Differential Regulation of Mitogen-activated Protein Kinase Kinase 4 (MKK4) and 7 (MKK7) by Signaling from G Protein βγ Subunit in Human Embryonal Kidney 293 Cells

Heterotrimeric G protein βγ subunit (Gβγ) mediates signals to two types of stress-activated protein kinases, c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase, in mammalian cells. To investigate the signaling mechanism whereby Gβγ regulates the activity of JNK, we transfected kinase-deficient mutants of two JNK kinases, mitogen-activated protein kinase kinase 4 (MKK4) and 7 (MKK7), into human embryonal kidney 293 cells. Gβγ-induced JNK activation was blocked by kinase-deficient MKK4 and to a lesser extent by kinase-deficient MKK7. Moreover, Gβγ increased MKK4 activity by 6-fold and MKK7 activity by 2-fold. MKK4 activation by Gβγ was blocked by dominant-negative Rho and Cdc42, whereas MKK7 activation was blocked by dominant-negative Rac. In addition, Gβγ-mediated MKK4 activation, but not MKK7 activation, was inhibited completely by specific tyrosine kinase inhibitors PP2 and PP1. These results indicate that Gβγ induces JNK activation mainly through MKK4 activation dependent on Rho, Cdc42, and tyrosine kinase, and to a lesser extent through MKK7 activation dependent on Rac.

Different G protein-coupled receptors stimulate the ERK pathway through different G protein subunits in various types of cells (11). In the case of the G q/11 -coupled m1 muscarinic acetylcholine and ␣ 1 -adrenergic receptors, the activation of ERK is mediated mainly by G␣ q/11 . G i -coupled m2 muscarinic acetylcholine and ␣ 2 -adrenergic receptors, and G s -coupled ␤-adrenergic receptor induce the ERK activation primarily through G␤␥. Many studies suggest that a signal transduction pathway from G␤␥ to ERK starts at the direct activation of phosphatidylinositol 3-kinase ␥, which increases the activities of Src family tyrosine kinases, in turn leading to tyrosine phosphorylation of Shc (11)(12)(13)(14). Subsequent recruitment of the Grb2-Sos complex to plasma membranes promotes the exchange of GDP to GTP on Ras and activates a sequential kinase cascade that includes Raf, MEK, and ERK (11)(12)(13)(14).
On the other hand, it has been demonstrated that JNK is also activated by an agonist stimulation of m1 and m2 muscarinic acetylcholine receptors expressed in NIH3T3, Rat-1, and COS-7 cells (15)(16)(17). JNK activation by muscarinic acetylcholine receptors has been shown to be mediated primarily by G␤␥ in COS-7 cells (17). However, the mechanism by which G␤␥ induces JNK activation has not been fully understood, although it has been suggested that phosphatidylinositol 3-kinase ␥, Ras and Rac, and STE20-like kinase Pak1 are involved in JNK activation by G␤␥ in COS-7 cells (17,18). Furthermore, overexpression of constitutively activated G␣ q , G␣ 16 , G␣ 12 , and G␣ 13 has been reported to induce the activation of JNK in some cells (19 -25).
During investigations on JNK activation by stimulation of the m1 muscarinic acetylcholine receptor, we found that its activation was mediated by both G␤␥ and G␣ q/11 in human embryonal kidney (HEK) 293 cells. We have reported recently that JNK activation by the m1 muscarinic acetylcholine receptor and G␣ q/11 partially involves the activation of protein kinase C and Src family tyrosine kinases (26). To clarify the signaling mechanism of JNK activation by G␤␥, we investigated whether G␤␥ stimulates the activities of two JNK kinases MKK4 and MKK7. In this paper, we describe that G␤␥ regulates MKK4 and MKK7 differentially through different signaling pathways.

