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

Junji Yamauchi, Yoshito Kaziro, and Hiroshi ItohDagger

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

    ABSTRACT
Top
Abstract
Introduction
References

Heterotrimeric G protein beta gamma subunit (Gbeta gamma ) 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 Gbeta gamma 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. Gbeta gamma -induced JNK activation was blocked by kinase-deficient MKK4 and to a lesser extent by kinase-deficient MKK7. Moreover, Gbeta gamma increased MKK4 activity by 6-fold and MKK7 activity by 2-fold. MKK4 activation by Gbeta gamma was blocked by dominant-negative Rho and Cdc42, whereas MKK7 activation was blocked by dominant-negative Rac. In addition, Gbeta gamma -mediated MKK4 activation, but not MKK7 activation, was inhibited completely by specific tyrosine kinase inhibitors PP2 and PP1. These results indicate that Gbeta gamma 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.

    INTRODUCTION
Top
Abstract
Introduction
References

Mitogen-activated protein kinases (MAPKs)1 are proline-directed serine/threonine kinases and play important functions as mediators of cellular responses to a variety of stimuli such as growth factors, cytokines, hormones, and environmental stresses (1-4). In mammalian cells, MAPKs have been classified into at least four subfamilies: ERK/MAPK, JNK/SAPK, p38 MAPK, and BMK1/ERK5. ERK is activated by many growth factors and cytokines and phosphorylates transcription factors, translational factors, cytoskeleton proteins, and other protein kinases (1, 2). ERK is implicated in cell growth, differentiation, and survival. Various stressors such as chemical agents and ultraviolet irradiation, tumor necrosis factor-alpha , interleukin-1, CD40 ligand, and Fas/CD95 ligand stimulate the activities of JNK and p38 MAPK, which appear to be involved in cell cycle arrest and apoptosis (2-4). JNK phosphorylates transcription factors including c-Jun, Elk-1, and ATF2, whereas p38 MAPK phosphorylates not only transcription factors such as CHOP/GADD153, MEF2C, Elk-1, and ATF2, but also MAPKAP kinases (2-4). BMK1 has been reported to be a redox-regulated kinase and phosphorylates a transcription factor MEF2C, although its physiological role has remained unclear (3).

MAPK cascades consist of three conserved components: a family of serine/threonine kinases called MAPKKK/MEKK, which activate MAPKK/MEK, which in turn activates MAPK by a simultaneous phosphorylation on threonine and tyrosine residues (1-4). Raf and Tpl-2 activate ERK through MEK1 and MEK2. On the other hand, it has been shown that overexpression of Tpl-2, MEKK1, MEKK2, MEKK3, MEKK4/MTK1, MAPKKK5/ASK1, TAK1, and MLK family protein kinases induce the activation of JNK and/or p38 MAPK cascade(s). JNK and p38 MAPK are phosphorylated and activated by MKK4/SEK1/JNKK1/SKK1 (2-4). More recently, MKK7/JNKK2/SKK4 has been cloned and shown to phosphorylate and activate only JNK (4). In contrast, MKK3/SKK2 and MKK6/SKK3 have been known to phosphorylate and activate specifically p38 MAPK (2-4). MKK5/MEK5 has been reported to phosphorylate BMK1 (3).

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are composed of alpha  and beta gamma subunits (Galpha and Gbeta gamma ) and transduce signals from G protein-coupled receptors to intracellular effectors (5-7). A GDP-bound Galpha forms a complex with Gbeta gamma . Upon ligand stimulation, the receptor stimulates the GDP-GTP exchange of Galpha , which leads dissociation of Galpha ·GTP from Gbeta gamma . Both the GTP-bound Galpha and the free Gbeta gamma regulate downstream effectors including adenylyl cyclases, phospholipase Cbeta isozymes, ion channels, phosphatidylinositol 3-kinase gamma  (8, 9), and Tec family tyrosine kinases (10).

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 Gq/11-coupled m1 muscarinic acetylcholine and alpha 1-adrenergic receptors, the activation of ERK is mediated mainly by Galpha q/11. Gi-coupled m2 muscarinic acetylcholine and alpha 2-adrenergic receptors, and Gs-coupled beta -adrenergic receptor induce the ERK activation primarily through Gbeta gamma . Many studies suggest that a signal transduction pathway from Gbeta gamma to ERK starts at the direct activation of phosphatidylinositol 3-kinase gamma , which increases the activities of Src family tyrosine kinases, in turn leading to tyrosine phosphorylation of Shc (11-14). 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 (11-14).

