The intracellular pathways by which interleukin-1 (IL-1) signals gene expression are not well understood. We have been interested in defining the IL-1 signal transduction pathways that trigger the kappa light chain immunoglobulin and the IL-2 gene expression in lymphocytes. IL-1 activates multiple membrane and cytoplasmic events including activation of protein kinases. At the nuclear level IL-1-induced kappa light chain immunoglobulin and IL-2 gene expression is regulated by the EkappaB transcriptional element which recognizes a number of different complexes including NF-kappaB. We have shown that IL-1 induced gene expression is associated with the activation of a nuclear kinase that is contained within a novel DNA-binding complex specifically recognized by the EkappaB transcriptional element. The EkappaB-associated complex that we have identified contains a number of subunits including a 65kD protein which is a substrate for the IL-1 responsive EkappaB-associated kinase. The objective of this proposal is to test a hypothesis that this IL-1 responsive protein kinase serves to link IL-1 induced cytoplasmic events with the kappa light chain immunoglobulin and the IL-2 gene expression. We will first attempt to identify which of the EkappaB-associated proteins contains the IL-1 responsive kinase. Sequential chromatography will be used to synthesize degenerate oligonucleotides for screening of lymphocytes cDNA libraries and the cloning of the IL-1-responsive EkappaB-associated protein kinase. Similar approach will be used for partial amino acid sequencing of the 65kD EkappaB-associated substrate. We will attempt to clone the 65kD EkappaB-associated protein only if the amino acid sequence is that of a novel protein and if it is itself a kinase. The IL-1-responsive kinase might regulate either the DNA-binding or the transcriptional activity or both of the EkappaB-binding complexes. Gel shift analysis and methylation interference will be sued to test whether phosphorylation of the EkappaB- associated proteins by the IL-1 responsive kinase alters the DNA-binding activity of factors recognized by the EkappaB transcriptional element. In vitro transcription assays will be attempted to assess the role of the IL- 1-responsive kinase in modulating the transcriptional activity of the EkappaB-binding proteins. The IL-1-responsive EkappaB-associated kinase is present in the nucleus but it also might be present in the cytoplasm. The entire pool or only a fraction of the IL-1-responsive kinase might be bound to the EkappaB-associated complex. To determine the cellular distribution of the IL-1-responsive kinase in relation to the other EkappaB-associated proteins we will attempt to produce antibodies to the EkappaB-associated proteins and use immunofluorescence for localization. The activation of the IL-1-responsive EkappaB-associated kinase might be mediated by a more proximal kinase of the IL-1-initiated """"""""phosphorylation cascade"""""""". In vitro phosphorylation assay will be attempted to determine whether the EkappaB- associated kinase is activated by another IL-1-responsive kinase. Our hypothesis would be supported if we show that a) the IL-1-responsive EkappaB-associated kinase stimulates gene expression by modifying the EkappaB-associated proteins, and b) that this kinase is itself stimulated by a more proximal IL-1-responsive kinase.
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