Interaction with Sug1 enables Ipaf ubiquitination leading to caspase 8 activation and cell death.
Activation of initiator caspases is dependent on interacting proteins, and Ipaf [ICE (interleukin-1beta-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-alpha (tumour necrosis factor alpha) 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.
Kumar
Yatender
Y
Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research (CSIR), Uppal Road, Hyderabad 500007, India.
Radha
Vegesna
V
Swarup
Ghanshyam
G
D000074183:ATPases Associated with Diverse Cellular Activities; D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D053468:CARD Signaling Adaptor Proteins / Q000235:genetics / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D053181:Caspase 8 / Q000378:metabolism*; D016923:Cell Death; D004789:Enzyme Activation; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D060588:LIM Domain Proteins; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D046988:Proteasome Endopeptidase Complex; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D054875:Ubiquitination
D048868:Adaptor Proteins, Signal Transducing; D053468:CARD Signaling Adaptor Proteins; D002135:Calcium-Binding Proteins; D060588:LIM Domain Proteins; C435692:NLRC4 protein, human; C092282:PSMC5 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; D053181:Caspase 8; D046988:Proteasome Endopeptidase Complex; D000074183:ATPases Associated with Diverse Cellular Activities