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Exon Skipping in Mcl-1 Results in a Bcl-2 Homology Domain 3 Only Gene Product That Promotes Cell Death*

Open AccessPublished:July 21, 2000DOI:https://doi.org/10.1074/jbc.M909572199
      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/ΔTM. 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/ΔTM 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/ΔTM 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.
      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
      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 (
      • Rudin C.M.
      • Thompson C.B.
      ). Apoptosis is controlled by evolutionarily conserved sets of genes with the Bcl-2 family playing a pivotal role (
      • Korsmeyer S.J.
      ,
      • Gross A.
      • McDonnell J.M.
      • Korsmeyer S.J.
      ). 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 (
      • Muchmore S.W.
      • Sattler M.
      • Liang H.
      • Meadows R.P.
      • Harlan J.E.
      • Yoon H.S.
      • Nettesheim D.
      • Chang B.S.
      • Thompson C.B.
      • Wong S.L.
      • Ng S.L.
      • Fesik S.W.
      ) and appear critical for anti-apoptotic function (
      • Yin X.M.
      • Oltvai Z.
      • Korsmeyer S.J.
      ). Whereas some death-inducing family members contain all three of these domains (e.g. Bax, Bak, and Bok (
      • Hsu S.Y.
      • Kaipia A.
      • McGee E.
      • Lomeli M.
      • Hsueh A.J.W.
      ,
      • Inohara N.
      • Ekhterae D.
      • Garcia I.
      • Carrio R.
      • Merino J.
      • Merry A.
      • Chen S.
      • Nunez G.
      ,
      • Oltvai Z.N.
      • Milliman C.L.
      • Korsmeyer S.J.
      )), others contain BH3 only (e.g. Bid, Bad, and Bim). This suggests that BH3 is a minimum requirement for pro-apoptotic function (
      • Kelekar A.
      • Thompson C.B.
      ,
      • O'Connor L.
      • Strasser A.
      • O'Reilly L.A.
      • Hausmann G.
      • Adams J.M.
      • Cory S.
      • Huang D.C.
      ), which probably relates to the fact that the exposed BH3 domain can bind to the hydrophobic cleft of pro-survival family members (
      • Sattler M.
      • Liang H.
      • Nettesheim D.
      • Meadows R.P.
      • Harlan L.E.
      • Eberstadt M.
      • Yoon H.S.
      • Shuker S.B.
      • Chang B.S.
      • Minn A.J.
      • Thompson C.
      ). 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 (
      • Nakai M.
      • Takeda A.
      • Cleary M.L.
      • Endo T.
      ,
      • Nguyen M.
      • Millar D.G.
      • Yong V.W.
      • Korsmeyer S.J.
      • Shore G.C.
      ,
      • Krajewski K.
      • Tanaka S.
      • Takayama S.
      • Schibler M.J.
      • Fenton W.
      • Reed J.C.
      ,
      • Yang T.
      • Kozopas K.M.
      • Craig R.W.
      ). Although this domain may aid in targeting certain family members for function (
      • Tanaka S.
      • Saito K.
      • Reed J.C.
      ,
      • Borner C.
      • Martinou I.
      • Mattman C.
      • Irmler M.
      • Schaerer E.
      • Martinou J.-C.
      • Tschopp J.
      ), a TM domain is not present in some death-inducing members, namely Bid and Bad (
      • Gross A.
      • McDonnell J.M.
      • Korsmeyer S.J.
      ). 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) (
      • Kozopas K.M.
      • Yang T.
      • Buchan H.L.
      • Zhou P.
      • Craig R.W.
      ). 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 (
      • Krajewski S.
      • Bodrug S.
      • Gascoyne R.
      • Berean K.
      • Krajewska M.
      • Reed J.C.
      ,
      • Krajewski S.
      • Bodrug S.
      • Krajewska M.
      • Shabaik A.
      • Gascoyne R.
      • Berean K.
      • Reed J.C.
      ). 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 (
      • Townsend K.J.
