Abstract
Activation of initiator caspases is dependent on interacting proteins, and Ipaf [ICE (interleukin-1β-converting enzyme)-protease activating factor] {NLRC4 [NLR (Nod-like receptor) family CARD (caspase activation and recruitment domain)-containing 4]} an inflammasome component, is involved in caspase 1 activation and apoptosis. Investigating the mechanisms of Ipaf activation, we found that the C-terminal LRR (leucine-rich repeat) domain of Ipaf, through intramolecular interaction, negatively regulates its apoptosis-inducing function. In A549 lung carcinoma cells, expression of Ac-Ipaf (LRR-domain-deleted Ipaf) induced cell death that was dependent on caspase 8, but not on caspase 1. A yeast two-hybrid screen using Ac-Ipaf as bait identified human Sug1 (suppressor of gal 1), a component of the 26S proteasome, as an interacting protein. In mammalian cells Sug1 interacts and co-localizes with Ipaf. Sug1 binds to amino acids 91–253 of Ipaf, which is also the region that the LRR domain binds to. It potentiates cell death induced by Ipaf and Ac-Ipaf, and co-expression of Sug1 and Ipaf induces caspase-8-dependent cell death. Cellular complexes formed by Ipaf and Sug1 contain caspase 8. Expression of Ac-Ipaf or co-expression of Sug1 with Ipaf results in the formation of cytoplasmic aggregates and caspase 8 activation. Sug1 co-expression enabled modification of Ipaf by ubiquitination. Tagging ubiquitin molecules to Ipaf led to aggregate formation, enhanced caspase 8 interaction and activation, resulting in induction of cell death. Using RNAi (RNA interference) and dominant-negative approaches, we have shown that cell death induced by Ac-Ipaf expression or by treatment with TNF-α (tumour necrosis factor α) or doxorubicin is dependent on Sug1. Our results suggest a role for ubiquitination of Ipaf that is enabled by its interaction with Sug1, leading to caspase 8 activation and cell death.
- aggresome
- caspase 8
- interleukin-1β-converting enzyme-protease-activating factor (Ipaf)
- leucine-rich repeat domain (LRR domain)
- suppressor of gal 1 (Sug1)
- ubiquitination
Abbreviations: aa, amino acid(s); Ac-Ipaf, LRR-deleted Ipaf; ASC, apoptosis-associated speck-like protein containing a CARD; BAG, Bcl-2-associated athanogene; CARD, caspase activation and recruitment domain; Cdk, cyclin-dependent kinase; c-FLIP, cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein; CMV, cytomegalovirus; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; FADD, Fas-associated death domain; GFP, green fluorescent protein; GST, glutathione transferase; HA, haemagglutinin; HDAC, histone deacetylase; HEK-293 cell, human embryonic kidney cell; IL-1β, interleukin 1β; Ipaf, ICE (IL-1β-converting enzyme)-protease-activating factor; LRR, leucine-rich repeat; mCaspase, dominant-negative caspase mutant; mSug1, Sug1(K196M); NA, numerical aperture; NBD, nucleotide-binding domain; PARP, poly(ADP-ribose) polymerase; RFP, red fluorescent protein; RIP, receptor-interacting protein; shRNA, small-hairpin RNA; Sug, suppressor of gal; TNF, tumour necrosis factor; Ub, ubiquitin; Ub1, Ub2 or Ub4, mono-, di- or tetra-Ub; UPS, Ub–proteasome system
- © The Authors Journal compilation © 2010 Biochemical Society