Functional interaction between the Fanconi Anemia D2 protein and proliferating cell nuclear antigen (PCNA) via a conserved putative PCNA interaction motif

Abstract

Fanconi Anemia (FA) is a rare recessive disease characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The FA proteins and the familial breast cancer susceptibility gene products, BRCA1 and FANCD1/BRCA2, function cooperatively in the FA-BRCA pathway to repair damaged DNA and to prevent cellular transformation. Activation of this pathway occurs via the mono-ubiquitination of the FANCD2 protein, targeting it to nuclear foci where it co-localizes with FANCD1/BRCA2, RAD51, and PCNA. The regulation of the mono-ubiquitination of FANCD2, as well as its function in DNA repair remain poorly understood. In this study, we have further characterized the interaction between the FANCD2 and PCNA proteins. We have identified a highly conserved, putative FANCD2 PCNA interaction motif (PIP-box), and demonstrate that mutation of this motif disrupts FANCD2-PCNA binding and precludes the mono-ubiquitination of FANCD2. Consequently, the FANCD2 PIP-box mutant protein fails to correct the mitomycin C hypersensitivity of FA-D2 patient cells. Our results suggest that PCNA may function as a molecular platform to facilitate the mono-ubiquitination of FANCD2 and activation of the FA-BRCA pathway.

FIGURE 1.

Co-immunoprecipitation of FANCD2 and PCNA. A, HeLa cells stably expressing HA-PCNA were untransfected or transiently transfected with the mammalian expression vector pMT5-FLAG-p21. HA-PCNA immune complexes were immunoprecipitated with anti-HA agarose, resolved, and immunoblotted with anti-FANCD2, -PCNA, -FLAG, and -p21 antibodies. B, PD20F pDEST40-FANCD2 cells, stably expressing 6xHis/V5-FANCD2, were treated with 20 J/m² UV irradiation and WCE prepared 4 h later. WCE were resolved and immunoblotted with anti-FANCD2 and anti-V5 antibodies. For the Ni²⁺ pull-down, WCE from PD20F pDEST40-FANCD2 cells were incubated with protein A/G-agarose or Ni²⁺ beads for 1 h at 4 °C, and bound proteins were immunoblotted with anti-PCNA and anti-V5 antibodies. C, HCT116 cells were treated with 40 J/m² UV irradiation and 4 h later lysed in NETN lysis buffer containing 100 mm or 420 mm NaCl without and with sonication to yield S100, S420, and P420 fractions, respectively. Lysates were incubated with anti-PCNA antibody or control mouse IgG. Immunoprecipitates were analyzed by immunoblotting with anti-FANCD2 and anti-PCNA antibodies. D, COS-7 cells were transiently transfected with the mammalian expression vectors pDEST40-FANCD2 or pMT5-FLAG-p21 and WCE incubated with GST-Empty and GST-PCNA recombinant proteins. Bound proteins were resolved and immunoblotted with anti-V5 and anti-FLAG antibodies. IP, immunoprecipitation. PD, pull-down. ND, no DNA.

FIGURE 2.

The FANCD2 PIP-box motif is required for the FANCD2-PCNA interaction. A, BLAST alignment of FANCD2 homologues demonstrating conservation of the human FANCD2 PIP-box, and mutation strategy. B, decreased association of the FANCD2 PIPΔ mutant with PCNA. WCE were incubated with anti-PCNA antibody, and immune complexes were resolved and immunoblotted with anti-FANCD2 and anti-PCNA antibodies. C, COS-7 cells were transiently transfected with the indicated pDEST40-FANCD2 constructs. 48 h later, WCE were prepared, incubated with anti-PCNA antibody, and immune complexes were resolved and immunoblotted with anti-V5 antibody. D, association of the FANCD2 PIPΔ mutant protein with FANCE. Cells were transiently transfected with the pEAK8-FLAG-FANCE construct (28) and 48 h later WCE incubated with anti-FLAG antibody. Immune complexes were resolved and immunoblotted with anti-FANCD2 and anti-FLAG antibodies. IP, immunoprecipitation.

