Involvement of Protein Kinase C and Src Family Tyrosine Kinase in Gαq/11-induced Activation of c-Jun N-terminal Kinase and p38 Mitogen-activated Protein Kinase*

Mitogen-activated protein kinases (MAPKs) are activated by various extracellular stimuli. The signaling pathways from G protein-coupled receptors to extracellular signal-regulated kinase have been partially elucidated, whereas the mechanisms by which G protein-coupled receptors stimulate c-Jun N-terminal kinase (JNK) and p38 MAPK activities remain largely unknown. We have recently demonstrated that the signal from Gq/11-coupled m1 muscarinic acetylcholine receptor to p38 MAPK is mediated by both Gαq/11 and Gβγ in HEK-293 cells (Yamauchi, J., Nagao, M., Kaziro, Y., and Itoh, H. (1997) J. Biol. Chem.272, 27771–27777). In the present study, we report that a constitutively activated mutant of Gα11(Gα11 Q209L) activated not only p38 MAPK, but also JNK, and the activation of JNK and p38 MAPK by Gα11 Q209L was partially inhibited by prolonged treatment with phorbol 12-myristate 13-acetate and calphostin C. In addition, the Gα11Q209L-stimulated activation of both kinases was blocked by a specific inhibitor of protein tyrosine kinases (PP2) and Csk (C-terminal Src kinase). Furthermore, we demonstrated that Gα11 Q209L stimulated Src family kinase activity and induced tyrosine phosphorylation of several proteins in HEK-293 cells. These results suggest that Gαq/11 stimulates JNK and p38 MAPK activities through protein kinase C- and Src family kinase-dependent signaling pathways.

Mitogen-activated protein kinases (MAPKs) 1 are important mediators of signal transduction from the cell surface to the nucleus. Regulation by MAPKs has been implicated in many cellular processes such as proliferation, differentiation, and apoptotic death. In mammals, MAPKs are divided into at least three subfamilies: ERK, JNK/stress-activated protein kinase, and p38 MAPK (1). ERK is activated in response to a variety of growth factors, cytokines, and mitogens. The ERK signaling pathways stimulated by receptor tyrosine kinases are well understood (2). JNK/stress-activated protein kinase and p38 MAPK are stimulated by cellular stresses such as heat shock, osmotic shock, and UV irradiation and by inflammatory cytokines such as tumor necrosis factor-␣ and interleukin-1 (3). JNK/stress-activated protein kinase phosphorylates and activates transcription factors that include c-Jun and ATF2 (3). p38 MAPK phosphorylates and activates transcription factors such as ATF2, CHOP, and MEF2C (3)(4)(5). It also activates MAPK-activated protein kinase-2 and -3 (3,6).
It has been reported that the stimulation of G protein-coupled receptors activates ERK (15). Recent studies have demonstrated that G protein-coupled receptor agonists such as carbachol (16 -19), angiotensin II (20), and thrombin (21,22) activate JNK or p38 MAPK in several kinds of cells. In addition, we and others have shown that constitutively activated G␣ q/11 can stimulate JNK (13,23) and p38 MAPK (18) activities. However, the signaling pathways from G␣ q/11 to JNK and p38 MAPK activation remain to be determined.
In this study, we investigated the mechanisms of JNK and p38 MAPK activation by G␣ q/11 using the transient expression of an activated G␣ 11 mutant in HEK-293 cells. Our data indicate that PKC and Src family kinase are involved in the G␣ q/ 11-stimulated activation of JNK and p38 MAPK.
Plasmids-Complementary DNAs coding for mouse G␣ 11 , CSBP1 (a human homologue of p38 MAPK), and the N terminus (amino acids 1-96) of ATF2 were isolated as described previously (18,25). A constitutively activated GTPase-deficient mutant of G␣ 11 (G␣ 11 Q209L) was made by primer-mediated mutagenesis as described previously (25). Wild-type G␣ 11 , G␣ 11 Q209L, and FLAG epitope-tagged p38 MAPK cDNAs were subcloned into mammalian expression vector pCMV5 (26). cDNA for the N terminus of ATF2 was subcloned into Escherichia coli expression vector pGEX-2T. The mammalian expression plasmid of HA-tagged JNK1 (pSR␣-HA-JNK1) and the E. coli expression plasmid of glutathione S-transferase (GST)-c-Jun (amino acids 1-79) (27) were kindly provided by M. Karin (University of California at San Diego, La Jolla, CA). Csk (28) and dominant-negative Ha-Ras(S17N) cDNAs, which were generous gifts from M. Okada (Institute for Protein Research, Osaka University, Osaka, Japan) and G. M. Cooper (Dana-Farber Cancer Institute, Boston, MA), respectively, were subcloned into pCMV5. The mammalian expression plasmid of human m1 muscarinic acetylcholine receptor was generously provided by E. M. Ross (University of Texas Southwestern Medical Center, Dallas, TX). The plasmids of v-Src and TAK1 were kindly provided by Y. Fukami (Kobe University, Kobe, Japan) and K. Matsumoto (Nagoya University, Nagoya, Japan), respectively. The isolated cDNAs and the mutations were confirmed by dideoxynucleotide sequencing.
