Vertebrate cells have adapted ancient signal transduction cascades that respond to changes in the environment. Ligand binding of receptor tyrosine kinases potently stimulates mitogen activated protein kinase (MAPK), whereas TNFalpha, IL-1, and UV-irradiation activates the stress- activated protein kinases (SAPK, also called JNK). The MAPK cascade consists of three kinases (raf/MEK/MAPK) that ultimately phosphorylate and activate transcription factors such as the ets-related TCF. The SAPK cascade leads to the phosphorylation and activation of c-jun. Recently, we isolated a cDNA, SEK1, from a murine erythroleukemia cell library that functions as an immediate upstream activator of SAPK. We also demonstrated that the kinase MEKK is an activator of SEK1, thus establishing the SAPK cascade (MEKK/SEK/SAPK). A third vertebrate cascade is stress-activated, and includes the kinase XMEK3 (also called MKK3), which activates a MAP kinase homolog called p38. We propose to determine which of these cascades are activated and/or required for normal erythropoiesis. Erythroid cells lines (i.e. MEL and Ba/F3) and normal erythroid progenitors will be transfected with vectors that express tagged MAPK, SAPK, or p38. Following extracellular stimulation to promote cell proliferation or differentiation (i.e. erythropoietin or DMSO), specific MAP kinase activity will be evaluated. Expression of dominant negative mutants of the specific activators (MEK, SEK, and XMEK3) will be used to define the requirement of each cascade for normal erythroid development and prevention of cell death. In yeast, ste20-like kinases function as upstream activators of MAPK cascades. We have isolated cDNAs encoding several distinct mouse homologs of ste20 that are expressed in erythroid cells and are likely to be upstream activators of vertebrate MAP kinase cascades. By constructing and expressing mutants of the ste20 kinase members, we plan to determine their function in erythroid cells. An analysis of the cascades activated by these ste20 family members and the upstream signals that lead to their activation will be undertaken. These studies will provide a better understanding of the role of the MAPK cascades in cell proliferation, death, or differentiation of hematopoietic cells. The pharmacologic manipulation of these pathways in vivo will be useful to treat anemia of chronic disease, sickle cell anemia, thalassemia and leukemia.
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