SMILE, a new orphan nuclear receptor SHP ... - Semantic Scholar

Biochem. J. (2008) 416, 463–473 (Printed in Great Britain)

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doi:10.1042/BJ20080782

SMILE, a new orphan nuclear receptor SHP-interacting protein, regulates SHP-repressed estrogen receptor transactivation Yuan-Bin XIE*, Ok-Hee LEE†, Balachandar NEDUMARAN*, Hyun-A SEONG‡, Kyeong-Min LEE§, Hyunjung HA‡, In-Kyu LEE§, Yungdae YUN† and Hueng-Sik CHOI*1 *Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea, †Department of Life Science, Ewha Woman’s University, Seoul 120-750, Republic of Korea, ‡Department of Biochemistry, School of Life Sciences, Chungbuk National University, Cheongju 361-763, Republic of Korea, and §Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu 700-721, Republic of Korea

SHP (small heterodimer partner) is a well-known NR (nuclear receptor) co-regulator. In the present study, we have identified a new SHP-interacting protein, termed SMILE (SHP-interacting leucine zipper protein), which was previously designated as ZF (Zhangfei) via a yeast two-hybrid system. We have determined that the SMILE gene generates two isoforms [SMILE-L (long isoform of SMILE) and SMILE-S (short isoform of SMILE)]. Mutational analysis has demonstrated that the SMILE isoforms arise from the alternative usage of initiation codons. We have confirmed the in vivo interaction and co-localization of the SMILE isoforms and SHP. Domain-mapping analysis indicates that the entire N-terminus of SHP and the middle region of SMILE-L are involved in this interaction. Interestingly, the SMILE isoforms counteract the SHP repressive effect on the transactivation of ERs (estrogen receptors) in HEK-293T cells (human embryonic

kidney cells expressing the large T-antigen of simian virus 40), but enhance the SHP-repressive effect in MCF-7, T47D and MDAMB-435 cells. Knockdown of SMILE gene expression using siRNA (small interfering RNA) in MCF-7 cells increases ERmediated transcriptional activity. Moreover, adenovirus-mediated overexpression of SMILE and SHP down-regulates estrogeninduced mRNA expression of the critical cell-cycle regulator E2F1. Collectively, these results indicate that SMILE isoforms regulate the inhibition of ER transactivation by SHP in a cell-typespecific manner and act as a novel transcriptional co-regulator in ER signalling.

INTRODUCTION

have been proposed: the blocking of DNA binding [17,19], coactivator competition [5,7] and the recruitment of co-repressors [7]. SHP is a key molecule in the regulation of bile-acid synthesis and also performs an important function in cholesterol, glucose, drug and steroidogenesis metabolism [2,3]. ERs include two separate gene-derived subtypes, ERα and ERβ [22,23], and belong to a family of steroid receptors. ERα and ERβ display quite different tissue-distribution patterns. In the rat, ERα was shown to be expressed in the uterus, testis, pituitary, ovary, kidney, epididymis and adrenal tissues, but ERβ was detected in the prostate, ovary, lung, bladder, brain, uterus and testis. ERα and ERβ have considerable aminoacid sequence similarity in both their DNA- and ligand-binding domains. Moreover, both ERs exhibit quite similar ligand-binding characteristics [24] and typically mediate their action via liganddependent binding to the ERE (estrogen-response element) of target genes, thereby resulting in their transcriptional regulation [25]. Many of the ER target gene products are directly involved in cancer-cell proliferation and survival, and in tumour progression. Examples include the cell cycle regulator cyclin D1, E2F1 and the anti-apoptotic factor Bcl-2 [26–28]. It has been determined that ERs play a pivotal role in tumour evolution and in the progression of breast cancer [29] and prostate cancer [30,31], as

SHP (small heterodimer partner) is a unique orphan NR (nuclear receptor) which lacks a conventional DBD (DNA-binding domain) and acts as an NR co-regulator [1–3]. Through a direct interaction, it represses the transcriptional activities of a variety of NRs, including the GR (glucocorticoid receptor), ERs (estrogen receptors), AR (androgen receptor), TR (thyroid receptor), RARα (retinoic acid receptor α), RXR (retinoid X receptor), PXR (pregnane X receptor), mCAR (constitutive androstane receptor), HNF (hepatocyte nuclear factor) 4, LRH-1 (liver receptor homologue-1), ERRγ (ER-related receptor γ ), Nur77 (nerve-growthfactor-inducible protein B) and LXR (liver X receptor) α and β [1,4–12]. It enhances the transactivation of NR PPAR (peroxisome-proliferator-activated receptor) α and γ [13,14]. It has also been demonstrated to interact with several other non-NR TFs (transcription factors), including ARNT [AHR (aryl hydrocarbon receptor) nuclear translocator protein], BETA2 (β-cell E box transactivator 2)/NeuroD (neurogenic differentiation), HNF3/ Foxa, FoxO1 (forkhead box O1) [also called FKHR (forkhead in rhabdosarcoma)], JunD, Smad3 and C/EBPα (CCAAT/enhancerbinding protein α) and down-regulate their transcriptional activities [15–21]. Thus three SHP-mediated repression mechanisms

Key words: alternative translation, E2F1, estrogen receptor (ER), small heterodimer partner (SHP), SHP-interacting leucine zipper protein (SMILE), Zhangfei (ZF).

