FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2029667265> ?p ?o ?g. }

• W2029667265 endingPage "16247" @default.
• W2029667265 startingPage "16240" @default.
• W2029667265 abstract "We have identified and characterized N-Bak, a neuron-specific isoform of the pro-apoptotic Bcl-2 family member Bak. N-Bak is generated by neuron-specific splicing of a novel 20-base pair exon, which changes the previously described Bak, containing Bcl-2 homology (BH) domains BH1, BH2, and BH3, into a shorter BH3-only protein. As demonstrated by reverse transcription-polymerase chain reaction and RNase protection assay, N-Bak transcripts are expressed only in central and peripheral neurons, but not in other cells, whereas the previously described Bak is expressed ubiquitously, but not in neurons. Neonatal sympathetic neurons microinjected with N-Bak resisted apoptotic death caused by nerve growth factor (NGF) removal, whereas microinjectedBak accelerated NGF deprivation-induced death. Overexpressed Bak killed sympathetic neurons in the presence of NGF, whereas N-Bak did not. N-Bak was, however, still death-promoting when overexpressed in non-neuronal cells. Thus, N-Bak is an anti-apoptotic BH3-only protein, but only in the appropriate cellular environment. This is the first example of a neuron-specific Bcl-2 family member. We have identified and characterized N-Bak, a neuron-specific isoform of the pro-apoptotic Bcl-2 family member Bak. N-Bak is generated by neuron-specific splicing of a novel 20-base pair exon, which changes the previously described Bak, containing Bcl-2 homology (BH) domains BH1, BH2, and BH3, into a shorter BH3-only protein. As demonstrated by reverse transcription-polymerase chain reaction and RNase protection assay, N-Bak transcripts are expressed only in central and peripheral neurons, but not in other cells, whereas the previously described Bak is expressed ubiquitously, but not in neurons. Neonatal sympathetic neurons microinjected with N-Bak resisted apoptotic death caused by nerve growth factor (NGF) removal, whereas microinjectedBak accelerated NGF deprivation-induced death. Overexpressed Bak killed sympathetic neurons in the presence of NGF, whereas N-Bak did not. N-Bak was, however, still death-promoting when overexpressed in non-neuronal cells. Thus, N-Bak is an anti-apoptotic BH3-only protein, but only in the appropriate cellular environment. This is the first example of a neuron-specific Bcl-2 family member. Bcl-2 homology postnatal day embryonic day reverse transcription-polymerase chain reaction green fluorescent protein superior cervical ganglion/ganglia/ganglial base pair(s) RNase protection assay nerve growth factor During development, two opposite processes, proliferation and naturally occurring cell death (apoptosis), regulate cell number in almost all tissues and organs (1Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2398) Google Scholar). In the developing nervous system, for example, 30–80% of the initially produced neurons die, mostly due to deficiency of neurotrophic factors that neutralize the death program in neurons (2Oppenheim R.W. Annu. Rev. Neurosci. 1991; 14: 453-501Crossref PubMed Scopus (2754) Google Scholar, 3Yuan J. Yankner B.A. Nature. 2000; 407: 802-809Crossref PubMed Scopus (1596) Google Scholar). The cells of an organism retain a potential to die apoptotically during their entire lifetime (4Vaux D.L. Korsmeyer S.J. Cell. 1999; 96: 245-254Abstract Full Text Full Text PDF PubMed Scopus (1361) Google Scholar). Apoptotic pathways are therefore delicately balanced and controlled positively and negatively at several levels (5Green D.R. Cell. 2000; 102: 1-4Abstract Full Text Full Text PDF PubMed Scopus (882) Google Scholar, 6Hengartner M.O. Nature. 2000; 407: 770-776Crossref PubMed Scopus (6233) Google Scholar). The life and death decisions of a cell are critically controlled by the proteins of the Bcl-2 family that are either anti-apoptotic or pro-apoptotic. When overexpressed, anti-apoptotic members protect cells against death stimuli, and their lack in vivo promotes developmental death or sensitivity to death stimuli. Conversely, pro-apoptotic Bcl-2 family members kill the cells even in the presence of life-promoting stimuli, and their deficiency reduces apoptotic death (7Antonsson B. Martinou J.