Immunosuppressant FK506 Activates NF-κB through the Proteasome-mediated Degradation of IκBα

Abstract

The immunosuppressant FK506 activates NF-B through IB␣ degradation in nonlymphoid cells. In the present study, we analyzed mechanisms by which FK506 induces IB␣ degradation. We found that FK506 induces the degradation of both IB␣ and IB␤ and that the time courses of the FK506-induced degradation are quite different from degradation induced by interleukin 1 (IL-1). Despite this difference, FK506-induced IB␣ degradation was dependent on the N-terminal Ser-32 and Ser-36 phosphorylation sites and was mediated by proteasomes, as is the case for IL-1-induced IB␣ degradation. We further showed that FK506 induces weak and slow phosphorylation of IB␣ at Ser-32. However, unlike IL-1-induced degradation, IKK-1 and IKK-2 were not activated significantly nor was FK506-induced IB␣ degradation dependent on the N-terminal ubiquitination sites (Lys-21 and Lys-22). These results therefore indicate that FK506 and IL-1 utilize similar but distinct mechanisms to induce the phosphorylation and degradation of IB␣. Nuclear factor B (NF-B)1 is a transcription factor that plays an important role in inducing the expression of diverse cellular genes, such as for various cytokines, cell surface receptors, and acute-phase proteins. It is a heterodimer mainly composed of the p50 and RelA proteins, but there might be a considerable heterogeneity in its composition in various cell types, because of the presence of p50/RelA-related proteins (p52, c-Rel, and RelB), which share extensive homology in their N-terminal DNA-binding/dimerization regions. These proteins are now known as the NF-B/Rel/Dorsal transcription factor family, as they are also related to the Drosophila maternal morphogen gene, dorsal. An unusual feature of this family is that they exist in the cytoplasm in an inactive form complexed with a family of inhibitor proteins termed IB (IB␣, IB␤, and IB⑀). A variety of stimuli, including virus infection, bacterial lipopolysaccharides, double-stranded RNA, phorbol esters, UV radiation, oxidative stress, and inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-␣ (TNF-␣) activate NF-B through the proteolytic degradation of IB and the subsequent translocation of NF-B to the nucleus, where it activates target genes (1-3).The prototypic and best-studied of the IBs is IB␣, which is phosphorylated at its N-terminal two serine residues (Ser-32 and Ser-36) prior to degradation, when cells are exposed to appropriate NF-B activators (4 -6). This phosphorylation triggers the ligation of multiple ubiquitin molecules to nearby lysine residues (Lys-21 and Lys-22), leading to the subsequent degradation of the protein by proteasomes (6 -9). The signalinduced phosphorylation of IB␣ is therefore a critical step in NF-B activation and has been investigated intensively. Recently, two closely related IB kinases (IKKs), termed IKK-1 and IKK-2, have been identified and cloned. Both kinases directly phosphorylate Ser-32 and Ser-36 of IB␣ and their activities are stimulated by IL-1 and TNF-␣ treatment (10 -14). In addition, pp90rsk kinase...

Anti‐inflammatory effect of astaxanthin in phthalic anhydride‐induced atopic dermatitis animal model

“…It was reported that systemic administration of cyclosporine in mice potentiated tributyltin‐induced skin irritation through activation of NF‐κB . Moreover, FK506 induced activation of NF‐κB without affecting activity of IKKs in fibroblasts . It was also reported that FK506 blocks NF‐κB activation in peripheral human T cells through anti‐CD3/28 activation .…”

ASC-mediated NF-κB Activation Leading to Interleukin-8 Production Requires Caspase-8 and Is Inhibited by CLARP

“…The cDNA encoding FLAG-tagged pyrin or RICK was generated in pCMV-Tag2B (Stratagene, La Jolla, CA) and then subcloned into pEF-Bos to generate pEF-FLAG-pyrin and pEF-FLAG-RICK. The pEF-IB␣-S32A/S36A carrying an IB␣ dominantnegative mutant cDNA (22) was kindly provided by Dr. Ken-ichi Yamamoto (Kanazawa University, Kanazawa, Japan).…”

Dynamic Receptor-dependent Activation of Inducible Nitric-oxide Synthase by ERK-mediated Phosphorylation of Ser745

“…In Vitro Kinase Assay-The generation and isolation of GST fusion proteins and in vitro kinase assay were done as described (18) with the following modifications. Induction with isopropyl 1-thio-␤-D-galactopyranoside was done at 30°C for 3 h, and 0.5 g of purified GST fusion protein was incubated with 50 ng of active or inactive ERK1 in kinase assay buffer containing 10 Ci of [␥-…”