Originally published In Press as doi:10.1074/jbc.M909572199 on April 13, 2000
J. Biol. Chem., Vol. 275, Issue 29, 22136-22146, July 21, 2000
Exon Skipping in Mcl-1 Results in a Bcl-2 Homology Domain 3 Only
Gene Product That Promotes Cell Death*
Colin D.
Bingle
§,
Ruth W.
Craig¶,
Brenka M.
Swales,
Vanessa
Singleton,
Ping
Zhou¶, and
Moira K. B.
Whyte
From the Respiratory Cell and Molecular Biology
Laboratory, Division of Molecular and Genetic Medicine, The University
of Sheffield Medical School, Sheffield, S10 2RX, United Kingdom and
the ¶ Department of Pharmacology and Toxicology, Dartmouth Medical
School, Hanover, New Hampshire 03755-3835
Received for publication, December 3, 1999, and in revised form, April 13, 2000
 |
ABSTRACT |
Mcl-1 is a member of the Bcl-2 family that is
regulated transcriptionally and post-transcriptionally, with expression
of the full-length Mcl-1-encoded gene product resulting in enhanced
cell survival. As reported here, the human Mcl-1 gene can also undergo differential splicing, which yields an internally deleted,
death-inducing gene product,
Mcl-1s/
TM. Whereas full-length Mcl-1
derives from three coding exons (instead of the two present in Bcl-2
and other anti-apoptotic members of this family), the
Mcl-1s/TM splice variant results from the
joining of the first and third exons with skipping of the central exon.
Because of the skipped exon and a shift in the reading frame
downstream, the Bcl-2 homology domain (BH3) remains intact, whereas the
BH1-, BH2-, and transmembrane-encoding domains do not.
Mcl-1s/TM thus has features similar to BH3 only, pro-apoptotic Bcl-2 family members and, accordingly, was found to
promote cell death. In addition to a variety of other types of
regulation, the Mcl-1 gene appears ideally designed for the generation
of either a Bcl-2-like viability promoting or, as reported here, a BH3
only death-inducing gene product.
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INTRODUCTION |
Apoptosis is a genetically determined cell death program that is
critical for the maintenance of tissue homeostasis in the healthy
organism. Alterations in apoptosis contribute to the pathological effects seen in a variety of diseases, including cancer and
inflammatory disease (1). Apoptosis is controlled by evolutionarily
conserved sets of genes with the Bcl-2 family playing a pivotal role
(2, 3). The members of this family fall into two groups with opposing functions. Pro-survival family members (e.g. Bcl-2,
Bcl-xL, and Mcl-1) inhibit apoptosis, whereas
death-inducing members (e.g. Bax, Bid, and Bad) have the
opposite effect. The family is characterized by domains of high
sequence conservation, termed the Bcl-2 homology (BH)1 domains. Bcl-2 and
other pro-survival family members contain BH1, BH2, and BH3 domains,
which form a hydrophobic cleft (4) and appear critical for
anti-apoptotic function (5). Whereas some death-inducing family members
contain all three of these domains (e.g. Bax, Bak, and Bok
(6-8)), others contain BH3 only (e.g. Bid, Bad, and Bim).
This suggests that BH3 is a minimum requirement for pro-apoptotic
function (9, 10), which probably relates to the fact that the exposed
BH3 domain can bind to the hydrophobic cleft of pro-survival family
members (11). In addition to the BH domains, many family members
contain a transmembrane (TM) domain at the C terminus. This anchors the
protein to intracellular membranes, such as to the outer surface of
mitochondria (12-15). Although this domain may aid in targeting
certain family members for function (16, 17), a TM domain is not
present in some death-inducing members, namely Bid and Bad (3). In sum,
the BH1/BH2/BH3 hydrophobic cleft and the TM anchor appear to
contribute to anti-apoptotic activity, whereas the BH3 domain is
critical for pro-apoptotic function.
Mcl-1 is a Bcl-2 family member that was originally identified as a gene
rapidly up-regulated early in the differentiation of a human myeloid
leukemia cell line (ML-1) upon induction with 12-O-tetradecanoylphorbol acetate (PMA) (18). Mcl-1
expression has since been found to exhibit differentiation
stage-specific expression in a variety of hematopoietic lineages as
well as in epithelial tissues (19, 20). In addition, Mcl-1 can be
rapidly induced through anti-apoptotic cytokine-mediated pathways, such as those stimulated by hematopoietic cell colony-stimulating factors and interleukins (21-25). For example, in neutrophils (26), external stimuli that prolong the neutrophil life span (such as
lipopolysaccharide and granulocyte-macrophage colony-stimulating
factor) cause an increase in Mcl-1 expression (23, 27). An increase in
Mcl-1 expression can be induced through a mitogen-activated protein kinase-dependent signal transduction pathway acting on
SRF/Elk-1(21, 28), as well as through an Akt/ cAMP-response
element-binding protein-regulated pathway (29). In addition to
regulation at a transcriptional level, the Mcl-1 mRNA can be
rapidly turned over (30). Furthermore, the upstream half of Mcl-1
contains PEST sequences, and the protein is likewise subject to rapid
turn-over (15). The downstream half of the protein contains the BH1,
BH2, and BH3 domains and terminates in a TM domain (15, 18). Overall, Mcl-1 is a highly regulated gene product and is frequently expressed during particular stages of cell differentiation and/or in response to
specific signals.
Increased expression of the endogenous full-length Mcl-1 protein is
associated with the maintenance of cell viability, and decreased
expression with cell death (22, 23, 25, 31). Accordingly, transfection
with a construct representing the full-length gene product results in
enhanced cell survival, as seen both in the FDC-P1 hematopoietic cell
line and in Chinese hamster ovary cells (32-34). Enhancement of cell
survival is seen in the presence of a variety of apoptosis-inducing
stimuli and at levels of the introduced protein that are readily
attainable endogenously. Conversely, loss of the expression of Mcl-1 is
associated with cell loss (28, 34, 35). Similarly, in mice that express
full-length Mcl-1 as a transgene in hematopoietic tissues (36),
lymphoid (B and T) and myeloid cells exhibit enhanced survival, and the
life span of mast cells and monocytes is dramatically prolonged.
In sum, expression of the full-length BH1/BH2/BH3/TM-containing Mcl-1 gene product results in enhanced cell survival.
Alternative splicing is increasingly being recognized to play a
significant role in the regulation of proteins involved in cell death,
occurring in a variety of death receptors, Bcl-2 family members, and
cell death effectors such as caspases (37). Bcl-x can undergo
alternative splicing to yield two forms (Bcl-xL and Bcl-xs) that have opposing biological functions. Bcl-x and
Bcl-2 contain two coding exons separated by a conserved intron in the BH2 domain near the C terminus (38-40). Bcl-xL, which is
anti-apoptotic, derives from both of the coding exons in their
entirety. Bcl-xS, which is pro-apoptotic, results from the
use of an alternate upstream splice donor site that does not lie at an
intron/exon border but rather lies within the first coding exon
(38-40). Other splice forms of Bcl-x also exist, and thus alternative
splicing to a variety of different isoforms can occur even with a
relatively simple gene structure (37, 41).
