From the Program of Molecular Neurobiology, Institute of Biotechnology, University of Helsinki, Viikki Biocenter, FIN-00014 Helsinki, Finland
Received for publication, November 16, 2000, and in revised form, January 26, 2001
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ABSTRACT |
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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 microinjected
Bak 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.
During development, two opposite processes, proliferation and
naturally occurring cell death (apoptosis), regulate cell number in
almost all tissues and organs (1). 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 (2, 3). The cells of an organism retain a potential to die
apoptotically during their entire lifetime (4). Apoptotic pathways are
therefore delicately balanced and controlled positively and negatively
at several levels (5, 6). 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 (7). 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) (7-10). Interestingly,
the recently discovered Bcl-G contains only BH3 and BH2 domains
(11).
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 (5, 6). Once activated above certain threshold levels, caspases irreversibly execute cell death (6, 12). 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 (7). BH3-only
proteins are believed to regulate or modulate the activity of multi-BH
domain members. Thus, Bid activates Bax (13, 14), whereas Bad
inactivates Bcl-xL (15), 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 (16). However,
BH3-only splice variants encoding proteins with pro-apoptotic activity
have been described for the anti-apoptotic Bcl-x (17) and Mcl-1
(18, 19) and also for Bcl-G (11). Here we describe a BH3-only splice
variant of the pro-apoptotic Bak (20-24) 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.
Cloning and Analysis of Bak Splice Variants--
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. 3A). The
peptide sequence used to generate these antibodies is present in both
Bak and N-Bak (25). 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 the
Bak- 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 M
urea. 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. 3A). 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 (26).
RT-PCR and RNase Protection--
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 (27). 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 (28). 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 (29). 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
Neuronal Cultures--
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.) (30), 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.).
Transfection of Cultured Primary Cells--
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
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.
N-Bak, a Novel Isoform of Bak, Is a BH3-only Protein--
When
verifying the nucleotide sequences of RT-PCR fragments of
Bak from mouse brain, we noticed a 20-bp sequence,
GCCAGCAGCAACATGCACAG (Fig.
1A), not present in the
published mouse Bak sequence (23). 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 Ulrich
et al. (23) or whether they represent polymorphisms or
mutations in the Bak gene of the 3T3 cells used by Ulrich
et 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. (23), followed by an additional alanine absent in the
published sequence (Fig. 1D).
The 20-bp sequence is inserted at position 344 (corrected nucleotide
numbering) of Bak cDNA (Fig. 1A), which
corresponds exactly with the junction of exons 4 and 5 of the mouse
Bak gene (23), 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.
1C). 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. 1C). 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 (31, 32). 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 mouse
Bak 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. 1D). 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.
1D) 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
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 Bak
cDNA, 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.
N-Bak Transcripts Are Expressed in Neurons, but Are Absent in
Non-neuronal Cells--
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. 2A). Identical
results were obtained when RNAs from human tissues were analyzed by
RT-PCR (data not shown). RPA also revealed expression of Bak
transcripts in all adult mouse tissues analyzed, whereas
N-Bak was detected only in brain. The levels of both
Bak and N-Bak transcripts were rather similar in
different brain regions (Fig. 2B). 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. 2C). In contrast to this, the levels of
Bak were high in E13 brain and decreased gradually during
development (Fig. 2C).
Both transcripts of Bak were detected in cultured cells of
rat cerebral cortex and hippocampus (Fig. 2A) 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. 2A). 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 of Bak 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.
2D). 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. 2C) 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 Bak
is expressed almost ubiquitously (20, 23), 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.
N-Bak Is a Survival-promoting Protein in Primary Neurons, but a
Death-promoting Protein in Non-neuronal Cells--
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 (33). The respective proteins
were produced from the corresponding expression plasmids in transiently
transfected COS-7 cells as shown by Western blotting (Fig.
3A). 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.
3A). 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.
3B). As shown previously (34), overexpressed Bak
rapidly killed the neurons even in the presence of NGF (Fig.
4A). In contrast, 93.7 ± 2.4% (mean ± S.E.) of the NGF-maintained N-Bak-expressing neurons remained healthy, which does not differ significantly from
vector-injected or uninjected neurons (Fig. 4A). The same
percentage of neurons survived when the concentration of injected
N-Bak-encoding plasmid was raised from 50 to 100 ng/µl (data not
shown). When deprived of NGF, 19.4 ± 6.7% of the uninjected
neurons and 17.8 ± 8.6% of the vector-injected neurons remained
alive 72-75 h after injection (Fig. 4B). By that time,
overexpressed Bak had killed almost all neurons (Fig. 4B),
as reported previously (21). In contrast, 78.0 ± 2.2% of the
N-Bak-expressing neurons resisted NGF removal, which is statistically
significantly different from the vector-injected (p < 0.001) and uninjected (p < 0.001) neurons (Fig.