Antibodies and Inhibitors-Mouse monoclonal antibody (B-14)
against Schistosoma japonicum GST was purchased from Santa Cruz Biotechnology, Inc. Mouse monoclonal antibodies M2 and 12CA5 against FLAG epitope and HA epitope were obtained from Eastman Kodak Co. and Boehringer Mannheim, Inc., respectively. Rabbit polyclonal antibodies 06-238 and T-20 against G␤ were obtained from Upstate Biotechnology, Inc. and Santa Cruz Biotechnology, Inc., respectively. Rabbit polyclonal antibodies C-14 and GC/2 against G␣ 11 and G␣ o were purchased from Santa Cruz Biotechnology, Inc. and New England Biolabs, respectively. Goat anti-mouse (NA931) and anti-rabbit (NA934) Ig antibodies conjugated with horseradish peroxidase were from Amersham Pharmacia Biotech. Tyrosine kinase inhibitors (PP2/ AG1879 and PP1/AG1872) were kindly provided by A. Levitzki (Hebrew University). Phosphatidylinositol 3-kinase inhibitors (wortmannin and LY294002) were purchased from Calbiochem-Novabiochem Co.
Construction of Mammalian and Bacterial Expression Plasmids-Complementary DNAs for human MKK4 (27) and human SKK4 (28), a human homolog of mouse MKK7, were amplified from a human fetal brain cDNA library (CLONTECH) by polymerase chain reaction using Ex Taq polymerase (TaKaRa). The DNAs were inserted into the BamHI restriction site of mammalian GST tag expression vector (pCMV-GST) which was generated by J. Suzuki, and into the BglII/BamHI restriction sites of mammalian FLAG tag expression vector (pCMV-FLAG). DNAs encoding kinase-deficient mutants of MKK4 and MKK7, MKK4K95R (27) and MKK7K63R (29), were produced by polymerase chain reactionmediated mutagenesis using Pfu polymerase (Stratagene) and inserted into pCMV-FLAG. The SR␣-HA-JNK1 and SR␣-HA-ERK2 were kindly provided by M. Karin (University of California, San Diego). pCMV-m1 muscarinic acetylcholine receptor was kindly provided by E. M. Ross (University of Texas Southwestern Medical Center). 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, and were subcloned into pCMV as described previously (30,31). pCMV-G␣ 11 Q209L and pCMV-G␣ o were generated as described before (32,33). cDNAs of H-RasS17N was inserted into pCMV as described previously (30). cDNAs of RhoA and Rac1 were kindly provided by K. Kaibuchi (Nara Institute of Science and Technology). pCMV-FLAG-RhoAT19N and pCMV-FLAG-Rac1T17N were constructed and kindly provided by Y. Yamazaki and H. Koide. Cdc42Hs cDNA was kindly provided by R. A. Cerione (Cornell University). pCMV-FLAG-Cdc42HsT17N was constructed and kindly provided by K. Nishida. JNK1 cDNA was amplified by polymerase chain reaction using SR␣-HA-JNK1 as a template and subcloned into the EcoRI restriction site of hexahistidine tag expression vector pET15b (Novagen, Inc.). Expression plasmid pGEX2T-c-Jun (amino acids 1-223) was kindly provided by M. Karin (University of California, San Diego) and digested by BamHI and EcoRI. The DNA fragment of c-Jun-(1-223) was subcloned into the BglII and EcoRI sites of Trx-tag expression vector pET32a (Novagen, Inc.). DNA sequences were confirmed by DNA sequencer (LI-COR 4000L) using thermo sequenase (Amersham Pharmacia Biotech).
MAPKK Assays-After 24 h of serum starvation, the cells transfected together with pCMV-GST-MKK4 or pCMV-GST-MKK7 were treated with or without 10 M carbachol at 37°C for 15 min and lysed in 600 l of lysis buffer A on ice. Aliquots (500 g) of the supernatants after centrifugation were mixed with glutathione-Sepharose 4B for 2 h at 4°C. GST-MKK4 or GST-MKK7 was precipitated by centrifugation and washed with lysis buffer A and reaction buffer A. The precipitates were incubated in 30 l of reaction buffer A containing 2 g of hexahistidine-JNK, 10 g of Trx-c-Jun-(1-223), 20 M ATP, and 5 Ci of [␥-32 P]ATP at 30°C for 20 min. The reaction was stopped by adding 10 l of 4 ϫ Laemmli sample buffer and boiling. Samples were subjected to SDS-polyacrylamide gel electrophoresis, and the radioactivity incorporated into Trx-c-Jun-(1-223) was measured by an imaging analyzer (Fuji BAS 2000) and detected by autoradiography. When the reaction was performed without hexahistidine-JNK, no obvious incorporation of radioactivity to Trx-c-Jun-(1-223) was observed.