Abbreviations used: ATF, activating transcription factor; bZIP, basic leucine zipper; C/EBPα, CCAAT/enhancer-binding protein α; CMV, cytomegalovirus; CREB, cAMP-response-element-binding protein; CREM, cAMP-response-element modulator; DAPI, 4 ,6-diamidino-2-phenylindole; E2 , 17β-estradiol; ER, estrogen receptor; ERE, estrogen-response element; ERRγ, ER-related receptor γ; GFP, green fluorescent protein;GST, glutathione transferase; HA, haemagglutinin; HCF-1, herpes simplex virus-related host-cell factor 1; HEK-293T cell, human embroynic kidney cell expressing the large T-antigen of simian virus 40; HNF, hepatocyte nuclear factor; NP-40, Nonidet P40; NR, nuclear receptor; ORF, open reading frame; RT–PCR, reverse transcription–PCR; RXR, retinoid X receptor; SHP, small heterodimer partner; hSHP, human SHP; mSHP, mouse SHP; SMILE, SHP-interacting leucine zipper protein; SMILE1Phe, SMILE M1F; SMILE-83Ile, SMILE M83I; SMILE-83Leu, SMILE M83L; SMILE-L, long isoform of SMILE; SMILE-S, short isoform of SMILE; siRNA, small interfering RNA; siSHP, SHP siRNA; siSMILE, SMILE siRNA; ZF, Zhangfei. 1 To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2008 Biochemical Society

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Y.-B. Xie and others

well as in uterine and ovarian cancers [32,33]. Therefore ERs constitute a crucial target in cancer therapy, and targeting of ERs has been demonstrated to be an effective strategy to inhibit the cell proliferation, survival and tumour growth in ER-related cancers via the regulation of ER signalling [31,33–35]. Previously, it has been shown that SHP can interact directly with agonistbound ERs and inhibit ER-mediated transcription [5], indicating that SHP might function as a tumour suppressor in ER-positive cancers. In the present study, we have identified SMILE (SHP-interacting leucine zipper protein), a new SHP-interacting protein which was previously referred to as Zhangfei (ZF) [36,37]. It has been identified as a protein which interacts with HCF-1 (herpes simplex virus-related host-cell factor 1) and has been proposed to play a role in inhibiting the replication of the herpes simplex virus [36,37]. SMILE harbours a conserved bZIP (basic leucine zipper) domain, which is significantly similar to other CREB (cAMP-response-element-binding protein)/ATF (activating transcription factor) bZIP family members. Although SMILE can homodimerize like other bZIP proteins, it lacks the ability to bind to DNA as a homodimer [36,38]. It has been reported recently that SMILE can augment the DNA-binding ability of ATF4 to the cAMP response element through SMILE–ATF4 association [39]. In the present study, we have described two isoforms of SMILE [SMILE-L (long isoform of SMILE) and SMILE-S (short isoform of SMILE), previously known as ZF] resulting from alternative usage of initiation codons in a single SMILE mRNA. Both the in vivo and in vitro results demonstrate that the two SMILE isoforms interact with SHP and regulate SHP function in repressing ER transactivation in a cell-type-specific manner. We therefore propose that SMILE is a novel transcriptional coregulator in ER signalling. MATERIALS AND METHODS Plasmid and DNA construction

The plasmids encoding LexA–hSHP (human SHP), LexA–mSHP (mouse SHP), mSHP deletion constructs [7,16,40] and pcDNA3HA-hSHP and pEBG-hSHP expression plasmids [16] have all been described previously. The reporter gene plasmid ERE–Luc (luciferase) was a gift from Dr Jae Woon Lee (Center for Ligand and Transcription, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea) [41]. The full ORF (open reading frame) of human SMILE was amplified by PCR from the IMAGE consortium (Integrated Molecular Analysis of Genomes and their Expression consortium) clone 30345789 (Open Biosystems). The purified PCR products were digested and subcloned into the EcoRI and XhoI sites of pcDNA3 and pcDNA3-FLAG vectors. The plasmid pcDNA3SMILE-S (ZF) was generated by inserting the digested SMILE-S fragments into the EcoRI/XhoI sites of pcDNA3. The SMILES fragments were obtained from pcDNA3-Flag-ZF, which was a gift from Professor Vikram Misra (Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada) [36]. The SMILE mutants, pcDNA3-SMILE-83Leu, pcDNA3-SMILE-83Ile and pcDNA3-SMILE-1Phe, were generated by PCR-mediated sitedirected mutagenesis. The plasmids pGEX4T-1-SMILE-L and pGEX4T-1-SMILE-S were constructed by subcloning EcoRIand XhoI-digested full-length SMILE or SMILE-S cDNA fragments into pGEX4T-1 (Amersham Biosciences). The constructs pEGFP-SMILE-L and pEGFP-SMILE-S were subcloned by inserting the fragments of full-length SMILE and SMILE-S into pEGFP-C1 (Clontech) between the XhoI and BamHI sites c The Authors Journal compilation c 2008 Biochemical Society

respectively. To generate B42AD-fused SMILE-L and SMILEL deletion mutants, the fragments were generated by PCR and subcloned into the EcoRI and XhoI sites of the pB42AD/pJG45 vector (Clontech). The pcDNA3-HA-ERα and pcDNA3-HAERβ plasmids were constructed by inserting PCR fragments of ERα and ERβ ORFs into the EcoRI and XhoI sites of a HA (haemagglutinin)-tagged pcDNA3 vector respectively. All plasmids were confirmed by DNA sequencing analysis. Cell culture, transient-transfection assay and luciferase assay

HEK-293T cells (human embryonic kidney cells expressing the large T-antigen of simian virus 40), HepG2 cells (human hepatoma cells), MCF-7 cells (human breast adenocarcinoma cells), T47D cells (human breast ductal carcinoma cells), MDA-MB-435 cells (human breast ductal carcinoma cells), HeLa cells (human cervix carcinoma cells), PC-3 cells (human prostate carcinoma cells), C2C12 cells (mouse myoblast cells), NIH 3T3 cells (mouse fibroblast cells), K28 cells (mouse Leydig cells), Y-1 cells (mouse adrenal cortex tumour cells) and TM4 cells (mouse sertoli cells) were obtained from the A.T.C.C. and cultured according to the manufacturer’s instructions. Transient transfection was performed using Superfect transfection reagent (Qiagen) in HEK-293T cells and LipofectamineTM 2000 reagent (Invitrogen) in MCF-7, T47D and MDA-MB-435 cells. HEK-293T, MCF-7, T47D and MDA-MB-435 cells were plated on to 24-well plates the day prior to transfection and were co-transfected with ERE–Luc coupled with various expression vectors. The plasmid expressing CMV (cytomegalovirus)–β-galactosidase was cotransfected as an internal control and the total DNA employed in each transfection was adjusted by the addition of an appropriate quantity of pcDNA3 vector. Approx. 24 h post-transfection, the cells were treated with or without 100 nM E2 (17β-estradiol) (Sigma) for 24 h at 37 ◦C, and then cells were harvested and the luciferase activity was measured and normalized against β-galactosidase activity as described previously [17]. The fold change in activity was calculated by considering the activity of reporter gene alone to be 1. GST (glutathione transferase) pull-down assay

