Báo cáo khoa học: MBP-1 is efficiently encoded by an alternative transcript of the ENO1 gene but post-translationally regulated by proteasome-dependent protein turnover doc

MBP-1 is efficiently encoded by an alternative transcript of the ENO1 gene but post-translationally regulated by proteasome-dependent protein turnover Jrhau Lung1, Ko-Jiunn Liu1, Jang-Yang Chang1, Sy-Jye Leu2 and Neng-Yao Shih1 National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan Department of Microbiology and Immunology, Taipei Medical University, Taiwan Keywords alternative transcription initiation; a-enolase; c-myc promoter-binding protein-1; protein stability; ubiquitination Correspondence N.-Y Shih, National Institute of Cancer Research, National Health Research Institutes, Tainan 704, Taiwan Fax: +886 208 3427 Tel: +886 700 0123 ext 65108 E-mail: jshih@nhri.org.tw or S.-J Liu, Department of Microbiology and Immunology, Taipei Medical University, Taipei 110, Taiwan Fax: +886 2377 862 Tel: +886 2736 1661 ext 3414 E-mail: cmbsycl@tmu.edu.tw (Received 28 June 2010, revised 16 August 2010, accepted 19 August 2010) The c-myc promoter-binding protein-1 (MBP-1) is a transcriptional suppressor of tumorigenesis and thought to be the product of alternative translation initiation of the a-enolase (ENO1) transcript In the present study, we cloned a 2552-bp novel cDNA with a putative coding sequence of MBP-1 and functionally examined its ability to encode the MBP-1 protein Similarly to ENO1, the obtained MBP-1 was widely and differentially expressed in a variety of normal tissues and cancer cells Experiments using MBP-1 promoter-driven luciferase reporter assays, biochemical cell fractionation followed by RT-PCR detection of the cytoplasmic mRNA, and transcription ⁄ translation-coupled reactions, consistently demonstrated that this novel transcript was alternatively transcribed from intron III of the ENO1 gene and was feasible for MBP-1 production Hypoxia treatments significantly increased the transcriptional activation of the MBP-1 gene Blocking the proteasomal degradation by MG132 stabilized the MBP-1 protein in cells Compared with the translation efficiency for production of the MBP-1 protein, the MBP-1 transcript was 17.8 times more efficient than the ENO1 transcript Thus, we suggest that this newly discovered transcript is a genuine template for the protein synthesis of MBP-1 in cells, and optimal expression of this gene in tumors may lead to effective clinical therapies for cancers doi:10.1111/j.1742-4658.2010.07819.x Introduction The role of c-myc promoter binding protein-1 (MBP-1) in tumor suppression has been demonstrated, in certain types of cancer, to be that of a general transcriptional repressor MBP-1 was originally identified from a human cervical carcinoma It has been reported to bind to a 5¢ sequence adjacent to the TATA box of the human c-myc P2 promoter and to negatively regulate transcription by preventing the formation of a transcription initiation complex [1] Exogenously expressed MBP-1 reportedly suppresses cell growth, and induces apoptosis and necrosis in breast [2], neuroblastoma [3], or non-small-cell lung cancer cells [4] via the transcriptional repression of c-myc or through physical interplay with its cellular partners [5,6] More recently, the results of an in vitro experiment suggested that the physiological level of MBP-1 is modulated by the concentration of glucose and that a change in the expression of MBP-1 leads to an alteration in cell proliferation [7] The antitumor activity of MBP-1 has also been demonstrated in human tumorxenografted mice [2,5] Thus, the expression level of MBP-1 appears to be a determining factor for cell Abbreviations CHX, cycloheximide; ENO1, a-enolase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; HIF1a, hypoxia-inducible factor 1a; MBP-1, c-myc promoter-binding protein-1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction 4308 FEBS Journal 277 (2010) 4308–4321 ª 2010 The Authors Journal compilation ª 2010 FEBS J Lung et al growth, and alterations in its level by tumor microenvironmental factors may affect cancer development Because MBP-1 shares a high sequence similarity with the glycolytic enzyme a-enolase (ENO1), this 37 kDa cellular protein is thought to be a short form of the 48 kDa ENO1 protein Sequence analysis of MBP-1 revealed 95% sequence identity with ENO1 cDNA in both the coding region and the 3¢-UTR [8] Coincidentally, both genes are close to the 1p36 region on chromosome [9,10] Ghosh et al [11] reported that the C-terminal MBP-1 protein, which is highly homologus to ENO1, exhibited transcriptional repression activity, and its activity was sufficient to stimulate regression of prostate tumor growth in nude mice [5] Additionally, work from Feo’s laboratory has shown that ectopic expression of the short form of ENO1, lacking the first 96 amino acids, functions in a manner similar to MBP-1 [12] They also showed that in vitro transcription and translation of the coding sequence of ENO1 can yield two polypeptides with apparent molecular masses of 48 and 37 kDa In an RNase protection assay, hybridization of the total RNA of HeLa cells with a cRNA antisense probe corresponding to ENO1 gave a single transcript, suggesting that the same transcript may encode both ENO1 and MBP-1 Moreover, site-directed mutagenesis of Met94 and Met97 on the ENO1 cDNA further supports this single-transcript hypothesis and suggests that MBP-1 is a product of alternative translation initiation of the ENO1 transcript [13] It is unclear, however, whether alternative translation-initiation codons of the ENO1 transcript are in the correct context for eukaryotic ribosomal scanning when a 5¢-UTR is present to mimic the endogenous ENO1 transcript In the present study, we report that a novel, naturally occurring, transcript is an alternative transcriptional product of ENO1 and is widely expressed in many normal tissues and cancer cells Hypoxia treatments induced significant levels of its mRNA in normal-type lung epithelial cells (BEAS2B), and blockade of the ubiquitin-dependent degradation pathways by MG132 markedly elevated the stability of MBP-1 Experiments using a full-length ENO1 cDNA (with intact 5¢- and 3¢-UTRs) for production of MBP-1 revealed that the amount of protein generated from the construct was 17.8 times lower than that produced from the full-length MBP-1 cDNA Therefore, we conclude that this newly discovered transcript is a genuine template for MBP-1 synthesis in cells In the future, it will be critical to elucidate potential factors or conditions to modulate its optimal expression in tumor cells in order to develop effective therapies for cancers Expression and regulation of MBP-1 Results Cloning of full-length putative MBP-1 cDNA MBP-1 protein is thought