XIAP Activity Dictates Apaf-1 Dependency for Caspase 9 Activation (original) (raw)
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Coupling of caspase-9 to Apaf1 in response to loss of pRb or cytotoxic drugs is cell-type-specific
The EMBO Journal, 2004
Inactivation of the tumor suppressor Rb in the mouse induces cell death, which depends entirely (in lens, CNS) and only partly (PNS, skeletal muscles) on Apaf1/Ced4, an apoptosomal factor thought to be required for processing procaspase-9 following mitochondrial permeabilization. Here, we report that in response to cytotoxic drugs, Apaf1 À/À primary myoblasts but not fibroblasts undergo bona fide apoptosis. Cell demise was associated with disruption of mitochondria but not endoplasmic reticulum. Processing of procaspase-9 occurred in Apaf1 À/À myoblasts but not fibroblasts, and ablation of Casp9 prevented drug-induced apoptosis in both cell types. Deregulation of the Rb pathway by overexpression of E2F1 also induced caspase-9-dependent, Apaf1-independent apoptosis in myoblasts. Despite its requirement for apoptosis in vitro, mutation in Casp9 abrogated cell death in the nervous system and lens but only partly in skeletal muscles of Rb-deficient embryos. In addition, developmental cell death in fetal liver and PNS was not inhibited in Casp9 À/À embryos. Therefore, loss of pRb elicits apoptosome-dependent and apoptosome-independent cell death, and the requirement and coupling of caspase-9 to Apaf1 are both context-dependent.
2007
During apoptosis, the initiator caspase 9 is activated at the apoptosome after which it activates the executioner caspases 3 and 7 by proteolysis. During this process, caspase 9 is cleaved by caspase 3 at Asp 330 , and it is often inferred that this proteolytic event represents a feedback amplification loop to accelerate apoptosis. However, there is substantial evidence that proteolysis per se does not activate caspase 9, so an alternative mechanism for amplification must be considered. Cleavage at Asp 330 removes a short peptide motif that allows caspase 9 to interact with IAPs (inhibitors of apoptotic proteases), and this event may control the amplification process. We show that, under physiologically relevant conditions, caspase 3, but not caspase 7, can cleave caspase 9, and this does not result in the activation of caspase 9. An IAP antagonist disrupts the inhibitory interaction between XIAP (X-linked IAP) and caspase 9, thereby enhancing activity. We demonstrate that the N-terminal peptide of caspase 9 exposed upon cleavage at Asp 330 cannot bind XIAP, whereas the peptide generated by autolytic cleavage of caspase 9 at Asp 315 binds XIAP with substantial affinity. Consistent with this, we found that XIAP antagonists were only capable of promoting the activity of caspase 9 when it was cleaved at Asp 315 , suggesting that only this form is regulated by XIAP. Our results demonstrate that cleavage by caspase 3 does not activate caspase 9, but enhances apoptosis by alleviating XIAP inhibition of the apical caspase.
Cell, 2000
cytochrome c complex then recruits the initiator caspase of this pathway, procaspase-9 and induces its autoactivation (Li et al., 1997b; Zou et al., 1999). Caspase-9 in turn activates downstream caspases including caspase-3, -6, and -7 (Li et al., 1997b; Srinivasula et al., ). University of Texas Southwestern Proteins of the Bcl-2 family are major regulators of Medical Center at Dallas the mitochondria-initiated caspase activation pathway Dallas, Texas 75235 (reviewed by Adams and Cory, 1998). The anti-apoptotic members of this family, including Bcl-2 and Bcl-X L , preserve mitochondrial integrity and prevent the release of Summary cytochrome c in the presence of apoptotic stimuli (Kluck et al., 1997; Yang et al., 1997). Conversely, the proapo-We report here the identification of a novel protein, ptotic members of this family such as Bad, Bax, Bid, Smac, which promotes caspase activation in the cytoand Bim move from other cellular compartments to mitochrome c/Apaf-1/caspase-9 pathway. Smac promotes chondria in response to apoptotic stimuli and promote caspase-9 activation by binding to inhibitor of apocytochrome c release (reviewed by Gross et al., 1999). Inhibitors of apoptosis proteins, IAPs, are another ptosis proteins, IAPs, and removing their inhibitory family of proteins that regulate the cytochrome c/Apaf-1 activity. Smac is normally a mitochondrial protein but caspase activating pathway (reviewed by Deveraux is released into the cytosol when cells undergo apoand Reed, 1999). Initially identified in the genome of a ptosis. Mitochondrial import and cleavage of its signal baculovirus as suppressors of apoptosis in host cells, peptide are required for Smac to gain its apoptotic endogenous IAPs have been found in a variety of organactivity. Overexpression of Smac increases cells' senisms