©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transforming G Protein-coupled Receptors Potently Activate JNK (SAPK)
EVIDENCE FOR A DIVERGENCE FROM THE TYROSINE KINASE SIGNALING PATHWAY (*)

(Received for publication, November 30, 1994)

Omar A. Coso Mario Chiariello Gilda Kalinec John M. Kyriakis (1) James Woodgett (2) J. Silvio Gutkind (§)

From the  (1)Molecular Signaling Unit, Laboratory of Cellular Development and Oncology, NIDR, National Institutes of Health, Bethesda, Maryland 20892, the Diabetes Unit, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, and the (2)Ontario Cancer Institute, Princess Margaret Hospital, Toronto M4X 1K9, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The expression of human muscarinic acetylcholine receptors (mAChRs) in NIH 3T3 cells has been used as a model for studying proliferative signaling through G protein-coupled receptors. In this biological system, the m1 class of mAChRs can effectively transduce mitogenic signals (Stephens, E. V., Kalinec, G., Brann, M. R., and Gutkind, J. S.(1993) Oncogene 8, 19-26) and induce [Medline] malignant transformation if persistently activated (Gutkind, J. S., Novotny, E. A., Brann, M. R., and Robbins, K. C.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4703-4708). Moreover, available evidence suggests that the m1-signaling pathway converges at the level of p21 with that emerging from tyrosine kinase receptors (Crespo, P., Xu, N., Simonds, W. F., and Gutkind, J. S.(1994) Nature 369, 418-420). To explore nuclear events involved in growth regulation by G protein-coupled receptors in this setting, we compared the effect of platelet-derived growth factor (PDGF) and the cholinergic agonist, carbachol, on the expression of mRNA for members of the jun and fos family of nuclear proto-oncogenes. We found that activation of m1 receptors by carbachol induces the expression of a distinct set of nuclear transcription factors. In particular, carbachol caused a much greater induction of c-jun mRNA and AP-1 activity. These responses did not correlate with protein kinase C stimulation nor with the activation of mitogen-activated protein (MAP) kinases. Recently, it has been shown that a novel family of kinases structurally related to MAP kinases, stress-activated protein kinases, or Jun kinases (JNKs), phosphorylate in vivo the amino-terminal transactivating domain of the c-Jun protein, thereby increasing its transcriptional activity. In view of our results, this observation prompted us to ask whether m1 and PDGF can differentially activate JNKs. Here, we show that m1 mAChRs can induce a remarkable increase in JNK activity, which was temporally distinct from that of MAP kinase and was entirely protein kinase C independent. In contrast, PDGF failed to activate JNK in these cells, although it stimulated MAP kinase to an extent even greater than that for carbachol. These findings demonstrate that G protein-coupled receptors can signal through pathways leading to the activation of JNK, thus diverging at this level with those signaling routes utilized by tyrosine kinase receptors.


INTRODUCTION

Critical molecules participating in the transduction of proliferative signals have just begun to be identified. One such example is the family of extracellular signal-regulated kinases (ERKs) (^1)or MAP kinases(7) . The enzymatic activity of these kinases increases in response to most mitogens, such as those acting on receptor-protein tyrosine kinases or on receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins)(3, 4, 7) . Activation of the tyrosine kinase class of receptors transmits signals to MAP kinases in a multistep process. For the EGF receptor, essential components of this process include the adaptor protein GRB2/SEM-5, a guanine nucleotide exchange protein such as SOS, p21, and a cascade of protein kinases defined sequentially as MAP kinase kinase kinase, represented by c-Raf-1 and MEKK, and MAP kinase kinase, such as MEK1 and MEK2(7, 8, 9) . MEKs ultimately phosphorylate MAP kinases in both threonine and tyrosine residues, thereby increasing their enzymatic activity(7, 8, 9) . In turn, MAP kinases phosphorylate and regulate the activity of key enzymes including the EGF receptor, phospholipase A(2), p90, and nuclear proteins such as c-Myc and p62 Elk1(10) , which ultimately regulate the expression of genes essential for proliferation.