Immunoblotting-Aliquots of cell lysates were boiled in Laemmli sample buffer. The boiled samples were separated by SDS-polyacrylamide gel electrophoresis, and the proteins were transferred to nitrocellulose membranes. After the membranes were blocked, the separated proteins were immunoblotted with various antibodies. The bound antibodies were visualized by an enhanced chemiluminescence detection system, using anti-rabbit or mouse Ig antibody conjugated with horseradish peroxidase as a secondary antibody.
Protein Assay-Protein concentrations were determined using Bradford reagent (Nacalai) with bovine serum albumin as the standard.

JNK Activation by the m1 Muscarinic Acetylcholine Receptor Is Mediated by Both G␤␥ and G␣ q/11 in HEK 293 Cells-To
confirm that the m1 muscarinic acetylcholine receptor stimulated JNK activity, we transfected plasmids encoding the receptor and the HA-tagged JNK into HEK 293 cells. Using an anti-HA antibody, HA-JNK was immunoprecipitated from lysates of the transfected cells, and the kinase activity was assayed using recombinant GST-c-Jun (amino acids 1-223) as a specific substrate. Fig. 1A shows the time course of JNK activation after stimulation by carbachol, which is an agonist of the receptor. Mock-transfected cells did not respond to carbachol (data not shown). In each experiment, we examined the expression level of HA-JNK by immunoblotting to monitor the transfection efficiency (see Figs. 1-3). The persistent activation of JNK was observed from 10 min to at least 30 min after the receptor stimulation. JNK activation by the m1 muscarinic acetylcholine receptor was dependent on the concentration of carbachol to maximum response at 10 M and decreased slightly at 100 M (Fig. 1B).
Next, we examined whether JNK activation by the m1 muscarinic acetylcholine receptor is mediated by G␤␥. It has been demonstrated previously that ERK phosphorylation (30) and p38 MAPK activation (33) induced by G protein-coupled receptors and G␤␥ are blocked by cotransfection of G␣ o , and G␣ o forms a complex with G␤␥ in HEK 293 cells (32). It is likely that exogenous G␣ o is able to sequester free endogenous G␤␥ dissociated from G␣ upon stimulation of the receptor, and free exogenous G␤␥ (33). The expression of endogenous G␣ o was below detectable level of immunoblotting in HEK 293 cells (Fig.  1, C-E). As shown in Fig. 1C, activation of JNK by the m1 muscarinic acetylcholine receptor was reduced to approximately 50% by cotransfection of G␣ o , indicating that both G␤␥ and G␣ q/11 may mediate the signal from the m1 muscarinic acetylcholine receptor to JNK. To verify that inhibition of the m1 muscarinic acetylcholine receptor-mediated JNK activation by G␣ o was due to the sequestration of G␤␥, G␣ o was cotransfected with G␤␥ or constitutively activated G␣ 11 (G␣ 11 Q209L) into the cells (Fig. 1, D and E). As reported previously (26,32), transfection of G␤␥ or G␣ 11 Q209L stimulated JNK activity by more than 5-fold in HEK 293 cells. JNK activation by G␤␥, but not by G␣ 11 Q209L, was blocked almost completely by cotransfection of G␣ o . These results indicate that JNK activation by the m1 muscarinic acetylcholine receptor is mediated by both G␤␥ and G␣ q/11 in HEK 293 cells.
Agonist Stimulation of the m1 Muscarinic Acetylcholine Receptor Activates JNK through MKK4 and MKK7-To explore whether two JNK kinases MKK4 and MKK7 are involved in JNK activation by the m1 muscarinic acetylcholine receptor, the cells were transfected with plasmids expressing the receptor and HA-JNK, and MKK4K95R or MKK7K63R. MKK4K95R and MKK7K63R are the kinase-deficient mutants in which a crucial lysine residue of ATP binding site is replaced by an arginine and act as dominant-interfering mutants by sequestering upstream components (27,29). As shown in Fig. 2, A and B, cotransfection of either MKK4K95R or MKK7K63R partially attenuated the m1 muscarinic acetylcholine receptor-mediated JNK activation, indicating that MKK4 and MKK7 are involved in this cascade.