to be an alternative translational product of the ENO1 transcript, produced by utilizing its internal AUG start codons [12,13] Here, we characterized an undefined cDNA (NCBI access number: AL833741), which comprises a putative ORF that encodes the MBP-1 protein Intriguingly, in addition to a 1486-nucleotide sequence spliced from exon IV to exon XII of ENO1, the AL833741 cDNA contains 789 nucleotides upstream of this sequence Because the latter, 789-nucleotide, sequence was located in the 3¢-end of intron III (Fig 1), the AL833741 may be an alternative transcription product of ENO1 To determine whether there were more sequences upstream of the AL833741 transcript, 5¢-RACE reactions were performed The cytoplasmic RNA was extracted from the CL1-5 lung adenocarcinoma cell line, then primed with oligo-d(T)18 primer and reverse transcribed into cDNA Three gene-specific reverse primers – P1, P2 and P3 (as indicated in Fig 1) – were used to align the putative ORF and 5¢-UTR sequences of AL833741 The resultant PCR product shown in Fig S1 was directly sequenced We identified 279 additional nucleotides upstream of this transcript and deposited this sequence in the NCBI database (access number: GU170215) To confirm this finding, amplification using primers specific to the 5¢-end of the 279nucleotide sequence and to the 3¢-end of the ENO1 cDNA was performed to clone out a 2552-bp fulllength cDNA Collectively, these data confirm the existence of this transcript in the cells Distribution of the MBP-1 transcript in normal tissues and cancers To determine the distribution of this putative MBP-1 transcript in normal tissues and cancer cells, commercially available cDNAs of a variety of normal tissues, and the cDNAs generated from normal lung primary cells as well as lung cancer and breast cancer cells, were analyzed by semiquantitative RT-PCR using gene-specific primers to align the putative MBP-1 or the ENO1 cDNAs The results in Fig 2A show wide expression of MBP-1 in many normal tissues In contrast to the ENO1 transcript, which was highly expressed in brain, liver and kidney tissues, the MBP-1 mRNA was exclusively highly abundant in spleen, and was rarely found in stomach and colon tissues These results suggest that the expressions of ENO1 and FEBS Journal 277 (2010) 4308–4321 ª 2010 The Authors Journal compilation ª 2010 FEBS 4309 Expression and regulation of MBP-1 J Lung et al A Fig The putative MBP-1 transcript is an alternative transcriptional product of the ENO1 gene locus (A) The AL833741 transcript comprises a 789-bp sequence transcribed from the intron III of the ENO1 gene and a sequence spliced from exons IV to XII The translational start codon for the ENO1 protein is located in exon II (gray arrow) and that for the MBP-1 protein is located in exon V (black arrow) The 5¢-RACE experiments using three genespecific reverse primers (P1, P2 and P3) cloned out an additional 279-nucleotide sequence upstream from the AL833741 sequence This nucleotide sequence is listed at the bottom of panel A The c-Myc and HIF1a binding sites are marked with solid ( ) and open (s) circles, respectively (B) The gene constructs transcribed into a transcript with intact 5¢- and 3¢-UTRs, as well as the coding sequences of ENO1 or MBP-1, were designated as ENO1 ⁄ FL or MBP-1 ⁄ FL, respectively The ENO1 ⁄ ORF and MBP-1 ⁄ ORF constructs contained only the coding regions of the ENO1 and MBP-1 genes, respectively The ENO1 cDNA with the first translational start codon mutated or lacking the 5¢-UTR, was named ENO1mut ⁄ FL or ENO1 ⁄ 3¢-UTR, respectively The MBP-1 ⁄ s-UTR lacked the rst 206 nucleotides in its 5Â-UTR B ã MBP-1 are differentially regulated in a tissue-dependent manner In various lung cancer and breast cancer cells it was found that although the MBP-1 transcript level was slightly lower in normal human lung tissue than in other cells, there were no significant differences between normal and lung cancer cells, between poorly and highly invasive cells, such as CL1-0 and CL1-5, respectively, or between benign and malignant breast cancer cells, such as MCF-7 and SK-BR3, respectively These data suggest that the MBP-1 transcript level is not related to cell malignancy under normal culture conditions Upregulation of the MBP-1 transcript level in hypoxia Upregulation of glycolytic enzymes under hypoxia has been previously documented [14] A hypoxia-inducible factor 1a (HIF1a)-binding site (GCGTG) is also 4310 found in a region 956 nucleotides upstream from the transcription start site of MBP-1, as indicated in Fig 1A To test whether the expressions of MBP-1 and ENO1 could be modulated by hypoxia, BEAS-2B and A549 cells were incubated in 2% O2 or treated with CoCl2 for 24 h to mimic hypoxic conditions The mRNA transcript and protein levels of MBP-1 and ENO1 were measured using reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) and immunoblotting analyses, respectively (Fig 3) In response to treatment with CoCl2 or 2% O2, HIF1a expression was markedly increased in BEAS-2B cells followed by upregulation of ENO1, as reflected by the increased levels of protein (Fig 3A) and of mRNA transcripts (Fig 3B) However, this phenomenon was not consistently observed in A549 cells, as elevated HIF1a resulted in an increased steady-state level of the ENO1 transcript, but barely affected the level of ENO1 protein Thus, induction of HIF1a by hypoxia FEBS Journal 277 (2010) 4308–4321 ª 2010 The Authors Journal compilation ª 2010 FEBS J Lung et al Expression and regulation of MBP-1 A A B Fig Abundance of putative MBP-1 and ENO1 transcripts in various tissues and cell lines (A) The ENO1 and putative MBP-1 transcript levels were determined by semiquantitative RT-PCR using a variety of commercially available normal tissue cDNAs as templates and resolved on 1% agarose gels containing ethidium bromide (B) Total RNA was extracted from normal lung bronchial epithelial (NHBE) cells and WI38 normal-like lung cells, as well as from lung cancer and breast cancer cells, as indicated After generation of the first-strain cDNA in the oligo-(dT)18-primed reverse-transcription reactions, the 5¢-UTRs of ENO1 and of putative MBP-1 cDNAs were amplified using their individual gene-specific primers (Table 1) The cDNA pool of normal lung tissue (N Lung) was used as a control for normal lung cells, and the b-actin gene served as a loading control was apparently insufficient to promote the production of ENO1 protein in this cell line By contrast, the expression pattern of the putative MBP-1 transcript was similar to that of the ENO1 transcript in both cell lines However, endogenous MBP-1 protein remained undetectable under these hypoxic conditions even though the transcriptional activation of its gene was markedly enhanced This may be a consequence of the remarkably