Proteins of the BCL2 family govern whether a cell lives or commits to apoptosis through the intrinsic apoptotic pathway. The bcl2 proto-oncogene is the founding member of the BCL2 family and was first identified at the t(14;18) chromosome translocation breakpoint in human B-cell follicular lymphoma, where its transcription was excessively driven by the immunoglobulin heavy chain gene promoter and enhancer on chromosome 14. Previously characterized oncogenes shared the ability to increase cellular proliferation, yet the over-expression of BCL2 did not promote cell proliferation but blocked cell death instead. This discovery thus introduced a new paradigm for carcinogenesis: inhibitors of apoptosis.

The cloning of bcl2 led to the identification of a family of at least 20 proteins related by amino acid sequence homology within conserved regions known as the BCL2 homology (BH) domains. The four BH domains (BH1-BH4) correspond to α-helical segments that functionally categorize the BCL2 family members into anti- and pro-apoptotic proteins and structurally define them as multi-domain or BH3-only proteins.

Anti-Apoptotic Proteins

The anti-apoptotic proteins BCL2, BCL-XL, and MCL1 contain all four BH domains. The three-dimensional structure of BCL-XL demonstrates that the protein is comprised of two central hydrophobic α-helices (α5 and α6) surrounded by five amphipathic helices (α1, α2, α3, α4 and α7) and arranged such that the BH1, BH2, and BH3 regions form a hydrophobic pocket to which a BH3 domain can bind. It should be noted that the NMR structure of BCL-XL complexed with a peptide derived from the BH3 domain of the pro-apoptotic protein BAK was obtained in solution. Considering that anti-apoptotic proteins have a hydrophobic C-terminal transmembrane domain that targets them to intracellular membranes, binding conformation may vary between a solution structure and a membrane-bound structure.

The BH4 domain of BCL-XL forms an amphipathic helix on the opposite face of the molecule and may further stabilize the hydrophobic pocket. Other anti-apoptotic members that share similar homology domains are predicted to have a similar tertiary structure. There is an unstructured, flexible loop, approximately 60 residues in length, between the BH3 and BH4 domains. This region is a site of negative regulation but is not essential for the antiapoptotic function of at least BCL-XL. Although the mechanism by which such regulation occurs is still unclear, phosphorylation of sites within the loop by serine/threonine kinases might be one way to modulate anti-apoptotic function. Alternatively, anti-apoptotic proteins may be converted to death promoters as a consequence of the loss of the BH4 domain by cleavage of putative caspase sites within the loop region. Loss of the BH4 domain in BCL2, for example, diminishes or abrogates its anti-apoptotic function and can impart death-promoting properties.

Anti-apoptotic proteins BCL2 and BCL-XL are similar in structure and size (~25 KDa), with a long protein half-life (more than 20 hours). MCL1 is a larger protein (37 KDa), with a short half-life (~30 min) that is regulated via the proteasome-dependent pathway. MCL1 is poly-ubiquitinated by the MCL1 ubiquitin ligase E3 (MULE) and subsequently targeted for proteasomal degradation. Interestingly, MULE contains a BH3 domain that allows it to selectively target MCL1 but not other anti-apoptotic proteins.

Phosphorylation of MCL1 may regulate its proteasome-dependent degradation. For example, GSK-3-mediated phosphorylation of MCL1 induces the ubiquitination and degradation of MCL1 and may impact recognition of MCL1 by MULE. Phosphorylation of MCL1 by ERK and JNK are thought to occur upstream of GSK-3 and act as an additional regulatory level of MCL1 stabilization. ERK phosphorylates MCL1 at threonine- 163 to slow the turnover rate of MCL1, thereby prolonging its protective effects. Phosphorylation of MCL1 at serine-121 by JNK, on the other hand, inactivates MCL1. These data suggest phosphorylation of MCL1 plays an important role in its half-life and its antiapoptotic activity.

BAX/BAK Pro-Apoptotic Proteins

BAX and BAK are the two multi-domain pro-apoptotic proteins responsible for permeabilizing the mitochondrial outer membrane. They are largely redundant in function, since the loss of either BAX or BAK has little effect in most cells and tissues. However, the absence of both proteins blocks apoptosis in many cell types, implicating the importance of BAX and BAK in the intrinsic apoptotic pathway.