-C. Exp. Cell Res. 2000; 256: 50-57Crossref PubMed Scopus (626) Google Scholar). All anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1, and Boo/DIVA) contain four conserved BH1 domains (BH1, BH2, BH3, and BH4), whereas pro-apoptotic members contain either three BH domains (BH1, BH2, and BH3) (Bax, Bak, and Mtd/Bok) or only the BH3 domain (Bad, Bid, Bik, Blk, Hrk, Bim, Rad9, and Noxa) (7Antonsson B. Martinou J.-C. Exp. Cell Res. 2000; 256: 50-57Crossref PubMed Scopus (626) Google Scholar, 8Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3245) Google Scholar, 9Komatsu K. Miyashita T. Hang H. Hopkins K.M. Zheng W. Cuddeback S. Yamada M. Lieberman H.B. Wang H.-G. Nat. Cell Biol. 2000; 2: 1-6Crossref PubMed Scopus (122) Google Scholar, 10Oda E. Ohki R. Murasawa H. Nemoto J. Shibue T. Yamashita T. Tokino T. Taniguchi T. Tanaka N. Science. 2000; 288: 1053-1058Crossref PubMed Scopus (1693) Google Scholar). Interestingly, the recently discovered Bcl-G contains only BH3 and BH2 domains (11Guo B. Godzik A. Reed J.C. J. Biol. Chem. 2001; 276: 2780-2785Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). How the Bcl-2 family proteins work is still poorly understood. According to the current understanding, pro-apoptotic members containing several BH domains (Bax and Bak), when activated by a death stimulus, generate pores to the mitochondrial outer membrane that lead to the release of cytochrome c and other mitochondrial molecules to the cytoplasm, where they trigger activation of caspases at the apoptosome (5Green D.R. Cell. 2000; 102: 1-4Abstract Full Text Full Text PDF PubMed Scopus (882) Google Scholar, 6Hengartner M.O. Nature. 2000; 407: 770-776Crossref PubMed Scopus (6233) Google Scholar). Once activated above certain threshold levels, caspases irreversibly execute cell death (6Hengartner M.O. Nature. 2000; 407: 770-776Crossref PubMed Scopus (6233) Google Scholar, 12Wolf B.B. Green D.R. J. Biol. Chem. 1999; 274: 20049-20052Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar). In addition, heterodimerization between certain pro- and anti-apoptotic Bcl-2 family proteins nullifies the activity of the partners, and the fate of the cell is determined by the member that is in excess (7Antonsson B. Martinou J.-C. Exp. Cell Res. 2000; 256: 50-57Crossref PubMed Scopus (626) Google Scholar). BH3-only proteins are believed to regulate or modulate the activity of multi-BH domain members. Thus, Bid activates Bax (13Desagher S. Osen-Sand A. Nichols A. Eskes R. Montessuit S. Lauper S. Maundrell K. Antonsson B. Martinou J.-C. J. Cell Biol. 1999; 144: 891-901Crossref PubMed Scopus (1092) Google Scholar, 14Eskes R. Desagher S. Antonsson B. Martinou J.-C. Mol. Cell. Biol. 2000; 20: 929-935Crossref PubMed Scopus (1014) Google Scholar), whereas Bad inactivates Bcl-xL (15Kelekar A. Chang B.C. Harlan J.E. Fesik S.W. Thompson C.B. Mol. Cell. Biol. 1997; 17: 7040-7046Crossref PubMed Scopus (270) Google Scholar), with the net result being cell death in both cases. Alternatively spliced transcripts have been described for many Bcl-2 family members, with the activity of the protein isoforms remaining unchanged in most cases (16Jiang Z.H. Wu J.Y. Proc. Soc. Exp. Biol. Med. 1999; 220: 64-72Crossref PubMed Google Scholar). However, BH3-only splice variants encoding proteins with pro-apoptotic activity have been described for the anti-apoptotic Bcl-x (17Boise L.H. González-Garcı́a M. Postema C.E. Ding L. Lindsten T. Turka L.A. Mao X. Núñez G. Thompson C.B. Cell. 1993; 74: 597-608Abstract Full Text PDF PubMed Scopus (2917) Google Scholar) and Mcl-1 (18Bae J. Leo C.P. Hsu S.Y. Hsueh A.J.W. J. Biol. Chem. 2000; 275: 25255-25261Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 19Bingle C.D. Craig R.W. Swales B.M. Singleton V. Zhou P. Whyte M.K.B. J. Biol. Chem. 2000; 275: 22136-22146Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) and also for Bcl-G (11Guo B. Godzik A. Reed J.C. J. Biol. Chem. 2001; 276: 2780-2785Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Here we describe a BH3-only splice variant of the pro-apoptotic Bak (20Chittenden T. Harrington E.A. O'Connor R. Flemington C. Lutz R.J. Evan G.I. Guild B.C. Nature. 1995; 374: 733-736Crossref PubMed Scopus (695) Google Scholar, 21Farrow S.N. White J.H.M. Martinou I. Raven T. Pun K.-T. Grinham C.J. Martinou J.-C. Brown R. Nature. 