Three Novel Bid Proteins Generated by Alternative Splicing of the Human Bid Gene*

Bid, a BH3-only Bcl-2 protein, is activated by proteolytic cleavage exposing the BH3 domain, which then induces apoptosis by interacting with pro-apoptotic Bcl-2 family proteins (e.g. Bax and Bak) at the mitochondrial surface. The arrangement of domains within Bid suggested that Bid function might be regulated in part by alternative splicing. We have determined the gene structure of human Bid and identified a number of novel exons. We have also demonstrated endogenous mRNA and protein expression for three novel isoforms of Bid, generated using these exons. BidS contains the N-terminal regulatory domains of Bid without the BH3 domain; BidEL corresponds to full-length Bid with additional N-terminal sequence; and BidES contains only the Bid sequence downstream of the BH3 domain. Expression of these isoforms is regulated during granulocyte maturation. In functional studies BidEL induces apoptosis, whereas BidS abrogates the pro-apoptotic effects of truncated Bid and inhibits Fas-mediated apoptosis. BidES induces apoptosis but is also able to partially inhibit the pro-apoptotic effects of truncated Bid. These three novel endogenously expressed isoforms of Bid are distinct in their expression, their cellular localization, and their effects upon cellular apoptosis. Differential expression of these novel Bid isoforms may regulate the function of Bid following cleavage and thus influence the fate of cells exposed to a range of pro-apoptotic stimuli.

Apoptosis, or programmed cell death, is an evolutionarily conserved program of changes in cell biochemistry and structure (1) leading to the loss of cellular functions and to engulfment and removal by phagocytes (2). This process is essential during normal development, in homeostasis, and also in disease pathogenesis (3). Apoptosis occurs following activation of specific caspases that amplify apoptotic signals in a cascade of proteolysis leading to cleavage of specific substrates. Caspase activation may be initiated in a number of ways, including ligation of cell surface death receptors (4) or activation of the apoptosome (a large multiprotein complex containing caspase-9) by cytochrome c released from mitochondria (5). Cytochrome c release from mitochondria is in turn regulated by the interaction of pro-and anti-apoptotic members of the Bcl-2 family of proteins at the mitochondrial surface (6).
Mammalian Bcl-2 family members share homology with the prototypic protein Bcl-2 and with the nematode CED-9 protein, across four conserved regions, termed the Bcl-2 homology domains (BH1-4). Bcl-2 proteins can be broadly divided into three groups according to their ability to either induce or inhibit apoptosis and their structural relationship to Bcl-2 (7). In addition to the BH3 domain, all anti-apoptotic Bcl-2 proteins described to date contain BH1, BH2, and sometimes BH4 domains. Pro-apoptotic family members either share the multidomain structure (e.g. Bax and Bak) or contain only the BH3 domain (e.g. Bim and Bid). Bcl-2 proteins containing only the BH3 domain have been suggested to play an important role in initiating mitochondrial-mediated apoptosis.
Bid is a 22-kDa "BH3-only" protein with similarity to other Bcl-2 family proteins only within the death-inducing BH3 region (8). Bid has a unique role in signaling of apoptosis, because it links the death receptor signaling pathway to the mitochondrial signaling pathway mediated by Bcl-2 proteins. Death receptor ligation activates caspase-8, which cleaves Bid, freeing the C-terminal moiety (t c Bid) to cooperate in the release of cytochrome c from mitochondria. Examination of the crystal structure of Bid reveals that the hydrophobic face of the BH3 domain, which is exposed in other family members, is enclosed by the N-terminal of Bid and only exposed following proteolytic cleavage of Bid (9,10). There appears to be a second functional domain in Bid, in the N terminus, termed the BH3B domain (11), which has activity to suppress the apoptogenic activity of the BH3 domain. The region between these two opposing domains is extremely sensitive to proteolytic cleavage, containing cleavage sites for caspases, granzyme B, and various lysosomal proteases. The action of these proteases leads to the creation of two cleavage products (termed t n Bid and t c Bid). t c Bid translocates to mitochondria, where it interacts with other Bcl-2 family proteins to bring about the release of cytochrome c (12,13).
We and others have shown that alternative gene splicing may regulate the function of other Bcl-2 family members (14 -17). The structure of Bid, with opposing Bcl-2 homology domains, would potentially be readily regulatable by alternative gene splicing. We therefore investigated the functional regulation of this gene at the molecular level.

