CaM kinase control of AKT and LNCaP cell survival

AKT and its substrate BAD have been shown to promote prostate cancer cell survival. Agonists, such as carbachol, and hormones that increase intracellular calcium concentration can activate AKT leading to cancer cell survival. The LNCaP prostate cancer cells express the carbachol-sensitive M(3) -subtype of G protein-coupled receptors that cause increases in intracellular calcium and activate the family of Ca(2+) /calmodulin-dependent protein kinases (CaM Ks). One type of CaM Kinase, CaM Kinase Kinase (CaM KK), phosphorylates several substrates including AKT on threonine 308. AKT phosphorylation and activation enhances cell survival through phosphorylation of BAD protein and the subsequent blockade of caspase activation. Our goals were to examine the mechanism of carbachol activation of AKT and BAD in LNCaP prostate cancer cells and evaluate whether CaM KK may be mediating carbachol's activation of AKT and cell survival. Our results suggest that carbachol treatment of LNCaP cells promoted cell survival through CaM KK and its phosphorylation of AKT. The bacterial toxin anisomycin triggered caspase-3 activation in LNCaP cells that was blocked by carbachol in a CaM KK- and AKT-dependent manner. AKT and BAD phosphorylation were blocked by the selective CaM KK inhibitor, STO-609, as well as siRNA directed against CaM KK. BAD phosphorylation was also blocked by treating cells with the AKT inhibitor, AKT-X, as well as siRNA to AKT. Additionally, epinephrine promoted LNCaP cell survival through activation of AKT that was insensitive to STO-609. Taken together these data suggest a survival role for CaM KK operating through AKT and BAD in LNCaP prostate cancer cells.     

Expression of the Bcl-2 Protein BAD Promotes Prostate Cancer Growth

BAD, a pro-apoptotic protein of the Bcl-2 family, has recently been identified as an integrator of several anti-apoptotic signaling pathways in prostate cancer cells. Thus, activation of EGFR, GPCRs or PI3K pathway leads to BAD phosphorylation and inhibition of apoptosis. Increased levels of BAD in prostate carcinomas have also been reported. It appears contradictory that instead of limiting expression of pro-apoptotic protein, prostate cancer cells choose to increase BAD levels while keeping it under tight phosphorylation control. Analysis of the effect of BAD on prostate cancer xenografts has shown that increased BAD expression enhances tumor growth, while knockdown of BAD expression by shRNA inhibits tumor growth. Tissue culture experiments demonstrated that increased BAD expression stimulates proliferation of prostate cancer cells. These results suggest that increased expression of BAD provides a proliferative advantage to prostate tumors, while BAD dephosphorylation increases sensitivity of prostate cancer cells to apoptosis. Combination of proliferative and apoptotic properties prompts prostate cancer cells to be “addicted” to increased levels of phosphorylated BAD. Thus, kinases that phosphorylate BAD are plausible therapeutic targets; while monitoring BAD phosphorylation could be used to predict tumor response to treatments.     

GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis

The chaperone glucose-regulated protein, 78/immunoglobulin binding protein (GRP78/Bip), protects cells from cytotoxicity induced by DNA damage or endoplasmic reticulum (ER) stress. In this study, we showed that GRP78 is a major inducible protein in human non-small cell lung cancer H460 cells treated with ER stress inducers, including A23187 and thapsigargin. AEBSF, an inhibitor of serine protease, diminished GRP78 induction, enhanced mitochondrial permeability, and augmented apoptosis in H460 cells during ER stress. Simultaneously, AEBSF promoted Raf-1 degradation and suppressed phosphorylation of Raf-1 at Ser338 and/or Tyr340 during ER stress. Coimmunoprecipitation assays and subcellular fractionations showed that GRP78 associated and colocalized with Raf-1 on the outer membrane of mitochondria, respectively. While treatment of cells with ER stress inducers inactivated BAD by phosphorylation at Ser75, a Raf-1 phosphorylation site; AEBSF attenuated phosphorylation of BAD, leading to cytochrome c release from mitochondria. Additionally, overexpression of GRP78 and/or Raf-1 protected cells from ER stress-induced apoptosis. Taken together, our results indicate that GRP78 may stabilize Raf-1 to maintain mitochondrial permeability and thus protect cells from ER stress-induced apoptosis.     