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 (15-17). JNK activation by muscarinic acetylcholine receptors has been shown to be mediated primarily by Gbeta gamma in COS-7 cells (17). However, the mechanism by which Gbeta gamma induces JNK activation has not been fully understood, although it has been suggested that phosphatidylinositol 3-kinase gamma , Ras and Rac, and STE20-like kinase Pak1 are involved in JNK activation by Gbeta gamma in COS-7 cells (17, 18). Furthermore, overexpression of constitutively activated Galpha q, Galpha 16, Galpha 12, and Galpha 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 Gbeta gamma and Galpha q/11 in human embryonal kidney (HEK) 293 cells. We have reported recently that JNK activation by the m1 muscarinic acetylcholine receptor and Galpha 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 Gbeta gamma , we investigated whether Gbeta gamma stimulates the activities of two JNK kinases MKK4 and MKK7. In this paper, we describe that Gbeta gamma regulates MKK4 and MKK7 differentially through different signaling pathways.

    MATERIALS AND METHODS

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 Gbeta were obtained from Upstate Biotechnology, Inc. and Santa Cruz Biotechnology, Inc., respectively. Rabbit polyclonal antibodies C-14 and GC/2 against Galpha 11 and Galpha 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 reaction-mediated mutagenesis using Pfu polymerase (Stratagene) and inserted into pCMV-FLAG. The SRalpha -HA-JNK1 and SRalpha -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 Gbeta 1 and Ggamma 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-Galpha 11Q209L and pCMV-Galpha 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 SRalpha -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).

Cell Culture and Transfection-- HEK 293 cells (ATCC CRL 1573) were maintained in Dulbecco's modified Eagle's medium (Sigma) containing 100 µg/ml kanamycin (Nacalai) with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.). The cells were cultured at 37 °C in humidified atmosphere containing 10% CO2. Plasmid DNAs were transfected into HEK 293 cells by the calcium phosphate precipitation method. The final amount of transfected DNA for a 60-mm dish was adjusted to 30 µg by empty vector, pCMV. Five µg of SRalpha -HA-JNK, SRalpha -HA-ERK, pCMV-GST-MKK4, or pCMV-GST-MKK7 was transfected with 0.3 µg of pCMV-m1 muscarinic acetylcholine receptor, 5 µg of pCMV-Gbeta 1, 5 µg of pCMV-Ggamma 2, 10 µg of pCMV-Galpha 11Q209L, 10 µg of pCMV-Galpha o, 10 µg of pCMV-FLAG-MKK4K95R, 10 µg of pCMV-FLAG-MKK7K63R, 15 µg of pCMV-RasS17N, 15 µg of pCMV-FLAG-RhoT19N, 15 µg of pCMV-FLAG-RacT17N, or 15 µg of pCMV-FLAG-Cdc42T17N. The cells were starved with serum-free medium containing 1 mg/ml bovine serum albumin (Nacalai) for 24 h post-transfection.

Purification of Recombinant Proteins from Escherichia coli-- E. coli strain BL21 (DE3) cells were transformed with pET15b-JNK, pET32a-c-Jun-(1-223), or pGEX2T-c-Jun-(1-223). An isolated colony was inoculated in 10 ml of LB medium containing 100 µg/ml carbenicillin and shaken at 30 °C overnight. This culture was diluted in 500 ml of medium and grown at 30 °C until A600 reached 0.2 and added to 0.4 mM isopropyl-1-thio-beta -D-galactopyranoside and further incubated for 3 h. The cells were harvested by centrifugation, washed with phosphate-buffered saline, and stored at -80 °C until use. The frozen cells were suspended and sonicated briefly in 10 ml of extraction buffer A (20 mM HEPES-NaOH (pH 8.0), 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 0.5% Nonidet P-40) for hexahistidine-JNK and Trx-c-Jun-(1-223) or extraction buffer B (20 mM HEPES-NaOH (pH 7.5), 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM EGTA, 0.5% Nonidet P-40) for GST-c-Jun-(1-223) on ice, and shaken for 20 min at 4 °C. Cell debris was removed by centrifugation at 150,000 × g for 30 min at 4 °C.