Transfection-HEK-293 cells on 60-mm dishes were transfected with each plasmid DNA using the calcium phosphate precipitation method. The total amount of plasmid DNA was adjusted to 30 g/60-mm dish with empty vector (pCMV5). The medium was replaced 24 h after transfection, and the cells were starved in the serum-free medium containing 1 mg/ml bovine serum albumin (Sigma) for 24 h and were harvested.
Src family kinase assay was performed as described previously (29) with some modifications. Cells were transfected with pCMV5-G␣ 11 (10 g). The transfected cells were harvested and lysed in 600 l of lysis buffer A after 48 h. The cell lysates were centrifuged at 15,000 rpm for 10 min at 4°C. Aliquots of the supernatants containing 250 g of protein were incubated for 1 h at 4°C with 0.2 g of anti-Src family kinase antibody (SRC2), which recognizes the C-terminal sequence conserved among Src, Yes, and Fyn, and mixed for 2 h at 4°C with protein A-Sepharose CL-4B. The immunoprecipitates were washed twice with lysis buffer A and twice with reaction buffer B (40 mM HEPES-NaOH (pH 7.5), 10 mM MgCl 2 , 3 mM MnCl 2 , 0.5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 g/ml leupeptin, 0.1 mM Na 3 VO 4 , 1 mM NaF, and 2 mM ␤-glycerophosphate). Kinase reactions were carried out in 30 l of reaction buffer B containing 4 g of acid-denatured enolase (Boehringer Mannheim), 10 M ATP, and 10 Ci of [␥-32 P]ATP at 22°C for 5 min. The reactions were stopped by adding 10 l of 4ϫ Laemmli sample buffer. The boiled samples were separated by 10% SDS-PAGE, and the radioactivity incorporated into the enolase was measured by an imaging analyzer (Fuji BAS2000) and detected by autoradiography.
Immunoprecipitation and Immunoblot Analysis-The transfected cells were lysed in 600 l of lysis buffer A, and the cell lysates were centrifuged at 15,000 rpm for 10 min at 4°C. The supernatants containing 350 g of protein were incubated for 1 h at 4°C with mouse monoclonal anti-phosphotyrosine antibody (PY20) (10 g) and mixed for 2 h at 4°C with protein A-Sepharose CL-4B preabsorbed with rabbit anti-mouse Ig antibody (20 g). The immunoprecipitates were washed twice with lysis buffer A and twice with reaction buffer A. The precipitated proteins were boiled in Laemmli sample buffer. The boiled samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked using 1% bovine serum albumin in phosphate-buffered saline containing 0.1% Tween 20 for PY20 or using blocking solution (50 mM Tris-HCl (pH 8.0), 2 mM CaCl 2 , 80 mM NaCl, 0.2% Nonidet P-40, 0.02% NaN 3 , and 5% nonfat dried milk) for other specific antibodies. Immunoreactive bands were visualized by using secondary horseradish peroxidase-conjugated antibodies and chemiluminescence (NEN Life Science Products). 11 Activates Not Only p38 MAPK, but Also JNK-We previously reported that a constitutively activated mutant of G␣ 11 (G␣ 11 Q209L) stimulates p38 MAPK activity in HEK-293 cells (18). In this study, we examined whether G␣ 11 Q209L can also stimulate JNK activity in the same cells. The cells were transfected with wild-type G␣ 11 or G␣ 11 Q209L together with HA epitope-tagged JNK1. HA-JNK1 was immunoprecipitated from the transfected cell lysate with anti-HA antibody, and the JNK1 activity was assessed by phosphorylation of GST-c-Jun (amino acids 1-79). Wild-type G␣ 11 did not significantly enhance the JNK1 activity, whereas G␣ 11 Q209L increased the JNK1 activity by 7-8fold in HEK-293 cells (Fig. 1A). Using FLAG epitope-tagged p38 MAPK and GST-ATF2, an ϳ4-fold stimulation of p38 MAPK by G␣ 11 Q209L was also observed (Fig. 1B). Immunoblot analysis indicated that HA-JNK1 and FLAG-p38 MAPK were expressed at similar levels in the transfected cells.