GST pull-down assays were performed following a method published previously [16]. Briefly, SHP was labelled with [35 S]methionine using the TNT® -coupled reticulocyte lysate system (Promega) according to the manufacturer’s instructions. GST alone, GST–SMILE-L and GST–SMILE-S proteins were prepared as described previously [16]. The GST proteins were pre-bound with glutathione–Sepharose beads (Amersham Biosciences) and then incubated with in vitro-translated [35 S]methionine-labelled SHP in binding buffer [25 mM Hepes (pH 7.6), 150 mM NaCl, 0.2 mM EDTA, 1 mM DTT (dithiothreitol), 20 % (v/v) glycerol and protease inhibitors] for 2–3 h at 4 ◦C. The beads were washed three times with the binding buffer, analysed by SDS/PAGE (10 % gels) and visualized by a phosphorimager analyser (BAS-1500; Fuji). Western blot analysis

The whole-cell extracts from cell lines were prepared using RIPA buffer [50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1 mM Na2 EDTA, 1 mM EGTA, 1 % NP-40 (Nonidet P40) and protease inhibitors]. Yeast protein extracts were prepared in TCA (tricholoroacetic acid) buffer [20 mM Tris/HCl (pH 8.0), 50 mM ammonium acetate, 2 mM EDTA and protease inhibitors] as stated in the Clontech yeast protocols handbook (PT3024-1). Western blot assays on 10–50 μg of protein were performed as described

Regulation of the repressive effect of SHP on estrogen receptors by SMILE

Figure 1

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SMILE isoforms arise from alternative translation initiation

(A) A schematic representation of SMILE-L (CREBZF) and SMILE-S (Zhangfei/ZF). The numbers in the Figure represent amino-acid residues. (B) Exogenous expression of human wild-type SMILE and ZF in HEK-293T cells using the pcDNA3 vector. Whole-cell extracts (10 μg) were analysed by Western blotting using a SMILE-specific antibody. (C) Schematic representation of the SMILE mutant constructs. The nucleotide sequences around the two in-frame methionine start codons (1 and 83) of the human SMILE cDNA are shown. The second ATG is underlined. The mutated nucleotides are indicated (䊉). Wt, wild-type SMILE; 83Leu, SMILE-83Leu (second ATG mutated to CTG); 83Ile, SMILE-83Ile (second ATG mutated to ATC); 1Phe, SMILE-1Phe (first ATG mutated to TTC). (D) Protein (10 μg) analysis of human SMILE mutants via transient transfection in HEK-293T cells, followed by Western blotting with a SMILE-specific antibody. Wt, wild-type SMILE; 83Leu, SMILE-83Leu (second ATG mutated to CTG); 83Ile, SMILE-83Ile (second ATG mutated to ATC); 1Phe, SMILE-1Phe (ATG mutated to TTC). (E) Expression of SMILE protein in cell lines (left-hand panel) and mouse tissues (right-hand panel). Protein samples (50 μg) were analysed by Western blotting using SMILE- or tubulin-specific antibodies. Results are representative of at least three independent experiments with similar results. 293T, HEK-293T.

previously [17]. The LexA fusion proteins, HA-tagged proteins, SMILE and tubulin proteins were detected using an anti-LexA antibody (1:1000 dilution; sc-1725, Santa Cruz Biotechnology), an anti-HA antibody (1:1000 dilution; 12CA5, Roche), an antiSMILE antibody (1:1000 dilution; ab28700, Abcam) and an anti-tubulin antibody (1:1000 dilution; #2146, Cell Signaling Technology) respectively. B42 fusion proteins were detected using the anti-HA antibody, as the B42 fusion proteins also have an HA tag.

previously [16]. Briefly, LexA only or LexA-fused hSHP, mSHP, mSHP deletion mutants and B42-AD or B42-fused SMILEL and B42-fused SMILE-L deletion mutants were transformed into the yeast EGY48 strain containing the β-galactosidase reporter plasmid 8H18-34, and the transformants were selected on agar plates using appropriate selection markers. The liquid β-galactosidase assay was then performed on the agar plates. In vivo interaction assay

Yeast two-hybrid interaction assay

Screening of a human fetal liver cDNA library (Invitrogen) using LexA–hSHP as the bait was carried out as described previously [16]. Yeast transformation was performed, positive clones were selected and the cDNA inserts were recovered and sequenced. Yeast two-hybrid interaction assays were performed as described

In vivo interaction experiments were performed as described previously [16]. In brief, HEK-293T cells were plated in 6well plates at a concentration of 2 × 105 cells per well the day prior to transfection. Each plasmid DNA (1 μg) was transfected into HEK-293T cells using the SuperFect transfection reagent and 48 h post-transfection, cells were solubilized with 100 μl of lysis buffer [20 mM Hepes (pH 7.9), 10 mM EDTA, 0.1 M c The Authors Journal compilation c 2008 Biochemical Society

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transfection, the cells were washed three times in cold PBS and fixed for 15 min in 2 % (w/v) formaldehyde. The fixed cells were then mounted on to glass slides with PBS and observed with a laser-scanning confocal microscope (Leica) at × 400 magnification. To detect HA–SHP and nuclei, the cells were incubated with an mouse monoclonal Alexa Fluor® 594conjugated anti-HA antibody (1:500 dilution; Invitrogen) for 1 h at room temperature (25 ◦C), washed three times in PBS and incubated with 0.1 mg/ml DAPI (4 ,6-diamidino-2-phenylindole) (Invitrogen) solution for 10 min at room temperature. After three further washes with PBS, the cells were examined. Preparation of recombinant adenovirus

The recombinant adenovirus encoding hSHP has been described elsewhere previously [17]. The adenovirus encoding human SMILE was constructed using the method described previously [17]. Briefly, the cDNA encoding human SMILE was cloned into the KpnI and XbaI sites of pAdTrack-CMV. Recombination of AdTrack-CMV-hSMILE (where hSMILE is human SMILE) with an adenoviral gene-carrier vector was performed by transformation into pre-transformed adEasy-BJ21 competent cells. RNA interference

