BID is cleaved by caspase-8 within a native complex on the mitochondrial membrane

Caspase-8 stably inserts into the mitochondrial outer membrane during extrinsic apoptosis. Inhibition of caspase-8 enrichment on the mitochondria impairs caspase-8 activation and prevents apoptosis. However, the function of active caspase-8 on the mitochondrial membrane remains unknown. In this study, we have identified a native complex containing caspase-8 and BID on the mitochondrial membrane, and showed that death receptor activation by Fas or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced the cleavage of BID (tBID formation) within this complex. tBID then shifted to separate mitochondria-associated complexes that contained other BCL-2 family members, such as BAK and BCL-X(L). We report that cells stabilize active caspase-8 on the mitochondria in order to specifically target mitochondria-associated BID, and that BID cleavage on the mitochondria is essential for caspase-8-induced cytochrome c release. Our findings indicate that during extrinsic apoptosis, caspase-8 can specifically target BID where it is mostly needed, on the surface of mitochondria.     

Caspase-3 feeds back on caspase-8, Bid and XIAP in type I Fas signaling in primary mouse hepatocytes

The TNF-R1 like receptor Fas is highly expressed on the plasma membrane of hepatocytes and plays an essential role in liver homeostasis. We recently showed that in collagen-cultured primary mouse hepatocytes, Fas stimulation triggers apoptosis via the so-called type I extrinsic signaling pathway. Central to this pathway is the direct caspase-8-mediated cleavage and activation of caspase-3 as compared to the type II pathway which first requires caspase-8-mediated Bid cleavage to trigger mitochondrial cytochrome c release for caspase-3 activation. Mathematical modeling can be used to understand complex signaling systems such as crosstalks and feedback or feedforward loops. A previously published model predicted a positive feedback loop between active caspases-3 and -8 in both type I and type II FasL signaling in lymphocytes and Hela cells, respectively. Here we experimentally tested this hypothesis in our hepatocytic type I Fas signaling pathway by using wild-type and XIAP-deficient primary hepatocytes and two recently characterized, selective caspase-3/-7 inhibitors (AB06 and AB13). Caspase-3/-7 activity assays and quantitative western blotting confirmed that fully processed, active p17 caspase-3 feeds back on caspase-8 by cleaving its partially processed p43 form into the fully processed p18 species. Our data do not discriminate if p18 positively or negatively influences FasL-induced apoptosis or is responsible for non-apoptotic aspects of FasL signaling. However, we found that caspase-3 also feeds back on Bid and degrades its own inhibitor XIAP, both events that may enhance caspase-3 activity and apoptosis. Thus, potent, selective caspase-3 inhibitors are useful tools to understand complex signaling circuitries in apoptosis.     

p38-mediated regulation of an Fas-associated death domain protein-independent pathway leading to caspase-8 activation during TGFbeta-induced apoptosis in human Burkitt lymphoma B cells BL41

On binding to its receptor, transforming growth factor beta (TGFbeta) induces apoptosis in a variety of cells, including human B lymphocytes. We have previously reported that TGFbeta-mediated apoptosis is caspase-dependent and associated with activation of caspase-3. We show here that caspase-8 inhibitors strongly decrease TGFbeta-mediated apoptosis in BL41 Burkitt's lymphoma cells. These inhibitors act upstream of the mitochondria because they inhibited the loss of mitochondrial membrane potential observed in TGFbeta-treated cells. TGFbeta induced caspase-8 activation in these cells as shown by the cleavage of specific substrates, including Bid, and the appearance of cleaved fragments of caspase-8. Our data show that TGFbeta induces an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and caspase-9 and -3 activation. Caspase-8 activation was Fas-associated death domain protein (FADD)-independent because cells expressing a dominant negative mutant of FADD were still sensitive to TGFbeta-induced caspase-8 activation and apoptosis. This FADD-independent pathway of caspase-8 activation is regulated by p38. Indeed, TGFbeta-induced activation of p38 and two different inhibitors specific for this mitogen-activated protein kinase pathway (SB203580 and PD169316) prevented TGFbeta-mediated caspase-8 activation as well as the loss of mitochondrial membrane potential and apoptosis. Overall, our data show that p38 activation by TGFbeta induced an apoptotic pathway via FADD-independent activation of caspase-8.     