      • Zhou P.
      • Qian L.
      • Bieszczad C.K.
      • Lowrey C.H.
      • Yen A.
      • Craig R.W.
      ,
      • Lomo J.
      • Blomhoff H.K.
      • Jacobsen S.E.
      • Krajewski S.
      • Reed J.C.
      • Smeland E.B.
      ,
      • Moulding D.A.
      • Quayle J.A.
      • Hart C.A.
      • Edwards S.W.
      ,
      • Druilhe A.
      • Arock M.
      • LeGoff L.
      • Pretolani M.
      ,
      • Chao J.R.
      • Wang J.M.
      • Lee S.F.
      • Peng H.W.
      • Lin Y.H.
      • Chou C.H.
      • Li J.C.
      • Huang H.M.
      • Chou C.K.
      • Kuo M.L.
      • Yen J.J.
      • Yang-Yen H.F.
      ). For example, in neutrophils (
      • Chuang P.I.
      • Yee E.
      • Karsan A.
      • Winn R.K.
      • Harlan J.M.
      ), external stimuli that prolong the neutrophil life span (such as lipopolysaccharide and granulocyte-macrophage colony-stimulating factor) cause an increase in Mcl-1 expression (
      • Moulding D.A.
      • Quayle J.A.
      • Hart C.A.
      • Edwards S.W.
      ,
      • Klampfer L.
      • Zhang J.
      • Nimer S.D.
      ). 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 (
      • Wang J.M.
      • Chao J.-R.
      • Chen W.
      • Kuo M.-L.
      • Yen J.J.Y.
      • Yang-Yen H.F.
      ). In addition to regulation at a transcriptional level, the Mcl-1 mRNA can be rapidly turned over (
      • Yang T.
      • Buchan H.L.
      • Townsend K.J.
      • Craig R.W.
      ). Furthermore, the upstream half of Mcl-1 contains PEST sequences, and the protein is likewise subject to rapid turn-over (
      • Yang T.
      • Kozopas K.M.
      • Craig R.W.
      ). The downstream half of the protein contains the BH1, BH2, and BH3 domains and terminates in a TM domain (
      • Yang T.
      • Kozopas K.M.
      • Craig R.W.
      ,
      • Kozopas K.M.
      • Yang T.
      • Buchan H.L.
      • Zhou P.
      • Craig R.W.
      ). 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 (
      • Lomo J.
      • Blomhoff H.K.
      • Jacobsen S.E.
      • Krajewski S.
      • Reed J.C.
      • Smeland E.B.
      ,
      • Moulding D.A.
      • Quayle J.A.
      • Hart C.A.
      • Edwards S.W.
      ,
      • Chao J.R.
      • Wang J.M.
      • Lee S.F.
      • Peng H.W.
      • Lin Y.H.
      • Chou C.H.
      • Li J.C.
      • Huang H.M.
      • Chou C.K.
      • Kuo M.L.
      • Yen J.J.
      • Yang-Yen H.F.
      ,
      • Altmeyer A.
      • Simmons R.C.
      • Krajewski S.
      • Reed J.C.
      • Bornkamm G.W.
      • Chen-Kiang S.
      ). 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 (
      • Reynolds J.E.
      • Yang T.
      • Qian L.
      • Jenkinson J.D.
      • Zhou P.
      • Eastman A.
      • Craig R.W.
      ,
      • Reynolds J.E.
      • Li J.
      • Craig R.W.
      • Eastman A.
      ,
      • Zhou P.
      • Qian L.
      • Kozopas K.M.
      • Craig R.W.
      ). 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 (
      • Townsend K.J.
      • Trusty J.L.
      • Traupman M.A.
      • Eastman A.
      • Craig R.W.
      ,
      • Zhou P.
      • Qian L.
      • Kozopas K.M.
      • Craig R.W.
      ,
      • Rinkenberger J.L.
      • Horning S.
      • Klocke B.
      • Roth K.