4B). A similar percentage of neurons were maintained alive
when the concentration of the N-Bak-encoding plasmid was raised to 100 ng/µl (data not shown). Thus, in neurons, alternative splicing
converts pro-apoptotic Bak into anti-apoptotic N-Bak. To our knowledge,
this is the first demonstration of the anti-apoptotic activity of a
BH3-only protein.
The survival-promoting activity of N-Bak was unexpected since we had
noted earlier that its overexpression triggers death of transfected
COS-7 cells (see "Experimental Procedures"). To address this
controversy, we studied the effects of overexpressed Bak isoforms in
non-neuronal cells more closely. To this end, we microinjected
expression plasmids encoding Bak, N-Bak, or empty vector into primary
non-neuronal cells of dissociated P1 mouse SCG. These cultures,
containing mostly glial cells, but also epithelial and fibroblastic
cells, expressed mRNA for Bak, but not for
N-Bak (Fig. 2D). 24 h after injection,
90.7 ± 1.2% (mean ± S.E.) of the vector-injected control
cells had survived, showing that these cells tolerate well the
procedure and ectopic DNA. As expected, only 10.6 ± 1.3% of the
Bak-injected cells remained alive. N-Bak was an equally
potent death inducer in these cells, as only 12.4 ± 0.5% of the
injected cells were living 24 h after injection (Fig.
4C). When COS-7 cells or non-neuronal cells from E18 mouse trigeminal ganglia were transfected with plasmid encoding Bak, N-Bak,
or empty vector together with a Bcl-xL-encoding plasmid and
stained the next day with anti-Bak antibodies, strongly immunopositive cells were found only in Bak- and
N-Bak-transfected cultures, but not in vector-transfected
cultures (data not shown). However, no Bak-immunopositive cells were
found when Bcl-xL was omitted (data not shown),
suggesting that Bak and N-Bak killed the cells in the absence of
Bcl-xL. Death of non-neuronal cells overexpressing N-Bak
was rapid, as already 1-2 h after transfection with expression plasmid
encoding GFP-N-Bak, the weakly fluorescent cells become round, detached
from the culture substrate, and died soon thereafter. Nuclei of the
GFP-N-Bak-transfected HeLa cells exhibited apoptotic features, being
fragmented and containing condensed chromatin, as shown by staining
with 4,6-diamidino-2-phenylindole, whereas the nuclei of the cells
overexpressing GFP were intact and contained diffuse chromatin (Fig.
3C). In addition, a fragmented DNA ladder typical for
apoptotic cells was induced in HeLa cells by forced expression of
N-Bak, but not GFP (data not shown). Thus, in non-neuronal cells,
ectopically expressed N-Bak is a potent apoptosis-promoting protein.
Overxpressed N-Bak Is Associated with Intracellular Membranes in
SCG Neurons--
Several pro-apoptotic proteins, e.g. Bax,
Bad, and Bid, have been shown to be inactive cytosolic proteins in
healthy cells, but are translocated to mitochondria during apoptosis
(7). To study the intracellular localization of N-Bak in neurons, we microinjected expression vector encoding N-Bak with N-terminally fused
GFP into SCG neurons. The N-terminal GFP moiety did not affect the
survival-promoting properties of N-Bak in microinjected NGF-maintained
or NGF-deprived neurons (data not shown). When examined 48 h after
injection, GFP-N-Bak had a clustered distribution both in
NGF-maintained and NGF-deprived neurons (Fig.
5), suggesting that GFP-N-Bak is
localized to intracellular membranes. Indeed, a similar distribution
pattern was observed for GFP-Bcl-2 (Fig. 5), which is known to be
associated with intracellular membranes (35). Deletion of the putative
transmembrane domain abolished the clustered localization of GFP-N-Bak.
Instead, a diffuse cytosolic pattern was observed that was similar to
the localization of GFP (Fig. 5). Thus, the predicted transmembrane
domain of N-Bak is used in SCG neurons to anchor N-Bak to intracellular
membranes. Green fluorescent clusters overlapped with Mitotracker Red
CMXRos in GFP-N-Bak- and GFP-Bcl-2-expressing neurons. However,
Mitotracker was not specifically concentrated in the clusters of
GFP-N-Bak and GFP-Bcl-2 (data not shown). More studies are necessary to ascertain the identity of neuronal membranes associated with clustered localization of GFP-N-Bak.
We describe here N-Bak, a splice variant of
Bak, that is generated by the use of a novel exon. Insertion
of this exon causes a translational frameshift in Bak
mRNA, thereby changing the C-terminal amino acid sequence of the
protein product. In N-Bak, only some structural elements of Bak,
including the BH3 domain, are preserved, whereas the BH1, BH2, and
transmembrane domains, as well as the predicted pore-forming
hydrophobic The change in function of Bcl-xS and Mcl-1S has
a structural explanation, as their domain arrangement is similar to
that of BH3-only proteins. Based on the determined three-dimensional
structures of Bid (36, 48) and Bcl-xL with or without a
bound BH3 domain of Bak (49-51), a ligand-receptor model has been
proposed for the heterodimerization of pro- and anti-apoptotic Bcl-2
family proteins (8, 36). The BH3-only proteins (and also activated Bax
and Bak) expose their BH3 domains at the surface (so-called ligand state), whereas in the anti-apoptotic family members, the BH1, BH2, and
BH3 domains form a hydrophobic surface groove (a receptor state).
