Ras Controls Tumor Necrosis Factor Receptor-associated Factor (TRAF)6-dependent Induction of Nuclear Factor-κB
2001; Elsevier BV; Volume: 276; Issue: 9 Linguagem: Inglês
10.1074/jbc.m006772200
ISSN1083-351X
AutoresChristopher J. Caunt, Endre Kiss-Tóth, Franço Carlotti, Robert M. Chapman, Eva E. Qwarnström,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoIn the present study, we show that Ras activity differentially controls interleukin (IL)-1 induced transcription factor activation by selective regulation of responses mediated by receptor complex components. Initial experiments revealed that stimulation with IL-1 caused a rapid, matrix-dependent activation of Ras. The effect was transient, peaking at 5 min and returning to base levels after 30 min. Activation correlated with pronounced changes in cell shape in EGFPH-Ras transfected cells. Transfection with the dominant negative mutant, RasAsn-17, inhibited IL-1 induced activation of the IL-8 promoter as well as of NF-κB and AP-1 synthetic promoters in transient transfection assays. Furthermore, overexpression of the IL-1 signaling proteins TRAF6 or MyD88 gave characteristic activation of IL-8, which was accentuated in the presence of IL-1. Co-transfection with RasAsn-17 gave a dose-dependent inhibition of TRAF6-induced responses in the presence and absence of IL-1, but had no effect on MyD88 mediated activity. Similarly, induction of NF-κB was abolished by RasAsn-17 only in TRAF6-transfected cells. In contrast, inhibiting Ras activity limited AP-1-mediated responses through both receptor complex proteins. Constitutively active RasVal-12increased the TRAF6 induced activity of the NF-κB pathway similar to the effect induced by IL-1, while the RasVal-12 induced activity was not inhibited by co-transfection with a dominant negative TRAF6. Our data show that activation of the Ras GTPase is an early, matrix-dependent response in IL-1 signaling which participates in structural regulation of IL-1-induced genes. In addition, they show that the Ras induced effect selectively regulates TRAF6-mediated activation of the NF-κB pathway, suggesting that Ras GTPase represents a convergence point in structural and cytokine responses, with distinct effects on a subset of downstream signaling events. In the present study, we show that Ras activity differentially controls interleukin (IL)-1 induced transcription factor activation by selective regulation of responses mediated by receptor complex components. Initial experiments revealed that stimulation with IL-1 caused a rapid, matrix-dependent activation of Ras. The effect was transient, peaking at 5 min and returning to base levels after 30 min. Activation correlated with pronounced changes in cell shape in EGFPH-Ras transfected cells. Transfection with the dominant negative mutant, RasAsn-17, inhibited IL-1 induced activation of the IL-8 promoter as well as of NF-κB and AP-1 synthetic promoters in transient transfection assays. Furthermore, overexpression of the IL-1 signaling proteins TRAF6 or MyD88 gave characteristic activation of IL-8, which was accentuated in the presence of IL-1. Co-transfection with RasAsn-17 gave a dose-dependent inhibition of TRAF6-induced responses in the presence and absence of IL-1, but had no effect on MyD88 mediated activity. Similarly, induction of NF-κB was abolished by RasAsn-17 only in TRAF6-transfected cells. In contrast, inhibiting Ras activity limited AP-1-mediated responses through both receptor complex proteins. Constitutively active RasVal-12increased the TRAF6 induced activity of the NF-κB pathway similar to the effect induced by IL-1, while the RasVal-12 induced activity was not inhibited by co-transfection with a dominant negative TRAF6. Our data show that activation of the Ras GTPase is an early, matrix-dependent response in IL-1 signaling which participates in structural regulation of IL-1-induced genes. In addition, they show that the Ras induced effect selectively regulates TRAF6-mediated activation of the NF-κB pathway, suggesting that Ras GTPase represents a convergence point in structural and cytokine responses, with distinct effects on a subset of downstream