Takahisa Sugita

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.

Suppression of arthritis by the inhibitors of dipeptidyl peptidase IV

Abstract Dipeptidyl peptidase IV (DP IV, CD26) is a serine exoprotease which selectively cleaves the penultimate proline residue of polypeptides. This enzyme is also expressed as a surface marker on activated T cells. In order to assess the relevance of DP IV in immunological disorders, we evaluated the in vivo effects of specific DP IV inhibitors using two arthritis models, one which was induced by collagen one by alkyldiamine. These animal models share several pathological features associated with rheumatoid arthritis. The transition state substrate analog of DP IV, (S)-Alanylpyrrolidine-boronic Acid (Ala-boroPro), suppressed hind paw swelling, which was associated with collagen-induced and alkyldiamine-induced arthritis. A competitive inhibitor of DP IV, Lys(Z(NO 2 ))-thiazolidide and an irreversible inhibitor, Ala-Pro-nitrobenzoylhydroxylamine also suppressed alkyldiamine-induced arthritis dose-dependently. We also analyzed the pharmacological effects of Lys(Z(NO 2 ))-thiazolidide on several immune responses in vitro, in order to determine its mode of action. This inhibitor suppressed mitogen-induced and antigen-induced proliferation of T cells. However, studies using splenic cells from DP IV deficient rats showed that the inhibition of lymphocyte proliferation was not exerted through the inhibition of DP IV.

Activation of transcription factor NF-κB in experimental glomerulonephritis in rats

Abstract NF-κB plays a pivotal role in cells of the immune system as an inducible transcriptional activator. NF-κB regulates the transcription of many genes of pro-inflammatory cytokines and cell adhesion molecules, which could be involved in the pathogenesis of glomerulonephritis. Using a gel shift assay, we investigated NF-κB DNA-binding activity in glomeruli of WKY rats injected with nephrotoxic serum (NTS). Kinetic analysis indicated that the NF-κB DNA-binding activity in glomeruli, composed of p50 subunit determined by a supershift assay, increased on day 1 after NTS injection and the maximal activation was observed on day 3 to 5. NF-κB activation persisted at least until day 14. Pyrrolidine dithiocarbamate (PDTC), a potent inhibitor of NF-κB activation, inhibited the NTS-induced increase of glomerular NF-κB DNA-binding activity, followed by the inhibition of mRNA expression of IL-1β, MCP-1, ICAM-1 and iNOS, which are known to be regulated by NF-κB. PDTC also prevented urinary protein excretion which is a pathophysiological parameter for glomerulonephritis. These results suggest that NF-κB activation causes the induction of pro-inflammatory factors in nephritic glomeruli, which may play significant roles in the pathogenesis of glomerulonephritis.

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.

Suppression of NF-κB and AP-1 activation by glucocorticoids in experimental glomerulonephritis in rats: molecular mechanisms of anti-nephritic action

Transcription factors nuclear factor-kappa B (NF-kappa B) and activator protein-1 (AP-1) play an important role in the induction of pro-inflammatory factors such as cytokines and cell adhesion molecules, which could be involved in the pathogenesis of glomerulonephritis. We have recently reported the pathogenic significance of NF-kappa B activation in experimental glomerulonephritis in rats. In this study, we investigated the pathogenic relevance of AP-1 activation in nephrotoxic serum (NTS)-induced glomerulonephritis. Increased AP-1 DNA-binding activity was detected in nephritic glomeruli by a gel shift assay. The kinetics of AP-1 activation was similar to that of NF-kappa B. Activation of both NF-kappa B and AP-1 preceded proteinuria, an important pathophysiological parameter for glomerulonephritis. Treatment with prednisolone, a glucocorticoid hormone, prevented activation of both NF-kappa B and AP-1 in glomeruli and subsequent mRNA expression of NF-kappa B- and AP-1-regulated genes. Prednisolone was also effective therapeutically and reduced DNA-binding activities of NF-kappa B and AP-1 which are already activated in nephritic glomeruli. These results suggest that activated NF-kappa B and AP-1 may play an important pathogenic role in glomerulonephritis and the anti-nephritic action of glucocorticoids may be mediated through the suppression of these transcription factors.

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.

A potent dipeptide inhibitor of dipeptidyl peptidase IV

A series of novel potent inhibitors of dipeptidyl peptidase IV (DPP-IV) has been developed. A brief structure-activity relationship of the inhibitors was investigated. The dipeptide TSL-225, tryptophyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, was identified with the critical structure for the inhibitory activity.

Alternative splicing and gene structure of the transforming growth factor β-activated kinase 1

We have identified a fourth splice variant of the TGF beta-activated kinase (TAK1), called TAK1-d, and identified an error in the previously published TAK1-c sequence. Our data shows that the c and d variants encode proteins whose carboxyl ends differ markedly from those of variants a and b. Analysis of the human TAK1 gene sequence, located at 6q16.1-q16.3, shows that the coding sequence is organised in 17 exons. The four splice variants result from alternative splicing of exons 12 and 16, the reading frame of exon 17 being determined by the presence or absence of exon 16. Study of the relative levels of expression of the four splice variants showed significant variations between tissues. Our evidence suggests that the alternative splicing of the TAK1 mRNA may have important functional implications.