EXPERIMENTAL PROCEDURES
Antibodies, Reagents, and Cell Lines-All chemicals were of analytical reagent grade and were purchased from Sigma unless stated otherwise. Culture media (Hanks' balanced salt solution, RPMI 1640, and  Dulbecco's modified Eagle's medium), LipofectAMINE, and Optimem were from Invitrogen. The antibody against Bid has been described previously (12) and was the kind gift of Dr. X. Wang (Howard Hughes Medical Institute, Dallas, TX). zVAD.fmk was from Bachem (St. Helen's, UK). Preprepared protein samples were from the BioChain Institute (Hayward, CA). CH-11 was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Cos, HeLa, Jurkat, HL 60, 293T, and HepG2 cells were from American Type Culture Collection (Manassas, VA) and were cultured in RPMI 1640 supplemented with 10% fetal calf serum (Invitrogen). mRNA from NB4 cells was kindly supplied to us by Dr. M. Lanotte (Paris, France) from cells cultured and stimulated as previously described (18). mRNA from neutrophil precursor populations was kindly supplied to us by Dr. J. Cowland (Copenhagen, Denmark) from samples purified from bone marrow and peripheral blood as previously described (19).
5Ј RACE was performed using the SMART TM RACE kit (BD Biosciences Clontech, Palo Alto, CA), using a modification of the protocol to include reduction of nonspecific base pair interaction (GC-Melt TM ; BD Biosciences Clontech). Gel extraction (Qiaquick; Qiagen), cloning (pC-RII-TOPO; Invitrogen), and isolation of plasmid DNA (Concert Rapid; Invitrogen) were performed using commercially available kits according to the manufacturers' instructions. Preprepared mRNA (Premium RNA) was from BD Biosciences Clontech, and cDNA was prepared using standard reverse transcription techniques.
Generation of Bid Expression Constructs-To generate the pCR3.1 (Invitrogen) expression constructs, PCR fragments were generated using the reverse primer, GGA TCC TCA GTC CAT CCC ATT TCT GGC TAA (exon 9), and the relevant forward primers, AAG CTT AGC CAC CAT GGA CTG TGA GGT CAA CAA C (Bid and Bid S , exon 4), AAG CTT AGC CAC CAT GTG CAG CGG TGC TGG GGT CA (Bid EL , exon 3) , and AAG CTT AGC CAC CAT GGA CCG TAG CAT CCC TCC GG (Bid ES , exon 7) using the appropriate construct in pCRII-TOPO as a template. The PCR product obtained was purified, and was cloned directly into pCR3.1. Correct orientation and reading frame were confirmed by sequencing. The inserts were subcloned by restriction enzyme digestion from these vectors into suitable pEGFP vectors (Living Colors; Clontech). pcDNA3.1.tBid.myc-His was generated by subcloning from pCR3.1.tBid into pcDNA3.1(ϩ)myc-His (Invitrogen). pCR3.1.FLAGBcl-2 and pCR3.1.FLAGBax were generated by subcloning from the appropriate pEF FLAG puro vector, kindly supplied to us by Dr. D. Huang (Walter and Eliza Hall Institute, Melbourne, Australia).
Transfection-A549 cells maintained in RPMI and 10% fetal calf serum were seeded at a concentration of 2 ϫ 10 5 /ml into 6-well plates and were transfected using LipofectAMINE in Optimem. The day after transfection, the presence of green fluorescent protein (transfection control) was examined by fluorescence microscopy.
HepG2 cells were maintained in Dulbecco's modified Eagle's medium and 10% fetal calf serum and seeded at a concentration of 2 ϫ 10 5 /ml into 6-well plates. Transfection was with the calcium phosphate method (20).
Western Blot Analysis-The cell lysates were prepared as previously described (21), and in vitro translation was performed using the TNT quick-coupled transcription/translation system (Promega UK, Southampton, UK) according to the manufacturer's instructions. The protein samples were loaded on polyacrylamide gels such that protein from 1 ϫ 10 6 cells (or in the case of preprepared samples, 50 g of protein) was loaded per lane. Electrophoresis, transfer, and probing were performed using standard methods.
Confocal Microscopy and Cell Staining-The cells were examined under magnifications in the range of 100ϫ to 1000-using a Molecular Dynamics CLSM 2010 coupled to a Nikon Diaphot microscope. Excita-tion was with the 488-or 568-nm line of the krypton-argon laser. Mitochondrial staining was with Mitotracker Red 580 (Molecular Probes, Eugene, OR) and was performed according to the manufacturer's instructions. Golgi staining was performed with BODIPY TR ceramide analogue (Molecular Probes) according to the manufacturer's instructions. Nuclear staining was performed using propidium iodide on formaldehyde fixed cells at a concentration of 1 g/ml. Endoplasmic reticulum staining was with rhodamine 6G chloride (Molecular Probes) and was performed according to the published protocol (22).
Statistical Analysis-Where appropriate the results are expressed as the means Ϯ S.E. of the number (n) of independent experiments, with each experiment performed in duplicate. Statistical analysis was performed by one-way analysis of variance with Bonferroni (see Figs. 5, A and C, and 7) and Dunnett (see Fig. 5B) post-test correction for multiple comparisons using GraphPad Prism (GraphPad Software Inc., San Diego, CA). Significance was assumed at levels of p Ͻ 0.05.

