Parallel Regulation of Mitogen-activated Protein Kinase Kinase 3 (MKK3) and MKK6 in Gq-signaling Cascade*

Heterotrimeric G protein Gq stimulates the activity of p38 mitogen-activated protein kinase (MAPK) in mammalian cells. To investigate the signaling mechanism whereby α and βγ subunits of Gq activate p38 MAPK, we introduced kinase-deficient mutants of mitogen-activated protein kinase kinase 3 (MKK3), MKK4, and MKK6 into human embryonal kidney 293 cells. The activation of p38 MAPK by Gαq and Gβγ was blocked by kinase-deficient MKK3 and MKK6 but not by kinase-deficient MKK4. In addition, Gαq and Gβγ stimulated MKK3 and MKK6 activities. The MKK3 and MKK6 activations by Gαq, but not by Gβγ, were dependent on phospholipase C and c-Src. Gαqstimulated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-dependent manner. On the other hand, Gβγ activated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-, Rac-, and Cdc42-dependent manner. Gβγ-induced MKK3 and MKK6 activations were dependent on a tyrosine kinase other than c-Src. These results suggest that Gαqand Gβγ stimulate the activity of p38 MAPK by regulating MKK3 and MKK6 through parallel signaling pathways.

Heterotrimeric G proteins are commonly used to transduce the signals across seven-transmembrane receptors. G proteins are composed of ␣, ␤, and ␥ subunits (G␣, G␤, and G␥) and activated by the G protein-coupled receptors, which respond to sensory signals, hormones, neurotransmitters, and chemokines in mammalian cells (5)(6)(7)(8). Many types of G protein-coupled receptors activate ERK in mammalian cells (8). G q -coupled m1 muscarinic acetylcholine and ␣1-adrenergic receptors have been reported to stimulate ERK activity mainly through G␣ q (8). G␣ q directly activates phospholipase C, leading to induction of PKC activation. The PKC activation is involved in the activation of some receptor and nonreceptor types of tyrosine kinases (9 -11). A tyrosine phosphorylation of Shc induces the translocation of Grb2-mSos complex to the plasma membranes, where mSos promotes the exchange of GTP for GDP bound on small GTPase Ras (10). Ras mediates the activation of c-Raf, which in turn activates MEK1 and MEK2. It is likely that MEK1 and MEK2 equally activate ERK.
Some types of G protein-coupled receptors have been reported to stimulate the activity of JNK (12,13). An agonist stimulation of m1 muscarinic acetylcholine receptor increases JNK activity through G␤␥ (14). Furthermore, it has been shown that G␤␥ stimulates JNK activity mainly through MKK4 dependent on a tyrosine kinase not in the Src family and Rho family small GTPases Rho and Cdc42 and to a lesser extent through MKK7 dependent on Rho family small GTPase Rac (15). Additionally, it has been reported that constitutively activated G␣ q/11 activates JNK (16), and its activation requires PKC and Src family tyrosine kinases (17).
In previous study, we found that m1 muscarinic acetylcholine receptor stimulates the activity of p38 MAPK, and its activation is mediated by both G␣ q and G␤␥ (18). However, it remains unclear how G protein-coupled receptors and G protein subunits activate p38 MAPK. Here we demonstrate that m1 muscarinic acetylcholine receptor induces p38 MAPK activation mediated by MKK3 and MKK6, but not by MKK4.
Furthermore, we show that G␣ q and G␤␥ differentially regulate MKK3 and MKK6 through parallel signaling pathways.

