¹Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona, AZ 85721-0207, USA

²Program in Molecular Signal Transduction, Division of Basic Sciences, National Jewish Medical and Research Center, Denver, Colorado, CO 80206, USA

Correspondence to: Richard R Vaillancourt, Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona, AZ 85721-0207, USA

^aCurrent address: Corixa Corporation, 1124 Columbia Street, Seattle, Washington, WA 98104, USA

Arsenate and arsenite activate c-Jun N-terminal kinase (JNK), however, the mechanism by which this occurs is not known. By expressing inhibitory mutant small GTP-binding proteins, p21-activated kinase (PAK) and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinases (MEKKs), we have identified specific proteins that are involved in arsenate- and arsenite-mediated activation of JNK. We observe a distinct difference between arsenate and arsenite signaling, which demonstrates that arsenate and arsenite are capable of activating unique proteins. Both arsenate and arsenite activation of JNK requires Rac and Rho. Neither arsenate nor arsenite signaling was inhibited by a dominant-negative mutant of Cdc42 or Ras. Arsenite stimulation of JNK requires PAK, whereas arsenate-mediated activation of JNK was unaffected by inhibitory mutant PAK. Of the four MEKKs tested, only MEKK3 and MEKK4 are involved in arsenate-mediated activation of JNK. In contrast, arsenite-mediated JNK activation requires MEKK2, MEKK3 and MEKK4. These results better define the mechanisms by which arsenate and arsenite activate JNK and demonstrate differences in the regulation of signal transduction pathways by these inorganic arsenic species.

arsenic; Rac; Rho; PAK; MEKK3; MEKK4

Introduction

c-Jun N-terminal kinase (JNK) is a member of the stress-activated protein kinase family and is activated by cellular stress, such as osmotic shock and irradiation [reviewed in (Treisman, 1996)]. JNK activation requires the small GTP-binding proteins Ras, Rac, Cdc42 and Rho (Coso et al., 1995; Derijard et al., 1994; Minden et al., 1995; Teramoto et al., 1996). These small GTP-binding proteins associate with and activate a variety of serine/threonine kinases that are important in JNK activation including p21-activated kinase (PAK), as well as mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase nnase kinases [MEKKs; (Fanger et al., 1997a)]. The MEKKs phosphorylate and activate JNK kinase (JNKK), which phosphorylates and activates JNK. Upon activation, JNK alters specific gene transcription events via phosphorylation of the transcription factor, c-Jun, which forms a heterodimer with c-Fos to form the AP-1 transacting factor. Activation of JNK is commonly associated with inhibition of cell growth and/or apoptosis (Kyriakis and Avruch, 1996). However, under certain circumstances, sustained JNK activation, as opposed to transient activation, is required for an apoptotic response (Guo et al., 1998).

Toxic doses of inorganic arsenicals, such as arsenite and arsenate, produce pleiotrophic effects. Using cultured macrophages as an in vitro system to study arsenic toxicity, it was observed that 80% of the dead cells were necrotic while 20% of the cells were apoptotic at the LD₅₀ dose of 5 M arsenite and 500 M arsenate (Sakurai et al., 1998). In addition to cell death, chronic arsenic exposure has been associated with malignant transformation and DNA hypomethylation of epithelial cells (Zhao et al., 1997). Furthermore, epidemiological studies demonstrate that arsenic is a human carcinogen (Smith et al., 1998). However, the poor mutagenicity of inorganic arsenicals (Jacobson-Kram and Montalbano, 1985; Lee et al., 1985; Rossman et al., 1980) suggests that the production of diverse physiological effects caused by arsenic are likely due to the activation of various signaling pathways and not a genotoxic effect. Consistent with this line of reasoning is the observation that arsenic activates multiple MAPK pathways (Liu et al., 1996). Consequently, the spectrum of physiological responses caused by arsenic range from carcinogenesis to cell death.

Both arsenate and arsenite activate JNK. However, the mechanism by which these arsenic species activate this pathway has not been characterized. One report suggests that arsenite activates JNK by inactivation of a JNK phosphatase (Cavigelli et al., 1996). Since arsenite has been shown to bind and inactivate proteins containing thiol functional groups, arsenite may bind to thiol groups in the active site of a JNK phosphatase. Because phosphorylation of JNK is critical for its activation, this mechanism does not explain how JNK is phosphorylated and thus activated in the first place. In addition, since arsenate does not bind to thiol groups, this mechanism does not explain how arsenate activates JNK. Thus, it is possible that arsenate and arsenite are capable of regulating specific signal transduction pathways associated with JNK activation.