Loss of caspase-9 reveals its essential role for caspase-2 activation and mitochondrial membrane depolarization

Caspase-9 plays an important role in apoptosis induced by genotoxic stress. Irradiation and anticancer drugs trigger mitochondrial outer membrane permeabilization, resulting in cytochrome c release and caspase-9 activation. Two highly contentious issues, however, remain: It is unclear whether the loss of the mitochondrial membrane potential DeltaPsi(M) contributes to cytochrome c release and whether caspases are involved. Moreover, an unresolved question is whether caspase-2 functions as an initiator in genotoxic stress-induced apoptosis. In the present study, we have identified a mutant Jurkat T-cell line that is deficient in caspase-9 and resistant to apoptosis. Anticancer drugs, however, could activate proapoptotic Bcl-2 proteins and cytochrome c release, similarly as in caspase-9-proficient cells. Interestingly, despite these alterations, the cells retained DeltaPsi(M). Furthermore, processing and enzyme activity of caspase-2 were not observed in the absence of caspase-9. Reconstitution of caspase-9 expression restored not only apoptosis but also the loss of DeltaPsi(M) and caspase-2 activity. Thus, we provide genetic evidence that caspase-9 is indispensable for drug-induced apoptosis in cancer cells. Moreover, loss of DeltaPsi(M) can be functionally separated from cytochrome c release. Caspase-9 is not only required for DeltaPsi(M) loss but also for caspase-2 activation, suggesting that these two events are downstream of the apoptosome.     

Real time analysis of tumor necrosis factor-related apoptosis-inducing ligand/cycloheximide-induced caspase activities during apoptosis initiation

Employing fluorescence resonance energy transfer (FRET) imaging, we previously demonstrated that effector caspase activation is often an all-or-none response independent of drug choice or dose administered. We here investigated the signaling dynamics during apoptosis initiation via the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor pathway to investigate how variability in drug exposure can be translated into largely kinetically invariant cell death execution pathways. FRET-based microscopy demonstrated dose-dependent responses of caspase-8 activation and activity within individual living HeLa cells. Caspase-8 on average was activated 45-600 min after TRAIL/cycloheximide addition. Caspase-8-like activities persisted for 15-60 min before eventually inducing mitochondrial outer membrane permeabilization. Independent of the TRAIL concentrations used or the resulting caspase-8-like activities, mitochondrial outer membrane permeabilization was induced when 10% of the FRET substrate was cleaved. In contrast, in Bid-depleted cells, caspase-8-like activity persisted for hours without causing immediate cell death. Our findings provide detailed insight into the intracellular signaling kinetics during apoptosis initiation and describe a threshold mechanism controlling the induction of apoptosis execution.     

