17β-Estradiol-induced cell proliferation requires estrogen receptor (ER) α monoubiquitination

Cellular Signalling 23 (2011) 1128–1135

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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g

17β-Estradiol-induced cell proliferation requires estrogen receptor (ER) α monoubiquitination Piergiorgio La Rosa, Valeria Pesiri, Maria Marino, Filippo Acconcia ⁎ Department of Biology, University Roma Tre, Viale Guglielmo Marconi, 446, I-00146, Rome, Italy

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Article history: Received 4 February 2011 Accepted 20 February 2011 Available online 26 February 2011 Keywords: 17β-Estradiol Estrogen receptor Monoubiquitination PI3K/AKT Signaling

a b s t r a c t Protein monoubiquitination (monoUbq) (i.e., the attachment of one single ubiquitin to the substrate) is a nonproteolytic reversible modification that controls protein functions. Among other proteins, the estrogen receptor α (ERα), which mediates the pleiotropic effects of the cognate hormone 17β-estradiol (E2), is a monoubiquitinated protein. Although it has been demonstrated that E2 rapidly reduces ERα monoUbq in breast cancer cells, the impact of monoUbq in the regulation of the ERα activities is poorly appreciated. Here, we show that mutation of the ERα monoUbq sites prevents the E2-induced ERα phosphorylation in the serine residue 118 (S118), reduces ERα transcriptional activity, and precludes the ERα-mediated extranuclear activation of signaling pathways (i.e., AKT activation) thus impeding the E2-induced cyclin D1 promoter activation and consequently cell proliferation. In addition, the interference with ERα monoUbq deregulates E2-induced association of ERα to the insulin like growth factor receptor (IGF-1-R). Altogether these data demonstrate an inherent role for monoUbq in ERα signaling and point to the physiological function of ERα monoUbq in the regulation of E2-induced cell proliferation. © 2011 Elsevier Inc. All rights reserved.

1. Introduction The cellular effects of the sex hormone 17β-estradiol (E2) are mediated by two estrogen receptor isoforms (i.e., ERα and ERβ) through different molecular mechanisms strictly dependent on the receptor intracellular localization. Indeed ERα nuclear localization is required for E2-induced gene transcription (i.e., nuclear mechanism). This mechanism dictates that E2 binding to the receptor induces ERα dimerization, its phosphorylation on the serine residue (S) 118, and its translocation to the nucleus where the transcription of estrogen responsive element (ERE)-containing genes occurs. On the other hand, ERα palmitoylation, which is required for receptor localization at the plasma membrane, allows E2 to trigger also the rapid (i.e., seconds to minutes) activation of several signaling kinase cascades (i.e., extranuclear mechanism). Through this mechanism E2 modulates, among others, the rapid ERα-dependent activation of the PI3K pathway allowing AKT activation, cyclin D1 transcription (an EREdevoid gene), and cell cycle progression [1–11]. Mounting evidence indicate that E2 can also modulate ERα activities (i.e., nuclear and extranuclear mechanisms) by either inducing or even reducing the amount of the post-translational-modified receptor [3,4,11–13]. Indeed, several E2-induced signaling cascades including the PI3K/AKT pathway control S118 phosphorylation [6,11,14]. On the other hand, ERα palmitoylation is reduced by E2

⁎ Corresponding author. Tel.: + 39 0657336320; fax: + 39 0657336321. E-mail address: [email protected] (F. Acconcia). 0898-6568/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2011.02.006

allowing receptor membrane relocalization and its association to different other signaling proteins [e.g., insulin like growth factor receptor (IGF-1-R)] [3,4]. Besides these and other post-translational modifications, ERα is a monoubiquitinated protein [16–18]. Ubiquitination, a post-translational modification involved in the modulation of a plethora of physiological processes, occurs through a cascade of complex enzymatic reactions which leads to the activation of the E3 ubiquitin (Ub)-ligase that covalently attaches Ub to the target protein. Several Ub modifications exist: polyubiquitination consists in the attachment of Ub chain(s) on the substrate while monoubiquitination (monoUbq) happens when Ub is appended to the target protein through one single lysine (K) residue. Remarkably, each Ub modification has a different signaling outcome. In particular, monoUbq of proteins represents a signaling intermediate for the transduction of diverse extracellular stimuli. Indeed, over the past ten years it has become clear that monoUbq works as nondegradative signal in many receptor-based transduction pathways (e.g., receptor tyrosine kinase, RTKs) and is required for regulating different physiological processes including cell proliferation [15]. At the present the possibility that ERα monoUbq could be a signaling modification also in the E2:ERα network required for the transduction of the E2-induced effects still remains to be understood. Our research group has recently demonstrated that the endogenous basal ERα monoUbq is rapidly reduced by E2 and the monoubiquitinated ERα localizes within the nucleus of breast cancer cells [18]. These observations sustain the hypothesis that the endogenous ERα monoUbq is involved in the regulation of ERα nuclear and extranuclear activities. Therefore, we investigated the impact of ERα monoUbq in E2-evoked

