Activation of NF-Kb by Oncogenic Raf in HEK 293 Cells Occurs Through Autocrine Recruitment of the Stress Kinase Cascade

Oncogene (1998) 17, 685 ± 690  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Activation of NF-kB by oncogenic Raf in HEK 293 cells occurs through autocrine recruitment of the stress kinase cascade

Jakob Troppmair, JoÈ rg Hartkamp and Ulf R Rapp

Institut fuÈr Medizinische Strahlenkunde und Zellforschung (MSZ), University of WuÈrzburg, Versbacher Str. 5, 97078 WuÈrzburg, Germany

Raf-1 kinase has been implicated in the induction of NF- kB activation by a variety of stimuli (Bruder et al., kB activity by serum growth factors, phorbol ester and 1993; Li and Sedivy, 1993; Finco and Baldwin, 1993). PTK oncogenes. Here we show that Raf activation of To study Raf dependent signaling pathways leading to NF-kB, as measured in reporter gene assays, occurs NF-kB activation we analysed the induction of a indirectly and requires the stress kinase cascade. The 6xNF-kB element driven luciferase reporter construct stress pathway presumably becomes activated through by a constitutively active form of Raf, Raf BXB induction of an autocrine loop by activated Raf (Raf- (Bruder et al., 1992). Our experiments show that this BXB) as suramin, the tyrphostin AG1478 and a pathway uses common Raf downstream signaling dominant negative mutant of the EGF-R blocked NF- elements, but is also sensitive to inhibition by kB activation. Raf-BXB synergizes with SAPKs and a dominant negative mutants of Ras and Raf. These dominant negative mutant of SEK signi®cantly reduces latter ®ndings suggest the involvement of membrane activation of NF-kB consistent with a role of this proximal events in the activation process. Presumably, signaling pathway in the activation of NF-kB. the mechanism of Raf-mediated NF-kB activation includes membrane receptors because inhibitors of Keywords: Raf; stress kinase pathway; NF-kB autocrine signaling loops block Raf-BXB eect. To test the potential involvement of protein kinase cascades that function in parallel to the Raf-MEK- MAPK pathway (Daum et al., 1994; Kyriakis and Introduction Avruch, 1996) in this process we examined the role of SAPKs (Kyriakis et al., 1994) and p38/RK (Han et al., NF-kB/Rel transcription factor dimers are constitu- 1994; Rouse et al., 1994). Over-expression of SAPKa in tively located within the nucleus of mature B cells 293 cells results in a strong transcriptional activation of whereas in other cell types this transcription factor the NF-kB response element, whereas no such eect is complex is bound to cytoplasmic inhibitor proteins of observed with p38RK. Furthermore, SAPK synergised the IkB family (Baeuerle and Baltimore, 1996; Wulczyn with Raf in transcription activation. A potential role et al., 1996; Schreck et al., 1997). Activation of for stress kinase pathways in the Raf induced cytoplasmic NF-kB requires degradation of IkBa, transcription activation