All purification steps were performed at 4 °C. For the purification of hexahistidine-JNK or Trx-c-Jun-(1-223), the supernatants were applied to NTA-agarose (Qiagen, Inc.) and washed with column buffer A (20 mM HEPES-NaOH (pH 8.0), 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin) containing 20 mM imidazole. Hexahistidine-JNK and Trx-c-Jun-(1-223) were eluted with column buffer A containing 200 mM imidazole. Hexahistidine-JNK was diluted to the concentration of 0.3 mg of protein/ml with column buffer A containing 200 mM imidazole before dialysis. For the purification of GST-c-Jun-(1-223), the supernatant was applied to glutathione-Sepharose 4B (Amersham Pharmacia Biotech) and washed with column buffer B (20 mM HEPES-NaOH (pH 7.5), 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM EGTA), and eluted with column buffer B containing 10 mM glutathione. The eluate was dialyzed against column buffer B and stored at -80 °C until use.

MAPK Assays-- After 24 h of serum starvation, the cells transfected together with SRalpha -HA-JNK or SRalpha -HA-ERK were treated with or without carbachol at 37 °C and lysed in 600 µl of lysis buffer A (20 mM HEPES-NaOH (pH 7.5), 3 mM MgCl2, 100 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml leupeptin, 1 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 20 mM beta -glycerophosphate, and 0.5% Nonidet P-40) on ice. The lysates were centrifuged at 14,000 rpm for 10 min at 4 °C. Aliquots (500 µg) of the supernatants were mixed with protein A-Sepharose CL-4B preabsorbed with a mouse anti-HA antibody for 1 h at 4 °C. The immune complexes were precipitated and washed twice with lysis buffer A and twice with reaction buffer A (20 mM HEPES-NaOH (pH 7.5), 10 mM MgCl2, 0.1 mM phenylmethanesulfonyl fluoride, 0.1 µg/ml leupeptin, 0.1 mM EGTA, 0.1 mM Na3VO4, and 0.2 mM beta -glycerophosphate). The precipitates were incubated in 30 µl of reaction buffer A containing 3 µg of GST-c-Jun-(1-223) for JNK assay or 7.5 µg of myelin basic protein (Sigma) for ERK assay, 20 µM ATP, and 5 µCi of [gamma -32P]ATP (NEN Life Science Products) at 30 °C for 10 min. The reaction was stopped by adding 10 µl of 4 × Laemmli sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 30 mM dithiothreitol, and 10% glycerol). The boiled samples were subjected to SDS-polyacrylamide gel electrophoresis, and the radioactivity incorporated into GST-c-Jun-(1-223) or myelin basic protein was measured by an imaging analyzer (Fuji BAS 2000), and detected by autoradiography.

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 µM carbachol at 37 °C for 15 min and lysed in 600 µl of lysis buffer A on ice. Aliquots (500 µg) 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 µl of reaction buffer A containing 2 µg of hexahistidine-JNK, 10 µg of Trx-c-Jun-(1-223), 20 µM ATP, and 5 µCi of [gamma -32P]ATP at 30 °C for 20 min. The reaction was stopped by adding 10 µl of 4 × 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.

    RESULTS

JNK Activation by the m1 Muscarinic Acetylcholine Receptor Is Mediated by Both Gbeta gamma and Galpha 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 µM and decreased slightly at 100 µM (Fig. 1B).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1.   JNK activation by stimulation of the m1 muscarinic acetylcholine receptor is mediated by Gbeta gamma and Galpha q/11. HEK 293 cells were transfected with plasmids carrying cDNAs for HA-JNK (pan els A-E), the m1 muscarinic acetylcholine receptor (panels A-C), Galpha o (alpha o, panels C-E), Gbeta 1 (beta 1, panel D), Ggamma 2 (gamma 2, panel D), and Galpha 11Q209L (alpha 11Q209L, panel E). JNK activity was measured as described under "Materials and Methods." JNK activity is shown at different time points after the addition of 10 µM carbachol (panel A), 15 min after the addition of increasing concentrations of carbachol (panel B), and 15 min after the addition of 10 µM carbachol (panel C). Values shown represent the mean ± S.E. from three separate experiments. The phosphorylation of GST-c-Jun and the expression of HA-JNK and G protein subunits in the cell lysates are shown.

