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 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

Gonadotropin-releasing hormone (GnRH) is a neuroendocrine peptide that plays a central role in the vertebrate hypothalamo-pituitary axis. The roles of GnRH in the control of vertebrate reproductive functions have been established, while its non-reproductive function has been suggested but less well understood. Here we show that the tunicate Ciona intestinalis has in its non-reproductive larval stage a prominent GnRH system spanning the entire length of the nervous system. Tunicate GnRH receptors are phylogenetically closest to vertebrate GnRH receptors, yet functional analysis of the receptors revealed that these simple chordates have evolved a unique GnRH system with multiple ligands and receptor heterodimerization enabling complex regulation. One of the gnrh genes is conspicuously expressed in the motor ganglion and nerve cord, which are homologous structures to the hindbrain and spinal cord of vertebrates. Correspondingly, GnRH receptor genes were found to be expressed in the tail muscle and notochord of embryos, both of which are phylotypic axial structures along the nerve cord. Our findings suggest a novel non-reproductive role of GnRH in tunicates. Furthermore, we present evidence that GnRH-producing cells are present in the hindbrain and spinal cord of the medaka, Oryzias latipes, thereby suggesting the deep evolutionary origin of a non-reproductive GnRH system in chordates.

We have previously identified a novel 130 kDa protein (p130) which binds Ins(1,4,5)P3 and shares 38% sequence identity with phospholipase C-delta 1 [Kanematsu, Misumi, Watanabe, Ozaki, Koga, Iwanaga, Ikehara and Hirata (1996) Biochem. J. 313, 319-325]. We have now transfected COS-1 cells with genes encoding the entire length of the molecule or one of several truncated mutants, in order to locate the region for binding of Ins(1,4,5)P3. Deletion of N-terminal residues 116-232, the region which corresponds to the pleckstrin homology (PH) domain of the molecule, completely abolished binding activity. This result was confirmed when the PH domain itself (residues 95-232), isolated from a bacterial expression system, was found to bind [3H]Ins(1,4,5)P3. We also found that Ins(1,4,5,6)P4 was as efficacious as Ins(1,4,5)P3 in displacing [3H]Ins(1,4,5)P3, suggesting that these two polyphosphates bind to p130 with similar affinity. This conclusion was confirmed by direct binding studies using [3H]Ins(1,4,5,6)P4 with high specific radioactivity which we prepared ourselves. Binding specificity was also examined with a variety of inositol phosphate derivatives. As is the case with other PH domains characterized to date, we found that the 4,5-vicinal phosphate pair was an essential determinant of ligand specificity. However, the PH domain of p130 exhibited some novel features. For example, the 3- and/or 6-phosphates could also contribute to overall binding; this contrasts with some other PH domains where these phosphate groups decrease ligand affinity by imposing a steric constraint. Secondly, a free monoester 1-phosphate substantially increased binding affinity, which is a situation so far unique to the PH domain of p130.

No abstract