Fig. 6: UBE2D3 promotes KAP1 phosphorylation and telomere NHEJ in a PP2A-dependent manner.
From: UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168
a Immunoblotting for pKAP1 (S824) in TRF2ts MEFs transduced with control, Ube2d3 and/or two independent Ppp2ca shRNAs at 32 °C or after 3 h at 37 °C. Representative blots from 2 independent experiments. b Quantification of chromosome fusions in TRF2ts MEFs transduced with control, Ube2d3 and two independent Ppp2ca shRNAs, upon 24 h of telomere uncapping. Two independent experiments are shown. c PP2A activity assays with immunoprecipitated PP2A from TRF2ts MEFs transduced as indicated and cultured at 32 °C or for 3 h at 37 °C to induce telomere uncapping (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). Immunoblots of input and immunoprecipitates are shown in Supplementary Fig. 8e. d PP2A phosphatase activity assay with immunoprecipitated PP2A-alpha (PP2Ac) from RNF168 mutant human cells (RIDDLE) with and without expression of ectopic HA-RNF168. Corrected for the amount of immunoprecipitated PP2A-alpha. Cells were untreated or harvested 30 min after irradiation with 3 Gy (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). The different symbols (dot, square and triangle) represent 3 biologically independent experiments. Immunoblots of input and immunoprecipitates are shown in Supplementary Fig. 8f. e Assessment of in vivo PP2A ubiquitination by immunoprecipitation of endogenous PP2A catalytic C subunit from HEK 293T cell lysates transfected with the indicated plasmids. See Supplementary Fig. 8i for input samples. Representative blots from 3 independent experiments. f Venn diagram showing the overlap in significantly altered phospho-substrates in 3 independent replicate phosphoproteomics analyses of UBE2D3-depleted (Ube2d3 sh1) or PP2A inhibitor-treated (5 μM LB100, 15 h) TRF2ts MEFs upon 3 h of telomere uncapping at 37 °C. Numbers indicate the phospho-substrates that were significantly reduced in UBE2D3-depleted cells or significantly increased in LB100-treated cells. Ube2d3 sh1 and LB100 were compared to control (shScramble) TRF2ts MEFs. g Quantification of pDNA-PKcs (S2056) foci in TRF2ts MEFs, transduced as indicated, at 32 °C (0 h) or upon telomere uncapping for 3 h at 39 °C (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). Ref reference. Source data are provided as a Source Data file.
