The PTEN phosphatase functions cooperatively with the Fanconi anemia proteins in DNA crosslink repair - PubMed (original) (raw)
The PTEN phosphatase functions cooperatively with the Fanconi anemia proteins in DNA crosslink repair
Elizabeth A Vuono et al. Sci Rep. 2016.
Abstract
Fanconi anemia (FA) is a genetic disease characterized by bone marrow failure and increased cancer risk. The FA proteins function primarily in DNA interstrand crosslink (ICL) repair. Here, we have examined the role of the PTEN phosphatase in this process. We have established that PTEN-deficient cells, like FA cells, exhibit increased cytotoxicity, chromosome structural aberrations, and error-prone mutagenic DNA repair following exposure to ICL-inducing agents. The increased ICL sensitivity of PTEN-deficient cells is caused, in part, by elevated PLK1 kinase-mediated phosphorylation of FANCM, constitutive FANCM polyubiquitination and degradation, and the consequent inefficient assembly of the FA core complex, FANCD2, and FANCI into DNA repair foci. We also establish that PTEN function in ICL repair is dependent on its protein phosphatase activity and ability to be SUMOylated, yet is independent of its lipid phosphatase activity. Finally, via epistasis analysis, we demonstrate that PTEN and FANCD2 function cooperatively in ICL repair.
Figures
Figure 1. PTEN−/− cells are hypersensitive to the clastogenic effects of mitomycin C.
HCT116 and MCF10A PTEN+/+ and PTEN−/− cells were incubated in the absence or presence of mitomycin C (MMC) for 24 h and metaphase spreads were analyzed for numerical and structural chromosome aberrations. (A) Representative images of the types of chromosome aberrations - including radial formations, telomere fusions, dicentrics, and complex aberrations - observed in PTEN−/− cells following MMC treatment. (B,C) Quantification of chromosome gaps and breaks (B) and total chromosome aberrations (C) observed in HCT116 PTEN+/+ and two independent clones of PTEN−/− cells incubated in the absence or presence of 20 nM MMC for 24 h. (D,E) Quantification of chromosome gaps and breaks (D) and total chromosome aberrations (E) observed in MCF10A PTEN+/+ and PTEN−/− cells incubated in the absence or presence of 24 nM MMC for 24 h. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 2. PTEN is required for efficient mitomycin C-inducible FANCD2 and FANCI nuclear foci formation.
(A) HCT116 PTEN+/+ and PTEN−/− cells were incubated in the absence or presence of 40 nM mitomycin C (MMC) for 18 h and FANCD2 and FANCI nuclear foci formation were analyzed by immunofluorescence microscopy. Representative images of FANCD2 and FANCI nuclear foci from cells exposed to MMC are shown. (B) Quantification of the percentage of PTEN+/+ and PTEN−/− nuclei displaying greater than five discrete FANCD2 or FANCI nuclear foci. Cells were incubated in the absence (NT) or presence of 40 nM MMC for 18 h and allowed to recover for up to 24 h. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Immunoblotting for FANCD2, FANCI, and RAD51 reveals no appreciable differences in levels of MMC-inducible FANCD2 and FANCI monoubiquitination or RAD51 between PTEN+/+ and PTEN−/− cells. Cells were incubated in the absence or presence of 200 nM MMC for 24 h and allowed to recover for up to 24 h. To improve clarity and conciseness, the presented blots have been cropped. All gels were run under the same experimental conditions.
Figure 3. Increased FANCM instability and defective chromatin recruitment of the FA core complex in PTEN-deficient cells.
(A) HCT116 PTEN+/+ and PTEN−/− cells were incubated in the absence or presence of 40 μg/mL cycloheximide (CHX) alone or 40 μg/mL CHX and 4 μM MG132 (CHX + MG) for the indicated times. Whole-cell lysates were prepared and immunoblotted with anti-FANCM, anti-p53, anti-PTEN, and anti-α-tubulin antibodies. RBI, Relative protein band intensity with respect to the untreated sample lane. (B) Chromatin fractionation analysis of MCF10A PTEN+/+ and PTEN−/− cells reveals a defect in the chromatin localization of FANCM and FANCA in the absence of PTEN. Cells were incubated in the absence (NT) or presence of 200 nM mitomycin C (MMC) for 24 h. W, unfractionated whole-cell lysate; S, soluble cytoplasmic and nuclear fraction; C, chromatin fraction. For (A,B), to improve clarity and conciseness, the presented blots have been cropped. All gels were run under the same experimental conditions. C:W, Ratio of protein in the chromatin fraction versus the whole-cell lysate. Immunoblotting experiments were performed multiple times with similar results. Protein band quantifications are from the immunoblots shown and are representative of results from several experiments. (C) Quantification of FANCM nuclear foci formation in MCF10A PTEN+/+ and PTEN−/− cells reveals a defect in MMC-inducible FANCM nuclear foci formation in PTEN−/− cells. Cells were incubated in the absence (NT) or presence of 200 nM MMC for 18 h. ***P < 0.001. (D) Quantification of FANCA nuclear foci formation in MCF10A PTEN+/+ and PTEN−/− cells reveals reduced FANCA nuclear foci formation in PTEN−/− cells both in the absence (NT) and presence of MMC. Cells were treated as described for (C). ***P < 0.001.
Figure 4. Inhibition of PLK1 rescues defective FANCD2 nuclear foci formation but is not sufficient to rescue the chromosome instability of PTEN−/− cells.
