Dependence of Activated Galpha 12-induced G1 to S Phase Cell Cycle Progression on Both Ras/Mitogen-activated Protein Kinase and Ras/Rac1/Jun N-terminal Kinase Cascades in NIH3T3 Fibroblasts*

(Received for publication, June 27, 1996, and in revised form, November 19, 1996)

Hiroshi Mitsui Dagger §, Noriko Takuwa , Kiyoshi Kurokawa §par , John H. Exton ** and Yoh Takuwa Dagger Dagger Dagger

From the Departments of Dagger  Cardiovascular Biology,  Physiology, and § Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan and the ** Howard Hughes Medical Institute, Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

We evaluated the roles of mitogen-activated protein kinase (MAPK) and Jun N-terminal kinase (JNK) signaling cascades in Galpha 12-induced G1 to S phase cell cycle progression in NIH3T3(M17) fibroblasts. Transient expression of a constitutively active mutant of Galpha 12, Galpha 12(R203C), resulted in a 2-fold increase in the number of bromodeoxyuridine-positive S phase cells over vector control level under serum-deprived conditions. Consistent with the ability of Galpha 12(R203C) to induce G1/S transition, its expression led to a 2-fold increase in cyclin A promoter activity, which showed a marked synergism with a low concentration of serum, resulting in up to a 15-fold elevation over the basal level. In addition, Galpha 12(R203C) caused a 2-fold stimulation in E2F-mediated transactivation. Wild type Galpha 12 showed similar stimulatory effects on cyclin A promoter activity and E2F-mediated transactivation, although of lesser magnitude. We observed a modest but constitutive activation of MAPK in cells transfected with Galpha 12(R203C), which was abolished by a dominant negative form of Ras. Galpha 12(R203C) also induced a 3-fold increase in JNK activity, which was abolished by dominant negative forms of either Rac1 or Ras. The expression of dominant negative forms of Ras, MAPK, Rac1, or JNK inhibited Galpha 12(R203C)-induced increases in bromodeoxyuridine-positive cells. Also, the dominant negative forms of Ras, MAPK, and JNK strongly inhibited Galpha 12(R203C)-induced stimulation of cyclin A promoter activity. These results demonstrate that both the Ras/MAPK and Ras/Rac1/JNK pathways convey necessary, if not sufficient, mitogenic signals induced by Galpha 12 activation.


INTRODUCTION

Heterotrimeric G proteins are critical components in transmembrane signaling via heptahelical receptors and regulate the activities of a variety of effector enzymes and ion channels. Heterotrimeric G proteins are composed of three polypeptides, an alpha  subunit, a beta  subunit, and a gamma  subunit, the latter two of which form a dimer (1). The Galpha subunits are a family of over 20 different proteins that share 45-95% amino acid identity. They have been divided into four classes: Gsalpha , Gialpha , Gqalpha , and Galpha 12 (1). The Galpha 12 class includes Galpha 12 and Galpha 13 (2, 3) and is ubiquitously expressed in mammalian tissues and cells (4). In contrast to Gs, Gi, and Gq class members for which functional roles have been well established (1), the biological activity of the Galpha 12 class is not fully clarified. However, accumulating evidence suggests that Galpha 12 is involved in cell growth and transformation (5). First, mutationally activated Galpha 12, Galpha 12(Q229L), stimulated cell proliferation and induced neoplastic transformation in NIH3T3 cells (3, 6, 7) and Rat-1 cells (8). Second, expression cloning of a transforming gene from a human sarcoma cDNA library resulted in isolation of wild type human Galpha 12, suggesting that overexpression of wild type Galpha 12 was sufficient to cause neoplastic transformation in the presence of serum growth factors (3). Third, DNA synthesis stimulated by thrombin and serum, but not basic fibroblast growth factor, was abrogated by microinjection of anti-Galpha 12 antibody in 1321N1 astrocytoma cells (9).

The molecular mechanisms by which activated Galpha 12 promotes cell growth and causes transformation have not yet been clearly understood. The expression of Galpha 12(Q229L) was shown to increase the activity of Na+/H+ exchanger in COS-1 cells (10). It was demonstrated for Rat-1 cells stably expressing Galpha 12(Q229L) that the basal activity of MAPK1 (extracellular signal-regulated kinase) under serum-starved conditions was not elevated, but MAPK activation induced by epidermal growth factor or serum was potentiated, as compared with cells transfected with an empty vector (8). Very recently, constitutive activation of JNK (stress-activated protein kinase), a new member of the MAPK family, has been demonstrated in Galpha 12(Q229L)-transformed NIH3T3 cells (11). However, it remains unknown whether activation of either of these signaling pathways contributes to Galpha 12-elicited cell growth or transformation.

Recent progress in the understanding of mammalian cell cycle regulation has elucidated the principal molecular mechanism of G1 to S phase progression (12, 13). A number of studies demonstrate that the E2F family transcription factors play pivotal roles for entry into the S phase (14). It was reported that microinjection of E2F1 expression plasmid into quiescent REF-52 fibroblasts is sufficient to induce DNA synthesis (15). In G0 and early G1 phases, E2F is complexed with pRb, the product of the retinoblastoma tumor suppressor gene, and the pRb-related proteins, p107 and p130. The E2F complex found during G0 and early G1 phases is inactive as a transcription activator for E2F target genes (12-14) or may have transcriptional repressor function (16). In middle to late G1 phase, activation of cyclin-dependent kinases occurs in a temporally ordered manner, and pRb and the pRb-related proteins become progressively phosphorylated by activated cyclin-dependent kinases, resulting in inactivation of their growth-suppressive function (12, 13). Consequently, E2F is liberated and transactivates a number of the late G1/S phase genes, including cyclin E, cyclin A, B-myb, cdc2, ribonucleotide reductase, thymidylate synthase, DNA polymerase alpha , and E2F1 itself, through consensus E2F binding sites in their promoter regions (14, 17). Cyclin A thus induced at the G1/S border forms complexes with its catalytic partner Cdk2. Accumulated evidence indicates that the cyclin A-Cdk2 complex mediates initiation and progression of the S phase (12, 13). It has also been demonstrated that MAPK is necessary for growth factor-stimulated G1/S progression (18). However, it is poorly understood how MAPK activation leads to activation of the critical events in late G1, i.e. E2F activation and induction of the E2F target genes that are necessary for G1 to S progression. Information about how JNK is involved in G1 to S progression is even more scanty.

