanism that is involved in the regulation of JNK are conﬁned to the monomeric form of GSTp. When ﬁrst activity before and after stress. In non-stressed cells, the identiﬁed, the Jun–JNK-associated protein had a mol. wt basal levels of JNK phosphorylation are low, in spite of of 23 kDa, the characteristic size for the monomeric form constitutive stimulation by growth factors and endogen- of GSTp. Immunoprecipitation reactions always identiﬁed ously formed ROS. Maintaining a low basal JNK activity the monomeric form of GSTp as the Jun–JNK-associated is believed to affect the half-life of JNK substrates, protein. Similarly, the ability of the monomer (but not the dimer) to elicit JNK inhibitory activity in vitro supports the role of the monomer GSTp in JNK inhibition in vivo. UV irradiation reduces GSTp–JNK association, probably as a result of the formation of GST–GST dimers and multimers; because of disulﬁde bond-induced steric con- straints, dimers/multimers cannot accommodate the Jun– JNK complex. ROS scavengers, such as NAC or eeGSH, inhibit the formation of GSTp multimers, prevent GSTp dissociation from Jun–JNK and maintain the low basal activity of JNK as a kinase. The switch from a monomer to a dimer/multimer form is likely to provide the underlying mechanism for GSTp’s ability to sense and transmit changes in redox potential as a regulator of JNK signaling. Thus, based on its cellular conformation, GSTp dictates the association and inhibition of JNK. Importantly, while our studies demonstrate the effect of ROS elicited by UV and H O on GSTp dimerization, certain type of stress 2 2 Fig. 6. (A) Expression of GST in GSTp null mice. Expression of GST GSTP1/P2(–/–) GSTwt in MEF (lanes A) and MEF (lanes B) was determined using speciﬁc antibodies to GSTP1-1 (Henderson et al., 1998). (B) Basal and induced JNK activity in GSTp null cells. Proteins prepared (–/–) from either GSTP1/P2 (null) or GST wt MEF (MEF wt) before (C) or after exposure to UV irradiation (UV; 40 J/m ), sorbitol (S; 0.6 M) or anisomycin (A; 10 μg/ml) were subjected to a solid-phase kinase reaction using c-Jun as substrate. Levels of phosphorylated Jun are shown. (C) Basal JNK activity in MEF cells of GSTp null mice can be reduced by forced expression of GSTp. JNK activity was monitored in MEF cells derived from wild- –/– type (MEF wt) or GST null (GST P1/P2 ) mice transfected via lipofection with either empty vector (4 μg; ﬁrst lane on left) or GSTp cDNA (0.5, 2 or 4 μg). JNK activity was determined 24 h after transfection into each of the MEF lines. (D) JNK phosphorylation is affected by GST expression. JNK phosphorylation was monitored in GSTwt GSTP1/P2(–/–) MEF and MEF cells before (C) and after UV irradiation (UV) as well as after transfection with the constitutively active form of MEKK1 (ΔMEKK1). The effect of the empty vector (I) or GSTp cDNA (II–IV) on overall JNK phosphorylation (I and II) is shown. The levels of JNK (III) and GSTp expression (IV) are also shown. JNK phosphorylation was determined 30 min after mock or UV irradiation (which was administered 24 h after transfection) using antibodies to phosphorylated residues 183 and 185 on JNK (P-JNK). The control reactions using antibodies that recognize non- phosphorylated forms are shown in (III). (E) Transactivation of the (–/–) Jun-driven luciferase construct in GSTP1/P2 cells. Forced expression of GSTp or the indicated mutant forms in MEFs of (–/–) GSTpwt or MEFs of GSTP1/P2 mice was used to determine the effect on Jun transcriptional activities. Each of the GSTp constructs was co-transfected with the Jun2-luciferase vector (consisting of ﬁve repeats of the Jun2 sequence TGACATCA). The amount of luciferase activity was quantitated 24 h after transfection. Values shown were normalized with respect to transfection efﬁciency. 