signaling events. nuclear factor-κB interleukin 1 receptor tumor necrosis factor receptor-associated factor IL-1R associated kinase cytomegalovirus epidermal growth factor enhanced green fluorescent protein Cellular responses to cytokines and growth factors are influenced by the extracellular milieu (1Lukashev M.E. Werb Z. Trends Cell Biol. 1998; 8: 437-441Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). These regulatory effects are mediated to a large extent by receptors of the integrin family and involve modulation of signaling pathways that link receptor activation and changes in cell shape, the cytoskeleton, and gene expression (2Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5184) Google Scholar, 3Lim L. Manser E. Leung T. Hall C. Eur. J. Biochem. 1996; 242: 171-185Crossref PubMed Scopus (273) Google Scholar). Ras acts cooperatively and/or hierarchically with the Rho subfamily of guanine nucleotide triphosphatases (GTPases) to regulate focal adhesion and polymerized actin turnover (4Nobes, C. D., and Hall, A. (1999) J. Cell Biol..Google Scholar). It is thought to be in their arrangement of signaling proteins and receptors via the cytoskeleton that the GTPases are able to channel a diverse range of stimuli, from growth factor and cytokines to cell attachment, into common downstream signaling cascades (5Scita G. Tenca P. Frittoli E. Tocchetti A. Innocenti M. Giardina G. Di Fiore P.P. EMBO J. 2000; 19: 2393-2398Crossref PubMed Google Scholar) such as the stress-activated protein kinase (6Coso O.A. Chiariello M., Yu, J.C. Teramoto H. Crespo P. Xu N.G. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar, 7Minden A. Lin A.N. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1443) Google Scholar) and nuclear factor-κB (NF-κB)1 pathways (8Perona R. Montaner S. Saniger L. SanchezPerez I. Bravo R. Lacal J.C. Genes Dev. 1997; 11: 463-475Crossref PubMed Scopus (532) Google Scholar,9Devary Y. Rosette C. Didonato J.A. Karin M. Science. 1993; 261: 1442-1445Crossref PubMed Scopus (574) Google Scholar).The membrane proximal molecular events that take place following interleukin-1 receptor (IL-1R) binding and leading to transcription factor activation involves initially the heterodimerization of the IL-1R with accessory protein (IL-1RAcP) (10Greenfeder S.A. Nunes P. Kwee L. Labow M. Chizzonite P.A. Ju G. J. Biol. Chem. 1995; 270: 13757-13765Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar), followed by binding of the adaptor protein MyD88 (11Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (973) Google Scholar). MyD88 in turn recruits the Ser/Thr kinases: IL-1R associated kinase (IRAK) 1, IRAK-2, and IRAK-3 or M (11Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (973) Google Scholar, 12Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z.D. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar, 13Wesche H. Gao X. Li X.X. Kirschning C.J. Stark G.R. Cao Z.D. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), which have recently been shown to be pre-associated with a further adaptor protein, Tollip (14Burns K. Clatworthy J. Martin L. Martinon F. Plumton C. Maschera B. Lewis A. Ray K. Tschopp J. Volpe F. Nat. Cell Biol. 2000; 2: 346-351Crossref PubMed Scopus (441) Google Scholar). Subsequently, the IL-1R associated kinase 1s are freed and interact with the tumor necrosis factor receptor-associated factor (TRAF) 6, followed by activation of the stress-activated protein kinase and NF-κB pathways (15Cao Z.D. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1111) Google Scholar).The majority of IL-1 receptors are located at focal adhesions (16Qwarnstrom E.E. Page R.C. Gillis S. Dower S.K. J. Biol. Chem. 1988; 263: 8261-8269Abstract Full Text PDF PubMed Google Scholar), and matrix attachment results in alterations in IL-1 receptor function (17Valles S. Tsoi C. Huang W.Y. Wyllie D. Carlotti F. Askari J.A. Humphries M.J. Dower S.K. Qwarnstrom E.E. J. Biol. Chem. 1999; 274: 20103-20109Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Subsequent IL-1-induced signal transduction and gene activation are regulated by cell attachment and the cytoskeleton (18Qwarnstrom E.E. Ostberg C.O. Turk