In the present study, we observed that the expression of the Galpha 12 mutant, Galpha 12(R203C), caused activation of both MAPK and JNK and stimulation of S phase entry in NIH3T3 fibroblasts. The mutation corresponding to Galpha 12(R203C) in Gsalpha , Gialpha 2, and Gqalpha renders them constitutively active and oncogenic (19-22). We evaluated the role of the MAPK and the JNK signaling cascades in Galpha 12(R203C)-induced G1 to S phase progression. We further explored the involvement of the Ras and Rho subfamilies of low molecular weight G proteins in Galpha 12(R203C)-induced activation of MAPK and JNK and tried to elucidate whether E2F-mediated transactivation and cyclin A gene expression, two major events occurring at the G1/S boundary, are downstream targets of these signaling molecules. The present results demonstrate that the expression of Galpha 12(R203C) leads to constitutive activation of MAPK and JNK in a small G protein-dependent manner and that both Ras/MAPK and Ras/Rac1/JNK pathways are indispensable for Galpha 12(R203C)-evoked activation of G1/S gene expression and S phase entry.


EXPERIMENTAL PROCEDURES

Molecular Cloning of Rat Galpha 12 cDNA

A 219-bp fragment (679-891 when the A residue of the initiation codon ATG is numbered as 1) of rat Galpha 12 was obtained from the cDNAs reverse-transcribed from rat liver poly(A)+ RNA by PCR amplification using two sets of degenerate oligonucleotide primers corresponding to the two six-amino acid sequences (182DVGGQR and 290FLNKQD in Gsalpha ) conserved between the Gsalpha and Gialpha class members that were designed by Strathmann et al. (24). The sense primer was GTCTAGAGA(C/T)GTC(A/C/G/T)GG(A/C/G/T)GG(A/G)(A/C)G, and the antisense primer was GGAATTC(A/G)TC(C/T)TT(C/T)TT(A/G)TT(A/C/G/T)AG(A/G)AA. The conditions for the PCR were 1 min at 94 °C, 1.5 min at 37 °C, and 2 min at 72 °C. A full-length Galpha 12 cDNA was isolated form a rat brain lambda ZAPII cDNA library (Stratagene) by hybridization screening using the 219-bp PCR fragment as a probe under high stringency condition (at 42 °C in the presence of 50% formamide and 0.9 M NaCl). The nucleotide sequence of the cloned cDNA insert was sequenced by the dideoxy chain termination method with Sequenase (U.S. Biochemical Corp.). The nucleotide sequence of rat Galpha 12 has been deposited in the GenBankTM/EMBL Data Bank with accession number D85760[GenBank]. A constitutively active mutant of Galpha 12, Galpha 12(R203C), was created with a site-directed mutagenesis kit (Muta-Gene M13, Bio-Rad) according to the manufacturer's instructions. The mutagenic primer was CATCCTGTTGGCATGCAAGGACACCAAG. The created mutation was confirmed by DNA sequencing. It was previously demonstrated for Gsalpha , Gialpha 2, and Gqalpha that substitution of the corresponding arginine with cysteine makes the Galpha proteins constitutively active (19-23). Wild type Galpha 12 and Galpha 12(R203C) were subcloned into the EcoRI site of pMT2 expression vector (a generous gift from Dr. Kaufman at Genetics Institute, Cambridge, MA).

Cell Culture, Plasmids, and Transfections

NIH3T3(M17) (a gift from Dr. G. M. Cooper at Harvard Medical School, Boston, MA) is a subclone of NIH3T3 fibroblasts in which the expression of the dominant negative mutant of Ras, Ras(N17), is induced by dexamethasone treatment (25). The cells were maintained in DMEM supplemented with 5% iron-enriched calf serum (25) and 200 µg/ml Geneticin at 37 °C in subconfluent states.

pSV-beta gal, the expression plasmid for beta -galactosidase, was purchased from Promega. pactEF-MAPK and pactEF-MAPK-DN, the expression vectors of Xenopus MAPK and its dominant negative form with Asp170 to Ala substitution, respectively, were kindly provided by Dr. Okazaki (26) (Kurume University Institute of Life Science, Kurume, Japan). The expression vector for a Myc epitope (EQKLISEEDL)-tagged MAPK (pME18S-Myc MAPK) was created by a PCR-based method (27). The cDNAs of human JNK1 with a Myc epitope tag at its N terminus and human Rac1 were obtained from a human WI-38 fibroblast cDNA library by PCR amplification. The cDNA of dominant negative JNK1 (JNK-DN) with Thr183 to Ala and Tyr185 to Phe substitutions (28) was generated by a PCR-based method (27). The cDNAs of Myc epitope-tagged MAPK and dominant negative Rac1 (Rac1(N17)) were also prepared by a PCR-based method (27). The nucleotide sequences of the cDNAs obtained by the PCR method were confirmed by sequencing with an ALFred DNA sequencer (Pharmacia Biotech Inc.). The cDNAs were subcloned into the EcoRI site or the BstX1 site of a mammalian expression vector, pME18S (generously provided by Dr. Maruyama at the Institute of Medical Science, University of Tokyo, Tokyo, Japan). A luciferase reporter vector, E2F-Luc, was created as described previously by Slansky et al. (29). The oligonucleotide 5'-CTAGCAGCTGCTGCGATTTCGCGCCAAACTTGACG-3', which contains a -20 to +9 from the dihydrofolate reductase promoter plus a PvuII site for screening and XhoI and NheI sites at the 5'- and 3'-ends, was inserted into the vector pGL3basic (Promega). A 2.5-kilobase pair BglII fragment of the cyclin A genomic DNA was isolated from a human leukocyte genomic library (Clontech). A luciferase reporter vector, CycA-Luc, was constructed by ligating the blunted 1.3-kilobase pair HindIII-SmaI fragment of cyclin A genomic DNA (30) to pGL2 basic vector (Promega) at the SmaI site. The bacterial expression plasmid of truncated c-Jun (amino acids 5-89) fused to glutathione S-transferase, pGEX-2T-c-Jun-(5-89), was kindly provided by Dr. A. Kraft (University of Alabama School of Medicine, Birmingham, AL). The plasmids were purified by two cycles of CsCl2 density gradient centrifugation and introduced into cells by the calcium phosphate precipitation procedure. The day after transfection, the cells were serum-deprived by incubating in DMEM containing 0.2% bovine serum albumin for 24 h. To induce Ras(N17), dexamethasone (5 × 10-7 M) was added into media at least 8 h prior to each experiment (25).

