Date Published: November 26, 2012
Publisher: Public Library of Science
Author(s): Abul Azad, Joanna Fox, Sabrina Leverrier, Alan Storey, Boris Zhivotovsky. http://doi.org/10.1371/journal.pone.0049601
The BCL-2 family protein BAK is a key regulator of mitochondrial apoptosis. BAK activation first involves N-terminal conformational changes that lead to the transient exposure of the BAK BH3 domain that then inserts into a hydrophobic groove on another BAK molecule to form symmetric dimers. We showed recently that post-translational modifications are important in the regulation of BAK conformational change and multimerization, with dephosphorylation at tyrosine 108 constituting an initial step in the BAK activation process. We now show that dephosphorylation of serine 117 (S117), located in the BAK hydrophobic groove, is also critical for BAK activation to proceed to completion. Phosphorylation of BAK at S117 has two important regulatory functions: first, it occludes the binding of BH3-containing peptides that bind to BAK causing activation and cytochrome c release from mitochondria; second, it prevents BAK-BH3:BAK-Groove interactions that nucleate dimer formation for subsequent multimerization. Hence, BH3-mediated BAK conformational change and subsequent BAK multimerization for cytochrome c release and cell death is intimately linked to, and dependent on, dephosphorylation at S117. Our study reveals important novel mechanistic and structural insights into the temporal sequence of events governing the process of BAK activation in commitment to cell death and how they are regulated.
Members of the BCL-2 family of proteins are the major regulators of the mitochondrial (or intrinsic) apoptotic pathway whose activity is exerted through a network of intermolecular interactions with other family members. BCL-2 proteins can have either anti- or pro-apoptotic functions, and are intimately involved in the permeabilization of the mitochondrial outer membrane (MOM) that permits the release of apoptotic factors such as cytochrome c in response to developmental cues or cytotoxic insults, including DNA damage , . A pivotal step in MOM permeabilization is the oligomerization of the pro-apoptotic proteins BAK or BAX, whose activation involves a number of conformational changes including the exposure of epitopes near the N-terminus  followed by homo-oligomerization to form pores in the outer mitochondrial membrane , , . Binding of BH3-only BCL-2-family proteins result in N-terminal conformational changes in the early stages of BAK/BAX activation. This was thought to occur via one of two mechanisms that may involve either an indirect activation – where BH3-only proteins bind to and neutralize anti-apoptotic BCL-2 family members that constitutively bind to BAK/BAX in healthy cells, or by the direct binding of ‘activator’ BH3-only proteins such as BID to BAK/BAX, reviewed in . Different modes of action of BCL-2-like proteins has been proposed in order to explain differences between the sequestration and direct activation models . In addition, p53 may act in an analogous way to activator BH3-only proteins by binding directly to either BAK or BAX , , but this occurs at a site on BAK distinct from that involved in binding of BH3-only proteins .
The present work has uncovered an important new step in our understanding of how signalling pathways are intimately linked to conformational changes in BAK that result in MOMP and cell death. Activation of BAX and BAK during the induction of apoptosis has been intensively studied, with details of the mechanisms underlying these processes emerging over the past few years that suggest some similarities, yet also clear fundamental differences between how the two proteins respond to apoptotic signals. It is apparent however that both proteins act at a nodal point during apoptotic commitment, in that their oligomerization generates pores in the mitochondrial membrane releasing apoptotic factors representing a ‘point-of-no-return’ in the apoptotic cascade. Together with our previous results our new data provide important insights into a novel and potentially physiologically significant post-translational mechanism regulating BAK activation.