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Transactivation by the P65 Subunit of NF-B in Response to Interleukin-1

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Transactivation by the P65 Subunit of NF-B in Response to Interleukin-1 MOLECULAR AND CELLULAR BIOLOGY, July 2001, p. 4544–4552 Vol. 21, No. 14 0270-7306/01/$04.00ϩ0 DOI: 10.1128/MCB.21.14.4544–4552.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved. Transactivation by the p65 Subunit of NF-␬B in Response to Interleukin-1 (IL-1) Involves MyD88, IL-1 Receptor-Associated Kinase 1, TRAF-6, and Rac1 CAROLINE JEFFERIES,1* ANDREW BOWIE,1 GARETH BRADY,1 EMMA-LOUISE COOKE,2 3 1 XIAOXIA LI, AND LUKE A. J. O’NEILL Department of Biochemistry and Biotechnology Institute, Trinity College, Dublin 2, Ireland1; Department of Cell Biology, Glaxo Wellcome Research and Development, Medicines Research Centre, Stevenage, Hertfordshire SG1 2NY, United Kingdom2; and Department of Molecular Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 441953 Received 30 August 2000/Returned for modification 2 October 2000/Accepted 19 April 2001 We have examined the involvement of components of the interleukin-1 (IL-1) signaling pathway in the transactivation of gene expression by the p65 subunit of NF-␬B. Transient transfection of cells with plasmids encoding wild-type MyD88, IL-1 receptor-associated kinase 1 (IRAK-1), and TRAF-6 drove p65-mediated transactivation. In addition, dominant negative forms of MyD88, IRAK-1, and TRAF-6 inhibited the IL-1- induced response. In cells lacking MyD88 or IRAK-1, no effect of IL-1 was observed. Together, these results indicate that MyD88, IRAK-1, and TRAF-6 are important downstream regulators of IL-1-mediated p65 transactivation. We have previously shown that the low-molecular-weight G protein Rac1 is involved in this response. Constitutively active RacV12-mediated transactivation was not inhibited by dominant negative MyD88, while dominant negative RacN17 inhibited the MyD88-driven response, placing Rac1 downstream of MyD88 on this pathway. Dominant negative RacN17 inhibited wild-type IRAK-1- and TRAF-6-induced trans- activation, and in turn, dominant negative IRAK-1 and TRAF-6 inhibited the RacV12-driven response, sug- gesting a mutual codependence of Rac1, IRAK-1, and TRAF-6 in regulating this pathway. Finally, Rac1 was found to associate with the receptor complex via interactions with both MyD88 and the IL-1 receptor accessory protein. A pathway emanating from MyD88 and involving IRAK-1, TRAF-6, and Rac1 is therefore involved in transactivation of gene expression by the p65 subunit of NF-␬B in response to IL-1. Regulation of the transcription factor nuclear factor kappa Tollip (Toll-interacting protein), which has been shown to be B (NF-␬B) following stimulation with the proinflammatory involved in IRAK-1 recruitment to the receptor complex via cytokine interleukin-1 (IL-1) occurs via activation of two inde- association of Tollip with IL-1RAcP (7). Once associated with pendent pathways (28). The first and to date best-characterized the receptor complex, IRAK-1 subsequently recruits the pathway regulates the release of NF-␬B (typically a het- adapter tumor necrosis factor (TNF) receptor-associated fac- erodimer comprising the p50 and p65 subunits) from its inhib- tor 6 (TRAF-6), an essential mediator of IL-1 signaling to ␬ ␬ itory protein I B, allowing NF- B to translocate to the nu- NF-␬B activation (10). Subsequent autophosphorylation of cleus. The second pathway, which has recently been described, IRAK-1 is thought to promote dissociation of IRAK-1 from regulates the transactivating ability of the p65 subunit of the receptor complex, thus enabling downstream signaling, re- ␬ NF- B once it is bound to its consensus sequence (3, 17). sulting in activation of the I␬B kinase (IKK) complex, respon- ␬ IL-1 signal transduction to I B degradation has been the sible for I␬B phosphorylation and subsequent ubiquitin-medi- subject of intense investigation (21). In response to IL-1 bind- ated degradation (12, 40). The upstream kinases involved in ing to its type I IL-1 receptor (IL-1RI), a complex is formed activating IKK1 and -2 are thought to belong to the mitogen- between IL-1RI and its accessory protein (IL-1RAcP). These activated protein kinase (MAPK) family of kinases. NF-␬B- proteins have a region of homology in their cytoplasmic do- interacting kinase (NIK) has been shown to phosphorylate and mains, the Toll/IL-1R (TIR) domain, which is characteristic of activate the IKKs when overexpressed in cells, and a role for the Toll/IL-1-like receptor superfamily and is also responsible the kinase TAK1 and its regulator TAB1, upstream of NIK, for signaling (27). The cytosolic adapter protein MyD88 (also has been indicated (23). However, the involvement of NIK in containing a C-terminal TIR domain) interacts with IL-1RAcP regulating IKK activation in response to either TNF or IL-1 via a homotypic interaction involving both TIR domains and is has recently been disputed (2). MEKK-1 (MAPK/ERK ki- a key regulator of IL-1 signal transduction (5, 8, 