P. La Rosa et al. / Cellular Signalling 23 (2011) 1128–1135

ERα nuclear (i.e., ERE-based transcription) activity as well as extranuclear (e.g., PI3K/AKT pathway activation) activities important for the proliferative effects of E2. Our results indicate that ERα monoUbq is required for the E2:ERαmediated events necessary for cell proliferation and show, for the first time, that monoUbq represents a signaling modification in the intricate network activated by the E2:ERα complex. 2. Materials and methods 2.1. Cell culture and reagents Wild type and stably transfected HEK293 cells were grown as previously described [18]. Specific antibodies against flag epitope (M2) and vinculin (Sigma, St. Louis, MO, USA); and IGF-1-R (C-20) and anti-ubiquitin (P4D1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. All other antibodies were purchased by Cell Signalling Technology Inc. (Beverly, MA, USA). All the products were from Sigma-Aldrich (St. Louis, MO, USA). Analytical or reagent grade products, without further purification, were used. 2.2. Plasmids The reporter plasmid 3× ERE TATA, protein complement 3 (pC3) and the pXP2-D1-2966 reporter plasmid (pD1) have been described elsewhere [3]. The pcDNA flag-ERα and was obtained by subcloning the ERα ORF from the pSG5-HE0 [3] into the pcDNA flag 3.1 C. Sitedirected mutagenesis of the ERα K302 and K303 residues was performed by using the QuikChange kit (Stratagene, La Jolla, CA) and the following oligonucleotide: 5′ATGATCAAACGCTCTAGGAGGAACAGCCTGGCC-3′ (bold underlined nucleotides differ from the wt ERα ORF). Plasmid was then sequenced to verify the introduction of the desired mutations. 2.3. Cellular and biochemical assays Before any cellular and biochemical assay, cells were grown in 1% DCC medium for 24 h and then stimulated with E2 (10−10 M) at the indicated time points. Cells were lysed in YY buffer (50 mM HEPES at pH 7.5, 10% glycerol, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA) plus protease and phosphatase inhibitors. Proteins were transferred on a nitrocellulose membrane (GE Healthcare Fairfield, CT, USA). After blocking (1 h at R.T. in 5% non-fat dry milk TBS-T solution), filters were incubated with the appropriate primary antibody o.n. at 4 °C, followed by three washes of 10 min each in TBS-T and then incubated with the anti-mouse or anti-rabbit horseradish peroxidaseconjugated secondary antibody diluted in TBS-T for 60 min at RT. After incubation with the secondary antibody, the filter was washed three times in TBS-T (5 min each) and the bound secondary antibody was revealed using the ECL (Enhanced Chemiluminescence) method (GE Healthcare Fairfield, CT, USA). Immunoprecipitation and growth curves were performed as previously reported [3,18]. 2.4. Stable transfection HEK293 cells were transfected using calcium chloride. Briefly, a total amount of 10 μg of DNA was mixed together with CaCl2 (0.25 M) in Hepes buffer (HBS, Hepes 25 mM, KCl 10 mM, Dextrose 12 mM, NaCl 280 mM Na2HPO4 × 7H2O 1.5 mM). Sixteen hours after trasfection medium was changed and the selection antibiotic was added. In particular, HEK293 cells stably expressing ERα were generated by using G418 (400 μg/ml), as previously reported [19]. For the pcDNAflag expressing cells three clones, which display the same growth rate, were selected on the basis of flag expression. For the wt ERα expressing HEK293 cells, three individual clones were selected for the same ability to respond to E2. For the ERα K302R, K303R three

