- Short communication
- Open Access
BAK multimerization for apoptosis, but not bid binding, is inhibited by negatively charged residue in the BAK hydrophobic groove
© Azad and Storey; licensee BioMed Central Ltd. 2013
- Received: 24 January 2013
- Accepted: 5 June 2013
- Published: 19 June 2013
BCL-2 family proteins BAK and BAX orchestrate outer mitochondrial membrane permeabilization (MOMP) during apoptosis by forming pores in the membrane to release apoptogenic factors that commits a cell to death. BAK and BAX therefore function as a ‘point of no return’ in the apoptotic cascade. BAK activation is a multi-step process involving conformational changes, mediated by BH3-only proteins or p53, which lead eventually to oligomerization and pore formation. Further, recent reports show that BAK activation is also linked to and dependent upon dephosphorylation of both tyrosine and serine residues.
We hypothesized that phosphorylation of BAK at tyrosine residue 110 (Y110) was functionally important during the BAK activation process. BAK/BAX double knockout HCT116 cells expressing a phosphor-mimetic BAK mutant (BAK Y110E), showed impaired dimerization and multimerization capacity when treated with either UV irradiation or etoposide when compared to cells reconstituted to express wild-type BAK. The Y110E mutant also showed decreased release of cytochrome c from isolated mitochondria challenged with tBid protein, resulting in a failure to activate caspase 3. Interestingly, co-immunoprecipitation experiments suggest that a negative charge at this residue may be important for the recruitment of Bid to BAK, but conversely that this also impairs BAK:BAK interactions.
These findings implicate dephosphorylation of Y110 as having an important mechanistic role in BAK activation, and underscores how post-translational modifications are intimately linked and coupled to the protein-protein interactions required for BAK activation during apoptosis.
- Cytochrome c
- Tyrosine Phosphorylation
- DNA damage
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 . 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 . 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 . 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 . Resultant homodimers then can form higher order structures via interaction between α6:α6 helices . 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)  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 .
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 . 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 .
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 , 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.
This work was supported by funding from Cancer Research UK. We also thank R. Youle for the gift of the HCT116bax−/− bak−/− double knockout cells.
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