16). The nase-1) has also been shown to phosphorylate and activate the interaction of MyD88 with the receptor complex mediates the IKKs (20), and recently an adapter protein, ECSIT (evolution- recruitment of the IL-1 receptor-associated kinases (IRAK) 1 and 2 (9, 26). Recently, a novel protein has been identified, arily conserved signaling intermediate in Toll pathways), has been described which regulates MEKK-1 and links TRAF-6 with MEKK-1 regulation (19). In contrast, little is known about the signaling components * Corresponding author. Mailing address: Department of Biochem- istry and Biotechnology Institute, Trinity College, Dublin 2, Ireland. involved in the pathway regulating the transactivating activity Phone: 353-1-6082449. Fax: 353-1-6772400. E-mail: [email protected]. of the p65 subunit of NF-␬B. Several reports have demon- 4544 VOL. 21, 2001 IL-1 REGULATION OF p65 TRANSACTIVATION 4545 strated that upon stimulation with either IL-1 or TNF, the dominant negative RacN17 were kind gifts from D. Cantrell (Imperial Cancer ability of the p65 subunit of NF-␬B to transactivate gene ex- Research Fund, London, United Kingdom) and have been described elsewhere (13). The cDNA for MyD88 was cloned into the bacterial expression vector pression is enhanced, possibly as a result of phosphorylation of pGEX, expressed in Escherichia coli BL21(DE3) as a fusion protein with gluta- multiple serine residues on p65 (3, 4, 17, 36). The kinases thione S-transferase (GST), and purified with glutathione-agarose beads (Sigma) involved in regulating this response following TNF stimulation by standard protocols. have, to some extent, been identified. Protein kinase A has Transient-transfection and reporter gene assays. EL4.NOB-1 cells (7 ϫ 106) been demonstrated to phosphorylate serine 276 in the Rel were transfected with plasmids as indicated in the figure legends in a final volume of 0.6 ml using DEAE-dextran. Following 16 to 18 h of recovery, cells were homology domain of p65, and casein kinase II has been shown seeded at a density of 106 viable cells (as determined by the trypan blue dye Ϫ Ϫ to be responsible for phosphorylating serine 529 in the C- exclusion method) prior to stimulation. 293-RI and 293-IRAK / cells (2 ϫ 104 terminal transactivation domain (37, 38, 41). In addition, re- per well) were seeded onto 96-well plates and transfected 24 h later with 5 ng of (1–551) ports have also indicated the ability of the IKKs to phosphor- Gal4-p65 , 25 ng of Gal-luciferase, and 40 ng of Renilla luciferase (used as an internal control) with FuGENE 6 (Roche) according to the manufacturer’s ylate p65 on serine 536 in the transactivation domain while the recommendations. MEK and MyD88-deficient cells (2.5 ϫ 104 per well) were protein is in the cytoplasm, adding another level of complexity seeded onto 24-well plates and transfected 24 h later with 100 ng of Gal4- to the regulation of transactivation of gene expression by p65 p65(1–551), 200 ng of Gal-luciferase, and 200 ng of Renilla luciferase with Fu- (30). Phosphorylation of either the Rel homology domain or GENE 6 (Roche) according to the manufacturers’ recommendations. In all the transactivation domain of p65 mediates the interaction of cases, the amount of DNA transfected was kept constant by the addition of various amounts of the appropriate empty vector plasmid. Cells were either left p65 with coactivators such as CREB-binding protein (CBP). A untreated or stimulated with IL-1 (10 ng/ml) as indicated following a period of role for phosphatidylinositol 3-kinase (PI3K) in the events recovery (16 to 18 h). To assay firefly luciferase activity, cells were lysed using leading to phosphorylation of p65 in response to IL-1 has been passive lysis buffer (Promega), and luciferase activity was determined by standard ␤ demonstrated (34). procedures. Renilla luciferase acivity and -galactosidase activity were deter- mined by standard protocols and used to normalize firefly luciferase activity in Despite the rapidly accumulating evidence for a role of var- relation to transfection efficiency. ious kinase pathways in regulating p65-mediated transactiva- Immunoprecipitation and Western blot analysis. Cells were treated as de- tion, much research is required into the signaling events reg- scribed in the figure legends for the times indicated, and treatment was termi- ulating the kinases responsible for phosphorylating p65. nated by the addition of 5 ml of ice-cold phosphate-buffered saline (PBS). Cells Previous studies in our laboratory have demonstrated the in- were lysed on ice (30 min) in buffer containing 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% NP-40, 0.2 mM phenylmethylsulfonyl fluoride, 0.2 mM Na3VO4 and volvement of the low-molecular-weight G protein Rac1 in reg- 1 ␮g of leupeptin per ml. Lysates were cleared by centrifugation, and following ulating these events in response to IL-1 stimulation, but exactly clearing for 30 min at 4°C with protein G-Sepharose (Sigma), AU1-tagged how Rac1 mediates this signal remains to be discovered.
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