The abrogation of apoptotic responses is a common feature of almost all cancer cells. Whether or not a cell undergoes apoptosis in response to stress depends on responses following the receipt of signals generated by cellular damage. The mitochondrial apoptotic pathway is regulated by BCL-2 proteins that can be divided into different pro- and anti-apoptotic groups depending on their structure and function [1]. Pro-apoptotic BH3-only proteins, such as Bid, Bim and Noxa, are required for the activation of multi-domain BCL-2 effector proteins BAK and BAX. BAK and BAX constitute a pre-requisite gateway for mitochondrial apoptosis to proceed, indeed cells lacking both BAK and BAX fail to undergo mitochondrial apoptosis [2]. The BAK responds to a myriad of death signals and plays a key role in executing mitochondrial apoptosis, effecting MOMP through oligomerization of the protein that releases apoptogenic factors including cytochrome c, that in turn lead to downstream activation of caspase 3. BAK activation is complex, involving a number of intra-molecular conformational changes leading to dimerization followed by oligomerization [2–4]. An early event during BAK activation is a conformational change towards to N-terminus of the protein [5]. This is followed by exposure of the BAK BH3 domain that then inserts into a hydrophobic groove on another BAK molecule leading to dimer formation [6]. Resultant homodimers then can form higher order structures via interaction between α6:α6 helices [7]. BAK N-terminal conformational change can be brought about by one of two mechanisms: first, by the transient binding of BH3-only proteins (such as tBid) [8] to the BAK hydrophobic groove [2, 9], or alternatively by the binding of p53 to residues near the BAK N-terminus [10–12]. Binding of BH3 proteins such as tBid to the BAK hydrophobic groove occurs with high affinity, but is necessarily transient as this same region of BAK is also required to nucleate BAK multimerization. Recently a model of BAK activation that tries to take into account the differing affinities of BH3 proteins for both pro- and anti-apoptotic BCL-2 proteins has been proposed [13].

In healthy, otherwise undamaged cells, BAK is present almost exclusively in a very heavily phosphorylated form. We recently demonstrated that BAK activation for apoptosis induction is closely linked to, and indeed dependent upon, specific dephosphorylation events on the protein [14]. The initial event in the BAK activation process is dephosphorylation at tyrosine 108 (Y108), an obligatory step that is required to permit conformational change by BH3 or p53 proteins [14, 15]. Further, we found that a subsequent PP2A-mediated dephosphorylation of BAK at serine 117 (S117) was required both for BH3 proteins to gain access to the BAK hydrophobic groove, and permit BAK dimerization via BAK-BH3:BAK-groove interactions [16].

During our investigations into the role of phosphorylation in regulating BAK activation, we reported in mass spectrometry analysis that BAK was also phosphorylated at residue Y110 [14]. Mutation of this residue to mimic either the dephosphorylated or phosphorylated forms of the protein (BAK mutants Y110F and Y110E respectively) did not affect the ability of BAK to undergo N-terminal conformational change [14]. However, modelling using PyMol based on BAK structure 2IMS, suggested that phosphorylation at Y110 might impinge upon the ability of BH3 proteins to bind BAK and may also inhibit BAK multimerization, as the Y110 side chain, like that of S117, may occlude access to the hydrophobic groove (Figure 1A). To test this idea, we performed multimerization assays using sub-cellular fractions enriched in mitochondria from HCT116^{bak−/−bax−/−} cells that had been reconstituted to express either wild-type (WT) or mutant BAK proteins (HCT-BAK cells). Cross-linking reactions were performed with the sulfhydryl-to-sulfhydryl crosslinker BMH (1, 6-bismaleimidohexane, Pierce) on sub-cellular fractions enriched in mitochondria, then analysed by western blotting to detected BAK multimers, as previously described [14, 16]. Following DNA damage by UV, BAK dimers, trimers and higher-order complexes were observed with WT BAK and the Y110F mutant, but multimer formation was impaired severely by the Y110E mutation (Figure 1B, upper panel). As we have noted previously, BAK cross-linking with BMH can be problematic with the formation of a faster migrating intra-molecularly cross-linked monomeric BAK protein (M_x) and generation of non-specific dimer bands without any DNA damage. A further cross-linking experiment a different sulfhydryl crosslinker, BMOE (Bismaleimidoethane), that generates only BAK dimers and at the same time minimizes the detection of both intra-molecularly cross-linked BAK monomers and non-specific dimer forms [16], supported the previous experiments with dimers being detected only when WT or the Y110F mutant was used (Figure 1B, lower panel). When the experiment was repeated using etoposide as the apoptotic inducer in place of UV, again dimers were readily detected in cells expressing the WT and Y110F proteins, but were not generated by the Y110E mutant (Figure 1C). The stronger dimer band present generated by the Y110F mutant compared to WT we suppose may be due to the mutant forming dimers more readily if the WT protein was not fully dephosphorylated following UV damage.

