The recognition of microbial pathogens by the innate immune system involves Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns. Different TLRs recognize different pathogen-associated molecular patterns, with TLR-4 mediating the response to lipopolysaccharide from Gram-negative bacteria. All TLRs have a Toll/IL-1 receptor (TIR) domain, which is responsible for signal transduction. MyD88 is one such protein that contains a TIR domain. It acts as an adapter, being involved in TLR-2, TLR-4 and TLR-9 signalling; however, our understanding of how TLR-4 signals is incomplete. Here we describe a protein, Mal (MyD88-adapter-like), which joins MyD88 as a cytoplasmic TIR-domain-containing protein in the human genome. Mal activates NF-kappaB, Jun amino-terminal kinase and extracellular signal-regulated kinase-1 and -2. Mal can form homodimers and can also form heterodimers with MyD88. Activation of NF-kappaB by Mal requires IRAK-2, but not IRAK, whereas MyD88 requires both IRAKs. Mal associates with IRAK-2 by means of its TIR domain. A dominant negative form of Mal inhibits NF-kappaB, which is activated by TLR-4 or lipopolysaccharide, but it does not inhibit NF-kappaB activation by IL-1RI or IL-18R. Mal associates with TLR-4. Mal is therefore an adapter in TLR-4 signal transduction.
Two receptors for the proinflammatory cytokine interleukin 1 (IL-1) have been cloned and characterized biochemically. While it has been well established that the type I (80-kDa) IL There are two known receptors for IL-1, which differ in both size and tissue distribution (2-4). The ligand-binding portions of the two receptors are similar, but whereas the cytoplasmic region of the type I (-80-kDa) receptor contains -215 amino acids, that of the type II (=60-kDa) receptor is only 29 amino acids long. It is well established that the type I receptor is capable of mediating biological responses (5, 6). In studies reported elsewhere, it has also been clearly shown that the type I and type II receptors are not subunits of a multimeric receptor complex but instead bind IL-1 independently of one another (7). This raises the possibility that each receptor might couple to different signal transduction pathways. In the studies reported here, however, we have been unable to find any evidence that the type II receptor signals at all.
We have identified a novel member of the interleukin-1 (IL-1) receptor family, which we have termed AcPL. In transient transfection assays, we were unable to demonstrate a role for AcPL in IL-1-induced activation of NFB. Interleukin-18 (interferon-␥-inducing factor) is another member of the IL-1 family of cytokines, and it has recently been shown that IL-18 has a weak affinity for IL-1R-rp1. We examined whether AcPL might function alone or in concert with IL-1R-rp1 to mediate IL-18 signaling. We found that both IL-1R-rp1 and AcPL expression were required for induction of NFB activity and for activation of c-Jun N-terminal kinase in response to IL-18. Furthermore, a dominant negative version of AcPL specifically inhibited IL-18 signaling. In vitro immunoprecipitation assays demonstrated that AcPL alone was unable to bind IL-18 with any appreciable affinity. We propose that although IL-1R-rp1 binds the cytokine, IL-1R-rp1 and AcPL proteins are both required for IL-18 signaling, analogous to the requirement for both IL-1R and IL-1RAcP in IL-1-mediated responses.
In fibroblasts and hepatoma cells, interleukin-1 (IL-1) treatment results in the rapid nuclear accumulation of the transcription factor NF-B, present largely as p65 (RelA)/p50 heterodimers. It is well established that this process is dependent in large part upon the phosphorylation and subsequent degradation of the cytosolic inhibitor IB. We looked for other IL-1-induced modifications of NF-B components and found that, in both cell types, IL-1 stimulation led, within minutes, to phosphorylation of both NF-B p65 and p50. Phosphorylation of p65 was sustained for at least 30 min after addition of the cytokine and occurred principally upon serine residues. Immunoprecipitates of NF-B complexes contained an associated protein kinase, the biochemical characteristics of which were indistinguishable from casein kinase II (CKII). Purified CKII efficiently phosphorylated p65 in vitro, apparently on the same major sites that became phosphorylated in intact IL-1-treated cells. Although IL-1 treatment caused little apparent stimulation of total cellular CKII activity, the fraction that was specifically associated with NF-B complexes was markedly elevated by the cytokine. The association of CKII with NF-B occurred in the cytoplasm, suggesting that this phosphorylation might be involved either in control of translocation of the activated complex or in modulation of its DNA binding properties. Interleukin-1 (IL-1)1 receptors are widely distributed on cells of many lineages (1). Thus IL-1 can exert effects on connective tissues, for example inducing mitogenesis through secretion of PDGF; effects on lymphoid cells, such as inducing up-regulation of surface immunoglobulin and secretion of other cytokines; and effects on hepatocytes, for example inducing secretion of acute phase proteins such as C-reactive protein. All of these responses are triggered through a single type of receptor (IL-1R1) (2), which is the archetype for a diverse family of proteins. Included in this group are the insect Toll proteins (3), which function in the establishment of dorsal-ventral polarity during embryogenesis, and the product of the tobacco N-gene (4), which mediates resistance to the pathogen tobacco mosaic virus. Many IL-1-responsive genes are regulated through activation of Rel family transcription factors, in particular NF-B p65/p50 heterodimers, and indeed, ligation of Toll ultimately signals the activation of Dorsal, a Rel-related protein (5).NF-B is a term used to refer to a group of transcription factors/DNA binding activities that recognize a consensus motif, 5Ј-GGGRNNYYCC-3Ј. All of these factors are dimers composed of subunits that share a common domain prototypically found in c-Rel (6). The transactivating activity of the NF-B factors is regulated by a distinct set of proteins termed IBs, which are characterized by a series of ankyrin repeats. IBs function by sequestering NF-B dimers in the cytosol. (7). A variety of stimuli can induce activation of NF-B (8), including viruses, lipopolysaccharides, and pro-inflammatory cytokines such as ...
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