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First published online 18 January 2005
doi: 10.1242/jcs.01641

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TRIP6 is a RIP2-associated common signaling component of multiple NF-B activation pathways

¹ Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing 100871, China
² Department of Immunology, National Jewish and Research Center, University of Colorado Health Sciences Center, 1400 Jackson Street, Denver, CO 80206, USA

View larger version (41K): [in a new window]   Fig. 1. RIP2 interacts with TRIP6. (A) The structures of RIP2, TRIP6 and their deletion mutants. The kinase and CARD domains of RIP2 and the LIM domains and nuclear export sequence (NES) of TRIP6 are indicated. (B) TRIP6 interacts with RIP2 and its individual domains. 293 cells (2x106) were transfected with 5 µg each of HA-TRIP6 and Flag-tagged RIP2 or its mutant plasmids. Cell lysates were immunoprecipitated with anti-Flag antibody (-F) or control mouse IgG (C). The immunoprecipitates were analysed by western blot with horseradish-peroxidase (HRP)-conjugated anti-HA antibody (top). Expression of the transfected proteins in the lysates was detected by western blot analysis with anti-Flag (middle) or anti-HA (bottom) antibody. (C) RIP2 interacts with the LIM domains of TRIP6. 293 cells (2x106) were transfected with 5 µg each of Flag-RIP2 and HA-tagged TRIP6 and its mutant plasmids. Cell lysates were immunoprecipitated with anti-Flag antibody (-F) or control mouse IgG (C). The immunoprecipitates and cell lysates (L) were analysed by western blot with a combination of anti-HA and anti-Flag antibodies. (D) RIP2 and TRIP6 interact in untransfected cells in a stimulation-dependent manner. 293 cells (2x107) were treated with TNF (10 ng ml-1) or IL-1 (10 ng ml-1), or left untreated for 5 minutes. Cells were lysed and the lysate was immunoprecipitated with goat anti-RIP2 antibody or control goat IgG. The immunoprecipitates were analysed by western blot with rabbit anti-TRIP6 antibody.

View larger version (27K): [in a new window]   Fig. 2. TRIP6 is involved in RIP2-mediated NF-B activation. (A) TRIP6 weakly activates NF-B in a dose-dependent manner. 293 cells (1x105) were transfected with 0.1 µg NF-B luciferase reporter plasmid and the indicated amounts of HA-tagged TRIP6 expression plasmid. Luciferase assays were performed 14 hours after transfection. TRIP6 expression levels in the transfected cells were analysed by western blot with anti-HA antibody (bottom). (B) TRIP6 synergizes with RIP to activate NF-B. 293 cells (1x105) were transfected with 0.1 µg NF-B luciferase reporter plasmid, 0.5 µg Flag-RIP2 expression plasmid and the indicated amounts of HA-TRIP6 expression plasmid. Luciferase assays were performed 14 hours after transfection. Increased TRIP6 expression did not affect RIP2 levels as suggested by western blots with anti-Flag and anti-HA antibodies (bottom). (C) Effects of TRIP6 deletion mutants on RIP2-mediated NF-B activation. 293 cells (1x105) were transfected with 0.1 µg NF-B luciferase reporter plasmid, 0.5 µg Flag-RIP2 expression plasmid (empty bars) or empty control plasmid (filled bars), and 0.5 µg of plasmids encoding the indicated HA-tagged TRIP6 mutant. Luciferase assays were performed 14 hours after transfection. Expression levels of RIP2 and the TRIP6 mutants were analysed by western blots with anti-Flag and anti-HA antibodies respectively (bottom).

View larger version (31K): [in a new window]   Fig. 3. TRIP6 interacts with TRAF2. 293 cells (2x106) were transfected with 5 µg each of plasmids encoding HA-TRIP6 or Flag-tagged TRADD, TRAF2 or IKKß. Cell lysates were immunoprecipitated with anti-Flag antibody (-F) or control mouse IgG (C). The immunoprecipitates were analysed by western blot with horseradish-peroxidase (HRP)-conjugated anti-HA antibody. Expression of the transfected proteins in the lysates was comparable, as indicated by western blot analysis with anti-Flag and anti-HA antibodies (bottom).