It has been well established that arsenite is an activator of the stress-activated protein kinase pathways (Cavigelli et al., 1996; Liu et al., 1996). We have determined that arsenate [As(V)], which is the oxidized precursor of arsenite [As(III)], can also activate JNK. Based on this observation, we wanted to determine whether arsenite and arsenate utilize the same signaling proteins to activate JNK. We set out to map these signaling pathways by using a series of inhibitory mutant proteins that are important for JNK activation by other stimuli. We have transfected inhibitory mutant small GTP-binding proteins, PAK, and MEKK1-4 into human embryonic kidney (HEK) 293 cells to determine which proteins were capable of blocking activation of JNK by either arsenate or arsenite. The data from our experiments show that arsenate and arsenite activate JNK through different signal transduction pathways, which require specific small GTP-binding proteins and serine/threonine kinases.

The goal of this study was to determine whether arsenate and arsenite activate JNK through the same upstream proteins. One feature that differentiates arsenate from arsenite is the relative permeability of each inorganic arsenic species across the plasma membrane. Arsenate must be transported into the cell by the organic anion transport protein, whereas arsenite freely crosses the plasma membrane (Kenney and Kaplan, 1988). In the presence of phosphate, arsenate is poorly transported into the cell since phosphate competes with arsenate for binding to the anion transport protein. Thus, the mechanism by which arsenic signal transduction is initiated by each arsenic species is potentially different.

Results

Arsenate is an activator of JNK

We used HEK 293 cells and transient transfection of cDNAs that encode candidate proteins as an approach to identify proteins that function upstream of JNK. We measured JNK activity in HEK 293 cells to establish that the signaling proteins were expressed in these cells. Since arsenate is transported into the cell by the anion transport protein (Kenney and Kaplan, 1988), a competition exists between arsenate and phosphate for the anion transport protein. To overcome this competition, HEK 293 cells were incubated in phosphate-free DMEM, which contained serum, immediately prior to treatment with increasing concentrations of arsenate.

A time course of JNK activity demonstrated that arsenate and arsenite activate JNK at different rates (Figure 1). The cells were treated with 300 M arsenate for various times and JNK activity was assayed by precipitation with GST-c-Jun (1 - 79). Cell lysates were prepared and incubated with GST-c-Jun, which is bound to Sepharose beads. GST-c-Jun associates with endogenous JNK and the protein complex is precipitated by centrifugation, washed with buffer, and then incubated with [-³²P]ATP. JNK phosphorylates its substrate, c-Jun, if it is activated by upstream kinases. Phosphorylated c-Jun was resolved by electrophoresis and detected by autoradiography. Maximal activation of JNK by arsenate was 40-fold over basal. Maximal levels of JNK activity were reached between 1 and 2 h. Arsenite activation of JNK followed a consistently different time course. Arsenite treatment resulted in nearly a 30-fold stimulation of JNK. Maximal stimulation with arsenite was measured between 30 and 60 min in contrast to the longer time required for maximal stimulation with arsenate. At 3 and 4 h, both arsenate and arsenite stimulated JNK activity was lower than the maximal stimulation, suggesting that activation of JNK is a reversible event. For both arsenic species, there were no detectable cytotoxic effects during the respective time courses (data not shown).

HEK 293 cells were treated with various concentrations of arsenate and arsenite. Both inorganic arsenic species activated JNK in a dose-dependent manner (Figure 2). At doses below 30 M of arsenate, there was no stimulation of JNK activity. However, a 100 M dose of arsenate produced a greater than sixfold stimulation of JNK activity. When the dose of arsenate was 300 M, JNK activity increased to almost 20-fold over basal levels of JNK activity. In parallel experiments, arsenite increased JNK activity to a similar level at a 100 M dose of arsenite as that observed for arsenate. At a 300 M dose, arsenite typically produced a greater activation of JNK than arsenate. Arsenite activated JNK 36-fold over basal levels while arsenate activated JNK 20-fold in this experiment. The level of JNK activity that we observed was maximal for both compounds at 300 M. We did not observe any increase in JNK activity at concentrations as high as 3 mM (data not shown). Thus, arsenate, like arsenite, activates JNK in HEK 293 cells, although arsenite exposure induced a consistently higher maximal response.

Dominant-negative Rac and Rho inhibit JNK activation by arsenite or arsenate

We transfected the cDNA encoding small GTP-binding proteins that are known to function upstream of JNK to better define the mechanisms by which arsenite and arsenate activate JNK. We used the inhibitory forms of Cdc42 (Cdc42^N17), Rac (Rac^N17), Ras (Ras^N17), and Rho (Rho^N19) to determine which small GTP-binding proteins inhibit the activation of JNK by arsenite or arsenate. Since the transfection efficiency was less than 100%, we co-transfected the cDNA encoding HA-JNK-1 with the cDNAs encoding the small GTP-binding proteins so that we could measure JNK-1 activity from only the transfected cells. Following stimulation with a dose of arsenite or arsenate that provides maximal JNK activity, HA-JNK-1 was immunoprecipitated from cells with the 12CA5 monoclonal antibody and an immune complex assay was performed where immunoprecipitated HA-JNK-1 was incubated with bacterially expressed GST-c-Jun and [-³²P]ATP. Phosphorylated GST-c-Jun was resolved by SDS - PAGE and detected by autoradiography.