is further supported by the triggered by phosphorylation of serines 32 and 36 ®nding that Raf-BXB induced NF-kB activation could (Baeuerle and Baltimore, 1988, 1996; Wulczyn et al., be blocked by a dominant negative mutant of the 1996; Schreck et al., 1997). Several kinases have been JNK/SAPK activating kinase SEK-1 (Sanchez et al., implicated in the phosphorylation of the serines at 1994). position 32 and 36. These protein kinases include p90rsk (Schouten et al., 1997), a MEKK-1 activated IkB kinase (Lee et al., 1997) which is part of a bigger Results 700 kDa protein complex (Chen et al., 1996) and the recently identi®ed kinase IKK/Chuck (DiDonato et al., Activation of NF-kB dependent transcription by 1997; Regnier et al., 1997). oncogenic Raf requires MEK/ERK and is blocked by Although several reports suggest that the PSK Raf-1 dominant negative Raf-1 mutants phosphorylates IkB (Li and Sedivy, 1993; Finco and Baldwin, 1993; Janosch et al., 1996) the sites of Oncogenic Raf activates NF-kB in 293 cells (Figure 1a) phosphorylation have not been identi®ed and indirect as detected in luciferase assays using a 6x NF-kB eects could not be excluded. Nevertheless, activtion of reporter gene and in EMSAs which used NIH3T3 cells NF-kB-dependent transcription has been observed that have no basal NF-kB activity (Figure 1b). These consistently following stimulation by Raf-1, PKC or data con®rm previously reported results (Bruder et al., PTK receptors (Bruder et al., 1992; Li and Sedivy, 1992; Li and Sedivy, 1993; Finco and Baldwin, 1993; 1993; Finco and Baldwin, 1993; Flory et al., 1998). In Flory et al., 1998). Induction is sensitive to IkBa as particular a dominant negative mutant of Raf-1, Raf- cotransfection completely abolished this eect (Figure C4B (Bruder et al., 1992), which interferes with Ras- 1a). dependent activation of Raf-1, completely blocks NF- We next investigated the signaling pathways employed by activated Raf in 293 cells for the stimulation of NF-kB. Figure 1a shows that a dominant negative mutant of ERK 2, C3 (Robbins et Correspondence: UR Rapp Received 14 October 1997; revised 16 March 1998; accepted 17 al., 1993), which has been shown previously to March 1998 completely block Raf-BXB induced ERK activation Stress kinase dependent NF-kB activation by Raf J Troppmair et al 686 (Troppmair et al., 1994) completely abolished tran- mutant blocks all Ras eectors by competing for Ras scription activation by Raf-BXB. This shows that binding (Bruder et al., 1992; Zhang et al., 1993). classical components of Raf downstream signaling are Surprisingly, Raf-BXB stimulation of the 6xNF-kB required for NF-kB activation. We also included in our reporter was sensitive to the inhibitory eect of Raf- experiments a dominant negative mutant of Raf-1, C4B (Figure 1a). The speci®city of this inhibition was Raf-C4B (Bruder et al., 1992). Over-expression of this demonstrated by use of a mutant of Raf-C4B, Raf-

