Regulation of Apoptosis by α-Subunits of G12 and G13 Proteins via Apoptosis Signal-regulating Kinase-1*

Many growth factors and G protein-coupled receptors activate mitogen-activated protein (MAP) kinase pathways. The MAP kinase pathways are involved in the regulation of the ubiquitous process of apoptosis or programmed cell death. Two related MAP kinase kinase kinases, apoptosis-signal regulating kinase 1 (ASK1) and MAP kinase kinase kinase 1 (MEKK1), stimulate c-Jun kinase (JNK) activity and induce apoptosis. Transient transfection of dominant negative and constitutively active components of the JNK pathway in COS-7 cells showed that two G protein subunits, Gα12 and Gα13, stimulated the JNK pathway in a ASK1- and MEKK1-dependent manner. Moreover, the mutationally activated Gα12 and Gα13 stimulated the kinase activity of ASK1. Both Gα12 and Gα13 employ small GTPases, Cdc42 and Rac1, to transduce signal to MEKK1 and, subsequently, to JNK. However, activation of JNK by Cdc42 and Rac1 did not require ASK1. Additionally, ASK1 and MEKK1 are involved in the apoptosis induced by Gα12 and Gα13. We conclude that Gα12 and Gα13 can induce apoptosis using two separate MAP kinase pathways; one is initiated by ASK1, and the other is initiated by MEKK1. Furthermore, Bcl-2 can block apoptosis induced by Gα12 and Gα13. This death-sparing function was associated with increased Bcl-2 phosphorylation, suggesting that phosphorylation of Bcl-2 may be a critical mechanism protecting cells from Gα12- and Gα13-induced apoptosis.

Mitogen-activated protein (MAP) 1 (1) kinases serve as a point of convergence for growth signals, including those generated from G protein-coupled receptors (1). The MAP kinase signaling pathway consists of three distinct members of the protein kinase family, including MAP kinase (MAPK), MAPK kinase (MAPKK), and MAPKK kinase (2). MAPKK kinase phosphorylates and activates MAPKK, and the activated form of MAPKK in turn phosphorylates and activates MAPK. Acti-vated MAPK may translocate to the cell nucleus and regulate the activities of transcription factors, thereby controlling gene expression. At least two defined MAP kinase signaling modules function in mammalian cells: the Raf-MEK-ERK, or ERK, pathway (3,4) and the MEKK1-JNK kinase-JNK, or JNK, pathway (5).
Apoptosis or programmed cell death is a highly conserved active cellular mechanism characterized by cell shrinkage, chromatin condensation, and nuclear fragmentation (6). Apoptosis occurs in many physiological and pathophysiological conditions and is a fundamental process for the normal development of multicellular organisms (6 -8). Apoptosis is controlled in part by a family of proteins whose prototype is Bcl-2. Bcl-2 protein blocks apoptosis mediated by many but not all mammalian physiological cell death stimuli (9). Phosphorylation is one of the mechanisms that may regulate the antiapoptotic function of Bcl-2 (10,11). Interestingly, Bcl-2 undergoes phosphorylation by JNK in the presence of constitutively activated member of Rho family G proteins, Rac1 (12).
The MAP kinase pathways, in particular the JNK pathway, participate in stress responses and apoptosis (13); the apoptosis signal-regulating kinase 1 (ASK1) corresponding to MAPKK kinase has been recently identified (14). ASK1 induces apoptosis and regulates the activity of JNK and p38 MAPK (14). Activated MEKK1 similarly induces apoptosis (15).
Among the important functions of G proteins, their involvement in the regulation of cell growth and mitogenesis contrasts with their signaling of apoptosis. Thus, activating mutations in G␣s and G␣i2 genes are found in specific subsets of human tumors (16,17). Additionally, mutationally activated ␣-subunits of G12 and G13 induce mitogenesis and neoplastic transformation in NIH3T3 cells and Rat-1 cells (18,19). In contrast, recent evidence demonstrates that heterotrimeric G proteins may be involved primarily in the signaling pathways that regulate apoptosis (20,21). In this study, we have analyzed the molecular mechanisms of signaling of apoptosis induced by G␣12 and G␣13. We found that both activation of JNK and apoptosis induced by G␣12 and G␣13 are mediated by two MAPKK kinases, MEKK1 and ASK1. We also observed that Bcl-2 inhibited apoptosis induced by G␣12 and G␣13. The results suggest that phosphorylation of Bcl-2 may be involved in the protective effect against apoptosis induced by G␣12 and G␣13.

