J Biol Chem, Vol. 275, Issue 11, 7633-7640, March 17, 2000

G_i-dependent Activation of c-Jun N-terminal Kinase in Human Embryonal Kidney 293 Cells^*

Junji Yamauchi, Takeharu Kawano, Motoshi Nagao, Yoshito Kaziro, and Hiroshi Itoh

From the Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Heterotrimeric G proteins stimulate the activities of two stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase in mammalian cells. In this study, we examined whether  subunits of G_i family activate JNK using transient expression system in human embryonal kidney 293 cells. Constitutively activated mutants of G_i1, G_i2, and G_i3 increased JNK activity. In contrast, constitutively activated G_o and G_z mutants did not stimulate JNK activity. To examine the mechanism of JNK activation by G_i, kinase-deficient mutants of mitogen-activated protein kinase kinase 4 (MKK4) and 7 (MKK7), which are known to be JNK activators, were transfected into the cells. However, G_i-induced JNK activation was not blocked effectively by kinase-deficient MKK4 and MKK7. In addition, activated G_i mutant failed to stimulate MKK4 and MKK7 activities. Furthermore, JNK activation by G_i was inhibited by dominant-negative Rho and Cdc42 and tyrosine kinase inhibitors, but not dominant-negative Rac and phosphatidylinositol 3-kinase inhibitors. These results indicate that G_i regulates JNK activity dependent on small GTPases Rho and Cdc42 and on tyrosine kinase but not on MKK4 and MKK7.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins)¹ are composed of , , and  subunits (G, G, and G), which are encoded by at least 15, 5, and 11 genes, respectively, in mammalian cells (1-4). In response to stimuli such as sensory signals, hormones, neurotransmitters, and chemokines, G protein-coupled receptors activate G proteins, which in turn modulate downstream effectors including adenylyl cyclases, phospholipase Cs, phosphatidylinositol 3-kinases, ion channels, and -adrenergic receptor kinases (1-4).

Mitogen-activated protein kinases (MAPKs) are serine/threonine kinases involved in cellular responses to various stimuli (5-8). MAPKs are grouped into four major classes: ERK/MAPK, BMK1/ERK5, JNK/stress-activated protein kinase, and p38 MAPK. ERK and BMK1 are activated mainly by growth factors and are involved in cell cycle progression and cell growth (5-8). Inflammatory cytokines and environmental stresses stimulate the activities of JNK and p38 MAPK, which appear to be implicated in cell cycle arrest and apoptosis (6-8). MAPKs are activated by dual phosphorylation on threonine and tyrosine residues catalyzed by MAPKK/MEK, which is phosphorylated and activated by serine/threonine kinases called MAPKKK/MEKK (5-8). Raf activates ERK via MEK1/MKK1 and MEK2/MKK2. MEK5/MKK5/SKK5 phosphorylates BMK1. JNK is phosphorylated and activated by MKK4/SEK1/JNKK1/SKK1 and MKK7/JNKK2/SKK4, while p38 MAPK is phosphorylated and activated by MKK3/SKK2, MKK6/SKK3, and MKK4. Since many MAPKKK/MEKKs including MEKK1, MEKK2, MEKK3, MEKK4, MAPKKK5, and MAPKKK6, induce the activation of JNK and/or p38 MAPK cascade(s), the linkage of MAPKKK/MEKK to MAPKK/MEK is more complicated than that of MAPKK to MAPK.

Several lines of evidence suggest that G protein-coupled receptors stimulate the ERK pathway through some G protein subunits in various cells (9, 10). It is likely that G_i-dependent ERK activation is mediated primarily by G (9, 10). G directly activates phosphatidylinositol 3-kinase  and -adrenergic receptor kinase 1, resulting in an increase of activities of Src family tyrosine kinases (9-14). Tyrosine-phosphorylated Shc permits the translocation of the Grb2-Sos complex to plasma membranes, leading to the promotion of the GDP-GTP exchange on Ras. Ras regulates the activity of Raf, which induces the activation of MEK1 and -2 and subsequently ERK.