Next, the cells were transfected with plasmids expressing the m1 muscarinic acetylcholine receptor and GST-MKK4 or GST-MKK7, which was tagged with GST at the NH 2 terminus (27,29). Using a glutathione-Sepharose 4B, GST-MKK4 or GST-MKK7 was affinity precipitated from lysates of the transfected cells, and the kinase activity was measured by the reconstitution assay using recombinant hexahistidine-tagged JNK and Trx-tagged c-Jun (amino acids 1-223). In each experiment, we examined the expression level of GST-MKK4 or GST-MKK7 by immunoblotting to monitor the transfection efficiency (see Figs. 2-7). Activation of the m1 muscarinic acetylcholine receptor with carbachol stimulated MKK4 and MKK7 activities (Fig. 2, C and D). These results suggest that the m1 muscarinic acetylcholine receptor activates JNK through at least two JNK kinases, MKK4 and MKK7.
MKK4 Is a Major Mediator for JNK Activation by G␤␥-Because JNK activation by the m1 muscarinic acetylcholine receptor appears to be mediated through MKK4 and MKK7, we investigated whether MKK4 and MKK7 may be involved in G␤␥-mediated JNK activation. As shown in Fig. 3, A and B, JNK activation by G␤␥ was reduced by about 90 and 50% by cotransfection of kinase-deficient MKK4 and MKK7, respectively, raising the possibility that either MKK4 or MKK7 may function as MAPKK in the pathway from G␤␥ to JNK, and MKK4 may play a major role in this cascade.
Next, we investigated whether G␤␥ activates MKK4 and MKK7. G␤␥ stimulated MKK4 activity by more than 5-fold (Fig. 3C). In contrast, G␤␥ activated MKK7 by about 2-fold (Fig. 3D). On the other hand, G␣ 11 Q209L only weakly activated MKK4 and MKK7 (data not shown). Together with data using dominant-interfering mutants, it is suggested that G␤␥ stimulates JNK activity mainly through MKK4 and to a lesser extent through MKK7.
G␤␥ Activates MKK4 and MKK7 in a Ras-independent Manner-Ras is known to be essential for the ERK activation by G␤␥ (11,30). It was reported that G␤␥-mediated JNK activation was blocked by dominant-negative Ras (17). To examine whether Ras is involved in the pathway from G␤␥ to MKK4 and MKK7, we utilized dominant-negative mutant of Ras (RasS17N), which inhibits the activation of endogenous Ras by sequestering guanine nucleotide exchange factors. Cotransfection of RasS17N completely inhibited the ERK activation by G␤␥ (Fig. 4C), whereas RasS17N had no effect on the activations of MKK4 and MKK7 by G␤␥ (Fig. 4, A and B).
MKK4 Activation by G␤␥ Is Dependent on Rho and Cdc42, whereas MKK7 Activation Is Dependent on Rac-It has been reported that Rho family GTPases are also involved in JNK activation upon various stimuli (34). Therefore, we investigated the role of these proteins on the G␤␥-mediated MKK4 and MKK7 activations by cotransfection of RhoT19N, RacT17N, or Cdc42T17N, which act as dominant-negative mutants analogous to RasS17N (35,36). The MKK4 activation by G␤␥ was blocked completely by RhoT19N and Cdc42T17N, but not RacT17N (Fig. 5). On the other hand, the MKK7 activation by G␤␥ was blocked by RacT17N, but not RhoT19N and Cdc42T17N (Fig. 5). These results suggest that G␤␥ regulates MKK4 and MKK7 differentially through different Rho family GTPases. We also examined the effect of dominant-negative mutants of Rho family GTPases on JNK1 activation by G␤␥ .  RhoT19N, Cdc42T17N, and RacT17N reduced JNK activation by 80, 60, and 30%, respectively. Data using dominant-negative mutants of Rho family GTPases also support that G␤␥ stimulates JNK activity mainly through MKK4.
