Phosphorylation of FANCD2 on two novel sites is required for mitomycin C resistance - PubMed (original) (raw)

Phosphorylation of FANCD2 on two novel sites is required for mitomycin C resistance

Gary P H Ho et al. Mol Cell Biol. 2006 Sep.

Abstract

The Fanconi anemia (FA) pathway is a DNA damage-activated signaling pathway which regulates cellular resistance to DNA cross-linking agents. Cloned FA genes and proteins cooperate in this pathway, and monoubiquitination of FANCD2 is a critical downstream event. The cell cycle checkpoint kinase ATR is required for the efficient monoubiquitination of FANCD2, while another checkpoint kinase, ATM, directly phosphorylates FANCD2 and controls the ionizing radiation (IR)-inducible intra-S-phase checkpoint. In the present study, we identify two novel DNA damage-inducible phosphorylation sites on FANCD2, threonine 691 and serine 717. ATR phosphorylates FANCD2 on these two sites, thereby promoting FANCD2 monoubiquitination and enhancing cellular resistance to DNA cross-linking agents. Phosphorylation of the sites is required for establishment of the intra-S-phase checkpoint response. IR-inducible phosphorylation of threonine 691 and serine 717 is also dependent on ATM and is more strongly impaired when both ATM and ATR are knocked down. Threonine 691 is phosphorylated during normal S-phase progression in an ATM-dependent manner. These findings further support the functional connection of ATM/ATR kinases and FANCD2 in the DNA damage response and support a role for the FA pathway in the coordination of the S phase of the cell cycle.

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Figures

FIG. 1.

FIG. 1.

ATR phosphorylates FANCD2 on T691 and S717 in vitro and in vivo. (A) Schematic showing GST fusion proteins containing variable regions of human FANCD2. The fusion proteins were incubated with immune complexes containing either wild-type ATR or a kinase-inactive form of ATR, and in vitro kinase reactions was performed. Three fragments of FANCD2 were phosphorylated by ATR in vitro (2-272, 570-880, and 1186-1451). (B) Sequence of the 570-880 region of FANCD2, with the five putative ATR phosphorylation sites indicated. (C) In vitro ATR kinase reactions, using GST fusion proteins with the indicated mutations.

FIG. 2.

FIG. 2.

In vivo phosphorylation of FANCD2 on T691 and S717 following DNA damage. (A) FA-D2 cells were stably transfected with the cDNAs encoding either wild-type FANCD2, FANCD2(T691A), or FANCD2(S717A). Cells were treated with UV irradiation or left untreated, as indicated, and proteins from whole-cell extracts were isolated, electrophoresed, and immunoblotted with the indicated antisera. (B) FA-D2 cells, stably transfected with wild-type FANCD2, were exposed to IR, as indicated, and total cellular proteins were immunoblotted with the indicated antisera. The anti-pS222 antiserum has been described (35). (C) FA-D2 cells were stably transfected with either wild-type FANCD2 or the K561R mutant. Cells were irradiated, and cell lysates were probed by immunoblotting with the indicated anti-FANCD2 antiserum.

FIG. 3.

FIG. 3.

Phosphorylation of T691 and S717 is required for optimal FANCD2 monoubiquitination and downstream functional activity. (A) FA-D2 fibroblasts were stably transduced with the cDNAs encoding the indicated wild-type (wt) or mutant FANCD2 proteins. Cells were treated as indicated, and whole-cell extracts were immunoblotted with anti-FANCD2 antiserum. The autoradiograph was scanned by densitometry, and the ratio of FANCD2-L (monoubiquitinated FANCD2 isoform) to FANCD2-S (unubiquitinated isoform) was calculated (L/S). (B) Cells were examined by the MMC cytotoxicity assay. The results are shown for vector alone (closed triangles), FANCD2 (closed squares), FANCD2(T691A,S717A) (closed circles), FANCD2(T691A) (open triangles), and FANCD2(S717A) (open circles).

FIG. 4.

FIG. 4.

FANCD2(T691A,S717A) fails to upregulate its monoubiquitination after MMC cross-linker damage. FA-D2 fibroblasts were stably transduced with the cDNAs encoding wild-type (wt) or mutant FANCD2 proteins. Cells were treated with MMC, as indicated, and whole-cell extracts were immunoblotted with anti-FANCD2 antiserum. The upper band is monoubiquitinated FANCD2 (FANCD2-L), and the lower band is unubiquitinated FANCD2 (FANCD2-S). The ratio of FANCD2-L to FANCD2-S (L/S) was calculated by densitometry.

FIG. 5.

FIG. 5.

FANCD2 is phosphorylated on S222, T691, and S717 by both ATR and ATM. (A) HeLa cells stably transfected with the cDNA encoding wild-type EGFP-tagged FANCD2 were exposed to siRNA specific for LacZ, ATM, ATR, or both ATM and ATR. After 48 h, whole-cell lysates were prepared and total cellular protein was analyzed by immunoblotting with antisera specific for the indicated proteins. (B) FA-D2 transfectants were subjected to the RDS assay.

FIG. 6.

FIG. 6.

NBS1 is not required for DNA damage-inducible phosphorylation of FANCD2 on T691. The indicated EBV-transformed human lymphoblast lines were exposed to either IR, MMC, or HU, as indicated. PD20 (FA-D2) fibroblasts, expressing wild-type FANCD2, were also analyzed. Proteins from whole-cell extracts were immunoblotted with antisera specific for either pT691 or unphosphorylated FANCD2.

FIG. 7.

FIG. 7.

ATM phosphorylates FANCD2 on T691 during normal S-phase progression. (A) HeLa cells were synchronized by double thymidine block and released into S phase. Whole-cell lysates were prepared from the synchronized cell populations at the indicated times, and FANCD2 was immunoblotted with either anti-pT691 or anti-FANCD2. Asynchronous cells (A), treated with IR or untreated, were also evaluated. DNA flow histograms corresponding to the synchronized cells are also shown. (B) HeLa cells were synchronized by the nocodazole block method. (C) ATM-deficient cells (vector control) and the isogenic ATM-corrected control (AT22IJE-T+ATM) were synchronized by double thymidine block and evaluated as described above.

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