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. 2015 Apr 23;11(5):704–714. doi: 10.1016/j.celrep.2015.03.058

Figure 4.

Figure 4

MLN4924 Blocks Ku Ubiquitylation after DNA Damage

(A) In vivo ubiquitylation assay. RPE-1 cells expressing Ku70 endogenously tagged with GFP (lanes 2–7) or RPE-1 cells stably expressing GFP (lane 1) were pre-treated with DMSO, 3 μM MLN4924, or 3 μM DNA-PK inhibitor for 1 hr prior to treatment with 500 μM phleomycin (Phleo) for 1 hr as indicated. Cell lysates were immunoprecipitated (IP) with GFP-specific antibody and immunoblotted (IB) with indicated antibodies. GFP-Ku70 IP was done under stringent conditions (see Experimental Procedures). Black arrows indicate GFP-Ku70 and GFP. Phosphorylated Ser824 of KAP1 is used as a DNA-damage marker.

(B) Identification of Ku ubiquitylation sites by quantitative LC-MS/MS. RPE-1 cells stably expressing Ku70 endogenously tagged with GFP were labeled with light, medium, or heavy SILAC isotopes and treated as indicated. Ku70 was enriched with GFP-Trap agarose under stringent washing conditions. Enriched proteins were resolved by SDS-PAGE and proteolysed in gel with trypsin. Peptides were extracted from gel and analyzed by LC-MS/MS. SILAC ratio M/L >2 represents induction upon DNA damage; SILAC ratio H/M < 0.5 represents inhibition by MLN4924 (see also Table S2).

(C) Schematic representation of Ku70 and Ku80 domains with neddylation-dependent ubiquitylation sites identified in (B). vWA, von Willebrand factor A; C-term, C terminus; SAP, SAF-A/B, Acinus, and PIAS.

(D) Positions of DNA-damage-induced neddylation-dependent ubiquitylation sites in the context of the structure of the Ku heterodimer (ID:1JEQ). Ku70 and Ku80 are in orange and red, respectively, and the ubiquitylated side chains are in black.

(E) Model. (1, 2) Ku and the NHEJ complex are recruited to sites of DSBs. (3) Neddylation-dependent ubiquitylation of Ku following completion of DNA repair. (4) Ku and NHEJ factors are released from sites of DNA damage. VCP might target ubiquitylated Ku to proteasome for degradation. N8, NEDD8; Ub, ubiquitin; P, phosphorylation (see text for details). See also Figure S4.