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Mol. Cells OS, 443-453, November 30, 2008 Molecules and Cells ©2008 KSMCB A Novel Human BTB-kelch Protein KLHL31, Strongly Expressed in Muscle and Heart, Inhibits Transcriptional Activities of TRE and SRE Weishi Yu1,2,4, Yongqing Li1,4, Xijin Zhou1, Yun Deng1, Zequn Wang1, Wuzhou Yuan1, Dali Li2, Chuanbing Zhu1, Xueying Zhao1, Xiaoyang Mo1, Wen Huang1, Na Luo1, Yan Yan1, Karen Ocorr1,3, Rolf Bodmer1,3,*, Yuequn Wang1,*, and Xiushan Wu1,* The Bric-a-brac, Tramtrack, Broad-complex (BTB) domain is a protein-protein interaction domain that is found in many zinc finger transcription factors. BTB containing proteins play important roles in a variety of cellular functions including regulation of transcription, regulation of the cytoskeleton, protein ubiquitination, angiogenesis, and apoptosis. Here, we report the cloning and characterization of a novel human gene, hieiPN, from a human embryonic heart cDNA library. The cDNA of hieiPN is 5743 bp long, encoding a protein product of 634 amino acids containing a BTB domain. The protein is highly conserved across different species. Western blot analysis indicates that the KLHL31 protein is abundantly expressed in both embryonic skeletal and heart tissue. In COS-7 cells, KLHL31 proteins are localized to both the nucleus and the cytoplasm. In primary cultures of nascent mouse cardiomyocytes, the majority of endogenous KLHL31 proteins are localized to the cytoplasm. KLHL31 acts as a transcription repressor when fused to GAL4 DNA-binding domain and deletion analysis indicates that the BTB domain is the main region responsible for this repression. Overexpression of KLHL31 in COS-7 cells inhibits the transcriptional activities of both the TPA-response element (TRE) and serum response element (SRE). KLHL31 also significantly reduces JNK activation leading to decreased phosphorylation and protein levels of the JNK target c-Jun in both COS-7 and Hela cells. These results suggest that KLHL31 protein may act as a new transcriptional repressor in MAPK/JNK signaling pathway to regulate cellular functions. INTRODUCTION The Bric-a-brac, Tramtrack, Broad-complex (BTB) or POZ (Poxvirus zinc finger) domain was originally identified as a motif present in the aêçëçéÜáä~= ãÉä~åçÖ~ëíÉê Tramtrack (Ttk) and Broad Complex (BR-C) zinc finger proteins (Couderc et al., 2002; Zollman et al., 1994). In most BTB containing proteins, the BTB domain acts as a protein-protein interaction module that is able to both self-associate and interact with non-BTB proteins (Geyer et alK, 2003). This evolutionarily conserved protein-protein interaction motif is often found at the NH2terminus of developmentally regulated zinc-finger transcription factors, as well as in some actin associated proteins bearing the kelch motif. The kelch motif is an ancient and evolutionarilywidespread sequence motif of 44-56 amino acids. It occurs as groups of five to seven repeats and has been identified in proteins of otherwise distinct molecular architecture, termed the kelch-repeat superfamily (Prag et al., 2003). Recently, the BACK domain (BTB and COOH-terminal kelch) has been described in several BTB-kelch proteins, however, its function is unknown (Stogios et al., 2004). The BTB-kelch motif is a highly evolutionarily conserved domain, the motif has been found in an increasing number of proteins including poxvirus, `~ÉåçêJ Ü~ÄÇáíáë=ÉäÉÖ~åë, wÉÄê~ÑáëÜ, and humans. It is generally found at the NH2 terminus of either actin-binding or, more commonly, nuclear transcriptional regulatory proteins (Li et al., 1999). Of the full-length human genes that encode a BTB domain, approximately two-thirds also encode C2H2 zinc finger modules; of the remaining one-third approximately 50% also contain the kelch motif (Carim-Todd et al., 2001). Diverse cellular functions have been identified for a few BTB containing proteins, including transcription regulation (Ahmad et al., 2003; Melnick et al., 2000), cytoskeleton regulation (Kang et al., 2004; Ziegelbauer et al., 2001), tetramerization and gating of ion channels (Kreusch et al., 1998; Minor et al., 2000), protein ubiquitination/degradation (Kobayashi et al., 2004; Stogios et al., 2005; Wilkins et al., 2004; Xu et al., 2003), tumorogenesis (Nakayama et al., 2006), and apoptosis (Qi et al., 2006). The BTB protein PLZF is a growth suppressor that blocks proliferation and myeloid differentiation through silencing of target genes, including cell cycle regulators (Shaknovich et al., 1998; Yeyati et al., 1999). KLHL10, like other BTB/kelch proteins, 1 The Center for Heart Development, Key Lab of Ministry of Education for Development Biology and Protein Chemistry, College of Life Sciences, Hunan Normal University, Changsha, 410081, Hunan, Peoples’ Republic of China, 2Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China, 3Burnham Institute for Medical Research, CA 92037, USA, 4These authors contributed equally to the work. *Correspondence: [email protected] (XW), [email protected] (YW), [email protected] (RB) Received December 8, 2007; revised August 3, 2008; accepted August 20, 2008; published online August 22, 2008 Keywords: BTB domain, deletion analysis, JNK, overexpression, transcriptional repressor, TRE 444 Suppression of MAPK/JNK Pathway by KLHL31 interacts with CUL3 to form a CUL3-based ubiquitin E3 ligase that functions specifically in the testis to mediate protein ubiquitination during spermiogenesis (Wang et al., 2006). KLHL6 plays an important role in BCR (B-cell antigen receptor) signal transduction and formation of the full germina center response (Kroll et al., 2005). The KRIP6 protein can directly regulate native kainate receptors and provides the first evidence for direct functional regulation by a BTB-kelch protein of a mammalian glutamate receptor (Laezza et al., 2007). Another