The goal of this study is to identify the early signal transduction mechanisms by which GM-CSF stimulates the differentiation of hematopoietic cells. These complex signals appear to be generated by the interaction of specific domains of the alpha and beta chains of the GM-CSF receptor (R) and associated protein kinases. The analysis of GM-CSF-induced differentiation will be made possible by a unique cell line FDCP-1/19WT that grows in IL-3, but differentiates in GM-CSF. This cell line will be used to (1) map the specific domains of the alpha and beta chains which regulate differentiation, (2) determine the role of specific phosphorylation sites in the beta chain in the differentiation process, and (3) examine the need for alphaPK and JAK2 protein kinases, as well as the Ras pathway in the differentiation. The preliminary results and unique reagents generated in our laboratory will make this analysis possible. We have found that (1) the sort 54 amino acid intracytoplasmic tail of the alpha chain of the GM-CSFR is necessary for GM-CSF mediated differentiation, (2) a newly cloned protein kinase, alphaPK, binds to this alpha intracytoplasmic domain, and (3) a CD16/JAK2 fusion protein encoding only the kinase domain of JAK2 is sufficient to stimulate cell growth of GM-CSF-dependent cell lines. Using these reagents and the FDCP-1 19WT cells, we will generate a unique understanding of how GM-CSF signals hematopoietic cells to differentiate.
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