      • Korsmeyer S.J.
      ). Similarly, in mice that express full-length Mcl-1 as a transgene in hematopoietic tissues (
      • Zhou P.
      • Qian L.
      • Bieszczad C.K.
      • Noelle R.
      • Binder M.
      • Levy N.B.
      • Craig R.W.
      ), 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 (
      • Jiang Z.H.
      • Wu J.Y.
      ). 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 (
      • Grillot D.A.M.
      • Gonzalez-Garcia M.
      • Ekhterae D.
      • Duan L.
      • Inohara N.
      • Ohta S.
      • Seldin M.F.
      • Nunez G.
      ,
      • Boise L.H.
      • Gonzalez-Garcia M.
      • Postema C.E.
      • Ding L.
      • Lindsten T.
      • Turka L.
      • Mao X.
      • Nunez G.
      • Thompson C.B.
      ,
      • Seto M.
      • Jaeger U.
      • Hockett R.D.
      • Graninger W.
      • Bennett S.
      • Goldman P.
      • Korsmeyer S.J.
      ). 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 (
      • Grillot D.A.M.
      • Gonzalez-Garcia M.
      • Ekhterae D.
      • Duan L.
      • Inohara N.
      • Ohta S.
      • Seldin M.F.
      • Nunez G.
      ,
      • Boise L.H.
      • Gonzalez-Garcia M.
      • Postema C.E.
      • Ding L.
      • Lindsten T.
      • Turka L.
      • Mao X.
      • Nunez G.
      • Thompson C.B.
      ,
      • Seto M.
      • Jaeger U.
      • Hockett R.D.
      • Graninger W.
      • Bennett S.
      • Goldman P.
      • Korsmeyer S.J.
      ). 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 (
      • Jiang Z.H.
      • Wu J.Y.
      ,
      • Fang W.
      • Rivard J.J.
      • Mueller D.L.
      • Behrens T.W.
      ).
      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) (
      • Whyte M.K.
      • Renshaw S.A.
      • Lawson R.
      • Bingle C.D.
      ) and in epithelial tissues. Mcl-1 is of particular interest because of its highly regulated pattern of expression in these tissues (
      • Krajewski S.
      • Bodrug S.
      • Krajewska M.
      • Shabaik A.
      • Gascoyne R.
      • Berean K.
      • Reed J.C.
      ,
      • Townsend K.J.
      • Zhou P.
      • Qian L.
      • Bieszczad C.K.
      • Lowrey C.H.
      • Yen A.
      • Craig R.W.
      ,
      • Lomo J.
      • Blomhoff H.K.
      • Jacobsen S.E.
      • Krajewski S.
      • Reed J.C.
      • Smeland E.B.
      ,
      • Moulding D.A.
      • Quayle J.A.
      • Hart C.A.
      • Edwards S.W.
      ,
      • Druilhe A.
      • Arock M.
      • LeGoff L.
      • Pretolani M.
      ,
      • Chao J.R.
      • Wang J.M.
      • Lee S.F.
      • Peng H.W.
      • Lin Y.H.
      • Chou C.H.
      • Li J.C.
      • Huang H.M.
      • Chou C.K.
      • Kuo M.L.
      • Yen J.J.
      • Yang-Yen H.F.
      ). In the research presented here, we describe Mcl-1s/ΔTM, 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/ΔTM 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/ΔTM-encoded gene product are thus reminiscent of BH3 only pro-apoptotic family members such Bid and Bad. Accordingly, transfection with Mcl-1s/ΔTM 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.

      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/ΔTM 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/ΔTM variant was found to induce cell death. Similarly, populations of cells that were largely healthy and viable exhibited low levels of Mcl-1s/ΔTM as compared with full-length wild-type Mcl-1.
      Other members of the Bcl-2 family can also undergo differential splicing (
      • Jiang Z.H.
      • Wu J.Y.
      ,
      • Boise L.H.
      • Gonzalez-Garcia M.
      • Postema C.E.
