©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of Jun Kinase/Stress-activated Protein Kinase by GTPase-deficient Mutants of G and G(*)

(Received for publication, March 30, 1995; and in revised form, May 19, 1995)

M. V. V. S. Vara Prasad (1)(§) Jonathan M. Dermott (1)(§) Lynn E. Heasley (2) Gary L. Johnson (3) N. Dhanasekaran (1)(¶)

From the  (1)Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, the (2)Department of Renal Medicine, University of Colorado Medical School, Denver, Colorado 80262, and the (3)Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Signal transduction pathways regulated by G and G heterotrimeric G proteins are largely unknown. Expression of activated, GTPase-deficient mutants of alpha and alpha alter physiological responses such as Na/H exchanger activity, but the effector pathways controlling these responses have not been defined. We have found that the expression of GTPase-deficient mutants of alpha (alphaQ229L) or alpha (alphaQ226L) leads to robust activation of the Jun kinase/stress-activated protein kinase (JNK/SAPK) pathway. Inducible alphaQ229L and alphaQ226L expression vectors stably transfected in NIH 3T3 cells demonstrated JNK/SAPK activation but not extracellular response/mitogen-activated protein kinase activation. Transient transfection of alphaQ229L and alphaQ226L also activated the JNK/SAPK pathway in COS-1 cells. Expression of the GTPase-deficient mutant of alpha(q) (alpha(q)Q209L) but not alpha(i) (alpha(i)Q205L) or alpha(s) (alpha(s)Q227L) was also able to activate the JNK/SAPK pathway. Functional Ras signaling was required for alphaQ229L and alphaQ226L activation of the JNK/SAPK pathway; expression of competitive inhibitory NRas inhibited JNK/SAPK activation in response to both alphaQ229L and alphaQ226L. The results describe for the first time a Ras-dependent signal transduction pathway involving JNK/SAPK regulated by alpha and alpha.


INTRODUCTION

The alpha- and the beta-subunits of heterotrimeric G proteins (^1)regulate the activity of diverse effectors in response to the activation of seven transmembrane receptors(1, 2, 3) . Galpha-subunits are grouped based on amino acid homology into four sub-families: G(s), G(i), G(q), and G(3) . Cellular signal transduction pathways regulated by the G members, alpha and alpha, remain most poorly defined. Several studies suggest alpha and alpha regulate signal pathways involved in controlling cell growth and differentiation (4, 5, 6, 7, 8, 9, 10) . For example, mutation of cta, a Drosophila homologue of alpha disrupts the ventral furrow formation in Drosophila embryos(4) ; alpha has been identified as a putative oncogene of soft tissue sarcomas(5) ; the overexpression of wild type alpha and alpha or the expression of activated GTPase-deficient mutants of alpha (alphaQ229L) and alpha (alphaQ226L) transform fibroblast cell lines(6, 7, 8, 9, 10) .

Activation of the extracellular response kinase/mitogen-activated protein kinase (ERK/MAPK) pathway has been strongly correlated with growth and transforming activities in fibroblasts(11) . Expression of alphaQ229L or alphaQ226L did not activate ERK/MAPK pathway but may enhance epidermal growth factor-stimulated ERK/MAPK activity in Rat1 cells(10) . Expression of an activated mutant of alpha also does not activate the ERK/MAPK pathway in COS-1 cells(12) . More recently, proline-directed protein kinases related to ERK/MAPK referred to as Jun kinase/stress-activated protein kinases (JNK/SAPK) have been identified(13, 14, 15, 16, 17) . Although the role of JNK/SAPKs in the control of cell growth and differentiation remains unclear at this time, they do appear to be activated in response to cellular stress and osmotic imbalance(13, 14, 15, 16, 17) .

The JNK/SAPKs phosphorylate and activate c-Jun, the basal transcription factor involved in the regulation of different phases of cell growth, differentiation, and other physiological responses(18) . Previously, it was demonstrated that alphaQ229L- and alphaQ226L-transformed NIH 3T3 cells had increased c-Jun expression(9) . Here, we demonstrate that the expression of alphaQ229L and alphaQ226L lead to the activation of JNK/SAPK. The results demonstrate a Ras-dependent signal transduction pathway controlled by alpha and alpha subunit proteins.