EXPERIMENTAL PROCEDURES
Materials-Protein A-agarose was obtained from Life Technologies, Inc. Antibodies to Bcl-2, G␣12, G␣13, MEKK1, and 9E10 antibody to c-Myc epitope were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). [␥-32 P]ATP and [ 3 H]adenine were obtained from NEN Life Science Products. Polyclonal ␤-galactosidase antibody and rabbit rhodamine-conjugated IgG were purchased from Cappel and Pierce, respectively. The Apoptag Plus kit was purchased from Oncor. The production of ASK1 antibody (DAV) was described previously (22).
Mounting medium with 4Ј,6-diamidino-2-phenylindole was obtained from Vector Laboratories. The ECL kit was obtained from Amersham Pharmacia Biotech. All other reagents were obtained from Sigma.
DNA Constructs-Plasmids used in this work are as follows. Murine G␣12 and G␣13 in the Bluescript vector were gifts from M. Simon (Caltech, CA). Constructs of constitutively active mutants of G␣12-(Q229L), G␣13(Q226L), G␣s(Q227L), hemagglutinin-tagged JNK (JNK-HA), and MEKK1 were described previously (23). Myc-Cdc42(V12) and Myc-Rac(V12) were provided by M. Symons (Onyx, CA). Wild type and dominant negative ASK1 and MKK6 were previously described (14). Bcl-2 was a gift from David Ucker (University of Illinois, Chicago). The C terminus of ASK1 was tagged with HA epitope. Briefly, ASK1 was excised from pcDNA3 using XhoI and BstBI restriction enzymes. Next, purified ASK1 DNA fragment was ligated with pcDNA3 in the presence of annealed oligonucleotides encoding the HA epitope. The sequences of the oligonucleotides encoding the HA epitope were as follows: CGAAG-TTATGATGTTCCTGATTATGCTTGAT (upper strand) and CTAGATC-AAGCATAATCAGGAACATCATAACTT (lower strand). The sequence of the resulting construct was confirmed using restriction and sequencing analysis.
Cell Culture and DNA Transfection-COS-7 cells and human kidney embryonic 293 (HEK293) cells were propagated and transfected using the DEAE-dextran/chloroquine method, as described previously (23,24) with an efficiency of transfection of 60 -70%. To maintain uniform amount of transfected DNA, empty vector was added to the transfection mixture when necessary. Forty-eight hours after transfection, cells were used in the experiments.
JNK Activity-JNK activity was determined as described previously (23). Briefly, HA-tagged JNK was transfected in the presence of various cDNA constructs as described under "Results." Forty-eight hours after transfection, cells were lysed, and JNK-HA was immunoprecipitated using 12CA5 antibody and protein A-agarose. The kinase activity of JNK-HA was measured using recombinant c-Jun as a substrate. Recombinant c-Jun was produced in Escherichia coli as a glutathione S-transferase fusion protein expressed from plasmid pGEX-c-Jun (23). The kinase reaction was terminated with Laemmli buffer, proteins were separated on SDS-polyacrylamide gel electrophoresis, phosphorylated c-Jun was visualized by autoradiography, and radioactivity was quantitated with a Molecular Imager System (Bio-Rad). Aliquots of whole cell lysates from the same experiments were subjected to immunoblotting analysis to confirm the appropriate expression of transfected proteins as described under "Results." ASK1 Activity-ASK1-HA was transfected in the presence of various cDNA constructs as described under "Results." Forty-eight hours after transfection, cells were lysed in lysis buffer containing 1% Triton X-100, 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, 1 M sodium orthovanadate with 0.1 mM phenylmethanesulfonyl fluoride, 1 g/ml leupeptine, and 1 M pepstatin for 30 min at 4°C. Insoluble material was removed by centrifugation at 12,000 ϫ g for 15 min at 4°C. Supernatants were immunoprecipitated with 12CA5 antibody and protein A-agarose for 2 h at 4°C. To measure kinase activity of ASK1-HA, 0.2 g of glutathione S-transferase-MKK6 was incubated with the immune complexes for 20 min at 30°C in buffer containing 40 mM HEPES (pH 8.0), 5 mM magnesium acetate, 2 mM dithiothreitol, 1 mM EGTA, 50 M ATP, and 1 Ci of [␥-32 P]ATP. Recombinant catalytically inactive MKK6 was produced in E. coli as a glutathione Stransferase fusion protein expressed from the plasmid pGEX-MKK6(KN). The kinase reaction was terminated with Laemmli buffer, proteins were separated on SDS-polyacrylamide gel electrophoresis, phosphorylated MKK6 was visualized by autoradiography, and radioactivity was quantitated with