PP2 and PP1, Specific Tyrosine Kinase Inhibitors, Significantly Inhibit G␤␥-induced MKK4 but Not MKK7 Activation-In a previous study (26), we demonstrated that JNK activation by the m1 muscarinic acetylcholine receptor was partially reduced by PP2 and PP1, which are known to be specific tyrosine kinase inhibitors (37). Therefore, we explored a possible involvement of tyrosine kinase in the signaling pathway from G␤␥ to MKK4 and MKK7. The transfected cells were incubated with various concentrations of PP2 or PP1. These inhibitors attenuated the MKK4 activation by G␤␥ in a dose-dependent manner (Fig. 6). The IC 50 value of PP2 and PP1 for the inhibition of G␤␥induced MKK4 activation is approximately 5 and 10 M, respectively. On the other hand, the MKK7 activation by G␤␥ was not inhibited by these inhibitors (Fig. 6). It is likely that G␤␥ regulates MKK4 in a tyrosine kinase-dependent manner and MKK7 in an independent manner.
G␤␥-induced MKK4 and MKK7 Activations Do Not Depend on Phosphatidylinositol 3-Kinase-To test the possibility that phosphatidylinositol 3-kinase is involved in the MKK4 and MKK7 activations by G␤␥, we investigated the effect of wortmannin and LY294002, which are specific inhibitors of phosphatidylinositol 3-kinase. The transfected cells were treated with 100 nM wortmannin or 100 M LY294002 as described previously (14,18). These inhibitors had no effect on the MKK4  RhoT19N (panels A and B), FLAG-RacT17N (panels C and D), and FLAG-Cdc42T17N (panels E and F). The activities of MKK4 and MKK7 were measured as described under "Materials and Methods." Values shown represent the mean Ϯ S.E. from three separate experiments. The phosphorylation of Trx-c-Jun and the expression of G␤ 1 and FLAG-tagged Rho family GTPases 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. and MKK7 activations (Fig. 7). In addition, we also observed that JNK activation by the m1 muscarinic acetylcholine receptor was not inhibited by the treatment of these inhibitors (data not shown). Under the same experimental conditions, the ERK activation by G␤␥ was effectively attenuated by these inhibitors, 2 being consistent with reports that G␤␥ activate ERK pathway via phosphatidylinositol 3-kinase (13,14). Therefore, phosphatidylinositol 3-kinase appears not to be necessary for the JNK pathway mediated by G␤␥ in HEK 293 cells. necrosis factor-␣ and interleukin-1 (27)(28)(29)38). However, the regulation of MKK4 and MKK7 by signaling through G proteincoupled receptors remained to be elucidated. In this paper, we showed that in HEK 293 cells, JNK activation by the m1 muscarinic acetylcholine receptor was mediated by G␤␥ as well as G␣ q/11 (26). We next demonstrated that the receptor-induced JNK activation required at least two JNK kinases, MKK4 and MKK7. Thus, we attempted to determine which signal component, including MKKs, small GTPases, tyrosine kinases, and phosphatidylinositol 3-kinases, is involved in the signaling pathway from G␤␥ to JNK. We found that JNK activation by G␤␥ was mediated mainly by MKK4 and partially by MKK7. Neither MKK4 nor MKK7 activation by G␤␥ was inhibited by dominant-negative Ras. However, G␤␥-induced MKK4 activation was blocked by dominant-negative Rho and Cdc42, whereas G␤␥-induced MKK7 activation was blocked by dominant-negative Rac. Furthermore, the MKK4 but not MKK7 activation by G␤␥ was inhibited by tyrosine kinase inhibitors. Finally, G␤␥-induced activations of MKK4 and MKK7 were independent on phosphatidylinositol 3-kinase activity. These results indicate that G␤␥ regulates MKK4 and MKK7 differentially through different signaling molecules.
Cotransfection of kinase-deficient MKK4 completely inhibited G␤␥-mediated JNK activation, whereas that of kinasedeficient MKK7 partially inhibited JNK activation (Fig. 3). However, we could not rule out the possibility that JNK kinase(s) other than MKK4 and MKK7 might be involved in the pathway. In fact, Moriguchi et al. (38) have reported that the activity of a JNK kinase rather than of MKK4 and MKK7 is detected in unabsorbed fraction of anion-exchange chromatography in the process of fractionating extracts from L5178Y and KB cells exposed to hyperosmolarity. Further studies are necessary for clarifying MAPKK respondent to the signal from G␤␥ in HEK 293 cells.
