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. 2023 Dec 14;14:8293. doi: 10.1038/s41467-023-43873-0

Fig. 4. A variant β-Trcp1 degron is necessary and sufficient for inducing Dbn1 and general protein degradation in response to DSBs.

Fig. 4

a Schematic representation of the conserved variant β-Trcp1 degron in the Dbn1 C-terminus. ADF-H, actin depolymerization factor homology; HCM, helical charged motif; ABD, actin-binding domain. b Extracts were untreated or supplemented with ATM inhibitor (“ATMi”), ubiquitin E1 inhibitor (“Ub E1i”), or neddylation E1 inhibitor (“Culi”), prior to addition of linearized plasmid DNA (“DSB”). Samples were analysed by western blot at the indicated timepoints. c Recombinant Dbn1 WT, S609A, or S609D were added to Dbn1-immunodepleted extracts. Samples were collected from Dbn1-immunodepleted extract prior to addition of recombinant protein, and at the indicated timepoints following addition of protein and linearized plasmid DNA. Samples were analysed by western blot. d Schematic representation of the recombinant proteins generated by insertion of WT or mutated variant β-Trcp1 degron at the M.HpaII C-terminus. e M.HpaII protein with or without the variant β-Trcp1 degron or buffer (“no M.HpaII”) was added to extracts prior to linearized plasmid DNA. Samples were analysed by western blot at the indicated timepoints. f M.HpaII protein with WT or AQ-mutated variant β-Trcp1 degron was added to extracts prior to undamaged- (“DNA”) or linearized plasmid DNA (“DSB”). Samples were analysed by western blot at the indicated timepoints. g In silico analysis of the human proteome revealed numerous proteins containing a potential ATM/ATR-activated β-Trcp1 degron, a subset of which are involved in the DNA damage response. *indicates proteins where the S/TQ site of the putative variant β-Trcp1 degron is known to be phosphorylated. h, i DNA damage, such as DSBs, activates the apical DDR kinase ATM, which mediates phosphorylation of the actin-organizing protein Dbn1 at the S609 SQ motif. This primes the connected conserved variant degron (ii) for recognition by the F-box protein β-Trcp1, resulting in ubiquitylation by the SCFβ-Trcp1 ubiquitin E3 ligase complex and subsequent proteasomal degradation of the Dbn1 protein. Source data are provided as a Source Data file. Figures a, d, g, and h were created with BioRender.com.