Activation of the death receptor pathway of apoptosis by the aldehyde acrolein

Reactive alpha,beta-unsaturated aldehydes such as acrolein are major components of common environmental pollutants. As a toxic by-product of lipid peroxidation, acrolein has been implicated as a possible mediator of oxidative damage to cells and tissues in a wide variety of disease states, including atherosclerosis and neurodegenerative and pulmonary diseases. Although acrolein can induce apoptotic cell death in various cell types, the biochemical mechanisms are not understood. This study investigates the implication of the death receptor pathway in acrolein-induced apoptosis. Exposure of Chinese hamster ovary cells to acrolein caused translocation of adaptor protein Fas associated with death domain to the cytoplasmic membrane and caspase-8 activation. Kp7-6, an antagonist of Fas receptor activation, blocked apoptotic events downstream of caspase-8, such as caspase-7 activation and nuclear chromatin condensation. Acrolein activated the cross-talk pathway between the death receptor and mitochondrial pathways. Bid was cleaved to truncated-Bid, which was translocated to mitochondria. Activation of the mitochondrial pathway by acrolein was confirmed by caspase-9 activation. Inhibition of activation of either the Fas receptor or caspase-8 partially decreased acrolein-induced caspase-9 activation. These findings indicate that acrolein activates the Fas receptor pathway, which occurs upstream of the mitochondrial pathway. Caspase-9 activation still occurred despite inhibition of the Fas receptor pathway, suggesting that acrolein could also trigger the mitochondrial pathway independent of the receptor pathway. These findings improve our understanding of mechanisms of toxicity of the reactive aldehyde acrolein, which has widespread implications in multiple disease states which seem to be mediated by oxidative stress and lipid peroxidation.     

The temporal relationship between protein phosphatase, mitochondrial cytochrome c release, and caspase activation in apoptosis

Apoptosis is mediated by members of the caspase family of proteases which can be activated by release of mitochondrial cytochrome c. Additional members of the caspase family are activated at the cell surface in response to direct stimulus from the external environment such as by activation of the Fas receptor. It has been suggested that these upstream caspases directly activate the downstream caspases which would obviate a role for cytochrome c in apoptosis induced by the Fas receptor. We demonstrate that cytochrome c is released from mitochondria of Jurkat cells in response to both staurosporine and an agonistic anti-Fas antibody and that only the latter is inhibited by the caspase inhibitor z-VAD-FMK. This suggests that an upstream caspase such as caspase-8 is required for the Fas-mediated release of mitochondrial cytochrome c. The protein phosphatase inhibitor calyculin A prevented cytochrome c release and apoptosis induced by both agents, suggesting that release of cytochrome c is required in both models. Zinc, once thought of as an endonuclease inhibitor, has previously been shown to prevent the activation of caspase-3. We show that zinc prevents the activation of downstream caspases and apoptosis induced by both insults, yet does not prevent release of mitochondrial cytochrome c. The ability of calyculin A and zinc to prevent DNA digestion implies that the mitochondrial pathway is important for induction of apoptosis by both agents. These results do not support an alternative pathway in which caspase-8 directly activates caspase-3. These results also demonstrate that a critical protein phosphatase regulates the release of cytochrome c and apoptosis induced by both insults.     

Transforming growth factor-beta 1 induces apoptosis through Fas ligand-independent activation of the Fas death pathway in human gastric SNU-620 carcinoma cells

To date, two major apoptotic pathways, the death receptor and the mitochondrial pathway, have been well documented in mammalian cells. However, the involvement of these two apoptotic pathways, particularly the death receptor pathway, in transforming growth factor-beta 1 (TGF-beta 1)-induced apoptosis is not well understood. Herein, we report that apoptosis of human gastric SNU-620 carcinoma cells induced by TGF-beta 1 is caused by the Fas death pathway in a Fas ligand-independent manner, and that the Fas death pathway activated by TGF-beta 1 is linked to the mitochondrial apoptotic pathway via Bid mediation. We showed that TGF-beta 1 induced the expression and activation of Fas and the subsequent caspase-8-mediated Bid cleavage. Interestingly, expression of dominant negative FADD and treatment with caspase-8 inhibitor efficiently prevented TGF-beta 1-induced apoptosis, whereas the treatment with an activating CH11 or a neutralizing ZB4 anti-Fas antibody, recombinant Fas ligand, or Fas-Fc chimera did not affect activation of Fas and the subsequent induction of apoptosis by TGF-beta 1. We further demonstrated that TGF-beta 1 also activates the mitochondrial pathway showing Bid-mediated loss of mitochondrial membrane potential and subsequent cytochrome c release associated with the activations of caspase-9 and the effector caspases. Moreover, all these apoptotic events induced by TGF-beta 1 were found to be effectively inhibited by Smad3 knockdown and also completely abrogated by Smad7 expression, suggesting the involvement of the Smad3 pathway upstream of the Fas death pathway by TGF-beta 1.     