Figure 1 (a) Activation of NF-kB dependent transcription by oncogenic Raf (Raf-BXB) is blocked by dominant negative mutants of ERK-2, Raf-1 (Raf-C4B) and Ras (Ras-N17). 0.6 mg of Raf-BXB and 0.5 mg 6xNF-kB luciferase reporter plasmid were cotransfected with 5 ± 7.5 mg of the plasmids indicated. These concentrations had given maximum inhibition in titration experiments. (Summary of results obtained in six independent experiments, three in the case of DN-ERK-2). Transfection of Raf-BXB routinely resulted in a 10 ± 20-fold induction of the reporter construct. Raf-C4BPM17 is a mutant of Raf-C4B with reduced ability to inhibit Ras dependent signaling. Luciferase activity was assayed 48 h after transfection. (b) NF-kB binding activity is increased in v-Raf transformed NIH3T3 cells. TNFa stimulation of control NIH3T3 cells was carried out for 2 h with 50 ng/ml. (c) Activation of NF- kB involves autocrine mechanisms which are responsive towards the inhibitory action of suramin, the tyrphostin AG1478 or a dominant negative EGF-R mutant. Following transfection cells were kept in the presence of suramin (10 mM) or AG1478 (1 mM). 0.6 mg of the reporter plasmid were transfected together with 1 ± 2 mg of Raf-BXB. In the case of the DN-EGF-R 2.5 ± 5.0 mgof mutant DNA were used. Presence of the solvent DMSO (for AG1478) had no eect. Results represent mean values from three experiments. Potential downregulating eects of the dominant negative mutants and of suramin and the tyrphostin AG1478 were excluded by demonstrating that the expression levels of Raf-BXB protein were unaltered under these circumstances Stress kinase dependent NF-kB activation by Raf J Troppmair et al 687 C4BPM17, which functions as a less eective Ras membrane by interfering with ligand-receptor interac- blocker (Bruder et al., 1992). These results suggest that tions (Garret et al., 1984; Betsholtz et al., 1986). As Raf-BXB stimulates NF-kB indirectly by a mechanism shown in Figure 1c presence of suramin signi®cantly that involves Ras. inhibits the Raf-BXB eect on reporter gene expression. To directly test for involvement of the EGF receptor, the target of both TGFa and hbEGF, we Ras is required for the activation of NF-kB dependent used a dominant negative mutant of the EGF-R transactivation by Raf-BXB (Redemann et al., 1992), comprizing the extracellular To directly test for a role of Ras in Raf stimulated NF- ligand binding domain of the receptor and a speci®c kB reporter gene expression the eect of the dominant inhibitor of the receptor kinase, tyrphostin AG1478 negative Ras mutant N17Ras (Farnsworth and Feig, (Fry et al., 1994; Levitzki and Gazit, 1995). This agent 1991) as well as oncogenic v-Ha-Ras were tested. has been shown to speci®cally block EGF-R autopho- Exchange of serine at codon 17 to asparagine of Ras sphorylation (Fry et al., 1994; Levitzki and Gazit, results in preferential binding of GDP over GTP. It is 1995). Moreover, presence of a related substance, PD speculated that the mutant protein retains high anity 153035, at a concentration of 0.3 micromolar for the GDP/GTP exchange factor (GEF) and thereby completely reverted ligand induced transformation of suppresses activtion of endogenous Ras by competing Swiss 3T3 cells transfected with a human EGF-R (Fry for available GEF (Farnsworth and Feig, 1991). As et al., 1994). As shown in Figure 1c presence of either shown in Figure 1a over-expression of N17Ras in 293 the dominant negative EGF-receptor mutant or cells completely blocks the Raf-BXB eect whereas AG1478 blocked reporter gene expression in 293 cells oncogenic v-Ha-Ras showed a sevenfold increase in (Figure 1c). We conclude that Raf-BXB activation of luciferase activity as compared to vector control (data the NF-kB dependent reporter involves induction of not shown). These ®ndings are consistent with a role ligand(s) for the EGF-R. for Ras in Raf-mediated NF-kB activation. Function of the SAPK/JNK cascade in the activation of Activation of NF-kB by oncogenic Raf involves autocrine NF-kB by Raf-BXB mechanisms EGF-R is known not only to activate the mitogenic As Raf does not directly aect Ras activity (Kyriakis et cascade but also the stress cascade especially when al., 1992) an indirect mechanism perhaps involving activated by hbEGF (McCarthy et al., 1995). To induction of an autocrine loop via TGFa and/or determine whether elements of the JNK/SAPK or hbEGF has to be considered (Heidecker et al., 1989; p38RK cascades could mediate NF-kB activation we McCarthy et al., 1995). To evaluate this possibility tested the relevant eector kinases in transient assays. suramin was added after transfection of the NF-kB Whereas p38RK was not active in these assays (Figure reporter gene and Raf-BXB DNA. Previous studies 2a), overexpression of SAPKa induced the reporter have demonstrated the suitability of this drug to block gene (Figure 2a). This induction could be further activation of growth factor receptors in the plasma increased by coexpression with suboptimal levels of