Little is known about the nature of proliferative pathways that participate in growth stimulation by G protein-coupled receptors. We have used the expression of human muscarinic receptors for acetylcholine (mAChRs) in NIH 3T3 cells as a model for studying proliferative signaling through this class of receptors(1) . The mAChR family consists of five distinct but highly related subtypes (m1-m5) (11, 12) . In this biological system, mAChR subtypes coupled to phosphatidylinositol biphosphate catabolism (m1, m3, and m5) can effectively transduce mitogenic signals (1) and, when persistently activated, can induce malignant transformation(2) . In contrast, those mAChRs coupled to the inhibition of adenylyl cyclase (m2 and m4) fail to induce the transformed phenotype(2) . Using this system, we have recently shown that triggering m1 receptors with the cholinergic agonist carbachol induces the activation of c-Raf and MAP kinases(3) . Moreover, using transient expression in COS-7 cells, we have found that activation of MAP kinases by muscarinic receptors involves beta subunits of G proteins acting on a Ras-dependent pathway(4) . These findings strongly suggest that the G protein-coupled receptor signaling pathway converges at the level of Ras with that emerging from receptors of the tyrosine kinase class. Thus, activation of either type of receptor would be expected to elicit a similar response at the level of nuclear transcription factors. Here, we present evidence that activation of m1 receptors in NIH 3T3 cells induces a distinct pattern of expression of immediate early genes of the jun and fos family and a much greater expression from an AP-1-responsive reporter construct. These responses did not correlate with the activation of MAP kinases. We found that triggering m1 mAChRs potently stimulate the activity of a novel family of enzymes closely related to MAP kinases, known as Jun kinases (JNKs) or stress-activated protein kinases (SAPKs)(5, 6) . In contrast, PDGF failed to activate JNK in these cells, although it stimulated MAP kinase to an even greater extent and for a more prolonged period of time than carbachol. These findings demonstrate that G protein-coupled receptors can signal through pathways leading to the activation of JNK, thus diverging at this level with those pathways utilized by receptors of the tyrosine kinase class.


EXPERIMENTAL PROCEDURES

Reagents and Cell Lines

NIH 3T3 cells expressing approximately 20,000 human m1 mAChRs per cell were used throughout the study. These cells, named NIH-m1.2, were obtained upon transfection of NIH 3T3 cells with an expression plasmid carrying the human m1 mAChR gene and a dominant selectable marker (gpt) as described (3) . NIH-m1.2 cells transfected with an expression vector carrying an ERK2 cDNA (13) mutated to harbor a tag of 9 amino acids representing influenza hemagglutinin HA1 protein (HA-ERK2) have been previously described(3) . Cultures were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% calf serum (Advanced Biotechnologies Inc.). PDGF was obtained from Upstate Biotechnology. All other chemicals were purchased from Sigma.

Preparation of RNA Samples (Northern Blots)

Cells were grown to confluence in 10-cm plates, washed twice with Dulbecco's modified Eagle's medium, and incubated for 18 h in Dulbecco's modified Eagle's medium without serum. Cultures were stimulated with the indicated experimental drugs for different periods of time at 37 °C and washed with cold PBS; total RNA was extracted from cells by homogenization in guanidinium thiocyanate as described(14) . For Northern blotting, samples containing 10 µg of total RNA were fractionated in 2% formaldehyde-agarose gels, transferred to nitrocellulose membranes, and then hybridized with P-labeled DNA probes prepared using a random priming kit (Boehringer Mannheim). DNA templates were full-length murine cDNAs for c-jun, junB, junD, c-fos, fosB, fra-1, and egr-1 kindly provided by R. Bravo(15) . Accuracy in gel-loading and transfer was confirmed by fluorescence under UV light upon ethidium bromide staining and by hybridization with a radiolabeled glyceraldehyde-3-phosphate dehydrogenase probe.

Reporter Gene Assays

NIH-m1.2 cells were transfected using the calcium phosphate precipitation technique with 2 µg of a reporter plasmid containing the chloramphenicol acetyltransferase gene (CAT) under the control of three AP-1 binding sites arranged in tandem (16) (kindly provided by Dr. M. Karin), together with 1 µg of pCMV-beta-gal DNA. 48 h later, the cells were serum starved overnight and then stimulated with agonists for an additional 4 h. Cells were then washed with cold PBS and lysed using reporter lysis buffer (Promega). CAT activity was assayed in cell extracts by incubation for 16 h in the presence of 0.25 µCi of [^14C]chloramphenicol (100 mCi/mmol) and 200 µg/ml butyryl-CoA (Sigma) in 0.25 M Tris-HCl, pH 7.4. Labeled butyrylated products were then extracted using a mixture of xylenes (Aldrich) and counted, as described(17) . beta-Galactosidase activity was measured by a colorimetric method (14) and used to normalize for transfection efficiency.

