PMC

The C terminus of NIK mediates heterotypic oligomerization with IKKα. 293 cells were cotransfected with IKKα-T7 and Myc-NIK or the series of deletion mutant forms of Myc-NIK. (A) Twenty-four hours after transfection, cell lysates were prepared, subjected to SDS-PAGE, and immunoblotted with anti-Myc antibodies or anti-T7 antibodies to assess the overall level of expression of the individual proteins. (B) Aliquots of the cell lysates were subjected to immunoprecipitation (IP) with anti-T7 antibodies conjugated to agarose beads. The immunoprecipitates were then subjected to SDS-PAGE and immunoblotted with anti-Myc antibodies.

(A) Expression of a C-terminal fragment (amino acids 735 to 947) of NIK inhibits the interaction of wild-type NIK and IKKα. 293 cells were cotransfected with IKKα-T7 and Myc-NIK in the presence of either N-terminal amino acids 1 to 220 or C-terminal amino acids 735 to 947 of NIK, as indicated. Twenty-four hours after transfection, cell lysates were prepared from the transfected cultures. (A) Aliquots of the cell lysates were subjected to immunoprecipitation (IP) with anti-T7 antibodies conjugated to agarose beads. The immunoprecipitates were then subjected to SDS-PAGE and immunoblotted with anti-Myc antibodies. (B) Aliquots of the cell lysates were subjected to SDS-PAGE and immunoblotted with anti-Myc antibodies to assess the expression of the individual proteins.

Multiple domains of NIK participate in homotypic oligomerization. (A) Schematic overview of the structural organization of NIK. (B) 293 cells were transiently transfected with plasmids encoding Myc-tagged NIK and the indicated series of deletion mutant forms of NIK similarly epitope tagged with Myc. After 24 h, cell lysates were prepared, subjected to SDS-PAGE, and blotted with anti-Myc or anti-T7 antibodies to verify protein expression levels. (C) To assess the ability of the mutant NIK proteins to form oligomers with NIK-T7, aliquots of the cell lysates were subjected to immunoprecipitation (IP) with anti-T7 antibody conjugated to agarose beads. The immunoprecipitates were subjected to SDS-PAGE and immunoblotted with anti-Myc antibodies.

The NIK-T559A mutant protein fails to phosphorylate IKKα and to undergo autophosphorylation. (A) 293 cells were seeded at 5 × 10⁵/well in six-well plates and transfected 24 h later with 2 μg of T7-tagged IKKα(K44M) expression vectors and 2 μg of Myc-tagged NIK expression vectors or vectors encoding the indicated mutant proteins. Twenty hours after transfection, in vitro kinase reactions were performed on anti-T7 immunoprecipitates from these cell lysates. The resulting kinase reactions were separated by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed by autoradiography. Phosphorylated IKKα(K44M) is indicated on the right in the upper part of the panel. The lower parts of the panel show the levels of immunoprecipitated IKKα and the levels of coimmunoprecipitated NIK proteins. (B) Two micrograms of Myc-tagged NIK expression vectors or the indicated mutants was transfected into 293 cells, and in vitro kinase reactions were performed on anti-Myc immunoprecipitates as described for panel A. Autophosphorylated NIK proteins are indicated on the right. The lower parts of the panel show the levels of immunoprecipitated NIK proteins present in the kinase reactions.

Biologically active NIK spontaneously forms homotypic oligomers in vivo. (A) HeLa cells were transiently transfected with plasmids encoding T7- and Myc epitope-tagged versions of NIK, as indicated. After 24 h of culture, a portion of the cells was treated with TNF-α for 5 or 10 min as indicated. Cells lysates were prepared from the cultures and subjected to immunoprecipitation (IP) with anti-T7 antibody conjugated to agarose beads. The immunoprecipitates were then analyzed by immunoblotting with anti-Myc antibodies. (B) Aliquots of the whole-cell lysates (10 μl) were subjected to SDS-PAGE and immunoblotted with anti-T7 or anti-Myc antibodies to determine the levels of Myc-NIK and NIK-T7 expression. In addition, the biological activity of the added TNF-α was confirmed by induced degradation of endogenous IκBα detected by immunoblotting with antibodies specific for the C terminus of this cytoplasmic inhibitor.