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individual clones were selected on the basis of the wt ERα expression levels. Experiments are shown for one of each HEK293 clone. 2.5. Transient transfection and luciferase assay Stable HEK293 cells were grown to 70% confluence and then transfected using Lipofectamine reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Three hours after transfection, the medium was changed, and 24 h after the cells were 24 h serum starved and then stimulated with E2 for 24 h. The cell lysis procedure as well as the subsequent measurement of luciferase gene expression was performed using the luciferase kit according to the manufacturer's instructions with a PerkinElmer Life and Analytical Sciences (Bad Wildbad, Germany) luminometer as previously described [3]. 2.6. GST pull-down assays GST fusion proteins were expressed, purified and performed as described [18]. All experiments were normalized by running 1/20 of the pull-down on an SDSPAGE gel. Proteins were detected by Comassie Brilliant Blue staining. 2.7. Confocal microscopy analysis HEK293 cells were stained with anti-flag (1:10,000) antibody or with phalloidin. In detail, cells were grown on 30-mm glass gelatinecoated coverslips in 6-well plates (5 × 105 cells/well). Cells were then fixed with paraformaldehyde (4%) for 1 h and permeabilized with Triton-X 100 (0.1%) for 5 min. After the permeabilization process, cells were incubated with bovine serum albumin (BSA) (2%) for 1 h and then stained with the anti-flag antibody for 1 h at room temperature in the presence of phalloidin. After that cells were rinsed three times in PBS for 5 min and incubated with Alexa Fluor 488 ® donkey antimouse secondary antibody (Invitrogen, Carlsbad, CA, USA) (1:400). Following extensive washes coverslips were mounted and confocal analysis was performed using LCS (Leica Microsystems, Heidelberg, Germany). 2.8. RNA isolation and quantitative RT-PCR analysis (qRT-PCR) The sequences for gene-specific forward and reverse primers were designed using the OligoPerfect Designer software program (Invitrogen, Carlsbad, CA, USA). The following primers were used: for human pS2 5′-CATCGACGTCCCTCCAGAAGAG-3′ (forward) and 5′-CTCTGGGACTAATCACCGTGCTG-3′ (reverse), for human GAPDH 5′-CGAGATCCCTCCAAAATCAA-3′ (forward) and 5′-TGTGGTCATGAGTCCTTCCA3′ (reverse). Total RNA was extracted from cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. To determine pS2 gene expression levels, cDNA synthesis and qPCR were performed using the GoTaq 2-step RT-qPCR system (Promega, Madison, MA, USA) in an ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Each sample was tested in triplicate, the experiment repeated twice and the gene expression normalized for GAPDH mRNA levels. 2.9. Statistical analysis A statistical analysis was performed using the ANOVA test with the InStat.3 software system (GraphPad Software Inc., San Diego, CA). In all analyses P values less than 0.01 were considered significant but for densitometric analyses P was b0.05. Data are means of three independent experiments ±S.D.

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3. Results 3.1. The K302 and K303 residues are the ERα endogenous monoUbq sites in HEK293 cells Experiment in vitro demonstrated that residues K302 and K303 represent the ERα monoUbq sites [16]. To begin unravel the role of the endogenous ERα monoUbq in ERα signaling, the ERα in vitro monoUbq sites (i.e., K302 and K303 residues) [16] were mutated to arginine (R) [i.e., ERα K302R and K303R (KKRR)] and stable clones expressing either the empty vector (pc), the wt ERα or the KKRR mutant receptor were generated in the ERα-devoid human embryonic kidney (HEK293) cells (Figs. 1A and 2A). Initially, we verified if ERα is monoubiquitinated also in HEK293 cells stably expressing the wt ERα. Proteins extracted from wt ERα HEK293 cells were subjected to a pull-down assay by using as baits both the S75 and the mutant S75 Y25F, A58G (YFAG), which displays a reduced ability to bind to ubiquitinated species [18]. Fig. 1B shows that the binding of S75 to ERα was prevented by the S75 Y25F, A58G mutation in HEK293 cells. Notably, the S75 Y25F, A58G pulled down a reduced amount of ubiquitinated species with respect to the wt S75 and equal amounts of all fusion proteins were used (Fig. 1B). These data further strengthen the notion that a fraction of the endogenous ERα is monoubiquitinated [18]. Next, the stable HEK293 cells expressing the wt ERα were characterized for their ability to respond to E2. As shown in Fig. 1, E2 was able to trigger the ERE-based promoter activity (Fig. 1C), a time-dependent ERα phosphorylation on the S residue 118 (Fig. 1C, inset) as well as the rapid AKT activation (Fig. 1D). In addition, E2 induced the proliferation of the wt ERα expressing HEK293 cells (Fig. 1E). Notably, as expected for an ERα-devoid cell line, the HEK293 cells stably expressing the empty vector (pc) did not respond to E2 stimulation (Fig. 1C–E). These data demonstrate that stable insertion

