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s t e r o i d s 7 3 ( 2 0 0 8 ) 853–858

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embrane association of estrogen receptor � and �

nfluences 17�-estradiol-mediated cancer cell proliferation

aria Marinoa,∗, Paolo Ascenzia,b

Department of Biology, University “Roma Tre”, Viale G. Marconi 446, I-00146 Roma, ItalyInterdepartmental Laboratory for Electron Microscopy, University ‘Roma Tre’, Via della Vasca Navale 79, I-00146 Roma, Italy

r t i c l e i n f o

ublished on line 14 December 2007

eywords:

strogen receptor �

strogen receptor �

strogen receptor palmitoylation

7�-Estradiol

a b s t r a c t

S-Palmitoylation is a widespread post-translational modification of integral and/or periph-

eral proteins occurring in all eukaryotic cells. The family of S-palmitoylated proteins is large

and diverse and recently, estrogen receptor isoforms (ER� and ER�) belonging to the nuclear

receptor superfamily have been added to the palmitoylproteoma. S-Palmitoylation allows

ER� and ER� localization at the plasma membrane, where they associate with caveolin-1.

Upon 17�-estradiol (E2) stimulation, ER� dissociates from caveolin-1 allowing the activation

of rapid signals relevant for cell proliferation. In contrast to ER�, E2 increases ER� associa-

apid signals

ancer cell proliferation

tion with caveolin-1 and activates p38 kinase and the downstream pro-apoptotic cascade

(i.e., caspase-3 activation and PARP cleavage). These data highlight the physiological role of

palmitoylation in modulating the ER� and ER� localization at the plasma membrane and

the regulation of different E2-induced non-genomic functions relevant for controlling cell

proliferation.

tates membrane association. Palmitoylation promotes protein

. Introduction

atty acid addition is a widespread post-translational mod-fication of integral and/or peripheral proteins occurring inll eukaryotic cells [1]. Proteins can be modified by fatty acidddition in several different ways. Co-translational modifica-ion with myristate (C14:0) (Fig. 1) occurs at the N-terminally residue after its appearance following the cleavage of

he initiator amino acid Met, this modification is knowns N-myristoylation. Palmitoylation refers to the linkage ofalmitate (C16:0) to Cys residues (Fig. 1). When palmitate

s added to the N-terminal Cys residue, a thioester inter-ediate occurs followed by a rearrangement to a more

table amide linkage, this modification is known as N-

almitoylation. More frequently, palmitate is attached to aon-N-terminal Cys residue through a thioester linkage, lead-

ng to S-palmitoylation [1,2].

∗ Corresponding author. Tel.: +39 06 55176345; fax: +39 06 55176321.E-mail address: m.marino@uniroma3.it (M. Marino).

039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved.oi:10.1016/j.steroids.2007.12.003

© 2007 Elsevier Inc. All rights reserved.

N-Palmitoylation results, like other lipid addition (e.g.,N-myristoylation and S-prenylation), as a permanent mod-ification, whereas S-palmitoylated proteins could encounterdifferent destinies. Proteins can be permanently modified withpalmitate or palmitate can be hydrolysed from the protein,as this is an essential step in the protein degradative process[1,2]. However, for many proteins, cycles of palmitoylation andde-palmitoylation occur throughout their lifetime rendering,thus, palmitoylation unique among lipid modifications in thatit is the only reversible lipid modification of proteins [1–4].

Palmitoylation exerts diverse effects on the protein struc-ture and function. Indeed, the attachment of this long chainfatty acid increases protein hydrophobicity and thereby facili-

targeting to membrane microdomains (e.g., lipid rafts andcaveolae) that are enriched in cholesterol and saturated fattyacid chains, allowing the lipid molecules to pack tightly

854 s t e r o i d s 7 3 ( 2 0 0 8 ) 853–858

Fig. 1 – Fatty acylation of proteins. S-Palmitoylcysteine(upper panel) is formed by the reversible addition of the16-carbon lipid, palmitate, to the thiol group of a Cysresidue. When this addition occurs at the N-terminal Cysresidue, there is a chemical rearrangement that results inthe attachment of the palmitate through a stable amidelinkage, a process known as N-palmitoylation (middlepanel). The 14-carbon lipid, myristate, can also be attachedto N-terminal Gly residues (N-myristoylation) (bottom