Jun Kinase Assay

A DNA fragment encoding the amino-terminal 79 amino acids of c-Jun was obtained by polymerase chain reaction using murine c-jun cDNA as a template and the following oligonucleotides: 5`-GGATCCACCATGACTGCAAAGATGGAA-3` and 5`-GTCGACTCAGAATTCCAGGCGCTCCAGCTCCGG-3`. The fragment was cloned between the BamHI and SalI site of pGEX4T-3 (Pharmacia Biotech Inc.), in frame with the GST gene. The resulting fusion protein (GSTc-jun79) was purified from bacterial lysates with the aid of glutathione-agarose beads (Thompson Instruments Co.) and used as a substrate (see below).

Confluent plates of transfected NIH 3T3 cells were incubated overnight in serum-free medium. Cells were then stimulated with agonists for different periods of time at 37 °C, washed with cold PBS, and lysed at 4 °C in a buffer containing 25 mM HEPES, pH 7.5, 0.3 M NaCl, 1.5 mM MgCl(2), 0.2 mM EDTA, 0.5 mM dithiothreitol, 20 mM beta-glycerophosphate, 1 mM vanadate, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 20 µg/ml leupeptin. Cleared lysates were rocked for 3 h at 4 °C in the presence of 1 µg of GST-cjun79 fusion protein bound to glutathione-agarose beads (5 mg of GST-cjun79 protein/25 ml of beads). Beads were washed three times with PBS containing 1% Nonidet P-40 and 2 mM vanadate, once with 100 mM Tris, pH 7.5, 0.5 M LiCl, and once in kinase reaction buffer (12.5 mM MOPS, pH 7.5, 12.5 mM beta-glycerophosphate, 7.5 mM MgCl(2), 0.5 mM EGTA, 0.5 mM sodium fluoride, 0.5 mM vanadate). Samples were then resuspended in 30 µl of kinase reaction buffer containing 1 µCi [-P]ATP per reaction and 20 µM of unlabeled ATP. After 20 min at 30 °C, the reactions were terminated by addition of 10 µl of 5 times Laemmli buffer. Samples were heated at 95 °C for 5 min and analyzed by SDS-gel electrophoresis on 12% acrylamide gels. Autoradiography was performed with the aid of an intensifying screen.

Immunocomplex JNK activity was determined upon immunoprecipitation with a JNK-specific polyclonal antibody(5) . Cleared lysates were incubated with 2 µl of antisera for 4 h at 4 °C. Immunocomplexes were recovered with the aid of protein G-Sepharose, and precipitates were processed as above. Kinase reaction was performed using 1 µg of purified GST-cjun79 protein as a substrate.

MAP Kinase Assay

MAP kinase activity in NIH-m1.2 cells stably expressing an epitope-tagged ERK2 protein was determined as described(3) . Briefly, upon stimulation cells were lysed by treating with a lysing buffer similar to that for JNK activity but containing 1% of Triton X-100. HA-ERK2 was immunoprecipitated from cleared lysates with the anti-epitope-specific antibody 12CA5 (Babco). Immunocomplex kinase assay was performed as above, using 1.5 mg/ml myelin basic protein (Sigma) as a substrate.


RESULTS AND DISCUSSION

One of the earliest nuclear events resulting from exposure of quiescent cells to mitogens is the induction of expression of genes of the jun and fos family(18) . Homodimers of the Jun protein product or Jun/Fos heterodimeric complexes form the AP-1 transcription factor, which binds the palindromic TRE sequence(19) , thereby controlling the expression of genes possessing this regulatory element. The Jun family consists of c-jun, junB, and junD while members of the Fos family include c-fos, fosB, fra-1, and fra-2(15) . To assess whether triggering m1 receptors in NIH 3T3 cells affects transcriptional activation mediated by AP-1, we used the transient expression of a reporter plasmid containing multiple TRE elements fused to the CAT gene (3XTRE-CAT)(16) . As shown in Fig. 1, carbachol addition to transfected NIH-m1.2 cells dramatically increased expression from the reporter gene construct. Interestingly, the AP-1-dependent response elicited by carbachol (6-fold increase) was much greater than that induced by activation of PDGF receptors or by direct stimulation of PKC by TPA (20) (approximately 2-fold increase for both cases).