Constitutively Activated Mutant of G␣
Activation of JNK and p38 MAPK by G␣ 11 Q209L Is PKCdependent-We next examined the signaling pathways from G␣ q/11 to JNK and p38 MAPK activation in HEK-293 cells. It is known that G␣ q/11 activates phospholipase C␤ to hydrolyze phosphatidylinositol 4,5-bisphosphate and thereby generates inositol 1,4,5-trisphosphate and diacylglycerol. The inositol 1,4,5-trisphosphate released into the cytoplasm mobilizes Ca 2ϩ from internal stores, whereas diacylglycerol activates some PKC isoforms (10). We therefore investigated whether PKC participates in the JNK1 and p38 MAPK activation by G␣ 11 Q209L. In HEK-293 cells, PMA caused an ϳ2-fold increase in JNK1 and p38 MAPK activities (Fig. 2A). The PMA-stimulated JNK1 and p38 MAPK activation was prevented by a 24-h pretreatment with PMA. In contrast, the prolonged PMA treatment did not diminish the anisomycin-induced activation of both kinases (data not shown). The cells transfected with G␣ 11 Q209L were treated with PMA for 24 h, and the effect of PKC inactivation on JNK1 and p38 MAPK activation was examined. The G␣ 11 Q209L-stimulated activation of JNK1 and p38 MAPK was partially inhibited by the prolonged PMA treatment (Fig.  2B). In addition, a PKC inhibitor (calphostin C) also blocked the JNK1 and p38 MAPK activation in a dose-dependent man-ner (Fig. 2C). These results indicate that PKC may be involved in part in the activation of JNK1 and p38 MAPK by G␣ 11 Q209L. The expression of G␣ 11 , HA-JNK1, or FLAG-p38 MAPK was not affected by treatment with PMA or calphostin C. Carbachol increased JNK1 and p38 MAPK activities by 3-fold in HEK-293 cells expressing G q/11 -coupled m1 muscarinic acetylcholine receptor, and calphostin C also reduced the activation by 50%.
To explore the role of Ca 2ϩ in JNK and p38 MAPK activation in HEK-293 cells, the cells were stimulated with A23187 or thapsigargin, which increases the intracellular Ca 2ϩ concentration. Activation of JNK1 and p38 MAPK by A23187 or thapsigargin was observed to a small extent (2-fold) (data not shown).
Activation of JNK and p38 MAPK by G␣ 11 Q209L Is Src Family Kinase-dependent-Recently, it has been shown that Src family kinase and Ras contribute to ERK1/2 activation by G q/11 -coupled ␣ 1B -adrenergic receptor in HEK-293 cells (30). To determine whether protein tyrosine kinase and Ras are implicated in the G␣ q/11 -mediated signaling pathways leading to JNK1 and p38 MAPK activation, we examined the effects of a protein tyrosine kinase inhibitor (PP2) (24), Csk, and dominant-negative Ras(S17N) on the G␣ 11 Q209L-stimulated JNK1 and p38 MAPK activation. The activation of both kinases by G␣ 11 Q209L was markedly attenuated by PP2 in a dose-dependent manner (Fig. 3, A and B). Similar inhibitory effects of another tyrosine kinase inhibitor (PP1) (24) and PP2 were observed in the JNK1 and p38 MAPK activation by m1 muscarinic acetylcholine receptor (Fig. 3, C and D). Western blots analysis indicated that the PP1 and PP2 treatment did not affect the expression of G␣ 11 , HA-JNK1, and FLAG-p38 MAPK.
Csk is a cytoplasmic protein tyrosine kinase that inactivates Src family kinases (28). The overexpression of Csk significantly blocked the G␣ 11 Q209L-stimulated JNK1 and p38 MAPK activation (Fig. 3E). Furthermore, the expression of v-Src effectively stimulated the JNK1 and p38 MAPK activities in HEK-293 cells (Fig. 3F). In contrast, Ras(S17N) failed to inhibit the G␣ 11 Q209L-induced JNK1 and p38 MAPK activation in the cells (Fig. 4). These data suggest that Src family kinases, but not Ras, are involved in the JNK1 and p38 MAPK activation by G␣ q/11 . Therefore, we tested whether G␣ 11 stimulates Src family kinase activity in HEK-293 cells.