G.L. Richardson C.A. Bomsztyk K. J. Biol. Chem. 1994; 269: 30765-30768Abstract Full Text PDF PubMed Google Scholar, 19Ostberg C.O. Zhu P. Wight T.N. Qwarnstrom E.E. FEBS Lett. 1995; 367: 93-97Crossref PubMed Scopus (27) Google Scholar, 20Zhu P. Xiong W.S. Rodgers G. Qwarnstrom E.E. Biochem. J. 1998; 330: 975-981Crossref PubMed Scopus (54) Google Scholar). Early alterations following IL-1 receptor binding in adherent cells include a rapid and transient phosphorylation of transmembrane linkage protein Talin and changes in the cytoskeleton (21Qwarnstrom E.E. Macfarlane S.A. Page R.C. Dower S.K. Proc. Natl. Acad. Sci. U. S.A. 1991; 88: 1232-1236Crossref PubMed Scopus (69) Google Scholar), suggesting an immediate effect on the turnover of structural components at these sites. Aspects of Ras, Rho, Rac, and Cdc42 involvement in downstream IL-1 signaling pathways have recently been elucidated (22Palsson E.M. Popoff M. Thelestam M. O'Neill L.A.J. J. Biol. Chem. 2000; 275: 7818-7825Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 23Singh R. Wang B. Shirvaikar A. Khan S. Kamat S. Schelling J.R. Konieczkowski M. Sedor J.R. J. Clin. Invest. 1999; 103: 1561-1570Crossref PubMed Scopus (120) Google Scholar, 24Jefferies C.A. O'Neill L.A.J. J. Biol. Chem. 2000; 275: 3114-3120Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 25Puls A. Eliopoulos A.G. Nobes C.D. Bridges T. Young L.S. Hall A. J. Cell Sci. 1999; 112: 2983-2992Crossref PubMed Google Scholar), suggesting they play a part in mediating these structural changes localized around the receptor complex. This may underpin the, as yet, unclear mechanism by which the GTPases interact with members of the classical IL-1 signaling cascade. So far, however, little is still known of how these effects are initiated, and how they may modulate IL-1 signaling and influence downstream cascades.We report here that IL-1-induced structural alterations in adherent cells involve activation of the Ras GTPase as an early signaling event, which regulates IL-1-induced NF-κB transactivation of inflammatory genes. In addition, we show that Ras regulation of NF-κB affects TRAF6-mediated activation, but not that induced through MyD88, indicating that IL-1 induced pathways leading to NF-κB activation diverge upstream of TRAF6. Furthermore, the data suggest that Ras represents a convergence of IL-1 and matrix-mediated signaling events induced downstream of the adaptor proteins.DISCUSSIONIn the present study, we demonstrate that IL-1 transiently activates Ras in a matrix-dependent fashion, correlating with translocation of EGFPH-Ras fusion protein and structural effects on IL-1 responses in transfected cells. We also show that the regulation of IL-1-mediated NF-κB activity by Ras is induced through TRAF6.The IL-1-induced Ras activation occurs during the initial steps of signal transduction involving heterodimerization of the receptor and the accessory protein and in matrix-attached cells, recruitment of a heparan sulfate to the receptor complex. Association of the adaptor protein MyD88 results in activation of the receptor IRAKs and association of TRAF6 (see Fig. 7). The observed Ras activity correlate with the similarly rapid, matrix-dependent IL-1 induced serine phosphorylation of the transmembrane linkage protein, Talin preceding the alterations in cell structure and the changes in the cytoskeleton (20Zhu P. Xiong W.S. Rodgers G. Qwarnstrom E.E. Biochem. J. 1998; 330: 975-981Crossref PubMed Scopus (54) Google Scholar, 21Qwarnstrom E.E. Macfarlane S.A. Page R.C. Dower S.K. Proc. Natl. Acad. Sci. U. S.A. 1991; 88: 1232-1236Crossref PubMed Scopus (69) Google Scholar). Such alterations are known to be directly associated with events related to GTPase activation (33Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3049) Google Scholar). These data agree with recent findings of essential roles for other members of the GTPase family in IL-1 signaling (22Palsson E.M. Popoff M. Thelestam M. O'Neill L.A.J. J. Biol. Chem. 2000; 275: 7818-7825Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 23Singh R. Wang B. Shirvaikar A. Khan S. Kamat S. Schelling J.R. Konieczkowski M. Sedor J.R. J. Clin. Invest. 