Some G protein-coupled receptors are able to activate JNK in certain types of cells (9). JNK is activated by agonist stimulation of G_i-coupled receptors in NIH3T3, Rat-1, and COS-7 cells (15-17). It has been reported that JNK activation by G_i-coupled m2 muscarinic acetylcholine receptor is mediated mainly by G in COS-7 cells (17). Small GTPases Ras and Rac and phosphatidylinositol 3-kinase  are involved in this G-induced JNK activation (17, 18). In the course of studying G_i-dependent JNK activation in human embryonal kidney (HEK) 293 cells, we found that its activation was mediated by both G_i and G. Here we show that constitutively activated G_i2 mutant as well as G (19) stimulates JNK activity. Furthermore, we investigate whether JNK kinases, Rho family GTPases, tyrosine kinases, and phosphatidylinositol 3-kinases participate in this signal transduction pathway.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Mastoparan and pertussis toxin were purchased from Calbiochem-Novabiochem Co. and Kaken Pharmaceutical Co., respectively. Tyrosine kinase inhibitors PP1/AG1872 and PP2/AG1879 were kindly provided by A. Levitzki (Hebrew University). Phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 were purchased from Calbiochem-Novabiochem Co. and BIOMOL, respectively. Mouse monoclonal antibodies M2, 12CA5, and 9E10 against FLAG, HA, and Myc epitopes were obtained from Eastman Kodak Co., Roche Molecular Biochemicals, and Babco, respectively. Mouse monoclonal antibody B-14 against Schistosoma japonicum GST was purchased from Santa Cruz Biotechnology, Inc. Rabbit polyclonal antibodies T-20 and 06-238 against G were obtained from Santa Cruz Biotechnology and Upstate Biotechnology, Inc., respectively. Rabbit polyclonal antibodies AS/7, EC/2, and GC/2 against G_i1/2, G_i3, and G_o, respectively, were purchased from NEN Life Science Products, Inc. Rabbit polyclonal antibodies against G_i1/2 and G_z (X264) were generously provided by T. Asano (Aichi Human Service Center) and P. C. Sternweis (University of Texas Southwestern Medical Center), respectively. Rabbit polyclonal anti-Csk antibody C-20 was purchased from Santa Cruz Biotechnology. Goat anti-mouse and anti-rabbit IgG antibodies conjugated with horseradish peroxidase were obtained from NEN Life Science Products.

Plasmids-- Complementary DNAs of G_i3, G_oQ205L, G_z, and G_zQ205L (20-22) were inserted into pCMV mammalian expression vector. pCMV-G_i1, pCMV-G_i1Q204L, pCMV-G_i2, pCMV-G_i2Q205L, pCMV-G_o, pCMV-G₁, pCMV-G₂, pCMV-carboxyl terminus of -adrenergic receptor kinase 1 (ARK1ct), pCMV-FLAG-RhoT19N, pCMV-FLAG-RacT17N, pCMV-FLAG-Cdc42T17N, pCMV-GST-MKK4, pCMV-FLAG-MKK4K95R, pCMV-GST-MKK7, and pCMV-FLAG-MKK7K63R were constructed as described previously (23-25). pCMV-G_i3Q204L was prepared by T. Yamaguchi and M. Tagaya (Tokyo University of Pharmacy and Life Science). cDNA of human orthologue (26) of mouse MKK7 (27) was inserted into a BamHI restriction site of pCMV-GST. SR-HA-JNK1 and SR-HA-ERK2 were kindly provided by M. Karin (University of California, San Diego). pEF-BOS-Clostridium botulinum C3 toxin (28) was generously provided by S. Narumiya (Kyoto University). Pak1 cDNA was a generous gift from L. Lim (National University of Singapore). A CRIB region, which interacts with active Cdc42 and Rac (29), was amplified by polymerase chain reaction using Pak1 cDNA as a template and was ligated into pCMV-Myc. Csk cDNA (30) was kindly provided by M. Okada (Institute for Protein Research, Osaka University). pCMV-Csk was constructed and generously provided by S. Mizutani. Hexahistidine tag expression plasmids, pET15b-JNK1 and pET32a-c-Jun (amino acids 1-223), were constructed as described before (19). pGEX2T-c-Jun (amino acids 1-223) was kindly provided by M. Karin (University of California, San Diego). All DNA sequences were confirmed by DNA sequencer L-4000L (LI-COR) according to the manufacturer's protocol.

Cell Culture-- HEK 293 cells (ATCC CRL 1573) were maintained in Dulbecco's modified Eagle's medium (Sigma) containing 100 µg/ml kanamycin (Nacalai) with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.). The cells were cultured at 37 °C in a humidified atmosphere containing 10% CO₂.

