A20 inhibits both the degradation and limited processing of the NF-κB p105 precursor: A novel additional layer to its regulator role.

p105 plays dual roles in NF-κB signaling: it generates the active subunit p50 and in its precursor form inhibits NF-κB activation. p105 processing occurs under basal conditions and increases following signaling. IκB kinase ß (IKKß) mediates phosphorylation at the C-terminal domain of p105, leads to accelerated processing and degradation of the precursor. A20 is a dual function deubiquitinating and ubiquitin ligating enzyme, involving in turning-off the NF-κB signaling pathway. Here we show that A20 suppresses TNFα-induced proteolysis of p105. We demonstrate that A20 inhibits both the signal induced processing and the degradation of p105 upstream to IKK stimulation. In addition, A20 represses the constitutive processing of the precursor protein in IKK independent manner. Silencing of A20 in cells by siRNA, restores p105 processing and the generation of p50. Functional analysis of A20, shows that the ubiquitin ligase activity mediated by its zinc finger domain (ZF), is required for the basal processing inhibitory effect. Its N-terminal ovarian tumor (OTU) domain, however, is not obligatory. We show that A20 inhibits p50 generation in cells by reducing the ubiquitination of its precursor, p105. Co-immunoprecipitation experiments show that A20 is immunoprecipitated by p105 only in the presence of its recently identified ligase, KPC1. Our data propose an additional novel mechanism to explain the known NF-κB inhibitory effects of A20: by affecting p105 ubiquitination and subsequently its degradation and limited processing.

ABIN-1 negatively regulates NF-kappaB by inhibiting processing of the p105 precursor.

p105 plays dual roles in NF-kappaB signaling: in its precursor form it inhibits NF-kappaB activation, but limited processing by the ubiquitin system generates the p50 active subunit of the transcription factor. Here we show that ABIN-1, an A20-binding protein that is also known to attenuate NF-kappaB activation, inhibits p105 processing. p105 and ABIN-1 physically interact with one another, but the binding is not necessary for inhibition of processing. Rather, it appears to stabilize ABIN-1 and to increase its level, which further augments its inhibitory effect. Deletion of the processing inhibitory domain (PID) of p105 abrogates the inhibition which also requires the ABIN homology domain (AHD)-2 of ABIN-1. Together, the effects of ABIN-1 on p105 processing and of p105 on stabilizing ABIN-1 act to potentiate the NF-kappaB inhibitory activity of ABIN-1.