G␣ 11 Q209L Stimulates Src Family Tyrosine Kinase Activity and Tyrosine Phosphorylation of Several Proteins-The cells
were transfected with wild-type G␣ 11 or G␣ 11 Q209L, and endogenous Src family kinases were immunoprecipitated with anti-Src family kinase antibody (SRC2), which recognizes the C-terminal sequence conserved among Src, Yes, and Fyn. Src family kinase activity was assessed by phosphorylation of aciddenatured enolase. As shown in Fig. 5, G␣ 11 Q209L stimulated an ϳ2-fold increase in Src family kinase activity over the basal level.
Next, we investigated whether the tyrosine phosphorylation of the intracellular proteins was induced by the expression of the activated mutant of G␣ 11 in HEK-293 cells. Lysates of the cells transfected with wild-type G␣ 11 or G␣ 11 Q209L were immunoprecipitated with anti-phosphotyrosine antibody (PY20). The immunoprecipitated proteins were separated by SDS-PAGE and subjected to immunoblot analysis with PY20. The expression of G␣ 11 Q209L promoted the tyrosine phosphorylation of several prominent bands with apparent molecular masses of 100, 105, and 120 -130 kDa (Fig. 6A). A similar pattern of protein tyrosine phosphorylation was observed when G q/11 -coupled m1 muscarinic receptor-expressed cells were stimulated by carbachol (data not shown). Some of the protein tyrosine phosphorylation enhanced by G␣ 11 Q209L were reduced by treatment with PP2 in a dose-dependent manner (Fig.  6B). The tyrosine phosphorylation of a 100-kDa protein was completely abolished by 10 M PP2, and that of 120 -130-kDa proteins was strongly inhibited by 50 M PP2. In contrast, PP2 had a very weak effect on the tyrosine phosphorylation of a 105-kDa protein. DISCUSSION We previously reported that both G␣ 11 Q209L and G␤ 1 ␥ 2 stimulate p38 MAPK activity and that the p38 MAPK activation by m1 muscarinic acetylcholine receptor is mediated by both G␣ q/11 and G␤␥ in HEK-293 cells (18). In addition, we showed that G␤␥ effectively stimulates JNK1 activity in the  11 (␣11WT) or G␣ 11 Q209L (␣11Q209L). JNK1 or p38 MAPK activity was measured by immune complex kinase assay using GST-c-Jun or GST-ATF2 as a substrate. The data are expressed as -fold stimulation in which the basal activity is defined as 1.0. Values represent the mean Ϯ S.E. from at least three independent experiments. A representative autoradiogram of GST-c-Jun or GST-ATF2 phosphorylation is shown. Cell lysates were resolved by SDS-PAGE, and G␣ 11 , HA-JNK1, and FLAG-p38 MAPK were detected by immunoblotting using anti-G␣ 11 , anti-HA, and anti-FLAG antibodies, respectively. cells (31). In the present study, we further found that G␣ 11 Q209L as well as G␤␥ can activate not only p38 MAPK, but also JNK1 in HEK-293 cells (Fig. 1). Although two reports (13,23) are consistent with our observation that G␣ 11 Q209L stimulates the JNK1 activity, other groups (32,33) have reported that a constitutively activated mutant of G␣ q fails to stimulate the JNK1 activity in COS-7 and HEK-293 cells. The discrepancy may be due to the difference of the expression level of constitutively activated G␣ q or the experimental conditions. It has been recently reported that Btk (Bruton's tyrosine kinase) is necessary for p38 MAPK activation by m1 muscarinic acetylcholine receptor in DT40 cells (19). However, the signal transduction pathways linking G␣ q/11 with JNK and p38 MAPK activation remain largely unknown. As shown in Fig. 2, G␣ 11 Q209L-stimulated JNK1 and p38 MAPK activation was partially inhibited by prolonged PMA treatment and calphostin C. These results suggest that a phorbol ester-sensitive PKC may be involved in part in the G␣ q/11 -induced JNK1 and p38 MAPK activation. It has been shown that PKC can activate