C-terminal Mutation of G Protein beta  Subunit Affects Differentially Extracellular Signal-regulated Kinase and c-Jun N-terminal Kinase Pathways in Human Embryonal Kidney 293 Cells*

(Received for publication, July 19, 1996, and in revised form, November 13, 1996)

Junji Yamauchi , Yoshito Kaziro and Hiroshi Itoh

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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

G protein beta  and gamma  subunits (Gbeta and Ggamma ) form a complex that is involved in various signaling pathways. We reported that the C-terminal 10 amino acids of Gbeta are required for association with Ggamma (Yamauchi, J., Kaziro, Y., and Itoh, H. (1995) Biochem. Biophys. Res. Commun., 214, 694-700). To evaluate further the significance of the C-terminal region of Gbeta in the formation of a Gbeta gamma complex and its signal transduction, we constructed several C-terminal mutants and expressed them in human embryonal kidney 293 cells. The mutant lacking the C-terminal 2 amino acids (Delta C2) failed to associate with Ggamma , whereas deletion of the C-terminal amino acid (Delta C1), replacement of Trp at -2 position by Ala (W339A), and addition of six histidines ((His)6) at the C terminus did not affect the association with Ggamma . We also studied the effect of these mutations on the activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). Co-expression of the Delta C2 or (His)6 mutant with Ggamma did not activate MAPK/ERK at all, whereas the Delta C1 or W339A mutant showed the MAPK/ERK activation. The JNK/SAPK activity was stimulated by the W339A, Delta C2, or (His)6 mutant, but not by the Delta C1 mutant. These results suggest that the C-terminal region of Gbeta participates differentially in the signaling for MAPK/ERK and JNK/SAPK activations in mammalian cells.


INTRODUCTION

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins)1 mediate signals from a variety of cell surface receptors to effector molecules (1-5). G proteins are composed of alpha , beta , and gamma  subunits. Binding of ligands to G protein-coupled receptors stimulates the dissociation of Galpha and Gbeta gamma , which regulate, independently or cooperatively, a variety of effector molecules.

Normally, Gbeta and Ggamma associate tightly and function as a complex. Gbeta contains seven WD repeating units, each of which consists of approximately 40 amino acids and ends mainly with Trp-Asp (WD) (6). Recently, Wall et al. (7) and Lambright et al. (8) reported the x-ray crystallographic structure of heterotrimer of Galpha i1beta 1gamma 2 and Galpha i/tbeta 1gamma 1, respectively, and the structure of Gbeta 1gamma 1 was analyzed by Sondek et al. (9). These reports have revealed that the structure of Gbeta gamma in the trimeric complex is not very much different from that in the dimeric complex. The WD repeat provides a rigid scaffold of beta -propeller structure, which is composed of seven beta -propeller blades containing four antiparallel beta -sheets. The N terminus of Ggamma forms an alpha -helical coiled coil structure with the N terminus of Gbeta , and the remainder of Ggamma stretches along the beta -propeller blades, forming multiple interaction sites with Gbeta . On the other hand, the N-terminal alpha -helix of Galpha binds with a beta -propeller blade of Gbeta , and a region designated switch II of Galpha fits into the top of the beta -propeller.

In mammalian cells, Gbeta gamma has been shown to modulate the activities of adenylyl cyclases (10), phospholipase Cbeta isozymes (11, 12), phosphatidylinositol 3-kinase gamma  (13), inward rectifier potassium channels (14, 15), and N-type and P/Q-type calcium channels (16, 17). Furthermore, it has been reported that Gbeta gamma binds directly with the C-terminal region of the pleckstrin homology domain to regulate beta -adrenergic receptor kinases (18, 19) and Tec family protein-tyrosine kinases (20, 21). More recently, Gbeta gamma was shown to stimulate the activities of the mitogen-activated protein kinase subfamilies MAPK/ERK (22-25) and JNK/SAPK (26). These studies have established a critical role of Gbeta gamma in the intracellular signal transduction, although little is known about the region involved in the effector regulation.

In the present study, we demonstrated that a few amino acids at the C terminus of Gbeta are involved in the complex formation with Ggamma . Furthermore, we found that mutations in C-terminal amino acids influence differentially the stimulation of MAPK/ERK and JNK/SAPK activities.