Expression of Rho^N19 inhibited greater than 90% of the arsenite-stimulated JNK activity (Figure 3a,b). In contrast, expression of Rac^N17 provided a modest statistically significant inhibition of arsenite-stimulated JNK activity, whereas expression of Ras^N17 and Cdc42^N17 had no effect. The data that are presented in Figure 3a represent the average of three separate experiments and are calculated relative to the arsenite-treated cells transfected with only the vector, pCMV5. In this figure, a representative experiment is shown in b and c. Similar levels of HA-JNK-1 were immunoprecipitated as determined by immunoblotting HA-JNK-1 with a JNK specific antibody (Figure 3c). These data demonstrate that endogenous Rho and Rac function upstream of JNK in the pathway for arsenite-stimulated JNK activity, while Ras and Cdc42 do not appear to function in arsenite-dependent signal transduction.

We next examined whether arsenate activates JNK via the same small GTP-binding proteins as were identified for arsenite. The cells were treated for 2 h since this time point yielded maximal activation of JNK (see Figure 1a). HA-JNK-1 was immunoprecipitated from cells that expressed inhibitory mutant forms of Cdc42, Rac, Ras, and Rho. We observed in multiple experiments that JNK activity was inhibited in cells expressing Rac^N17 and Rho^N19, but not Ras^N17 and Cdc42^N17 (Figure 4a). The data from a representative experiment demonstrate that endogenous Rac and Rho function in arsenate signaling to JNK (Figure 4b,c). Moreover, these data demonstrate that arsenate and arsenite utilize similar GTP binding proteins to activate JNK.

Dominant-negative PAK inhibits arsenite-dependent JNK activity

Heterologous expression of PAK results in the activation of JNK, suggesting that PAK functions upstream of JNK (Bagrodia et al., 1995). We expressed kinase-inactive inhibitory PAK (KM-PAK) to determine if the activity of this kinase was required for arsenite- and arsenate-mediated JNK activation. HEK 293 cells were treated with arsenite or arsenate, as described above, and HA-JNK-1 was immunoprecipitated. KM-PAK inhibited ~60% of the arsenite-stimulated JNK activity (Figure 5, compare lanes c and f). However, even though KM-PAK was effective at inhibiting arsenite-stimulated JNK activation there was no effect on arsenate-mediated JNK activity (Figure 5, compare lanes b and e). From these data we conclude that arsenite, but not arsenate, requires PAK to activate JNK, which provides evidence that arsenate and arsenite activate JNK through different signaling mechanisms.

Dominant-negative MEKK2, MEKK3 and MEKK4 inhibit arsenite-dependent JNK activity: Dominant-negative, MEKK3 and MEKK4 inhibit arsenate-dependent JNK activity

Since the MEK kinase (MEKK) family members regulate JNK activity, we examined the role of all four MEKKs in arsenite and arsenate signaling to JNK. Given the differences that we found between these two arsenic species at the level of PAK, we predicted that the MEKK proteins would differentially affect arsenate and arsenite signaling to JNK. HEK 293 cells were transfected with kinase-inactive inhibitory mutants of the different MEKK family members (KM-MEKK1-4) and HA-JNK-1. Following treatment with arsenite, HA-JNK-1 was immunoprecipitated and phosphorylation of GST-c-Jun was determined. We consistently found that expression of KM-MEKK2, KM-MEKK3 and KM-MEKK4 significantly inhibited arsenite-stimulated Jun kinase activity by greater than 60% (Figure 6b). As in Figure 3, the data were calculated relative to the arsenite-stimulated Jun kinase activity in cells transfected with pCMV5. In similar experiments using arsenate, we found that expression of KM-MEKK3 and KM-MEKK4 significantly inhibited arsenate-stimulated JNK activity by greater than 80% (Figure 7b). In contrast to our findings with arsenite, KM-MEKK2 did not inhibit arsenate-stimulated JNK activity, which suggests that this kinase is not involved in arsenate-mediates JNK activation. Neither arsenite nor arsenate-stimulated JNK activity was significantly inhibited by a dominant negative MEKK1. Thus, arsenite appears to require the activity of MEKK2, MEKK3 and MEKK4 to activate JNK while arsenate appears to require the activity of only MEKK3 and MEKK4.

