The long term goal of this project is to characterize the mechanism of nuclear export of a protein as a means of transcriptional regulation. This project aims to define the region of the yeast transcriptional activator, Pho4, that mediates nuclear export of Pho4 and to identify proteins involved in cdk phosphorylation-dependent nuclear export of Pho4. In our model, rapid export of Pho4 from the nucleus requires a nuclear export signal (NES) that is masked when Pho4 is un-phosphorylated. Phosphorylation of pho4 unmasks or creates the NES or results in binding of a protein containing an NES. Nuclear export of Pho4 is mediated by an association between Pho4 and a protein or proteins involved in nuclear export. Phosphorylation of Pho4 promotes association with a nuclear export protein(s). The first part of our model predicts that Pho4 contains a nuclear export signal or that Pho4 binds a protein containing an NES. The second part of our model predicts that the phosphorylation status of Pho4 dictates its interaction with a nuclear export carrier protein. These models lead to the specific aims, respectively, of this proposed research. The first specific aim is to determine the region(s) of Pho4 required for nuclear export. Additionally, a novel in vivo export assay will be employed to identify and characterize the region on Pho4 that confers nuclear export. Additionally, a noel in vivo export assay will be employed to identify and characterize the region on Pho4 that confers nuclear export. The second specific aim is to identify nuclear export proteins that bind differentially to phosphorylated versus un-phosphorylated Pho4. Biochemical methodologies will be employed to achieve this specific aim. These studies bear on health issues, specifically cancer, in that loss of control of appropriate subcellular localization of oncoproteins can result in unregulated cell growth and in development of cancer.