Caspase-3 is a component of Fas death-inducing signaling complex in lipid rafts and its activity is required for complete caspase-8 activation during Fas-mediated cell death

Since its discovery, caspase-8 has been placed at the apex of the proteolytic cascade triggered by death receptor (DR) cross-linking. Because of its capacity to interact with the cytoplasmic portion of DR, it has been suggested that caspase-8 acts independently of other caspases in the initiation of Fas and other DR signaling. In this study, we demonstrate that in Jurkat cells, caspase-3 cleavage is an early step during Fas-induced apoptosis. We show that caspase-3 processing into its p20 occurs rapidly after Fas cross-linking, in the absence of mitochondrial depolarization and caspase-9 activation. Moreover, caspase-3 is present in lipid rafts of untreated Jurkat cells and peripheral T lymphocytes. Caspase-3, caspase-8, and Fas-associated death domain are further recruited to lipid rafts of Jurkat cells following anti-Fas treatment. Fas immunoprecipitation reveals that caspase-3 is a component of the death-inducing signaling complex, suggesting that this cysteine protease is in close proximity to caspase-8. Furthermore, transduction of Jurkat cells with a caspase-3 dominant-negative form inhibits caspase-8 processing and results in inhibition of apoptosis, suggesting that caspase-3 activity is required for caspase-8 activation. Overall, these findings support a model whereby caspase-3 is a component of the death-inducing signaling complex located in lipid rafts, and as such, is involved in the amplification of caspase-8 activity by the mitochondrion.     

Loss of the Fanconi anemia group C protein activity results in an inability to activate caspase-3 after ionizing radiation

Fanconi anemia (FA) is a human genetic disease featuring cancer predisposition, genetic instability and DNA damage hypersensitivity. Although abnormalities in DNA repair and cell cycle checkpoint have been proposed as the underlying defect in this syndrome, these hypotheses did not provide full explanations of the complex phenotype. Although not exclusive of such possibilities, alterations in the control of apoptosis might account for the pleiotropic phenotype of this syndrome. We and others have previously reported a deregulation of the apoptotic response to mitomycin C, suggesting that the products of the Fanconi anemia group C protein (FANCC) contribute to the regulation of apoptosis. To explore the functional importance of the apoptotic alterations in FA we analyzed biochemical steps of the execution phase of apoptosis stimulated by another DNA damaging agent, the gamma-ray using FA cell lines derived from complementation group C (FA-C) independent patients. It is shown that the poly(ADP-ribose) polymerase, a target of caspase-3, is not cleaved in FA-C after ionizing radiation (IR). Moreover, caspase-3 is not processed in its active form and, its activity is not increased by IR in FA-C cells compared to normal cells. Altogether, these results demonstrate that loss of the FANCC activity results in a deficiency of the IR-induced apoptosis which is due to an inability to activate caspase-3. Our work suggests that apoptosis signaling induced by mitomycin C and IR is subject to common regulation involving the FANCC protein.     

Mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in activated T cells abrogates TRAIL-induced apoptosis upstream of the mitochondrial amplification loop and caspase-8

Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) induce apoptosis in many different cell types. Jurkat T cells die rapidly by apoptosis after treatment with either ligand. We have previously shown that mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) can act as a negative regulator of apoptosis mediated by the Fas receptor. In this study we examined whether MAPK/ERK can also act as a negative regulator of apoptosis induced by TRAIL. Activated Jurkat T cells were efficiently protected from TRAIL-induced apoptosis. The protection was shown to be MAPK/ERK dependent and independent of protein synthesis. MAPK/ERK suppressed TRAIL-induced apoptosis upstream of the mitochondrial amplification loop because mitochondrial depolarization and release of cytochrome c were inhibited. Furthermore, caspase-8-mediated relocalization and activation of Bid, a proapoptotic member of the Bcl family, was also inhibited by the MAPK/ERK signaling. The protection occurred at the level of the apoptotic initiator caspase-8, as the cleavage of caspase-8 was inhibited but the assembly of the death-inducing signaling complex was unaffected. Both TRAIL and Fas ligand have been suggested to regulate the clonal size and persistence of different T cell populations. Our previous results indicate that MAPK/ERK protects recently activated T cells from Fas receptor-mediated apoptosis during the initial phase of an immune response before the activation-induced cell death takes place. The results of this study show clearly that MAPK/ERK also participates in the inhibition of TRAIL-induced apoptosis after T cell activation.     