BTBkelch protein KLEIP is a novel regulator of endothelial function during angiogenesis that controls the VEGF-induced activation of Rho GTPases (Tanju et al., 2007). However, the physiological and biochemical functions of numerous BTB-kelch proteins remain uncharacterized. With the aim of identifying genes with transcription regulatory activity involved in human heart development and diseases, a novel BTB-kelch gene hieiPN was cloned from an embryonic cDNA heart library. KLHL31 encodes a predicted protein of 634 amino acids containing a BTB domain at the NH2-terminus, six kelch repeats at the COOH-terminus, and a BACK domain between the BTB and Kelch domains. Western blot analysis shows that a 70 kDa transcript product is expressed in several early human embryonic tissues, with an especially strong level of expression in skeletal muscle and heart. We show that the KLHL31 protein has strong transcriptional suppression activity when fused to GAL4 DNA-binding domain in COS-7 cells. Overexpression of KLHL31 in COS-7 inhibits the transcriptional activities of TRE/AP-1 (TPA-response element) and SRE (serum response element) in a dose dependent fashion. We show that the KLHL31 protein inhibits the transcriptional activity of TRE in the presence of c-jun overexpression. Our data also suggest that KLHL31 may play a role in mediating UV-induced phosphorylation of JNK in the MAPK signal pathway. MATERIALS AND METHODS Construction of cDNA library of human embryonic heart Total RNA from 20-week human embryo heart was extracted using standard methods as previously described (Ai et al., 2007). Embryos were obtained with the consent of the patients and according to the guidelines approved by Hunan Nomal University of Ethics Committee, and with the approval of the Changsha Women and Children’s Hospital, People’s Republic of China. Isolated RNA was pretreated with DNase I (RNase free) to eliminate DNA contamination. mRNA preparation and reverse transcription reactions were performed using a cDNA PCR Library Kit and cDNA Synthesis kit according to manufacturer’s protocol (TaKaRa Biotechnology, China). Briefly, 5 μg mRNA was purified from 500 μg total RNA using Rapid mRNA™ purification Kit (Amresco, Solon, USA). Reverse transcription reactions were performed with the purified embryonic heart mRNA and oligo dT-RA primer according to the cDNA Synthesis kit protocol. After Cassette Adaptor Ligation reactions using cDNA PCR Library Kit, cDNA amplification reactions were performed with RA (5′-CTGATCTAGACCTGCAGGCTC3′), CA primer (5′-CGTGGTACCATGGTCTAGAGT-3′), and Ex Taq (TaKaRa Biotechnology, China). Blast searching and bioinformatics analysis The nucleotide sequence of human hieiPN (NCBI Gene ID: 401265) was obtained from the NCBI website (http://www.ncbi. nlm.nih.gov). BLASTn program was used to search a human EST database. The Blastn program was applied to identify the cytological locus of genes and to look for exons and introns. The homologs of KLHL31 were found with BLASTp, and the sequence alignment and phylogenetic tree analysis were performed with MegAlign program (BGI Life Tech, China) (Cai et al., 2006). Cloning of KLHL31 and rapid amplification of 5′ cDNA ends (5′RACE) PCR was performed using a PCRSPRINT reactor (Thermo Fisher Scientific Inc, Shanghai, China) with one pair of degenerate oligonucleotide primers P1 and P2 (Table 1) corresponding to the DNA sequence of KLHL31 ORF (open reading frame) (Accession number: NM_001003760). Amplification was carried out at 94°C, 4 min; 94°C, 30 s; 60°C, 30 s; and 72°C, 2 min for 35 cycles; then 72°C, 10 min. The amplification products were separated by agarose gel and the bands were cloned into a pMD18-T vector (TaKaRa Biotechnology, China). Transformants were randomly chosen and sequenced with DNA Sequencer (ABI PRISM 3730, USA) according to the manufacturer’s procedures. 5′-RACE was performed using a SMART™ RACE cDNA Amplification Kit (TaKaRa Biotechnology, China). The primer specific for the 5′ end of the cDNA was PR1, and the nested primer was PR2 (Table 1). All the PCR products were then cloned into pMD18-T-vector (TaKaRa Biotechnology, China) and sequenced. Sequence analysis was performed using the BLASTn program from NCBI. Finally, they were assembled into contigs to complete the full-length cDNA. Expression of GST fusion proteins and preparation of KLHL31 polyclonal antibody For GST fusion proteins expresion, the PCR products of KLHL31 and KLHL31 (170-410 aa) were cloned into the pGEX4T-1 vector at the EÅçRI sites, and the insertion was confirmed by DNA sequencing. The primers for KLHL31 and KLHL31 (170-410aa) were P2S, P2AS and PE2S, PE2AS, respectively (Table 1). The recombinant fusion protein vector was transformed and expressed in= bKÅçäá BL21 (Invitrogen, Carlsbad, US). Bacteria were grown at 37°C until the culture reached an OD600 of 0.4-0.6, and expression was induced by adding 0.2 mM isopropyl-beta-a-thiogalactopyranoside (IPTG) for 6 h at 26°C. After sonication for 15 min on ice, the bacteria were centrifuged at 12,000 rpm= for 10 min at 4°C to obtain supernatants containing the GST-KLHL31 and GST-KLHL31 (170-410 aa)=proteins. The soluble fusion protein was purified on a glutathione-Sepharose column (Amersham Biosciences UK, UK) and subjected to preparative scale SDS-PAGE. The major band was excised and finally eluted with 10 mM glutathione in 50 mM Tris-HCl (pH 8.0). The GST-KLHL31 (170410 aa)=fusion protein was used to immunize rabbits for polyclonal antiserum production and the polyclonal antibody was purified from the sera using 60% (NH4)2SO4 salt-out and NHSactivated Sepharose 4 Fast Flow (Amersham Biosciences UK, UK). The antibody specificity was confirmed by Western blotting using recombinant KLHL31 protein expressed in bacterial and eukaryotic cells. Tissue protein extraction Human tissues from therapeutically