MATERIALS AND METHODS

Antibodies

Rabbit polyclonal anti-Galpha o antibody was produced against amino acids 94-108 of Galpha o and purified by a peptide affinity column. Rabbit polyclonal antibody (06-238) that recognizes a peptide spanning amino acids 127-139 identical in Gbeta 1 and Gbeta 2 was purchased from Upstate Biotechnology, Inc. Mouse monoclonal antibodies (M2 and 12CA5) against FLAG epitope (8 amino acids, EYKEEEEK) and HA epitope (9 amino acids, YPYDVPDYA) were from Eastman Kodak Co. and Boehringer Mannheim, respectively. Rabbit anti-mouse Ig antibody (55480) was from Cappel.

cDNA Construction

Rat Galpha o-1 cDNA (27, 28) was inserted into mammalian expression vector pCMV5 (29). cDNAs of bovine Gbeta 1 (30) and bovine Ggamma 2 (31, 32) were generously provided by M. I. Simon (California Institute of Technology) and T. Nukada (Tokyo Institute of Psychiatry), respectively. Wild type Gbeta 1 and Ggamma 2 were subcloned into pCMV5 as described before (25). To construct mutants of Gbeta , the coding region of Gbeta 1 was amplified by PCR using 5' sense primer containing an EcoRI recognition site and 3' antisense primer containing a HindIII site. PCR was carried out for 30 cycles, each at 94 °C for 1 min, 50 °C for 2 min, and 72 °C for 3 min, using Pfu polymerase (Stratagene). The following oligonucleotides were used as PCR primers: primer 1, 5'-CCGGAATTC<UNL>ATG</UNL>AGTGAACTTGACCAGTTA-3'; primer 2, 5'-TTAAAAGCTTGCGGCCG<UNL>CTA</UNL>GCTGTCCCAGGATCCCGT-3'; primer 3, 5'-CGCCAAGCT<UNL>TCA</UNL>GATTTTGAGGAAGCT-3'; primer 4, 5'-CACCAAGCTTTGCGCA<UNL>TCA</UNL>CCAGATTTTGAGGAAGCT-3'; and primer 5, 5'-CCCAAGCTTCTCGAG<UNL>TTA</UNL>GTTAGCGATTTTGAGGAAGCTGTCCCA-3' (start and stop codons are underlined). Primer 1 was used to construct all Gbeta mutants as a 5' sense primer. Primers 2, 3, 4, and 5 were used for the Delta C6, Delta C2, Delta C1, and W339A mutants, respectively. PCR products were double-digested by EcoRI and HindIII and inserted into pCMV5. cDNAs for Gbeta 1 tagged with six histidines ((His)6) at the C terminus, the N-terminal 38-amino acid deletion mutant (Delta N38), and Ggamma 2 tagged with FLAG epitope at the N terminus (FLAG-Ggamma ) were prepared and inserted into pCMV5 as described previously (33). The DNA sequence of amplified inserts was confirmed by the dideoxy method and the chemiluminescence detection system (New England Biolabs). The pLNCX-RafFH6 plasmid, which expresses c-Raf-1 with FLAG epitope and six histidines at the C terminus, was a gift from M. McMahon (DNAX Research Institute) (25). Escherichia coli expression plasmids of six-histidine-tagged MEK and kinase-negative GST-fused MAPK were kindly provided by E. Nishida (Kyoto University) (34, 35). The SRalpha -HA-ERK2 and SRalpha -HA-JNK1 plasmids, which express ERK2 and JNK1, respectively, with HA epitope at the N terminus, and the E. coli expression plasmid of GST-c-Jun (amino acids 1-79) were kindly provided by M. Karin (University of California, San Diego).

Cell Culture and Transfection

Human embryonal kidney (HEK) 293 cells were maintained in Dulbecco's modified Eagle's medium (Sigma) containing 1 µg/ml kanamycin (Sigma) 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 technique. The final amount of transfected DNA per 60-mm dish was adjusted to 30 µg by adding empty vector pCMV5. The medium was replaced 24 h after transfection, and the cells were harvested at 48 h posttransfection.

