Interleukin-1 (IL-1) is a proinflammatory cytokine that elicits its pleiotropic effects through activation of the transcription factors NF-kappaB and AP-1. Binding of IL-1 to its receptor results in rapid assembly of a membrane-proximal signalling complex that consists of two different receptor chains (IL-1Rs), IL-1RI and IL-1RAcP, the adaptor protein MyD88, the serine/threonine kinase IRAK and a new protein, which we have named Tollip. Here we show that, before IL-1beta treatment, Tollip is present in a complex with IRAK, and that recruitment of Tollip-IRAK complexes to the activated receptor complex occurs through association of Tollip with IL-1RAcP. Co-recruited MyD88 then triggers IRAK autophosphorylation, which in turn leads to rapid dissociation of IRAK from Tollip (and IL-1Rs). As overexpression of Tollip results in impaired NF-kappaB activation, we conclude that Tollip is an important constituent of the IL-1R signalling pathway.
The nucleotide sequence of a folk acid synthesis (fax) gene from Pneumocystis curinii contains an open reading frame (ORF) that predicts a protein of 740 amino acids with an M, of 83979. A recombinant baculovirus was constructed which directed expression of the predicted Fas740 polypeptide in cultured Spodoptera frugiperdu (SF9) insect cells. The overexpressed 'full-length' protein migrated anomalously in sodium dodecyl sulfate/polyacrylamide gels, with an apparent molecular mass of 71.5 kDa. An abundant 69-kDa species was also recognized by polyclonal sera specific for the Fas protein in immunoblotting analyses. Dihydroneopterin aldolase, dihydropterin pyrophosphokinase and dihydropteroate synthase activities were readily detected in SF9 extracts in which the 71.5/69-kDa immunoreactive species were overproduced, demonstrating that three enzyme functions involved in catalysing three sequential steps of the folate biosynthetic pathway are encoded by a single gene in R curinii. Importantly, the polyclonal sera recognize a single 69-kDa species in R curinii extracts suggesting that the three activities are indeed properties of a single polypeptide, although the nature of the suggested post-translational modification is unknown. Location of the individual enzyme domains with the Fas polypeptide based upon amino acid sequence similarity to their bacterial counterparts is discussed. Futhermore, expression of various truncated fus gene constructs demonstrates that the complete fus ORF, including the N-terminus of the predicted polypeptide (FasA domain) whose enzyme function is unknown, must be expressed for maximum dihydroneopterin aldolase (FasB domain) and dihydropteroate synthase (FasD domain) activites. This suggests interactions between the domains within the larger polypeptide to stabilize the functions of these two enzymes. The FasC domain, which contains 6-hydroxymethyl-7,s-dihydropterin pyrophosphokinase activity, is able to fold and function independently of the other domains. The requirement by mammalian cells for preformed folates, and the absence of dihydroneopterin aldolase, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase and dihydropteroate synthase from these tissues opens up the possibility of designing higly selective drugs which inhibit these unique targets.Reduced folate cofactors are essential for the syntheses of purines, thymidylate, glycine, methionine, pantothenic acid, and N-formylmethionyl-tRNA in all cells [ 11. Most microbes must synthesise folates de n o w since they lack the Correspondence to C . J.
Upon interleukin 1 (IL-1) stimulation, the IL-1-receptor (IL-1R)-associated kinase (IRAK) is rapidly recruited to the IL-1R complex and undergoes phosphorylation. Here we demonstrate that recombinant wild-type IRAK (IRAK-WT), but not a kinase-defective mutant with Asp340 replaced by an asparagine residue (IRAK-Asp340Asn), is highly phosphorylated and is capable of auto-phosphorylation in vitro. Overexpression of both IRAK-WT and IRAK-Asp340Asn caused activation of nuclear factor kappaB, suggesting that the kinase activity of IRAK is not required outside of the IL-1R complex.
Following interleukin-1 (ILl) stimulation, an ILl receptor associated kinase (IRAK) is rapidly recruited to the receptor complex. However, it is not understood if IRAK is able to interact directly with the intracellular portion of the IL1-RI or if its recruitment is mediated by a different molecule. Using the yeast two-hybrid system, we have analysed possible proteinprotein interactions between IRAK, IL1-RI and ILl-RAcP. We found that IRAK is able to interact with the equivalent cytoplasmic region of the ILl-RAcP but is unable to interact with the cytoplasmic region of the IL1-RI. Immunoprecipitation of the ILl-RAcP followed by Western blot analysis using anti-IRAK antibodies revealed that IRAK co-precipitated with the ILl-RAcP. We propose that, in non-stimulated cells, IRAK is bound to the ILl-RAcP and therefore, following ILl stimulation, both molecules are recruited simultaneously to the IL1-RI complex.
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