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.

泛素广泛修饰蛋白质，从而调节大多数细胞功能。泛素信号传导的复杂性需要能够对动态蛋白质泛素化进行全局检测的公正方法。在这里，我们描述了 UBIMAX（通过X enopus卵提取物中的质谱法鉴定UB iquitin 靶点I ），它富集了泛素缀合蛋白，并在精确和适应性条件下量化了蛋白质泛素化的调节。我们通过研究 DNA 双链断裂响应泛素化事件、识别先前已知的靶标并揭示肌动蛋白组织蛋白 Dbn1 作为 DNA 损伤诱导的泛素化的主要靶标来对 UBIMAX 进行基准测试。我们发现，Dbn1 是 SCF ^β-Trcp1泛素连接酶蛋白酶体降解的目标，其机制是由 ATM 介导的包含 SQ 基序的先前未表征的 β-Trcp1 降解决定子的磷酸化驱动的。我们进一步表明，这种降解决定子足以诱导模型底物的 DNA 损伤依赖性蛋白质降解。总的来说，我们证明了 UBIMAX 能够识别刺激调节的泛素化靶标，并揭示由顶端 DNA 损伤反应激酶直接控制的 SCF ^β-Trcp1介导的泛素化机制。