Western Blot Analysis

Two or three days after transfection, as indicated in the figure legends, the cells were washed twice with Ca2+- and Mg2+-free Dulbecco's phosphate-buffered saline and lysed in 2 × SDS sample buffer. Gel-loaded sample volumes were adjusted on the basis of protein concentration (5) determined on parallel dishes so that an equal amount of total cellular protein was loaded onto gels per well. Proteins were separated on SDS-10% polyacrylamide gel electrophoresis and transferred onto Immobilon P membranes (Millipore Corp.). Western blot analysis was performed by probing with anti-Galpha 12 antibody (Santa Cruz) or a mouse monoclonal anti-Myc epitope antibody (9E10), which recognized the N-terminal amino acid sequence (EQKLISEEDL) of c-Myc, and respective alkaline phosphatase-conjugated secondary antibody (Zymed).

BrdUrd Incorporation

NIH3T3 cells, seeded at 1 × 105 cells/35-mm dish, were co-transfected with pSV-beta gal and various expression plasmids at a total of 3 µg of DNA/dish as indicated in the figure legends. The cells were serum-deprived in the presence or absence of dexamethasone. Then 10 mM of BrdUrd (Boehringer Mannheim) was added and further incubated for 17 h in the presence or absence of dexamethasone. The cells were washed with Ca2+- and Mg2+-free Dulbecco's phosphate-buffered saline, fixed in 3.7% formaldehyde, and permeabilized in 0.25% Triton X-100. Cells were first incubated with a rabbit polyclonal anti-beta -galactosidase antibody (Cappel) and then with a rhodamine-conjugated goat anti-rabbit IgG antibody (Cappel). After fixation in 3.7% formalin and treatment in 1.5 N HCl (31), BrdUrd was probed sequentially with a mouse monoclonal anti-BrdUrd antibody (Sigma) and a fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG antibody (Zymed). Each incubation was performed according to the manufacturers' recommendations. More than 200 beta -galactosidase-positive (transfected) cells were examined, and BrdUrd-positive cells were counted under a fluorescent microscope (Olympus, Tokyo, Japan).

Luciferase Assay

NIH3T3(M17) cells (seeded at 5 × 104/well in 12-well plates) were co-transfected with expression plasmids and either CycA-Luc or E2F-Luc (a total of 2 µg of DNA/well), serum-deprived for 24 h, and then incubated in DMEM containing 0.2% bovine serum albumin with or without calf serum (0.5%) and dexamethasone for 17 h. Cell lysates were prepared, and luciferase activity was measured with a Lumat LB95001 luminometer (Berthold) using the luciferase assay system (Promega). Protein concentrations of the same samples were determined using Bio-Rad protein assay reagent, and luciferase activity was normalized for protein content.

Measurement of MAPK Activity

NIH3T3(M17) cells in 35-mm dishes were co-transfected with pME18S-Myc-MAPK and either pMT2-Galpha 12(R203C) or pMT2 empty vector (a total of 3 µg of DNA/dish). After incubation for 24 h in DMEM containing 0.2% bovine serum albumin in the presence or absence of dexamethasone (5 × 10-7 M), the cells were lysed in a lysis buffer containing 50 mM Tris (pH 8.0), 1 mM EDTA, 150 mM NaCl, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 0.5% sodium deoxycholate, 0.1% SDS, and 1% NP-40. Myc-tagged MAPK was immunoprecipitated by using 9E10 anti-Myc epitope antibody. MAPK activity associated with the immune complex was assayed in vitro at 30 °C for 10 min using myelin basic protein (Sigma) as a substrate as described (32). The reaction was terminated by adding 4 × SDS sample buffer, and samples were analyzed on SDS-15% polyacrylamide gel electrophoresis followed by autoradiography. The radioactivity in the band corresponding to myelin basic protein was determined with a Fuji BAS 2000 Bio-Image Analyzer (Fuji Film Co., Tokyo, Japan).

Measurement of JNK Activity

NIH3T3(M17) cells in 35-mm dishes were co-transfected with pME18S-Myc-JNK and indicated expression plasmids (a total of 3 µg/dish). The cells were deprived of serum for 24 h and then an immune complex JNK assay was performed as described by Derijard et al. (33) with a slight modification. Briefly, cells were lysed in an ice-cold lysis buffer (25 mM HEPES (pH 7.5), 1% Triton X-100, 20 mM Tris (pH 7.6), 0.5% Nonidet P-40, 500 mM NaCl, 50 mM NaF, 5 mM EDTA, 3 mM EGTA, 1 mM Na3VO4, 10 µg/ml each of leupeptin and aprotinin, and 1 mM phenylmethylsulfonyl fluoride). A soluble fraction was obtained by centrifugation and precleared with protein A-conjugated Sepharose 4B beads (Pharmacia). The supernatant was incubated with 9E10 antibody and subsequently with rabbit anti-mouse IgG (Zymed)-bound protein A-Sepharose beads. The immunoprecipitates were washed three times with the lysis buffer and twice with a kinase assay buffer (20 mM HEPES (pH 7.6), 20 mM MgCl2, 20 mM beta -glycerophosphate, 20 mM p-nitrophenyl phosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4). The pelleted beads were incubated with 30 µl of the kinase assay buffer containing 3 µg of GST-c-Jun-(5-89), 20 µM ATP, and 5 µCi of [gamma -32P]ATP at 30 °C for 20 min. The reaction was terminated by adding 10 µl of 4 × Laemmli's SDS-sample buffer and boiled. The samples were analyzed by SDS-12% polyacrylamide gel electrophoresis, and the radioactivity in the band corresponding to GST-c-Jun-(5-89) was measured.


RESULTS

Molecular Cloning and Expression of Rat Galpha 12 cDNA

A 2.1-kilobase pair cDNA clone containing the entire coding region of rat Galpha 12 was isolated. Sequence analysis of this clone revealed that the predicted amino acid sequence was identical to that of mouse Galpha 12 except for one residue (341), where serine in mouse Galpha 12 is changed to glycine in rat Galpha 12. NIH3T3(M17) cells were transiently transfected with expression plasmids carrying either wild type Galpha 12 or the constitutively active mutant Galpha 12(R203C). The expression of either form was confirmed by Western blot analysis using anti-Galpha 12 antibody (Fig. 1), which detected expression of 45-kDa proteins corresponding to Galpha 12 and Galpha 12(R203C) proteins. A trace level of endogenous Galpha 12 was detected in empty vector-transfected control cells.


