Abstract
Fanconi anemia is a cancer-prone inherited bone marrow failure and cancer susceptibility syndrome with at least 13 complementation groups (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, and FANCN). Our laboratory has previously described several regulatory phosphorylation events for core complex member proteins FANCG and FANCA by phosphorylation. In this study, we report a novel phosphorylation site serine 331 (S331) of FANCD2, the pivotal downstream player of the Fanconi anemia pathway. Phosphorylation of S331 is important for its DNA damage–inducible monoubiquitylation, resistance to DNA cross-linkers, and in vivo interaction with FANCD1/BRCA2. A phosphomimetic mutation at S331 restores all of these phenotypes to wild-type. In vitro and in vivo experiments show that phosphorylation of S331 is mediated by CHK1, the S-phase checkpoint kinase implicated in the Fanconi anemia DNA repair pathway. [Cancer Res 2009;69(22):8775–83]
Introduction
Fanconi anemia is a rare genetic disorder characterized by bone marrow failure, developmental abnormalities, and increased susceptibility to cancer (1). The cellular hallmark of Fanconi anemia is hypersensitivity and chromosomal breakage on exposure to DNA interstrand cross-linkers such as mitomycin C, suggesting a defect in DNA damage response (2). Thus far, 13 subtypes of Fanconi anemia have been identified and corresponding genes were cloned, including three breast cancer–related genes, FANCD1/BRCA2 (3), FANCJ/BRIP1 (4), and FANCN/PALB2 (5). Eight of the Fanconi anemia proteins [A-C, E-G, and L (6, 7) and M (8, 9)] associate to form a nuclear multiprotein “core” complex, which is necessary for monoubiquitylation of FANCD2 on lysine 561, a critical step for activation of the Fanconi anemia pathway in response to S-phase progression or DNA damage (10).
Cell cycle checkpoint kinases play important roles in various DNA damage responses including the Fanconi anemia pathway. Ataxia telangiectasia and Rad3–related protein kinase (ATR), involved in the DNA damage response, interacts with the Fanconi anemia pathway and couples monoubiquitylation of FANCD2 to DNA damage (11). ATR has been reported to directly phosphorylate serine 717 and threonine 691 on FANCD2 to promote monoubiquitylation and resistance to DNA cross-linking agents (12). Furthermore, CHK1, a substrate of ATR, is also required for initiation of interstrand cross-linker–induced S-phase checkpoint (13) and monoubiquitylation of FANCD2 (14). CHK1 directly phosphorylates the FANCE subunit of Fanconi anemia core complex on two conserved sites (threonine 346 and serine 374), which is functionally important though dispensable for FANCD2 monoubiquitylation (15).
In an attempt to further understand the extensive interactions between ATR-CHK1 and the Fanconi anemia pathway, we have identified a novel DNA damage–inducible FANCD2 phosphorylation site. Here, we show that this DNA damage–inducible phosphorylation site on FANCD2, serine 331 (S331), is essential for normal cellular resistance to mitomycin C and for DNA damage–inducible monoubiquitylation of FANCD2. Furthermore, phosphorylation at S331 occurs independently of the Fanconi anemia core complex and is necessary for interaction of FANCD2 with BRCA2. A phosphomimetic mutation at S331 restores all of these wild-type functions to FANCD2. Phosphorylation at this site is dependent on CHK1, signifying the importance of the S-phase checkpoint in the activation of Fanconi anemia pathway.
Materials and Methods
Cell culture
Cells were maintained at 37°C in a CO2 incubator. HeLa cells were cultured in DMEM supplemented with 10% fetal bovine serum. FANCD2-deficient PD20 cells and its derived cells were grown in DMEM plus 15% fetal bovine serum. Lymphoblast cell lines BD180 (wild-type), HSC72 (FA-A), HSC536 (FA-C), EUFA143 (FA-G), and EUFA868 (FA-L), fibroblast cell line GM6914 (FA-A), and their cDNA complemented derivatives were grown as described previously (16, 17). EUFA868 cell lines were generously provided by Ruhikanta Meetei. For stable transduction, PD20 cells corrected with pMMP-derived wild-type and mutant FANCD2 constructs were selected under 1 μg/mL puromycin for 1 week. 293GPG producer cells were grown as described previously (18).
Generation of phosphospecific antibody
Phosphospecific antibodies for human FANCD2 were generated by immunization of rabbits with keyhole limpet hemocyanin–conjugated phosphopeptide KSKGRAS[P]SSGNQESS with S331 of FANCD2 in the middle (Proteintech Group). Antibody was affinity-purified using the phosphorylated peptide-conjugated gels and nonspecificity was removed by absorption with unphosphorylated peptide-conjugated gels. Preparation of peptide-coupled affinity gel was carried out with AminoLink coupling gel (Pierce Group) according to the manufacturer's instruction.
Clonogenic survival and growth inhibition assays
Clonogenic survival assays were done by plating 500 cells in log growth phase on a 10 cm dish and allowing them to spread for 8 h, at which point cells were treated with varying concentration of mitomycin C in DMEM + 15% fetal bovine serum for 1 h. Then, cells were washed with PBS two times and allowed to grow in fresh medium for 10 to 12 days into visible colonies. Colonies with >50 cells were stained with 0.1% crystal violet in methanol and counted. Growth inhibition by mitomycin C was as described previously (17).
Immunoprecipitation, immunoblotting, and phosphatase treatment
Cell pellets were lysed in buffer containing 350 mmol/L NaCl, 50 mmol/L Tris (pH 7.4), 0.5% NP-40, 5 mmol/L EDTA, 2 mmol/L sodium pyrophosphate, and 1 mmol/L β-glycerophosphate plus proteinase inhibitors followed by high-speed centrifugation. To pull down Flag-tagged FANCD2, 30 μL of 50% anti-Flag M2 agarose (Sigma) slurry was added to 1 mg total cellular lysate and incubated in 4°C for 4 h. Beads were then washed in extraction buffer once and TBS three times. To pull down endogenous FANCD2, 1 mg total lysate was incubated with 2 μg FANCD2 antibody H-300 (Santa Cruz Biotechnology) on ice or 1 h followed by addition of 40 μL of 40% slurry of protein A beads (Sigma). Immunoblotting in Fanconi anemia core complex mutant cell lines and immunoprecipitation of FANCD2 and BRCA2 were as described previously (16, 17, 19).