Fig. 2 – Amino acid sequence alignment of human ER� andER�. GenBank accession codes are ER�, NP 000116; andER�, NP 001428. The amino acid sequence alignment wasobtained with Clustal [50]. The diamond, the circle, and thehorizontal line indicate the palmitoylable Cys residue, the

panel).

together and form a “liquid ordered” phase. This enhancesprotein/protein and protein/lipid interactions which appearrelevant for efficient signal transduction. Palmitoylation alsoinfluences intracellular protein trafficking and protein activity[3–7].

Palmitoylation is a widespread modification found almostexclusively in membrane proteins. The family of pro-teins modified with thioester-linked palmitate is large anddiverse. It includes transmembrane-spanning proteins andsoluble proteins such as ion channels, neurotransmitterreceptors, heterotrimeric guanine nucleotide-binding protein(G-protein)-coupled receptors, and integrins. In addition, hor-mone receptor signalling relies on protein palmitoylation atmany levels, including palmitoylated co-receptors, Src familykinases, and adaptor or scaffolding proteins. The localizationand signalling capacities of Ras and G-proteins are modulatedby dynamic protein palmitoylation [1,5].

Recently, estrogen receptor (ER) isoforms belonging to thenuclear receptor superfamily have been added to the palmi-toylproteoma [8]. Here, we review the role of ER� and ER�

S-palmitoylation on 17�-estradiol (E2)-induced non-genomicfunctions committed to the control of cell proliferation.

2. ERs are palmitoylated proteins

Although dozens of palmitoylproteins have been described,few strong predictors of whether a protein will be a sub-

hydrophilic positively charged Lys residue, and thehydrophobic patch, respectively. For details, see [11].

strate for palmitoylation have emerged. In fact, palmitoylatedproteins have no clear consensus sequence(s) for this post-translational modification although protein palmitoylation isthought to be an enzymatic reaction, mediated by a palmi-toyltransferase(s) (PAT). The common denominator for mostpalmitoylated proteins is a membrane targeting sequenceencompassing the target Cys, it consists of positively chargedresidue(s) and lipid anchors or transmembrane domains [9].Based on multiple sequence alignment, we postulated thatthe amino acid sequence encompassing the solvent-exposedCys447 and Cys399 residues present in the ligand-bindingdomain (LBD) of ER� and ER�, respectively, are the uniquewhich follow this requirement. In fact, an hydrophobic patch(Leu zipper-like) and an hydrophilic positively charged Lysresidue are present close to the S-palmitoylable Cys447and Cys339 residues of ER� and ER�, respectively (Fig. 2)[10]. Remarkably, this sequence appears to occur also inother hormone receptors, representing a general rule for S-palmitoylation of this class of proteins [11].

The experimental evidence that ERs are palmitoylated pro-teins derived later from experiments performed with labeledpalmitate [12,13]. In cervix carcinoma cells (HeLa cells) tran-siently transfected with ER� or ER� expression vectors, bothreceptors undergo S-palmitoylation. Remarkably, palmitateincorporation in both ERs is not a spontaneous reaction, beingcatalyzed by PAT. In fact, low levels of S-palmitoylated ER�

or ER� were found in cells pre-treated with the PAT inhibitor2-bromo-hexadecanoic acid (2-Br) [12,13]. The half time forPAT catalyzed incorporation of palmitate in ER�- and ER�-transfected HeLa cells is ∼7 min and ∼60 min, respectively.This suggests that ER� is a poorer substrate than ER� for PAT[13].

3. ER� and ER� palmitoylation is aligand-dependent process

Palmitoylation must be considered more than a simple way ofmembrane association of otherwise soluble proteins. In fact,cycles of palmitoylation and de-palmitoylation affect protein

activation allowing their movement(s) within membrane sub-domains. De-palmitoylation of the endothelial nitric oxidesynthase increases in response to cell treatment with theactivator bradykinin [14]. Moreover, de-palmitoylation has a

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ubtle effect on membrane distribution of G-proteins affectingheir partitioning within membrane sub-domains [1].

