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c-Jun N-Terminal Kinase Cascade
Biochemical and Biophysical Research Communications 288, 1087–1094 (2001) doi:10.1006/bbrc.2001.5891, available online at http://www.idealibrary.com on
G␣q-Dependent Activation of Mitogen-Activated Protein Kinase Kinase 4/c-Jun N-Terminal Kinase Cascade Junji Yamauchi,* Hiroshi Itoh,* Hitomi Shinoura,† Yuki Miyamoto,* Keiko Tsumaya,† Akira Hirasawa,† Yoshito Kaziro,‡ and Gozoh Tsujimoto† ,1 *Department of Cell Biology, Graduate School of Biological Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma-shi, Nara 630-0101, Japan; †Department of Molecular Cell Pharmacology, National Children’s Medical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8509, Japan; and ‡Sanyo-Gakuen University and College, 1-14-1 Hirai, Okayama-shi, Okayama 703-8501, Japan
Received October 11, 2001
G-protein-coupled receptors (GPCRs) typically activate c-Jun N-terminal kinase (JNK) through the G protein ␥ subunit (G␥), in a manner dependent on Rho family small GTPases, in mammalian cells. Here we show that JNK activation by the prototypic Gqcoupled ␣1B-adrenergic receptor is mediated by the ␣ subunit of Gq (G␣q), not by G␥, using a transient transfection system in human embryonic kidney cells. JNK activation by the ␣1B-adrenergic receptor/G␣q was selectively mediated by mitogen-activated protein kinase kinase 4 (MKK4), but not MKK7. Also, MKK4 activation by the ␣1B-adrenergic receptor/G␣q required c-Src and Rho family small GTPases. Furthermore, activation of the ␣1B-adrenergic receptor stimulated JNK activity through Src family tyrosine kinases and Rho family small GTPases in hamster smooth muscle cells that natively express the ␣1Badrenergic receptor. Together, these results suggest that the ␣1B-adrenergic receptor/G␣q may up-regulate JNK activity through a MKK4 pathway dependent on c-Src and Rho family small GTPases in mammalian cells. © 2001 Academic Press Key Words: G␣q; ␣1B-adrenergic receptor; MKK4; JNK; Rho family small GTPase; c-Src.
Gprotein-coupled receptors (GPCRs), which respond to sensory signals, hormones, neurotransmitters, and chemokines, activate heterotrimeric G proteins (1, 2), and in turn induce various intracellular signaling cascades including the mitogen-activated protein kinases (MAPKs) (3, 4). Many mitogenic GPCRs, such as the lysophosphatidic acid and thrombin receptors, stimulate the activity of extracellular signal-regulated proTo whom correspondence should be addressed. Fax: ⫹81-3-34191252. E-mail: [email protected]. 1
tein kinase (ERK), which appears to contribute to growth in cultured cells (3, 4). It has been shown that GPCR-induced ERK activation is mediated by the G protein ␥ subunit (G␥) rather than the ␣ subunit (G␣) (3, 4). In this scenario, GPCR/G␥ activates effector molecules, such as phosphatidylinositol 3-kinases, leading to activation of Src family tyrosine kinases. Activation of Src family tyrosine kinases results in a promotion of GDP-GTP exchange reaction in small GTPase Ras (3, 4). The GTP-bound Ras induces upregulation of Raf-1 activity, following equal activation of ERK kinases MKK1 and MKK2, and in turn ERK. It has been shown that the GPCR stimulation increases the activity of c-Jun N-terminal kinase (JNK), a subfamily of MAPKs, through G␥ (3). However, it is likely that the GPCR-induced JNK activation is mediated not only by G␥ (3, 5) but also by G␣, because constitutively activated forms of G␣i1/i2/i3 (6, 7), G␣q/ 11/16 (8, 9), and G␣12/13 (10 –14) have been reported to stimulate JNK activity. Additionally, JNK activation by constitutively activated mutants of G␣i2 and G␣12/ 13, and G␥ has been shown to involve Rho family small GTPases (3, 5, 7), although the mechanism by which G␣q family increases JNK activity remains largely unknown. We previously reported that JNK activation by ␣1Badrenergic receptor—which is the prototypic Gq-coupled receptor and widely distributed (15)—is involved in inhibition of cell growth (16). In the course of a study of the JNK signaling pathway in human embryonic kidney cells transiently expressing the ␣1B-adrenergic receptor, we found that the receptor-induced JNK activation was mediated by G␣q, not by G␥. Additionally, we investigated how the ␣1B-adrenergic receptor/ G␣q stimulates the activity of JNK. Furthermore, we examined a pathway linking the ␣1B-adrenergic receptor to JNK, in hamster smooth muscle cells that natively express the ␣1B-adrenergic receptor.
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MATERIALS AND METHODS Antibodies. Mouse monoclonal antibodies M2, 12CA5, and 9E10 against FLAG-, HA-, and Myc-peptides were purchased from Sigma Chemical Co. (St. Louis, MO), Roche Diagnosticks Co. (Tokyo, Japan), and Babco (Richmond, CA), respectively. A mouse monoclonal antibody B-14 against Schistosoma japonicum glutathione-Stransferase (GST) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A rat polyclonal antibody against Aequorea victoria green fluorescence protein (GFP) was kindly provided by K. Hirata (Mitsubishi Institute of Life Science). Rabbit polyclonal antibodies C-19, T-20, and C-20 against G␣q/11, G, and Csk, respectively, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A mouse monoclonal anti-c-Src antibody GD11 was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Rabbit polyclonal antibodies SRC2 and N-16 against c-Src were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A rabbit polyclonal anti-JNK1 antibody C-17 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), as was a mouse monoclonal antibody 26C4 against RhoA. Mouse monoclonal antibodies 102 and 44 against Rac1 and Cdc42, respectively, were purchased from BD PharMingen (San Diego, CA). Goat anti-mouse and anti-rabbit IgG antibodies conjugated with horseradish peroxidase were obtained from Amersham-Pharmacia Co. (Buckinghamshire, UK). An anti-rat IgG antibody conjugated with horseradish peroxidase was kindly provided by K. Hirata (Mitsubishi Institute of Life Science). Inhibitors. PP1 and PP2 were kindly provided by A. Levitzki (Hebrew University). Ro-31-8220 was purchased from Merk Japan Ltd. (Tokyo, Japan). Clostridium botulinum C3 exoenzyme was purchased from Biomol (Plymouth Meeting, PA) and Sigma Chemical Co. (St. Louis, MO). Plasmids. The pEGFP-N3-␣1B-adrenergic receptor plasmid was constructed as described previously (17). EGFP-tagged ␣1B-adrenergic receptor binds to agonists and antagonists and stimulates phosphatidylinositol/Ca 2⫹ signaling in a similar fashion to the wild type receptor (17). cDNAs encoding G1 and G␥2 were generously provided by M. I. Simon (California Institute of Technology) and T. Nukada (Tokyo Institute of Psychiatry), respectively. RhoA and Rac1 cDNAs were kindly provided by K. Kaibuchi (Nagoya University). Cdc42Hs cDNA was a generous gift from R. A. Cerione (Cornell University). pCMV-G␣qQ209L, pCMV-G1, pCMV-G␥2, pCMVARK1ct, pCMV-GST-MKK4, pCMV-GST-MKK7, pCMV-FLAGMKK4K95R, pCMV-FLAG-MKK7K63R, pCMV-FLAG-RhoAT19N, pCMV-FLAG-Rac1T17N, pCMV-FLAG-Cdc42HsT17N, pCMV-Myc␣PakCRIB, and pCMV-Csk were constructed as described previously (5, 7, 18, 19). The Rho-binding domain (RBD) sequence of Rhotekin (20) was amplified from a mouse spleen cDNA library, constructed using a cDNA synthesis kit (Takara, Kyoto, Japan). pET42amDia1RBD and pET42a-␣PakCRIB (the Rac- and Cdc42-binding domain of ␣Pak) were constructed as described previously (19, 21, 22). pUSE-c-Src and pUSE-DN-Src (a dominant-negative mutant of c-Src) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). pME18S-DN-Fyn and pME18S-DN-Lyn were generously provided by T. Yamamoto (Institute of Medical Science, University of Tokyo). SR␣-HA-JNK1 and pGEX2T-c-Jun (amino acids 1-223) were generously provided by M. Karin (University of California, San Diego). A hexahistidine-tag Escherichia coli expression plasmid encoding a kinase-deficient form of Mpk2, the Xenopus orthologue of mouse p38␣, was kindly provided by E. Nishida (Kyoto University). pET15b-JNK1 and pET32a-c-Jun (amino acids 1-223) were constructed as described previously (5, 7). All DNA sequences were confirmed using a DNA sequencer (MegaBASE 1000). Recombinant proteins. Recombinant hexahistidine-tagged JNK, thioredoxin (Trx)-c-Jun, hexahistidine-tagged kinase-deficient Mpk2, GST-c-Jun, GST-mDia1RBD, and GST-PakCRIB were purified using E. coli BL21 (DE3) pLysS (Takara, Kyoto, Japan) as described previously (5, 7, 18, 19).
