D2 Complex - PubMed (original) (raw)

Ubiquitination-Linked Phosphorylation of the FANCI S/TQ Cluster Contributes to Activation of the Fanconi Anemia I/D2 Complex

Ronald S Cheung et al. Cell Rep. 2017.

Abstract

Repair of interstrand crosslinks by the Fanconi anemia (FA) pathway requires both monoubiquitination and de-ubiquitination of the FANCI/FANCD2 (FANCI/D2) complex. In the standing model, the phosphorylation of six sites in the FANCI S/TQ cluster domain occurs upstream of, and promotes, FANCI/D2 monoubiquitination. We generated phospho-specific antibodies against three different S/TQ cluster sites (serines 556, 559, and 565) on human FANCI and found that, in contrast to the standing model, distinct FANCI sites were phosphorylated either predominantly upstream (ubiquitination independent; serine 556) or downstream (ubiquitination-linked; serines 559 and 565) of FANCI/D2 monoubiquitination. Ubiquitination-linked FANCI phosphorylation inhibited FANCD2 de-ubiquitination and bypassed the need to de-ubiquitinate FANCD2 to achieve effective interstrand crosslink repair. USP1 depletion suppressed ubiquitination-linked FANCI phosphorylation despite increasing FANCI/D2 monoubiquitination, providing an explanation of why FANCD2 de-ubiquitination is important for function of the FA pathway. Our work results in a refined model of how FANCI phosphorylation activates the FANCI/D2 complex.

Keywords: ATR; DNA damage response; DNA repair; FANCD2; FANCI; Fanconi anemia; USP1; interstrand crosslink; monoubiquitination; phosphorylation.

PubMed Disclaimer

Figures

Figure 1. Characterization of Phospho-Specific Antibodies Against Human FANCI S/TQ Cluster Sites

(A) Schematic of human FANCI (hFANCI), indicating the six putative phosphorylation sites within the S/TQ cluster domain. Sites whose phosphorylation was confirmed by mass spectrometry in this study, and sites against which phospho-specific antibodies were generated, are indicated in the table. The FANCI monoubiquitination site (K523) is also shown. (B) Schematic of the immunogenic phospho-peptides used to generate hFANCI phospho-specific antibodies. (C) Immunoblot with hFANCI phospho-S556, phospho-S559 and phospho-S565 antibodies of U2OS cells that were either untreated (Utx), or treated with 60 ng/mL MMC for the indicated times. An ATR inhibitor (VE821) was used to test the ATR-dependence of the FANCI phosphorylation sites. (D) Immunoblot of FANCI-deficient (F010191) cells complemented with either MYC-tagged wild-type FANCI (FANCIwt), or MYC-tagged FANCI mutants that are alanine-substituted at the relevant sites. Cells were either untreated, or treated with 60 ng/mL MMC for 24 hours. Ub refers to the monoubiquitinated form of FANCI, and non-Ub refers to the non-ubiquitinated form. The asterisk (*) indicates a non-specific band. See also Figure S1.

Figure 2. FANCI Phosphorylation Occurs During S-Phase of the Cell Cycle and Occurs Predominantly on Chromatin

(A and B) Immunoblot of U2OS cells that were synchronized by, and released from, double thymidine block (A) or nocodazole block (B). Cell cycle phases at each time point were determined by flow cytometry of DNA content. Asynchronous cells were included for comparison. (C) Immunoblot of soluble and pellet (chromatin-containing) fractions prepared from U2OS cells that were either untreated (Utx), or treated with 60 ng/mL MMC for 24 hours. Ub refers to the monoubiquitinated forms of FANCI and FANCD2, and non-Ub refers to the non-ubiquitinated forms. The asterisk (*) indicates a non-specific band. (D) Indirect immunofluorescence with FANCI phospho-S565 and FLAG antibodies of U2OS 3xFLAG-FANCD2 cells that were either untreated (Utx), or treated with 60 ng/mL MMC for 24 hours. Cells siRNA-depleted of FANCI, or λ phosphatase-treated on coverslips prior to staining, were included as negative controls for phospho-S565 FANCI antibody staining. See also Figure S2.

Figure 3. Ubiquitination-Independent and Ubiquitination-Linked FANCI Phosphorylation

(A) Immunoblot of FANCA-deficient (GM6914), FANCF-deficient (TOV21G) and FANCG-deficient (326SV) cells, and their complemented counterparts, that were either untreated (Utx), or treated with 60 ng/mL MMC for 24 hours. (B) Immunoblot of FANCD2-deficient (PD20F) cells, complemented with either wild-type FANCD2 (FANCD2wt) or a monoubiquitination-deficient (K561R) mutant, that were either untreated, or treated with 60 ng/mL MMC for 24 hours. (C) Immunoblot of pellet (chromatin containing) fractions prepared from FANCI-deficient (F010191) cells complemented with either wild-type FANCI (FANCIwt) or a monoubiquitination-deficient (K523R) mutant, that were either untreated, or treated with 60 ng/mL MMC for 24 hours. Irrelevant lanes have been removed from these blots. (D) Immunoblot of FANCI-deficient (HCT116 FANCI−/−) cells complemented with an empty vector, wild-type FANCI, or the indicated mutants. Cells were then either untreated, or treated with 60 ng/mL MMC for 24 hours. Ratios of monoubiquitinated to non-ubiquitinated FANCD2 (D2 Ub:non-Ub) are indicated beneath the corresponding lanes. Ub refers to the monoubiquitinated forms of FANCI and FANCD2, and non-Ub refers to the non-ubiquitinated forms. The asterisk (*) indicates a non-specific band. See also Figures S3 and S4.