      • Ding L.
      • Lindsten T.
      • Turka L.
      • Mao X.
      • Nunez G.
      • Thompson C.B.
      ,
      • Fang W.
      • Rivard J.J.
      • Mueller D.L.
      • Behrens T.W.
      ,
      • Hsu S.Y.
      • Hsueh A.J.W.
      ,
      • Ban J.
      • Eckhart L.
      • Weninger W.
      • Mildner M.
      • Tschachler E.
      ,
      • Yang X.F.
      • Weber G.F.
      • Cantor H.
      ,
      • Shiraiwa N.
      • Inohara N.
      • Okada S.
      • Yuzaki M.
      • Shoji S.I.
      • Ohta S.
      ). In terms of anti-apoptotic family members, the splicing of Bcl-xL to Bcl-xSinvolves the use of an alternate splice site within the coding sequence (
      • Boise L.H.
      • Gonzalez-Garcia M.
      • Postema C.E.
      • Ding L.
      • Lindsten T.
      • Turka L.
      • Mao X.
      • Nunez G.
      • Thompson C.B.
      ). Bcl-w can also undergo alternate splicing, and in this case splicing is to an adjacent gene (
      • Gibson L.
      • Holmgreen S.P.
      • Huang D.C.
      • Bernard O.
      • Copeland N.G.
      • Jenkins N.A.
      • Sutherland G.R.
      • Baker E.
      • Adams J.M.
      • Cory S.
      ). With both Bcl-x and Bcl-2, unspliced transcripts that read through into the intron have also been reported (
      • Jiang Z.H.
      • Wu J.Y.
      ). 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) (
      • Grillot D.A.M.
      • Gonzalez-Garcia M.
      • Ekhterae D.
      • Duan L.
      • Inohara N.
      • Ohta S.
      • Seldin M.F.
      • Nunez G.
      ,
      • Seto M.
      • Jaeger U.
      • Hockett R.D.
      • Graninger W.
      • Bennett S.
      • Goldman P.
      • Korsmeyer S.J.
      ,
      • Gibson L.
      • Holmgreen S.P.
      • Huang D.C.
      • Bernard O.
      • Copeland N.G.
      • Jenkins N.A.
      • Sutherland G.R.
      • Baker E.
      • Adams J.M.
      • Cory S.
      ,
      • Hatakeyama S.
      • Hamasaki A.
      • Negishi I.
      • Loh D.Y.
      • Sendo F.
      • Nakayama K.
      • Nakayama K.
      ), multiple coding exons (
      • Yin X.M.
      • Oltvai Z.
      • Korsmeyer S.J.
      ,
      • Hsu S.Y.
      • Kaipia A.
      • McGee E.
      • Lomeli M.
      • Hsueh A.J.W.
      ) are present in pro-apoptotic family members. This is because of the presence of 3–5 introns in the coding region (
      • Jiang Z.H.
      • Wu J.Y.
      ,
      • Choi S.S.
      • Park S.H.
      • Kim U.J.
      • Shin H.S.
      ,
      • Herberg J.A.
      • Phillips S.
      • Beck S.
      • Jones T.
      • Sheer D.
      • Wu J.J.
      • Prochazka V.
      • Barr P.
      • Kiefer M.C.
      • Trowsdale J.
      ,
      • Hsu S.Y.
      • Hsueh A.J.W.
      ,
      • Ulrich E.
      • Kauffmann-Zeh A.
      • Hueber A.O.
      • Williamson J.
      • Chittenden T.
      • Ma A.
      • Evan G.
      ,
      • Wang K.
      • Yin X.M.
      • Copeland N.G.
      • Gilbert D.J.
      • Jenkins N.A.
      • Keck C.L.
      • Aimonjic D.B.
      • Popescu N.C.
      • Korsmeyer S.J.
      ), 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 Baxδ, where exon 4 is spliced to exon 2 (in frame) with skipping of exon 3, as well as Baxγ, where exon 3 is spliced to exon 1 (with a change in reading frame) with skipping of exon 2 (
      • Oltvai Z.N.