Figure 2 Overexpression of SAPKa but not p38RK results in the activation of NF-kB-dependent transcription (a) and synergizes with Raf-BXB (b). a: decreasing amounts of Raf-BXB (0.6, 0.3, and 0.15 mg) or SAPKa (5 and 2.5 mg) were cotransfected with 0.5 mg of 6xNF-kB luciferase reporter plasmid. b: 0.5 mg of 6xNF-kB reporter plasmid were transfected with 0.2 mg of Raf-BXB, 5 mg of SAPKa or a combination thereof. Luciferase activity was assayed 48 h after transfection. Experiments were performed twice with identical results Stress kinase dependent NF-kB activation by Raf J Troppmair et al 688

Figure 4 Raf-BXB induces activation of SAPKb in 293 cells via MEK- and Ras dependent pathways. 293 cells were transiently transfected with the DNAs indicated and activity of immunopre- cipitated p54-HA-SAPKb was measured 48 h following transfection. pcDNA LIDA is a dominant negative mutant of MEK (Cowley et al., 1994), Raf-C4B is a dominant negative mutant of Raf-1 (Bruder et al., 1992). This experiment was performed twice with identical results

cotransfection of a dominant negative mutant of MEK (pcDNA-LIDA) or Raf-1 (Raf-C4B) abolished JNK activation (Figure 4). These data thus show that Raf- activated pathways leading to activation of NF-kB overlap with those that mediate SAPK activation.

Material and methods

Figure 3 Raf-BXB induced activation of NF-kB dependent Plasmids transcription can be blocked by a dominant negative Sek mutant Constructs for the expression of constitutively active or (pEBG-SEKK-R). 1 mg of Raf-BXB DNA was cotransfected with dominant negative mutants of Raf, Ras, MEK and ERK2 5 mg of pEBG-SEKK-R or the vector control (pEBG). Luciferase have been described previously (Bruder et al., 1992; activity was assayed 48 h after transfection. Summary of three experiments Farnsworth and Feig, 1991; Cowley et al., 1994; Robbins et al., 1993; Troppmair et al., 1994). The dominant negative ERK2 C3 mutant was recloned into the KRSPA vector (Bruder et al., 1992), the dominant negative MEK Raf-BXB (Figure 2b). SEK-1 is the physiological mutant (LIDA) was expressed from the vector pcDNA3 activator of SAPKa (Sanchez et al., 1994). In order (Invitrogen). Plasmid pEBG-SEK (Sanchez et al., 1994) to test the requirement of the stress cascade for NF-kB was kindly provided by Dr John Kyriakis. The kinase

activation we co-transfected the kinase dead inhibitory inactive mutant pEBG-SEKK-R was generated by Dr S

SEK-1 mutant (pEBG-SEKK-R) together with Raf- Ludwig as previously described (Sanchez et al., 1994). A

