©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ceramide Activates the Stress-activated Protein Kinases (*)

(Received for publication, June 28, 1995; and in revised form, August 2, 1995)

John K. Westwick (§) Alicja E. Bielawska (1) Ghassan Dbaibo (1) Yusuf A. Hannun (1) David A. Brenner (¶)

From the Departments of Medicine and Biochemistry and Biophysics, Center for Gastrointestinal Biology and Disease, University of North Carolina, Chapel Hill, North Carolina 27599-7038 and the Departments of Medicine and Cell Biology, Duke University, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tumor necrosis factor alpha (TNFalpha) activates the stress-activated protein kinases (SAPKs, also known as Jun nuclear kinases or JNKs) resulting in the stimulation of AP-1-dependent gene transcription and induces the translocation of NFkappaB to the nucleus resulting in the stimulation of NFkappaB-dependent gene transcription. A potential second messenger for these signaling pathways is ceramide, which is generated when TNFalpha activates sphingomyelinases. We show that treatment of HL-60 human promyelocytic cells with exogenous sphingomyelinase leads to rapid stimulation of JNK/SAPK activity, an effect not mimicked by treatment with phospholipase A(2), C, or D. Further, JNK/SAPK activity is stimulated 2.7- and 2.8-fold, respectively, in cells exposed to C(2)-ceramide (5 µM) or TNFalpha (10 ng/ml). The prolonged stimulation of this kinase activity by C(2)-ceramide is similar to that previously reported for TNFalpha. In contrast, the related mitogen-activated protein kinases ERK1 and ERK2 are weakly stimulated following TNFalpha treatment (1.5-fold) and are inhibited by C(2)-ceramide treatment. TNFalpha also potently stimulates NF-kappaB DNA binding activity and transcriptional activity, but these effects are not mimicked by addition of C(2)-ceramide or sphingomyelinase to intact cells. Furthermore, TNFalpha, sphingomyelinase, and C(2)-ceramide induce c-jun, a gene that is stimulated by the ATF-2 and c-Jun transcription factors. These data suggest that ceramide may act as a second messenger for a subset of TNFalpha's biochemical and biological effects.


INTRODUCTION

TNFalpha (^1)is a multifunctional cytokine involved in inflammation, infection, and cancer(1, 2) . TNFalpha has potent biological effects on cultured cells, including induction of apoptosis and differentiation(3, 4) . TNFalpha signals via unknown second messengers leading to activation of NFkappaB (5, 6) and JNKs/SAPKs(7, 8, 9) and a weaker stimulation of MAP kinase activity(7, 8) . Each of these pathways leads to activation of a specific set of transcription factors; JNK/SAPK phosphorylates and activates c-Jun (10, 11) and ATF-2 (12) , NF-kappaB translocates to the nucleus where it functions as a transcription factor, and MAP kinase phosphorylates and activates Elk-1 (13, 14) and other transcription factors.

TNFalpha stimulates sphingomyelinase activity, which results in the generation of ceramide(15, 16) . Treatment of cells with the cell-permeable C(2)-ceramide mimics TNFalpha in the induction of apoptosis(4) , differentiation(15) , and in the activation of a ceramide-activated protein phosphatase activity similar to PP2A(17, 18) . A ceramide-activated protein kinase that is proline-directed has been described, but its specific target(s) is unknown(19) . Thus, many of the biological and biochemical effects of TNFalpha are mimicked by ceramide treatment, but the molecular links between TNFalpha-induced ceramide production and transcriptional activation are not clear.


EXPERIMENTAL PROCEDURES

Reagents

All reagents were obtained from Sigma unless otherwise specified. Recombinant human TNFalpha (for HL-60 cells) and murine TNFalpha (for L929 cells) were obtained from R& Systems, Minneapolis, MN. Sphingomyelinase (Bacillus cereus) was obtained from Boehringer Mannheim. (2S,3R)-D-erythro-N-Acetylsphingosine (D-e-C(2)) was prepared by acetylation of (2S,3R)-sphingosine as described (20) . DL-erythro-Dihydro-C(2)-ceramide was prepared by acetylation of DL-erythro-dihydrosphingosine (20) (obtained from Sigma). Aliquots were resuspended at 20 mM in ethanol before use. Radionucleotides were obtained from ICN Biomedicals (Irvine, CA). [-P]ATP used for in vitro kinase assays was >4000 Ci/mmol.

