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. 2015 Sep 29;290(46):27545–27556. doi: 10.1074/jbc.M115.672626

ATM-dependent Phosphorylation of the Fanconi Anemia Protein PALB2 Promotes the DNA Damage Response*

Yingying Guo ‡,1, Wanjuan Feng ‡, Shirley M H Sy ‡,§,2, Michael S Y Huen ‡,§,¶,3
PMCID: PMC4646007  PMID: 26420486

Background: The ATM kinase orchestrates DNA damage responses by protein phosphorylation.

Results: PALB2 becomes phosphorylated in an ATM-dependent manner.

Conclusion: PALB2 phosphorylation is important for cell responses to ionizing radiation-induced DNA damage.

Significance: The ATM-PALB2 axis promotes cellular responses to DNA damage.

Keywords: BRCA1, DNA damage, DNA damage response, phosphorylation, protein phosphorylation

Abstract

The Fanconi anemia protein PALB2, also known as FANCN, protects genome integrity by regulating DNA repair and cell cycle checkpoints. Exactly how PALB2 functions may be temporally coupled with detection and signaling of DNA damage is not known. Intriguingly, we found that PALB2 is transformed into a hyperphosphorylated state in response to ionizing radiation (IR). IR treatment specifically triggered PALB2 phosphorylation at Ser-157 and Ser-376 in manners that required the master DNA damage response kinase Ataxia telangiectasia mutated, revealing potential mechanistic links between PALB2 and the Ataxia telangiectasia mutated-dependent DNA damage responses. Consistently, dysregulated PALB2 phosphorylation resulted in sustained activation of DDRs. Full-blown PALB2 phosphorylation also required the breast and ovarian susceptible gene product BRCA1, highlighting important roles of the BRCA1-PALB2 interaction in orchestrating cellular responses to genotoxic stress. In summary, our phosphorylation analysis of tumor suppressor protein PALB2 uncovers new layers of regulatory mechanisms in the maintenance of genome stability and tumor suppression.

Introduction

PALB2, originally identified as Partner and Localizer of BRCA2, plays pivotal roles in maintaining the stability and chromatin association of the breast and ovarian susceptible gene product (1). Together with BRCA2, the PALB2-BRCA2 heterodimeric complex initiates high-fidelity homologous recombination (HR)4 DNA repair by promoting the assimilation of the recombinase RAD51 onto resected 3′ single-strand overhangs (1–3). Inactivation of PALB2 compromised RAD51 loading onto DNA breaks, greatly diminished HR repair, and hyper-sensitized cells to genotoxic stress (4, 5). Consistent with the intimate links between dysregulated DNA repair and human tumorigenesis, PALB2 mutations predispose individuals to early onset of a number of cancers, including those of breast and pancreatic origins (6–10). Biallelic mutations of PALB2 also lead to Fanconi anemia (11, 12), an autosomal recessive blood disorder characterized by genome instability and extreme sensitivity to inter-strand cross-linking agents.

Aside its established role in HR repair, more recent studies have uncovered in the tumor suppressor PALB2 a wide repertoire of genome integrity protection protocols, including control of cell cycle checkpoints (1, 13), repair of damaged replication forks (14–16), transcription control (17), and regulation of cellular redox homeostasis (18). PALB2 also supports spermatogenesis, as PALB2 mutant animals suffered from reduced fertility, presumably due to defects in meiosis (19).

Although PALB2 has emerged as a crucial component of the mammalian DNA damage response (DDR) toolbox, it has remained unclear how PALB2 functionally connects to the DDR protein network. Although PALB2 physically interacts with BRCA1 to facilitate HR repair (20–22), exactly how BRCA1 communicates with PALB2 to enforce Rad51/DMC1-dependent recombination remains obscure.