The number of MAPKKK involved in JNK pathway is currently growing, and the regulation of MAPKKK is very divergent and complicated (3,4). Several groups reported recently that MEKK1, MEKK4, MLK2, and MLK3 specifically associate with Cdc42 and Rac and may mediate JNK activation through Cdc42 and Rac (39 -41). Because Cdc42 and Rac were involved in the activations of MKK4 and MKK7 by G␤␥, respectively (Fig. 5), MEKK1, MEKK4, MLK2, and/or MLK3 might act upstream of MKK4 and MKK7 in these pathways.
We have shown previously that G␤␥ increases the level of the GTP-bound form of Ras in HEK 293 cells (30). Furthermore, it has been reported that oncogenic Ras is a potent activator of the JNK pathway (35,36). We thought that G␤␥ might activate MKK4 and MKK7 through a Ras-dependent pathway. However, neither MKK4 nor MKK7 activation by G␤␥ was blocked by cotransfection of dominant-negative Ras (Fig. 4). Collins et al. (22) reported that constitutively activated G␣ 12 stimulates JNK activity in a Ras-independent manner, although G␣ 12 is able to activate Ras in HEK 293 cells. It appears that Ras is not essential for the JNK pathway mediated by G␣ 12 and G␤␥ in HEK 293 cells.
We found differential involvement of Rho family GTPases in G␤␥-induced MKK4 and MKK7 activation (Fig. 5). Because MKK4 activation by G␤␥ is blocked by both dominant-negative Rho and Cdc42, it is possible that the MKK4 activation is mediated by a guanine nucleotide exchange factor specific for Rho and Cdc42, e.g. Dbl and Ost (34). On the other hand, MKK7 activation by G␤␥ is blocked only by dominant-negative Rac. Thus, G␤␥ may activate MKK7 through a guanine nucleotide exchange factor specific for Rac, e.g. Tiam1 (34).
It is likely that tyrosine kinase is required for the MKK4 but not MKK7 activation by G␤␥ (Fig. 6). We observed that G␤␥ and the m1 muscarinic acetylcholine receptor induced tyrosine phosphorylation of intracellular proteins, and tyrosine-phosphorylated proteins were reduced by the treatment with PP2. 3 Many lines of evidence suggest that Src family tyrosine kinases act downstream of G␤␥ in various cells (11,12). In addition, PP2 and PP1 preferentially inhibit Src family tyrosine kinases, and the IC 50 value of PP2 for the inhibition of Src is 15 M in intact cells. 4 Thus, we considered that Src family tyrosine kinases might contribute to G␤␥-mediated MKK4 activation, and we transfected plasmids encoding a negative regulator of Src family tyrosine kinases (Csk) and kinase-negative Fyn or Lyn into the cells. However, these plasmids did not affect the MKK4 pathway. 5 It was also reported that G␤␥ directly activated Tec family tyrosine kinases Tsk and Btk in the presence of plasma membrane fractions in vitro (10), and Btk regulated JNK activity in vivo (42). This is unlikely to be a general mechanism for JNK regulation by G␤␥ because Tsk and Btk appear to be expressed in very limited tissue distribution. However, another Tec family tyrosine kinase may be involved in the MKK4 activation.
Crespo et al. (43) reported recently that tyrosine phosphorylation of Vav guanine nucleotide exchange factor promoted the exchange of GDP to GTP on Rac1 and to a lesser extent on Cdc42. It is conceivable that G␤␥ may induce the tyrosine phosphorylation of guanine nucleotide exchange factors exerted on Rho family GTPases and may increase the intrinsic exchange activity, leading to the MKK4 activation.
Pretreatment of specific phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 failed to inhibit MKK4 and MKK7 activation by G␤␥ in HEK 293 cells (Fig. 7). Very recently, Lopez-Ilasaca et al. (18) demonstrated that in COS-7 cells, JNK stimulation induced by G␤␥ was effectively suppressed by wortmannin or LY294002 and partially blocked by coexpression of a kinase-deficient mutant of phosphatidylinositol 3-kinase ␥. This discrepancy may be caused by the difference of cell types.
The present study presents some hints for elucidating the mechanism by which G␤␥ induces MKK4 and MKK7 activations. In conclusion, G␤␥ activates JNK through at least two distinct pathways: one pathway is dependent on Rho and Cdc42 and tyrosine kinase, and the other is dependent on Rac. Further studies are needed to prove how G␤␥ differentially regulates the activities of MKK4 and MKK7.