EXPERIMENTAL PROCEDURES

Plasmids, Cell Culture, and Transfections

COS-1 and NIH 3T3 cells (ATCC) were maintained by serial passage in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) containing 10% newborn calf serum (Life Technologies, Inc.), 50 units/ml penicillin, and 50 µg/ml streptomycin at 37 °C in a 5% CO(2) incubator. pcDNA3-alphaQ229L plasmid was constructed by ligating the EcoRI-XbaI (1.8 kilobase) fragment from pcDNA1-alphaQ229L into the EcoRI-XbaI site of pcDNA3 vector (Invitrogen). pcDNA3-alphaQ226L was constructed by ligating the BamHI-XbaI fragment (2.4 kilobases) into the BamHI-XbaI site of pcDNA3. Plasmids were purified using a cesium chloride gradient. The procedures for the transfection and transformation of NIH 3T3 cells have been previously described(9) . NIH 3T3 cell lines expressing inducible alphaQ229L and alphaQ226L were established using the Lac Switch inducible expression system (Stratagene). Blunt-ended 1.8-kilobase HindIII-XbaI fragment of alphaQ229L or the 2.4-kilobase BamHI-XbaI fragment of alphaQ226L excised from the respective pcDNA3 vectors was ligated into the blunted NotI site of pOPRSVI plasmid following the published procedures(19) . The orientation of the constructs was verified by restriction analysis, and the plasmids were purified. NIH 3T3 cells (0.75 10^6) were cotransfected with p3`SS plasmid vector expressing the Lac repressor and pOPRSVI-alphaQ229L or pOPRSVI-alphaQ226L vector (5 µg each) by electroporation. A control NIH 3T3 cell line was established by cotransfecting p3`SS and pOPRSVI vectors alone. Electroporation was carried out using a Bio-Rad gene pulser as follows: 4-mm electroporation cuvette containing 0.75 10^6 cells in 0.5 ml of DMEM with 10% FBS and 5 µg of plasmid DNA was placed in a gene pulser chamber, and a pulse was delivered at 0.2 kV and 960 microfarads. After the pulse, the cells were incubated at room temperature for 5 min, resuspended in 10 ml of DMEM containing 10% FBS, and plated in a 100-mm culture dish. After 48 h, the cells were split into 1:4 ratio, and hygromycin (50 µg/ml, Calbiochem) and G-418 (400 µg/ml, Life Technologies, Inc.) were added to select the transformed cells. The selection was carried out for 3 weeks with biweekly feedings, and individual clones were isolated and propagated. The expression of alphaQ229L and alphaQ226L following induction with 1 mM IPTG was determined by Northern blot analyses using total RNA from these cells. Clones having the lowest expression of the mutant Galpha subunit in the absence of IPTG were selected and used for further experiments. Transfection of COS-1 cells was carried out using lipofectamine reagent (Life Technologies, Inc.) following the manufacturer's protocol. COS-1 cells (2 10^5) were seeded in a 35-mm dish with 2 ml of DMEM containing 10% FBS. 24 h later, the cells were washed with 2 ml of serum-free medium. Meanwhile, the respective plasmids (0.5 µg each) along with 0.5 µg of the reporter plasmid pSVbetaGal (Promega) were taken in 100 µl of DMEM and mixed with 100 µl of DMEM containing 6 µl of lipofectamine (Life Technologies, Inc.). The DNA-lipid complexes were allowed to form at room temperature for 30 min. The complex was supplemented with 0.8 ml of DMEM and was added to the cells. The dishes were incubated at 37 °C for 6 h, after which 1 ml of DMEM with 20% FBS was added, and the incubation was continued. The medium was replaced at 24 h, and the cells were harvested for analysis at 60 h post-transfection.