In elucidating how PALB2 promotes genome stability and suppresses tumorigenesis, we found that the tumor suppressor protein becomes hyperphosphorylated in response to ionizing radiation (IR)-induced DNA damage, raising the intriguing possibility that post-translational modification of the PALB2 polypeptide may represent a means via which the DDR orchestrates PALB2 functions in protecting genome integrity. That PALB2 had also been identified as one of the proteins that undergoes phosphorylation at consensus sites recognized by the master DNA damage response kinase ATM (Ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) (23) further prompted us to systematically examine the genetic requirements and functional regulation of PALB2 phosphorylation events.

Materials and Methods

Cell Culture

HeLa, 293T, and U2OS cell lines from ATCC were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS at 37 °C in 5% CO2. For generation of cell lines stably expressing PALB2 (wild type and mutants), BOSC23 cells were transfected with pEF1A-HA-Flag-PALB2 expression constructs together with pCL-Ampho using polyethylenimine. Viral supernatants were collected and filtered 48 h after transfection, and were subsequently applied to recipient cells. Stable integrants were selected by supplementing culture media with 2 μg/ml of puromycin.

Site-directed Mutagenesis of PALB2

PALB2 phospho-mutations (Ser to Ala missense mutations or Ser to Glu/Asp phospho-mimicking mutations) and COSMIC mutations were introduced using the QuikChange site-directed mutagenesis kit. Primer pairs were as follows: S157A sense: 5′-acatttattgcacaggagagagactgtgtctttg-3′; S157A antisense: 5′-agtctctctcctgtgcaataaatgtcctcttctgctg-3′; S376A sense: 5′-agtgagattctagctcaacctaagagtcttagcctg-3′; S376A antisense: 5′-actcttaggttgagctagaatctcactttcctgaag-3′;S157D sense: 5′-agaaagaagcagcagaagaggacatttattgatcaggagagagactgtg-3′; S157D antisense: 5′-cacagtctctctcctgatcaataaatgtcctcttctgctgcttctttct-3′; S376D sense: 5′-aatgaaaatcttcaggaaagtgagattctagatcaacctaagagtcttagcc-3′; S376D antisense: 5′-ggctaagactcttaggttgatctagaatctcactttcctgaagattttcatt-3′; S157E sense: 5′-agaaagaagcagcagaagaggacatttattgagcaggagagagactgtg-3′; S157E antisense: 5′-cacagtctctctcctgctcaataaatgtcctcttctgctgcttctttct-3′; S376E sense: 5′-aaatcttcaggaaagtgagattctagagcaacctaagagtcttagcctggaag-3′; S376E antisense: 5′-cttccaggctaagactcttaggttgctctagaatctcactttcctgaagattt-3′; Q377E sense: 5′-tgagattctaagtgaacctaagagtcttagcctgg-3′; Q377E antisense: 5′-agactcttaggttcacttagaatctcactttcctg-3′; K379N sense: 5′-taagtcaacctaacagtcttagcctggaagcaac-3′; K379N antisense: 5′-ccaggctaagactgttaggttgacttagaatctcac-3′; K152N sense: 5′-agaagcagcagaacaggacatttatttcacaggag-3′; K152N antisense: 5′-aaataaatgtcctgttctgctgcttctttcttctg-3′.

To generate siRNA-resistant PALB2 expression constructs, site-directed mutagenesis was performed using primers 5′-agctgcataaacacagtgtagaacagactgaaacagc-3′ and 5′-ttcagtctgttctacactgtgtttatgcagctcctg-3′. All desirable mutations were verified by sequencing.

Plasmids and siRNAs

HA-FLAG, Myc, and streptavidin binding peptide-FLAG (SFB)-tagged PALB2 or BRCA1 expression constructs were previously described (21, 24, 25). For RNAi-mediated depletion experiments, cells were transfected twice with either non-targeting control or target siRNAs (Dharmacon) using Oligofectamine according to the manufacturer's instructions (Invitrogen). Sequences for siRNAs targeting PALB2, BRCA1, ATM, and ATR are as follows: siPALB2: 5′-GCAUAAACAUUCCGUCGAAdTdT-3′; siBRCA1: 5′-GGAACCTGTCTCCACAAAGdTdT-3′; siATM: 5′-GCCUCCAGGCAGAAAAAGAdTdT-3′; and siATR: 5′-CCUCCGUGAUGUUGCUUGAdTdT-3′.