The failure of the BAK Y110E mutant to dimerize or multimerize we reasoned may be due to interference with the binding of BH3 proteins, as observed previously for the S117E phosphor-mimic [16]. We therefore tested the ability of the BH3-activator protein tBid to release cytochrome c from mitochondrial preparations using a previously established method [16, 17]. When purified tBid protein was incubated with mitochondrial preparations from cells expressing either the WT or Y110F BAK proteins, cytochrome c was readily released into the supernatant (Figure 2A). Consistent with the multimerization assays, tBid was unable to release cytochrome c from mitochondrial preparations from the BAK-Y110E mutant cells compared to the HCT116-BAK or HCT116-BAKY110F cells (Figure 2A). The small quantity of cytochrome c that was detected in the supernatant fraction derived from the Y110E mutant might be due to the mutant being very inefficient at releasing cytochrome c since the substituted amino acid may not correctly mimic a phosphor-tyrosine residue, however we have noted that processing of the samples leads to a degree of leakiness of the mitochondrial preparations where mitochondria expressing BAK mutants we find to be more fragile compared to cells expressing WT BAK, as previously noted [16]. Likewise, the Y110F mutant showed amounts of cytochrome c retained in the pellet fraction decreased somewhat when mitochondrial preparations were treated with increasing amounts of tBid, but by comparison cytochrome c was readily detected in the supernatant fraction even in the absence of tBid, and did not increase to the same extent as observed for the WT BAK protein. To investigate further whether the cytochrome c release was likely to be functionally significant and bypass potential problems of mitochondrial fragility, we performed caspase 3 activation assays using a FITC-conjugated antibody that recognizes only activated caspase 3 using FACS on intact cells. This revealed that UV irradiation resulted in an increase in caspase 3 activation in BAK WT or Y110F cells, but in marked contrast no caspase 3 activation was detected in UV-irradiated Y110E cells, findings in-line with and supporting the cytochrome c release experiment (Figure 2B). We conclude that the Y110E mutation interferes with the ability of BAK to form dimers and multimers, and that this results in the failure to release cytochrome c and activate caspase 3.

In previous studies, we found that neither the BAK Y110F nor Y110E mutations had any effect on BAK being able to undergo the N-terminal conformational change associated with an early step in BAK activation, indicating that the mutations did not perturb the overall BAK structure [14]. Since either p53 or BH3 proteins bring about BAK conformational change by different routes [16], we next asked whether the BAK mutants recruited p53 to the mitochondria as efficiently as the WT protein. The p53 binding site on BAK is located near the N-terminus of the protein involving residues E24/E25 [12, 16], and p53 recruitment to the mitochondria is dependent on BAK expression [10, 18, 19]. Following DNA damage by UV irradiation, we found that p53 was readily recruited to the mitochondria as was detected by western blot in mitochondrial extracts of cells expressing the WT or either of the two mutant BAK Y110F or Y110E (Figure 3A). As the Bid BH3 sequence interacts with the BAK hydrophobic groove, we next examined whether Bid was able to Co-IP BAK following UV irradiation. As expected, Bid was able to pull down BAK protein following UV irradiation from whole cell extracts of cells expressing either the WT or Y110F mutant BAK proteins (Figure 3B). Somewhat surprisingly, Bid was able to pull down more BAK from the Y110E cells when compared to cells expressing either WT or the Y110F mutant (Figure 3B). To explore further the role of a negative charge at Y110 on the recruitment of Bid to BAK, we performed similar IP/western blot experiments using mitochondrial enriched preparations. We found that similar levels of Bid were able to be immunoprecipitated in the three BAK cell lines expressing either WT, Y110F or Y110E mutants, irrespective of whether the cells has been treated with UV or not (Figure 3C). As before, WT or Y110F BAK proteins were only co-precipitated with Bid following UV damage, and that a greater amount of the Y110E BAK mutant we again found was readily pulled down by Co-IP with Bid following UV treatment (Figure 3C). However, in these mitochondrial enriched subcellular fractions we were also now able to detect BAK that had been pulled down by the Bid antibody even in the absence of UV treatment. Together these findings provides the first evidence that a negative charge located in the BAK hydrophobic surface groove may be a factor important in the recruitment of BH3 proteins such as Bid to BAK.

Blockade of the BAK hydrophobic groove by phosphorylation of S117, or an S117E mutant, each impair binding of either BH3 peptides or proteins to BAK. Located further along the same helix as S117 but also facing the hydrophobic BAK surface pocket, we now identify a role for Y110 in the BAK activation process. Taken together, our results identify for the first time a novel and important bi-functional role for post-translational modifications at Y110. Binding of BH3 proteins to BAK has been reported to occur with high affinity yet the interaction must be transient [20], as the same hydrophobic surface interface is required to nucleate BAK dimerization. We recently reported that phosphorylation of BAK at S117 blocks access of BH3 sequences of Bid and BAK to the hydrophobic pocket on BAK. In contrast, these findings suggest that while the Y110E mutant interferes with the ability to form stable BAK-BH3:BAK-groove interactions for dimerization, a negative charge at this site may also be a positive factor that is involved in the recruitment of BH3-containing proteins, such as Bid, to BAK. This implies that dephosphorylation of S117 must occur prior to dephosphorylation of Y110 in order to permit Bid recruitment, however the exact timing of these events and whether binding of other BH3-containing BCL-2 family proteins is also affected by this modification remains to be established.

Findings presented here further underscore the importance of post-translational modifications in regulating different steps in BAK activation, and may reveal new avenues to either potentiate or inhibit BAK activity through modulation of the phosphorylation status of the protein.