Tissue Culture

HL-60 cells were maintained at a density of <1 times 10^6 cells/ml for less than 1 month before discarding. Cells were cultured in RPMI 1640 (Life Technologies, Inc.) with 10% heat-inactivated fetal bovine serum (Gemini Bioproducts, Calabasas, CA) in a humidified atmosphere of 5% CO(2), 95% air. For treatment with agonists, cells were resuspended in serum-free medium as described (18) at a density of 250,000 cells/ml and incubated for at least 2 h before treatment. L929 fibrosarcoma cells (21) were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 5% fetal bovine serum (Gemini).

Extract Preparation and in Vitro Kinase Assays

Whole cell extracts were prepared as described previously(22) . For assessment of JNK/SAPK activity, 50 µg of extract was used in a solid state in vitro kinase assay as described (23) with GST-c-Jun (10) as the substrate. For assessment of MAP kinase activity, whole cell extracts were immunoprecipitated with antisera to ERK2, which cross-reacts with ERK1 (SC-94, Santa Cruz Biotechnology, Santa Cruz, CA). Immune complexes were collected with protein A/G-agarose (Oncogene Science, Uniondale, NY), washed extensively, and used in an in vitro kinase assay with 50 µM ATP (5 µCi of [-P]ATP), 10 mM MgCl(2), 1 mM dithiothreitol, 0.1 mM Na(3)VO(4), 25 mM beta-glycerophosphate, 10 mMp-nitrophenyl phosphate for 20 min at 30 °C. 2 µg of GST-ElkC (a glutathione S-transferase fusion protein containing the C terminus of Elk-1, which contains multiple MAP kinase phosphorylation sites) was used as substrate(14, 24) . Proteins were fractionated on 12.5% SDS-polyacrylamide gel electrophoresis and subjected to autoradiography.

Gel Mobility Shift Assays

NF-kappaB gel mobility shift assays were performed as described previously(3) . Antibodies used in binding assays (1 µl/reaction) were obtained from Santa Cruz Biotechnology.

Plasmids and Transfection Analysis

Transient transfection of L929 cells was performed by calcium phosphate coprecipitation as described previously(25) . Jun-luciferase consists of -1.1 to +740 of human c-jun(26) linked to luciferase. Gal-ElkC (13) is cotransfected with 5 times Gal-luciferase as described(25) . (KB)3-luciferase consists of three consensus NF-kappaB sites (27) linked to luciferase. Luciferase assays were performed as previously described (28) .

Northern Blot Analysis

Total RNA was prepared from HL-60 cells and electrophoresed as described previously(29) . After blotting to nitrocellulose, the RNA was probed with a HindIII-PstI fragment of human c-jun(30) labeled by random priming (Stratagene, San Diego, CA) with [alpha-P]dCTP (3000 Ci/mmol, ICN, Irvine, CA). Final wash stringency was 0.1 times SSC, 0.1% SDS at 55 °C. The blot was stripped and reprobed with a glyceraldehyde-phosphate dehydrogenase cDNA probe (pHcGAP, ATCC 57090) to assess sample loading.


RESULTS AND DISCUSSION

We investigated the role of ceramide in TNFalpha-stimulated signal transduction by comparing the effects of TNFalpha, sphingomyelinase, and C(2)-ceramide on JNK/SAPK activity, NFkappaB activity, and MAP kinase activity. Exogenous bacterial sphingomyelinase catalyzes the hydrolysis of cell membrane sphingomyelin and the formation of ceramide when added to intact HL-60 human myelocytic leukemia cells(31, 32) . Treatment of cells with exogenous sphingomyelinase potently activates JNK/SAPK activity, while equivalent amounts of the lipases phospholipase C, phospholipase A(2), or phospholipase D do not (Fig. 1A). Exogenously added C(2)-ceramide or TNFalpha stimulates JNK/SAPK activity in HL-60 cells to comparable levels (Fig. 1A). The activation of JNK/SAPK activity by C(2)-ceramide is first detected in cell extracts after 10 min (Fig. 1B). In contrast to agents that transiently stimulate JNK/SAPK activity, such as epidermal growth factor and phorbol esters(8) , ceramide elicits a prolonged activation (more than 2-fold over background levels) at least 2 h after stimulation. The kinetics of JNK/SAPK activation by ceramide parallel those of TNFalpha stimulation (data not shown and (8) ).