1995; 374: 731-733Crossref PubMed Scopus (470) Google Scholar, 22Kiefer M.C. Brauer M.J. Powers V.C. Wu J.J. Umansky S.R. Tomei L.D. Barr P.J. Nature. 1995; 374: 736-739Crossref PubMed Scopus (497) Google Scholar, 23Ulrich E. Kauffmann-Zeh A. Hueber A.O. Williamson J. Chittenden T. Ma A. Evan G. Genomics. 1997; 44: 195-200Crossref PubMed Scopus (23) Google Scholar, 24Herberg J.A. Phillips S. Beck S. Jones T. Sheer D. Wu J.J. Prochazka V. Barr P.J. Kiefer M.C. Trowsdale J. Gene ( Amst. ). 1998; 211: 87-94Crossref PubMed Scopus (25) Google Scholar) and show that it is expressed exclusively in neurons and encodes a protein isoform that is anti-apoptotic in neurons, but promotes death in non-neuronal cells. Full-length N-Bak and Bak cDNAs were generated by RT-PCR from P1 mouse brain RNA, inserted into the pCR3.1 expression vector (Invitrogen, Groningen, The Netherlands), and verified by sequencing. To check whether the expression plasmids produce the respective proteins in cells, COS-7 cells in semiconfluent 10-cm dishes were transfected with Bak- or N-Bak-encoding expression plasmids or with the empty pCR3.1 vector (5 μg/dish) using Fugene transfection reagent (Roche Molecular Biochemicals). To protect cells against death caused by overexpression of pro-apoptotic proteins, plasmid encoding human Bcl-xL (5 μg/dish) was cotransfected. Also, plasmid pGreenLantern-1, encoding green fluorescent protein (GFP) (0.5 μg/dish; Life Technologies, Inc.), was included to reveal transfection efficiency. The next day, cells were lysed in phosphate-buffered saline containing 10 mm KCl, 2 mm EDTA, 1% SDS, and protease inhibitors (Roche Molecular Biochemicals) and analyzed by Western blotting with anti-Bak antibodies (65606E; Pharmingen, San Diego, CA) (see Fig. 3 A). The peptide sequence used to generate these antibodies is present in both Bak and N-Bak (25Krajewski S. Krajewska M. Reed J.C. Cancer Res. 1996; 56: 2849-2855PubMed Google Scholar). Some of the transfected cells were fixed with 4% paraformaldehyde in phosphate-buffered saline, permeabilized with 0.5% Triton X-100, and stained with the same anti-Bak antibodies. Both theBak- and N-Bak-transfected cultures, but not the vector-transfected or untransfected cultures, contained strongly Bak-immunoreactive cells (data not shown). To demonstrate endogenous N-Bak protein, newborn mouse brain, cultured SCG or hippocampal neurons, or Neuro2A neuroblastoma cells were lysed in buffer containing either 2% Triton X-100 and 1% SDS or 1% SDS and 8 murea. Either Bak proteins were immunoprecipitated from the lysates with the anti-Bak antibodies (Pharmingen 65606E), or the crude lysate was used; and the filter was probed with the same antibody. In all samples, only Bak (but not N-Bak) protein was visible (data not shown), whereas both N-Bak and Bak proteins were detected in control COS-7 cells transiently overexpressing N-Bak and Bcl-xL (similar to the results shown in Fig. 3 A). The same result was obtained with anti-Bak antibodies from Oncogene Research Products (AM04; Darmstadt, Germany) or from Santa Cruz Biotechnology (H-211; Santa Cruz, CA) (data not shown). To show that the non-neuronal cells overexpressing N-Bak die apoptotically, HeLa cells were transiently transfected with expression vector for GFP-N-Bak or with the empty pEGFP-C1 vector. 2 h after transfection, GFP-N-Bak-transfected cells exhibited weak fluorescence, became round, and began to detach from the substrate, whereas GFP-expressing cells remained healthy and flat. The cells were fixed with 4% paraformaldehyde in phosphate-buffered saline and stained with 1 μm 4,6-diamidino-2-phenylindole. Images were acquired on an Olympus AX 70 Provis microscope and analyzed by Adobe Photoshop software. For the DNA ladder assay, HeLa cells were transiently transfected with expression plasmids encoding N-Bak or GFP. After 3 h, DNA was prepared and analyzed for the presence of internucleosomal degradation fragments according to a published protocol (26Kaufmann S.H. Mesner P.W. Samejima K. Toné S. Earnshaw W.C. Methods Enzymol. 2000; 322: 3-15Crossref PubMed Google Scholar). Total RNAs from different rat and mouse tissues or from cultured purified neurons or non-neuronal cells were isolated with Trizol reagent (Life Technologies, Inc.). RNAs from human tissues were obtained from CLONTECH. Rat cortical and hippocampal neurons were cultured as described (27O'Malley E.K. Sieber B.