MATERIALS AND METHODS
Antibodies-Mouse monoclonal antibodies M2, 12CA5, and 9E10 against FLAG, HA, and Myc epitopes were obtained from Sigma, Roche Molecular Biochemicals, and Babco, respectively. A mouse monoclonal antibody B-14 against Schistosoma japonicum GST was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A rabbit polyclonal antibody C-19 against G␣ q/11 was obtained from Santa Cruz Biotechnology. Rabbit polyclonal antibodies T-20 and 06 -238 against G␤ were purchased from Santa Cruz Biotechnology and Upstate Bio-technology, Inc. (Lake Placid, NY), respectively. Rabbit polyclonal anti-Ras (C-20) and anti-Csk (C-20) antibodies were obtained from Santa Cruz Biotechnology. A mouse monoclonal anti-c-Src antibody (05-184) and a rabbit polyclonal anti-phosphorylated c-Src (Tyr(P) 418 ) antibody  were purchased from Upstate Biotechnology, Inc. and BIO-SOURCE International, respectively. Goat anti-mouse and anti-rabbit IgG antibodies conjugated with horseradish peroxidase were obtained from PerkinElmer Life Sciences.
Plasmids-A complementary DNA of G␣ q (GenBank TM accession number U40038) was isolated by polymerase chain reaction using hu-FIG. 1. p38 MAPK activation by m1 muscarinic acetylcholine receptor is mediated through MKK3 and MKK6, but not MKK4. HEK 293 cells were transfected with the plasmids encoding m1 muscarinic acetylcholine receptor (A-C), HA-p38 MAPK (A-C), FLAG-MKK3K64R (A), FLAG-MKK4K95R (B), and FLAG-MKK6K82R (C). The p38 MAPK activity was measured at 10 min after the addition of 10 M carbachol as described under "Materials and Methods." Cells were transfected with the plasmids encoding m1 muscarinic acetylcholine receptor (D and E), GST-MKK3 (D), and GST-MKK6 (E). The MKK3 and MKK6 activities were measured at 10 min after the addition of 10 M carbachol as described under "Materials and Methods." Values shown represent the mean Ϯ S.E. from three or four separate experiments. The phosphorylation of GST-ATF2 and KD-p38 MAPK and the expression of HA-p38 MAPK, FLAG-MKK3K64R, FLAG-MKK4K95R, and FLAG-MKK6K82R in the cell lysates are shown. GST-MKK3 and GST-MKK6 were precipitated with glutathione-Sepharose 4B from the cell lysates and immunoblotted with anti-GST antibody.
FIG. 2. G␣ q -induced p38 MAPK activation is mediated through MKK3 and MKK6, but not MKK4. Cells were transfected with the plasmids encoding G␣ q Q209L (A-C), HA-p38 MAPK (A-C), FLAG-MKK3K64R (A), FLAG-MKK4K95R (B), and FLAG-MKK6K82R (C). The p38 MAPK activity was measured as described under "Materials and Methods." Cells were transfected with the plasmids encoding G␣ q Q209L (D and E), GST-MKK3 (D), and GST-MKK6 (E). The MKK3 and MKK6 activities were measured as described under "Materials and Methods." Values shown represent the mean Ϯ S.E. from three or four separate experiments. The phosphorylation of GST-ATF2 and KD-p38 MAPK and the expression of G␣ q Q209L, HA-p38 MAPK, FLAG-MKK3K64R, FLAG-MKK4K95R, and FLAG-MKK6K82R in the cell lysates are shown. GST-MKK3 and GST-MKK6 were precipitated with glutathione-Sepharose 4B and immunoblotted with anti-GST antibody. man fetal brain cDNA library (CLONTECH) and inserted into the EcoRI restriction site of mammalian expression vector pCMV. G␣ q Q209L and G␣ q Q209L(R256A, T257A) mutants were constructed by the method of overlapping polymerase chain reaction using isolated G␣ q cDNA as a template and ligated into the EcoRI restriction site of pCMV. cDNAs of G␤1 and G␥2 were generously provided by M. I. Simon (California Institute of Technology) and T. Nukada (Tokyo Institute of Psychiatry), respectively. G␤1T143A was constructed by overlapping polymerase chain reaction using G␤1 