Ligand (e.g., E2) binding modulates ER� and ER� palmi-oylation [13,15]. E2 binding to ER� and ER� decreases theR palmitoylation rate (t1/2 ∼ 30 min) and induces structuralodification(s) impairing PAT recognition (i.e., ER palmitoyla-

ion) [13,15]. Note that, upon E2 binding, the Cys447 residuef ER� is not yet able to react with iodoacetic acid, a Cys-eacting reagent [16]. This is in agreement with the solventnaccessibility of Cys447 in the ER�- and ER�-ligand adductssee [17]). Furthermore, E2 affects the localization of the sta-ly integrated membrane targeted ER (MT-ER) in breast cancerell lines [18]. MT-ER is myristoylated at the N-terminus andalmitoylated at the C-terminus. In the absence of E2, MT-ER is

ocalized primarily to the cell membrane with some cytoplas-ic localization. After E2 treatment, the localization of MT-ER

t the plasma membrane decreases showing a punctuate pat-ern with localization in the cytoplasm [18].

ER� and ER� bind a wide variety of compounds withemarkable structural and functional diversity. Partial-gonists (e.g., flavonoids, 4-hydroxytamoxifen, and raloxifene)nd antagonists (e.g., ICI 164,384) bind to LBD with a geom-try reminiscent that of natural agonists (i.e., E2). However,heir large side chain substituents are not accommodatedithin the confines of the binding cavity, this results inultiple ligand-dependent conformationals of ER� and ER�

17] which, in turn, affects ER� and ER� palmitoylationtatus. Remarkably, the flavonoid naringenin (Nar, 5,7,4′-rihydroxyflavanone), a weak ER� antagonist [19], induces aaster ER� de-palmitoylation than E2 (t1/2 ∼ 8 min and ∼30 min,espectively) (Marino, unpublished results). These data raisehe possibility that different ligands, which induce differentonformational changes in ER� and ER�, could differentlyffect receptor palmitoylation status.

. Palmitoylation impacts on ER� and ER�ssociation to membrane proteins

urrent evidence indicates that the small population of ER�

nd ER� localized at the plasma membrane exists withinaveolar rafts, interacting with specific membrane proteins10,20–22]. ER� can further associate with specialized proteinsncluding the modulator of the non-genomic activity of estro-en receptor (MNAR) (also named proline-, glutamic acid-, andeucine-rich protein-1; PELP1), Shc, epidermal growth factorEGF) and insulin-like growth factor-1 (IGF-1) receptors, andtriatin [23–29]. Shc and striatin have been reported to inter-ct with the A/B domain of ER� [30], while MNAR acts as andapter coupling ER to Src [28].

Although E2-induced ER� and ER� de-palmitoylation dis-lays similar kinetics (t1/2 ∼ 30 min), the E2 stimulationromotes different ER/protein recognition. Palmitoylationnables ER� to reside at the plasma membrane and to inter-ct with caveolin-1. Upon E2 binding, ER� undergoes slowe-palmitoylation and dissociates from caveolin-1, facilitating

R� movement to other membrane micro-domains [15]. Thus,R� could be re-located by docking to other partner proteinsi.e., Shc/IGF-1 receptor and Src/p85) [31,32]. E2-stimulationf HeLa cells transfected with the un-palmitoylable ER�

8 ) 853–858 855

Cys447Ala mutant does not increase ER� association toMNAR or c-Src, impairing ER� ability to activate downstreamkinases (Marino, personal communication). Correspondingly,the rapid Nar-induced ER� de-palmitoylation (t1/2 ∼ 8 min)induces the dissociation of the ER�-caveolin-1 complex andimpairs receptor association with adaptors and/or signalingproteins (i.e., MNAR and c-Src) (Marino, personal communica-tion).