-A. Morrison R.S. Black I.B. Dreyfus C.F. Brain Res. 1994; 647: 83-90Crossref PubMed Scopus (55) Google Scholar). First strand cDNAs were synthesized using oligo(dT)15 or dN6 random primers (Roche Molecular Biochemicals) with enhanced avian reverse transcriptase (Sigma) or Superscript II (Life Technologies, Inc.). 2-μl cDNA aliquots were processed by PCR with the High Fidelity PCR system (Roche Molecular Biochemicals), and Bak-specific primers (forward, 5′-CCACCATGGCATCTGGACAAGGACCAG; and reverse, 5′-TCATGATCTGAAGAATCTGTGTACC) were used to amplify full-length N-Bak and Bak cDNAs. For nested PCR, a different pair of N-Bak-specific primers was used after amplifications with the primers shown above: forward, 5′-TTGCCCAGGACACAGAGGAGGT; and reverse, 5′-GAATTGGCCCAACAGAACCACACC. Both primers were localized to different exons to distinguish amplification products from cDNA and genomic DNA. In addition, with either pair of primers, fragments of the size of Bak or N-Bak cDNAs were not amplified from mouse genomic DNA (data not shown). PCR was performed for 35 cycles with the following program: 95 °C for 45 s, 60° for 45 s, and 72 °C for 45 s. PCR products (10–20 μl) were separated on 1.5% agarose gel to reveal the 20-bp difference in the Bak fragments. Fragments of interest were excised and cloned into the pCR2.1 vector or the pCR3.1 expression vector. Nucleotide sequences were verified by sequencing. To prepare neurons completely devoid of non-neuronal cells, 1000–2000 neurons dissociated from neonatal mouse SCG were seeded onto a restricted area on the culture dish, and all non-neuronal cells were manually killed by a micromanipulator-driven needle. Absence of non-neuronal cells was then checked for several days, and the few non-neuronal cells that remained in the culture were killed. This approach was not feasible for hippocampal cultures. Therefore, neurons from 5-day cultures of E16 mouse hippocampi were individually picked up with glass pipettes with the help of a micromanipulator under visual control with an inverted microscope essentially as described (28Moshnyakov M. Arumäe U. Saarma M. Mol. Brain Res. 1996; 43: 141-148Crossref PubMed Scopus (26) Google Scholar). About 30 neurons were individually picked up with separate glass pipettes with an aperture slightly bigger than the cell diameter and collected into Trizol reagent. RNA isolation and RT-PCR were performed as described above. No fragments were amplified from the culture medium collected in the same way as the neurons (data not shown). The RNase protection assay (RPA) was performed as described (29Timmusk T. Belluardo N. Metsis M. Persson H. Eur. J. Neurosci. 1993; 5: 605-613Crossref PubMed Scopus (230) Google Scholar). Full-length N-Bak cDNA was used to generate 32P-labeled antisense RNA probe. The assay resulted in one fragment of 572 bp for N-Bak and two fragments of 322 and 230 bp for Bak. Only the 572- and 230-bp fragments are shown in Fig. 2. For a positive control, the mouse %-actin riboprobe (Ambion Inc., Austin, TX) was used. P1–P2 mouse SCG were digested with collagenase (2.5 mg/ml; Worthington), dispase (5 mg/ml; Roche Molecular Biochemicals), and trypsin (10 mg/ml; Worthington) for 45 min at 37 °C and dissociated mechanically with a siliconized glass Pasteur pipette. Non-neuronal cells were removed by extensive preplating. Almost pure neurons were cultured in polyornithine/laminin (Sigma)-coated 35-mm plastic dishes at a 1:1 ratio of nutrient mixture F-12 to Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 3% fetal calf serum (Hyclone, Cramlington, United Kingdom), serum substitute containing 0.35% bovine serum albumin (Pathocyte-4, ICN Pharmaceuticals, Inc.), 60 ng/ml progesterone, 16 μg/ml putrescine, 400 ng/ml L-thyroxine, 38 ng/ml sodium selenite, and 340 ng/ml triiodothyronine (all from Sigma Chemical Co.) (30Davies A.M. Cohen J. Wilkin G.P. Neural Cell Culture. Oxford University Press, Oxford1995: 153-175Google Scholar), and 30 ng/ml mouse 2.5 S nerve growth factor (NGF) (Promega, Madison, WI) at 37 °C in a humid atmosphere containing 5% CO2. Neither antibiotics nor antimitotic drugs were included in the culture medium. Hippocampi from E16 mice were dissociated with trypsin (0.25%) for 15 min at 37 °C in Hanks' balanced salt solution containing 1 mg/ml