Solid Phase JNK/SAPK Assay

Assay of Jun kinase activity was carried out following previously published procedures(20, 21) . Actively growing NIH 3T3 cells (1 10^6) were seeded in 100-mm dishes containing DMEM with 10% newborn calf serum. 24 h later, the cells were serum starved by replacing them in DMEM containing 0.2% bovine serum albumin. The cells were allowed to reach quiescence for 16-24 h, and they were stimulated with or without growth factors. After growth factor stimulation, the cells were rinsed with ice-cold phosphate-buffered saline and harvested in 400 µl of lysis buffer containing 25 mM HEPES (pH 7.6), 0.1% Triton X-100, 300 mM NaCl, 1.5 mM MgCl(2), 20 mM beta-glycerophosphate, 100 µM Na(3)VO(4), 0.2 mM EDTA, 0.5 mM dithiothreitol, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 100 µM phenylmethylsulfonyl fluoride, and 1 mM benzamidine. The cell lysates were centrifuged for 10 min in a microcentrifuge (16,000 g), and the supernatants were normalized for protein content. The solid phase kinase assay was carried out using GST-c-Jun(1-79) bound glutathione-Sepharose (Pharmacia Biotech Inc.) beads as substrate. GST-c-Jun(1-79)-bound glutathione-Sepharose beads were prepared as previously described(20) . 10 µl of 50% GST-c-Jun(1-79)-bound glutathione-Sepharose beads were washed with JNK lysis buffer and incubated with 100 µg of lysate protein (in 100 µl) for 2 h at 4 °C in a rotator. At the end of the incubation, the beads were washed twice in JNK lysis buffer followed by two washes in JNK buffer containing 20 mM HEPES, pH 7.6, 20 mM beta-glycerophosphate, 10 mM MgCl(2), and 100 µM Na(3)VO(4). The kinase reaction was carried out by resuspending the beads in 40 µl of JNK buffer containing 20 µM [-P]ATP (5000 cpm/pmol) and incubating them for 20 min at 30 °C. After stopping the reaction with the addition of Laemmli's buffer followed by boiling the samples for 3 min, the phosphorylated GST-c-Jun was separated on 12% SDS-PAGE. The gel was dried, and an autoradiogram was developed. The radioactive GST-c-Jun band was excised out and quantitated in a liquid scintillation counter.

Immune Complex JNK/SAPK Assay

COS-1 cells transfected with HA-JNK1 along with test plasmids were subjected to immune complex JNK assay according to the previously published procedures(20, 21) . The transfected COS-1 cells were lysed in 300 µl of JNK lysis buffer, and the lysate was prepared as described in the case of NIH 3T3 cells. The cell lysates were normalized for transfection using the expression of beta-galactosidase reporter activity(22) . The HA epitope-tagged JNK1 in 100 µg of cell extract was immunoprecipitated by incubating the lysate with 1 µg of monoclonal (12CA5) HA antibodies (Boehringer Mannheim) for 1 h. This was followed by an additional incubation with 20 µl of protein A-Sepharose (Pharmacia) for 1 h. The protein A-Sepharose beads were washed twice with JNK lysis buffer followed by two washes with JNK reaction buffer. The beads were resuspended in 40 µl of JNK buffer containing 20 µM [-P]ATP (5000 cpm/pmol), and the kinase reaction was carried out for 20 min at 30 °C using 3 µg of purified GST-c-Jun(1-79) as substrate. The reaction was stopped, and the radioactivity in the phosphorylated GST-c-Jun was quantitated as described above.

ERK1/2 Assay

Cell extracts were prepared as described above using ERK lysis buffer containing 20 mM HEPES (pH 7.4), 50 mM beta-glycerophosphate, 0.5% Triton X-100, 2 mM MgCl(2), 1 mM EGTA, 1 mM dithiothreitol, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 100 µM phenylmethylsulfonyl fluoride, and 1 mM benzamidine. ERK1 or ERK2 in 100 µg of cell lysate was immunoprecipitated by incubating 100 µg of lysate protein with 1 µg of the respective polyclonal antibodies (antibodies sc-19 and sc-154 to ERK1 and ERK2, respectively, are from Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h followed by an additional incubation with 20 µl of protein A-Sepharose (Pharmacia) for 1 h. The immune complex-bound protein A-Sepharose beads were washed twice with lysis buffer and twice with ERK/MAPK buffer containing 20 mM HEPES (pH 7.4), 50 mM beta-glycerophosphate, 10 mM MgCl(2), 1 mM EGTA, 1 mM dithiothreitol, and 100 µM Na(3)VO(4). The beads were resuspended in 40 µl of MAPK buffer containing 100 µM [-P]ATP (5000 cpm/pmol), and the kinase assay was carried out for 20 min at 30 °C using 5 µg of myelin basic protein as substrate. The reaction was stopped by the addition of Laemmli's sample buffer followed by the boiling of the samples for 3 min. The proteins were resolved on 12% SDS-PAGE; the gel was dried, and an autoradiogram was developed.

