Volume 47, Issue 3, Pages 444-456 (August 2012) Sequential Posttranslational Modifications Program FEN1 Degradation during Cell-Cycle Progression  Zhigang Guo, Julie Kanjanapangka, Na Liu, Songbai Liu, Changwei Liu, Zhenxing Wu, Yingjie Wang, Tiffany Loh, Claudia Kowolik, Joonas Jamsen, Mian Zhou, Khue Truong, Yuan Chen, Li Zheng, Binghui Shen  Molecular Cell  Volume 47, Issue 3, Pages 444-456 (August 2012) DOI: 10.1016/j.molcel.2012.05.042 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 47, 444-456DOI: (10.1016/j.molcel.2012.05.042) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 FEN1 Is Degraded at G2/M Phase via the Proteasome Pathway (A) Endogenous FEN1 levels during the cell cycle. HeLa cells were synchronized at G2/M and then released and collected at the indicated time points. FEN1 levels in total cell lysates were determined by western blotting using an anti-FEN1 antibody. (B) Dynamics of FEN1 modifications during cell cycle. HeLa cells were synchronized and released as shown in Figure 1A. Immunoprecipitates by anti-FEN1 antibody were tested by blotting with different anti-FEN1-modification antibodies as indicated. (C) MG132 inhibits FEN1 degradation in G2/M phase. HeLa cells with or without MG132 treatment were synchronized at the S and G2/M phases. FEN1 levels in total cell lysates were determined by western blotting and quantified. (D) Degradation of His-tagged FEN1 by cell extracts. Purified recombinant His-FEN1 was incubated with extracts of cells in S or G2/M phase. Aliquots were taken at the indicated times and His-FEN1 detected by western blotting using an anti-His antibody. Quantitative results are shown in the bottom panel. (E) His-FEN1 was 32P labeled in vitro and incubated with G2/M cell extract for the indicated times. His-FEN1 was pulled down with Ni-NTA beads and the radioactivity in solution measured by liquid scintillation. Radioactivity from 32P-FEN1 in G2/M cell extracts, ■; radioactivity from 32P-FEN1 in G2/M cell extracts +MG132, ●. The error bars in all panels represent mean ± SD from three independent experiments. See also Figure S1. Molecular Cell 2012 47, 444-456DOI: (10.1016/j.molcel.2012.05.042) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 SUMO3 Modification Mediates FEN1 Degradation (A) Exogenous expression of SUMO3 in HeLa cells reduces FEN1 levels. Western blotting of FEN1 levels in HeLa cells overexpressing SUMO1, SUMO2, or SUMO3 with and without UBC9 and/or SENP1. (B) Validation of SUMO3 modification site in vitro. SUMOylation was conducted in vitro with purified SUMO3 protein and WT or K168R FEN1 and then the reactions analyzed by western blot using an anti-FEN1 antibody. (C) Validation of SUMO3 modification site in cells. HeLa cells were cotransfected with His-tagged SUMO3 and c-Myc-tagged WT or K168R FEN1. Shown is the western blot of the total cell lysates with or without Ni-NTA purification. (D) Degradation of WT and K168R FEN1 in G2/M cell extracts. WT or K168R His-FEN1 was incubated with G2/M cell extract for the indicated times. His-FEN1 was analyzed by western blotting using an anti-His antibody. (E) 32P-labeled WT or K168R FEN1 was incubated with G2/M cell extract for the indicated times with or without SENP1 or MG132. His-FEN1 was pulled down by Ni-NTA beads and the radioactivity measured by liquid scintillation. WT FEN1 incubated in G2/M cell extract, ■; K168R FEN1 incubated in G2/M extract, ▿; WT FEN1 incubated with G2/M extract and SENP1, ▴; and WT FEN1 incubated with G2/M extract and MG132 proteasome inhibitor, ●. The error bars in the (D) and (E) represent mean ± SD from three independent experiments. See also Figure S2. Molecular