Identification of Novel Bid-related Proteins in Human
Cells-A search of the known sequence for human Bid against the genomic sequence of chromosome 22 identified five coding exons analogous to those coding for murine Bid (23). In addition, a sixth noncoding exon is present, which lies ϳ25 kilobase pairs 5Ј of the Bid gene, which we have designated as exon 2 ( Fig. 1). This exon has been previously recognized to be part of the Bid gene (24). A second previously undescribed 5Ј exon of the Bid gene exists within an expressed sequence tag (accession number AA338833) that maps to chromosome 22 just 5Ј of exon 2 and that we have designated exon 1. To identify mRNA species utilizing exon 1 or 2 in association with other bid exons, 5Ј RACE was performed. Neutrophil mRNA was chosen as the source of transcripts, because of our interest in death receptor signaling in these cells (25,26) and because transcripts of alternatively spliced products were abundant in myeloid cell lines in preliminary experiments (data not shown). Using 5Ј RACE, a number of alternatively spliced transcripts were isolated, including transcripts utilizing two further previously undescribed exons. The third of these novel exons we refer to as exon 3, the genomic locus of which is located 3Ј of exon 2. mRNA transcripts contained exon 3 as the most 5Ј exon, and this was identified either in continuity with exons 4, 5, 7, 8, and 9, or in alternatively spliced isoforms "missing" exon 4 or exons 4 and 5. The fourth novel exon, which we refer to as exon 6 was isolated between sequences corresponding to exons 2, 4, and 5 and exons 7, 8, and 9. Translation of these novel transcripts creates three potential novel proteins, which we have named Bid S (accession number AY005151) for "short," Bid EL (accession number AF250233) for "extra long," and Bid ES for "extra short." Bid ES is generated from a number of splice variants that share the common feature of loss of the first AUG together with the potential for translation from an internal AUG within exon 7 of the Bid gene. A summary of all the splice variants of bid isolated by PCR, RACE, and searching the GenBank TM data base is shown in Fig. 1A. For the sake of clarity, we will use "Bid L " to refer specifically to the previously described gene product of the Bid gene (8) and "Bid" where the reference is to a more general property of the gene and its products.
Putative protein products of these splice variants are shown in Fig. 1B. Bid EL is predicted to have an additional 45 amino acids at the N terminus that do not contain any recognized conserved domains. Bid S contains only the unopposed regulatory BH3-B domain, omitting the pro-apoptotic BH3 domain. There are an additional 63 amino acids at the C terminus of Bid S that are entirely novel and that contain no recognized conserved domains. Bid ES contains the final 99 amino acids of Bid L , beginning with the terminal amino acids of the BH3 domain and including the sequence likely to be involved in mitochondrial targeting of Bid (27).
Differential Expression of Bid Isoforms-Using specific primers, as described under "Experimental Procedures," PCR was performed using a commercially available RNA panel (Premium RNA; Clontech) to screen a variety of tissues. Using primers in exons 2 and 9 of bid (BidF4, BidR2), a three-banded pattern was seen in all of the tissues studied ( Fig. 2A). Sequencing of cDNA extracted from these bands confirmed that these correspond to cDNA for Bid L and two transcripts for Bid ES (designated Bid ES (2) and Bid ES (3) in Fig. 1A). Using primers in exons 3 and 9 (BidF7 and BidR2), transcripts encoding Bid EL were detected predominantly in tissues with large numbers of hemopoetic cells (e.g. spleen and bone marrow). Bid EL was also expressed at high levels in cerebral and cerebellar cortex, as might be expected given the importance of Bid in regulating neuronal cell death (28). Transcripts corresponding to cDNA for Bid ES (4) and Bid ES (5) were also detected. PCR using primers to exon 6 and 9 (BidF10 and BidR2) shows expression of cDNA for Bid S in a similar distribution to cDNA for Bid EL . In addition, a range of transformed cell lines was screened for the presence of mRNA for Bid isoforms. mRNA for Bid L , Bid ES (2), and Bid ES (3) were seen in Cos, HeLa, HL60, Jurkat, and 293T cells. mRNA for Bid S was seen in all of the above cell lines, and mRNA for Bid EL was seen only in Jurkat cells (Fig. 2B). There is therefore evidence of endogenous mRNAs expressing all of the isoforms identified by RACE.