Next, we examined whether JNK activation by the m1 muscarinic acetylcholine receptor is mediated by Gbeta gamma . It has been demonstrated previously that ERK phosphorylation (30) and p38 MAPK activation (33) induced by G protein-coupled receptors and Gbeta gamma are blocked by cotransfection of Galpha o, and Galpha o forms a complex with Gbeta gamma in HEK 293 cells (32). It is likely that exogenous Galpha o is able to sequester free endogenous Gbeta gamma dissociated from Galpha upon stimulation of the receptor, and free exogenous Gbeta gamma (33). The expression of endogenous Galpha 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 Galpha o, indicating that both Gbeta gamma and Galpha 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 Galpha o was due to the sequestration of Gbeta gamma , Galpha o was cotransfected with Gbeta gamma or constitutively activated Galpha 11 (Galpha 11Q209L) into the cells (Fig. 1, D and E). As reported previously (26, 32), transfection of Gbeta gamma or Galpha 11Q209L stimulated JNK activity by more than 5-fold in HEK 293 cells. JNK activation by Gbeta gamma , but not by Galpha 11Q209L, was blocked almost completely by cotransfection of Galpha o. These results indicate that JNK activation by the m1 muscarinic acetylcholine receptor is mediated by both Gbeta gamma and Galpha 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.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 2.   JNK activation by stimulation of the m1 muscarinic acetylcholine receptor is mediated by MKK4 and MKK7. The cells were transfected with plasmids carrying cDNAs for HA-JNK (panels A and B), GST-MKK4 (panel C), GST-MKK7 (panel D), m1 muscarinic acetylcholine receptor (panels A-D), FLAG-MKK4K95R (panel A), and FLAG-MKK7K63R (panel B). The activities of JNK, MKK4, and MKK7 were measured at 15 min after the addition of 10 µM carbachol as described under "Materials and Methods." Values shown represent the mean ± S.E. from three or four separate experiments. The phosphorylation of GST-c-Jun and Trx-c-Jun and the expression of HA-JNK, FLAG-MKK4K95R, and FLAG-MKK7K63R in the cell lysates are shown. GST-MKK4 and GST-MKK7 were precipitated with glutathione-Sepharose 4B from the cell lysates and immunoblotted with anti-GST antibody.

Next, 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 NH2 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 Gbeta gamma -- 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 Gbeta gamma -mediated JNK activation. As shown in Fig. 3, A and B, JNK activation by Gbeta gamma 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 Gbeta gamma to JNK, and MKK4 may play a major role in this cascade.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   MKK4 is a major mediator of Gbeta gamma -induced JNK activation. The cells were transfected with plasmids carrying cDNAs for HA-JNK (panels A and B), GST-MKK4 (panel C), GST-MKK7 (panel D), Gbeta 1 (beta 1, panels A-D), Ggamma 2 (gamma 2, panels A-D), FLAG-MKK4K95R (panel A), and FLAG-MKK7K63R (panel B). The activities of JNK, MKK4, and MKK7 were measured as described under "Materials and Methods." Values shown represent the mean ± S.E. from three or four separate experiments. The phosphorylation of GST-c-Jun and Trx-c-Jun and the expression of HA-JNK, FLAG-MKK4K95R, FLAG-MKK7K63R, and Gbeta 1 in the cell lysates are shown. GST-MKK4 and GST-MKK7 were precipitated with glutathione-Sepharose 4B from the cell lysates and immunoblotted with anti-GST antibody.

Next, we investigated whether Gbeta gamma activates MKK4 and MKK7. Gbeta gamma stimulated MKK4 activity by more than 5-fold (Fig. 3C). In contrast, Gbeta gamma activated MKK7 by about 2-fold (Fig. 3D). On the other hand, Galpha 11Q209L only weakly activated MKK4 and MKK7 (data not shown). Together with data using dominant-interfering mutants, it is suggested that Gbeta gamma stimulates JNK activity mainly through MKK4 and to a lesser extent through MKK7.

Gbeta gamma Activates MKK4 and MKK7 in a Ras-independent Manner-- Ras is known to be essential for the ERK activation by Gbeta gamma (11, 30). It was reported that Gbeta gamma -mediated JNK activation was blocked by dominant-negative Ras (17). To examine whether Ras is involved in the pathway from Gbeta gamma 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 Gbeta gamma (Fig. 4C), whereas RasS17N had no effect on the activations of MKK4 and MKK7 by Gbeta gamma (Fig. 4, A and B).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 4.   Gbeta gamma activates MKK4 and MKK7 in a Ras-independent manner. The cells were transfected with plasmids carrying cDNAs for GST-MKK4 (panel A), GST-MKK7 (panel B), HA-ERK (panel C), Gbeta 1 (beta 1, panels A-C), Ggamma 2 (gamma 2, panels A-C), and RasS17N (panels A-C). The activities of MKK4, MKK7, and ERK were measured as described under "Materials and Methods." Values shown represent the mean ± S.E. from three separate experiments. The phosphorylation of Trx-c-Jun and myelin basic protein (MBP) and the expression of HA-ERK, Gbeta 1, and Ras 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.