Pro-apoptotic members insert their BH3 domains into the surface pocket
of the anti-apoptotic partners, thereby inactivating them and favoring
death. In Bcl-xS and Mcl-1S, hydrophobic
grooves cannot form due to loss of the BH1 and BH2 domains. Instead,
Bcl-xS is predicted to take a ligand conformation with an
exposed BH3 domain (36). Although we do not know the spatial structure
of N-Bak, it probably has a conformation similar to that of
Bcl-xS and should be intrinsically pro-apoptotic. Indeed,
N-Bak was strongly pro-apoptotic in non-neuronal cells. This is,
however, an artificial situation, as N-Bak is not
endogenously expressed in these cells.
In neurons, in which N-Bak is endogenously expressed, it has no
intrinsic apoptosis-promoting activity. Apparently, something in the
neuronal environment blocks the killing activity of N-Bak. In neurons
that would otherwise die due to the absence of NGF signaling,
overexpressed N-Bak actively neutralized the death program. A
survival-promoting effect of N-Bak in neurons was surprising, as to our
knowledge, there are no other examples where a BH3-only protein
protects the cells against apoptosis. Actually, the BH3 domain has
intrinsic apoptosis-promoting properties, being called a minimal
death domain. Indeed, oligopeptides encompassing little more than the
BH3 domain of Bak are sufficient to release cytochrome c
from isolated mitochondria, to activate caspases, and to promote cell
death (52-55). The same was shown for the BH3 domains of Bax, Bid,
Bad, and Bik (15, 52, 54, 56, 57). Moreover, the BH3 domains of the
anti-apoptotic Bcl-2 and Bcl-xL also facilitate apoptosis
(58). How then can overexpressed N-Bak with an intact BH3 domain
protect neurons from apoptotic death? Binding to and neutralizing the
long Bak isoform, as shown for Bcl-xL/Bcl-xS and Mcl-1L/Mcl-1S (18, 19, 58), are not
probable in SCG neurons, as they do not express Bak (although very low
levels of Bak transcripts may still remain undetected by our
assay). Heterodimerization with other Bcl-2 family members,
e.g. neutralizing Bax or releasing Bcl-xL from
some blocking constraint, cannot be excluded. However, we have not
found any difference in the expression of other Bcl-2 family members in
neurons and non-neuronal cells of cultured mouse SCG. Instead, almost
all the known Bcl-2 family members were found in both cell
types.2 Therefore, we do not
believe that simple heterodimerization with known Bcl-2 family members
can explain the opposite behavior of N-Bak in neurons and non-neuronal
cells. We propose that N-Bak is intrinsically apoptosis-promoting,
as predicted by its domain structure, but that in neurons, it protects
against apoptosis indirectly via some other neuron-specific molecules.
The molecular interactions of N-Bak in neurons are currently being
studied in our laboratory. There are also other examples where some
Bcl-2 family members do not behave according to the ligand-receptor model described above. In NGF-deprived chick sensory neurons, overexpressed Bax protected the neurons, although it killed
NGF-maintained neurons (59). Similarly, overexpressed Bak inhibited
apoptosis in a serum-deprived Epstein-Barr virus-transformed
lymphoblastoid cell line (22), whereas Bax protected central neurons
against Sindbis virus-induced apoptotic death in vivo
(60). Also, Bax- We are aware that the study of a protein overexpressed at high levels
does not necessarily reflect the functional properties of the
endogenous protein. Moreover, our failure to demonstrate endogenous
N-Bak protein suggests that the levels of the protein may be very low,
in contrast to highly overexpressed N-Bak in our microinjection
experiments. Therefore, we cannot claim that endogenous N-Bak is also
anti-apoptotic in neurons.
N-Bak is expressed strictly in the nervous tissue and, at
least in SCG and hippocampus, strictly in neurons and not in
non-neuronal cells. This is, to our knowledge, the first
neuron-specific member of the Bcl-2 family. Our results call for
caution when interpreting functional studies of Bcl-2 family proteins
in an inappropriate cellular environment. Indeed, overexpressed N-Bak
had an opposite effect in different cell types. Several other Bcl-2
family members regulate life and death preferentially in some (but not
all) tissues. Bid is the main mediator of Fas-induced death in
hepatocytes, but not in thymocytes (66), whereas the primary cells
affected by the absence of Bim are T and B lymphocytes (67). Bax- The function of N-Bak in the nervous system remains elusive, but a
specific role is anticipated by its strict neuron-specific expression.
