Proapoptotic Bcl-2 members

Most of the responses which lead to cytochrome c release are triggered by activated proapoptotic members of the Bcl-2 family which are directed to mitochondria, where they insert into the outer membrane. What mediates the specific targeting of these proteins to mitochondria, hi a recent work, Lutter et al. (2000) investigated this question using the proapoptotic Bcl-2 member (tBid) as a tool. These authors estabhshed a hposome model for cytochrome c release and recreated the lipid constitution of mitochondrial outer... 

The precise mechanism how proapoptotic Bcl-2 members regulate the exit of cytochrome c and how phospholipids participate in this process is still controversial. There are two prevailing models (Figure 3) In model 1 Bcl-2 members insert into the lipid bilayer of the mitochondrial outer membrane and form channels that facilitate exit of cytochrome c and other apoptogenic... 

Figure 3. Possible mechanisms of actions of Bcl-2 members. Two prevailing models through which Bcl-2 membas trigger cytochrome c release have been suggested. In both models phospholipids in the bilayer stnicture either individually and/or collectively induce a conformational change in Bcl-2 members, allowing them to insert into the outer mitochondrial membrane. In model 1 proapoptotic proteins destabilize the outer mitochondrial membrane, oligomerize and form channels through which cytochrome c and other proteins of the intermembrane space can escape.BcI-2 proteins such as Bax or tBid act in concert with other proteins of the BcI-2 family to form channels. In model 2 Bcl-2 members such as Bax interact with residoit proteins in the outer membrane (OM) such as the voltage-dependent anion...

However, not all survival-promoting signals act via Akt. For example, cAMP-mediated neuronal survival occurs in an Akt-independent manner (Li et al., 2000). The mechanisms of Akt-induced survival are only now falling in place. Akt phosphorylates the proapoptotic Bcl-2 family member, Bad. Phosphorylation of Bad results in decreased formation of the Bad-Bcl-XL heterodimer. This allows free Bcl-xL to protect the cell from apoptosis (Cantley, 2002 Vivanco and Sawyers, 2002). Although this appears to be an important mechanism for PI-3-K-induced cell survival, alternate mechanisms might exist. Bad is expressed in RPE cells (Mukherjee et al., 2004). In certain cell types where Bad is not expressed, PI-3-K/Akt still prevents apoptosis. Possible alternate mechanisms by which PT3-K may promote cell survival are as follows (1) Akt also phosphorylates the Forkhead-related transcription factor (FKHR) in the cytoplasm. 

Bouillet P, Strasser A. BH3-only proteins - evolutionarily conserved proapoptotic Bcl-2 family members essential for initiating programmed cell death. J Cell Sci 2002 115 1567-1574. 

In response to ischemia and reperfusion, mitochondrial membranes undergo depolarization and elevated levels of Ca in the intermembrane space. The control of mitochondrial membrane depolarization depends, in part, on Bcl-2 family member proteins. The proapoptotic group of Bcl-2 members consists of the Bax-subfamily (Bax, Bak, and Bok) and the BH3-only proteins (Bid, Bim, Bik, Bad, Bmf, Hrk, Noxa, Puma, Blk, BNIP3, and Spike) (Cory and Adams, 2002 Mund et al., 2003). It appears that the main function of the Bcl-2 family proteins is to guard mitochondrial integrity and to control the release of mitochondrial proteins into the cytoplasm (Cory and Adams, 2002). It is believed that the proapoptotic Bax and Bak provoke or contribute to the perme-abilization of the outer mitochondrial membrane, either by forming channels by themselves (Antonsson et al., 2000) or by interacting with components such as VDAC (Tsujimoto and Shimizu, 2000). 

A balance between proapoptotic (BAX, BID, BAK, or BAD) and anti-apoptotic (Bcl-Xl and Bcl-2) members of the Bcl-2 family is recognized. The proapoptotic members are required to make the mitochondrial membrane permeable for the release of caspase activators such as cytochrome c. Control of proapoptotic proteins under normal cell conditions of non-apoptotic cells is incompletely understood, generally Bax or Bak are activated by the activation of BH3 proteins [23]. 