aborted fetuses (16 weeks) were obtained under the approval of Changsha Women and Children’s Hospital, People’s Republic of China, with the consent of the patients, and with the approval of Hunan Nomal University of Ethics Committee. The following fetal tissues were used: brain, kidney, heart, liver, lung, muscle, stomach and small intestine. Protein extracts were prepared from frozen human tissues (0.2 g) pulverized in liquid nitrogen to fine powders. Extraction buffer [1 ml: 7 M Urea, 2 M Thiourea, 60 mM Weishi Yu et al. Table 1. Sets of specific oligonuleotide primers Primer Orientation Nucleotide sequence P1 Sense P2 Anitsense 5′ TCTACCTGGGCTCAGATACTGA 3′ 5′ AACAGTGTATTTGCCAACATGG 3′ PR1 Antisense 5′ TTCTCAACACTCATCTCCCGTAT 3′ PR2 Antisense 5′ CTGAAGATAAACAGCAGCAGAAAT 3′ P2S Sense 5′ TGAATTCGCCAACATGGCACCCAAA 3′ P2AS Antisense 5′ CGGAATTCGATCTACCTGGGCTCAGATACT 3′ PE2S Sense 5′ GTGAATTCCTGATACGGGAGATGAGTG 3′ PE2AS Antisense 5′ ATGAATTCGCTCAGGCTGAAGTGC 3′ P3 Antisense 5′ TAGTCGACCGCAGATACTGACTGGCACAG 3′ P4 Antisense 5′ GGAATTCGTCAAGGGAGTATGTTTCAGCA 3′ P5 Sense 5′ GGGAATTCAGTCCTCGTCACTGTTGG 3′ DTT, 4% CHAPS, 2% pharmalyte 3-10, 1.4 mg/ml PMSF] was added to the powdered tissue and the mixture was incubated on ice for 30 min with repeated shaking and vortex-mixing every 10 min. Mixtures were centrifuged at 14,000 rpm at 4°C for 30 min. The supernatants containing the tissue extracts were collected and stored at -80°C until use. Sample protein content was estimated using the Thermo Scientific Pierce BCA Protein Assay (Thermo Scientific, USA). Plasmid construction To generate a fusion protein between KLHL31 and enhanced green fluorescent protein (EGFP), the KLHL31 ORF was amplified by PCR (primer P2S, P3) (Table 1) and then subcloned into the bÅçRI and p~äI sites of the pEGFP-N2 vector with the TGC codon replacing the TGA stop codon in the KLHL31 coding sequence. To generate a fusion protein of KLHL31 with GAL4 or FLAG tag, the KLHL31 ORF was amplified by PCR with primer P2S, P2AS (Table 1) and then subcloned in-frame to the bÅçRI site of the pCMV-BD and pCMV-Tag2B, respectively. To identify which domain(s) of KLHL31 contribute to its transcriptional activity, we generated three GAL4-BD fusion proteins corresponding to the different functional domains of KLHL31 for our deletion analysis. The BACK fragment was cleaved from pGEX4T-1-KLHL31 (170-410aa) with bÅçRI and inserted into the pCMV-BD vector producing GAL4-BD-BACK. The BTB and Kelch-repeat fragments were amplified by PCR with primers P2S, P4 and P5, P2AS (Table 1) for the GAL4BD-BTB and GAL4-BD-Kelch constructs, respectively, and then subcloned inframe into the bÅçRI site of the pCMV-BD expression vector. Cell culture, transient transfection, and subcellular localization analysis COS-7 and Hela cells were maintained and passaged according to standard methods in Dulbecco’s Modified Eagle Medium (DMEM, Gibco BRL, US) supplemented with 10% fetal calf serum (FCS) in an humidified atmosphere of 95% air and 5% CO2. Neonatal mouse cardiomyocytes were cultured as described previously with minor modifications (Fu et alK, 2005). Briefly, left ventricles were isolated in ice-cold ADS buffer and cultured by a series of digestions with a mixture of pancreatin (0.5 mg/mL) and collagenase (0.6 mg/ml) at 37°C. Non myocyte contamination was minimized by preplating cells onto tissue culture dishes (Falcon) at 37°C for 30 min. The cells were plated at a density of 6 million per 10 cm primeria plate. The cardiomyocytes were re-suspended in 5% myocyte media con- 445 taining BrdU (0.1 mM). COS-7 Cells were transfected with pEGFP-N2-KLHL31 using LipofectAMINE (Invitrogen, USA) according to the method described previously (Cai et al., 2006). The 24 h after transfection, cells were fixed with 4% paraformaldehyde for 15 min and washed with PBS three times and nuclei were stained with 4′, 6′-diamidino-2-phenylindole hydrochloride (DAPI, Roche, Basel, Switzerland). Subcellular localization of the EGFP-KLHL31 fusion protein was detected using an inverted fluorescence microscopy (E400, Nikon). Since KLHL31 is highly conserved between mouse and human (identity 92%), affinity purified rabbit polyclonal anti-human KLHL31 (1:100) was applied as the primary antibody to explore distribution of endogenous KLHL31 protein in primary nenonatal mouse cardiomyocytes. F(ab’)2-PE-Cy3, goat anti-rabbit IgG (Santa Cruz Biotechnology Inc., USA) was used as secondary antibody. Immunostaining was performed as previously described (Kim et al., 2003). Briefly, cells cultured 48 hours were fixed with cold methanol and were then blocked for 20 min in PBS, 1% bovine serum albumin, 10% goat serum, and 0.05% Triton X-100. Next, the cells were incubated in primary antibody diluted in PBS, 5% goat serum, and 0.2% Triton X-100 for 1.5 h followed by secondary antibodies for 1 h. Cells were stained with Hoechst 33258 (Molecular Probes, USA) to visualize nuclei and mounted with Prolong mounting medium (Invitrogen, USA), and examined using a fluorescence microscope. Transient expression reporter gene assay The pL8G5-Luc and pLexA-VP16 constructs were used for mammalian cell transfection in the luciferase reporter assay (Cai et al., 2006). To examine the potential transcriptional activity of KLHL31, the pCMV-BD-KLHL31, pCMV-BD-BTB, pCMVBD-BACK, pCMV-BD-Kelch, and pCMV-BD were transiently cotransfected into COS-7 cells along with the pL8G5-Luc reporter and pLexA-VP16 using LipofectAMINE as described above (Cao et al., 2005). In order to test the transcriptional regulation of KLHL31 in the MAPK signaling pathway, the PathDetect AP-1 cis-Reporting System (pTRE-Luc) and the PathDetect SRE cisReporting System (pSRE-Luc) from Stratagene were used. pFA2-cJun or pFA2-cfos was co-transfected with pTRE-Luc or pSRE-Luc as shown in Fig. 5. The luciferase activity assay