We are interested in the role that the Bcl-2 family plays in
controlling cell life span in differentiating tissues, such as in the
myeloid lineage (both monocytic and granulocytic branches) (42) and in
epithelial tissues. Mcl-1 is of particular interest because of its
highly regulated pattern of expression in these tissues (20-25). In
the research presented here, we describe
Mcl-1s/TM, a splice variant of Mcl-1 that
derives from exon skipping. Although the full-length human Mcl-1 gene
product was found to consist of three coding exons (rather that the two
coding exons seen in Bcl-2 and Bcl-x), the
Mcl-1s/TM variant represented joining of the
first and third exons. This does not affect the BH3 domain in the first
exon but does affect the BH1, BH2, and TM domains downstream because of
the skipped second exon and a change in the reading frame downstream.
The features of the Mcl-1s/TM-encoded gene
product are thus reminiscent of BH3 only pro-apoptotic family members
such Bid and Bad. Accordingly, transfection with
Mcl-1s/TM was found to result in cell death.
In addition to regulation at the level of both transcription and
turn-over, the Mcl-1 gene appears ideally designed for the generation
of either a Bcl-2-like viability promoting or a BH3 only death-inducing
gene product.
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MATERIALS AND METHODS |
Isolation and Culture of Human Cells and Cell Lines--
Human
peripheral blood neutrophils and mononuclear cells were isolated from
normal healthy donors by dextran sedimentation and plasma-Percoll
gradient centrifugation as described previously (43) and cultured in
RPMI and 10% fetal calf serum in Teflon pools. Monocyte-derived
macrophages were obtained by purification of peripheral blood
neutrophils and mononuclear cells and adherence to tissue culture
plasticware in serum-free medium for 1 h followed by culture for
4-5 days in Iscove's modified Dulbecco's medium and 10% autologous
serum (44). Established cell lines, ML-1, BL41-3, K562, Jurkat, A549,
H441, HepG2, and MCF-7 were cultured in RPMI with 10% fetal calf
serum, 10 mM glutamine, and 50 µg/ml streptomycin and
penicillin. PMA stimulation of BL41-3 cells was performed using 1-50
ng/ml PMA applied for 2 or 6 h.
RT-PCR Detection of Mcl-1s/TM in Human Cells and
Cell Lines--
Total RNA was isolated from peripheral blood cells and
cell lines by the RNAeasy system (Qiagen). First strand cDNA was
generated by oligo(dT)-primed reverse transcription using avian
myeloblastosis virus reverse transcriptase (Promega) and under standard
conditions (1 µg of RNA in a total volume of 50 µl). Aliquots (5 µl) were subjected to amplification with one of several sets of
primers (Mcl-1-F3, 5'-GTT GGT CGG GGA ATC TGG TA; Mcl-1-F4, 5'-ATC TCT CGG TAC CTT CGG GA; Mcl-1-F5, 5'-GTA AGG AGT CGG GGT CTT CCC; Mcl-1-R4,
5'-AAA TTA ATG AAT TCG GCG GG; Mcl-1-R7, 5'-TCC TCT TGC CAC TTG CTT
TTC). Reactions used the following primer combinations F3 and R4, F4
and R4, or F5 and R7. PCR was performed in a PCT2000 thermal cycler (MJ
Research) for 30 cycles with denaturation at 94 °C for 60 s,
annealling at 60 °C for 60 s, and extension at 72 °C for
60 s. Aliquots of the reaction products were subjected to
electrophoresis in 1 or 2% agarose gels (Tris-acetate-EDTA buffer) and
visualized by staining with ethidium bromide. The bands obtained with
the F4/R4 and F5/R7 primer combinations were excised and cloned using
the TOPO pCR II and bidirectional eukaryotic (pCR3.1) TA cloning
systems (Invitrogen). Representative minipreps were chosen for sequence
analysis using an ABI 373 automatic sequencer. A complete sequence was
obtained from multiple clones.
Cloning, Mapping, and Sequencing of the Human Mcl-1 Genomic
Locus--
A 32P-labeled probe containing the entire Mcl-1
coding region (p3.2; (18)) was used to screen a human leukocyte genomic
in the EMBL-3 lambda phage vector (CLONTECH catalog
no. HL1006d (Palo Alto, CA)), using standard techniques. The resulting
positive clones were characterized by restriction enzyme mapping.
Selected restriction enzyme fragments were isolated from agarose gels, subcloned into the pBluescript SKII+ vector (Stratagene), and sequenced
on both strands.
Identification of Mcl-1 Transcriptional Start Sites--
Primer
extension was carried out using an oligonucleotide primer complementary
to the Mcl-1 coding region in its upstream portion (primer #105C
primer, 5'-CCCCACAGTAGAGGTTGAGTCCGATTACCG-3'). Counting the ATG of the
initiator methionine as the first through third nucleotides, the primer
used represents nucleotides 23-52 (or nucleotides 77-106 of the p3.2
Mcl-1 cDNA (18, 21)). After 5'-end-labeling with 32P
using T4 polynucleotide kinase, the primer (~2 × 105 cpm) was hybridized to total RNA (20 µg) from ML-1
cells that had been treated with PMA to increase expression of the
Mcl-1 mRNA (18, 21). As a control, the primer was hybridized in parallel to yeast tRNA (20 µg). Hybridization was carried out at
30 °C for 14 h in 10 mM Tris-Cl buffer, pH 8.3, containing 150 mM KCl and 1 mM EDTA. Reverse
transcription with avian myeloblastosis virus reverse transcriptase
(Promega) was carried out at 42 °C for 90 min in 50 mM
Tris-Cl buffer, pH 8.3, containing 10 mM MgCl2, 50 mM KCl, 10 mM dithiothreitol,
deoxynucleotide triphosphates (0.25 mM each), and 0.15 µg/µl actinomycin D. After stopping the reaction by the addition of
EDTA (to 20 µM), samples were treated with RNase A
(Sigma) at 37 °C for 30 min, extracted with phenol/chloroform, precipitated with ethanol, and subjected to gel electrophoresis.
The S1 nuclease protection assay was carried out using a
32P-labeled single-stranded DNA probe complementary to
Mcl-1 in the upstream portion of the coding sequence and extending into
the 5'-flanking region immediately upstream. This probe was prepared using a template representing single-stranded Mcl-1 genomic DNA derived
from an M13 subclone containing the transcript strand of a 567 base
SacI-NotI Mcl-1 genomic subclone from this region (clone SN0.6, which extends from the NotI site within Mcl-1
exon 1 (see Fig. 2) to a SacI site ~0.6 kb upstream). The
32P-labeled #105C oligonucleotide primer used above was
annealed to this template, and the extension reaction was carried out. The double-stranded DNA product obtained was then cleaved at a DraI site 153 nucleotides upstream from the initiator
methionine (18, 21) (see also Fig. 3B). After
alkaline-denaturing gel electrophoresis, phenol extraction, and ethanol
precipitation, the resultant 5'-end-labeled single-stranded probe,
205 bases in length (2 × 105 CPM), was hybridized
with total RNA (30 µg) from ML-1 cells treated with PMA (18). As a
control, the probe was hybridized in parallel to yeast tRNA (20 µg).