BXB. pEBG-SEKK-R speci®cally blocked Raf-BXB dominant negative EGF-R mutant was kindly provided by induced NF-kB activation (Figure 3). Dr Axel Ullrich (Redemann et al., 1992) and recloned into Stimulation of cells with growth factors which the expression vector KRSPA (Bruder et al., 1992). p38RK activate Raf-1 results in a modest activation of SAPK was a gift of Dr J Han and was recloned for these (Kyriakis and Avruch, 1996) whereas expression of experiments into the expression vector KRSPA (Bruder et al., 1992). Plasmids pMT2-SAPKa and b were a generous constitutively active forms of Raf in ®broblasts leads to gift of Dr Jim Woodgett. The 6xNF-kB motif driven pronounced and prolonged activation of stress kinases luciferase reporter construct and the IkBa expression via an autocrine pathway (McCarthy et al., 1995; construct (Haskill et al., 1991; Pahl and Baeurele, 1995) Kerkho and Rapp, 1997). To test whether expression were provided by Dr Patrick Bauerle. of Raf-BXB in 293 cells also leads to the activation of SAPK, an HA-tagged version of p54SAPKb was EMSA cotransfected with activated Raf (Raf-BXB) and SAPK activity was measured 48 h after transfection Whole cell extracts were prepared and mobility shift assays in an immune-complex kinase assay using GST-c-Jun (EMSAs) were performed as reported previously (Wirth as substrate. As shown in Figure 4 transfection of Raf- and Baltimore, 1988; Lernbecher et al., 1993). BXB resulted in increased SAPK activity. The degree of activation was comparable to the eect of Cells and transfection anisomycin stimulation (data not shown). Further- 293 human embryonic kidney cells and NIH3T3 more, blockage of Raf-1 downstream signaling by ®broblasts were routinely grown in DMEM medium Stress kinase dependent NF-kB activation by Raf J Troppmair et al 689 supplemented with 10% heat inactivated fetal calf serum, signaling. Two compounds were used: suramin, which 2mML-glutamine and 100 U/ml of penicillin/streptomy- blocks ligand receptor interaction (Garret et al., 1984; cin. Cells were seeded at a density of 2.56105 cells per Betsholtz et al., 1986), and a speci®c inhibitor of the 2 ml in six well plates and transfected 48 h later using EGF-R PTK activity, the tyrphostin AG 1478 (Fry et al., the modi®ed calcium phosphate method (Chen and 1994; Levitzki and Gazit, 1995). Both substances eciently Ohayama, 1987). 0.5 mg of reproter plasmid were blocked Raf-BXB induced NF-kB activation. To further transfected together with expression plasmids as indi- establish this point we employed a dominant negative cated in the ®gure legends. Total amount of DNA mutant of the EGF-R. Expression of this mutant blocked transfected was kept constant by ®lling up with vector Raf-BXB induced NF-kB reporter gene expression. DNA. 6 ± 12 h after transfection precipitates were The signal from the EGF-R that mediates NF-kB removed by washing with phosphate buered saline activation is presumably propagated via Ras because (PBS) and cells were refed with medium containing dominant negative mutants of Ras as well as the Ras 0.3% serum. Forty-eight hours after transfection cells blocking mutant of Raf (Raf-C4B) inhibit induced reporter were washed with PBS and cell pellets were either shock gene expression. We postulate therefore a branchpoint in frozen in liquid nitrogen and stored at 7808Cor signal transduction cascade from the EGF-R at the level of analysed directly. Ras. The role of Ras in regulation of stress kinase pathways is not fully understood. The most potent physiological activators of JNK/SAPK do not seem to Luciferase assay require Ras (Kyriakis and Avruch, 1996). On the other Cell pellets were lysed in 400 ml of harvesting buer hand, overexpression of Ras in PC12 cells results in the (50 mM Tris, 50 mM MES, pH 8.7, 1 mM DTT, 0.1% activation of MEKK1, a kinase functioning in the Triton X-100), precleared by centrifugation (15 000 g, activation cascade of JNK/SAPK, and stimulation of 10 min at 48C), standardized for protein concentration MEKK1 activity by EGF is blocked by a dominant and 50 ml of lysate were analysed together with 50 mlof negative Ras mutant (Lange-Carter and Johnson, 1994). assay buer (125 mM TRIS, 125 mM MES, pH 7.8, Nevertheless, comparison of Ras with two small GTPases of 25 mM magnesium acetate, 5 mM ATP) and 50 mlofD- the Rho family, Rac and Cdc42, showed that Ras only luciferin (1 mM in 5 mM KHPO4,pH7.8)ina poorly induced JNK/SAPK activity (Minden et al., 1995; luminometer (Microlumat LB 96P, Berthold AG, Ger- Coso et al., 1995). Experiments using dominant interfering many). alleles of Rac1 position Rac1 between Ha-Ras and MEKK1 in the signaling cascade leading from growth factor receptors