View larger version (29K): [in a new window]   Fig. 4. TRIP6 is involved in TNF-induced NF-B activation. (A) TRIP6 potentiates TNF-induced NF-B activation. 293 cells (1x105) were transfected with 0.1 µg NF-B luciferase reporter plasmid and the indicated amounts of TRIP6 expression plasmid. 14 hours after transfection, cells were treated with TNF (10 ng ml-1) (filled bars) or left untreated (empty bars) for 6 hours before luciferase assays were performed. (B) TRIP6 has no significant effect on IFN--induced IRF-1 activation. 293 cells (1x105) were transfected with 0.1 µg IRF-1 luciferase reporter plasmid and 1 µg TRIP6-encoding or control plasmid. 14 hours after transfection, cells were treated with IFN- (100 ng ml-1) (filled bars) or left untreated (empty bars) for 6 hours before luciferase assays were performed. (C) TRIP6 mutants inhibit TNF-induced NF-B activation. 293 cells (1x105) were transfected with 0.1 µg NF-B luciferase reporter plasmid and 0.5 µg TRIP6-(280-480)-encoding (filled bars), TRIP-(1-279)-encoding (dashed bars) or empty control (empty bars) plasmid. 14 hours after transfection, cells were treated with TNF (10 ng ml-1) or left untreated, as indicated, for 6 hours before luciferase assays were performed. (D) TRIP6 mutants inhibit TRAF2- and TRADD-induced NF-B activation. 293 cells (1x105) were transfected with 0.1 µg NF-B luciferase reporter plasmid, 0.5 µg HA-tagged TRIP6-(280-480)-encoding (filled bars), HA-tagged TRIP6-(1-279)-encoding (dashed bars) or empty control (empty bars) plasmid, and 0.5 µg of Flag-tagged TRAF2 or TRADD expression plasmid, as indicated. Luciferase assays were performed 14 hours after transfection. Expression levels of the transfected proteins were analysed by western blot with anti-Flag and anti-HA antibodies (bottom).

View larger version (37K): [in a new window]   Fig. 5. TRIP6 interacts with signaling components in the IL-1, TLR2 and Nod1 pathways. (A) TRIP6 interacts with IL-1 receptors, TLR2 and Nod1 but not MEKK3. 293 cells (2x106) were transfected with 5 µg each of HA-TRIP6 and Flag-tagged IL-1Rs (IL-1R+IL-1RAcP), TLR2, Nod1 or MEKK3. Cell lysates were immunoprecipitated with anti-Flag antibody (F) or control mouse IgG (C). The immunoprecipitates were analysed by western blots with horseradish-peroxidase (HRP)-conjugated anti-HA antibody (right). Expression of the transfected proteins in the lysates was detected by western blot analysis withanti-Flag and anti-HA antibodies (left). (B) TRIP6 interacts with MyD88, Tollip, IRAK and TRAF6. 293 cells (2x106) were transfected with 5 µg each of plasmids encoding HA-TRIP6 and Flag-tagged IRAK, Tollip or TRAF6, or Myc-tagged MyD88, as indicated. Before immunoprecipitation, cells were treated with IL-1 (+) or left untreated (-), as indicated, for 2 minutes. Cell lysates were immunoprecipitated with anti-Flag antibody (F), anti-Myc (M) or control mouse IgG (C). The immunoprecipitates were analysed by western blots with HRP-conjugated anti-HA antibody (top). The same blots were detected again with anti-Myc or anti-Flag antibody as indicated (middle). Expression of transfected HA-TRIP6 in the lysates was detected by western blot analysis with anti-HA antibody (bottom).