Figure 1: Stimulation of JNK/SAPK activity by sphingomyelinase (SM) and D-e-C(2)-ceramide. A, HL-60 cells were cultured as described (18) and treated with D-e-C(2)-ceramide (C2, 5 µM, lane2) or ethanol only (con, lane1) or the enzymes indicated (300 milliunits/ml, lanes4-7) or vehicle (con, lane3) or TNFalpha (10 ng/ml, lane8) for 20 min. The single prominent band visible following 10% SDS-polyacrylamide gel electrophoresis and autoradiography represents phosphorylated GST-c-Jun. Equal substrate loading was confirmed in all cases by Coomassie Blue staining of the gels prior to autoradiography. PL, phospholipase. B, time course of JNK/SAPK induction following D-e-C(2)-ceramide addition (5 µM) to HL-60 cells. The substrate (sub) for the kinase assays was GST-cJun or GST alone as control. C, response of SAPK activity to D-e-C(2)-ceramide treatment. All cells received the indicated concentration in an equal volume of ethanol or ethanol alone 5(di), 5 µM dihydro-C(2)-ceramide. Extracts were prepared after 10 min of treatment. The substrate for the kinase assay was wild-type GST-cJun (lanes 2-9) or GST-cJun(AA) (lane1) in which Ser-63 and Ser-73 are mutated to Ala(10) .



Increases in JNK/SAPK activity are observed at C(2)-ceramide concentrations of 1 µM and above (Fig. 1C). 5 µM C(2)-ceramide stimulates JNK/SAPK activity 2.7-fold over untreated cell levels (average of seven experiments), while TNFalpha (10 ng/ml) stimulates JNK activity 2.8-fold (average of four experiments). Uptake of C(2)-ceramide applied at 3 µM(16, 20) results in an intracellular C(2)-ceramide concentration that is equimolar to the concentration of ceramides generated endogenously following cell treatment with TNFalpha(15) .

Because C(2)-ceramide is an amphiphilic lipid analog that may have nonspecific activities, it was important to establish the specificity of its activation of JNK/SAPK, a family of kinases activated by a wide variety of stress stimuli(7) . DL-erythro-dihydro-C(2)-ceramide is a close structural analog of C(2)-ceramide lacking the trans-unsaturated bond in the sphingosine moiety. Previous studies (20) demonstrated that the uptake and metabolism of radiolabeled C(2)-ceramide and dihydro-C(2)-ceramide are similar. DL-erythro-dihydro-C(2)-ceramide fails to activate JNK/SAPK activity (Fig. 1C, lane8). Therefore, the effects of C(2)-ceramide on JNK/SAPK are specific and suggest a specific interaction of ceramide with a component of the JNK/SAPK pathway.

The effect of ceramide on the induction of NFkappaB activity is unresolved, in that ceramide treatment of intact cells induces binding activity in one report (31) but not others(3, 33, 34) . TNFalpha potently stimulated NF-kappaB DNA binding activity (Fig. 2A, lane7), with the predominant form of NFkappaB consisting of p65/p50 heterodimers (Fig. 2B, lane3). However, treatment with bacterial sphingomyelinase (Fig. 2A, lanes5 and 6) or C(2)-ceramide (Fig. 2A, lanes 2-4) failed to induce NFkappaB binding activity.