Fig. 1. Expression of Galpha 12 and Galpha 12(R203C) in transiently transfected cells. NIH3T3(M17) cells in 35-mm dishes were transfected with 3 µg of pMT2 vector, pMT2-Galpha 12, or pMT2-Galpha 12(R203C). Three days later, the cell lysate of 60 µg of protein was analyzed by Western blotting.
[View Larger Version of this Image (15K GIF file)]


Galpha 12(R203C) Elicits G1/S Cell Cycle Progression, E2F-mediated Transactivation, and Cyclin A Promoter Activation

Previous studies demonstrated that expression of Galpha 12(Q229L), another constitutively active mutant with deficient GTPase activity, resulted in an increased thymidine incorporation into DNA (8). To evaluate whether Galpha 12(R203C) has a similar stimulatory effect on cell cycle progression, cells were transfected with pMT2-Galpha 12(R203C), pMT2-Galpha 12, or pMT2 empty vector together with a beta -galactosidase expression plasmid, and BrdUrd incorporation into nuclei in a beta -galactosidase-positive cell population was determined (Fig. 2). Under serum-deprived conditions, the expression of Galpha 12(R203C) resulted in an approximately 2-fold increase in BrdUrd-positive cells over the vector control level. The stimulatory effect of Galpha 12(R203C) on S phase progression was also evident in the presence of a low concentration of serum (0.5%). The expression of wild type Galpha 12 tended to increase BrdUrd-positive cells in the presence and absence of serum but without statistical significance.


Fig. 2. Stimulation of DNA synthesis in Galpha 12(R203)-expressing cells. NIH3T3(M17) cells were co-transfected with pSV-beta gal (1.5 µg) and pMT2 vector (open bars), pMT2-Galpha 12 (stippled bars), or pMT2-Galpha 12(R203C) (1.5 µg/dish) (filled bars). After serum deprivation for 24 h, the cells were incubated with BrdUrd in the presence or absence of 0.5% calf serum for 17 h and fixed. After processing for immunofluorescence, the percentage of BrdUrd-positive cells in beta -galactosidase-positive cell population was scored under fluorescent microscopy. Values shown represent means ± S.E. from four separate experiments in each of which over 200 cells were inspected. * and NS, statistically significant difference (p < 0.05) and statistically not significant difference, respectively, as compared with vector control.
[View Larger Version of this Image (26K GIF file)]


Recent investigations have elucidated that G1 to S phase progression requires activation of E2F family transcription factors, a process dependent on cyclin-dependent kinase-mediated phosphorylation and inactivation of pRb family proteins (12-14). It is also known that S phase entry is associated with transcriptional activation of the cyclin A gene (12, 13). To investigate whether or not activation of Galpha 12 elicits these events, we performed a series of co-transfection experiments using luciferase-reporter plasmids. As shown in Fig. 3 (left), under serum-deprived conditions, transfection of Galpha 12(R203C) resulted in a 2-fold increase in transactivation of a luciferase gene that is under the control of a consensus E2F binding site in the 5'-upstream region. It was of note that transfection of wild type Galpha 12 also resulted in stimulation of E2F activity, although to a smaller extent. In the presence of a low concentration of serum, the basal luciferase activity was slightly stimulated as compared with serum-deprived cells. Under this condition, wild type Galpha 12 and Galpha 12(R203C) also caused stimulation of luciferase activity over the vector control level. Shown in Fig. 3 (right) is Galpha 12-induced stimulation of cyclin A promoter activity. Transfection of Galpha 12(R203C) resulted in a weak but significant increase in luciferase activity in the absence of serum stimulation, which was potentiated by a low concentration of serum in a synergistic manner. Again, the wild type Galpha 12 showed stimulatory effects, although to a lesser extent. These results demonstrate that Galpha 12(R203)-induced G1/S transition is associated with stimulation of E2F activity and up-regulation of cyclin A gene transcription.


Fig. 3. Effects of expression of Galpha 12 and Galpha 12(R203C) on E2F-mediated transactivation (left) and cyclin A promoter activation (right). NIH3T3(M17) cells were co-transfected with pMT2 vector (open bars), pMT2-Galpha 12 (stippled bars), or pMT2-Galpha 12(R203C) (filled bars) (1 µg/well) and either of the reporter vectors (1 µg/well), i.e. pGL3-E2F (left) or pGL2-cycA (right). After serum deprivation, the cells were incubated for 17 h in the presence or absence of 0.5% calf serum, and the luciferase activity in cell lysate was measured as described under "Experimental Procedures." The data shown represent means ± S.E. from three separate experiments each performed in triplicate and are expressed as -fold increase over the mean value for vector control in the absence of serum. *, **, and NS, statistically significant differences (**, p < 0.01; *, p < 0.05) and statistically not significant difference, respectively, as compared with vector controls.
[View Larger Version of this Image (19K GIF file)]


Galpha 12(R203C) Induces Ras-dependent Activation of MAPK

We evaluated whether Galpha 12(R203C) activates MAPK in a transient co-transfection assay with a Myc epitope-tagged MAPK (Fig. 4). In cells transfected with Galpha 12(R203C), MAPK activity showed a 1.5-fold increase over the vector control in the absence of external growth factors. This increase was statistically significant (p < 0.01) and reproducible. By taking advantage of inducible expression of the dominant negative mutant of Ras(N17) by dexamethasone treatment, we examined whether Galpha 12(R203C)-induced activation of MAPK is dependent on Ras. When Ras(N17) was induced, MAPK activity in Galpha 12(R203C)-transfected cells was not significantly different from the vector control level. Western blot analysis using anti-Myc epitope antibody revealed that the expression level of a Myc epitope-tagged MAPK was not changed by co-expression of Ras(N17) and/or Galpha 12(R203C). The results demonstrate that Galpha 12(R203C) induces stimulation of MAPK in a Ras-dependent manner.


Fig. 4. Galpha 12(R203C) induces Ras-dependent activation of epitope-tagged MAPK in a transient co-expression system. NIH3T3(M17) cells were co-transfected with pME18S-Myc-MAPK (0.75 µg/dish) and either pMT2-Galpha 12(R203C) or pMT2 empty vector (2.25 µg/dish). Where indicated, dexamethasone was added to induce Ras(N17). After serum deprivation for 24 h, Myc epitope-tagged MAPK was immunoprecipitated by using 9E10 anti-Myc tag antibody, and associated MAPK activity was measured in vitro. A (upper panel), autoradiograms of the band corresponding to myelin basic protein from a representative experiment performed in duplicate. Lower panel, quantitation of MAPK activity. The MAPK activity was quantitated using a Fuji Bioimage analyzer, as described under "Experimental Procedures." The data are expressed as percentages of the mean value for vector-transfected, untreated control and represent the means ± S.E. from four separate experiments each performed in triplicate or quadruplicate. *, the difference is statistically significant (p < 0.01). B, a portion of each cell lysate was analyzed by Western blotting, showing comparable expression of Myc-MAPK under each condition.
[View Larger Version of this Image (25K GIF file)]


Galpha 12(R203C) Induces JNK Activation in a Ras- and Rac1-dependent Manner

Very recently Prasad et al. (11) have reported that Galpha 12(Q229L) activates JNK. We examined whether Galpha 12(R203C) activated JNK and, if so, whether Galpha 12(R203C)-induced JNK activation was dependent on the small G proteins, Ras and Rac. As shown in Fig. 5A, co-transfection of Galpha 12(R203C) and a Myc epitope-tagged JNK resulted in an approximately 2.5-fold increase in JNK activity over the vector control level. Galpha 12(R203C)-induced activation of JNK was completely abolished when Ras(N17) was expressed. In addition, Galpha 12(R203C)-induced JNK activation was entirely dependent on Rac1, as demonstrated by the inhibition of JNK activation by the dominant negative Rac1(N17). Western blot analysis using anti-Myc epitope antibody revealed that the expression level of a Myc epitope-tagged JNK was not changed by co-expression of either Rac1(N17) or Ras(N17) (Fig. 5B).


