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. 2018 Sep 8;46(20):10724–10739. doi: 10.1093/nar/gky810

Figure 3.

Figure 3.

CtIP is phosphorylated by PLK1. (A) CtIP phosphorylation by PLK1 was analyzed by mass spectrometry and the potential PLK1 target sites on CtIP were identified. PLK (+): purified CtIP protein was incubated with PLK1 in vitro and then subjected to mass spectrometry. NOC (+): U2OS cells stably expressing Flag-CtIP were incubated with nocodazole (NOC) for 16 h before collection. Flag-CtIP protein was immunopurified from the cell lysate using an anti-Flag antibody and subjected to mass spectrometry. (B) Purified GST-CtIP fragments (600-897) and the indicated mutants were incubated with [γ-32P] ATP in the presence of PLK1 for the in vitro kinase assay. The radio-labeled proteins were visualized following SDS-PAGE. Coomassie Blue staining indicates the protein loading. (C) Sequence alignment of the CtIP S723 surrounding region, showing a canonical PLK1 target sequence and the PLK1 target residue S723. (D) 293T cells were co-transfected with Flag-tagged CtIP and HA-tagged PLK1 expression constructs, and treated with or without 10 μM BI2536 for 1 h before harvesting. Cell extracts were subjected to immunoprecipitation followed by western blotting with the indicated antibodies. (E) 293T cells were co-transfected with Myc-tagged CtIP and Flag-tagged PLK1 WT or PLK1-KD (kinase dead K82M/D176N) expression constructs. Cell extracts were subjected to immunoprecipitation followed by western blotting with the indicated antibodies. (F) 293T cells were co-transfected with Flag-tagged CtIP and HA-tagged PLK1 WT or analog-sensitive PLK1 mutant (PLK1-AS) expression constructs, and treated with or without the indicated amounts of 3-MB-PP1 for 16 h. The cell extracts were subjected to immunoprecipitation followed by western blotting with the indicated antibodies. (G) U2OS cells stably expressing Myc-tagged CtIP were treated with nocodazole for 16 h before harvesting. Cell extracts were subjected to immunoprecipitation followed by western blotting with the indicated antibodies. (H) CtIP-knockout (KO) HCT116 cells stably expressing Flag-CtIP WT were synchronized by double thymidine-block release; CtIP S723 phosphorylation was analyzed using pS723 antibody. Cell-cycle progression was analyzed by FACS.