Figure 2

In vitro and in vivo interactions between SHP and SMILE isoforms

(A) In vitro GST pull-down assays. Equivalent amounts of bead-bound proteins were used. The input lane represents 10 % of the total volume of in vitro -translated proteins used for the binding assay. (B, C) In vivo interaction of SMILE-L (B) and SMILE-S (C) with SHP. HEK-293T cells were co-transfected with expression vectors for FLAG–SMILE-L or FLAG–SMILE-S with pEBG-SHP (GST–SHP) or pEBG alone (GST). The complex formation (top panel, GST puri) and the amount of FLAG–SMILE-L used for the in vivo binding assay (bottom panel, lysate) were determined by Western blotting with an anti-Flag antibody. The same blot was stripped and reprobed with an anti-GST antibody (middle panel) to confirm the expression levels of the GST fusion protein (GST–SHP) and the GST control (GST). (D) SMILE isoforms and SHP are co-localized in the nucleus. Hela cells were transiently transfected with pEGFP-SMILE-L or pEGFP-SMILE-S along with pcDNA3-HA-SHP. Results are representative of at least three independent experiments with similar results.

KCl and 0.3 M NaCl] containing 0.1 % NP-40, and protease inhibitors. The cleared lysates (80 μl) were mixed with 15 μl of glutathione–Sepharose beads and rotated for 2 h at 4 ◦C. The beads were washed three times with the lysis buffer. The bound proteins were eluted by boiling in SDS sample buffer for 5 min and then subjected to SDS/PAGE (10% gels) and Western blotting. GST- and FLAG-fusion proteins were detected using anti-GST (1:1000 dilution; sc-33614, Santa Cruz Biotechnology) and anti-FLAG (1:1000 dilution; #200472-21, Stratagene) antibodies respectively. Confocal microscopy

The confocal microscopy assays were carried out as described previously [16] with minor modifications. Briefly, Hela cells were grown on gelatin-coated coverslips and transfected with pEGFP-SMILE-L or pEGFP-SMILE-S and pCDNA3-HA-SHP using the Effectene transfection reagent (Qiagen) and, 24 h after c The Authors Journal compilation c 2008 Biochemical Society

The ablation of SHP and SMILE was performed using the pSuper vector system [42]. pSuper vector and pSuper vector encoding siSHP [SHP siRNA (small interfering RNA)] were gifts from Dr Jongsook Kim Kemper (Department of Molecular and Integrative Physiology, School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, IL, U.S.A.) [43]. The siSMILE (SMILE siRNA) constructs were constructed by ligation of a 64-mer double-stranded oligonucleotide containing + 656 to + 676 (siSMILE-I) or + 1038 to + 1058 (siSMILE-II) of the human SMILE cDNA sequence into the pSUPER vector digested with BglII and Xho I. MCF-7 and T47D cells were transfected with siRNA constructs using LipofectamineTM 2000 (Invitrogen) according to the manufacturer’s guidelines. siRNA-treated cells were subjected to RT–PCR (reverse transcription–PCR), Western blot analysis or a second transfection step as indicated in the Figure legends. RT–PCR analysis

Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions. The mRNAs of SHP, SMILE and E2F1 were analysed by RT–PCR as described previously [17], and the mRNA levels of β-actin were used as an internal control for RT–PCR. The sequences for the oligonucleotide primers used are as follows: SHP, forward primer, 5 -CTTCCTCAGGAACCT3 , and reverse primer, 5 -CCCAGTGAGCCTCCT-3 ; E2F1, forward primer, 5 -TGCCAAGAAGTCCAAGAACC-3 , and reverse primer, 5 -CTCAGGGCACAGGAAAACAT-3 ; SMILE, forward primer, 5 -AAAAGAGGCGGA GAAAGTCC-3 , and reverse primer, 5 -CTCTGAAGAGCGAGGTGGTC-3 ; and βactin forward primer, 5 -GTCATCACCATTGGCAATGAG3 ; and reverse primer, 5 -CGTCATACTCCTGCTTGCTG-3 . Amplifications of 20–22, 22–24, 23–25 and 30–34 cycles were performed for β-actin, E2F1, SHP and SMILE respectively. Statistical analysis

The Student’s t test was performed using GraphPad Prism version 3.0 for Windows and results were considered to be statistically significant when P < 0.05.


Figure 3

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Determination of the interaction domains within SHP and SMILE-L

(A) Schematic representation of mSHP wild-type (WT) and deletion mutants. INT, NR-interaction domain; REP, NR-represssion domain. Values underneath indicate the amino-acid residues. (B) The N-terminal region (amino acids 1–159) of mSHP is required for the interaction with SMILE in the yeast two-hybrid assay. Plasmids encoding LexA–mSHP wild-type (WT) or deletion mutants as shown in (A) were co-transformed with B42AD and B42–SMILE into yeast cells. A liquid β-galactosidase assay was performed as described in the Materials and methods section. Results are means + − S.D. (n = 3). (C) Protein expression of B42–SMILE-L, LexA–mSHP wild-type (WT) and mSHP deletion mutants [see (A) for details] in the yeast co-transformants. LexA-fusion proteins and B42–SMILE-L were detected by anti-LexA and anti-HA antibodies respectively. Values underneath indicate the amino-acid residues. (D) Schematic representation of SMILE-L wild-type and the SMILE deletion mutants. (E) The middle region (amino acids 113–267) of SMILE is involved in the interaction with SHP in the yeast two-hybrid assay. The plasmids for LexADBD (DNA-binding domain) or LexA–hSHP were transformed with the plasmids encoding B42 fusions of SMILE-L (WT), or the SMILE mutants detailed in (D). A liquid β-galactosidase assay was performed as described in the Materials and methods section. Results are means + − S.D. (n = 3). (F) Western blot analysis of LexA–hSHP, B42–SMILE-L WT and its deletion mutants [see (D) for details] in the yeast co-transformants. LexA–hSHP and B42–SMILE were detected using anti-LexA and anti-HA antibodies respectively. Tubulin expression was detected using an anti-tubulin antibody and is shown as a loading control.