cDNA as a template and inserted into the BamHI restriction site of pCMV. pCMV-m1 muscarinic acetylcholine receptor was generously provided by E. M. Ross (University of Texas Southwestern Medical Center). RhoA and Rac1 cDNAs were kindly provided by K. Kaibuchi (Nagoya University). Cdc42 cDNA was kindly provided by R. A. Cerione (Cornell University). Pak1 cDNA was a generous gift from L. Lim (National University of Singapore). pCMV-G␤1, pCMV-G␥2, pCMV-RasS17N, pCMV-FLAG-RhoT19N, pCMV-FLAG-RacT17N, pCMV-FLAG-Cdc42T17N, pCMV-FLAG-RhoG14V, pCMV-FLAG-RacG12V, pCMV-FLAG-Cdc42G12V, pCMV-Myc-Pak1C-RIB, and pCMV-FLAG-MKK4K95R were constructed as described previously (15,(17)(18)(19). cDNAs of MKK3 (20) and MKK6 (21)(22)(23)(24) were amplified from human fetal brain cDNA library and inserted into the BamHI restriction site of pCMV-GST and the BglII/BamHI sites of pCMV-FLAG. pCMV-FLAG-MKK3K64R and pCMV-FLAG-MKK-6K82R were generated by polymerase chain reaction-mediated mutagenesis. The sequence (GSSYPYDVPDYASSG) containing HA epitope was inserted between Met 1 and Ser 2 of p38␣ using the synthetic primer. HA-tagged p38 MAPK was ligated into the BamHI restriction site of pCMV. pEFBOS-Clostridium botulinum C3 exoenzyme was generously provided by S. Narumiya (Kyoto University). Csk cDNA was kindly provided by M. Okada (Osaka University), and pCMV-Csk was constructed by S. Mizutani (Gunma University). pEFBOS-v-Src was generously provided by Y. Fukami (Kobe University). A hexahistidine tag expression plasmid encoding the kinase-deficient form of Mpk2, a Xenopus orthologue of mouse p38␣, was kindly provided by E. Nishida (Kyoto University). pGEX2T-ATF2 (amino acids 1-96) was constructed as described previously (18). All DNA sequences were confirmed by DNA sequencer L-4000L (LI-COR).
Cell Culture and Transfection-Human embryonal kidney (HEK) 293 cells (ATCC CRL 1573) were maintained in Dulbecco's modified Eagle's medium containing 100 g/ml kanamycin and 10% heat-inactivated fetal bovine serum. The cells were cultured at 37°C in humidified atmosphere containing 10% CO 2 . 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 g by empty vector, pCMV. Three g of pCMV-HA-p38 MAPK, pCMV-GST-MKK3, or pCMV-GST-MKK6 was cotransfected with 0.3 g of pCMV-m1 muscarinic acetylcholine receptor, 10 g of plasmids encoding G␣ q wild type or mutants, 5 g of pCMV-G␤1 or pCMV-G␤1T143A and 5 g of pCMV-G␥2, 10 g of pCMV-FLAG-MKK3K64R, 10 g of pCMV-FLAG-MKK4K95R, 10 g of pCMV-FLAG-MKK6K82R, 10 g of plasmids encoding dominant negative Ras and Rho family small GTPases, 10 g of pEFBOS-C3 exoenzyme, 10 g of pCMV-Myc-Pak1CRIB, 10 g of plasmids encoding constitutively activated Rho family small GTPases, 3 g of pCMV-Csk, or 10 g of pEFBOS-v-Src. The medium was replaced with serum-free medium containing 1 mg/ml bovine serum albumin 24 h after transfection, and the cells were starved in serum-free medium for 24 h.
Kinase Assays-The transfected 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 3 VO 4 , 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 the p38 MAPK assay, aliquots (50 g of protein) of the supernatants were mixed with protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) preabsorbed with a mouse anti-HA antibody for 2 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  Immunoprecipitation-The cells transfected with the plasmids encoding Myc-Pak1CRIB were lysed in lysis buffer A. Aliquots (500 g of protein) of the cell lysates were mixed with protein A-Sepharose CL-4B preabsorbed with a mouse anti-Myc antibody for 2 h at 4°C. The immune complexes were precipitated by centrifugation and washed three times with lysis buffer A.
