Estrogen receptor alpha transcriptional activity is regulated by distinct conformational states that are the result of ligand binding. Phage display was used to identify peptides that interact specifically with either estradiol- or tamoxifen-activated estrogen receptor alpha. When these peptides were coexpressed with estrogen receptor alpha in cells, they functioned as ligand-specific antagonists, indicating that estradiol-agonist and tamoxifen-partial agonist activities do not occur by the same mechanism. The ability to regulate estrogen receptor alpha transcriptional activity by targeting sites outside of the ligand-binding pocket has implications for the development of estrogen receptor alpha antagonists for the treatment of tamoxifen-refractory breast cancers.

Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 proteins. We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation of SEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW domain-containing hect (WW-hect) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.Ubiquitin-dependent proteolysis involves the covalent ligation of ubiquitin to substrate proteins, which are then recognized and degraded by the 26S proteasome. While many of the components involved in catalyzing protein ubiquitination have been identified and characterized biochemically, we are only beginning to understand how the system specifically recognizes appropriate substrates. At least three classes of activities, known as E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin-protein ligase) enzymes, cooperate in catalyzing protein ubiquitination (34). The enzymatic mechanisms and functions of the E1 and E2 proteins have been well characterized. In contrast, the E3 enzymes are a diverse and less-well-characterized group of activities, and many lines of evidence indicate that E3 activities play a major role in determining the substrate specificity of the ubiquitination pathway (14,28,34).The hect (homologous to E6-AP carboxyl terminus) domain defines a family of E3 proteins that were discovered through the characterization of human E6-AP (17). The interaction of E6-AP with the E6 protein of the cervical cancer-associated human papillomavirus types causes E6-AP to associate with and ubiquitinate p53, suggesting that E6 functions in promoting cellular immortalization by, at least in part, stimulating the destruction of this important tumor suppressor protein (16). The hect E3 molecular masses range from 92 to over 500 kDa, with the hect domain comprising the approximately 350 carboxyl-terminal amino acids (17, 34). Exactly five hect E3s are encoded by the Saccharomyces cerevisiae genome, and over 30 have been identified so far in mammalian species. An obligatory intermediate in the ubiquitination reactions catalyzed by hect E3s is a ubiquitin-thioester formed between the thiol group of an absolutely conserved cysteine within the hect domain and the terminal carboxyl group of ubiquitin (33). E3 becomes "charged" with ubiquitin vi...

Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302, and 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.14. Subject Terms (keywords previously assigned to proposal abstract or terms which apply to this award) Reportable Outcomes 8-9Conclusions 10References 11Appendices 12-43 IntroductionOur longstanding interest in cellular proteins and pathways which influence the transcriptional activity of the progesterone receptor led to the identification of yeast RSP5and the its human homolog hRPFl/Nedd4 as modulators of progesterone receptordependent transcription [1]. Given the homology of hRSP5 and hRPFl/Nedd4 with the 'hect' family of E3 ubiquitin ligases, a class of enzymes implicated in the covalent attachment of ubiquitin to substrate molecules [2], we proposed initially that the ubiquitin-dependent degradation of a substate of hRPFl/Nedd4 might explain these observed effects upon PR-dependent and p53 dependent transcription. However, work from this and other laboratories [3], have demonstrated that the effects of hRPFl/Nedd4 upon transcription by steroid receptors is not dependent upon the ubiquitin ligase activity of hRPFl/Nedd4. Nonetheless, we have remained interested in the identification and characterization of novel substrates of the ubiquitination ac...