Systems Modeling of Anti-apoptotic Pathways in Prostate Cancer: Psychological Stress Triggers a Synergism Pattern Switch in Drug Combination Therapy

Prostate cancer patients often have increased levels of psychological stress or anxiety, but the molecular mechanisms underlying the interaction between psychological stress and prostate cancer as well as therapy resistance have been rarely studied and remain poorly understood. Recent reports show that stress inhibits apoptosis in prostate cancer cells via epinephrine/beta2 adrenergic receptor/PKA/BAD pathway. In this study, we used experimental data on the signaling pathways that control BAD phosphorylation to build a dynamic network model of apoptosis regulation in prostate cancer cells. We then compared the predictive power of two different models with or without the role of Mcl-1, which justified the role of Mcl-1 stabilization in anti-apoptotic effects of emotional stress. Based on the selected model, we examined and quantitatively evaluated the induction of apoptosis by drug combination therapies. We predicted that the combination of PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and inhibition of BAD phosphorylation at S112 would produce the best synergistic effect among 8 interventions examined. Experimental validation confirmed the effectiveness of our predictive model. Moreover, we found that epinephrine signaling changes the synergism pattern and decreases efficacy of combination therapy. The molecular mechanisms responsible for therapeutic resistance and the switch in synergism were explored by analyzing a network model of signaling pathways affected by psychological stress. These results provide insights into the mechanisms of psychological stress signaling in therapy-resistant cancer, and indicate the potential benefit of reducing psychological stress in designing more effective therapies for prostate cancer patients.     

Diverse antiapoptotic signaling pathways activated by vasoactive intestinal polypeptide, epidermal growth factor, and phosphatidylinositol 3-kinase in prostate cancer cells converge on BAD

It has been demonstrated that vasoactive intestinal polypeptide, epidermal growth factor, and chronic activation of phosphatidylinositol 3-kinase can protect prostate cancer cells from apoptosis; however, the signaling pathways that they use and molecules that they target are unknown. We report that vasoactive intestinal polypeptide, epidermal growth factor, and phosphatidylinositol 3-kinase activate independent signaling pathways that phosphorylate the proapoptotic protein BAD. Vasoactive intestinal polypeptide operated via protein kinase A, epidermal growth factor required Ras activity, and effects of phosphatidylinositol 3-kinase were predominantly mediated by Akt. BAD phosphorylation was critical for the antiapoptotic effects of each signaling pathway. None of these survival signals was able to rescue cells that express BAD with mutations in phosphorylation sites, whereas knockdown of BAD expression with small hairpin RNA rendered cells insensitive to apoptosis. Taken together, these results identify BAD as a convergence point of several antiapoptotic signaling pathways in prostate cells.     

Estradiol abrogates apoptosis in breast cancer cells through inactivation of BAD: Ras-dependent nongenomic pathways requiring signaling through ERK and Akt

Estrogens such as 17-beta estradiol (E(2)) play a critical role in sporadic breast cancer progression and decrease apoptosis in breast cancer cells. Our studies using estrogen receptor-positive MCF7 cells show that E(2) abrogates apoptosis possibly through phosphorylation/inactivation of the proapoptotic protein BAD, which was rapidly phosphorylated at S112 and S136. Inhibition of BAD protein expression with specific antisense oligonucleotides reduced the effectiveness of tumor necrosis factor-alpha, H(2)O(2), and serum starvation in causing apoptosis. Furthermore, the ability of E(2) to prevent tumor necrosis factor-alpha-induced apoptosis was blocked by overexpression of the BAD S112A/S136A mutant but not the wild-type BAD. BAD S112A/S136A, which lacks phosphorylation sites for p90(RSK1) and Akt, was not phosphorylated in response to E(2) in vitro(.) E(2) treatment rapidly activated phosphatidylinositol 3-kinase (PI-3K)/Akt and p90(RSK1) to an extent similar to insulin-like growth factor-1 treatment. In agreement with p90(RSK1) activation, E(2) also rapidly activated extracellular signal-regulated kinase, and this activity was down-regulated by chemical and biological inhibition of PI-3K suggestive of cross talk between signaling pathways responding to E(2). Dominant negative Ras blocked E(2)-induced BAD phosphorylation and the Raf-activator RasV12T35S induced BAD phosphorylation as well as enhanced E(2)-induced phosphorylation at S112. Chemical inhibition of PI-3K and mitogen-activated protein kinase kinase 1 inhibited E(2)-induced BAD phosphorylation at S112 and S136 and expression of dominant negative Ras-induced apoptosis in proliferating cells. Together, these data demonstrate a new nongenomic mechanism by which E(2) prevents apoptosis.     

