Article Figures & Data
Figures
- Fig. 1.
IKKα regulates the basal IκB kinase activity of IKKβ. 293 cells were transfected with 0.6 μg of IKKβ-Flag expression vector alone or with increasing doses of either IKKαK44M-HA or IKKα-HA expression plasmids (0.6 μg, 1.2 μg, and 2.4 μg or 0.6 μg, 1.2 μg, 2.4 μg, and 3.6 μg, respectively). After 24 or 48 h, cell lysates were immunoprecipitated with anti-Flag M2-agarose. Immunoprecipitated complexes were assayed for kinase activity by incubation with 0.5 μg of GST-IκBα and [γ-32P]ATP. The resultant products were separated by SDS-PAGE (7.5% gels), transferred to nitrocellulose membranes, and subjected to autoradiography. The levels of IKKβ and IKKα in each lysate were determined by immunoblotting with Flag-specific or HA-specific antibodies (lower panels).
- Fig. 2.
NIK-induced phosphorylation of IKKs. (A) HeLa cells were transfected with 1 μg of IKKβK44A-Flag expression vector alone or in combination with 1 μg of IKKα-HA or IKKαK44M-HA with and without 1 μg of Myc-NIK expression plasmids. (B) HeLa cells were transfected with 1 μg of IKKβK44A-Flag or IKKβK44A-STS/AAA-Flag and NIK expression vectors alone or in combination with 1 μg of each IKKα-HA construct as indicated (2 μg of IKKα was used in lanes 6 and 12). Cells were harvested 48 h after transfection, and IKKβK44A was immunoprecipitated with anti-Flag M2-agarose (A) or anti-IKKγ/NEMO (B) antibodies. Immunoprecipitated complexes were subjected to in vitro kinase assay in the presence of [γ-32P]ATP. The products were separated by SDS-PAGE (7.5% gels), transferred to nitrocellulose membranes, and subjected to autoradiography. The level of IKKβ in each lysate was detected by immunoblotting with Flag-specific antibodies (lower panel).
- Fig. 3.
NIK-induced phosphorylation of IKKα is not blocked by catalytically inactive IKKβ. 293 cells were transfected with 1 μg of IKKαK44M-HA or IKKαS176A alone or in combination with IKKβK44-Flag and Myc-NIK as indicated. Each transfection was supplemented with empty vector to a final total of 4 μg of DNA. Cells were harvested, and signalsomes were immunoprecipitated with anti-IKKγ/NEMO antibodies. Following an in vitro kinase assay and heat dissociation, the tagged IKKα constructs were reimmunoprecipitated with anti-HA-Sepharose. The products were separated by SDS-PAGE (7.5% gels), transferred to nitrocellulose membranes, and subjected to autoradiography. The level of IKKα in each lysate was detected by immunoblotting with HA-specific antibodies (lower panel).
- Fig. 4.
NIK-induced phosphorylation of IKKβ requires catalytically active IKKα. (A) HeLa cells were transfected with IKKβK44A alone or with either wild-type or kinase-inactive NIK in combination with wild-type or kinase-inactive IKKα. Cells were lysed 48 h posttransfection. Signalsomes were immunoprecipitated with anti-IKKγ/NEMO antibodies and subjected to an in vitro kinase assay followed by heat dissociation in 10% SDS. IKKβK44A substrates were selectively immunoprecipitated from the disrupted complexes by a second immunoprecipitation with anti-Flag M2-agarose. (B) Unstimulated and TNF-α-stimulated (5 min) HeLa cell lysates were subjected to FPLC size fractionation on a Superose 6 column. Fractions were collected, separated by SDS-PAGE, and immunoblotted with anti-IKKα antibodies to identify fractions containing the endogenous signalsome (fractions 12 to 17, ∼900 kDa). (C) HeLa cells, transfected with Flag-tagged, kinase-inactive IKKβ, NIK, and either kinase-proficient or kinase-defective IKKα, were lysed and size fractionated by FPLC. Fractions were separated by SDS-PAGE followed by immunoblotting with anti-Flag or anti-IKKγ antibodies. (D) Fractions corresponding to those containing the endogenous IKK signalsome, as identified by anti-IKKα and anti-IKKγ antibodies, were collected, pooled, immunoprecipitated, and subjected to an in vitro kinase assay as described for Fig. 2. The level of phosphorylated IKKβ-K44A is shown in the upper panel; the levels of protein as determined by anti-Flag immunoblotting are shown in the lower panel.
- Fig. 5.
IKKβ phosphorylation induced by TNF-α in the presence and absence of IKKα. (A) HeLa cells were transfected with 2 μg of IKKβK44A-Flag and 2 μg of IKKα-HA or 2 μg of IKKαK44M-HA expression vector. Forty-eight hours after transfection, cells were stimulated with TNF-α (20 ng/ml) for 1, 5, and 10 min and lysed. Lysates were immunoprecipitated with anti-Flag M2-agarose and analyzed as for Fig. 4. Levels of IKKβK44A were evaluated by immunoblotting (lower panel). (B) HeLa cells were transfected with 0.5 μg of IKKβK44A-Flag and 1 μg IKKα-HA with increasing amounts of IKKα-as (0.5, 1, 2, and 4 μg). Forty-eight hours after transfection, cells were stimulated with TNF-α (20 ng/ml) for 10 min and lysed. Lysates were immunoprecipitated with anti-IKKγ/NEMO antibodies and analyzed as for Fig. 4. Levels of IKKβK44A-Flag and IKKα-HA were evaluated by immunoblotting (lower panel).
- Fig. 6.
IKKβ phosphorylation induced by HTLV-1 Tax. (A) Approximately 3 × 105 293 cells were transfected with 1 μg of kinase-deficient IKKβ (IKKβK44A-Flag) in combination with 1 μg of IKKα-HA or IKKαK44M-HA expression construct in the presence of wild-type Tax (1 μg) or the M22 Tax mutant (2 μg) as indicated. Cell lysates were then immunoprecipitated with anti-Flag M2-agarose and subjected to an in vitro kinase assay with [γ-32P]ATP. The reaction products were separated by SDS-PAGE (7.5% gel), transferred to a nitrocellulose membrane, and analyzed by autoradiography. The amount of IKKβK44A-Flag in each reaction is shown in the lower panel. (B) The levels of wild-type amd mutant Tax proteins in the cell lysates were assessed by immunoblotting with Tax-specific antiserum.
- Fig. 7.
Kinase-deficient IKKα blocks MEKK1- and Cot/Tpl-2-induced, but not PKCθ-induced, phosphorylation of IKKβ. HeLa cells and 293 cells were transfected with 1 μg of IKKβK44A-Flag expression plasmid and 1 μg of HA-tagged wild-type or kinase-deficient IKKα in the presence or absence of the Myc-Cot (A and B), HA-MEKK1 (C and D), and NIK and PKCθ(A/E) (E) expression vectors. After 24 h (293) and 48 h (HeLa), cells were harvested and lysates were immunoprecipitated with anti-Flag M2-agarose (A to D) or with IKKγ-specific antibodies (E). The immunoprecipitated complexes were subjected to an in vitro kinase assay and analyzed as for Fig. 4. The levels of phosphate incorporated into Flag-tagged, kinase-deficient IKKβ are shown in the upper panel, and the levels of Flag-tagged IKKβ are shown in the lower panels.