      • Milliman C.L.
      • Korsmeyer S.J.
      ,
      • Jiang Z.H.
      • Wu J.Y.
      ). 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 (
      • Hsu S.Y.
      • Hsueh A.J.W.
      ) and, in Bim, alternate splicing can alter the N terminus of the encoded gene product (
      • O'Connor L.
      • Strasser A.
      • O'Reilly L.A.
      • Hausmann G.
      • Adams J.M.
      • Cory S.
      • Huang D.C.
      ). 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 (
      • Oltvai Z.N.
      • Milliman C.L.
      • Korsmeyer S.J.
      ). Other pro-apoptotic family members, namely Bid and Bak, also contain an intron downstream of BH3 (
      • Choi S.S.
      • Park S.H.
      • Kim U.J.
      • Shin H.S.
      ,
      • Herberg J.A.
      • Phillips S.
      • Beck S.
      • Jones T.
      • Sheer D.
      • Wu J.J.
      • Prochazka V.
      • Barr P.
      • Kiefer M.C.
      • Trowsdale J.
      ,
      • Wang K.
      • Yin X.M.
      • Copeland N.G.
      • Gilbert D.J.
      • Jenkins N.A.
      • Keck C.L.
      • Aimonjic D.B.
      • Popescu N.C.
      • Korsmeyer S.J.
      ). 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-xslikewise lies downstream of BH3, although this site is again not located at a position identical to that of the intron in Mcl-1 (
      • Boise L.H.
      • Gonzalez-Garcia M.
      • Postema C.E.
      • Ding L.
      • Lindsten T.
      • Turka L.
      • Mao X.
      • Nunez G.
      • Thompson C.B.
      ). 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 (
      • Morishi K.
      • Huang D.C.
      • Cory S.
      • Adams J.M.
      ). 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 (
      • Zha J.
      • Harada H.
      • Yang E.
      • Jockel J.
      • Korsmeyer S.J.
      ). 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 (
      • Puthalakath H.
      • Huang D.C.
      • O'Reilly L.A.
      • King S.M.
      • Strasser A.
      ). 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 (
      • Li H.
      • Zhu H.
      • Xu C.J.
      • Yuan J.
      ). 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/ΔTM 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 (
      • Herberg J.A.
      • Phillips S.
      • Beck S.
      • Jones T.
      • Sheer D.
      • Wu J.J.
      • Prochazka V.
      • Barr P.
      • Kiefer M.C.
      • Trowsdale J.
      ) and which underwent gene duplication (
      • Hatakeyama S.
      • Hamasaki A.
      • Negishi I.
      • Loh D.Y.
      • Sendo F.
      • Nakayama K.
      • Nakayama K.
      ). 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 (
      • Herberg J.A.
      • Phillips S.
      • Beck S.
      • Jones T.
      • Sheer D.
      • Wu J.J.
      • Prochazka V.
      • Barr P.
      • Kiefer M.C.
      • Trowsdale J.
      ). 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 (
      • Evans D.L.
      • Mansel R.E.
      ). This possibility and the findings described here could relate to previous observations concerning the BH3 domain of Mcl-1 (
      • Kelekar A.
      • Thompson C.B.
      ). 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 (
      • Sattler M.
      • Liang H.
      • Nettesheim D.
      • Meadows R.P.
      • Harlan L.E.
      • Eberstadt M.
      • Yoon H.S.
      • Shuker S.B.
      • Chang B.S.
      • Minn A.J.
      • Thompson C.
      ). 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.

      Acknowledgments

      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.

      REFERENCES

        • Rudin C.M.
        • Thompson C.B.
        Annu. Rev. Med. 1997; 48: 267-281
        • Korsmeyer S.J.
        Cancer Res. 1999; 59: 1693-1700
        • Gross A.
        • McDonnell J.M.