For dephosphorylation, beads with immunoprecipitated FANCD2 were dried by fine-needle aspiration and then resuspended in 30 μL λ-phosphatase reaction buffer with or without 400 units λ-phosphatase or with λ-phosphatase with or without phosphatase inhibitor (1 mmol/L sodium pyrophosphate) and incubated at 30°C for 30 min.
Densitometric quantification
ImageJ was used for densitometric analysis.7
7ImageJ software. Washington (DC): NIH, 2009. Available from: http://rsbweb.nih.gov/ij/download.html.
Mass spectrometry analysis
The Mass Spectrometry Core at the University of Virginia performed all mass spectrometry work. Briefly, FANCD2-deficient cells corrected with Flag-tagged wild-type FANCD2 were treated with 0.5 μmol/L mitomycin C for 18 h and 1 μmol/L okadaic acid 20 min before cell lysate preparation. FANCD2 was then immunoprecipitated as described above and separated on SDS-PAGE gel. The silver-stained bands (SilverQuest; Invitrogen) corresponding to FANCD2 were trypsin-digested and subjected to liquid chromatography-mass spectrometry on an ion-spray mass spectrometer. Spectra were analyzed and identified using the Sequest search algorithm as described previously (19).
Production of wild-type and mutant FANCD2 constructs
Single-point mutagenesis was carried out by employing PCR-ligation-PCR method (20). The mutated PCR products were then purified and used as templates to generate full-length FANCD2 by PCR-mediated ligation. Final PCR products were sequenced to verify accuracy before being subcloned into Flag- or Flag-EGFP pMMP vector.
The resulting pMMP constructs were transfected into 293GPG producer lines with Lipofectamine (Amersham Biosciences), and viral supernatants were collected daily between 3 and 5 days after transfection. Retroviral supernatants were used for subsequent transduction (19).
In vitro CHK1 kinase assay
Peptide-based in vitro kinase assays were carried out as described previously (19). Briefly, FANCD2 peptides encompassing amino acids 324 to 338 (KSKGRASSSGNQESS) or with singly mutated peptide S331A (KSKGRASASGNQESS) were synthesized by Keck Facility Center of Yale University. In vitro kinase reaction was carried out under room temperature in a volume of 25 μL with indicated amount of synthesized peptide [10 mmol/L HEPES (pH 7.5), 10 mmol/L MgCl2, 0.5 mmol/L EGTA, 0.2 mmol/L ATP, 10 μCi [γ-32P]ATP (3,000 Ci/mmol), 50 ng recombinant GST-CHK1, 0.1 mmol/L DTT]. The reaction was stopped after 30 min by adding 45 μL ice-cold 10% trichloroacetic acid. After centrifugation at 10,000 rpm for 2 min, 30 μL supernatants were spotted onto Whatman P81 filter circles, which were then washed with cold 0.5% phosphoric acid and acetone. The filter circles were analyzed in a scintillation counter (Beckman).
Results
S331 is a novel phosphorylation site
To examine the regulatory role of FANCD2 phosphorylation in the Fanconi anemia pathway, we investigated the phosphorylation of FANCD2 following genotoxic stress by mass spectrometry analysis as described above. The result revealed several modification sites (Fig. 1A), among which the previously unreported phosphorylation event at S331 had a relative abundance of 20%, second only to monoubiquitylation at lysine 561. In addition, sequence alignment of FANCD2 homologues from human, mouse, and zebra fish showed that the three serine residues from 330 to 332 are highly conserved during evolution, suggesting functional importance (Fig. 1A).
S331 is a novel phosphorylation site. A, mass spectroscopy analysis reveals a novel phosphorylation site of FANCD2 at S331. FANCD2 cells corrected with Flag-FANCD2 were treated with mitomycin C, and Flag immunoprecipitated FANCD2 was subjected to mass spectrometry analysis. Phosphorylated peptides or monoubiquitylated peptides are listed with relative abundance (modified peptide fragment/total peptide fragment). S331 is a previously unreported phosphorylation site, which is highly conserved during evolution as shown by sequence alignment (vertical bar, identical residues). B, endogenous phosphorylation of S331 of FANCD2 is abolished by λ-phosphatase experiment. HeLa whole-cell lysate was immunoprecipitated with anti-FANCD2 antiserum, and the immunoprecipitates were treated either with (lane 1) or without (lane 2) phosphatase inhibitor sodium pyrophosphate (Na4P2O7) or λ-phosphatase with (lane 3) or without (lane 4) the inhibitor sodium pyrophosphate. Dephosphorylation by λ-phosphatase abolishes phosphospecific signals detected by affinity-purified anti–phospho-S331 antibody (top), which is preserved by the phosphatase inhibitor sodium pyrophosphate. Immunoblotting against total FANCD2 (bottom) shows equal amount of FANCD2 present. C, phosphorylation of S331 is not dependent on monoubiquitylation. Total cell lysates (2 mg) made from FANCD2-deficient PD20 cells corrected with Flag-EGFP–tagged wild-type FANCD2, FANCD2 (K561R), or FANCD2 (S331A) were immunoprecipitated and blotted against phospho-S331–specific antibody. Top, phosphorylation of S331 in K561R mutant as well as wild-type FANCD2 but not in S331A mutant; bottom, total FANCD2 equal in all lanes.