Northern Blot Analysis

Northern analysis was performed following previously published methods(9, 22) . Total RNA from NIH 3T3 cells was prepared using 10^8 cells, and the alpha and alpha probes (50 ng) were labeled with [P]dCTP as previously described(9, 22) . Total RNA (20 µg) was resolved on a denaturing 1% agarose and 2.2 M formaldehyde gel. The RNA was blotted onto a zeta probe-GT membrane using a vacuum blotter and cross-linked to the membrane by UV; the blot was then probed with the cDNA probes. Other general analytical procedures were carried out as previously described (9, 22) .


RESULTS AND DISCUSSION

Expression of GTPase-inhibited mutants of alpha-subunit results in the constitutive activation of the signaling pathway regulated by the respective alpha-subunit. Hence, the cells expressing the GTPase-deficient mutants of alpha (alphaQ229L) and alpha (alphaQ226L) were analyzed for the constitutive activation of any of the known G protein-coupled signaling pathways. Stable expression of either of these mutant alpha-subunits readily confers transforming ability to fibroblast cell lines such as NIH 3T3 or Rat1. However, these studies failed to identify a constitutively activated alpha- or alpha-specific signaling pathway(6, 7, 8, 9, 10) . To investigate whether the expression of alphaQ229L or alphaQ226L activates JNK/SAPK pathway, we determined JNK/SAPK activity in vector-transfected and alphaQ229L- and alphaQ226L-transformed NIH 3T3 cells. JNK/SAPK activities were determined in the lysates from these cells by a solid phase kinase assay using GST-c-Jun(1-79) fusion protein immobilized on glutathione-Sepharose beads(21) . Our results using this assay indicated that the JNKs were constitutively activated in both alpha- and alpha-transformed 3T3 cells (Fig. 1). JNK responses to growth factors in alphaQ229L and alphaQ226L transformants were analyzed by stimulating cells with 10% FCS or FGF (5 ng/ml). FGF or serum stimulation of JNK/SAPK was only weak in control NIH 3T3 cells. FGF and FCS only modestly or not at all enhanced JNK/SAPK activation over that observed with the expression of alphaQ229L or alphaQ226L alone (Fig. 1). JNK/SAPK activation was found in eight of eight alphaQ229L- and six of six alphaQ226L-transformed clones, indicating this is a response reproducibly correlated with expression of activated alpha and alpha (Fig. 2, A and B). The differences in JNK/SAPK activation between alphaQ229L and alphaQ226L clones in the presence of FCS may be indicative of the subtle differences in the signaling pathways regulated by these alpha-subunits ( Fig. 1and Fig. 2).


Figure 1: Constitutive activation of JNK/SAPK in alphaQ229L- and alphaQ226L-transformed NIH 3T3 cells. The cell lysates from vector control, alphaQ229L- and alphaQ226L-transformed NIH 3T3 cells, were subjected to a solid phase JNK assay using glutathione-Sepharose-bound GST-c-Jun(1-79) as described under ``Experimental Procedures.'' Similar sets of cells were either stimulated with 10% FBS or 5 ng/ml basic FGF. The phosphorylated proteins were resolved on SDS-PAGE and visualized by autoradiography (upper panel). The radioactive bands were cut and counted to quantify the JNK activity. JNK activity is presented as the cpm in the radioactive GST-c-Jun(1-79) band (lower panel). The experiment was repeated a minimum of six times, and the results are from a typical experiment.




Figure 2: Constitutive activation of JNK/SAPK in multiple clones of alphaQ229L- and alphaQ226L-transformed NIH 3T3 cells. Different clonal isolates of alphaQ229L (A) and alphaQ226L transformants (B) were analyzed for JNK activities. Cells were processed for JNK solid phase JNK assay as described under ``Experimental Procedures.'' Following 24 h serum starvation, these cells were challenged with (openbars) or without (hatchedbars) 10% FCS. The phosphorylated protein was separated by SDS-PAGE, an autoradiogram was developed, and the radioactive bands were cut and counted. The JNK activity was presented as fold stimulation over non-FCS-stimulated control groups. This experiment was repeated three times.



In contrast to the constitutive activation of JNK/SAPK, the ERK/MAPK pathway was not activated. Our results indicated that the ERK1 and ERK2 activities of NIH 3T3 cells expressing alphaQ229L or alphaQ226L were not altered (Fig. 3). Their activities determined in alphaQ229L or alphaQ226L transformants were comparable to the control cells. Both serum and FGF stimulated the basal ERK/MAPK activities in all the cells, suggesting that the alpha or alpha pathway does not have a significant role in stimulating or inhibiting the ERK/MAPK pathway in NIH 3T3 cells (Fig. 3).