1999; 103: 1561-1570Crossref PubMed Scopus (120) Google Scholar, 24Jefferies C.A. O'Neill L.A.J. J. Biol. Chem. 2000; 275: 3114-3120Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 25Puls A. Eliopoulos A.G. Nobes C.D. Bridges T. Young L.S. Hall A. J. Cell Sci. 1999; 112: 2983-2992Crossref PubMed Google Scholar), and together with the known interdependence between members of the Ras and Rho subfamilies suggest that their roles in IL-1-mediated responses to be interconnected.Our data further suggest that the IL-1 effect involves an increase in the concentration of the GTPase at the plasma membrane during activation. This is likely, to a significant extent, to be a consequence of the reduction in cell shape. However, Ras isoforms have been shown to have the ability to rapidly diffuse through the plasma membrane (34Niv H. Gutman O. Henis Y.I. Kloog Y. J. Biol. Chem. 1999; 274: 1606-1613Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) and an increase in local concentration at the cell surface could reflect recruitment to specific areas to facilitate signal transduction. This could involve a mechanism similar to that demonstrated for Ras, Rac, and Rho during integrin and growth factor signaling, when localization to caveolin-rich regions in pre-assembled complexes is a prerequisite for signal transduction (35Roy S. Luetterforst R. Harding A. Apolloni A. Etheridge M. Stang E. Rolls B. Hancock J.F. Parton R.G. Nat. Cell Biol. 1999; 1: 98-105Crossref PubMed Scopus (124) Google Scholar, 36Wary K.K. Mariotti A. Zurzolo C. Giancotti F.G. Cell. 1998; 94: 625-634Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 37Michaely P.A. Mineo C. Ying Y.S. Anderson R.G.W. J. Biol. Chem. 1999; 274: 21430-21436Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar).The rapid induction of Ras activation suggests that it is induced through an immediate, receptor-associated event, and could be a direct consequence of the early IRAKs and TRAF6 association. This likely involves receptor-associated regulators of the GTPases such as the putative Rap/GTPase activating protein (GAP), IIP-1 (38Sims, J. E., Bird, T. A., and Mitcham, J. L. (1995)Cytokine 324.Google Scholar). Furthermore, the attachment dependence suggests that activation could be induced as a consequence of recruitment of the matrix-dependent, accessory receptor component to the IL-1 receptor complex (17Valles S. Tsoi C. Huang W.Y. Wyllie D. Carlotti F. Askari J.A. Humphries M.J. Dower S.K. Qwarnstrom E.E. J. Biol. Chem. 1999; 274: 20103-20109Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) and thus result from co-regulation through integrin mediated activities. Such effects could be induced following selective activation of signaling components by structural events, as suggested by our data demonstrating specificity for the activities mediated through receptor-associated proteins MyD88 and TRAF6. This type of collaboration would thus be similar to that reported for MyD88 and Tollip during recruitment to the IL-1 receptor (14Burns K. Clatworthy J. Martin L. Martinon F. Plumton C. Maschera B. Lewis A. Ray K. Tschopp J. Volpe F. Nat. Cell Biol. 2000; 2: 346-351Crossref PubMed Scopus (441) Google Scholar).We show that IL-1-induced NF-κB mediated transcription of IL-8 is dependent on Ras activity, demonstrated by using wild type and dominant negative mutant forms of Ras in transient transfection reporter assays. This agrees with previous findings from this laboratory, showing a direct dependence of IL-1 induced inflammatory genes (19Ostberg C.O. Zhu P. Wight T.N. Qwarnstrom E.E. FEBS Lett. 1995; 367: 93-97Crossref PubMed Scopus (27) Google Scholar) and NF-κB activation (18Qwarnstrom E.E. Ostberg C.O. Turk G.L. Richardson C.A. Bomsztyk K. J. Biol. Chem. 1994; 269: 30765-30768Abstract Full Text PDF PubMed Google Scholar, 20Zhu P. Xiong W.S. Rodgers G. Qwarnstrom E.E. Biochem. J. 1998; 330: 975-981Crossref PubMed Scopus (54) Google Scholar) on cell architecture and attachment. These effects of Ras could be mediated through MEKK (39Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1010) Google Scholar) with subsequent effects on the NF-κB