Immunoblotting-Aliquots of the cell lysates and immune complexes were boiled in Laemmli sample buffer. The boiled samples were separated by 8 or 15% SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to nitrocellulose membranes. After the membranes were blocked with phosphate-buffered saline containing 0.1% Tween 20 and 5 mg/ml bovine serum albumin, the membranes were subjected to immunoblotting with various antibodies. The bound antibodies were detected using Renaissance (PerkinElmer Life Sciences) with anti-mouse or -rabbit IgG antibody conjugated with horseradish peroxidase.

RESULTS
The Pathway from m1 Muscarinic Acetylcholine Receptor, G␣ q , and G␤␥ to p38 MAPK Is Mediated by MKK3 and MKK6, but Not by MKK4 -To examine a signal transduction pathway linking m1 muscarinic acetylcholine receptor to p38 MAPK, we transfected plasmids encoding HA-tagged p38 MAPK with various cDNAs into human embryonal kidney 293 cells. Using an anti-HA antibody, the epitope-tagged p38 MAPK was immunoprecipitated from the cell lysate, and the in vitro kinase activity was assessed as the radioactivity incorporated into recombinant ATF2. The expression level of HA-p38 MAPK was confirmed by immunoblotting in each experiment to compare each transfection efficiency (see Figs. 1-3). The p38 MAPK activation by stimulating m1 muscarinic acetylcholine receptor with an agonist, carbachol, was specifically blocked by co-transfection of MKK3K64R or MKK6K82R but not by that of MKK4K95R (Fig. 1, A-C). MKK3K64R, MKK4K95R, and MKK6K82R are kinase-deficient constructs and act as dominant negative mutants by sequestering upstream components (25).
Next, we attempted to measure the kinase activities of MKK3 and MKK6. The cells were co-transfected with plasmids encoding GST-MKK3 or GST-MKK6, which are tagged with GST at the N terminus. GST-MKK3 and GST-MKK6 were precipitated from the cell lysate using glutathione-resin, and the kinase activities were assessed as the radioactivity incorporated into recombinant kinase-deficient p38 MAPK (KD-p38 MAPK). In each experiment, we examined the expression level of GST-MKK3 or GST-MKK6 to monitor each transfection efficiency (see Figs. 1-11). As shown in Fig. 1, D and E, m1 muscarinic acetylcholine receptor stimulated the activities of MKK3 and MKK6. These results indicate that m1 muscarinic acetylcholine receptor stimulates the activity of p38 MAPK through two p38 MAPK kinases, MKK3 and MKK6.
G␣ q and G␤␥ Induce the Activations of MKK3 and MKK6 in a Ras-independent Manner-Oncogenic Ras (RasG12V) activates p38 MAPK (26 -28). Additionally, RasG12V transfection resulted in increase of the MKK3 and MKK6 activities (data not shown). Furthermore, signal-dependent p38 MAPK activation has been effectively blocked by co-transfection of dominant negative Ras (RasS17N) (29). In order to examine whether m1 muscarinic acetylcholine receptor stimulates the activities of MKK3 and MKK6 via Ras, we co-transfected the plasmid encoding RasS17N into the cells. However, co-transfection of RasS17N did not block MKK3 and MKK6 activations by m1 muscarinic acetylcholine receptor (Fig. 4, A and B). Similarly, RasS17N failed to suppress G␣ q Q209L-and G␤␥-induced activations of MKK3 and MKK6 (data not shown). These results suggest that Ras is not required for the pathway from G␣ q and G␤␥ to MKK3 and MKK6.