Cell culture and transfection. Human embryonic kidney 293T and hamster smooth muscle DDT1 MF-2 cells were maintained in Dulbecco’s modified Eagle medium containing 50 g/ml streptomycin, 50 unit/ml penicillin, and 10% heat-inactivated fetal bovine serum. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO 2. Plasmid DNAs were transfected into 293T cells by the calcium phosphate precipitation method. The final amount of the transfected DNA for a 60-mm dish was adjusted to 25 g by empty vector, pCMV. Three micrograms of SR␣-HA-JNK, pCMVGST-MKK4, or pCMV-GST-MKK7, or 1g of pUSE-c-Src was cotransfected with 0.3 g of pEGFP-N3-␣1B-adrenergic receptor, 10 g of pCMV-G␣qQ209L, 5 g of pCMV-G1, 5 g of pCMV-G␥2, 3 g of pCMV-Myc-ARKct, 10 g of pCMV-FLAG-MKK4K95R, 10 g of pCMV-FLAG-MKK7K63R, 10 g of plasmids encoding dominantnegative mutants of Rho family small GTPases, 10 g of pCMVRhotekinRBD, 10 g of pCMV-␣PakCRIB, 3 g of pCMV-Csk, 10 g of pUSE-DN-Src, 10 g of pME18S-DN-Fyn, and 10 g of pME18SDN-Lyn. The medium was replaced 24 h after transfection, and 293T cells were starved in serum-free medium for 24 h. DDT1 MF-2 cells were also starved in serum-free medium for 24 h before the addition of phenylephrine. Immuno-(affinity-) precipitation and immunoblotting. After the addition of phenylephrine, 293T and DDT1 MF-2 cells were lysed in 600 l of lysis buffer A (20 mM Hepes-NaOH (pH 7.5), 3 mM MgCl 2, 100 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 g/ml leupeptin, 1 mM EGTA, 1 mM Na 3VO 4, 10 mM NaF, 20 mM -glycerophosphate, and 0.5% NP-40) and centrifuged, as described previously (5, 7, 18, 19). Aliquots (500 g) of the supernatants were mixed with protein A-(or protein G-)Sepharose CL-4B preabsorbed with anti-HA, anti-c-Src, anti-myc, or anti-JNK antibody. The immune-complexes were precipitated by centrifugation and washed. GST-tagged MKKs were affinity-precipitated with glutathione-Sepharose 4B from aliquots (500 g) of the supernatants and washed. To compare the expression level of various transfected cDNAs, the immune-(or affinity-)complexes and aliquots of the cell lysates were boiled in Laemmli sample buffer, and then separated on 8 or 15% SDS–polyacrylamide electrophoresis gels. The separated proteins were transferred to a PVDF membrane. The membranes were blocked with Block Ace and immunoblotted with various antibodies. The bound antibodies were detected using the ECL or ECL Plus systems (Amersham-Pharmacia Co., Buckinghamshire, UK) with anti-mouse, rabbit, or rat IgG antibodies conjugated with horseradish peroxidase, according to the manufacturer’s protocol. Images of kinases were captured using Adobe Photoshop 5.0 plug-in software for Macintosh and EPSON GT-7000U scanner. The band intensities were semi-quantified using NIH Image 1.61. Assays of MKK4, MKK7, and JNK. 293T and DDT1 MF-2cells were lysed in 600 l of lysis buffer A and centrifuged. Aliquots (500 g) of the supernatants were used to assay JNK or MKKs activities. JNK and MKKs activities were measured as the radioactivities incorporated into GST-c-Jun and Trx-c-Jun, respectively, as described previously (5, 7, 18, 19). MKK4 activity was also measured as the radioactivity incorporated into kinase-deficient Mpk2, using an imaging analyzer (Fuji BAS 2000). JNK and MKKs activities were normalized to the amounts of kinases in the immuno-(or affinity-)precipitates. c-Src assay. 293T cells transfected with pUSE-c-Src were lysed in 600 l of lysis buffer A and centrifuged. Aliquots (500 g) of the supernatants were mixed with protein G-Sepharose CL-4B preabsorbed with an anti-c-Src antibody for 2 h at 4°C. The immunecomplexes were washed twice with lysis buffer A and twice with reaction buffer B (20 mM Hepes-NaOH (pH 7.5), 10 mM MgCl 2, 3 mM MnCl 2, 0.1 mM phenylmethanesulfonyl fluoride, 0.1 g/ml leupeptin, 0.1 mM EGTA, 10 M Na 3VO 4, and 2 mM -glycerophosphate). The immobilized c-Src was incubated in 30 l of reaction buffer B containing 20 M ATP and 5 Ci of [␥- 32P]ATP at 25°C for 15 min. The reactions were stopped by adding 10 l of 4⫻ Laemmli
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sample buffer. After boiled, the samples were subjected to 15% SDS–polyacrylamide gel electrophoresis. Autophosphorylation of c-Src was detected using an imaging analyzer. c-Src activity was normalized to the amount of kinase in the immunoprecipitate. Detection of active Rho family small GTPases. 293T cells transfected with pEGFP-N3-␣1B-adrenergic receptor and DDT1 MF-2 cells were lysed in 600 l of lysis buffer B (50 mM Hepes-NaOH (pH 7.5), 20 mM MgCl2, 150 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 g/ml leupeptin, 1 mM EDTA, 1 mM Na3VO 4, 10 mM NaF, and 0.5% NP-40) and centrifuged, and the supernatants were recovered. The pull-down assays to detect active Rho family small GTPases were performed as described previously (21, 22).
RESULTS AND DISCUSSION To investigate whether the Gq-coupled ␣1B-adrenergic receptor stimulated the activity of JNK, we cotransfected plasmids encoding HA-tagged JNK with the ␣1B-adrenergic receptor into 293T cells. Using an anti-HA antibody, HA-JNK was immunoprecipitated from the cell lysate. The in vitro kinase activity of HA-JNK was assayed. As shown in Fig. 1A, JNK activity was increased by stimulation with phenylephrine, an agonist of the ␣1-adrenergic receptor. Pretreatment of the cells with prazosin, an antagonist of the ␣1-adrenergic receptor, abolished JNK activation by phenylephrine (data not shown). Next, we attempted to determine which subunit of Gq was involved in JNK activation by the ␣1Badrenergic receptor. We cotransfected a plasmid encoding the C-terminus of -adrenergic receptor kinase (ARKct), which is known to bind to G␥ and to inhibit the cellular function of G␥ (4, 7). Cotransfection of ARKct inhibited JNK activation induced by G␥, but not by constitutively activated G␣q (G␣qQ209L) (Figs. 1B and 1C). As shown in Fig. 1A, ARKct failed to inhibit JNK activation by the ␣1B-adrenergic receptor, suggesting that the ␣1B-adrenergic receptor activates JNK via G␣q. To clarify whether JNK activation by the ␣1Badrenergic receptor involves JNK kinases MKK4 and MKK7, we cotransfected the plasmids encoding MKK4K95R or MKK7K63R, which are kinase-deficient mutants and sequestrate upstream MAPKKKs. As shown in Figs. 2A and 2B, JNK activation by the ␣1B-adrenergic receptor was inhibited by cotransfection with MKK4K95R, but not with MKK7K63R. Similarly, G␣qQ209L-induced JNK activation was blocked by cotransfection with MKK4K95R, but not with MKK7K63R (Figs. 2C and 2D). Next, we tried to measure the intrinsic kinase activities of MKK4 and MKK7. As shown in Figs. 2E and 2F, stimulation of the ␣1B-adrenergic receptor activated MKK4, but not MKK7. Additionally, G␣qQ209L stimulated only MKK4 activity (Figs. 2G and 2H). These results indicate that JNK activation by the ␣1Badrenergic receptor/G␣q depends only on MKK4. It has been shown that Rho family small GTPases Rho,
FIG. 1. JNK activation by the ␣1B-adrenergic receptor is mediated by G␣q, but not G␥. (A) 293T cells were transfected with plasmids encoding ␣1B-adrenergic receptor, ARKct, and JNK. JNK activity was measured at 10 min after the addition of 20 M phenylephrine. (B) Cells were transfected with plasmids encoding G␣qQ209L, ARKct, and JNK. (C) Cells were transfected with plasmids encoding G1, G␥2, ARKct, and JNK. JNK activity was measured as described under Materials and Methods. Values shown represent the mean ⫾ SE from three or four separate experiments. Expression of ␣1B-adrenergic receptor, G protein subunits, and ARKct are shown. HA-tagged JNK was immunoprecipitated with an anti-HA antibody from the cell lysates and immunoblotted with an anti-HA antibody.
