Phosphorylation is involved in the activation of metal-regulatory transcription factor 1 in response to metal ions.

We have studied the role of phosphorylation in the activation of metal-regulatory transcription factor-1 (MTF-1) and metallothionein (MT) gene expression. We showed that MTF-1 is phosphorylated in vivo and that zinc stimulates MTF-1 phosphorylation 2-4-fold. Several kinase inhibitors were used to examine the possible involvement of kinase cascades in the activation of MTF-1. Metal-induced MT gene expression was abrogated by protein kinase C (PKC), c-Jun N-terminal kinase (JNK), phosphoinositide 3-kinase, and tyrosine-specific protein kinases inhibitors, as assayed by Northern analysis and by cotransfection experiments using a metal regulatory element-luciferase reporter plasmid. The extracellular signal-activated protein kinase and the p38 kinase cascades did not appear to be essential for the activation of MT gene transcription by metals. By using dominant-negative mutants of PKC, JNK, mitogen-activated kinase kinase 4 (MKK4), and MKK7, we provide further evidence supporting a role for PKC and JNK in the activation of MTF-1 in response to metals. Notably, increased MTF-1 DNA binding in response to zinc and MTF-1 nuclear localization was not inhibited in cells preincubated with the different kinase inhibitors despite strong inhibition of MTF-1-mediated gene expression. This suggests that phosphorylation is essential for MTF-1 transactivation function. We hypothesize that metal-induced phosphorylation of MTF-1 is one of the primary events leading to increased MTF-1 activity. Thus, metal ions such as cadmium could activate MTF-1 and induce MT gene expression by stimulating one or several kinases in the MTF-1 signal transduction pathway.

Metallothioneins (MTs) 1 are small cysteine-rich multifunc-tional stress proteins that bind with high affinity transition metal ions such as cadmium, zinc, and copper (1,2). All vertebrates examined contain two or more distinct MTs that are grouped into four families, MT-1 through MT-4. The expression of MT-1 and MT-2 is ubiquitous, while that of MT-3 and MT-4 is restricted to specific tissues. MTs play important roles in zinc and copper homeostasis and detoxification of toxic metals. MT can also confer protection against reactive oxygen species, electrophilic antineoplastic agents, various mutagens, ionizing radiation, and nitric oxide.
MT genes are universally inducible at the transcriptional level by metals and a variety of stress conditions such as reactive oxygen species, hypoxia, and UV irradiation. The ability of MT genes to be induced by metals is controlled by a short DNA sequence (metal regulatory element; MRE), which is present in six nonidentical copies (MREa through MREf) in the 5Ј-flanking region of the mouse MT-1 gene, and by the capacity of metal-regulatory transcription factor-1 (MTF-1) to bind to the MREs in the presence of zinc and induce transcription (3). MTF-1 is a zinc finger transcription factor of the Cys 2 His 2 family (4) that is essential for both basal and metal-inducible MT gene expression (5). MTF-1 is not only essential for induction by zinc, but it also mediates the response to all metals that activate MT gene expression (5,6). MTF-1 is also a central cellular component of the stress signal transduction machinery activated in response to oxidative stress (7,8) and hypoxia (9). Furthermore, MTF-1 is essential for liver development (5) and mediates metal regulation of the genes encoding ␥-glutamylcysteine synthetase (10), zinc transporter-1 (11), and possibly cadmium/zinc superoxide dismutase (12).
The molecular mechanisms by which metals exert their action on MTF-1 still remain unclear. On the one hand, MTF-1 was initially described as a constitutive activator responsible for basal MT gene expression (4). It was also suggested that MTF-1 availability was regulated by a zinc-sensitive inhibiting factor, called MTI, similar to the NF-B-IB system (6). However, the isolation of such an inhibitor has not yet been reported. On the other hand, MTF-1 DNA binding activity rapidly increases in cells treated with zinc (4,5,(13)(14)(15)(16)(17)(18). Similarly, MTF-1 DNA binding activity is rapidly activated in vitro by zinc in assays using recombinant MTF-1 synthesized in a reticulocyte lysate (15,18) or glutathione S-transferase (GST)-MTF-1 fusion protein expressed in bacteria (19). This suggests that MTF-1 is reversibly activated to bind to DNA and to enhance MT gene transcription in response to increased free zinc levels. Zinc would lead to an allosteric change in MTF-1 causing exposure of zinc fingers involved in DNA binding (for a review, see Giedroc et al. (20)). All of the other metals capable of inducing MT would do so by displacing zinc from the weakly bound pool, and this displaced zinc would activate MTF-1 DNA-binding (6). In addition to regulating DNA binding, metals appear to modulate nuclear translocation, since zinc promotes the rapid transport of MTF-1 into the nucleus (21).
However, cadmium, a potent inducer of MT gene expression, has no major effect on the DNA binding activity of MTF-1 either in vivo or in vitro (13,(15)(16)(17)(18). Furthermore, low nontoxic concentrations of cadmium exert only a small effect on MTF-1 nuclear translocation despite strong induction of MT gene expression (21). This suggests that cadmium may activate MT gene expression by increasing the transactivation potential of MTF-1. It has been suggested that this could be accomplished by post-translational modifications such as phosphorylation (16,18). Indeed, a survey of the MTF-1 sequence for kinase consensus sites indicated that the protein contains numerous putative casein kinase-II, protein kinase C (PKC), glycogen synthetase kinase-3, and tyrosine protein kinase sites.