Figure 1: Induction of AP-1 activity in NIH 3T3 cells expressing m1 mAChRs (effect of PKC blockade). NIH-m1.2 cells were cotransfected with 3xTRE-CAT and pCMV-beta-gal plasmids. 48 h later, cells were serum starved for 18 h and left untreated or treated for 30 min with 1 µM of the PKC inhibitor GF 109203X. Cells were then exposed for 4 h to 100 µM carbachol, 10 ng/ml PDGF or 50 ng/ml TPA, as indicated. CAT activity was measured and data normalized by beta-galactosidase activity present in each sample. Data represent the average ± S.E. of triplicate samples from a typical experiment expressed as -fold induction with respect to unstimulated cells (control). Similar results were obtained in four independent experiments.



Activation of m1 receptors induces the hydrolysis of PIP(2) and consequently activates PKC(1, 3, 20) . To explore whether PKC mediates the AP-1 response to carbachol, we pretreated cells with the non-toxic PKC-specific inhibitor GF 109203X and then challenged these cells with the various agonists. Under these conditions, we have previously shown that this PKC inhibitor effectively blocks phosphorylation of endogenous PKC substrates as well as biological responses induced by phorbol esters(3) . Accordingly, GF 109203X pretreatment abolished the CAT activity elicited by phorbol esters. However, PKC inhibition reduced only about 40% of the AP-1 response to carbachol (Fig. 1). In contrast, stimulation of CAT activity by PDGF was not affected by GF 109203X. Thus, m1 signaling pathways induce AP-1-dependent transcriptional activation more effectively than those pathways triggered by tyrosine kinases or phorbol esters. In addition, m1 receptors appear to induce AP-1 activity through both PKC-dependent as well as PKC-independent pathways.

The activity of AP-1 is regulated at the level of jun and fos gene transcription and by post-translational modification of their protein products(18, 19, 21) . Because of the remarkable effect of m1 on AP-1 activity, we next examined the ability of carbachol, PDGF, and TPA to induce expression of jun and fos family members. As shown in Fig. 2, all agonists rapidly affected the expression of mRNA for these immediately early genes but to different extents. Carbachol induced a much greater expression of c-jun, junD, c-fos, and fosB mRNA, whereas PDGF preferentially induced junB and egr-1 (another immediate early gene unrelated to jun and fos)(16) . TPA was less effective in most cases, although it potently induced expression of egr-1 (Fig. 2). PDGF and TPA not only failed to increase levels of junD mRNA but instead they decreased its expression (Fig. 2). All agonists induced fra-1 messages much later than for the other genes (Fig. 2).


Figure 2: Expression of immediate early genes in response to agonists. Quiescent NIH-m1.2 cells were stimulated with 100 µM carbachol, 10 ng/ml PDGF, or 50 ng/ml TPA for the indicated period of time. Cells were lysed, and total RNAs were extracted as described under ``Experimental Procedures.'' Samples containing 10 µg of total RNA per lane were fractionated and analyzed by Northern blotting, using the indicated P-labeled DNA probes. Material present in each lane was determined to be equivalent by ethidium bromide staining of ribosomal RNAs and by hybridization with a radiolabeled glyceraldehyde-3-phosphate dehydrogenase probe (data not shown). Similar results were obtained in at least three independent experiments.



We next explored which of these responses were mediated by PKC. Treatment of cells with the PKC inhibitor abolished all responses induced by TPA, demonstrating the effectiveness of this procedure (Table 1). In contrast, responses induced by PDGF were not affected by this treatment except for c-fos, which was slightly reduced. For carbachol, the picture appears to be more complex. The induction of egr-1 was completely abolished, and the expression of c-fos and fosB was reduced by 50 and 25%, respectively. In contrast, PKC blockade did not have any demonstrable effect on m1-mediated induction of c-jun and junD. Thus, the most striking difference between carbachol and the other agonists is the potent induction of c-jun and junD mRNA, in both cases involving PKC-independent pathways.