Figure 3: Effect of alphaQ229L and alphaQ226L expression on ERK/MAPK activity. The pcDNA3 transfected NIH 3T3 cells along with alphaQ229L and alphaQ226L transformed cells were processed for ERK/MAPK immune complex kinase assay as described under ``Experimental Procedures'' using myelin basic protein (MBP) as a substrate. The basal and serum- and FGF-stimulated activities were assayed as indicated. The phosphorylated myelin basic protein was separated on 12% SDS-PAGE and visualized by autoradiography.



Further demonstrating that the activation of JNK/SAPK is mediated by the expression of the activated alpha and alpha polypeptides, NIH 3T3 cells stably transfected with IPTG-inducible alphaQ229L and alphaQ226L expression vectors were utilized. Addition of 1 mM IPTG to the culture medium induced the transcription of alphaQ229L or alphaQ226L within 30 min (Fig. 4). Analysis of JNK/SAPK activity in cell lysates at different time points following IPTG induction demonstrated that JNK/SAPK was activated by 1 h following the induction of either alphaQ229L or alphaQ226L (Fig. 4). These results indicate that alpha and alpha activation of JNK/SAPK is an acute response and not due to an adaptive response produced by the long term expression of the respective mutant alphaor alpha polypeptides in NIH 3T3 cells. Reproducibly, induction of alphaQ226L gave an earlier activation of JNK/SAPK relative to alphaQ229L. This was most likely due to higher levels of alphaQ226L expression compared to alphaQ229L. Alternatively, this could be due to the differences in the potency of alphaQ229L and alphaQ226L in activating the JNK/SAPK pathway.


Figure 4: Activation of JNK/SAPK by alphaQ229L and alphaQ226L in NIH 3T3 cells. 1 mM IPTG was added to the culture dishes, and the cells were harvested at various times after IPTG induction as indicated. Cells were harvested for RNA isolation to monitor the respective alpha-subunit expression. Northern blots were developed using the respective cDNA probes. Cells were also harvested at these time points and processed for JNK assay. JNK was assayed using solid phase kinase assay using GST-c-Jun(1-79)-bound glutathione-Sepharose beads. Protein was separated by SDS-PAGE, and a 1-h autoradiogram was developed. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.



In addition to the inducible vector system, we also transiently expressed alphaQ229L or alphaQ226L in COS-1 cells to demonstrate the ability of alpha and alpha to activate the JNK/SAPK pathway. The expression vectors containing alphaQ229L or alphaQ226L inserts were cotransfected with an expression vector containing HA epitope-tagged JNK1(21) . The cells were lysed after 60 h, and HA-JNK1 was immunoprecipitated using the monoclonal antibodies to HA epitope tag. An immune complex JNK/SAPK assay was carried out using GST-c-Jun as substrate(21) . The results clearly indicated that the expression of alphaQ229L or alphaQ226L stimulated the activity of JNK/SAPK (Fig. 5). It is worth noting that the expression of wild type alpha or alpha failed to activate JNK/SAPK in contrast to the GTPase-deficient mutants (data not shown). Although the fold increases in JNK activity mediated by alphaQ229L and alphaQ226L in COS-1 cells were less than those observed with NIH 3T3 cells, the results suggested that the transient expression system can be effectively used to dissect the signaling components involved in the activation of JNK/SAPK by alpha. To examine whether the activation of JNK is specific to alpha and alpha alone, we tested the ability of GTPase-deficient alpha(s), alpha, and alpha(q) to stimulate the activity of JNK in COS cells. Constitutively activated mutants of these alpha-subunits were cotransfected with HA-JNK1, and 60 h later, JNK activities of the transfectants were determined by immune complex JNK assay. The results indicated that only alpha(q)Q209L activated JNK/SAPK in the COS cell transient expression system with equal potency as alpha (Fig. 6). Expression of alpha(s)Q227L and alphaQ205L was largely without effect on JNK activity.


Figure 5: Effect of transient expression of alphaQ229L and alphaQ226L on JNK/SAPK activity of COS-1 cells. Respective alpha-subunits (0.5 µg) contained in pcDNA3 expression vector were cotransfected with HA-JNK1 (0.5 µg) using lipofectamine (Life Technologies, Inc.). After 48 h, the HA-tagged JNK was immunoprecipitated with anti-HA antibodies (Boehringer Mannheim), and an immune complex kinase assay was carried out using GST-c-Jun(1-79) as a substrate. The phosphorylated proteins were separated by SDS-PAGE and visualized by autoradiography. The experiment was repeated at least four times with similar results. Upperpanel, lane 1, control; lane 2, alphaQ229L; and lane 3, alphaQ226L. Lowerpanel, the radioactive bands were excised and counted in a scintillation counter. JNK activity was expressed as % of control.