The intact A/B domain and the Tyr537 residue present inthe E domain are both required for ER� interaction with Src inthe MNAR-ER�-Src complex [23,28]. No association betweenER�, MNAR, and Src was observed before and after E2 stimula-tion [13]. On the other hand, E2 induces ER� de-palmitoylationand increases ER� level and its association with caveolin-1 [13].As a whole these data raise the intriguing possibility that theshort A/B domain of ER� could facilitate the E2-induced asso-ciation between ER� and caveolin-1, impairing its associationwith MNAR and Src. However, E2 increases the associationof ER� to a cytosolic kinase, p38 kinase, a member of themitogen-activated protein kinase (MAPK) family, inducing p38activation [13].

5. Palmitoylation is necessary forE2-induced effects

The mechanism(s) by which E2 exerts proliferative effectsis assumed to be exclusively mediated by rapid membrane-starting actions [33–39]. E2 treatment of mammary-derivedMCF-7 cells triggers the association of ER� to Src and p85, theregulatory subunit of PI3K, leading to DNA synthesis [36]. Inhepatoma cell line (HepG2), multiple and parallel membrane-starting pathways are rapidly activated by E2 binding to ER�

[33,38,39]. The blockade of phospholipase C/protein kinase C(PLC/PKC), extracellular regulated kinase/MAPK (ERK/MAPK),and phosphoinositide 3 kinase/AKT (PI3K/AKT) pathwayscompletely prevents the E2-induced DNA synthesis [38,39].ERK/MAPK and PI3K/AKT pathways, rapidly activated by theER�–E2 complex, also have a critical role in E2 action as asurvival agent. In fact, these pathways enhance the expres-sion of the anti-apoptotic protein Bcl-2, block the activation ofthe p38 kinase, reduce the pro-apoptotic caspase-3 activation,and promote G1-to-S phase transition via the enhancementof the cyclin D1 expression [12,38,39]. S-Palmitoylation is aprerequisite of these E2-induced rapid events. In fact, theun-palmitoylable ER� Cys447Ala mutant or PAT inhibitiondo not support the E2-induced proliferative signaling viathe ERK/MAPK and PI3K/AKT pathways in ER� containingHeLa and HepG2 cells [15]. Similar results have been recentlyreported in other tumor cell lines [40].

What is the contribution of ER� to E2-induced cell pro-liferation? ER� appears to act as a dominant regulator ofE2 signaling. When co-expressed with ER�, ER� causes areduction of the ER�-mediated transcriptional activation andthe repression of ER�-mediated effects, including cell pro-liferation [41,42]. Consistent with this notion, E2 increases

proliferation of ER�-expressing MCF-7 cells [42]. On the otherhand, ER� inhibits the E2-induced proliferation in trans-fected MCF-7 cells and prevents tumor formation in a mousexenograft model in response to E2 [43]. These findings seem to

856 s t e r o i d s 7 3 ( 2 0 0 8 ) 853–858

Fig. 3 – Schematic model illustrating the localization at and maintenance to the plasma membrane of ER� and ER�. Understeady-state condition (upper panel, left), ER� is palmitoylated (black triangle) and localized at the plasma membraneassociated to caveolin-1 (cav1). Upon E2 stimulation (upper panel, right) ER� is de-palmitoylated allowing its association tosignaling proteins to trigger cell functions. Palmitoylated ER� (bottom panel, left) resides at the plasma membraneassociated to caveolin-1 (cav1) and p38. Upon E2 stimulation (bottom panel, right) ER� undergoes de-palmitoylation, thisincreases ER� association to cav1, p38, and membrane. This association impairs the ER� re-allocation at the plasmamembrane and its association with other signaling proteins. However, ER� association to p38 increases the kinase activity

; G-pdylin

triggering cell functions. ERK, extracellular regulated kinaseof non-genomic activity of ER; p85�, �-subunit of phosphati

be linked to the ER� repressive effect on ER�-induced indirectgene transcription by binding to other transcription factors(e.g., AP-1 and Sp1) [42]. However, in ER� transfected HeLacells E2 has been reported to rapidly induce a persistentmembrane-initiated activation of p38 without any interfer-ence on survival proliferative pathways [12]. ER-dependentcaspase-3 activation and poly(ADP-ribose)polymerase (PARP)cleavage are some of the downstream events triggered byE2-induced p38 activation in ER� containing colon cancercells (DLD1) [13]. Accordingly, E2 induces the cleavage of thecaspase-3 proform, resulting in the production of the proteaseactive subunit. In turn, the active enzyme catalyzes the inac-tivation of PARP [13]. These findings indicate that the plasmamembrane localized ER� is important for anti-proliferativeeffects of E2 [12].

