Recent investigations have elucidated the cytokineinduced NF-B activation pathway. IB kinase (IKK) phosphorylates inhibitors of NF-B (IBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-B. We have examined the possibility that IKK can phosphorylate the p65 NF-B subunit as well as IB in the cytokine-induced NF-B activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF-␣ in a time dependent manner similar to IB 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 IKK␤ 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-Binducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-B⅐IB complex.The transcription factor nuclear factor-B (NF-B) 1 plays a pivotal role in inflammatory and immune responses (1-3). NF-B is composed of a heterodimer of p65 and p50 subunits in most cell types and is sequestered in the cytoplasm by its inhibitor proteins, the IBs (4 -8). Several NF-B-activating agents, including pro-inflammatory cytokines, phorbol esters, and bacterial lipopolysaccaride, induce the phosphorylation of IBs at two NH 2 -terminal Ser residues. The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, thereby releasing NF-B to enter the nucleus for gene expression (9 -15).Recent investigations have focused on the phosphorylation of IBs and clearly elucidated the molecular mechanisms of the phosphorylation. In brief, two closely related kinases, designated IB kinase (IKK) ␣ and IKK␤, have been identified as components of the multiprotein IKK complex (500 -900 kDa) that directly phosphorylates the critical Ser residues of . IKK␣ and IKK␤ together form a heterodimer through their COOH-terminal leucine zipper motifs, and the functional IKK complex contains both IKK subunits. NF-B-inducing kinase (NIK), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, interacts with and activates the IKK complex (21). Other MAP3Ks, including transforming growth factor-␤ activated kinase 1 (TAK1) (22-24), MAPK/ extracellular signal-regulated kinase kinase kinases (MEKK1-3) (25-28), and Cot/Tpl2 (29), have been shown to be involved in the IKK activation pathways, indicating the important roles of MAP3K family kinases in the IKK activation by diverse extracellular stimuli.The activity of several inducible transcription factors, including cAMP response element-binding protein (CREB) (30) and c-Jun (31), has been shown to be regulated by phosphorylation. It has been shown that the p65 NF-B subun...

Several mitogen-activated protein kinase kinase kinases play critical roles in nuclear factor-B (NF-B) activation. We recently reported that the overexpression of transforming growth factor-␤-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-B activation. Here we investigated the molecular mechanism of TAK1-induced NF-B activation. Dominant negative mutants of IB kinase (IKK) ␣ and IKK␤ inhibited TAK1-induced NF-B activation. TAK1 activated IKK␣ and IKK␤ in the presence of TAB1. IKK␣ and IKK␤ were coimmunoprecipitated with TAK1 in the absence of TAB1. TAB1-induced TAK1 activation promoted the dissociation of active forms of IKK␣ and IKK␤ from active TAK1, whereas the IKK mutants remained to interact with active TAK1. Furthermore, tumor necrosis factor-␣ activated endogenous TAK1, and the kinase-negative TAK1 acted as a dominant negative inhibitor against tumor necrosis factor-␣-induced NF-B activation. These results demonstrated a novel signaling pathway to NF-B activation through TAK1 in which TAK1 may act as a regulatory kinase of IKKs. Transcription factor nuclear factor B (NF-B)1 is composed of homodimers and heterodimers of Rel family proteins and plays a pivotal role in the gene expression involved in inflammatory and immune responses (1-3). NF-B is sequestered in the cytoplasm by inhibitory proteins such as IB␣, IB␤, and IB⑀, which mask the nuclear localization signal of NF-B (4 -8). The phosphorylation of two Ser residues at an N-terminal regulatory domain of IB proteins triggers polyubiquitination of IB proteins, which targets them for rapid degradation through a proteasome-dependent pathway, thereby releasing NF-B to enter the nucleus (9 -15). Diverse extracellular stimuli such as tumor necrosis factor (TNF)-␣ and interleukin-1␤, phorbol esters, and environmental stresses lead to NF-B activation utilizing the common mechanism for the IB degradation, suggesting the diversity of the upstream signaling pathways for phosphorylation of IB proteins.Several regulatory kinases involved in the signal-induced phosphorylation of IB proteins have recently been reported. Two closely related kinases designated IB kinase (IKK) ␣ and IKK␤ have been identified as components of the multiprotein IKK complex (500 -900 kDa) that directly phosphorylates the critical Ser residues of IB proteins (16 -20). Together, IKK␣ and IKK␤ form a heterodimer through their C-terminal leucine zipper motifs, and the functional IKK complex contains both IKK subunits. NF-B-inducing kinase (NIK) is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, which was first identified as a TNF-␣ receptor-associated factor (TRAF) 2-interacting protein (21). The ligand-mediated trimerization of the TNF-␣ receptor triggers the recruitment of NIK to TRAF2, and this association results in the activation of NIK, which in turn phosphorylates and activates IKKs. NIK also interacts with TRAF6, another member of the...

TAK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that is involved in the c-Jun N-terminal kinase/ p38 MAPKs and NF-U UB 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.z 2000 Federation of European Biochemical Societies.

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We recently cloned a c-Jun amino-terminal kinase (JNK) sequence from the C6/36 cell line, derived from the mosquito Aedes albopictus. We showed that SP600125, an inhibitor of JNK proteins, inhibits phagocytosis by C6/36 cells, suggesting that the JNK-like protein regulates phagocytosis. Here, we show that C6/36 cells constitutively express low levels of mRNA encoding the antibacterial peptides, cecropin and defensin, but that these mRNAs were up-regulated upon stimulation by lipopolysaccharide (LPS). Thus, the C6/36 cells have properties similar to those of mammalian macrophages. To characterize further the functional properties of C6/36 cells, we have assayed the role of the JNK-like protein in phagocytosis, endocytosis, and viral infection. C6/36 cells phagocytosed bacteria and artificial beads, and this was only slightly up-regulated following LPS stimulation, suggesting that newly stimulated JNK-like protein was not necessary for phagocytosis. SP600125 inhibited the acidification of intracellular compartments, including those involved in the endocytic pathway. Pretreatment of C6/36 cells with SP600125 or bafilomycin A1, but not cytochalasin D, inhibited the entry of West Nile virus (WNV), suggesting that WNV is internalized mainly by endocytosis, and that the JNK signalling pathway is important for endocytic entry. These findings indicate that the JNK-like protein regulates basic physiological functions, including phagocytosis and endocytosis and infection of WNV.