Hybridization (20 µl/reaction) was carried out at 30 °C in 40 mM PIPES, pH 6.4, containing 80% formamide, 400 mM NaCl, and 1 mM EDTA. After 14 h, the
hybridization reaction mixture was diluted by the addition of 300 µl
of S1 nuclease buffer (50 µM sodium acetate, pH 4.5, containing 0.28 M NaCl and 4.5 mM
ZnSO4) to which denatured calf thymus DNA (0.02 µg/µl)
had been added. Digestion with S1 nuclease (300 units) was carried out
at 30 °C for 60 min and was terminated by the addition of 80 µl of
stop buffer (4 M ammonium acetate, pH 8.0, containing 20 mM EDTA and 40 µg/ml yeast tRNA). After ethanol
precipitation and resuspension in formamide, the size of the protected
fragments was assessed by gel electrophoresis on a DNA sequencing gel
alongside a sequencing reaction carried out with the 105C primer and
the SN0.6 M13 clone as template. The products of the primer extension reaction (above) were analyzed on the same gel.
RNase Protection Analysis--
RNase protection analysis was
performed using the multiprobe RNase protection analysis system from
Pharmingen. A specific probe set was generated which contained probes
to Bcl-x, A1, and Mcl-1. The Mcl-1 probe corresponded to positions 961 to 1107 in the human Mcl-1 cDNA sequence; this spans a region of
the cDNA containing sequence from exons 2 and 3, part of which is
deleted in Mcl-1s/TM. Full-length wild-type Mcl-1 transcripts would be expected to yield a protected product of 146 bp, and the deleted Mcl-1s/TM transcript a
protected product of 109 bp. For positive controls in the hybridization reactions, mRNA was transcribed from the TOPO pCR II constructs containing wild-type Mcl-1 and Mcl-1s/TM.
tRNA was used as a negative control. As a control for RNA loading,
samples were also hybridized with the hAPO-2 probe set (Pharmingen).
The RNase protection analysis reactions were carried out as described
in the protocol provided by the manufacturer, using 1-3 µg of total cell RNA and dilutions of the positive control RNAs. The reaction products were resolved on 5% sequencing gels in 0.5×
Tris-borate-EDTA, which were then dried and exposed to film at
80 °C.
Western Blotting and in Vitro Translation--
For Western
blotting, cell extracts were prepared using a bromphenol blue lysis
buffer (containing 80 mM Tris/HCl, pH 7.5, 2% SDS, 20%
glycerol, 0.001% bromphenol blue, 0.1 M dithiothreitol, 1 µM leupeptin, and 2 mM phenylmethylsulfonyl
fluoride). Electrophoresis (12% SDS-polyacrylamide gel
electrophoresis) was carried out with the equivalent of 1-3 × 105 cells being loaded in each lane, prestained rainbow
markers (Amersham Pharmacia Biotech) being run with each gel. Following
electrophoresis, samples were blotted onto Trans-blot nitrocellulose
membranes (Bio-Rad) using a semi-dry blotter, and protein transfer was
confirmed by staining with Ponceau S. A rabbit polyclonal antibody
raised against a peptide representing amino acids 121-139 of human
Mcl-1 (Santa Cruz, SC-819) was used at a final dilution of 1:200, and detection was with ECL (Amersham Pharmacia Biotech). The specificity of
this antibody was confirmed using specific blocking peptides corresponding to the peptide used as the initial immunogen (Santa Cruz,
SC-819P). To prepare authentic Mcl-1 proteins as standards for Western
blots, in vitro transcription and translation were carried
out using the wild-type Mcl-1 and Mcl-1s/TM
constructs in TOPO pCR II. A coupled transcription/translation system
(Promega) was used according to the manufacturer's instructions with
2-µl aliquots of the reaction products being loaded on the Western blots.
Transient Transfection with Mcl-1s/TM Expression
Constructs to Assess Effects on Cell Death--
Two types of
expression constructs were prepared for use in transfection into
mammalian cells; in one case Mcl-1 or the
Mcl-1s/TM variant were fused to green
fluorescent protein and in the other case they were expressed in the
pCR-3.1 vector. The Mcl-1 RT-PCR reaction products from the 4F/4R
primer pair reactions (cloned in pCR3.1) were digested with
KpnI (a KpnI site is present in the 4F primer)
and HindIII, as was full-length Mcl-1 (Mcl-1 clone p3.2 in
pBluescript II SK (18)), and the resulting 5'-region of the cDNA
was isolated and ligated into pCR3.1 to yield full-length pCR3.1-Mcl-1
and pCR3.1-Mcl-1s/TM. C-terminal green
fluorescent protein fusion protein expression constructs were made by
digesting the two Mcl-1 fragments from the pCR3.1 clones using
HindIII and PstI, followed by subcloning in frame
into pEGFP-C1 (CLONTECH), to yield pEGFP-C1-Mcl-1
and pEGFP-C1-Mcl-1s/TM. The resulting clones
were sequenced using flanking and internal oligonucleotides.
The pEGFP-based vectors (pEGFP-C1-Mcl-1,
pEGFP-C1-Mcl-1s/TM, and insertless pEGFP-C1;
0.25 µg each) were transiently transfected into subconfluent
monolayers of the A549 human airway epithelial cell line in chamber
slides using LipofectAMINE. Following overnight culture, transfected
cells expressing the fusion proteins were examined for the
morphological features of cell death using confocal microscopy. The
pCR3.1-based Mcl-1 clones were transiently transfected into
subconfluent monolayers of either A549 or HeLa cells (in 12 well
plates) using Superfect (Qiagen). The constructs to be tested
(pCR3.1-Mcl-1, pCR3.1-Mcl-1s/TM,
pcDNABax as a positive control, or insertless pCR3.1 vector as a
negative control; 1 µg each) were transfected along with
pCMV-
-galactosidase (1 µg; Promega) as a marker of transfection.
The use of a total of 2 µg of DNA/well was based on a series of
initial transfections. Forty hours after transfection, cells were
fixed, stained for -galactosidase activity, and examined as above
for the morphologic features of cell death. This analysis was performed
in a double blind manner, and cells were scored either as having a
normal, viable morphology (flat, healthy cells) or an abnormal
morphology characteristic of dying cells (dense, rounded, detaching
cells). All blue staining cells in each dish were counted, and the
results were expressed as percentages of cells with viable
versus dying phenotypes.
| |
RESULTS |
A Variety of Cells Contain an Internally Deleted Variant,
Mcl-1s/TM, Along with the Full-length Mcl-1
Transcript--
Several Mcl-1-encoding cDNA clones in the
GenBankTM human EST data base were noted to contain an
internal 248-bp deletion corresponding to nucleotides 749-996 of the
full-length Mcl-1 cDNA. This suggested that Mcl-1 might be capable
of undergoing some form of alternative splicing, as does Bcl-x. Several
such internally deleted Mcl-1 cDNAs were present in three
distinct cDNA libraries, lending credence to this possibility
(accession AA457098, AA749362, AA521010, and AI435426). While a
distinct transcript representing such a deletion had not been
distinguished previously on Northern blots, this is not surprising as a
248-bp deletion would represent a decrease in MW of only ~7% as
compared with the full-length 3.8-kb Mcl-1 transcript (18, 30), and
probes recognizing the putative deleted transcript would also recognize
full-length Mcl-1. We thus tested for the existence of such an
internally deleted Mcl-1 by using primers from either side of the
putative deletion in RT-PCR. Two distinct cDNA products were
obtained, one of which corresponded to the size expected for
full-length Mcl-1 and the other of which was ~250 bp shorter (Fig.