Characterization of the nervous system of Bak-deficient mice has not
yet been reported (70). Transcripts of N-Bak are expressed
at the highest levels in perinatal and newborn mouse brain, at the time
of active neurogenesis and programmed cell death. Presumably, N-Bak is
part of the molecular network controlling the delicate processes that
regulate neuronal number in the developing nervous system, but its
explicit role remains to be studied.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin riboprobe (Ambion Inc., Austin, TX) was used.
= 0.05.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Generation and structure of the isoforms of
mouse Bak. A, schematic representation of the mouse
Bak gene. The insert is the nucleotide sequence of exon N. B, two transcripts (Bak and N-Bak)
generated by alternative use of exon N. Shown are the positions of the
translation start (ATG) and stop (TGA) sites as well as the region in
N-Bak with a changed reading frame (gray boxes).
C, partial intronic sequence containing exon N
(uppercase letters). Shown are the sequences of donor and
acceptor splice sites (boldface). D, aligned
amino acid sequences of two mouse Bak isoforms, Bak and N-Bak. Shown
are the novel sequence in N-Bak (boldface) generated by
frameshift due to use of exon N; the BH3, BH1, and BH2 domains
(boxed); the transmembrane domain (underlined);
and amino acids different from those published by Ulrich et
al. (23) (lowercase letters).
-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.
1D). 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.
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Fig. 2.
Expression of N-Bak and
Bak transcripts in rat and mouse tissues and
cells. A, RT-PCR analysis of Bak transcripts
in rat tissues and cultured cells. B, RPA analysis of
Bak transcripts in non-neuronal tissues and brain regions of
adult mouse. Shown are 572-bp fragments of N-Bak and 230-bp
fragments of Bak. C, RPA analysis of Bak
transcripts in mouse brain of different ages. Shown are 572-bp
fragments of N-Bak and 230-bp fragments of Bak.
The lower panels in B and C show the
levels of -actin transcripts in the RNA samples analyzed. Note that
-actin mRNA levels decreased during postnatal brain development,
as has been reported (71). D, RT-PCR analysis of
Bak and N-Bak expression in cultured neonatal
mouse SCG and hippocampal neurons free of non-neuronal cells,
non-neuronal SCG and hippocampal cells free of neurons, or
undissociated hippocampal tissue (Total). Note that the
RT-PCR analyses were not quantitative and show only the presence or
absence of the transcripts analyzed. ctx, cortex; ctx
g, cortical non-neuronal cells; hip, hippocampus;
sp c, spinal cord; sc n, sciatic nerve of adult
rat; ad, adult; hrt, heart; kid,
kidney; liv, liver; mus, skeletal muscle;
spl, spleen; tes, testis; thyr,
thyroid; thym, thymus; olf, olfactory bulb;
str, striatum; tha, thalamus; col,
colliculi superior and inferior; v midbr, ventral midbrain;
cbl, cerebellum; med, medulla; total,
whole P60 brain; tRNA, yeast tRNA.
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Fig. 3.
Transfection of cells with
expression plasmid encoding N-Bak. A, COS-7
cells were transfected with pCR3.1 vectors encoding Bak, N-Bak, or
empty vector together with Bcl-xL- and GFP-encoding
plasmids. Cell lysates were analyzed by Western blotting with anti-Bak
antibodies. Molecular mass markers are shown on the left. The anti-Bak
antibodies recognize the introduced Bak (~28 kDa) (first
lane), which comigrated with the endogenous Bak of the COS-7
cells, and the introduced N-Bak (~22 kDa) (second lane).
Note that direct comparison of the intensities of Bak bands between
Bak-transfected and mock-transfected or untransfected cells
is inappropriate, as Bak and N-Bak killed many cells despite the
presence of Bcl-xL. B, shown is the expression
of the introduced N-Bak protein in microinjected SCG neurons. Cultured
neonatal mouse SCG neurons were injected with a mixture of expression
plasmids encoding N-Bak and GFP or empty vector and GFP, grown 3 days
with NGF, and stained with anti-Bak antibodies. Bak immunoreactivity
was visualized by rhodamine-conjugated secondary antibodies. Three
images (phase-contrast, green fluorescent for GFP, and red fluorescent
for Bak immunoreactivity) were captured for every neuron. Shown are one
typical Bak/GFP-injected neuron, one typical vector/GFP-injected
neuron, and one typical uninjected neuron. Faint red fluorescence of
control cells is a nonspecific background. C, overexpression
of GFP-N-Bak (but not GFP) in HeLa cells caused nuclear fragmentation
and chromatin condensation, as shown by staining with
4,6-diamidino-2-phenylindole. In the upper panels, green
fluorescence of the two GFP-N-Bak-transfected cells is weak, as the
cells died rapidly. Fragmented nuclei with condensed chromatin are
above the nuclei of the flat untransfected cells because the apoptotic
cells became round. In the lower panels, two cells are
shown, one of which overexpressed GFP. The nuclei of both cells are
healthy with diffuse chromatin.
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Fig. 4.
Effect on survival of overexpression of
Bak splice variants in primary SCG cells.