a Molecular Imager System (Bio-Rad). The HA epitope did not alter the signaling functions of ASK1 based on the following observations. Titration experiments showed that comparable activation of the endogenous JNK can be achieved after transient transfection of either wild type ASK1 or HA-tagged ASK1 (data not shown). Aliquots of whole cell lysates from the same experiments were subjected to immunoblotting analysis to confirm the appropriate expression of transfected proteins as described under "Results." Bcl-2 Phosphorylation-Bcl-2 was transfected in the presence of various cDNA constructs as described under "Results," and each transfection was performed on 2 ϫ 10 6 cells in a 60-mm dish. Twenty-four hours after transfection, the cells were transferred into two wells of a six-well plate. For each transfection, one well was used for Bcl-2 phosphorylation assay, and the other was used for immunoblotting analysis to confirm the appropriate expression of transfected proteins. Cells were labeled with 0.5 mCi/ml [ 32 P]orthophosphoric acid in phosphate-free medium for 16 h. Cells were lysed with lysis buffer, and Bcl-2 protein was immunoprecipitated with 5 g of Bcl-2 antibody for 4 h at 4°C. The proteins were separated on SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and visualized by autoradiography, and radioactivity was quantitated with a Molecular Imager System (Bio-Rad). The same blot was then probed with Bcl-2 antibody and developed using the ECL kit.
Analysis of Apoptotic Phenotype-For apoptosis analysis, cells were co-transfected with cDNA encoding ␤-galactosidase. Twenty-four hours after transfection the cells were transferred into two wells of a six-well plate. Cells were grown on coverslips and fixed 48 h after transfection with 2% paraformaldehyde for 15 min. Cells were then permeabilized with 0.1% Triton X-100 for 10 min. Staining for fragmented genomic DNA with the Apoptag Plus kit was performed according to manufac-FIG. 1. ASK1 stimulates JNK activity. COS-7 cells were transiently transfected with 1 g of JNK-HA and the indicated doses of ASK1 and ASK1(K709R). To maintain an equal amount of DNA in each transfection, empty vector was used when necessary. Forty-eight hours after transfection, JNK activity was measured using exogenous substrate. A, JNK activity is expressed as -fold increase over basal level. Basal level of phosphorylation was defined as the amount of [␥-32 P]ATP incorporated into c-Jun by JNK-HA expressed in the cells alone. Data represent mean Ϯ S.E. of triplicate determinations obtained from five experiments. B, a representative experiment. Cell lysates were divided and immunoprecipitated (IP) with 12CA5 antibody, and kinase activity was measured using recombinant c-Jun as a substrate (top). The presence of ASK1 and ASK1(K709R) proteins was determined by immunoblotting with ASK1 antibody DAV (middle). The presence of JNK-HA was determined with HA antibody 12CA5 (bottom).
turer's instructions. ␤-Galactosidase was stained with ␤-galactosidase antibody (15 g/ml) for 1 h followed by a rabbit rhodamine-conjugated IgG for 30 min. Coverslips were mounted on glass slides using mounting medium with 4Ј,6-diamidino-2-phenylindole to visualize cell nuclei. Cells were counted using a Nikon inverted model 200 microscope; 200 -600 cells were counted in each experiment. Aliquots of whole cell lysates from the same transfections were subjected to immunoblotting analysis to confirm the appropriate expression of transfected proteins as described under "Results." cAMP Accumulation-cAMP accumulation was determined as described previously (19). Briefly, 24 h after transfection, cells were reseeded onto 24-well plates and labeled with [ 3 H]adenine (2 Ci/ml) for 24 h. For measuring cAMP accumulation, cells were washed once with Dulbecco's modified Eagle's medium and incubated (37°C for 30 min) in the same medium containing 1 mM 1-methyl-3-isobutylxanthine. Reactions were terminated by aspiration and the immediate addition of 5% ice-cold trichloroacetic asid. Acid-soluble nucleotides were separated on ion exchange columns as described (19), and results are expressed as (cAMP/cAMP ϩ ATP) ϫ 10 3 . (23,25,26) have shown previously that mutationally activated G␣12 and G␣13 stimulate JNK activity. In addition, both ␣-subunits employ small GTPases, Cdc42 and Rac (23,26), and serine-threonine kinase MEKK1 (23) to transduce signal to JNK.