MKK4 Activation by Gbeta gamma 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 Gbeta gamma -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 Gbeta gamma was blocked completely by RhoT19N and Cdc42T17N, but not RacT17N (Fig. 5). On the other hand, the MKK7 activation by Gbeta gamma was blocked by RacT17N, but not RhoT19N and Cdc42T17N (Fig. 5). These results suggest that Gbeta gamma 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 Gbeta gamma . 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 Gbeta gamma stimulates JNK activity mainly through MKK4.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 5.   MKK4 activation by Gbeta gamma is dependent on Rho and Cdc42, whereas MKK7 activation by Gbeta gamma is dependent on Rac. The cells were transfected with plasmids carrying cDNAs for GST-MKK4 (panels A, C, and E), GST-MKK7 (panels B, D, and F), Gbeta 1 (beta 1, panels A-F), Ggamma 2 (gamma 2, panels A-F), FLAG-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 ± S.E. from three separate experiments. The phosphorylation of Trx-c-Jun and the expression of Gbeta 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.

PP2 and PP1, Specific Tyrosine Kinase Inhibitors, Significantly Inhibit Gbeta gamma -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 Gbeta gamma to MKK4 and MKK7. The transfected cells were incubated with various concentrations of PP2 or PP1. These inhibitors attenuated the MKK4 activation by Gbeta gamma in a dose-dependent manner (Fig. 6). The IC50 value of PP2 and PP1 for the inhibition of Gbeta gamma -induced MKK4 activation is approximately 5 and 10 µM, respectively. On the other hand, the MKK7 activation by Gbeta gamma was not inhibited by these inhibitors (Fig. 6). It is likely that Gbeta gamma regulates MKK4 in a tyrosine kinase-dependent manner and MKK7 in an independent manner.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 6.   Activation of MKK4 but not MKK7 by Gbeta gamma is dependent on tyrosine kinase. The cells were transfected with plasmids carrying cDNAs for GST-MKK4 (panels A and C), GST-MKK7 (panels B and D), Gbeta 1 (beta 1, panels A-D), and Ggamma 2 (gamma 2, panels A-D) and treated with or without increasing concentrations of PP2 (panels A and B) and PP1 (panels C and D) for 18 h after 24 h post-transfection. The activities of MKK4 and MKK7 were measured as described under "Materials and Methods." Values shown represent the mean ± S.E. from three separate experiments. The phosphorylation of Trx-c-Jun and the expression of Gbeta 1 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.

Gbeta gamma -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 Gbeta gamma , 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 µM LY294002 as described previously (14, 18). These inhibitors had no effect on the MKK4 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 Gbeta gamma was effectively attenuated by these inhibitors,2 being consistent with reports that Gbeta gamma activate ERK pathway via phosphatidylinositol 3-kinase (13, 14). Therefore, phosphatidylinositol 3-kinase appears not to be necessary for the JNK pathway mediated by Gbeta gamma in HEK 293 cells.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 7.   MKK4 and MKK7 activation by Gbeta gamma does not depend on phosphatidylinositol 3-kinase. The cells were transfected with plasmids carrying cDNAs for GST-MKK4 (panels A and C), GST-MKK7 (panels B and D), Gbeta 1 (beta 1, panels A-D), and Ggamma 2 (gamma 2, panels A-D) and treated with or without 100 nM wortmannin (panels A and B) and 100 µM LY294002 (panels C and D) for 20 min after 48 h post-transfection. The activities of MKK4 and MKK7 were measured as described under "Materials and Methods." Values shown represent the mean ± S.E. from three separate experiments. The phosphorylation of Trx-c-Jun and the expression of Gbeta 1 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.