low abundance of the MBP-1 mRNA transcripts in those cells compared with the mRNA transcripts of ENO1 (Fig 3A) Surprisingly, we later found that the MBP-1 protein was also undetected in cells transfected with an exogenous MBP-1 ⁄ FL-HA construct, suggesting that MBP-1 is a stability-labile protein Thus, failure to detect the MBP-1 protein in BEAS-2B and A549 cells under normoxic and hypoxic conditions can be explained by the low transcript abundance and the low stability of the MBP-1 protein in these cells B Fig Increasing expression of MBP-1 transcription in hypoxia BEAS-2B lung normal-like epithelial or A549 lung adenocarcinoma cells were seeded onto six-well plates, incubated at 37 °C overnight and subjected to incubation in 20% O2 and 5% CO2 normoxia (Nm) or 2% and 5% CO2 hypoxia (Hp) conditions, or to treatment with 150 mM CoCl2 (Co) at 37 °C for 24 h (A) The total RNA was extracted from each type of cell and reverse transcribed into cDNA The levels of endogenous MBP-1 and ENO1 gene transcripts were quantified by RT-qPCR, normalized to the b-actin control levels and calculated as )4Ct = )[Ct (ENO1 or MBP-1) ) Ct (b-actin)] The ratios of ENO1 or MBP-1 mRNA to b-actin mRNA were then expressed as 2ÀDC t  K (K, constant) Data obtained from three independent experiments are shown as the mean ± SEM of the relative levels of MBP-1 or ENO1 to the b-actin control level, as indicated (B) The lysates were resolved on 10% SDS ⁄ PAGE, and the protein levels of endogenous MBP-1 and ENO1 were determined by western blot analyses using an antibody cross-reactive to ENO1 and MBP-1 After antibody stripping, the same blots were reprobed with antiHIF1a or b-actin IgG, as indicated The immunocomplexes were detected using the SuperSignal chemiluminescent system The MBP-1 transcript is functionally translatable Initially, we constructed a luciferase reporter driven by the kb proximal promoter of MBP-1 containing the HIF-1a-binding site to determine whether the promoter was transcriptionally functional BEAS-2B cells were transfected with the MBP-p-Luc plasmid, and 12 h later the cells were treated with or without CoCl2 for an additional 24 h then with luciferase reporter assays As shown in Fig 4A, the luciferase activity driven by the MBP-1 promoter was significantly induced by 4.3-fold compared with its vector control The FEBS Journal 277 (2010) 4308–4321 ª 2010 The Authors Journal compilation ª 2010 FEBS 4311 Expression and regulation of MBP-1 J Lung et al A B C D Fig The MBP-1 transcript is functionally translatable (A) The 1-kb promoter sequence of the putative MBP-1 gene was amplified by PCR and cloned into the pGL3-Basic reporter vector The resultant MBP-1-p-Luc reporter plasmid (1 lg) and pRL-TK plasmid (0.05 lg), which was used to correct the differences in transfection efficiency between experiments, were co-transfected into BEAS-2B cells and cultured for 12 h The transfected cells were treated with (+) or without ()) 150 mM CoCl2 for 24 h The luciferase activity of each lysate was determined using a dual-luciferase reporter system (Promega) Data are the mean ± SEM luciferase activities of four independent experiments VC, control vector (B) The cytoplasmic (C) and nuclear (N) fractions of BEAS-2B and A549 cells were fractionated as described in the Materials and methods Western blot analysis of one-tenth of the original volume of each fractionated lysate was carried out to examine the fractionation efficiency by probing with antibody specific to a cytosolic or a nuclear protein marker (GAPDH or Histone 3, respectively) (C) The total RNA of the remaining lysates was extracted and reverse transcribed into cDNA using the oligo-(dT)18 primer The MBP-1 and ENO1 cDNAs were detected by PCR amplification, and then the amplicons were resolved on 1% agarose gels containing ethidium bromide A blank control with no cDNA is marked ‘B’ (D) In vitro transcription ⁄ translation-coupled reactions were performed using the putative fulllength MBP-1 (MBP-1 ⁄ FL) plasmid or a control vector (VC) as templates (lower panel) The inserted gene was transcribed by T7 RNA polymerase, expressed in a [35S] methionine-containing reticulocyte lysate system with a protease inhibitor cocktail, and visualized by autoradiography activity was further augmented in cells treated with CoCl2, indicating that the MBP-1 promoter is a functional element and can be modulated by hypoxic conditions To determine whether the MBP-1 transcript could be exported from the nucleus to the cytoplasm, the MBP-1 and ENO1 transcripts were analyzed by biochemical cell fractionation followed by RT-PCR Western blot analyses of the fractionated lysates of BEAS-2 and A549 cells were first carried out using antibodies against the cytosolic and nuclear markers, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Histone 3, respectively, to show that the cytoplasmic fractions were clearly separated from the nuclear fractions (Fig 4B) Subsequently, total RNAs of the fractionated lysates of both cell lines were extracted and reverse transcribed into cDNAs using 4312 the oligo-(dT)18 primer The results shown in Fig 4C demonstrate that both MBP-1 and ENO1 mRNA species are present in the cytoplasm and nucleus, indicating that the nuclear export of MBP-1 mRNA is similar to that of other mature mRNA species, such as that from COX-2 [15] To determine whether the putative MBP-1 transcript was translationally functional, experiments using transcription ⁄ translation-coupled reactions were performed To mimic endogenous mRNA, we constructed a recombinant plasmid, MBP-1 ⁄ FL, which contains putative MBP-1 cDNA along with its 5¢- and 3¢-UTRs as well as its ORF fused with the hemagglutinin (HA)tag sequence Using T7 RNA polymerase in the coupled reticulocyte lysate system, we detected a radiolabeled protein with an apparent molecular mass of 37 kDa (Fig 4D), indicating that this transcript was FEBS Journal 277 (2010) 4308–4321 ª 2010 The Authors Journal compilation ª 2010 FEBS J Lung et al Expression and regulation of MBP-1 A B C E D Fig MBP-1 is a stability-liable protein regulated by ubiquitin-dependent degradation (A) Cos-7 or HEK293 cells were transfected with the MBP-1 ⁄ FL gene and incubated for 24 h Then, the transfected cells were treated with tosyl-L-lysine chloromethyl ketone (TL; 100 lM), tosyl)1 L-phenylalanine chloromethyl ketone (TP; 20 lM), leupeptin (LU; 10 lgỈmL ), chloroquine (CQ; 150 lM), MG132 (MG; 10 lM), lactacystin (LC; 10 lM), or dimethylsulfoxide vehicle control (C), for 24 h (B) Cos-7 cells were transfected with the MBP-1 ⁄ ORF plasmid After 24 h of incubation, the transfected cells were treated with various doses of MG132 or dimethylsulfoxide vehicle control (C), or untreated ()), as indicated, for 12 h The lysates were resolved on 10% SDS ⁄ PAGE, blotted