G␣12 and G␣13 Stimulate JNK via ASK1-We and others
To examine if, in addition to MEKK1, other members of the MAP kinase family can mediate the regulation of JNK activity by G␣12 and G␣13, we used expression vectors encoding wild type and dominant negative ASK1. The substitution of Lys 709 to arginine makes ASK1 dominant negative; this mutant can prevent both activation of JNK and apoptosis induced by tumor necrosis factor-␣ and actinomycin D in lung epithelial cells (14). To investigate the involvement of ASK1 in the signaling pathways regulated by G␣12 and G␣13, JNK-HA activity was measured in COS-7 cells expressing different amounts of wild type ASK1 and dominant negative ASK1(K709R) (Fig. 1). In this model system, transient expression of wild type ASK1 dramatically stimulated JNK activity in a dose-dependent manner (Fig. 1). Titration experiments indicated that a 5-fold stimulation of JNK activity was observed with 100 ng of ASK1 plasmid. JNK activity was not changed with 200 ng of ASK1(K709R) plasmid. A further increase of ASK1(K709R) cDNA resulted in modest activation of JNK (Fig. 1). To dissect the regulation of ASK1 activity, we tested in our subsequent experiments the effect of upstream molecules by cotransfection with 100 ng/transfection of ASK1 cDNA or 200 ng/transfection of ASK1(K709R) cDNA. . JNK activity is expressed as -fold increase over control. Data represent mean Ϯ S.E. of triplicate determinations. Graphs, a representative experiment. Cell lysates were divided and immunoprecipitated with 12CA5 antibody, and kinase activity was measured using recombinant c-Jun as a substrate. The presence of JNK-HA was determined with HA antibody 12CA5. The presence of ASK1 and ASK1(K709R) proteins was determined by immunoblotting with ASK1 antibody DAV. The presence of G␣12 or G␣13 proteins was determined by immunoblotting with G␣12 or G␣13 antibody, respectively. Two additional experiments gave similar results.
FIG. 3. Effect of G␣12 and G␣13 on ASK1 activity. A, effect of G␣12 and G␣13 on ASK1 activity. COS-7 cells were transiently transfected with 0.1 g of ASK1-HA, 1 g of the indicated ␣-subunits, and 0.2 g of ASK1(K709R) as indicated. Forty-eight hours after transfection, cells were lysed, ASK1-HA was immunoprecipitated, and kinase activity of ASK1 was determined. Phosphorylation of recombinant MKK6 by ASK1 is expressed as -fold increase over control. The control level of phosphorylation was defined as the amount of [␥-32 P]ATP incorporated into the MKK6 by ASK1-HA expressed alone. Data represent mean Ϯ S.E. of triplicate determinations. B, a representative experiment. Cell lysates were divided and immunoprecipitated with 12CA5 antibody, and kinase activity was measured. The presence of ASK1-HA was determined by immunoblotting with 12CA5 antibody. The presence of G␣12 or G␣13 proteins was determined by immunoblotting with G␣12 or G␣13 antibody, respectively. The presence of ASK1 and ASK1(K709R) proteins was determined by immunoblotting with DAV antibody. The experiment was repeated with similar results.
To evaluate how ASK1 is involved in the activation of JNK by G␣12 and G␣13, we measured JNK-HA activity in cells expressing wild type and constitutively activated G␣12 and G␣13 and wild type and dominant negative ASK1 (Fig. 2). Consistent with previous results showing that G␣12 and G␣13 activate the JNK pathway (23,25,26), constitutively activated forms of both proteins stimulated JNK-catalyzed phosphorylation of c-Jun in COS-7 cells (Fig. 2). Co-expression of the ␣-subunits with ASK1 resulted in additive increases in JNK activity. In contrast, dominant negative ASK1(K709R) inhibited JNK activation induced by both G␣12 and G␣13 (Fig. 2, graphs). Expression of JNK-HA protein remained unchanged in all experimental conditions as judged by the immunoblotting analysis (Fig. 2, graphs). This result indicates that changes in the JNK-HA activity are not due to changes in JNK-HA expression. Similarly, expression of G␣12, G␣13, ASK1, or ASK1(K709R) proteins remained unchanged after co-expression with indicated cDNA constructs as it was determined by the immunoblotting analysis with polyclonal antibodies to G␣12, G␣13, or ASK1 (Fig. 2, graphs). Thus, in addition to MEKK1 (23) G␣12 and G␣13 appear to stimulate JNK via ASK1.