Epidermal growth factor protects prostate cancer cells from apoptosis by inducing BAD phosphorylation via redundant signaling pathways

Protection from apoptosis by receptor tyrosine kinases, resistant to the inhibition of phosphatidylinositol 3 '-kinase/Akt and Ras/MEK pathways, has been reported in several cell types, including fibroblasts and epithelial prostate cancer cells; however, mechanisms of this effect were not clear. Here we report that in prostate cancer cells, epidermal growth factor activates two antiapoptotic signaling pathways that impinge on the proapoptotic protein BAD. One signaling cascade operates via the Ras/MEK module and induces BAD phosphorylation on Ser112. Another pathway predominantly relies on Rac/PAK1 signaling that leads to BAD phosphorylation on Ser136. Each of these two pathways is sufficient to protect cells from apoptosis, and therefore both have to be inhibited simultaneously to block epidermal growth factor-dependent survival. Redundancy of antiapoptotic signaling pathways should be considered when therapies targeting antiapoptotic mechanisms are designed.     

BAD contributes to RAF-mediated proliferation and cooperates with B-RAF-V600E in cancer signaling

BAD (Bcl-2 antagonist of cell death) belongs to the proapoptotic BH3-only subfamily of Bcl-2 proteins. Physiological activity of BAD is highly controlled by phosphorylation. To further analyze the regulation of BAD function, we investigated the role of recently identified phosphorylation sites on BAD-mediated apoptosis. We found that in contrast to the N-terminal phosphorylation sites, the serines 124 and 134 act in an antiapoptotic manner because the replacement by alanine led to enhanced cell death. Our results further indicate that RAF kinases represent, besides PAK1, BAD serine 134 phosphorylating kinases. Importantly, in the presence of wild type BAD, co-expression of survival kinases, such as RAF and PAK1, leads to a strongly increased proliferation, whereas substitution of serine 134 by alanine abolishes this process. Furthermore, we identified BAD serine 134 to be strongly involved in survival signaling of B-RAF-V600E-containing tumor cells and found that phosphorylation of BAD at this residue is critical for efficient proliferation in these cells. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation and its role in cancer signaling.     

Activation of BAD by therapeutic inhibition of epidermal growth factor receptor and transactivation by insulin-like growth factor receptor

Novel cancer chemotherapeutics are required to induce apoptosis by activating pro-apoptotic proteins. Both epidermal growth factor (EGF) and insulin-like growth factor (IGF) provide potent survival stimuli in many epithelia, and activation of their receptors is commonly observed in solid human tumors. Here we demonstrate that blockade of the EGF receptor by a new drug in phase III clinical trails for cancer, ZD1839, potently induces apoptosis in mammary epithelial cell lines and primary cultures, as well as in a primary pleural effusion from a breast cancer patient. We identified the mechanism of apoptosis induction by ZD1839. We showed that it prevents cell survival by activating the pro-apoptotic protein BAD. Moreover, we demonstrate that IGF transactivates the EGF receptor and that ZD1839 blocks IGF-mediated phosphorylation of MAPK and BAD. Many cancer therapies kill tumor cells by inducing apoptosis as a consequence of targeting DNA; however, the threshold at which apoptosis can be triggered through DNA damage is often different from that in normal cells. Our results indicate that by targeting a growth factor-mediated survival signaling pathway, BAD phosphorylation can be manipulated therapeutically to induce apoptosis.     

