Fig. 1. MEKK3–TAK1 association in stably transfected cells. (A) Expression levels of MEKK3 in parental (lane 1) and stably transfected HEK293T cells (lane 2). In the upper panel, MEKK3 was immunoprecipitated and immunoblotted with anti-MEKK3 antibody. In the bottom panel, MEKK3 was precipitated with anti-FLAG antibody and immunoblotted with anti-HA. (B) TAP assay detects MEKK3 associated proteins. Silver-stained SDS-PAGE gel of tandem affinity purified MEKK3 complexes from parental HEK293T cells, FLAG–MEKK3–HA stably transfected cells, and TNFα-treated FLAG–MEKK3–HA cells. M1–M5 represent gel slices submitted for analysis by mass spectrometry and the major protein identified in each slice. (C) TAP detects association of MEKK3 with endogenous TAK1. Tandem affinity purified MEKK3 complexes from parental 293 cells, FLAG–MEKK3–HA stably transfected cells, and TNF-treated FLAG–MEKK3–HA cells were Western blotted and stained with anti-TAK1 antibody.

Fig. 2. FRET analysis of MEKK3–TAK1 interactions. CFP–MEKK3 and YFP–TAK1 tagged proteins were transiently transfected into HEK293T cells. 48 h later cells were washed and fixed. (A) Distribution of CFP–MEKK3 and YFP–TAK1 proteins before bleaching. Dashed red line indicates sections used for FRET analysis. The fluorescence intensity of CFP and YFP before (B) and after (C) photobleaching.

Fig. 3. TAK1 inhibits MEKK3-induced NF-κB activity and MEKK3 phosphorylation. (A) Effects of MEKK3, inactive MEKK3, and TAK1 expression on NF-κB transcriptional activity. FLAG–MEKK3 or FLAG–MEKK3(S526A/K391M) were transfected into HEK293T cells with or without TAK1-Myc plus a NF-κB luciferase reporter gene. The indicated groups were stimulated for 6 h with TNFα. Relative luciferase activity was determined and normalized to Renilla luciferase activity. Experiments were performed in triplicate. Graphs include data from three independent experiments. An asterisk () indicates statistical significance (P < 0.01) between the indicated groups. (B) TAK1 regulates MEKK3 phosphorylation. FLAG–MEKK3 was transfected into HEK293T cells with or without Myc-TAK1, 48 h later, cell lysates were prepared and some groups were incubated with calf intestinal alkaline phosphatase (CIP) for 30 min at 37 °C. Protein samples were immunoblotted and stained for MEKK3 with anti-FLAG antibody.

Fig. 4. TAB1 reverses TAK1-mediated inhibition. (A) TAK1 modulates MEKK3 phosphorylation. FLAG–MEKK3 was transfected into HEK293T cells with TAK1 or TAK1 plus TAB1, 48 h later, cell lysates were prepared. Protein samples were immunoblotted and MEKK3 was detected with anti-FLAG antibody. Further details are included in Fig. 6A. (B) In vitro phosphorylation of TAK1. FLAG–MEKK3, FLAG–MEKK3(S526A/K391M), and Myc–TAK1 proteins were expressed in HEK293T cells. The wild type and mutant MEKK3 proteins were purified and eluted with 3×FLAG peptide. The TAK1 and TAB1 proteins were isolated on anti-Myc agarose beads. After the kinase assay the beads were washed and phospho-TAK1, TAK1, and TAB1 were detected by Western blot. Input levels of MEKK3 are presented. (C) TAB1 reverses TAK1 inhibition of MEKK3-induced NF-κB activity. HEK293T cells were transfected with the indicated constructs. Groups were treated with medium or TNFα for 6 h. Relative luciferase activity was determined and normalized to Renilla luciferase activity. Graphs depict data from three independent experiments. An asterisk () indicates statistical significance (P < 0.01) between groups.

Fig. 5. Effects of TAK1 and TAB1 on MEKK3-induced NF-κB reporter activity in TAK1−/−MEF. The indicated combinations of FLAG–MEKK3, Myc–TAK1, and Myc–TAB1 were transfected into TAK1−/−MEF cells along with the NF-κB luciferase reporter gene. 48 h later, cells were serum starved for 4 h then treated with medium or 10 ng/ml TNFα for 6 h. Relative luciferase activity was determined and normalized to Renilla luciferase activity. Experiments were performed in triplicate. An asterisk () indicates statistical significance (P < 0.01), a triangle (Δ) indicates (P < 0.05).

Fig. 6. Association between non-phosphorylated MEKK3 and non-phosphorylated TAK1. (A) TAB1 regulates TAK1 phosphorylation and MEKK3–TAK1 interaction. The indicated epitope-tagged constructs were transfected into HEK293T cells. After 48 h cells were treated with medium or TNFα for 10 min. Cell lysates were prepared and the TAK1–Myc complex was immunoprecipitated with monoclonal anti-TAK1 antibody. Phosphorylated (Thr184/187) TAK1 was detected with anti-phosphoTAK1 antibody, MEKK3 associated with TAK1 was detected with anti-FLAG antibody, TAK1 and TAB1 were detected with anti-Myc antibody. (B) Reciprocal immunoprecipitation of MEKK3–TAK1 complexes. The indicated constructs were transfected into HEK293T cells. After 48 h some groups were treated with TNFα for 15 min. FLAG–MEKK3 and FLAG–MEKK3(S526A/K391M) complexes were immunoprecipitated with anti-FLAG antibody. (C) MEKK3 interacts with non-phosphorylated TAK1. The indicated constructs were transfected into HEK293T cells. After 48 h cell lysates were prepared and immunoprecipitated with the indicated antibodies.

Fig. 7. Model of MEKK3 interactions. MEKK3 interacts with endogenous TAK1 in unstimulated cells to prevent basal NF-κB signaling. Following receptor activation or ectopic expression of MEKK3 or TAK1–TAB1 the MEKK3–TAK1 complex dissociates favoring bifurcation of independent MEKK3 and TAK1 signaling pathways. Complexes containing either MEKK3 oligomers or TAK1–TAB1 are autophosphorylated. The latter phosphorylated complexes directly or indirectly phosphorylate components of the IKK complex leading to NF-κB transcriptional activity.