Discussion

Arsenite has been shown to activate the ERK, JNK, and p38 MAP kinases (Adler et al., 1995; Cavigelli et al., 1996; Liu et al., 1996; Meier et al., 1996; Rouse et al., 1994; Trigon and Morange, 1995). In addition, arsenite induces the expression of heat shock proteins including heme oxygenase (Elbirt et al., 1998). For historical reasons, arsenite has been the inorganic arsenic species that has received the most study because it is considered one of the most toxic forms of arsenic. However, human exposure to arsenate frequently occurs due to contaminated water supplies (Stoner et al., 1977). We show for the first time that arsenate, like arsenite is capable of activating JNK. Consequently, arsenate is another inorganic chemical species of arsenic that deserves attention with regard to the signal transduction pathways that are regulated by arsenic. This is particularly important since there is epidemiological data to link inorganic arsenic, such as arsenite and arsenate, to multiple forms of cancer in people who drink and bathe in arsenic contaminated water (Smith et al., 1998; Yeh et al., 1968), Yet, the molecular mechanism by which inorganic arsenic species cause cancer is unknown.

It is clear that arsenate must enter the cell to have its effect on JNK, likely via an anion transport protein (Kenney and Kaplan, 1988). In fact, our data indicate that the effects of arsenate are limited when the cells are dosed in phosphate containing media (data not shown). This suggests that there is a competition for the transporter between phosphate and arsenate. Upon entry of arsenate and arsenite into the cellular milieu, the proteins that transduce the signal to the MAP kinases as well as transcription factors have not been described. Therefore, we determined if arsenate and arsenite were capable of regulating specific signal transduction pathways that are typically associated with JNK activation.

Using inhibitory small GTP-binding protein constructs including Cdc42, Rac, Ras and Rho, we show that specific GTP-binding proteins are involved in arsenate- and arsenite-mediated JNK activation. Interestingly, the data indicate that there is no involvement of Cdc42 in either arsenate or arsenite activation of JNK, although Cdc42 has been shown in some cases to be involved in JNK activation (Coso et al., 1995; Minden et al., 1995). Rac and Rho both play a role in arsenite- and arsenate-mediated JNK activation, although Rho was the most effective inhibitory mutant small GTP-binding protein that inhibited JNK activation by arsenite and arsenate.

The inositol phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP₂), is a precursor for the production of second messengers, such as inositol trisphosphate, diacylglycerol, and 3,4,5-PIP₃, by phospholipase C (PLC) and phosphatidyinositol 3-kinase (PI3-K). It is interesting to note that Rho and Rac associate with phosphatidyinositol 4-phosphate 5-kinase [(PIP5K); reviewed in (Ren and Schwartz, 1998)] and regulate the production of PIP₂ (Ren et al., 1996). Thus, Rho and Rac may provide a link between oxidative stress signaling and inositol phosphate production. It has recently been shown that osmotic stress induces the expression of PIP5K mRNA in Arabidopsis thaliana (Mikami et al., 1998). It is tempting to speculate that oxidative stress caused by inorganic arsenic species may also induce mammalian PIP5K through a Rho or Rac-dependent pathway.

The exact role for Rac in arsenite and arsenate signal transduction to JNK is unclear. It has been shown that Rac associates with cytosolic proteins to form the multicomponent respiratory burst of NADPH oxidase in neutrophils (Freeman et al., 1996). We have found that HEK 293 cells chemically reduce arsenate to arsenite (data not shown), which may result in oxidative stress in the cell as glutathione stores are depleted. It is not clear if these cells are capable of producing the methylated products of arsenite. Thus, the involvement of small GTP-binding proteins such as Rac in arsenite- and arsenate-mediated signal transduction may be due to oxidative stress mechanisms (Figure 4). Consistent with this hypothesis is the observation that the activation of all three MAP kinases was prevented by the free radical scavenger, N-acetyl-L-cysteine (Liu et al., 1996). It is possible that the Rac and Rho small GTP-binding proteins that are required for arsenite or arsenate signaling to JNK also play a role in the metabolism of arsenate to arsenite.

Rac may also play a role in arsenic-induced carcinogenesis. Activated Rac (Rac^V12) and its effector, Tiam1, will activate JNK (Michiels et al., 1997) and both proteins are essential for malignant transformation of NIH3T3 and lymphoma cells (Habets et al., 1994; Michiels et al., 1995; Qiu et al., 1995; van Leeuwen et al., 1995). In addition, the effector site of Rac has been implicated in the control of mitogenesis through superoxide production (Joneson and Bar-Sagi, 1998). These observations have significant public health implications as arsenate is the most prevalent arsenic species in surface or oxygenated water (Braman and Foreback, 1973), which means that arsenate, [As(V)], in addition to arsenite [As(III)] are carcinogens that humans are likely to encounter in their lifetime. Thus, a better understanding of the Rac pathway may characterize a mechanism to describe how arsenic causes cancer, which may ultimately provide us with therapeutic targets to treat arsenate-induced cancer.