Figure 2: TNFalpha, but not sphingomyelinase or D-e-C(2)-ceramide, activates NF-kappaB binding activity. A, HL-60 cells were treated with vehicle, 0.1, 1, or 10 µM C(2)-ceramide (lanes 1-4), or with 10 or 100 milliunits/ml sphingomyelinase (SM) (B. cereus, Boehringer Mannheim) (lanes5 and 6) or recombinant TNFalpha (T, 10 nM, lane7) for 30 min prior to nuclear extract preparation and gel mobility shift analysis using a consensus NF-kappaB binding site probe. ns, nonspecific shifted complex; p50-65, heterodimeric NF-kappaB. B, extracts from TNFalpha-treated HL-60 cells were used in binding assays, with antibodies to p50 (lane2), p65 (lane3), or c-Rel (lane4), or with a 50-fold excess of unlabeled wild-type (wt, lane5) or mutant NF-kappaB binding site (mut, lane6) included in the reaction. After incubation on ice for 1 h, products were resolved on non-denaturing polyacrylamide gels. SS, supershifted NF-kappaB; ns, nonspecific shifted complex (not competed by cold excess oligonucleotide).



TNFalpha is a weak inducer of MAP kinase (ERK1 and ERK2) activity relative to its induction of JNK/SAPK or relative to the induction of MAP kinases by growth factors such as epidermal growth factor(7, 8) . In HL-60 cells, high concentrations of TNFalpha weakly activate MAP kinase activity using recombinant GST-Elk activation domain as a substrate in an immune complex kinase assay (1.5-fold over unstimulated levels) (Fig. 3, lane5). As reported elsewhere(35) , high concentrations of exogenous sphingomyelinase activate MAP kinase activity (Fig. 3, lane7). However, treatment of cells with C(2)-ceramide almost completely attenuates basal MAP kinase activity in a concentration-dependent manner (Fig. 3, lanes 2, 3, and 8), and this effect was not observed with dihydro-C(2)-ceramide-treated cells (Fig. 3, lanes4 and 9). Similar results are obtained using myelin basic protein as a substrate for MAP kinase in an immune complex kinase assay or using an in-gel MAP kinase assay (36) with MBP incorporated into the gel (data not shown).


Figure 3: MAP kinase activity is stimulated by sphingomyelinase but not ceramide. An immune complex MAP kinase assay was performed with HL-60 cell extracts prepared following cell treatment (Tx) with the indicated agonists for 10 min (lanes 1-5) or 2 min (lanes 6-9). C5 and C10, 5 and 10 µMD-e-C(2)-ceramide; C5(di), 5 µMDL-erythro-dihydro-C(2)-ceramide; SM, 100 milliunits/ml Staphylococcus aureus sphingomyelinase; TNF, 50 ng/ml recombinant human TNFalpha; con, control. GST-ElkC was used as the substrate. Equal substrate loading was confirmed in all cases by Coomassie Blue staining of the gels prior to autoradiography.



The ability of ceramide to activate JNK/SAPK predicts that ceramide would activate downstream targets of this kinase cascade. The c-jun gene contains two non-consensus AP-1 binding sites in its promoter, which are recognized with high affinity by protein complexes containing c-Jun and ATF-2(37, 38) . The transcriptional activation domains of both c-Jun (11) and ATF-2 (12) are phosphorylated and activated by JNK/SAPK. TNFalpha, C(2)-ceramide, and sphingomyelinase stimulate c-jun mRNA levels in HL-60 cells (Fig. 4A). Thus, all three agents that stimulate JNK/SAPK activity also lead to enhanced expression of the c-jun gene. The heightened ability of sphingomyelinase to activate c-jun transcription may be due to its activation of Elk-1 (Fig. 3), which can stimulate c-fos transcription and hence increase total AP-1 activity.


Figure 4: Stimulation of c-jun transcriptional activity. A, c-jun mRNA levels are increased following TNFalpha, sphingomyelinase, and C(2)-ceramide treatment. HL-60 cells were incubated with TNFalpha (TNF, 20 ng/ml), sphingomyelinase (SM, 100 milliunits/ml), C(2)-ceramide (CER, 10 µM), or untreated (NONE) for 1 h, and total RNA was prepared followed by electrophoresis and Northern blotting. The blot was hybridized first with a radioactively labeled human c-jun probe (upperpanel) and then with a labeled human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. Blots were quantitated by phosphor image analysis, and -fold induction of c-jun mRNA normalized to GAPDH mRNA (FOLD) is indicated. B, activity of luciferase reporter genes in the L929 fibrosarcoma cell line. Jun-luciferase (Luc), or 5XGal-luciferase plus Gal-ElkC, or (KB)3-luciferase (three copies of the consensus NF-kappaB response element linked to luciferase) was transfected into L929 cells. Cells were then treated with TNFalpha (20 ng/ml) or sphingomyelinase (100 milliunits/ml) for 5 h, and extracts were prepared for determination of luciferase activity. -Fold activation represents reporter activity relative to cells treated with vehicle only. Results from one representative experiment performed in duplicate are shown (±S.E.). Results were consistent over at least three experiments.



