Regulation of Bcl-2 Proteins Independent of BH3 Figure 7. Co-immunoprecipitation analyses of Bcl-xAK with Bcl-2 family members. (A) SK-Mel-13 melanoma cells were transiently transfected with each 5 mg of pcDNA3 plasmids encoding Bcl-xL, Bcl-xAK, Bax or empty vector (Mock). Cells lysates were immunoprecipitated with microbeads covered with anti-Myc antibody, and immunoprecipitates were analysed by Western blotting. Non-bound supernatants (S) were compared with the immunoprecipitated pellet fractions (P). Antibodies for immunodetection: anti- Myc, Bcl-2, Bax and Bad. The complete experiment was performed two times, which both gave the same result. (B) A model for apoptosis induction by Bcl-xAK is suggested. It is based on mitochondrial translocation of Bcl-xAK and activation of Bax/Bak. Bcl-2/Bcl-xL prevent Bax/Bak activation but not Bcl-xAK translocation. BH3-only proteins may mediate a BH3 domain-dependent pathway via inactivation of antiapoptotic Bcl-2 proteins and may also drive a BH3-independent pathway analogous to Bcl-xAK (see discussion part). doi:10.1371/journal.pone.0034549.g007 Nbk and tBid go via Bax [9,18] and Bcl-xS goes via Bak [34]. For Bcl-xAK, however, Bak expression could compensate for loss of Bax and vice versa, and apoptosis induction was abolished only in Bax/Bak double deficient cells. This suggests that Bcl-xAK may may drive more general changes at the mitochondrial membrane rather than selectively targeting a specific protein. Importantly, after transduction all melanoma cells were responsive to Bcl-xAK, as the whole cell population showed reduced Dym, increased ROS as well as activated Bax and Bak. However, certain thresholds may prevent full apoptosis induction in the majority of cells. This may be related to the activity of antiapoptotic Bcl-2 family members, which may block Bax and Bak. Thus, overexpression of Bcl-2 abrogated apoptosis induced in melanoma cells by Bik/Nbk [35,36], and Bcl-xL inhibited Bax- induced apoptosis in mouse embryonic fibroblasts [37]. These antiapoptotic activities had been described as dependent on BH3- mediated heterodimerization. However, also the proapoptotic effects of Bcl-xAK were completely inhibited by Bcl-2 or Bcl-xL. This may depend on the inhibition of Bax and Bak by the antiapoptotic proteins, rather than on direct inhibition of Bcl-xAK. In agreement, Bcl-2 could not prevent Bcl-xAK mitochondrial translocation. Highly characteristic for Bcl-xAK-induced apoptosis was a time delay of 48 h, whereas other Bcl-2 proteins as Bik/Nbk and Bcl-xS induced apoptosis in melanoma cells already at 24 h [35,36]. In general, proapoptotic signaling as mutual regulation of Bcl-2 proteins, cytochrome c release and caspase activation are rather quick cellular events [38]. The time delay of Bcl-xAK in contrast to other proapoptotic Bcl-2 proteins is indicative for an indirect mechanism enclosing a time-consuming step. No relation was seen to the expression of other Bcl-2 proteins. Rather, Bcl-xAK mitochondrial localization appeared as a critical step, and membrane transport may play a regulatory role therein. Whereas Bcl-xAK was cytosolic at 24 h, it translocated to mitochondria at 48 h, when apoptosis was induced. Also other proapoptotic Bcl-2 proteins have to translocate to mitochondria to exert their proapoptotic activities, as shown for tBid and Bax [5,39]. Thus, apoptosis by Bcl-xAK appeared as tightly linked to its presence in mitochondria, where it resulted in Bax and Bak activation. An interesting finding was that loss of Dym preceded translocation of Bcl-xAK and MOMP. The relation between Dym and MOMP is still a matter of discussion; one effect may precede the other or they may even occur independently of each other [40,41]. Loss of Dym may result from uncoupling of the mitochondrial electron transport chain which may lead to Bax and Bak oligomerization [42]. Mitochondrial dynamics appears as another important level, which may be influenced by Bcl-xAK overexpression. Mitochondrial dynamics may contribute to the control of MOMP, which is further dependent on Bax [43]. Formation of large Bax/Bak clusters has been suggested, which may translocate to mitochondrial constriction sites, to drive MOMP [23]. Clustering of Bax and Bak was clearly induced in response to Bcl-xAK, thus further relations to mitochondrial fission and fusion may be expected. For BH3-only proteins, different mechanisms have been suggested to explain their proapoptotic activities. In the neutral- ization/displacement model, BH3-only proteins bind antiapopto- tic family members, to release Bax or Bak [5]. This activity is based on BH3, which binds to the hydrophobic groove of antiapoptotic Bcl-2 proteins [44]. According to a second model, BH3-only proteins may also directly bind and activate Bax or Bak, which has been shown for tBid, Bim and Puma [38,45,46]. This activity is also regarded as BH3-dependent. Thus, direct, although week binding of Bim to Bax has been shown, which was abrogated by the replacement of the Bim BH3 [30]. Also peptides of the BH3 domains of Bid, Bim and Puma were able to drive direct activation of Bax [45]. Both ways of apoptosis induction can not apply to Bcl- xAK, due to its lack of BH3. A third way of apoptosis induction has been recently suggested. It is explained by a general remodelling of the mitochondrial outer PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e34549