        • Korsmeyer S.J.
        Genes Dev. 1999; 13: 632-640
        • Muchmore S.W.
        • Sattler M.
        • Liang H.
        • Meadows R.P.
        • Harlan J.E.
        • Yoon H.S.
        • Nettesheim D.
        • Chang B.S.
        • Thompson C.B.
        • Wong S.L.
        • Ng S.L.
        • Fesik S.W.
        Nature. 1996; 381: 335-341
        • Yin X.M.
        • Oltvai Z.
        • Korsmeyer S.J.
        Nature. 1994; 369: 321-323
        • Hsu S.Y.
        • Kaipia A.
        • McGee E.
        • Lomeli M.
        • Hsueh A.J.W.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12401-12406
        • Inohara N.
        • Ekhterae D.
        • Garcia I.
        • Carrio R.
        • Merino J.
        • Merry A.
        • Chen S.
        • Nunez G.
        J. Biol. Chem. 1998; 273: 8705-8710
        • Oltvai Z.N.
        • Milliman C.L.
        • Korsmeyer S.J.
        Cell. 1993; 74: 609-619
        • Kelekar A.
        • Thompson C.B.
        Trends Cell Biol. 1998; 1998: 324-330
        • O'Connor L.
        • Strasser A.
        • O'Reilly L.A.
        • Hausmann G.
        • Adams J.M.
        • Cory S.
        • Huang D.C.
        EMBO J. 1998; 17: 384-395
        • Sattler M.
        • Liang H.
        • Nettesheim D.
        • Meadows R.P.
        • Harlan L.E.
        • Eberstadt M.
        • Yoon H.S.
        • Shuker S.B.
        • Chang B.S.
        • Minn A.J.
        • Thompson C.
        Science. 1997; 275: 983-986
        • Nakai M.
        • Takeda A.
        • Cleary M.L.
        • Endo T.
        Biochem. Biophys. Res. Commun. 1993; 196: 233-239
        • Nguyen M.
        • Millar D.G.
        • Yong V.W.
        • Korsmeyer S.J.
        • Shore G.C.
        J. Biol. Chem. 1993; 268: 25265-25268
        • Krajewski K.
        • Tanaka S.
        • Takayama S.
        • Schibler M.J.
        • Fenton W.
        • Reed J.C.
        Cancer Res. 1993; 53: 4701-4714
        • Yang T.
        • Kozopas K.M.
        • Craig R.W.
        J. Cell Biol. 1995; 128: 1173-1184
        • Tanaka S.
        • Saito K.
        • Reed J.C.
        J. Biol. Chem. 1993; 268: 10920-10926
        • Borner C.
        • Martinou I.
        • Mattman C.
        • Irmler M.
        • Schaerer E.
        • Martinou J.-C.
        • Tschopp J.
        J. Cell Biol. 1994; 126: 1059-1068
        • Kozopas K.M.
        • Yang T.
        • Buchan H.L.
        • Zhou P.
        • Craig R.W.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3516-3520
        • Krajewski S.
        • Bodrug S.
        • Gascoyne R.
        • Berean K.
        • Krajewska M.
        • Reed J.C.
        Am. J. Pathol. 1994; 145: 515-525
        • Krajewski S.
        • Bodrug S.
        • Krajewska M.
        • Shabaik A.
        • Gascoyne R.
        • Berean K.
        • Reed J.C.
        Am. J. Pathol. 1995; 146: 1309-1319
        • Townsend K.J.
        • Zhou P.
        • Qian L.
        • Bieszczad C.K.
        • Lowrey C.H.
        • Yen A.
        • Craig R.W.
        J. Biol. Chem. 1999; 274: 1802-1813
        • Lomo J.
        • Blomhoff H.K.
        • Jacobsen S.E.
        • Krajewski S.
        • Reed J.C.
        • Smeland E.B.
        Blood. 1997; 89: 4415-4424
        • Moulding D.A.
        • Quayle J.A.
        • Hart C.A.