To further assess the functional sequela of stimulation by TNFalpha or bacterial sphingomyelinase, we measured transcriptional activities with reporter gene assays in L929 fibrosarcoma cells, which are efficiently transfected and are responsive to TNFalpha(21) . TNFalpha and sphingomyelinase are equally effective in stimulating the reporter gene driven by the c-jun promoter (Jun-Luc, Fig. 4B, toppanel), consistent with their stimulation of JNK/SAPK activity (Fig. 1) and c-jun mRNA level (Fig. 4A). The transcriptional activity of a fusion protein consisting of the Gal4 DNA binding domain and the Elk-1 transactivation domain (Gal-ElkC(14) ; a MAP kinase substrate), measured by a reporter plasmid containing a luciferase gene with Gal4 binding sites (5XGal-luciferase), is activated by sphingomyelinase and to a lesser degree by TNFalpha (Fig. 4B, middlepanel), in accord with their stimulation of MAP kinase activity in vitro (Fig. 3). TNFalpha but not sphingomyelinase markedly stimulated an NF-kappaB-responsive reporter gene ((KB)3-luciferase, Fig. 4B, bottompanel), consistent with the effect of these agents on NF-kappaB DNA binding activity (Fig. 2).

TNFalpha signals through two cell surface receptors, TNF-R1 (p55) and TNF-R2 (p75), which contain no apparent catalytic activity and whose intracellular domains are not homologous to characterized signaling proteins. Two proteins, TRAF1 and TRAF2, bind to the cytoplasmic domain of TNF-R2(39) . An unrelated protein, TRADD, binds to the TNF-R1-associated death domain and might be involved in TNF-induced apoptosis and NF-kappaB activation(40) . Most of TNFalpha's biological effects are mediated by TNF-R1(41, 42, 43) , but the mechanisms by which second messengers are recruited are unknown. Several of TNFalpha's second messengers are activated via TNF-R1, including the activation of a sphingomyelinase, protein kinase C, and phospholipase A(2)(41) . By using differences in the species specificity of TNFalpha(44, 45) , we have found that TNFalpha also activates JNK/SAPK through TNF-R1 (data not shown).

Recent studies have delineated the steps immediately upstream of JNK/SAPK activation(46, 47, 48) , but connections between these cytoplasmic and nuclear kinases and upstream messengers leading to their activation are unresolved. This study demonstrates that ceramide is a link between binding of TNFalpha to TNFR1 and activation of a cytoplasmic kinase cascade that results in stimulation of JNK/SAPK activity and c-jun expression. TNFalpha and C(2)-ceramide activate JNK/SAPK to the same degree (Fig. 1) and stimulate activity with similar kinetics in HL-60 cells. We have previously demonstrated that JNK1 (also known as SAPK) is a major component of TNFalpha-activated JNK/SAPK(8) . By activating JNK/SAPK, TNFalpha and ceramide activate a subset of AP-1 transcription factors, such as c-Jun and ATF-2, which in turn will preferentially induce genes with specific non-consensus AP-1 binding sites, such as the c-jun gene itself (Fig. 4). Therefore, this study provides strong evidence that ceramide functions as the second messenger in TNFalpha signaling resulting in the activation of JNK/SAPK and c-jun.

TNFalpha is not a potent activator of MAP kinases(7, 8) , and we demonstrate here that C(2)-ceramide actually decreases MAP kinase activity in HL-60 cells. Inhibition of MAP kinase activity may result from the activity of a ceramide-activated protein phosphatase (17, 18) acting on a component of the MAP kinase cascade. Ceramide-activated protein phosphatase is a phosphatase of the PP2A class, and PP2A activity on both MAP kinases and MEKs has been described (reviewed in (49) ). The weak activation of MAP kinase activity by TNFalpha may be the result of ceramide-independent activation of a component (MEK or MAP kinase itself) downstream of Raf-1 and/or by activation of protein kinase C(50) .