To examine how G␣12 and G␣13 affect the kinase activity of ASK1, COS-7 cells were transiently transfected with ASK1 tagged with the HA epitope (ASK1-HA) at the C terminus. After immunoprecipitation with 12CA5 antibody and kinase assay with an exogenous substrate, we found that constitutively activated forms of both G proteins stimulated ASK1catalyzed phosphorylation of MKK6 in COS-7 cells (Fig. 3, A  and B). In agreement with previous observations (14), stimulation of the cells with tumor necrosis factor-␣ for 30 min also resulted in ASK1 activation. Dominant negative ASK1(K709R) inhibited ASK1 activation induced by G␣12, G␣13, and tumor necrosis factor-␣ (Fig. 3). Control immunoblotting from the same lysates confirmed appropriate expression from transfected plasmids (Fig. 3), indicating that G␣12 and G␣13 stimulate kinase activity of ASK1 in vivo.
Cdc42 and Rac1 Stimulate JNK via MEKK1 but Not via ASK1-Small GTPases, Rac1, Cdc42, and RhoA regulate the activity of the JNK pathway (27,28). Because G␣12 and G␣13 regulate JNK activity via Cdc42 and Rac1 (23,26), we investigated if Cdc42 and Rac1 employ the serine/threonine kinase ASK1 to stimulate JNK. Co-expression of constitutively activated Cdc42(V12) with ASK1 resulted in a synergistic increase of JNK activity (Fig. 4A). In addition, dominant negative ASK1(K709R) did not affect JNK activation induced by Cdc42(V12) (Fig. 4A). Similarly, co-expression of constitutively activated Rac1(V12) with ASK1 resulted in a synergistic increase of the JNK activity (Fig. 4A). Finally, dominant negative ASK1(K709R) did not affect the JNK activation induced by Rac1(V12) (Fig. 4A). The expression of JNK-HA protein remained unchanged in all experimental conditions as it was judged by immunoblotting analysis (Fig. 4A, graph), indicating that observed changes in the JNK-HA activity are not due to changes in JNK-HA expression. Control immunoblotting from the same lysates confirmed appropriate expression of the wild type and mutant ASK1, Cdc42(V12), and Rac(V12) from trans- Graph, a representative experiment. Cell lysates were divided and immunoprecipitated with 12CA5 antibody, and kinase activity was measured using recombinant c-Jun as a substrate. The presence of JNK-HA was determined by immunoblotting with 12CA5 antibody. The presence of ASK1 and ASK1(K709R) proteins was determined by immunoblotting with DAV antibody. Expression of Myc-Cdc42(V12) or Myc-Rac(V12) was determined by immunoblotting with 9E10 antibody, which recognizes the c-Myc epitope. The experiment was repeated with similar results. B, ASK1 activity in the cells expressing Cdc42 and Rac. COS-7 cells were transiently transfected with 0.1 g of ASK1-HA, 1 g of indicated Cdc42(V12) or Rac1(V12) as indicated. Cell lysates were divided and immunoprecipitated with 12CA5 antibody, and kinase activity was measured using recombinant MKK6 as a substrate. The presence of ASK1-HA was determined by immunoblotting with 12CA5 antibody. Expression of Myc-Cdc42(V12) or Myc-Rac(V12) was determined by immunoblotting with 9E10 antibody. C, effect of MEKK1 on JNK activated by Cdc42 and Rac1. COS-7 cells were transiently transfected with 1 g of JNK-HA, 1 g of Cdc42(V12) or Rac1(V12), 1 g MEKK1(K432A) as indicated. Data represent mean Ϯ S.E. of triplicate determinations. Graph, a representative experiment. Cell lysates were divided and immunoprecipitated with 12CA5 antibody, and kinase activity was measured using recombinant c-Jun as a substrate. The presence of JNK-HA was determined by immunoblotting with 12CA5 antibody. Expression of Myc-Cdc42(V12) or Myc-Rac(V12) was determined by immunoblotting with 9E10 antibody. The presence of MEKK1 protein was determined by immunoblotting with MEKK1 antibody. The experiment was repeated with similar results. fected plasmids (Fig. 4A, graph), suggesting that small G proteins Cdc42 and Rac1 and serine/threonine kinase ASK1 use different pathways to stimulate JNK.