S331 is a novel phosphorylation site. A, mass spectroscopy analysis reveals a novel phosphorylation site of FANCD2 at S331. FANCD2 cells corrected with Flag-FANCD2 were treated with mitomycin C, and Flag immunoprecipitated FANCD2 was subjected to mass spectrometry analysis. Phosphorylated peptides or monoubiquitylated peptides are listed with relative abundance (modified peptide fragment/total peptide fragment). S331 is a previously unreported phosphorylation site, which is highly conserved during evolution as shown by sequence alignment (vertical bar, identical residues). B, endogenous phosphorylation of S331 of FANCD2 is abolished by λ-phosphatase experiment. HeLa whole-cell lysate was immunoprecipitated with anti-FANCD2 antiserum, and the immunoprecipitates were treated either with (lane 1) or without (lane 2) phosphatase inhibitor sodium pyrophosphate (Na4P2O7) or λ-phosphatase with (lane 3) or without (lane 4) the inhibitor sodium pyrophosphate. Dephosphorylation by λ-phosphatase abolishes phosphospecific signals detected by affinity-purified anti–phospho-S331 antibody (top), which is preserved by the phosphatase inhibitor sodium pyrophosphate. Immunoblotting against total FANCD2 (bottom) shows equal amount of FANCD2 present. C, phosphorylation of S331 is not dependent on monoubiquitylation. Total cell lysates (2 mg) made from FANCD2-deficient PD20 cells corrected with Flag-EGFP–tagged wild-type FANCD2, FANCD2 (K561R), or FANCD2 (S331A) were immunoprecipitated and blotted against phospho-S331–specific antibody. Top, phosphorylation of S331 in K561R mutant as well as wild-type FANCD2 but not in S331A mutant; bottom, total FANCD2 equal in all lanes.
To confirm this finding, we raised a rabbit polyclonal antiserum against a synthesized 12-mer peptide flanking phospho-S331. Immunoblotting with our phosphospecific antibody detected a specific signal in PD20 + Flag-FANCD2, which was abolished by λ-phosphatase treatment (Fig. 1B), showing the specificity of this phospho-antiserum.
To test in vivo phosphorylation of FANCD2 at S331, stable FANCD2-deficient PD20 cells expressing Flag-EGFP–tagged wild-type FANCD2 or its derivative mutants K561R or S331A were made by infection with respective retroviral pMMP constructs. Cell lysate was prepared and subjected to Flag affinity immunoprecipitation and subsequent immunoblotting using the affinity-purified phospho-antiserum. Expression of phospho-S331 was detected in the PD20 cell line corrected with wild-type FANCD2, but not mutant FANCD2, with substitution of S331A, confirming the specificity of the antiserum (Fig. 1C). This phosphorylation event is not dependent on monoubiquitylation as evidenced by phosphospecific signal present in FANCD2 mutant K561R, suggesting that phosphorylation of S331 is upstream of activation of FANCD2.
Phosphorylation of S331 is essential for conferring resistance to mitomycin C
The current paradigm of the Fanconi anemia pathway highlights the pivotal role of FANCD2 in the Fanconi anemia pathway: on replication fork stalling by DNA damage, FANCD2 is activated by monoubiquitylation mediated by the Fanconi anemia core complex and deposited onto chromatin, where it is proposed to promote the bypass and repair of DNA lesions by translesion synthesis and homologous recombination repair (21, 22). To address the functional significance of the phosphorylation of S331, we constructed stable PD20 cells transduced with wild-type Flag-EGFP-FANCD2, Flag-EGFP-FANCD2 (S331A), or empty vector and compared their sensitivity to the DNA cross-linking agent mitomycin C by clonogenic survival assay. Abolishing phosphorylation of S331 resulted in complete loss of resistance to mitomycin C (Fig. 2A), because PD20 cells expressing the point mutation S331A behaved essentially like the uncorrected parental PD20 cell line.
Phosphorylation of S331 is essential for conferring resistance to mitomycin C. A, FANCD2 (S331A) fails to correct mitomycin C sensitivity in FANCD2 cells. Resistance of FANCD2-deficient cell PD20 + vector, PD20 + wild-type Flag-EGFP-FANCD2, or PD20 + Flag-EGFP-D2 (S331A) cells against mitomycin C was measured by clonogenic survival assays. Points, mean; bars, SE (n = 3). B, disruption of phosphorylation at S331 abolishes inducible monoubiquitylation. FANCD2-deficient PD20 cells corrected with Flag-EGFP–tagged wild-type FANCD2, FANCD2 (K561R), or FANCD2 (S331A) were treated with or without 0.5 μmol/L mitomycin C for 20 h before being analyzed by immunoblotting against total FANCD2. The result shows loss of monoubiquitylated FANCD2 (L-isoform) in the cases of S331A and K561R compared with wild-type FANCD2 (top). Immunoblot for Ku70 served as a loading control (bottom). C, hydroxyurea (HU) also induces monoubiquitylation in a phospho-S331–dependent fashion. FANCD2 mutant cells transduced with either Flag-FANCD2 or Flag-FANCD2 (S331A) were treated with 1 mmol/L hydroxyurea or 0.1 μmol/L mitomycin C for 24 h. Immunoblotting revealed that hydroxyurea induced monoubiquitylation of FANCD2 only in wild-type FANCD2 but not in FANCD2 (S331A). D, FANCD2 (S331A) localizes to the nucleus. FANCD2 mutant cells expressing Flag-GFP-FANCD2 wild-type or S331A mutant were plated and examined by direct fluorescent microscopy. The micrographs show that Flag-EGFP–tagged FANCD2 (S331A) has similar nuclear cellular distribution as that of wild-type FANCD2.
