Regulation of cell cycle genes by K protein

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DESCRIPTION (provided by applicant) This is a grant application for the Fogarty International Research Collaboration Award (FIRCA) to supplement presently funded NIH project, grant number R01GM 45134-09. The proposed research will be done primarily in Prof. Jerzy Ostrowski's Laboratory in Warsaw, Poland, the foreign collaborator of this FIRCA proposal. The heterogeneous nuclear ribonucleoprotein K, hnRNP K, protein interacts with a diversity of molecules including kinases, transcription and translation factors, RNA and DNA. Many of these molecular interactions are regulated by extracellular signals, such as growth factors. K protein has been shown to regulate rates of DNA transcription and mRNA translation through direct interaction with CT- and CU-rich nucleotide sequences, respectively. It has been suggested that K protein, acting as a docking platform, serves to link signal transduction pathways to sites of nucleic acid-directed processes. K protein binds mRNAs that encode cell cycle regulators, is a substrate for mitogen-responsive kinases, and has an expression pattern correlated with cell proliferation. Based on these observations we postulate that in response to mitogenic signals K protein regulates expression of cell cycle gene(s) at both DNA- and RNA-dependent tiers. The following aims will this hypothesis. Aim#1. We will identify the repertoire of K protein-binding mRNAs that encode cell cycle regulators. mRNAs immunoprecipitated with K protein from quiescent and serum-treated cells will be used to generate a complex probe to profile cDNA microarrays. Computer-based analysis and gel-shift assay will be used to identify RNA sequences that are responsible for binding K protein to the target transcripts. Aim#2. We will identify the repertoire of cell cycle DNA loci that recruit K protein. Chromatin immunoprecipitation with anti-K protein antibody and profiling of microarrays will be used to identify DNA loci that recruit K protein in cells entering cell cycle. Computer-based analysis and gel-shift assays will be used to identify DNA sequences that recruit K protein to the target loci. Aim#3. We will define the role of K protein in the regulation of cognate cell cycle DNA loci and mRNAs. Protein levels (Western blots) of the selected (Aim#1-2) cell cycle regulators and their mRNAs (Northern blots) will be assessed in serum stimulated cells that express wild type and a dominant negative K protein mutant. Identification of cell cycle genes whose expression is K protein-dependent will set the stage for future work to define the precise molecular mechanisms by which K protein regulates and coordinates cell cycle gene DNA- and mRNA-directed processes. The proposed studies will explain the need of proliferating and tumor cells to alter K protein expression.