of the ERα in HEK293 cells determines an experimental model, which responds to E2 like other endogenously expressing ERα cell lines (e.g., MCF-7 cells) and further indicate that this model system is useful to evaluate the impact of specific ERα mutations in E2:ERα signaling without the interference of the endogenous ERα. Therefore, in order to follow an unbiased approach in analyzing the impact of the KKRR mutation in E2:ERα signaling, HEK293 cells were stably transfected with the KKRR mutant ERα and selected both to express the same amount of receptor (Fig. 2A) and to display the same receptor intracellular localization (i.e., nuclear, cytosolic, and membrane-tethered) (Fig. 2B) as the wt ERα expressing HEK293 clone. Next, the ERα monoUbq status was studied in the context of the KKRR mutation. Proteins extracted from exponentially growing wt and KKRR mutant ERα HEK293 clones were subjected to a pull-down assay using the S75 [18]. Moreover, the HEK293 clone expressing the empty vector (pc) was also included in the experiment as a negative control. Fig. 2C shows that the ability of S75 to pull down the ERα was reduced to 50% when the receptor was mutated in the in vitro monoUbq sites (i.e., KKRR) (Fig. 2C). Notably, no ERα association to GST was detected, equal amounts of both GST and GST-S75 were used (Fig. 2C) and the S75 efficiently bound ubiquitinated proteins (data not shown). These data demonstrate that mutation of K302 and K303 residues reduces the endogenous ERα monoUbq in HEK293 cells. 3.2. Reduction in ERα monoUbq decreases ERE-containing gene transcription The presence of monoubiquitinated ERα into the nucleus [18] prompted us to determine whether the reduction in ERα monoUbq could affect ERα transcriptional activity. This analysis was undertaken in stable HEK293 cells transiently transfected with two well known gene reporter constructs, which contain the ERE sequence either in an artificial promoter (i.e., 3× ERE-TATA, pERE) or in a natural promoter

Fig. 1. Stable wt ERα expressing HEK293 cells respond to E2. (A) Western blot analysis was performed in HEK293 cells stably expressing the pcDNA (pc) or the pcDNA flag-ERα (wt) for the presence of the transgene. (B) Pull-down assay was done using GST as negative control and the GSTRUZ:MIU fragment of the Rabex5 (S75) or the GST-RUZ:MIU fragment of the Rabex5 mutated in Y25F, A58G (YFAG) immobilized on GSH beads as baits. Fusion proteins were incubated with proteins extracted from HEK293 cells stably expressing the pcDNA flag-ERα wt and then analyzed by either an anti-flag or an anti-ubiquitin immunoblot. 1/20 of each pull down was run into different gels and either probed with antiubiquitin antibody or stained with Blue Comassie (Com.) for normalization. (C) Luciferase assay detection on the HEK293 stable clones expressing pcDNA (pc), and the pcDNA flagERα (wt) transfected with the 3× ERE TATA reporter plasmid and then treated 24 h with E2. (C, inset) Time course analysis of ERα S118 phosphorylation in HEK293 cells stably expressing the pcDNA flag-ERα in the presence of E2. (D) Western blot analysis of AKT phosphorylation was performed in HEK293 cells stably expressing the pcDNA (pc) and the pcDNA flag-ERα (wt) in the presence of E2. (E) Number of the HEK293 stable cells expressing pcDNA flag (pc), and pcDNA flag-ERα (wt) was assayed both in the presence or in the absence of E2 (48 h). * indicates significant differences with respect to the relative C sample (P b 0.01).

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Fig. 2. Endogenous monoubiquitination of the ERα Lys302 and Lys302 residues. (A) Western blot analysis was performed in HEK293 cells stably expressing the pcDNA (pc), the pcDNA flag-ERα (wt) or the pcDNA flag-ERα K302R, K303R (KKRR) mutant. Graph shows quantification of the flag ERα. (B) Confocal microcopy analysis of the HEK293 stable clones expressing pcDNA flag (pc), pcDNA flag-ERα (wt), and the pcDNA flag-ERα K302R, K303R (KKRR) mutant kept in growing conditions and stained for flag. Arrows indicate membrane-ERα. (C) Pull-down assay was done using GST as negative control and the GSTRUZ:MIU fragment of the Rabex5 (S75) immobilized on GSH beads as baits. Fusion proteins were incubated with proteins extracted from HEK293 cells stably expressing the pcDNA (pc), the pcDNA flag-ERα (wt) and the pcDNA flag-ERα K302R, K303R (KKRR) mutant kept in growing conditions and then analyzed by immunoblot. Graph shows quantification of the monoubiquitinated fraction of the ERα. 1/20 of each pull down was run into a different gel and stained with Blue Comassie (Com.) for normalization. * indicates significant differences with respect to the relative wt ERα sample (P b 0.05).