To date, however, there has been no clear indication of any specific cellular components serving as kinase targets in the putative signaling cascade leading to MTF-1 activation. Previous studies showed that zinc-and cadmium-induced MT gene expression is abrogated by the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) or chelerythrine, as assayed by Northern analysis (22). Furthermore, inhibition of PKC by prolonged exposure to phorbol ester completely blocked cadmium-mediated activation of the MRE in HepG2 cells and significantly reduced zinc-mediated activation (23). These data have been interpreted as indicating that PKC is involved in the activation of MT gene expression in response to metals (22). However, prolonged exposure to phorbol ester can affect many cellular processes in addition to inhibiting PKC. Furthermore, H7 markedly reduces the cellular accumulation of zinc (22) and inhibits phosphorylation of RNA polymerase II in vivo (24). Thus, the inhibitory effect of H7 on MT gene expression could be due to the blocking of zinc transport or the inhibition of elongation of RNA transcripts.
We therefore undertook the present study to ascertain whether MTF-1 itself is phosphorylated in vivo and to characterize the kinase signaling pathway involved in MTF-1 activation and MT gene expression in response to metals. In mammalian cells, a variety of extracellular stimuli generate intracellular signals that converge on a limited number of protein kinase cascades, commonly referred to as mitogenactivated protein kinase (MAPK) pathways (25,26). There are three distinct classes of MAPK, the extracellular signal-activated protein kinase (ERK), the Jun N-terminal kinase (JNK), and the p38 MAP kinases. While the ERK MAPKs are activated by mitogenic stimuli, the two other types of MAPKs, JNK and p38, are in general activated by antimitogenic stimuli such as environmental or genotoxic stress, transforming growth factor-␤, tumor necrosis factor-␣, and interleukin-1. In addition, there is significant cross-talk between MAPK transduction and other kinase signaling pathways such as those involving the PKC and the phosphoinositide 3-kinase (PI3K). By using several kinase inhibitors, mitogen extracellular signal-regulated kinase kinase 1 (MEK1)-null mutant cells and dominant negative mutants of PKC, JNK, and its upstream activators MAP kinase kinase 4 (MKK4) and MKK7, we provide evidence supporting a role for PKC, PI3K, and JNK, but not for ERK or p38, in the activation of MTF-1 and the induction of MT gene expression in response to metal ions.

EXPERIMENTAL PROCEDURES
Materials-PD98059, SB202190, and LY294002 inhibitors were purchased from Calbiochem. The PKC inhibitors GF109203X, Ro-31-8220, and H7 and the JNK inhibitor dicoumarol were obtained from ICN Pharmaceuticals (Costa Mesa, CA). The transfection reagent SuperFect was purchased from Qiagen (Mississauga, Canada) and recombinant heat shock protein 27 (HSP27) was obtained from J. Landry (Center de Recherche, l'Hôtel-Dieu de Québec). All other chemicals were purchased from Sigma.
Plasmids-The luciferase (LUC) reporter plasmid 1843MT1-LUC contains 1843-bp 5Ј-flanking and 68-bp 5Ј-untranslated regions and was described elsewhere (27). To construct plasmid (MREa) 6 -LUC, a synthetic DNA fragment containing six mouse MT-1 MREa elements (28) (five elements in direct tandem orientation and the sixth in the opposite orientation) was cloned in front of a minimal mouse MT-1 promoter DNA fragment (positions Ϫ35 to ϩ68) into the LUC reporter plasmid pGL2-Basic (Promega, Madison, WI). The plasmid pcDNA-His-HA-MTF-1 was created by first inserting a polymerase chain reaction fragment encoding a hemagglutinin (HA) epitope provided with an ATG initiation codon into the NcoI site (nucleotide ϩ1, relative to the transcription start point) of the mouse MTF-1 cDNA and then by cloning the resulting HA-MTF-1 DNA fragment into the plasmid pCDNA3-His (Invitrogen, Carlsbad, CA). The mouse MTF-1 cDNA was generously provided by Dr. Walter Schaffner (University of Zurich, Zurich, Switzerland) (4). For all of the constructs, the identity of the fragments was confirmed by DNA sequencing using a T7 sequencing kit (Amersham Pharmacia Biotech). The PKC-KR kinase-dead dominant negative mutant was described previously (29), while the JNK1-APF and MKK4-AA mutants were obtained from Dr. Roger Davis (30) and MKK7-KL was obtained from Dr. Eisuke Nishida (31).
Cells, Northern Blot Analysis, and Transfections-Mouse L cells were obtained from D. H. Hamer (National Institutes of Health, Bethesda, MD), human HepG2 cells were from A. Anderson (Center de Recherche, l'Hôtel-Dieu de Québec), and monkey kidney COS-7 cells were from A. Ruiz-Carrillo (Center de Recherche, l'Hôtel-Dieu de Québec). The MEK1-null mutant mouse embryo fibroblasts were derived from MEK1-null mutant embryos (32). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS). Where indicated, protein kinase inhibitors and the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid were added 30 -45 min prior to the direct addition of metals, as indicated in the figure legends, and maintained during the remainder of the incubation period. For metal induction, ZnCl 2 (final concentration 100 M) or CdCl 2 (2.5 M) was added directly to the medium, and the cells were harvested 6 -8 h later. ZnSO 4 was also tested instead of ZnCl 2 and gave similar results. Anisomycin (10 g/ml) was added to the culture medium 6 h before harvesting. Protein kinase inhibitors were dissolved in sterile water or Me 2 SO as a 1000ϫ stock solution and stored at Ϫ80°C. Total RNA was extracted with guanidium isothiocyanate, and Northern analyses of MT-1 mRNA were performed (33), using as the probe the cDNA insert for the mouse MT-1 or the rat glyceraldehyde 6-phosphate dehydrogenase. The mouse MT-1 cDNA hybridizes equally with both MT-1 and MT-2 transcripts. Northern analysis data were quantified using a PhosphorImager 860 and ImageQuant 4.2 software (Molecular Dynamics, Inc., Sunnyvale, CA). Cells were transfected with the different reporter plasmids by the calcium phosphate method (34). Briefly, cells were seeded (ϳ4 ϫ 10 5 / 6-cm plate) 16 h prior to transfection. Cells were exposed to the DNA-CaPO 4 precipitate for 3 h, shocked for 3 min at 37°C with 15% glycerol in HEPES-buffered saline, incubated for 12 h in growth medium, and then treated or not with protein kinase inhibitors and metals. The plasmid pTK-rLUC or pCMV-rLUC (Promega) was used as an internal standard to monitor transfection efficiency. For transfections with the dominant negative mutants, cells were transfected with 7.5 g of the reporter plasmid (MREa) 6 -LUC, 1-5 g of various expression vectors, and 0.5 g of pTK-rLUC. PCDNA3 plasmid DNA was added to the transfections as needed to achieve the same total amount of plasmid DNA per transfection. The preparation of cell extract and measurement of LUC activities were carried out with a Dual-LUC assay kit (Promega) according to the manufacturer's instructions.