Transfection-- Plasmid DNAs were transfected into HEK 293 cells by the calcium phosphate precipitation method. The final amount of the transfected DNA for a 60-mm dish was adjusted to 25 µg by empty vector, pCMV. Three µg of SR-HA-JNK1, SR-HA-ERK2, pCMV-GST-MKK4, pCMV-GST-MKK7, or pCMV-GST-MKK7 was cotransfected with 3 µg of pCMV-Myc-ARK1ct, 10 µg of each G wild type or QL mutant plasmid, 5 µg of pCMV-G₁, and 5 µg of pCMV-G₂, 10 µg of pCMV-FLAG-MKK4K95R, 10 µg of pCMV-FLAG-MKK7K63R, 10 µg of dominant negative Rho family plasmid, 10 µg of pEF-BOS-C3 toxin, 10 µg of pCMV-Myc-Pak1CRIB, or 3 µg of pCMV-Csk. The medium was replaced 24 h after transfection, and the cells were starved in the serum-free medium containing 1 mg/ml bovine serum albumin (Nacalai) for 24 h.

Recombinant Proteins-- Recombinant hexahistidine-JNK, Trx-c-Jun, and GST-c-Jun were purified from the transformed E. coli strain BL21 (DE3) cells as described before (19). Briefly, E. coli cells treated with isopropyl-1-thio--D-galactopyranoside were harvested by centrifugation and sonicated in extraction buffer A (20 mM HEPES-NaOH (pH 8.0), 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 0.5% Nonidet P-40) for hexahistidine-JNK and Trx-c-Jun or extraction buffer B (20 mM HEPES-NaOH (pH 7.5), 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM EGTA, 0.5% Nonidet P-40) for GST-c-Jun. The cell extracts were centrifuged at 150,000 × g for 30 min. All purification steps were performed at 4 °C. To purify hexahistidine-JNK or Trx-c-Jun, the supernatants were subjected to nickel-nitrilotriacetic acid-agarose (Qiagen, Inc.), and the resin was washed with column buffer A (20 mM HEPES-NaOH (pH 8.0), 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 200 mM NaCl) containing 20 mM imidazol. Hexahistidine-JNK and Trx-c-Jun were eluted with column buffer A containing 200 mM imidazol. For the purification of GST-c-Jun, the supernatant was applied to glutathione-Sepharose 4B (Amersham Pharmacia Biotech), and the resin was washed with column buffer B (20 mM HEPES-NaOH (pH 7.5), 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM EGTA). GST-c-Jun was eluted with column buffer B containing 10 mM glutathione. Hexahistidine-JNK was dialyzed against column buffer B containing 200 mM NaCl and stored at 80 °C until use. Under these storage conditions, hexahistidine-JNK retained the catalytic activity within at least 6 months. The eluate of GST-c-Jun and Trx-c-Jun was dialyzed against column buffer B and stored at 80 °C until use.

Kinase Assays-- After 24 h of serum starvation, the cells transfected with SR-HA-JNK1, SR-HA-ERK2, pCMV-GST-MKK4, pCMV-GST-MKK7, or pCMV-GST-MKK7 were lysed in 600 ml of lysis buffer A (20 mM HEPES-NaOH (pH 7.5), 3 mM MgCl₂, 100 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml leupeptin, 1 mM EGTA, 1 mM Na₃VO₄, 10 mM NaF, 20 mM -glycerophosphate, and 0.5% Nonidet P-40) on ice. The lysates were centrifuged at 14,000 rpm for 10 min at 4 °C. For JNK and ERK assay, aliquots (500 µg) of the supernatants were mixed with protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) preabsorbed with a mouse anti-HA antibody for 12 h at 4 °C. The immune complexes were washed twice with lysis buffer A and twice with reaction buffer A (20 mM HEPES-NaOH (pH 7.5), 10 mM MgCl₂, 0.1 mM phenylmethanesulfonyl fluoride, 0.1 µg/ml leupeptin, 0.1 mM EGTA, 10 mM Na₃VO₄, and 2 mM -glycerophosphate) and incubated in 30 µl of reaction buffer A containing 3 µg of GST-c-Jun for JNK assay or 5 µg of myelin basic protein for ERK assay, 20 µM ATP, and 5 µCi of [-³²P]ATP (Amersham Pharmacia Biotech) at 30 °C for 10 min. For MKK4, MKK7, or MKK7 assay, aliquots (500 µg) of the supernatants were mixed with glutathione-Sepharose 4B for 12 h at 4 °C and centrifuged. The precipitate was washed with lysis buffer A and with reaction buffer A and was incubated in 30 µl of reaction buffer A containing 2 µg of hexahistidine-JNK, 10 µg of Trx-c-Jun, 20 µM ATP, and 5 µCi of [-³²P]ATP at 30 °C for 20 min. The reaction was stopped by adding 10 µl of 4 × Laemmli sample buffer and boiling, and the sample was subjected to SDS-polyacrylamide gel electrophoresis. The radioactivity incorporated into GST-c-Jun, Trx-c-Jun, and myelin basic protein was measured by an imaging analyzer (FUJI BAS 2000) and detected by autoradiography.