and v-Src to JNK activation (Minden et al., SAPK assays and immunoblotting 1994). Such a position of Rac relative to Ras has also been An HA-tagged version of the pMT2-p54SAPKb (Kyriakis shown for Ras transformation (Qui et al., 1995). It is thus et al., 1994) was used for the analysis of SAPK activation conceivable that Ras dependent activation of Rac connects in transiently transfected 293 cells. Immunoprecipitation receptor PTK signaling with the activation of JNK/SAPK. and assay of kinase activity were performed as previously Ras requirement for the activation of JNK/SAPK has also described for the analysis of ERK-1 (Troppmair et al., been demonstrated for the cytoplasmic PTK Bcr-abl 1994) using puri®ed GST-c-jun (1 ± 135) as substrate. (Raitano et al., 1995). Our results support published data Immunoblot analysis was carried out as described suggesting a role of Ras in the activation of JNK/SAPK previously (Kerkho and Rapp, 1997). under conditions where PTKs function as activators. Data relating to the role of stress kinase pathways in the activation of NF-kB are limited and controversial. Over- expression of MEKK1, an activator for the JNK/SAPK Discussion upstream kinase SEK/MKK4, induced transcription from the HIV-LTR as well as the IL2Ra promoter in a NF-kB Use of dominant negative Raf mutants has implicated dependent manner (Lee et al., 1997). Furthermore, JNK1 Raf-1 PSK in the activation of NF-kB by a variety of synergized with MEKK1 in this process. More recently it has stimuli including phorbol esters, serum, or PTK oncogenes been demonstrated that addition of puri®ed MEKK1 protein (Bruder et al., 1993; Li and Sedivy, 1993; Finco and to the multi subunit containing IkBa kinase complex (Lee et Baldwin, 1993). Furthermore, transfection of oncogenic al., 1997) resulted in the activation of IkBa kinase in vitro Raf as well as Ras resulted in enhanced transcription from and phosphorylation of IkBa on serines 32 and 36. MEKK1 NF-kB driven reporter constructs (Bruder et al., 1993; induction of IkBa kinase activity was independent of Finco and Baldwin, 1993; Finco et al., 1997) and ubiquitination and thus may constitute a separate pathway transfection of gag-v-raf into NIH3T3 cells led to elevated for NF-kB activation. However, this report failed to detect nuclear levels of the p65 subunit of NF-kB (Li and any requirement for MKK4 or JNK in the activation of NF- Sedivy, 1993). Since activation of NF-kB requires kB. It is therefore possible that a separate MEKK1 initiated phosphorylation dependent degradation of its cytoplasmic signaling pathway is involved. A MEKK1 independent inhibitor IkB several reports have addressed the question pathway for the activation of NF-kB has been suggested by whether Raf itself might function as IkB kinase. These the analysis of TNF receptor I (TNF-RI) signaling (Liu et al., reports, however, have contradictory results. Whereas Raf 1996). Thus the precise link between JNK/SAPK pathways was initially presented as an IkB kinase (Li and Sedivy, and signaling pathways leading to NF-kB activation remains 1993), subsequent work has pointed to an indirect to be determined. mechanism involving casein kinase II (CKII), thought to be speci®cally Raf-associated (Janosch et al., 1996). As none of the reports demonstrated phosphorylation of serines 32 and 36 on IkB which are critical for NF-kB activation other eector kinases might have intervened. Abbreviations The data in the present paper suggest a role for Raf-1 far PTK, protein tyrosine kinase; PSK, protein serine kinase; more distal to the phosphorylation of IkB. Dominant EGF, epidermal growth factor; hbEGF, heparin binding negative mutants of Raf and Ras blocked activation of EGF; EGF-R, EGF receptor; EMSA, electrophoretic gel NF-kB dependent transcription by oncogenic Raf (Raf- mobility shift essay; HIV, human immunode®ciency virus; BXB). We therefore tested the possible involvement of LTR, long terminal repeat, SAPK, stress activated protein autocrine loops by using drugs that interfere with receptor kinase. Stress kinase dependent NF-kB activation by Raf J Troppmair et al 690 Acknowledgements PfraÈ nger for her help in the preparation of ®gures. We are We thank Barbara Bauer and Petra Lorenz for excellent grateful to Drs P Baeuerle, J Kyriakis, J Han, A Ullrich technical assistance, Drs Roland Houben, Angelika and J Woodgett for providing expression plasmids. This Homeyer, Eugen Kerkho, Barbara Kistler, Christoph work was supported by a grant from the DFG (Sonder- Weber, Joseph Slupsky, Bernd Baumann and Thomas forschungsbereich 172, C13). Wirth for helpful discussions and suggestions and Silvia

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