The mechanism by which arsenic causes cancer is unknown. Mutations in the ras gene have been associated with many human cancers, including skin melanoma [reviewed in (Bos, 1989)]. Based on those observations, Ras and Ras-dependent pathways have been implicated as important mediators for human carcinogens. UV light is an example of a potent skin carcinogen that activates JNK through Ras (Derijard et al., 1994). It is interesting to note, however, that Ras plays little role in the activation of JNK by either arsenate or arsenite. It is likely that Ras plays a role in the regulation of the ERKs, as arsenite induces anchorage-independent growth of mouse epidermal cells in soft agar (Huang et al., 1999). Our result is consistent with previous studies indicating that Ras does not play a role in the activation of JNK or p38 MAPK (Liu et al., 1996). From our results we can conclude that there are mechanistic differences between UV light and arsenic-mediated activation of JNK.

The involvement of PAK in arsenite- and not arsenate-mediated JNK activation is an indication that there are differences in signaling pathways between arsenate and arsenite in the cell. The precise role that PAK plays in mediating the biological effects of arsenite is not yet entirely clear. PAK1 has been shown to activate p38, another member of the stress activated protein kinase family (Bagrodia et al., 1995). In addition, a link between PAK and the ERK pathway has been established as PAK3 phosphorylates Raf-1 in vitro and in vivo (King et al., 1998). PAK is also important in mediating the morphological changes of the cytoskeleton that occur during apoptosis (Zhang et al., 1995). With regard to JNK regulation, it has been shown that Rho regulates JNK in HEK 293 cells independent of PAK (Teramoto et al., 1996). Therefore, another effector kinase, other than PAK, is required for JNK activation by arsenite (Figure 5). Additional effector kinases that are known activators of JNK and may be activated by arsenite include GCK (germinal center kinase), Tpl-2 (tumor progression locus 2), MLK3 (mixed lineage kinase), DLK (dual leucine zipper bearing kinase), and TAK1 [(TGF--activated protein kinase); reviewed in (Fanger et al., 1997a)]. Alternatively, arsenite may by-pass the MAP4K level of the signaling cascade. Consistent with this notion is the work of Fanger et al. (1997b) who showed that Rac and Rho physically associate with MEKK4. Collectively, these data demonstrate a direct link between small GTP binding proteins and the MEKK family of proteins in the regulation of JNK by arsenate and arsenite.

There are differences in the involvement of the MEKKs in arsenate and arsenite signal transduction as well. Dominant-negative MEKK2, MEKK3 and MEKK4 block arsenite activation of JNK. Only MEKK3 and MEKK4 block arsenate activation of JNK. The physiological role that the four MEKKs perform is just beginning to emerge. For example, MEKK4 interacts with the stress-inducible GADD4S proteins (Takekawa and Saito, 1998), which positions MEKK4 to regulate cellular responses to environmental stress such as arsenite and arsenate.

An analysis of the amino acid sequence of these proteins provides us with some insight as to the function of these proteins. A similarity in amino acid sequence suggests a similarity in function, as all four MEKK proteins activate JNK (Blank et al., 1996; Gerwins et al., 1997; Lange-Carter et al., 1993; Minden et al., 1994; Xu et al., 1996; Yan et al., 1994). However, although the MEKKs have similar protein kinase domains at the carboxyl-terminus (Hanks et al., 1988), there are differences in the substrate specificity for each of these kinases. For example, MEKK4 activates the JNK (Gerwins et al., 1997) and p38 (Takekawa et al., 1997) pathways but not the ERKs, differentiating it from MEKK1, 2 and 3, which are capable of also activating the ERK pathway. MEKK2 and MEKK3 share a 94% amino acid sequence homology in their kinase domain (Blank et al., 1996), while there is much less homology with MEKK1 and MEKK4 (Gerwins et al., 1997). There is even less homology between MEKKs when MEKK2 and 3 are compared to MEKK1 and 4. A comparison of amino acid sequence in the putative regulatory domain, in particular, the sequence outside the kinase domain, shows a high level of dissimilarity between the MEKKs, which suggests that these kinases are regulated by different proteins and that they regulate different pathways. Thus, it is not surprising to observe that arsenate and arsenite utilize two dissimilar MEKKs, MEKK3 and MEKK4, to activate JNK.

In summary, we have shown that arsenate and arsenite activate JNK by utilizing different, as well as overlapping, signal transduction pathways (Figure 8). Each of the proteins that we have identified as participating in the activation of JNK may also regulate other signal transduction pathways and may account for the many effects of arsenic. Activation of these pathways likely contributes to some of the biological effects observed from chronic low-dose arsenic exposures frequently associated with arsenic toxicity. Our results provide a framework for future studies to characterize the early events in arsenic signaling.

Materials and methods

Materials

Sodium arsenate, ACS certified, was purchased from JT Baker Chemical Co. (Phillipsburg, NJ, USA). Sodium arsenite, ACS certified, was purchased from Fisher Scientific (Tustin, CA, USA) and chemicals for protein electrophoresis were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Anti-HA mouse monoclonal antibody was purchased from Boehringer Mannheim (Indianapolis, IN, USA). Recombinant GST-c-Jun (1 - 79) was expressed in the JM109 strain of E. coli and purified using glutathione Sepharose (Amersham Pharmacia Biotech Inc) as described previously (Hibi et al., 1993).