To demonstrate endogenous protein expression, we screened a panel of primary tissues by Western blotting. Immunoreactive bands of appropriate size were seen for Bid EL in spleen, placenta, and pancreas; for Bid S in lung, pancreas, and spleen; and for Bid ES in lung and pancreas (Fig. 3A). Transformed cell lines with high levels of mRNA expression of various Bid isoforms were also screened for Bid protein expression. Both HeLa cells and 293T cells contain an immunoreactive band that migrates at the same size as in vitro translated Bid S (Fig. 3B). Jurkat cells contain a low intensity protein band of ϳ27 kDa on Western blotting with an anti-Bid antibody that migrates at an identical size to Bid EL overexpressed in the 293T cell line (Fig. 3B) and to in vitro translated Bid EL (data not shown). We were thus able to identify protein bands on Western blotting that correspond to either in vitro translated or transfected Bid EL and Bid S , confirming endogenous expression of these proteins.
Recently, a role for Bid L in regulation of myeloid homeostasis, including peripheral blood neutrophil numbers, has been demonstrated using BidϪ/Ϫ mice (29). We therefore examined expression of Bid isoforms in two models of neutrophil maturation. NB4 cells are members of a myeloid leukemia cell line that, following treatment with retinoic acid, undergo biochemical and functional differentiation along the neutrophil lineage, such that by 24 h these cells share many characteristics with mature neutrophils (18,30). With progressive maturation, NB4 cells expressed higher levels of Bid EL (Fig. 4). Bid S levels did not consistently change in these experiments. In addition, we studied Bid L , Bid EL , and Bid S expression in myeloid precursors extracted from bone marrow by density gradient centrifugation (19,31). Cells extracted in this way represent progressively more mature neutrophil precursors and are here compared with circulating neutrophils from the same donor. Mature neutrophils express more of both Bid EL and Bid S (Fig. 4), in part paralleling the data from NB4 cells. Thus, not only are transcripts for Bid EL and Bid S expressed in a variety of primary  (3) and stop (͉) codons are indicated, and the predicted protein products are named to the right of the illustration. None of the mRNA structures shown have been previously described, with the exception of Bid L and Bid ES (1) (accession number AA338833). All splice variants are capable of encoding the putative 10-kDa protein Bid ES , by translation from an internal AUG in exon 5. The 5Ј extent of all the mRNAs shown has not been unequivocally determined. B, potential protein species generated by the mRNA species shown in A. The location of the pro-apoptotic BH3 (black box) and inhibitory BH3B (gray box) domains are shown, along with the cleavage site for caspase-8 (vertical line). Novel protein sequence is shown as a shaded area. The number of amino acids and predicted molecular masses are indicated. tissues, they are differentially regulated between tissue types and, during maturation of a single cell type, the neutrophil granulocyte.
Effects of Bid Isoforms on Cellular Apoptosis-Based on the predicted protein structure determined for the Bid isoforms (Fig. 1B), each of the three novel isoforms would be predicted to behave in different ways to modulate the activity of Bid following cleavage and activation. We hypothesized that Bid S , containing only the inhibitory BH3B domain, could act as a naturally occurring inhibitor of t c Bid-induced apoptosis. Bid EL is identical to Bid L at the C terminus, contains the BH3 domain, and differs only in the N-terminal fragment. Bid EL might therefore induce apoptosis in a similar way to Bid L , but the additional N-terminal sequence might influence the subcellular localization of Bid EL or perhaps alter the cleavage and activation of Bid EL . Because Bid ES , which again lacks a BH3 domain, corresponds to the portion of Bid that localizes to the mitochondrial surface, we hypothesized that Bid ES might interrupt the action of cleaved Bid L at the mitochondria. To test these hypotheses we first assessed the potential of Bid S , Bid EL , and Bid ES to modulate apoptosis in an overexpression assay. Apoptotic morphology was assessed in A549 cells transfected with the following constructs: pCR3.1.empty vector, pCR3.1.Bid EL , pCR3.1.Bid S , pCR3.1.Bid L , or pCR3.1.Bid ES as described under "Experimental Procedures" (16). In keeping with our predictions, transfection with constructs expressing Bid L or Bid EL induced high levels of apoptotic morphology, whereas Bid S did not (Fig. 5A). Bid S did not protect against cell death caused by the transfection process. In contrast to the hypothesis based on predicted structure, Bid ES , which contains no functional BH3 homology domains, appeared to induce apoptosis, albeit to a lesser degree than Bid L and Bid EL .
We then tested the ability of the novel isoforms to interfere with Bid L signaling. To simulate the specific effects of Bid activation, the cells were transfected with a construct expressing t c Bid L (pCR3.1.t c Bid L ). This mimics the effects of isolated Bid L cleavage, without direct activation of caspase pathways, as might occur with death receptor ligation. The ability of Bid S and Bid ES to inhibit t c Bid L -induced cell death was tested in comparison with a construct expressing the N terminus of Bid L (pCR3.1.t n Bid L ), which has previously been shown to inhibit the effects of t c Bid L (32). Bid S and Bid ES both significantly inhibited t c Bid L -induced apoptosis to a similar degree to t n Bid L (Fig. 5B). These studies also included an investigation of a further construct, corresponding to the N-terminal fragment of Bid EL , to assess the effects of the additional N-terminal sequence on the ability of the cleavage product t n Bid EL to inhibit t c Bid L -induced apoptosis. t n Bid EL , unlike t n Bid L, did not significantly inhibit t c Bid L -induced apoptosis (Fig. 5B).