Phosphorylation of S331 is essential for conferring resistance to mitomycin C. A, FANCD2 (S331A) fails to correct mitomycin C sensitivity in FANCD2 cells. Resistance of FANCD2-deficient cell PD20 + vector, PD20 + wild-type Flag-EGFP-FANCD2, or PD20 + Flag-EGFP-D2 (S331A) cells against mitomycin C was measured by clonogenic survival assays. Points, mean; bars, SE (n = 3). B, disruption of phosphorylation at S331 abolishes inducible monoubiquitylation. FANCD2-deficient PD20 cells corrected with Flag-EGFP–tagged wild-type FANCD2, FANCD2 (K561R), or FANCD2 (S331A) were treated with or without 0.5 μmol/L mitomycin C for 20 h before being analyzed by immunoblotting against total FANCD2. The result shows loss of monoubiquitylated FANCD2 (L-isoform) in the cases of S331A and K561R compared with wild-type FANCD2 (top). Immunoblot for Ku70 served as a loading control (bottom). C, hydroxyurea (HU) also induces monoubiquitylation in a phospho-S331–dependent fashion. FANCD2 mutant cells transduced with either Flag-FANCD2 or Flag-FANCD2 (S331A) were treated with 1 mmol/L hydroxyurea or 0.1 μmol/L mitomycin C for 24 h. Immunoblotting revealed that hydroxyurea induced monoubiquitylation of FANCD2 only in wild-type FANCD2 but not in FANCD2 (S331A). D, FANCD2 (S331A) localizes to the nucleus. FANCD2 mutant cells expressing Flag-GFP-FANCD2 wild-type or S331A mutant were plated and examined by direct fluorescent microscopy. The micrographs show that Flag-EGFP–tagged FANCD2 (S331A) has similar nuclear cellular distribution as that of wild-type FANCD2.
Monoubiquitylation of FANCD2 at lysine 561 has been shown to be a pivotal event in the activation of the Fanconi anemia pathway in response to stalling of DNA replication forks. Thus, hypersensitivity to mitomycin C observed in phosphodeficient mutant S331A might be explained by an impairment of inducible monoubiquitylation. Indeed, immunoblotting revealed that expression of monoubiquitylated FANCD2 (L-isoform) in PD20 cells transduced with Flag-EGFP-FANCD2 (S331A) failed to increase in response to mitomycin C treatment (Fig. 2B), suggesting that phosphorylation of S331 is important for inducible monoubiquitylation of FANCD2 in response to DNA damage. This failure of induction of FANCD2 monoubiquitylation could also be seen in the same cells treated with hydroxyurea (Fig. 2C).
The deficiency of inducible monoubiquitylation of FANCD2 (S331A) is unlikely explained by sequestering FANCD2 away from its E3 ubiquitin ligase as a result of improper nuclear transportation or retention of FANCD2, because direct fluorescence microscopy showed that the cellular distribution of Flag-EGFP-FANCD2 (S331A) is similar to that of wild-type FANCD2 (Fig. 2D): both localize to the nucleus as reported previously (23).
Phosphomimetic mutation S331D restores the monoubiquitylation of FANCD2 and resistance to mitomycin C
It is conceivable that the dysfunctions of mutant FANCD2 (S331A) were caused by gross structural alterations introduced by this specific point substitution. To exclude this possibility, we constructed a stable PD20 cell line transduced with Flag-tagged phosphomimetic mutant FANCD2 construct with S331 mutated to aspartic acid (S331D). Interestingly, immunoblotting showed inducible monoubiquitylation of the phosphomimetic mutant FANCD2 (S331D) in response to mitomycin C (Fig. 3A).
Phosphomimetic mutation S331D restores monoubiquitylation of FANCD2 and resistance to mitomycin C. A, phosphomimetic mutation S331D restores inducible monoubiquitylation. Whole-cell lysates were prepared from FANCD2 cells expressing wild-type or S331D mutant FANCD2 and treated with mitomycin C. FANCD2 immunoblotting revealed that FANCD2 (S331D) was monoubiquitylated following mitomycin C treatment as wild-type FANCD2. B, phosphomimetic mutant FANCD2 (S331D) confers resistance against mitomycin C. Clonogenic survival assay was conducted in triplicate for FANCD2-deficient PD20 + vector, PD20 + Flag-tagged wild-type FANCD2, or phosphomimetic S331D cell line. The phosphomimetic mutant S331D conferred near-full mitomycin C resistance. Points, mean; bars, SE (n = 3). C, growth inhibition by mitomycin C confirms clonogenic survival assay. Cells (106) were plated into 75 cm2 flasks in growth medium containing mitomycin C, grown until near-confluence was reached in control cultures, and trypsinized and cell numbers were determined by hemocytometer. The response of the phosphomimetic FANCD2 (S331D) cell line is indistinguishable from wild-type, whereas the FANCD2 (S331A) cell line is considerably much more mitomycin C hypersensitive. Points, mean; bars, SE (n = 3).
Phosphomimetic mutation S331D restores monoubiquitylation of FANCD2 and resistance to mitomycin C. A, phosphomimetic mutation S331D restores inducible monoubiquitylation. Whole-cell lysates were prepared from FANCD2 cells expressing wild-type or S331D mutant FANCD2 and treated with mitomycin C. FANCD2 immunoblotting revealed that FANCD2 (S331D) was monoubiquitylated following mitomycin C treatment as wild-type FANCD2. B, phosphomimetic mutant FANCD2 (S331D) confers resistance against mitomycin C. Clonogenic survival assay was conducted in triplicate for FANCD2-deficient PD20 + vector, PD20 + Flag-tagged wild-type FANCD2, or phosphomimetic S331D cell line. The phosphomimetic mutant S331D conferred near-full mitomycin C resistance. Points, mean; bars, SE (n = 3). C, growth inhibition by mitomycin C confirms clonogenic survival assay. Cells (106) were plated into 75 cm2 flasks in growth medium containing mitomycin C, grown until near-confluence was reached in control cultures, and trypsinized and cell numbers were determined by hemocytometer. The response of the phosphomimetic FANCD2 (S331D) cell line is indistinguishable from wild-type, whereas the FANCD2 (S331A) cell line is considerably much more mitomycin C hypersensitive. Points, mean; bars, SE (n = 3).
We further tested whether the phosphomimetic mutation S331D also restored resistance to DNA cross-linkers in FANCD2-deficient PD20 cells. In a clonogenic survival assay, FANCD2 (S331D) conferred near wild-type resistance against mitomycin C (Fig. 3B). Complementation of mitomycin C hypersensitivity by FANCD2 (S331D) was confirmed in a growth inhibition assay (Fig. 3C). Here, the response of PD20 + FANCD2 (S331D) was indistinguishable from the wild-type corrected cell line.