Immunoprecipitation and Immunoblotting-- Aliquots (250 µg) of cell lysates were mixed with protein A-Sepharose CL-4B preabsorbed with a mouse anti-Myc antibody for 12 h at 4 °C. The immune complexes were precipitated by centrifugation and washed four times with lysis buffer A. Aliquots of cell lysates and immune complexes were boiled in Laemmli sample buffer. The boiled samples were separated by SDS-polyacrylamide gel electrophoresis, and the proteins were transferred to nitrocellulose membranes (BA85; Schneider & Schnell). After the membranes were blocked with phosphate-buffered saline containing 0.1% Tween 20 and 5 mg/ml bovine serum albumin, the separated proteins were immunoblotted with various antibodies. The bound antibodies were detected using anti-rabbit or mouse IgG antibody conjugated with horseradish peroxidase.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mastoparan-induced JNK Activation Is Mediated by both G_i and G in HEK 293 Cells-- We introduced plasmids encoding HA-tagged JNK with various cDNAs into HEK 293 cells. Using anti-HA antibody, the epitope-tagged JNK was immunoprecipitated from lysates of the transfected cells. The JNK activity was assessed as the radioactivity incorporated into recombinant GST-c-Jun. The expression level of HA-JNK was confirmed by immunoblotting in each experiment to compare the transfection efficiency (see Figs. 1-7). To examine G_i-dependent JNK activation, we searched an endogenous G_i-coupled receptor in HEK 293 cells. However, we could not find a candidate for the receptor suitable to this study. Then we transfected a plasmid encoding G_i-coupled m2 muscarinic acetylcholine receptor into the cells, but JNK was activated only weakly with an agonist stimulation. The reason may be that the ectopic expression level of m2 muscarinic acetylcholine receptor is not so high (24). As shown in Fig. 1A, mastoparan, a peptide that directly activates G proteins by a mimic of G protein-coupled receptor (31), stimulated the activity of JNK by approximately 4-fold.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   JNK activation by mastoparan is mediated by both Gi and G. HEK 293 cells were transfected with plasmids carrying cDNAs for HA-JNK (A and B) and Myc-ARK1ct (ARK1ct) (B) and treated with or without 200 ng/ml pertussis toxin for 24 h after transfection (A). The JNK activity was measured at 15 min after the addition of 50 µM mastoparan as described under "Experimental Procedures." Values shown represent the mean ± S.E. from three or four separate experiments. The phosphorylation of GST-c-Jun and the expression of HA-JNK and ARK1ct in the cell lysates are shown.

Next, we investigated whether G_i is involved in mastoparan-induced JNK activation. The cells were treated with pertussis toxin, which ADP-ribosylates G_i/G_o and inhibits the coupling of G_i/G_o with the receptors, for 24 h before the addition of mastoparan (Fig. 1A). The activation was almost completely inhibited by the pretreatment of pertussis toxin. Since there is no G_o in HEK 293 cells as described below, the inhibition by pertussis toxin indicated that mastoparan increases JNK activity via G_i.

To determine whether mastoparan-induced JNK activation is mediated by G_i and/or G, a plasmid encoding carboxyl-terminal peptide of -adrenergic receptor kinase 1 (ARK1) was cotransfected. It has been shown that ARK1ct associates with G and inhibits G-mediated ERK and JNK activations by a G protein-coupled receptor (11, 17). Mastoparan-induced JNK activation was reduced approximately 50% by cotransfection of ARK1ct (Fig. 1B), suggesting that the JNK activation is mediated by both G_i and G in HEK 293 cells.

G_i2Q205L Stimulates the Activity of JNK in HEK 293 Cells-- We explored which  subunit of the G_i family increases JNK activity. As shown in Fig. 2A, constitutively activated mutants of G_i1, G_i2, and G_i3 stimulated JNK activity by approximately 3.5-, 5-, and 3.5-fold, respectively. On the other hand, constitutively activated G_o and G_z mutants did not stimulate JNK activity. The activation of JNK by Gi2Q205L was comparable with that by G (Fig. 2A). The expression of endogenous G_o and G_z in HEK 293 cells was not detected by immunoblotting (Fig. 2A).