was performed according to the protocols of Stratagene (Cao et al., 2005). The luciferase activity was normalized for transfection efficiency by co-transfection with pCMV-lacZ. The data presented are the mean of three individually transfected wells. The experiments were performed in triplicate. Western blotting analysis The expression pattern of KLHL31 was analyzed using Western blotting analysis. The 50 μg protein of each tissue sample extract was separated by 10% SDS polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% skimmed milk in TBS-T [150 mM NaCl, 10 mM Tris-HCl (pH 7.5) and 0.1% tween 20], the membrane was incubated with the first antibody, anti-KLHL31 prepared in this study or with anti-β-actin antibodies from Santa Cruz (Cell Signaling Technology Inc., USA). The secondary antibody was a horseradish peroxidase-conjugated (HRP) goat anti-rabbit IgG antibody (Chemicon International Inc., USA), and detection was performed using the DAB and H2O2. For cell total protein western analysis, COS-7 and Hela cells transfected with pCMV-Tag2B or pCMV-Tag2B-KLHL31 were lyzed in RIPA-lysis buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 50 mM NaF,2 mM EDTA, 1 mM Na3VO4, 1% NP-40, 1 μg/ml Leupeptin,1 μg/ml Pepstatin,1 mM PMSF). PVDF mem- 446 Suppression of MAPK/JNK Pathway by KLHL31 brane was incubated with the first antibody of anti-Flag or antic-Jun antibodies from Cell Signaling Technology (Cell Signaling Technology Inc., USA). UV irradiation and starvation treatment UV irradiation was performed as described previously (Rozek et al., 1993). The 24 h after transfection, cells were transferred to DMEM without serum and immediately treated with 100 J/m2 of UV irradiation using a calibrated UV light source (General Electric, UVP UVGL-25). After treatment with the aforementioned agents, the cells were incubated for 40 min at 37°C for JNK/cJun phosphorylation assays. PVDF membrane was incubated with the first antibody: c-Jun, c-fos, or phospho-c-Jun (Ser73), and SAPK/JNK or phospho-SAPK/JNK (Thr183/Tyr185) antibodies from Cell Signaling Technology (CST, US). The secondary antibody was an HRP goat anti-rabbit IgG antibody (Chemicon International Inc., Canada), and detection was performed using the Amersham ECL Plus Western Blotting Detection System (GE Healthcare, UK). RESULTS Molecular characterization and evolutionary conservation of the KLHL31 gene We screened the human EST database with the partial nucleotide sequence of the human hieiPN gene. To confirm the cDNA sequences identified from the database, one pair of primers (P1 and P2, Table 1) based on the mRNA sequences (NM_001003760) was used to carry out standard PCR using the human embryonic heart cDNA library as template. A single 1905 bp fragment was obtained under standard PCR conditions and subcloned into a T-vector. Sequence analysis indicated that the PCR product included the partial hieiPN cDNA sequence. To obtain the full-length cDNA of hieiPN, 5′RACE was performed using 5′-gene specific nested primers: 5′ TTCTCAACACTCATCTCCCGTAT 3′ and 5′CTGAAGATAAACAGCAGCAGAAAT 3′ (Supplementary Fig. 1). These procedures yielded approximately a 600-bp DNA for 5′ RACE fragment, the product was then cloned and sequenced. An analysis of the fragment suggested that it was a cDNA fragment from the human gene hieiPN. A sequence of 5743 bp constituting the fulllength gene was assembled, and the nucleotide sequence reported here is available in GenBank with accession number EF633513. The complete sequence of the hieiPN cDNA is 5743-bp in length and contains a putative open-reading frame of 1905 nucleotides. There is a Kozak sequence, AACATGG, in the translational start site and there is a potential polyadenylation signal, AATAAA, in the 3′ untranslated region (UTR). The protein predicted from the open-reading frame contains 634 amino acids and has a calculated relative molecular mass of 70.2 kDa. Furthermore, analysis of the KLHL31 using the SMART program indicated the presence of a BTB domain at the NH2terminus and six-kelch motifs in the COOH-terminus of KLHL31 protein (Fig. 1A). Comparison of the KLHL31 sequence with the genomic sequence shows that KLHL31 maps to chromosome region 6p12.1 and spans approximately 18 kb on the genome. The KLHL31 gene consists of three exons and two introns, and all exon-intron junctions contain the gt/ag consensus splice site (Table 2). BLAST searches using the KLHL31 sequence identified closely related sequences in `K=ÉäÉÖ~åë, aêçëçéÜáä~, zebrafish, uK=íêçéáÅ~äáë, chicken, and mouse. Sequence comparison of the BTB motif among its orthologs indicates that the degree of Table 2. Genomic structure of the hieiPN gene Intron Exon Number Size Intron Size I 108 AAGGAGGgtaaggagc 10290 tctttttagCAAGCTC II 1205 TCTGCAGgtacttgatct catttccagATACGAT III 4430 1765 Genomic organization of the KLHL31 gene from 6p12.1. Exon and intron sizes are given in base pairs. Intronic and exonic sequences are shown in lower-case and upper-case characters, respectively. The acceptorsplice site ~Ö and the donor-splice site Öí are shown in bold. conservation in this domain is quite high among the different family members, especially in vertebrates (Fig. 1B-a). We then aligned the sequence containing the BACK and Kelch-repeats with their orthologues. This comparison shows that BACK and Kelch domains are also conserved during evolution from `K=ÉäÉJ Ö~åë to aêçëçéÜáä~, and mouse to human (Figs. 1B-b and 1B-c). A phylogenetic tree analysis using DNAstar software suggests that KLHL31 is an evolutionarily conserved gene (Fig. 1C). Preparation and identification of anti-KLHL31 antibody In order to examine the expression of the KLHL31 gene, we generated antibodies using the GST-KLHL31-(170-410aa) fusion protein, which encodes a segment (aa 170-410) between the BTB domain and kelch motifs of KLHL31. After induction