We further explored the ability of Bid S to inhibit apoptosis by investigating whether Bid S could inhibit Fas-mediated apoptosis in a "type II" cell, which requires Bid activity to complete the apoptotic program following Fas ligation. HepG2 cells are a hepatocellular line, with a type II phenotype (23). Transient transfection with pCR3.1.Bid S protected HepG2 cells from CH-11 (an anti-Fas agonistic antibody)-induced apoptosis (Fig.  5C), consistent with a role for Bid S in direct inhibition of t c Bid L -induced apoptosis.
Subcellular Distribution of Bid Isoforms-Because the function of Bid is thought to relate to the ability of the active C-terminal cleavage fragment to translocate from cytoplasm to mitochondria, we investigated the subcellular localization of the various Bid isoforms using GFP fusion proteins. Transfection of a construct expressing GFP-Bid L into A549 cells showed the expected diffuse distribution (Fig. 6a). When the cells were transfected with constructs for GFP-Bid S , the fluorescence was similarly distributed throughout the cell (Fig. 6b), suggesting that the additional sequence present in Bid S does not encode an additional functional localization motif. Cells expressing GFP-Bid ES showed co-localization of fluorescence with mitochondrial stains, in keeping with the predicted function of this region of the Bid molecule as a mitochondrial-targeting region.
Following transfection, GFP-Bid EL localized to distinct regions within the cell (Fig. 6, c-f, middle panels). Further staining of organelles demonstrated co-localization with Golgi staining (BODIPY TR ceramide analogue; Fig. 6d) but not with nuclear structures (propidium iodide staining; Fig. 6c), mitochondrial structures (Mitotracker Red staining; Fig. 6e), or endoplasmic reticulum staining (rhodamine; Fig. 6f). This localization is confined to GFP fusion proteins to the N terminus of Bid EL . When constructs expressing Bid EL -GFP (i.e. a fusion of GFP to the C terminus of Bid EL ) were transfected, a significant part of the fluorescence is localized to the mitochondria (Fig. 6g), as is seen with GFP-Bid ES (Fig. 6h). In addition, Western blotting of lysates from transfected cells shows partial cleavage of GFP-Bid EL but not GFP-Bid L (Fig. 6j). This suggested that Bid EL is cleaved constitutively in transfected cells, with the N terminus localizing with the Golgi and the C terminus translocating to the mitochondria. This is supported by the observation that the broad spectrum caspase inhibitor, zVAD.fmk, is able to prevent both apoptosis and the localization of GFP-Bid EL to Golgi, whereas an inhibitor of proteosomal degradation, M132, had no effect on the appearance of GFP-Bid EL transfected cells.
The observed distribution of GFP-Bid EL was not solely due to the onset of apoptosis because apoptotic cells from transfections with the other GFP fusion vectors did not show the same pattern of distribution as was seen consistently with GFP-Bid EL (data not shown). Transfection of these cells with PCR3.1Bid EL and a vector expressing GFP alone (pEGFPC1) does not have the same distribution (data not shown), suggesting that the observed subcellular distribution was not an artifact of GFP degradation in apoptotic cells. However, GFP constructs containing only the N-terminal cleavage fragment of Bid EL (t n Bid EL ) did not localize to the Golgi (Fig. 6i), which may indicate that further modification of Bid EL during apoptosis is required for this observed localization.
Functional Interaction of Bid Isoforms with Other Bcl-2 Family Members-Bid was initially identified by its ability to interact with both pro-and anti-apoptotic Bcl-2 family members (8), and this interaction is thought to be dependent upon the BH3 domain and to be important in the function of Bcl-2 proteins. The ability of the Bid isoforms to interact with pro-and antiapoptotic members of the Bcl-2 family was therefore assessed. It has been well documented that there is considerable potential for unphysiological heterodimerization of Bcl-2 family proteins in buffer systems (33)(34)(35), and moreover, because Bid ES and Bid S lack a BH3 domain, they would not be anticipated to heterodimerize with other Bcl-2 proteins. To establish whether the novel Bid isoforms would functionally interact with other pro-or anti-apoptotic Bcl-2 proteins, functional assays were devised. In these assays we assessed both the ability of Bid isoform-induced apoptosis to be inhibited by Bcl-2 and the ability of Bax induced apoptosis to be inhibited by anti-apoptotic Bid isoforms. Apoptosis induced by Bid L , Bid EL , but not Bid ES was inhibited by Bcl-2 overexpression (Fig. 7A). This is consistent with the predicted BH3-dependent (Bid L and Bid EL ) and -independent (Bid ES ) mechanisms of cell death. Bax-induced cell death was inhibitable by Bcl-2 but not by Bid S , and Bid ES was also without effect upon Bax-induced apoptosis (Fig. 7B).