Differential Regulation of the Signaling Pathways from G␣ q and G␤␥ to MKK3 and MKK6 by Rho Family Small GTPases-Rho family small GTPases Rac and Cdc42 have been reported to function upstream of p38 MAPK signaling cascade (1-4, 26 -28). We investigated the involvement of Rho family small GTPases in m1 muscarinic acetylcholine receptor-induced MKK3 and MKK6 activations by using the dominant negative mutants, RhoT19N, RacT17N, and Cdc42T17N. Co-transfection of RacT17N or Cdc42T17N, but not that of RhoT19N, reduced the receptor-induced MKK3 activation (Fig. 5, A, C, and E), while co-transfection of RhoT19N, RacT17N, or Cdc42T17N reduced the receptor-induced MKK6 activation (Fig. 5, B, D, and F). Next, we tested whether MKK3 and MKK6 activations mediated by G␣ q and G␤␥ involve Rho family small GTPases. Co-transfection of RacT17N or Cdc42T17N, but not of RhoT19N, inhibited G␣ q Q209L-induced MKK3 activation (Fig. 5, G, I, and K). Similarly, G␤␥-induced MKK3 activation was also blocked by RacT17N or Cdc42T17N, but not by RhoT19N (Fig. 5, M, O, and Q). In contrast, G␣ q Q209Linduced MKK6 activation was blocked only by co-transfection of RhoT19N (Fig. 5, H, J, and L), while G␤␥-induced MKK6 activation was blocked by three dominant negative mutants (Fig. 5, N, P, and R). These results suggest that G␣ q and G␤␥ activate MKK3 in a Rac-and Cdc42-dependent manner, whereas G␣ q activates MKK6 in a Rho-dependent manner and G␤␥ activates MKK6 in a Rho-, Rac-, and Cdc42-dependent manner.
To confirm that Rho family small GTPases are involved in MKK3 and MKK6 activations mediated by G␣ q and G␤␥, we further utilized C. botulinum C3 exoenzyme and Pak1CRIB. The C3 exoenzyme ADP-ribosylates Rho and inhibits its cellular function (30). The Pak1CRIB binds to an active form of Rac or Cdc42 (31) and inhibits the interaction of Rac or Cdc42 with the effectors. G␣ q Q209L-induced MKK3 activation was reduced by co-transfection of Pak1CRIB (Fig. 6C) but not by C3 exoenzyme (Fig. 6A). Similarly, G␤␥-induced MKK3 activation was inhibited by Pak1CRIB ( Fig. 6G) but not by C3 exoenzyme (Fig. 6E). On the other hand, G␣ q Q209L-induced MKK6 activation was blocked by C3 exoenzyme (Fig. 6B) but not by Pak1CRIB (Fig. 6D). G␤␥-induced MKK6 activation was blocked by C3 exoenzyme (Fig. 6F) and Pak1CRIB (Fig. 6H). Again, these results suggest that G␣ q and G␤␥ activate MKK3 in a Rac-and Cdc42-dependent manner, whereas G␣ q activates MKK6 in a Rho-dependent manner and G␤␥ activates MKK6 in a Rho-, Rac-, and Cdc42-dependent manner.
Differential Involvement of Tyrosine Kinases in MKK3 and MKK6 Activations by G␣ q and G␤␥-p38 MAPK activation by m1 muscarinic acetylcholine receptor was suppressed by PP1 and PP2 (17), which are known to be tyrosine kinase inhibitors preferential for Src family tyrosine kinases. MKK3 and MKK6 activations by m1 muscarinic acetylcholine receptor were inhibited by treatment with PP1 (Fig. 7, A and B). Next, we investigated the involvement of tyrosine kinase in MKK3 and MKK6 activations induced by G␣ q and G␤␥. Treatment of the cells with PP1 inhibited MKK3 and MKK6 activations induced by G␣ q Q209L and G␤␥ in a dose-dependent manner (Fig. 7, C-F). PP2 also inhibited the activations in the same manner (data not shown). It is likely that G␣ q and G␤␥ activate MKK3 and MKK6 via tyrosine kinase.
