Regulation of Bcl-2 Proteins Independent of BH3 Figure 6. Bcl-2 blocks Bcl-xAK-mediated cytochrome c release and Bax translocation. Mel-2a, A375-Mock and A375-Bcl-2 cells were transduced with AdV-AK (MOI = 50) and were kept under OFF and ON conditions. At 24 h and 48 h, cytosolic fractions (Cyto) and mitochondrial fractions (Mito) were isolated and analysed by Western blotting. Non-transfected controls (2) are shown as controls. The whole experiment was performed two times, resulting in highly comparable results. (A) Cytosolic extracts were analyzed for showing expression of Bcl-xAK and release of cytochrome c. Mitochondrial extracts serve as positive controls, the mitochondrial protein VDAC ruled out any contaminations of cytosolic extracts with mitochondria, and b-actin served as loading control. (B) Mitochondrial extracts were analyzed for showing mitochondrial translocation of Bcl-2 proteins. Here, cytosolic extracts served as controls, equal protein loading was confirmed by VDAC and the relative purity of mitochondrial extracts was examined by GAPDH. 5% of the total mitochondrial fractions and 2% of the total cytosolic fractions had been loaded on the gels. doi:10.1371/journal.pone.0034549.g006 pathways [5]. Their already high number is further increased by differential splicing, leading to an enhanced complexity. Thus, up to 10 splice products have been reported for the bim gene, of which BimS, BimL and BimEL have been characterized. Also eight splice products with different domain structures have been reported for the bax gene, of which Bax-a is best characterized [24,25]. Another example is given by the bcl-x gene, which is expressed in four reported isoforms with different activities. Besides Bcl-xL (long), antiapoptotic functions have also been reported for Bcl-xES (extra short) [26,27]. In contrast, Bcl-xS (short) and Bcl-xAK (atypical killer) exert proapoptotic functions [14,15]. Alternative splicing is a target of specific regulations. Thus, the switch from Bcl-xL to Bcl-xS in response to genotoxic stress was related to an ATM/CHK2/p53-dependent pathway [28]. The pathway, which triggers Bcl-xAK expression, is not yet defined. Bcl-2 proteins are categorized in three subfamilies according to different domain structures, enclosing antiapoptotic proteins (BH 1–4), the Bax/Bak group (BH 1–3) and BH3-only proteins [9]. The bcl-x splice products, however, reveal unique structures. Thus, Bcl-xS encloses BH3 and BH4 [24], whereas Bcl-xAK encloses BH2 and BH4 [15]. Despite the BH3 domain has been regarded as indispensible for proapoptotic functions [12], we had previously categorized Bcl-xAK as proapoptotic based on a moderate induction of apoptosis in melanoma cells (two-fold), after plasmid transfection [15]. For unraveling Bcl-xAK-mediated pathways, we have constructed an adenoviral vector, which drives its high and conditional expression under Tet-OFF control. With this efficient expression system, Bcl-xAK induced apoptosis in up to 40% of melanoma and in 50% of non-melanoma cells. In its efficacy, Bcl- xAK was comparable to the BH3-only protein Bik/Nbk, which was available in the same adenoviral backbone [19]. Under AdV-AK-mediated high expression of Bcl-xAK, signifi- cant caspase activation became evident, in contrast to previous findings under moderate expression of Bcl-xAK [15]. Thus, caspase activation by Bcl-xAK in melanoma cells appeared as dependent on its expression level. Initiator caspases of both extrinsic and intrinsic pathways (caspase-8, and 29) were cleaved. However, caspase-8 may also be activated downstream of caspase-3 in a described amplification loop [29], which is suggestive for Bcl-xAK. Bcl-2 family proteins are particularly involved in the control of mitochondrial apoptosis pathways, which can be induced by overexpression of BH3-only proteins as well as by overexpression of Bax or Bak [18,30,31]. Also, Bcl-xAK resulted in significant decrease of mitochondrial membrane potential and in cytochrome c release, thus clearly indicating parallels to other proapoptotic Bcl-2 proteins. Although Bax/Bak-independent mechanisms were also discussed [32], mitochondrial activation is mainly related to Bax or Bak function [9]. Here again, Bcl-xAK revealed typical characteristics of proapoptotic Bcl-2 proteins, namely a strong dependency on either Bax or Bak. Both proteins share a similar structure and related functions [33]. Some proapoptotic Bcl-2 proteins show preference for activating either Bax or Bak, as Bik/ PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e34549