The heterogeneous nuclear fibonucleoprotein K (hnRNP K) protein is involved in multiple processes that compose gene expression including transcription and RNA processing. K protein is inducibly phosphorylated by several kinase cascades. K protein is comprised of multiple domains that serve to engage DNA, RNA and protein factors involved in gene expression and signal transduction. K protein interactions are regulated by phosphorylation. We have shown that treatment of cells with serum, a model for mitogenic stimulation, causes recruitment of K protein to inducibly transcribed loci, including c-myc, egr-1 and other immediate-early genes. Based on these observations we propose a hypothesis that K protein serves to link kinase cascades to inducibly transcribed c-myc and egr-1 gene loci by facilitating phosphorylation of factors involved in transcription and pre-mRNA processing. The current proposal represents a continuation of our research on K protein. We will identify serum inducible kinase cascades that direct K protein to distinct domains within the c-myc and egr-1 loci that include not only DNA elements but also pre-mRNA and protein complexes. Chromatin immunoprecipitation (CHIP) assays, computer-based analysis and in vitro binding will be used to determine if K protein binds to the promoter, transcribed and other regions within c-myc and egr-1 loci. Specific kinase inhibitors, constitutively active and dominant negative kinase mutants will be used to define upstream signals responsible for K protein recruitment to these sites. We will explore the mechanisms responsible for the recruitment of K protein to the inducible c-myc and egr-1 loci. Mutation and deletion analysis will be used to map K protein modules that are responsible for its recruitment to DNA, RANA, and protein sites within a target gene locus. Pharmacologic and genetic strategies will be used to define which cascades regulate these K protein domains that mediate its recruitment. We will define the role of K protein recruitment in the inducible expression of the c-myc and egr-I loci. Northern blot analysis, RT-PCR, transcription and pre-mRNA splicing assays will be used to assess the effects of K protein on the expression of target gene loci. Kinase assays, pharmacologic and genetic strategies will be used to test the role of K protein in regulating both phosphorylation and activity of components of transcription and splicing machinery. Each stage of gene expression provides a potential point for regulation by signal transduction pathways triggered by mitogens. K protein represents a conceptually novel class of nucleic acid-binding factors that may provide an avenue for kinase cascades to regulate distinct processes that compose gene expression. Exploring the mechanisms and rote of K protein mitogen-induced recruitment to inducible gene loci will provide new insight into the regulation of gene expression.
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