Antibodies and Chemicals

Antibodies against PALB2, BRCA1, Rad51, and γH2AX were described previously (21, 24–27). Anti-FLAG (M2) and anti-Actin antibodies were from Sigma. Anti-Myc (9E10) and anti-ATR (N-19) were purchased from Santa Cruz Biotechnology. Anti-KAP1-pS826 (A300-767A) was from Bethyl. Anti-ATM (D2E2; number 2873S), anti-ATM-pS1981 (number 4526L), anti-CHK1-pS345 (number 2341L), and anti-histone H3-pS10 (number 9701S) were purchased from Cell Signaling. Anti-HA (MMS-101R) was from Covance. Anti-KAP1 (610680) and anti-PLK-pT210 (558400) antibodies were from BD Biosciences. Anti-CHK1 (sc-8408) was from Santa Cruz and anti-DNA-PK-pS2056 (ab18192) was from Abcam. Rabbit polyclonal phosphoantibodies against PALB2 Ser(P)-157 and Ser(P)-376 were raised by immunizing rabbits with corresponding synthetic phosphopeptides. Sequences for PALB2 phosphopeptides were PALB2 Ser(P)-157 (QKRTFIpSQERDC) and PALB2 Ser(P)-376 (QESEILpSQPKSLS). Chemical inhibition of DNA damage response kinases was carried out using KU55933 (ATM inhibitor), VE821 (ATR inhibitor), or NU7441 (DNA-PK inhibitor). All chemicals were purchased from Selleckchem.

Immunoprecipitation Experiments

For detection of PALB2 phosphorylation at Ser-157 and Ser-376, HeLa cells expressing HA-FLAG epitope-tagged PALB2 were lysed with denaturing lysis buffer (20 mm Tris·HCl, pH 8.0, 50 mm NaCl, 0.5% Nonidet P-40, 0.5% deoxycholate, 0.5% SDS, 1 mm EDTA) on ice for 15 min, followed by sonication until the cell lysate turned clear. Cell lysate was then incubated with anti-FLAG M2-agarose (Sigma) for 2 h at 4 °C. Beads were washed 4 times and subsequently boiled with SDS sample buffer. The Western blotting experiment was then performed for the detection of PALB2 phosphorylation with the indicated phosphoantibodies. For co-immunoprecipitation experiments, 24 h after transfection of the indicated expression constructs, cells were lysed with NETN Buffer (20 mm Tris·HCl, pH 8, 100 mm NaCl, 0.5% Nonidet P-40, 1 mm EDTA) on ice for 15 min, followed by centrifugation (13,000 × g, 10 min). Supernatants were incubated with streptavidin beads (GE Healthcare) for 2 h at 4 °C. Protein complexes were separated by SDS-PAGE and detected by Western blotting experiments using the indicated antibodies.

Gene Conversion Assay

U2OS cells stably expressing DR-GFP (DR-U2OS) were pre-treated with controls siRNAs (siCTR) or PALB2-targeting siRNAs (siPALB2) twice at 24-h intervals. 24 h post-siRNA transfection, cells were electroporated with pCBASce (5 μg) and various PALB2-expressing constructs (5 μg; wild type and mutants) at 250 V, 950 microfarads by using Gene Pulser XCell (Bio-Rad). Cells recovered for 48 h before they were subjected to FACS analyses using a BD Biosciences LSR Fortessa Analyzer. Two-tailed Student's t tests were performed where necessary to determine the statistical significance of test and control samples.

Immunofluorescence Staining

HeLa cells were transfected with HA-FLAG-tagged PALB2 (wild type or various phospho-mutants). 48 h after transfection, cells were irradiated with 10 Gy. 4 h post-IR, cells were permeabilized with 0.5% Triton X-100 solution on ice for 1 min, followed by fixation with 3% paraformaldehyde at room temperature for 15 min. Cells were subsequently processed for immunostaining experiments using the indicated antibodies. Images were acquired using an Olympus BX51 fluorescence microscope. One-way analysis of variance followed by post hoc tests were performed where appropriate to determine whether the percentage of cells positive for 53BP1 or γH2AX IRIF differed among control, PALB2-depleted, and reconstituted cells.

