Our long-term goal is to elucidate the role of kinases in mediating interleukin-1 (IL-1)-induced gene expression. The kappaB motif is a ubiquitous transcriptional element that participates in IL-1-induced expression of many genes. To explore the role of protein phosphorylation in cytokine-induced gene expression we set out to identify kinases that interact with factors recognized by the kappaB transcriptional element. This search led to the identification of a kappaB element-binding 65kD phosphoprotein that is associated with an IL-1-responsive serine/threonine kinase. In the current funding period we purified this protein and isolated its cDNA. Sequencing and expression of the cDNA demonstrated that this kappaB motif-binding phosphoprotein is the murine hnRNP K protein and is not a kinase. We also demonstrated that the associated kinase is a novel enzyme that phosphorylates K protein in a DNA- and RNA- dependent manner. The K protein is a highly interactive molecule that binds RNA and DNA, interacts with protein tyrosine kinases and Vav, and regulates c-myc promoter activity. The objective of the present proposal is to explore how the interaction of K protein with its molecular partners is regulated by phosphorylation mediated by the K protein-associated IL-1- responsive kinases. The proposed studies represent a logical extension of the currently funded project. FIRST, we will define phosphorylation and dephosphorylation of K protein. (i) Edman degradation and mass spectrometry will be used to map the K protein serine/threonine residues that are phosphorylated in vivo and in vitro in response to treatment of cells with IL-1. (ii) Deletion analysis and site directed mutagenesis will be used to map the K protein kinase- binding domains. (iii) Phosphatases and phosphatase inhibitors will be used to define the class of enzymes that dephosphorylate protein. SECOND, we will define the mechanisms of the IL-1-mediated K protein phosphorylation and define the role of phosphorylation in modulating K protein interaction with RNA and DNA. (i) We will use K protein deletion mutants to map DNA- and RNA-binding domains and determine whether these domains contain phosphorylation sites that regulate K interaction with its cognate nucleic acid motifs. (ii) We will determine whether tyrosine kinases and Vav are activated and/or bind to the K protein in response to IL-1 and whether the mechanism is responsible for the IL-1-induced phosphorylation of K protein by the associated serine/threonine kinase(s). THIRD, we will use several approaches to identify, purify and clone IL-1- responsive kinases that bind and phosphorylate K protein. (i) A two hybrid and cDNA library screening will be used to directly isolate the K protein- binding kinases(s). (ii) Sequential chromatography will be used to purify K protein-associated kinases to obtain partial amino acid sequences. These sequences will be used to design oligonucleotides to generate PCR probes to clone genes from cDNA libraries that encode K protein kinase(s). (iii) Isolated cDNA will be used to define tissue distribution of the K protein kinase(s), and produce antibodies to define subcellular localization of the kinase(s) in response to IL-1 treatment. The recent discovery of K protein and its interactions with diverse types of molecules suggests that novel pathways participate in the IL-triggered gene expression. This project will provide new insight into how the function of K protein is regulated by IL-1-induced phosphorylation, and therefore will contribute to the understanding of the action of this key cytokine.
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