Electrophoretic Mobility Shift Assays (EMSA)-Nuclear extracts were prepared according to Schreiber et al. (35) with minor modification (18). EMSA was performed (18) using end-labeled MRE-s oligonucleotide (4), a synthetic MRE consensus sequence, as the probe. Reaction mixtures were incubated for 10 min on ice, and protein-DNA complexes were separated by polyacrylamide gel electrophoresis.
Protein Kinase Assays-Cells were serum-starved for 2 h before the addition of the protein kinase inhibitors and the different inducers. For metal induction, ZnCl 2 (100 M) or CdCl 2 (2.5 M) was added to the medium, and the cells were harvested 30 min later. Phorbol 12-myristate 13-acetate (100 nM), anisomycin (10 g/ml), and H 2 O 2 (200 M) were added to the culture medium 15, 20, and 10 min, respectively, before harvesting. For UVC irradiation, exponentially growing cells were serum-starved for 60 min, washed twice with phosphate-buffered saline, irradiated with a UV germicidal lamp ( max ϭ 254 nm; 100 J/m 2 ), and cultured in the original culture serum-free medium for 1 h. For FBS induction, cells were serum-starved for 2 h and then incubated in the presence of 10% FBS for 1 h. H 2 O 2 was prepared fresh in water as a 250ϫ concentrated stock and used immediately.
Cells were washed in cold phosphate-buffered saline buffer, scraped in lysis buffer (36), and clarified by centrifugation at 15,000 ϫ g for 20 min. Lysates were normalized for protein content. The clarified lysates were diluted in buffer A (36), immunoprecipitated with limiting concentrations of anti-ERK2 (obtained from J. Grose, Center de Recherche, l'Hôtel-Dieu de Québec) or anti-MAPK-activated protein-K2 (MAPKAP-K2) antibodies (obtained from J. Landry) for 2 h and incubated with protein A-Sepharose beads. Samples were centrifuged, and immunocomplexes were washed three times in buffer A, incubated with [␥-32 P]ATP and the appropriate substrate (myelin basic protein (MBP) or recombinant HSP27) in buffer K (36). Kinase reactions were stopped by boiling in SDS sample buffer. JNK activity was measured using a solid state kinase assay in which GST-Jun-(1-79) (obtained from J. Landry) bound to glutathione-Sepharose 4B beads was used to affinitypurify JNK. Then JNK activity was measured in an in vitro kinase assay using the Sepharose-bound GST-Jun as a substrate, as described (37), except that buffer K was used for the kinase reaction. The PKC immune complex assay was performed (29) using L cell extracts, anti-PKC monoclonal antibodies (clone MC5; Sigma), and MBP as the substrate. Signal intensities of protein-DNA complexes were quantified using a PhosphorImager.
Metabolic Labeling-Metabolic 32 P i labeling was performed as described (38). Briefly, COS-7 cells were transfected with the pcDNA-His-HA-MTF-1 plasmid using the SuperFect reagent (Qiagen), according to the manufacturer's instructions. As a control, some cells were transfected with the expression plasmid pCMV-HA-LacZ (Invitrogen), which encodes for an HA-␤-galactosidase fusion protein with a molecular mass of ϳ115 kDa. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FBS for 48 h, serumand phosphate-starved for 1 h by incubation in phosphate-free Dulbecco's modified Eagle's medium, and incubated with 3 mCi of 32 P i /ml in the same medium for 90 min. Cells were treated or not with 100 M ZnCl 2 for 20 min. Cells were then washed in cold medium, resuspended in radioimmune precipitation buffer, and clarified by centrifugation at 15,000 ϫ g for 20 min. The clarified cell lysates were preabsorbed on protein A-Sepharose beads for 30 min at 4°C and centrifuged, and His-HA-MTF-1 or HA-LacZ was immunoprecipitated with an anti-HA monoclonal antibody (HA.11; Babco, Richmond, CA). Immunocomplexes were washed three times in radioimmune precipitation buffer and resuspended in SDS sample buffer. Proteins were resolved on 7.5% SDS gels and transferred onto a 0.45-m Hybond C nitrocellulose membrane, and, after 32 P detection by autoradiography, the membrane was reacted with anti-HA or anti-Xpress antibody (Invitrogen) according to the manufacturer's instructions. Anti-Xpress antibodies are directed against the Xpress epitope located next to the His tag. The relative radioactivity was quantitated using a PhosphorImager.