(i.e., promoter of protein complement 3, pC3) [3]. As shown in Fig. 3A, E2 was able to trigger the artificial promoter activation (pERE) both in wt and KKRR receptor expressing HEK293 cells. The pERE promoter activation was significantly reduced in cells containing the KKRR mutant receptor with respect to the wt ones both in the presence and

in the absence of E2; however, the extent of the E2-dependent pERE promoter activation was greatly enhanced (i.e., 17 vs 3 folds) in the KKRR endowed HEK293 clone. On the contrary, E2 was able to activate the natural promoter (pC3) in wt but not in the KKRR mutant receptor expressing HEK293 cells while both in the presence and in the

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Fig. 3. Reduction in ERα monoUbq decreases ERE-containing gene transcription. Luciferase assay detection on the HEK293 stable clones expressing the pcDNA flag-ERα (wt) and the pcDNA flag-ERα K302R, K303R (KKRR) mutant transfected with the 3× ERE TATA reporter plasmid (A) and the pC3 reporter plasmid (B) and then treated 24 h with E2. (C) RT-qPCR analysis of pS2 mRNA expression normalized on the GAPDH mRNA expression in the HEK293 stable clones expressing the pcDNA flag-ERα (wt) and the pcDNA flag-ERα K302R, K303R (KKRR) mutant treated with E2 for 24 h. * indicates significant differences with respect to the relative C sample (P b 0.01). # indicates significant differences with respect to the relative wt ERα C sample (P b 0.01). ° indicates significant differences with respect to the relative wt ERα E2-treated sample (P b 0.01).

absence of E2 pC3 promoter activation was significantly decreased in the KKRR HEK293 cells (Fig. 3B). In order to analyze the ERα ERE-based transcriptional activity in a more physiologically relevant context, we investigated the expression of the endogenous E2-responsive ERE-containing gene presenelin 2 (pS2) in HEK293 stable clones. Real-time qPCR analysis revealed that E2 induces a 12 fold and a 0.45 fold (i.e., 45%) increase in the amount of pS2 mRNA levels in the presence of wt and KKRR receptor, respectively (Fig. 3C). However, the pS2 mRNA cellular content was significantly lower in the mutant receptor containing HEK293 cells when compared with the wt counterparts (Fig. 3C). Taken together, these data indicate that the KKRR mutation determines a functional receptor, which is still able to respond to E2 and demonstrate that the reduction in ERα monoUbq results in an overall lower ERE-based ERα transcriptional activity.

3.3. Reduction in ERα monoUbq prevents ERα and AKT phosphorylation It is well known that full ERα transcriptional activity towards the ERE-containing genes is acquired in response to E2 when the receptor becomes rapidly phosphorylated on the S residue 118 (S118) [14]. Indeed, lack of ERα S118 phosphorylation results in a significant reduction in E2-induced gene transcription [14]. As a consequence, the reduction in the ERα transcriptional activity observed when the receptor is mutated in the monoubiquitination sites (i.e., K302 and K303 residues) (Fig. 3) may be due to a defect in the E2-dependent signaling modulation of ERα S118 phosphorylation. Therefore, the ERα S118 phophorylation status was evaluated in the wt and KKRR mutant receptor expressing HEK293 cells. As shown in Fig. 4A, the E2-induced rapid (30 min) ERα S118 phosphorylation observed in the presence of wt ERα was not detected in the presence of KKRR mutant ERα. Moreover, since several lines of evidence indicate that the E2-induced ERα S118 phosphorylation is regulated by several extranuclear ERα-activated kinases [6], our data further suggest that reduction in ERα monoUbq may influence also the E2evoked activation of the ERα-mediated extranuclear signaling kinase pathways. Among others, the extranuclear E2-activated PI3K/AKT pathway is emerging as a key signaling modulator of the E2-induced S118 phosphorylation [6,11,14]. Therefore, the E2-induced AKT activation was next evaluated in both the wt ERα and the KKRR mutant receptor HEK293 clones. Time course analysis revealed that E2 induces a rapid increase in AKT phosphorylation in the wt ERα expressing clone while the hormone fails to trigger it in the KKRR mutant receptor expressing cells (Fig. 4B). Surprisingly, the basal AKT

