PIASy mediates NEMO sumoylation and NF-κB activation in response to genotoxic stress

Abstract

Protein modification by SUMO (small ubiquitin-like modifier) is an important regulatory mechanism for multiple cellular processes. SUMO-1 modification of NEMO (NF-kappaB essential modulator), the IkappaB kinase (IKK) regulatory subunit, is critical for activation of NF-kappaB by genotoxic agents. However, the SUMO ligase, and the mechanisms involved in NEMO sumoylation, remain unknown. Here, we demonstrate that although small interfering RNAs (siRNAs) against PIASy (protein inhibitor of activated STATy) inhibit NEMO sumoylation and NF-kappaB activation in response to genotoxic agents, overexpression of PIASy enhances these events. PIASy preferentially stimulates site-selective modification of NEMO by SUMO-1, but not SUMO-2 and SUMO-3, in vitro. PIASy-NEMO interaction is increased by genotoxic stress and occurs in the nucleus in a manner mutually exclusive with IKK interaction. In addition, hydrogen peroxide (H2O2) also increases PIASy-NEMO interaction and NEMO sumoylation, whereas antioxidants prevent these events induced by DNA-damaging agents. Our findings demonstrate that PIASy is the first SUMO ligase for NEMO whose substrate specificity seems to be controlled by IKK interaction, subcellular targeting and oxidative stress conditions.

A Cytosolic ATM/NEMO/RIP1 Complex Recruits TAK1 To Mediate the NF-κB and p38 Mitogen-Activated Protein Kinase (MAPK)/MAPK-Activated Protein 2 Responses to DNA Damage

“…The reaction samples were separated by SDS-PAGE, and MKK6 phosphorylation determined by immunoblotting with a phospho-specific MKK6 antibody (Cell Signaling). In vitro sumoylation of RIP1 and determination of the effects of PIASy on RIP1 sumoylation were performed by in vitro sumoylation assays described previously (16).…”

“…Upon recognition of DNA double-strand breaks, PARP-1 catalyzes the attachment of PAR chains to glutamic acid residues on PARP-1 itself, as well as other substrates. PAR-modified PARP-1 recruits the ATM kinase and the inhibitor of activated STATy (PIASy), which sumoylates the IKK regulatory subunit NEMO at lysines K277 and K309 (16,22,27). DNA damage also stimulates interactions in the nucleus between NEMO, receptor-interacting protein 1 (RIP1), and p53-induced protein with death domain (PIDD) (11).…”

p53-induced protein with a death domain (PIDD) isoforms differentially activate nuclear factor-kappaB and caspase-2 in response to genotoxic stress

“…Activation of NF-kB in response to DSB depends on two parallel signaling cascades, namely those orchestrated by the PI3 kinase like ataxia-telangectasia mutated (ATM), which is activated by DSB or higher order chromatin changes (Bakkenist and Kastan, 2003) and those regulated by PIDD, which is activated by an as yet unknown signal (Janssens et al, 2005). In response to DSB, NEMO (also known as IKKg) is sumoylated on K277 and K309 by PIASg in a PIDD-dependent fashion, and SUMO-NEMO accumulates in the nucleus (Huang et al, 2003;Janssens et al, 2005;Mabb et al, 2006). Activated ATM phosphorylates SUMO-NEMO to allow its desumoylation and subsequent monoubiquitination on the same lysine residues (Huang et al, 2003).…”

Paulownia tomentosa (Princess Tree) Extract Reduces DNA Damage and Induces DNA Repair Processes in Skin Cells

“…These diseases are characterized at the level of the skin by extreme sensitivity to sunlight, resulting in sunburn, pigmentation changes, an early onset of the appearance of skin aging signs and a greatly elevated incidence of skin cancers in particular for XP disorder [12]. These changes can be explained by long lasting DNA damages that induces prolonged cellular inflammation through the activation of the NF-κB pathway [2,13,14,15,16] and an acquired immune deficiency [17] as well as rapid accumulation of mutation leading to cell apoptosis, senescence and cell tumorigenesis [18,19,20,21].…”