Estrogen receptor-α hinge-region lysines 302 and 303 regulate receptor degradation by the proteasome

Nicholas B. Berry, Meiyun Fan, Kenneth Nephew

Research output: Contribution to journalArticle

62 Citations (Scopus)

Abstract

Cellular levels of estrogen receptor-α (ERα) protein are regulated primarily by the ubiquitin-proteasome pathway. Dynamic interactions between ERα and the protein degradation machinery facilitate the down-regulation process by targeting receptor lysine residues for polyubiquitination. To date, the lysines that control receptor degradation have not been identified. Two receptor lysines, K302 and K303, located in the hinge-region of ERα, serve multiple regulatory functions, and we examined whether these might also regulate receptor polyubiquitination, turnover, and receptor-protein interactions. We used ERα-negative breast cancer C4-12 cells to generate cells stably expressing wild-type (wt)ERα or ERα with lysine-to-alanine substitutions at K302 and K303 (ERα-AA). In the unliganded state, ERα-AA displayed rapid polyubiquitination and enhanced basal turnover, as compared with wtERα, due to its elevated association with the ubiquitin ligase carboxy terminus of Hsc70-interacting protein (CHIP) and the proteasome-associated cochaperone Bag1. Treatment of C4-12 cells with either 17β-estradiol (E2) or the pure antiestrogen ICI 182,780 (ICI) induced rapid degradation of wtERα via the ubiquitin-proteasome pathway; however, in the presence of these ligands, ERα-AA was less efficiently degraded. Furthermore, ERα-AA was resistant to ICI-induced polyubiquitination, suggesting that these lysines are polyubiquitinated in response to the antiestrogen and demonstrate a novel role for these two lysines in the mechanism of action of ICI-induced receptor down-regulation. The reduced stability of ERα-AA in the unliganded state and the increased stability of ERα-AA in the liganded state were concordant with reporter gene assays demonstrating that ERα-AA has lower basal activity but higher E2 inducibility than wtERα. These data provide the first evidence that K302/303 protect ERα from basal degradation and are necessary for efficient E2- and ICI-induced turnover in breast cancer cells.

Original languageEnglish
Pages (from-to)1535-1551
Number of pages17
JournalMolecular Endocrinology
Volume22
Issue number7
DOIs
StatePublished - Jul 2008

Fingerprint

Proteasome Endopeptidase Complex
Estrogen Receptors
Lysine
Ubiquitin
Estrogen Receptor Modulators
Down-Regulation
HSC70 Heat-Shock Proteins
Breast Neoplasms
Ligases
Reporter Genes
Alanine
Proteolysis
Estradiol
Ligands

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

Cite this

Estrogen receptor-α hinge-region lysines 302 and 303 regulate receptor degradation by the proteasome. / Berry, Nicholas B.; Fan, Meiyun; Nephew, Kenneth.

In: Molecular Endocrinology, Vol. 22, No. 7, 07.2008, p. 1535-1551.

Research output: Contribution to journalArticle

@article{5ca958f77d354551abb3611686370c49,
title = "Estrogen receptor-α hinge-region lysines 302 and 303 regulate receptor degradation by the proteasome",
abstract = "Cellular levels of estrogen receptor-α (ERα) protein are regulated primarily by the ubiquitin-proteasome pathway. Dynamic interactions between ERα and the protein degradation machinery facilitate the down-regulation process by targeting receptor lysine residues for polyubiquitination. To date, the lysines that control receptor degradation have not been identified. Two receptor lysines, K302 and K303, located in the hinge-region of ERα, serve multiple regulatory functions, and we examined whether these might also regulate receptor polyubiquitination, turnover, and receptor-protein interactions. We used ERα-negative breast cancer C4-12 cells to generate cells stably expressing wild-type (wt)ERα or ERα with lysine-to-alanine substitutions at K302 and K303 (ERα-AA). In the unliganded state, ERα-AA displayed rapid polyubiquitination and enhanced basal turnover, as compared with wtERα, due to its elevated association with the ubiquitin ligase carboxy terminus of Hsc70-interacting protein (CHIP) and the proteasome-associated cochaperone Bag1. Treatment of C4-12 cells with either 17β-estradiol (E2) or the pure antiestrogen ICI 182,780 (ICI) induced rapid degradation of wtERα via the ubiquitin-proteasome pathway; however, in the presence of these ligands, ERα-AA was less efficiently degraded. Furthermore, ERα-AA was resistant to ICI-induced polyubiquitination, suggesting that these lysines are polyubiquitinated in response to the antiestrogen and demonstrate a novel role for these two lysines in the mechanism of action of ICI-induced receptor down-regulation. The reduced stability of ERα-AA in the unliganded state and the increased stability of ERα-AA in the liganded state were concordant with reporter gene assays demonstrating that ERα-AA has lower basal activity but higher E2 inducibility than wtERα. These data provide the first evidence that K302/303 protect ERα from basal degradation and are necessary for efficient E2- and ICI-induced turnover in breast cancer cells.",
author = "Berry, {Nicholas B.} and Meiyun Fan and Kenneth Nephew",
year = "2008",
month = "7",
doi = "10.1210/me.2007-0449",
language = "English",
volume = "22",
pages = "1535--1551",
journal = "Molecular Endocrinology",
issn = "0888-8809",
publisher = "The Endocrine Society",
number = "7",

}

TY - JOUR

T1 - Estrogen receptor-α hinge-region lysines 302 and 303 regulate receptor degradation by the proteasome

AU - Berry, Nicholas B.