MTF-1 Is Phosphorylated in Vivo-
The mechanisms by which metals control MTF-1 activity are still the subject of some controversy. While zinc is essential for MTF-1 DNA binding activity, other post-translational mechanisms may be involved in this regulation. We (18) and others (16) have proposed that phosphorylation may be involved in the regulation of MTF-1 activity. To ascertain whether MTF-1 is phosphorylated in vivo, we transfected COS cells with a CMV His-HA-tagged mouse MTF-1 expression plasmid and performed metabolic labeling of the transfected COS cells with [ 32 P]orthophosphate, followed by immunoprecipitation with the anti-HA antibody of the cell lysates. The immunoprecipitated proteins were applied on a denaturing polyacrylamide gel, transferred to a nitrocel-lulose membrane, and subjected to autoradiography. Both in control untreated and zinc-stimulated cells, a radioactive protein with an apparent molecular weight around 100,000 was immunoprecipitated with the anti-HA antibody (Fig. 1A, lane 2, and data not shown). Upon exposure to zinc for 20 min, an ϳ4-fold stimulation of MTF-1 phosphorylation was observed or an anti-Express (data not shown) antibody. The His-HA epitopes did not modify the properties of MTF-1, since DNA-binding and transactivation activities of the His-HA-MTF-1 fusion protein were indistinguishable from those of the wild type protein, as assessed by EMSA and co-transfection experiments (data not shown). These data clearly show that MTF-1 is phosphorylated in vivo. This prompted us to investigate the kinase signaling pathway involved in this process.
Zinc and Cadmium Activate PKC and ERK, JNK, and p38 MAPKs-Metal ions have been shown to stimulate the activity of several kinases, including ERK-1 and -2 (39,40), JNK (40), p38 (40), p70 S6 kinase (41), PI3K (41), and PKC (42,43). Thus, we wished to determine whether metals could activate PKC or any of the three major kinase signaling pathways in L cells. We examined the effects of CdCl 2 and ZnCl 2 on PKC and MAPK activities by assessing the activity of PKC and of kinases specifically involved in the signal transduction of a given MAPK cascade, namely ERK2 for the ERK pathway, JNK1 for the JNK pathway, and MAPKAP-K2 for the p38 pathway. L cells were serum-starved for 2 h and then treated with 100 M ZnCl 2 or 2.5 M CdCl 2 for 30 min, and cell extracts were analyzed for kinase activities by specific immune complex kinase assays.
The protein kinase activity of ERK2 was detected in control untreated cells ( Fig. 2A, ERK pathway) and was induced ϳ3fold by ZnCl 2 , while activity was stimulated 5-7-fold in re- sponse to H 2 O 2 , phorbol 12-myristate 13-acetate and FBS treatments. ERK activity was not induced by CdCl 2 at this time point after treatment. However, a 2-fold induction was observed 5-10 min after the addition of CdCl 2 (data not shown).
P38 and PKC activities were also detected in untreated cells, and their levels increased ϳ6and 3-fold, respectively, above control values in response to ZnCl 2 ( Fig. 2A, p38 and PKC pathways). In comparison, FBS induced p38 activity ϳ4-fold. P38 activity was stimulated ϳ1.5-fold by CdCl 2 , whereas that of PKC was maintained at control values (data not shown).
JNK activity was barely detectable in control cells, and activity 4 -15-fold above control values was attained after 30 min in response to ZnCl 2 (Fig. 2, A (JNK pathway) and B). In comparison, anisomycin and UV irradiation, two strong JNK activators, stimulated JNK kinase activity ϳ3-20-fold. JNK activity levels increased ϳ3-fold above the control value in response to CdCl 2 . Phosphorylation activity of the MAPKs and of PKC was not altered in cells exposed to vehicle (H 2 O and Me 2 SO) for up to 1 h (data not shown). Overall, these results shows that metal ions can activate the MAPK and PKC in L cells.
PKC, JNK, and PI3K Inhibitors Abrogate Metal-induced MT Gene Expression-To address the role of individual MAPK pathways and of PKC and PI3K signaling cascades in MT gene regulation by metals, we examined the effects of PD98059, an ERK pathway inhibitor; SB203580, an inhibitor of p38; H7, GF109203X, and Ro-31-8220, three PKC inhibitors; and wortmannin and LY294002, two PI3K inhibitors, on MT mRNA accumulation in L cells by Northern analysis. To assess the JNK pathway, we used dicoumarol, a quinone reductase inhibitor that inhibits JNK signaling (44) and its activation by zinc (Fig. 2B, upper panel) as well as selenite (45), a molecule that suppressed JNK (Fig. 2B, lower panel) and p38 activity.
Treatment of cells with up to 50 M PD98059 (a dose that inhibits both MEK1 and MEK2) and 30 M SB203580 had no effect on either basal or zinc and cadmium-induced MT transcript accumulation ( Fig. 3A and Table I). We also analyzed MT gene expression in established mouse embryo fibroblasts derived from MEK1 null embryos (32) by Northern analysis. MEK1 and MEK2 are the MAPK kinases of the ERK pathway, and, consistent with the results obtained with the ERK inhibitory drugs, both constitutive and metal-induced MT gene transcription were unchanged in mutant cells (data not shown).
Interestingly, H7, GF109203X, Ro-31-8220, dicoumarol, and selenite (Fig. 3, B and C, and Table I), and wortmannin and LY294002 (data not shown) completely abrogated metal induction of MT mRNA. While dicoumarol and selenite had no effect on basal MT mRNA levels, H7, GF109203X, and Ro-31-8220 inhibited basal levels ϳ2-fold. Diminution of metal-induced MT transcripts was dose-dependent. Doses of 25 M GF109203X or 20 M Ro-31-8220 were sufficient to reach almost complete inhibition of zinc-induced levels, while 50 M H7, 25 M GF109203X, or 10 M Ro-31-8220 completely inhibited cadmium induction (Table I). For dicoumarol and selenite, maximal inhibitory effects were observed when cells were treated with doses of 300 and 100 M, respectively. The involvement of JNK in metal-regulated MT gene transcription was further inferred by the fact that anisomycin, a potent activator of the JNK pathway and of p38 kinase activity (46), also activated MT transcription as assayed by Northern analysis (Fig. 3E and Table I).