Phosphorylation of BAD at Ser-128 during mitosis and paclitaxel-induced apoptosis

Phosphorylation of BCL-2 family member BAD at different residues triggers different physiological effects, either inhibiting or promoting apoptosis. The recently identified phosphorylation site at Ser-128 enhances the apoptotic activity of BAD. We here show that BAD becomes phosphorylated at Ser-128 in the mitotic phase of the cell cycle in NIH3T3 cells. We also show that BAD-S128 is phosphorylated in taxol-treated mouse fibroblasts and MDA-MB-231 human breast cancer cells. However, expression of a phosphorylation-defective dominant negative BAD mutant did not block taxol-induced apoptosis. These data support the view that the phosphorylation of BAD Serine 128 exerts cell-specific effects on apoptosis. Whereas the BAD Serine 128 phosphorylation induces apoptosis in neuronal cells, it does not appear to promote apoptosis in proliferating non-neural cells during mitosis or upon exposure to the antineoplastic agent taxol.     

Inhibition of phosphorylation of BAD and Raf-1 by Akt sensitizes human ovarian cancer cells to paclitaxel

We studied the roles of the phosphatidylinositol 3-kinase (PI-3K)-Akt-BAD cascade, ERK-BAD cascade, and Akt-Raf-1 cascade in the paclitaxel-resistant SW626 human ovarian cancer cell line, which lacks functional p53. Treatment of SW626 cells with paclitaxel activates Akt and ERK with different time frames. Interference with the Akt cascade either by treatment with PI-3K inhibitor (wortmannin or LY294002) or by exogenous expression of a dominant negative Akt in SW626 cells caused decreased cell viability following treatment with paclitaxel. Interference with the ERK cascade by treatment with an MEK inhibitor, PD98059, in SW626 cells also caused decreased cell viability following treatment with paclitaxel. Treatment of cells with paclitaxel also stimulated the phosphorylation of BAD at both the Ser-112 and Ser-136 sites. The phosphorylation of BAD at Ser-136 was blocked by treatment with wortmannin or cotransfection with the dominant negative Akt. On the other hand, the phosphorylation of BAD at Ser-112 was blocked by PD98059. We further examined the role of BAD in the viability following paclitaxel treatment using BAD mutants. Exogenous expression of doubly substituted BAD2SA in SW626 cells caused decreased viability following treatment with paclitaxel. Moreover, because paclitaxel-induced apoptosis is mediated by activated Raf-1 and the region surrounding Ser-259 in Raf-1 conforms to a consensus sequence for phosphorylation by Akt, the regulation of Raf-1 by Akt was examined. We demonstrated an association between Akt and Raf-1 and showed that the phosphorylation of Raf-1 on Ser-259 induced by paclitaxel was blocked by treatment with wortmannin or LY294002. Furthermore, interference with the Akt cascade induced by paclitaxel up-regulated Raf-1 activity, and expression of constitutively active Akt inhibited Raf-1 activity, suggesting that Akt negatively regulates Raf-1. Our findings suggest that paclitaxel induces the phosphorylation of BAD Ser-112 via the ERK cascade, and the phosphorylation of both BAD Ser-136 and Raf-1 Ser-259 via the PI-3K-Akt cascade, and that inhibition of either of these cascades sensitizes ovarian cancer cells to paclitaxel.     

Tumor necrosis factor induces phosphorylation and translocation of BAD through a phosphatidylinositide-3-OH kinase-dependent pathway.