View larger version (43K): [in this window] [in a new window]   Fig. 2.   JNK is activated by constitutively activated forms of Gi and G. Cells were transfected with plasmids carrying cDNAs for HA-JNK (A and B), Gi1 (i1) (A), Gi1Q204L (i1Q204L) (A), Gi2 (i2) (A), Gi2Q205L (i2Q205L) (A and B), Gi3 (i3) (A), Gi3Q204L (i3Q204L) (A), Go (o) (A), GoQ205L (oQ205L) (A), Gz (z) (A), GzQ205L (zQ205L) (A), G1 (1) (A and B), G2 (2) (A and B), and Myc-ARK1ct (ARK1ct) (B). The JNK activity was measured as described under "Experimental Procedures." Values shown represent the mean ± S.E. from three or six separate experiments. The phosphorylation of GST-c-Jun and the expression of HA-JNK, G protein subunits, and Myc-ARK1ct in the cell lysates are shown. WT, wild type; QL, QL mutant.

To confirm that the inhibitory effect of ARK1ct on mastoparan-induced JNK activation results from the sequestration of G, ARK1ct was cotransfected with Gi2Q205L or G (Fig. 2B). The activation of JNK by G, but not Gi2Q205L, was blocked completely by cotransfection of ARK1ct.

Mastoparan-induced JNK Activation Is Dependent Partially on MKK4 and MKK7-- To clarify whether mastoparan activates JNK through two JNK kinases, MKK4 and MKK7, we cotransfected a plasmid of MKK4K95R or MKK7K63R. MKK4K95R and MKK7K63R are kinase-deficient mutants that sequester upstream components such as MEKK1. Mastoparan-induced JNK activation was suppressed partially by cotransfection of MKK4K95R or MKK7K63R (Fig. 3, A and B).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Partial involvement of MKK4 and MKK7 in mastoparan-induced JNK activation. Cells were transfected with plasmids carrying cDNAs for HA-JNK (A and B), GST-MKK4 (C), GST-MKK7 (D), FLAG-MKK4K95R (MKK4K95R) (A), and FLAG-MKK7K63R (MKK7K63R) (B). The activities of JNK, MKK4, and MKK7 were measured at 15 min after the addition of 50 µM mastoparan as described under "Experimental Procedures." Values shown represent the mean ± S.E. from three or four separate experiments. The phosphorylation of GST-c-Jun and Trx-c-Jun and the expression of HA-JNK, GST-MKK4, GST-MKK7, FLAG-MKK4K95R, and FLAG-MKK7K63R in the cell lysates are shown. GST-MKK4 and GST-MKK7 were precipitated with glutathione-Sepharose 4B from the cell lysates and immunoblotted with anti-GST antibody.

Next, we analyzed the activities of MKK4 and MKK7. The cells were cotransfected with a plasmid encoding GST-fused MKK4 or MKK7. GST-MKK4 or -MKK7 was precipitated from lysates of the transfected cells using glutathione-Sepharose 4B and incubated with recombinant JNK and c-Jun, and the radioactivity incorporated into c-Jun was measured. Mastoparan activated only weakly MKK4 and MKK7 (Fig. 3, C and D). This result, considered together with results of kinase-deficient mutants, indicates that JNK activation by mastoparan is dependent partially on MKK4 and MKK7.

G_i2Q205L Fails to Activate MKK4 and MKK7-- We reported previously that G activates JNK mainly through MKK4 and to a lesser extent through MKK7 (19). Because JNK activation by mastoparan was dependent partially on MKK4 and MKK7, we considered that MKK4 and MKK7 might be involved in JNK activation mediated by G_i as well as that by G. However, cotransfection of MKK4K95R failed to attenuate G_i2Q205L-induced JNK activation (Fig. 4A). Furthermore, G_i2Q205L failed to activate MKK4 (Fig. 4C). Although cotransfection of MKK7K63R inhibited partially G_i2Q205L-induced JNK activation, MKK7 activity was not stimulated by G_i2Q205L (Fig. 4, B and D). A human MKK7 gene appears to generate some alternative splicing forms. A MKK7 isoform has 86 extra amino acid residues at the N terminus of MKK7 (26) and is expressed mainly in HEK 293 cells (data not shown). We thought that G_i might activate a MKK7 isoform. However, G_i2Q205L failed to stimulate MKK7 activity (Fig. 4E). In contrast, G increased the activities of MKK4, MKK7, and MKK7 by approximately 6-, 2.5-, and 2-fold, respectively (Fig. 4, C-E). These results indicate that G_i2 regulates JNK activity through MKK4- and MKK7-independent pathway. On the other hand, G activates JNK through MKK4- and MKK7-dependent pathways (19). It is likely that mastoparan induces MKK4 and MKK7 activation through G but not G_i.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Gi2Q205L activates JNK through an MKK4- and MKK7-independent pathway. Cells were transfected with plasmids carrying cDNAs for HA-JNK (A and B), GST-MKK4 (C), GST-MKK7 (D), GST-MKK7 (E), Gi2Q205L (i2Q205L) (A-E), G1 (1) (C-E), G2 (2) (C-E), FLAG-MKK4K95R (MKK4K95R) (A), and FLAG-MKK7K63R (MKK7K63R) (B). The activities of JNK, MKK4, MKK7, and MKK7 were measured as described under "Experimental Procedures." Values shown represent the mean ± S.E. from at least three separate experiments. Statistical analysis was performed using Student's t test. *, p < 0.01 (n = 6) compared with Gi2Q205L without MKK7K63R. The phosphorylation of GST-c-Jun and Trx-c-Jun and the expression of HA-JNK, GST-MKK4, GST-MKK7, GST-MKK7, G protein subunits, FLAG-MKK4K95R, and FLAG-MKK7K63R in the cell lysates are shown. GST-MKK4, GST-MKK7, and GST-MKK7 were precipitated with glutathione-Sepharose 4B from the cell lysates and immunoblotted with anti-GST antibody.

