Hidetaka Miyoshi

IκB Kinases Phosphorylate NF-κB p65 Subunit on Serine 536 in the Transactivation Domain

Recent investigations have elucidated the cytokine-induced NF-κB activation pathway. IκB kinase (IKK) phosphorylates inhibitors of NF-κB (IκBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-κB. We have examined the possibility that IKK can phosphorylate the p65 NF-κB subunit as well as IκB in the cytokine-induced NF-κB activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF-α in a time dependent manner similar to IκB phosphorylation. In vitro phosphorylation with GST-fused p65 showed that a p65 phosphorylating activity was present in the cytoplasmic fraction and the target residue was Ser-536 in the carboxyl-terminal transactivation domain. The endogenous IKK complex, overexpressed IKKs, and recombinant IKKβ efficiently phosphorylated the same Ser residue of p65 in vitro. The major phosphorylation site in vivo was also Ser-536. Furthermore, activation of IKKs by NF-κB-inducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-κB·IκB complex.

Functional Interactions of Transforming Growth Factor β-activated Kinase 1 with IκB Kinases to Stimulate NF-κB Activation

Several mitogen-activated protein kinase kinase kinases play critical roles in nuclear factor-κB (NF-κB) activation. We recently reported that the overexpression of transforming growth factor-β-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase family, together with its activator TAK1-binding protein 1 (TAB1) stimulates NF-κB activation. Here we investigated the molecular mechanism of TAK1-induced NF-κB activation. Dominant negative mutants of IκB kinase (IKK) α and IKKβ inhibited TAK1-induced NF-κB activation. TAK1 activated IKKα and IKKβ in the presence of TAB1. IKKα and IKKβ were coimmunoprecipitated with TAK1 in the absence of TAB1. TAB1-induced TAK1 activation promoted the dissociation of active forms of IKKα and IKKβ from active TAK1, whereas the IKK mutants remained to interact with active TAK1. Furthermore, tumor necrosis factor-α activated endogenous TAK1, and the kinase-negative TAK1 acted as a dominant negative inhibitor against tumor necrosis factor-α-induced NF-κB activation. These results demonstrated a novel signaling pathway to NF-κB activation through TAK1 in which TAK1 may act as a regulatory kinase of IKKs.

Phosphorylation-dependent activation of TAK1 mitogen-activated protein kinase kinase kinase by TAB1

TAK1 is a mitogen‐activated protein kinase kinase kinase (MAP3K) that is involved in the c‐Jun N‐terminal kinase/p38 MAPKs and NF‐κB signaling pathways. Here, we characterized the molecular mechanisms of TAK1 activation by its specific activator TAB1. Autophosphorylation of two threonine residues in the activation loop of TAK1 was necessary for TAK1 activation. Association with TAK1 and induction of TAK1 autophosphorylation required the C‐terminal 24 amino acids of TAB1, but full TAK1 activation required additional C‐terminal Ser/Thr rich sequences. These results demonstrated that the association between the kinase domain of TAK1 and the C‐terminal TAB1 triggered the phosphorylation‐dependent TAK1 activation mechanism.

TAK1–TAB1 fusion protein: a novel constitutively active mitogen-activated protein kinase kinase kinase that stimulates AP-1 and NF-κB signaling pathways

TAK1 mitogen-activated protein kinase kinase kinase (MAP3K) is activated by its specific activator, TAK1-binding protein 1 (TAB1). A constitutively active TAK1 mutant has not yet been generated due to the indispensable requirement of TAB1 for TAK1 kinase activity. In this study, we generated a novel constitutively active TAK1 by fusing its kinase domain to the minimal TAK1-activation domain of TAB1. Co-immunoprecipitation assay demonstrated that these domains interacted intra-molecularly. The TAK1-TAB1 fusion protein showed a significant MAP3K activity in vitro and activated c-Jun N-terminal kinase/p38 MAPKs and IkappaB kinase in vivo, which was followed by increased production of interleukin-6. These results indicate that the fusion protein is useful for characterizing the physiological roles of the TAK1-TAB1 complex.

Pathophysiological role of nitric oxide in rat experimental colitis

Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) may contribute to the pathophysiology of ulcerative colitis. A 2,4,6-trinitrobenzenesulfonic acid sodium salt (TNBS) colitis model was established to examine the effect of selective iNOS inhibition, by S-(2-aminoethyl) isothiouronium bromide (ITU), on colonic mucosal cell damage and inflammation. Rats, killed 7 days after TNBS, had increased colonic mucosal levels of iNOS and interleukin-8 (IL-8), in addition to severe colonic inflammation which was characterized by significantly increased colon weight, damage score and colonic myeloperoxidase activity (MPO) (a marker of neutrophil influx). TNBS-treated rats had markedly decreased body weight and thymus weight. Administration of colitic rats with ITU significantly inhibited iNOS activity/expression and tended to reduce mucosal levels of IL-8, but no effect on MPO activity was observed. Following ITU therapy, colitic rats had reduced colonic damage and losses in body weight and thymus weight were reversed. Improvement of TNBS colitis by ITU suggested that excess NO, produced by iNOS, may have contributed to the initiation/amplification of colonic disease, by mechanisms including enhancement of IL-8 release. NO-mediated enhancement of pro-inflammatory cytokine release was further investigated in vitro. Lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) stimulated release of nitrite, lactate dehydrogenase (LDH), TNF alpha, IL-1 beta and IL-8 from rat peritoneal macrophages, all of which were significantly reduced by ITU. This suggests that NO-mediated cell damage enhances pro-inflammatory mediator release from macrophages. In addition, enhancement of IL-8 and TNF alpha release was also partially NO-dependent in activated peritoneal neutrophils. Therefore, the amelioration of TNBS colitis by ITU could include inhibition of NO-mediated pro-inflammatory cytokine release.

Immunosuppressive effects of the heat shock protein 90‐binding antibiotic geldanamycin

Ansamycin antibiotic, geldanamycin has a unique pharmacological effect to bind to heat shock protein 90 (hsp90) and deplete hsp90 substrates. We investigated the immunopharmacological effects of geldanamycin. Geldanamycin depleted cellular Raf‐1 of rat splenic cells without affecting the steady state levels of hsp90 and downstream mitogen activated protein (MAP) kinases, ERK1 and ERK2. In parallel, it inhibited mitogen‐induced nuclear factor‐kappa B (NF‐κB) activation in these cells. Geldanamycin also had a potent suppressive effect on recall antigen‐induced T cell proliferation, with an IC50 value of 1 nM. In vivo, geldanamycin suppressed the progression of adjuvant‐induced arthritis dose‐dependently. These results suggest that geldanamycin exerts an immunosuppressive effect partly through destabilizing Raf‐1, and raise a new strategy for the prevention of inflammatory diseases.