A and B, neonatal mouse sympathetic neurons were
microinjected with expression plasmids encoding Bak, N-Bak, or the
empty pCR3.1 vector (all 50 ng/µl) together with pGreenLantern-1 (10 ng/µl) and grown further with (A) or without
(B) NGF. Living fluorescent neurons, counted 72-75 h after
injection, are expressed as percentage of initial cells counted 3-5 h
after injection. C, non-neuronal cells from neonatal mouse
SCG were injected as described for A and B, and
living fluorescent cells were counted 24 h after injection.
Means ± S.E. of three independent experiments are shown for each
condition. Statistical significance between means was estimated by
one-way analysis of variance. *, p < 0.05; ***,
p < 0.001.
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Fig. 5.
GFP-N-Bak is anchored to intracellular
membranes. SCG neurons overexpressing GFP-N-Bak, GFP-Bcl-2, GFP,
or GFP-N-Bak lacking the predicted transmembrane domain
(GFP-N-Bak- TM) were grown for 48 h in the presence or absence
of NGF. Shown are confocal microscopic images of a typical neuron for
each condition. A lower level of fluorescence was common for
NGF-deprived neurons not protected by overexpressed anti-apoptotic
proteins.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5- and
6-helices (36), are missing due to a
translational frameshift and premature stop codon. Splice variants
encoding different protein isoforms have been described for several
other Bcl-2 family members. In most cases, as for Bcl-x
(37),
Bcl-x
(37), Bcl-x
TM (38), Bax-
(39), Bax-
(40), Bax-
(41), Bcl-2
(42), and Bad-
(43), the changes involve the
C-terminal regions of the proteins, leaving the BH domains unaltered.
In other splice variants, regions encoding one or more BH domains are
missing. For example, Bax-
(44) lacks the BH2 domain; Bax-
lacks
the BH3 domain (45); and, BokS contains only the BH2 domain
and a composite domain consisting of half of the BH3 domain and
half of the BH1 domain (46). The functional activity of these splice
variants remains mainly unchanged. The pro-apoptotic activity of three
splice variants of Bim is increased as the protein is shortened (47).
There are, however, a few cases where alternative splicing generates
shorter BH3-only variants of multi-BH domain Bcl-2 family proteins,
whose activity is converted opposite to that of the longer isoform. So
far, such splice variants have been described only for the
anti-apoptotic proteins Bcl-x (17) and Mcl-1 (18, 19). Thus, the
multi-BH domain Bcl-xL and Mcl-1L isoforms
protect against apoptosis, whereas their short BH3-only variants,
Bcl-xS (which, however, possesses also a BH4 domain) and
Mcl-1S, promote death. The recently described Bcl-G has
also two splice variants: Bcl-GL, which contains the BH3
and BH2 domains and which has only minor effect on apoptosis, and
Bcl-GS, a BH3-only protein with strong death-promoting
activity (11). In contrast, N-Bak, a BH3-only variant of the
pro-apoptotic protein Bak described here, is anti-apoptotic in cell
types in which it is endogenously expressed. Thus, the change of
activity to the opposite direction may be a general phenomenon when a
multi-BH domain Bcl-2 family protein is converted into a BH3-only
protein by alternative splicing.
protected cells against tumor necrosis
factor-induced apoptosis, although it promoted cell death in other
circumstances (41). Currently, there are no good explanations for these
results. Our results also show that the presence of four BH domains is
not always necessary for a Bcl-2 family protein to be anti-apoptotic. Indeed, the activity of N-Bak in neurons is indistinguishable from that
of Bcl-w, Bcl-2, or Bcl-xL microinjected into sympathetic or sensory neurons deprived of neurotrophic factors (43, 61-65).
, a pro-apoptotic splice variant of Bax, is expressed mostly in immune organs and plays a critical role in T cell survival after T cell receptor activation (37). Expression of Bcl-xS is more
restricted than that of Bcl-xL (68). Overexpressed
Bcl-xS promotes apoptosis in 3T3 cells (69), whereas in 293 kidney epithelial cells or yeast cells, it only neutralizes the
survival-promoting effect of Bcl-xL, but is not apoptotic
by itself (58). Interestingly, whereas Bcl-GL is expressed
in many tissues, its BH3-only isoform is exclusively testis-specific
(11).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Alexei Titievsky and
Mihail Paveliev for assistance with confocal microscopy, Dr. Deyin
Guo for preparing the GFP-N-Bak-TM construct, Dr. Susanne
Hamnér (Biomedical Center, Uppsala) for providing the
GFP-Bcl-2 construct, and Dr. Matti Airaksinen for critical reading of
the manuscript.