Fig. 5. Galpha 12(R203) induces Ras- and Rac1-dependent activation of Myc epitope-tagged JNK in a transient co-expression system. NIH3T3(M17) cells were co-transfected with pME18S-Myc-JNK (1 µg/dish), together with pMT2-Galpha 12(R203C) (1 µg/dish) and pME18S-Rac1(N17) (1 µg/dish) or the respective empty vector. After serum deprivation for 24 h in the presence or absence of dexamethasone, the cells were lysed, and an immune complex JNK assay was carried out by using 9E10 anti-Myc tag antibody. A (upper panel), autoradiogram of the band corresponding to GST-c-Jun-(5-89) from a representative experiment performed in duplicate. Lower panel, quantitation of JNK activity. The data are expressed as means ± S.E. from three separate experiments each performed in duplicate. *, the difference is statistically significant (p < 0.01) as compared with vector control. B, a portion of each cell lysate was analyzed by Western blotting, showing comparable expression of Myc-JNK under each condition.
[View Larger Version of this Image (36K GIF file)]


Galpha 12(R203C)-elicited Activation of Cyclin A Gene Transcription Is Completely Abolished by the Dominant Negative Forms of Ras, MAPK, and JNK

We investigated whether Ras, MAPK, and JNK are involved in G1/S cell cycle progression evoked by Galpha 12. To this end, we studied the effects of expression of dominant negative forms of these signaling molecules on Galpha 12(R203C)-induced activation of cyclin A promoter and BrdUrd incorporation into DNA. As shown in Fig. 6A, stimulation of cyclin A promoter activity by activated Galpha 12 was completely abolished by induced expression of Ras(N17), both in the presence and absence of a low concentration of serum. The dominant negative MAPK also strongly inhibited the stimulatory effect of Galpha 12(R203C) down to the serum-starved vector control level (Fig. 6B). Similarly, co-expression of the dominant negative JNK potently inhibited Galpha 12(R203C)-induced cyclin A promoter activation (Fig. 6C).


Fig. 6. Effects on cyclin A promoter activity of co-expression of Galpha 12(R203C) and dominant negative forms of Ras, MAPK, and JNK. A, NIH3T3(M17) cells were transfected with pGL2-cycA (1 µg/well) and either pMT2-Galpha 12(R203C) or pMT2 vector (1 µg/well). Dexamethasone was added to the cells to induce Ras(N17) (closed bars), or the cells were left untreated (open bars). B and C, the cells were transfected with 1 µg/well of pGL2-cycA, 0.5 µg/well of pMT2-Galpha 12(R203C), and 0.5 µg/well of pactEF-MAPK-DN (B) or pME18S-JNK-DN (C). For control, the respective empty vector was included instead of the expression plasmid. The cyclin A promoter activity was measured, and data were expressed as described in the legend to Fig. 3.
[View Larger Version of this Image (21K GIF file)]


Galpha 12(R203C)-mediated S Phase Entry Is Inhibited by the Dominant Negative Forms of Ras, Rac1, MAPK, and JNK

As described in Fig. 2, BrdUrd-positive cells among the beta -galactosidase-positive population were monitored (Table I). The expression of either dominant negative MAPK or Ras(N17) strongly inhibited basal and Galpha 12(R203C)-induced BrdUrd incorporation into nuclei. Both dominant negative JNK and Rac1(N17) also inhibited basal and Galpha 12(R203C)-induced BrdUrd incorporation; however, they were less potent as compared with dominant negative MAPK and Ras(N17). We confirmed the expression of the dominant negative proteins by Western blot analysis of samples from parallel cultures using specific antibodies (data not shown). These results clearly indicate that activation of Ras/MAPK and Ras/Rac1/JNK pathways are both necessary for Galpha 12(R203C)-induced G1 to S phase progression.

Table I.

Effects on Galpha 12(R203C)-induced stimulation of BrdUrd incorporation of dominant negative forms of MAPK and JNK, Ras(N17), and Rac1(N17)

NIH3T3(M17) cells were transfected with a combination of pSV-beta gal, pMT2-Galpha 12(R203C) and one of the expression plasmids carrying dominant negative forms of MAPK (MAPK-DN) and JNK (JNK-DN), Rac1(N17), or an empty vector. Expression of Ras(N17) was induced by dexamethasone treatment as described under "Experimental Procedures." The cells were incubated with BrdUrd in the presence of 0.5% calf serum for 17 h. Percentage of BrdUrd-positive cells of the beta -galactosidase-positive cell population was scored as described for Fig. 2.
Vector MAPK-DN Ras(N17)

%
Exp. 1a Vector 19.5 7.8 7.0
Galpha 12(R203C) 29.3 8.5 5.0
Exp. 2 Vector 13.5 8.9 4.9
Galpha 12(R203C) 23.3 6.8 6.9
Vector JNK-DN Rac1(N17)

%
Exp. 1 Vector 23.8 17.6 20.0
Galpha 12(R203C) 33.0 23.5 24.5
Exp. 2 Vector 12.5 7.8 7.8
Galpha 12(R203C) 22.8 14.0 15.8

a  Exp., experiment.


DISCUSSION

In the present work we studied the mitogenic signaling in Galpha 12(R203C)-induced G1 to S phase cell cycle progression. The mutation corresponding to Galpha 12(R203C) in Gsalpha renders it constitutively active and oncogenic (gsp) (20). It is also the site of ADP-ribosylation by cholera toxin, which activates it (19). The corresponding mutation in Gialpha 2 also activates it (21) and creates the gip2 oncogene (22). Finally, the corresponding mutation in Gqalpha renders it constitutively active (23). As a general mechanism, the mutation causes activation by decreasing the GTPase activity (19, 20). In view of these findings, we think it is reasonable to infer that the R203C mutation in Galpha 12 would also render it constitutively active, particularly since the data are consistent with this conclusion. The present study demonstrated that Galpha 12(R203C) stimulated G1 to S phase cell cycle progression in NIH3T3 cells in a manner dependent upon both MAPK and JNK cascades. Consistent with the ability of Galpha 12(R203C) to promote G1 to S progression, we observed that the expression of Galpha 12(R203C) led to stimulations of E2F-mediated transactivation and the cyclin A promoter activity, the latter of which was also dependent upon both MAPK and JNK. In addition, we found that the small G protein Ras was required for Galpha 12(R203C)-induced activation of MAPK, while both Ras and Rac were required for JNK activation and S phase entry. The results clearly indicate the critically important roles of both Ras/MAPK and Ras/Rac/JNK cascades in activated Galpha 12-induced G1/S cell cycle progression.