To ensure that the protein kinase inhibitors were effective in inhibiting the corresponding kinases, in vitro kinase assays were performed with extracts prepared from induced cells treated with PD98059, SB203580, dicoumarol, and selenite. At the concentration of inhibitors used, the ERK ( Fig. 2A (ERK  panel), compare lanes H 2 O 2 and PDϩH 2 O 2 ), p38 ( Fig. 2A (p38  panel), compare lanes FBS and SBϩFBS), and JNK (Fig. 2B, Dicoumarol and Selenite lanes) pathways were efficiently inhibited. We also determined whether the JNK inhibitors interfered with the PKCs. In fact, at the higher dose utilized (300 M) dicoumarol partially inhibited the activation of PKC by zinc ( Fig. 2A, PKC panel, Dicoumarol 300 lanes). However, 150 M dicoumarol did not inhibit zinc-induced PKC activity ( Fig.  2A, PKC panel) but reduced zinc-induced MT mRNA levels by ϳ70% (Fig. 3B and Table I) and metal-induced MRE-LUC expression by almost 100% (Fig. 5). We also verified whether the PKC inhibitor Ro-31-8220 affected JNK activity, because in rat 1 fibroblasts Ro-31-8220 activates JNK (47). However, in L cells, it did not stimulate JNK activity; nor did it prevent its activation by zinc (Fig. 2B, upper panel). FBS, or ZnCl 2 , as indicated. Cells were lysed and assayed for kinase activity using an anti-ERK2 antibody and MBP as the substrate for the ERK pathway, an anti-MAPKAP-K2 and HSP27 for p38, and an anti-PKC and MBP for PKC, as indicated. JNK activity was measured using a solid state kinase assay in which GST-Jun-(1-79) was used to affinitypurify JNK. Phosphorylated substrates (MBP, recombinant HSP27, or c-Jun) were visualized by autoradiography. The results shown are representative of five independent experiments. B, to confirm the inhibitory action of dicoumarol and selenite on JNK activity and assess the stimulatory action of Ro-31-8220 on JNK activity, L cells were serumstarved, pretreated for 45 min with 150 or 300 M dicoumarol, 10 M Ro-31-8220, 100 M selenite, or the vehicle alone and then incubated in the presence of 100 M ZnCl 2 for 60 min before harvesting. Some cells were incubated with anisomycin or exposed to UV irradiation as described in Fig. 2A. Cells were lysed and assayed for JNK activity. The results shown are representative of three independent experiments.
Previous reports showed that calcium is a potent inducer of MT mRNA in EC3 cells (48) and that perturbations in cytosolic calcium ion concentrations are involved in the signal transduction pathway governing MT gene expression. In cells treated with 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid, a cytoplasmic calcium chelator, metal activation of MT gene expression was inhibited to basal levels as assayed by Northern analysis (Fig. 3D, and Table I) (48). This indicates that an increase in intracellular calcium is a key event in the activation of MT gene expression by metals and suggests that conventional PKC isoforms are involved in this induction. Consistent with these observations, constitutive and metal-induced MT promoter activity were not inhibited by the PKC␦-selective inhibitor rottlerin, as assayed by transfection analyses (data not shown).
Interestingly, genistein (100 M), a specific inhibitor of tyrosine-specific protein kinases (49), stimulated basal levels ϳ4fold and brought zinc-induced levels to control values, i.e. those measured in cells treated with genistein only (Fig. 3E, lanes 0, and Table I). Taken together, these results suggest that metalmediated MT gene induction involves a complex signal transduction pathway that includes a conventional PKC, PI3K, JNK, and a tyrosine-specific protein kinase. However, the ERK and the p38 pathways do not appear to be essential.  6 -LUC (containing six tandem copies of the MREa with the MT-1 minimal promoter). In control cells, the 1843MT1-LUC reporter plasmid was activated 8-fold in response to zinc induction and 25-fold by cadmium (Fig. 4), while the MREa-LUC plasmid was activated 6-fold by zinc and ϳ30-fold by cadmium (Fig. 5). Treatment of cells with 5 M Ro-31-8220 (data not shown) or 50 M GF109203X (Figs. 4 and 5) reduced basal MT1-LUC expression by ϳ60% and completely abrogated zinc-and cadmium-stimulated expression of both MT1-LUC and (MREa) 6 -LUC. Dicoumarol also completely abolished zincand cadmium-mediated activation the MRE plasmid in a dosedependent manner (Fig. 5), whereas LY294002 and genistein (Fig. 5) diminished metal-induced (MREa) 6 -LUC expression by 60 -70%. Genistein also strongly inhibited metal-induced MT1-LUC expression (Fig. 4). LY294002 reduced basal MRE-LUC expression by 60% (data not shown), whereas dicoumarol (data not shown) and genistein (Fig. 5) had no effect on this activity. Expression of the pTK-rLUC and pCMV-rLUC internal control plasmids was not modified by any of the kinase inhibitors, thus indicating that the inhibition did not represent a nonspecific effect on RNA polymerase II or general transcription factors. It is also worth noting that the decreases in metal-stimulated MT1-LUC and (MREa) 6 Table I. are equally potent inhibitors of MAPKAP kinase-1␤, p70 S6 kinase, and glycogen synthetase kinase-3 (50,51), while dicoumarol is a quinone reductase inhibitor that blocks both JNK and NF-B signaling (44). Moreover, 300 M dicoumarol inhibited the activation of PKC by zinc ( Fig. 2A, PKC pathway  panel). Because of the possible nonspecific effects of kinase inhibitors (52), the role of PKC and JNK in metal-dependent MT gene regulation was further examined using kinase-dead dominant-negative mutants