pathway (40Hirano M. Osada S. Aoki T. Hirai S. Hosaka M. Inoue J. Ohno S. J. Biol. Chem. 1996; 271: 13234-13248Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 41Meyer C.F. Wang X. Chang C. Templeton D. Tan T.H. J. Biol. Chem. 1996; 271: 8971-8976Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 42Karin M. Delhase M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9067-9069Crossref PubMed Scopus (204) Google Scholar) resulting from induction of IκBα phosphorylation (43Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar). However, the lack of effect of nuclear translocation of RelA could reflect that Ras, as has been suggested for Rac1 (24Jefferies C.A. O'Neill L.A.J. J. Biol. Chem. 2000; 275: 3114-3120Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), may regulate subsequent transactivation events such as phosphorylation of nuclear NF-κB (44Zhong H.H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1021) Google Scholar). In addition, the burgeoning evidence of complexity of NF-κB activity, involving association of nuclear IκBα·NF-κB complexes subsequent to independent translocation (45Johnson C. VanAntwerp D. Hope T.J. EMBO J. 1999; 18: 6682-6693Crossref PubMed Google Scholar) and regulation through RelA shuttling 2F. Carlotti, S. K. Dower, and E. E. Qwarnstrom, submitted for publication. suggests that significant activation of the pathway may be induced without any net effect on the level of nuclear RelA. This is also supported by results from studying the endogenous protein demonstrating that activation of the pathway does not necessarily correlate with an absolute increase in nuclear NF-κB. 3E. E. Qwarnstrom and S. K. Dower, unpublished data. Finally, it is possible that the lack of RelA translocation reflects that structural induction activates only a subset of NF-κB-dependent genes, as has been shown for glycogen synthase kinase-3β (46Hoeflich K.P. Luo J. Rubie E.A. Tsao M.S. Jin O. Woodgatt J.R. Nature. 2000; 406: 86-90Crossref PubMed Scopus (1207) Google Scholar).This type of specificity is also in agreement with the suggested divergence of the pathway upstream of TRAF6, and indicate that MyD88 can activate NF-κB via a TRAF6 independent pathway. The effect on both TRAF6 and MyD88 induced AP-1 regulation indicate that Ras may be able to influence activation of specific genes through selectivity of adaptor protein function, reflected in distinct sets of upstream regulators. The total lack of effect of the dominant negative TRAF6 on NF-κB activation induced through transfection with RasVal-12 suggests that structural regulation through Ras feeds into the NF-κB pathway downstream of TRAF6 or is induced through second messengers totally separate from that regulated by TRAF6. Thus, activation through TRAF6 via TAB-2 and TAK-1 (47Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1010) Google Scholar) constitutes an IL-1-regulated pathway controlling NF-κB, while other regulators, such as NIK and NAK (48Tojima Y. Fujimoto A. Delhase M. Chen Y. Hatakeyama S. Nakayama K. Kaneko Y. Nimura Y. Motoyama N. Ikeda K. Karin M. Nakanishi M. Nature. 2000; 404: 778-782Crossref PubMed Scopus (312) Google Scholar) have been shown not to directly mediate IL-1 induced NF-κB activity. The indicated synergy in the observed response suggests, however, that whatever the upstream discrepancies in signaling, the Ras-induced effect on the NF-κB pathway is not totally distinct from the IL-1 mode of activation but rather that, the two activities converge upstream of induction of transcription.This type of receptor proximal divergence of TRAF6/MyD88-regulated pathways has been reported in in vitro studies on Toll signaling (49Muzio M. Natoli G. Saccani S. J. Exp. Med. 1998; 187: 2097-2101Crossref PubMed Scopus (525) Google Scholar). Studies on TRAF6 and MyD88 knockout mice have also shown a degree of compensation and/or redundancy and partial overlapping at the level of the adaptor proteins in transcriptional activation by IL-1 and lipopolysaccharide (50Lomaga M.A. Yeh W.C. Sarosi I. Duncan G.S. Furlonger C. Ho A. Morony S. Capparelli C. Van G. Kaufman S. vanderHeiden A. Itie A. Wakeham A. Khoo W. Sasaki T. Cao Z.D. Penninger J.M. Paige C.J. Lacey D.L. Dunstan C.R. Boyle W.J. Goeddel D.V. Mak T.W. Genes Dev. 1999; 13: 1015-1024Crossref PubMed Scopus (1068) Google Scholar, 51Kawai T. Adachi O. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 115-122Abstract Full Text Full Text PDF PubMed Scopus (1703) Google Scholar). Other examples are signaling proteins ECSIT (evolutionarily conserved signaling intermediate in toll signaling) (52Kopp E. Medzhitov R. Carothers J. Xiao C.C. Douglas I. Janeway C.A. Ghosh S. Genes Dev. 1999; 13: 2059-2071Crossref PubMed Scopus (269) Google Scholar) and glycogen synthase kinase-3β (46Hoeflich K.P. Luo J. Rubie E.A. Tsao M.S. Jin O. Woodgatt J.R. Nature. 2000; 406: 86-90Crossref PubMed Scopus (1207) Google Scholar) which provide selective options for cross-talk between kinase pathways in increasingly complex regulatory networks. By analogy with these systems, the type of model applied to our data suggests that IL-1 mediated responses may be "tailored" by GTPase activation and regulated by events such as cell attachment induced by selective involvement of receptor complex components (see Fig. 7). Cellular responses to cytokines and growth factors are influenced by the extracellular milieu (1Lukashev M.E. Werb Z. Trends Cell Biol. 1998; 8: 437-441Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). These regulatory effects are mediated to a large extent by receptors of the integrin family and involve modulation of signaling pathways that link receptor activation and changes in cell shape, the cytoskeleton, and gene expression (2Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5184) Google Scholar, 3Lim L. Manser E. Leung T. Hall C. Eur. J. Biochem. 1996; 242: 171-185Crossref PubMed Scopus (273) Google Scholar). Ras acts cooperatively and/or hierarchically with the Rho subfamily of guanine nucleotide triphosphatases (GTPases) to regulate focal adhesion and polymerized actin turnover (4Nobes, C. D., and Hall, A. (1999) J. Cell Biol..Google Scholar). It is thought to be in their arrangement of signaling proteins and receptors via the cytoskeleton that the GTPases are able to channel a diverse range of stimuli, from growth factor and cytokines to cell attachment, into common downstream signaling cascades (5Scita G. Tenca P. Frittoli E. Tocchetti A. Innocenti M. Giardina G. Di Fiore P.P. EMBO J. 2000; 19: 2393-2398Crossref PubMed Google Scholar) such as the stress-activated protein kinase (6Coso O.A. Chiariello M., Yu, J.C. Teramoto H. Crespo P. Xu N.G. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar, 7Minden A. Lin A.N. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1443) Google Scholar) and nuclear factor-κB (NF-κB)1 pathways (8Perona R. Montaner S. Saniger L. SanchezPerez I. Bravo R. Lacal J.C. Genes Dev. 1997; 11: 463-475Crossref PubMed Scopus (532) Google Scholar,9Devary Y. Rosette C. Didonato J.A. Karin M. Science. 1993; 261: 1442-1445Crossref PubMed Scopus (574) Google Scholar). The membrane proximal molecular events that take place following interleukin-1 receptor (IL-1R) binding and leading to transcription factor activation involves initially the heterodimerization of the IL-1R with accessory protein (IL-1RAcP) (10Greenfeder S.A. Nunes P. Kwee L. Labow M. Chizzonite P.A. Ju G. J. Biol. Chem. 1995; 270: 13757-13765Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar), followed by binding of the adaptor protein MyD88 (11Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (973) Google Scholar). MyD88 in turn recruits the Ser/Thr kinases: IL-1R associated kinase (IRAK) 1, IRAK-2, and IRAK-3 or M (11Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (973) Google Scholar, 12Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z.D. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar, 13Wesche H. Gao X. Li X.X. Kirschning C.J. Stark G.R. Cao Z.D. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), which have recently been shown to be pre-associated with a further adaptor protein, Tollip (14Burns K. Clatworthy J. Martin L. Martinon F. Plumton C. Maschera B. Lewis A. Ray K. Tschopp J. Volpe F. Nat. Cell Biol. 2000; 2: 346-351Crossref PubMed Scopus (441) Google Scholar). Subsequently, the IL-1R associated kinase 1s are freed and interact with the tumor necrosis factor receptor-associated factor (TRAF) 6, followed by activation of the stress-activated protein kinase and NF-κB pathways (15Cao Z.D. