Many growth factors affect the decision of a cell to divide or differentiate by binding to a specific receptor tyrosine kinase (RTK) in the cell membrane. Activation of RTKs has been found to lead, through a series of common intermediates, to activation of MAP (mitogen-activated protein) kinases. The specificity of effect of different RTKs may be due, at least in part, to the diversity of transcription factors modulated by these MAP kinases. Very few transcription factors have been shown to be direct targets of an activated RTK pathway; even less is known about athe genes that these activated transcription factors regulate. Our goal is to identify and characterize the transcription factor target of a genetically well described RTK. The pathway activated by the torso (tor) RTK, which functions to establish fate at the two poles of the early Drosophila embryo, is uniquely suited for studies to identify a novel transcription factor modulated by an RTK. This pathway has been studied in detail genetically and most of its components identified; staged Drosophila embryos can be collected in quantities suitable for protein purification. We have identified a small (<14 bp) tor Response Element (tor-RE) in the promoter of the tailless (tll) gene (the key gene regulated by the tor pathway). The tor-RE is unusual in that it acts as a repressor element; its function is inactivated in cells in which the tor RTK is active, allowing transcription of tll. To identify the protein (the tor-REB) binding to the tor-RE, and characterize its function in the tor RTK pathway and possibly in other pathways, we propose to do the following; 1.) further determine the presumptive tor-RE by site-directed mutagenesis and transformation analysis, 2.) purify the transcription factor (the tor-REB) that binds to the tor-RE, 3.) clone and sequence the gene encoding the tor- REB, 4.) characterize the interaction between the tor-REB and components of the tor system (particularly mAP kinase), 5.) characterize tor-REB RNA and protein expression during development in Drosophila, 6.) carry out genetic studies on the tor-REB gene to confirm that it functions in the terminal pathway, and to assess its role in different pathways and developmental processes. 7.) Identify the mammalian homolog of the tor-REB and test whether it is modulated by RTK activated pathways in vertebrates. These studies are expected to provide novel insights into the molecular mechanisms by which RTK activated pathways affect cell differentiation and proliferation, and may identify genetic targets for diagnostic screening in cancer.
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