including seven in mammals so far (Crook et al., sitivity to apoptotic stimuli. Smac is the second mito-1993; Deveraux and Reed, 1999). The antiapoptotic acchondrial protein, along with cytochrome c, that protivity of IAPs has been attributed to the conserved bacumotes apoptosis by activating caspases. lovirus IAP repeat (BIR) domain (Takahashi et al., 1998). Three human IAPs, XIAP, c-IAP-1, and c-IAP-2 have Introduction been shown to bind procaspase-9 and prevent its activation (Deveraux et al., 1998). These IAPs can also di-One of the key regulatory steps for apoptosis is the rectly bind and inhibit active caspases (Deveraux et al., activation of caspases, the intracellular cysteine prote-1998). In Drosophila, the anti-apoptotic activity of IAPs ases that cleave substrates after aspartic acid residues is countered by Reaper, Hid, and Grim (Vucic et al., (reviewed by Thornberry and Lezebnik, 1998). Existing 1998; McCarthy and Dixit, 1998; Goyal et al., 2000). as inactive zymogens in living cells, apoptotic caspases Mammalian homologs of Reaper, Hid, and Grim have become activated during apoptosis either through autonot been identified. catalysis or cleavage by other caspases. Active caspases Despite significant progress in dissecting the cytothen cleave many important intracellular substrates, chrome c-mediated apoptotic pathway, several puzleading to the characteristic morphological changes aszling experimental observations remain to be explained. sociated with apoptotic cells. These changes include First of all, certain types of cells are responsive to michromatin condensation, DNA fragmentation into nucleocroinjected cytochrome c while others are not (Li et somal fragments, nuclear membrane break down, exteral., 1997a). Second, healthy neurons do not respond nalization of phosphatidylserine, and formation of apoto microinjected cytochrome c unless they have been ptotic bodies that are readily taken up by phagocytosis subjected to NGF withdrawal for a certain period of time, gaining the status of "competent to die" (Deshmukh and (reviewed by Thornberry and Lezebnik, 1998).
The IAP-antagonist ARTS initiates caspase activation upstream of cytochrome C and SMAC/Diablo
Cell Death and Differentiation, 2012
ARTS (Sept4_i2) is a pro-apoptotic tumor suppressor protein that functions as an antagonist of X-linked IAP (XIAP) to promote apoptosis. It is generally thought that mitochondrial outer membrane permeabilization (MOMP) occurs before activation of caspases and is required for it. Here, we show that ARTS initiates caspase activation upstream of MOMP. In living cells, ARTS is localized to the mitochondrial outer membrane. In response to apoptotic signals, ARTS translocates rapidly to the cytosol in a caspase-independent manner, where it binds XIAP and promotes caspase activation. This translocation precedes the release of cytochrome C and SMAC/Diablo, and ARTS function is required for the normal timing of MOMP. We also show that ARTS-induced caspase activation leads to cleavage of the pro-apoptotic Bcl-2 family protein Bid, known to promote MOMP. We propose that translocation of ARTS initiates a first wave of caspase activation that can promote MOMP. This leads to the subsequent release of additional mitochondrial factors, including cytochrome C and SMAC/Diablo, which then amplifies the caspase cascade and causes apoptosis.
Protein Kinase A Regulates Caspase-9 Activation by Apaf-1 Downstream of Cytochrome c
Journal of Biological Chemistry, 2005
The cyclic AMP signal transduction pathway modulates apoptosis in diverse cell types, although the mechanism is poorly understood. A critical component of the intrinsic apoptotic pathway is caspase-9, which is activated by Apaf-1 in the apoptosome, a large complex assembled in response to release of cytochrome c from mitochondria. Caspase-9 cleaves and activates effector caspases, predominantly caspase-3, resulting in the demise of the cell. Here we identified a distinct mechanism by which cyclic AMP regulates this apoptotic pathway through activation of protein kinase A. We show that protein kinase A inhibits activation of caspase-9 and caspase-3 downstream of cytochrome c in Xenopus egg extracts and in a human cell-free system. Protein kinase A directly phosphorylates human caspase-9 at serines 99, 183, and 195. However, mutational analysis demonstrated that phosphorylation at these sites is not required for the inhibitory effect of protein kinase A on caspase-9 activation. Importantly, protein kinase A inhibits cytochrome c-dependent recruitment of procaspase-9 to Apaf-1 but not activation of caspase-9 by a constitutively activated form of Apaf-1. These data indicate that extracellular signals that elevate cyclic AMP and activate protein kinase A may suppress apoptosis by inhibiting apoptosome formation downstream of cytochrome c release from mitochondria.