We previously showed that G␣ q/11 -mediated p38 MAPK activation is attenuated by co-transfection of Csk (17), a negative regulator of Src family tyrosine kinases. In order to test whether Src family tyrosine kinases mediate the pathway from G␣ q and G␤␥ to MKK3 and MKK6, we co-transfected the plasmid encoding Csk into the cells. Csk resulted in attenuation of MKK3 and MKK6 activations induced by G␣ q Q209L (Fig. 8, A  and B), but not by G␤␥ (Fig. 8, C and D).
Next, we attempted to evaluate G q -mediated c-Src activation using an antibody that recognizes a phosphorylated state of tyrosine 418 on c-Src. The tyrosine 418 is known to be an autophosphorylation site essential for its activation (32). Fig.  8E shows that G␣ q Q209L transfection induced c-Src phosphorylation. Treatment with PP1 reduced G␣ q Q209L-induced c-Src phosphorylation (Fig. 8E). We observed the weak but inconsistent tyrosine phosphorylation of c-Src by overexpression of G␤␥ (Fig. 8F). These results suggested that G␣ q may activate MKK3 and MKK6 through Src family tyrosine kinases, and G␤␥ may activate MKK3 and MKK6 through tyrosine kinases not in the Src family.
c-Src Functions Upstream of Rho Family Small GTPases in MKK3 and MKK6 Signaling Pathways-To determine whether c-Src acts as an upstream or downstream component of Rho family small GTPases in the pathway from G␣ q to MKK3 and MKK6, we co-transfected the plasmids encoding v-Src and dominant negative mutants of Rho family small GTPases into the cells. Co-transfection of RacT17N or Cdc42T17N suppressed the v-Src-induced MKK3 activation (Fig. 9, A and B), and RhoT19N co-transfection blocked MKK6 activation by v-Src (Fig. 9C), indicating that c-Src acts upstream of Rac and Cdc42 in the MKK3 signaling pathway and upstream of Rho in the MKK6 signaling pathway.
RhoG14V, RacG12V, and Cdc42G12V are constitutively activated mutants of Rho family small GTPases (26 -28). As shown in Fig. 9, D-H, RacG12V and Cdc42G12V activated MKK3, whereas RhoG14V, RacG12V, and Cdc42G12V activated MKK6. In contrast, RhoG14V failed to activate MKK3 (data not shown). Since Rho family small GTPases might function downstream of c-Src (Fig. 9, A-C), we investigated whether MKK3 and MKK6 activations by the constitutively activated mutants of Rho family small GTPase are inhibited by PP1. Pretreatment of this inhibitor failed to attenuate MKK3 and MKK6 activations induced by RhoG14V, RacG12V or Cdc42G12V (Fig. 9, D-H), indicating that Rho family small GTPases function downstream of tyrosine kinases in MKK3 and MKK6 signaling cascades.
MKK3 and MKK6 Activations by G␣ q , but Not by G␤␥, Involve Phospholipase C-Phospholipase C is known to be an effector of G q (7). To explore whether phospholipase C is involved in MKK3 and MKK6 activations mediated by G␣ q and G␤␥, we utilized G␣ q and G␤ mutants incapable of activating phospholipase C. Mutations of amino acid residues 256 and 257 in G␣ q retain the ability to bind GTP but impair the capability to activate phospholipase C in HEK 293 cells (33). These mutations dramatically reduced the ability of constitutively activated G␣ q to stimulate the activities of MKK3 and MKK6 (Fig.  10, A and B). A mutation of amino acid residue 143 in G␤ results in impairment of its ability to stimulate phospholipase C (34). This mutation in G␤ had no effect on G␤␥-induced MKK3 and MKK6 activations (Fig. 10, C and D). Furthermore, a phospholipase C inhibitor edelfosine, 1-O-octadecyl-2-Omethyl-rac-glycero-3-phosphocholine (ET-18-OCH 3 ), inhibited the activation of MKK3 and MKK6 by G␣ q Q209L but not by G␤␥ (data not shown). These results suggested that phospholipase C may be involved in MKK3 and MKK6 activations mediated by G␣ q but not by G␤␥.