Rac, and Cdc42 participate in various signal transduction pathways, including JNK activation (23, 24). Furthermore, G␥-mediated MKK4 and MKK7 activations depend on Rho family small GTPases (5). Thus, we examined whether Rho family small GTPases are involved in MKK4 activation by the ␣1B-adrenergic receptor/G␣q. As shown in Figs. 3A–3C, MKK4 activation by the ␣1B-adrenergic receptor was inhibited by cotransfection with RhoT19N, RacT17N, or Cdc42T17N. RhoT19N, RacT17N, and Cdc42T17N are dominantnegative mutants which sequestrate guanine-nucleotide exchange factors for Rho family small GTPases. G␣qQ209L-induced MKK4 activation was also blocked by these dominant-negative mutants (Figs. 3D–3F). Next, we utilized RhotekinRBD and PakCRIB. It is known that RBD specifically binds to Rho (20), while CRIB specifically interacts with Rac and Cdc42 (22). Cotransfection of RBD or CRIB blocked MKK4 activation by G␣qQ209L (Figs. 3G and 3H). Together, these results suggest that Rho family small GTPases are
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FIG. 2. JNK activation by the ␣1B-adrenergic receptor/G␣q is mediated by MKK4, but not MKK7. (A) 293T cells were transfected with plasmids encoding ␣1B-adrenergic receptor, MKK4K95R, and JNK. JNK activity was measured at 10 min after the addition of 20 M phenylephrine. (B) Cells were transfected with plasmids encoding ␣1B-adrenergic receptor, MKK7K63R, and JNK. JNK activity was measured at 10 min after the addition of 20 M phenylephrine. (C) Cells were transfected with plasmids encoding G␣qQ209L, MKK4K95R, and JNK. (D) Cells were transfected with plasmids encoding G␣qQ209L, MKK7K63R, and JNK. JNK activity was measured as described under Materials and Methods. (E) Cells were transfected with plasmids encoding ␣1B-adrenergic receptor and MKK4. MKK4 activity was measured at 10 min after the addition of 20 M phenylephrine. (F) Cells were transfected with plasmids encoding ␣1B-adrenergic receptor and MKK7. MKK7 activity was measured at 10 min after the addition of 20 M phenylephrine. (G) Cells were transfected with plasmids encoding G␣qQ209L and MKK4. (H) Cells were transfected with plasmids encoding G␣qQ209L and MKK7. MKK4 and MKK7 activities were measured as described under Materials and Methods. Values shown represent the mean ⫾ SE from three or four separate experiments. Expression of ␣1B-adrenergic receptor, G␣qQ209L, MKK4K95R, and MKK7K63R are shown. HA-tagged JNK was immunoprecipitated with an anti-HA antibody from the cell lysates and immunoblotted with an anti-HA antibody. GST-tagged MKK4 and MKK7 were precipitated with glutathione–Sepharose 4B from the cell lysates and immunoblotted with an anti-GST antibody.
involved in MKK4 activation by the ␣1B-adrenergic receptor/G␣q. It has been reported that Src family tyrosine kinases mediate JNK activation by G␣11, a member of G␣q family (9). We therefore investigated whether the ␣1Badrenergic receptor activates MKK4 through c-Src. Transfected cells were treated with PP1, a specific inhibitor of Src family tyrosine kinases. As shown in Fig. 4A, MKK4 activation by the ␣1B-adrenergic receptor was inhibited by treatment with PP1. G␣qQ209Linduced MKK4 activation was also inhibited by PP1
and another Src family tyrosine kinase inhibitor PP2 (Figs. 4B and 4C). In addition, cotransfection of Csk, a negative regulator of Src family tyrosine kinases, blocked G␣qQ209L-induced MKK4 activation (Fig. 4D). Furthermore, G␣qQ209L-induced MKK4 activation was almost completely inhibited by cotransfection of DN-Src (Fig. 4E). In contrast, G␣qQ209L-induced MKK4 activation was less than 30% inhibited by cotransfection of DN-Fyn or DN-Lyn (data not shown). Together, it is likely that c-Src primarily participates in G␣q-mediated MKK4 activation.
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FIG. 3. MKK4 activation by the ␣1B-adrenergic receptor/G␣q is mediated by Rho family small GTPases. (A–C) 293T cells were transfected with plasmids encoding ␣1B-adrenergic receptor, each dominant-negative Rho family small GTPase, and MKK4. MKK4 activity was measured at 10 min after the addition of 20 M phenylephrine. (D–F) Cells were transfected with plasmids encoding G␣qQ209L, each dominant-negative Rho family small GTPase, and MKK4. (G and H) Cells were transfected with plasmids encoding G␣qQ209L, RhotekinRBD or PakCRIB, and MKK4. MKK4 activity was measured as described under Materials and Methods. Values shown represent the mean ⫾ SE from three or four separate experiments. Expression of ␣1B-adrenergic receptor, G␣qQ209L, dominant-negative Rho family small GTPases, and PakCRIB are shown. The transcription levels of RhotekinRBD and -actin (control) were demonstrated by RT-PCR. GST-tagged MKK4 was precipitated with glutathione–Sepharose 4B from the cell lysates and immunoblotted with an anti-GST antibody.
Next, we tried to measure c-Src activity. c-Src immunoprecipitated from cell lysates was incubated with [ 32P]ATP. As shown in Fig. 4F, c-Src was autophosphorylated when c-Src was cotransfected with G␣qQ209L. In addition, PP1 treatment almost completely inhibited c-Src autophosphorylation by G␣qQ209L. Furthermore, we have confirmed that the Tyr 418 autophosphorylation of c-Src was induced by G␣qQ209L using its phosphorylated Tyr 418-specific antibody (18). We investigated the effect of PP1 or Clostridium botulinum C3 exoenzyme, which ADP-ribosylates Rho and weakly Rac and inhibits those cellular function (24), on the ␣1B-adrenergic receptor-induced endogenous JNK activation in 293T cells expressing the ␣1Badrenergic receptor and DDT1 MF-2 cells. DDT1 MF-2 cells natively express the ␣1B-adrenergic receptor (25,
26). Additionally, we examined the effect of Ro-318220, an inhibitor of PKC, on JNK activation by the ␣1B-adrenergic receptor in these cells, because PKC was a downstream molecule of G␣q family (9). As shown in Figs. 5A and 5B, Ro-31-8220, PP1, or C3 exoenzyme blocked JNK activation induced by the ␣1B-adrenergic receptor in 293T cells expressing its receptor and DDT1 MF-2 cells, suggesting the involvement of PKC, Src family tyrosine kinases, and Rho family small GTPases in a pathway linking the ␣1Badrenergic receptor to endogenous JNK. Next, we examined the effect of Ro-31-8220 or PP1 on the ␣1B-adrenergic receptor-induced endogenous Rho family small GTPase activation in 293T cells expressing the ␣1B-adrenergic receptor and DDT1 MF-2 cells. As shown in Figs. 5C and 5D, active GTP-bound
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FIG. 4. MKK4 activation by the ␣1B-adrenergic receptor/G␣q is mediated by c-Src. (A) 293T cells transfected with plasmids encoding ␣1B-adrenergic receptor and MKK4 were pretreated with 15 M PP1 for 1 h. MKK4 activity was measured at 10 min after the addition of 20 M phenylephrine. (B and C) Cells transfected with plasmids encoding G␣qQ209L and MKK4 were pretreated with the indicated concentration of PP1 or PP2 for 18 h. (D) Cells were transfected with plasmids encoding G␣qQ209L, Csk, and MKK4. (E) Cells were transfected with plasmids encoding G␣qQ209L, DN-Src, and MKK4. MKK4 activity was measured as described under Materials and Methods. (F) Cells transfected with plasmids encoding G␣qQ209L and c-Src were pretreated with 15 M PP1 for 18 h. c-Src autophosphorylation was measured as described under Materials and Methods. Values shown represent the mean ⫾ SE from three or four separate experiments. Expression of ␣1B-adrenergic receptor, G␣qQ209L, and Csk are shown. GST-tagged MKK4 was precipitated with glutathione– Sepharose 4B from the cell lysates and immunoblotted with an anti-GST antibody. c-Src was immunoprecipitated with an anti-c-Src antibody from the cell lysates and immunoblotted with an anti-c-Src antibody.
