Regulation of Estrogen Receptor α N-Terminus Conformation and Function by Peptidyl Prolyl Isomerase Pin1

Abstract

Estrogen receptor alpha (ER␣), a key driver of growth in the majority of breast cancers, contains an unstructured transactivation domain (AF1) in its N terminus that is a convergence point for growth factor and hormonal activation. This domain is controlled by phosphorylation, but how phosphorylation impacts AF1 structure and function is unclear. We found that serine 118 (S118) phosphorylation of the ER␣ AF1 region in response to estrogen (agonist), tamoxifen (antagonist), and growth factors results in recruitment of the peptidyl prolyl cis/trans isomerase Pin1. Phosphorylation of S118 is critical for Pin1 binding, and mutation of S118 to alanine prevents this association. Importantly, Pin1 isomerizes the serine118-proline119 bond from a cis to trans isomer, with a concomitant increase in AF1 transcriptional activity. Pin1 overexpression promotes ligand-independent and tamoxifeninducible activity of ER␣ and growth of tamoxifen-resistant breast cancer cells. Pin1 expression correlates with proliferation in ER␣-positive rat mammary tumors. These results establish phosphorylation-coupled proline isomerization as a mechanism modulating AF1 functional activity and provide insight into the role of a conformational switch in the functional regulation of the intrinsically disordered transactivation domain of ER␣.E strogen receptor alpha (ER␣), a member of the nuclear receptor superfamily of transcription factors, mediates the actions of estrogen in normal physiology and disease (17). ER␣ is expressed in the normal mammary gland and in 70% of human breast cancers and is a key driver of breast cell proliferation (16,26,83). Directed overexpression of ER␣ in the mammary gland is sufficient to induce hyperplasia, and blockade of ER␣ activity by hormonal therapies (aromatase inhibitors, tamoxifen, and fulvestrant) reduces recurrence and improves clinical outcomes of ER␣-positive breast cancer patients (19,22). Two activation functions mediate the transcriptional activity of ER␣, a C-terminal liganddependent AF2 and an N-terminal ligand-independent AF1 (89). Regulation of ER␣ activity via the C-terminal AF2 has been wellcharacterized through biochemical and crystallographic studies and forms the basis for our understanding of hormonal therapy for breast cancer (10,34,84). In the canonical activation pathway, ligand binding initiates C-terminal structural rearrangements that facilitate downstream events, including dimerization, DNA binding, and coregulator interactions, ultimately engaging the basal transcriptional machinery to regulate gene expression. However, ER␣ can also be activated by growth factors and kinases, which phosphorylate the receptor N terminus and other domains to regulate transcription in the absence of direct ligand engagement (for reviews, see references 27, 47, 75, 94, and 95). In contrast to the C-terminal AF2 domain, biochemical and structural mechanisms that control N-terminal AF1 remain poorly understood (48,91).Multiple challenges have hindered molecular dissection of AF1 regulation. First, AF1 resid...

Prolyl isomerase Pin1 in cancer

“…This isomerization stabilizes ERα protein by blocking ERα interaction with the E3 ligase, E6AP, and thus inhibiting the E6AP-mediated ubiquitylation and degradation of ERα. In addition, Pin1 and ERα levels are positively correlated in human breast carcinoma specimens [100,101].…”

Peptidylprolyl Isomerase Pin1 Directly Enhances the DNA Binding Functions of Estrogen Receptor α

“…Although ID domains lack a specific identifying motif or secondary structure, they play key roles in post-translational modifications, protein-protein interactions, and allosteric control (19 -21). By virtue of their unstructured characteristics, ID domains are difficult to study, yet AF1 has been shown to provide an interaction surface for coregulator proteins (10,(22)(23)(24)(25). An understanding of the interactions in ID domains could provide a mechanistic window to better understand the allosteric role of unstructured domains in transcriptional signaling (26).…”

“…Phosphorylation-dependent isomerization by Pin1 affects diverse cellular processes, such as protein-protein interactions, subcellular localization, dephosphorylation, and transcription (30). We previously identified cis-trans isomerization of the Ser(P) 118 -Pro 119 bond in AF1 by Pin1 as a novel post-translational modification regulating ER␣ conformation, protein stability, and transcriptional function (25,31). Ser 118 in the AF1 region of ER␣ is a major site of phosphorylation by proline-directed kinases in response to estrogen, growth factors, and anti-estrogens, such as tamoxifen (11)(12)(13)(14)(15).…”

Minute Time Scale Prolyl Isomerization Governs Antibody Recognition of an Intrinsically Disordered Immunodominant Epitope

“…1A) by standard ELISAs. We found no reactivity for either the complete E7 N-terminal (E7(1-40)) or C-terminal (E7(40 -98)) domains or for the fragments E7 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and E7 (16 -40) spanning the conserved regions CR1 and CR2 from the N-terminal domain (Fig. 1B).…”

“…Cis-trans isomerization thus provides a molecular switch that works in the minute time scale and can expand the conformational repertoire of proteins. Recent evidence shows that proline isomerization in both disordered and globular domains can regulate the kinetics of processes such as phage infection (15), antibody folding (16), estrogen receptor signaling (17), self-inhibition of a signaling protein (18), and aggregation dynamics of the intrinsically disordered Tau protein (19). It has been proposed that some antibodies do recognize the less populated cis isomer of the peptide bond (20,21).…”