activation was also reduced in the presence of the KKRR mutant receptor with respect to the wt ERα (Fig. 4B). It is now accepted that the activation of the ERα extranuclear signaling arises from pool of the receptor that localizes to the plasma membrane [3] and requires, at least in part, the E2-induced physical interaction of ERα to several signaling proteins including growth factor receptors such as the IGF-1-R [10]. Since our data indicate that the mutation of the monoUbq sites does not influence the ERα localization to the plasma membrane (Fig. 2B) but prevents rapid AKT activation (Fig. 4B), we next evaluated the involvement of receptor monoUbq in the ERα:IGF-1-R association process. Co-immunoprecipitation analysis confirms that E2 triggers the rapid interaction between wt ERα and the IGF-1-R [10] also in stable HEK293 cells; however, the constitutive KKRR mutant ERα association with the IGF1-R was reduced upon E2 administration (Fig. 4C), thus indicating that the reduction in ERα monoUbq alters the basal and E2-induced ERα:IFG-1-R interactions. Moreover, these data demonstrate that ERα monoUbq is required for the physiological modulation of the E2induced extranuclear signaling. 3.4. Reduction in ERα monoUbq prevents E2-induced proliferative effect We and others have firmly demonstrated that the E2-induced cyclin D1 (i.e., an ERE-devoid gene) transcription is dependent on the extranuclear activity of the ERα [3,5,21]. Therefore, the ability of E2 to trigger the activation of the cyclin D1 promoter was studied in the HEK293 clones transfected with the reporter gene plasmid containing the cyclin D1 promoter region (pD1) [3,5,21]. In HEK293 cells stably expressing the wt ERα, E2 induced a significant increase in the cyclin D1 promoter activity with respect to unstimulated cells (Fig. 5A) while the hormone failed to stimulate it in empty vector (pc) HEK293 clone (data not shown). On the contrary, the cyclin D1 promoter activity was not affected by E2 administration to the HEK293 cells containing the KKRR ERα mutant (Fig. 5A), thus confirming the critical role for monoUbq in this ERα-dependent extranuclear activity. Since the E2-induced AKT activation is sufficient and necessary for the induction of the cyclin D1 transcription, which in turn is important for the E2-induced cell cycle progression [3,5,21], we next determined whether ERα monoUbq could impact on the E2-induced cell proliferation. E2 treatment was able to induce a significant increase in the cell number with respect to unstimulated cells in the stable wt ERα HEK293 clone. On the contrary, E2 did not trigger cell proliferation in the KKRR mutant receptor expressing clone (Fig. 5B). In addition, E2 induced and increase in the mitotic index of the HEK293 cells stably transfected with wt ERα while the hormone did not affect it in the

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Fig. 5. Reduction in ERα monoUbq prevents E2-induced cell proliferation. (A) Luciferase assay detection on the HEK293 stable clones expressing the pcDNA flag-ERα (wt) and the pcDNA flag-ERα K302R, K303R (KKRR) mutant transfected with the pXP2-D1-2966luciferase reporter plasmid (pD1) then treated 24 h with E2. Number (B) and mitotic index (C) of the HEK293 stable cells expressing the pcDNA flag-ERα (wt) and the pcDNA flagERα K302R, K303R (KKRR) mutant was assayed both in the presence or in the absence of E2 (48 h). * indicates significant differences with respect to the relative C sample; ° indicates significant differences with respect to the wt ERα E2 sample (P b 0.01).

Fig. 4. Reduction in ERα monoUbq prevents ERα and AKT phosphorylation. Western blot analysis of ERα S118 phosphorylation (A) and of AKT phosphorylation (B) in HEK293 cells stably expressing the pcDNA flag-ERα (wt) and the pcDNA flag-ERα K302R, K303R (KKRR) treated with E2 at the indicated time points. (C) ERα:IGF-1-R coimmunoprecipitation analysis in the HEK293 stable clones expressing pcDNA flag (pc), the pcDNA flag-ERα (wt) and the pcDNA flag-ERα K302R, K303R (KKRR) treated with E2 at the indicated time points. * indicates significant differences with respect to the relative C sample; # indicates significant differences with respect to the wt ERα C sample (P b 0.05). Blots and the relative densitometric analysis are representative of two independent experiments.

KKRR mutant receptor expressing cells (Fig. 5C), thus strongly revealing a critical role of monoUbq in the E2:ERα-mediated cell proliferation.

4. Discussion E2 is a steroid hormone critical for the control of a plethora of biological responses, which strongly influence several aspects of male and female physiology including regulation of cell proliferation. Indeed, it is now clear from epidemiological, clinical and basic research studies that E2 is, at the same time, a risk factor for the initiation and the progression of the breast cancer and a protective molecule for the development of the colon cancer [1]. These contrasting effects relate