1A). Two such cDNA
products were obtained using three different combinations of primers
(F3 and R4, F4 and R4, and F5 and R7). This result was seen using RNA
from a variety of different types of cells, including primary hematopoietic cells (e.g. neutrophils) as well as cell lines
of both hematopoietic and nonhematopoietic (e.g. epithelial)
origin. The smaller of the cDNA products consistently stained less
intensely than the longer product, suggesting that it could represent a transcript of lower abundance as is seen with Bcl-xs, which
is generally less abundant than Bcl-xL.
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Fig. 1.
A variety of cells express the
Mcl-1s/TM variant in which the
region encoding BH3 remains intact but those encoding the BH1, BH2, and
TM domains do not. A, Mcl-1 cDNA products obtained by
RT-PCR using primers spanning the internally deleted segment. The RNA
used was from a variety of primary human hematopoietic (human buffy
coat cells (lanes 2 and 3), monocyte-derived
macrophages (lane 4), peripheral blood mononuclear cells
(lane 5), peripheral blood neutrophils (lanes 6,
15, and 16)) or from hematopoietic and
nonhematopoietic cell lines (Jurkat (lane 7), K562
(lane 8), A549 (lane 9), H441 (lane
10), MCF-7 (lane 11), and Hep G2 (lane 12)
cells). RT-PCR was performed using primer pairs as follows: F3 and R4
(lane 2); F4 and R4 (lanes 3-12, 15);
F5 and R7 (lane 16), where the latter pair spans the entire
Mcl-1 coding sequence. A 1-kb ladder (Bioline) was run in lanes
1,13, and 14. Lanes 1-13 were resolved on a
1% Tris-acetate-EDTA gel, and lanes 14-16 were resolved on
a 2% Tris-acetate-EDTA gel. All primer pairs were shown to amplify a
single larger band in reactions with human genomic DNA (results not
shown). B, the predicted amino acid sequence of the
Mcl-1s/TM variant as compared with
full-length Mcl-1 (Mcl-1WT). In B, the positions
of the BH1, BH2, and BH3 domains are indicated by the boxes.
The PEST region is marked with dashed lines. The
vertical line indicates the position at which the predicted
Mcl-1s/TM gene product diverges from
Mcl-1WT, and the novel sequence is indicated by
italics. The C-terminal transmembrane domain present in wild
type Mcl-1 is indicated by double overlines and
underlines. C, schematic representation of the
predicted Mcl-1WT and Mcl-1s/TM
protein isoforms.
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Cloning and sequencing of the above cDNA products demonstrated that
the longer product matched the full-length Mcl-1 sequence, whereas the
shorter product contained the internal deletion of nucleotides
749-996. This deletion lies within the protein coding sequence, which
is encoded by nucleotides 61-1110 of the full-length Mcl-1 cDNA
(#L08246). Both the shorter and the longer product contained the Mcl-1
start site of translation (18) as well as a stop codon, as seen upon
sequencing of the products obtained with primers (F5 and R7)
representing regions upstream (F5) and downstream (R7) of the Mcl-1
coding sequence. In sum, the shorter cDNA appeared to represent a
variant Mcl-1 containing a 248-bp deletion within the coding sequence.
Except for the deletion, the nucleotide sequence of the variant Mcl-1
matches that of the full-length cDNA; however, the sequence downstream of the deletion is in a different reading frame in the
variant. Therefore, the amino acid sequence predicted for the variant
is identical to that of full-length Mcl-1 upstream of the deletion but
differs at the C terminus. Specifically, the first 229 (out of 271)
amino acid residues of the internally deleted variant are identical to
full-length Mcl-1, whereas the C-terminal 42 residues are not (Fig.
1B). The deletion thus does not affect the upstream portion
of Mcl-1 encoding the PEST sequences and the BH3 domain but does affect
the downstream portion encoding the BH1, BH2, and TM domains (Fig. 1,
B and C). Not only does the C terminus of the
deleted gene product differ from full-length Mcl-1, but it also does
not match other proteins in the data bases. Because the internally
deleted variant encodes a shorter protein with an altered C terminus,
we term it Mcl-1s/TM, where s designates
short and 
TM designates lack of the TM domain. This variant is in
some respects similar to Bcl-xs, which retains the BH3 but
not the BH1 and BH2 domains. A difference is that the splicing that
yields Bcl-xs does not affect the reading frame and
therefore does not affect the C-terminal TM domain. However, other
pro-apoptotic family members, namely Bid and Bad, contain BH3 but not
the BH1, BH2, or TM domains. Overall, in addition to the full-length
Mcl-1 transcript, a variety of cells contain a variant,
Mcl-1s/TM, which has characteristics
reminiscent of death-promoting Bcl-2 family members.
The Mcl-1s/TM Variant Derives from Skipping of a
Central Coding Exon Delineated by an Intron Downstream of BH3 along
with the Conserved Intron in BH2--
The splicing that yields
Bcl-xs results from the use of an alternate upstream splice
donor site lying within the first coding exon (39), and Bcl-x, like
other anti-apoptotic family members, contains a single intron within
the protein-coding region (near the C terminus) (38-40, 45, 46). An
intron at this position is a highly conserved feature of the Bcl-2
family (37, 47-51). For clues as to how the
Mcl-1s/TM variant arises, we characterized ~30 kb of the Mcl-1 human genomic locus. In contrast to other anti-apoptotic family members, Mcl-1 was found to consist of three coding exons (Fig. 2A). Thus,
Mcl-1 contains an intron just downstream of BH3, in addition to the
conserved intron further downstream in BH2 (Table
I). Exon 1 is G/C-rich and encodes the
first 229 amino acid residues containing the PEST sequences and BH3;
exon 2 encodes BH1 and a portion of BH2, and exon 3 encodes the
remainder of BH2 and the C-terminal TM domain. The
Mcl-1s/TM variant represents exon 1 joined
to exon 3 with skipping of exon 2 (Fig. 2C). Exons 2 and 3 are not in the same phase (Table I), which accounts for the shift in
reading frame at the C terminus. Whereas Bcl-2 and other anti-apoptotic
family members do not contain an intron comparable to a Mcl-1 intron 1, an intron is present at this location in pro-apoptotic Bax (Bax intron
3) (8).
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Fig. 2.