1330 Regulation of JNK signaling by GSTp Fig. 7. Model of GST inhibition of JNK signaling. Based on our ﬁndings, the following model is proposed: under non-stressed conditions, GSTp can be free or part of a complex with Jun–JNK. Upon stress, in which ROS are formed, GSTp forms dimers and larger aggregates which cannot accommodate Jun–JNK, thus enabling JNK phosphorylation of c-Jun, which as a result is a stable and active transcription factor. (i.e. certain cytokines) are not expected to cause the same as forced expression of GSTp did not affect ERK1/2 degree of ROS, and yet are strong inducers of JNK. The phosphorylation. Our data also imply that MEKK1 is not latter suggests that dissociation of GSTp from JNK may a target for GSTp inhibition; other MKK4 upstream also occur due to changes other than disulﬁde bond-based kinase(s), which include ASK1, TAKs, MLKs and GCKs, dimerization. The effect of post-translational modiﬁcations are currently being examined. Of interest is the observation of GSTp on its association with JNK is being investigated. that UV doses (10 J/m ) which decrease GST inhibitory The ﬁnding that GSTp transfection reduces basal and activities are insufﬁcient for JNK activation, which UV-inducible levels of JNK phosphorylation in vivo pro- requires doses 20 J/m . This suggests that conversion vides further insight into the mechanism by which GSTp of the monomer to the dimer may precede JNK ability to elicits inhibition of JNK signaling. It is possible to rescue elicit kinase activity MKK4/7, and is in agreement with GSTp-mediated inhibition of JNK phosphorylation by the ﬁnding that GSTp efﬁciently reduces JNK phospho- transfecting a constitutively active form of MEKK1. rylation of c-Jun in vivo, even when its upstream kinase Independently, under physiological conditions, GSTp MKK4 is active. inhibition of JNK takes place in spite of activated MKK4, That speciﬁc inhibitors of GSTp efﬁciently decrease possibly through direct inhibition of JNK-mediated Jun GSTp inhibition of JNK both in vitro (TER-117, TER-287) phosphorylation, supporting our original in vitro observa- and in vivo (TER-199) suggests that their binding to GSTp tion where GSTp inhibited c-Jun phosphorylation by an is within the domain which is also required for association activated form of JNK. The ability of GSTp to block JNK with Jun–JNK or, alternatively, that they alter the con- activity in spite of MKK4 and JNK phosphorylation formation of GSTp, affecting its association with JNK. by MEKK1 suggests that the nature of the GST–JNK Our results suggest that GSTp does not require transferase association disables JNK activity as a kinase. That endo- activity to mediate inhibition of JNK, in as much as genous GSTp does not affect the MKK4 phosphorylation bacterially produced GST, as well as GSTp mutated on level in non-stressed cells provides further evidence for Tyr7, which is essential for its catalytic activity, efﬁciently selective inhibition of the Jun–JNK module by physio- inhibited JNK enzyme and Jun transcriptional activities, logical levels of GSTp. That high expression of GSTp respectively. also reduces MKK4 phosphorylation could be attributed Important conﬁrmation of the ﬁnding that GSTp is a to possible inclusion of MKK4 with the GST–JNK module. regulator of JNK kinase activity comes from the use of (–/–) Our data suggest that the interplay between upstream cells from GSTP1/P2 -null mice (Henderson et al., (–/–) MKK4 signaling and the amount of monomer GSTp may 1998). GSTP1/P2 -derived MEFs revealed a noticeably determine the effect of GSTp on MKK4. Interplay between higher basal level of JNK activity, which was also reﬂected MEKK1–MKK4 and MKK4–JNK modules (Xia et al., in JNK’s phosphorylation and Jun-driven transcriptional GSTP1/P2(–/–) 1998) and their existence in complex with scaffold protein, activities. Transfection of GSTp into MEF cells which determines the speciﬁcity of these signaling cas- caused a decrease in high basal JNK phosphorylation, cades (Whitmarsh et al., 1998), may explain the nature kinase activity and Jun-mediated transactivation, further of changes elicited with different expression levels of supporting the role of GSTp as an inhibitor of JNK GSTp. The speciﬁcity of