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Signal Transduction Pathways Regulated by Arsenate and Arsenite

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Signal Transduction Pathways Regulated by Arsenate and Arsenite Oncogene (1999) 18, 7794 ± 7802 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc Signal transduction pathways regulated by arsenate and arsenite Amy C Porter1, Gary R Fanger2,3 and Richard R Vaillancourt*,1 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 Arsenate and arsenite activate c-Jun N-terminal kinase c-Jun, which forms a heterodimer with c-Fos to form (JNK), however, the mechanism by which this occurs is the AP-1 transacting factor. Activation of JNK is not known. By expressing inhibitory mutant small GTP- commonly associated with inhibition of cell growth binding proteins, p21-activated kinase (PAK) and and/or apoptosis (Kyriakis and Avruch, 1996). mitogen-activated protein kinase/extracellular signal- However, under certain circumstances, sustained JNK regulated kinase kinase kinases (MEKKs), we have activation, as opposed to transient activation, is identi®ed speci®c proteins that are involved in arsenate- required for an apoptotic response (Guo et al., 1998). and arsenite-mediated activation of JNK. We observe a Toxic doses of inorganic arsenicals, such as arsenite distinct dierence between arsenate and arsenite signal- and arsenate, produce pleiotrophic eects. Using ing, which demonstrates that arsenate and arsenite are cultured macrophages as an in vitro system to study capable of activating unique proteins. Both arsenate and arsenic toxicity, it was observed that 80% of the dead arsenite activation of JNK requires Rac and Rho. cells were necrotic while 20% of the cells were Neither arsenate nor arsenite signaling was inhibited by apoptotic at the LD50 dose of 5 mM arsenite and a dominant-negative mutant of Cdc42 or Ras. Arsenite 500 mM arsenate (Sakurai et al., 1998). In addition to stimulation of JNK requires PAK, whereas arsenate- cell death, chronic arsenic exposure has been associated mediated activation of JNK was unaected by inhibitory with malignant transformation and DNA hypomethy- mutant PAK. Of the four MEKKs tested, only MEKK3 lation of epithelial cells (Zhao et al., 1997). Further- and MEKK4 are involved in arsenate-mediated activa- more, epidemiological studies demonstrate that arsenic tion of JNK. In contrast, arsenite-mediated JNK is a human carcinogen (Smith et al., 1998). However, activation requires MEKK2, MEKK3 and MEKK4. the poor mutagenicity of inorganic arsenicals (Jacob- These results better de®ne the mechanisms by which son-Kram and Montalbano, 1985; Lee et al., 1985; arsenate and arsenite activate JNK and demonstrate Rossman et al., 1980) suggests that the production of dierences in the regulation of signal transduction diverse physiological eects caused by arsenic are likely pathways by these inorganic arsenic species. due to the activation of various signaling pathways and not a genotoxic eect. Consistent with this line of Keywords: arsenic; Rac; Rho; PAK; MEKK3; MEKK4 reasoning is the observation that arsenic activates multiple MAPK pathways (Liu et al., 1996). Conse- quently, the spectrum of physiological responses caused by arsenic range from carcinogenesis to cell death. Introduction Both arsenate and arsenite activate JNK. However, the mechanism by which these arsenic species activate c-Jun N-terminal kinase (JNK) is a member of the this pathway has not been characterized. One report stress-activated protein kinase family and is activated suggests that arsenite activates JNK by inactivation of by cellular stress, such as osmotic shock and a JNK phosphatase (Cavigelli et al., 1996). Since irradiation [reviewed in (Treisman, 1996)]. JNK arsenite has been shown to bind and inactivate proteins activation requires the small GTP-binding proteins containing thiol functional groups, arsenite may bind Ras, Rac, Cdc42 and Rho (Coso et al., 1995; Derijard to thiol groups in the active site of a JNK phosphatase. et al., 1994; Minden et al., 1995; Teramoto et al., Because phosphorylation of JNK is critical for its 1996). These small GTP-binding proteins associate with activation, this mechanism does not explain how JNK and activate a variety of serine/threonine kinases that is phosphorylated and thus activated in the ®rst place. are important in JNK activation including p21- In addition, since arsenate does not bind to thiol activated kinase (PAK), as well as mitogen-activated groups, this mechanism does not explain how arsenate protein kinase/extracellular-signal-regulated kinase ki- activates