The long-term objectives of this project are to determine the mechanisms by which interleukin 3 (IL-3) regulates the growth and differentiation of hematopoietic cells. The research focuses on the initial biochemical events that occur following IL-3 binding to its receptor and are based on studies which initially demonstrated a critical role for IL-3-induced tyrosine phosphorylation. Based on these data, and the absence of intrinsic kinase activity associated with the receptor, approaches and reagents were developed to examine the potential role of various cytoplasmic protein tyrosine kinases in IL-3 signaling. These studies demonstrated that the Janus kinase Jak2 and to a lesser extent Jak1, associates with the IL-3 receptor and is activated following ligand binding. More recently, it has been found that, in general, members of the Jak family of kinases are involved in signaling through receptors of the cytokine receptor superfamily. In the first specific aim, approaches are outlined which will establish the functional role for Jak1 and Jak2 in IL-3 signaling through the use of various cell systems and dominant negative approaches. In addition, targeted gene disruption will be used to assess the requirement for Jak1 and Jak2 in normal development and hematopoiesis. In the second specific aim, the applicant will define the functional domains of Jak2 and relate these domains to specific biological responses. These studies will use point mutations, deletions and Jak chimeric proteins in various biological assays. Approaches are also proposed to identify gain of function mutations and to determine if such mutations are oncogenic. Lastly, the functional interplay between various members of the Jak family will be examined. One important consequence of IL-3 signaling is hypothesized to be the activation of genes that participate in both cellular function and mitogenesis. A novel family of transcription factors, termed signal transducers and activators of transcription (Stats), has recently been implicated in many cytokine responses and its members are substrates of the Jaks. In IL-3 signaling, Stat5, and possibly Stat6, are specifically tyrosine phosphorylated and activated. Based on this, the studies proposed in the third specific aim would address the role of Stat5 and Stat6 in IL-3 responses. Functional domains would be identified by mutations, deletions or chimeric proteins. The role of Stat5 and Stat6 in specific aspects of the response to IL-3 will be assessed with dominant negative mutants and targeted gene disruptions. Approaches are also proposed that will begin to identify the genes that are specifically regulated by Stat5 or Stat6. Together, the studies address fundamental questions regarding the biochemical mechanisms by which IL-3 regulates the growth and differentiation of hematopoietic cells.
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