Rho family small GTPases were detected following stimulation of the ␣1B-adrenergic receptor, using the pull-down assay. Either Ro-31-8220 or PP1 inhibited Rho family small GTPase activation induced by the ␣1B-adrenergic receptor in 293T cells expressing its receptor and DDT1 MF-2 cells, suggesting the involvement of PKC and Src family tyrosine kinases in a pathway linking the ␣1B-adrenergic receptor to endogenous Rho family small GTPases. We have previously shown that G␣i2 activates JNK through a putative MKK other than MKK4 and MKK7 (7). In addition, G␣i2-mediated JNK activation was dependent on c-Src, Rho, and Cdc42 (7). On the other hand, G␥ activated JNK through the MKK4 pathway dependent on tyrosine kinase other than c-Src, Rho, and Cdc42, and to a lesser extent the MKK7 pathway dependent on Rac (5). In this study, we demonstrated that G␣q up-regulates only MKK4 activity and in turn JNK activity through c-Src and three Rho family small GTPases in 293T cells. Also, we reached basically the same results with DDT1 MF-2 cells that natively express the ␣1B-adrenergic receptor, as we had in the
transient transfection system. It is likely that signaling components of the MAPK cascade are organized into the module called scaffold protein in vivo (27). The differences in JNK signaling pathways by the G protein subunits may be caused by a difference in scaffold proteins. There are more than fifteen MAPK kinase kinases (MAPKKKs); MEKK1 appears to induce JNK activation through MKK4 in vivo (27). Additionally, MEKK1 harbors a CRIB domain which specifically interacts with active Rac and Cdc42 (28). MKK4 activation by the ␣1B-adrenergic receptor/G␣q depended on Rho family small GTPases Rac and Cdc42 (Fig. 3). Taken with the observation that Rac and Cdc42 activate JNK (3, 29) and MKK4 (data not shown), it is possible that MEKK1 is a MAPKKK in a pathway linking the ␣1Badrenergic receptor/G␣q to MKK4. On the other hand, MKK4 activation by ␣1B-adrenergic receptor/G␣q also involved Rho (Fig. 3). In addition, Rho increases the activities of JNK (3, 29) and MKK4 (data not shown) in 293T cells. Thus, a putative Rho-dependent MAPKKK (29) might contribute to JNK activation by the ␣1Badrenergic receptor/G␣q in these cells.
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FIG. 5. Involvement of PKC, Src family tyrosine kinases, and Rho family small GTPases in the pathway from the ␣1B-adrenergic receptor to JNK in 293T and DDT1 MF-2 cells. (A) 293T cells transfected with plasmids encoding ␣1B-adrenergic receptor were pretreated with 10 M Ro-31-8220 for 30 min, 15 M PP1 for 1 h, or 1 g/ml C3 exoenzyme for 18 h. JNK activity was measured at 10 min after the addition of 20 M phenylephrine. Endogenous JNK was immunoprecipitated with an anti-JNK antibody from the cell lysates and assayed using GST-c-Jun as a substrate. (B) DDT1 MF-2 cells were pretreated with 10 M Ro-31-8220 for 30 min, 15 M PP1 for 1 h, or 1 g/ml C3 exoenzyme for 18 h. JNK activity was measured at 20 min after the addition of 20 M phenylephrine. Endogenous JNK was immunoprecipitated with an anti-JNK antibody from the cell lysates and assayed. Values shown represent the mean ⫾ SE from three or four separate experiments. JNK was immunoprecipitated with an anti-JNK antibody from the cell lysates and immunoblotted with an anti-JNK antibody. Values shown represent the mean ⫾ SE from five separate experiments. (C) 293T cells transfected with plasmids encoding ␣1B-adrenergic receptor were pretreated with 10 M Ro-31-8220 for 30 min or 15 M PP1 for 1 h. The activities of Rho family small GTPases were measured at 10 min after the addition of 20 M phenylephrine. Active GTP-bound form of Rho family small GTPases was affinity-precipitated with GST-mDia1RBD or GST-PakCRIB from the cell lysates and immunoblotted with antibodies against Rho family small GTPases. (D) DDT1 MF-2 cells were pretreated with 10 M Ro-31-8220 for 30 min or 15 M PP1 for 1 h. The activities of Rho family small GTPases were measured at 20 min after the addition of 20 M phenylephrine. Active GTP-bound form of Rho family small GTPases was affinityprecipitated with GST-mDia1RBD or GST-PakCRIB from the cell lysates and immunoblotted with antibodies against Rho family small GTPases. Total Rho family small GTPases in the cell lysates were detected by immunoblotting with antibodies against Rho family small GTPases. Immunoblots of Rho family small GTPases are representative of three separate experiments.
It has been reported that JNK activation by GPCR is involved in the expression of various genes (3). Ramirez et al. reported that ␣1-adrenergic receptorinduced JNK activation up-regulates expression of atrial natriuretic factor in cardiac myocytes (30). Also, in the course of this study, Clerk et al. reported that stimulation of ␣1-adrenergic receptor activates Rho family small GTPases in cardiac myocytes (31). However, it remains unclear whether ␣1B-adrenergic receptor activates JNK through Rho family small GTPases in cardiac myocytes. On the other hand, the ␣1B-adrenergic receptor inhibits cell growth through JNK in 293T cells (19). It is possible that this antimitogenic pathway involves c-Src, Rho family small GTPases, and MKK4. Further investigation of the pathway from the ␣1B-adrenergic receptor to JNK may
shed light on our understanding of intracellular signal transduction and various cellular functions regulated by Gq-coupled receptor. ACKNOWLEDGMENTS This work was supported by grants from the Japan Science and Technology Corporation (JST), the Organization for Pharmaceutical Safety and Research (OPSR), and the Japanese Ministry of Education, Science, Sports, and Culture.