S331 phosphorylation is inducible in response to DNA damage
Previously, phosphorylation of FANCD2 on T691 and S717 was shown to increase dynamically following DNA damage (12). Thus, we tested kinetics of phosphorylation of FANCD2 at S331 to determine whether this phosphorylation event is constitutive or temporally regulated. HeLa cells were treated with 1 μmol/L mitomycin C for different amounts of time before cellular extracts were prepared and analyzed by SDS-PAGE. Immunoblotting against phospho-S331–specific antiserum showed that expression of phospho-FANCD2 increases substantially by 8 h (Fig. 4A). Interestingly, total levels of FANCD2 rose concomitantly with phosphorylation, suggesting that phosphorylation may be a part of protein level regulation. Densitometric quantification showed that not only the expression level of FANCD2 but also the relative abundance of phospho-FANCD2 increased over time (Fig. 4B). Surprisingly, phosphorylation of S331 only appeared in the S-isoform but not in L-isoform, suggesting coordinated dephosphorylation at S331 on addition of the ubiquitin moiety.
S331 phosphorylation is inducible in response to DNA damage. A, temporal pattern of phosphorylation at S331 is similar to that of inducible monoubiquitylation. HeLa cells were treated with 0.5 μmol/L mitomycin C for the indicated time interval followed by immunoblotting. Expression of phospho-FANCD2 was increased by 8 h following mitomycin C treatment. The temporal pattern of inducible phosphorylation is similar to that of monoubiquitylation of FANCD2. Furthermore, the phospho-FANCD2 comigrated with unmonoubiquitylated FANCD2 (S-isoform). Ku70 immunoblot served as a loading control. B, percentage of ratio of the phosphospecific signals to corresponding total FANCD2 signals. C, To quantify the amount of phospho-FANCD2 relative to total FANCD2 following DNA damage, the immunoblot images above were analyzed further by ImageJ to calculate the ratio of the intensity of phosphospecific signal to corresponding intensity of S-isoform. The results confirmed that not only the total level but also the proportion of phospho-FANCD2 were increased and peaked ∼8 to 16 h following mitomycin C treatment. Asterisk, data not applicable.
S331 phosphorylation is inducible in response to DNA damage. A, temporal pattern of phosphorylation at S331 is similar to that of inducible monoubiquitylation. HeLa cells were treated with 0.5 μmol/L mitomycin C for the indicated time interval followed by immunoblotting. Expression of phospho-FANCD2 was increased by 8 h following mitomycin C treatment. The temporal pattern of inducible phosphorylation is similar to that of monoubiquitylation of FANCD2. Furthermore, the phospho-FANCD2 comigrated with unmonoubiquitylated FANCD2 (S-isoform). Ku70 immunoblot served as a loading control. B, percentage of ratio of the phosphospecific signals to corresponding total FANCD2 signals. C, To quantify the amount of phospho-FANCD2 relative to total FANCD2 following DNA damage, the immunoblot images above were analyzed further by ImageJ to calculate the ratio of the intensity of phosphospecific signal to corresponding intensity of S-isoform. The results confirmed that not only the total level but also the proportion of phospho-FANCD2 were increased and peaked ∼8 to 16 h following mitomycin C treatment. Asterisk, data not applicable.
Phospho-S331 FANCD2 is expressed in Fanconi anemia core complex mutant cell lines
To establish whether phosphorylation of FANCD2 at S331 is also dependent on the integrity of FANC core complex like monoubiquitylation, we analyzed expression of phospho-S331 FANCD2 in several human Fanconi anemia cell lines (Fig. 5A). Immunoblotting with the S331 phosphospecific FANCD2 antibody showed that similar levels of protein were detectable in FA-A, FA-C, FA-G, and FA-L cell lines as well as their cDNA complemented counterparts. Phospho-S331 FANCD2 was also present in PD20-315 and PD20-K651R. These results indicate that neither an intact core complex nor monoubiquitylation of FANCD2 is required for efficient phosphorylation at S331.
Phosphorylation of S331 is independent of the Fanconi anemia core complex and required for FANCD2/BRCA2 interaction. A, phospho-S331 FANCD2 is expressed in Fanconi anemia core complex mutant cell lines. FANCD2-deficient PD20 cell and its derived cells expressing Flag-tagged FANCD2 (S331A; PD20-S331A), FANCD2 (K561R; PD20-K561R), or wild-type FANCD2 (PD20-315), lymphoblast cell lines BD180 (wild-type), HSC72 (FA-A), HSC536 (FA-C), EUFA143 (FA-G), and EUFA868 (FA-L), fibroblast cell line GM6914 (FA-A), and their respective cDNA complemented derivatives were analyzed by immunoblotting. Expression of phospho-FANCD2 was present in each of the uncomplemented core complex mutant cell lines and wild-type cells, except PD20 and PD20-S331A. B, FANCD1/BRCA2 is coimmunoprecipitated with FANCD2. Cell lysates were immunoprecipitated with antibody to FANCD2 and probed either with an anti-BRCA2 COOH-terminal antibody (amino acids 3245-3418) to determine FANCD2-BRCA2 interaction (top) or with the same FANCD2 antibody used for immunoprecipitation (bottom). BRCA2 coprecipitated with FANCD2 in cell lines expressing wild-type (PD20-315; PD20 + FANCD2) and FANCD2 (K561R) proteins. (PD20-315 cells are derived from complementing FANCD2-deficient PD20 cells with human chromosome 3p.) Coprecipitation was absent in FANCD2-deficient PD20 cells and expression of FANCD2 (S331A) failed to restore this interaction. In contrast, in PD20 cells expressing the phosphomimetic mutation S331D, interaction between FANCD2 and BRCA2 was restored. The input lane represents 1/50 of the protein extract used for immunoprecipitation in the cell lines indicated. C, FANCD2 is coimmunoprecipitated with FANCD1/BRCA2. The reciprocal immunoprecipitation with anti-BRCA2 antibody was done using the same cells as in B and subsequently immunoblotted with either FANCD2 antibody (top) or BRCA2 antibody (bottom). Blotting with FANCD2 antibody yielded two adjacent FANCD2 protein bands in PD20-315, PD20 + FANCD2, and PD20 + FANCD2 (S331D) cell lines, indicating that both isoforms of FANCD2 interact with BRCA2. No interaction was detected in PD20 cells. Neither isoform was coprecipitated in PD20 + FANCD2 (S331A) cells, indicating that prior phosphorylation of FANCD2 at S331 is a requirement for interaction with BRCA2 regardless of monoubiquitylation status. BRCA2 is expressed in all cell lines (bottom). The input lane represents 1/50 of the protein extract used for immunoprecipitation in the cell lines indicated.
