ABSTRACT
As part of the genotoxic stress response, cells activate the transcription factor NF-κB. The DNA strand break sensor poly(ADP-ribose)-polymerase-1 (PARP-1) and the kinase ataxia telangiectasia mutated (ATM) act as proximal signal mediators. PARP-1 assembles a nucleoplasmic signalosome, which triggers PIASy-mediated IKKγ SUMOylation. ATM-dependent IKKγ phosphorylation and subsequent ubiquitination were implicated to activate the cytoplasmic IκB kinase (IKK) complex by unknown mechanisms. We show that activated ATM translocates in a calcium-dependent manner to cytosol and membrane fractions. Through a TRAF-binding motif, ATM activates TRAF6, resulting in Ubc13-mediated K63-linked polyubiquitin synthesis and cIAP1 recruitment. The ATM-TRAF6-cIAP1 module stimulates TAB2-dependent TAK1 phosphorylation. Both nuclear PARP-1- and cytoplasmic ATM-driven signaling branches converge at the IKK complex to catalyze monoubiquitination of IKKγ at K285. Our data indicate that exported SUMOylated IKKγ acts as a substrate. IKKγ monoubiquitination is a prerequisite for genotoxic IKK and NF-κB activation, but also promotes cytokine signaling.
Subject(s)
Cell Cycle Proteins/metabolism , Cytosol/enzymology , DNA Damage , DNA-Binding Proteins/metabolism , Inhibitor of Apoptosis Proteins/metabolism , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , TNF Receptor-Associated Factor 6/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitin/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Ataxia Telangiectasia Mutated Proteins , Calcium/metabolism , Cell Cycle Proteins/genetics , Cell Nucleus/enzymology , DNA Breaks, Double-Stranded , DNA-Binding Proteins/genetics , HeLa Cells , Hep G2 Cells , Humans , I-kappa B Kinase/metabolism , Inhibitor of Apoptosis Proteins/genetics , MAP Kinase Kinase Kinases/metabolism , Mutation , NF-kappa B/genetics , Phosphorylation , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/metabolism , Protein Binding , Protein Serine-Threonine Kinases/genetics , Protein Transport , RNA Interference , Recombinant Proteins/metabolism , TNF Receptor-Associated Factor 6/genetics , Time Factors , Transfection , Tumor Necrosis Factor-alpha/metabolism , Tumor Suppressor Proteins/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases , UbiquitinationABSTRACT
Upon genotoxic stresses, cells activate IkappaB kinases (IKKs) and the transcription factor NF-kappaB to modulate apoptotic responses. The SUMO-1 ligase PIASy and the kinase ataxia talengiectasia mutated (ATM) have been implicated to SUMOylate and phosphorylate nuclear IKKgamma (NEMO) in a consecutive mode of action, which in turn results in activation of cytoplasmic IKK holocomplexes. However, the nuclear signals and scaffold structures that initiate IKKgamma recruitment and activation are unknown. Here, we show that poly(ADP-ribose)-polymerase-1 (PARP-1) is the DNA proximal regulator, which senses DNA strand breaks and, through poly(ADP-ribose) (PAR) synthesis, assembles IKKgamma, PIASy, and ATM in a dynamic manner. Signalosome formation involves direct protein-protein interactions and binding to ADP-ribose polymers through PAR binding motifs (PARBM). Activated PARP-1 and a PARBM in PIASy are required to trigger IKKgamma SUMOylation, which in turn permits IKK and NF-kappaB activation, as well as NF-kappaB-regulated resistance to apoptosis.