RESULTS SMILE gene encodes two isoforms by alternative initiation of translation

In an effort to identify new proteins that might be involved in the repressive function of SHP, the yeast two-hybrid method was employed in the screening of a human fetal liver cDNA library using full-length hSHP as bait. One of the positive clones corresponded to clone BC060807 (CREBZF, IMAGE 30345789) and clone AF039942 (ZF). Amino-acid alignment showed that CREBZF and ZF harboured the same amino acids, except that CREBZF has an extra 82 amino acids at its N-terminus (Figure 1A and results not shown). A BLAST search using the AF039942 nucleotide sequence against the GenBank® human genome database showed that a cytosine nucleotide was missing at position 227 in the AF039942 clone (results not shown), which resulted in the misidentification of the ZF start codon described previously [36]. This showed that ZF is only a partial clone of CREBZF, which encodes a 354-amino-acid protein with a predicted molecular mass of 37.2 kDa. Since ZF is a member of the bZIP family [36], we named CREBZF as SMILE. To determine the apparent molecular mass of the SMILE protein, we overexpressed SMILE in HEK-293T cells by transient transfection, and Western blot analysis was performed using the whole-cell extracts. In HEK-293T cells transfected with

control vector (Figure 1B; lane 1), no bands were detected by an anti-SMILE antibody, which recognizes the C-terminal peptide of SMILE. When full-length human SMILE was transfected into HEK-293T cells, the antibody detected two SMILE bands (Figure 1B, lane 3) with apparent molecular masses of 50–60 kDa (SMILE-L) and 40 kDa (SMILE-S) respectively. Interestingly, an overexpressed ZF protein co-migrated at exactly the same mass as the SMILE-S band (Figure 1B). These result compelled us to hypothesize that both ZF and SMILE-S initiated within the same reading frame at the second AUG start codon at amino acid position 83 in SMILE. To verify this hypothesis, Western blot analysis was performed after transfecting pcDNA3 vectors encoding wild-type SMILE, SMILE-83Leu (SMILE M83L), SMILE-83Ile (SMILE M83I) and SMILE-1Phe (SMILE M1F) into HEK-293T cells. As shown in Figure 1(D), wild-type SMILE gave rise to both SMILE-L and SMILE-S bands. However, when the second ATG codon was mutated into CTG (Figure 1C; SMILE-83Leu construct) or ATC (Figure 1C; SMILE-83Ile construct), the SMILE-S band disappeared (Figure 1D, lanes 3 and 4). Moreover, when the first ATG codon was mutated to TTC (Figure 1C; SMILE-1Phe construct), only the SMILE-S band was detected (Figure 1D, lane 5). These results indicate that the second ATG codon represents the start codon for SMILE-S, meaning that SMILE-S and ZF proteins are exactly identical. On the basis of the observation that the SMILE gene harbours only one exon and c The Authors Journal compilation c 2008 Biochemical Society

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that SMILE-L is ubiquitously expressed, which is consistent with the expression pattern of SMILE mRNA [37], whereas SMILE-S is both liver- and cell-type-specific. SHP directly interacts with SMILE isoforms in vitro and in vivo

We identified SMILE as an SHP-interacting protein from a yeast two-hybrid library screening. To further confirm the interaction between SHP and SMILE, in vitro and in vivo GST pull-down assays were performed. For the in vitro GST pull-down assay, bacterially expressed GST only, GST–SMILE-L or GST–SMILES were incubated with in vitro translated 35 S-labelled SHP. We found that 35 S-labelled SHP was able to bind to GST-tagged SMILE-L and SMILE-S (Figure 2A), indicating that SHP can interact with both SMILE isoforms in vitro. For the in vivo GST pull-down assay, mammalian expression vectors encoding either GST alone or GST–SHP together with pcDNA3-FLAGSMILE-L were co-transfected into HEK-293T cells. As shown in Figure 2(B), SMILE-L was coimmunoprecipitated only when it was co-expressed with GST–SHP, but not with GST alone (Figure 2B). The expression levels of GST–SHP and FLAG– SMILE-L were confirmed by Western blot analysis (Figure 2B, middle and bottom panels respectively). Similar results were obtained when an expression plasmid encoding FLAG–SMILE-S was used (Figure 2C). These results indicated that SHP interacts with the SMILE isoforms in vivo. In an effort to further confirm this interaction, we examined the potential co-localization of SHP with the SMILE isoforms. HeLa cells were co-transfected with the expression plasmids pEGFP-SMILE-L or pEGFP-SMILE-S along with pcDNA3-HASHP, stained with Alexa Fluor® 594-conjugated anti-HA antibody and DAPI, and analysed by confocal microscopy. As shown in Figure 2(C), GFP (green fluorescent protein)–SMILE-L and GFP–SMILE-S were localized predominantly in the nucleus, but also weakly detected in the cytoplasm. Consistent with previous reports [16,17], SHP was detected principally in the nucleus. The merged image suggested that SHP and the SMILE isoforms were co-localized in the nucleus. In summary, these results demonstrate that SHP interacts and co-localizes with SMILE isoforms in vivo. Figure 4 SMILE releases the inhibition of transactivation of ERs by SHP in HEK-293T cells Reporter assays (A–C) were performed as described in the Materials and methods section, and the activity of the reporter gene alone was considered to be 1. (A) SMILE opposes the repressive effect of SHP on ERα/β-mediated transactivation in HEK-293T cells (293T). (B, C) Both SMILE-83Leu and SMILE-1Phe counteract the inhibitory effect of SHP on the transcriptional activity of ERα (B) and ERβ (C) in HEK-293T cells. Results are means + − S.D. (n = 3). ∗ P < 0.05, using Student’s t test.