Phosphorylation of S331 is independent of the Fanconi anemia core complex and required for FANCD2/BRCA2 interaction. A, phospho-S331 FANCD2 is expressed in Fanconi anemia core complex mutant cell lines. FANCD2-deficient PD20 cell and its derived cells expressing Flag-tagged FANCD2 (S331A; PD20-S331A), FANCD2 (K561R; PD20-K561R), or wild-type FANCD2 (PD20-315), lymphoblast cell lines BD180 (wild-type), HSC72 (FA-A), HSC536 (FA-C), EUFA143 (FA-G), and EUFA868 (FA-L), fibroblast cell line GM6914 (FA-A), and their respective cDNA complemented derivatives were analyzed by immunoblotting. Expression of phospho-FANCD2 was present in each of the uncomplemented core complex mutant cell lines and wild-type cells, except PD20 and PD20-S331A. B, FANCD1/BRCA2 is coimmunoprecipitated with FANCD2. Cell lysates were immunoprecipitated with antibody to FANCD2 and probed either with an anti-BRCA2 COOH-terminal antibody (amino acids 3245-3418) to determine FANCD2-BRCA2 interaction (top) or with the same FANCD2 antibody used for immunoprecipitation (bottom). BRCA2 coprecipitated with FANCD2 in cell lines expressing wild-type (PD20-315; PD20 + FANCD2) and FANCD2 (K561R) proteins. (PD20-315 cells are derived from complementing FANCD2-deficient PD20 cells with human chromosome 3p.) Coprecipitation was absent in FANCD2-deficient PD20 cells and expression of FANCD2 (S331A) failed to restore this interaction. In contrast, in PD20 cells expressing the phosphomimetic mutation S331D, interaction between FANCD2 and BRCA2 was restored. The input lane represents 1/50 of the protein extract used for immunoprecipitation in the cell lines indicated. C, FANCD2 is coimmunoprecipitated with FANCD1/BRCA2. The reciprocal immunoprecipitation with anti-BRCA2 antibody was done using the same cells as in B and subsequently immunoblotted with either FANCD2 antibody (top) or BRCA2 antibody (bottom). Blotting with FANCD2 antibody yielded two adjacent FANCD2 protein bands in PD20-315, PD20 + FANCD2, and PD20 + FANCD2 (S331D) cell lines, indicating that both isoforms of FANCD2 interact with BRCA2. No interaction was detected in PD20 cells. Neither isoform was coprecipitated in PD20 + FANCD2 (S331A) cells, indicating that prior phosphorylation of FANCD2 at S331 is a requirement for interaction with BRCA2 regardless of monoubiquitylation status. BRCA2 is expressed in all cell lines (bottom). The input lane represents 1/50 of the protein extract used for immunoprecipitation in the cell lines indicated.
Phosphorylation of FANCD2 at S331 is required for its in vivo interaction with BRCA2
We and others have shown previously that FANCD1/BRCA2 and FANCD2 can be coimmunoprecipitated in mammalian cells (24). BRCA2 has been shown to have a role in homologous recombination repair mediated via direct interaction with RAD51 (25, 26). It was reported previously that FANCD2 binds to a highly conserved COOH-terminal site of BRCA2 using yeast two-hybrid analysis (24, 27). Interestingly, a region encompassing S331 (amino acid residues 248-359) is required for association between FANCD2 and BRCA2 (24). In addition, we more recently showed that the in vivo interaction of FANCD2 and BRCA2 takes place in core complex mutants such as FA-A and FA-C (16), as does the expression of phospho-S331 FANCD2 (Fig. 5A). These observations prompted us to investigate the effect of phosphorylation at S331 on FANCD2 interaction with BRCA2. Immunoprecipitation-immunoblotting experiments showed that interaction of FANCD2 with BRCA2 was abolished in FANCD2-deficient PD20 cells expressing mutant FANCD2 (S331A; Fig. 5B). Analogous to monoubiquitylation and survival assays, expression of the phosphomimetic FANCD2 mutation S331D restored the physical interaction between FANCD2 and BRCA2. When immunoprecipitating with anti-BRCA2 antibody (Fig. 5C), we confirmed that both isoforms of FANCD2 interact in PD20 cells expressing wild-type FANCD2 (16). These interactions are also present in PD20 + FANCD2 (S331D), whereas both interactions are absent in PD20 + FANCD2 (S331A). The absence of interaction between both isoforms of FANCD2 in PD20 + FANCD2 (S331A) suggests that prior phosphorylation of S331 is required for interaction with BRCA2 irrespective of monoubiquitylation status. This further shows that phosphorylation at S331 is required for the functional interplay between BRCA2 and FANCD2.
Phosphorylation at S331 is mediated by CHK1
The sequence flanking S331 (KSKGRASSS) is highly conserved among mammalian species and is a partial fit for phosphorylation motif of CHK1 (Arg-X-X-Ser; ref. 28), an ATR substrate that has been implicated in regulating cell cycle checkpoint in the Fanconi anemia pathway-mediated genomic stress response (13, 29). Furthermore, CHK1 also directly regulates the Fanconi anemia pathway by phosphorylation of Fanconi anemia member protein FANCE (15). Thus, we examined whether phosphorylation of S331 might be regulated by CHK1.
To test this idea, we synthesized two peptides of 15 amino acid residues around S331 either wild-type or with the S331A substitution. In vitro CHK1 kinase experiments were done to measure (a) if CHK1 could phosphorylate the peptide and (b) the substrate-dependent nature of the phosphorylation. Marked specific CHK1 phosphorylation occurred on the wild-type peptide compared with S331A peptide (Fig. 6A).