        • Edwards S.W.
        Blood. 1998; 92: 2495-2502
        • Druilhe A.
        • Arock M.
        • LeGoff L.
        • Pretolani M.
        Am. J. Respir. Cell Mol. Biol. 1998; 18: 315-322
        • Chao J.R.
        • Wang J.M.
        • Lee S.F.
        • Peng H.W.
        • Lin Y.H.
        • Chou C.H.
        • Li J.C.
        • Huang H.M.
        • Chou C.K.
        • Kuo M.L.
        • Yen J.J.
        • Yang-Yen H.F.
        Mol. Cell. Biol. 1998; 18: 4883-4898
        • Chuang P.I.
        • Yee E.
        • Karsan A.
        • Winn R.K.
        • Harlan J.M.
        Biochem. Biophys. Res. Commun. 1998; 249: 361-365
        • Klampfer L.
        • Zhang J.
        • Nimer S.D.
        Cytokine. 1999; 11: 849-855
        • Townsend K.J.
        • Trusty J.L.
        • Traupman M.A.
        • Eastman A.
        • Craig R.W.
        Oncogene. 1998; 17: 1223-1234
        • Wang J.M.
        • Chao J.-R.
        • Chen W.
        • Kuo M.-L.
        • Yen J.J.Y.
        • Yang-Yen H.F.
        Mol. Cell. Biol. 1999; 19: 6195-6206
        • Yang T.
        • Buchan H.L.
        • Townsend K.J.
        • Craig R.W.
        J. Cell. Physiol. 1996; 166: 523-536
        • Altmeyer A.
        • Simmons R.C.
        • Krajewski S.
        • Reed J.C.
        • Bornkamm G.W.
        • Chen-Kiang S.
        Immunity. 1997; 7: 667-677
        • Reynolds J.E.
        • Yang T.
        • Qian L.
        • Jenkinson J.D.
        • Zhou P.
        • Eastman A.
        • Craig R.W.
        Cancer Res. 1994; 54: 6348-6352
        • Reynolds J.E.
        • Li J.
        • Craig R.W.
        • Eastman A.
        Exp. Cell Res. 1996; 225: 430-436
        • Zhou P.
        • Qian L.
        • Kozopas K.M.
        • Craig R.W.
        Blood. 1997; 89: 630-643
        • Rinkenberger J.L.
        • Horning S.
        • Klocke B.
        • Roth K.
        • Korsmeyer S.J.
        Genes Dev. 2000; 14: 21-27
        • Zhou P.
        • Qian L.
        • Bieszczad C.K.
        • Noelle R.
        • Binder M.
        • Levy N.B.
        • Craig R.W.
        Blood. 1998; 92: 3226-3239
        • Jiang Z.H.
        • Wu J.Y.
        Proc. Soc. Exp. Biol. Med. 1999; 220: 64-72
        • Grillot D.A.M.
        • Gonzalez-Garcia M.
        • Ekhterae D.
        • Duan L.
        • Inohara N.
        • Ohta S.
        • Seldin M.F.
        • Nunez G.
        J. Immunol. 1997; 158: 4750-4757
        • Boise L.H.
        • Gonzalez-Garcia M.
        • Postema C.E.
        • Ding L.
        • Lindsten T.
        • Turka L.
        • Mao X.
        • Nunez G.
        • Thompson C.B.
        Cell. 1993; 74: 597-608
        • Seto M.
        • Jaeger U.
        • Hockett R.D.
        • Graninger W.
        • Bennett S.
        • Goldman P.
        • Korsmeyer S.J.
        EMBO J. 1988; 7: 123-131
        • Fang W.
        • Rivard J.J.
        • Mueller D.L.
        • Behrens T.W.
        J. Immunol. 1994; 153: 4388-4398
        • Whyte M.K.
        • Renshaw S.A.
        • Lawson R.
        • Bingle C.D.
        Biochem. Soc. Trans. 1999; 27: 802-807
        • Haslett C.