FIG. 3. Identification of endogenously occurring Bid isoforms by Western blotting.
Tissue protein or cell lysates were subjected to Western blotting, and the membranes were probed with an anti-Bid polyclonal antibody, as described under "Experimental Procedures." The protein bands corresponding to Bid L and Bid EL and Bid S are indicated. A, a commercially available protein panel was screened by Western blotting. Bid L is seen in spleen, placenta, and transfected HeLa cells and was visible in other lanes on longer exposures. Bid S was seen in lung, pancreas, and spleen. Bid EL was seen in spleen, placenta, and pancreas. A band of similar size to that predicted for Bid ES was seen in lung and spleen. B, the cell lines indicated were lysed as described under "Experimental Procedures." An immunoreactive band of identical size to in vitro translated Bid S was seen in HeLa cells and 293T cells. Jurkat cells express a protein band of identical size to that seen in 293T cells transfected with pCR3.1.Bid EL . This band was not seen in untransfected 293T cells and was of an identical size to in vitro translated Bid EL .

DISCUSSION
Bcl-2 family proteins are important regulators of cellular apoptosis. As such, their activity is highly regulated. In addition to regulation at the level of transcription and translation, their activity is regulated by post-translational modification. For example, Bad is regulated by phosphorylation/dephosphorylation (36); Bid, Bcl-2, and Bcl-X L are regulated by caspasemediated proteolysis (12,13,(37)(38)(39), which in turn may be mediated by phosphorylation of Bid (40); Bax is cleaved by calpains (41), and t c Bid undergoes N-myristoylation (42). Bcl-2 proteins are also regulated by homodimerization and heterodimerization (43) and differential subcellular localization (44). In addition, many are known to be regulated by alternative splicing (15), which can regulate the apoptogenic potential (e.g. Bim (45)) or alter its function entirely (e.g. Bcl-xL and Bcl-xS (14)).
Bid acts to convert proteolytic signals from Granzyme B, caspases (predominantly caspase-8), and lysosomal proteases into apoptotic signals. Cleavage of a protease-sensitive domain of Bid yields an active fragment (t c Bid) and an inhibitory fragment (t n Bid). We have identified three novel isoforms of Bid that may modulate the function of cleaved Bid. These proteins have a restricted and distinct tissue distribution and also differ in their intracellular localization. Importantly, they show distinct differences in their ability to modulate cellular apoptosis. These isoforms may therefore exist to provide an additional layer of control of Bid function.
Bid EL exists in many cell types at mRNA level, as we have shown, and other groups have recently also deposited sequence corresponding to Bid EL in the GenBank TM data base (e.g. (47)). Cells transfected with expression vectors for Bid EL readily express protein, and an endogenous protein of identical size is seen in a number of primary tissue types and in Jurkat cell lysates. Overexpression of Bid EL in A549 cells leads to apoptosis at levels indistinguishable from those produced by Bid L overexpression. There is therefore good evidence to suggest that this protein species exists in vivo and that it may have functional relevance to the control of apoptosis.
The subcellular localization of Bid EL is complicated by the cleavage of this protein. The N-terminal fragment localizes to the Golgi apparatus following onset of apoptosis, whereas the C-terminal fragment, as predicted, traffics to mitochondria. The findings could be an artifact of overexpression but are not seen with overexpression of other closely related isoforms of Bid or with the same protein when labeled with GFP at the C terminus. The localization of the N-terminal fragment to the Golgi apparatus may remove the inhibitory effect of this portion of Bid EL , potentially regulating the activity of Bid EL following cleavage in vivo. In the co-transfection studies shown in FIG. 4. Modulation of Bid isoform expression during myeloid differentiation. mRNA extracted from NB4 cells that had been induced to undergo differentiation (left panel) was used as a template for reverse transcription-PCR as described under "Experimental Procedures." Levels of the control mRNA, GAPDH, remained constant as did those for Bid L , Bid ES (2), and Bid ES (3), whereas mRNA for Bid EL was only seen in differentiated NB4 cells. Levels of Bid S mRNA were not consistently different in repeated experiments. The figure shown is representative of three independent experiments. mRNA extracted from bone marrow myeloid precursors that had been separated into fractions of progressively increasing maturation (fractions 3, 2, and 1) was used as a template for reverse transcription-PCR as described under "Experimental Procedures." Mature peripheral blood neutrophils were extracted from the same subjects and are indicated by N. Levels of GAPDH consistently decline during neutrophil maturation, but levels of Bid L , Bid ES (1), and Bid ES (2) remain constant. Bid S and Bid EL increase dramatically with progressive maturation to the levels seen in peripheral blood neutrophils. The experiment shown using two independent subjects is representative of three separate experiments. Fig. 5B, rates of apoptosis in cells treated with t c Bid L and t n Bid EL were not significantly different from those seen with t c Bid L alone. These results may indicate that t n Bid EL differs from t n Bid L in its ability to act as an apoptosis inhibitor, perhaps as a consequence of its sequestration within the Golgi. However, Bid EL is indistinguishable from Bid L with regard to its pro-apoptotic effect, which is inhibited by Bcl-2.