Besides well studied growth factor ligands for receptor protein-tyrosine kinases (34), certain agonists for G protein-coupled receptors, including thrombin, lysophosphatidic acid, and gastrin-releasing peptide/bombesin, are capable of stimulating mitogenesis (35-37). Several previous studies demonstrated that pertussis toxin caused substantial inhibition of thrombin- and lysophosphatidic acid-induced mitogenesis in fibroblasts, indicating a role for a heterotrimeric G protein of the Gi or Go classes (36-38). Subsequent studies demonstrated that activation of pertussis toxin-sensitive G proteins by these ligands led to activation of Ras, which was shown to mediate stimulation of DNA synthesis (39-41). On the other hand, there are also many reports describing that agonists for G protein-coupled receptors stimulate DNA synthesis through pertussis toxin-insensitive G proteins (1, 42). In addition, more recent studies demonstrated that thrombin and angiotensin II can activate Ras through a pertussis toxin-insensitive G protein in astroglial cells (9, 43) and cardiac myocytes (44). A very recent study by Aragay et al. (9) using microinjection of anti-Galpha 12 antibody indicated that Galpha 12 functions as a pertussis toxin-insensitive G protein that mediates thrombin-induced, Ras-dependent mitogenesis in human astroglial cells. Consistent with this report, the present results directly document that Galpha 12 causes stimulation of DNA synthesis in a Ras-dependent manner (Table I). In view of the ubiquitous expression of Galpha 12 in various tissues and cell types (4), it is a likely possibility that Galpha 12 is involved in the pertussis toxin-insensitive, Ras-dependent mitogenesis evoked by G protein-coupled receptor agonists in a variety of cell types.

There is now much evidence for the notion that Ras activates multiple signaling pathways for cell proliferation and differentiation. The Raf/MEK/MAPK cascade is the best characterized example of Ras-dependent signaling. The activation of the MAPK cascade is critically important for growth factor-induced G1/S progression, because expression of dominant negative forms of MEK (18) and a synthetic inhibitor of MEK (45) were shown to inhibit growth factor-induced DNA synthesis, and, conversely, expression of a constitutively active MEK induced cell proliferation and transformation (18, 46). It is known that activation of MAPK can be brought about by both Ras-dependent and -independent mechanisms in response to various external stimuli (18, 39-41, 43, 44, 47). The present study demonstrated that the expression of Galpha 12(R203C) led to activation of MAPK via a Ras-dependent mechanisms (Fig. 4). Galpha 12(R203C)-induced activation of MAPK is not as potent as that observed with acute growth factor stimulation but is very likely sustained, i.e. still evident 2 days after transfection. A previous study demonstrated that in fibroblasts stably expressing activated Galpha 12, MAPK activity was not increased under serum-deprived conditions as compared with cells transfected with an empty vector, although epidermal growth factor-stimulated MAPK activation was enhanced in Galpha 12-transformed cells (8). The reason for the discrepancy between the previous report and the present result is not clear. However, one reason might be that we adopted a transient transfection method with the Galpha 12(R203C) expression vector and the Myc-tagged MAPK expression vector. This may have allowed for the detection of a small increase in MAPK in the present study. The activation of MAPK in Galpha 12(R203C)-transfected cells appears to be critically important for G1/S cell cycle progression, because the dominant negative MAPK totally abolished Galpha 12(R203C)-induced DNA synthesis in the present study (Table I). It is poorly understood how the MAPK functions in mitogenic signaling, although the MAPK cascade was shown to be associated with activation of several different transcription factors including the Ets family proteins (48, 49). In the present study we demonstrated for the first time that MAPK is involved in activation of cyclin A promoter. It is now established that E2F functions as a crucial cell cycle-specific transcription factor to activate a number of genes (including cyclin A) that control cell cycle progression (14, 17). Therefore, Galpha 12(R203C)-induced activation of E2F is likely an important mechanism underlying Galpha 12(R203C)-evoked G1/S progression. Accumulated evidence indicates that E2F activation is brought about by phosphorylation of pRb and its related proteins by cyclin-dependent kinases (12-14). In middle to late G1, cyclin D-Cdk4 or cyclin D-Cdk6 complex is first activated to initiate phosphorylation of pRb family proteins. With regard to the activation mechanisms of Cdk4 or Cdk6, a very recent study demonstrated that a dominant negative MAPK inhibited epidermal growth factor-induced activation of cyclin D1 promoter (50). Since the induction of cyclin D1 in middle to late G1 is a prerequisite for activation of Cdk4 and/or Cdk6 (12-14), which in turn activates E2F, MAPK-mediated cyclin D1 induction may be an upstream mechanism for MAPK-mediated cyclin A induction at the G1/S boundary.

Recent evidence suggests that Ras may activate a second signaling pathway for cell proliferation and transformation, which involves Rac, a member of the Rho subfamily of small G proteins. Several studies demonstrated that expression of a dominant negative form of Rac1 blocked transformation by oncogenic Ras, suggesting that Rac functions downstream of Ras (51, 52). It was also shown that an activated form of Rac1 markedly synergized with activated Raf to enhance transformation (52). Further, it was demonstrated that microinjection of activated forms of Rac into quiescent fibroblasts stimulated G1/S cell cycle progression, whereas expression of a dominant negative form of Rac1 blocked serum-induced DNA synthesis (53). These observations suggest that in addition to the Raf/MEK/MAPK cascade, the Rac1 signaling cascade is required for Ras-mediated cell proliferation and transformation. In the present study we have shown that Galpha 12(R203C)-induced G1 to S progression is also dependent upon Rac (Table I). Recent studies identified JNK as a downstream effector of Rac (54, 55). JNK phosphorylates and activates the transcription factors c-Jun and ATF-2, which results in induction of c-Jun itself via two AP-1 sites in its promoter region (28, 54, 55). We demonstrated by employing a dominant negative form of JNK (Figs. 4 and 5, Table I) that JNK is actually involved in the Rac-dependent mitogenic signaling. Previous studies demonstrated that c-Jun, in conjunction with several related AP-1 proteins, promotes G1 phase progression and S phase entry (56, 57). The important AP-1 target genes implicated in G1 to S progression include cyclin D1 (50, 56). Therefore, Galpha 12(R203C)-induced, JNK-mediated cyclin A promoter activation (Fig. 4) may be mediated partly by cyclin D1 induction via the AP-1 site. Thus, both MAPK and JNK appear to be involved in sequential induction of cyclins and resultant activation of cyclin-dependent kinases at G1 and S.