GSTp-mediated JNK inhibition signaling. was demonstrated via comparison of different protein It is well documented that GSTp levels vary between kinases in a solid-phase reaction, as well as in vivo, different cell types (Tew, 1994). This, together with 1331 V.Adler et al. previously (Ramgamathan and Tew, 1991; Henderson et al., 1998). variable cellular compartmentalization and the expression Antibodies to the phosphorylated form of MKK4, ERK1/2 were obtained of other detoxiﬁcation enzymes, will contribute to vari- from New England Biolabs. ability of GSTp inhibition of JNK activity. Indeed, Immunoprecipitations were carried out using 1 mg of protein extracts comparison of 3T3 ﬁbroblasts and MEFs revealed that and 500 ng of the antibodies, for 16 h at 4°C. Protein A/G beads (Gibco- BRL) were added (15 μl) for 30 min at room temperature before while GSTp transfection causes 100% inhibition in MEFs, washes were carried out in PBS supplemented with Tween-100 (0.5%). it is limited to 50% in the 3T3 ﬁbroblasts. Immunoblot analysis was performed using 50 μg of whole-cell extract Overexpression of GSTp has been associated with that had been separated on SDS–PAGE (10%) followed by electrotransfer transformation to malignancy (Sato, 1989) and acquired to PVDF membrane. Ponceau staining was carried out to conﬁrm equal resistance to electrophilic anticancer drugs (Nakagawa loading, followed by blocking (5% non-fat milk) and reaction with the appropriate antibodies (diluted 1:3000) for 16 h at 4°C. Reactions et al., 1990; Tew, 1994). The ﬁnding that GSTp is a were visualized using enhanced chemiluminesence (ECL) reagents modulator of JNK inhibitor and the relationship between (Amersham). Analysis of GSTp migration was performed as indicated expression of this protein and JNK inhibition suggests (Shen et al., 1993), using material that had been immunoprecipitated that cancer cells prone to overexpress GSTp may exhibit (0.3 μg of GSTp antibodies and 1 mg of proteins) from cells. Where indicated, separation under non-reducing conditions was performed in high intrinsic JNK inhibitory activity. In this model, tumor SDS–PAGE without including the reducing agent β-mercaptoethanol in cells overexpressing GSTp may escape apoptosis, which sample buffer. has been implicated as one of the end-points of JNK activity (Xia et al., 1995; Kasibhatla et al., 1998). Peptides and inhibitor The emerging model suggests that through its associ- Selective inhibitors of GST, including TER-117, TER-199, TER-291 and TER-317, were synthesized, puriﬁed to 93% purity, and kindly ation with the Jun–JNK complex under non-stressed condi- provided by Telik Inc. (San Francisco, CA). Peptides corresponding to tions, GSTp inhibits JNK phosphorylation and activity ﬂexible domains on GST [amino acids 194–201, sequence SSPEHVNR (Figure 7). Changes in the level of ROS elicited by UV (P1), and amino acids 36–50, sequence TIDTWMQGLLKPTCL (P2)] irradiation or H O treatments decrease the amount of were synthesized (Peptide Technologies Corp., Gaithersburg, MD) and 2 2 puriﬁed by HPLC to 98.5%. monomeric GSTp, resulting in a reduction of the GST– JNK complex, thereby enabling JNK phosphorylation UV irradiation and H O treatment 2 2 (Figure 7). The fact that transcriptionally active p53 and Cells in the logarithmic growth phase were exposed to UV-C (60 J/m , c-Jun directly affect transcription of radical scavenging which requires a 15 s exposure in PBS with the lids off), followed by enzymes including GST (Polyak et al., 1997; Komarova addition of medium and incubation for 45 min. When indicated, NAC (10 mM) was added to the cells 1 h prior to UV irradiation. For H O et al., 1998) points to the possible existence of an 2 2 treatment, medium from the culture dish was mixed with freshly diluted autoregulatory loop for GSTp regulation of JNK. Under H O (10 μM) and immediately applied to the ﬁbroblasts (10 cells). 