NF-kappaB regulates the expression of Nucling, a novel apoptosis regulator, with involvement of proteasome and caspase for its degradation.
Nucling is identified as a novel regulator of apoptosis. In this study, we investigated the nuclear factor-κB (NF-κB)-dependent mechanism of Nucling expression, as well as the degradation pathways mediated by proteasome system and caspase activation. Using chromatin immunoprecipitation assay in wild-type mouse embryonic fibroblasts (WT MEFs), we found that NF-κB p65 could bind to the κB motifs in the promoter regions of Nucling gene at four putative-binding sites. By real-time PCR and immunoblot, we confirmed that Nucling expression was up-regulated by tumour necrosis factor-α (TNF-α) stimulation in WT MEFs, but not in NF-κB p50 knockout MEFs. On the other hand, we investigated the degradation of Nucling in connection with proteasome and caspase by using cycloheximide chase. The results showed that Nucling is a short-lived protein, and its degradation was recovered by proteasome inhibitor MG132. Moreover, under TNF-α stimulation, degradation of Nucling was recovered by pan-caspase inhibitor zVAD-fmk. Taken together, we propose a mechanism of Nucling intracellular metabolism. Nucling expression is induced by canonical NF-κB signalling pathway, whereas Nucling is undergoing proteasome degradation, as well as being cleaved by caspase system under stress conditions. This opens a new perspective for studying the NF-κB dependent regulation mechanism of cell death and survival.
Tran
Nam Hoang
NH
The Institute for Enzyme Research, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima, 770-8503, Japan.
Sakai
Takashi
T
Kim
Sun Mi
SM
Fukui
Kiyoshi
K
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis* / Q000378:metabolism; D020169:Caspases / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D016328:NF-kappa B / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation
D000590:Amino Acid Chloromethyl Ketones; D051017:Apoptosis Regulatory Proteins; D007976:Leupeptins; D008565:Membrane Proteins; D016328:NF-kappa B; D061988:Proteasome Inhibitors; D014409:Tumor Necrosis Factor-alpha; C483107:Uaca protein, mouse; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D020169:Caspases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde