Sequence and expression of ECSIT. (A) Multiple tissue Northern blot. The clone retrieved from yeast two-hybrid assay was labeled with ³²P and used to probe a murine blot containing mRNA from the indicated tissues. (B) Clones identified in hybridization screening of murine cDNA libraries. The yeast two-hybrid activation domain clone (amino acids 21–435) is compared with ECSIT retrieved from a mouse liver cDNA library (clone 5) (Clontech) and to clones retrieved in a murine pre-B cell-library (left). The alternate carboxy-terminal sequences in the 9/11 and 14/15 cDNAs are represented by the hatched boxes. In vitro translation products of the three cDNAs are shown (right). (C) Comparison of sequences of murine ECSIT (mECSIT) and dECSIT. Identical and similar amino acids are indicated by shading.

Activation of NF-κB by overexpression of ECSIT. (A) 293 cells stably transfected with an NF-κB /luciferase reporter gene (293κB–LUC) were transiently transfected with increasing amounts of ECSIT. Twenty-four hours after transfection, cells were lysed and assayed for luciferase activity. Background (untransfected) was subtracted, and values shown are per microgram of total protein in each sample. (B) 293κB–LUC cells were transfected with 1 μg of the indicated ECSIT mutants. Twenty-four hours after transfection, cells were lysed and assayed for luciferase activity relative to activity achieved with the ECSIT wild-type protein (ECSITwt). ECSIT (261–435) is referred to as ECSITΔ. ECSIT–AD corresponds to the original clone isolated from the yeast two-hybrid library.

Inhibition of MAPKKK activity by dominant-negative ECSITΔ. (A) Inhibition of NF-κB and AP-1 activity induced by MEKK-1 but not NF-κB activity induced by NIK. MEKK-1 (0.5 μg) or NIK (0.5 μg) was transfected into 293κB–LUC cells where indicated (left). Alternately, a luciferase reporter vector for AP-1 (AP-1–LUC) was transfected (0.4 μg) in all wells (right). ECSITΔ or vector alone (−) was cotransfected in increasing amounts. Cells were assayed for luciferase activity after 24 hr, and the results are reported as RLU/ μg of protein. (B) Inhibition of NF-κB and AP-1 activity induced by TRAF6. The experiment was performed similar to A, except that 0.5 μg of TRAF6 was transfected as indicated.

ECSIT induces the processing of MEKK-1, and ECSITΔ inhibits this processing. (A) 293 cells were transfected with MEKK-1 and vector DNA (lane 3), increasing amounts of ECSITΔ (lanes 1,2), or increasing amounts of ECSIT wild type (lanes 4–6). Twenty-four hours after transfection, lysates were prepared and analyzed by SDS-PAGE. Gel was immunoblotted (IB) with an antibody to the carboxyl terminus of MEKK-1. The full-length and processed forms of MEKK-1 are indicated by asterisks (*). (B) Differential inhibition of NF-κB activity induced by MEKK-1 and MEKK-1Δ. 293 κB–LUC cells were transfected with either MEKK-1 wild type (full length) or MEKK-1Δ in the presence of increasing amounts of ECSITΔ or vector control as indicated. Twenty-four hours after transfection, lysates were prepared and assayed for luciferase activity. (C) Differential inhibition of AP-1 activity induced by MEKK-1 and MEKK-1Δ. 293 cells were transfected with 0.4 μg of AP-1–LUC reporter gene and MEKK-1 wild type (wt) or MEKK-1Δ, or vector alone as indicated. Twenty-four hours after transfection, lysates were prepared and assayed for luciferase activity.

A model of how ECSIT may participate in Toll/IL-1 signaling. The major signaling pathways leading to NF-κB and AP-1 from TNF and IL-1/Toll receptors are indicated. The different proteins indicated do not form multiprotein complexes, probably reflecting the dynamic nature of the signaling pathways; for example, IRAK is rapidly degraded upon signaling. The order of the different proteins in the pathways has been deduced from a combination of experimental approaches, including use of dominant-negative mutants. It is important to note that this figure does not represent the totality of information available about these signaling pathways but is a selective depiction of the key molecules discussed in the paper.