Tumor necrosis factor (TNF) induced the phosphorylation of BAD at serine 136 in HeLa cells under conditions that are not cytotoxic. BAD phosphorylation by TNF was dependent on phosphatidylinositide-3-OH kinase (PI3K) and was accompanied by the translocation of BAD from the mitochondria to the cytosol. Blocking the phosphorylation of BAD and its translocation to the cytosol with the PI3K inhibitor wortmannin activated caspase-3 and markedly potentiated the cytotoxicity of TNF. Transient transfection with a PI3K dominant negative mutant or a dominant negative mutant of the serine-threonine kinase Akt, the downstream target of PI3K and the enzyme that phosphorylates BAD, similarly potentiated the cytotoxicity of TNF. By contrast, transfection with a constitutively active Akt mutant protected against the cytotoxicity of TNF in the presence of wortmannin. Phosphorylation of BAD prevents its interaction with the antiapoptotic protein Bcl-XL. Transfection with a Bcl-XL expression vector protected against the cytotoxicity of TNF in the presence of wortmannin. The mechanism by which the inhibition of the phosphorylation of BAD is likely linked to the induction of lethal mitochondrial damage in TNF-intoxicated cells is discussed.     

Phosphorylation of the pro-apoptotic protein BAD on serine 155, a novel site, contributes to cell survival

Phosphorylation of BAD, a pro-apoptotic member of the Bcl-2 protein family, on either Ser112 or Ser136 is thought to be necessary and sufficient for growth factors to promote cell survival. Here we report that Ser155, a site phosphorylated by protein kinase A (PKA), also contributes to cell survival. Ser112 is thought to be the critical PKA target, but we found that BAD fusion proteins containing Ala at Ser112 (S112A) or Ser136 (S136A) or at both positions (S112/136A) were still heavily phosphorylated by PKA in an in vitro kinase assay. BAD became insensitive to phosphorylation by PKA only when both Ser112 and Ser136, or all three serines (S112/136/155) were mutated to alanine. In HEK293 cells, BAD fusion proteins mutated at Ser155 were refractory to phosphorylation induced by elevation of cyclic AMP(cAMP) levels. Phosphorylation of the S112/136A mutant was >90% inhibited by H89, a PKA inhibitor. The S155A mutant induced more apoptosis than the wild-type protein in serum-maintained CHO-K1 cells, and apoptosis induced by the S112/136A mutant was potentiated by serum withdrawal. These data suggest that Ser155 is a major site of phosphorylation by PKA and serum-induced kinases. Like Ser112 and Ser136, phosphorylation of Ser155 contributes to the cancellation of the pro-apoptotic function of BAD.     

Overexpression of BAD potentiates sensitivity to tumor necrosis factor-related apoptosis-inducing ligand treatment in the prostatic carcinoma cell line LNCaP

Here we show that LNCaP, which is resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, becomes sensitive to TRAIL after overexpression of full-length, wild-type BAD (BAD WT). TRAIL induces caspase-dependent cleavage of BAD WT that results in generation of a M(r) 15,000 protein. LNCaP stably expressing truncated BAD (tBAD) and cells expressing mutated BAD at the caspase cleavage site were less sensitive to TRAIL treatment when compared to LNCaP expressing BAD WT. Cytochrome c and Smac/DIABLO release from mitochondria into cytosol was found after TRAIL treatment only in cells overexpressing BAD WT. Furthermore, differences in phosphorylation of serine residues for BAD WT and tBAD were identified. BAD WT was phosphorylated at positions S136 and S155, whereas tBAD was phosphorylated at positions S112, S136, and S155. LNCaP stably expressing BAD mutated at serine 112 to alanine was less sensitive to TRAIL treatment when compared to LNCaP expressing BAD WT. Lastly, recombinant BAD cleaved by caspase-3 is a more potent inducer of cytochrome c and Smac/DIABLO release than BAD WT. In summary, BAD-mediated sensitivity of LNCaP to TRAIL depends on the phosphorylation status of BAD WT and tBAD.     