B cell receptor cross-linking triggers a caspase-8-dependent apoptotic pathway that is independent of the death effector domain of Fas-associated death domain protein.

We have previously reported that B cell receptors, depending on the degree to which they are cross-linked, can promote apoptosis in various human B cell types. In this study, we show that B cell receptors can trigger two apoptotic pathways according to cross-linking and that these pathways control mitochondrial activation in human Burkitt's lymphoma cells. Whereas soluble anti-mu Ab triggers caspase-independent mitochondrial activation, cross-linked anti-mu Ab induces an apoptotic response associated with a caspase-dependent loss of mitochondrial transmembrane potential. This B cell receptor-mediated caspase-dependent mitochondrial activation is associated with caspase-8 activation. We show here that caspase-8 inhibitors strongly decrease cross-linking-dependent B cell receptor-mediated apoptosis in Burkitt's lymphoma BL41 cells. These inhibitors act upstream from the mitochondria as they prevented the loss of mitochondrial membrane potential observed in B cell receptor-treated BL41 cells. Caspase-8 activation in these cells was also evident from the detection of cleaved fragments of caspase-8 and the cleavage of specific substrates, including Bid. Our data show that cross-linked B cell receptors induced an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and the activation of caspase-9 and caspase-3. Cells expressing a dominant negative mutant of Fas-associated death domain protein were sensitive to cross-linked B cell receptor-induced caspase-8 activation and apoptosis; therefore, this caspase-8 activation was independent of the death effector domain of Fas-associated death domain protein.     

The Temporal Relationship between Protein Phosphatase, Mitochondrial CytochromecRelease, and Caspase Activation in Apoptosis

Apoptosis is mediated by members of the caspase family of proteases which can be activated by release of mitochondrial cytochromec.Additional members of the caspase family are activated at the cell surface in response to direct stimulus from the external environment such as by activation of the Fas receptor. It has been suggested that these upstream caspases directly activate the downstream caspases which would obviate a role for cytochromecin apoptosis induced by the Fas receptor. We demonstrate that cytochromecis released from mitochondria of Jurkat cells in response to both staurosporine and an agonistic anti-Fas antibody and that only the latter is inhibited by the caspase inhibitor z-VAD-FMK. This suggests that an upstream caspase such as caspase-8 is required for the Fas-mediated release of mitochondrial cytochromec.The protein phosphatase inhibitor calyculin A prevented cytochromecrelease and apoptosis induced by both agents, suggesting that release of cytochromecis required in both models. Zinc, once thought of as an endonuclease inhibitor, has previously been shown to prevent the activation of caspase-3. We show that zinc prevents the activation of downstream caspases and apoptosis induced by both insults, yet does not prevent release of mitochondrial cytochromec.The ability of calyculin A and zinc to prevent DNA digestion implies that the mitochondrial pathway is important for induction of apoptosis by both agents. These results do not support an alternative pathway in which caspase-8 directly activates caspase-3. These results also demonstrate that a critical protein phosphatase regulates the release of cytochromecand apoptosis induced by both insults.     

Activation of the c-Jun N-terminal kinase/stress-activated protein kinase pathway by overexpression of caspase-8 and its homologs.