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FOOTNOTES |
---|
* This work was supported by Academy of Finland Programs 44896 (Finnish Center of Excellence Program 2000-2005) and 43679, European Union Biotech Grant BIO4-98-0293, and a Sigrid Jusélius Foundation grant.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Program of Molecular Neurobiology, Inst. of Biotechnology, University of Helsinki, P. O. Box 56, Viikki Biocenter, FIN-00014 Helsinki, Finland. Tel.: 358-9-19159369; Fax: 358-9-19159366; E-mail: urmas.arumae@helsinki.fi.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M010419200
2 Y.-F. Sun, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are: BH, Bcl-2 homology; P, postnatal day; E, embryonic day; RT-PCR, reverse transcription-polymerase chain reaction; GFP, green fluorescent protein; SCG, superior cervical ganglion/ganglia/ganglial; bp, base pair(s); RPA, RNase protection assay; NGF, nerve growth factor.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Jacobson, M. D., Weil, M., and Raff, M. C. (1997) Cell 88, 347-354[Medline] [Order article via Infotrieve] |
2. | Oppenheim, R. W. (1991) Annu. Rev. Neurosci. 14, 453-501[CrossRef][Medline] [Order article via Infotrieve] |
3. | Yuan, J., and Yankner, B. A. (2000) Nature 407, 802-809[CrossRef][Medline] [Order article via Infotrieve] |
4. | Vaux, D. L., and Korsmeyer, S. J. (1999) Cell 96, 245-254[Medline] [Order article via Infotrieve] |
5. | Green, D. R. (2000) Cell 102, 1-4[Medline] [Order article via Infotrieve] |
6. | Hengartner, M. O. (2000) Nature 407, 770-776[CrossRef][Medline] [Order article via Infotrieve] |
7. | Antonsson, B., and Martinou, J.-C. (2000) Exp. Cell Res. 256, 50-57[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Gross, A.,
McDonnell, J. M.,
and Korsmeyer, S. J.
(1999)
Genes Dev.
13,
1899-1911 |
9. | Komatsu, K., Miyashita, T., Hang, H., Hopkins, K. M., Zheng, W., Cuddeback, S., Yamada, M., Lieberman, H. B., and Wang, H.-G. (2000) Nat. Cell Biol. 2, 1-6[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Oda, E.,
Ohki, R.,
Murasawa, H.,
Nemoto, J.,
Shibue, T.,
Yamashita, T.,
Tokino, T.,
Taniguchi, T.,
and Tanaka, N.
(2000)
Science
288,
1053-1058 |
11. |
Guo, B.,
Godzik, A.,
and Reed, J. C.
(2001)
J. Biol. Chem.
276,
2780-2785 |
12. |
Wolf, B. B.,
and Green, D. R.
(1999)
J. Biol. Chem.
274,
20049-20052 |
13. |
Desagher, S.,
Osen-Sand, A.,
Nichols, A.,
Eskes, R.,
Montessuit, S.,
Lauper, S.,
Maundrell, K.,
Antonsson, B.,
and Martinou, J.-C.
(1999)
J. Cell Biol.
144,
891-901 |
14. |
Eskes, R.,
Desagher, S.,
Antonsson, B.,
and Martinou, J.-C.
(2000)
Mol. Cell. Biol.
20,
929-935 |
15. | Kelekar, A., Chang, B. C., Harlan, J. E., Fesik, S. W., and Thompson, C. B. (1997) Mol. Cell. Biol. 17, 7040-7046[Abstract] |
16. | Jiang, Z. H., and Wu, J. Y. (1999) Proc. Soc. Exp. Biol. Med. 220, 64-72[Abstract] |
17. | Boise, L. H., González-García, M., Postema, C. E., Ding, L., Lindsten, T., Turka, L. A., Mao, X., Núñez, G., and Thompson, C. B. (1993) Cell 74, 597-608[Medline] [Order article via Infotrieve] |
18. |
Bae, J.,
Leo, C. P.,
Hsu, S. Y.,
and Hsueh, A. J. W.
(2000)
J. Biol. Chem.
275,
25255-25261 |
19. |
Bingle, C. D.,
Craig, R. W.,
Swales, B. M.,
Singleton, V.,
Zhou, P.,
and Whyte, M. K. B.
(2000)