G_i2-mediated JNK Activation Is Dependent on Rho and Cdc42 but Not on Rac-- Rho family GTPases have been shown to be implicated in the activation of JNK by various stimuli (32). In addition, JNK activation by G depends on Rho family GTPases (17-19). To test the possibility that Rho family GTPases are involved in the pathway from G_i to JNK, we cotransfected each plasmid of dominant-negative Rho family GTPases. Mastoparan-induced JNK activation was inhibited by RhoT19N and Cdc42T17N but not RacT17N (Fig. 5, A-C). In addition, JNK activation by G_i2Q205L was also blocked by the dominant negative mutants of Rho and Cdc42 (Fig. 5, D-F), indicating that G_i2 regulates JNK activity through Rho and Cdc42 in HEK 293 cells. To confirm that Rho and Cdc42 are involved in G_i2Q205L-induced JNK activation, we utilized C3 toxin and Pak1CRIB. C3 toxin ADP-ribosylates Rho and inhibits the Rho functions (28). Pak1CRIB is associated with active Rac or Cdc42 and inhibits the interaction of active Rac or Cdc42 with the effectors (29, 32). As shown in Fig. 5, G and H, JNK activation by G_i2Q205L was blocked by cotransfection of C3 toxin or Pak1CRIB. These results suggest that Rho and Cdc42 participate in the JNK pathway from G_i.

View larger version (44K): [in this window] [in a new window]   Fig. 5.   Involvement of Rho and Cdc42 but not Rac in Gi-mediated JNK activation. Cells were transfected with plasmids carrying cDNAs for HA-JNK (A-H), Gi2Q205L (i2Q205L) (D-H), FLAG-RhoT19N (RhoT19N) (A and D), FLAG-RacT17N (RacT17N) (B and E), FLAG-Cdc42T17N (Cdc42T17N) (C and F), C3 toxin (G), and Myc-Pak1CRIB (Pak1CRIB) (H). Cells were treated with 50 µM mastoparan for 15 min (A-C). The JNK activity was measured as described under "Experimental Procedures." Values shown represent the mean ± S.E. from three or four separate experiments. The phosphorylation of GST-c-Jun and the expression of HA-JNK, Gi2Q205L, FLAG-tagged dominant-negative Rho family GTPases, and Myc-Pak1CRIB in the cell lysates are shown. Myc-Pak1CRIB was immunoprecipitated with anti-Myc antibody and immunoblotted with anti-Myc antibody.

Effect of Tyrosine Kinase Inhibitors on G_i-mediated JNK Activation-- To investigate whether tyrosine kinases are involved in G_i-mediated activation of JNK, we used PP1 and PP2, which are tyrosine kinase inhibitors preferential for Src family tyrosine kinases (33), and Csk, which is a negative regulator of Src family tyrosine kinases (30). As shown in Fig. 6A, mastoparan-induced JNK activation was attenuated by treatment with PP1. Moreover, the activation of JNK by G_i2Q205L was inhibited by PP1 and PP2 in a dose-dependent manner (Fig. 6, B and C). Next, a plasmid encoding cDNA of Csk was transfected. As shown in Fig. 6, D and E, cotransfection of Csk suppressed the JNK activation by G_i2Q205L, but not by G. We further examined whether G_i2Q205L-induced JNK activation is inhibited by dominant-negative Fyn. Cotransfection of dominant-negative Fyn resulted in a 60% decrease of JNK activation by G_i2Q205L (data not shown). These results indicate the involvement of Src family tyrosine kinases in G_i-mediated JNK activation.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Gi-mediated JNK activation depends on Src family tyrosine kinases. Cells were transfected with plasmids carrying cDNAs for HA-JNK (A-E), Gi2Q205L (i2Q205L) (B-D), G1 (1) (E), G2 (2) (E), and Csk (D and E). Transfected cells were treated with 50 µM mastoparan for 15 min in the presence or absence of 30 µM PP1 (A) or treated with increasing concentrations of PP1 (B) and PP2 (C) for 18 h. The JNK activity was measured as described under "Experimental Procedures." Values shown represent the mean ± S.E. from three or four separate experiments. The phosphorylation of GST-c-Jun and the expression of HA-JNK, G protein subunits, and Csk in the cell lysates are shown.