Phosphorylation at S331 is mediated by CHK1. A, recombinant GST-CHK1 specifically phosphorylates wild-type peptide encompassing S331. In vitro CHK1 kinase assay was conducted with indicated concentrations of either wild-type or S331A peptides. Radioactivity of blank controls (reaction systems without peptide substrate) was subtracted from counts to calculate the mole amount of phosphate group incorporated by peptides. Only wild-type FANCD2 peptide was significantly phosphorylated. Points, mean; bars, SE (n = 3). B, CHK1 specifically phosphorylates peptide substrates compared with CHK2. In vitro CHK1 or CHK2 kinase assay was conducted with wild-type FANCD2 peptide substrates at a concentration of 0.2 mmol/L or 30 μmol/L CHKtide substrates as the positive control. The kinase activity of CHK1 with wild-type FANCD2 peptide substrate was adjusted for differing enzymatic potency between CHK1 and CHK2 by multiplying the ratio of CHKtide phosphorylated by CHK2 to that by CHK1. The result indicates that CHK1 displays ∼8-fold more relative activity than CHK2 at the FANCD2 peptide. Columns, mean; bars, SE (n = 3). C, in vivo inhibition of CHK1 eliminates phosphorylation of FANCD2 at S331. HeLa whole-cell extracts made from cells undergoing the indicated treatment (16 h) were separated by SDS-PAGE and immunoblotted against phospho-S331 FANCD2, FANCA, and phospho-S1449 FANCA. Lane 1, HeLa cells without treatment; lane 2, HeLa cells treated with 1 μmol/L mitomycin C; lane 3, HeLa treated with 1 μmol/L mitomycin C plus 5 μmol/L CHK1 inhibitor, SB-218078; lane 4, HeLa cells treated with 1 μmol/L mitomycin C plus 1 mmol/L CHK1 inhibitor. CHK1 inhibitor effectively inhibited inducible expression of phospho-FANCD2, whereas phospho-FANCA remains intact.
Phosphorylation at S331 is mediated by CHK1. A, recombinant GST-CHK1 specifically phosphorylates wild-type peptide encompassing S331. In vitro CHK1 kinase assay was conducted with indicated concentrations of either wild-type or S331A peptides. Radioactivity of blank controls (reaction systems without peptide substrate) was subtracted from counts to calculate the mole amount of phosphate group incorporated by peptides. Only wild-type FANCD2 peptide was significantly phosphorylated. Points, mean; bars, SE (n = 3). B, CHK1 specifically phosphorylates peptide substrates compared with CHK2. In vitro CHK1 or CHK2 kinase assay was conducted with wild-type FANCD2 peptide substrates at a concentration of 0.2 mmol/L or 30 μmol/L CHKtide substrates as the positive control. The kinase activity of CHK1 with wild-type FANCD2 peptide substrate was adjusted for differing enzymatic potency between CHK1 and CHK2 by multiplying the ratio of CHKtide phosphorylated by CHK2 to that by CHK1. The result indicates that CHK1 displays ∼8-fold more relative activity than CHK2 at the FANCD2 peptide. Columns, mean; bars, SE (n = 3). C, in vivo inhibition of CHK1 eliminates phosphorylation of FANCD2 at S331. HeLa whole-cell extracts made from cells undergoing the indicated treatment (16 h) were separated by SDS-PAGE and immunoblotted against phospho-S331 FANCD2, FANCA, and phospho-S1449 FANCA. Lane 1, HeLa cells without treatment; lane 2, HeLa cells treated with 1 μmol/L mitomycin C; lane 3, HeLa treated with 1 μmol/L mitomycin C plus 5 μmol/L CHK1 inhibitor, SB-218078; lane 4, HeLa cells treated with 1 μmol/L mitomycin C plus 1 mmol/L CHK1 inhibitor. CHK1 inhibitor effectively inhibited inducible expression of phospho-FANCD2, whereas phospho-FANCA remains intact.
CHK1 and CHK2 share some substrates such as CDC25C (30, 31) but have different roles in DNA repair control and cell cycle arrest: ATR and ATM phosphorylate CHK1 and CHK2, respectively, to activate the checkpoint as well as DNA damage responses (32, 33). To determine the FANCD2 peptide substrate preference between CHK1 and CHK2, the kinase activity of CHK1 and CHK2 was first adjusted for differing enzyme potency by normalizing the ratio of each respective kinase activity on a control substrate, CHKtide (Millipore). In accordance with the fact that ATR and not ATM is required for resistance to mitomycin C, the result showed that CHK1 phosphorylated the peptide substrate specifically compared to CHK2, further supporting that CHK1 targets FANCD2 at S331 (Fig. 6B).
To confirm the importance of CHK1 for phosphorylation of FANCD2 on S331 in vivo, we measured intensity of phosphorylation of FANCD2 at this site in HeLa cells in the presence of a CHK1 inhibitor, SB-218078 (34), following mitomycin C treatment. Treatment with CHK1 inhibitor SB-218078 completely blocked the phosphorylation of S331 in FANCD2, whereas phosphorylation at S1449 of FANCA was preserved, which we have shown to be an ATR phosphorylatable site (Fig. 6C; ref. 17). Taken together, these data suggest that CHK1 mediates the activation of the Fanconi anemia pathway by direct phosphorylation of S331 on FANCD2.
Discussion
Several groups, ours included, have shown that phosphorylation of Fanconi anemia proteins such as FANCA, FANCD2, and FANCG plays an important functional role in regulating the Fanconi anemia DNA repair pathway (16, 19, 35, 36). In this study, we have identified a novel phosphorylation site at S331 on FANCD2, the pivotal player downstream of the Fanconi anemia core complex.