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4. Luttrell, L. M., Daaka, Y., and Lefkowitz, R. J. (1999) Curr. Opin. Cell Biol. 11, 177–183. 5. Yamauchi, J., Kaziro, Y., and Itoh, H. (1999) J. Biol. Chem. 274, 1957–1965. 6. Guo, J. H., Wang, H., and Malbon, C. C. (1998) J. Biol. Chem. 273, 16487–16493. 7. Yamauchi, J., Kawano, T., Nagao, M., Kaziro, Y., and Itoh, H. (2000) J. Biol. Chem. 275, 7633–7640. 8. Heasley, L. E., Storey, B., Fanger, G. R., Butterfield, L., Zamarripa, J., Blumberg, D., and Maue, R. A. (1996) Mol. Cell. Biol. 16, 648 – 656. 9. Nagao, M., Yamauchi, J., Kaziro, Y., and Itoh, H. (1998) J. Biol. Chem. 273, 22892–22898. 10. Vara Prasad, M. V. V. S., Dermott, J. M., Heasley, L. E., Johnson, G. L., and Dhanasekaran, N. (1995) J. Biol. Chem. 270, 18655–18659. 11. Collins, L. R., Minden, A., Karin, M., and Brown, J. H. (1996) J. Biol. Chem. 271, 17349 –17353. 12. Voyno-Yasenetskaya, T. A., Faure, M. P., Ahn, N. G., and Bourne, H. R. (1996) J. Biol. Chem. 271, 21081–21087. 13. Mitsui, H., Takuwa, N., Kurokawa, K., Exton, J. H., and Takuwa, Y. (1997) J. Biol. Chem. 272, 4904 – 4910. 14. Jho, E.-H., Davis, R. J., and Malbon, C. C. (1997) J. Biol. Chem. 272, 24468 –24474. 15. Zhong, H., and Minneman, K. P. (1999) Eur. J. Pharmacol. 375, 261–276. 16. Yamauchi, J., Itoh, H., Shinoura, H., Miyamoto, Y., Hirasawa, A., Kaziro, Y., and Tsujimoto, G. (2001) Biochem. Biophys. Res. Commun. 281, 1019 –1023. 17. Awaji, T., Hirasawa, A., Kataoka, M., Shinoura, H., Nakayama,
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G␣q-Dependent Activation of Mitogen-Activated Protein Kinase Kinase 4/c-Jun N-Terminal Kinase Cascade Junji Yamauchi,* Hiroshi Itoh,* Hitomi Shinoura,† Yuki Miyamoto,* Keiko Tsumaya,† Akira Hirasawa,† Yoshito Kaziro,‡ and Gozoh Tsujimoto† ,1 *Department of Cell Biology, Graduate School of Biological Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma-shi, Nara 630-0101, Japan; †Department of Molecular Cell Pharmacology, National Children’s Medical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8509, Japan; and ‡Sanyo-Gakuen University and College, 1-14-1 Hirai, Okayama-shi, Okayama 703-8501, Japan
Received October 11, 2001
G-protein-coupled receptors (GPCRs) typically activate c-Jun N-terminal kinase (JNK) through the G protein ␥ subunit (G␥), in a manner dependent on Rho family small GTPases, in mammalian cells. Here we show that JNK activation by the prototypic Gqcoupled ␣1B-adrenergic receptor is mediated by the ␣ subunit of Gq (G␣q), not by G␥, using a transient transfection system in human embryonic kidney cells. JNK activation by the ␣1B-adrenergic receptor/G␣q was selectively mediated by mitogen-activated protein kinase kinase 4 (MKK4), but not MKK7. Also, MKK4 activation by the ␣1B-adrenergic receptor/G␣q required c-Src and Rho family small GTPases. Furthermore, activation of the ␣1B-adrenergic receptor stimulated JNK activity through Src family tyrosine kinases and Rho family small GTPases in hamster smooth muscle cells that natively express the ␣1Badrenergic receptor. Together, these results suggest that the ␣1B-adrenergic receptor/G␣q may up-regulate JNK activity through a MKK4 pathway dependent on c-Src and Rho family small GTPases in mammalian cells. © 2001 Academic Press Key Words: G␣q; ␣1B-adrenergic receptor; MKK4; JNK; Rho family small GTPase; c-Src.
Gprotein-coupled receptors (GPCRs), which respond to sensory signals, hormones, neurotransmitters, and chemokines, activate heterotrimeric G proteins (1, 2), and in turn induce various intracellular signaling cascades including the mitogen-activated protein kinases (MAPKs) (3, 4). Many mitogenic GPCRs, such as the lysophosphatidic acid and thrombin receptors, stimulate the activity of extracellular signal-regulated proTo whom correspondence should be addressed. Fax: ⫹81-3-34191252. E-mail: [email protected]. 1
tein kinase (ERK), which appears to contribute to growth in cultured cells (3, 4). It has been shown that GPCR-induced ERK activation is mediated by the G protein ␥ subunit (G␥) rather than the ␣ subunit (G␣) (3, 4). In this scenario, GPCR/G␥ activates effector molecules, such as phosphatidylinositol 3-kinases, leading to activation of Src family tyrosine kinases. Activation of Src family tyrosine kinases results in a promotion of GDP-GTP exchange reaction in small GTPase Ras (3, 4). The GTP-bound Ras induces upregulation of Raf-1 activity, following equal activation of ERK kinases MKK1 and MKK2, and in turn ERK. It has been shown that the GPCR stimulation increases the activity of c-Jun N-terminal kinase (JNK), a subfamily of MAPKs, through G␥ (3). However, it is likely that the GPCR-induced JNK activation is mediated not only by G␥ (3, 5) but also by G␣, because constitutively activated forms of G␣i1/i2/i3 (6, 7), G␣q/ 11/16 (8, 9), and G␣12/13 (10 –14) have been reported to stimulate JNK activity. Additionally, JNK activation by constitutively activated mutants of G␣i2 and G␣12/ 13, and G␥ has been shown to involve Rho family small GTPases (3, 5, 7), although the mechanism by which G␣q family increases JNK activity remains largely unknown. We previously reported that JNK activation by ␣1Badrenergic receptor—which is the prototypic Gq-coupled receptor and widely distributed (15)—is involved in inhibition of cell growth (16). In the course of a study of the JNK signaling pathway in human embryonic kidney cells transiently expressing the ␣1B-adrenergic receptor, we found that the receptor-induced JNK activation was mediated by G␣q, not by G␥. Additionally, we investigated how the ␣1B-adrenergic receptor/ G␣q stimulates the activity of JNK. Furthermore, we examined a pathway linking the ␣1B-adrenergic receptor to JNK, in hamster smooth muscle cells that natively express the ␣1B-adrenergic receptor.
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0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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MATERIALS AND METHODS Antibodies. Mouse monoclonal antibodies M2, 12CA5, and 9E10 against FLAG-, HA-, and Myc-peptides were purchased from Sigma Chemical Co. (St. Louis, MO), Roche Diagnosticks Co. (Tokyo, Japan), and Babco (Richmond, CA), respectively. A mouse monoclonal antibody B-14 against Schistosoma japonicum glutathione-Stransferase (GST) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A rat polyclonal antibody against Aequorea victoria green fluorescence protein (GFP) was kindly provided by K. Hirata (Mitsubishi Institute of Life Science). Rabbit polyclonal antibodies C-19, T-20, and C-20 against G␣q/11, G, and Csk, respectively, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A mouse monoclonal anti-c-Src antibody GD11 was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Rabbit polyclonal antibodies SRC2 and N-16 against c-Src were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A rabbit polyclonal anti-JNK1 antibody C-17 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), as was a mouse monoclonal antibody 26C4 against RhoA. Mouse monoclonal antibodies 102 and 44 against Rac1 and Cdc42, respectively, were purchased from BD PharMingen (San Diego, CA). Goat anti-mouse and anti-rabbit IgG antibodies conjugated with horseradish peroxidase were obtained from Amersham-Pharmacia Co. (Buckinghamshire, UK). An anti-rat IgG antibody conjugated with horseradish peroxidase was kindly provided by K. Hirata (Mitsubishi Institute of Life Science). Inhibitors. PP1 and PP2 were kindly provided by A. Levitzki (Hebrew University). Ro-31-8220 was purchased from Merk Japan Ltd. (Tokyo, Japan). Clostridium botulinum C3 exoenzyme was purchased from Biomol (Plymouth Meeting, PA) and Sigma Chemical Co. (St. Louis, MO). Plasmids. The pEGFP-N3-␣1B-adrenergic receptor plasmid was constructed as described previously (17). EGFP-tagged ␣1B-adrenergic receptor binds to agonists and antagonists and stimulates phosphatidylinositol/Ca 2⫹ signaling in a similar fashion to the wild type receptor (17). cDNAs encoding G1 and G␥2 were generously provided by M. I. Simon (California Institute of Technology) and T. Nukada (Tokyo Institute of Psychiatry), respectively. RhoA and Rac1 cDNAs were kindly provided by K. Kaibuchi (Nagoya University). Cdc42Hs cDNA was a generous gift from R. A. Cerione (Cornell University). pCMV-G␣qQ209L, pCMV-G1, pCMV-G␥2, pCMVARK1ct, pCMV-GST-MKK4, pCMV-GST-MKK7, pCMV-FLAGMKK4K95R, pCMV-FLAG-MKK7K63R, pCMV-FLAG-RhoAT19N, pCMV-FLAG-Rac1T17N, pCMV-FLAG-Cdc42HsT17N, pCMV-Myc␣PakCRIB, and pCMV-Csk were constructed as described previously (5, 7, 18, 19). The Rho-binding domain (RBD) sequence of Rhotekin (20) was amplified from a mouse spleen cDNA library, constructed using a cDNA synthesis kit (Takara, Kyoto, Japan). pET42amDia1RBD and pET42a-␣PakCRIB (the Rac- and Cdc42-binding domain of ␣Pak) were constructed as described previously (19, 21, 22). pUSE-c-Src and pUSE-DN-Src (a dominant-negative mutant of c-Src) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). pME18S-DN-Fyn and pME18S-DN-Lyn were generously provided by T. Yamamoto (Institute of Medical Science, University of Tokyo). SR␣-HA-JNK1 and pGEX2T-c-Jun (amino acids 1-223) were generously provided by M. Karin (University of California, San Diego). A hexahistidine-tag Escherichia coli expression plasmid encoding a kinase-deficient form of Mpk2, the Xenopus orthologue of mouse p38␣, was kindly provided by E. Nishida (Kyoto University). pET15b-JNK1 and pET32a-c-Jun (amino acids 1-223) were constructed as described previously (5, 7). All DNA sequences were confirmed using a DNA sequencer (MegaBASE 1000). Recombinant proteins. Recombinant hexahistidine-tagged JNK, thioredoxin (Trx)-c-Jun, hexahistidine-tagged kinase-deficient Mpk2, GST-c-Jun, GST-mDia1RBD, and GST-PakCRIB were purified using E. coli BL21 (DE3) pLysS (Takara, Kyoto, Japan) as described previously (5, 7, 18, 19).