to the spectacular complexity of the E2 intracellular signaling, which is triggered by the activation of ERs. Nowadays there is a general agreement that both nuclear and extranuclear ERα activities are essential for the E2 signaling devoted to the progression of cell proliferation. However, present challenge remains to identify how these ERα-based molecular mechanisms are regulated by specific E2triggered receptor post-translational modifications. Elucidating these mechanisms is critical to understanding the basic molecular circuitries of E2 cellular actions and, in turn, to uncovering potential mechanisms at the root of the E2-sensitive cancer development. As supported by mass spectrometry data, endogenous ERα phosphorylation, palmitoylation, methylation and acetylation impact on both nuclear and extranuclear ERα activities [3,6,12–14]. On the contrary, the functions of ERα ubiquitination are not completely understood. Indeed, while it is known that ERα polyubiquitination is the signal for the E2-induced ERα degradation [2], there is a lack of information for the implication of the recently discovered ERα monoUbq in E2 signaling. However, it is now accepted that ubiquitination of proteins can serve both degradative (i.e., proteasome-dependent) and non-degradative (i.e., non-proteolitic) purposes depending on the topology of the Ub-based protein modification [15]. In particular, monoUbq is a non-proteolitic signaling modification, which modulates the activities of proteins involved in diverse cellular processes [15]. Interestingly, we previously found that E2 regulates ERα monoUbq [18] and now we extend these discoveries by demonstrating that monoUbq is a regulatory nodule in the ERα nuclear and extranuclear signals required for the E2-induced cellular processes (e.g., cell proliferation). The functions of ERα monoUbq were analyzed by expressing the ERα in the ERα-devoid HEK293 cells. Although introduction of ERα into ER-negative cells often results in a model that lacks E2 responsiveness [22], our results demonstrate that stable insertion of the receptor in HEK293 cells determines a cell line, which acquires the ability to respond to E2 by activating the ERα nuclear and extranuclear signaling. In turn, the roles of receptor monoUbq on ERα signaling in response to E2 could then be evaluated in the absence of any interference with the endogenous ERα.

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Remarkably, in this cellular context, we report that mutation of the recently identified [16] ERα in vitro monoUbq sites (i.e., K302R and K303R; KKRR) reduces endogenous ERα monoUbq, thus indicating that the lysine residues 302 and 303 within the ERα ligand binding domain (LBD) are ERα monoubiquitination sites in vivo. However, the question arises of whether the monoUbq mutant ERα specifically recapitulates the lack of ERα monoUbq. Since ubiquitination enzymes do not display sufficient fidelity to ubiquitinate a particular lysine residue [16] (i.e., a consensus ubiquitination sequence has not yet been identified), it is most likely that if the primary ubiquitinated lysine in ERα is mutated, another lysine residue can be ubiquitinated, possibly because the experimental system is saturated with the components required for the Ub-based reactions (i.e., ligases, Ub, and ERα) [23]. Indeed, individual in vitro mutagenesis analysis of all 13 lysines to arginine within the ERα LBD as well as of only the K302,303A mutant does not result in a blockade of ERα monoUbq [16]. Furthermore, this phenomenon can be observed also in cells: under condition in which both ERα and BRCA1 (i.e., the ERα E3 Ub ligase [16]) are overexpressed in DU-145 cells, the K302,303A mutation strongly reduces ERα monoUbq [17] whereas under condition in which both ERα and Ub are overexpressed in HeLa cells, the K302,303A mutation greatly enhances ERα ubiquitination [24]. Taken together these evidence strengthen the notion that another ERα lysine residue can be targeted for ubiquitination when the major ERα ubiquitination site is absent and confirm that interfering with the endogenous components of the Ub-network can produce contrasting results [18,23]. Therefore, since the decrease in ERα monoUbq we observed occurs under experimental conditions which minimize the saturation of the Ub-system (i.e., neither Ub nor any E3 ligase are exogenously supplied) [18], it is not surprising that the 50% reduction in the monoUbq of the KKRR mutant ERα translates in a dramatic impairment of ERα activities. In particular, reduction in ERα monoUbq determines a receptor with an overall reduced sensitivity to E2. NMR-analyzed folding of KKRR mutant showed that this receptor is in native-like form [16], furthermore, ERα K303R binds to E2 with the same affinity as the wt ERα [25]. Finally, although strongly reduced, the KKRR mutant ERα still maintained a significant E2-evoked induction of the EREcontaining gene transcription. Thus, the introduction of the KKRR mutation does not affect the receptor structure. Interestingly, the analysis of the effects of the mutation in the ERα K302 and K303 residues on E2-mediated ERα gene transcription has given contrasting results. Indeed, the mutant receptors show either an increased or a decreased transcriptional activity depending on the cell type used [17,24,26,27]. Our data, which have been obtained in stable transfected HEK293 cells, further show that the observed differences can be ascribed also to the method by which ERα transcriptional activity is assessed. In fact, the KKRR mutant receptor displays an increased efficiency in triggering the E2-dependent activation of an artificial ERE-containing promoter (i.e., 3× ERE-TATA) while it strongly prevents the E2-induced natural promoter (i.e., pC3) activation or the endogenous pS2 gene transcription. Although we did not define the mechanistic reasons for these discrepancies, our results suggest a dual role for monoUbq in ERα ERE-driven gene transcription. Indeed, it is tempting to speculate that after E2 stimulation the reduction in ERα monoUbq [18] would address the receptor to the sites where transcription needs to efficiently take place [20] (e.g., EREcontaining promoters) and at the same time limit the ERα transcriptional activity. This hypothesis is further supported by the discovery that the reduction in ERα monoUbq prevents the ERα S118 phosphorylation. This phosphorylation event, which is required for full ERα transcriptional activity [14], is under the control of several extranuclear E2activated signaling cascades. In particular, the E2-induced activation of the PI3K axis is required for the phosphorylation of ERα on the S118 residue [6,11]. In line with this evidence, we found that reduction of

