Analysis of the Mechanism of Action of RPF1: Potentiator of Progesterone Receptor and p53-Dependent Transcriptional Activity

摘要

The E3 ubiquitin ligase, hRPFl/Nedd4 has been previously been described as a potentiator of progesterone receptor (PR)- and p53-dependent transcriptional activity. Given the observation that hRPFl/Nedd4 shares amino acid sequence homology with the hect' family of E3 ligases, we proposed to identify substrates of hRPFl/Nedd4 ubiquitination activity with the goal of elucidating the mechanism for these observed PR and p53 transcriptional effects. Using a yeast two-hybrid approach, we have identified hPRTB (proline-rich protein, brain expressed) as a nuclear protein which interacts with and is a ubiquitination substrate of hRPFl/Nedd4 in vitro and in cultured cells. Furthermore, with the identification of a rev-like nuclear export sequence in HRPFl/Nedd4, we suggest that nuclear import/export of distinct hect' family members will contribute to the regulation of enzyme/substrate specificity within a cell. It is now apparent the potentiative effects of hRPFl/Nedd4 upon PR- and p53-dependent transcription are independent of ubiquitination activity of this enzyme. Nonetheless, the localization of hPRTB, a ubiquitination substrate of HRPFl/Nedd4, in splicing factor-rich nuclear speckles remains suggestive of a potential link between ubiquitination and the general transcriptional machinery.