Mutual Regulation of Bcl-2 Proteins Independent of the BH3 Domain as Shown by the BH3-Lacking Protein Bcl-xAK Michael Plo¨ tz1, Amir M. Hossini1, Bernhard Gillissen2, Peter T. Daniel2, Eggert Stockfleth1, Ju¨ rgen Eberle1* 1 Department of Dermatology and Allergy, Skin Cancer Center, University Medical Center Charite´, Berlin, Germany, 2 Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charite´, Berlin, Germany Abstract The BH3 domain of Bcl-2 proteins was regarded as indispensable for apoptosis induction and for mutual regulation of family members. We recently described Bcl-xAK, a proapoptotic splice product of the bcl-x gene, which lacks BH3 but encloses BH2, BH4 and a transmembrane domain. It remained however unclear, how Bcl-xAK may trigger apoptosis. For efficient overexpression, Bcl-xAK was subcloned in an adenoviral vector under Tet-OFF control. The construct resulted in significant apoptosis induction in melanoma and nonmelanoma cell lines with up to 50% apoptotic cells as well as decreased cell proliferation and survival. Disruption of mitochondrial membrane potential, and cytochrome c release clearly indicated activation of the mitochondrial apoptosis pathways. Both Bax and Bak were activated as shown by clustering and conformation analysis. Mitochondrial translocation of Bcl-xAK appeared as an essential and initial step. Bcl-xAK was critically dependent on either Bax or Bak, and apoptosis was abrogated in Bax/Bak double knockout conditions as well by overexpression of Bcl-2 or Bcl-xL. A direct interaction with Bcl-2, Bax, Bad, Noxa or Puma was however not seen by immunoprecipitation. Thus besides BH3-mediated interactions, there exists an additional way for mutual regulation of Bcl-2 proteins, which is independent of the BH3. This pathway appears to play a supplementary role also for other proapoptotic family members, and its unraveling may help to overcome therapy resistance in cancer. Citation: Plo¨ tz M, Hossini AM, Gillissen B, Daniel PT, Stockfleth E, et al. (2012) Mutual Regulation of Bcl-2 Proteins Independent of the BH3 Domain as Shown by the BH3-Lacking Protein Bcl-xAK. PLoS ONE 7(4): e34549. doi:10.1371/journal.pone.0034549 Editor: Dhyan Chandra, Roswell Park Cancer Institute, United States of America Received November 3, 2011; Accepted March 2, 2012; Published April 10, 2012 Copyright: 2012 Plo¨ tz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The study was supported by the Sonnenfeld-Stiftung, Berlin. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: juergen.eberle@charite.de Introduction Apoptosis is a defined genetic death program that leads to ordered destruction of cellular components while membrane integrity is preserved [1]. It also represents a safeguard mechanism against tumor formation, due to the elimination of altered and mutated cells. Thus, apoptosis resistance is characteristic for tumor cells, and therapeutic strategies aim to overcome this resistance [2]. Two major apoptosis pathways (extrinsic and intrinsic) have been described in detail. Extrinsic pathways are initiated by binding of death ligands (TNF-a, CD95L and TRAIL) to cell surface receptors, leading to the formation of death-inducing signaling complexes, where initiator caspases 8 and 10 are activated [3,4]. On the other hand, intrinsic/mitochondrial apoptosis pathways are triggered by intracellular signals such as by cellular or DNA damage. Key events are depolarization of the mitochondrial membrane potential (Dym) and mitochondrial outer membrane permeabilisation (MOMP) resulting in cyto- chrome c release and subsequent activation of initiator caspase 9 [5]. Initiator caspases cleave and activate downstream effector caspases, which target a large number of death substrates to set apoptosis into work [6,7]. Mitochondrial activation is critically controlled by the family of pro- and antiapoptotic Bcl-2 proteins [8]. These proteins share homology in four conserved regions termed Bcl-2 homology domains (BH) and in a transmembrane domain (TM). Anti- apoptotic proteins as Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and Bfl-1/A1 enclose all four BH domains whereas proapoptotic Bcl-2 homologues subdivide in the Bax/Bak group characterized by BH 1–3, and the BH3-only group enclosing several proteins i.e. Bad, Bid, Bik/Nbk, Bim, Noxa and Puma. In present models, Bax and Bak drive MOMP and are neutralized by antiapoptotic family members. The BH3-only proteins contribute to the regulation either as sensitizers through inhibition of antiapoptotic Bcl-2 proteins or as direct activators of Bax and Bak [8,9]. Mutual regulation and neutralization has been described as based on the formation of heterodimers between Bcl-2 family members. Thus, the BH3 domain of proapoptotic Bcl-2 proteins encloses an amphipathic a helix, which binds to a hydrophobic groove formed by BH1, BH2 and BH3 of antiapoptotic members [10]. In a rheostat model, the balance of pro- and antiapoptotic Bcl-2 proteins determines the fate of a cell [11]. In melanoma, apoptosis deficiency has been attributed to high expression of antiapoptotic Bcl-2 proteins [12,13]. PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e34549