DNase I (Sigma) and 10 mm glucose, triturated with a siliconized glass Pasteur pipette, plated onto polyornithine (Sigma)-coated dishes, and grown further in neurobasal medium (Life Technologies, Inc.) containing B-27 serum substitute (Life Technologies, Inc.). Nuclei of the SCG neurons, cultured for 5–6 days with 30 ng/ml NGF, were pressure-injected under direct visual control with expression plasmids encoding Bak or N-Bak or with the empty pCR3.1 vector, all 50 ng/μl. All injection solutions contained also 10 ng/μl pGreenLantern-1. A Model MMO-220 micromanipulator (Narishige International Ltd., London) and a Model 5246 Transjector (Eppendorf Scientific, Westbury, NY) were used for injection. Neurons were grown further with NGF or in NGF-free medium with function-blocking anti-NGF antibodies (Roche Molecular Biochemicals). Initial neurons surviving the procedure were counted 3–4 h later. 50–100 neurons were successfully injected with each plasmid combination in every experiment. To later follow all injected neurons individually, the positions of the injected neurons were mapped according to the grid scratched in the bottom of the dish. Healthy fluorescent neurons with phase-bright cytoplasm and an intact neuritic tree, identified according to the fluorescence and the map, were counted daily and expressed as a percentage of the uninjected cells. Few neurons that lost fluorescence during experiment were subtracted from the initial neurons. Uninjected neurons were counted from one dish. Non-neuronal cells from dissociated P1 mouse SCG were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum without neurotrophic factors until all neurons were dead and injected as described above for neurons. Initial cells were counted 2 h (40–50 cells for every plasmid) and living fluorescent cells 18–20 h after injection. During that period, injected cells neither moved nor divided significantly, so they were recognizable according to the fluorescence and the map. Experiments were repeated three times on independent cultures. In every repeat, all treatment groups presented in Fig. 4 (A–C) were studied collectively in the same culture. Statistical significance of the data was analyzed by one-way analysis of variance, followed by Tuckey's post hoc test at a significance level of α = 0.05. For intracellular localization studies, GFP was fused to the N terminus of full-length N-Bak using the pEGFP-C1 vector (CLONTECH). N-Bak cDNA without a putative transmembrane domain (lacking amino acids 125–150) was generated by PCR and cloned into the pEGFP-C1 vector. Functional properties of this deletion mutant together with other mutated forms of N-Bak are currently being studied in our laboratory. Neurons microinjected with these constructs (50 ng/μl) were grown with or without NGF for 48 h and fixed with 4% paraformaldehyde in phosphate-buffered saline. In some experiments, neurons were treated with 1 μm Mitotracker Red CMXRos (Molecular Probes, Inc.) for 15 min and washed extensively before fixation. Although a classical punctate pattern of Mitotracker staining was obtained with the non-neuronal cells in culture, the neurons showed more broad and diffuse staining where individual mitochondria were difficult to discern. Images were acquired on an OZ confocal laser scanning microscope (Noran Instruments Inc., Middleton, WI) and processed with Adobe Photoshop software. When verifying the nucleotide sequences of RT-PCR fragments ofBak from mouse brain, we noticed a 20-bp sequence, GCCAGCAGCAACATGCACAG (Fig.1 A), not present in the published mouse Bak sequence (23Ulrich E. Kauffmann-Zeh A. Hueber A.O. Williamson J. Chittenden T. Ma A. Evan G. Genomics. 1997; 44: 195-200Crossref PubMed Scopus (23) Google Scholar). We cloned and sequenced full-length Bak cDNA from mouse brain. The 20-bp insert was found in several sequenced cDNA clones. Also some minor differences from the published Bak sequence were detected in all clones: there is an additional guanine between positions 147 and 148 as well as two additional cytidines between positions 153 and 154 of the published mouse Bak sequence. We do not know whether these minor differences result from sequencing errors of Ulrichet al. (23Ulrich E. Kauffmann-Zeh A. Hueber A.O. Williamson J. Chittenden T. Ma A. Evan G. Genomics. 