TNFalpha is a pleiotropic cytokine that induces either cell proliferation or cell death. Inhibition of NF-kappaB activation increases susceptibility to TNFalpha-induced death, concurrent with sustained JNK activation, an important contributor to the death response. Sustained JNK activation in NF-kappaB-deficient cells was suggested to depend on reactive oxygen species (ROS), but how ROS affect JNK activation was unclear. We now show that TNFalpha-induced ROS, whose accumulation is suppressed by mitochondrial superoxide dismutase, cause oxidation and inhibition of JNK-inactivating phosphatases by converting their catalytic cysteine to sulfenic acid. This results in sustained JNK activation, which is required for cytochrome c release and caspase 3 cleavage, as well as necrotic cell death. Treatment of cells or experimental animals with an antioxidant prevents H(2)O(2) accumulation, JNK phosphatase oxidation, sustained JNK activity, and both forms of cell death. Antioxidant treatment also prevents TNFalpha-mediated fulminant liver failure without affecting liver regeneration.

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Here, we used a reductant, N-acetyl-L-cysteine (NAC), to investigate the redox-sensitive step(s) in the signalling pathway from the tumor necrosis factor (TNF) receptor to nuclear factor U UB (NF-U UB). We found that NAC suppressed NF-U UB activation triggered by TNF or by overexpression of either the TNF receptor-associated death domain protein, TNF receptor-associated factor 2, NF-U UB-inducing kinase (NIK), or IU UB kinases (IKKK K and IKKL L). NAC also suppressed the TNFinduced activation of IKKK K and IKKL L, phosphorylation and degradation of IU UB, and nuclear translocation of NF-U UB. Furthermore, NAC suppressed the activation of IKKK K and IKKL L triggered by the overexpression of NIK. These results indicate that IKKK K and IKKL L are subject to redox regulation in the cells, and that NAC inhibits NF-U UB activation through the suppression of these kinases.z 2000 Federation of European Biochemical Societies.

The cellular redox state regulates nuclear factor-U UB (NF-U UB) signaling systems. We investigated the effects of H 2 O 2 on inhibitor of NF-U UB (IU UB) kinases (IKKK K and IKKL L), which phosphorylate IU UB leading to its degradation and NF-U UB activation. Tumor necrosis factor (TNF) stimulation increased IKK activity within 10 min, and then IKK activity decreased gradually within 30 min in HeLa cells. Stimulation of the cells with H 2 O 2 induced a slight activation of IKK within 30 min. Furthermore, co-stimulation with TNF suppressed the downregulation of IKK and sustained the activation for more than 30 min. H 2 O 2 also markedly activated IKK in cells that were pretreated with TNF or phorbol myristate acetate. Electrophoretic mobility shift assay revealed that H 2 O 2 enhanced TNFinduced NF-U UB activation. Studies using IKK mutants and an antibody against phosphorylated IKK proteins revealed that phosphorylation of serine residues, Ser180 of IKKK K and Ser181 of IKKL L, in the activation loops was essential for the H 2 O 2 -mediated activation of IKK. H 2 O 2 -induced activation of IKKK K and IKKL L was reduced by IKKL L and IKKK K kinase-negative mutants, respectively, indicating that IKKK K and IKKL L were stimulated by H 2 O 2 in an interdependent manner. These results suggest that oxidative radical stress has stimulatory effects on NF-U UB through the activation of IKK, which is mediated by the phosphorylation of serine residues in the activation loops. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

The pleckstrin homology (PH) domain of phosphatidylinositol-specific phospholipase C-␦1 (PLC-␦1) binds to both D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) with high affinities. We have previously identified a region rich in basic amino acids within the PH domain critical for ligand binding (Yagisawa, H., Hirata, M., Kanematsu, T., Watanabe, Y., Ozaki, S., Sakuma, K., Tanaka, The pleckstrin homology (PH) 1 domain has been initially identified as a region of sequence similarity of about 120 amino acid residues (3, 4). At the last count, more than 100 proteins have been reported to have this sequence motif; many of these proteins are involved in cellular signaling and cytoskeletal functions (5-8). Studies of several PH domains using x-ray crystallography or NMR (9 -12) revealed a conserved structural module containing a seven-stranded ␤-sandwich formed by two orthogonal antiparallel ␤-sheets and a C-terminal amphiphilic ␣-helix. The loops between the ␤-strands, particularly the ␤1/ ␤2, ␤3/␤4, and ␤6/␤7, differ greatly in length and sequence. Each PH domain is electrostatically polarized, and the most variable loops coincide with the positively charged face.By analogy with other conserved structural modules (e.g. SH2 and SH3 domains), it has been proposed that the PH domain could be involved in signaling by mediating intermolecular interactions. Consequently, a great effort has been made to identify ligand(s) for this domain. Although there are examples of PH domains involved in protein-protein interactions (e.g. binding of G␤␥ by ␤-adrenergic receptor kinase PH domain (13) or recognition of phosphotyrosine by Sch PH/PTB domain (14)) there is an increasing evidence that many PH domains interact with different inositol lipids and inositol phosphates (15,16). In this respect, the PH domain of phospholipase C-␦1 (PLC-␦1) has been studied most extensively. Determination of association constants for different inositol lipids and their head groups (1, 2, 17), and relative abundance of these phospholipids in the cell identified PtdIns(4,5)P 2 as a potentially important physiological ligand (18,19). Ins(1,4,5)P 3 can bind to the same binding pocket as the head group of