Figure 6: Effect of constitutively activated alpha-subunits on JNK/SAPK activity. Respective alpha-subunits contained in pcDNA3 expression vector (0.5 µg each) were cotransfected with HA-JNK1 (0.5 µg) into COS-1 cells using lipofectamine reagent. After 48 h, the HA-tagged JNK was immunoprecipitated, and an immune complex kinase assay was carried out using GST-c-Jun(1-79) as a substrate. The phosphorylated proteins were separated by 12% SDS-PAGE and visualized by autoradiography. The experiment was repeated at least four times with similar results.



Diverse stimuli appear to activate ERKs and JNKs through different signaling pathways(11, 14, 15, 16, 17, 20, 21, 23) . Ras appears to play a pivotal role in the activation of both ERK/MAPK and JNK/SAPKs in response to specific growth factors and hormones(13, 15, 21) . Ras is capable of activating JNK/SAPK through its activation of MAPK/ERK kinase kinase 1(21) . MAPK/ERK kinase kinase 1 (MEKK1) in turn activates a dual specificity kinase MAP kinase kinase 4 (MKK4)/SAPK/ERK kinase 1 (SEK1) that consequently activates JNK(24, 25) . Cytokines such as TNFalpha and IL1 activate JNK/SAPK pathways independent of Ras (21, 26) . Thus, alpha activation of JNK may involve a Ras-dependent or -independent mechanism. To investigate whether alpha activation of JNK/SAPK is dependent or independent of Ras, inhibitory Ras (NRas) was coexpressed with alphaQ229L and alphaQ226L. NRas blocked the ability of alphaQ229L and alphaQ226L to stimulate JNK/SAPK activity (Fig. 7). The results demonstrate alphaQ229L and alphaQ226L require Ras to activate JNK/SAPK. While our results presented here provide evidence that alphaQ229L and alphaQ226L communicate to Ras in activating JNK/SAPK in the COS cells, it is likely that alpha and alpha may also interact with additional signaling pathways in different cell types. In fact, we have evidence indicating that alphaQ229L and alphaQ226L activation of JNK/SAPK may involve a Rho-dependent signaling cascade in Swiss 3T3 cells. (^2)At present, the nature of the molecular communication between alpha and Ras as well as other Ras-like GTPases is unknown.


Figure 7: alphaQ229L and alphaQ226L require functional Ras for the activation of JNK/SAPK. JNK activities in the respective COS cell transfectants were determined using an immune complex kinase assay with immunoprecipitated HA epitope-tagged JNK1. Recombinant GST-c-Jun(1-79) was used as a substrate. The phosphorylated GST-c-Jun was separated by 12% SDS-PAGE, and an autoradiogram was developed (upper panel). The radioactive bands were cut and quantitated by liquid scintillation counting. JNK activity is expressed as percent of control (lower panel). Lane 1, vector-transfected COS cells; lane 2, alphaQ229L transfectant; lane 3, alphaQ226L transfectant; lane 4, Nras transfectants; lane 5, alphaQ229L plus Nras transfectant; lane 6, alphaQ226L plus Nras transfectant. The experiment was repeated four times with similar results.