    DISCUSSION

MKK4 and MKK7 have been shown to respond to environmental stresses and inflammatory cytokines such as tumor necrosis factor-alpha and interleukin-1 (27-29, 38). However, the regulation of MKK4 and MKK7 by signaling through G protein-coupled 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 Gbeta gamma as well as Galpha 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 Gbeta gamma to JNK. We found that JNK activation by Gbeta gamma was mediated mainly by MKK4 and partially by MKK7. Neither MKK4 nor MKK7 activation by Gbeta gamma was inhibited by dominant-negative Ras. However, Gbeta gamma -induced MKK4 activation was blocked by dominant-negative Rho and Cdc42, whereas Gbeta gamma -induced MKK7 activation was blocked by dominant-negative Rac. Furthermore, the MKK4 but not MKK7 activation by Gbeta gamma was inhibited by tyrosine kinase inhibitors. Finally, Gbeta gamma -induced activations of MKK4 and MKK7 were independent on phosphatidylinositol 3-kinase activity. These results indicate that Gbeta gamma regulates MKK4 and MKK7 differentially through different signaling molecules.

Cotransfection of kinase-deficient MKK4 completely inhibited Gbeta gamma -mediated JNK activation, whereas that of kinase-deficient 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 Gbeta gamma 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 Gbeta gamma , respectively (Fig. 5), MEKK1, MEKK4, MLK2, and/or MLK3 might act upstream of MKK4 and MKK7 in these pathways.

We have shown previously that Gbeta gamma 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 Gbeta gamma might activate MKK4 and MKK7 through a Ras-dependent pathway. However, neither MKK4 nor MKK7 activation by Gbeta gamma was blocked by cotransfection of dominant-negative Ras (Fig. 4). Collins et al. (22) reported that constitutively activated Galpha 12 stimulates JNK activity in a Ras-independent manner, although Galpha 12 is able to activate Ras in HEK 293 cells. It appears that Ras is not essential for the JNK pathway mediated by Galpha 12 and Gbeta gamma in HEK 293 cells.

We found differential involvement of Rho family GTPases in Gbeta gamma -induced MKK4 and MKK7 activation (Fig. 5). Because MKK4 activation by Gbeta gamma 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 Gbeta gamma is blocked only by dominant-negative Rac. Thus, Gbeta gamma 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 Gbeta gamma (Fig. 6). We observed that Gbeta gamma 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 Gbeta gamma in various cells (11, 12). In addition, PP2 and PP1 preferentially inhibit Src family tyrosine kinases, and the IC50 value of PP2 for the inhibition of Src is 15 µM in intact cells.4Thus, we considered that Src family tyrosine kinases might contribute to Gbeta gamma -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 Gbeta gamma 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 Gbeta gamma 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 Gbeta gamma 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 Gbeta gamma in HEK 293 cells (Fig. 7). Very recently, Lopez-Ilasaca et al. (18) demonstrated that in COS-7 cells, JNK stimulation induced by Gbeta gamma was effectively suppressed by wortmannin or LY294002 and partially blocked by coexpression of a kinase-deficient mutant of phosphatidylinositol 3-kinase gamma . This discrepancy may be caused by the difference of cell types.

The present study presents some hints for elucidating the mechanism by which Gbeta gamma induces MKK4 and MKK7 activations. In conclusion, Gbeta gamma 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 Gbeta gamma differentially regulates the activities of MKK4 and MKK7.

    ACKNOWLEDGEMENTS

We thank Drs. E. M. Ross, M. I. Simon, T. Nukada, M. Karin, R. A. Cerione, and K. Kaibuchi for supplying the plasmids. We are grateful to Dr. A. Levitzki for providing PP2 and PP1. We also thank Dr. K. Tago for advice regarding recombinant protein expression and purification, and Dr. H. Koide, K. Nishida, J. Kato, M. Nagao, J. Suzuki, and Y. Yamazaki for helpful discussions and plasmid construction.

    FOOTNOTES

* This work was supported in part by grants from Core Research for Evolutional Science and Technology and from the Ministry of Education, Science, Sports, and Culture. Our laboratory at the Tokyo Institute of Technology is supported by funding from the Schering-Plogh Corporation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

The abbreviations used are: MAPK(s), mitogen-activated protein kinase(s); ERK, extracellular signal-regulated kinase; JNK/SAPK, c-Jun NH2-terminal kinase/stress-activated protein kinase; MAPKK/MEK, MAPK kinase/MAPK or ERK kinase; MAPKKK/MEKK, MAPKK kinase/MEK kinase; MKK, MAPK kinase; MLK, mixed lineage kinase; SKK1, SAPK kinase; G protein, heterotrimeric guanine nucleotide-binding regulatory protein; Galpha , G protein alpha  subunit; Gbeta gamma , G protein beta gamma subunit; HEK, human embryonal kidney; GST, glutathione S-transferase; HA, hemagglutinin; CMV, cytomegalovirus; Trx, thioredoxin.