The Mcl-1 human genomic locus contains an
intron downstream of BH3, in addition to the conserved intron further
downstream in BH2. A, intron/exon structure and G/C
content of the human Mcl-1 genomic locus. A >8-kb region of human
genomic DNA that included the Mcl-1 gene was sequenced, using genomic
subclones as shown in B. The three Mcl-1 coding exons are
shaded gray, with the 3'-untranslated region being
white. The two introns are also white, and the
5'- and 3'-flanking regions are hatched. The regions
encoding the BH1, BH2, BH3, and TM domains are indicated, where BH2 is
encoded in exons 2 and 3. A GGCCCC repeat and two initiator sequences
that lie upstream of exon 1 are indicated (black dot and
upward pointing arrow; see also Fig. 3). Also indicated are
two polyadenylation signals in the long 3'-untranslated region; these
correspond to two transcripts previously reported (3.8 and 2.5 kb
(18)), the former representing the full-length cDNA and the latter
polyadenylation at nucleotide 2425 of the cDNA sequence (see
accession no. AF118124). Alu sequences are present in the 5'- and
3'-flanking regions of Mcl-1 and embedded within the upstream Alu
sequence is a retrovirus long terminal repeat with the properties of a
3'-long terminal repeat; these sequences are shown as heavily
hatched regions. The C+G and CpG content is diagrammed below
the sequence. The complete genomic sequence has the accession number
AF198614. B, two lambda phage clones that span the Mcl-1
genomic locus (B3 and B5 in EMBL-3) were subcloned and used for
restriction enzyme mapping and sequencing. Restriction enzyme digestion
sites are indicated at the top, as follows: B,
BamHI; E, EcoRI; N,
NotI; X, XhoI; Xb,
XbaI; S, SalI (the latter from the
EMBL-3 vector). The region represented in A is the region
between the Xb1 and Xb3 sites, as indicated.
Selected genomic subclones (in pBluescript) are shown at the bottom,
where these are designated according to the restriction enzyme sites at
their 5'- and 3'-ends (abbreviated as above except that X
designates XbaI and K designates KpnI
and where the numbers indicate the subclone size (in kb)).
C, the Mcl-1s/TM variant
represents juxtaposition of exon 1 to exon 3 with the skipping of exon
2, as compared with full-length Mcl-1 in which exons 1, 2, and 3 are
represented.
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Table I
Mcl-1 contains an intron downstream of the BH3 domain in a position
conserved in pro-apoptotic Bax but not other anti-apoptotic family
members, along with the conserved intron in BH2 further downstream
The positions of Mcl-1 introns 1 and 2 are shown. Intron 1 lies just
downstream of the BH3 domain and intron 2 lies within the BH2 domain
(see also Fig. 2). A terminal intron in BH2 is a highly conserved
feature of the Bcl-2 family. An intron at a position similar to that of
Mcl-1 intron 1 is present in the Bax gene (intron 3), but not in Bcl-2.
The total number of nucleotides in Mcl-1 introns 1 and 2 is indicated
in parentheses. Dashes were inserted to optimize sequence alignment
(4). Subscripts indicate the numbering of the full-length Mcl-1
cDNA and amino acid sequence (the latter in bold).
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Mcl-1 Transcriptional Initiation Sites Lie Directly Upstream of the
First Coding Exon without an Intervening 5'-Untranslated
Exon--
Both Bcl-2 and Bcl-x contain upstream untranslated exons in
addition to the two coding exons (38, 40). To look for such upstream
exons and to identify the start site(s) of transcription in Mcl-1, we
carried out primer extension analysis and S1 nuclease mapping. In
primer extension, the most abundant of the products obtained extended
to about 70 bp upstream of the Mcl-1 translation start site (Fig.
3A, lane 3);
additional products extended 10 bp further upstream (these two
positions are indicated with arrows to the left of the
sequencing ladder in Fig. 3A). Transcription thus appeared
to initiate in the region of two tandem initiator sequences (52)
present at this location in the immediate 5'-flank of Mcl-1 (Fig.
3A left column of letters and Fig. 3B,
double underlines). The primer extension analysis did not
specify initiation exclusively at a single nucleotide within the region
containing these tandem sequences. Instead, initiation appears to occur
at at least two major sites within an ~10-bp stretch, possibly
reflecting the presence of two initiator sequences. S1 nuclease mapping
also yielded a protected fragment indicative of transcriptional
initiation in the region of the initiator sequences (Fig. 3, lane
2). The longest fragment obtained upon S1 nuclease mapping
corresponded most closely to the more downstream of the sites indicated
by primer extension. Additional shorter fragments were also seen, which
could reflect incomplete protection from S1 nuclease digestion. The
reason that the more upstream site was not detected by S1 nuclease
mapping could relate to the fact that it was associated with less
abundant primer extension products. It could also relate to the
presence of a repeated sequence in the tandem initiators (i.e. CACTTC), which could allow the formation of a loop
upon binding to the probe. Whatever the case, both mapping methods suggested that a majority of Mcl-1 transcripts initiated from the more
downstream of the two initiator sequences; this location was therefore
designated as position +1 (indicated with an asterisk in
Fig. 3).
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Fig. 3.
Mcl-1 transcription initiates adjacent to
tandem initiator sequences directly upstream of the first coding
exon. A, Mcl-1 transcriptional start sites were mapped
using primer extension analysis (products in lane 3, yeast
tRNA control in lane 4) and S1 nuclease digestion (protected
fragments in lane 2, yeast tRNA control in lane
1, probe in lane 5). The sequencing ladder (lanes
A, T, G, and C) was generated
using the #105C primer employed in the mapping (see "Materials and
Methods"). The RNA used was from PMA-treated ML-1 human myeloid
leukemia cells, the cell line from which Mcl-1 was originally cloned
(18). Arrows to the left of the sequencing ladder indicate
the two major positions where primer extension products were seen (120 and 130 bp upstream from the 5'-end of the #105C primer). The more
abundant primer extension products were found at the position of the
lower arrow, as opposed to the upper arrow 10 bp
upstream. The lower position was thus designated +1. This position is
indicated with an asterisk on the Mcl-1 coding sequence,
which is listed in the column at the left. The sequence as
listed includes the two tandem initiator sequences at 69-82 bp
upstream of the start site of Mcl-1 translation (see B
below). The sequence of these initiators is CCACTTCTCACTTC, where the
consensus for initiator sequences is YYAX(T/A)YY. The
sequence in the right column is that of the complementary
strand, which was that sequenced. The distance (bp) indicated at the
right side of the gel represents the distance from 5'-end of the #105C
primer. B, the sequence of the immediate 5'-flank of the
human Mcl-1 gene is shown in comparison to the comparable region of the
mouse Mcl-1 5'-flank. The tandem initiator sequences located 69-82 bp
upstream of the start site of translation of the human gene are
double underlined, where a similar sequence is present in
the mouse sequence. The site designated as +1 in human Mcl-1 (see
A and under "Results") is in bold and is
indicated with an asterisk. The +1 site for mouse Mcl-1 is
similarly indicated. The tandem initiator sequences lie in break
between an upstream CpG-rich area (labeled and indicated with an
arrows) and the G/C-rich first exon downstream (see Fig.
2A). The GGCCCC repeat region is dot underlined
and CpG pairs are in italics. The numbering in
bold (in parentheses) indicates the position relative to the
+1 site. The numbering that is not in bold refers to the numbering of
the 8253 kb of sequenced human genomic Mcl-1 (diagrammed in Fig.
2A) or the corresponding sequenced area of the mouse Mcl-1
gene (25, 29).