To confirm that ASK1 is not involved in the pathway that leads from Cdc42 and Rac to stimulation of JNK, the ASK1 activity was measured in the cells expressing activated Cdc42 and Rac (Fig. 4B). Our results showed that co-expression of either Cdc42(V12) or Rac(V12) with ASK1-HA did not result in increased catalytic activity of the tested kinase (Fig. 4B). At the same time, immunoblotting analysis of the total cell lysates showed the appropriate expression of the proteins from the transfected plasmids (Fig. 4B, graph).
Apoptosis Induced by G␣12 and G␣13 Is Mediated by both ASK1 and MEKK1-To study the apoptosis induced by G␣12 and G␣13, ␤-galactosidase cDNA was co-transfected with the various ␣-subunits. Twenty-four and 48 h after transfection, the extent of cell death was estimated measuring the number of cells with fragmented DNA using the Apoptag Plus kit (Fig. 5, A-C). Constitutively activated G␣12 and G␣13 induced apoptosis in 25-30% of the cells (Fig. 5D). Apoptosis induced by wild type G␣12 and G␣13 was less pronounced. To study the specificity of apoptotic response induced by G␣12 and G␣13, constitutively activated G␣s(Q227L) was used. G␣s(Q227L) did not induce apoptosis when transiently transfected into COS-7 cells (Fig. 5D). In the same cells, G␣s(Q227L) induced a 6-fold increase in cAMP accumulation (Fig. 5E), confirming the functional activity of the expressed protein.
We next examined if serine/threonine kinases ASK1 and MEKK1 are involved in the apoptosis induced by G␣12 and G␣13. Co-expression of the tested ␣-subunits with ASK1 resulted in an additive increase in apoptosis (Fig. 6, A and B). Moreover, dominant negative ASK1(K709R) inhibited apoptosis induced by G␣12 and G␣13 by 50 -60% (Fig. 6, A and B). Similarly, co-expression of the tested ␣-subunits with MEKK1 resulted in additive increase in apoptosis (Fig. 6, A and B). Additionally, dominant negative MEKK1(K432A) inhibited apoptosis induced by G␣12 and G␣13 by 50 -60% (Fig. 6, A and  B). Immunoblotting analysis from the same lysates confirmed appropriate expression of the proteins from transfected plasmids (Fig. 6, A and B, graphs), indicating that apoptosis induced by G␣12 and G␣13 is mediated by both ASK1 and MEKK1.
Bcl-2 Prevents Apoptosis and Undergoes Phosphorylation in Cells Expressing G␣12 and G␣13-The protooncogene Bcl-2 functions as an apoptosis suppresser in many systems (29 -33), although its mechanism of action is not yet known. Emerging data imply that members of the Bcl-2 gene family are targets for phosphorylation, suggesting one potential mechanism of control (10,12). Co-expression of Bcl-2 with constitutively activated G␣12 and G␣13 almost completely abolished apoptosis induced by these ␣-subunits (Fig. 7A). Immunoblotting analysis revealed endogenously expressed G␣12 and G␣13 in COS-7 FIG. 5. G␣12 and G␣13 induce apoptosis. In the micrographs, chromatin condensation is shown morphologically by staining with Apoptag Plus of COS-7 cells induced to undergo apoptosis after transfection with G␣12 and G␣13. A, phase contrast micrograph of COS-7 cells at ϫ 100 magnification. B, the same field stained with Apoptag Plus conjugated with fluorescein showing the fragmented nucleus. C, staining of the cells expressing ␤-galactosidase with ␤-galactosidase antibody conjugated with rhodamine. Cells with fragmented nuclei were counted among cells expressing ␤-galactosidase. D, effect of different G␣ subunits on apoptosis in COS-7 cells. One microgram of the indicated G␣ subunits was transfected into COS-7 cells, and the number of cells that underwent apoptosis was estimated 24 or 48 h after transfection. In each experiment, 200 -600 cells were counted. Data represent mean Ϯ S.E. of triplicate independent experiments. Transient transfection of G␣12 and G␣13 in HEK293 cells also resulted in apoptosis; the percentage of apoptotic cells was similar to that found in COS-7 cells (data not shown). E, cAMP accumulation in the cells expressing G␣s(Q227L). After transfection, cells were divided for apoptosis and cAMP assays. The data represent the mean Ϯ S.E. of triplicate determinations in a representative experiment; two additional experiments gave similar results.
cells. The amount of the G protein ␣-subunits was apparently increased in the cells transfected with cDNAs encoding G␣12 or G␣13. At the same, the commercial antiserum for Bcl-2 failed to detect endogenous Bcl-2 protein; however, the transfected Bcl-2 protein was readily detectable (Fig. 7A, graph).