2 A. Ito, Y. Kaziro, and H. Itoh, unpublished results.

3 M. Nagao, Y. Kaziro, and H. Itoh, unpublished results.

4 A. Levitzki, personal communication.

5 J. Yamauchi, Y. Kaziro, and H. Itoh, unpublished results.

    REFERENCES
Top
Abstract
Introduction
References

  1. Nishida, E., and Gotoh, Y. (1993) Trends Biochem. Sci. 18, 128-131[CrossRef][Medline] [Order article via Infotrieve]
  2. Cobb, M. H., and Goldsmith, E. J. (1995) J. Biol. Chem. 270, 14843-14846[Free Full Text]
  3. Kyriakis, J. M., and Avruch, J. (1996) BioEssays 18, 567-577[Medline] [Order article via Infotrieve]
  4. Ip, Y. T., and Davis, R. (1998) Curr. Opin. Cell Biol. 10, 205-219[CrossRef][Medline] [Order article via Infotrieve]
  5. Gilman, A. G. (1987) Annu. Rev. Biochem. 56, 615-649[CrossRef][Medline] [Order article via Infotrieve]
  6. Kaziro, Y., Itoh, H., Kozasa, T., Nakafuku, M., and Satoh, T. (1991) Annu. Rev. Biochem. 60, 349-400[CrossRef][Medline] [Order article via Infotrieve]
  7. Neer, E. J. (1995) Cell 80, 249-257[Medline] [Order article via Infotrieve]
  8. Stoyanov, B., Volinia, S., Hanck, T., Rubio, I., Loubtchenkov, M., Malek, D., Stoyanova, S., Vanhaesebroeck, B., Dhand, R., Nürnberg, B., Gierschik, P., Seedorf, K., Hsuan, J. J., Waterfield, M. D., and Wetzker, R. (1995) Science 269, 690-693[Medline] [Order article via Infotrieve]
  9. Stephens, L. R., Eguinoa, A., Erdjument-Bromage, H., Lui, M., Cooke, F., Coadwell, J., Smrcka, A. S., Thelen, M., Cadwallader, K., Tempst, P., and Hawkins, P. T. (1997) Cell 89, 105-114[Medline] [Order article via Infotrieve]
  10. Lahghans-Rajasekaran, S. A., Wan, Y., and Huang, X.-Y. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8601-8605[Abstract]
  11. Gutkind, J. S. (1998) J. Biol. Chem. 273, 1839-1842[Free Full Text]
  12. Luttrell, L. M., Hawes, B. E., van Biesen, T., Luttrel, D. K., Lansing, T. J., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 19443-19450[Abstract/Free Full Text]
  13. Hawes, B. E., Luttrell, L. M., van Biesen, T., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 12133-12136[Abstract/Free Full Text]
  14. Lopez-Ilasaca, M., Crespo, M., Pellici, P., Gutkind, J. S., and Wetzker, R. (1997) Science 275, 394-397[Abstract/Free Full Text]
  15. Coso, O. A., Chiariello, M., Kalinec, G., Kyriakis, J. M., Woodgett, J. P., and Gutkind, J. S. (1995) J. Biol. Chem. 270, 5620-5624[Abstract/Free Full Text]
  16. Mitchell, F. M., Russell, M., and Johnson, G. L. (1995) Biochem. J. 309, 381-384[Medline] [Order article via Infotrieve]
  17. Coso, O. A., Teramoto, H., Simonds, W. F., and Gutkind, J. S. (1996) J. Biol. Chem. 271, 3963-3966[Abstract/Free Full Text]
  18. Lopez-Ilasaca, M., Gutkind, J. S., and Wetzker, R. (1998) J. Biol. Chem. 273, 2505-2508[Abstract/Free Full Text]
  19. 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[Abstract]
  20. Higasita, R., Li, L., Van Putten, V., Yamamura, Y., Zarinetchi, F., Heasley, L., and Nemenoff, R. A. (1997) J. Biol. Chem. 272, 25845-25850[Abstract/Free Full Text]
  21. 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[Abstract/Free Full Text]
  22. Collins, L. R., Minden, A., Karin, M., and Brown, J. H. (1996) J. Biol. Chem. 271, 17349-17353[Abstract/Free Full Text]