Cell Lysis and Immunoprecipitation

To analyze the association of Gbeta mutants with FLAG-Ggamma , cells were transfected with 10 µg of Gbeta and 10 µg of Ggamma DNAs. The transfected cells were washed twice with phosphate-buffered saline and suspended in lysis buffer A (20 mM HEPES-NaOH (pH 7.5), 5 mM MgCl2, 150 mM NaCl, 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml leupeptin, 1 mM EDTA, and 1% Lubrol-PX) in a total volume of 600 µl and incubated for 10 min on ice. To analyze the interaction of Gbeta gamma complex with Galpha o, 10 µg of Gbeta , 10 µg of Ggamma , and 10 µg of Galpha o DNAs were transfected. The transfected cells were lysed in lysis buffer A containing 100 µM GDP. The cell lysates were centrifuged at 14,000 rpm for 10 min at 4 °C in a microcentrifuge. The supernatants were incubated at 4 °C for 1 h with mouse anti-FLAG antibody (1 µg) and mixed gently at 4 °C for 1 h with protein A-Sepharose CL-4B (Pharmacia Biotech Inc.), which was preabsorbed with rabbit anti-mouse Ig antibody (1 µg). The immune complexes were precipitated by centrifugation and washed four times with lysis buffer A.

SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting

Samples were boiled in Laemmli sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 30 mM dithiothreitol, and 10% glycerol). The boiled samples were separated by SDS-polyacrylamide gel electrophoresis, and the proteins were transferred to BA81 nitrocellulose membranes (Schleicher & Schuell). After blocking the membranes, the separated proteins were immunoblotted with rabbit anti-common Gbeta or Galpha o antibody. The bound antibodies were visualized by the enhanced chemiluminescence detection system using anti-Ig antibody conjugated with horseradish peroxidase as a secondary antibody (Amersham Life Science Inc.).

Guanine Nucleotide Exchange of Immunoprecipitated Galpha beta gamma

The cells were transfected with cDNAs of Galpha o, Gbeta mutants, and FLAG-Ggamma and lysed in buffer A. The cell lysates were used for immunoprecipitation with mouse anti-FLAG antibody as described above, and the complexes were incubated in buffer A containing 500 µM GDP or GTPgamma S at 30 °C for 2 h. The reaction was stopped by chilling on ice. The complexes were washed with buffer A, boiled in Laemmli sample buffer, and subjected to immunoblot analysis using rabbit anti-Galpha o or anti-common Gbeta antibody.

Kinase Assays

The MEK kinase activity of Raf was measured as described previously (25). The cells were transfected with 10 µg of pLNCX-RafFH6, 10 µg of Gbeta DNA, and 10 µg of Ggamma DNA. The transfected cells were starved in the serum-free medium containing 1 mg/ml bovine serum albumin for 24 h. The cells were lysed in 600 µl of lysis buffer B (20 mM HEPES-NaOH (pH 7.5), 3 mM MgCl2, 100 mM NaCl, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 20 mM beta -glycerophosphate, and 0.5% Nonidet P-40). After centrifugation at 14,000 rpm for 10 min in a microcentrifuge, the epitope-tagged c-Raf-1 was immunoprecipitated from aliquots of the supernatants with mouse anti-FLAG antibody (1 µg) and protein A-Sepharose CL-4B preabsorbed with rabbit anti-mouse Ig antibody (1 µg). The immunoprecipitates were washed twice with lysis buffer B and twice with reaction buffer A (20 mM HEPES-NaOH (pH 7.5), 5 mM MgCl2, 0.5 mM MnCl2, 0.2 µg/ml aprotinin, 0.1 µg/ml leupeptin, and 0.1 mM EGTA). The precipitates were incubated at 30 °C for 20 min in reaction buffer A with 3.3 µg of recombinant MEK, 6.6 µg of recombinant kinase-negative MAPK, 200 µM ATP, and 5 µCi of [gamma -32P]ATP. Recombinant (His)6 MEK and kinase-negative GST-fused MAPK were produced in E. coli and purified as described before (34, 35). The reaction was stopped by adding 4 × Laemmli sample buffer and heating at 95 °C for 5 min. The boiled samples were separated by SDS-polyacrylamide gel electrophoresis, and the incorporation of radioactive phosphate into the MAPK was measured by an imaging analyzer (Fuji BAS 2000).

