p105 plays dual roles in NF-x13 signaling: it generates the active subunit p50 and in its precursor form inhibits NF-kappa B activation. p105 processing occurs under basal conditions and increases following signaling. lid3 kinase 13 (IKK(3) 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-kappa B signaling pathway. Here we show that A20 suppresses TNF alpha-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. Coimmunoprecipitation 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-KB inhibitory effects of A20: by affecting p105 ubiquitination and subsequently its degradation and limited processing. (C) 2017 Elsevier Inc. All rights reserved.