of these enzymes. Consistent with the results obtained using PKC and JNK inhibitors, co-expression of PKC-KR, JNK1-APF, MKK4-AA, and MKK7-KL mutants reduced basal and zinc-and cadmium-stimulated (MREa) 6 -LUC activity by 50 -70% (Fig. 6, A and B). A reporter plasmid, TRE-LUC, which carries a 12-O-tetradecanoylphorbol-13-acetate response element (TRE) was used to assess the efficiency of the dominant negative mutants of the JNK pathway. TRE contains AP-1 binding sites, which are targets for AP-1 transcription factors whose activation is controlled by JNK. JNK, MKK4, and MKK7 mutants reduced TRE-LUC activity by 70 -80%, thus indicating that inhibition of MRE-  Northern analyses were performed using total RNA prepared from mouse L cells treated with the indicated protein kinase inhibitors in the absence (basal) or presence of inducing doses of ZnCl 2 or CdCl 2 . Data were quantified using a PhosphorImager. Data represent the average Ϯ S.D. of two or three independent experiments. The number of experiments performed for each treatment is indicated in parenthesis. Relative signal intensities are expressed as a percentage relative to that of the zinc-treated control, which is taken as 100. Numbers on the right of each inhibitor indicate the dose (g) used. SB, SB209190; PD, PD 98059; GF, GF109203X; Ro, Ro-31-8220; Sel, selenite; Dic, dicoumarol; NA, not available; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈtetraacetic acid. LUC activity by the same mutants corresponds to nearly the maximal inhibition achievable in this experimental system. Altogether, these transfection experiments show that the kinase signaling cascade involved in the regulation of MT gene expression in response to metals converges on MTF-1.

MTF-1 Is Involved in the
PKC, PI3K, and JNK Inhibitors Have No Effect on MTF-1 DNA Binding Activity-Phosphorylation may be involved in the regulation of MTF-1 DNA binding activity (18), its transactivation function, or its nuclear localization. Thus, we next examined the effects of the PKC, JNK, and PI3K inhibitors on MTF-1 DNA binding activity and nuclear localization by EMSA analysis. L cells pretreated or not with GF109203X, dicoumarol, selenite, or LY294002 were grown in the presence or absence of zinc or cadmium, and nuclear and cytoplasmic extracts were prepared and analyzed by EMSA. As we reported earlier (18), extracts prepared from control untreated cells contained MTF-1 DNA binding activity, of which ϳ80% was detected in the nuclear fractions (Fig. 7, A, B, and D). Zinc induction caused a 3-7-fold increase in the levels of MTF-1 DNA binding activity in the nuclear fractions and reduced activity in the cytosolic fraction to background values (Figs. 7, A, B, and D). As previously shown (13,(15)(16)(17)(18), cadmium had little effect on MTF-1 DNA binding activity (Fig. 7, A-C). Interestingly, in extracts prepared from cells preincubated with GF109203X (Fig. 7A), selenite (Fig. 7, B and C), dicoumarol (Fig. 7C), or LY294002 (Fig. 7D), but not treated with metals, most of the MTF-1 DNA binding activity was still detected in the nuclear fractions. Most importantly, MTF-1 DNA binding activity in nuclear extracts prepared from cells treated with the inhibitors and induced with zinc was not diminished compared with control extracts prepared from zinc-treated cells. Control experiments using an Sp1 binding site in the EMSA showed that the cytosol extracts were completely devoid of Sp1 activity, thus showing that they were not significantly contaminated with nuclear proteins. Furthermore, the kinase inhibitors and metal induction had no effects on Sp1 DNA binding. These results suggest that the protein kinase inhibitors repressed a kinase signaling transduction pathway(s) that regulates MTF-1 transcriptional activity. Indeed, if MTF-1 nuclear localization and DNA binding activity are not changed following treatments with the inhibitors, they strongly suggest that PKC-, PI3K-, and JNK-mediated MTF-1 phosphorylation modulates the transactivation function of MTF-1. DISCUSSION MTF-1 is required for basal as well as zinc-and cadmiuminduced MT gene expression. MTF-1 is considered to be a cytoplasmic zinc sensor, characterized by increased DNA binding activity upon zinc treatment (5,15). While activation of MTF-1 DNA binding activity is dependent on the interaction of zinc with the zinc fingers of the protein (53, 54), it does not appear sufficient for zinc induction of MT gene expression in vivo. Analysis of MTF-1 zinc finger mutants indicated that a functional domain responsible for zinc regulation resides outside of the zinc finger domain in the N-terminal region (54) and that MTF-1 zinc fingers cooperate with the transactivation domains to produce a zinc-sensing metalloregulatory transcription factor (53). MTF-1 activity can be modulated by mechanisms independent of the zinc-dependent increase in DNA binding activity. Indeed, MTF-1 binding in nuclear extracts from zinc-pretreated cells is always higher than from untreated cells, even if saturating amounts of zinc are added to the binding reaction (5,18). This suggests that zinc induction in vivo induces other changes in MTF-1 enhancing its activity, distinct from the structural changes directly induced by zinc. Zinc may simply be a constitutive structural component of MTF-1 but may also act as a regulatory molecule. In addition to the direct interaction of zinc with MTF-1, other signal transduction cascades may be involved in the regulation of MTF-1 activity in response to metal ions.