Regulation of BAD protein by PKA, PKCdelta and phosphatases in adult rat cardiac myocytes subjected to oxidative stress

H2O2, as an example of oxidative stress, induces cardiac myocyte apoptosis. Bcl-2 family proteins are key regulators of the apoptotic response while their functions can be regulated by post-translational modifications including phosphorylation, dimerization or proteolytic cleavage. In this study, we examined the role of various protein kinases in regulating total BAD protein levels in adult rat cardiac myocytes undergoing apoptosis. Stimulation with 0.1 mM H2O2, which induces apoptosis, resulted in a marked down-regulation of BAD protein, which is attributed to cleavage by caspases since it can be restored in the presence of a general caspase inhibitor. Inhibition of PKC, p38-MAPK, ERK1/2 and PI-3-K did not influence the reduced BAD protein levels observed after stimulation with H2O2. On the contrary, inhibition of PKA or specifically PKCdelta resulted in up-regulation of BAD. Decreased caspase 3 activity was observed in H2O2 treated cells after inhibition of PKA or PKCdelta whereas inhibition of PKA also resulted in improved cell survival. Furthermore, addition of okadaic acid to inhibit selected phosphatases resulted in enhanced BAD cleavage. These data suggest that, during oxidative stress-induced cardiac myocyte apoptosis, there is a caspase-dependent down-regulation of BAD protein, which seems to be regulated by coordinated action of PKA, PKCdelta and phosphatases.     

Caspase cleavage enhances the apoptosis-inducing effects of BAD

The function of BAD, a proapoptotic member of the Bcl-2 family, is regulated primarily by rapid changes in phosphorylation that modulate its protein-protein interactions and subcellular localization. We show here that, during interleukin-3 (IL-3) deprivation-induced apoptosis of 32Dcl3 murine myeloid precursor cells, BAD is cleaved by a caspase(s) at its N terminus to generate a 15-kDa truncated protein. The 15-kDa truncated BAD is a more potent inducer of apoptosis than the wild-type protein, whereas a mutant BAD resistant to caspase 3 cleavage is a weak apoptosis inducer. Truncated BAD is detectable only in the mitochondrial fraction, interacts with BCL-X(L) at least as effectively as the wild-type protein, and is more potent than wild-type BAD in inducing cytochrome c release. Human BAD, which is 43 amino acids shorter than its mouse counterpart, is also cleaved by a caspase(s) upon exposure of Jurkat T cells to anti-FAS antibody, tumor necrosis factor alpha (TNF-alpha), or TRAIL. Moreover, a truncated form of human BAD lacking the N-terminal 28 amino acids is more potent than wild-type BAD in inducing apoptosis. The generation of truncated BAD was blocked by Bcl-2 in IL-3-deprived 32Dcl3 cells but not in Jurkat T cells exposed to anti-FAS antibody, TNF-alpha, or TRAIL. Together, these findings point to a novel and important role for BAD in maintaining the apoptotic phenotype in response to various apoptosis inducers.     

The herpes simplex virus 1 US3 protein kinase blocks caspase-dependent double cleavage and activation of the proapoptotic protein BAD

An earlier report showed that the U(S)3 protein kinase blocked the apoptosis induced by the herpes simplex virus 1 (HSV-1) d120 mutant at a premitochondrial stage. Further studies revealed that the kinase also blocks programmed cell death induced by the proapoptotic protein BAD. Here we report the effects of the U(S)3 protein kinase on the function and state of a murine BAD protein. Specifically, (i) in uninfected cells, BAD was processed by at least two proteolytic cleavages that were blocked by a general caspase inhibitor. The untreated transduced cells expressed elevated caspase 3 activity. (ii) In cells cotransduced with the U(S)3 protein kinase, the BAD protein was not cleaved and the caspase 3 activity was not elevated. (iii) Inasmuch as the U(S)3 protein kinase blocked the proapoptotic activity and cleavage of a mutant (BAD3S/A) in which the codons for the regulatory serines at positions 112, 136, and 155 were each replaced with alanine codons, the U(S)3 protein kinase does not act by phosphorylation of these sites nor was the phosphorylation of these sites required for the antiapoptotic function of the U(S)3 protein kinase. (iv) The U(S)3 protein kinase did not enable the binding of the BAD3S/A mutant to the antiapoptotic proteins 14-3-3. Finally, (v) whereas cleavage of BAD at ASP56 and ASP61 has been reported and results in the generation of a more effective proapoptotic protein with an M(r) of 15,000, in this report we also show the existence of a second caspase-dependent cleavage site most likely at the ASP156 that is predicted to inactivate the proapoptotic activity of BAD. We conclude that the primary effect of U(S)3 was to block the caspases that cleave BAD at either residue 56 or 61 predicted to render the protein more proapoptotic or at residue 156, which would inactivate the protein.     