We investigated if the phosphorylation state of Bcl-2 was affected by G␣12 and G␣13. Following metabolic labeling with inorganic 32 P, Bcl-2 could be identified as a phosphoprotein (Fig. 7B). Incorporation of labeled phosphate into Bcl-2 in the cells expressing G␣12 and G␣13 was significantly higher. However, the amount of Bcl-2 protein expressed in the same experimental conditions remained unchanged as tested with Western blotting analysis (Fig. 7B), indicating a net increase in Bcl-2 phosphorylation. These data show that G protein-induced apoptosis was suppressed by Bcl-2 and that this sparing effect was associated with Bcl-2 phosphorylation.

DISCUSSION
Our results allow us to draw three new conclusions with respect to the effects of G␣12 and G␣13 on downstream signaling pathways. These proteins (a) activate MAPKK kinase ASK1 in a Cdc42-and Rac1-independent manner; (b) induce apoptosis by a mechanism that involves MEKK1 and ASK1; and (c) induce the phosphorylation of the antiapoptotic Bcl-2 protein.
Activation of ASK1 by G␣12 and G␣13-In the present investigation, we characterized a new signaling pathway that is regulated by G␣12 and G␣13. Recently identified MAPKK kinase ASK1 activates MKK4 and MKK6, which in turn activate JNK and p38 MAPK, respectively (14). In addition, ASK1 is activated by tumor necrosis factor-␣ and induces apoptosis (14). We tested if ASK1 is regulated by ␣-subunits of heterotrimeric G12 and G13 proteins. Our data show that in transient transfection experiments, constitutively activated mutants of G␣12 and G␣13 proteins stimulate the kinase activity of ASK1 (Figs. 3 and 8). In our previous work, we showed that G␣12 and G␣13 stimulate JNK via MEKK1 (23). We now show that G␣12 and G␣13 stimulate JNK via ASK1 in addition to MEKK1 (Figs. 2 and 8).
The MAP kinase pathways (ERK and JNK) are typically regulated by small G proteins: Ras in the case of the ERK pathway (34,35) and Cdc42, Rac, and Rho (27,28) in the case of the JNK pathway. Moreover, regulation of these MAP kinase pathways by heterotrimeric G proteins seems to require activation of the small G proteins mentioned above. Thus, G i -dependent activation of ERK requires Ras (36), and G12/G13-dependent regulation of JNK requires Cdc42 (23) or Rac (26). We therefore investigated if (a) Cdc42 and Rac stimulate JNK via ASK1 and (b) if G12/G13-dependent activation of ASK1 requires Cdc42 or Rac1. Our results show that G␣12 and G␣13 stimulate ASK1 independently of Cdc42 or Rac1 (Fig. 4). Finally, stimulation of the JNK by Cdc42 and Rac1 occurs by a mechanism that requires MEKK1 but not ASK1 (Figs. 4 and 8). Whether small G proteins can regulate ASK1 activity remains to be investigated.
G␣12 and G␣13 Induce Apoptosis by a Mechanism That Involves MEKK1 and ASK1-Several lines of evidence suggest that heterotrimeric G proteins are involved in the regulation of apoptosis in mammalian cells as follows. (a) G protein-coupled receptors can either prevent or induce apoptosis. Somatostatin receptor type 3 and purinergic receptor P2Y2 induce apoptosis in different cell systems (37,38). In contrast, activation of muscarinic cholinergic receptors blocks apoptosis of cultured cerebellar neurons (39). (b) Small G proteins, such as Ha-Ras, R-Ras, Rho, and Rac, which are regulated by heterotrimeric G proteins (among other stimuli), induce apoptosis in certain cell systems. For example, two small G proteins, R-Ras and Rho, induce apoptosis in cultured cells via a Bcl-2-suppressible mechanism (40,41). Additionally, in human leukemic Jurkat cells, apoptosis induced by ceramide and Fas ligand was blocked after inhibition of Ras, Rac1, or JNK/p38 MAPK (42,43). Finally, genetic inhibition of Ras and Rac by dominant negative N17Ras and N17Rac, antisense oligonucleotides to Rac, or cellular treatment with Botulinus C3 exoenzyme prevent Ras stimulation as well as apoptosis after Fas receptor stimulation (43). (c) The JNK/p38 MAPK pathways, which are regulated by heterotrimeric G proteins (among other stimuli), cause apoptosis in certain cell systems. Thus, persistent activation of JNK is involved in the initiation of the apoptosis (13,44). (d) Apoptosis induced by familial Alzheimer's disease-associated mutants of the amyloid precursor protein is mediated by the Go protein (20). A recent report by Althoefer (21) shows that G␣q and G␣13 induce apoptosis that is Bcl-2 suppressible and does not require serum deprivation (21).