Recent data demonstrate that beta gamma subunits of heterotrimeric G proteins can mediate activation of both MAPK and JNK in a Ras-dependent manner (58, 59). However, the present study demonstrated that the alpha  subunit of Galpha 12 alone is able to activate MAPK and JNK in Ras- and Ras/Rac-dependent manners, respectively. The Ras dependence of Galpha 12- or Galpha 13-induced JNK activation was also recently shown by another group (11). These observations provide compelling evidence that there is a Ras-dependent pathway activated by the alpha  subunits of G12 and G13. The direct effector of Galpha 12 in terms of Ras activation is presently unknown. Recent studies have demonstrated that Gq-coupled receptor agonists activate Ras via activation of nonreceptor tyrosine kinases through a mechanism involving tyrosyl phosphorylation of Shc, a SH2 domain-containing adaptor protein (44, 60). Shc thus phosphorylated at specific tyrosyl residues mediates recruitment of the Ras GDP/GTP exchange factor mSOS to the plasma membrane (61). Rac1 activation that appears to be required for Galpha 12-induced JNK activation is probably caused by recruitment and activation of a GDP/GTP exchange factor for Rac1. The activation of Ras might be necessary for the activation of the Rac1 GDP/GTP exchange factor. Further work will be required to understand the Galpha 12 effector pathway more thoroughly.


FOOTNOTES

*   This work was supported by grants from the Ministry of Education, Science and Culture of Japan, funds for cardiovascular research from Tsumura Co., and funds from the Japan Heart Foundation and Japan Research Foundation for Clinical Pharmacology. 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.
par    Present address: Tokai University School of Medicine, Bouseidai, Isehara, Kanagawa, 259-11, Japan.
Dagger Dagger    To whom all correspondence should be addressed: Dept. of Cardiovascular Biology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113, Japan. Tel: 81-3-3812-2111 (ext. 3469); Fax: 81-3-5800-6845.
1    The abbreviations used are: MAPK, mitogen-activated protein kinase; JNK, Jun N-terminal kinase; pRb, retinoblastoma protein; DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; BrdUrd, bromodeoxyuridine; bp, base pair(s); MEK, mitogen-activated protein kinase kinase.

Acknowledgments

We thank R. Nakanishi, F. Iwase, and W. Zhou for technical and secretarial assistance. We also thank Drs. G. M. Cooper, A. Kraft, and K. Okazaki for giving us NIH3T3 cells (M17), pGEX-2T-c-Jun-(5-89), and pactEF-MAPK and pactEF-MAPK-DN, respectively.