View larger version (26K): [in a new window]   Fig. 6. TRIP6 is involved in IL-1-, TLR2- and Nod1-induced NF-B activation. (A) TRIP6 potentiates IL-1-induced NF-B activation. 293 cells (1x105) were transfected with 0.1 µg NF-B luciferase reporter plasmid and the indicated amounts of TRIP6 expression plasmid. 14 hours after transfection, cells were treated with IL-1 (10 ng ml-1) (filled bars) or left untreated (empty bars) for 6 hours before luciferase assays were performed. (B) TRIP6 potentiates TLR2-induced NF-B activation. 293 cells (1x105) were transfected with 0.1 µg NF-B luciferase reporter plasmid, 0.5 µg TLR2-encoding (filled bars) or empty control (empty bars) plasmid, and the indicated amounts of TRIP6 expression plasmid. Luciferase assays were performed 14 hours after transfection. (C) TRIP6 potentiates Nod1-induced NF-B activation. The experiments were done as in B except that TLR2 was replaced with 0.1 µg Nod1 expression plasmid. (D,E) A TRIP6 mutant inhibits IL-1-, TLR2- and Nod1-induced NF-B activation. 293 cells (1x105) were transfected with 0.1 µg NF-B luciferase reporter plasmid and 0.5 µg TRIP6-(280-480)-encoding (filled bars) or empty control (empty bars) plasmid. 14 hours after transfection, cells were treated with IL-1 (10 ng ml-1) or left untreated for 6 hours before luciferase assays were performed (D). Alternatively, 293 cells (1x105) were transfected with 0.1 µg NF-B luciferase reporter plasmid, 0.5 µg TRIP6-(280-480)-encoding (filled bars) or empty control (empty bars) plasmid, and 0.5 µg of TLR2 (D) or Nod1 (E) expression plasmid. Luciferase assays were performed 14 hours after transfection. (F) TRIP6 dominant-negative mutant has no effect on IFN--induced IRF-1 activation. 293 cells (1x105) were transfected with 0.1 µg IRF-1 luciferase reporter plasmid and 1 µg TRIP6-(280-476)-encoding (filled bars) or control (empty bars) plasmid. 14 hours after transfection, cells were treated with IFN- (100 ng ml-1) or left untreated for 6 hours before luciferase assays were performed.

View larger version (27K): [in a new window]   Fig. 7. Effects of TRIP6 RNAi on NF-B activation by various stimuli. (A) TRIP6 RNAi inhibits the expression of transfected TRIP6. 293 cells (2x105) were transfected with 1 µg of an expression plasmid for Flag-TRIP6, 1 µg of an expression plasmid for Flag-IKK and the indicated amounts of TRIP6 RNAi plasmid (pSuper-TRIP6). 14 hours after transfection, cell lysates were analysed by western blot with anti-Flag antibody. (B) TRIP6 RNAi inhibits expression of endogenous TRIP6. 293 cells were transfected with pSuper or pSuper-TRIP6. Transfected cells were selected with puromycin (10 µg ml-1) for 1 week. Cell lysates were analysed by western blot with a rabbit anti-TRIP6 and a monoclonal anti-ß-actin antibody. (C) Effects of TRIP6 RNAi on NF-B activation by various stimuli. NF-B reporter gene assays with the cell lines stably transfected with TRIP6 RNAi or control vector were performed as in Figs 4, 6.

View larger version (12K): [in a new window]   Fig. 8. TRIP6 potentiates RIP2- and Nod1-mediated Elk1 activation. (A) Effects of TRIP6 and its mutants on Elk1 activation. The detection of ERK activity was measured by Elk1 activity according to the PathDetect in vivo signal transduction pathway trans reporting systems protocols (Stratagene, La Jolla, CA). 293 cells (2x105) were transfected with 0.5 µg pFR plasmid, 0.05 µg the trans-activating plasmid pFA-Elk1, 0.2 µg Rous sarcoma virus (RSV)/ß-galactosidase plasmid (as transfection control) and 0.2 µg plasmid encoding the indicated TRIP6 or its mutant. Luciferase and ß-galactosidase reporter assays were performed 16 hours after transfection. Luciferase activities were normalized on the basis of ß-galactosidase activities. (B) TRIP6 potentiates RIP2- and Nod1-mediated Elk1 activation. 293 cells (2x105) were transfected with 0.5 µg pFR plasmid, 0.05 µg the trans-activating plasmid pFA-Elk1, 0.2 µg RSV/ß-galactosidase plasmid (as transfection control), 0.2 µg RIP2 or Nod1 plasmid and the indicated amount of TRIP6 expression plasmid. Luciferase and ß-galactosidase reporter assays were performed 16 hours after transfection. Luciferase activities were normalized on the basis of ß-galactosidase activities.

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