Interleukin-3 (IL-3) is a growth factor that supports the proliferation and differentiation of early hematopoietic stem cells as well as committed progenitors of the myeloid lineages. The long term goal of this proposal is to determine the mechanisms by which IL-3 regulates growth and differentiation by using a series of IL-3-dependent myeloid cell lines. The cells require IL-3 for maintenance of viability, progression through the cell cycle and for the expression of genes including c-myc, c-fos, c- pim-1 and ornithine decarboxylase. The studies proposed in this application will identify genes that are involved in the early events in signal transduction. In the first specific aim, we will pursue our studies which demonstrated that IL-3 binds to cause the rapid tyrosine phosphorylation of a cell surface 140 kDa protein. Using large scale purifications with anti-phosphotyrosine monoclonal antibody resins we propose to purify, sequence and obtain cDNAs clones for pp140. The cDNA clones would be used to study IL-3 bindings, the ability to mediate IL-3 specific signal transduction and the possible role of pp140 in transformation. In the second specific aim we will purify, sequence and obtain cDNA clones for other substrates of IL-3 induced tyrosine phosphorylation. The products of these cDNAs will be characterized for their role in IL-3 signal transduction and transformation of hematopoietic cells. In the last specific aim, we propose to clone unique early hematopoietic lineage specific protein tyrosine kinases by using oligonucleotide probes to conserved domains. Experiments are proposed to determine whether they encode the receptors for known hematopoietic growth factors or contribute to the mechanisms by which IL-3 regulates growth or gene expression. Together these approaches will identify new genes that are involved in the growth regulation of hematopoietic cells and better our understanding of the growth and differentiation of myeloid cells.
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