Cell culture and transfection. Human embryonic kidney 293T and hamster smooth muscle DDT1 MF-2 cells were maintained in Dulbecco’s modified Eagle medium containing 50 g/ml streptomycin, 50 unit/ml penicillin, and 10% heat-inactivated fetal bovine serum. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO 2. Plasmid DNAs were transfected into 293T cells by the calcium phosphate precipitation method. The final amount of the transfected DNA for a 60-mm dish was adjusted to 25 g by empty vector, pCMV. Three micrograms of SR␣-HA-JNK, pCMVGST-MKK4, or pCMV-GST-MKK7, or 1g of pUSE-c-Src was cotransfected with 0.3 g of pEGFP-N3-␣1B-adrenergic receptor, 10 g of pCMV-G␣qQ209L, 5 g of pCMV-G1, 5 g of pCMV-G␥2, 3 g of pCMV-Myc-ARKct, 10 g of pCMV-FLAG-MKK4K95R, 10 g of pCMV-FLAG-MKK7K63R, 10 g of plasmids encoding dominantnegative mutants of Rho family small GTPases, 10 g of pCMVRhotekinRBD, 10 g of pCMV-␣PakCRIB, 3 g of pCMV-Csk, 10 g of pUSE-DN-Src, 10 g of pME18S-DN-Fyn, and 10 g of pME18SDN-Lyn. The medium was replaced 24 h after transfection, and 293T cells were starved in serum-free medium for 24 h. DDT1 MF-2 cells were also starved in serum-free medium for 24 h before the addition of phenylephrine. Immuno-(affinity-) precipitation and immunoblotting. After the addition of phenylephrine, 293T and DDT1 MF-2 cells were lysed in 600 l of lysis buffer A (20 mM Hepes-NaOH (pH 7.5), 3 mM MgCl 2, 100 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 g/ml leupeptin, 1 mM EGTA, 1 mM Na 3VO 4, 10 mM NaF, 20 mM -glycerophosphate, and 0.5% NP-40) and centrifuged, as described previously (5, 7, 18, 19). Aliquots (500 g) of the supernatants were mixed with protein A-(or protein G-)Sepharose CL-4B preabsorbed with anti-HA, anti-c-Src, anti-myc, or anti-JNK antibody. The immune-complexes were precipitated by centrifugation and washed. GST-tagged MKKs were affinity-precipitated with glutathione-Sepharose 4B from aliquots (500 g) of the supernatants and washed. To compare the expression level of various transfected cDNAs, the immune-(or affinity-)complexes and aliquots of the cell lysates were boiled in Laemmli sample buffer, and then separated on 8 or 15% SDS–polyacrylamide electrophoresis gels. The separated proteins were transferred to a PVDF membrane. The membranes were blocked with Block Ace and immunoblotted with various antibodies. The bound antibodies were detected using the ECL or ECL Plus systems (Amersham-Pharmacia Co., Buckinghamshire, UK) with anti-mouse, rabbit, or rat IgG antibodies conjugated with horseradish peroxidase, according to the manufacturer’s protocol. Images of kinases were captured using Adobe Photoshop 5.0 plug-in software for Macintosh and EPSON GT-7000U scanner. The band intensities were semi-quantified using NIH Image 1.61. Assays of MKK4, MKK7, and JNK. 293T and DDT1 MF-2cells were lysed in 600 l of lysis buffer A and centrifuged. Aliquots (500 g) of the supernatants were used to assay JNK or MKKs activities. JNK and MKKs activities were measured as the radioactivities incorporated into GST-c-Jun and Trx-c-Jun, respectively, as described previously (5, 7, 18, 19). MKK4 activity was also measured as the radioactivity incorporated into kinase-deficient Mpk2, using an imaging analyzer (Fuji BAS 2000). JNK and MKKs activities were normalized to the amounts of kinases in the immuno-(or affinity-)precipitates. c-Src assay. 293T cells transfected with pUSE-c-Src were lysed in 600 l of lysis buffer A and centrifuged. Aliquots (500 g) of the supernatants were mixed with protein G-Sepharose CL-4B preabsorbed with an anti-c-Src antibody for 2 h at 4°C. The immunecomplexes were washed twice with lysis buffer A and twice with reaction buffer B (20 mM Hepes-NaOH (pH 7.5), 10 mM MgCl 2, 3 mM MnCl 2, 0.1 mM phenylmethanesulfonyl fluoride, 0.1 g/ml leupeptin, 0.1 mM EGTA, 10 M Na 3VO 4, and 2 mM -glycerophosphate). The immobilized c-Src was incubated in 30 l of reaction buffer B containing 20 M ATP and 5 Ci of [␥- 32P]ATP at 25°C for 15 min. The reactions were stopped by adding 10 l of 4⫻ Laemmli
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sample buffer. After boiled, the samples were subjected to 15% SDS–polyacrylamide gel electrophoresis. Autophosphorylation of c-Src was detected using an imaging analyzer. c-Src activity was normalized to the amount of kinase in the immunoprecipitate. Detection of active Rho family small GTPases. 293T cells transfected with pEGFP-N3-␣1B-adrenergic receptor and DDT1 MF-2 cells were lysed in 600 l of lysis buffer B (50 mM Hepes-NaOH (pH 7.5), 20 mM MgCl2, 150 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 g/ml leupeptin, 1 mM EDTA, 1 mM Na3VO 4, 10 mM NaF, and 0.5% NP-40) and centrifuged, and the supernatants were recovered. The pull-down assays to detect active Rho family small GTPases were performed as described previously (21, 22).