MEKK1 in vivo (34). MEKK1 was originally identified as a FIG. 2. Effects of prolonged PMA treatment and calphostin C on G␣ 11 Q209L-stimulated JNK1 and p38 MAPK activation. A, cells were transfected with the plasmid of HA-JNK1 or FLAG-p38 MAPK. The transfected cells were pretreated with (ϩ) or without (Ϫ) 1.6 M PMA for 24 h. After stimulation with 160 nM PMA for 60 min (for JNK1 activation) or 30 min (for p38 MAPK activation), each kinase activity was measured by immune complex kinase assay using GST-c-Jun or GST-ATF2. The data are presented as -fold stimulation in which the basal activity is defined as 1.0. Values are the mean Ϯ S.E. from at least three separate experiments. B and C, cells were transfected with the plasmid of HA-JNK1 or FLAG-p38 MAPK together with the plasmid of G␣ 11 Q209L (␣11Q209L). The transfected cells were treated with (ϩ) or without (Ϫ) 1.6 M PMA for 24 h in B and with the indicated concentrations of calphostin C for 3 h in C. JNK1 or p38 MAPK activity was measured as described above. The data are expressed as the percent of G␣ 11 Q209L-stimulated JNK1 or p38 MAPK activation in the cells not treated with PMA or calphostin C. Values represent the mean Ϯ S.E. from at least three separate experiments. The expression level of G␣ 11 Q209L, HA-JNK1, or FLAG-p38 MAPK shown in A-C was determined by immunoblotting using each specific antibody.

FIG. 3. Effects of tyrosine kinase inhibitors and v-Src on JNK1 and p38 MAPK activities.
A and B, cells were transfected with the plasmid of HA-JNK1 or FLAG-p38 MAPK together with the plasmid of G␣ 11 Q209L (␣11Q209L). The transfected cells were treated with the indicated concentrations of PP2 for 24 h. JNK1 or p38 MAPK activity was measured by immune complex kinase assay using GST-c-Jun or GST-ATF2. The data are expressed as the percent of G␣ 11 Q209L-stimulated JNK1 or p38 MAPK activation in the absence of PP2. C and D, cells were transfected with the plasmid of HA-JNK1 or FLAG-p38 MAPK together with the plasmid of m1 muscarinic acetylcholine receptor (m1 MAPK kinase kinase that has the ability to activate ERK1/2 (35), but recent findings indicated that MEKK1 functions as an activator of the JNK pathway (36,37). Although MEKK1 is not directly phosphorylated by PKC, MEKK1 activity is stimulated by PMA treatment in COS cells (34). Therefore, G␣ q/11 may regulate the MEKK1-JNK pathway through a PKC-dependent mechanism. In addition to MEKK1, other MAPK kinase kinases such as MEKK2-4 (38,39), TAK1 (40), and ASK1 (41) have been shown to be involved in JNK or p38 MAPK activation. G␣ q/11 may also stimulate these MAPK kinase kinases activity through PKC-dependent and -independent signaling pathways and thereby induce JNK and p38 MAPK activation.
It has been reported that PP1 and PP2 are selective inhibitors for Src family kinases (24). We found that the activation of JNK1 and p38 MAPK by G␣ 11 Q209L was inhibited by PP2 (Fig. 3, A and B). In HEK-293 cells, overexpression of TAK1 markedly stimulated JNK1 and p38 MAPK activities, and the activation by TAK1 was not inhibited by 50 M PP2 (data not shown). This demonstrates that PP2 does not inhibit JNK1 and p38 MAPK themselves or their MAPK kinases. In addition, the overexpression of Csk attenuated the G␣ 11 Q209L-stimulated JNK1 and p38 MAPK activation (Fig. 3E). Furthermore, we demonstrated that G␣ 11 Q209L can stimulate Src family ki-nase activity (Fig. 5). These results suggest that Src family kinases are implicated in G␣ q/11 -induced JNK and p38 MAPK signaling pathways. Since it has been reported that phorbol esters activate Src family kinases in Swiss 3T3 cells (42), G␣ q/11 may partly stimulate Src family kinase activity through a phorbol ester-sensitive PKC.