G␤␥-mediated MKK3 and MKK6 Activations Do Not Depend on Phosphatidylinositol 3-Kinase-It has been shown that G␤␥ participates in activating phosphatidylinositol 3-kinase (35,36). To examine the involvement of phosphatidylinositol 3-ki-nase in MKK3 and MKK6 activations by G␤␥, the transfected cells were treated with phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 as described previously (37,38). Wortmannin had no effect on carbachol-and G␤␥-induced MKK3 and MKK6 activations (Fig. 11). Similarly, LY294002 did not inhibit the MKK3 and MKK6 activations (data not shown). These results suggested that phosphatidylinositol 3-kinase is not implicated in MKK3 and MKK6 activations by G␤␥. DISCUSSION MKK3 and MKK6 have been shown to be activated by physical and chemical stresses and inflammatory cytokines such as tumor necrosis factor-␣ and interleukin-1 (21,24), although only limited information is known about how signals from G protein-coupled receptors are linked to the activation of these kinases. In the present study, we showed that m1 muscarinic acetylcholine receptor and G protein subunits, G␣ q and G␤␥, stimulated p38 MAPK activity dependent on MKK3 and MKK6, but not on MKK4. G␣ q induced MKK3 and MKK6 activations in a phospholipase C-dependent manner. In addi- tion, G␣ q induced c-Src activation that is involved in MKK3 and MKK6 activations. There were two distinct signaling pathways from G␣ q to MKK3 and MKK6 at the level of Rho family small GTPases; one was the MKK3 pathway dependent on Rac and Cdc42, and the other was the MKK6 pathway dependent on Rho. In contrast, G␤␥ activated MKK3 and MKK6 via tyrosine kinases not in the Src family. G␤␥ also activated p38 MAPK through two distinct signaling pathways; one was the MKK3 pathway dependent on Rac and Cdc42, and the other was the MKK6 pathway dependent on Rho, Rac, and Cdc42. On the basis of these results, we summarized the signaling pathways from the m1 muscarinic acetylcholine receptor to p38 MAPK (Fig. 12).
Many MAPKKKs induce the activation of the p38 MAPK cascade, although it is likely that MAPKKK selects specific MAPKK as its partner. For example, MEKK1 preferentially activates MKK3, MKK4, and MKK6 rather than MKK7 (39), whereas MEKK4 activates MKK3 and MKK6 rather than MKK4 (40). In addition, MEKK3 couples to MKK6 and MKK7 but not to MKK3 and MKK4 (41). Since MKK3 and MKK6 activations by G q signaling are mediated through distinct pathways, different MAPKKKs might be implicated in the regulation of MKK3 and/or MKK6.
MEKK1 and MEKK4 contain a CRIB domain that interacts with Rac and Cdc42 (42). Additionally, constitutively activated mutants of Rac and Cdc42 stimulate p38 MAPK activity (26 -28). Rac and Cdc42 were involved in G␣ q -mediated MKK3 activation and G␤␥-mediated MKK3/6 activations (Figs. 5 and 6). Therefore, MEKK1 and MEKK4 might be candidates of MAPKKKs in these signaling pathways. On the other hand, MKK6 activation mediated by G␣ q and G␤␥ was dependent on Rho (Figs. 5 and 6). A recent report has indicated that a transcription factor, SRF, is phosphorylated through a p38 MAPK-dependent mechanism (43), and Rho has been sug-gested to be involved in SRF phosphorylation (44). Although MAPKKK regulated by Rho has not yet been clarified, a putative Rho-regulated MAPKKK may participate in the MKK6 signaling pathway mediated by G␣ q and G␤␥.