TNFalpha is a potent inducer of NFkappaB (5, 51) ( Fig. 2and Fig. 4), perhaps through the activation of protein kinase C (52) , and ceramide potentiates the stimulation of NFkappaB by TNFalpha(3) . Ceramide stimulates NF-kappaB binding activity in permeabilized cells, which has been attributed to the activation of an acidic sphingomyelinase(32) . However, extracts derived from intact HL-60 cells treated with exogenous sphingomyelinase or C(2)-ceramide have elevated JNK/SAPK activity but not NF-kappaB DNA binding activity. Perhaps these different results reflect the existence of separate ceramide pools, such that C(2)-ceramide and the ceramide generated by exogenous bacterial sphingomyelinase are in a different cellular compartment than the ceramide generated by acidic sphingomyelinase.

The evidence is pointing to the existence of a novel signal transduction pathway that may be specifically involved in the stress response. A number of agents of systemic stress (e.g. TNFalpha) and tissue injury (hypoxia, UV irradiation, and chemotherapeutic agents) activate both a neutral sphingomyelinase and JNK/SAPK. This study provides a link between the sphingomyelinase/ceramide pathway and the JNK/SAPK pathway by demonstrating that ceramide functions upstream of JNK/SAPK, resulting in activation of nuclear downstream effects.


FOOTNOTES

*
This work was supported by Grant DK34987 from the University of North Carolina Center for Gastrointestinal Biology and Disease (to J. K. W.) and by National Institutes of Health Grants CA 50528 and GM 41084 (to D. A. B.) and GM 43825 (to Y. A. H.). 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.

§
Present address: C.B. 7365, Dept. of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7365. jwesty{at}email.unc.edu.

To whom correspondence should be addressed: C.B. 7080, Dept. of Medicine, University of North Carolina, Chapel Hill, NC 27599-7038. Tel.: 919-966-7885; Fax: 919-966-7468; dab{at}med.unc.edu.

(^1)
The abbreviations used are: TNFalpha, tumor necrosis factor alpha; JNK, Jun nuclear kinase; SAPK, stress-activated protein kinase; MAP, mitogen-activated protein; PP2A, protein phosphatase 2A; D-e-C(2), (2S,3R)-D-erythro-N-acetylsphingosine; GST, glutathione S-transferase; MEK, MAP kinase kinase.


ACKNOWLEDGEMENTS

We thank G. Franzoso for advice on extract preparation, C. Hauser for providing the (KB)3-luciferase reporter plasmid, R. Treisman for Gal-ElkC, A. Lin for GST-ElkC, and C. Bradham for comments on the manuscript.