REFERENCES

  1. Neer, E. J. (1995) Cell 80, 249-257 [Medline] [Order article via Infotrieve]
  2. Strathmann, M. P., and Simon, M. I. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5582-5586 [Abstract]
  3. Chan, A. M.-L., Fleming, T. P., McGovern, E. S., Chedid, M., Miki, T., and Aaronson, S. T. (1993) Mol. Cell. Biol. 13, 762-768 [Abstract]
  4. Spicher, K., Kalkbrenner, F., Zobel, A., Harhammer, R., Nürnberg, B., Soling, A., and Schultz, G. (1994) Biochem. Biophys. Res. Commun. 198, 906-914 [CrossRef][Medline] [Order article via Infotrieve]
  5. Offerman, S., and Schultz, G. (1994) Mol. Cell. Endocrinol. 100, 71-74 [CrossRef][Medline] [Order article via Infotrieve]
  6. Xu, N., Bradley, L., Ambdukar, I., and Gutkind, J. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6741-6745 [Abstract]
  7. Jiang, H., Wu, D., and Simon, M. I. (1993) FEBS Lett. 330, 319-322 [CrossRef][Medline] [Order article via Infotrieve]
  8. Voyno-Yasenetskaya, T. A., Pace, A. M., and Bourne, H. R. (1994) Oncogene 9, 2559-2565 [Medline] [Order article via Infotrieve]
  9. Aragay, A. M., Collins, L. R., Post, G. R., Watson, A. J., Feramisco, J. R., Brown, J. H., and Simon, M. I. (1995) J. Biol. Chem. 270, 20073-20077 [Abstract/Free Full Text]
  10. Dhanasekaran, N., Prasad, M. V. V. S. V., Wadsworth, S. J., Dermott, J. M., and van Rossum, G. (1994) J. Biol. Chem. 269, 11802-11806 [Abstract/Free Full Text]
  11. Prasad, M. V. V. S. V., Dermott, J. M., Heasley, L. E., Johnson, G. L., and Dhanasekaran, N. (1995) J. Biol. Chem. 270, 18655-18659 [Abstract/Free Full Text]
  12. Sherr, C. J. (1994) Cell 76, 551-555
  13. Morgan, D. O. (1995) Nature 374, 131-134 [CrossRef][Medline] [Order article via Infotrieve]
  14. Nevins, J. R. (1992) Science 258, 424-429 [Medline] [Order article via Infotrieve]
  15. Johnson, D. G., Schwarz, J. K., Cress, W. D., and Nevins, J. R. (1993) Nature 365, 349-352 [CrossRef][Medline] [Order article via Infotrieve]
  16. Lam, E. W., and Watson, R. J. (1993) EMBO J. 12, 2705-2713 [Abstract]
  17. DeGregori, J., Kowalik, T., and Nevins, J. R. (1995) Mol. Cell. Biol. 15, 4215-4224 [Abstract]
  18. Cowley, S., Paterson, H., Kemp, P., and Marshall, C. J. (1994) Cell 77, 841-852 [Medline] [Order article via Infotrieve]
  19. Freissmuth, M., and Gilman, A. G. (1989) J. Biol. Chem. 264, 21907-21914 [Abstract/Free Full Text]
  20. Landis, C. A., Masters, S. B., Spada, A., Pace, A. M., Bourne, H. R., and Vallar, L. (1989) Nature 340, 692-696 [CrossRef][Medline] [Order article via Infotrieve]
  21. Wong, Y. H., Federman, A., Pace, A. M., Zachary, I., Evans, T., Pouysségur, J., and Bourne, H. R. (1991) Nature 351, 63-65 [CrossRef][Medline] [Order article via Infotrieve]
  22. Pace, A. M., Wong, Y. H., and Bourne, H. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7031-7035 [Abstract]
  23. Conklin, B. R., Chabre, O., Wong, Y. H., Federman, A. D., and Bourne, H. R. (1992) J. Biol. Chem. 267, 31-34 [Abstract/Free Full Text]
  24. Strathmann, M., Wilke, T. M., and Simon, M. I. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7407-7409 [Abstract]
  25. Cai, H., Szeberenyi, J., and Cooper, G. M. (1990) Mol. Cell. Biol. 10, 5314-5323 [Medline] [Order article via Infotrieve]
  26. Okazaki, K., and Sagata, N. (1995) Oncogene 10, 1149-1157 [Medline] [Order article via Infotrieve]
  27. Sells, M. A., and Chernoff, J. (1995) Gene (Amst.) 152, 187-189 [CrossRef][Medline] [Order article via Infotrieve]
  28. Gupta, S., Campbell, D., Derijard, B., and Davis, R. J. (1995) Science 267, 389-393 [Medline] [Order article via Infotrieve]
  29. Slansky, J. E., Li, Y., Kaelin, W. G., and Farnham, P. J. (1993) Mol. Cell. Biol. 13, 1610-1618 [Abstract]
  30. Henglein, B., Chenivesse, X., Wang, J., Eick, D., and Brechot, C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5490-5494 [Abstract]
  31. Baldin, V., Lukas, J., Marcote, M. J., Pagano, M., and Draetta, G. (1993) Gene. & Dev. 7, 812-821 [Abstract]
  32. Lubinus, M., Meier, K. E., Smith, E. A., Gause, K. C., LeRoy, E. C., and Trojanowska, M. (1994) J. Biol. Chem. 269, 9822-9825 [Abstract/Free Full Text]
  33. Derijard, B., Hibi, M., Wu, I.-H., Barrett, T., Su, B., Deng, T., Karin, M., and Davis, R. J. (1994) Cell 76, 1025-1037 [Medline] [Order article via Infotrieve]
  34. Ullrich, A., and Schlessinger, J. (1990) Cell 61, 203-212 [Medline] [Order article via Infotrieve]
  35. Takuwa, N., Takuwa, Y., Bollag, W. E., and Rasmussen, H. (1987) J. Biol. Chem. 262, 182-188 [Abstract/Free Full Text]
  36. Chambard, J. C., Paris, S., L'Allemain, G., and Pouysségur, J. (1987)
  37. Moolenaar, W. H. (1995) J. Biol. Chem. 270, 12949-12952 [Free Full Text]
  38. van Corven, E. J., Groenink, A., Jalink, K., Eichholtz, T., and Moolenaar, W. H. (1989) Cell 59, 45-54 [Medline] [Order article via Infotrieve]
  39. van Corven, E. J., Hordijk, P. L., Medema, R. H., Bos, J. L., and Moolenaar, W. H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1257-1261 [Abstract]
  40. Alblas, J. A., van Corven, E. J., Hordijk, P. L., Milligan, G., and Moolenaar, W. H. (1993) J. Biol. Chem 268, 22235-22238 [Abstract/Free Full Text]
  41. Winitz, S., Russel, M., Qian, N.-X., Gardner, A., Dwyer, L., and Johnson, G. L. (1993) J. Biol. Chem. 268, 19196-19199 [Abstract/Free Full Text]
  42. Takuwa, Y., Yanagisawa, M., Takuwa, N., and Masaki, T. (1989) Prog. Growth Factor Res. 1, 195-206 [Medline] [Order article via Infotrieve]
  43. LaMorte, V. J., Kennedy, E. D., Collins, L. R., Goldstein, D., Harootunian, A. T., Brown, J. H., and Feramisco, J. R. (1993) J. Biol. Chem. 268, 19411-19415 [Abstract/Free Full Text]
  44. Sadoshima, J., and Izumo, S. (1996) EMBO J. 15, 775-787 [Abstract]
  45. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7686-7689 [Abstract]
  46. Mansour, S. J., Matten, W. T., Hermann, A. S., Candia, J. M., Rong, S., Fukasawa, K., Wounde, G. F. V., and Ahn, N. G. (1994) Science 265, 966-970 [Medline] [Order article via Infotrieve]
  47. Koch, W., Hawes, B. E., Allen, L. F., and Lefkowitz, R. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12706-12710 [Abstract/Free Full Text]
  48. Davis, R. J. (1993) J. Biol. Chem. 268, 14553-14556 [Free Full Text]
  49. Marais, R., Wynne, J., and Treisman, R. (1993) Cell 73, 381-393 [Medline] [Order article via Infotrieve]
  50. Albanese, C., Johnson, J., Watanabe, G., Eklund, N., Vu, D., Arnold, A., and Pestell, R. G. (1995) J. Biol. Chem. 270, 23589-23597 [Abstract/Free Full Text]
  51. Qiu, R-G., Chen, J., Kirn, D., McCormick, F., and Symons, M. (1995) Nature 374, 457-459 [CrossRef][Medline] [Order article via Infotrieve]
  52. Khosravi-Far, R., Solski, P. A., Clark, G. J., Kinch, M. S., and Der, C. J. (1995) Mol. Cell. Biol. 15, 6443-6453 [Abstract]
  53. Olson, M. F., Ashworth, A., and Hall, A. (1995) Science 269, 1270-1272 [Medline] [Order article via Infotrieve]
  54. 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]
  55. Minden, A., Lin, A., Claret, F-X., Abo, A., and Karin, M. (1995) Cell 81, 1147-1157 [Medline] [Order article via Infotrieve]
  56. Karin, M. (1992) FASEB J. 6, 2581-2590 [Abstract/Free Full Text]
  57. Kovary, K., and Bravo, R. (1991) Mol. Cell. Biol. 11, 4466-4472 [Medline] [Order article via Infotrieve]
  58. Crespo, P., Xu, N., Simonds, W. F., and Gutkind, J. S. (1994) Nature 369, 418-420 [CrossRef][Medline] [Order article via Infotrieve]
  59. Coso, O. A., Teramoto, H., Simonds, W. F., and Gutkind, J. S. (1996) J. Biol. Chem. 271, 3963-3966 [Abstract/Free Full Text]
  60. Wan, Y., Kurosaki, T., and Huang, X. (1996) Nature 380, 541-544 [CrossRef][Medline] [Order article via Infotrieve]
  61. Pelicci, G., Lanfrancone, L., Grignani, F., McGlade, J., Cavallo, F., Forni, G., Nicoletti, I., Grignani, F., Pawson, T., and Pelicci, P. G. (1992) Cell 70, 93-104 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.