In viable cells, BAX and BAK exist as monomers. Inactive BAX resides in the cytosol or is loosely attached to the membrane with its C-terminal tail folded back into the BAX hydrophobic groove formed by the BH1, BH2, and BH3 domains. Upon an apoptotic stimulation, BAX undergoes a conformational change that releases the C-terminal tail, allowing BAX to insert into the mitochondria outer membrane. Unlike BAX, BAK monomers are mitochondria-resident proteins. BAK also undergoes an allosteric conformational activation in response to death signals.

Once activated, both BAX and BAK homo-oligomerize to form pores in the mitochondrial outer membrane. Cytochrome c is released into the cytosol through these pores, ultimately leading to the activation of the caspase cascade.

BH3-Only Proteins

BH3-only proteins are pro-apoptotic and function as initial sensors of apoptotic signals that arise from various cellular processes. Their BH3 domain consists of 9-16 amino acids and is critical for their pro-apoptotic functions. BH3-only proteins interact with the hydrophobic groove formed by their anti-apoptotic partners to promote apoptosis.

The pro-apoptotic activity of BH3-only proteins is stringently regulated via three main mechanisms: transcriptional up-regulation, post-translational modification, and subcellular localization. Growth factor deprivation induces HRK and BIM mRNA expression in neurons via a JNK-dependent mechanism. The induction of bim and noxa is regulated by the class O forkhead box transcription factor 3A (FOXO3A). The noxa gene was first discovered as a phorbol-12-myristate-13-acetate (PMA)-inducible gene. It was not until both NOXA and PUMA were found to be up-regulated by the p53 pathway upon DNA damage that these two proteins were considered for their roles in apoptosis. NOXA and PUMA are generally expressed at low levels and need to be induced upon cellular stress. Due to their low level of expression, many studies involving NOXA and PUMA have been done in systems that either over-expressed or knocked down/out the two proteins. To date, whether PUMA is regulated at the protein level is not known, though NOXA can be targeted for proteasomal degradation by forming a complex with its antiapoptotic partner MCL1.

Subcellular localization of BH3-only proteins is often co-regulated with posttranscriptional modification, allowing these proteins to relay apoptotic signals to and subsequently initiate apoptosis at the mitochondria. A large number of signaling pathways target BAD for phosphorylation. BAD can be phosphorylated by AKT, PKA, PKCΦ, JNK, CDC2, and p90rsk. Phosphorylated BAD is then bound and sequestered away from the mitochondria by protein 14-3-3 in the cytosol. BIMEL and BIML, the two most abundantly expressed isoforms of the bim gene, are generally thought to be tethered to the microtubular dynein motor complex by binding to the dynein light chain LC8 but have also been reported to be bound by the antiapoptotic proteins BCL2, BCL-XL, and MCL1 at the mitochondria. These two BIM isoforms can be phosphorylated on multiple sites by members of the MAP kinase family, including ERK, JNK, and p38. Phosphorylation of BIM has been implicated to alter its stability and sensitivity to apoptotic stimuli. Apoptotic stimuli that activate the intrinsic pathway (e.g., taxol) but not the extrinsic pathway (e.g., TNFα plus cycloheximide) can cause the release of BIM from the cytoskeleton, allowing it to translocate to mitochondria where it exerts its pro-apoptotic activity. BMF is regulated in a similar way by binding to dynein light chain-2 that sequesters BMF to myosin V motors on the actin cytoskeleton. This difference in subcellular localization may be why BMF is mobilized by cellular detachment, whereas BIM is mobilized by the disruption of the microtubule network.

The number and type of BH3-only proteins found within a cell is indicative of specialization rather than redundancy. Their unique subcellular localization, protein associations, and mechanisms of activation suggest that each acts as a sentinel for a distinct damage signal, such as DNA damage, growth factor withdrawal, UV irradiation, or chemotherapy.

BCL2 gene family reference

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2. Vaux D, Cory S, Adams J. Bcl-2 and cell survival[J]. Nature, 1988, 335: 440-442.

3. Cheng E H Y, Kirsch D G, Clem R J, et al. Conversion of Bcl-2 to a Bax-like death effector by caspases[J]. Science, 1997, 278(5345): 1966-1968.

4. Antignani A, Youle R J. How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane?[J]. Current opinion in cell biology, 2006, 18(6): 685-689.

5. Puthalakath H, Villunger A, O'Reilly L A, et al. Bmf: a proapoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis[J]. Science, 2001, 293(5536): 1829-1832.

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