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1111) Google Scholar). The majority of IL-1 receptors are located at focal adhesions (16Qwarnstrom E.E. Page R.C. Gillis S. Dower S.K. J. Biol. Chem. 1988; 263: 8261-8269Abstract Full Text PDF PubMed Google Scholar), and matrix attachment results in alterations in IL-1 receptor function (17Valles S. Tsoi C. Huang W.Y. Wyllie D. Carlotti F. Askari J.A. Humphries M.J. Dower S.K. Qwarnstrom E.E. J. Biol. Chem. 1999; 274: 20103-20109Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Subsequent IL-1-induced signal transduction and gene activation are regulated by cell attachment and the cytoskeleton (18Qwarnstrom E.E. Ostberg C.O. Turk G.L. Richardson C.A. Bomsztyk K. J. Biol. Chem. 1994; 269: 30765-30768Abstract Full Text PDF PubMed Google Scholar, 19Ostberg C.O. Zhu P. Wight T.N. Qwarnstrom E.E. FEBS Lett. 1995; 367: 93-97Crossref PubMed Scopus (27) Google Scholar, 20Zhu P. Xiong W.S. Rodgers G. Qwarnstrom E.E. Biochem. J. 1998; 330: 975-981Crossref PubMed Scopus (54) Google Scholar). Early alterations following IL-1 receptor binding in adherent cells include a rapid and transient phosphorylation of transmembrane linkage protein Talin and changes in the cytoskeleton (21Qwarnstrom E.E. Macfarlane S.A. Page R.C. Dower S.K. Proc. Natl. Acad. Sci. U. S.A. 1991; 88: 1232-1236Crossref PubMed Scopus (69) Google Scholar), suggesting an immediate effect on the turnover of structural components at these sites. Aspects of Ras, Rho, Rac, and Cdc42 involvement in downstream IL-1 signaling pathways have recently been elucidated (22Palsson E.M. Popoff M. Thelestam M. O'Neill L.A.J. J. Biol. Chem. 2000; 275: 7818-7825Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 23Singh R. Wang B. Shirvaikar A. Khan S. Kamat S. Schelling J.R. Konieczkowski M. Sedor J.R. J. Clin. Invest. 1999; 103: 1561-1570Crossref PubMed Scopus (120) Google Scholar, 24Jefferies C.A. O'Neill L.A.J. J. Biol. Chem. 2000; 275: 3114-3120Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 25Puls A. Eliopoulos A.G. Nobes C.D. Bridges T. Young L.S. Hall A. J. Cell Sci. 1999; 112: 2983-2992Crossref PubMed Google Scholar), suggesting they play a part in mediating these structural changes localized around the receptor complex. This may underpin the, as yet, unclear mechanism by which the GTPases interact with members of the classical IL-1 signaling cascade. So far, however, little is still known of how these effects are initiated, and how they may modulate IL-1 signaling and influence downstream cascades. We report here that IL-1-induced structural alterations in adherent cells involve activation of the Ras GTPase as an early signaling event, which regulates IL-1-induced NF-κB transactivation of inflammatory genes. In addition, we show that Ras regulation of NF-κB affects TRAF6-mediated activation, but not that induced through MyD88, indicating that IL-1 induced pathways leading to NF-κB activation diverge upstream of TRAF6. Furthermore, the data suggest that Ras represents a convergence of IL-1 and matrix-mediated signaling events induced downstream of the adaptor proteins. DISCUSSIONIn the present study, we demonstrate that IL-1 transiently activates Ras in a matrix-dependent fashion, correlating with translocation of EGFPH-Ras fusion protein and structural effects on IL-1 responses in transfected cells. We also show that the regulation of IL-1-mediated NF-κB activity by Ras is induced through TRAF6.The IL-1-induced Ras activation occurs during the initial steps of signal transduction involving heterodimerization of the receptor and the accessory protein and in matrix-attached cells, recruitment of a heparan sulfate to the receptor complex. Association of the adaptor protein MyD88 results in activation of the receptor IRAKs and association of TRAF6 (see Fig. 7). The observed Ras activity c
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