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 ( UB iquitin target Identification by M ass spectrometry in X enopus 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-organising protein Dbn1 as a novel 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 novel targets of stimulus-regulated ubiquitylation and reveal an SCF β-Trcp1 -mediated ubiquitylation mechanism controlled directly by the apical DNA damage response kinases.

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

Profiling ubiquitin signaling with UBIMAX reveals DNA damage- and SCFβTRCP-dependent ubiquitylation of the actin-organizing protein Dbn1