onto nitrocellulose membrane and probed with antibody against HA-tagged MBP-1 The protein b-actin served as a loading control (C) After transfection with MBP-1 ⁄ FL or ENO1 ⁄ FL genes for 24 h, the cells were treated with (+) or without ()) 10 lM MG132 for an additional 24 h, lysed, then immunoblotted for HA-tagged MBP-1 or ENO1, respectively MBP-1 (37 kDa), alternatively translated from the ENO1 ⁄ FL gene, is marked with an arrow at the right (D) The half-life of MBP-1 was measured following treatment with CHX Cos-7 cells were transfected with the MBP-1 ⁄ ORF gene, incubated for 20 h, then pretreated with (+) or without ()) MG132 (10 lM) for an additional h After washing, translation of the MG132-pretreated cells was blocked with 100 lgỈmL)1 of CHX for the designated periods of time in the presence or absence of MG132, as indicated at the top The levels of MBP-1-HA and endogenous ENO1 proteins were determined by western blotting analyses using antibodies specific to HA or ENO1, respectively (E) Cos-7 cells were co-transfected with MBP-1 ⁄ ORF, or the vector control (VC), and the pcDNA-Flag-Ub plasmid encoding flag-tagged ubiquitin After 24 h of incubation, the transfected cells were treated (+) or untreated ()) with 10 lM MG132 for an additional 24 h, as indicated, and lysed The ubiquitinization of MBP-1 was determined by precipitating with an anti-HA IgG, followed by resolution of the precipitants in SDS ⁄ PAGE, and immunoprobing for flag-tagged ubiquitin The heavy chain of the antibody loaded for immunoprecipitation is indicated by an arrow on the right translatable in vitro Collectively, our data confirm that the MBP-1 transcript is a product of alternative transcriptional initiation of ENO1 and is transcribed from the intron III of the gene The transcript can be exported to the cytoplasm, and can be translated in vitro Susceptibility of the MBP-1 protein to ubiquitin-dependent degradation Surprisingly, when the same construct was transfected into Cos-7 and HEK293 cells, there was no immunoreactivity against HA To investigate whether this outcome was because of the poor stability of the MBP-1 protein, the transfected cells were treated with a variety of protease and proteasome inhibitors Only the ubiquitin-associated proteasome inhibitor, MG132, could effectively stabilize MBP-1 protein in the cells; lactacystin, a 20S proteasome inhibitor, provided only a minor protective effect (Fig 5A), and chymotrypsinand trypsin-like serine protease inhibitors (tosyl-llysine chloromethyl ketone and tosyl-l-phenylalanine chloromethyl ketone), a lysosome protease inhibitor (chloroquine), and a serine or cysteine protease inhibitor (leupeptin) had no effect on MBP-1 stability Thus, although the MBP-1 transcript is translatable in vitro, the MBP-1 protein is susceptible to proteolysis, requiring the presence of the proteasome inhibitor MG132 for stability in cells To further verify the poor protein stability of MBP-1 under normal culture conditions, its half-life and its ubiquitination state in cells were determined by FEBS Journal 277 (2010) 4308–4321 ª 2010 The Authors Journal compilation ª 2010 FEBS 4313 Expression and regulation of MBP-1 J Lung et al treatment with cycloheximide (CHX) and immunoprecipitation of ubiquitinated MBP-1 protein, respectively Previous studies have shown that MBP-1 protein can be expressed in cells and function as a c-myc promoter-binding protein [2,16] by ectopic expression of its ORF conjugated with the 5¢-Kozak sequence (MBP-1 ⁄ ORF) In Fig 5B, we demonstrated that the MBP-1 level could be further elevated fivefold following treatment with ‡ lm MG132 when compared with the level in cells treated with a control vehicle The same observations were obtained from cells transfected with newly discovered full-length MBP-1 (MBP1 ⁄ FL) (Fig 5C, left panel) or with ENO1 (ENO1 ⁄ FL) (Fig 5C, right panel), showing that MG132 could stabilize MBP-1 protein generated by either of these constructs To measure the half-life of MBP-1, cells were transfected with MBP-1 ⁄ ORF for 20 h, then pretreated, or not, with MG132, as indicated in Fig 5D After removal of MG132, the transfected cells were incubated in medium containing either a combination of MG132 + CHX or CHX alone, for different periods of time The half-life of MBP-1 in cells treated with CHX alone was about 2.5 h, whereas the half-life was > h in cells treated with a combination of MG132 and CHX By contrast, no change was found in the endogenous ENO1 protein level during those treatments To determine whether the stabilizing effect of MG132 occurred through the blockade of ubiquitin-mediated degradation pathways, the MBP-1-transfected cells were treated or untreated with MG132 The ubiquitinated MBP-1 was detected exclusively in cells treated with MG132 (Fig 5E) Collectively, the data confirm that MBP-1 is a stability-labile protein and is susceptible to ubiquitin-dependent degradation pathways; however, its long form, ENO1, is substantially stable under normal culture conditions was decreased by as much as 14.8-fold when compared with that of cells transfected with MBP-1 ⁄ ORF (Fig 6) Moreover, deletion of the first 206 nucleotides in the 5¢-UTR entirely ablated the ability of the MBP-1 transcript to encode its protein, suggesting that this sequence is required for ribosome recognition or binding Thus, the low abundance of MBP-1 in cells appears to be a result of its high turnover rate and the tightly regulated translation control of the 5¢-UTR of its transcript The ENO1 transcript preferentially encodes ENO1 protein in cells The results of previous reports showed that ectopic expression of the ENO1 ORF in cells produced the 48 kDa ENO1 and its short form, the 37 kDa protein MBP-1 [7,12,13] To examine whether ENO1 containing intact UTRs could efficiently encode MBP-1, we expressed an ENO1 transcript with its 5¢- and 3¢-UTRs (ENO1 ⁄ FL) to mimic its endogenous native form, with the first coding AUG mutated (ENO1-mut ⁄ FL), or with the 3¢-UTR alone (ENO1 ⁄ 3¢-UTR) The results in Fig basically agree with previous reports, showing Protein synthesis of MBP-1 is tightly regulated In addition to protein stability, the low abundance of a protein has been attributed to translational regulation of its transcript via the 5¢-UTR [17] To investigate whether this might be a cause of the low abundance of MBP-1 in cells, we generated three plasmids: one with the MBP-1 ORF insert only (MBP1 ⁄ ORF); one with the full-length MBP-1 cDNA (MBP-1 ⁄ FL); and one with the full-length cDNA lacking the first 206 nucleotides (MBP-1 ⁄ s-UTR) The MBP-1 ⁄ ORF construct contains the Kozak sequence followed by the AUG translation start codon Western blot analysis of the MBP-1 protein level in cells transfected with each plasmid demonstrated that the translation