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The role of the estrogen receptor (ER) in breast cancer has been suggested both by its ability to stimulate cell proliferation as well as the observation that ER is expressed ir 60% of primary breast tumor biopsies but only in 6% of normal breast tissue. Drugs which interfere with ER activity such as the antiestrogen Tamoxifen have been only partially successful in the treatment of breast cancers emphasizing the need for new targets as well as new pharmacological agents against these targets. The observation that antiprogestins such as RU486 could function as antiestrogens suggested that the progesterone receptor (PR) could be a potential target in the treatment of breast cancers. The goal of this project was to elucidate the mechanism of hPR-A transdominant repression by characterizing potential hPR-A-interacting partners which are necessary for ER transcriptional activation. We anticipate that new pharmacological agents against these targets could be used to treat breast cancers which currently escape endocrine intervention.

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.

HRPF1/Nedd4 was originally identified in our laboratory as a potentiator of progesterone-receptor transcriptional activity, and subsequently demonstrated to similarly modulate p53-dependent transcription. As a hect' E3 ubiquitin ligase, hRPFl/Nedd4's domain structure suggests that it is able to target substrate proteins for ubiquitination. We have been interested in identifying nuclear substrates of hRPF1/Nedd4's ubiquitination activity with a view to understanding the mechanism by which hRPFl/Nedd4 is able to modulate such transcriptional events. We present compelling evidence that hPRTB, a novel proline-rich protein which colocalizes with splicing machinery in nuclear speckles, is a bona fide' nuclear substrate of the WW hect E3 ubiquitin ligase, hRPFl/Nedd4. In addition to providing the first description of a nuclear substrate of mammalian Nedd4, these observations underscore the potential for regulation of splicing proteins by the ubiquitination. Lastly, with the identification of a leucine-rich rev-like nuclear export sequence within hRPFI/Nedd4, we propose that nuclear import/export is an important component of the regulation between the primarily cytoplasmic E3 enzyme, hRPFl/Nedd4 and its nuclear substrate, hPRTB. Thus, we have firmly established a role for hRPFl/Nedd4 within the nucleus, and identified a substrate protein which may explain in part our observed effects upon activated transcription., Original contains color plates: All DTIC reproductions will be in black and white.