2 2 such autoregulation, stress-mediated GSTp dimerization/ When indicated, H O was added in the presence of 1 mM eeGSH 2 2 multimerization could enable JNK activation, yielding (Sigma). transcriptionally active p53/Jun and, in turn, newly synthe- Protein kinase assays sized GSTp that re-forms a complex with JNK to limit Protein kinase assays were carried out using a fusion protein, GST–Jun the degree and duration of JNK kinase activity. Our studies (amino acids 5–89; Adler et al., 1992) or c-Jun (full-length; Treier his support this model, which provides a possible underlying et al., 1994) as a substrate. JNK2 was puriﬁed from UV-treated 3T3-4A mechanism for the regulation of stress kinases by altered cells as previously described (Adler et al., 1995a). The purity of bacterially produced c-Jun and of 3T3-4A-derived JNK was conﬁrmed redox potential (Adler et al., 1996; Gomez del Arco, by silver-stained SDS–PAGE. Solid-phase JNK assays were carried out 1996; Kuo et al., 1996; Wilhelm et al., 1997; reviewed as previously described (Adler et al., 1995b, 1996). Brieﬂy, the GST– by Finkel, 1998). Thus, as a guardian of JNK activities 5–89 full-length Jun or Jun fusion proteins (0.5 μg/assay) were bound to his in normally growing cells, GSTp may serve as a sensor glutathione–Sepharose or nickel beads, before incubation with the puriﬁed form of JNK (0.5 μg/assay) in the presence of kinase buffer of intracellular changes in redox potential that are elicited [20 mM HEPES, pH 7.6, 1 mM EGTA, 1 mM dithiothreitol (DDT), by various forms of stress. 2 mM MgCl , 2 mM MnCl , 5 mM NaF, 1 mM NaVO , 50 mM NaCl] 2 2 3 at room temperature for 15 min. The beads were pelleted and washed extensively with PBST [150 mM NaCl, 16 mM sodium phosphate, Materials and methods pH 7.5, 1% Triton X-100, 2 mM EDTA, 0.1% β-mercaptoethanol, 0.2 mM phenylmethylsulfonyl ﬂuoride (PMSF) and 5 mM benzamidine], Cells and protein preparation 32 before they were incubated with [γ- P]ATP (50 c.p.m./fmol; Amersham) The mouse ﬁbroblast cell line 3T3-4A (Adler et al., 1995a), kindly in the presence of kinase buffer. Following extensive washing, the provided by Dr Claudio Basilico, was maintained in Dulbecco’s modiﬁed phosphorylated Jun was boiled in SDS sample buffer and the eluted Eagle’s medium (DMEM) supplemented with 10% calf serum and proteins were run on a 15% SDS–polyacrylamide gel. The gel was antibiotics (Gibco-BRL). Cells were grown at 37°C with 5% CO . MEFs dried, and phosphorylation of the Jun substrate was determined by (–/–) from GSTP1/P2 cells and wild-type control animals were prepared autoradiography, followed by quantiﬁcation with a phosphoimager using standard protocols. MEFs were maintained in DMEM supple- (Bio-Rad). mented with 10% fetal bovine serum (FBS) for no longer than 2 weeks. To assay for the presence of the JNK inhibitor, we used Jun–JNK as Proteins were prepared from cells as previously described (Adler et al., a pre-formed complex. GST–Jun or Jun (0.5 μg) was ﬁrst pre-incubated his 1995a). In all cases, buffers contained a protease ‘cocktail’ (1 μg/ml of with the puriﬁed form of active JNK (0.5 μg), thus forming a Jun–JNK pepstatin, leupeptin and aprotinin) and phosphatase inhibitors (1 mM complex which subsequently was incubated with protein extracts (for sodium orthovanadate and 5 mM sodium ﬂuoride). 30 min) before addition of [γ- P]ATP (for 10 min) at room temperature. Important for the formation of these pre-formed complexes is the ratio Chemicals between Jun and JNK molecules (1:1 molar ratio) and the saturation of H O , eeGSH, GST and NAC were purchased fom Sigma. 