Cell 2012 47, 444-456DOI: (10.1016/j.molcel.2012.05.042) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 SUMO3 Modification of FEN1 Is Phosphorylation Dependent (A) Phosphorylation enhances SUMO3 modification of FEN1 in vitro. Wild-type or in vitro phosphorylated FEN1 were in vitro SUMOylated with SUMO3. Shown is the western blot of the reaction mixtures probed with anti-FEN1 antibody. (B) Elimination of phosphorylation disrupts SUMO3 conjugation. HeLa cells were cotransfected with plasmids encoding His-tagged SUMO3 and c-Myc-tagged WT, S187A, or S187D FEN1. Cell lysates were analyzed directly by western blotting or were pulled down by Ni-NTA and then analyzed by western blotting. (C) Olomoucine treatment inhibits FEN1 modification by SUMO3. HeLa cells overexpressing His-SUMO3- and c-Myc-tagged FEN1 were treated with or without olomoucine and lysed. Cell lysates were directly analyzed by western blotting or were pulled down with Ni-NTA and then analyzed by western blotting. (D) Olomoucine inhibits SUMO3-induced FEN1 degradation in cells. HeLa cells overexpressing His-SUMO3 were treated with olomoucine or MG132. Cells were lysed and analyzed by western blotting. Quantitation is shown in the bottom panel. (E) Degradation of WT, S187A, or S187D FEN1 in G2/M cell extract. WT or mutant His-FEN1 was incubated with G2/M cell extract for the indicated times. Shown is the western blotting of His-FEN1 using an anti-His antibody. (F) Interaction of the SUMOylation ligase UBC9 with WT, S187A, or S187D FEN1 in cells. HeLa cells overexpressing c-Myc-tagged WT, S187A, or S187D FEN1 were lysed. C-Myc-tagged proteins were purified with agarose resin coupled with anti-c-Myc antibody and western blotted with UBC9 and FEN1. The error bars in the (D) and (E) represent mean ± SD from three independent experiments. See also Figure S3. Molecular Cell 2012 47, 444-456DOI: (10.1016/j.molcel.2012.05.042) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Identification of the FEN1 Ubiquitin Ligase (A) In vitro ubiquitination. His-tagged WT or K354R FEN1 and ubiquitin protein were incubated with a HeLa cell fraction that served as a mixture of ubiquitination enzymes (Boston Biochem Inc, K960). The reaction mixture was purified by Ni-NTA resin and analyzed by western blotting using an anti-ubiquitin antibody. (B) Ubiquitination of FEN1 in cells. Lysates of HeLa cells overexpressing HA-tagged Ubi, His-tagged WT, or K354R FEN1 were analyzed directly by western blot or were purified by Ni-NTA followed by western blot. (C) Western blot analysis of FEN1 levels in cell lysates from HeLa cells transfected with plasmids encoding Ubi. (D) PYR-41 inhibits SUMO3-induced FEN1 degradation in cells. HeLa cells overexpressing His-SUMO3 were treated with the ubiquitination inhibitor PYR-41 or proteasome inhibitor MG132. Cells were lysed and analyzed by western blotting. (E) 32P-labeled WT or K354R FEN1 was incubated with G2/M cell extract for the indicated time with or without PYR-41 or MG132. His-FEN1 was pulled down by Ni-NTA beads, and the radioactivity in solution was measured by liquid scintillation. WT FEN1 incubated with G2/M cell extract, ■; K354R FEN1 incubated with G2/M extract, ▿; WT FEN incubated with G2/M extract and PYR-41, ▴; and WT FEN1 incubated with G2/M extract and MG132, ●. The error bars represent mean ± SD from three independent experiments. (F) Coimmunoprecipitation assay from HeLa cell lysates using a FEN1-specific antibody and then western blotting using antibodies against UBE1, UBE2M, PRP19, and UBQLN4. (G) Antibody depletion and in vitro ubiquitination assay. His-tagged FEN1 was incubated with ubiquitin and