1997; 44: 195-200Crossref PubMed Scopus (23) Google Scholar) or whether they represent polymorphisms or mutations in the Bak gene of the 3T3 cells used by Ulrichet al. to clone it. In the predicted Bak protein deduced from the corrected nucleotide sequence, residues 50 and 51 are both alanines, but not arginine and proline, as published by Ulrich et al. (23Ulrich E. Kauffmann-Zeh A. Hueber A.O. Williamson J. Chittenden T. Ma A. Evan G. Genomics. 1997; 44: 195-200Crossref PubMed Scopus (23) Google Scholar), followed by an additional alanine absent in the published sequence (Fig. 1 D). The 20-bp sequence is inserted at position 344 (corrected nucleotide numbering) of Bak cDNA (Fig. 1 A), which corresponds exactly with the junction of exons 4 and 5 of the mouseBak gene (23Ulrich E. Kauffmann-Zeh A. Hueber A.O. Williamson J. Chittenden T. Ma A. Evan G. Genomics. 1997; 44: 195-200Crossref PubMed Scopus (23) Google Scholar), suggesting that the 20-bp insert is a hitherto undescribed exon. We determined a partial sequence of the intron between exons 4 and 5 of the Bak gene. The 20-bp sequence was found in the intronic sequence flanked by intron-exon junction sequences corresponding to the GT-AG rule (Fig.1 C). In addition, the 3′-splice site of the 20-bp exon deviated from the respective consensus sequence (Pyr)12CAG (where Pyr is pyrimidine), with several purines interrupting the polypyrimidine tract (Fig. 1 C). The majority of the alternatively spliced exons have weak 3′-splice sites with higher purine content compared with the 3′-sites of constitutively spliced exons (31Stamm S. Zhang M.Q. Marr T.G. Helfman D.M. Nucleic Acids Res. 1994; 22: 1515-1526Crossref PubMed Scopus (87) Google Scholar, 32Lou H. Gagel R.F. J. Endocrinol. 1998; 156: 401-405Crossref PubMed Scopus (42) Google Scholar). We designated this exon as exon N (for neuron-specific exon; see below) and the transcript using this exon as N-Bak. The corrected genomic organization of the mouseBak gene and the two transcripts generated from it by alternative use of exon N are schematically presented in Fig. 1. Use of exon N would cause a translational frameshift, resulting in a changed amino acid sequence and a truncated protein due to a premature stop codon (Fig. 1 D). The predicted protein translated from N-Bak contains 150 amino acids, with a calculated molecular mass of 16.4 kDa and a calculated pI of 4.48. The previously described Bak isoform has 208 residues, with a calculated molecular mass of 23.3 kDa and a calculated pI of 6.08. The novel C-terminal amino acid stretch RPAATCTAYLRVASAGAAWWLSWALATVWPCTSTSVV of N-Bak (Fig.1 D) has no homology to any of the known proteins. As predicted by the Sosui and TopPred2 programs, 24 C-terminal residues of this novel sequence (VASAGAAWWLSWALATVWPCTSTS) may form a transmembrane α-helix. No other known structural motifs were found in this novel sequence by the PSORT II program. Use of exon N would lead to the change of the BH1 domain of the Bak protein into a different amino acid sequence. The BH2 as well as transmembrane domains would not be translated, whereas the BH3 domain would remain unchanged (Fig.1 D). Thus, use of exon N would convert the three BH domain-containing Bak protein into a BH3-only protein with a novel putative transmembrane domain. We designated the short protein isoform (encoded by N-Bak) as N-Bak and the previously known longer protein isoform as Bak. A similar 20-bp sequence (GCCAGCAGCAACACCCACAG) was found in four independent Bak cDNA clones from human brain with two nucleotide differences from mouse exon N. This human sequence is inserted at the position identical to that in mouse BakcDNA, leading to the same changes in amino acid sequence as in mouse N-Bak: a truncated protein of 150 amino acids, a novel C-terminal amino acid sequence (RPAATPTACLRVASIGAVWWLFWASATVWPYTSTSMA) with a predicted transmembrane domain, and a lack of BH1 and BH2 domains. Expression of Bak splice variants in different tissues was studied by RT-PCR and RPA. RT-PCR analysis showed that Bak was expressed in all studied rat tissues, whereas N-Bak was expressed exclusively in the nervous tissue (Fig. 2 A). Identical results were obtained when RNAs from human tissues were analyzed by RT-PCR (data not shown). RPA also revealed expression of Baktranscripts in all adult mouse tissues analyzed, whereas N-Bak was detected only