The activities of MAPK/ERK and JNK/SAPK were measured as described before (36). Cells were transfected with 5 µg of SRalpha -HA-ERK2 or 5 µg of SRalpha -HA-JNK1 together with 10 µg of Gbeta and 10 µg of Ggamma DNAs or 5 µg of Gbeta and 5 µg of Ggamma DNAs, respectively. The transfected cells were lysed in lysis buffer C (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). Aliquots of the supernatants were mixed with mouse anti-HA antibody (1 µg). HA-ERK2 or HA-JNK1 was precipitated with protein A-Sepharose CL-4B and washed twice with lysis buffer C and twice with reaction buffer B (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, 1 mM NaF, and 2 mM beta -glycerophosphate). The washed immunoprecipitates were incubated in reaction buffer B with 7.5 µg of myelin basic protein (Sigma) for ERK2 assay or 1.5 µg of affinity-purified GST-c-Jun (amino acids 1-79) (36) for JNK1 assay, 20 µM ATP, and 5 µCi of [gamma -32P]ATP at 30 °C for 20 min, and the reaction was stopped by adding 4 × Laemmli sample buffer. The boiled samples were subjected to SDS-polyacrylamide gel electrophoresis, and the radioactivity incorporated into myelin basic protein or GST-c-Jun was measured by an imaging analyzer (Fuji BAS 2000).


RESULTS

In a previous study, we found that the C-terminal region of Gbeta is involved in the complex formation with Ggamma (33). To identify which amino acid residue(s) within the last 10 amino acids in the C-terminal region is required for the association with Ggamma , we constructed several C-terminal deletion mutants that lack the last six (Delta C6), two (Delta C2), and one (Delta C1) amino acid(s) (Fig. 1). Since Ggamma tagged with FLAG epitope at the N terminus can be co-immunoprecipitated with Gbeta , FLAG-Ggamma was utilized to analyze the complex formation with the C-terminal mutants of Gbeta (33). The Gbeta mutants and FLAG-Ggamma were expressed at a similar level in HEK 293 cells (Fig. 2B). Fig. 2A shows that the Delta C6 and Delta C2 mutants failed to associate with FLAG-Ggamma , whereas the Delta C1 mutant could form a Gbeta gamma complex. Next, we constructed a mutant (W339A) in which the second amino acid (Trp) from the C terminus was replaced by Ala (Fig. 1). The W339A mutant could form a complex with FLAG-Ggamma (Fig. 2A), suggesting that the presence of an amino acid residue at the -2 position of Gbeta appeared to be important for the complex formation with Ggamma . The addition of six histidines ((His)6) to the C terminus of Gbeta was not inhibitory to the interaction of Gbeta with Ggamma (Fig. 2A).


Fig. 1. The C-terminal amino acid sequences of wild type and mutants of Gbeta . The cDNAs encoding Gbeta mutants were constructed by PCR and placed under the control of the pCMV5 promoter as described under "Materials and Methods."
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Fig. 2. Association of the C-terminal mutants of Gbeta with Ggamma . HEK 293 cells were transfected with vector alone (Mock) or plasmids carrying cDNA for wild type Gbeta (wt), Delta C1, Delta C2, Delta C6, W339A, (His)6, and FLAG-Ggamma (gamma ), as indicated. The cells were lysed as described under "Materials and Methods." Aliquots of the cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-common Gbeta antibody (A). Other aliquots were immunoblotted with anti-common Gbeta antibody (B, upper) or anti-FLAG antibody (B, lower). The results shown are representative of three or five independent experiments.
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The results of x-ray crystal structure analysis suggest that the C terminus of Gbeta may be located near the N terminus of Galpha (7, 8). We analyzed the effect of the C-terminal mutations of Gbeta on the interaction with Galpha . Addition of the FLAG sequence to the N terminus of Ggamma has no effect on either the association with Gbeta or the formation of a Galpha beta gamma complex (33). In the presence of FLAG-Ggamma , the Delta C1, W339A, and (His)6 mutants retained their full ability to interact with Galpha o (Fig. 3). Furthermore, we examined the effect of the C-terminal mutations on the GTP-dependent dissociation of Galpha from Gbeta gamma . The cells were transfected with cDNAs of Galpha o, C-terminal mutants of Gbeta , and FLAG-Ggamma . The cells were lysed, and the ternary complexes were immunoprecipitated with anti-FLAG antibody. The immune complexes were incubated with a buffer containing GDP or GTPgamma S (Fig. 4). The ternary complexes formed with Gbeta mutants of Delta C1, W339A, and (His)6 showed the GTPgamma Sdependent dissociation similar to the one formed with wild type Gbeta .