with IPTG, we found GST-KLHL31 (170-410aa) was expressed in bacteria BL21, and we subsequently purified the GST fusion protein (Supplementary Fig. 2). To characterize the anti-KLHL31 antiserum, Western blotting was performed using recombinant protein and áå=îáíêç=translated KLHL31, and nuclear extracts prepared from COS-7= cells (Fig. 2A). The results show that affinity-purified anti-KLHL31 antibodies specifically recognized the translated KLHL31 in COS-7 cells and the recombinant protein GST-KLHL31 (lanes 3 and 4); no bands were resolved in the control reaction using rabbit pre-immune serum and empty vector as template (lanes 1 and 2). Expression of KLHL31 protein in human embryos In an effort to understand the expression pattern of the KLHL31 in early human embryogenesis, we used the polyclonal antibody of KLHL31 as a probe to examine expression in multiple tissues during human embryo development. As shown in Fig. 2B, Western blotting analysis detected a 70 kDa protein product in a few embryonic tissues, with strong expression levels in skeletal muscle and heart. KLHL31 was also expressed in brain, liver, and kidney, although at much lower levels. Previously, KLHL31 has been reported to be expressed only in adult skeletal and cardiac muscles (Wu et al., 2004). The expression pattern suggests that KLHL31 might be involved in human embryonic development. KLHL31 is a nuclear and cytoplasmic protein To examine the subcellular location of KLHL31, the pEGFP-N2KLHL31 was transfected into COS-7 cells, and 24 h after the transfection, the cells were visualized with epifluorescence microscope after labeling with the nuclear stain DAPI. EGFPKLHL31 protein is distributed in both the nucleus and cytoplasm of COS-7 cells (Fig. 3A). To explore the distribution of endogenous KLHL31 in cardiomyocytes, immunocytochemistry in neonatal mouse primary cardiomyocytes was performed using affinity purified rabbit polyclonal anti-human KLHL31 as the primary antibody and F(ab’)2-PE-Cy3, goat anti-rabbit IgG Weishi Yu et al. A B a b c 447 Fig. 1. KLHL31 is conserved BTB-kelch protein during evolution. (A) The domain structure of KLHL31. Schematic diagram of a BTB (amino acids 73-167), a BACK (amino acids 172-273), and six consecutive kelch repeats (KH; amino acids 317614) of the KLHL31 protein. (B-a) Alignment of BTB domain, (B-b) BACK domain and six kelch domains with other orthologues. Highly conserved amino acids are indicated in deep grey. DDBJ/ EMBL/GenBank Accession Nos. for the sequences are mouse Klhl31 (NM_ 172925); rat (XM_236428); aêçëçéÜáä~ (NM_080250); dK= Ö~ääìë (XM_419909); zebrafish (NM_001003727); uK=íêçéáÅ~äáë (BC077340); `K= ÉäÉÖ~åë= (NM_058921). The consensus sequence of the domain is calculated and colored using Chroma. (C) Un-rooted phylogenetic tree analysis of KLHL31 and other homologue proteins. In addition to the proteins shown in (B), Loc481846 (Canis), F7A7_180 (Arabidopsis thaliana), yjhT (Coli), and SPCC 1223 (yeast) were also used in the analysis. C as secondary antibody. As shown Fig. 3B, the endogenous KLHL31 is also located in both the cytoplasm and nucleus of mouse cardiomyocytes, however, most of the KLHL31 signal is localized to the cytoplasm. KLHL31 is a transcriptional repressor To examine the potential function of KLHL31 in transcriptional regulation, we constructed a fusion protein of KLHL31 with the DNA binding domain (BD) of the yeast transcription factor GAL4 under the control of the CMV promoter (Ai et al., 2007). This construct, pCMV-BD-KLHL31, was co-transfected with pL8G5-Luc reporter gene and pLexA-VP16 into COS-7 cells. The pL8G5-Luc reporter contains eight copies of the LexA DNA binding sites and five copies of the GAL4 DNA binding sites linked to the luciferase reporter gene. pLexA-VP16 increases luciferase expression through binding to the LexA-binding site of pL8G5-Luc. When co-transfected with the pL8G5-Luc plasmid, the GAL4 BD-KLHL31 fusion protein inhibited the endogenous luciferase activity by 75%, and co-expression of GAL4BD-KLHL31 with pLexA-VP16 significantly inhibited the VP16activated luciferase activity by 66% (Fig. 4A), suggesting that KLHL31 functions as a transcriptional repressor. To further identify potential transcriptional regulatory domains in KLHL31, we constructed three deletion mutants of GAL4KLHL31, pCMV-BD-BTB (1-184aa), pCMV-BD-BACK (170410aa) and pCMV-BD-Kelch-repeat (317-634aa). As shown in Fig. 4B, pCMV-BD-BTB inhibited the luciferase activity of reporter gene by 60% and the pCMV-BD-Kelch-repeat inhibited 448 Suppression of MAPK/JNK Pathway by KLHL31 A B Fig. 2. The tissue distribution of KLHL31 transcript. (A) Identification of polyclonal antibodies against KLHL31. få= îáíêç= translated products Flag-KLHL31 detected with the rabbit pre-immune serum (lane 1). få=îáíêç=translated products using either the Flag-vector (lane 2) or Flag-KLHL31 (lane 3) as template detected with purified antiKLHL31 antibodies. The recombinant protein GST-KLHL31 (lane 4) detected with purified anti-KLHL31 antibodies was used as a positive control. The ~êêçï=indicates Flag-KLHL31; the ~êêçïÜÉ~Ç=indicates GST-KLHL31 protein. (B) Expression of KLHL31 in human embryonic tissues (16 weeks) analyzed by Western blot. A protein product of approximately 70 kDa was detected in most tissues examined, with highest levels in skeletal muscle and heart. β-actin was used to confirm equal loading of total protein. luciferase activity by 30%. These results indicate that both the NH2-terminal BTB domain and the COOH-terminal Kelchrepeat motif contribute to transcriptional repression by KLHL31, and that the BTB domain is the main target for the transcriptional repressor activity of KLHL31. KLHL31 suppresses TRE and SRE-mediated transcriptional activation In order to investigate the role of KLHL31 in cell signal transduction, we examined whether