  23. Voyno-Yasenetskaya, T. A., Faure, M. P., Ahn, N. G., and Bourne, H. R. (1996) J. Biol. Chem. 271, 21081-21087[Abstract/Free Full Text]
  24. Mitsui, H., Takuwa, N., Kurokawa, K., Exton, J. H., and Takuwa, Y. (1997) J. Biol. Chem. 272, 4904-4910[Abstract/Free Full Text]
  25. Jho, E.-H., Davis, R. J., and Malbon, C. C. (1997) J. Biol. Chem. 272, 24468-24474[Abstract/Free Full Text]
  26. Nagao, M., Yamauchi, J., Kaziro, Y., and Itoh, H. (1998) J. Biol. Chem. 273, 22892-22898[Abstract/Free Full Text]
  27. Sánchez, I., Hughes, R. T., Mayer, B. J., Yee, K., Woodgett, J. R., Avruch, J., Kyriakis, J. M., and Zon, L. I. (1994) Nature 372, 794-798[Medline] [Order article via Infotrieve]
  28. Lawler, S., Cuenda, A., Goedert, M., and Cohen, P. (1997) FEBS Lett. 414, 153-158[CrossRef][Medline] [Order article via Infotrieve]
  29. Yao, Z., Diener, K., Wang, X. S., Zukowski, M., Matsumoto, G., Zhou, G., Mo, R., Sasaki, T., Nishina, H., Hui, C. C., Tan, T.-H., Woodgett, J. P., and Penninger, J. M. (1997) J. Biol. Chem. 272, 32378-32383[Abstract/Free Full Text]
  30. Ito, A., Satoh, T., Kaziro, Y., and Itoh, H. (1995) FEBS Lett. 368, 183-187[CrossRef][Medline] [Order article via Infotrieve]
  31. Yamauchi, J., Kaziro, Y., and Itoh, H. (1995) Biochem. Biophys. Res. Commun. 214, 694-700[CrossRef][Medline] [Order article via Infotrieve]
  32. Yamauchi, J., Kaziro, Y., and Itoh, H. (1997) J. Biol. Chem. 272, 7602-7607[Abstract/Free Full Text]
  33. Yamauchi, J., Nagao, M., Kaziro, Y., and Itoh, H. (1997) J. Biol. Chem. 272, 27771-27777[Abstract/Free Full Text]
  34. Van Aelst, L., and D'Souza-Schorey, C. (1997) Genes Dev. 11, 2295-2322[Free Full Text]
  35. Coso, O. A., Chiariello, M., Yu, J.-C., Teramoto, H., Crespo, P., Xu, N., Miki, T., and Gutkind, J. S. (1995) Cell 81, 1137-1146[Medline] [Order article via Infotrieve]
  36. Minden, A., Lin, A., Claret, F.-X., Abo, A., and Karin, M. (1995) Cell 81, 1147-1157[Medline] [Order article via Infotrieve]
  37. Hanke, J. H., Gardner, J. P., Dow, R. L., Changelian, P. S., Brissette, W. H., Weringer, E. J., Pollok, B. A., and Connelly, P. A. (1996) J. Biol. Chem. 271, 695-701[Abstract/Free Full Text]
  38. Moriguchi, T., Toyoshima, F., Masuyama, N., Hanafusa, H., Gotoh, Y., and Nishida, E. (1997) EMBO J. 16, 7045-7053[Abstract/Free Full Text]
  39. Fanger, G. R., Johnson, N. L., and Johnson, G. L. (1997) EMBO J. 16, 4961-4972[Abstract/Free Full Text]
  40. Nagata, K., Puls, A., Futter, C., Aspenstorm, P., Schaefer, E., Nakata, T., Hirokawa, N., and Hall, A. (1998) EMBO J. 17, 149-158[Abstract/Free Full Text]
  41. Teramoto, H., Coso, O. A., Miyata, H., Igishi, T., Miki, T., and Gutkind, J. S. (1996) J. Biol. Chem. 271, 27225-27228[Abstract/Free Full Text]
  42. Kawakami, Y., Miura, T., Bissonnette, R., Hata, D., Khan, W. N., Kitamura, T., Maeda-Yamamoto, M., Hartman, S. E., Yao, L., Alt, F. W., and Kawakami, T. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3938-3942[Abstract/Free Full Text]
  43. Crespo, P., Schuebel, K. E., Ostrom, A. A., Gutkind, J. S., and Bustelo, X. R. (1997) Nature 385, 169-172[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.