Fig. 3. Interaction of Galpha o with the C-terminal mutants of Gbeta and Ggamma . Cells were transfected with vector alone (Mock) or plasmids carrying cDNA for Galpha o (alpha ), wild type Gbeta (wt), Delta C1, Delta C2, Delta C6, W339A, (His)6, and FLAG-Ggamma (gamma ), as indicated. The cells were lysed as described under "Materials and Methods." Aliquots of the cell lysates were immunoprecipitated with anti-FLAG antibody, immunoblotted with anti-Galpha o antibody (A, upper), and reimmunoblotted with anti-common Gbeta antibody (A, lower). Other aliquots were immunoblotted with anti-Galpha o antibody (B, upper), anti-common Gbeta antibody (B, middle), or anti-FLAG antibody (B, lower). The results shown are representative of three independent experiments.
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Fig. 4. Guanine nucleotide-dependent dissociation of Galpha o from Gbeta gamma . Cells were transfected with cDNAs for Galpha o (alpha ), wild type Gbeta (wt), and FLAG-Ggamma (A); Galpha o, Delta C1, and FLAG-Ggamma (B); Galpha o, W339A, and FLAG-Ggamma (C); and Galpha o, (His)6, and FLAG-Ggamma (D). The cells were lysed as described under "Materials and Methods." The lysates were divided into two halves and immunoprecipitated with anti-FLAG antibody. The immune complexes were incubated with a buffer containing GDP (left) or GTPgamma S (right). The washed immune complexes were immunoblotted with anti-Galpha o antibody (upper) and reimmunoblotted with anti-common Gbeta antibody (lower). The results shown are representative of three independent experiments.
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Gbeta gamma has been shown to stimulate the MAPK/ERK signaling pathway (22-25). To explore the effect of the C-terminal mutations of Gbeta on MAPK/ERK activation, cDNAs of Gbeta mutants, wild type Ggamma , and HA-ERK2 were co-transfected into HEK 293 cells. HA-ERK2 was immunoprecipitated with anti-HA antibody, and the kinase activity was assessed using myelin basic protein as a substrate. It has been shown that MAPK/ERK activation by Gi-coupled receptors is mediated by Gbeta gamma , whereas the activation by Gq/11 coupled receptors is mediated by Gq/11 (22-25). Carbachol stimulated the HA-ERK2 activity 2- and 5-fold in HEK 293 cells transfected with m2-muscarinic and m1-muscarinic acetylcholine receptors, respectively (data not shown). Crespo et al. (22) and Faure et al. (23) demonstrated that the ERK2 activity is stimulated 4- and 2-fold, respectively, by overexpression of Gbeta gamma in COS cells. In HEK 293 cells, overexpression of Gbeta gamma induces a weak phosphorylation of endogenous ERK2 (25). As shown in Fig. 5A, co-expression of wild type Gbeta and Ggamma activated HA-ERK2 about 1.5-fold, whereas the Delta C1 and W339A mutants had a relatively weak effect on ERK2 activation, and the Delta C2, Delta C6, and (His)6 mutants could not activate ERK2 at all.


