Protein Interactions Regulating Pituitary Signaling

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DESCRIPTION (provided by applicant): Transcriptional activity is dependent not only on the binding of transcription factors to cis-acting elements, but also on the ability of those factors to recruit a complex hierarchy of proteins to stabilize the basal transcriptional machinery. Oncogenic Ras signaling to the rat prolactin (rPRL) promoter is dependent on the interaction of the proto-oncoprotein c-Ets-1, a member of the Ets family of transcription factors, with the pituitary-specific transcription factor, Pit-1, at a composite Ets/Pit-1 DNA binding element (RRE) in the rPRL promoter. This signaling cascade utilizes a tripartite code comprised of Pit-1 and Ets-1 binding to the unique RRE element, the physical interaction of the Ets-1 Rill TAD with the Pit-1 homeodomain, and Ras-stimulated MAPkinase phosphorylation of threonine 82 in Ets-1. Together this code creates a structural platform for the binding of a transcriptional regulatory complex through which Ras can mediate activation of the rPRL promoter. Recent studies suggest that the phosphorylation of Pit-1 can modulate both Ras signaling and Ets-1/Pit-1 binding. Thus, I hypothesize that the Pit-1/Ets-1 complex binding to the RRE mediates the oncogenic Ras response of the rPRL promoter by recruiting specific transcription regulatory proteins to a unique interaction face. The goals of this proposal are to determine the mechanism by which phosphorylation of Pit-1 inhibits Ras-activation of the rPRL promoter, and to use liquid chromatography/mass spectrometry methods to identify the protein components that are recruited by Ras signaling to the Ets-1/Pit-1 complex. Therefore, these studies are important to my career development as they will allow me to gain expertise in the cutting edge field of proteomics and protein structure-function relationships, which I can then utilize to establish my career as an independent investigator.