Restoration of NF-kappaB activation by tumor necrosis factor alpha receptor complex-targeted MEKK3 in receptor-interacting protein-deficient cells

Abstract

Receptor-interacting protein (RIP) plays a critical role in tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB activation. However, the mechanism by which RIP mediates TNF-alpha-induced signal transduction is not fully understood. In this study, we reconstituted RIP-deficient Jurkat T cells with a fusion protein composed of full-length MEKK3 and the death domain of RIP (MEKK3-DD). In these cells, MEKK3-DD substitutes for RIP and directly associates with TRADD in TNF receptor complexes following TNF-alpha stimulation. We found that TNF-alpha-induced NF-kappaB activation was fully restored by MEKK3-DD in these cells. In contrast, expression of a fusion protein composed of NEMO, a component of the IkappaB kinase complex, and the death domain of RIP (NEMO-DD) cannot restore TNF-alpha-induced NF-kappaB activation in RIP-deficient cells. These results indicate that the role of RIP is to specifically recruit MEKK3 to the TNF-alpha receptor complex, whereas the forced recruitment of NEMO to the TNF-alpha receptor complex is insufficient for TNF-alpha-induced NF-kappaB activation. Although MEKK2 has a high degree of homology with MEKK3, MEKK2-DD, unlike MEKK3-DD, also fails to restore TNF-alpha-induced NF-kappaB activation in RIP-deficient cells, indicating that RIP-dependent recruitment of MEKK3 plays a specific role in TNF-alpha signaling.

FIG. 1.

MEKK3 physically associates with RIP. (A) HEK293 cells (7 × 10⁵) were transiently transfected with 1 μg of Flag-tagged MEKK3 and cotransfected with 1.5 μg of Myc-tagged RIP. The cells were lysed 24 h later, and the lysates were precipitated with anti-Flag M2-agarose affinity gel. Immunocomplexes (top and middle) and whole-cell lysates (bottom) were subjected to SDS-PAGE and analyzed by Western blotting (WB) with the indicated antibodies. IP, immunoprecipitation. (B) Schematic diagram of MEKK3-DD, MEKK2-DD, and NEMO-DD fusion proteins. The RIP DD was fused to full-length MEKK3, MEKK2, or NEMO. RD, regulatory domain.

FIG. 2.

MEKK3-DD, but not the kinase-deficient mutant version of it, restores TNF-α-induced NF-κB activation in RIP-deficient cells. (A) Levels of MEKK3-DD and MEKK3(K391A)-DD fusion protein expression. RIP-deficient Jurkat cells (2 × 10⁷) stably transfected with Flag-tagged MEKK3-DD (M3-DD cell line) or MEKK3(K391A)-DD[M3(K391A)-DD cell line] were lysed and immunoprecipitated (IP) with anti-Flag M2-agarose affinity gel. Immunoprecipitates (top) and whole-cell lysates (middle and bottom) were subjected to SDS-PAGE and probed with anti-Flag antibodies or anti-RIP and anti-β-tubulin antibodies, respectively. WB, Western blotting. (B) Levels of endogenous MEKK3 in Jurkat, M3-DD, and M3(K391A)-DD cells. The cells were either untreated or stimulated with TNF-α (20 ng/ml) for 5 min. Lysates were subjected to SDS-PAGE and probed with anti-MEKK3 or anti-β-tubulin antibodies. (C) NF-κB binding activity was analyzed by EMSA. Jurkat, RIP-deficient [RIP(−)], M3-DD, and M3(K391A)-DD cells were either untreated or stimulated with TNF-α (10 ng/ml) or phorbol myristate acetate (PMA; 10 ng/ml) plus a CD28 monoclonal antibody (CD28Ab; 1 μg/ml) for 30 min. Nuclear extracts (10 μg) from these cells were incubated with ³²P-labeled probes containing NF-κB binding sites for 15 min and then subjected to electrophoresis in 5% polyacrylamide gel and autoradiography. ns, nonspecific. (D) Time course of IκBα degradation in TNF-α (10 ng/ml)-stimulated cells. The level of IκBα expression was determined by Western blotting with anti-IκBα antibodies. The membrane was reprobed with anti-β-tubulin antibodies (a loading control).

FIG. 3.

The MEKK2-DD fusion protein does not rescue TNF-α-mediated NF-κB activation in RIP-deficient cells. (A) Jurkat and RIP⁻ cells and cells expressing MEKK2-DD (M2-DD) were stimulated as described for Fig. 2C, and NF-κB binding activity was analyzed by EMSA. ns, nonspecific. (B) The expression of IκBα in TNF-α (10 ng/ml)-stimulated cells was determined by Western blotting with anti-IκBα antibodies. The membrane was reprobed with anti-β-tubulin antibodies (a loading control).

FIG. 4.

Differential effects of MEKK2-DD and MEKK3-DD expression on TNF-α-induced NF-κB activation in RIP-deficient cells. (A) Jurkat, RIP⁻, M2-DD, and M3-DD cells were stimulated with TNF-α (10 ng/ml) for the indicated times, and EMSA was performed using ³²P-labeled probes containing NF-κB or Oct-1 binding sites. ns, nonspecific. (B) Levels of MEKK2-DD and MEKK3-DD fusion protein expression. Cells (2 × 10⁷) were lysed and immunoprecipitated (IP) with anti-Flag M2-agarose affinity gel. Immunoprecipitates (top) and whole-cell lysates (bottom) were subjected to SDS-PAGE and probed with anti-Flag, anti-RIP, or anti-β-tubulin antibodies. WB, Western blotting.

FIG. 5.

The NEMO-DD fusion protein is incorporated into the IKK complex, but its recruitment to the TNF-R1 complex is not sufficient for NF-κB activation in the absence of RIP. (A) Jurkat, RIP⁻, and RIP⁻ cells expressing NEMO-DD (NE-DD) were either untreated or stimulated with TNF-α (10 ng/ml). The cells were lysed and subjected to SDS-PAGE and Western blotting (WB) with anti-RIP antibodies that recognize the DD of RIP. (B) Jurkat, RIP⁻, and NE-DD cells were either untreated or stimulated with TNF-α (20 ng/ml) for 5 min. The cells were lysed and precipitated with anti-Flag M2-agarose affinity gel. Immunocomplexes (left) and whole-cell lysates (right) were subjected to SDS-PAGE and analyzed by Western blotting with antibodies against IKKα, IKKβ, NEMO, Flag, or Bcl10 as a loading control. IP, immunoprecipitation. (C) Jurkat, RIP⁻, and NE-DD cells were stimulated as described for Fig. 2C, and NF-κB binding activity was analyzed by EMSA. PMA, phorbol myristate acetate; ns, nonspecific. (D) The expression of IκBα in TNF-α (10 ng/ml)-stimulated cells was determined by Western blotting with anti-IκBα antibodies. The membrane was reprobed with anti-β-tubulin antibodies (a loading control).

FIG. 6.

MEKK3-DD, MEKK2-DD, and NEMO-DD fusion proteins associate with TRADD. HEK293 cells (7 × 10⁵) were transiently transfected with 1.3 μg of expression plasmids encoding several constructs: Myc-tagged TRADD, Flag-tagged MEKK3, or MEKK3-DD (A), MEKK2 or MEKK2-DD (B), or NEMO or NEMO-DD (C) or with control vectors pRK6 and pcDNA3. Cells were lysed and precipitated with anti-Flag M2-agarose affinity gel. The immunocomplexes were subjected to SDS-PAGE and analyzed by Western blotting (WB) with anti-Myc or anti-Flag antibodies. The levels of expression of TRADD were assessed with anti-Myc immunoblotting of total cell lysates. IP, immunoprecipitation.

FIG. 7.

NEMO-DD and MEKK3-DD fusion proteins are recruited to the TNF-R1 complex in TNF-α-treated cells. (A) Jurkat or NE-DD cells (2 × 10⁷) were either untreated or stimulated with TNF-α (20 ng/ml) for the indicated times. These cells were lysed and precipitated with mouse monoclonal anti-TNF-R1 antibodies. The immunocomplexes (top) and whole-cell lysates (middle and bottom) were subjected to SDS-PAGE and analyzed by Western blotting (WB) with anti-Flag or anti-TNF-R1 antibodies. IP, immunoprecipitation; Ig, immunoglobulin. (B) HEK293 cells (7 × 10⁵) were transiently transfected with 1 μg of empty vector (pcDNA3) or Flag-tagged MEKK3-DD. Twenty-four hours later the cells were stimulated with TNF-α (20 ng/ml) for 10 min and lysates were tested as described for panel A.

FIG. 8.

MEKK3 and MEKK3-DD associate with IKKβ. (A) HEK293 cells (7 × 10⁵) were transiently transfected with 1.3 μg of empty vector (pcDNA3) (lane 1) or expression plasmids encoding Myc-tagged MEKK3 (lanes 2 to 4), MEKK3(K391A) (lanes 5 to 7), MEKK2 (lanes 8 to 10), or MEKK2(K395M) (lanes 11 to 13) and cotransfected with 1 μg of Flag-tagged IKKβ (lanes 3, 6, 9, and 12) or NEMO (lanes 4, 7, 10, and 13). The cells were lysed and precipitated with anti-Flag M2-agarose affinity gel. The immunocomplexes were subjected to SDS-PAGE and analyzed by Western blotting (WB) withanti-Myc antibodies. The levels of expression of the transfected products were assessed with anti-Myc and anti-Flag immunoblots of total cell lysates. IP, immunoprecipitation. (B) HEK293 cells (7 × 10⁵) were transiently transfected with 1.3 μg of Myc-tagged MEKK3 (lanes 2, 5, and 6) and cotransfected with Flag-tagged IKKβ or NEMO. The cells were lysed and tested as described for panel A. (C) HEK293 cells (7 × 10⁵) were transiently transfected with Flag-tagged MEKK3, MEKK3-DD, NEMO, or NEMO-DD expression vectors, and the cells were lysed 24 h later. The lysates were precipitated with anti-Flag M2-agarose affinity gel. Immunocomplexes (top and middle) and whole-cell lysates (bottom) were subjected to SDS-PAGE and analyzed by Western blotting with antibodies against IKKβ or Flag.

FIG. 9.

Kinase-active form of MEKK3 phosphorylates IKKβ. HEK293 cells (7 × 10⁵) were transiently transfected with 1.3 μg of empty vector (pcDNA3) (lane 1) or expression plasmids encoding Myc-MEKK3 (lane 2) and Myc-MEKK3(K391A) (lane 3). The cells were lysed 24 h later and precipitated with anti-c-Myc-agarose affinity gel. The immunocomplexes were subjected to an in vitro kinase assay. Whole-cell lysates were analyzed by Western blotting (WB) with anti-phospho-IKKβ and anti-IKKβ antibodies. IP, immunoprecipitation.

FIG. 10.

Working model of TNF-α-induced NF-κB activation in RIP-deficient cells expressing MEKK3-DD or NEMO-DD fusion proteins.