In our experiments, we found that constitutively activated G␣12 and G␣13 induce apoptosis in COS-7 (Fig. 5) and HEK293 cells (data not shown). In addition, we found that apoptosis induced by G␣12 and G␣13 was executed by a MEKK1-and ASK1-dependent mechanism (Fig. 6). We also found that ASK1 is a more potent inducer of apoptosis than MEKK1 (Fig. 6); to induce an equal amount of apoptotic cells, 10 times more MEKK1 was required than ASK1 (Fig. 6). However, the mechanism by which ASK1 induces apoptosis is unknown. One possibility is that, similarly to MEKK1 (45), ASK1 activation by caspase cleavage will be required for apoptotic response.
G␣12 and G␣13 Induced Apoptosis by a Mechanism That Involves Phosphorylation of Bcl-2-In the Bcl-2 family of proteins, both repressors and promoters of apoptosis are found (46,47). Bcl-2, Bcl-xl, Mcl-1, and A1 inhibit apoptosis, whereas Bax, Bcl-xs, Bak, and Bad promote apoptosis. These proteins can form homo-and heterodimers that are important in determining sensitivity to apoptotic signals (46,48). Additionally, phosphorylation of Bcl-2 is required for its antiapoptotic function in some instances (10). Thus, removal of putative protein kinase C phosphorylation sites eliminates the ability of Bcl-2 to protect cells from apoptosis induced by growth factor deprivation (10). Recently, it was shown that Bcl-2 can be phosphorylated both in vivo and in vitro by JNK (12). Interestingly, mutationally activated Rac1 induces Bcl-2 phosphorylation (12).
In our experiments, Bcl-2 protected cells from apoptosis induced by G␣12 and G␣13 (Figs. 7 and 8). Moreover, G␣ subunits induced phosphorylation of Bcl-2 ( Figs. 7 and 8). In conclusion, G␣12 and G␣13 can induce either mitogenesis and neoplastic transformation (18,19) or apoptosis (Ref. 21; this paper), depending on the cell type. However, the mechanisms that allow these two G proteins to regulate such different events are not known. We now know several intracellular molecular steps that connect G␣12 and G␣13 with either mitogen- FIG. 7. Apoptosis induced by G␣12 and G␣13 is inhibited by Bcl-2. A, Bcl-2 inhibited apoptosis induced by G␣12 and G␣13. COS-7 cells were transiently transfected with 1 g of ␣-subunits and 1 g of Bcl-2 as indicated. The number of cells that underwent apoptosis was estimated 48 h after transfection. In each experiment, 400 cells were scored. Data represent mean Ϯ S.E. of triplicate independent experiments. Graph, expression of the transfected cDNAs. After transfection, cells were divided for apoptosis and immunoblotting assays. Immunoblotting of aliquots of whole cell lysate with G␣12 (top) or G␣13 (middle) antibody is shown. Expression of Bcl-2 protein was determined by immunoblotting with Bcl-2 antibody (bottom). B, incorporation of inorganic 32 P into Bcl-2 in cells overexpressing G␣12 and G␣13. After transfection, cells were divided for phosphorylation and immunoblotting assays. Immunoblotting of aliquots of whole cell lysate with Bcl-2, G␣12, or G␣13 antibody is shown. The experiment was performed twice with similar results.
FIG. 8. Signaling pathways connecting G␣12 and G␣13 with activation of JNK (A) and apoptosis (B). G␣12 and G␣13 stimulate JNK using two independent pathways; one pathway connects G␣ subunits to JNK via small G proteins, Cdc42 and Rac, and kinase MEKK1. Another pathway is using ASK1 to stimulate JNK. However, both MEKK1 and ASK1 are involved in the apoptosis induced by G␣12 and G␣13 proteins. Question marks show unknown participants in the G␣12 and G␣13 signaling pathway. JNKK, JNK kinase. esis or apoptosis (18,19,21,26). One possibility is that different composition of signaling components that can be regulated by G␣12 and G␣13 will determine the cellular response initiated by these G proteins.