J. Biol. Chem.
275,
22136-22146 |
20. | Chittenden, T., Harrington, E. A., O'Connor, R., Flemington, C., Lutz, R. J., Evan, G. I., and Guild, B. C. (1995) Nature 374, 733-736[CrossRef][Medline] [Order article via Infotrieve] |
21. | Farrow, S. N., White, J. H. M., Martinou, I., Raven, T., Pun, K.-T., Grinham, C. J., Martinou, J.-C., and Brown, R. (1995) Nature 374, 731-733[CrossRef][Medline] [Order article via Infotrieve] |
22. | Kiefer, M. C., Brauer, M. J., Powers, V. C., Wu, J. J., Umansky, S. R., Tomei, L. D., and Barr, P. J. (1995) Nature 374, 736-739[CrossRef][Medline] [Order article via Infotrieve] |
23. | Ulrich, E., Kauffmann-Zeh, A., Hueber, A. O., Williamson, J., Chittenden, T., Ma, A., and Evan, G. (1997) Genomics 44, 195-200[CrossRef][Medline] [Order article via Infotrieve] |
24. | Herberg, J. A., Phillips, S., Beck, S., Jones, T., Sheer, D., Wu, J. J., Prochazka, V., Barr, P. J., Kiefer, M. C., and Trowsdale, J. (1998) Gene (Amst.) 211, 87-94[CrossRef][Medline] [Order article via Infotrieve] |
25. | Krajewski, S., Krajewska, M., and Reed, J. C. (1996) Cancer Res. 56, 2849-2855[Abstract] |
26. | Kaufmann, S. H., Mesner, P. W., Samejima, K., Toné, S., and Earnshaw, W. C. (2000) Methods Enzymol. 322, 3-15[CrossRef][Medline] [Order article via Infotrieve] |
27. | O'Malley, E. K., Sieber, B.-A., Morrison, R. S., Black, I. B., and Dreyfus, C. F. (1994) Brain Res. 647, 83-90[Medline] [Order article via Infotrieve] |
28. | Moshnyakov, M., Arumäe, U., and Saarma, M. (1996) Mol. Brain Res. 43, 141-148[CrossRef][Medline] [Order article via Infotrieve] |
29. | Timmusk, T., Belluardo, N., Metsis, M., and Persson, H. (1993) Eur. J. Neurosci. 5, 605-613[Medline] [Order article via Infotrieve] |
30. | Davies, A. M. (1995) in Neural Cell Culture (Cohen, J. , and Wilkin, G. P., eds) , pp. 153-175, Oxford University Press, Oxford |
31. | Stamm, S., Zhang, M. Q., Marr, T. G., and Helfman, D. M. (1994) Nucleic Acids Res. 22, 1515-1526[Abstract] |
32. |
Lou, H.,
and Gagel, R. F.
(1998)
J. Endocrinol.
156,
401-405 |
33. | Deckwerth, T. L., and Johnson, E. M. (1993) J. Cell Biol. 123, 1207-1222[Abstract] |
34. | Martinou, I., Missotten, M., Fernandez, P. A., Sadoul, R., and Martinou, J.-C. (1998) Neuroreport 9, 15-19[Medline] [Order article via Infotrieve] |
35. |
Wolter, K. G.,
Hsu, Y.-T.,
Smith, C. L.,
Nechushtan, A.,
Xi, X.-G.,
and Youle, R. J.
(1997)
J. Cell Biol.
139,
1281-1292 |
36. | McDonnell, J. M., Fushman, D., Milliman, C. L., Korsmeyer, S. J., and Cowburn, D. (1999) Cell 96, 625-634[Medline] [Order article via Infotrieve] |
37. | Yang, X.-F., Weber, G. F., and Cantor, H. (1997) Immunity 7, 629-639[Medline] [Order article via Infotrieve] |
38. |
Fang, W.,
Rivard, J. J.,
Mueller, D. L.,
and Behrens, T. W.
(1994)
J. Immunol.
153,
4388-4398 |
39. | Oltvai, Z. N., Milliman, C. L., and Korsmeyer, S. J. (1993) Cell 74, 609-619[Medline] [Order article via Infotrieve] |
40. | Schmitt, E., Paquet, C., Beauchemin, M., Dever-Bertrand, J., and Bertrand, R. (2000) Biochem. Biophys. Res. Commun. 270, 868-879[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Zhou, M.,
Demo, S. D.,
McClure, T. N.,
Crea, R.,
and Bitler, C. M.
(1998)
J. Biol. Chem.
273,
11930-11936 |
42. | Negrini, M., Silini, E., Kozak, C. A., Tsujimoto, Y., and Croce, C. M. (1987) Cell 49, 455-463[Medline] [Order article via Infotrieve] |
43. | Hamnér, S., Arumäe, U., Yu, L.-Y., Y., Sun, Y.-F., Saarma, M., and Lindholm, D. (2001) Mol. Cell. Neurosci. 17, 97-106[CrossRef][Medline] [Order article via Infotrieve] |
44. | Shi, B., Triebe, D., Kajiji, S., Iwata, K. K., Bruskin, A., and Mahajna, J. (1999) Biochem. Biophys. Res. Commun. 254, 779-785[CrossRef][Medline] [Order article via Infotrieve] |
45. | Apte, S. S., Mattei, M. G., and Olsen, B. R. (1995) Genomics 26, 592-594[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Hsu, S. Y.,
and Hsueh, A. J.
(1998)
J. Biol. Chem.
273,
30139-30146 |
47. |
O'Connor, L.,
Strasser, A.,
O'Reilly, L. A.,
Hausmann, G.,
Adams, J. M.,
Cory, S.,
and Huang, D. C. S.
(1998)
EMBO J.