Cell culture and transfection

Human embryonic kidney (HEK) 293 cells were maintained in Dulbecco's modified Eagle's medium with 5% newborn calf serum, 5% calf serum, 100 units/ml penicillin and 100 g/ml streptomycin at 37°C under 5% CO₂. Transient transfection of HEK 293 cells was performed using calcium phosphate, followed by DMSO shock (Cullen, 1987). cDNA constructs encoding pGEX-c-Jun (1 - 79), pCMV5-Ras^N17, pcDNA3-Rho^N19, pSR3-HA-JNK1, pcDNA3-MEKK1 (KM), pCMV5-MEKK2 (KM), pCMV5-HA-MEKK3 (KM), pCMV5-MEKK4, and pcDNA3-HA-PAK (KM) were gifts of GL Johnson. Constructs encoding pCMV5-Rac^N17 and pCMV5-Cdc42^N17 were gifts from N Dhanasekaran.

To characterize the role of small GTP binding proteins in the activation of JNK, cells were transfected with 10 g of epitope-tagged HA-JNK-1 and 20 g dominant-negative small GTP-binding proteins [Rac^N17, Cdc42^N17, Ras^N17, or Rho^N19]. To characterize the role of the MEKKs in the activation of JNK, cells were transfected with 10 g of epitope-tagged HA-JNK-1 and 20 g of dominant-negative MEKKs [(KM)-MEKK1, (KM)-MEKK2, (KM)-HA-MEKK3, or (KM)-MEKK4]. Finally, to characterize the role of PAK in the activation of JNK, cells were transfected with 10 g of epitope-tagged HA-JNK-1 and 20 g of epitope-tagged, dominant-negative PAK [(KM)-HA-PAK]. Cells were plated on 100-mm dishes 1 - 2 days prior to transfection. Two days later, the cells were treated with 300 M arsenate or arsenite and harvested for JNK activity.

Solid phase JNK assay

HEK cells were treated with arsenate or arsenite in DMEM containing serum. Stock solutions of 300 mM sodium arsenate or sodium arsenite were prepared on the day of the experiment in 100 mM HEPES, pH 7.2. The cells were then washed with ice-cold PBS and scraped into 600 l of lysis buffer (20 mM Tris, pH 7.6, 0.5% NP-40, 0.25 M NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM PMSF, 2 mM Na₃VO₄, 5 g/ml leupeptin and 1 mM DTT). Cell debris was removed by centrifugation at 14 000 r.p.m. for 10 min at 4°C. The protein concentration was determined as described previously, using BSA as a standard (Bradford, 1976). A solid-phase kinase assay was performed in which 40 g of protein from cell lysates was incubated at 4°C with 5 l of a 1 : 1 slurry of fusion protein, GST-c-Jun, which was bound to Sepharose beads (Hibi et al., 1993). Following an incubation of 1.5 h with rotation, the beads were collected at 2000 r.p.m. for 1 min. The precipitated proteins were washed twice with 1 ml of lysis buffer and once with kinase buffer (20 mM HEPES, pH 7.2, 20 mM -glycerophosphate, 10 mM p-nitrophenylphosphate (pNpp), 10 mM MgCl₂, 1 mM DTT and 50 M Na₃VO₄). The activity of precipitated JNK was assayed with 1 l of [-³²P]ATP (10 Ci/ml) in 40 l of kinase buffer at 30°C for 20 min. The reaction was stopped by the addition of 40 l of 2´SDS - PAGE Laemmli sample buffer. Phosphorylated GST-c-Jun was separated from unincorporated radioactivity by SDS - PAGE using a 10% gel (Fling and Gregerson, 1986), then identified by autoradiography and quantified by using a Packard Instant Imager Electronic Autoradiography System^TM (Packard Instrument Co, Meriden, CT, USA).