It has been proposed that JNK/SAPKs may be more involved in the regulation of transmitting growth inhibition rather than mediating mitogenic responses(24, 25) . However, our studies reported here present an apparent paradox to this paradigm. The GTPase-deficient mutants of alpha and alpha activate a mitogenic pathway(6, 7, 8, 9, 10) . However, they also constitutively activate JNK/SAPKs, whose activation has been proposed to be associated with growth arrest (24, 25) . It is likely that the role of JNK/SAPK in cell proliferation or growth arrest is cell-type specific, and thus the activation of JNK/SAPK by alpha and alpha may have different consequences in different cell types. Consistent with this notion is the observation that c-Jun is essential for the transformation of 3T3 cells by Ha-Ras (27) and the overexpression of c-Jun itself can transform rat fibroblasts(28, 29) . Presumably, the opposing effects of c-Jun in different cells (and sometimes in the same cell in a different context) may be due to its interaction with different transactivators. c-Jun can form heterodimers with at least nine different proteins(18, 20, 30) . Each of these heterodimers along with c-Jun homodimers can potentially transactivate different sets of genes by binding to distinct DNA sites leading to diverse functional responses(18) . In this context, it is interesting to note that E1A transformation of 3T3 cells is stimulated by Jun-ATF heterodimer while inhibited by Jun-Jun homodimers(31) . The relative abundance of transcription factors capable of dimerizing to c-Jun can thus dictate cell responses. Alternatively, alpha and alpha activation of JNK/SAPK may be indicative of a signaling cascade independent of cell proliferation. It has been shown that JNK/SAPK can be activated upon hyperosmolar shock(16) . In addition, JNK1 has been demonstrated to complement yeast cells lacking the hyper osmolarity glycerol response gene 1 (HOG1) in regulating cell volume in response to hyperosmotic shock(16) . Based on these observations, it has been proposed that JNK1 may be involved in the cell volume regulation of higher eukaryotes. However, the upstream ``osmotic sensor,'' the transducer that stimulates JNK/SAPK, and the effector that is activated by JNK/SAPK activity are not known. It is significant to note that the regulation of Na/H exchanger activity is considered to play an important role in the regulatory volume increase following hyperosmotic shock in eukaryotic cells(32) . Our observation that alpha and alpha acutely activate JNK/SAPK pathway in NIH 3T3 and COS-1 cells becomes more significant in this context since the G family of G proteins has been shown to stimulate Na/H exchanger(22, 33, 34) . The findings are suggestive that the regulation of the JNK/SAPK pathway and Na/H exchanger may be an integrated response controlled by G and G. This observation associates volume regulation with specific gene transcription, a question that has long eluded an explanation. Experiments are now possible to directly test the integration of Na/H exchanger activity with the JNK/SAPK pathway.


FOOTNOTES

*
This work was supported by NCI Core Program on Carcinogenesis Grant 5-P30-CA 12227 (to N. D.) and National Institutes of Health Grants GM 30324, CA 58187, and DK 37871 (to G. L. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Contributed equally to this paper.

To whom correspondence should be addressed. Tel.: 215-707-1941; Fax: 215-707-2102.

^1
The abbreviations used are: G protein, heterotrimeric guanine nucleotide binding protein; JNK/SAPK, Jun kinase/stress-activated protein kinase; ERK/MAPK, extracellular response/mitogen-activated protein kinase; FGF, fibroblast growth factor; FCS, fetal calf serum; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; HA, hemagglutinin; IPTG, isopropyl-1-thio-beta-D-galactopyranoside.

^2
A. M. Buhl, N. Dhanasekaran, and G. L. Johnson, unpublished observation.


ACKNOWLEDGEMENTS

We thank Drs. Scott K. Shore and E. Premkumar Reddy for valuable suggestions and encouragement.