RESULTS AND DISCUSSION To investigate whether the Gq-coupled ␣1B-adrenergic receptor stimulated the activity of JNK, we cotransfected plasmids encoding HA-tagged JNK with the ␣1B-adrenergic receptor into 293T cells. Using an anti-HA antibody, HA-JNK was immunoprecipitated from the cell lysate. The in vitro kinase activity of HA-JNK was assayed. As shown in Fig. 1A, JNK activity was increased by stimulation with phenylephrine, an agonist of the ␣1-adrenergic receptor. Pretreatment of the cells with prazosin, an antagonist of the ␣1-adrenergic receptor, abolished JNK activation by phenylephrine (data not shown). Next, we attempted to determine which subunit of Gq was involved in JNK activation by the ␣1Badrenergic receptor. We cotransfected a plasmid encoding the C-terminus of -adrenergic receptor kinase (ARKct), which is known to bind to G␥ and to inhibit the cellular function of G␥ (4, 7). Cotransfection of ARKct inhibited JNK activation induced by G␥, but not by constitutively activated G␣q (G␣qQ209L) (Figs. 1B and 1C). As shown in Fig. 1A, ARKct failed to inhibit JNK activation by the ␣1B-adrenergic receptor, suggesting that the ␣1B-adrenergic receptor activates JNK via G␣q. To clarify whether JNK activation by the ␣1Badrenergic receptor involves JNK kinases MKK4 and MKK7, we cotransfected the plasmids encoding MKK4K95R or MKK7K63R, which are kinase-deficient mutants and sequestrate upstream MAPKKKs. As shown in Figs. 2A and 2B, JNK activation by the ␣1B-adrenergic receptor was inhibited by cotransfection with MKK4K95R, but not with MKK7K63R. Similarly, G␣qQ209L-induced JNK activation was blocked by cotransfection with MKK4K95R, but not with MKK7K63R (Figs. 2C and 2D). Next, we tried to measure the intrinsic kinase activities of MKK4 and MKK7. As shown in Figs. 2E and 2F, stimulation of the ␣1B-adrenergic receptor activated MKK4, but not MKK7. Additionally, G␣qQ209L stimulated only MKK4 activity (Figs. 2G and 2H). These results indicate that JNK activation by the ␣1Badrenergic receptor/G␣q depends only on MKK4. It has been shown that Rho family small GTPases Rho,
FIG. 1. JNK activation by the ␣1B-adrenergic receptor is mediated by G␣q, but not G␥. (A) 293T cells were transfected with plasmids encoding ␣1B-adrenergic receptor, ARKct, and JNK. JNK activity was measured at 10 min after the addition of 20 M phenylephrine. (B) Cells were transfected with plasmids encoding G␣qQ209L, ARKct, and JNK. (C) Cells were transfected with plasmids encoding G1, G␥2, ARKct, and JNK. JNK activity was measured as described under Materials and Methods. Values shown represent the mean ⫾ SE from three or four separate experiments. Expression of ␣1B-adrenergic receptor, G protein subunits, and ARKct are shown. HA-tagged JNK was immunoprecipitated with an anti-HA antibody from the cell lysates and immunoblotted with an anti-HA antibody.
Rac, and Cdc42 participate in various signal transduction pathways, including JNK activation (23, 24). Furthermore, G␥-mediated MKK4 and MKK7 activations depend on Rho family small GTPases (5). Thus, we examined whether Rho family small GTPases are involved in MKK4 activation by the ␣1B-adrenergic receptor/G␣q. As shown in Figs. 3A–3C, MKK4 activation by the ␣1B-adrenergic receptor was inhibited by cotransfection with RhoT19N, RacT17N, or Cdc42T17N. RhoT19N, RacT17N, and Cdc42T17N are dominantnegative mutants which sequestrate guanine-nucleotide exchange factors for Rho family small GTPases. G␣qQ209L-induced MKK4 activation was also blocked by these dominant-negative mutants (Figs. 3D–3F). Next, we utilized RhotekinRBD and PakCRIB. It is known that RBD specifically binds to Rho (20), while CRIB specifically interacts with Rac and Cdc42 (22). Cotransfection of RBD or CRIB blocked MKK4 activation by G␣qQ209L (Figs. 3G and 3H). Together, these results suggest that Rho family small GTPases are
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FIG. 2. JNK activation by the ␣1B-adrenergic receptor/G␣q is mediated by MKK4, but not MKK7. (A) 293T cells were transfected with plasmids encoding ␣1B-adrenergic receptor, MKK4K95R, and JNK. JNK activity was measured at 10 min after the addition of 20 M phenylephrine. (B) Cells were transfected with plasmids encoding ␣1B-adrenergic receptor, MKK7K63R, and JNK. JNK activity was measured at 10 min after the addition of 20 M phenylephrine. (C) Cells were transfected with plasmids encoding G␣qQ209L, MKK4K95R, and JNK. (D) Cells were transfected with plasmids encoding G␣qQ209L, MKK7K63R, and JNK. JNK activity was measured as described under Materials and Methods. (E) Cells were transfected with plasmids encoding ␣1B-adrenergic receptor and MKK4. MKK4 activity was measured at 10 min after the addition of 20 M phenylephrine. (F) Cells were transfected with plasmids encoding ␣1B-adrenergic receptor and MKK7. MKK7 activity was measured at 10 min after the addition of 20 M phenylephrine. (G) Cells were transfected with plasmids encoding G␣qQ209L and MKK4. (H) Cells were transfected with plasmids encoding G␣qQ209L and MKK7. MKK4 and MKK7 activities were measured as described under Materials and Methods. Values shown represent the mean ⫾ SE from three or four separate experiments. Expression of ␣1B-adrenergic receptor, G␣qQ209L, MKK4K95R, and MKK7K63R are shown. HA-tagged JNK was immunoprecipitated with an anti-HA antibody from the cell lysates and immunoblotted with an anti-HA antibody. GST-tagged MKK4 and MKK7 were precipitated with glutathione–Sepharose 4B from the cell lysates and immunoblotted with an anti-GST antibody.
involved in MKK4 activation by the ␣1B-adrenergic receptor/G␣q. It has been reported that Src family tyrosine kinases mediate JNK activation by G␣11, a member of G␣q family (9). We therefore investigated whether the ␣1Badrenergic receptor activates MKK4 through c-Src. Transfected cells were treated with PP1, a specific inhibitor of Src family tyrosine kinases. As shown in Fig. 4A, MKK4 activation by the ␣1B-adrenergic receptor was inhibited by treatment with PP1. G␣qQ209Linduced MKK4 activation was also inhibited by PP1
and another Src family tyrosine kinase inhibitor PP2 (Figs. 4B and 4C). In addition, cotransfection of Csk, a negative regulator of Src family tyrosine kinases, blocked G␣qQ209L-induced MKK4 activation (Fig. 4D). Furthermore, G␣qQ209L-induced MKK4 activation was almost completely inhibited by cotransfection of DN-Src (Fig. 4E). In contrast, G␣qQ209L-induced MKK4 activation was less than 30% inhibited by cotransfection of DN-Fyn or DN-Lyn (data not shown). Together, it is likely that c-Src primarily participates in G␣q-mediated MKK4 activation.
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FIG. 3. MKK4 activation by the ␣1B-adrenergic receptor/G␣q is mediated by Rho family small GTPases. (A–C) 293T cells were transfected with plasmids encoding ␣1B-adrenergic receptor, each dominant-negative Rho family small GTPase, and MKK4. MKK4 activity was measured at 10 min after the addition of 20 M phenylephrine. (D–F) Cells were transfected with plasmids encoding G␣qQ209L, each dominant-negative Rho family small GTPase, and MKK4. (G and H) Cells were transfected with plasmids encoding G␣qQ209L, RhotekinRBD or PakCRIB, and MKK4. MKK4 activity was measured as described under Materials and Methods. Values shown represent the mean ⫾ SE from three or four separate experiments. Expression of ␣1B-adrenergic receptor, G␣qQ209L, dominant-negative Rho family small GTPases, and PakCRIB are shown. The transcription levels of RhotekinRBD and -actin (control) were demonstrated by RT-PCR. GST-tagged MKK4 was precipitated with glutathione–Sepharose 4B from the cell lysates and immunoblotted with an anti-GST antibody.