KLHL31 was directly or indirectly involved in regulating the activity of transcription factors, specificially MAPK-mediated transcriptional regulation. MAPK signal transduction pathways are the most widespread mechanisms of eukaryotic cell regulation (Reszka et al., 1995). To examine the effect of KLHL31 on this cell-signaling pathway, we performed pathway-specific reporter gene assays to meas- A a b c B d e f ure the transcription of TRE and SRE by KLHL31 in the COS-7 cells. Firstly, using pTRE-Luc, designed for monitoring induction of TRE, we tested the effect of KLHL31 on the transcriptional activity of TRE. Expression of KLHL31 significantly inhibited TRE transcriptional activity by approximately 55% alone, by 80% in presence of c-Jun, by 25% in presence of c-fos, and by 68% in presence of c-jun and c-fos (Fig. 5A). Furthermore, using a pSRE-Luc reporter designed for monitoring the induction of SRE, we demonstrated that KLHL31 also strongly inhibited SRE transcriptional activity by 59% alone and by 50% in presence of c-Jun (Fig. 5B). In a further experiment, different amounts of pCMV-Tag2B-KLHL31 were co-transfected with a fixed amount of pTRE-Luc or pSRE-Luc into COS-7 cells. As shown in Figs. 5C and 5D, KLHL31 caused dose-dependent repression of TRE and SRE activity. To confirm the suppressive effect of KLHL31 in the MAPK pathway, we examined protein levels of the downstream transcriptional factor c-Jun by Western blot in COS-7 and Hela cells. Overexpression of FLAG-KLHL31 significantly reduced c-Jun protein levels in both COS-7 and Hela cells (Figs. 6A and 6B), and KLHL31 caused a dose-dependent repression of c-Jun protein levels. However, c-fos protein levels were not affected in Hela cells after 12 h of starvation (Fig. 7A). KLHL31 blocks UV-induced activation of JNK and c-Jun phosphorylation The transcription factor c-Jun is one of the major targets of the JNK-signaling pathway. JNK phosphorylates c-Jun at Ser-63 and Ser-73 leading to an increase in its transcriptional activity (Davis, 2000). To determine whether the activation of c-Jun is affected by KLHL31, we examined the protein level of phosphoc-Jun in COS-7 cells. We found that the levels of phospho-cJun were significantly reduced in cells where KLHL31 is overexpressed (Fig. 7B, lanes 1-3). It is possible that KLHL31 action is exerted through the dephosphorylation of JNK/SAPK resulting in a suppression of c-Jun activation. To determine whether KLHL31 dephosphorylates JNKs, we then examined the protein levels of JNK/SAPK and phospho-JNK/SAPK in these cells. As shown in lanes 1-3 of Fig. 7B, the overall protein levels of JNK/SAPK were not affected by KLHL31, but the phosphorylated form of the protein could not be detected. JNK was identified almost 10 years ago as a group of MAP kinases that are activated by UV exposure and other external stresses (Hibi et al., 1993). Consequently, we determined c-Jun levels following JNK activation in response to UV irradiation. As shown in Fig. 7B (lanes 4 and 5), UV-treated COS-7 cells exhibit relatively high levels of phosphorylated c-Jun and JNK, We then determined whether this phosphorylation was affected by Fig. 3. Cellular localization of KLHL31 protein in cells. (A) Showing subcellular localization of KLHL31 when overexpressed in COS-7 cells. (a) EGFP-KLHL31 is localized both in the cell nucleus and cytoplasm of COS-7 cells. (b) Cell nuclei stained with DAPI. (c) The combined image of (a) and (b). (B) Cellular localization of endogenous KLHL31 protein in primary neonatal mouse cardiomyocytes. (d) The majority of KLHL31 is localized in the cytoplasm of the cells; only a relatively small amount KLHL31 signal is observed in the nuclei. (e) Cell nuclei stained with Hochest. (f) The combined image of (d) and (e). Weishi Yu et al. A 449 B Fig. 4. KLHL31 is a repressive regulator of transcription. (A) pCMV-BD-KLHL31 is transiently transfected into COS-7 cells along with the pL8G5-Luc reporter, the GAL4-KLHL31 fusion protein inhibited the luciferase activity by 75%. After cotransfection with pLexA-VP16, the VP16activated luciferase activity was significantly inhibited by 66%. (B) pCMV-BD-KLHL31-BTB, pCMV-BD-KLHL31-BACK, pCMV-BD-Kelch and pCMV-BD-KLHL31 are transiently transfected into COS-7 cells along with the pL8G5-Luc reporter and pLexA-VP16. Control, no GAL4 fusion protein was transfected. The BTB domain represses the reporter gene transcriptional activity as well as the full-length of KLHL31 by 60%. The data are the mean of three repeats in a single transfection experiment after normalization for β-galactosidease activity. Each experiment was repeated at least three times. A C B Fig. 5. Overexpression of KLHL31 inhibits transcriptional activities of TRE and SRE. (A) Repression of TRE-Luc transcriptional activity by the overexpression of KLHL31. (B) Repression of SRE-Luc transcriptional activity by the overexpressing of KLHL31. (C, D) Dose-dependent activation of the reporter genes TRE-Luc and SRE-Luc activity by pCMV-Tag2B-KLHL31. COS-7 cells transfected with individual reporter plasmid and the corresponding plasmids are shown in the figures. The data are the mean of three repeats in a single transfection experiment after normalization for β-galactosidease activity. Each experiment was performed at least three times. D KLHL31. As shown in Fig. 7B overexpression of KLHL31 was associated with a significant reduction in phsophorylatioin of cJun and JNK (Fig. 7B, compare lanes 5 and 6), similar results were obtained in Hela cells (Fig. 7B, lanes 7-9). Taken together, these results provide evidence that KLHL31 may interact with MAP kinase to down-regulate JNK signaling, leading to reduced phosphorylation of the JNK target c-Jun. DISCUSSION Recent studies of BTB-kelch family proteins suggest their extensive involvement in development and disease. For example, the