In the case of Bid EL , regulation of activity may be at the level of mRNA. We have not isolated mRNAs containing both exon 3 and exon 2, which may suggest different promoter usage. If reliant on the same promoter, the differing 5Ј sequence of mRNA species for Bid L and Bid EL may well alter relative stability or translation efficiency, leading to altered levels of protein present (48). In addition, the additional upstream reading frame seen in the mRNA for Bid EL may function to regulate expression of Bid L transcribed from this mRNA. It is recognized that transcripts with additional upstream reading frames (such as Bid EL ) may exist to tightly regulate the expression of translations beginning at the intended downstream AUG (49).
Bid S is expressed in a number of cell types at mRNA and protein level, although no expressed sequence tags are present in the GenBank TM data base that code for Bid S . In vitro translated Bid S migrates at the same size as endogenous immunoreactive bands seen in a variety of human tissues and in HeLa and 293T cells. In addition, cells transfected with expression vectors for Bid S express a protein band of appropriate size on Western blotting (data not shown). Bid S remains cytoplasmic when transfected as a GFP fusion protein, as does Bid L . Transfection of A549 cells with GFP-Bid S does not induce apoptosis, as suggested by the predicted presence of a BH3B but not a BH3 domain. Bid S transfection is, however, able to inhibit apoptosis caused by truncated Bid L and to prevent CH-11/Fasinduced cell death. Bid S does not, however, appear to directly interact with Bcl-2 or Bax in functional assays. Thus, Bid S has the potential to act as a naturally occurring inhibitor of t c Bid Linduced cell death and to modulate the response of a cell to a variety of death-inducing stimuli, including death receptor ligation. There is therefore evidence to suggest that Bid S exists in vivo and that it has functional relevance to the control of apoptosis.
The recent publication of data suggesting that Bid is essential for normal terminal differentiation of myeloid cells (29), together with the original derivation of Bid S and Bid EL from human neutrophils, suggested to us that expression of Bid isoforms may be of interest in neutrophil precursors. Using parallel strategies we identified increases in levels of mRNA for Bid EL and Bid S during myeloid differentiation. No changes in the levels of Bid L were seen in these experiments. This suggests that Bid S and Bid EL may be involved differentially in the regulation of Bid-induced death in developing myeloid cells. In particular, they may influence the sensitivity of these cells to death receptor-induced apoptosis. Neutrophils contain high levels of lysosomal proteases, and Bid S may be present to prevent inadvertent cell death following proteolytic cleavage of Bid (50). The parallel increases in Bid S and Bid EL , which initially appear conflicting in effect, may suggest a degree of subtlety in the regulation of Bid-induced apoptosis that is not revealed in these in vitro experiments.
A 10-kDa protein, Bid ES , is potentially generated from all of the Bid transcripts identified and corresponds to the C-terminal fragment of Bid, downstream of the BH3 domain. This protein would be the only product derived from a number of alternatively spliced mRNA species whose expression is detected during PCR analysis of the Bid gene (indicated in Fig.  1A), and this may explain the existence of these mRNAs, whose purpose is otherwise unknown. A protein band of appropriate size was identified on Western blotting, although this has not been completely characterized. The apparent actions of Bid ES as both a pro-apoptotic agent and as an inhibitor of t c Bid-FIG. 5. Modulation of apoptosis by Bid isoforms. A, A549 cells were transfected with a PCR3.1 vector containing the constructs shown, a GFP reporter, and carrier DNA to a total of 5 g. 24 h following transfection, the plates were examined, and the cells expressing the GFP reporter were assessed for apoptotic morphology. The results are shown as the means Ϯ S.E. of four independent experiments performed in duplicate. The rates of apoptosis seen with Bid, Bid EL , and Bid ES are significantly larger than control or Bid S (p Ͻ 0.01). Bid ES rates of apoptosis are significantly lower than those seen with either Bid or Bid EL (p Ͻ 0.01). B, A549 cells were transfected as above with pCR3.1.t c Bid (50 ng), pEGFPc1 (1 g), test vector (500 ng), and carrier DNA to a total of 5 g. Compared with Bcl-2 and Bcl-x L , nBid L , Bid S , and Bid ES were less efficient inhibitors of apoptosis. In this series of experiments, inhibition of tBid by nBid EL was not statistically significant. *, p Ͻ 0.05; **, p Ͻ 0.01 compared with tBid transfected cells alone. C, HepG2 cells were transfected with pCR3.1.empty vector control or pCR3.1.Bid S (50 ng), with pEGFPc1 (1 g) as a marker of transfection. After 24 h, CH-11 (an agonistic anti-Fas antibody) was added at a concentration of 500 ng/ml. A further 24 h later, transfected cells were assessed for apoptotic morphology, as above. Bid S was able to completely inhibit CH-11-induced apoptosis (p Ͻ 0.05).
