Activated BH3-only proteins were thought to bind indiscriminately to all their pro-survival counterparts until quantitative studies revealed marked differences [10**
]. Bim, Puma and tBid (the activated, truncated form of Bid) do bind avidly to all the pro-survival proteins, but the others associate only with subsets [10**
]. For example, Noxa engaged only Mcl-1 and A1, and Bad only Bcl-2, Bcl-xL
and Bcl-w. Importantly, the promiscuous binders killed much more potently than the selective binders, but the combination of the complementary binders Noxa and Bad readily induced cell death [10**
]. These findings indicate that efficient apoptosis requires neutralization of multiple pro-survival proteins.
The BH3-only proteins clearly act upstream of Bax and Bak, because they cannot induce apoptosis in cells lacking both Bax and Bak [6
]. Their role in the activation of Bax and Bak, however, is highly controversial. In the direct activation
model (), a sub-group of BH3-only proteins, termed activators
, are proposed to bind directly to Bax and Bak to promote their activation [11
]. The putative activators include Bim and tBid [11
], and perhaps also Puma [16*
], although that has been disputed [12*
]. In this model, the remaining BH3-only proteins, termed sensitisers
, function by binding to the pro-survival proteins and freeing any bound Bim or tBid to directly activate Bax and Bak.
Figure 1 Two models for how BH3-only proteins activate Bax and Bak. (A) In the direct activation model , the indicated activator BH3-only proteins, via their BH3 domain (red triangle), directly engage Bax and Bak and activate them, whereas sensitizer BH3-only (more ...)
The indirect activation
model () proposes instead that all BH3-only proteins function solely by binding to their pro-survival relatives, thereby preventing those guardians of cell survival from inhibiting Bax and Bak [10**
]. In this model, Bim, tBid and Puma are the most potent BH3-only proteins simply because they can engage all the pro-survival proteins [10**
Several recent findings strongly challenge the direct activation
]. Firstly, no Bak co-immunoprecipitated with any BH3-only protein. Secondly, no Bax bound detectably to the physiologically relevant forms of Bim (BimEL
), and none co-immunoprecipitated with Bim in dying cells. Thirdly, although tBid and a minor Bim isoform (BimS
) bound Bax weakly, tBid and BimS
bearing BH3 mutations that prevented binding to Bax but retained normal binding to pro-survival proteins remained just as potent as killers. Most tellingly, in response to several apoptotic stimuli, or enforced co-expression of Noxa and Bad, cells lacking both Bid and Bim died as readily as wild-type cells, and even down-regulation of Puma by RNAi in these cells did not impair apoptosis driven by several stimuli [20**
]. Thus, none of the putative activator BH3-only proteins appears to be essential for apoptosis.
Mouse genetic studies also favor the indirect activation
model. Mice lacking both Bim and Bid [20**
], or both Bim and Puma [22
], appear normal, and their cells remain normally sensitive to certain apoptotic signals. On the direct activation
model, the absence of these activators
should have blocked apoptosis and promoted severe developmental abnormalities, as in mice lacking both Bax and Bak [23
Some viral Bcl-2 homologs, which may have been selected for resistance to inhibition by BH3-only regulators, also act by binding to and restraining Bax and Bak [24
], further supporting the indirect activation
model. Remarkably, M11L of myxoma virus, an anti-apoptotic protein lacking sequence homology to Bcl-2, has an extremely similar 3D fold [25*
], and structure-based mutagenesis suggested that its critical binding targets were Bax and Bak rather than BH3-only proteins.
Collectively, these findings demonstrate that apoptosis does not rely upon direct activation of Bax or Bak by any known BH3-only protein (). Nevertheless, direct activation might occur in some circumstances. Protein-free liposomes can be lysed by cooperation of Bax with tBid [26
] or with BH3 peptides from Bim or Bid at high (eg >10 micromolar) concentrations [12*
], or at much lower levels if these peptides are chemically ‘stapled’ into an α helix and/or artificially targeted to membranes [14*
]. Perhaps direct binding by Bim or tBid contributes to an amplification step in apoptosis.