Fig. 5. Effects of the C-terminal mutants of Gbeta on c-Raf-1 and MAPK/ERK stimulations. Cells were co-transfected with plasmid DNAs of HA-ERK2 (A) or RafFH6 (B) plus vector alone (Mock) or cDNA for wild type Gbeta (wt), Delta C1, Delta C2, Delta C6, W339A, (His)6, and Ggamma (gamma ), as indicated. A, the immune complexes of the cell lysates were assayed by measuring the incorporation of radioactive phosphate into myelin basic protein as described under "Materials and Methods." B, the immunoprecipitates were incubated with recombinant MEK and recombinant kinase-negative MAPK in the presence of [gamma -32P]ATP, and the radioactivities incorporated into the MAPK were measured as described under "Materials and Methods." Values shown represent the mean ± S.E. from three or four separate experiments.
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Furthermore, we examined the effect of the C-terminal mutations of Gbeta on MEK kinase activity of c-Raf-1. Cells were co-transfected with cDNAs of various Gbeta mutants, wild type Ggamma , and RafFH6, which is c-Raf-1 tagged with FLAG epitope and six histidines. The RafFH6 was immunoprecipitated with anti-FLAG antibody, and its MEK kinase activity was assayed using recombinant MEK and recombinant kinase-negative MAPK. The MEK kinase activity of RafFH6 was stimulated more than 1.5-fold by wild type Gbeta and Ggamma . On the other hand, the activation of RafFH6 by the Delta C1 and W339A mutants was less potent than that by the wild type Gbeta , and the Delta C2, Delta C6, and (His)6 mutants failed to activate RafFH6 (Fig. 5B). Effects of the C-terminal mutations on the stimulation of c-Raf-1 activity were comparable to those on the stimulation of ERK2 activity. Transfection of Gbeta or Ggamma alone fails to activate ERK2 (see Fig. 8A, and Refs. 22, 23, and 25) and c-Raf-1 (see Fig. 8B). It is noteworthy that the (His)6 mutant could stimulate neither ERK2 nor c-Raf-1.


Fig. 8. Effect of transfection with Gbeta or Ggamma alone on the MAPK/ERK, c-Raf-1, and JNK/SAPK stimulations. Cells were co-transfected with plasmid carrying DNAs for HA-ERK2 (A), RafFH6 (B), or HA-JNK1 (C) together with vector alone (Mock) or DNAs for wild type Gbeta (beta ) and/or Ggamma (gamma ), as indicated. The cells were lysed, and the tagged kinases were immunoprecipitated with anti-HA antibody (A and C) or anti-FLAG antibody (B). The immune complexes were used to measure the kinase activities as described under "Materials and Methods." Values shown represent the mean ± S.E. from three separate experiments.
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Since the N-terminal region of c-Raf-1 associates physically with the C-terminal region of Gbeta (37), we tested whether the C-terminal mutants of Gbeta are able to interact with c-Raf-1. As shown in Fig. 6, the (His)6 mutant could not bind with c-Raf-1, but other mutants could bind. It is likely that six histidine residues added at the C terminus of Gbeta may sterically inhibit the association of Gbeta with c-Raf-1. It is suggested that the association of Gbeta with c-Raf-1 by itself is not sufficient for activation of c-Raf-1, although the association may be required for its activation.


Fig. 6. Association of the C-terminal mutants of Gbeta with c-Raf-1. Cells were co-transfected with vector alone (Mock) or plasmid carrying cDNA for wild type beta  (wt), Delta C1, Delta C2, Delta C6, W339A, (His)6, Ggamma (gamma ), and RafFH6 (Raf), as indicated. The cells were lysed as described under "Materials and Methods." Aliquots of the cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-common Gbeta antibody (A). Other aliquots were immunoblotted with anti-common Gbeta antibody (B, upper) or anti-FLAG antibody (B, lower). The results shown are representative of three independent experiments.
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It has recently been reported that the signaling from G protein-coupled receptors to JNK/SAPK involves Gbeta gamma and that overexpression of Gbeta gamma enhances JNK/SAPK activity (26). We examined the effect of the Gbeta C-terminal mutants on JNK/SAPK stimulation. The cells were co-transfected with cDNAs of each Gbeta mutant and wild type Ggamma together with HA-JNK1. After lysis, HA-JNK1 was immunoprecipitated, and its kinase activity was assayed using GST-c-Jun as a specific substrate. Wild type Gbeta and Ggamma increased the activity of JNK1 approximately 5-fold (Fig. 7A). In contrast to the results of MAPK/ERK and c-Raf-1 activations (Fig. 5), the Delta C1 mutant had little ability to stimulate JNK1, whereas the Delta C2, Delta C6, and (His)6 mutants showed moderate stimulatory effect. The stimulatory effect of the W339A mutant on JNK1 was almost indistinguishable from that of the wild type Gbeta (Fig. 7A). Although the Delta C2 and Delta C6 mutants had little ability to associate with Ggamma , they activated JNK1 significantly. In order to examine whether Gbeta may activate JNK1 in the absence of interaction with Ggamma in the cells, we utilized a N-terminal deletion mutant of Gbeta . Since the N-terminal region of Gbeta is essential to form an alpha -helical coiled coil structure with the N terminus of Ggamma , the deletion of this region of Gbeta completely prevents the dimer formation with Ggamma (9, 33, 38). As shown in Fig. 7B, the Delta N38 mutant, which lacks N-terminal 38 amino acids, stimulated the activity of JNK1 approximately 3-fold. Furthermore, we transfected with Gbeta or Ggamma alone and measured the JNK1 activity. Fig. 8C shows that the transfection of Gbeta alone caused the activation of JNK1. These results suggested that the overexpression of Gbeta alone can stimulate the JNK1 activity in the cells.