encodes a single 4.6 kb mRNA [37], we finally conclude that the SMILE-L and SMILE-S proteins are translated from a single mRNA by alternative usage of initiation codons. To assess the expression of endogenous SMILE proteins, Western blot analysis was performed using lysates from different cell lines and mouse tissues. As shown in Figure 1(E), both SMILEL and SMILE-S isoforms were detected in HepG2, MCF-7, HeLa, PC-3, NIH 3T3 and Y1 cells, but not in K28 and TM4 cells, whereas only SMILE-L was detected in HEK-293T and C2C12 cells. In mouse tissues, SMILE-L was detected in all the examined tissues, including brain, lung, heart, liver, kidney, spleen, prostate and testis. However, SMILE-S was detected only in the liver. Notably, SMILE-L was the predominant form in all of the cells and tissues tested (Figure 1E). These results indicate c The Authors Journal compilation c 2008 Biochemical Society

Mapping of the interaction domains between SHP and SMILE

To identify the interaction domain of SHP involved in this interaction, a yeast two-hybrid assay was performed using a series of SHP deletion mutants (see Figure 3A for details). As shown in Figure 3(B), B42–SMILE-L was shown to interact with LexAfused wild-type SHP and SHP W160X (amino acids 1–159), which harbours the entire N-terminus including the NR-interacting domain. However, B42–SMILE-L interacted slightly with the LexA-fused SHP N (amino acids 1–3 and 72–260) and SHP N148 (amino acids 1–3 and 72–148), which harbour the interaction domain for the NRs including ERRγ [10] and RXR [40]. Moreover, no interactions were observed between SMILE and other SHP deletion constructs E1 (amino acids 1–71 and 92– 260), E1X (amino acids 1–71 and 91–93), 120 (amino acids 120–260) and 210 (amino acids 210–260), which lack the entire N-terminus domain (Figures 3A and 3B). Western blot analysis showed that all the LexA–mSHP fusion proteins and B42– SMILE-L were expressed properly in the yeast co-transformants (Figure 3C), indicating that the differences in the interaction between the SHP deletion mutants and SMILE-L are not the result of differences in protein expression. Taken together, these results indicate that the full-length N-terminus of SHP is required for a


Figure 5

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SMILE enhances the inhibition of SHP on ER-mediated transactivation in MCF-7 cells

Reporter assays in (A–C, F) were performed as described in the Materials and methods section and the activity of reporter gene alone was considered to be 1. Results are means + − S.D. (n = 3) ∗ P < 0.05, using Student’s t test. (A) SMILE increases the inhibitory effect of SHP on endogenous ER-mediated transcriptional activity in MCF-7 cells. (B, C) Both SMILE-83Leu and SMILE-1Phe reinforce the inhibition of SHP on the transcriptional activity of ERα (B) and ERβ (C) in MCF-7 cells. (D, E) The effect of overexpressed SMILE on the protein levels of HA–ERα, HA–ERβ and HA–SHP. MCF-7 cells were co-transfected with various plasmids as indicated. Whole-cell extracts (50 μg) from the transient transfection assay were subjected to Western blot analysis. The proteins of HA–ERα, HA–ERβ, HA–SHP, SMILE and tubulin were detected as described in the Materials and methods section. Tubulin expression was detected using an anti-tubulin antibody and is shown as a loading control. (F) SMILE siRNA induces endogenous ER-mediated transactivation in MCF-7 cells. MCF-7 cells were transfected with the indicated siRNA expression vector and after 24 h, the cells were co-transfected with ERE–Luc and the SMILE expression vector together with pCMV-β-gal vector. After a 24 h transfection, cells were treated with or without E2 for 24 h before luciferase activity was measured. (G) Effect of siRNA on the expression of SHP and SMILE. MCF-7 cells were transfected with pSUPER siSHP, pSUPER siSMILE-I or pSUPER siSMILE-II or pSUPER [control (con)], and after 72 h, total RNA (upper panel) or protein (lower panel) was isolated. The mRNA expression of SHP and SMILE was measured by RT–PCR. The protein expression of SMILE was detected by Western blot analysis . Results are representative of three experiments. wt, wild-type.

full interaction with SMILE, and amino acids 72–92 on SHP form a core binding site for SMILE. To map the regions of SMILE required for the SHP–SMILE interaction, a variety of B42-fused SMILE-L or SMILE-L deletion mutants (Figure 3D) were co-transformed into yeast with LexA– hSHP. As shown in Figure 3(E), LexA–hSHP interacted with B42fused SMILE-L, and SMILE-L mutants NC (amino acids 113– 202) and 202 (amino acids 203–354), but not with another two SMILE-L mutants, N (amino acids 1–112) and 268 (amino acids 269–354) (see Figure 3D for details). Our Western blot analysis (Figure 3F) indicates that the distinct pattern of interaction between SHP and the SMILE mutants is not due to the differences in the protein level. These findings suggest that the middle region (amino acids 113–268) of SMILE-L, which is equal to the region spanning amino acids 31–186 of SMILE-S, is involved in this interaction.

SMILE isoforms regulate the inhibition of ER transactivation by SHP

On the basis of the fact that SHP functions as a co-repressor of NRs [1–3] and that the SMILE isoforms interact physically with SHP (Figure 3), we attempted to determine whether the SMILE proteins regulate the repressive function of SHP. Using reportergene assays, we investigated whether SMILE proteins influence

the inhibition of ER-dependent transcription by SHP, as no direct interaction was observed between ERs and SMILE isoforms (results not shown). To investigate this, the ERE–Luc reporter construct was used. As shown in Figure 4(A), in HEK-293T cells ERα and ERβ activated the reporter ERE–Luc in the presence of the ligand E2 , and SHP inhibited ER-mediated transcriptional activity as described previously [5]. Interestingly, wild-type SMILE alone did not show any effect on the transactivation of ERs, but SMILE relieved the inhibition of ERα- and ERβdependent transactivation by SHP in a dose-dependent manner. Because the results above suggest that wild-type SMILE cDNA can generate two SMILE isoforms (Figures 1B and 1D), SMILEL and SMILE-S, we attemped to determine which isoform of SMILE contributes most profoundly to counteracting the repressive effect of SHP. To address this issue, we used SMILE mutants SMILE-83Leu and SMILE-1Phe, which are able to generate only SMILE-L or SMILE-S respectively (Figure 1D). As shown in Figure 4(B), both SMILE-83Leu and SMILE-S were able to counteract SHP in repressing ER-mediated transcription, and similar results were observed with ERβ (Figure 4C). These results suggest that SMILE-L and SMILE-S exert identical effects by releasing the inhibition of ER transactivation by SHP in HEK293T cells. To determine whether SMILE isoforms counteract SHP in regulating ER-dependent transcription in a cell-type-specific c The Authors Journal compilation c 2008 Biochemical Society