Caspase-8 is the most proximal caspase in the caspase cascade and possesses a prodomain consisting of two homologous death effector domains (DEDs). We have discovered that caspase-8 and its homologs can physically interact with tumor necrosis factor receptor-associated factor family members and activate the c-Jun N-terminal kinase (JNK, or stress-activated protein kinase) pathway. This ability resides in the DED-containing prodomain of these proteins and is independent of their role as cell death proteases. A point mutant in the first DED of caspase-8 can block JNK activation induced by several death domain receptors. Inhibition of JNK activation blocks apoptosis mediated by caspase-10, Mach-related inducer of toxicity/cFLIP, and Fas/CD95, thereby suggesting a cooperative role of this pathway in the mediation of caspase-induced apoptosis.     

Chemotherapy-induced apoptosis of S-type neuroblastoma cells requires caspase-9 and is augmented by CD95/Fas stimulation

Stromal or S-type tumor cells are a distinct lineage found in neuroblastoma tumors and have an important role in the biology of this disease. Anticancer agents induce apoptosis through death receptor- and mitochondria-initiated pathways. The object of this work was to determine the involvement of these pathways in the response to doxorubicin (Dox) and cisplatin (CDDP) in S-type neuroblastoma cells. Both drugs activated caspase-9 and caspase-3 but not caspase-8. Caspase-9-specific inhibition blocked S-type cell death induced by Dox. SH-EP1 cells transfected to express dominant negative mutant caspase-9, but not those expressing DN caspase-8, were resistant to Dox- and CDDP-induced apoptosis. The lack of caspase-8 involvement in chemotherapy-induced death was not the result of an intrinsic inability of these cells to activate this enzyme because when they were treated with tumor necrosis factor-related apoptosis-inducing ligand, caspase-8 was activated. We also found that both drugs up-regulated CD95/Fas expression but that CD95/Fas signaling was not necessary for cell killing. Experiments testing the response of chemotherapy-treated cells to agonists of the CD95/Fas receptor established that Dox and CDDP treatment sensitizes cells to CD95/Fas killing. Together, these results are consistent with a model in which caspase-9 is of central importance in the death mechanism utilized by these drugs in S-type cells. Although the death response is not dependent on CD95/Fas, concomitant stimulation of this receptor amplifies the death response in drug-treated cells.     

Autophagosomal membrane serves as platform for intracellular death-inducing signaling complex (iDISC)-mediated caspase-8 activation and apoptosis

Autophagy and apoptosis are two evolutionarily conserved processes that regulate cell fate in response to cytotoxic stress. However, the functional relationship between these two processes remains far from clear. Here, we demonstrate an autophagy-dependent mechanism of caspase-8 activation and initiation of the apoptotic cascade in response to SKI-I, a pan-sphingosine kinase inhibitor, and bortezomib, a proteasome inhibitor. Autophagy is induced concomitantly with caspase-8 activation, which is responsible for initiation of the caspase cascade and the mitochondrial amplification loop that is required for full execution of apoptosis. Inhibition of autophagosome formation by depletion of Atg5 or Atg3 results in a marked suppression of caspase-8 activation and apoptosis. Although caspase-8 self-association depends on p62/SQSTM1, its self-processing requires the autophagosomal membrane. Caspase-8 forms a complex with Atg5 and colocalizes with LC3 and p62. Moreover, FADD, an adaptor protein for caspase-8 activation, associates with Atg5 on Atg16L- and LC3-positive autophagosomal membranes and loss of FADD suppresses cell death. Taken together, these results indicate that the autophagosomal membrane serves as a platform for an intracellular death-inducing signaling complex (iDISC) that recruits self-associated caspase-8 to initiate the caspase-8/-3 cascade.     