This phosphorylation event is essential for activation of the Fanconi anemia pathway because single-point substitution S331A diminishes the inducible monoubiquitylation of FANCD2 and fails to complement the mitomycin C hypersensitivity of FANCD2-deficient PD20 cells. Our time course indicates that phosphorylation at S331 increases by 4 h following drug treatment, just preceding the expression of monoubiquitylated FANCD2, further supporting the notion that phosphorylation of S331 is independent of, but necessary for, inducible FANCD2 monoubiquitylation. Contrary to monoubiquitylation at lysine 561, the phosphorylation of FANCD2 at S331 does not require an intact Fanconi anemia core complex.
FANCD2 has been shown to directly interact with BRCA2, which is involved in homologous recombination via direct binding with RAD51 (37). We recently showed that the two proteins are components of a protein complex that also contains FANCG and the RAD51 paralogue XRCC3 (16, 38). S331 lies in the domain essential for FANCD2-BRCA2 interaction as evidenced in yeast two-hybrid system (24), and we now show that phosphorylation of S331 is required for their in vivo interaction. The effect on inducible monoubiquitylation and binding with BRCA2 is significant and specific because phosphomimetic mutation (S331D) complements both phenotypes. Thus, our findings corroborate earlier studies on FANCD2 interacting partners by providing a mechanistic link between FANCD2 and BRCA2. We showed previously that phosphorylation of FANCG at S7 is also required for interaction between FANCD2 and BRCA2 (16), and it will be of great interest to determine how these two phosphorylation events are coordinated mechanistically. At present, it is not precisely understood how the interaction of FANCD2 with BRCA2 modulates homologous recombination repair in response to DNA damage. Given that CHK1 phosphorylation of both RAD51 and BRCA2 regulates the binding of RAD51 to BRCA2 and the ensuing recruitment of RAD51 to sites of DNA damage (39), it is tempting to speculate that CHK1-mediated phosphorylation of FANCD2 at S331 may also affect on this process. BRCA2 has been thought of as a downstream effector in a linear Fanconi anemia pathway, because monoubiquitylation of FANCD2 is independent of BRCA2. Given that phosphorylation precedes monoubiquitylation at lysine 331 in time-course experiments, coimmunoprecipitation of FANCD2 and BRCA2 suggests that BRCA2 may act in concert with FANCD2 in the Fanconi anemia pathway at least in part. That phosphorylation of S331 is simultaneously required for FANCD2-BRCA2 interaction and promotion of FANCD2 monoubiquitylation implicates a complexity of response that is inconsistent with a simple linear pathway. Interestingly, the coprecipitation of FANCD2 by BRCA2 reveals both short and long isoforms of FANCD2 in a phospho-S331–dependent fashion, although phosphorylation was detectable only on the short form. This observation may be due to a regulated dephosphorylation event at the time of monoubiquitylation. Alternatively, our phosphospecific antibody may simply be unable to detect the S331 phosphorylated species of FANCD2 when in the monoubiquitylated state.
Cell cycle checkpoint machinery plays a significant role in genomic stress responses by allowing various DNA repair pathways enough time to repair DNA lesions. Previous studies show that ATR is the DNA damage sensor involved in the Fanconi anemia pathway: ATR acts upstream of the Fanconi anemia pathway in the interstrand cross-linker–induced intra-S-phase checkpoint (13), and silencing of ATR by small interfering RNA knocks down monoubiquitylated FANCD2 expression and sensitizes cells to DNA cross-linkers, probably mediated by direct phosphorylation of FANCD2 at S717 and T691 (12). In this study, we further extend the interactions between cellular checkpoint machinery and DNA repair pathways by establishing the links between phosphorylation of FANCD2 at S331 and CHK1, the substrate of ATR. This is consistent with the finding that loss of CHK1 impedes FANCD2 monoubiquitylation and resistance to mitomycin C (14). Furthermore, CHK1 is also required for FANCD2-independent Fanconi anemia functions mediated by phosphorylation of FANCE at T346 and S374 (15). Although it may be impossible to rule out a role for CHK2 phosphorylation at S331, as we have not examined FANCD2 S331 phosphorylation after ionizing radiation, these published reports and our in vitro data suggest an exclusive role for CHK1.
The mechanism by which CHK1-dependent phosphorylation of S331 promotes FANCD2 monoubiquitylation may be mediated by regulating the interactions of FANCD2 with its binding partners. There are several possible mechanisms. First, phosphorylation of FANCD2 by CHK1 may enhance its affinity to the FANCL subunit of the Fanconi anemia core complex, the putative ubiquityl E3 ligase of FANCD2 (6). Second, the phosphorylation event at S331 may facilitate monoubiquitylation by blocking access to the deubiquitylation enzyme USP1 (40). Third, it is recently reported that CHK1 and its binding partner claspin are required for DNA damage–induced ubiquitylation of proliferating cell nuclear antigen by RAD18 (41). Because proliferating cell nuclear antigen shares the same deubiquitylation enzyme USP1 with FANCD2 (42, 43) and proliferating cell nuclear antigen and FANCD2 have been shown to colocalize in foci (24), it is possible that they share the same regulation through CHK1 as well. Given the general proximity of these key enzymes, it is certainly a possibility that phosphorylation regulates monoubiquitylation of FANCD2 in a coordinated way, allowing a dephosphorylation of FANCD2 at the same time, consistent with our data. Regardless of the mechanisms that promote FANCD2 monoubiquitylation and the interaction of FANCD2 with BRCA2, our study highlights the regulation of the Fanconi anemia/BRCA pathway by checkpoint kinases via phosphorylation.
FANCI, the paralogue of FANCD2, associates with FANCD2 to form so-termed ID complex (44). Both FANCD2 and FANCI undergo interdependent monoubiquitylation and relocalization to nuclear foci following DNA damage or replication fork stress (45). Recently, it was reported that monoubiquitylation of FANCI is dependent on phosphorylation at six conserved and clustered Ser/Thr-Gln motifs and phosphomimetic mutations induce persistent monoubiquitylation and foci formation (46). We are interested to investigate whether FANCI association is disrupted in phosphomutant FANCD2 (S331A) as well.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Grant support: NIH grant R01-HL063776 (G.M. Kupfer).
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