Keap1/Cullin3 Modulates p62/SQSTM1 Activity via UBA Domain Ubiquitination

Abstract

Summary p62/SQSTM1 (p62) is a scaffolding protein that facilitates the formation and degradation of ubiquitinated aggregates via its self-interaction and ubiquitin binding domains. The regulation of this process is unclear but may relate to the post-translational modification of p62. In the present study, we find that Keap1/Cullin3 ubiquitinates p62 at lysine 420 within its UBA domain. Substitution of lysine 420 with an arginine diminishes p62 sequestration and degradation activity similar to that seen when the UBA domain is deleted. Overexpression of Keap1/Cullin3 in p62-WT expressing cells increases ubiquitinated inclusion formation, p62’s association with LC3 and rescues proteotoxicity. This effect is not seen in cells expressing a mutant p62 that fails to interact with Keap1. Interestingly, p62 disease mutants have diminished or absent UBA domain ubiquitination. These data suggest that the ubiquitination of p62’s UBA domain at lysine 420 may regulate p62’s function and be disrupted in p62 associated disease.

p62/SQSTM1: ‘Jack of all trades’ in health and cancer

“…Considering the analogy of APE1 transport into vacuole through selective autophagy in yeast and the cases of parkin‐mediated mitophagy and CCPG1‐mediated ER‐phagy , upstream autophagy factors such as the ULK1‐kinase complex as well as p62 binders such as Keap1 might also be translocated onto the droplets. In agreement with this hypothesis, Keap1, a p62‐interacting protein involved in Nrf2 signaling , has been found to localize to these droplets . Once the pathway is activated, p62‐positive structures together with the recruited cargo are degraded by autophagy.…”

“…Then, the variant form stabilizes Keap1 and suppresses the expression of Nrf2 targets (including p62) . Keap1 can also downregulate the p62‐Keap1‐Nrf2 pathway by promoting the ubiquitination and subsequent autophagic degradation of p62 . Although Keap1 degradation is mainly achieved through autophagy , possibly in a p62‐dependent manner , it is not clear whether the ubiquitination of p62 by Keap1 affects the autophagic degradation of Keap1 or not.…”

p62 filaments capture and present ubiquitinated cargos for autophagy

“…We next asked how dynamically p62 exchanges with the puncta observed in cells. We therefore performed FRAP experiments with p62 puncta similar to previous studies (Matsumoto et al , ; Lee et al , ), but for the first time employing cells with endogenously GFP‐tagged p62 (Figs A and B, and B). In general, p62 showed higher recovery in cells than in vitro, but the recovery of the puncta was slow and plateaued below 40% (Fig A and B).…”

p62-mediated phase separation at the intersection of the ubiquitin-proteasome system and autophagy

“…Phosphorylation of Ser409 in the UBA domain by ULK1 occurs upon proteotoxic stress and increases the binding to ubiquitin by destabilizing the UBA-dimer interface (Lim et al, 2015). Additionally, the UBA domain is subject to ubiquitylation at Lys 420; and this modification increases its ability to form condensates, probably because it interferes with the inhibitory homodimerization of the UBA domain, thereby activating ubiquitin binding (Peng et al, 2017;Lee et al, 2017). In contrast, ubiquitylation of Lys 7 in the PB1 domain negatively regulates oligomerization and cargo sequestration (Pan et al, 2016).…”