ARK, the Apaf-1 related killer in Drosophila, requires diverse domains for its apoptotic activity

Abstract

In mammals and Drosophila, apoptotic caspases are under positive control of the CED-4-like proteins Apaf-1 and ARK, respectively. In an EMS-mutagenesis screen, we isolated 33 ark mutants as recessive suppressors of hid-induced apoptosis. The ark mutants are loss-of-function alleles characterized by reduced developmental apoptosis. Using the phenotypic series of these alleles, we identified helical domain I in the nucleotide oligomerization domain as critical for ARK's apoptotic activity. Interestingly, the WD40 region may also have an unanticipated positive requirement for the apoptotic activity of ARK. Considering structural information, we discuss the roles of these domains for assembly and activity of the ARK apoptosome, and propose that the WD40 region is anti-apoptotic in the absence of apoptotic signals, and pro-apoptotic in the presence of such signals. Furthermore, a defined null allele reveals that ark is required for most, but not all apoptosis suggesting the existence of an ARK-independent apoptotic pathway. Subsequently, the CED-4-like genes Apaf-1 (apoptotic protease activating factor-1) in mammals and ARK (Apaf-1-related killer, also known as Dark, D-Apaf-1 and Hac-1) in Drosophila were discovered.3-6 CED-4-like proteins are characterized by the presence of a N-terminal caspase activation and recruitment domain (CARD), and a nucleotide oligomerization domain (NOD, also referred to as CED-4 homology domain and nucleotide-binding domain). Based on structural analysis of inactive, ADP-bound WD40-depleted Apaf-1, the NOD was divided into several distinct domains: a/b domain, helical domain I (HD1), winged-helix domain (WHD), and helical domain II (HD2).7 Apaf-1 and ARK also contain in their C-terminal half a series of WD40 repeats which are not found in CED-4 (reviewed in Cain et al. 8 ). WD40 repeats are short B40 amino-acid motifs, often terminating in a Trp-Asp (WD) dipeptide, and are thought to form a circularized b-propeller structure. WD40-repeat containing proteins coordinate multi-protein complex assemblies, where the repeating units serve as a rigid scaffold for protein interactions. 9Among the CED-4-like proteins, Apaf-1 is best characterized both biochemically and structurally. Apaf-1 functions to multimerize and activate Caspase-9, an initiator caspase that triggers apoptosis when activated. 8 The CARD of Apaf-1 is required for homotypic interaction with the CARD of Caspase-9.10 However, inactive Apaf-1 is a globular monomer that is kept in an auto-inhibited state by complex formation between the CARD and the WD40 repeats, 11 thus preventing CARD-CARD interactions. Hence, the WD40 repeats have an inhibitory function for Apaf-1 activity in the absence of apoptotic signals. Consistently, deletion of the WD40 repeats results in the activation of Apaf-1. 12,13 In response to apoptotic stimuli, cytochrome c is released from mitochondria and binds to the WD40 repeats, thus displacing the CARD from WD40 inhibition. This displacement allows binding of dATP/ATP to the NOD, which then promotes assem...

Physiological apoptosis of polar cells during Drosophila oogenesis is mediated by Hid-dependent regulation of Diap1

“…18,24,25 Our analysis of dronc and dark null mutations indicates that both are necessary for PC apoptosis cell autonomously in PCs destined to die. The partnership between Dronc and Dark thus seems maintained in the case of PC apoptosis, further supporting the idea that Dronc and Dark are universal partners for induction of apoptosis.…”

Caspase function in programmed cell death

“…41 Loss-of-function mutations in ark block most developmentally programmed cell death, resulting in extra cells in the embryos, hyperplasia of the central nervous system and other abnormalities. [60][61][62] ark mutants essentially phenocopy dronc mutants and show a block in the removal of larval salivary glands, but the histolysis of midgut appears normal in ark-null animals. [60][61][62] Caspase activation is severely impaired in the absence of ARK, suggesting that ARK is required for DRONC activation.…”

“…[60][61][62] ark mutants essentially phenocopy dronc mutants and show a block in the removal of larval salivary glands, but the histolysis of midgut appears normal in ark-null animals. [60][61][62] Caspase activation is severely impaired in the absence of ARK, suggesting that ARK is required for DRONC activation. 60 Structurally, ARK is more similar to its mammalian counterpart Apaf-1 than to CED-4, in that it consists of several WD40 repeats not found in CED-4.…”

Genetic characterization of two gain-of-function alleles of the effector caspase DrICE in Drosophila

“…[12][13][14] In fact, mutants of diap1, dronc and drICE genes were isolated in genetic screens searching for modifiers of the eye ablation phenotypes caused by reaper or hid overexpression. [21][22][23][24][25][26][27] Mammalian IAP antagonists are Smac/Diablo and HtrA2/Omi, which function similarly to the RHG proteins. 28 Abbreviations: ALPS, autoimmune lymphoproliferative syndrome; APF, after puparium formation; Ced-3, cell death defective 3; CyO, curly of Oster; Dcp-1, death caspase 1; DIAP1, death-associated inhibitor of apoptosis 1; DNA, desoxyribonucleic acid; drICE, death-related ICE (interleukin converting enzyme); dronc, death regulator Nedd2-like caspase; EMS, ethyl methanesulfonate; Ey, eyeless; Flp, flippase; FRT82B, flippase recombination target at 82B; GFP, green fluorescent protein; Gh, GMR-hid; Hid, head involution defective; GMR, glass multimer reporter; HtrA2, high-temperature-required protein A2; IAP, inhibitor of apoptosis protein; PCR, polymerase chain reaction; R8, photoreceptor 8; RHG, reaper, hid, grim; Smac/Diablo, second mitochondria-derived activator of caspases/direct IAP binding protein with low pI; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; Ubi, ubiquitous; wt, wild-type; XIAP, X-linked inhibitor of apoptosis…”

“…[12][13][14] In fact, mutants of diap1, dronc and drICE genes were isolated in genetic screens searching for modifiers of the eye ablation phenotypes caused by reaper or hid overexpression. [21][22][23][24][25][26][27] Mammalian IAP antagonists are Smac/Diablo and HtrA2/Omi, which function similarly to the RHG proteins. [28][29][30][31] Both IAPs and IAP antagonists are under tight control by various mechanisms to ensure proper regulation of caspase activity.…”