The Family of Bcl-2 Proteins

The Bcl-2 protein was first identified as an oncoprotein coded by a gene affected by translocations of chromosomes 14 and 18 in B cell lymphomas. It was soon shown, however, that the Bcl-2 protein is not involved in regulation of the cell cycle, in contrast to many other oncoproteins, and thus does not fit into the classical oncogene picture. Furthermore, homology was estabhshed with the Ced9 protein of C. elegans, which has an antiapoptotic function in this organism. 

The Bcl-2 protein is a member of a protein family involved in regulation of the apoptotic program in mammalian cells (review Reed, 1997 Adams and Cory, 1998). At present, at least 15 members are known of the Bcl-2 family, which can have a negative or a positive effect on initiation of the apoptotic program. All Bcl-2 family members have at least one copy of a so-called BH motif (BH, Bcl-2 homolog), of which there are four types (BHl - BH4). 

The antiapoptotic members of the Bcl-2 family (Bcl-2, BclX, BclW, Al, Mcl-l) inhibit apoptosis by various cytotoxic effects. At a minimum, they contain the motives BHl and BH2. Bcl-2 protein contains all fom BH motives. For the mechanism of the antiapoptotic function, see 15.3.4 and 15.4. 

The antiapoptotic function of the Bcl-2 protein has been clearly shown experimentally. Its overexpression can prevent initiation of the apoptotic program in various cell types. The oncogenic function of Bcl-2 protein, observed in association with its overex- 

One family includes the Bax, Bak and Bok proteins that have a similar structure to the Bcl-2 protein. There is a link via the Bax protein to the tumor suppressor protein p53 (see 15.6). 

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Fig. 15.9. Antiapoptotic signalling by the PI3-kinase/Akt kinase pathway The PI3 kinase/Akt kinase pathway influences apoptosis via phosphorylation of the Bad protein, which is a member of the family of Bcl-2 proteins. Activation of the PI3-kinase pathway leads to Akt-kinase-catalyzed phosphorylation of Bad protein. Bad protein in its unphosphorylated form participates in activation of initiator caspases and thus has a proapoptotic effect. Phosphorylation of Bad protein by Akt kinase (or related kinases) has an antiapoptotic effect since phosphoryla-ted Bad protein is a binding substrate of 14-3-3 proteins. Bad is thus sequestered in an inactive state and is not available for triggering of apoptosis.

An important transcriptional target of the p53 protein that can induce apoptosis is the bax gene. The Bax protein belongs to the family of Bcl-2 proteins (see 15.3.2) and has a proapototic effect. There is speculation that the p53-induced increase in Bax concentration leads to formation of ion pores in mitochondria and that cytochrome c is released into the cytosol via these pores. Cytochrome c then functions as a cofactor which, together with Apafl protein, activates procaspase 8 and initiates the apoptotic program. 

Buggins AG, Pepper Cl (2010) The role of Bcl-2 family proteins in chronic lymphocytic leukaemia. Leuk Res 34 837-842... 

Crocin blocks the release of cytochrome c from mitochondria by modulating the expression of Bcl-2 family protein mRNAs... 

L-Digitoxose (53L) and its 4-(9-acetyl derivative are present in a tetrasaccha-ride glycosidically linked in tetrocarcin A, an antibiotic inhibitor of the anti-poptotic Bcl-2 protein family.123 Other tetrocarcins have been isolated from the culture of Micromonospora chalcea KY 11091.124 From another strain of Micromonospora, analogues of tetrocarcin A, called arisostatins A and B, containing isobutanoyldigitoxose instead of acetyldigitoxose were isolated.125... 

