Caspase-Mediated Cleavage, IAP Binding, and Ubiquitination: Linking Three Mechanisms Crucial for Drosophila NF-κB Signaling

Abstract

Innate immune responses are critical for the immediate protection against microbial infection. In Drosophila, infection leads to the rapid and robust production of antimicrobial peptides, through two NF-κB signaling pathways - IMD and Toll. The IMD pathway is triggered by DAP-type peptidoglycan, common to most Gram-negative bacteria. Signaling downstream from the peptidoglycan receptors is thought to involve K63-ubiquitination and caspase-mediated cleavage, but the molecular mechanisms remain obscure. We now show that PGN-stimulation causes caspase-mediated cleavage of the imd protein, exposing a highly conserved IAP-binding motif (IBM) at its neo-N-terminus. A functional IBM is required for the association of cleaved-IMD with the ubiquitin E3 ligase DIAP2. Through its association with DIAP2, IMD is rapidly conjugated with K63-linked polyubiquitin chains. These results mechanistically connect caspase-mediated cleavage and K63-ubiquitination in immune-induced NF-κB signaling.

Inhibitor of Apoptosis (IAP) Proteins-Modulators of Cell Death and Inflammation

“…diap2 mutant flies fail to activate NF-kB-mediated expression of antibacterial peptide genes and, consequently, rapidly succumb to bacterial infection (Leulier et al 2006a;Huh et al 2007). DIAP2-mediated signaling to NF-kB critically depends on its Ub-E3 ligase activity (Leulier et al 2006a;Huh et al 2007;Paquette et al 2010;Meinander et al 2012). In conjunction with the E2 Ub-conjugating enzymes Effete (UBC5) and UEV1a/ Bendless (UEV1a/Ubc13) (Zhou et al 2005;Paquette et al 2010), DIAP2 promotes the conjugation of K63-linked Ub chains on IMD and DREDD (also referred to as DCP-2) (Paquette et al 2010;Meinander et al 2012), the Drosophila ortholog of caspase-8.…”

“…Recruitment of DIAP2 targets DREDD for K63-linked ubiquitylation, which allows Ub-mediated aggregation and activation of DREDD (Meinander et al 2012). Active DREDD subsequently cleaves IMD (Paquette et al 2010). Upon cleavage, IMD exposes an IBM at its neo-NH 2 terminus, which binds to the BIR2/3 of DIAP2.…”

“…Upon cleavage, IMD exposes an IBM at its neo-NH 2 terminus, which binds to the BIR2/3 of DIAP2. This provides DIAP2 with an additional docking site, reinforcing complex stability and allowing DIAP2-mediated ubiquitylation of IMD, and quite possibly other components of the signaling complex (Paquette et al 2010). The Ub chains on IMD and DREDD appear to serve as scaffolds for the recruitment of dTAK1, IKK, and the precursor form of the NF-kB transcription factor Relish (Rutschmann et al 2000(Rutschmann et al , 2002Silverman et al 2000Silverman et al , 2003Lu et al 2001;Vidal et al 2001;Kanayama et al 2004;Kleino et al 2005;Zhuang et al 2006;Ferrandon et al 2007).…”

Serine/threonine acetylation of TGFβ-activated kinase (TAK1) by Yersinia pestis YopJ inhibits innate immune signaling

“…We have shown that the cleavage and subsequent ubiquitination of the imd protein are crucial events during signaling (16). Because YopJ was proposed to act as a ubiquitin-protein protease (7,8), we examined whether either of these IMD modifications were altered in stable cell lines expressing YopJ.…”

“…Cleaved-IMD is then K63-polyubiquitinated through its association with the E3 ligase DIAP2 (16). These K63-polyubiquitin chains are proposed to then recruit and activate the downstream MAP3 kinase dTAK1, as reported for mammalian NF-κB signaling pathways (17,18).…”