The ability of multicellular organisms to maintain cellular homeostasis is critically dependent on a balance between cell survival and cell death (apoptosis). The responsiveness of individual cells to death signals varies greatly depending on the presence of continuous survival cues from the extracellular environment. The perturbation of normal cell survival mechanisms, leading to an increase in cell survival or cell death plays an important role in the development of a number of disease states, including cancer. BAD (BCL2 Associated Death Promoter) is a pro-apoptotic critical regulatory component of the intrinsic cell death machinery that exerts its death-promoting effect upon heterodimerization with the antiapoptotic proteins of the BCL2 family defined by conserved regions of identity called BH (BCL2 homology) domains (Ref.1).
Members of the BCL2 (B-Cell CLL/Lymphoma-2) family are intracellular proteins that can either promote survival (BCL2, BCLXL, Mcl1, and A1) or augment cell death (BAD, BID, BAX and BAK). BCL2 family proteins homo- and heterodimerize, and susceptibility to cell death is dictated by the relative levels and interactions of BCL family members. The association of BAD with BCLXL is mediated through dimerization of BH3 domains. The phosphorylation of BAD at specific residues (Ser112, Ser136 and Ser155), results in loss of the ability of BAD to heterodimerize with the survival proteins BCLXL or BCL2 (Ref.1). Ser136 phosphorylation of BAD is concordant with the activation of Akt and Ser112 phosphorylation requires activation of the Ras-MAPK (Mitogen-Activated Protein Kinase) pathway. Ras-mediated activation of PI3K triggers the phosphorylation of Akt through the functions of PDKs (Phosphoinositide-Dependent Kinases), such as PDK1 and PDK2. Akt then acts as a central regulator of cell survival, phosphorylating BAD, leading to displacement from the mitochondria and sequestration by chaperone 14-3-3Tau. Concomitantly with the activation of the Akt pathway and Integrin ligation via SHC (Src Homology) and FAK (Focal Adhesion Kinase) leads to the activation of the Ras-Raf-MEK1-ERK1/2 cascade.
Ser155 is a major site of phosphorylation for BAD following stimulation by
GF (Growth Factors) including IGF1 (Insulin-like GF1), EGF (Epidermal Growth Factor), PDGF (Platelet-Derived Growth Factor), NGF (Nerve Growth Factor), TGF-Beta (Transforming Growth Factor-Beta), GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor), and under noncytotoxic conditions, even TNF (Tumor Necrosis Factor), Integrin and many Cytokines (such as Interleukin-3 [IL-3]) (Ref.2). These survival factors promote cell survival through their ability to stimulate PI3K (Phosphatidylinositol 3-Kinase) and GRB2 (Growth Factor Receptor-Bound Protein-2)-SOS phosphorylation of Ras, which inactivate BAD. The PI3K-sensitive pathway activates Akt or PKB (Protein Kinase-B) and direct phosphorylation of BAD at Ser136 (Ref.3) resulting in its dissociation from complexes with BCL2/BCLXL and its subsequent association with 14-3-3Tau. The uncomplexed BCLXL is then capable of suppressing cell death responses by blocking the release of mitochondrial CytoC (Cytochrome-C), but release APAF1 (Apoptotic Protease Activating Factor-1) apoptosome or regulate other BCLXL activities resulting in a Caspase9 initiated cascade of proteolysis and induction of apoptosis (Ref.4). Upon calcium influx increase or GF deprivation, phosphorylated BAD is rapidly dephosphorylated by the specific serine-phosphatase Calcineurin or PP1Alpha, which then translocates to the mitochondrion outer membrane, where, through its BH3 domain, it interacts with anti-apoptotic BCL2 and BCLXL antibodies (Ref.2). Such dephosphorylation of BAD by Calcineurin, reverses the cycle back to an unphosphorylated, death-promoting agonist during Ca2+ induced apoptosis (Ref.5). In addition to phosphorylation at Ser136 mediated by PKB, BAD undergoes cAMP-dependent PKA (Protein Kinase-A) and MAPK-activated RSK (Ribosomal S6 Kinases) mediated phosphorylation on Ser112 following stimulation by GPCR (G-protein Coupled Receptors) and IL-3, respectively (Ref.6).
The regulation of programmed cell death, apoptosis, is an exceptionally complicated process that involves a myriad of proteins (Ref.3). BAD represent an important bridge between survival signaling by GF receptors and the prevention of apoptosis (Ref.4). BAD phosphorylation also protects cells from the deleterious effects of apoptotic stimuli and attenuates death pathway signaling by raising the threshold at which mitochondria release CytoC to induce cell death.