Fig. 7. The C-terminal mutants of Gbeta influence the stimulation of JNK/SAPK activity. Cells were co-transfected with plasmid carrying DNAs for HA-JNK1 plus vector alone (Mock) or plasmid carrying cDNAs for wild type Gbeta (wt), Delta C1, Delta C2, Delta C6, W339A, (His)6, Delta N38, and Ggamma (gamma ), as indicated (A and B). The immune complexes of the cell lysates were incubated with GST-c-Jun in the presence of [gamma -32P]ATP and assayed by measuring the incorporation of radioactive phosphate into GST-c-Jun as described under "Materials and Methods." Values shown represent the mean ± S.E. from three separate experiments (upper). The autoradiogram of GST-c-Jun is representative of three experiments (middle). Aliquots of the cell lysates were immunoblotted with anti-HA antibody to detect HA-JNK1 (lower).
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DISCUSSION

The mutant (Delta C1) lacking the C-terminal amino acid of Gbeta retained the full ability to associate with Ggamma and to form a ternary complex with Galpha (Figs. 2, 3, 4). Although the C-terminal Asn-340 of Gbeta 1 participates in the specific interaction with Asn-62 of Ggamma 1 (corresponding to Asn-59 of Ggamma 2) (9), the deletion of Asn-340 of Gbeta 1 did not affect the association with Ggamma 2. The interaction does not seem to be necessary for the complex formation. Removal of the two amino acid residues from the Gbeta C terminus dramatically abolished the binding of Gbeta with Ggamma (Fig. 2). Since Gbeta associates with Ggamma at multiple sites (9), it was unexpected that the truncation of the last two amino acid residues resulted in the loss of the ability to form a Gbeta gamma complex. We first thought that the Trp-339 may be important for the complex formation, but the replacement of Trp-339 by Ala (W339A) did not show any effect on the association of Gbeta with Ggamma (Fig. 2). It appears that the presence of an amino acid residue at the -2 position of Gbeta is important for the Gbeta gamma complex formation.

Genetic studies of the pheromone response pathway in Saccharomyces cerevisiae suggested that two regions of the Ste4 protein (S. cerevisiae Gbeta ) may be involved in the effector activation (39). The first region is localized in the alpha -helical structure of the N terminus and far from the binding sites with Galpha (7, 8, 39). If this region is involved in the binding with an effector, it is unclear how the activation of effector by Gbeta gamma is inhibited by Galpha . The second region is found in the third WD repeat (39). We made a substitution mutant using Gly at Val-135 that was well conserved between mammalian Gbeta and yeast Ste4. However, the V135G mutant of Gbeta retained the ability to stimulate the MAPK/ERK and JNK/SAPK activities to an extent similar to that of the wild type Gbeta in HEK 293 cells (data not shown). It is possible that the V135G mutation may affect other signaling pathways in mammalian cells or that other mutations in the second region of Gbeta may affect ERK/MAPK and JNK/SAPK activities.