in brain. The levels of bothBak and N-Bak transcripts were rather similar in different brain regions (Fig. 2 B). Furthermore,Bak and N-Bak transcripts were differently regulated during mouse brain development. Low levels of N-Bak were present at E13, and the levels increased in the late embryogenesis and early postnatal days, with the peak being around birth (Fig. 2 C). In contrast to this, the levels ofBak were high in E13 brain and decreased gradually during development (Fig. 2 C). Both transcripts of Bak were detected in cultured cells of rat cerebral cortex and hippocampus (Fig. 2 A) that contained neurons as well as non-neuronal cells. In contrast, the non-neuronal cells from rat cerebral cortical culture expressed only Bak, but not N-Bak. Similarly, the rat sciatic nerve, known to contain mostly Schwann cells, expressed Bak, but not N-Bak (Fig. 2 A). To clarify the cell-type specificity of expression of the two Bak transcripts, we manually separated cultured neonatal mouse SCG or E16 mouse hippocampal neurons from all non-neuronal cells (see “Experimental Procedures”). Neuron-free cultures of non-neuronal cells from both sources were also prepared. We then analyzed expression ofBak splice variants in purified cell populations by RT-PCR. Both SCG and hippocampal neurons expressed only N-Bak, but not Bak. Conversely, only Bak (but not N-Bak) was expressed in non-neuronal cells (Fig.2 D). The experiments with purified SCG neurons were repeated three times with identical results. Absence of Bak in neurons was further verified with totally non-neuronal cell-free SCG neurons by nested PCR. Bak message was still not detected (data not shown). Although we did not analyze other neuronal populations, it is probable that the Bak transcript in brain tissues (Fig. 2 C) may be of glial origin. Thus, expression of N-Bak is strictly neural tissue-specific and, at least in SCG and hippocampus, strictly neuron-specific, whereas Bakis expressed almost ubiquitously (20Chittenden T. Harrington E.A. O'Connor R. Flemington C. Lutz R.J. Evan G.I. Guild B.C. Nature. 1995; 374: 733-736Crossref PubMed Scopus (695) Google Scholar, 23Ulrich E. Kauffmann-Zeh A. Hueber A.O. Williamson J. Chittenden T. Ma A. Evan G. Genomics. 1997; 44: 195-200Crossref PubMed Scopus (23) Google Scholar), but is absent (or below the detection limit) in the neurons. Exon N, as well as neuron-specific expression of N-Bak, is conserved in the mouse, rat, and human species. However, as our attempts to demonstrate endogenous N-Bak protein failed (see “Experimental Procedures”), we do not have evidence that endogenous N-Bak mRNA is translated into protein in neurons. To study the functional activity of N-Bak in its natural cellular environment, we microinjected cultured neonatal mouse sympathetic SCG neurons with the expression plasmid encoding N-Bak or Bak and maintained the neurons further with or without NGF. Cultured neonatal SCG neurons are known to die apoptotically when deprived of NGF (33Deckwerth T.L. Johnson E.M. J. Cell Biol. 1993; 123: 1207-1222Crossref PubMed Scopus (515) Google Scholar). The respective proteins were produced from the corresponding expression plasmids in transiently transfected COS-7 cells as shown by Western blotting (Fig.3 A). An ∼28-kDa band was greatly enhanced in Bak-transfected cells, comigrating with the endogenous Bak in COS-7 cells, whereas a smaller band of ∼22 kDa was revealed only in N-Bak-transfected cells (Fig.3 A). For some unknown reason, both proteins migrated somewhat slower than predicted by their amino acid sequences. N-Bak was also produced by the injected expression plasmid in the neurons, as shown by staining the injected neurons with anti-Bak antibodies (Fig.3 B). As shown previously (34Martinou I. Missotten M. Fernandez P.A. Sadoul R. Martinou J.-C." @default.
• W2029667265 created "2016-06-24" @default.
• W2029667265 creator A5001221244 @default.
• W2029667265 creator A5014174188 @default.
• W2029667265 creator A5026170248 @default.
• W2029667265 creator A5053193606 @default.
• W2029667265 creator A5080305063 @default.
• W2029667265 date "2001-05-01" @default.
• W2029667265 modified "2023-10-08" @default.
• W2029667265 title "Neuron-specific Bcl-2 Homology 3 Domain-only Splice Variant of Bak Is Anti-apoptotic in Neurons, but Pro-apoptotic in Non-neuronal Cells" @default.