Figure 4. USP1 is Required for Optimal Ubiquitination-Linked FANCI Phosphorylation

(A) Immunoblot of U2OS cells transfected with the indicated siRNAs and either untreated (Utx), or treated with 60 ng/mL MMC for the indicated times. (B) Immunoblot of U2OS cells that were either untreated, or treated with 60 ng/mL MMC for the indicated times, either in the presence or absence of 30 μM ML323. Ub refers to the monoubiquitinated forms of FANCI and FANCD2, and non-Ub refers to the non-ubiquitinated forms. The asterisk (*) indicates a non-specific band.

Figure 5. Phosphomimetic Mutations at Ubiquitination-Linked FANCI Sites Bypass the Need to De-Ubiquitinate FANCD2 for ICL Repair

(A) Outline of experiment to determine the relationship between FANCI de-phosphorylation and FANCI/D2 de-ubiquitination. Whole cell lysates for immunoblot were prepared at the various points during the experiment (triangles). (B) Immunoblots of U2OS cells subjected to the experiment described in Figure 5A. Ratios of monoubiquitinated to non-ubiquitinated FANCD2 (D2 Ub:non-Ub) are indicated beneath the corresponding lanes. (C) Immunoblot of HCT116 FANCI −/− cells complemented with either wild-type FANCI (FANCIwt) or a ubiquitination-linked phosphomimetic FANCI mutant (S559D-S565D), and subjected to the VE821-only component of the experiment described in Figure 5A. Ratios of monoubiquitinated to non-ubiquitinated FANCD2 (D2 Ub:non-Ub) are indicated beneath the corresponding lanes. Ub refers to the monoubiquitinated forms of FANCI and FANCD2, and non-Ub refers to the non-ubiquitinated forms. The asterisk (*) indicates a non-specific band. (D) Quantification of FANCD2 foci formation in HCT116 FANCI−/− cells complemented with either wild-type FANCI (FANCIwt) or a ubiquitination-linked phosphomimetic FANCI mutant (S559D-S565D), and either untreated, or treated with 60 ng/mL MMC for 24 hours. (E) MMC cell survival assay of HCT116 FANCI−/− cells complemented with the indicated FANCI constructs, or with an empty vector. (F) MMC cell survival assay of HCT116 FANCI−/− cells complemented with wild-type FANCI, either in the presence or absence of 10 μM ML323. Cells complemented with monoubiquitination-deficient FANCI (K523R) were included in this experiment for comparison. (G) Quantification of fold decrease in cell survival caused by USP1 inhibition with 10 μM ML323 in HCT116 FANCI−/− cells complemented with either wild-type FANCI or S559D-S565D FANCI, and treated with 2 different concentrations of MMC. All error bars represent S.E.M in experiments that were performed at least in triplicate, n.s. = not statistically significant. (H) Model of how ubiquitination-independent and ubiquitination-linked phosphorylation regulates FANCD2 monoubiquitination and downstream function of the FA pathway. (I) Increased FANCI/D2 monoubiquitination in the context of USP1 deficiency leads to hypophosphorylation of ubiquitination-linked FANCI sites and inefficient ICL repair. See also Figure S5 and Table S1.

References

1. Boisvert RA, Howlett NG. The Fanconi anemia ID2 complex: dueling saxes at the crossroads. Cell Cycle. 2014;13:2999–3015. -PMC -PubMed
1. Castella M, Jacquemont C, Thompson EL, Yeo JE, Cheung RS, Huang JW, Sobeck A, Hendrickson EA, Taniguchi T. FANCI Regulates Recruitment of the FA Core Complex at Sites of DNA Damage Independently of FANCD2. PLoS genetics. 2015;11:e1005563. -PMC -PubMed
1. Ceccaldi R, Sarangi P, D’Andrea AD. The Fanconi anaemia pathway: new players and new functions. Nature reviews Molecular cell biology. 2016;17:337–349. -PubMed
1. Chen YH, Jones MJ, Yin Y, Crist SB, Colnaghi L, Sims RJ, 3rd, Rothenberg E, Jallepalli PV, Huang TT. ATR-mediated phosphorylation of FANCI regulates dormant origin firing in response to replication stress. Molecular cell. 2015;58:323–338. -PMC -PubMed
1. Dexheimer TS, Rosenthal AS, Liang Q, Chen J, Villamil MA, Kerns EH, Simeonov A, Jadhav A, Zhuang Z, Maloney DJ. Probe Reports from the NIH Molecular Libraries Program. Bethesda (MD): 2010. Discovery of ML323 as a Novel Inhibitor of the USP1/UAF1 Deubiquitinase Complex. -PubMed

Ubiquitination-Linked Phosphorylation of the FANCI S/TQ Cluster Contributes to Activation of the Fanconi Anemia I/D2 Complex - PubMed (original) (raw)