2 2 the nickel or glutathione beads with EDTA or glutathione to prevent non-speciﬁc binding of cellular proteins. In all cases, the buffers used Antibodies, immunoprecipitations and immunoblots contained a cocktail of protease and phosphatase inhibitors (Adler et al., Antibodies to c-Jun, PKA and ERK1 were purchased from Santa Cruz. 1995c, 1997). Antibodies to JNK were obtained from PharMingen. Antibodies to CKII Other kinase assays were performed with peptides that are known to were purchased from Upstate Biotechnology and antibodies to phospho- serve as speciﬁc substrates for Src tyrosine kinases, MAPKs and for JNK were purchased from Promega. Antibodies to GSTs were as decribed PKA, all of which were purchased from Santa Cruz Biotech. Co. (Santa 1332 Regulation of JNK signaling by GSTp Cruz, CA). Histone H1 (Sigma) was used as a substrate for CKII. In Adler,V., Pincus,M.R., Brandt-Rauf,P. and Ronai,Z. (1995a) Complexes ras all cases, the substrates were covalently bound to beads using the of p21 with jun N-terminal kinase and Jun proteins. Proc. Natl immunolink kit (Pierce) according to the manufacturer’s recommenda- Acad. Sci. USA, 92, 10585–10589. tions. In these assays, 20 μl of bead-coupled peptides (1 μg) were Adler,V., Fuchs,S.Y., Kim,J., Kraft,A., King,M.P., Pelling,J. and Ronai,Z. incubated with the source of kinase in the presence or absence of the (1995b) Jun-NH -terminal kinase activation mediated by UV-induced inhibitor fraction, followed by extensive washing and quantiﬁcation. DNA lesions in melanoma and ﬁbroblast cells. Cell Growth Differ., 6, 1437–1446. Puriﬁcation of JNK inhibitor Adler,V., Shaffer,A., Kim,J., Dolan,L. and Ronai,Z. (1995c) UV- Protein extracts prepared from 3T3-4A mouse ﬁbroblast cells (4 mg/ml) irradiation and heat shock mediate JNK activation via alternate were precipitated with ammonium sulfate (50%). The precipitated pathways. J. Biol. Chem., 270, 26071–26077. material was dialyzed against kinase buffer and subjected to fractionation Adler,V., Polotskaya,A., Kim,J., Dolan,L., Davis,R., Pincus,M. and on small concentrators with cut-off membranes of 100 and 30 kDa Ronai,Z. (1995d) Dose rate and mode of exposure are key factors in (Millipore, Bedford, MA). Material that passed through the 100 kDa JNK activation by UV-irradiation. Carcinogenesis, 17, 2073–2076. membrane was placed on a 30 kDa column and the ﬁltrate (30 kDa) Adler,V., Pincus,M.R., Polotskaya,A., Montano,X., Friedman,F. and was concentrated further to a volume of 50 μl using a 10 kDa membrane Ronai,Z. (1996) Activation of c-Jun NH -kinase by UV irradiation is (Amicon). Concentrated material that was 10–30 kDa in size was ras dependent on p21 . J. Biol. Chem., 271, 23304–23309. separated on a Superdex 75 column (SMART system; Pharmacia) pre- Adler,V. et al. (1997) Conformation-dependent phosphorylation of p53. calibrated with respect to the position of the expected molecular weight Proc. Natl Acad. Sci. USA, 94, 1686–1691. using a combination of protein standards. Using kinase buffer and a Baichwal,V.R., Park,A. and Tjian,R. (1991) Control of c-Jun activity by ﬂow rate of 40 μl/min, fractions (200 μl) were collected and tested for interaction of a cell speciﬁc inhibitor with regulatory domain delta, inhibitor activity. In these assays, 20 μl fractions were added to pre- differences between c-jun and v-jun. Nature, 352, 165. formed Jun–JNK complex before addition of [γ- P]ATP in the his Cavigelli,M., Li,W.W., Lin,A., Su,B., Yoshioka,K. and Karin,M. (1996) presence of kinase buffer and a cocktail of protease and phosphatase The tumor promoter arsenite stimulates AP1 activity by inhibiting a inhibitors. Active fractions were pooled and applied to a MonoQ column JNK phosphatase. EMBO J., 15, 6269–6279. with a gradient of 20–500 mM NaCl in kinase buffer at a ﬂow rate of Ciaccio,P.J., Tew,K.D. and La Creta,F.P. (1991) Enzymatic conjugation 75 μl/min. MonoQ fractions were tested for inhibitory activity, and of chlorambucil with glutathione by human glutathione S-transferases positive fractions were pooled and loaded on a phenyl-Sepharose column and inhibition by ethacrynic acid. Biochem. Pharmacol., 42, 1504– in a buffer consisting of 20 mM K HPO pH 7.5, 10% glycerol and 2 4 0.6 M ammonium sulfate. Using a gradient elution scheme, the inhibitory Coso,O.A, Chiariello,M., Yu,J.C., Teramoto,H., Crespo,P., Xu,N., Miki,T. component was found in the 0.2 M ammonium sulfate fraction. To adjust and Gutkind,J.S. (1995) The small GTP-binding proteins Rac1 and for kinase buffer and to concentrate the fraction that contained