17,
384-395 |
48. | Chou, J. J., Li, H., Salvesen, G. S., Yuan, J., and Wagner, G. (1999) Cell 96, 615-624[Medline] [Order article via Infotrieve] |
49. | Muchmore, S. W., Sattler, M., Liang, H., Meadows, R. P., Harlan, J. E., Yoon, H. S., Nettesheim, D., Chang, B. S., Thompson, C. B., Wong, S. L., Ng, S. L., and Fesik, S. W. (1996) Nature 381, 335-341[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Aritomi, M.,
Kunishima, N.,
Inohara, N.,
Ishibashi, Y.,
Ohta, S.,
and Morikawa, K.
(1997)
J. Biol. Chem.
272,
27886-27892 |
51. |
Sattler, M.,
Liang, H.,
Nettesheim, D.,
Meadows, R. P.,
Harlan, J. E.,
Eberstadt, M.,
Yoon, H. S.,
Shuker, S. B.,
Chang, B. S.,
Minn, A. J.,
Thompson, C. B.,
and Fesik, S. W.
(1997)
Science
275,
983-986 |
52. | Chittenden, T., Flemington, C., Houghton, A. B., Ebb, R. G., Gallo, G. J., Elangovan, B., Chinnadurai, G., and Lutz, R. J. (1995) EMBO J. 14, 5589-5596[Abstract] |
53. | Cosulich, S. C., Worrall, V., Hedge, P. J., Green, S., and Clarke, P. R. (1997) Curr. Biol. 7, 913-920[Medline] [Order article via Infotrieve] |
54. |
Narita, M.,
Shimizu, S.,
Ito, T.,
Chittenden, T.,
Lutz, R. J.,
Matsuda, H.,
and Tsujimoto, Y.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14681-14686 |
55. |
Holinger, E. P.,
Chittenden, T.,
and Lutz, R. J.
(1999)
J. Biol. Chem.
274,
13298-13304 |
56. | Simonen, M., Keller, H., and Heim, J. (1997) Eur. J. Biochem. 249, 85-91[Abstract] |
57. |
Shimizu, S.,
and Tsujimoto, Y.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
577-582 |
58. |
Chang, B. S.,
Kelekar, A.,
Harris, M. H.,
Harlan, J. E.,
Fesik, S. W.,
and Thompson, C. B.
(1999)
Mol. Cell. Biol.
19,
6673-6681 |
59. |
Middleton, G.,
Núñez, G.,
and Davies, A. M.
(1996)
Development
122,
695-701 |
60. | Lewis, J., Oyler, G. A., Ueno, K., Fannjiang, Y.-R., Chau, B. N., Vornov, J., Korsmeyer, S. J., Zou, S., and Hardwick, J. M. (1999) Nat. Med. 5, 832-835[CrossRef][Medline] [Order article via Infotrieve] |
61. | Allsopp, T. E., Wyatt, S., Paterson, H. F., and Davies, A. M. (1993) Cell 73, 295-307[Medline] [Order article via Infotrieve] |
62. | Borner, C., Martinou, I., Mattmann, C., Irmler, M., Schaerer, E., Martinou, J.-C., and Tschopp, J. (1994) J. Cell Biol. 126, 1059-1068[Abstract] |
63. | Greenlund, L. J. S., Korsmeyer, S. J., and Johnson, E. M. (1995) Neuron 15, 649-661[Medline] [Order article via Infotrieve] |
64. | González-García, M., García, I., Ding, L., O'Shea, S., Boise, L. H., Thompson, C. B., and Núñez, G. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4304-4308[Abstract] |
65. |
Vekrellis, K.,
McCarthy, M. J.,
Watson, A.,
Whitfield, J.,
Rubin, L. L.,
and Ham, J.
(1997)
Development
124,
1239-1249 |
66. | Yin, X.-M., Wang, K., Gross, A., Zhao, Y., Zinkel, S., Klocke, B., Roth, K. A., and Korsmeyer, S. J. (1999) Nature 400, 886-891[CrossRef][Medline] [Order article via Infotrieve] |
67. |
Bouillet, P.,
Metcalf, D.,
Huang, D. C. S.,
Tarlinton, D. M.,
Kay, T. W. H.,
Kööntgen, F.,
Adams, J. M.,
and Strasser, A.
(1999)
Science
286,
1735-1738 |
68. | Krajewski, S., Krajewska, M., Shabaik, A., Wang, H.-G., Irie, S., Fong, L., and Reed, J. C. (1994) Cancer Res. 54, 5501-5507[Abstract] |
69. |
Fridman, J. S.,
Benedict, M. A.,
and Maybaum, J.
(1999)
Cancer Res.
59,
5999-6004 |
70. |
Wei, M. C.,
Lindsten, T.,
Mootha, V. K.,
Weiler, S.,
Gross, A.,
Ashiya, M.,
Thompson, C. B.,
and Korsmeyer, S. J.
(2000)
Genes Dev.
14,
2060-2071 |
71. | Lazarini, F., Deslys, J. P., and Dormont, D. (1991) Mol. Brain Res. 10, 343-346[Medline] [Order article via Infotrieve] |