AU - Fan, Meiyun

AU - Nephew, Kenneth

PY - 2008/7

Y1 - 2008/7

N2 - Cellular levels of estrogen receptor-α (ERα) protein are regulated primarily by the ubiquitin-proteasome pathway. Dynamic interactions between ERα and the protein degradation machinery facilitate the down-regulation process by targeting receptor lysine residues for polyubiquitination. To date, the lysines that control receptor degradation have not been identified. Two receptor lysines, K302 and K303, located in the hinge-region of ERα, serve multiple regulatory functions, and we examined whether these might also regulate receptor polyubiquitination, turnover, and receptor-protein interactions. We used ERα-negative breast cancer C4-12 cells to generate cells stably expressing wild-type (wt)ERα or ERα with lysine-to-alanine substitutions at K302 and K303 (ERα-AA). In the unliganded state, ERα-AA displayed rapid polyubiquitination and enhanced basal turnover, as compared with wtERα, due to its elevated association with the ubiquitin ligase carboxy terminus of Hsc70-interacting protein (CHIP) and the proteasome-associated cochaperone Bag1. Treatment of C4-12 cells with either 17β-estradiol (E2) or the pure antiestrogen ICI 182,780 (ICI) induced rapid degradation of wtERα via the ubiquitin-proteasome pathway; however, in the presence of these ligands, ERα-AA was less efficiently degraded. Furthermore, ERα-AA was resistant to ICI-induced polyubiquitination, suggesting that these lysines are polyubiquitinated in response to the antiestrogen and demonstrate a novel role for these two lysines in the mechanism of action of ICI-induced receptor down-regulation. The reduced stability of ERα-AA in the unliganded state and the increased stability of ERα-AA in the liganded state were concordant with reporter gene assays demonstrating that ERα-AA has lower basal activity but higher E2 inducibility than wtERα. These data provide the first evidence that K302/303 protect ERα from basal degradation and are necessary for efficient E2- and ICI-induced turnover in breast cancer cells.

AB - Cellular levels of estrogen receptor-α (ERα) protein are regulated primarily by the ubiquitin-proteasome pathway. Dynamic interactions between ERα and the protein degradation machinery facilitate the down-regulation process by targeting receptor lysine residues for polyubiquitination. To date, the lysines that control receptor degradation have not been identified. Two receptor lysines, K302 and K303, located in the hinge-region of ERα, serve multiple regulatory functions, and we examined whether these might also regulate receptor polyubiquitination, turnover, and receptor-protein interactions. We used ERα-negative breast cancer C4-12 cells to generate cells stably expressing wild-type (wt)ERα or ERα with lysine-to-alanine substitutions at K302 and K303 (ERα-AA). In the unliganded state, ERα-AA displayed rapid polyubiquitination and enhanced basal turnover, as compared with wtERα, due to its elevated association with the ubiquitin ligase carboxy terminus of Hsc70-interacting protein (CHIP) and the proteasome-associated cochaperone Bag1. Treatment of C4-12 cells with either 17β-estradiol (E2) or the pure antiestrogen ICI 182,780 (ICI) induced rapid degradation of wtERα via the ubiquitin-proteasome pathway; however, in the presence of these ligands, ERα-AA was less efficiently degraded. Furthermore, ERα-AA was resistant to ICI-induced polyubiquitination, suggesting that these lysines are polyubiquitinated in response to the antiestrogen and demonstrate a novel role for these two lysines in the mechanism of action of ICI-induced receptor down-regulation. The reduced stability of ERα-AA in the unliganded state and the increased stability of ERα-AA in the liganded state were concordant with reporter gene assays demonstrating that ERα-AA has lower basal activity but higher E2 inducibility than wtERα. These data provide the first evidence that K302/303 protect ERα from basal degradation and are necessary for efficient E2- and ICI-induced turnover in breast cancer cells.

UR - http://www.scopus.com/inward/record.url?scp=46349108800&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=46349108800&partnerID=8YFLogxK

U2 - 10.1210/me.2007-0449

DO - 10.1210/me.2007-0449

M3 - Article

C2 - 18388150

AN - SCOPUS:46349108800

VL - 22

SP - 1535

EP - 1551

JO - Molecular Endocrinology

JF - Molecular Endocrinology

SN - 0888-8809

IS - 7

ER -