JNK assay

Stock solutions of 300 mM sodium arsenate or sodium arsenite were prepared on the day of the experiment in 100 mM HEPES, pH 7.2. HEK cells were treated with a final concentration of 300 M arsenate for 2 h in phosphate free media with serum or 300 M arsenite in media containing serum for 30 min. The cells were then washed twice with ice-cold PBS and lysed in 20 mM Tris, pH 7.6, 0.5% NP-40, 0.25 M NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM PMSF, 2 mM Na₃VO₄, 5 g/ml leupeptin and 1 mM DTT. Soluble proteins were collected after centrifugation at 15 000 r.p.m. in an Eppendorf centrifuge. The cell lysate (1 - 3 mg) was incubated with a monoclonal antibody (0.4 mg/ml), clone 12CA5, that recognizes the HA epitope (Boehringer Mannheim, Indianapolis, IN, USA) at a dilution of 1 : 250. After 90 min, 10 l of Protein A Sepharose (1 : 1 slurry, Sigma Chemical Co., St. Louis, MO, USA) was added and the samples were maintained at 4°C with rotation for at least 1.5 h. The immune complexes were collected by centrifugation at 2000 r.p.m. for 1 min in an Eppendorf centrifuge. Then, the immune complexes were washed twice with 1 ml of lysis buffer and once with kinase buffer (20 mM HEPES, pH 7.2, 20 mM -glycerophosphate, 10 mM pNpp, 10 mM MgCl₂, 1 mM DTT and 50 M Na₃VO₄). The activity of precipitated JNK was assayed with 1 l of [-³²P]ATP (10 Ci/ml) and 1 g of GST-jun in 40 l of kinase buffer at 30°C for 20 min. The reaction was terminated by the addition of 40 l of 2´Laemmli sample buffer (Fling and Gregerson, 1986). Phosphorylated GST-c-Jun was separated from unincorporated radioactivity by SDS - PAGE using a 10% gel and identified by autoradiography, then quantified as described above.

Statistical analysis

Statistical analysis was performed using pair-wise one-way analysis of variance (ANOVA). The data were analysed using the SigmaStat (SPSS, Inc., Chicago, IL, USA) program and results were considered significant at P<0.05. The vertical bars that are starred in each graph represent the standard deviations of three separate experiments. Triplicate determinations were significantly different from the pCMV5 control by one way analysis of variance with a secondary Bonferroni's test.

Immunoblotting

In order to determine whether equal amounts of HA-JNK-1 were immunoprecipitated for each condition in a particular experiment, Western blots were performed as follows. The Protein A Sepharose beads were washed once as described above, and then 1 ml of lysis buffer was added to the beads. A volume of buffer, which was equivalent to 100 g of protein, based on the protein concentration that was used at the beginning of the experiment, was removed from the second 1 ml wash. The sample was resolved by SDS - PAGE and the proteins were transferred to nitrocellulose. The blot was incubated with JNK antibody (catalog number sc-571, Santa Cruz Biotechnology, Santa Cruz, CA, USA), at a dilution of 1 : 1000 for 1 h at room temperature. After washing three times with T-TBS, the blot was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG, at a dilution of 1 : 1000 for 45 min at room temperature. The blot was washed three times with T-TBS and the presence of HA-JNK-1 was visualized by enhanced chemiluminescence followed by autoradiography.

Acknowledgements

This work was supported, in part, by grants from the American Cancer Society (IRG 110T), the Southwest Environmental Health Sciences Center (ES 06694), and the Arizona Disease Control Research Commission.

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Figure 1 Time course of JNK activation by arsenate and arsenite. HEK 293 cells were treated with 300 M arsenate (a) or arsenite (b) for the times indicated. GST-c-Jun that was bound to Sepharose conjugated with glutathione was incubated with 40 g of cell lysate. Endogenous JNK was precipitated by centrifugation and the protein complex was incubated with [-³²P]ATP. Phosphorylated GST-c-Jun was resolved by electrophoresis and the autoradiogram is shown (in lower panels). Activity is expressed graphically as fold stimulation over basal, quantified using a Packard Instant Imager Electronic Autoradiography System^TM (Packard Instrument Co, Meriden, CT, USA). This is a representative experiment of three

Figure 2 (a) Dose response curve of JNK activation by arsenate and arsenite. HEK 293 cells were treated with arsenate for 2 h or arsenite for 30 min. Endogenous JNK was precipitated with GST-c-Jun that was bound to Sepharose and a solid-phase kinase assay was performed in the presence of [-³²P]ATP. Phosphorylated GST-c-Jun was separated by SDS - PAGE using a 10% gel (b). Activity is expressed graphically as fold stimulation over basal. Phosphorylated GST-c-Jun was identified by autoradiography (b) and quantitated as described above. This is a representative experiment of at least three separate experiments

Figure 3 Inhibitory mutant small GTP-binding proteins inhibit arsenite-stimulated JNK activity. HEK 293 cells were transfected with HA-tagged JNK-1 and mutant small GTP-binding proteins as described in the Materials and methods. Two days after transfection, cells were treated (+) with 300 M arsenite or with vehicle (-) for 30 min. After treatment the cells were lysed and JNK-1 was immunoprecipitated with the 12CA5 monoclonal antibody. Immunoprecipitated JNK activity was assayed by using GST-c-Jun as a substrate as described above. The graph shows arsenite-stimulated JNK1 activity from three separate experiments (a). Basal activity was defined as JNK1 activity from untreated cells transfected with pCMV5. Activity from treated cells transfected with pCMV5 was arbitrarily set at 100%. The data represent the mean±s.d. from three separate experiments. *P<0.05 versus control. In b and c, a representative experiment of at least three separate experiments shows phosphorylated GST-c-Jun (b) and a Western blot of immunoprecipitated JNK1 protein (c)