induced apoptosis are of interest. t c Bid L has previously been shown to target mitochondria in a BH3-independent manner (27) and does not rely on the BH3 domain for trimerization and cytochrome c release (51). The same mechanisms that underly these processes could also account for the action of Bid ES in inducing apoptosis. The absence of a BH3 domain in Bid ES presumably accounts for its lack of direct interaction with either Bcl-2 or Bax in functional assays. The effects of Bid ES on FIG. 6. Subcellular localization of GFP-Bid isoform fusion constructs. A549 cells were transfected with the following constructs: pEGFPC2.Bid L (a), pEGFPC2.Bid S (b), pEGFPC2.Bid EL (c-f), pEGFPN3.Bid EL (g), pEGFPC2.Bid ES (h), and pEGFPC2.t n Bid EL (i). The cells were counterstained as indicated, as described under "Experimental Procedures." At 24 h post-transfection, confocal scanning images were taken at 200ϫ, and a composite image was assembled. The images shown for each were chosen as being representative of the experiment shown. j, A549 cells transfected with GFP-bid L , GFP-Bid S , and GFP-Bid EL were lysed and subjected to Western blot analysis, as described under "Experimental Procedures." Bands of sizes corresponding to the transfected proteins were detected using an anti-Bid polyclonal antibody. In addition, a band of corresponding length for cleaved GFP-Bid EL was seen. No bands of appropriate size for known caspase cleavage fragments of GFP-Bid L were seen. k and l, A549 cells were transfected with pEGFPC2.Bid EL and cultured with the broad spectrum caspase inhibitor, zVAD.fmk (k), or the proteosome inhibitor, M132 (l). At 24 h post-transfection, confocal scanning images were taken at 200ϫ. The images shown for each were chosen as being representative of the experiment shown. apoptosis must therefore relate to effects other than the ability to interact with other BH3 domain-containing proteins. Targeting of t c Bid to mitochondria requires binding to cardiolipin (27) or related sites on the mitochondria surface (46,52). The effect of Bid ES in inhibiting t c Bid-induced apoptosis may relate to its ability to inhibit binding of t c Bid to the mitochondria. Thus, Bid ES might act in different ways to modulate apoptosis depending on the relative levels of pro-apoptotic and antiapoptotic Bid moieties in the cell. In this way Bid ES could provide flexible and dynamic regulation of Bid-mediated apoptosis.
The important role of BH3 proteins in initiating apoptosis in response to a varied range of stimuli is becoming clear. The existence of a family of Bid proteins suggests that the fate of a cell following Bid cleavage may not be as simple as previously thought and may be influenced by the relative expression of the different Bid isoforms. The novel isoforms described here are endogenously expressed and perform distinct functions, as evidenced by differences in expression, cellular localization, and functional effects upon cellular apoptosis. This suggests that they have an important place in the complex network of pathways used by cells to determine their fate following a proapoptotic stimulus. FIG. 7. Interaction of Bid-isoforms with other Bcl-2 family proteins. A, A549 cells transfected with the vectors expressing the inserts shown were assessed for rates of apoptosis, as described under "Experimental Procedures". Bid L and Bid EL significantly increased rates of apoptosis compared with empty vector control (p Ͻ 0.001 in each case), that is inhibited by co-transfection with Bcl-2 (p Ͻ 0.001 compared with either isoform alone), consistent with functional interaction between these proteins in vivo. In contrast, the pro-apoptotic effects of Bid ES were not significantly abrogated by Bcl-2. B, A549 cells transfected with the vectors expressing the inserts shown were assessed for rates of apoptosis, as described under "Experimental Procedures." Bax shows significantly increased rates of apoptosis compared with empty vector control (p Ͻ 0.01). This can be abrogated by co-transfection with Bcl-2 (p Ͻ 0.05 compared with Bax alone) but not by co-transfection with Bid S or Bid ES (p Ͼ 0.5 compared with Bax alone).