Ubiquitin-interaction motifs of RAP80 are critical in its regulation of estrogen receptor α

Abstract

In this study, we demonstrate that receptor-associated protein 80 (RAP80) interacts with estrogen receptor alpha (ERα) in an agonist-dependent manner. The interaction is specific for ERα as ERβ and several other nuclear receptors tested did not interact with RAP80. Interaction between RAP80 and ERα was supported by mammalian two-hybrid, GST pull-down, and co-immunoprecipitation analyses. The hinge/ligand-binding domain of ERα is sufficient for interaction with RAP80. RAP80 overexpression reduces ERα polyubiquitination, increases the level of ERα protein, and enhances ERα-mediated transactivation. Knockdown of endogenous RAP80 expression by small-interfering RNA (siRNA) reduced ERα protein level and the E2-dependent induction of pS2. In this study, we also demonstrate that RAP80 contains two functional ubiquitin-interaction motifs (UIMs) that are able to bind ubiquitin and to direct monoubiquitination of RAP80. Deletion of these UIMs does not affect the ability of RAP80 to interact with ERα, but eliminates the effects of RAP80 on ERα polyubiquitination, the level of ERα protein, and ERα-mediated transcription. These data indicate that the UIMs in RAP80 are critical for the function of RAP80. Our study identifies ERα as a new RAP80-interacting protein and suggests that RAP80 may be an important modulator of ERα activity.

A Regulatory Loop Composed of RAP80-HDM2-p53 Provides RAP80-enhanced p53 Degradation by HDM2 in Response to DNA Damage

“…As part of a genome wide search for similar motifs, 5 we found that the region around p53-RE5 as well as p53-RE3 and -4 is surrounded by several putative estrogen response elements. Interestingly, RAP80 has been reported to interact directly with ER␣ and to positively modulate ER␣-mediated transactivation (30). Whether there is any functional link between the interactions of RAP80 with ER␣ and p53 requires further study.…”

“…This was stimulated by the observation that RAP80 interacts with estrogen receptor ␣ (ER␣) affecting its stability/ activity (30) and that HDM2, which is regulated by p53, also determines p53 stability. We first examined whether RAP80 and p53 interact.…”

“…Moreover, GST-p53 fusion protein effectively pulled down in vitro translated 35 S-labeled RAP80, whereas GST alone did not bind RAP80. ER␣, previously shown to bind p53 (30), was used as a positive control (Fig. 2B).…”

“…RAP80 Promotes HDM2-mediated Ubiquitination of p53-Given that RAP80 affects the stability of another protein, ER␣ (30) and that p53 ubiquitination and stability is controlled by HDM2, the major E3 ubiquitin ligase of p53, we examined whether RAP80 was associated with HDM2. Co-immunoprecipitation analysis using U2OS cells transfected with FLAG-RAP80 and Myc-HDM2 expression plasmids demonstrated that RAP80 was able to pull down HDM2 (Fig.…”

NBA1, a new player in the Brca1 A complex, is required for DNA damage resistance and checkpoint control

“…Abraxas associates with Rap80 through the N-terminal domain . Rap80 contains two UIM (ubiquitin-interacting motif) domains that have been shown to bind K63-linked ubiquitin chains (Sobhian et al 2007;Yan et al 2007), and are required for the localization of the BRCA1 A complex to ionizing radiation (IR)-induced foci (IRIFs) Sobhian et al 2007;). Abraxas binds BRCC36 through the coiled-coil domains on both proteins .…”