Effect of Phosphatidylinositol 3-Kinase Inhibitors on G_i-mediated JNK Activation-- It has been shown that G_i1 directly activates phosphatidylinositol 3-kinase  in vitro (34), and overexpression of this enzyme in the cells induces the activation of JNK (18). To examine the involvement of phosphatidylinositol 3-kinase in the JNK pathway from G_i, the transfected cells were treated with wortmannin and LY294002, phosphatidylinositol 3-kinase inhibitors (13, 18). Wortmannin inhibited mastoparan-induced activation of ERK, but not JNK, as shown in Fig. 7, A-D. Furthermore, neither wortmannin nor LY294002 inhibited G_i2Q205L-induced JNK activation (Fig. 7, E and F), indicating that phosphatidylinositol 3-kinase is not implicated in G_i-mediated JNK activation in HEK 293 cells.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Gi-mediated JNK activation is independent of phosphatidylinositol 3-kinase. Cells were transfected with plasmids carrying cDNAs for HA-JNK (A, C, E, and F), HA-ERK (B and D), and Gi2Q205L (i2Q205L) (E and F). A and B, cells 48 h after transfection were treated with 50 µM mastoparan for the indicated time in the presence (open circle) or absence (closed circle) of 1 µM wortmannin. C and D, the transfected cells were pretreated with the indicated concentrations of wortmannin for 20 min and were treated with 50 µM mastoparan for 15 min. E and F, the cells were pretreated with 100 nM wortmannin and 100 µM LY294002 for 20 min. Kinase activities of HA-JNK and HA-ERK were measured as described under "Experimental Procedures." Values shown represent the mean ± S.E. from three or four separate experiments. The phosphorylation of GST-c-Jun and the expression of HA-JNK and Gi2Q205L in the cell lysates are shown.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

JNK activation induced by constitutively activated mutant of G_i2 has been shown in two systems including G_i2Q205L-overexpressing mice (35) and Rat-1 cells inducibly expressing G_i2Q205L (36), although the mechanism by which G_i2 regulates JNK activity remained to be characterized. In the present study, we first found that mastoparan-induced JNK activation was dependent on G_i and mediated by both G_i and G in HEK 293 cells. In addition, constitutively activated mutants of G_i1, G_i2, and G_i3 induced JNK activation. But JNK was not stimulated by activated G_o and G_z mutants. Moreover, G_i2 appeared to regulate JNK activity through a MKK4- and MKK7-independent pathway. Furthermore, JNK activation by G_i2 was dependent on small GTPases Rho and Cdc42 and Src family tyrosine kinases but not on phosphatidylinositol 3-kinase.

Two JNK kinase genes, MKK4 and MKK7, have been cloned to date. We showed previously that G activates JNK via mainly MKK4 and to a lesser extent MKK7 (19). In contrast, JNK activation by G_i2Q205L was not inhibited effectively by cotransfection of kinase-deficient MKK4 and MKK7 (Fig. 4). Moreover, transfection of G_i2Q205L into the cells failed to stimulate the activities of MKK4, MKK7, and MKK7, an alternative splicing form of MKK7 (Fig. 4). It has been reported that there is a JNK kinase other than MKK4 and MKK7 at the level of the fractionation by column chromatography with lysates of stress-stimulated cells (37). G_i2 may regulate JNK activity through an unidentified JNK kinase.

A Rac and Cdc42-binding region is found in MAPKKKs including MEKK1, MEKK4, MLK2, and MLK3. MEKK1 activates primarily JNK pathway and appears to mediate JNK activation by Rac or Cdc42 (38). It has been shown that MEKK1 and ASK1 mediate JNK activation induced by G₁₂ and G₁₃ in COS-7 and HEK 293 cells (39). Additionally, Collins et al. (40) have reported that G₁₂-induced JNK activation partially involves Cdc42 in HEK 293 cells. It appears that a Cdc42-MEKK1 signaling unit functions upstream of JNK. Because G_i2-induced JNK activation depended on Cdc42 (Fig. 5), MEKK1 might be a candidate of MAPKKK in the JNK pathway from G_i. JNK activation by G_i depended on Rho (Fig. 5). Although a MAPKKK regulated by Rho has not yet been identified, it is expected that there may exist a MAPK cascade linking Rho with a transcription factor SRF in c-fos promoter activation induced by lysophosphatidic acid, a G_i-coupled receptor ligand (41).