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Fig. 3B shows the position of the initiator sequences
relative to other features of the immediate 5'-flank of Mcl-1. Upstream from the initiator sequences lie the Ets and SRF binding elements previously found to have a role in the control of transcription of the
human Mcl-1 gene (underlined in Fig. 3B (21)).
Elements similar to these transcription factor binding sites, as well
as a potential initiator sequence, are present in the 5'-flank of mouse
Mcl-1(25, 29), although, elsewhere, the latter does not demonstrate the
extensive identity to the 5'-flank of human Mcl-1 (~56% in the
region shown in Fig. 3B). Elements in this conserved region
appear to function in both species as recent experiments show that
mutations affecting this region also have an effect on transcription
from the mouse Mcl-1 promoter (29). Further upstream in the human gene
lies a GGCCCC repeat region (dot underlined in Fig.
3B) within a G/C-rich upstream area. Thus, the initiator sequences that mark the sites of Mcl-1 transcriptional initiation lie
in a break between a G/C-rich, repeat-containing area upstream and the
G/C-rich first coding exon downstream (Fig. 2A and
3B).
One further point is that, with the primer extension method, a very
faint product extended beyond the two initiator sequences (approximately another 25 nucleotides beyond the most upstream of the
other 2 sites, to ~155 bp from the #105C primer; faintly visible in
Fig. 3A, lane 3). This may mean that
transcription can also initiate further upstream. Consistent with this
possibility is the fact that, although several EST clones present in
the data bases extend to near the initiator sequences (e.g.
accession AI204385, AI202072, AA776756, and AI340205), some clones
contain an additional upstream sequence (e.g. AI439001,
AA884201, and AA453505). However, in all of the latter, the upstream sequence is co-linear with Mcl-1 genomic DNA, rather than containing a
large gap. Thus, although the sequence of upstream portion of Mcl-1
contains regions that could be considered as candidate splice donor and
acceptor sequences, there is at present no evidence suggestive of an
upstream untranslated exon. Overall, human Mcl-1 can be transcribed
from a region containing tandem initiator sequences that lie 69-82 bp
directly upstream of the coding sequence.
Transfection with the Mcl-1s/TM Variant Results in
Cell Death--
Because the internally deleted
Mcl-1s/TM variant displays features
reminiscent of the BH3 only pro-apoptotic family members Bid and Bad,
we wondered whether it might similarly have death-promoting activity.
We tested this in epithelial cells, where dead cells are readily
detectable as they shrink, round up, and detach from the substratum
while viable cells grow as a flat monolayer. We first used expression
constructs in which Mcl-1s/TM was fused to
EGFP (in the vector pEGFP-C1), as this provides a rapid assay. With
this method, cells expressing the introduced fusion protein are
identified based on green fluorescence and can then be examined for the
morphologic features of cell death (53, 54). We found that A549 airway
epithelial cells expressing
pEGFP-C1-Mcl-1s/TM, as detected by green
fluorescence, exhibited cell shrinkage (Fig. 4B) as well as nuclear
condensation (Fig. 4C) and blebbing (Fig. 4D),
hallmarks of cell death by apoptosis (53, 54). In contrast, cells
expressing the pEGFP-C1 vector or full-length pEGFP-C1-Mcl-1 (Fig.
4A and legend) remained morphologically normal and viable, as expected based on previous experiments (32-34).
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Fig. 4.
Transfection with the
Mcl-1s/TM variant results in cell
death. A D, A549 cells were transfected with either
pEGFP-C1-Mcl-1, in which full-length Mcl-1 is fused to EGFP
(A), or with the
pEGFP-C1-Mcl-1s/TM variant
(B-D). Cells were examined microscopically after overnight
culture. Those that were transfected with pEGFP-C1-Mcl-1 (or with
pEGFP-C1 vector, not shown) and that expressed EGFP exhibited the flat
morphology of normal viable cells (A). Those that were
transfected with pEGFP-C1-Mcl-1s/TM and
expressed EGFP rounded up and exhibited cell shrinkage (B).
On higher magnification, some of the latter exhibited the nuclear
condensation (C) and blebbing (D) that is
characteristic of apoptosis. Cells that did not express the EGFP
transfection marker could be seen as adherent, flat cells.
E, A549 cells were either mock-transfected (lane
2) or transiently transfected with pCR3.1-Mcl-1 (lane
3) or pCR3.1-Mcl-1s/TM (lane
4) and assayed after 40 h for Mcl-1 expression by Western
blotting. In vitro translation products representing a
control reaction with firefly luciferase (lane 5),
full-length wild-type Mcl-1 (lane 6), and
Mcl-1s/TM (lane 7) were used as
standards. The position of the immunoreactive protein products are
indicated by the arrows along with the position of a 30-kDa
protein standard (lane 1). The specificity of the assay was
confirmed by the addition of the Mcl-1 blocking peptide, which
abolished detection of the bands representing Mcl-1 (not shown). The
electrophoretic mobility of Mcl-1s/TM was
noted to be retarded compared with its predicted molecular mass (29.4 kDa), a phenomenon which is also seen with Mcl-1 and other members of
this family (18). FH, HeLa cells were transiently
transfected with the full-length pCR3.1-Mcl-1 expression construct or
with pCR3.1-Mcl-1s/TM, along with
pCMV--galactosidase as a transfection marker. The pCR3.1 vector
served as a negative control and pcDNA-Bax served as a positive
control. Forty hours after transfection, cells were washed extensively
and stained in situ for expression of -galactosidase.
Cells that expressed the transfection marker were then scored for
viability as described under "Materials and Methods." Upon
transfection with the full-length pCR3.1-Mcl-1, cells that stained blue
exhibited the flat morphology of normal viable cells (F).
Identical results were obtained using the pCR3.1 vector. Upon
transfection with pCR3.1-Mcl-1s/TM, cells
that stained blue were rounded up and exhibited cell shrinkage
(G), whereas nonstaining cells could be seen as adherent,
flat, elongated cells. These results were summarized as the percentage
of viable cells ± S.E. of 6-9 individual wells
(H).
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To confirm the above observations, we used the
pCR3.1-Mcl-1s/TM construct that expresses
Mcl-1s/TM itself rather than a fusion
protein. A549 cells transfected with this construct (but not
mock-transfected controls) expressed the
Mcl-1s/TM-encoded protein, detected by
anti-Mcl-1 antibodies as a protein corresponding in size to authentic
in vitro translated Mcl-1s/TM
(Fig. 4E, lane 4). Endogenous full-length Mcl-1
was also detectable in these cells, but its level of expression was not
altered in the presence of Mcl-1s/TM.
Although the level of expression of
Mcl-1s/TM appears lower, the relative levels
of the variant and full-length Mcl-1 cannot be determined as the former
is present only in transfected cells, whereas the latter represents in
the entire cell population. Whatever the relative levels, it was clear that the introduced Mcl-1s/TM protein was
expressed in this system. We therefore performed co-transfection
experiments with pCR3.1-Mcl-1s/TM, along
with pCMV--galactosidase as a marker for transfected cells, to test
the effect of expression on cell death. Transfected (i.e.