It was first observed in type Type 2 B-cell lymphoma that the translocation of the Bcl-2 gene leads to radical overexpression of Bcl-2 (10). The overproduction of Bcl-2 has been shown to be transforming (10). Members of the Bcl-2 protein family contain as many as four characteristically helical Bcl-2 homology motifs (BH1-BH4). Bcl-2 proteins are divided into classes, as prosurvival proteins (Bcl-2, Bc1-xl, Mc1-1, A-1, and Bcl-w), proapoptotic proteins (Bak and Bax), and proapoptotic proteins that contain only the BH3 motif (Bim, Bid, Puma, and Noxa) (3, 11). The manner in which the different Bcl-2 family members interact and the mechanisms by which cytochrome c release is initiated are unclear, but the central role of the Bcl-2 family in apoptosis, and the therapeutic potential of regulating these proteins, is well established. 

Insights into the functions of Bcl-2 family proteins have come from studies of the BH domains of Bc1-Xl and its interactions with the pro-apoptotic protein, Bak. The BHl, 2 and 3 domains of Bc1-Xl form a hydrophobic pocket, into which the BH3 domain of Bak (and perhaps that of other pro-apoptotic proteins) may fit, thereby preventing it from activating the downstream events (e.g. caspase activation) that trigger the death pathway [77]. However, molecular modelling indicates that in some pro-apoptotic proteins, the BH3 domain is normally not exposed on the periphery of the protein, but rather is buried within the core of the molecule. Such proteins may therefore require some form of activation step (e.g. phosphorylation or proteolytic cleavage) to expose the BH3 domain, thereby activating the molecule. 

Activated MAPKs have been found to modulate mitochondria-mediated apoptotic pathways, JNK in particular [71]. In fact, it appears that MAPK signaling may represent a potential cross-link between die different apoptotic pathways. JNK activation is not required for death-receptor-mediated apoptosis but is required for caspase-9 activation by the mitochondrial pathway, induced by a variety of proapoptotic stimuli [72,73]. JNK activation and c-Jun phosphorylation were found to be necessary to promote cytochrome c release from mitochondria, with the sequential assembly of the apoptosome and caspase-3 activation. The molecular mechanism of this effect was unclear, but it appeared to involve regulation of the expression and phosphorylation state of the Bcl-2 protein family and their recruitment to the mitochondrial outer membrane (Fig. 4). In a number of apoptotic models, JNK activation was associated with a downregulation of the antiapoptotic Bcl-2 and Bc1-Xl and upregulation of the proapoptotic Bax and Bad [74—77]. Two cell culture studies provided very strong evidence that JNK activation resulted in the phosphorylation of Bcl-2 and Bc1-Xl ex vivo, with the induction of apoptosis [76,77]. In other words, Bcl-2 and Bc1-Xl appear to be substrates of JNK, and phosphorylation results in their inactivation, thereby abolishing their ability to prevent cytochrome c release. 

Omi/Htra2, which lead to the activation of caspases— cysteine proteases that cleave a variety of intracellular contents. Through autoproteolytic activation, caspases create a feed-forward loop of proteolysis that inevitably leads to cell death. Accordingly, the cell contains the Bcl-2 family of proteins, which regulate mitochondrial permeability and caspase activity. A detailed description of apoptosis and its regulation by Bcl-2 proteins is beyond the scope of this chapter. Readers interested in additional details about the regulatory role of Bcl-2 proteins in apoptosis should read the work by Youle and Strasser (2008). 

Whether a cell undergoes apoptosis appears to be determined by the balance of Bcl-2 family proapoptotic (e.g., Bax, Bak) and anti-apoptotic (e.g., Bcl-2, Bc1-Xl) proteins [534], Bax itself induces cytochrome c release from isolated mitochmidria [535] and proapoptotic members bind to VDAC to form a large cytochrome c conducting channel [536]. In Cd -exposed cell lines, generally antiapoptotic Bcl-2 is decreased and/or proapoptotic Bax translocates to the mitochondria where it can release propapoptotic proteins [528,537,538] but a lack of effect of Cd " oti Bcl-2 family proteins has also been reported [523,539]. Addition of Bcl-2 can protect cells from undergoing Cd -induced apoptosis [539]. Furthermore, Cd " exposure can increase Bcl-2 as part of a survival mechanism. 

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