The activity of c-Jun appears to be controlled by a novel family of enzymes structurally related but clearly distinct from MAP kinases. These enzymes, named JNKs (6) or SAPKs(5) , selectively phosphorylate the amino-terminal transactivating domain of the c-Jun protein, thereby increasing its transcriptional activity(5, 6) . In light of our results, we decided to compare the ability of carbachol, PDGF, and TPA to induce MAP kinase and JNK (SAPK) activity. The former was determined in NIH-m1.2 cells expressing an epitope-tagged ERK2 cDNA(3) . The latter was assayed by first retaining activated JNK using bacterially expressed recombinant GST-c-jun79 protein bound to glutathione-agarose and then assessing its phosphorylating activity on GST-c-jun79. Alternatively, JNK activity was measured in an immunocomplex kinase assay upon immunoprecipitation of SAPKs with a specific antiserum, using purified GST-c-jun79 as a substrate(5) . As previously reported (3) , carbachol potently induced MAP kinase activation, which peaked after 3-5 min, and remained slightly above unstimulated levels for more than 2 h. PDGF and TPA also induced a marked increase in MAP kinase activity; however, its activity remained higher for a more prolonged period of time (Fig. 3). In contrast, only carbachol induced JNK activation and, to an extent, similar to that caused by cycloheximide, which was used as a control (5) (Fig. 3). It is noticeable that m1-mediated activation of JNK was delayed with respect to MAP kinase. It peaked between 10-15 min upon carbachol addition and remained higher than unstimulated cells for approximately 40 min. Furthermore, JNK activity in response to carbachol in NIH-m1.2 cells was similar or even greater than that induced by known JNK activators, such as tumor necrosis factor-alpha, interleukin-1, or heat shock (5) (data not shown). On the other hand, TPA or agonists acting on tyrosine kinase receptors, such as epidermal growth factor or fibroblast growth factor, have been shown to poorly induce JNK activity, and only in a few cell types(5, 22) . The lack of JNK activation by PDGF and TPA in NIH 3T3 cells is in line with those observations. We next determined whether PKC activation plays a role in JNK activation through m1 G protein-coupled receptors. We found that neither blockade of PKC nor depletion of this enzyme by prolonged treatment with TPA (1, 3) affect the JNK response to carbachol in m1-expressing cells (Fig. 4), thus demonstrating that signaling from m1 to JNK involves PKC-independent pathways.


Figure 3: The cholinergic agonist carbachol induces MAP kinase and JNK (SAPK) activity in NIH 3T3 cells expressing m1 mAChRs. Confluent plates of NIH-m1.2 cells were serum starved for 18 h and treated with 100 µM carbachol, 10 ng/ml PDGF, or 50 ng/ml TPA for the indicated times. Treatment with 180 µM cycloheximide (Cx) for 1 h was used as a control. Cells were lysed, and kinase activity present in cell extracts was recovered by immunoprecipitation using the monoclonal antibody 12CA5 for MAP kinase (MAPK) or affinity precipitated using recombinant GST-cjun79 fusion protein bound to glutathione beads for JNK. Kinase reactions were performed as described under ``Experimental Procedures.'' The products of kinase reactions were fractionated in 12% SDS-polyacrylamide gel electrophoresis gels. Position of labeled myelin basic protein (MBP) or GST-jun79 are indicated. Similar results were obtained in four independent experiments. For JNK, almost identical results were obtained by immunoprecipitation of endogenous JNK using a specific antibody (data not shown and Fig. 4).




Figure 4: Effect of PKC depletion or blockade on JNK activity. NIH-m1.2 cells were serum starved for 18 h(-), depleted of PKC by treatment with 1 µg/ml TPA in serum-free conditions (TPA O/N), or serum starved for 18 h and treated for the last 30 min with 1 µM of the specific PKC inhibitor GF 109203X, as indicated. Cells were treated with 100 µM carbachol, 10 ng/ml PDGF, or 50 ng/ml TPA for 20 min and lysed. Cell extracts were processed as described under ``Experimental Procedures'' for immunocomplex JNK assay. Results are representative from three independent experiments. Near identical results were obtained using the GST-jun79 affinity precipitation technique (data not shown).



