Our broad goal is to identify cytokine-responsive nuclear protein kinases in lymphocytes, explore mechanisms by which they are activated and define their role in lymphocyte nuclear events. We have recently identified a novel 85kD nuclear kinase that is activated by treatment of cells with cytokines and other growth factors. The 85kD kinase is activated by phosphorylation suggesting that the 85kD kinase is linked to a cytokine-induced phosphorylation cascade. The objective of this proposal is to clone this protein kinase, and then to test a hypothesis that this 85kD kinase links a cytoplasm-to-nucleus phosphorylation cascade that regulates gene expression. First, we will purify and clone this 85kD nuclear protein kinase. (i) Small scale preparation of nuclear extracts will be used to develop a scheme to purify the 85kD protein. (ii) Scaled up procedures will then be used to obtain sufficient amounts of the protein for proteolytic digestion and sequencing of peptide fragments. (iii) The amino acid sequence of the proteolytic fragments will be used to design synthetic degenerate oligonucleotides for the screening of cDNA libraries and cloning of the 85kD nuclear kinase gene. Second, we will explore whether or not the 85kD enzyme is linked to a cytokine-induced phosphorylation cascade and search for the 85kD kinase effector. (i) We will determine whether the in vivo state of phosphorylation of the 85kD protein is modulated by cytokines. (ii) We will test known kinases and cytoplasmic and nuclear extracts from cytokine-treated cells for their ability to phosphorylate and activate the 85kD nuclear kinase. (iii) Cells transformed by negative or activated dominant mutants of ras and other key transducers will be used to assess whether or not the 85kD kinase is linked to a known growth factor-triggered signal transduction pathway. Third, we will define the role of the 85kD kinase in cytokine-induced nuclear events. (i) The activity of the 85kD kinase will be correlated with defined cellular responses in wild-type and mutant cell lines. (ii) We will determine the effects of overexpression of the wild-type and mutated 85kD kinase. (iii) Mutant cells will be transfected with constitutively active 85kD kinase in an attempt to complement their signal transduction defect. (iv) We will test the effect of targeted disruption of the 85kD kinase gene in cell cultures and in """"""""knockout mouse."""""""" Many of the protein kinases participating in signal transduction are distributed throughout subcellular compartments and are multifunctional. In contrast, the 85kD kinase that we have identified is exclusively nuclear. Demonstration of involvement of the 85kD kinase in cytokine- induced lymphocytes gene expression, proliferation or apoptosis would open up the way to use the 85kD kinase as a model system to devise strategies to abrogate signal transduction exclusively in the nucleus. In cytokine-mediated diseases therapeutic abrogation of signals at the nuclear level would spare membrane, cytoplasmic and other vital non- nuclear processes.
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