-galactosidase expressing) cells were found to undergo cell death
exactly as described above with
pEGFP-C1-Mcl-1s/TM, an effect that was not
seen with the insertless pCR3.1 vector or full-length pCR3.1-Mcl-1
(results not shown). An identical effect was seen in another epithelial
cell line, HeLa. Upon transfection with
pCR3.1-Mcl-1s/TM, HeLa cells expressing the
-galactosidase marker rounded up and exhibited shrinkage/blebbing
(Fig. 4G), as did cells transfected with a Bax expression
vector. In contrast, in parallel cultures transfected with the
insertless vector or full-length pCR3.1-Mcl-1 (Fig. 4F and
legend), the majority of cells remained morphologically normal and
viable. Cells expressing -galactosidase were scored as having either
a normal, viable morphology (flat, healthy cells) or the altered
morphology characteristic of cells dying by apoptosis (dense, rounded,
detaching cells). This analysis showed that expression of the
Mcl-1s/TM isoform was nearly as effective as
Bax in inducing cell death (Fig. 4H). In two different cell
types, in sum, transfection with Mcl-1s/TM resulted in cell death.
Endogenous Mcl-1s/TM Is Expressed at Lower Levels
than Full-length Mcl-1 in Viable Cells--
The initial RT-PCR assays
above suggested that Mcl-1s/TM was expressed
at lower abundance than full-length Mcl-1. To confirm this, we used an
RNase protection assay in which the probe contains sequence from exons
2 and 3 and thus protects fragments of different sizes for
Mcl-1s/TM and full-length Mcl-1. We found
the protected fragment corresponding to
Mcl-1s/TM to be detectable at low levels in
an epithelial cell line (A549; Fig.
5A, lane 1) and
also in cells of the myeloid lineage including monocyte-derived
macrophages and polymorphonuclear neutrophils (Fig. 5A,
lanes 5 and 6, where authentic in
vitro transcribed Mcl-1 and Mcl-1s/TM
are in lanes 7 and 8). The alternately spliced
variant was indeed present at much lower abundance than the full-length
Mcl-1 transcript, a finding that is perhaps not surprising in view of
the fact that the analysis was performed using healthy, viable
cells.
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Fig. 5.
Endogenous
Mcl-1s/TM is expressed at low
levels relative to full-length Mcl-1 in viable cells.
A, RNase protection analysis was performed using a
32P-labeled cRNA probe that detects both full-length
wild-type Mcl-1 and the Mcl-1s/TM variant.
The total cellular RNA used was from A549 cells (3 µg; lane
1), BL41-3 cells (3 µg; lanes 2-4),
monocyte-derived macrophages (1 µg; lane 5), or PMNs (1 µg; lane 6). In the case of BL41-3, cells were either
left untreated (lane 2) or treated for 2 h with 5 or 50 ng/ml PMA (lanes 3 and 4, respectively). Positive
control RNA transcribed from plasmids containing full-length wild-type
Mcl-1 or Mcl-1s/TM is in lanes 7 and 8, respectively. Identical samples were hybridized with
the hAPO-2 probe set to confirm equivalent loading (not shown).
B, BL41-3 cells were either not exposed (lane 2)
or exposed (lane 3) to 10 ng/ml PMA and assayed for
expression of Mcl-1 by Western blotting. In vitro translated
Mcl-1s/TM was used as a standard (lane
1). The band traveling with an electrophoretic mobility just
slightly more rapid than wild-type Mcl-1 is also specifically detected
with anti-Mcl-1 antibodies, although its identity has not yet been
determined.
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Expression of Mcl-1 is markedly induced by PMA in a variety of cells
(18, 30), and it seemed possible that
Mcl-1s/TM might similarly be increased with
this agent. We examined this possibility using a Burkitt lymphoma cell
subline, BL41-3, in which the Mcl-1 genomic locus is amplified and
overexpressed (55). Our rationale was that the presence of multiple
copies of the Mcl-1 gene might optimize the possibility of observing
increased expression. The Mcl-1s/TM
transcript, although not detectable in untreated BL41-3 cells, was
detectable in cells exposed to PMA (Fig. 5A, lane 2 versus lanes 3-4). The
Mcl-1s/TM-encoded protein was likewise not
detected in untreated BL41-3 cells but was detected at low levels in
cells treated with PMA (Fig. 5B). The fact that the levels
of the alternately spliced variant were lower than those of full-length
Mcl-1, as in the other cells tested above, could be the reason that
BL41-3 cells remain viable upon PMA treatment. Although the studies
performed to date are limited in that they have examined cells that are
largely viable and that also express full-length Mcl-1, these studies
demonstrate endogenous cellular expression of the
Mcl-1s/TM splice variant. They thus open the
way to future studies aimed at monitoring the expression of the two
isoforms during the course of cell death and at manipulating the
expression of the isoforms as recently described for Bcl-x (56) as an
approach to inducing the death of cancer cells.
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DISCUSSION |
Mcl-1 is emerging as a highly regulated member of the Bcl-2
family, and the results reported here demonstrate an additional, heretofore unrecognized form of regulation, alternative splicing. The
mechanism of generation of the novel
Mcl-1s/TM isoform involves exon skipping and
thus differs from that seen in the case of Bcl-xs. This
mechanism depends upon the presence of three coding exons in Mcl-1
instead of the two coding exons seen in other anti-apoptotic members of
this family. These three coding exons arise because of the presence of
an additional intron just downstream of the BH3 domain of Mcl-1, along
with the conserved intron in BH2 near the C terminus. The elimination
of the central second exon along with a shift in reading frame
downstream results in the elimination of the BH1, BH2, and TM-encoding
domains. In accordance with the resemblance to BH3 only pro-death
family members, exogenous introduction of the
Mcl-1s/TM variant was found to induce cell
death. Similarly, populations of cells that were largely healthy and
viable exhibited low levels of Mcl-1s/TM as
compared with full-length wild-type Mcl-1.
Other members of the Bcl-2 family can also undergo differential
splicing (37, 39, 41, 49, 57-59). In terms of anti-apoptotic family
members, the splicing of Bcl-xL to Bcl-xS
involves the use of an alternate splice site within the coding sequence
(39). Bcl-w can also undergo alternate splicing, and in this case
splicing is to an adjacent gene (45). With both Bcl-x and Bcl-2,
unspliced transcripts that read through into the intron have also been
reported (37). The mechanism involved in the splicing of Mcl-1 differs from that seen with these other anti-apoptotic family members in that
it involves exon skipping, where the subsequent exon is placed in an
altered reading frame. It is the presence of an additional intron in
Mcl-1 downstream of BH3, along with the conserved intron further
downstream in BH2, that allows for this ability to skip an exon and
eliminate BH domains critical for anti-apoptotic effects.