We found the G␣ 11 Q209L-dependent tyrosine phosphorylation of the 100-, 105-, and 120 -130-kDa proteins in HEK-293 cells (Fig. 6A). In addition, these proteins were tyrosine-phosphorylated upon the stimulation of m1 muscarinic acetylcholine receptor-expressed cells (data not shown). Since G␣ 11 stim-mAChR). The transfected cells were pretreated with the indicated concentrations of PP1 or PP2 for 30 min before stimulation with 10 M carbachol for 10 min. The data are expressed as the percent of carbachol-stimulated JNK1 or p38 MAPK activation in the absence of PP1 or PP2. E, cells were transfected with the plasmid of HA-JNK1 or FLAG-p38 MAPK together with the plasmids of G␣ 11 Q209L and Csk. The data are expressed as the percent of G␣ 11 Q209L-stimulated JNK1 or p38 MAPK activation in the absence of Csk. F, cells were transfected with the plasmid of HA-JNK1 or FLAG-p38 MAPK together with an empty expression vector (Mock) or the v-Src plasmid. The data are presented as -fold stimulation in which the basal activity is defined as 1.0. All values represent the mean Ϯ S.E. from at least three separate experiments. Representative autoradiograms of GST-c-Jun and GST-ATF2 phosphorylation are shown. The expression level of G␣ 11 Q209L, HA-JNK1, FLAG-p38 MAPK, Csk, or v-Src shown in A-F was determined by immunoblotting using each specific antibody.
FIG. 4. Effect of dominant-negative Ras(S17N) on G␣ 11 Q209Lstimulated JNK1 and p38 MAPK activation. Cells were transfected with the plasmid of HA-JNK1 or FLAG-p38 MAPK together with the plasmids of G␣ 11 Q209L (␣11Q209L) and Ras(S17N). JNK1 or p38 MAPK activity was measured by immune complex kinase assay. The data are expressed as the percent of G␣ 11 Q209L-stimulated JNK1 or p38 MAPK activation in the absence of Ras(S17N). Values represent the mean Ϯ S.E. from at least three separate experiments. The expression level of G␣ 11 Q209L, HA-JNK1, FLAG-p38 MAPK, or Ras(S17N) was determined by immunoblotting using each specific antibody.
FIG. 5. G␣ 11 Q209L stimulates Src family kinase activity in HEK-293 cells. Cells were transfected with an empty expression vector (Mock) or the plasmid of wild-type G␣ 11 (␣11WT) or G␣ 11 Q209L (␣11Q209L). Endogenous Src family kinases were immunoprecipitated from the transfected cell lysates with anti-Src family kinase antibody (SRC2), and Src family kinase activity was assessed by phosphorylation of acid-denatured enolase. The data are presented as -fold stimulation with respect to mock-transfected cells. Values are the mean Ϯ S.E. from at least three separate experiments. A representative autoradiogram of enolase phosphorylation is shown. Cell lysates were resolved by SDS-PAGE, and G␣ 11 and Src family kinases were detected by immunoblotting using anti-G␣ 11 and anti-Src family kinase antibodies, respectively. ulated Src family kinase activity and the Src family kinase inhibitor attenuated the tyrosine phosphorylation, it is likely that these proteins are Src family kinase substrates. Many Src family kinase substrates have been reported, and they are involved not only in mitogenic signaling pathways, but also in cytoskeletal organization, cell-matrix interaction, cell-cell interaction, and cell-cell communication (50). Therefore, we expect that G␣ q/11 may regulate the focal adhesion proteins and the cytoskeletal proteins as well as JNK and p38 MAPK through Src family kinases.
In summary, we have demonstrated that G␣ q/11 can stimulate both JNK and p38 MAPK activities and that PKC and Src family kinase contribute to the activation by G␣ q/11 in HEK-293 cells. We previously reported that G␤␥, as well as G␣ q/11 , stimulates JNK and p38 MAPK activities (18,31). The signaling pathways from G␤␥ to JNK and p38 MAPK and the regulatory mechanism of Src family kinase activation by G␣ q/11 remain to be clarified. FIG. 6. G␣ 11 Q209L-induced protein tyrosine phosphorylation and its inhibition by PP2. A, HEK-293 cells were transfected with an empty expression vector (Mock) or the plasmid of wild-type G␣ 11 (␣11WT) or G␣ 11 Q209L (␣11Q209L). The transfected cell lysates were immunoprecipitated with anti-phosphotyrosine antibody (PY20). The immunoprecipitated proteins were separated by 7.5% SDS-PAGE and immunoblotted with PY20. B, cells were transfected with the plasmid of G␣ 11 Q209L and treated with the indicated concentrations of PP2 for 24 h. Immunoprecipitation and immunoblotting with PY20 were performed as described above. Molecular mass markers are shown on the left. Arrows indicate the tyrosine phosphorylation induced by G␣ 11 Q209L. The results shown in A and B are representative of three independent experiments.