REFERENCES

  1. Johnson, G. L., and Dhanasekaran, N.(1989)Endocr. Rev.10,317-331 [Medline] [Order article via Infotrieve]
  2. Hepler, J. R., and Gilman, A. G.(1992)Trends Biochem. Sci. 17,383-387 [CrossRef][Medline] [Order article via Infotrieve]
  3. Strathman, M. P., and Simon, M. I.(1991)Proc. Natl. Acad. Sci. U. S. A. 88,5582-5586 [Abstract]
  4. Parks, S., and Wieschaus, E.(1991)Cell64,447-458 [Medline] [Order article via Infotrieve]
  5. Chan, A. N.-L., Fleming, T. P., McGovern, E. S., Chedid, M., Miki, T., and Aaronson, S. A. (1993)Mol. Cell. Biol.13,762-768 [Abstract]
  6. Xu, N., Bradley, L., Ambudkar, 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. Xu, N., Voyno-Yasenetskaya, T., and Gutkind, J. S.(1994) Biochem. Biophys. Res. Commun.201,603-609 [CrossRef][Medline] [Order article via Infotrieve]
  9. Vara Prasad, M. V. V. S., Shore, S. K., and Dhanasekaran, N.(1994)Oncogene 9,2425-2429 [Medline] [Order article via Infotrieve]
  10. Voyno-Yasenetskaya, T. A., Pace, A. M., and Bourne, H. R.(1994)Oncogene 9,2559-2569 [Medline] [Order article via Infotrieve]
  11. Johnson, G. L., and Vaillancourt, R. R.(1994)Curr. Opin. Cell Biol. 6,230-238 [Medline] [Order article via Infotrieve]
  12. Crespo, P., Xu, N., Simmons, W. F., and Gutkind, S. J.(1994)Nature 369,418-420 [CrossRef][Medline] [Order article via Infotrieve]
  13. Davis, R. J. (1994)Trends Biochem. Sci.19,470-473 [CrossRef][Medline] [Order article via Infotrieve]
  14. Derijard, B., Hibi, M., Wu, I-H., Barrett, T., Su, B., Deng, T., Karin, K., and Davis, R. J. (1994)Cell76,1025-1037 [Medline] [Order article via Infotrieve]
  15. Kyriakis, J. M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E. A., Ahmad, M. F., Avruch, J., and Woodgett, J. R.(1994)Nature 369,156-160 [CrossRef][Medline] [Order article via Infotrieve]
  16. Galcheva-Gargova, Z., Derijard, B., Wu, I-H., and Davis, R. J.(1994) Science265,806-808 [Medline] [Order article via Infotrieve]
  17. Han, J., Lee, J.-D., Bibbs, L., and Ulevitch, R. J.(1994)Science 265,808-811 [Medline] [Order article via Infotrieve]
  18. Angel, P., and Karin, M. (1991)Biochim. Biophys. Acta1072,129-157 [CrossRef][Medline] [Order article via Infotrieve]
  19. Sambrook, J., Fritsch, E., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  20. Hibi, M., Lin, A., Smeal, T., Minden, A., and Karin, M.(1993)Genes & Dev.7,2135-2148
  21. Minden, A., Lin, A., Mcmahon, M., Lange-Carter, C., Derijard, B., Davis, R. J., Johnson, G. L., and Karin, M.(1994)Science266,1719-1722 [Medline] [Order article via Infotrieve]
  22. Dhanasekaran, N., Vara Prasad, M. V. V. S., Wadsworth, S. J., Dermott, J. M., and van Rossum, G.(1994)J. Biol. Chem.269,11802-11806 [Abstract/Free Full Text]
  23. Minden, A., Lin, A., Smeal, T., Derijard, B., Cobb, M., Davis, R., and Karin, M. (1994)Mol. Cell. Biol.10,6683-6688
  24. Yan, M., Dai, T., Deak, J. C., Kyriakis, J. M., Zon, L. I., Woodgett, J. R., and Templeton, D. J.(1994)Nature372,798-800 [Medline] [Order article via Infotrieve]
  25. Derijard, B., Raingeaud, J., Barrett, T., Wu, I-H., Han, J., Ulevitch, R. J., and Davis, R. J. (1995)Science267,682-685 [Medline] [Order article via Infotrieve]
  26. Bird, T. A., Kyriakis, J. M., Tyshler, L., Gayle, M., Milne, A., and Virca, G. D.(1994) J. Biol. Chem.269,31836-31844 [Abstract/Free Full Text]
  27. Smeal, T., Binetruy, B., Mercola, D. A., Birrer, M., and Karin, M.(1991) Nature354,494-496 [CrossRef][Medline] [Order article via Infotrieve]
  28. Schutte, J., Minna, J., and Birrer, M.(1989)Proc. Natl. Acad. Sci. U. S. A. 86,2257-2261 [Abstract]
  29. Binetruy, B., Smeal, T., and Karin, M.(1991)Nature351,122-127 [CrossRef][Medline] [Order article via Infotrieve]
  30. Hai, T., and Curran, T. (1991)Proc. Natl. Acad. Sci. U. S. A.88,3720-3724 [Abstract]
  31. Hagmeyer, B. M., Konig, H., Herr, I., Offringa, R., Zantema, A., van der Eb, A. J., Herrlich, P., and Angel, P.(1993)EMBO J.12,3559-3572 [Abstract]
  32. Grinstein, S., Woodside, M., Goss, G. G., and Kapus, A.(1994)Biochem. Soc. Trans.22,512-516 [Medline] [Order article via Infotrieve]
  33. Voyno-Yasenetskaya, T., Conklin, B. R., Gilbert, R. L., Hooley, R., Bourne, H. R., and Barber, D. L.(1994)J. Biol. Chem.269,4721-4724 [Abstract/Free Full Text]
  34. Kitamura, K., Singer, W., Cano, A., and Miller, R. T.(1995)Am. J. Physiol. 268,C101-C110

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