Growth factors inactivate the cell death promoter BAD by phosphorylation of its BH3 domain on Ser155

The Bcl-2 family protein BAD promotes apoptosis by binding through its BH3 domain to Bcl-x(L) and related cell death suppressors. When BAD is phosphorylated on either Ser(112) or Ser(136), it forms a complex with 14-3-3 in the cytosol and no longer interacts with Bcl-x(L) at the mitochondria. Here we show that phosphorylation of a distinct site Ser(155), which is at the center of the BAD BH3 domain, directly suppressed the pro-apoptotic function of BAD by eliminating its affinity for Bcl-x(L). Protein kinase A functioned as a BAD Ser(155) kinase both in vitro and in cells. BAD Ser(155) was found to be a major site of phosphorylation induced following stimulation by growth factors and prevented by protein kinase A inhibitors but not by inhibitors of the phosphatidylinositol 3-kinase/Akt pathway. Growth factors inhibited BAD-induced apoptosis in both a Ser(112)/Ser(136)- and a Ser(155)-dependent fashion. Thus, growth factors engage an anti-apoptotic signaling pathway that inactivates BAD by direct modification of its BH3 cell death effector domain.     

The Bcl-2-associated death promoter (BAD) lowers the threshold at which the Bcl-2-interacting domain death agonist (BID) triggers mitochondria disintegration

The Bcl-2-associated death promoter (BAD) protein, like many other BH3-only proteins, is known to promote apoptosis through the intrinsic mitochondrial pathway. Unlike the BH3-interacting domain death agonist (BID) protein, BAD cannot directly trigger apoptosis but, instead, lowers the threshold at which apoptosis is induced. In many mathematical models of apoptosis, BAD is neglected or abstracted. The work presented here considers the incorporation of BAD and its various modifications in a model of the tBID-induction of BAK (Bcl-2 homologous antagonist killer) or the tBID-induction of BAX (Bcl-2-associated X protein). Steady state equations are used to develop an explicit formula describing the total concentration level of tBID, guaranteed to trigger apoptosis, as a bilinear function of the total BAD concentration level and the total anti-apoptotic protein concentration level (usually Bcl-2 or Bcl-x_L). In particular, the formula explains how the pro-apoptotic protein BAD lowers the threshold at which tBID induces BAK/BAX activation—reducing the level of total Bcl-2/Bcl-x_L available to inhibit tBID signaling in the mitochondria. Attention is then turned to the experimental data surrounding BAD phosphorylation, a process known to inhibit the pro-apoptotic effects of BAD. To address this data, the phosphorylation process is modeled following two separate kinetics in which either free unbound BAD is the assumed substrate or Bcl-x_L/Bcl-2-bound BAD is the assumed substrate. Bifurcation analysis and further analysis of the bilinear equation validate experiments, which suggest that BAD phosphorylation prevents irreversible BAK/BAX-mediated apoptosis, even when phosphorylation-induced dissociation of Bcl-x_L/Bcl-2-bound BAD is blocked. It is also shown that a cooperative, even synergistic, removal of mitochondrial BAD is seen when both types of phosphorylation are assumed possible. The presented work, however, reveals that the balance between BAD phosphorylation and dephosphorylation modulates the degree to which BAD influences the signaling from tBID to BAK/BAX. Our model shows that both the mode(s) of phosphorylation and the BAD dephosphorylation rate become important factors in determining whether BAD influences the activation of the BAK/BAX signal or not. Such potential variations in the pro-apoptotic effects of BAD are used to explain some of the inconsistent experimental data surrounding BAD phosphorylation. Nonetheless, our model serves to evaluate BAD and its sensitizing effects on the tBID-induction of BAK/BAX and thus aid in predicting when the incorporation of BAD in an apoptosis signaling model is important and when it is not.