Mammalian guanine-nucleotide exchange factors (GEFs) for Rho family small GTPases have been grouped (45). Some members of Rho family GEFs exhibit the exchange activity for a broad range of Rho family small GTPases, whereas others are more specific. We found differential involvement of Rho family small GTPases in the activations of MKK3 and MKK6 mediated by G protein subunits (Figs. 5 and 6). The differential involvement of Rho family small GTPases in the pathways might be due to the different Rho family GEFs. Since the MKK3 activation mediated by G␣ q and G␤␥ is dependent on Rac and Cdc42 (Figs. 5 and 6), it is possible that its MKK3 activation is mediated by Rho family GEF specific for Rac and Cdc42, e.g. Vav and ␣Pix/Cool-2. However, it must be noted that MKK3 activation by G␣ q involves c-Src, whereas MKK3 activation by G␤␥ involves a tyrosine kinase not in the Src family ( Figs. 7 and 8). It is conceivable that diverse Rho family GEFs may function downstream of G␣ q and G␤␥ in the MKK3 signaling pathway. On the other hand, MKK6 activation by G␣ q only depended on Rho (Figs. 5 and 6). Thus, Rho-specific GEF (e.g. Lbc and Lfc) may play a role in G␣ q -mediated MKK6 activation. In contrast, G␤␥-mediated MKK6 activation required Rho, Rac, and Cdc42 (Figs. 5 and 6). It is possible that G␤␥ activates MKK6 through Rho family GEFs (e.g. Abr and Bcr) that have a broad specificity for Rho family small GTPases.
G␣ q -mediated MKK3 and MKK6 activations involved c-Src (Figs. 7 and 8). In addition, G␣ q activated MKK3 and MKK6 pathways via phospholipase C (Fig. 10) and PKC (17). 2 Since Pyk2/CAK␤/RAFTK/CADTK/FAK2 is associated with c-Src and activated upon the stimulation of G q -coupled bradykinin and ␣ 1 -adrenergic receptors (9, 10), Pyk2 might connect G␣ q with c-Src to lead to MKK3 and MKK6 activations. A recent report that Pyk2 increases p38 MAPK activity (46) is consistent with this possibility. On the other hand, it is likely that G␤␥ activates MKK3 and MKK6 through a tyrosine kinase not in the Src family (Figs. 7 and 8). It has been reported that G␤␥ directly activates Btk in the presence of plasma membrane fraction (47). We tried to introduce a Btk construct with G␤␥ into the cells and measure Btk activity. Although G␤␥ failed to activate Btk (data not shown), we could not rule out the involvement of another Tec family tyrosine kinase in the pathway from G␤␥ to MKK3 and MKK6.
Tyrosine kinases appear to act as the upstream regulator not only of Ras but also of Rho family small GTPases (48). For example, the Vav group of Rho family GEF has been shown to be activated by its tyrosine phosphorylation (49 -51). Rho subfamily-specific GEFs Vav-2 and Vav-3 have been reported to be activated directly by Src family tyrosine kinases (50,51). This is in agreement with our result that tyrosine kinases including c-Src function upstream of Rho family small GTPases in the signaling pathway linking G protein subunits to MKK3 and MKK6 (Fig. 9).
The signaling components of MAPK cascade may be organized into a module in vivo (52). It has been reported that JSAP1 provides a scaffold of the JNK signaling module containing only MKK4 as JNK kinase and facilitates JNK activation in mammalian cells (53). In contrast, JIP family scaffold proteins such as JIP1, JIP2, and JIP3 (an alternative splicing form of JSAP1) are specifically associated with another JNK kinase MKK7 and modulate JNK activation (54 -56). In the present study, we demonstrated that G␣ q and G␤␥ differentially regulate MKK3 and MKK6. It is conceivable that such a scaffold protein may contribute to the communication between each G protein subunit and MAPKKs.
p38 MAPK pathway has been implicated in various cellular functions such as gene expressions, cytoskeletal regulations, and morphological changes by stimuli through some G q -coupled receptors (57)(58)(59). In this study, we presented some clues for solving the mechanism whereby G q activates MKK3 and MKK6 and in turn p38 MAPK. Further studies are needed to elucidate how G q stimulates tyrosine kinases and regulates Rho family small GTPases.