470

Figure 6


SMILE enhances the inhibition of SHP on ER-mediated transactivation in T47D and MB-435 cells

Reporter assays (A–D) were performed as described in the Materials and methods section and the activity of the reporter gene alone was considered to be 1. Results are means + − S.D. (n = 3) ∗ P < 0.05, using Student’s t test. (A, B) Both SMILE-83Leu and SMILE-1Phe reinforce the inhibition of SHP on the transcriptional activity of ERα (A) and ERβ (B) in T47D cells. (C, D) Both SMILE-83Leu and SMILE-1Phe reinforce the inhibitory effect of SHP on the transcriptional activity of ERα (C) and ERβ (D) in MDA-MB-435 cells.

manner, we performed transfection experiments in breast cancer cell lines, including ER-positive MCF-7 and T47D cells, and ERnegative MDA-MB-435 cells. As shown in Figure 5(A) and Supplementary Figure S1A (http://www.BiochemJ.org/bj/416/ bj4160463add.htm), overexpressing SHP down-regulated endogenous ER-mediated reporter activity significantly, consistent with a previous report [5]. Interestingly, both SMILE-83Leu and SMILE-1Phe alone were able to down-regulate the reporter activity slightly but significantly. Moreover, SMILE-83Leu and SMILE-1Phe augmented the inhibition of the reporter activity by SHP in a dose-dependent manner, thereby indicating that SMILEL and SMILE-S are capable of reinforcing the repressive effect of SHP on endogenous ERs. Furthermore, the SMILE-83Leu and SMILE-1Phe mutants enhanced the inhibitory effect of SHP on agonist-activated ERα without affecting the protein expression of transfected ERα and SHP (Figures 5B, 5D, 6A and 6C), and similar results were observed with ERβ (Figure 5C, 5E, 6B and 6D). Taken together, these results indicate that SMILE-L and SMILE-S function as enhancers of SHP in repressing agonist-activated ERs in breast cancer cells. To explore the role of endogenous SMILE in regulating the transactivation of ERs, we examined the consequences of SMILE and SHP suppression on E2 -mediated transcriptional activity of ERs in MCF-7 and T47D cells. Endogenous gene expression of SMILE was efficiently silenced at the mRNA level by siSMILEII, but not by siSMILE-I, and the gene expression of SHP was c The Authors Journal compilation c 2008 Biochemical Society

efficiently knocked down by siSHP in MCF-7 cells (Figure 5G and Supplementary Figure S1C). E2 -mediated transactivation of ERs increased significantly upon silencing of SMILE by siSMILE-II, but no changes were observed in siSMILE-I-treated treated cells (Figure 5F and Supplementary Figure S1B). As a positive control, the silencing of SHP by siSHP increased significantly E2 -mediated transactivation of ERs. Interestingly, with the silencing of SHP, overexpression of SMILE failed to down-regulate E2 -mediated transactivation of ERs. These results indicate that SMILE may regulate endogenous transactivation of ERs in a SHP-dependent manner in ER-positive MCF-7 and T47D cells. Adenovirus-mediated overexpression of SHP and SMILE inhibits E2 -induced gene expression of E2F1 in MCF-7 cells

Downstream targets of ER signalling include the cell-cycle regulator cyclin D1 and the transcription factor E2F1 [26,27]. To confirm whether SMILE is able to co-operate with SHP in repressing ER target-gene expression, we performed RT–PCR analysis to measure the E2 -induced E2F1 mRNA level in MCF-7 and T47D cells after adenovirus-mediated overexpression of SMILE and SHP. As expected, E2 treatment clearly induced E2F1 at the mRNA level, and the induction of E2F1 was significantly decreased by overexpression of SMILE, as well as by overexpression of SHP (Figure 7). Moreover, SMILE synergized with SHP significantly in down-regulating the E2 -mediated induction


Figure 7

471

Regulation of E2F1 mRNA levels by SMILE and SHP

MCF-7 cells (A) and T47D cells (C) were infected with an adenoviral vector (Ad) expressing hSHP, human SMILE or GFP as indicated. After 24 h of infection, the cells were stimulated with 100 nM E2 for 24 h. Total RNA was isolated from cells and the mRNA levels of E2F1, SMILE, SHP, and β-actin were analysed by RT–PCR. Values (0, 50 and 100) indicate the multiplicity of adenovirus ∗ infection. (B, D) RT–PCR results were quantified using densitomeric analysis. E2F1 mRNA levels were normalized against the mRNA levels of β-actin. Results are means + − S.D. P < 0.05, using Student’s t test.

of E2F1 (Figure 7). Hence, these results suggest that SMILE may co-operate with SHP in the negative regulation of estrogen signalling in ER-positive MCF-7 and T47D cells.

DISCUSSION

In the present study, we have determined that SMILE is a novel SHP-interacting protein. We present evidence suggesting that at least two SMILE isoforms arise from a single SMILE transcript by the alternative usage of translation initiation sites. SMILE-L and SMILE-S differ with regard to the presence or absence of 82 amino acids in the N-terminal regions. SMILE-L is expressed ubiquitously in mouse tissues and tumour-derived cells. However, SMILE-S exhibits a liver- and cell-type-specific expression pattern. This discrepancy may be the result of the differences between normal cells and tumour-derived cells. The mechanisms involved in the control of the liver- and cell-type-specific expression of SMILE-S remain to be clearly elucidated. Although SMILE isoforms could be generated via a proteolytic cleavage mechanism, we have excluded this possibility on the basis of our mutation analysis results and the cell-type-specific, tissue-specific expression pattern of SMILE-S. Interestingly, SMILE-L displays a broad band with an apparent molecular mass of 50–60 kDa, and even appears as two bands in some cases (Figures 1D and 1E). This could possibly be the result of post-translational modifications,