REFERENCES

  1. Vassalli, P. (1992) Annu. Rev. Immunol. 10,411-452 [CrossRef][Medline] [Order article via Infotrieve]
  2. Tracey, K. J., and Cerami, A. (1993) Annu. Rev. Cell Biol. 9,317-343 [CrossRef]
  3. Dbaibo, G. S., Obeid, L. M., and Hannun, Y. A. (1993) J. Biol. Chem. 268,17762-17766 [Abstract/Free Full Text]
  4. Obeid, L. M., Linardic, C. M., Karolak, L. A., and Hannun, Y. A. (1993) Science 259,1769-1771 [Medline] [Order article via Infotrieve]
  5. Osborne, L., Kunkel, S., and Nabel, G. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,2336-2340 [Abstract]
  6. Poli, G., Kinter, A., Justement, J. S., Kehrl, J. H., Bressler, P., Stanley, S., and Fauci, A. S. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,782-785 [Abstract]
  7. Kyriakis, J. M., Banerjee, P., Nikolakaki, E., Dai, T., Ruble, E. A., Ahmad, M. F., Avruch, J., and Woodgett, J. R. (1994) Nature 369,156-160 [CrossRef][Medline] [Order article via Infotrieve]
  8. Westwick, J. K., Weitzel, C., Minden, A., Karin, M., and Brenner, D. A. (1994) J. Biol. Chem. 269,26396-26401 [Abstract/Free Full Text]
  9. Sluss, H. K., Barrett, T., Derijard, B., and Davis, R. J. (1994) Mol. Cell. Biol. 14,8376-8384 [Abstract]
  10. Hibi, M., Lin, A., Smeal, T., Minden, A., and Karin, M. (1993) Genes & Dev. 7,2135-2148
  11. Derijard, B., Hibi, M., Wu, I.-H., Barrett, T., Su, B., Deng, T., Karin, M., and Davis, R. J. (1994) Cell 76,1025-1038 [Medline] [Order article via Infotrieve]
  12. Gupta, S., Campbell, D., Derijard, B., and Davis, R. J. (1995) Science 267,389-393 [Medline] [Order article via Infotrieve]
  13. Hill, C. S., Marais, R., John, S., Wynne, J., Dalton, S., and Treisman, R. (1993) Cell 73,395-406 [Medline] [Order article via Infotrieve]
  14. Marais, R., Wynne, J., and Treisman, R. (1993) Cell 73,381-393 [Medline] [Order article via Infotrieve]
  15. Kim, M.-Y., Linardic, C., Obeid, L., and Hannun, Y. (1991) J. Biol. Chem. 266,484-489 [Abstract/Free Full Text]
  16. Hannun, Y. A. (1994) J. Biol. Chem. 269,3125-3128 [Free Full Text]
  17. Dobrowsky, R. T., Kamibayashi, C., Mumby, M. C., and Hannun, Y. A. (1993) J. Biol. Chem. 268,15523-15530 [Abstract/Free Full Text]
  18. Wolff, R. A., Dobrowsky, R. T., Bielawska, A., Abeid, L. M., and Hannun, Y. A. (1994) J. Biol. Chem. 269,19605-19609 [Abstract/Free Full Text]
  19. Joseph, C. E., Byun, H.-S., Bittman, R., and Kolesnick, R. N. (1993) J. Biol. Chem. 268,20002-20006 [Abstract/Free Full Text]
  20. Bielawska, A., Crane, M. M., Liotta, D., Obeid, L. M., and Hannun, Y. A. (1993) J. Biol. Chem. 268,26226-26232 [Abstract/Free Full Text]
  21. Tsujimoto, M., Yip, Y. K., and Vilcek, J. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,7226-7230
  22. Franzoso, G., Biswas, P., Poli, G., Carlson, L. M., Brown, K. D., Tomita-Yamaguchi, M., Fauci, A. S., and Siebenlist, U. K. (1994) J. Exp. Med. 180,1445-1456 [Abstract]
  23. Westwick, J. K., and Brenner, D. A. (1995) Methods Enzymol. 255,342-359 [Medline] [Order article via Infotrieve]
  24. Smith, S. B., and Johnson, K. S. (1988) Gene (Amst.) 67,31-40 [CrossRef][Medline] [Order article via Infotrieve]
  25. Westwick, J. K., Cox, A. D., Der, C. J., Cobb, M. H., Hibi, M., Karin, M., and Brenner, D. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,6030-6034 [Abstract]
  26. Hattori, K., Angel, P., Le Beau, M. M., and Karin, M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,9148-9152 [Abstract]
  27. Galang, C. K., Der, C. J., and Hauser, C. A. (1994) Oncogene 9,2913-2921 [Medline] [Order article via Infotrieve]