efficiency of MBP-1 ⁄ FL with the 5¢-UTR 4314 Fig Translational regulation of MBP-1 synthesis Cells were transfected with MBP-1 ⁄ ORF, MBP-1 ⁄ FL, the full-length MBP-1 construct lacking the first 206 nucleotides in the 5¢-UTR (MBP-1 ⁄ sUTR), or a control vector (VC), in the presence or absence of 10 lM MG132, as indicated The sample lysates (80 lgỈsample)1) were resolved by 10% SDS ⁄ PAGE, blotted onto nitrocellulose membrane and probed with antibody against HA-tag The protein bands were quantified using QUANTITY ONE software (Bio-Rad Laboratories) Data obtained from three independent experiments were normalized to b-actin and are expressed as mean ± SEM of the MBP-1 protein levels compared to cells transfected with MBP-1 ⁄ FL FEBS Journal 277 (2010) 4308–4321 ª 2010 The Authors Journal compilation ª 2010 FEBS J Lung et al Expression and regulation of MBP-1 Additionally, a full-length ENO1 cDNA construct with a mutated first ATG codon (ENO1 ⁄ mut-FL) served as a control Co-transfection of green fluorescence protein into the cells was used to examine the transfection efficiency of each construct No significant differences regarding transfection were found for these constructs, with their transfection efficiencies ranging from 68.3% to 76.8% The mRNA and protein levels of ENO1 and MBP-1 were examined using RT-PCR and western blot analyses, respectively The results in Fig demonstrate that, in the presence of similar levels of each transcript, 17.8 times more MBP-1 protein is synthesized from the newly discovered MBP-1 transcript (MBP-1 ⁄ FL) than from the ENO1 ⁄ FL transcript Hence, we suggest that this newly discovered transcript is a genuine template for the synthesis of the MBP-1 tumor suppressor in cells Discussion Fig The native ENO1 transcript preferentially encodes its own protein Cells were transfected with a plasmid containing full-length ENO1 cDNA (ENO1 ⁄ FL), full-length ENO1 with the first ATG start codon mutated (ENO1-mut ⁄ FL), its ORF plus the 3¢-UTR (ENO1 ⁄ 3¢UTR) with Kozak’s sequence, or its ORF alone with Kozak’s sequence (ENO1 ⁄ ORF), in the presence of 10 lM MG132 The levels of exogenous ENO1 and its translational alternative MBP-1 proteins were measured by western blotting analysis using anti-HA IgG Cells transfected with MBP-1 ⁄ ORF or a control vector (VC) served as positive and negative controls, respectively The protein bands were quantified using QUANTITY ONE software (Bio-Rad Laboratories) Data obtained from three independent experiments were normalized to b-actin and are presented as means ± SEM of the protein levels of MBP-1-HA compared to cells transfected with ENO1 ⁄ ORF (lower panel) that MBP-1 can be synthesized from a transcript with the ORF of ENO1 alone (ENO1 ⁄ ORF) Comparable MBP-1 expression was also observed in cells transfected with ENO1 ⁄ 3¢-UTR or ENO1-mut ⁄ FL plasmids However, a very low amount of MBP-1 was synthesized in the ENO1 ⁄ FL-transfected cells, indicating that the endogenous ENO1 transcript preferentially encodes the 48 kDa ENO1, and not the 37 kDa MBP-1 The MBP-1 transcript generates MBP-1 protein more efficiently To determine which transcript generated the MBP-1 protein more efficiently, cells were transfected with full-length ENO1 (ENO1 ⁄ FL) or MBP-1 (MBP-1 ⁄ FL) cDNA containing their intact UTR sequences The role of MBP-1 in cell death and tumor suppression has been previously demonstrated, and MBP-1 is believed to be produced through an alternative translation process that utilizes the internal translation-initiation sites located 374 and 383 nucleotides downstream on the ENO1 cDNA [13] In the present study, we found a novel transcript containing the MBP-1 ORF, an alternative transcriptional product of ENO1 that is expressed in a variety of tissues After cloning its fulllength gene and examining its expression in vitro and in vivo, we demonstrated that the transcript is functional and has 17.8-fold higher efficiency for coding the MBP-1 protein than the full-length ENO1 cDNA with intact UTRs Moreover, the ENO1 transcript preferentially generates its own protein rather than MBP-1, indicating that the first AUG codon of the ENO1 transcript is optimized for ribosome scanning, while the alternative start codons are not Thus, our data suggest that this transcript could be a genuine template for the MBP-1 protein in cells Most mammalian genes not appear to conform to a simple model in which a TATA box directs transcription from a single defined nucleotide position, but instead they contain multiple transcription-initiation sites Intriguingly, it appears that these alternative sites are generally used in different cell contexts or tissues The UDP-glucuronosyltransferase locus, for example, has at least seven promoters with different tissueexpression profiles [18] The human platelet-derived growth-factor receptor gene has two transcription-initiation sites [19] One is in the intron 1, and the resulting transcript contains 363 bp in its 5¢-UTR Similarly, the MBP-1 transcript described here contains a sequence FEBS Journal 277 (2010) 4308–4321 ª 2010 The Authors Journal compilation ª 2010 FEBS 4315 Expression and regulation of MBP-1 A J Lung et al B C Fig The translational efficiency of the MBP-1 transcript to generate MBP-1 protein is markedly higher than that of the ENO1 transcript Cells were transfected with ENO1 ⁄ FL, ENO1-mut ⁄ FL, MBP-1 ⁄ FL, or vector control (VC) in the presence of 10 lM MG132, as indicated Each sample was divided into two aliquots One was used for RT-PCR analysis and the other was used for western blot analysis Three independent experiments were carried out One representative result is shown here (A) For RT-PCR analysis, the cytoplasmic RNA of the transfected cells was reverse transcribed into cDNA The level of each exogenous gene transcript was determined by PCR amplification using a pair of primers probing for the common region of ENO1 and MBP-1 cDNA as well as for the HA-tag sequence The ENO1-HA and MBP-1-HA PCR amplicons was quantified using QUANTITY ONE software (Bio-Rad Laboratories) (B) For western blot analysis, the lysate of each transfected cell type was resolved by 10% SDS ⁄ PAGE, blotted onto nitrocellulose membrane and probed with anti-HA IgG The level of b-actin protein served as a control (C) After being quantified and normalized to b-actin levels, data were expressed as fold increase in the protein ⁄ mRNA ratio of MBP-1-HA compared to cells transfected with ENO1 ⁄ FL of 1063 nucleotides located in the intron III of the ENO1 gene locus, indicating that this transcript is an alternative transcription initiation product of ENO1 Although the MBP-1 transcript is constitutively expressed in a variety of cell types, its encoded protein was barely detectable Previous studies have demonstrated the role of MBP-1 in