since post-translational modifications can change the mobility of proteins [44,45]. Such an alternative usage of initiation codons has been reported in association with other genes, including those encoding GATA1, GATA-6, C/EBPα and β, CREM (cAMP-response-element modulator) and JunD [46–50]. The last four proteins and SMILE are members of the same bZIP protein family. In some of the reported cases, the two proteins produced through alternative translation exhibit quite different functions and regulate each other functionally [48,49]. For example, CREMγ and CREMS, derived from the CREM gene by alternative translation, act as an agonist and an antagonist of cAMP-induced transcription respectively [49]. However, in the case of SMILE, both SMILE-L and SMILE-S associate physically with SHP within the nucleus. Functional analyses of SMILE-L and SMILE-S using SMILE mutants showed that the two SMILE isoforms exhibit similar regulatory effects on SHP in repressing transactivation of ERs, similar to the case of JunD [50]. It is certainly possible that the two forms exhibit different regulatory functions in certain specific cellular contexts. As the short form of SMILE, ZF was reported to be involved in the herpesvirus infection cycle and associated cellular processes through ZF–HCF and ZF–CREB3 association [36,37,51]. However, whether wild-type SMILE has the same function in vivo still needs to be determined because SMILE-L was the dominant isoform in all cells and mouse tissues tested. c The Authors Journal compilation c 2008 Biochemical Society

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SMILE isoforms do not interact directly with ERs, and exert no direct effect on ER-mediated transcription in HEK-293T cells. However, SMILE isoforms regulate the inhibition of transactivation of ERs by SHP in a cell-type-specific manner. Both SMILE isoforms counteract the SHP repressive effect for inhibiting the transactivation of ERs in HEK-293T cells, but enhance the SHP repressive effect in both ER-positive MCF-7 and T47D cells, and ER-negative MDA-MB-435 cells. Interestingly, the effect of SMILE-L and SMILE-S on the repressive function of SHP on ERs in ER-positive cells is quite similar to that in ERnegative cells, which suggests that the basal expression of ERs in ER-positive cells does not affect the response of ERs to SMILE in transient transfection conditions. Knockdown of SMILE gene expression by pSuper vector-mediated RNA interference was sufficient to increase the transactivation of ERs in ER-positive MCF-7 and T47D cells, indicating that SMILE was functionally silenced. Importantly, SMILE fails to inhibit the transactivation of ERs with silencing of SHP. These results clearly suggest that the inhibitory effect of SMILE on the transactivation of ERs depends on SHP in MCF-7 and T47D cells. Moreover, our RT–PCR results demonstrated that SMILE and SHP are able to down-regulate the gene expression of the ER target E2F1 in MCF-7 and T47D cells. There is growing evidence suggesting that co-repressors play important roles in regulating ER-dependent tumorigenesis [5,52,53]. Therefore the fact that SMILE isoforms enhance the inhibition of SHP on the transactivation of ERs in ER positive MCF-7 and T47D cells clearly indicates that SMILE may play an important role in ER-dependent tumorigenesis by regulating the gene expression of some cell-cycle-related ER targets, such as E2F1 and cyclin D1. Certainly, the mechanisms involved in the enhancement of SMILE on the inhibition of transactivation of ERs by SHP remain to be elucidated clearly. In conclusion, our results suggests that the SMILE isoforms derived from alternative translation regulate SHP function in repressing transcriptional activity mediated by ERs in a cell-type-specific manner and SMILE acts as a novel regulator of ER signalling. In light of our new findings, further work will be necessary to determine whether SMILE isoforms regulate the function of other NRs via the SMILE–SHP association. We thank Dr H. J. Lee and K. S. Kim for their technical assistance. This work was supported by a National Research Laboratory grant (ROA-2005-000-10047-0) and by the KRF (Korea Research Foundation) (2006-005-J03003).

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Received 15 April 2008/7 July 2008; accepted 25 July 2008 Published as BJ Immediate Publication 25 July 2008, doi:10.1042/BJ20080782

c The Authors Journal compilation c 2008 Biochemical Society

Biochem. J. (2008) 416, 463–473 (Printed in Great Britain)

doi:10.1042/BJ20080782

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SMILE, a new orphan nuclear receptor SHP-interacting protein, regulates SHP-repressed estrogen receptor transactivation Yuan-Bin XIE*, Ok-Hee LEE†, Balachandar NEDUMARAN*, Hyun-A SEONG‡, Kyeong-Min LEE§, Hyunjung HA‡, In-Kyu LEE§, Yungdae YUN† and Hueng-Sik CHOI*1 *Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea, †Department of Life Science, Ewha Woman’s University, Seoul 120-750, Republic of Korea, ‡Department of Biochemistry, School of Life Sciences, Chungbuk National University, Cheongju 361-763, Republic of Korea, and §Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu 700-721, Republic of Korea

Figure S1

SMILE enhances the inhibition of SHP on ER-mediated transactivation in T47D cells

Reporter assays in (A, B) were performed as described in the Materials and methods section of the main text and the activity of the reporter gene alone was considered to be 1. Results are means + − S.D. (n = 3). *P < 0.05, using Student’s t test. (A) SMILE increases the inhibitory effect of SHP on endogenous ER-mediated transcriptional activity in T47D cells. (B) SMILE siRNA induces endogenous ER-mediated transactivation in T47D cells. T47D cells were transfected with the indicated siRNA expression vector and after 24 h, the cells were co-transfected with ERE–Luc and the SMILE expression vector together with pCMV-β-gal vector. After a 24 h transfection, cells were treated with or without E2 for 24 h before luciferase activity was measured. (C) Effect of siRNAs on the expression of SHP and SMILE in T47D cells. T47D cells were transfected with pSUPER siSHP, pSUPER siSMILE-I, pSUPER siSMILE-II or pSUPER [control (con)], and after 72 h total RNA or protein were isolated. The mRNA expression of SHP and SMILE was measured by RT–PCR analysis (upper panel), with β-actin shown as a control. The protein expression of SMILE was detected by Western blot analysis (lower panel), with tubulin shown as a control. Results are representative of three experiments. wt, wild-type.

Received 15 April 2008/7 July 2008; accepted 25 July 2008 Published as BJ Immediate Publication 25 July 2008, doi:10.1042/BJ20080782

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To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2008 Biochemical Society