FIGURE 3.

The FANCD2 PIP-box motif is required for both spontaneous and DNA damage-inducible FANCD2 mono-ubiquitination. A, cells were untreated (NT) or exposed to 1.0 μm APH for 24 h (APH), and WCE were immunoblotted with the indicated antibodies. The catalytic subunit of DNA Pol δ was used as a protein loading control. B, cells were untreated (NT) or exposed to 60 J/m² UV irradiation (UV), and 4 h later whole-cell lysates were prepared and immunoblotted with the indicated antibodies. C, cells were untreated (NT) or exposed to 15 Gy X-irradiation (X-IR). Whole-cell lysates were prepared 3 h post-irradiation and immunoblotted with the indicated antibodies. D, PD20F pMMP-FANCD2 wt and -FANCD2 PIPΔ were untreated (NT) or exposed to 60 J/m² UV irradiation (UV), and 4 h later, whole-cell lysates were prepared and incubated in the presence or absence of λ phosphatase (λ-P Pase), prior to immunoblotting with the indicated antibodies.

FIGURE 4.

The FANCD2 PIP-box mutant localizes to chromatin yet fails to assemble into nuclear foci. A, cells were incubated in the absence and presence of 120 nm MMC for 18 h, fractionated into cytoplasmic (S1), soluble nuclear (S2), and chromatin-associated fractions (S3), and immunoblotted with antibodies to FANCD2, α-tubulin, PCNA, and H2A. B, FANCD2 PIP-box mutant fails to assemble into nuclear foci. PD20F pMMP-FANCD2 wt and pMMP-FANCD2 PIPΔ cells were incubated in the absence or presence of 1.0 μm APH for 24 h, fixed, and stained with rabbit polyclonal anti-FANCD2 E35 (green) antibody, and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue).

FIGURE 5.

The mono-ubiquitination of FANCD2 promotes its chromatin stabilization. A, cells were treated with cycloheximide (CHX) and whole-cell lysates immunoblotted with antibodies against FANCD2, PCNA, and α-tubulin. B, FANCD2 protein band intensities from Fig. 6A were quantified using ImageJ and plotted. FANCD2-Ub (○), FANCD2 wt (non-ubiquitinated) (●), FANCD2 K561R (□), FANCD2 PIPΔ (■).

FIGURE 6.

The FANCD2 PIP-box mutant fails to rescue the cellular phenotypes of PD20F (FA-D2) cells. A, PD20F (FA-D2) pMMP-Empty (○), -FANCD2 wt (●), -FANCD2 K561R (□), and -FANCD2 PIPΔ (■) were treated with MMC for 10–14 days. Error bars represent the S.E. from triplicate measurements, and are smaller than the dimensions of the symbols for several points. This experiment was performed three times with similar findings. B, same cells were untreated (white bars) or exposed to 0.2 μm APH (gray bars) or 30 nm MMC (black bars) for 24 h, and chromosome aberrations quantified. At least 30 metaphases were scored for each treatment. Error bars indicate the S.E. This experiment was performed three times with similar findings. C, for the G2 accumulation assay cells were untreated (NT) or exposed to 75 nm MMC for 48 h, fixed, stained with propidium iodide, and analyzed by flow cytometry.

FIGURE 7.

A speculative model depicting mono-ubiquitination-dependent and independent functions of the FA-BRCA pathway in DNA replication-associated repair processes. In response to DNA damage encountered by the DNA replisome, the FA core complex (a) promotes the formation of REV1 nuclear foci in a rapid error-prone TLS pathway (11) and (b) promotes the mono-ubiquitination of FANCD2 and FANCI, which, together with mono-ubiquitinated PCNA (42), may promote a post-replicative, HR-dependent DSG repair mechanism.