cell apoptosis and tumor suppression in an overexpression system [2–4] Hence, the level of MBP-1 appears to have an impact on cell fate During normal cell culture, cells grow continually, and a low level of MBP-1 is expected Ray and Miller [1] characterized this MBP-1 protein as 37 kDa in HeLa and HL-60 cells using a radiolabeled c-myc P2 promoter DNA fragment as a probe in a Southwestern blot Consistently, endogenous MBP-1 protein has not been detected in the cell lines examined so far, such as A549, HeLa, CL1-0 and CL1-5, as well as in the breast 4316 cancer cells used in the present study, with our previously published ENO1-specific antibody [20], antiserum raised against C-terminal ENO1, or with a commercially available mAb in our chemiluminescence system (data not shown) Experiments using MG132 and CHX, as well as immunoprecipitation of MBP-1 and blotting to determine its polyubiquitinization (Fig 5), confirmed that MBP-1 was a stability-liable protein with a half-life of about 2.5 h in a cell In addition to its susceptibility to ubiquitin-dependent degradation processes, the 5¢-UTR of the MBP-1 transcript also tightly regulated the expression of its protein, resulting in a low abundance of MBP-1 under normal culture conditions Although a detectable level of endogenous MBP-1 has been shown in some cell lines [12], the two factors contributing to its low abundance (i.e labile protein stability and tightly regulated translation of its transcript) may vary among cell FEBS Journal 277 (2010) 4308–4321 ª 2010 The Authors Journal compilation ª 2010 FEBS J Lung et al types Furthermore, sensitivity of the antibody and detection systems used in different laboratories may also affect apparent differences in abundance The MBP-1 protein can be produced from at least two templates that are generated through distinct cellular processes One template is derived from alternative translation initiation of the ENO1 transcript, and the other is transcribed from an alternative transcription start site in the ENO1 gene locus In a comparison of their relative mRNA abundance in the same cell, the RT-qPCR data in Fig support those in a previous study [13] and show that the level of the MBP-1 transcript is much lower than that of the ENO1 transcript Using the radiolabeled ENO1 riboprobe in an RNase protection assay, Subramanian et al [13] also detected only very low levels of the endogenous MBP-1 transcript in HeLa cells At that time, they did not find evidence of a transcript for MBP-1, and suggested that MBP-1 was exclusively synthesized by utilization of the alternative AUG codons of the ENO1 transcript In the present study, we demonstrated that this newly discovered MBP-1 transcript has a translation efficiency 17.8-fold higher for MBP-1 than that of the ENO1 transcript, suggesting that a subtle change in the cellular level of the MBP-1 transcripts may have a more profound effect on cell-growth regulation than that of the ENO1 Furthermore, our data indicate that the endogenous ENO1 transcript appears to preferentially produce its own protein in cells, rather than the MBP-1 protein (Figs and 8) Despite the results discussed above, the MBP-1 transcript is probably still a minor template for MBP-1 protein production in growing cells because its high translation efficiency is insufficient to compensate for its low abundance In general, the transcript level is normally more than 100-fold lower than that of ENO1 in a variety of normal and cancer cells (Figs and 3) In response to hypoxia, the MBP-1 transcript level can be elevated by up to 4.3-fold (Fig 3A) Therefore, it is reasonable to speculate whether an as-yet-undefined physiological condition, individually or in combination with hypoxia, favors the activation of MBP-1 gene transcription The increased levels of this high translation-efficient MBP-1 transcript may, in turn, become one of major templates responsible for MBP-1 synthesis in the cells Undoubtedly, this hypothesis will be an interesting subject of future study For optimal expression of MBP-1 in cells, attenuation of its degradation is prerequisite In the present study we showed that the level of MBP-1 was dramatically elevated in cells transfected with MBP-1 ⁄ ORF or MBP-1 ⁄ FL plasmids in the presence of the ubiquitin-dependent proteasome inhibitor, MG132 (Figs and 6), suggesting the presence of a high MBP-1 Expression and regulation of MBP-1 protein-turnover rate in cells Similarly to our observation with MBP-1, HIF1a is also susceptible to ubiquitin-mediated degradation under normoxic conditions, in which key proline residues of HIF1a are hydroxylated and then bound by von Hipple-Lindau protein, leading to its ubiquitination and degradation [21,22] However, it is stabilized in hypoxic conditions Therefore, we examined whether MBP-1 could also be stabilized under hypoxia Experiments using 2% O2 or CoCl2 to mimic hypoxia showed that both treatments failed to rescue the endogenous MBP-1 protein in BEAS-2B and A549 cells (Fig 3B) More recently, researchers from Miller’s laboratory reported that MBP-1 expression was increased in cells grown in low-glucose concentrations [7] However, our preliminary studies using nm glucose or serum-free medium for MCF7 and MBP-1 ⁄ ORF-transfected cells did not support their results (data not shown) In spite of those inconsistencies, it is reasonable that the expression of MBP-1 may be modulated by unique environmental factors, which are different from those modulating HIF1a expression, presumably because of their distinct roles in tumorigenesis Thus, identification of physiological factors that increase MBP-1 protein synthesis and attenuate the MBP-1 turnover rate is an important challenge for future research related to cancer therapy Materials and methods Cell culture and cell transfection Normal human bronchial epithelium primary cells were grown in bronchial ⁄ tracheal epithelial growth medium supplemented with essential growth factors, as recommended by the supplier (Cell Applications, San Diego, CA, USA) WI-38 was cultured in Eagle’s-modified minimum essential medium (Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (Hyclone) and · penicillin ⁄ streptomycin ⁄ glutamine (Gibco, Carlsbad, CA, USA) NCI-H23 and A549 lung adenocarcinoma cells were cultured in RPMI1640 (Hyclone) and F12K (Invitrogen, Carlsbad, CA, USA) media containing 5% or 10% fetal bovine serum, respectively HEK293, Cos-7, BEAS-2B, MCF7, BT-474, MDA-MB-231 and SK-BR3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone) containing 25 mm glucose and 10% fetal bovine serum (FBS) CL1-0 and CL1-5 lung adenocarcinoma cells, generously provided by Dr Pan-Chyr Yang [23], were cultured in 10% FBS-containing RPMI-1640 All cell lines were grown in a humidified atmosphere at 37 °C with 5% CO2 Transfection