KLHL10 interacts with CUL3 and mediates protein ubiquiti- nation during spermiogenesis (Wang et al., 2006). The vaccinia virus kelch-like protein C2L affects calcium-independent adhesion to the extracellular matrix and inflammation in a murine intradermal model (Pires de Miranda et al., 2003). KLHL7 antibodies are associated with various cancers, and also with neurological disease in some patients (Bredholt et al., 2006). Gigaxonin is a novel and distinct cytoskeletal BTB protein that may represent a general pathological target for neurodegenerative disorders (Bomont et al., 2000). The PLZF (promyelocytic leukemia zinc finger) transcriptional repressor, when fused to retinoic acid receptor alpha (RARalpha), causes a refractory form of acute promyelocytic leukemia, and the protein inhibits cell growth and expression of cyclin A. PLZF lacking the BTB 450 A Suppression of MAPK/JNK Pathway by KLHL31 B A B Fig. 6. Western blot analysis of c-Jun. Western blot was performed using the proteins of COS-7 cells (A) and Hela cells (B) transfected with pCMV-Tag2B and pCMV-Tag2B-KLHL31(1 μg or 4 μg plasmids were transfected into cells in 60-mm dish). Overexpression of KLHL31 significantly reduced c-Jun protein levels. Anti-Flag antibody was used to measure the KLHL31 protein level, and β-actin was used as internal control. domain interacts with a Wilson disease protein ATP7B to positively regulate ERK signal transduction (Ko et al., 2006; Melnick= et al., 2002; Yeyati et al., 1999). Finally, the gene encoding the BTB-protein BCL-6, is a proto-oncogene specifically involved in the pathogenesis of large-cell lymphoma (Shaffer et al., 2000; Ye et al., 1993). Here, we report the identification of a novel member of the human BTB-kelch transcriptional factor family, hieiPN, from an embryonic cDNA heart library. This gene has an NH2terminal BTB domain and six COOH-terminal kelch motifs. Like other BTB-kelch family proteins, KLHL31 is highly conserved during evolution. Most BTB family members have been proposed to be transcriptional repressors (Ahmad et al., 2003; Melnick et al., 2000; Qi et al., 2006; Shaffer et al., 2000; Yeyati et al., 1999) and we now show that the BTB protein KLHL31 exerts a strong repressive effect on transcription mediated through TRE and SRE. We also show that the BTB domain alone exerts significant repression on reporter gene activity, although somewhat weaker than that intact KLHL31 (Fig. 4). Interestingly, the kelch motifs also exert a weak repressive effect suggesting that both domains are required for full repressor activity. The MAPK family is an important mediator of signal transduction and is activated by a variety of stimuli, such as growth factors and cellular stresses (Davis, 1994). MAPK are major components of pathways involved in embryogenesis including cell differentiation, cell proliferation, and cell death. One of the most explored functions of MAPK signaling is the regulation of gene expression by direct or indirect phosphorylation and subsequent activation of transcription factors (Whitmarsh et al., 2000). MAPK pathways are also involved in multiple cellular processes through phosphorylation of specific endpoint targets. For example, the persistent elevation of phosphorylated extracellular-signal-regulated kinase (ERK) regulates cell proliferation (Huang et al., 2003). Phosphorylation and activation of JNK and c-Jun, may contribute to uncontrolled cell-cycle progression and oncogenesis (Leventaki et al., 2007). The JNK-dependent phosphorylation of the transcription factor, SRF, plays a crucial role for ÅóêSN expression during neuronal cell death, and the SRE-like CArG domain in the upstream ÅóêSN promoter is necessary for its induction by etoposide (Kim et al., 2003). Some proteins of the BTB family have been shown to regulate the MAPK signaling pathway. For example, overexpression Fig. 7. Overexpression of KLHL31 blocks JNK-mediated c-Jun activity not c-fos. (A) c-fos is induced with serum starvation, overexpression of KLHL31 has no apparent effect on c-fos activation (lane 4). (B) Expression vector or KLHL31 were transfected into cells, and treated 2 with 100 J/m ultraviolet light (UV); lysates were prepared 40 min after stimulation and were assayed for their ability to phosphorylate MAPK signal molecules. Lanes 1-3 shows the protein levels for the untreated COS-7 cells, KLHL31 reduces the phosphorylation of c-Jun. Lanes 46 shows that KLHL31 inhibits UV-induced JNK phosphorylation, and leads to reduced protein levels and reduced phosphorylation of the JNK target c-Jun in COS-7 cells. The UV-irradiation-induced JNK and c-Jun activation are also significantly blocked by expression of KLHL31 in Hela cells (compare lanes 7-9). The experiment was repeated twice with similar results. of Mayven results in an induction of c-Jun protein levels, as well as increased AP-1 transcriptional activity in MCF-7 and T47D breast cancer cells. Furthermore, Mayven activated the c-Jun N-terminal kinase in breast cancer cells (Bu et al., 2005). Using transient transfection and reporter assays, we have shown that KLHL31 represses transcription via both SRE and TRE. KLHL31 also strongly enhances the transcriptional repression exerted by c-Jun in luciferase reporter assays (Fig. 5A). Interestingly, KLHL31 inhibits UV-induced JNK phosphorylation, leading to reduced protein levels and reduced phosphorylation of the JNK target c-Jun (Fig. 7B). KLHL31 had only a weak repressive effect on c-fos (Fig. 5A), implying that the activity of c-fos is not regulated by KLHL31; In addition, overexpression of KLHL31 had no effect on the stress-induced c-fos expression (Fig. 7A). Recent studies suggest that MAPK pathways are critical not only to the response of cardiovascular cells to extracellular stress but also to developmental cues that regulate cardiovascular