JNK. Thus, it is possible that arsenate and nase kinases [MEKKs; (Fanger et al., 1997a)]. The arsenite are capable of regulating speci®c signal MEKKs phosphorylate and activate JNK kinase transduction pathways associated with JNK activation. (JNKK), which phosphorylates and activates JNK. It has been well established that arsenite is an Upon activation, JNK alters speci®c gene transcription activator of the stress-activated protein kinase path- events via phosphorylation of the transcription factor, ways (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 *Correspondence: RR Vaillancourt whether arsenite and arsenate utilize the same signaling 3 Current address: Corixa Corporation, 1124 Columbia Street, Seattle, Washington, WA 98104, USA proteins to activate JNK. We set out to map these Received 3 December 1998; revised 2 September 1999; accepted signaling pathways by using a series of inhibitory 7 September 1999 mutant proteins that are important for JNK activation Arsenic regulates Rac, Rho, PAK, MEKK3 and MEKK4 AC Porter et al 7795 by other stimuli. We have transfected inhibitory for the anion transport protein. To overcome this mutant small GTP-binding proteins, PAK, and competition, HEK 293 cells were incubated in MEKK1-4 into human embryonic kidney (HEK) 293 phosphate-free DMEM, which contained serum, cells to determine which proteins were capable of immediately prior to treatment with increasing con- blocking activation of JNK by either arsenate or centrations of arsenate. arsenite. The data from our experiments show that A time course of JNK activity demonstrated that arsenate and arsenite activate JNK through dierent arsenate and arsenite activate JNK at dierent rates signal transduction pathways, which require speci®c (Figure 1). The cells were treated with 300 mM arsenate small GTP-binding proteins and serine/threonine for various times and JNK activity was assayed by kinases. precipitation with GST-c-Jun (1 ± 79). Cell lysates were The goal of this study was to determine whether prepared and incubated with GST-c-Jun, which is arsenate and arsenite activate JNK through the same bound to Sepharose beads. GST-c-Jun associates with upstream proteins. One feature that dierentiates endogenous JNK and the protein complex is pre- arsenate from arsenite is the relative permeability of cipitated by centrifugation, washed with buer, and each inorganic arsenic species across the plasma then incubated with [g-32P]ATP. JNK phosphorylates membrane. Arsenate must be transported into the cell its substrate, c-Jun, if it is activated by upstream by the organic anion transport protein, whereas kinases. Phosphorylated c-Jun was resolved by arsenite freely crosses the plasma membrane (Kenney electrophoresis and detected by autoradiography. and Kaplan, 1988). In the presence of phosphate, Maximal activation of JNK by arsenate was 40-fold arsenate is poorly transported into the cell since over basal. Maximal levels of JNK activity were phosphate competes with arsenate for binding to the reached between 1 and 2 h. Arsenite activation of anion transport protein. Thus, the mechanism by JNK followed a consistently dierent time course. which arsenic signal transduction is initiated by each Arsenite treatment resulted in nearly a 30-fold arsenic species is potentially dierent. 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 Results and arsenite stimulated JNK activity was lower than the maximal stimulation, suggesting that activation of Arsenate is an activator of JNK JNK is a reversible event. For both arsenic species, We used HEK 293 cells and transient transfection of there were no detectable cytotoxic eects during the cDNAs that encode candidate proteins as an approach respective time courses (data not shown). to identify proteins that function upstream of JNK. We HEK 293 cells were treated with various concentra- measured JNK activity in HEK 293 cells to establish tions of arsenate and arsenite. Both inorganic arsenic that the signaling proteins were expressed in these cells. species activated JNK in a dose-dependent manner Since arsenate is transported into the cell by the anion (Figure 2). At doses below 30 mM of arsenate, there transport protein (Kenney and Kaplan, 1988), a was no stimulation of JNK activity. However, a competition exists between arsenate and phosphate 100 mM dose of arsenate produced a greater than Figure 1 Time course of JNK activation by arsenate and arsenite. HEK 293 cells were treated with 300 mM arsenate (a) or arsenite (b) for the times indicated. GST-c-Jun that was bound to Sepharose conjugated with
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