Abstract
The NF-κB transcription factors control many physiological processes, including inflammation, immunity, and apoptosis. Its activity contributes to the development of various cell malignancies. NF-κB-inducing kinase (NIK) plays a pivotal role in NF-κB activation. However, the molecular mechanism to stabilize and activate NIK remains elusive, although it is known that cIAP1/2 (cellular inhibitor of apoptosis 1 and 2) ubiquitinate NIK for degradation. Here, we report a novel NF-κB-related zinc finger protein 91 (ZFP91) that stabilizes and activates NIK in a ubiquitination-dependent manner. We show that ZFP91 interacts with and promotes the Lys(63)-linked ubiquitination of NIK and subsequent processing of p100 to p52. The results of in vitro biochemical assays indicate that ZFP91 functions as an E3 ligase directly to NIK. Remarkably, the ubiquitination of NIK coincides with its Thr(559) phosphorylation. Furthermore, knockdown of ZFP91 expression by RNA interference inhibits the CD40 ligation-induced activation of NIK and p100 processing as well as the expression of noncanonical NF-κB target genes. These data clearly indicate that ZFP91 is an important regulator of the noncanonical NF-κB pathway.
Highlights
The transcription factor NF-B plays a central role in immune responses, development, and cell proliferation [1]
zinc finger protein 91 (ZFP91) increased the NF-B-inducing kinase (NIK) level and p100 processing to p52 dose-dependently (Fig. 1C, top two panels), whereas it did not affect the expression level of p50 (Fig. 1C, third panel), suggesting that ZFP91 may be associated with the noncanonical NF-B pathway
We showed that ZFP91 ulation and decreased to basal level over time. siRNA-mediated directly ubiquitinated NIK with Lys63-only ubiquitin (Fig. 5D). knockdown of ZFP91, did not alter the IL-6, IL-8, These results demonstrate that ZFP91 is a novel E3 ligase that MCP1, and Tumor necrosis factor (TNF)-␣ mRNA levels induced by CD154 (Fig. 6E)
Summary
Cell Culture, Transfection, and Luciferase Reporter Assay— HEK293 and MDA-MB231 cells were grown in Dulbecco’s modified Eagle’s medium with penicillin (100 units/ml) plus streptomycin (100 units/ml) (Invitrogen) and 10% heat-inactivated fetal bovine serum (Hyclone). We generated the anti-ZFP91 polyclonal antibody by immunizing mice with full-length ZFP91, which was purified by a Ni2ϩ-NTA chelating agarose column (Peptron, Daejeon, Korea) from lysates of Sf21 insect cells transfected with recombinant baculovirus expressing full-length ZFP91. The resulting mixture was immunoprecipitated with anti-NIK antibody and protein A/G PLUS-agarose beads, and the resultant precipitates were incubated in sample buffer and separated on SDS-PAGE as described. ZFP91-specific double-stranded siRNA oligonucleotides were obtained from Qiagen Inc. The siRNA duplexes prepared in diethyl pyrocarbonate-treated water at a 20 M concentration were transfected in subconfluent cells with Lipofectamine RNAiMAX (Invitrogen) following the manufacturer’s instructions and incubated for 24 – 48 h before analysis
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