G_i2Q205L-induced JNK activation was inhibited by tyrosine kinase inhibitors PP1 and PP2 in a dose-dependent manner (Fig. 6). These inhibitors preferentially inhibit Src family tyrosine kinases, and the IC₅₀ value of PP2 for the inhibition of Src is 15 µM in intact cells.² Furthermore, cotransfection of Csk attenuated G_i2Q205L-induced JNK activation (Fig. 6). Src family tyrosine kinases are likely to be involved in the signaling pathway from G_i to JNK.

We reported previously that G stimulates MKK4 activity in a Rho- and Cdc42-dependent manner (19). In contrast, G_i2Q205L failed to activate MKK4, but activated JNK in a Rho- and Cdc42-dependent manner. This difference may be due to the difference of downstream signaling components of G_i and G. It must be noted that JNK activation by G_i2Q205L, but not by G, was reduced by Csk (Fig. 6) and dominant-negative Fyn. These results suggest that the relationships among tyrosine kinases, Rho, and Cdc42 may be more complex than a single sequential cascade.

Fig. 8 shows a proposed pathway from G_i to JNK. In the present study, we used mastoparan as an activator of G_i. JNK activation by mastoparan, as well as G_i2Q205L and G (19), was almost completely inhibited by dominant negative mutants of Rho and Cdc42 (Fig. 5). Moreover, mastoparan-induced JNK activation was blocked partially by Src family inhibitor PP1 and the kinase-deficient mutant of MKK4 (Figs. 3 and 6). These results are consistent with the model in which G_i and G participate equivalently in the signaling pathway from G_i to JNK.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Schematic model for signal transduction pathways from Gi to JNK in HEK 293 cells. Details are described under "Discussion." PTK, a protein-tyrosine kinase other than Src family; MKK-X, an unidentified JNK kinase.

Activating mutations of G_i2, which were denoted as the gip2 oncogene, have been found in some types of tumor including ovarian sex cord stromal tumors, adrenal cortex tumors, and nonfunctional pituitary tumors (42). In addition, ectopic expression of the gip2 oncogene has been shown to cause oncogenic transformation of Rat-1 fibroblast cells. However, it appears that the gip2 oncogene fails to transform other fibroblast cells such as NIH3T3 (42). Recently, we reported that conditional expression of active G_i2 mutant in Rat-1 cells induces the colony formation on soft agar (36). We found that JNK is activated with the expression of active G_i2 mutant in the cells (36). On the other hand, JNK activity was not stimulated in NIH3T3 cells expressing active G_i2 mutant (43). Further elucidation of the G_i2-JNK pathway may clarify the relationship between the activation of JNK and the mechanism of oncogenic transformation by the gip2 oncogene.

	ACKNOWLEDGEMENTS

We thank Drs. T. Asano, R. A. Cerione, K. Kaibuchi, M. Karin, L. Lim, S. Narumiya, T. Nukada, M. Okada, M. I. Simon, P. C. Sternweis, M. Tagaya, H. Umemori, and T. Yamaguchi for supplying the antibodies and plasmids. We are grateful to Dr. A. Levitzki for providing PP1/AG1872 and PP2/AG1879. We also thank N. Mizuno, S. Mizutani, J. Kato, K. Nishida, and J. Suzuki for plasmid constructions and helpful discussion.

	FOOTNOTES

^* This work was supported by grants from CREST and from the Ministry of Education, Science, Sports, and Culture. Our laboratory is funded by Schering-Plough Corporation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-45-924-5746; Fax: 81-45-924-5822; E-mail: hitoh@bio.titech.ac.jp.

² A. Levitzki, personal communication.

	ABBREVIATIONS

The abbreviations used are: G protein, heterotrimeric guanine nucleotide-binding regulatory protein; G, G protein  subunit; G, G protein subunit; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; MAPKK, MAPK kinase; MEK, MAPK/ERK kinase; MAPKKK, MAPKK kinase; MEKK, MEK kinase; GST, glutathione S-transferase; Trx, thioredoxin; HA, hemagglutinin; HEK, human embryonal kidney; ARK1, -adrenergic receptor kinase 1.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES