(A) Predicted amino acid sequence of CIKS. (B) Detection of the ubiquitous 2.5-kB CIKS mRNA transcript on a Multiple Tissue Northern Blot (CLONTECH) (Upper). Actin mRNA is a control for RNA loading (Lower). The tissue source of the RNA is indicated.

Physical interaction between CIKS and NEMO/IKKγ. (A) GST pull-down assay: GST- NEMO/IKKγ or GST (negative control) (Upper, second and third lanes, respectively) was incubated with in vitro-translated CIKS* (amino acids 88–574) (Upper, first lane). (Lower) Aliquots of GST or GST-NEMO/IKKγ stained with Coomassie blue are shown. Molecular weight markers (in kDa) are indicated. (B) Anti-Flag immunoprecipitates (IP) of HeLa cell extracts transfected with FLAG-NEMO/IKKγ and CIKS were Western-blotted with anti-CIKS antibodies to detect coimmunoprecipitated CIKS (Top, lane 4). (Middle and Bottom) Western blots with anti-Flag (anti-NEMO) and anti-CIKS antibodies on whole-cell extracts are shown, respectively. (C) Coimmunoprecipitation of endogenous CIKS with endogenous NEMO/IKKγ. Immunoprecipitates of Jurkat cells with anti-NEMO antibodies were subjected to Western blot (WB) analysis with anti-CIKS antibodies. Jurkat cells were treated with TNF for the times indicated or left untreated. Extracts also were immunoprecipitated with an unrelated antibody (CTRL) raised against CD4. Ig Hch, Ig heavy chain.

CIKS interacts with IKKα and IKKβ, but not with NIK or MEKK1 in transfected cells. HeLa cells (2 × 10⁶) were cotransfected with an expression vector encoding FLAG-tagged CIKS (5 μg) together with expression vectors encoding HA-MEKK1 (3 μg), HA-NIK (3 μg), HA-IKKα (5 μg), or HA-IKKβ (3 μg). Anti-FLAG immunoprecipitates (IP) from cell lysates were analyzed by Western blotting (WB) with anti-HA antibodies to detect coprecipitated proteins (Top). Western blots with the anti-HA antibody (Middle) or anti-FLAG antibody (Bottom) were performed to check for expression of the constructs.

CIKS stimulates IKK and NF-κB activity. (A) Relative luciferase activity observed in HeLa cells transfected in triplicate with 0.5 μg of Ig-kB luciferase reporter plasmid, with or without CIKS and NEMO/IKKγ expression vectors, as indicated. Values shown (in arbitrary units) represent the means (± S.D) of at least three independent experiments, normalized for β-galactosidase activity of a cotransfected Rous sarcoma virus-β-galactosidase plasmid. (B) HeLa cells (4 × 10⁵) were transfected in duplicate with 0.5 μg of CRE-luciferase reporter plasmid together with increasing amounts of CIKS expression vector, as shown. Twenty four hours after transfection, cells were either stimulated for 5 h with cAMP (1 mM final concentration) or left untreated and then harvested. Measurements were normalized for β-galactosidase activity and data (± SD) are shown as fold induction. (C) Analysis of the effects of wild-type or kinase-deficient mutants (mut) of NIK, MEKK1, IKKα, or IKKβ on CIKS-dependent NF-κB activation. HeLa cells (4 × 10⁵) were transfected in triplicate with 0.5 μg of Ig-κB luciferase reporter plasmid, together with CIKS (0.5 μg) and NIK (0.2 μg), MEKK1 (0.4 μg), IKKα (0.4 μg), IKKβ (0.4 μg), NIK mut (0.2 μg), MEKK1 mut (0.2 μg), IKKα mut (0.4 μg), or IKKβ mut (0.4 μg). Data from a representative experiment, normalized for β-galactosidase activity, are shown as relative luciferase activity (± SD). Similar results were obtained in three independent experiments. (D) HeLa cells (3 × 10⁶) were left untransfected, or transfected with an expression vector encoding Flag-IKKα (2 μg), alone or together with expression vectors encoding HA-CIKS (4 μg), HA-NIK (3 μg), or HA-MEKK1 (5 μg), as indicated. Twenty four hours after transfection cells were harvested and IKKα was immunoprecipitated with anti-FLAG. IKK kinase activity was assayed as described in Materials and Methods with GST-IkBα (amino acids 1–54) as substrate. Also shown are an immunoblot for transfected IKKα present in the various extracts (anti-FLAG) and an immunoblot for endogenous, coprecipitated IKKβ present in the in vitro kinase assays.

Transfected CIKS activates SAPK/JNK activity. (Upper) HeLa cells (2 × 10⁶) were cotransfected with an expression vector encoding HA-SAPK/JNK (2 μg) and an expression vector encoding CIKS (4 μg) together with, or without, an expression vector for a kinase-deficient mutant of SEK/MKK4 (2 μg). Twenty four hours after transfection, cells were stimulated with TNF (2,000 units/ml) for 15 min, or left untreated, and then harvested. SAPK/JNK activity was assayed as described in Materials and Methods with GST–c-JUN (amino acids 1–79) as substrate. (Lower) An HA immunoblot is shown to document the relative amounts of HA-SAPK/JNK present in the cell extracts.

Distinct domains of CIKS required for activation of NF-κB and SAPK/JNK. CIKS mutants ΔN 301, ΔC 200, and ΔC 300 are truncations lacking the amino-terminal 301 aa, the carboxyl-terminal 200 aa, or the carboxyl-terminal 300 aa, respectively. The ability of these deletion mutants to interact with NEMO/IKKγ, IKKα, and IKKβ was assayed in coimmunoprecipitation experiments of transfected HeLa cells, as described for Fig. 3. The ability of these deletion mutants to activate NF-κB and SAPK/JNK was assayed with luciferase assays (as described for Fig. 4) and by immunecomplex kinase assay (as described in Fig. 5), respectively. +, positive assay; −, negative assay; *, a marginal effect in at least some assays.