ERα monoUbq impairs the E2-triggered AKT activation. The mechanism underlying this phenomenon appears to involve the interaction of the ERα with membrane growth factor receptors rather than affecting the ERα plasma membrane localization. Indeed, the ERα KKRR mutation strongly influences the ability of ERα to associate to IGF-1-R. In the absence of E2 the monoUbq mutant receptor is constitutively associated with the IGF-1-R as it occurs in the case of the E2-actiaved wt ERα. Since E2 rapidly reduces ERα monoUbq [18] and triggers ERα:IGF-1-R association, the modulation of ERα monoUbq could affect receptor function by influencing ERα tri-dimensional structure. Accordingly, in response to E2, the KKRR mutant receptor, unlike the wt ERα, dissociates from IGF-1-R. Since monoUbq regulates internalization and intracellular trafficking of activated growth factor receptors (e.g., IGF-1-R) [15], the reduction of ERα monoUbq [18] together with the activation of IGF-1-R [10] as a consequence of E2: ERα binding may be a mean to allow ERα intracellular trafficking. Remarkably, the apparent paradox for which the monoUbq mutant is constitutively associated to the IGF-1-R and the AKT activation basal levels are lower with respect to the wt ERα could be reconciled by considering that the ERα-mediated activation of the PI3K/AKT pathway occurs only via the E2-dependent recruitment of the ERα:p85 (the PI3K subunit) complex to the activated IGF-1-R [9,10], a process that the KKRR mutant receptor is not able to sustain and thus is incapable to signal through AKT. Therefore, since the PI3K axis is important for the transduction of the E2-dependent proliferative signals [3,5,21], the reduction of ERα monoUbq further prevents E2-induced cyclin D1 promoter activity and consequently cell proliferation. Accumulating evidence is indicating that the spontaneous mutation of one of the two monoUbq sites (i.e., K303R) occurs in the majority of primary breast cancers [28,29]. However, the oncogenic role of this ERα natural point mutation is manifested only when it is coexpressed with the wild type ERα [28,29], which is still able to drive the proliferative signals (e.g., PI3K/AKT pathway activation) [3]. Therefore, the discovery that the reduction in ERα monoUbq blocks the E2-dependent proliferative effects opens the possibility that the growth advantage of the cells expressing the non-modified ERα may differentially reside in their ability to resist to stress stimuli. Nonetheless, direct cross-talk between the wild type and mutant receptor cannot also be excluded. Finally, it should be noted here that the phenotype observed when ERα monoUbq is reduced mimics the ones demonstrated for the lack of ERα palmitoylation [3,18, and unpublished results]. Thus, since upon E2 binding to receptor both monoUbq and palmitoylation are reduced [3,18] whereas ERα phosphorylation is increased [14], a cross-talk between these different post-translational modifications (i.e., monoUbq, phosphorylation and palmitoylation) may occur and be critical for the fine-tuning of ERα activities. In conclusion, the data presented here demonstrate for the first time that the endogenous ERα monoUbq is critical for the E2dependent ERα activities, thus allowing to refine the proposed model for the integration of the nuclear and extranuclear ERα signaling in the modulation of the E2-induced cell proliferation [3]. On E2 binding, ERα monoUbq is removed [18]. As a consequence, the extent of the E2-activated ERα extranuclear signaling is limited and in parallel the nuclear ERα effects are synchronized. Furthermore, the present discoveries indicate that monoUbq should be considered as a new signaling modifier for the E2:ERα-based transduction pathway required to elicit the regulation of cell proliferation.

Acknowledgements This study was supported by grants from Ateneo Roma Tre to FA. The authors wish to thank Dr. Maurizio Bocchetta, Loyola University Chicago, USA for the generous gift of the anti-IGF-1-R antibody and Dr. Simona Polo, IFOM, Milan, Italy for the generous gift of pcDNA 3.1 flag frame B and C, plasmids.

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