Furthermore, the rapid and prolonged E2-induced p38 acti-vation is also fundamental both for the rapid increase of ER�

mRNA translation and for the slow ER� gene transcription[44]. The consequence is the E2-induced increase of ER� lev-els, in DLD1 cells which further increases the E2 protectiveeffect against the cancer cell growth [13,45]. Indeed, p38 inhi-bition impairs the increase of the ER� levels and prevents theE2-induced DLD-1 cell number reduction [44]. These resultsreinforce the interpretation of a role for ER� levels as a nega-tive regulator of colon tumor growth [13,44 and literature citedtherein].

Even though the membrane localization of ER� and ER�

and the associated non-genomic actions are an area of activeresearch, nuclear actions of membrane ERs has not receivedmuch attention [26,46]. Note that ER� and ER� palmitoylationis required for E2-induced gene transcription [15]. In fact, in

rot, guanine nucleotide-binding protein; MNAR, modulatorositol 3 kinase. For details, see text.

HeLa cells co-transfected with the estrogen responsive ele-ment (ERE) containing complement 3 (pC3) promoter or withthe ERE-devoid cyclin D1 (pD1) promoter constructs and ER�

expression vector, the E2 treatment induces the increase ofpC3 and pD1 promoter activities, respectively [13]. On theother hand, in ER�-transfected HeLa cells, E2 induces theincrease of the pC3 promoter activity only [13]. Notably, thepre-treatment of ER� or ER�-transfected HeLa cells with 2-Br reduces the E2-mediated pC3 promoter activity [13]. Asexpected, ER� palmitoylation has a powerful effect on the E2-induced cyclin D1 promoter activity which is totally impairedby treatment with 2-Br, whereas ER� was unable to mediatecyclin D1 promoter activity, both in the presence or absenceof 2-Br [13]. Thus, E2-induced rapid signaling could affect thenuclear activities of ER� and ER�. Both rapid and genomicpathways synergize each other to provide plasticity for cellresponse to sex steroids.

6. Conclusion and perspectives

The mechanism(s) underling the mitogenic role played by E2in different target tissues are now better understood based onthe studies reported by different laboratories, including ourown. These studies designate a strict relationship between theE2-induced non-genomic functions and cell proliferation. Thenon-genomic functions are thought to require plasma mem-

brane ER� and ER� which are dependent on S-palmitoylation.

S-Palmitoylation localizes ER� and ER� at the plasmamembrane directing several and distinct E2 effects on cell pro-liferation (Fig. 3). The molecular mechanism(s) which rapidly

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ollows E2 entry in ER�-containing cells further sustainshe tumor suppressor function played by this ER isoform.lthough ER� undergoes palmitoylation, the outcome effects

n cell physiology are opposite to those reported for ER�. Thus,he expression of each ER isoform and/or their co-expressionn the cells could account for the different E2-dependent mod-lation of cell proliferation (Fig. 3).

Considering that all classes of steroid hormones possesseceptors able to generate rapid responses [18,47,48], a newrea of investigation focusing the role played by chemicalodification(s) on steroid receptor activity and localization

ould be open. Intriguingly, several members of the steroidormone receptor family, displaying an amino acid sequenceighly homologous to that encompassing the Cys447 andys399 residues of ER� and ER�, respectively, could undergo-palmitoylation [11]. Recently, mutational analysis of thisequence present in the LBD of the progesterone receptornd of the androgen receptor confirmed that S-palmitoylationediates rapid signalling [49]. These studies will aid the

evelopment of specific agonist and antagonists with well-haracterized effects able to modify discrete E2-inducedellular functions.

cknowledgments

he authors wish to thank past and present members ofheir laboratories who contributed to the ideas presented herehrough data and discussions. This work was supported byrants from the Ministry of University and Research of ItalyPRIN-COFIN 2006 to M.M.).

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