(A) HCT116 PTEN+/+ and PTEN−/− cells were incubated in the absence (−) and presence (+) of 100 nM BI2536 and 500 nM mitomycin C (MMC) for 18 h, and whole-cell lysates were immunoblotted with the indicated antibodies. L:S, Ratio of phosphorylated to unphosphorylated FANCM. (B) PTEN+/+ and PTEN−/− cells were incubated in the absence and presence of 200 nM MMC for 24 h. Whole-cell lysates were then incubated in the absence (−) or presence (+) of 10 U/μg λ-phosphatase for 4 h at 30 °C, followed by immunoblotting with the indicated antibodies. For (A,B), to improve clarity and conciseness, the presented blots have been cropped. All gels were run under the same experimental conditions. RBI, Relative unmodified FANCM protein band intensity. Immunoblotting experiments were performed multiple times with similar results. Protein band quantifications are from the immunoblots shown and are representative of results from several experiments. (C) PTEN+/+ and PTEN−/− cells were incubated in the absence or presence of 2 nM BI2536, 20 nM MMC, or both BI2536 and MMC for 24 h and metaphase spreads were analyzed for the presence of numerical and structural chromosome aberrations. (D) PTEN−/− cells stably expressing empty vector or wild-type PTEN were incubated in the absence or presence of 5 nM BI2536, 200 nM MMC, or both BI2536 and MMC for 24 h, and allowed to recover for 8 h, and FANCD2 nuclear foci formation were analyzed by immunofluorescence microscopy. *P < 0.05; ***P < 0.001.
Figure 5. Increased γH2AX, 53BP1, and DNA-PKcs pS2056 nuclear foci formation in PTEN-deficient cells.
(A) Quantification of γH2AX nuclear foci formation in HCT116 PTEN+/+ and PTEN−/− cells reveals persistent elevated levels of γH2AX nuclear foci in PTEN−/− cells following mitomycin C (MMC) treatment. Cells were incubated in the absence (NT) or presence of 40 nM MMC for 18 h and allowed to recover for up to 24 h. ***P < 0.001. (B) Quantification of 53BP1 nuclear foci formation in PTEN+/+ and PTEN−/− cells reveals persistently elevated levels of 53BP1 nuclear foci in PTEN−/− cells following MMC treatment. Cells were treated as described for (A). (C) Quantification of DNA-PKcs pS2056 nuclear foci formation in PTEN+/+ and PTEN−/− cells reveals persistently elevated levels of DNA-PKcs pS2056 nuclear foci in PTEN−/− cells. Cells were treated as described for (A and B). (D) Quantification of FK2 nuclear foci formation reveals no overt differences in levels of mono-, multi-, or poly-ubiquitin conjugates between PTEN+/+ and PTEN−/− cells. Cells were treated as described for (A–C). *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 6. PTEN function in ICL repair is protein phosphatase and SUMOylation-dependent.
(A) HCT116 PTEN−/− cells were stably transduced with pLenti6.2-LacZ, -PTEN-WT, -PTEN-C124S, -PTEN-G129E, and -PTEN-K254R. Whole-cell lysates were prepared and immunoblotted for PTEN, AKT, AKT pS473, RAD51, and α-tubulin. To improve clarity and conciseness, the presented blots have been cropped. All gels were run under the same experimental conditions. (B) PTEN-C124S and PTEN-K254R fail to rescue the sensitivity of PTEN−/− cells to the clastogenic effects of mitomycin C (MMC), in contrast to PTEN-WT or PTEN-G129E. Cells were incubated in the absence (NT) or presence of 20 nM MMC for 24 h and metaphase spreads were analyzed for structural chromosome aberrations. ***P < 0.001. (C,D) Defective FANCD2 (C) and FANCI (D) nuclear foci formation in PTEN−/− cells expressing LacZ, PTEN-C124S, and PTEN-K254R. Cells were incubated in the absence (NT) or presence of 200 nM MMC for 18 h. ***P < 0.001.
Figure 7. PTEN functions epistatically with FANCD2 in ICL repair.
(A) siRNA-mediated knockdown of PTEN in FA-D2 (_FANCD2_−/−) and FA-D2 + FANCD2 cells. Increased levels of AKT S473 phosphorylation confirmed the functional depletion of PTEN. To improve clarity and conciseness, the presented blots have been cropped. All gels were run under the same experimental conditions. (B) Knockdown of PTEN (siPTEN) in FA-D2 + FANCD2 cells leads to increased sensitivity to mitomycin C (MMC) cytotoxicity. In contrast, knockdown of PTEN in FA-D2 (_FANCD2_−/−) cells does not lead to a further increase in sensitivity to MMC cytotoxicity. siCtrl, non-targeting siRNA. (C and D) Metaphase chromosome analysis of FA-D2 (_FANCD2_−/−) cells reveals that knockdown of PTEN does not lead to a further increase in the levels of chromosome gaps and breaks (C) or total chromosome aberrations (D), including radial formations, dicentrics, and complex aberrations.
References
- Fanconi Anemia Research Fund, Inc. Fanconi Anemia: Standards for Clinical Care. Second edn, Vol. 1 (Fanconi Anemia Research Fund, Inc., 2003).
- Walden H. & Deans A. J. The Fanconi anemia DNA repair pathway: structural and functional insights into a complex disorder. Annu Rev Biophys 43, 257–278 (2014). -PubMed
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