Dataset Information

Profiling ubiquitin signalling with UBIMAX reveals DNA damage- and SCF^β-Trcp1-dependent ubiquitylation of the actin-organizing protein Dbn1.

ABSTRACT: Ubiquitin widely modifies proteins, thereby regulating most cellular functions. The complexity of ubiquitin signalling necessitates unbiased methods enabling global detection of dynamic protein ubiquitylation. Here, we describe UBIMAX (UBiquitin target Identification by Mass spectrometry in Xenopus egg extracts), which enriches ubiquitin-conjugated proteins and quantifies regulation of protein ubiquitylation under precise and adaptable conditions. We benchmark UBIMAX by investigating DNA double-strand break-responsive ubiquitylation events, identifying previously known targets and revealing the actin-organizing protein Dbn1 as a major target of DNA damage-induced ubiquitylation. We find that Dbn1 is targeted for proteasomal degradation by the SCF^β-Trcp1 ubiquitin ligase, in a conserved mechanism driven by ATM-mediated phosphorylation of a previously uncharacterized β-Trcp1 degron containing an SQ motif. We further show that this degron is sufficient to induce DNA damage-dependent protein degradation of a model substrate. Collectively, we demonstrate UBIMAX's ability to identify targets of stimulus-regulated ubiquitylation and reveal an SCF^β-Trcp1-mediated ubiquitylation mechanism controlled directly by the apical DNA damage response kinases.

SUBMITTER: Colding-Christensen CS

PROVIDER: S-EPMC10721886 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Profiling ubiquitin signalling with UBIMAX reveals DNA damage- and SCF<sup>β-Trcp1</sup>-dependent ubiquitylation of the actin-organizing protein Dbn1.

Colding-Christensen Camilla S CS Kakulidis Ellen S ES Arroyo-Gomez Javier J Hendriks Ivo A IA Arkinson Connor C Fábián Zita Z Gambus Agnieszka A Mailand Niels N Duxin Julien P JP Nielsen Michael L ML

Nature communications 20231214 1

Ubiquitin widely modifies proteins, thereby regulating most cellular functions. The complexity of ubiquitin signalling necessitates unbiased methods enabling global detection of dynamic protein ubiquitylation. Here, we describe UBIMAX (UBiquitin target Identification by Mass spectrometry in Xenopus egg extracts), which enriches ubiquitin-conjugated proteins and quantifies regulation of protein ubiquitylation under precise and adaptable conditions. We benchmark UBIMAX by investigating DNA double- ...[more]

PMID: 38097601

Similar Datasets

Project description:TRAIL-R2 (DR5) is a clinically-relevant therapeutic target and a key target for immune effector cells. Herein, we identify a novel interaction between TRAIL-R2 and the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2). We find that SCFSkp2 can interact with both TRAIL-R2's pre-ligand association complex (PLAC) and ligand-activated death-inducing signalling complex (DISC). Moreover, Cullin-1 interacts with TRAIL-R2 in its active NEDDylated form. Inhibiting Cullin-1's DISC recruitment using the NEDDylation inhibitor MLN4924 (Pevonedistat) or siRNA increased apoptosis induction in response to TRAIL. This correlated with enhanced levels of the caspase-8 regulator FLIP at the TRAIL-R2 DISC, particularly the long splice form, FLIP(L). We subsequently found that FLIP(L) (but not FLIP(S), caspase-8, nor the other core DISC component FADD) interacts with Cullin-1 and Skp2. Importantly, this interaction is enhanced when FLIP(L) is in its DISC-associated, C-terminally truncated p43-form. Prevention of FLIP(L) processing to its p43-form stabilises the protein, suggesting that by enhancing its interaction with SCFSkp2, cleavage to the p43-form is a critical step in FLIP(L) turnover. In support of this, we found that silencing any of the components of the SCFSkp2 complex inhibits FLIP ubiquitination, while overexpressing Cullin-1/Skp2 enhances its ubiquitination in a NEDDylation-dependent manner. DISC recruitment of TRAF2, previously identified as an E3 ligase for caspase-8 at the DISC, was also enhanced when Cullin-1's recruitment was inhibited, although its interaction with Cullin-1 was found to be mediated indirectly via FLIP(L). Notably, the interaction of p43-FLIP(L) with Cullin-1 disrupts its ability to interact with FADD, caspase-8 and TRAF2. Collectively, our results suggest that processing of FLIP(L) to p43-FLIP(L) at the TRAIL-R2 DISC enhances its interaction with co-localised SCFSkp2, leading to disruption of p43-FLIP(L)'s interactions with other DISC components and promoting its ubiquitination and degradation, thereby modulating TRAIL-R2-mediated apoptosis.

Dataset Information

Profiling ubiquitin signalling with UBIMAX reveals DNA damage- and SCF^β-Trcp1-dependent ubiquitylation of the actin-organizing protein Dbn1.

Publications

Profiling ubiquitin signalling with UBIMAX reveals DNA damage- and SCF<sup>β-Trcp1</sup>-dependent ubiquitylation of the actin-organizing protein Dbn1.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Profiling ubiquitin signalling with UBIMAX reveals DNA damage- and SCFβ-Trcp1-dependent ubiquitylation of the actin-organizing protein Dbn1.

Publications

Profiling ubiquitin signalling with UBIMAX reveals DNA damage- and SCF&lt;sup&gt;β-Trcp1&lt;/sup&gt;-dependent ubiquitylation of the actin-organizing protein Dbn1.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Profiling ubiquitin signalling with UBIMAX reveals DNA damage- and SCF^β-Trcp1-dependent ubiquitylation of the actin-organizing protein Dbn1.

Profiling ubiquitin signalling with UBIMAX reveals DNA damage- and SCF<sup>β-Trcp1</sup>-dependent ubiquitylation of the actin-organizing protein Dbn1.