Yeast RSP5 and Its Human Homolog hRPF1 Potentiate Hormone-Dependent Activation of Transcription by Human Progesterone and Glucocorticoid Receptors

Abstract

We have developed a system in Saccharomyces cerevisiae in which agonist-dependent transcriptional activity of the human progesterone receptor (hPR) is elevated to the point that it compromises cell growth. Screens for suppressors of this phenotype led to the demonstration that RSP5 is involved in hPR transactivation. Expression of RSP5 in yeast cells potentiated hPR and human glucocorticoid receptor (hGR) transcriptional activity and increased the efficacy of weak agonists of these receptors. Remarkably, expression of this yeast protein in mammalian cells had a similar effect on PR and GR transcriptional activity. Importantly, a human homolog of RSP5, hRPF1, functioned identically in mammalian cells. Previously, it has been demonstrated that RSP5 overexpression in yeast cells suppressed mutations within SPT3, a protein which interacts with the TATAbox-binding protein (TBP), suggesting that RSP5 and SPT3 operate in the same regulatory pathway. In support of this observation, we have shown that SPT3 enhances the activity of RSP5 on GR and PR when tested in yeast or mammalian cells. We conclude from these experiments that the regulatory pathways in which RSP5 and SPT3 operate in yeast cells are conserved in higher eukaryotes. Additionally, since SPT3 has been shown to contact yeast TBP directly and is the likely homolog of human TBP-associated factor TAF II 18, we propose that RSP5/hRPF1 and SPT3 establish a functional link between activated PR and GR and the general transcription apparatus.Steroid hormone receptors are ligand-inducible transcription factors belonging to a superfamily of nuclear proteins (16, 17) which also includes receptors for vitamins and thyroid hormone as well as orphan receptors for which no ligands have yet been identified. Within this superfamily, the steroid receptors represent a subgroup of proteins which have considerable sequence similarity and are closely related mechanistically. In the absence of hormone, the steroid receptors are transcriptionally inert and reside in a large macromolecular docking complex consisting of heat shock protein 90 (hsp90), hsp70, hsp56, and other, less well characterized proteins. Upon interaction with their cognate ligands, they undergo a conformational change, displacing the associated heat shock proteins and spontaneously forming receptor homodimers. Subsequently, the homodimeric receptor complex can interact with high affinity with specific DNA response elements located within the regulatory regions of target genes. It is the ability to cooperate with additional cell specific transcription factors, or other trans-acting factors located on the same promoter, which determines the biological activity of the DNA-bound receptor (44, 51). The precise mechanism by which steroid receptors alter transcription by RNA polymerase II is unknown. However, it has been shown that transcriptionally active progesterone receptor (PR) can potentiate target gene transcription, in a reconstituted system in vitro, by stabilizing the preinitiation complex (34). Additionally, ...

EDD, the Human Hyperplastic Discs Protein, Has a Role in Progesterone Receptor Coactivation and Potential Involvement in DNA Damage Response

“…Recently, the HECT domain proteins yeast Rsp5 and its human orthologue hRPF1 (30) and E6-AP (31) were identified as having PR coactivation activity. The mechanism for this activity is unknown, but, like SRC-1, E6-AP interacts directly with PR.…”

The Co-repressor Hairless Protects RORα Orphan Nuclear Receptor from Proteasome-mediated Degradation

“…A number of these co-factors have been identified as Ubproteasome or Ub-like pathway enzymes, with a role in both proteasomal degradation and transcriptional activation. The E3 Ub-ligases RSP5/RPF1 and the E6-associated protein and the ATPase subunit of the 26 S proteasome SUG1 (suppressor of Gal4) all participate in nuclear receptor transactivation while simultaneously mediating their degradation (17)(18)(19)(20)(21)(22). Recently, p300/CREB-binding protein has been shown to mediate the polyubiquitination of the p53 transcription factor through its intrinsic Ub-ligase activity in addition to its function as a transcriptional co-activator (23).…”

Coactivator AIB1 links estrogen receptor transcriptional activity and stability

“…Ubiquitination of proteins involves the action of three classes of enzymes, namely the ubiquitin-activating enzyme (UBA), the ubiquitin-conjugating enzyme (UBC), and the ubiquitin ligase (19). Interestingly, several steroid receptor-interacting proteins have been identified as components of the ubiquitin-proteasome degradation system, including SUG1͞Trip1, an ATPase subunit of the 26S proteasome complex (20); uba3, the catalytic subunit of the NEDD8-activating enzyme (21); UBC9 (22); and RSP5͞RPF1 and E6-AP, both ubiquitin ligases (23,24). Not only do these proteins interact with the steroid receptors, they also modulate the receptor activity, suggesting that mechanisms targeting receptor for degradation are linked to those regulating receptor activation.…”