  28. Hauser, C. A., Westwick, J. K., and Quilliam, L. A. (1995) Methods Enzymol. 255,412-426 [Medline] [Order article via Infotrieve]
  29. Hattori, M., Tugores, A., Westwick, J., Veloz, L., Leffert, H. L., Karin, M., and Brenner, D. A. (1993) Am. J. Physiol. 264,G95-G103
  30. Angel, P., Hattori, K., Smeal, T., and Karin, M. (1988) Cell 55,875-885 [Medline] [Order article via Infotrieve]
  31. Yang, Z., Costanzo, M., Golde, D. W., and Kolesnick, R. N. (1993) J. Biol. Chem. 268,20520-20523 [Abstract/Free Full Text]
  32. Schutze, S., Potthoff, K., Hachiedt, T., Berkovic, D., Wiegmann, K., and Kronke, M. (1992) Cell 71,765-776 [Medline] [Order article via Infotrieve]
  33. Betts, J. C., Agranoff, A. B., Nabel, G. J., and Shayman, J. A. (1994) J. Biol. Chem. 269,8455-8458 [Abstract/Free Full Text]
  34. Johns, L. D., Sarr, T., and Ranges, G. E. (1994) J. Immunol. 152,5877-5882 [Abstract/Free Full Text]
  35. Raines, M. A., Kolesnick, R. N., and Golde, D. W. (1993) J. Biol. Chem. 268,14572-14575 [Abstract/Free Full Text]
  36. Finney, R. E., Robbins, S. M., and Bishop, J. M. (1993) Curr. Biol. 3,805-812
  37. van Dam, H., Duyndam, M., Rottier, R., Bosch, A., de Vries-Smits, L., Herrlich, P., Zantema, A., Angel, P., and van der Eb, A. J. (1993) EMBO J. 12,479-487 [Abstract]
  38. Herr, I., van Dam, H., and Angel, P. (1994) Carcinogenesis 15,1105-1113 [Abstract]
  39. Rothe, M., Wong, S. C., Henzel, W. J., and Goeddel, D. V. (1994) Cell 78,681-692 [Medline] [Order article via Infotrieve]
  40. Hsu, H., Xiong, J., and Goeddel, D. V. (1995) Cell 81,495-504 [Medline] [Order article via Infotrieve]
  41. Wiegmann, K., Schutze, S., Kampen, E., Himmler, A., Machleidt, T., and Kronke, M. (1992) J. Biol. Chem. 267,17997-18001 [Abstract/Free Full Text]
  42. Smith, C. A., Farrah, T., and Goodwin, R. G. (1994) Cell 76,959-962 [Medline] [Order article via Infotrieve]
  43. Erickson, S. L., de Sauvage, F. J., Kikly, K., Carver-Moore, K., Pitts-Meek, S., Gillett, N., Sheehan, K. C. F., Schreiber, R. D., Goeddel, D. V., and Moore, M. W. (1994) Nature 372,560-563 [Medline] [Order article via Infotrieve]
  44. Vandenabeele, P., Declercq, W., Vercammen, D., Van de Craen, M., Grooten, J., Loetscher, H., Brockhaus, M., Lesslauer, W., and Fiers, W. (1992) J. Exp. Med. 176,1015-1024 [Abstract]
  45. Lewis, M., Tartaglia, L. A., Bennett, G. L., Rice, G. C., Wong, G. H. W., Chen, E. Y., and Goeddel, D. V. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,2830-2834 [Abstract]
  46. Yan, M., Dai, T., Deak, J. C., Kyriakis, J. M., Zon, L. I., Woodgett, J. R., and Templeton, D. J. (1994) Nature 372,798-800 [Medline] [Order article via Infotrieve]
  47. Sanchez, A., Hughes, R. T., Mayer, B. J., Yee, K., Woodgett, J. R., Avruch, J., Kyriakis, J. M., and Zon, L. I. (1994) Nature 372,794-798 [Medline] [Order article via Infotrieve]
  48. Lin, A., Minden, A., Martinetto, H., Claret, F.-X., Lange-Carter, C., Mercurio, F., Johnson, G. L., and Karin, M. (1995) Science 268,286-290 [Medline] [Order article via Infotrieve]
  49. Mumby, M. C., and Walter, G. (1993) Physiol Rev. 73,673-699 [Abstract/Free Full Text]
  50. Brenner, D. A., O'Hara, M., Angel, P., Chojkier, M., and Karin, M. (1989) Nature 337,661-663 [CrossRef][Medline] [Order article via Infotrieve]
  51. Duh, E. J., Maury, W. J., Folks, T. M., Fauci, A. S., and Rabson, A. B. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,5974-5978 [Abstract]
  52. Lozano, J. B. E., Municia, M. M., Diaz-Meco, M. T., Dominguez, I., Sanz, L., and Moscat, J. (1994) J. Biol. Chem. 269,19200-19202 [Abstract/Free Full Text]

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