• W2029667265 cites W103053889 @default.
• W2029667265 cites W1481679077 @default.
• W2029667265 cites W1501566043 @default.
• W2029667265 cites W1518101530 @default.
• W2029667265 cites W1530428200 @default.
• W2029667265 cites W1540436551 @default.
• W2029667265 cites W1593219141 @default.
• W2029667265 cites W172276776 @default.
• W2029667265 cites W1745634660 @default.
• W2029667265 cites W1913661233 @default.
• W2029667265 cites W1965217295 @default.
• W2029667265 cites W1965566566 @default.
• W2029667265 cites W1979782947 @default.
• W2029667265 cites W1980837484 @default.
• W2029667265 cites W1981231641 @default.
• W2029667265 cites W1982687089 @default.
• W2029667265 cites W1985683891 @default.
• W2029667265 cites W1986678922 @default.
• W2029667265 cites W1996524350 @default.
• W2029667265 cites W1997619705 @default.
• W2029667265 cites W2003301112 @default.
• W2029667265 cites W2010336934 @default.
• W2029667265 cites W2011799913 @default.
• W2029667265 cites W2012892271 @default.
• W2029667265 cites W2024996523 @default.
• W2029667265 cites W2034609335 @default.
• W2029667265 cites W2039355845 @default.
• W2029667265 cites W2040792935 @default.
• W2029667265 cites W2040945511 @default.
• W2029667265 cites W2041822578 @default.
• W2029667265 cites W2043350035 @default.
• W2029667265 cites W2054669186 @default.
• W2029667265 cites W2055186588 @default.
• W2029667265 cites W2060982272 @default.
• W2029667265 cites W2061273150 @default.
• W2029667265 cites W2061833772 @default.
• W2029667265 cites W2063176993 @default.
• W2029667265 cites W2064127449 @default.
• W2029667265 cites W2070640811 @default.
• W2029667265 cites W2072355782 @default.
• W2029667265 cites W2073028327 @default.
• W2029667265 cites W2075226068 @default.
• W2029667265 cites W2075278727 @default.
• W2029667265 cites W2075749235 @default.
• W2029667265 cites W2077270970 @default.
• W2029667265 cites W2077312805 @default.
• W2029667265 cites W2078458079 @default.
• W2029667265 cites W2081690888 @default.
• W2029667265 cites W2084533871 @default.
• W2029667265 cites W2088187860 @default.
• W2029667265 cites W2091483784 @default.
• W2029667265 cites W2106697922 @default.
• W2029667265 cites W2106799894 @default.
• W2029667265 cites W2132411546 @default.
• W2029667265 cites W2134872883 @default.
• W2029667265 cites W2134979684 @default.
• W2029667265 cites W2136269288 @default.
• W2029667265 cites W2146983373 @default.
• W2029667265 cites W2154745705 @default.
• W2029667265 cites W2157740207 @default.
• W2029667265 cites W2158552404 @default.
• W2029667265 cites W2162560719 @default.
• W2029667265 cites W2164799217 @default.
• W2029667265 cites W2169746454 @default.
• W2029667265 cites W2244063038 @default.
• W2029667265 cites W2275275778 @default.
• W2029667265 doi "https://doi.org/10.1074/jbc.m010419200" @default.
• W2029667265 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11278671" @default.
• W2029667265 hasPublicationYear "2001" @default.
• W2029667265 type Work @default.
• W2029667265 sameAs 2029667265 @default.
• W2029667265 citedByCount "80" @default.
• W2029667265 countsByYear W20296672652012 @default.
• W2029667265 countsByYear W20296672652013 @default.
• W2029667265 countsByYear W20296672652014 @default.
• W2029667265 countsByYear W20296672652015 @default.
• W2029667265 countsByYear W20296672652016 @default.
• W2029667265 countsByYear W20296672652017 @default.
• W2029667265 countsByYear W20296672652019 @default.
• W2029667265 countsByYear W20296672652020 @default.
• W2029667265 countsByYear W20296672652021 @default.
• W2029667265 countsByYear W20296672652022 @default.
• W2029667265 crossrefType "journal-article" @default.
• W2029667265 hasAuthorship W2029667265A5001221244 @default.
• W2029667265 hasAuthorship W2029667265A5014174188 @default.
• W2029667265 hasAuthorship W2029667265A5026170248 @default.
• W2029667265 hasAuthorship W2029667265A5053193606 @default.
• W2029667265 hasAuthorship W2029667265A5080305063 @default.

• next