Recent findings have demonstrated that mitogens acting on a large variety of cell surface receptors converge at the level of Ras to induce a cascade of serine-threonine kinases leading to the activation of MAP kinases. In turn, MAP kinases phosphorylate a number of intracellular substrates that are important for cell proliferation (10) . For example, MAP kinases phosphorylate the transcription factor ternary complex factor, known as p62 or Elk-1(23) , which represents a critical event in controlling the expression of c-fos(24) . Interestingly, this observation might account for the decreased c-fos and AP-1 response to carbachol upon blockade of PKC, as we have recently shown that MAP kinase activation through m1 mAChRs is partially dependent on PKC(3) . However, other nuclear events in response to environmental stimuli might involve additional members of the MAP kinase family, such as JNK and the mammalian homolog of HOG kinase(25) . In particular, JNK is thought to be responsible for phosphorylating in vivo the transactivating domain of c-Jun protein (and probably Jun D)(^2)(5, 6, 22) . Phosphorylated c-Jun homodimers have potent AP-1 activity, and this complex appears to control the expression of c-jun mRNA(27, 28) . Consistent with this idea, activation of JNK by m1 mAChRs correlated well with both the potent induction of AP-1 activity and the remarkable expression of c-jun mRNA in response to carbachol. Conversely, induction of AP-1 and c-jun expression by PDGF and TPA might involve alternate mechanisms, including MAP kinase-dependent events or as the result of dephosphorylation of inhibitory phosphorylated sites in the c-Jun protein, as previously suggested(28) .

Our findings also have implications with regard to activating pathways for JNK. Although it was initially suggested that these enzymes were located downstream from Ras, this hypothesis is in conflict with recently available data(26) , including the lack of activation of JNK by PDGF (this study) or by other agonists acting on receptors that are known to couple to the ras pathway(5, 22) . Furthermore, agonists such as tumor necrosis factor-alpha and interleukin-1 potently induce JNK, but they activate MAP kinase poorly ( (5) and data not shown). Taken together, these observations suggest the existence of parallel pathways leading to the activation of either MAP kinase or JNK. Based upon our results, both pathways can be effectively stimulated by transforming G protein-coupled receptors. Although the precise role of JNK activation by m1 mAChRs is not known, it correlates well with agonist-induced expression of genes important for cell growth. Thus, JNK is a likely candidate to be an integral part of the mitogenic signaling pathway utilized by these G protein-coupled receptors. Whether that is the case warrants further investigation.


FOOTNOTES

*
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.

§
To whom correspondence should be addressed: Molecular Signaling Unit, Laboratory of Cellular Development and Oncology, NIDR, National Institutes of Health, 9000 Rockville Pike, Bldg. 30, Rm. 212, Bethesda, MD 20892-4330. Tel.: 301-496-6259; Fax: 301-402-0823.

(^1)
The abbreviations used are: ERK, extracellular signal-regulated kinases; MAChR, muscarinic acetylcholine receptors; PDGF, platelet-derived growth factor; MOPS, 4-morpholinepropanesulfonic acid; MAP, mitogen-activated protein; GST, glutathione S-transferase; TPA, 12-O-tetradecanoylphorbol-13-acetate; SAPK, stress-activated protein kinases; JNK, Jun kinases; PBS, phosphate-buffered saline; CAT, chloramphenicol acetyltransferase; AP-1, activator protein-1.

(^2)
O. A. Coso and J. S. Gutkind, unpublished observation.


ACKNOWLEDGEMENTS

We thank M. Karin for the gift of 3XTRE-CAT plasmid DNA, R. Bravo for the gift of immediate early gene cDNAs, and J. Avruch for SAPK antibodies. We thank L. Vitale and A. Yeudall for critically reviewing this manuscript.