Although two coding exons are present in anti-apoptotic family members
so far characterized (other than Mcl-1) (38, 40, 45, 46), multiple
coding exons (5-6) are present in pro-apoptotic family members. This
is because of the presence of 3-5 introns in the coding region (37,
47-51), where the terminal intron is that which is conserved
throughout the family. For example, additional introns in the Bax gene
place BH1, BH2, and BH3 on separate exons. A variety of splice variants
have been identified; this includes Bax
, where exon 4 is spliced to
exon 2 (in frame) with skipping of exon 3, as well as Bax
, where
exon 3 is spliced to exon 1 (with a change in reading frame) with
skipping of exon 2 (8, 37). The activity of these various variants has
not been completely elucidated. Differential splicing is also seen in
other pro-apoptotic family members; in Bok/Mtd, exon 2 can be skipped
(49) and, in Bim, alternate splicing can alter the N terminus of the
encoded gene product (10). Although the altered splicing of Bok results in retention of a similar function, the different variants of Bim
exhibit differences in pro-apoptotic efficacy. One of the introns in
pro-apoptotic Bax lies downstream of BH3 in a position analogous to the
additional (first) intron in Mcl-1 (8). Other pro-apoptotic family
members, namely Bid and Bak, also contain an intron downstream of BH3
(47, 48, 51). However, in the latter two genes these introns are not at
positions identical to that of intron 1 in Mcl-1 and the analogous
intron in Bax. The alternate upstream splice site within the Bcl-x
first coding exon that is used in the production of Bcl-xs
likewise lies downstream of BH3, although this site is again not
located at a position identical to that of the intron in Mcl-1 (39).
Overall, the multiple introns in pro-apoptotic family members provide a
variety of possibilities in terms of differential splicing. Moreover
whereas differential splicing through exon skipping, as seen here with Mcl-1, has not been reported for other anti-apoptotic family members, it is common among pro-apoptotic members.
Whereas these studies demonstrate that the human Mcl-1 gene can undergo
differential splicing into two distinct isoforms, it remains to be
determined how these isoforms function and are coordinated in
vivo. It has been suggested that anti-apoptotic members of the
Bcl-2 family prevent cell death by inhibiting the action of adaptor
proteins involved in the initiation of the caspase cascade, although
this is controversial (60). Pro-apoptotic Bcl-2 proteins may then bind
to anti-apoptotic family members and neutralize the block on the
adaptor proteins to allow the death cascade to develop. The
pro-apoptotic activity of several BH3 only Bcl-2 family members has
been shown to be regulated in a post-translational manner by
protein-protein interactions, which make these proteins unavailable to
initiate the death pathway. For example Bad is bound in a
phosphorylation-dependent manner to 14-3-3 proteins, which
sequesters the protein in a form that is unable to associate with Bcl-2
and Bcl-xL (61). Upon dephosphorylation, Bad translocates
to bind Bcl-2 and Bcl-xL and induces the cell death
cascade. Bim, another BH3 only protein, is sequestered in the
cytoskeletal associated motor complex bound to LC8, a cytoplasmin dynein light chain protein. Apoptotic stimuli disrupt this interaction, and the freed Bim then binds to Bcl-2, neutralizing its anti-apoptotic activity and promoting cell death (62). A third BH3 only protein, Bid,
is sequestered in the cytosol in an inactive form and is cleaved by
caspase-8 to yield two distinct fragments (63). The larger fragment can
relocalize to the mitochondria where it acts to antagonize
anti-apoptotic Bcl-2 proteins and so promotes cell death. Therefore
through distinctly different mechanisms it has been shown that BH3 only
proteins are normally inactive. Following a pro-apoptotic stimulus, the
protein is released and acts as a death inducer by antagonizing the
anti-apoptotic activity of Bcl-2-like proteins. In the case of Mcl-1
with two isoforms with opposite functions being present in the same
cell, a mechanism may exist for coordinated regulation. By analogy with
BH3 only pro-apoptotic family members, the
Mcl-1s/TM variant may be sequestered in an
inactive form in viable cells.
Because the conserved intron within the BH2 domain near the C terminus
is common to anti- and pro-apoptotic family members, it has been
hypothesized that both sides of the family may have arisen from a
primordial member, which contained a single intron in the coding region
(48) and which underwent gene duplication (46). The addition of introns
upstream of the conserved intron could then have occurred during the
evolution of pro-apoptotic family members. The fact that the position
of the additional upstream introns is not precisely conserved among
pro-apoptotic family members led to the hypothesis that these
additional introns were added after the divergence of the primordial
gene (48). Mcl-1 contains a single additional upstream intron along
with the conserved intron near the C terminus. It thus differs from
other anti-apototic family members, which do not contain such introns,
as well as from pro-apoptotic family members, which contain multiple
upstream introns. Mcl-1 might then represent an intermediate in the
development of the pro- and anti-apoptotic family members. This
possibility is consistent with evolutionary analysis, which suggests
that Mcl-1 and A1 represent a very ancient branch of this family
different from the branch containing Bcl-2 and Bcl-x and from that
containing Bax (64). This possibility and the findings described here
could relate to previous observations concerning the BH3 domain of
Mcl-1 (9). Specifically, the core of the BH3 domain of Bcl-2, Bcl-x, and Bcl-w contains an alanine residue at position 4, whereas
pro-apoptotic family members contain bulkier domains (e.g.
isoleucine or valine). These bulky residues appear important for
binding to the hydrophobic cleft of anti-apoptotic family members (11).
Mcl-1 contains a valine at this position, which is more typical of the
pro-apoptotic family members. In addition, at position 3 from the BH3
core, the other three anti-apoptic family members have a charged
residue (histidine or lysine), whereas pro-apoptotic members have
uncharged residues; in this aspect Mcl-1 is also similar to the latter
in that this residue is a leucine. The presence in Mcl-1 of residues in
BH3 that are more typical of pro-apoptotic family members may relate to
the fact that Mcl-1 can be alternately spliced to a pro-apoptotic form.
Taking into account these aspects of its structure, Mcl-1 appears to be
ideally designed to be capable of undergoing differential splicing to
either a viability-promoting or a BH3 only death-promoting gene product.
| |
ACKNOWLEDGEMENTS |
We thank Dr. Gabriel Nunez for the human Bax
expression plasmid, Drs. Alan Eastman and Raymond Perez for their
thoughtful reading of the manuscript, Dr. Stephen Renshaw for his help
with the figures, and Dr. Julie Vrana and Aaron Domina for many helpful discussions.
| |
FOOTNOTES |
*
This work was supported by grants from the Special Trustees
for the Former United Sheffield Hospitals and Grant CA57359 from the
National Institutes of Health.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF198614.
§
To whom correspondence should be addressed. Division of Molecular
and Genetic Medicine, The University of Sheffield Medical School, M128,
The Royal Hallamshire Hospital, Glossop Rd., Sheffield S10 2RX, UK.
Tel.: 44 (0)114 271-2809; Fax: 44 (0)114 272-1104; E-mail:
c.d.bingle@sheffield.ac.uk.
Published, JBC Papers in Press, April 13, 2000, DOI 10.1074/jbc.M909572199
| |
ABBREVIATIONS |
The abbreviations used are:
BH, Bcl-2 homology;
TM, transmembrane;
ML, myeloid leukemia;
PMA, 12-O-tetradecanoylphorbol acetate;
RT, reverse
transcriptase;
PCR, polymerase chain reaction;
kb, kilobase;
PIPES, 1,4-piperazinediethanesulfonic acid;
bp, base pairs.
| |
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Jiang, Z. H.,
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TOP
ABSTRACT
INTRODUCTION