We found differential effects of the C-terminal mutations of Gbeta on the MAPK/ERK and JNK/SAPK pathways in mammalian cells. The Delta C1 mutant of Gbeta , together with Ggamma , could stimulate the activity of c-Raf-1 and MAPK/ERK but showed only a slight activation of JNK/SAPK. On the other hand, the (His)6 mutant of Gbeta failed to stimulate c-Raf-1 and MAPK/ERK activities in the presence of wild type Ggamma , and the mutant retained the ability to activate JNK/SAPK (Figs. 5 and 7). The C-terminal amino acid residue of Gbeta is localized on the outside of a Gbeta gamma complex (9). It is possible that the C terminus of Gbeta is involved in binding with effector molecule(s). We speculate that the differential effects of these mutations on the activation of MAPK/ERK and JNK/SAPK might be due to the difference of their direct effector(s). The putative effector molecule(s) of Gbeta gamma in the MAPK/ERK and JNK/SAPK cascades are as yet unidentified, although phosphatidylinositol 3-kinase has been reported to be involved in the pathway from Gbeta gamma to MAPK/ERK (40). Phosphatidylinositol 3-kinase gamma  (13) may be a candidate for its effector. It has been reported that calcium ion is important in Gi-coupled receptor-mediated stimulation of JNK/SAPK (41). The direct regulation of phospholipase Cbeta isozymes by Gbeta gamma (11, 12) may be involved in the signaling pathway.

Recently, Coria et al. (42) reported that the C-terminal region of yeast Ste4 is essential in triggering the yeast pheromone response cascade. Ste4 has four extra amino acid residues at the C terminus compared with mammalian Gbeta . The extra amino acid residues may be important for the function of Ste4.

It has been shown that overexpression of Ste4 alone causes an increased response to the pheromone in yeast (43-45). We found that the Delta C2 and Delta C6 mutants, which were unable to associate with FLAG-Ggamma , could stimulate JNK/SAPK but not MAPK/ERK activity (Figs. 5 and 7). Therefore, we examined whether Gbeta alone can stimulate the activity of JNK/SAPK in HEK 293 cells. It was observed that the transfection of Gbeta alone could stimulate JNK/SAPK to the same extent as co-transfection of Gbeta and Ggamma (Fig. 8C). As shown in Fig. 7B, Ggamma -incompetent Gbeta mutant (Delta N38), which lacks N-terminal alpha -helical structure for coiled coil interaction with Ggamma , has the ability to activate JNK/SAPK in the cells. Taken together, it is suggested that Gbeta plays an essential role in the JNK/SAPK pathway in HEK 293 cells. Coso et al. (26) have reported that overexpression of Gbeta alone does not stimulate the JNK/SAPK activity in COS cells. The discrepancy may be due to the difference of cell type.

In the course of this study, two groups have reported the regions of Gbeta involved in the interaction with effector. Yan and Gautam (46), using a yeast two-hybrid system, showed that the N-terminal 100-amino acid fragment of Gbeta associated with adenylyl cyclase type II and G protein-coupled inward rectifier potassium channel 1. Zhang et al. (47) demonstrated that the Gbeta mutation in the C-terminal region prevented the stimulation of phospholipase Cbeta 2 in COS cells. We obtained C-terminal mutants of Gbeta that retained the ability to interact with Ggamma and Galpha yet exhibited dramatic decreases in the MAPK/ERK or JNK/SAPK activation in mammalian cells. Further biological studies using these mutants should throw more light on the role of Gbeta in the distinct cellular signaling pathways.


FOOTNOTES

*   This work was partly supported by grants in aid for scientific research from the Ministry of Education, Science, Sport, and Culture of Japan. Our laboratory is supported by funding from Schering-Plough 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.
   To whom correspondence should be addressed: Tel.: 81-45-924-5746; Fax: 81-45-924-5822; E-mail: hitoh{at}bio.titech.ac.jp.
1   The abbreviations used are: G protein, heterotrimeric guanine nucleotide-binding regulatory protein; Galpha , G protein alpha  subunit; Gbeta , G protein beta  subunit; Ggamma , G protein gamma  subunit; Gbeta gamma , G protein beta gamma subunit; MAPK/ERK, mitogen-activated protein kinase/extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; JNK/SAPK, c-Jun N-terminal kinase/stress-activated protein kinase; GST, glutathione S-transferase; HEK, human embryonal kidney; PCR, polymerase chain reaction; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate.

Acknowledgments

We thank M. I. Simon, T. Nukada, M. McMahon, E. Nishida, and M. Karin for supplying the plasmids. We also thank S. Mizutani, K. Tago, and K. Nishida for advice regarding kinase assays.


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