REFERENCES

  1. Stephens, E. V., Kalinec, G., Brann, M. R. & Gutkind, J. S. (1993) Oncogene 8, 19-26
  2. Gutkind, J. S., Novotny, E. A., Brann, M. R. & Robbins, K. C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4703-4708 [Abstract]
  3. Crespo, P., Xu, N., Daniotti, J. L., Troppmair, J., Rapp, U. R. & Gutkind, J. S. (1994) J. Biol Chem. 269, 21103-21109 [Abstract/Free Full Text]
  4. Crespo, P., Xu, N., Simonds, W. F. & Gutkind, J. S. (1994) Nature 369, 418-420 [CrossRef][Medline] [Order article via Infotrieve]
  5. Kyriakis, J., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E., Ahmad, M., Avruch, J. & Woodgett, J. (1994) Nature 369, 156-160 [CrossRef][Medline] [Order article via Infotrieve]
  6. Derijard, B., Hibi, M., Wu, I., Barrett, T., Su, B., Deng, T., Karin, M. & Davis, R. (1994) Cell 76, 1025-1037 [Medline] [Order article via Infotrieve]
  7. Pelech, S. L. (1993) Curr. Biol. 3, 513-516 [Medline] [Order article via Infotrieve]
  8. McCormick, F. (1993) Nature 363, 15-16 [CrossRef][Medline] [Order article via Infotrieve]
  9. Schlessinger, J. (1993) Trends Biochem. Sci. 18, 273-275 [CrossRef][Medline] [Order article via Infotrieve]
  10. Davis, R. J. (1993) J. Biol. Chem. 268, 14553-14556 [Free Full Text]
  11. Bonner, T. I., Burkley, N. J., Young, A. C. & Brann, M. R. (1987) Science 237, 527-532 [Medline] [Order article via Infotrieve]
  12. Peralta, E. G., Ashkenazi, A., Winslow, J. W., Smith, D. H., Ramachandran, J. & Capon, D. J. (1987) EMBO J. 6, 3923-3929 [Abstract]
  13. Her, J., Wu, J., Rall, T., Sturgill, T. & Weber, M. (1991) Nucleic Acids Res. 19, 3743 [Medline] [Order article via Infotrieve]
  14. Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J. & Struhl, K. (eds) (1994) Current Protocols in Molecular Biology , pp. 4.2.1-4.2.8, Greene Publishing Associates. Brooklyn, NY
  15. Bravo, R. (1990) Cell Growth & Differ. 1, 305-309
  16. Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra, R., Rahmsdorf, H., Jonat, C., Herrlich, P. & Karin, M. (1987) Cell 49, 729-739 [Medline] [Order article via Infotrieve]
  17. Seed, B. & Sheen, J. (1988) Gene (Amst.) 67, 271-277 [CrossRef][Medline] [Order article via Infotrieve]
  18. Herschman, H. (1991) Annu. Rev. Biochem. 60, 281-319 [CrossRef][Medline] [Order article via Infotrieve]
  19. Angel, P. & Karin, M. (1991) Biochim. Biophys. Acta 1072, 129-157 [CrossRef][Medline] [Order article via Infotrieve]
  20. Nishizuka, Y. (1988) Nature 334, 661-665 [CrossRef][Medline] [Order article via Infotrieve]
  21. Hunter, T. & Karin, M. (1992) Cell 70, 375-387 [Medline] [Order article via Infotrieve]
  22. Westwick, J., Weitzel, C., Minden, A., Karin, M. & Brenner, D. (1994) J. Biol. Chem. 269, 26396-26401 [Abstract/Free Full Text]
  23. Marais, R., Wynne, J. & Treisman, R. (1993) Cell 73, 381-393 [Medline] [Order article via Infotrieve]
  24. Hipskind, R., Rao, V., Mueller, C., Reddy, E. & Nordheim, A. (1992) Nature 354, 19-26
  25. Han, J., Lee, J.-D., Bibbs, L. & Ulevitch, R. J. (1994) Science 265, 808-811 [Medline] [Order article via Infotrieve]
  26. Sun, H., Tonks, N. & Bar Sagi, D. (1994) Science 266, 285-288 [Medline] [Order article via Infotrieve]
  27. Angel, P., Hattori, K., Smeal, T. & Karin, M. (1988) Cell 55, 875-885 [Medline] [Order article via Infotrieve]
  28. Boyle, W., Smeal, T., Defize, L., Angel, P., Woodgett, J., Karin, M. & Hunter, T. (1991) Cell 64, 573-584 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.