of HEK293 and Cos-7 cells was performed using Lipofectamine and Lipofectamine 2000 (Invitrogen), respectively, according to the manufacturers’ instructions FEBS Journal 277 (2010) 4308–4321 ª 2010 The Authors Journal compilation ª 2010 FEBS 4317 Expression and regulation of MBP-1 J Lung et al Gene cloning and plasmid construction We used 5¢-RACE to clone additional sequences upstream of the AL833741 site CL1-5 cells were lysed in lysis buffer [20 mm Tris ⁄ HCl, 100 mm KCl, mm MgCl2, 0.3% Nonidet P-40, 100 unitsỈmL)1 of RNase inhibitor (Invitrogen) and a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA), pH 7.5] and incubated on ice for The cytoplasmic fraction was obtained by centrifugation (10 000 g, 10 min, °C) Then, the cytoplasmic polyA+-RNA was purified with Oligtex dT beads (Qiagen, Valencia, CA, USA) and reverse transcribed into a 5¢- and 3¢-adaptor-tagged cDNA pool using the SMART RACE kit (Clontech, Palo Alto, CA, USA) The sequence upstream of AL833741 was obtained via a sequential nesting-PCR using P1, P2 and P3 reverse primers (Fig and Table 1) and cloned into a pCR4TOPO vector (Invitrogen) To verify the existence of the full-length cDNA in the cells, PCR amplification used a 5¢-RACE adaptor forward primer as well as a reverse primer to align the 3¢ end of the ENO1 cDNA (GDB access number: NM_001428) Following gel purification, the fragment was cloned and sequenced This construct was designated as pCR-MBP1 ⁄ FL The MBP-1 and ENO1 cDNA constructs, with or without their individual UTRs, were engineered as follows First, to express full-length ENO1, a construct was generated from CL1-5 cDNA using primers that annealed with the 5¢ and 3¢ ends of the ENO1 complete cDNA (the primer sequences are shown in Table 1) After amplification with PfuUltra DNA polymerase (Stratagene, La Jolla, CA, USA), the PCR product was digested with BamHI and EcoRV and ligated into the pcDNA3.1 vector The insertion of an HA-tag sequence into the 3¢ end of the ENO1 ORF was performed by PCR-based site-directed mutagenesis to obtain the ENO1 ⁄ FL plasmid Second, using this plasmid as a template, the first ATG codon of the ENO1 cDNA was mutated to AAG by site-directed mutagenesis using two antiparallel primers (Table 1) The resulting construct was designated ENO1-mut ⁄ FL Third, after PCR amplification, the fragment lacking the 5¢-UTR of ENO1 was digested with BamHI and EcoRV and cloned into pcDNA3.1 to obtain the ENO1 ⁄ 3¢-UTR plasmid Fourth, using the ENO1 ⁄ FL plasmid, created earlier, as a template, the ORF of ENO1 was amplified, digested with BamHI and NotI, and ligated into a vector to obtain the ENO1 ⁄ ORF plasmid Fifth, the MBP-1 ⁄ FL plasmid was constructed by PCR amplification using the pCR-MBP-1 ⁄ FL plasmid as a template, which was followed by ligation of the 5¢-HindIII and 3¢-EcoRV-cut fragment into pcDNA3.1 Sixth, the MBP-1 ⁄ s-UTR plasmid was engineered similarly, but lacked the first 206 nucleotides in the 5¢-UTR of MBP-1 Seventh, after PCR amplification and restriction enzyme digestion, the BamHI and NotI-digested fragment containing the ORF of MBP-1 was cloned into the pcDNA3.1 4318 vector to obtain MBP-1 ⁄ ORF Eighth, the insert encoding N-terminal flag-tagged ubiquitin was generated by PCR using HeLa cDNA as template, as described by Sadeh et al [24], and cloned into the 5¢-BamHI and 3¢-EcoRV sites of pcDNA3.1 The resultant plasmid was designated as pcDNA-Flag-Ub Finally, the kb sequence upstream of the newly discovered MBP-1 transcript was amplified by PCR, using BEAS-2B genomic DNA as the template, and cloned into the 5¢-BamHI and 3¢-EcoRV sites of the pGL3Basic vector (Promega, Madison, WI, USA) The resultant plasmid was designated as MBP-1-p-Luc reporter All gene constructs were verified by direct sequencing Cell fractionation and RNA isolation BEAS-2B and A549 cells were treated with 150 lm CoCl2 or left untreated After washing with NaCl ⁄ Pi, the cells were centrifuged at 250 g for at room temperature, resuspended in RSB buffer (10 mm NaCl, mm MgCl2, 10 mm Tris ⁄ HCl, pH7.5) containing protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany) and 1000 unitsỈmL)1 of RNaseOUT (Invitrogen) and incubated on ice for 15 Igepal CA630 (Sigma, St Louis, MO, USA) was then added to a final concentration of 0.05%, mixed by inversion and the cells were placed on ice for an additional 10 Cell fractionation was carried out by centrifugation at 200 · g for at room temperature to separate the cytoplasm from the nuclear fraction The pellet containing the nuclear fraction was washed twice with RSB buffer and resuspended in a volume of buffer equal to that of the cytoplasmic fraction One-tenth of the original volume was analyzed by western blotting to confirm the purity of each fractionation The total RNA of the remaining cytoplasmic and nuclear fractions was extracted using the RNeasy purification kit (Qiagen), primed with oligo-(dT)18 primer and reverse transcribed into cDNA RT-PCR analysis Cells were lysed in the aforementioned lysis buffer and incubated on ice for The cytoplasmic polyA+-RNA was purified with Oligtex dT beads and reverse transcribed into cDNA using SuperScript III reverse transcriptase according to the manufacturer’s instructions (Invitrogen) To detect the endogenous putative MBP-1 and ENO1 transcripts, semi- and RT-qPCR analyses of the cDNA pools generated from various cell lines, obtained as indicated above or purchased from Clontech Laboratories (Mountain View, CA, USA), were performed using the primers shown in Table 1, specific for their corresponding 5¢-UTR sequences Normal lung tissue cDNA was purchased from ResGen, Invitrogen (Huntsville, AL, USA) For semi-qPCR analyses, the resultant amplicons were resolved in 1% agarose gels containing ethidium bromide and quantified using quantity one software in the Bio-Rad Gel Doc system FEBS Journal 277 (2010) 4308–4321 ª 2010 The Authors Journal compilation ª 2010 FEBS J Lung et al Expression and regulation of MBP-1 Table Primer sequences for the gene construct or detection AS, antisense; S, sense Construct or gene ¢-RACE AL833741 ENO1 ⁄ FL ENO1 ⁄ 3¢-UTR ENO1 ⁄ ORF MBP-1 ⁄ FL MBP-1 ⁄ s-UTR MBP-1 ⁄ ORF Flag-Ub Mutagenesis ENO1-mut ⁄ FL HA-tag insertion Semi-qPCR b-actin ENO1 Putative MBP-1 ENO1-HA or MBP-1-HA Cytoplasmic and nuclear MBP-1 and ENO1 mRNA levels Real-time qPCR b-actin ENO1 Putative MBP-1 Reporter assay MBP-1-p-Luc Sequence P1 AS: 5¢-GCCAAGTCAGCGATGTGGCGG-3¢ P2 AS: 5¢-GGCTGTTCAATCAGTTACCTGAGT-3¢ P3 AS: 5¢-TCAAGGCACAGCATCATCCTGCTCC-3¢ S: 5¢-AAGGATCCTAGCTAGGCAGGAAGTCGGCG-3¢ AS: 5¢-CTCATGGGTCACTGAGGCTTTTTATTTTG-3¢ S: 5¢-AAGGATCCGTTCACCATGTCTATTCTCAAGATCCATG-3¢ AS: 5¢-CTCATGGGTCACTGAGGCTTTTTATTTTG-3¢ S: 5¢-AAGGATCCGTTCACCATGTCTATTCTCAAGATCCATG-3¢ AS: 5¢-CACAGCGGCCGCTTAAGCGTAATCTGGAACATCG-3¢ S: 5¢-CCCAAGCTTAACTAAAGAAAAGTTTCCCCATCTCCC-3¢ AS: 5¢-CTCATGGGTCACTGAGGCTTTTTATTTTG-3¢ S: 5¢-CCCAAGCTTTATTCAAATTGACTTAGTGTGTGTG-3¢ AS: 5¢-CTCATGGGTCACTGAGGCTTTTTATTTTG-3¢ S: 5¢-AAGGATCCGTTCACCATGGATGGAACAGAAAATAAATC-3¢ AS: 5¢-CACAGCGGCCGCTTAAGCGTAATCTGGAACATCG-3¢ S: 5¢-CCCGGATCCATGGATTACAAGGATGACGACGATAAGATGCAAATCTTCGTGAAGACCCTGACTG-3¢ AS: 5¢-TTAACCCCCCCTCAAGCGCAGGACCAAG-3¢ S: 5¢-AAGTCTATTCTCAAGATCCATGC-3¢ AS: 5¢-GGTGAACTTCTAGCCACTGG-3¢ S: 5¢-AGTACCCATACGATGTTCCAGATTACGCTTAAGCTG TGGGCAGGCAAGCC-3¢ AS: 5¢-TAAGCGTAATCTGGAACATCGTATGGGTACTTGGCC AAGGGGTTTCTGAA-3¢ S: 5¢-GGTCACCCACACTGTGCCCATCTA-3¢ AS: 5¢-GAAGCATTGCGGTGGACGATGGAG-3¢ S: 5¢-CACAGTGACCAACCCAAAGAG-3¢ (exon 9) AS: 5¢-CTCATGGGTCACTGAGGCTTTTTATTTTG-3¢ (exon 12) S: 5¢-GTCTCTCACTCGCAGTCCTTAATC-3¢ (intron 3) AS: 5¢-GCCAAGTCAGCGATGTGGCGG-3¢ (exon 6) S: 5¢-ACTGATGATCGAGATGGATGGAACA-3¢ (exon 5) AS: 5¢-TTAAGCGTAATCTGGAACATCG-3¢ S: 5¢-GTCTCTCACTCGCAGTCCTTAATC-3¢ (in 5¢-UTR of MBP-1) S: 5¢-AAGGATCCTAGCTAGGCAGGA AGTCGGCG-3¢ (in 5¢-UTR of ENO1) As: 5¢-AATGAATTCTTACTTGGCCAAGGGGTTTCT-3¢ S: 5¢-CCCTTTTTGTCCCCCAAC-3¢ AS: 5¢-CTGGTCTCAAGTCAGTGTACAGGT-3¢ S: 5¢-GATCTCTTCACCTCAAAAGG-3¢ (exon 2) AS: 5¢-TTCCATCCATCTCGATCATC-3¢ (exon 5) S: 5¢-CAACAGTGGTTCTCTCTAAC-3¢ (intron 3) AS: 5¢-TTCCATCCATCTCGATCATC-3¢ (exon 5) S: 5¢-GAAGGTACCGACCAGCCTGGCCAACATGG-3¢ AS: 5¢-AACAGGCCCTGTTCTCATGC-3¢ (Bio-Rad Laboratories, Hercules, CA, USA) b-actin was used as a loading control For RT-qPCR analyses, the transcript levels of putative MBP-1 and ENO1 in each sample were determined using SRBR green qPCR master mix (Stratagene) in the Bio-Rad DNA Engine OPTICON2 detection system All reactions were performed under the following PCR conditions: denaturation at 95 °C for 10 min, followed immediately by 40 cycles at 95 °C for 10 s and at 60 °C for After being normalized to expression of the b-actin control gene, the changes in the comparative threshold (Ct) data were calculated as )4Ct = )[Ct (ENO1 or MBP-1) ) Ct (b-actin)] The ratio of ENO1 or MBP-1 mRNA to the b-actin mRNA was expressed as 2ÀDCt  K (K, constant) [25] and the results are shown as mean ± SEM The means and SEM values were derived from three independent experiments FEBS Journal 277 (2010) 4308–4321 ª 2010 The Authors Journal compilation ª 2010 FEBS 4319 Expression and regulation of MBP-1 J Lung et al Western blotting analysis and immunoprecipitation HEK293 or Cos-7 cells were transfected for 24 h with ENO1 or MBP-1 tagged with an HA sequence, then treated with MG132 or other protease inhibitors, as indicated, for an additional 24 h The transfected cells were harvested and lysed in 1% Triton X-100, 0.5% sodium deoxycholate, 1% SDS, 0.15 m NaCl, 10 mm Na2HPO4 and 10 mm NaF (pH 7.2), supplemented with a protease inhibitor cocktail (Roche) Approximately 100 lg of each lysate was loaded onto and resolved in 10% SDS-containing polyacrylamide gels, and then blotted onto nitrocellulose membranes (Whatman Schleicher and Schuell, London, UK) Those membranes were incubated with monoclonal anti-HA.11 (Covance, Princeton, NJ, USA) (1 : 1500) and probed with a secondary antibody conjugated with horseradish peroxidase To detect the cytosolic protein marker GAPDH and the nuclear marker Histone 3, antibodies from Santa Cruz (Santa Cruz, CA, USA) and Cell Signaling (Danvers, MA, USA), respectively, were used As a loading control, b-actin was detected by an antibody purchased from Sigma To detect ubiquitinized MBP-1, Cos-7 cells (4–5 · 105 cells per well) were seeded into six-well plates day before transfection The cells were co-transfected with lg of MBP-1 ⁄ ORF, or with lg of pcDNA-Flag-Ub and lg of the control vector using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions, and incubated for 24 h The transfected cells were treated with 10 lm MG-132, or untreated, for 24 h and then lysed in lysis buffer [50 mm Tris (pH 7.4), 150 mm NaCl, 0.1% SDS, 1% Triton X-100, mm EDTA, protease inhibitor cocktail (Roche), 10 lm MG-132 and proteasome inhibitor IV (Calbiochem, San Diego, CA, USA)] Twenty micrograms of each sample was resolved in 10% SDS-containing polyacrylamide gels, blotted onto nitrocellulose membranes and immunodetected with antibodies against HA-tagged MBP-1 or b-actin The lysates (450 lg per sample) were precleaned with 50% protein A ⁄ G slurry (Pierce Biotechnology, Rockford, IL, USA) and immunoprecipitated with anti-HA.11 IgG (Covance) Subsequently, the precipitants were resolved on SDS-containing polyacrylamide gels, blotted onto nitrocellulose membranes and probed for flag-tagged ubiquitin The antigen–antibody immunocomplexes were visualized using SuperSignal enhanced chemiluminescence (Pierce Biotechnology) In vitro-coupled transcription and translation Coupled transcription and translation reaction systems (Promega) were used to detect the translational product of the putative MBP-1 transcript in vitro The gene product was generated from the MBP-1 ⁄ FL plasmid or from a control vector in a reticulocyte lysate system containing T7 RNA polymerase and [35S] methionine (> 1000 CiỈmmole)1 4320 at 10 mCiỈmL)1; Amersham Biosciences, Piscataway, NJ, USA) Subsequently, the radiolabeled proteins were resolved using 10% SDS ⁄ PAGE and visualized by autoradiography Protein stability of MBP-1 The half-life of MBP-1 was determined by treatment with CHX Briefly, Cos-7 cells were transfected with the MBP1 ⁄ ORF construct using Lipofectamine 2000 (Invitrogen) and incubated at 37 °C for 20 h Then, the transfected cells were, or were not, pretreated with 10 lm MG132 for an additional h After washing in NaCl ⁄ Pi, the translational activity of the transfected cells pretreated with MG-132 was blocked with 100 lgỈmL)1 of CHX for specific periods of time, in the presence or absence of MG132, as indicated in Fig The levels of MBP-1-HA and endogenous ENO1 were determined by western blotting using antibodies specific to HA-tag or ENO1 Acknowledgements We thank Dr Peter Goedegebuure and Dr Julia Wang at Washington University, St Louis, MO, USA for their critical appraisal of the manuscript and are also grateful to Dr Pan-Chyr Yang for providing the CL1-5 cells for this study This work was supported by the Intramural 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4321 ... Collectively, these data confirm the existence of this transcript in the cells Distribution of the MBP-1 transcript in normal tissues and cancers To determine the distribution of this putative MBP-1 transcript. .. The putative MBP-1 transcript is an alternative transcriptional product of the ENO1 gene locus (A) The AL833741 transcript comprises a 789-bp sequence transcribed from the intron III of the ENO1. .. of the ENO1 transcript, and the other is transcribed from an alternative transcription start site in the ENO1 gene locus In a comparison of their relative mRNA abundance in the same cell, the RT-qPCR