HeLa cell fractions, with or without depletion by the indicated antibodies. The reaction mixture was then analyzed by western blotting using anti-ubiquitin antibody. (H) In vitro ubiquitination of FEN1 with purified UBE1, UBE2M, and PRP19. Shown is the western blotting of reaction products using an anti-FEN1 antibody. See also Figure S4. Molecular Cell 2012 47, 444-456DOI: (10.1016/j.molcel.2012.05.042) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 SUMO3 Modification Induces FEN1 Ubiquitination (A) SUMO3 enhances ubiquitination of FEN1 in cells. HeLa cells were cotransfected with plasmids encoding c-Myc FEN1, HA-Ubi, and/or His-SUMO1/2/3. c-Myc-FEN1 was pulled down by beads precoated with anti-c-Myc antibody, followed by western blotting analysis using an anti-HA antibody. Total cell lysate was also analyzed by western blotting using anti-c-Myc or anti-actin antibodies. (B) The K168R FEN1 mutation is resistant to SUMO3-stimulated ubiquitination in cells. HeLa cells overexpressing His-SUMO3, HA-Ubi, or/and c-Myc-tagged WT or K168R FEN1 were lysed and pulled down by anti-c-Myc antibody, followed by western blotting using an anti-HA antibody. (C) In vitro ubiquitination of SUMOylated FEN1. FEN1 SUMOylated in vitro with SUMO1, SUMO2, or SUMO3 was ubiquitinated with purified UBE1, UBE2M, PRP19, and biotinylated ubiquitin. The ubiquitination mixture was then purified by streptavidin agarose, followed by western blotting using an anti-FEN1 antibody. (D) PRP19 preferentially binds to SUMO3-FEN1 in cells. Lysates of HeLa cells cotransfected with c-Myc-FEN1 and His-SUMO1/2/3 were pulled down with c-Myc antibody and detected by western blotting using anti-PRP19 antibody. Quantitative results are shown in the bottom panel. The error bars represent mean ± SD from three independent experiments. Molecular Cell 2012 47, 444-456DOI: (10.1016/j.molcel.2012.05.042) Copyright © 2012 Elsevier Inc. Terms and Conditions

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. Molecular Cell 2012 47, 444-456DOI: (10.1016/j.molcel.2012.05.042) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 Defective FEN1 Degradation Results in Cell-Cycle Delay (A) Western blot analysis of cell lines with endogenous FEN1 depletion and exogenous FEN1 overexpression. (B) Western blot analysis of FEN1, Cyclin B, and Cyclin E expression during the cell cycle. Parental cells (Ctrl) or cells overexpressing WT, K168R, or K354R FEN1 were synchronized at G2/M phase and released. Ectopically expressed FEN1 was detected by anti-Flag antibody. (C) FACS profile of cells examined in (B). (D) FACS analysis of cell-cycle phase of unsynchronized cells overexpressing WT, K168R, or K354R FEN1 is shown in the left panel, while the quantification of FACS analysis is shown in right panel. (E) Chromosome number counting. (F) Inhibition of ubiquitination of Cyclin B by FEN1. Cyclin B was ubiquitinated in vitro using a ubiquitination kit (Boston Biochemical) and biotin-ubiquitin in the presence of gradient amounts of FEN1 or BSA. Ubiquitinated samples were pulled down by Stratavidin beads, followed by western blotting using anti-Cyclin B antibody. The relative amount of ubiquitinated Cyclin B was quantified in the bottom panel. (G) Inhibition of degradation of Cyclin B by FEN1. Degradation assay of Cyclin B was carried out in vitro using HeLa S100 fraction (Boston Biochemical). Cyclin B was determined by western blotting and quantified in the bottom panel. The error bars in (E)–(G) represent mean ± SD from three independent experiments. Molecular Cell 2012 47, 444-456DOI: (10.1016/j.molcel.2012.05.042) Copyright © 2012 Elsevier Inc. Terms and Conditions