Figure 6. Sequential PTMs lead to programmed degradation of FEN1.

(A) Phosphorylation, SUMOylation and ubiquitination of FEN1 in sequence. Cells were treated with MG132 to inhibit ubiquitination-mediated protein degradation. Exogenous FEN1 was immunoprecipitated from lysates of cells overexpressing c-Myc-tagged WT, S187A, K168R, and K354R FEN1, followed by Western blotting with indicated antibodies. (B) Mutation of FEN1 phosphorylation, SUMOylation or ubiquitination sites prevents FEN1 degradation. HeLa cells overexpressing exogenous WT, S187A, K168R or K354R FEN1 were synchronized at the S and G2/M phases, lysed and analyzed by Western blotting with anti-FEN1 antibodies. (C) FEN1 degradation is blocked by inhibitors of phosphorylation (olomoucine), SUMOylation (ginkgolic acid), ubiquitination (PYR-41) and the proteasome (MG132). His-tagged FEN1 was incubated with HeLa cell lysates from cells at G2/M phase and different inhibitors for 6 hr. The mixture was then analyzed by Western blotting using anti-His and anti-actin antibodies. (D) His-tagged FEN1 labeled with ³²P was subjected to in vitro SUMOylation and ubiquitination. SUMO3-³²P-His-FEN1 and ubiquitin-³²P-His-FEN1 were purified by Ni-NTA resin, followed by anti-SUMO3 and anti-Ubiquitin antibody resins, respectively. The modified species of FEN1 immobilized on the Ni-NTA or antibody resins were incubated with extracts of cells from G2/M phase in the presence of indicated inhibitors. After 6 hr, the radioactivity in solution was quantified by liquid scintillation. The error bars represent mean ± SD from three independent experiments. (E) A model of sequential modifications to degrade FEN1. In late S phase, FEN1 is phosphorylated, resulting in dissociation from PCNA and the DNA replication fork. Once phosphorylated FEN1 is released from the DNA replication fork, it is then SUMOylated, which triggers ubiquitination by PRP19 and ultimately its degradation. See also Figure S5.