development (Aggeli et al., 2004; Tanoue et al., 2003). The DNA-binding activity of AP-1/c-Jun was found to be dramatically increased in failing hearts, an increase that was not observed in compensatory cardiac hypertrophy (Freire et al., 2007). In addition, AP-1/c-Jun activation in cardiomyocytes resulted in reduction of α-MHC mRNA (Freire=et al., 2007). cJun is a key component of AP-1 transcription factor complex, and its transcriptional activity is potentiated by phosphorylation of serines 63 and 73 within its transactivation domain by the cJun kinase family of MAPKs (JNK1-3) (Xia et al., 1998). Phos- Weishi Yu et al. phorylation of pre-existing and newly synthesized c-Jun proteins can further enhance their transcriptional activities and inhibit its ubiquitination and degradation (Fuchs et al., 1996; Musti et alK, 1997), supporting a strong autoregulatory loop for c-jun expression and induction of TRE target genes (Angel et alK, 1988; Jeanmougin et alK,=1998; Whitmarsh et alK,=1996). The phosphorylation of transcription factors by MAPK signaling pathways regulates their activities in a number of ways including (i) their intracellular location, (ii) their protein levels, (iii) their binding to DNA, and (iv) their interactions with regulatory proteins (Yang=et al., 2003). With respect to cellular localization, MAPK signaling pathways can either stimulate the translocation of transcription factors to the nucleus to promote their activity or conversely, stimulate the export of transcription factors from the nucleus and hence facilitate their inactivation (Hood et alK, 1999). The efficient phosphorylation of transcription factors by MAPKs requires that these proteins colocalize in cells. Therefore, in addition to mechanisms that control the localization of transcription factors, there are also mechanisms to control the cellular distribution of MAPKs to ensure that the appropriate transcriptional responses to extracellular stimuli are elicited. In unstimulated cells, ERK is predominantly cytoplasmic, but upon activation, it accumulates in the nucleus (reviewed in Lewis et al., 1998). Transfected KLHL31 protein distributes in both the nucleus and cytoplasm of COS-7 cells, but in neonatal mouse primary cardiomyocytes, most of the KLHL31 signal is localized in the cytoplasm (Fig. 3), suggesting the native KLHL31 protein may reduce the phosphorylation of JNK, and block the translocation of JNK to the nucleus to activate the substrate c-Jun. SRE is one of several cis-elements that mediate c-fos induction and is recognized by a dimer of the serum response factor (SRF), which recruits monomeric ternary complex factors, whose members include Elk-1, SAP-1 or SAP-2 (Herrera et al., 1989). SRF function appears to be important for heart development as SRF mutant embryos show defects in both the cardiac compact layer and in the trabeculations, in addition several critical cardiac transcriptional factors were downregulated in these embryos (Parlakian et al., 2004). The absence of SRF in cardiomyocytes and smooth muscle cells (SMC) leads to ultrastructural defects in contractile/cytoskeletal assembly (Miano=et al., 2004). SRF controls SMC gene transcription via binding to CArG box DNA sequences found within genes that exhibit SMC-restricted expression. The SRF-CArG interaction is a critical convergence point for signals that either activate SMC gene expression to promote normal SMC differentiation or repress SMC gene expression during pathophysiological conditions (McDonald et al., 2006). Interestingly, the promoter region of KLHL31 contains a CArG box. Thus, SRF may bind to the CArG boxes of KLHL31 gene and regulate KLHL31 gene function. In conclusion, we have identified and characterized a novel human gene, KLHL31, from a human embryonic heart cDNA library. KLHL31 is highly expressed in human embryonic skeletal and cardiac muscle where it is located in both the cell cytoplasm and in the nucleus. Overexpression of KLHL31 inhibits the transcriptional activities of both TRE and SRE, and downregulates JNK signaling, leading to reduced phosphorylation of the JNK target c-Jun. These results suggest that KLHL31 may act, directly or indirectly, as a negative transcriptional regulator in MAPK-mediated signaling pathways. It is likely that KLHL31 protein acts in synergy with other transcription factors to integrate information from multiple extracellular signals to induce the necessary cellular changes required for tissue specification and morphogenesis and it may also play roles in the regulation of heart development and in heart disease. 451 kçíÉW= pìééäÉãÉåí~êó= áåÑçêã~íáçå= áë= ~î~áä~ÄäÉ= çå= íÜÉ= jçäÉJ ÅìäÉë=~åÇ=`Éääë=ïÉÄëáíÉ=EïïïKãçäÅÉääëKçêÖFK ACKNOWLEDGMENTS We are grateful to all members of the Center for Heart Development, College of Life Sciences in Hunan Normal University for their excellent technical assistance and encouragement. This study was supported in part by the National Natural Science Foundation of China (No. 90508004, 30570934, 30671054, 30671053, 30671171, 30670274, 30671137, 30771146, 30771170, 30871340, 30871417, 30800627), PCSIRT of Education Ministry of China (IRT0445), National Basic Research Program of China (2005CB522505), New Century Excellent Talents in University (NCET-05-0713), a China Postdoctoral Science Foundation (20060400260), the Foundation of Hunan Province (05J2007, 06JJ4120), and Hunan Science and Technology Project (2008NK3108, 2008 RS4011). This work is also partially supported by the Research Platform of Cell Signaling Networks from the Science and Technology Commission of Shanghai Municipality (06DZ22923). REFERENCES Aggeli, I.K., Gaitanaki, C., Lazou, A., and Beis, I. (2002). Hyperosmotic and thermal stresses activate p38-MAPK in the perfused amphibian heart. J. Exp. Biol.=OMR, 443-454. 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