In an effort to further explore the mechanisms controlling MTF-1 activity, we wished to determine whether MTF-1 is a FIG. 6. Co-expression of kinase-deficient mutants of PKC␣, JNK, MKK7, and MKK4 inhibits basal and metal-induced (MREa) 6 -LUC expression. A, HepG2 cells were transfected with a plasmid mixture containing 7.5 g of (MREa) 6 -LUC, 100 ng of pTK-LUC, and 2.5 or 5 g of the PKC␣ dominant negative mutant, PKC-KR. PCDNA3 plasmid DNA was added to provide the same total concentration of DNA. Cells were cultured for 24 h in complete medium and then treated or not with 100 M ZnCl 2 or 2.5 M CdCl 2 for 8 h before measurement of LUC activity as described in the legend of Fig. 4. Data represent the average Ϯ S.D. of three independent experiments performed in duplicate. B, HepG2 cells were transfected with a plasmid mixture containing 7 g of (MREa) 6 -LUC (upper panel) or TRE-LUC (lower panel), 100 ng of pTK-LUC, and 1 or 5 g of the indicated MKK or JNK dominant-negative mutants. PCDNA3 plasmid DNA was added to provide the same total concentration of DNA. Cells transfected with (MREa) 6 -LUC were cultured for 24 h in complete medium and then treated or not with 100 M ZnCl 2 for 8 h before measurement of LUC activity as described in the legend of Fig. 4. Data represent the average Ϯ S.D. of three independent experiments performed in duplicate.
phosphoprotein. Metabolic labeling experiments showed that MTF-1 is phosphorylated in vivo both in the uninduced and induced state and that zinc treatment led to a 2-4-fold increase in the overall phosphorylation status of the protein.
Using different kinase inhibitors and kinase-dead dominant negative mutants, we showed that MTF-1 activity and MT gene expression are controlled by a complex kinase signaling transduction pathway that includes PKC, PI3K, JNK, and a tyrosine-specific protein kinase. The ERKs and the p38 pathways do not appear to be essential for the activation of MT gene expression in response to metals. Notably, increased MTF-1 DNA binding in response to zinc and MTF-1 nuclear localization was not inhibited in cells preincubated with the different kinase inhibitors despite strong inhibition of MTF-1-mediated gene expression. Thus, despite the fact that MTF-1 is localized in the nucleus in a zinc-activated DNA binding conformation, as assayed by EMSA, it did not activate MRE-mediated transcription. This strongly suggests that phosphorylation is essential for MTF-1 transactivation function. These results allow us to hypothesize that metalinduced phosphorylation of MTF-1 is one of the primary events leading to increased MTF-1 activity. Metals may trigger a protein kinase cascade(s) that results in the phosphorylation by a specific MTF-1 kinase(s) (MTFK) of regulatory site(s) required for transcriptional activity of MTF-1 (Fig. 8).
Interactions of zinc with the zinc finger domain of MTF-1 would remain, in cooperation with the MTF-1 kinase cascade, an important regulatory mechanism controlling MTF-1 DNA binding activity and, possibly, nuclear translocation.
Zinc is an essential trace nutrient for all life forms and participates in a variety of enzymatic reactions. Zinc has been identified as a central component of over 300 metalloenzymes as well as numerous zinc finger transcription factors (55). The biological essentiality of zinc implies the existence of homeostatic mechanisms that tightly regulate its absorption, distribution, cellular uptake, and excretion. Because of its central role in the regulation of key molecules involved in zinc homeostasis, such as MT and ZnT-1, control of MTF-1 activity by zinc is likely to play an important role in these homeostatic mechanisms. Our results show that regulation of MTF-1 activity involves a specific metal-activated kinase signaling cascade; thus, mechanisms regulating this cascade are also likely to be crucial for zinc homeostasis. We postulate the existence of a zinc-responsive pathway that functions as a reversible activated sensor of the free zinc pool in the cell. A component(s) in this pathway would be reversibly activated in response to increased intracellular free zinc levels and would control the activity of the kinase cascade converging on MTFK and MTF-1 and, possibly, on other transcription factors and molecules involved in zinc homeostasis and stress response. Cadmium (Fig. 7, B and C), as well as other transition metals, have little effect on MTF-1 DNA binding activity in vivo and in vitro despite strong MTF-1-dependent induction of MT genes (16,18). These data were interpreted as suggesting that cadmium activation of MT gene expression by MTF-1 could be modulated by indirect mechanisms independent of those used by zinc (3,16). However, cadmium appears to use the same signaling cascade as that used by zinc to activate MT gene expression, since the kinase inhibitors used in this study abrogated the activation of MT gene transcription in response to both zinc and cadmium. Cadmium is a toxic transition metal with no known physiological function, which activates the expression of several genes such as those encoding HSPs, heme oxygenase, c-Fos, c-Jun, and Erg-1 (56). In mammalian cells, several different sequence-specific DNA-binding proteins can function as "cadmium-responsive" factors. These include USF (57), AP-1 (58), Nrf2 (40), and MTF-1 (5). Alam et al. (40) suggested that cadmium could regulate target genes by modulating the activity of transcription factors that normally regulate the response to other physiological stimuli. In the case of the MT genes, this would be by interfering with the mechanisms controlling MTF-1 activity in response to zinc. Indeed, cadmium can activate several kinases (40,43,59), of which some may overlap with the MTF-1 kinase transduction path- way, thus stimulating MTF-1 transcriptional activity and MT gene expression without increasing MTF-1 DNA binding activity. While zinc is required for MTF-1 DNA binding activity, in normal physiological conditions, cells presumably contain enough endogenous zinc to keep MTF-1 in a transcriptionally inducible form that can undergo transcriptional activation through phosphorylation without further increase in DNA binding activity. Thus, in this hypothesis, cadmium and other metals do not simply cause the redistribution of zinc but alter the transactivation potential of MTF-1 by phosphorylation (Fig. 8).