ECSIT specifically interacts with TRAF6. (A) Coprecipitation of endogenous TRAF6 with transfected ECSIT. Lysates were prepared from untransfected and HA-tagged ECSIT-transfected 293 cells (left) and immunoprecipitated overnight with 20 μl of anti-HA monoclonal antibody and 25 μl of protein G–Sepharose. Sepharose beads were centrifuged and washed extensively, and bound endogenous protein was removed from the beads by addition of SDS-containing running buffer and boiling. Samples were divided in two, separated by SDS-PAGE, and immunoblotted with either an antibody to TRAF6 or an antibody to TRAF2. Lysates (Ly) that had not been immunoprecipitated were also assayed for the presence of transfected ECSIT (HA antibody) or for the presence of endogenous TRAF2 or TRAF6. COS cells (right) were assayed similarly for the ability of HA–ECSIT to immunoprecipitate TRAF5 (antibody from Santa Cruz). (IP) Immunoprecipitation; (Ly) lysates; (Ig) immunoglobulin. (B) In vivo mapping of the TRAF6 binding site on ECSIT. 293 cells were transfected with amino-terminal Flag epitope-tagged deletion constructs of ECSIT (schematic shown at right) and immunoprecipitated (IP) with TRAF6 antibody. Unprecipitated lysates were separated by SDS-PAGE (top) and assayed by immunoblotting (IB) with an antibody to Flag (M5). Immunoprecipitates were washed, denatured with SDS running buffer, boiled, and analyzed by SDS-PAGE (bottom). Membrane was blotted in Flag (M5) antibody to reveal interacting proteins. As a control the immunoprecipitated blot was reprobed with an antibody to TRAF6. (Ig) Immunoglobulin; (ECSIT–AD) ECSIT 21–435 originally cloned from yeast two-hybrid library; (ECSITΔ) ECSIT 261–435 deletion mutant. (C) In vitro mapping of the ECSIT binding site on TRAF6. A GST fusion protein of ECSIT (21–435) was prepared in bacteria and incubated with ³⁵S-labeled in vitro translation products of TRAF6 including full-length TRAF6, the amino-terminal ring and zinc fingers of TRAF6 (T6 R+Z), and the carboxy-terminal TRAF domain (T6 N+C). Proteins binding to GST–ECSIT (21–435) were bound to glutathione–Sepharose beads, washed, and analyzed by PAGE (right). Two micrograms of GST alone was also incubated with full-length TRAF6 (GST+TRAF6) as a control.

Inhibition of Toll and IL-1 receptor pathway members by a dominant-negative ECSIT mutant. (A) 293κB–LUC cells were transfected with 0.5 μg of the constitutively active human Toll mutant CD4/TLR4 and either vector control DNA (−) or indicated amounts of ECSITΔ. DNA concentration in all transfections was equilibrated to 2 μg with vector. Twenty-four hours after transfection, lysates were assayed for luciferase activity. (B) 293κB–LUC cells were transfected with the indicated DNAs and assayed as above. The different constructs were RIP wild type, IRAK cytoplasmic domain fused to CD4 transmembrane and extracellular domain, and different amounts of ECSITΔ as indicated.

MEKK-1 interacts with ECSIT, and transfection of MEKK-1 with ECSIT induces a post-translational modification of ECSIT. (A) Immunoprecipitation of ECSIT with MEKK-1. 293 cells were transfected with HA–ECSIT, HA–ECSIT, and HA–MEKK-1 (wild type), or HA–ECSIT and HA–MEKK-1 DN, (dominant negative, a point mutant in the kinase domain), and lysates were analyzed by PAGE followed by immunoblotting (left). Alternatively, MEKK-1 was immunoprecipitated from the lysates, and the precipitated proteins were analyzed by PAGE followed by immunoblotting (right). In both instances, immunoblotting was performed using HA antibody (12CA5). (NS) Nonspecific; (Ig) immunoglobulin. The asterisk (*) specifies the upshifted ECSIT band that appears upon cotransfection with MEKK-1. (B) ECSIT dominant negative (ECSITΔ) interacts with MEKK-1. 293 cells were cotransfected with Flag–ECSITΔ, Flag–ECSIT (234–435), Flag–ECSIT (137–435), or Flag–ECSIT (21–435) and HA–MEKK-1 or HA–MEKK-1 alone as indicated. Twenty-four hours after transfection, lysates were made, and a portion of the lysate was used for immunoblotting (IB) with the HA antibody. The rest of the lysate was immunoprecipitated (IP) with Flag (M2) beads and analyzed by SDS-PAGE. Immunoprecipitated proteins were visualized with the HA antibody and with the Flag (M5) antibody. (Bottom) Flag-tagged proteins were expressed and immunoprecipitated in the appropriate samples. (C) Cotransfection of ECSIT with TRAF6 and kinases of the NF-κB signal transduction pathway. HA–ECSIT was cotransfected with the indicated proteins (see Materials and Methods) and analyzed by SDS-PAGE. Gels were immunoblotted with the HA antibody (12CA5).

dECSIT plays a role in innate immunity in Drosophila. (A). Association of dECSIT with dTRAF6. Schneider insect cells were untransfected or transfected with V5 epitope-tagged dTRAF6 and Flag-tagged dECSIT as indicated. Lysates were prepared after 24 hr and immunoprecipitated with α-Flag-conjugated beads. Precipitates were analyzed by SDS-PAGE. (B) Activation of the diptericin promoter by dECSIT. Schneider cells were transfected with dECSIT, Toll^10b dominant-active mutant, or vector alone at the concentrations indicated. The diptericin–luciferase promoter was cotransfected in all samples. Thirty-six hours after transfection, lysates were prepared and assayed for luciferase activity. (C) Induction of defensin and attacin peptide transcription by dECSIT. l2mbn insect cells were transfected with dECSIT, dominant-active Toll^10b mutant, or vector. One sample was treated with LPS at 20 ng/ml as indicated. After 36 hr, cDNA was prepared from each sample, and RT–PCR was performed using oligos to RP49 (control, bottom), defensin, or attacin.