Next, we tried to measure c-Src activity. c-Src immunoprecipitated from cell lysates was incubated with [ 32P]ATP. As shown in Fig. 4F, c-Src was autophosphorylated when c-Src was cotransfected with G␣qQ209L. In addition, PP1 treatment almost completely inhibited c-Src autophosphorylation by G␣qQ209L. Furthermore, we have confirmed that the Tyr 418 autophosphorylation of c-Src was induced by G␣qQ209L using its phosphorylated Tyr 418-specific antibody (18). We investigated the effect of PP1 or Clostridium botulinum C3 exoenzyme, which ADP-ribosylates Rho and weakly Rac and inhibits those cellular function (24), on the ␣1B-adrenergic receptor-induced endogenous JNK activation in 293T cells expressing the ␣1Badrenergic receptor and DDT1 MF-2 cells. DDT1 MF-2 cells natively express the ␣1B-adrenergic receptor (25,
26). Additionally, we examined the effect of Ro-318220, an inhibitor of PKC, on JNK activation by the ␣1B-adrenergic receptor in these cells, because PKC was a downstream molecule of G␣q family (9). As shown in Figs. 5A and 5B, Ro-31-8220, PP1, or C3 exoenzyme blocked JNK activation induced by the ␣1B-adrenergic receptor in 293T cells expressing its receptor and DDT1 MF-2 cells, suggesting the involvement of PKC, Src family tyrosine kinases, and Rho family small GTPases in a pathway linking the ␣1Badrenergic receptor to endogenous JNK. Next, we examined the effect of Ro-31-8220 or PP1 on the ␣1B-adrenergic receptor-induced endogenous Rho family small GTPase activation in 293T cells expressing the ␣1B-adrenergic receptor and DDT1 MF-2 cells. As shown in Figs. 5C and 5D, active GTP-bound
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FIG. 4. MKK4 activation by the ␣1B-adrenergic receptor/G␣q is mediated by c-Src. (A) 293T cells transfected with plasmids encoding ␣1B-adrenergic receptor and MKK4 were pretreated with 15 M PP1 for 1 h. MKK4 activity was measured at 10 min after the addition of 20 M phenylephrine. (B and C) Cells transfected with plasmids encoding G␣qQ209L and MKK4 were pretreated with the indicated concentration of PP1 or PP2 for 18 h. (D) Cells were transfected with plasmids encoding G␣qQ209L, Csk, and MKK4. (E) Cells were transfected with plasmids encoding G␣qQ209L, DN-Src, and MKK4. MKK4 activity was measured as described under Materials and Methods. (F) Cells transfected with plasmids encoding G␣qQ209L and c-Src were pretreated with 15 M PP1 for 18 h. c-Src autophosphorylation was measured as described under Materials and Methods. Values shown represent the mean ⫾ SE from three or four separate experiments. Expression of ␣1B-adrenergic receptor, G␣qQ209L, and Csk are shown. GST-tagged MKK4 was precipitated with glutathione– Sepharose 4B from the cell lysates and immunoblotted with an anti-GST antibody. c-Src was immunoprecipitated with an anti-c-Src antibody from the cell lysates and immunoblotted with an anti-c-Src antibody.
Rho family small GTPases were detected following stimulation of the ␣1B-adrenergic receptor, using the pull-down assay. Either Ro-31-8220 or PP1 inhibited Rho family small GTPase activation induced by the ␣1B-adrenergic receptor in 293T cells expressing its receptor and DDT1 MF-2 cells, suggesting the involvement of PKC and Src family tyrosine kinases in a pathway linking the ␣1B-adrenergic receptor to endogenous Rho family small GTPases. We have previously shown that G␣i2 activates JNK through a putative MKK other than MKK4 and MKK7 (7). In addition, G␣i2-mediated JNK activation was dependent on c-Src, Rho, and Cdc42 (7). On the other hand, G␥ activated JNK through the MKK4 pathway dependent on tyrosine kinase other than c-Src, Rho, and Cdc42, and to a lesser extent the MKK7 pathway dependent on Rac (5). In this study, we demonstrated that G␣q up-regulates only MKK4 activity and in turn JNK activity through c-Src and three Rho family small GTPases in 293T cells. Also, we reached basically the same results with DDT1 MF-2 cells that natively express the ␣1B-adrenergic receptor, as we had in the
transient transfection system. It is likely that signaling components of the MAPK cascade are organized into the module called scaffold protein in vivo (27). The differences in JNK signaling pathways by the G protein subunits may be caused by a difference in scaffold proteins. There are more than fifteen MAPK kinase kinases (MAPKKKs); MEKK1 appears to induce JNK activation through MKK4 in vivo (27). Additionally, MEKK1 harbors a CRIB domain which specifically interacts with active Rac and Cdc42 (28). MKK4 activation by the ␣1B-adrenergic receptor/G␣q depended on Rho family small GTPases Rac and Cdc42 (Fig. 3). Taken with the observation that Rac and Cdc42 activate JNK (3, 29) and MKK4 (data not shown), it is possible that MEKK1 is a MAPKKK in a pathway linking the ␣1Badrenergic receptor/G␣q to MKK4. On the other hand, MKK4 activation by ␣1B-adrenergic receptor/G␣q also involved Rho (Fig. 3). In addition, Rho increases the activities of JNK (3, 29) and MKK4 (data not shown) in 293T cells. Thus, a putative Rho-dependent MAPKKK (29) might contribute to JNK activation by the ␣1Badrenergic receptor/G␣q in these cells.
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FIG. 5. Involvement of PKC, Src family tyrosine kinases, and Rho family small GTPases in the pathway from the ␣1B-adrenergic receptor to JNK in 293T and DDT1 MF-2 cells. (A) 293T cells transfected with plasmids encoding ␣1B-adrenergic receptor were pretreated with 10 M Ro-31-8220 for 30 min, 15 M PP1 for 1 h, or 1 g/ml C3 exoenzyme for 18 h. JNK activity was measured at 10 min after the addition of 20 M phenylephrine. Endogenous JNK was immunoprecipitated with an anti-JNK antibody from the cell lysates and assayed using GST-c-Jun as a substrate. (B) DDT1 MF-2 cells were pretreated with 10 M Ro-31-8220 for 30 min, 15 M PP1 for 1 h, or 1 g/ml C3 exoenzyme for 18 h. JNK activity was measured at 20 min after the addition of 20 M phenylephrine. Endogenous JNK was immunoprecipitated with an anti-JNK antibody from the cell lysates and assayed. Values shown represent the mean ⫾ SE from three or four separate experiments. JNK was immunoprecipitated with an anti-JNK antibody from the cell lysates and immunoblotted with an anti-JNK antibody. Values shown represent the mean ⫾ SE from five separate experiments. (C) 293T cells transfected with plasmids encoding ␣1B-adrenergic receptor were pretreated with 10 M Ro-31-8220 for 30 min or 15 M PP1 for 1 h. The activities of Rho family small GTPases were measured at 10 min after the addition of 20 M phenylephrine. Active GTP-bound form of Rho family small GTPases was affinity-precipitated with GST-mDia1RBD or GST-PakCRIB from the cell lysates and immunoblotted with antibodies against Rho family small GTPases. (D) DDT1 MF-2 cells were pretreated with 10 M Ro-31-8220 for 30 min or 15 M PP1 for 1 h. The activities of Rho family small GTPases were measured at 20 min after the addition of 20 M phenylephrine. Active GTP-bound form of Rho family small GTPases was affinityprecipitated with GST-mDia1RBD or GST-PakCRIB from the cell lysates and immunoblotted with antibodies against Rho family small GTPases. Total Rho family small GTPases in the cell lysates were detected by immunoblotting with antibodies against Rho family small GTPases. Immunoblots of Rho family small GTPases are representative of three separate experiments.
It has been reported that JNK activation by GPCR is involved in the expression of various genes (3). Ramirez et al. reported that ␣1-adrenergic receptorinduced JNK activation up-regulates expression of atrial natriuretic factor in cardiac myocytes (30). Also, in the course of this study, Clerk et al. reported that stimulation of ␣1-adrenergic receptor activates Rho family small GTPases in cardiac myocytes (31). However, it remains unclear whether ␣1B-adrenergic receptor activates JNK through Rho family small GTPases in cardiac myocytes. On the other hand, the ␣1B-adrenergic receptor inhibits cell growth through JNK in 293T cells (19). It is possible that this antimitogenic pathway involves c-Src, Rho family small GTPases, and MKK4. Further investigation of the pathway from the ␣1B-adrenergic receptor to JNK may
shed light on our understanding of intracellular signal transduction and various cellular functions regulated by Gq-coupled receptor. ACKNOWLEDGMENTS This work was supported by grants from the Japan Science and Technology Corporation (JST), the Organization for Pharmaceutical Safety and Research (OPSR), and the Japanese Ministry of Education, Science, Sports, and Culture.
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