inhibitory Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell, activity, the phenyl-Sepharose eluate was concentrated ona3kDa 81, 1137–1146. column and applied to a pre-formed Jun–JNK complex. This fraction his Cui,X.L. and Douglas,J.G. (1997) Arachidonic acid activates c-jun inhibited JNK activity (Table I). Material that was bound to the Jun– his N-terminal kinase through NADPH oxidase in rabbit proximal tubular JNK complex revealed a single protein on silver-stained SDS–PAGE. epithelial cells. Proc. Natl Acad. Sci. USA, 94, 3771–3776. Dhar,V., Adler,V., Lehman,A. and Ronai,Z. (1996) Impaired jun-NH - Microsequencing A large-scale preparation of the JNK inhibitor (Table I) was subjected terminal kinase activation by ultraviolet irradiation in ﬁbroblasts of to multiple separation steps as described above, followed by SDS–PAGE patients with Cockayne syndrome complementation group B. Cell and blotting onto a PVDF membrane. The purity of the single protein Growth Differ., 7, 841–846. identiﬁed by Ponceau staining on the PVDF membrane was conﬁrmed Fanger,G.R., Gerwins,P., Widmann,C., Jarpe,M.B., Johnson,G.L. (1997) in parallel by silver staining of the same material. The band identiﬁed MEKKs, GCKs, MLKs, PAKs, TAKs and tpls: upstream regulators on the PVDF membrane was excised and subjected to N-terminal of the c-Jun amino terminal kinases? Curr. Opin. Genet. Dev., 7, 67–74. analysis on an ABI protein sequencer Model 494 equipped with a Model Finkel,T. (1998) Oxygen radicals and signaling. Curr. Opin. Cell Biol., 140C phenylthiohydantoin microanalyzer. The sequence Pro-Pro-Tyr- 10, 248–253. Thr-Val-Val-Tyr-Phe-Pro-Val-Arg-Gly, which was obtained at the Flartaard,J.E., Bauer,K.E. and Kauvar,L.M. (1993) Isozyme speciﬁcity 10 pmol level, has 100% homology to the human GST P1-1. of novel glutathione S-transferase inhibitors. Cancer Chemother. Pharmacol., 33, 63–70. Puriﬁcation of GSTp Fuchs,S.Y. Dolan L.R., Davis,R.J. and Ronai,Z. (1996) JNK targets GSTp was puriﬁed from human placenta. GSTα and GSTμ were puriﬁed the ubiquitination of c-Jun in a phosphorylation-dependent manner. from human liver as described (Ciaccio et al., 1991), followed by Oncogene, 13, 1531–1535. Superdex 75 gel ﬁltration (SMART). Purity was conﬁrmed by silver- Fuchs,S.Y., Xie,B., Adler,V., Fried,V.A., Davis,R.J. and Ronai,Z. (1997) stained SDS–PAGE. c-Jun NH -terminal kinases target the ubiquitination of their associated transcription factors. J. Biol. Chem., 272, 32163–32168. Transcription and ubiquitination assays Fuchs,S.Y., Adler,V., Pincus,M.R. and Ronai,Z. (1998a) MEKK1/JNK Transcriptional analysis of Jun was carried out using the 5 Jun2-driven stabilizes and activates p53. Proc. Natl Acad. Sci. USA, 95, 10541– luciferase construct as previously reported (van Dam et al., 1998). In all cases, values were normalized with respect to transfection efﬁciency. Fuchs,S.Y., Adler,V., Buschmann,T., Yin,Z., Wu,X., Jones,S.T. and In vivo ubiquitination assays were carried out by transfection of Jun and Ronai,Z. (1998b) JNK targets p53 ubiquitination and degradation in HA-tagged ubiquitin into the 3T3 cells as indicated elsewhere (Treier nonstressed cells. Genes Dev., 12, 2658–2663. et al., 1994). Fuchs,S.Y., Fried,V. and Ronai,Z. (1998c) Stress activated kinase regulates protein stability. Oncogene, 17, 1483–1490. Acknowledgements Galcheva-Gargova,Z., Derijard,B., Wu,I.-H. and Davis,R.J. (1994) An osmosensing signal transducing pathway in mammalian cells. Science, We are grateful to Andras Schaffer, Mark Bluth, Zheng Dong and 265, 806–808. 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The EMBO Journal – Springer Journals

Published: Mar 1, 1999

Keywords: GSTp; JNK; signaling; stress kinase

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Regulation of JNK signaling by GSTp

Regulation of JNK signaling by GSTp

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Regulation of JNK signaling by GSTp

Regulation of JNK signaling by GSTp

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