Figure 4 Inhibitory mutant small GTP-binding proteins inhibit arsenate-stimulated JNK1 activity. HEK 293 cells were transfected as described above and 2 days after transfection, cells were treated (+) with 300 M arsenate or with vehicle (-) for 2 h. After treatment the cells were lysed and JNK1 was immunoprecipitated with the 12CA5 monoclonal antibody. Immunoprecipitated JNK1 activity was assayed by using GST-c-Jun as a substrate as described above. The graph shows arsenate-stimulated JNK1 activity from three separate experiments (a). Basal activity was defined as JNK1 activity from untreated cells transfected with pCMV5. Activity from treated cells transfected with pCMV5 was arbitrarily set at 100%. The data represent the mean±s.d. from three separate experiments. *P<0.05 versus control. In b and c, a representative experiment of at least three separate experiments shows phosphorylated GST-c-Jun (b) and a Western blot of immunoprecipitated JNK1 protein (c)

Figure 5 Dominant-negative PAK inhibits arsenite-stimulated JNK1 activity. HEK 293 cells were transfected with HA-tagged JNK1 and dominant-negative PAK. Two days after transfection, cells were treated with 300 M arsenate or 300 M arsenite. After treatment the cells were lysed and HA-JNK-1 was immunoprecipitated with the 12CA5 monoclonal antibody. GST-c-Jun was used as a JNK substrate. Basal (hatched bars), arsenite-stimulated (black bars), and arsenate-stimulated (open bars) JNK activity was determined either in the absence of KM-PAK (lanes a - c) or in the presence of KM-PAK (lanes d - f). Activity is expressed as relative JNK1 activity. The autoradiogram is shown below the graph. The Western blot, showing immunoprecipitated JNK1, is shown at the bottom of the figure. This is a representative experiment of at least three separate experiments

Figure 6 Inhibition of arsenite-stimulated JNK1 activity by dominant-negative MEKKs. HEK 293 cells were transfected with HA-tagged JNK1 and dominant negative MEKK1-4. Two days after transfection, cells were treated (+) with 300 M arsenite or with vehicle (-) for 30 min. After treatment the cells were lysed and JNK1 was immunoprecipitated with the 12CA5 monoclonal antibody. Immunoprecipitated JNK activity was assayed by using GST-c-Jun as a substrate. The graph shows arsenite-stimulated JNK1 activity from three separate experiments (a). Basal activity was defined as JNK1 activity from untreated cells transfected with pCMV5. Activity from treated cells transfected with pCMV5 was arbitrarily set at 100%. The data represent the mean±s.d. from three separate experiments. *P<0.05 versus control. In b and c, a representative experiment of at least three separate experiments shows phosphorylated GST-c-Jun (b) and a Western blot of immunoprecipitated JNK1 protein (c)

Figure 7 Inhibition of arsenate-stimulated JNK1 activity by dominant-negative MEKKs. HEK 293 cells were transfected with HA-tagged JNK1 and dominant negative MEKK1-4. Two days after transfection, cells were treated (+) with 300 M arsenate or with vehicle (-) for 2 h. After treatment the cells were lysed and JNK1 was immunoprecipitated with the 12CA5 monoclonal antibody. Immunoprecipitated JNK activity was assayed by using GST-c-Jun as a substrate. The graph shows arsenate-stimulated JNK1 activity from three separate experiments (a). Basal activity was defined as JNK1 activity from untreated cells transfected with pCMV5. Activity from treated cells transfected with pCMV5 was arbitrarily set at 100%. The data represent the mean±s.d. from three separate experiments. *P<0.05 versus control. In b and c, a representative experiment of at least three separate experiments shows phosphorylated GST-c-Jun (b) and a Western blot of immunoprecipitated JNK1 protein (c)

Figure 8 Summary of signal transduction pathways activated by arsenate and arsenite. Arsenate and arsenite activate different proteins to regulate JNK, which functions in the stress-activated protein kinase pathway (SAPK). A SAPK pathway is a sequential protein kinase cascade where a protein, generically referred to as a MAP Kinase Kinase Kinase Kinase (MAP4K), phosphorylates and activates a MAP Kinase Kinase Kinsase (MAP3K), which repeats the cycle by phosphorylating and activating the next kinase in the cascade. The small GTP binding proteins are localized upstream of the sequential protein kinase cascade. The anion transport protein regulates entry of arsenate into the cell, while arsenite, which is an uncharged arsenic species, enters the cell by diffusion. The small GTP binding proteins that are regulated by arsenate and arsenite include Rac and Rho. Cdc42 and Ras do not appear to play a significant role in arsenite and arsenate signaling to JNK. PAK plays a role in arsenite-dependent JNK activity. MEKK3 and MEKK4 are involved in both arsenate and arsenite activation of JNK, while MEKK2 may be involved in the activation of JNK by arsenite