If metals activate MTF-1 by phosphorylation as suggested in the above model, MTF-1 should be phosphorylated on at least one additional site in response to metal induction. Indeed, phosphorylation of MTF-1 increased 2-4-fold in response to zinc induction in vivo (Fig. 1). This extent of enhanced phosphorylation of MTF-1 in zinc-treated cells compared with the control is compatible with the proposed model despite stronger MTF-1-dependent induction of MT genes in vivo. Indeed, since MTF-1 is already phosphorylated in control cells, the phosphorylation of an extra amino acid in response to metals could easily produce a relatively modest increase in the overall phosphorylation state of the protein. However, the phosphorylation of only one or two additional amino acid residues can have a major effect on protein activity. For example, the phosphorylation of serine 392 can efficiently activate p53's DNA binding and transcriptional function (60). Similarly, phosphorylation of c-Jun serine 63 and 73 dramatically stimulates the transcriptional activity of the protein (61). Use of MTF-1 deletion mutants should facilitate the identification of the amino acid residue(s) phosphorylated in response to metal induction.
Because JNK and PKC can directly phosphorylate and activate transcription factors such as NF-E2 (62) and SAF (63), the signaling pathways(s) utilized for regulation of MT gene expression in response to metals may ultimately depend on the ability of MTF-1 to serve as a substrate for JNK and PKC. Although kinase-dead mutants and PKC and JNK inhibitors can inhibit MTF-1 activity, direct phosphorylation of MTF-1 by PKC and JNK remains to be tested. PI3K controls key target kinases such as 3-phosphoinositide-dependent protein kinase-1 and protein kinase B/AKT (AKT) and is apparently not directly involved in the phosphorylation of transcription factors. AKT, however, has been shown to phosphorylate some transcription factors including NF-B (64) and the forkhead-related transcription factor FKHRL1 (65). Because AKT and PKC are two possible immediate downstream components of PI3K 3 3-phosphoinositidedependent protein kinase-1, PKC may be downstream of PI3K and upstream of JNK in the MTF-1 signal transduction cascade. In fact, a 150 M concentration of the JNK inhibitor dicoumarol completely inhibited MRE-LUC expression (Fig. 5) and did not prevent the activation of PKC by zinc ( Fig. 2A, PKC pathway), thus indicating that PKC is indeed upstream of JNK. However, the PKC inhibitor Ro-31-8220 did not prevent the activation of JNK in response to zinc (Fig. 2B), thus suggesting that JNK is upstream of PKC, which would be quite surprising and unexpected. However, in mammals there are three JNK genes, and elaborate splicing generates at least 10 different JNK isoforms, which might differ in their substrate specificity (66). The assay used in this study to measure JNK activity does not allow us to distinguish each JNK family member. Thus, it is possible that inhibition by Ro-31-8220 of a specific JNK isoform involved in regulation of MT gene expression could have gone undetected due to the imprecision of the assay. Another pos- Under normal physiological conditions, zinc (q) and cadmium (ࡗ) ions would enter the cell through yet unidentified transporters (T? and Tcd, respectively) and be metabolized through a variety of metalloproteins and enzymes, including MT. ZnT1 is located at the plasma membrane and functions as an exporter for zinc efflux. In control (basal) cells, there is presumably enough endogenous zinc to promote the transport of some MTF-1 into the nucleus and cause its conversion from an inactive (non-DNA-binding) state to an active, DNA-binding configuration. In zinc-induced cells, MTF-1 nuclear localization and transcriptional activity would be enhanced. In this model, the activity of MTFK, and ultimately the transcriptional activity of MTF-1, is controlled by a kinase transduction cascade (large arrows) involving a tyrosine-specific protein kinase (TyrK), PI3K, PKC, and JNK. The MTF-1 kinase signaling transduction pathway would be controlled by a yet uncharacterized zinc-sensing metalloregulatory pathway in which a component(s) plays the role of zinc sensor in cooperation with the metal-sensing zinc finger domain of MTF-1. The precise order in which each kinase is positioned in the cascade is still unknown. It is also possible that multiple pathways converge on MTF-1 at the same time and that more than one kinase can directly phosphorylate MTF-1 in response to metals. Because JNK and PKC can directly phosphorylate and activate transcription factors, one or both may correspond to MTFK. Cadmium and other metal ions would activate MTF-1 and induce MT gene expression by stimulating one or several kinases in the MTF-1 kinase pathway. The dashed arrow indicates putative action of cadmium on the MTF-1 kinase cascade. ✡, phosphate group. sible way to reconcile these apparently contradictory observations is to assume that PKC and JNK are in two different MTF-1regulating signal transduction pathways and that both cascades are required for the activation of MTF-1 and of MT gene expression in response to metals. This would imply that two events of phosphorylation are required to fully activate MTF-1 transcriptional potential and that MTFK is in fact composed of two kinases. Other transcription factors have been shown to be regulated by multiple pathways. For example, cadmium induces c-fos in part through activation of the ERK pathway and through an as yet unidentified kinase cascade (67). Similarly, MEF2A is a nuclear target for both the p38 pathway and PKC (68), and several stress response kinases have been shown to phosphorylate p53 and control its activity (60).
In conclusion, regulation of MTF-1 activity appears to require multiple hierarchical phosphorylations involving several different kinases. A thorough analysis of MTF-1 phosphorylation and the identification of MTFK will be essential for a complete understanding of MTF-1 signal transduction.