Changes in intracellular calcium levels are known to activate the mitochondrial pathway of apoptosis. A key regulator of Ca -dependent proteins is calmodulin. SM has been shown to cause a time-dependent induction of calmodulin in keratinocytes (Simbulan-Rosenthal et al., 2006). Moreover, depletion of calmodulin using antisense probes attenuated SM-induced activation of caspases involved in the mitochondrial pathway of apoptosis. Both antisense and pharmacological inhibition of calmodulin prevented SM-induced nuclear fragmentation in the keratinocytes. Bad, a proapoptotic Bcl-2 family member present in an inactive phosphorylated form in viable cells, was also activated by SM. Furthermore, cyclosporine A, a selective inhibitor of calcineurin, a Bad phosphatase, inhibited SM-induced keratinocyte apoptosis. These results suggest that calcium-dependent activation of Bad may be a mechanism by which SM induces apoptosis in keratinocytes. 

Proapototic signals direct these proteins to mitochondria where they compete with antiapoptotic members of the Bcl-2 family to regulate the cytochrome c release and to determine the fate of the cell life or death (Cosulich et al., 1999). Unlike proapoptotic proteins, the antiapoptotic Bcl-2 proteins reside in the outer mitochondrial membrane, anchored by a hydrophobic stretch of amino acids located at the COOH-termini,... 

It has been well established that Bcl-2 prevents most forms of apoptotic cell death as well as certain forms of necrotic cell death (T6). A large number of Bcl-2-related proteins have been isolated and divided into three categories (T6) (1) antiapoptotic members such as Bcl-2, BcI-Xl, Bcl-w, Mcl-l, A1 (Bfl-1), and Boo, all of which exert anti-cell death activity (2) proapoptotic members including Bax, Bak, Bad, Mtd, and Diva and (3) proapoptotic proteins, which include Bik, Bid, Bim, Hrk, Blk, Bnip3, and Bnip3L and share sequence homology only in Bcl-2 homology (BH)3. 

The biochemical basis of the various activities of the Bcl-2 family members is only partially imderstood. The antiapoptotic Bcl-2 proteins may function by directly inhibiting the activation of the caspases (see 15.3.3). It is assumed that proapoptotic proteins interact with antiapoptotic proteins and halt their inhibition of apoptosis. 

Various members of the Bcl-2 family can interact with another via the BH domains and form hetero-oligomeric complexes in which the different activities neutralize one another. The relationship of proapoptotic and antiapoptotic Bcl-2 family proteins thus helps to determine a cell s susceptibility to apoptosis. 

The antiapoptotic Bcl-2 family members control apoptosis by various mechanisms without directly binding to the caspases. Bcl-2 proteins can interact with cofactors and inhibit their activity. They can also act antiapoptotically by binding to mitochondria and interfering with release of cytochrome c. Furthermore, they can interact with other proapoptotic proteins, e.g. with propapoptotic members of their own family. 

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.

PI3-kinase (see 6.6) can mediate antiapoptotic signals, in addition to growth-promoting signals (Fig. 14.9). The antiapoptotic signal conduction starts at PI3-kinase to Akt kinase, which is activated by the messenger substance PtdInsPs formed by PI3-kinase. The Bad protein has been identified as a substrate of Akt kinase. The Bad protein is a proapoptotic member of the Bcl-2 family. It is phosphorylated by Akt kinase at several Ser residues and its proapoptotic effect is thus inhibited (Datta et al., 1997). Experimental evidence exists that the 14-3-3 proteins are involved in this inhibition these bind to phosphoserine residues of Bad protein and thus inactivate its proapoptotic fim-ction. 

Members of the Bcl-2 family share one or more Bcl-2 homology (BFI) domains, named BFI1, BFI2, BFI3, and BFI4 (Adams and Cory, 1998). It is not yet clear which structural features determine if these proteins possess pro- or anti-apoptotic activities. However, some studies revealed that the BH3 domain is a critical domain for the proapoptotic members (Chittenden et al., 1995). Besides BH domains, some contain a hydrophobic domain in the C-terminal region, which is essential for the attachment to intracellular membranes, like the outer mitochondrial, nuclear, and endoplasmic reticulum membranes (Krajewski et al., 1993 Nguyen et al., 1993). 

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