Analysis of the functional effects of various deletion mutant forms of NIK in the presence and absence of TNF-α (A) or wild-type NIK (B). (A) Three micrograms of expression vectors encoding the indicated NIK mutant proteins were cotransfected into 293 cells together with κB-luciferase and β-galactosidase reporter plasmids. Twenty hours after transfection, the cultures were stimulated with or without TNF-α (20 ng/ml) for 6 h. Cell lysates were then prepared from the cultures, and the luciferase activities of these lysates were determined. The β-galactosidase activities of these lysates were measured for normalization of differences in transfection efficiency occurring in the cultures. (B) Three micrograms of expression vectors encoding the indicated NIK mutant proteins in the absence or presence of 0.3 μg of a wild-type NIK expression vector were cotransfected into 293 cells together with κB-luciferase and β-galactosidase reporter plasmids. Twenty-four hours after transfection, cell lysates were prepared from the cultures. The luciferase and β-galactosidase activities of these lysates were determined. (C) Analysis of the inhibitory effect of the C terminus of NIK (amino acids 735 to 947) on TNF-α (left graph) and NIK (right graph) induction of κB-luciferase activity. Experiments were performed as described for panels A and B for the smaller fragments of NIK. All data are presented as mean fold induction of luciferase activity ± the standard deviation derived from independent triplicate transfections. The closed bars show the biological activity of wild-type or mutant NIK alone, while the open bars show the effect of mutant NIK on TNF-α or wild-type NIK stimulation.

The NIK-T559A mutant protein fails to activate IKKα- and NF-κB-dependent transcription. (A) 293 cells were seeded at 5 × 10⁵/well in six-well plates and transfected 24 h later with 4 μg of plasmid DNA encoding Myc-tagged NIK or the indicated mutant proteins. Twenty hours after transfection, in vitro kinase reactions were performed by using anti-IKKα immunoprecipitates prepared from these cell lysates; GST–IκBα(1-62) was added as an exogenous substrate. The kinase reactions were analyzed by SDS-PAGE, followed by transfer to a nitrocellulose membrane and autoradiography. The phosphorylated GST–IκBα(1-62) substrate is indicated on the right. The lower panels show the amounts of immunoprecipitated IKKα and expressed NIK present in each of the cell lysates. One microgram of expression vector DNA encoding wild-type NIK or the indicated mutants form of NIK (B) or 1, 2, or 3 μg of an expression vector encoding NIK-T559A (C) was cotransfected into 293 cells together with 200 ng of κB-luciferase and 100 ng of β-galactosidase reporter plasmids. The total DNA concentration was held constant at 4 μg by supplementation with the parental pRK vector. Twenty hours after transfection, the cultures were stimulated with or without TNF-α (20 ng/ml) for 6 h, as indicated in panels B and C. Cell lysates were then prepared from the cultures, and the luciferase activity present in these lysates was determined. The β-galactosidase activities of these lysates were also measured for normalization of differences in transfection efficiency occurring in the cultures.

(A) Alignment of the amino acid sequences of the activation loop between subdomains VII (DFG) and VIII (M(A/S)PE) of NIK and other MAP kinases. Asterisks denote residues shown to be phosphorylated and/or implicated in the activation of these kinases. (B) Biological function of wild-type NIK and kinase domain mutant forms of NIK. Expression vectors encoding wild-type NIK or the indicated mutant forms of NIK were cotransfected into 293 cells together with κB-luciferase and β-galactosidase reporter plasmids. Twenty hours later, the cultures were stimulated with or without TNF-α (20 ng/ml) for 6 h. Cell lysates were then prepared, and the luciferase activities of these lysates were determined. β-Galactosidase activities in these lysates were measured for normalization of differences in transfection efficiency occurring in the cultures. Data are presented as fold induction of luciferase activity ± the standard deviations derived from independent triplicate transfections. The closed bars show the biological activity of wild-type NIK or the mutant forms of NIK, while the open bars show the effect of these mutants in the presence of TNF-α stimulation. (C) In vivo phosphorylation of wild-type NIK and the mutant forms of NIK. 293 cells were transfected with wild-type NIK or the indicated mutant forms of NIK containing an N-terminal Myc epitope tag. These cells were then incubated in phosphate-free medium for 1 h, radiolabeled for 2 h with ³²P-labeled orthophosphoric acid, and either cultured in medium alone or stimulated with TNF-α (20 ng/ml) for 15 min, and then NIK was immunoprecipitated with anti-Myc antibodies. The resulting immunoprecipitates were subjected to SDS-PAGE, followed by transfer to a nitrocellulose membrane and autoradiography. The lower part of the panel shows the levels of the wild-type and mutant NIK proteins present in the samples.