Role of caspase-8 in thymus function

The thymus is the primary organ responsible for de novo generation of immunocompetent T cells that have a diverse repertoire of antigen recognition. During the developmental process, 98% of thymocytes die by apoptosis. Thus apoptosis is a dominant process in the thymus and occurs through either death by neglect or negative selection or through induction by stress/aging. Caspase activation is an essential part of the general apoptosis mechanism, and data suggest that caspases may have a role in negative selection; however, it seems more probable that caspase-8 activation is involved in death by neglect, particularly in glucocorticoid-induced thymocyte apoptosis. Caspase-8 is active in double-positive (DP) thymocytes in vivo and can be activated in vitro in DP thymocytes by T-cell receptor (TCR) crosslinking to induce apoptosis. Caspase-8 is a proapoptotic member of the caspase family and is considered an initiator caspase, which is activated upon stimulation of a death receptor (e.g., Fas), recruitment of the adaptor molecule FADD, and recruitment and subsequent processing of procaspase-8. The main role of caspase-8 seems to be pro-apoptotic and, in this review, we will discuss about the involvement of caspase-8 in (1) TCR-triggered thymic apoptosis; (2) death receptor-mediated thymic apoptosis; and (3) glucocorticoid-induced thymic apoptosis. Regarding TCR triggering, caspase-8 is active in medullary, semi-mature heat-stable antigen^hi (HAS^hi SP) thymocytes as a consequence of strong TCR stimulation. The death receptors Fas, FADD, and FLIP are involved upstream of caspase-8 activation in apoptosis; whereas, Bid and HDAC7 are involved downstream of caspase-8. Finally, caspase-8 is involved in glucocortocoid-induced thymocyte apoptosis through an activation loop with the protein GILZ. GILZ activates caspase-8, promoting GILZ sumoylation and its protection from proteasomal degradation.     

c-Abl tyrosine kinase regulates caspase-9 autocleavage in the apoptotic response to DNA damage

Activation of the initiator caspase-9 is essential for induction of apoptosis by developmental signals, oncogenic transformation, and genotoxic stress. The c-Abl tyrosine kinase is also involved in the apoptotic response to DNA damage. The present results demonstrate that c-Abl binds directly to caspase-9. We show that c-Abl phosphorylates caspase-9 on Tyr-153 in vitro and in cells treated with DNA damaging agents. Moreover, inhibition of c-Abl with STI571 blocked DNA damage-induced autoprocessing of caspase-9 to the p35 subunit and activation of caspase-3. Caspase-9(Y153F) also attenuated DNA damage-induced processing of caspase-9 to p35, activation of caspase-3, and apoptosis. These findings indicate that caspase-9 autoprocessing is regulated by c-Abl in the apoptotic response to genotoxic stress.     

The small heat shock protein alpha B-crystallin negatively regulates cytochrome c- and caspase-8-dependent activation of caspase-3 by inhibiting its autoproteolytic maturation

Caspases are universal effectors of apoptosis. The mitochondrial and death receptor pathways activate distinct apical caspases (caspase-9 and -8, respectively) that converge on the proteolytic activation of the downstream executioner caspase-3. Caspase-9 and -8 cleave procaspase-3 to produce a p24 processing intermediate (composed of its prodomain and large subunit), which then undergoes autoproteolytic cleavage to remove the prodomain from the active protease. Recently, several heat shock proteins have been shown to selectively inhibit the mitochondrial apoptotic pathway by disrupting the activation of caspase-9 downstream of cytochrome c release. We report here that the small heat shock protein alphaB-crystallin inhibits both the mitochondrial and death receptor pathways. In S-100 cytosolic extracts treated with cytochrome c/dATP or caspase-8, alphaB-crystallin inhibits the autoproteolytic maturation of the p24 partially processed caspase-3 intermediate. In contrast, neither the closely related small heat shock protein family member Hsp27 nor Hsp70 inhibited the maturation of the p24 intermediate. We also demonstrate that alphaB-crystallin co-immunoprecipitates with the p24 partially processed caspase-3 in vivo. Taken together, our results demonstrate that alphaB-crystallin is a novel negative regulator of apoptosis that acts distally in the conserved cell death machinery by inhibiting the autocatalytic maturation of caspase-3.