        • Guthrie L.A.
        • Kopaniak M.A.
        • Johnston R.B.
        • Hennson P.M.
        Am. J. Pathol. 1985; 119: 101-110
        • Savill J.S.
        • Wyllie A.H.
        • Henson J.E.
        • Walpoort M.J.
        • Henson P.M.
        • Haslett C.
        J. Clin. Invest. 1989; 83: 865-875
        • Gibson L.
        • Holmgreen S.P.
        • Huang D.C.
        • Bernard O.
        • Copeland N.G.
        • Jenkins N.A.
        • Sutherland G.R.
        • Baker E.
        • Adams J.M.
        • Cory S.
        Oncogene. 1996; 13: 665-675
        • Hatakeyama S.
        • Hamasaki A.
        • Negishi I.
        • Loh D.Y.
        • Sendo F.
        • Nakayama K.
        • Nakayama K.
        Int. Immunol. 1998; 10: 631-637
        • Choi S.S.
        • Park S.H.
        • Kim U.J.
        • Shin H.S.
        Mamm. Genome. 1997; 8: 781-782
        • Herberg J.A.
        • Phillips S.
        • Beck S.
        • Jones T.
        • Sheer D.
        • Wu J.J.
        • Prochazka V.
        • Barr P.
        • Kiefer M.C.
        • Trowsdale J.
        Gene (Amst.). 1998; 211: 87-94
        • Hsu S.Y.
        • Hsueh A.J.W.
        J. Biol. Chem. 1998; 273: 30139-30146
        • Ulrich E.
        • Kauffmann-Zeh A.
        • Hueber A.O.
        • Williamson J.
        • Chittenden T.
        • Ma A.
        • Evan G.
        Genomics. 1997; 44: 195-200
        • Wang K.
        • Yin X.M.
        • Copeland N.G.
        • Gilbert D.J.
        • Jenkins N.A.
        • Keck C.L.
        • Aimonjic D.B.
        • Popescu N.C.
        • Korsmeyer S.J.
        Genomics. 1998; 53: 235-238
        • Liston D.R.
        • Johnson P.J.
        Mol. Cell. Biol. 1999; 19: 2380-2388
        • Wyllie A.H.
        Br. J. Cancer. 1993; 67: 205-208
        • Wyllie A.H.
        • Kerr J.F.R.
        • Currie A.R.
        Int. Rev. Cytol. 1980; 68: 251-305
        • Bieszczad C.K.
        • Craig R.W.
        Proc. Am. Assoc. Cancer Res. 1997; 38: 134
        • Taylor J.K.
        • Zhang Q.Q.
        • Wyatt J.R.
        • Dean N.M.
        Nature Biotechnol. 1999; 17: 1097-1100
        • Ban J.
        • Eckhart L.
        • Weninger W.
        • Mildner M.
        • Tschachler E.
        Biochem. Biophys. Res. Commun. 1998; 248: 147-152
        • Yang X.F.
        • Weber G.F.
        • Cantor H.
        Immunity. 1997; 7: 629-639
        • Shiraiwa N.
        • Inohara N.
        • Okada S.
        • Yuzaki M.
        • Shoji S.I.
        • Ohta S.
        J. Biol. Chem. 1996; 271: 13258-13265
        • Morishi K.
        • Huang D.C.
        • Cory S.
        • Adams J.M.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 17: 9683-9688
        • Zha J.
        • Harada H.
        • Yang E.
        • Jockel J.
        • Korsmeyer S.J.
        Cell. 1996; 87: 619-628
        • Puthalakath H.
        • Huang D.C.
        • O'Reilly L.A.
        • King S.M.
        • Strasser A.
        Mol. Cell. 1999; 3: 287-296
        • Li H.
        • Zhu H.
        • Xu C.J.
        • Yuan J.
        Cell. 1998; 94: 491-501
        • Evans D.L.
        • Mansel R.E.
        J. Mol. Evol. 1995; 41: 775-783