Mass Spectrometric Profiling of PALB2 Phosphorylation

293T cells stably expressing FLAG-tagged PALB2 were treated with or without IR (10 Gy). Cells were lysed under denaturing conditions (20 mm Tris·HCl, pH 8.0, 50 mm NaCl, 0.5% Nonidet P-40, 0.5% deoxycholate, 0.5% SDS, 1 mm EDTA) on ice for 15 min, followed by sonication until the cell lysate turned clear. FLAG-PALB2 was then precipitated with anti-FLAG M2-agarose (Sigma) for 2 h at 4 °C. Precipitated PALB2 proteins were then analyzed for phosphopeptides by mass spectrometry (Taplin biological mass spectrometry facility, Harvard University, Cambridge, MA).

Results

PALB2 Is Phosphorylated in Response to IR-induced DNA Damage

Protein phosphorylation underlies the functional connectivity of the mammalian DDR network (28, 29). Indeed, proteome-wide profiling of DNA damage-induced protein phosphorylation events revealed hundreds of protein factors that become post-translationally modified in response to IR (23, 30, 31). Although many of the IR-induced phosphorylation events targeted consensus sequences that are recognized by the master DDR kinases ATM and ATR, regulation and functional significance of most of these modifications remain undefined.

In examining how the DDR orchestrates PALB2 in DNA repair processes, we found that IR treatment, in a dose-dependent manner, triggered a change in the electrophoretic mobility of PALB2 proteins (Fig. 1A). Similar mobility shifts of PALB2 proteins were also reported after hydroxyurea treatment (32). Notably, this was reproducibly seen across different cell lines, including 293T, HeLa, and U2OS cells, thus excluding cell-specific effects (data not shown). The slower migrating species of PALB2 resembled that of BRCA1, and were reversible by λ phosphatase treatment (Fig. 1B), strongly suggesting that PALB2 is subjected to phosphorylation in response to IR.

FIGURE 1.

FIGURE 1.

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Phosphorylation analysis of the Fanconi anemia protein PALB2. A, PALB2 is post-translationally modified in response to IR. 293T cells were irradiated with the indicated doses of IR. 4 h post-IR, cells were lysed and whole cell extracts were separated by SDS-PAGE and immunoblotted with the indicated antibodies; B, post-translational modifications of PALB2 is reversed by λ phosphatase treatment; C, schematic illustration depicting the experimental procedures to profile PALB2 phosphorylation; D, list of PALB2 phosphopeptides recovered from cells challenged with IR. *, highlights potential ATM/ATR substrates -(S/T)Q motif; E, IR-induced mobility shift is hampered in PALB2 S157A/S376A (S/A) mutant. Cells expression WT PALB2 or its phospho-mutant (S/A) were left untreated or irradiated. HA-tagged PALB2 proteins were immunoprecipitated and immunoblotted to evaluate its IR-induced migration properties on SDS-PAGE.

To profile PALB2 phosphorylation, we affinity purified FLAG epitope-tagged PALB2 proteins from control and irradiated (10 Gy) cells under denaturing conditions, and subjected the immunoprecipitates to mass spectrometric analysis (Harvard Taplin; Fig. 1C). Among the list of IR-induced PALB2 phosphopeptides we recovered (Fig. 1D), we were particularly interested in Ser-157 and Ser-376, as both of these putative phosphorylation sites resided within consensus sequences that are recognized by the master DDR kinases ATM and ATR (Fig. 2A), and were previously identified in a proteome-wide analysis for ATM/ATR-dependent phosphorylation events (23). Notably, PALB2 mutated at both Ser-157 and Ser-376 hampered its mobility shift on SDS-PAGE (Fig. 1E), highlighting their potential roles in connecting the DDR network with the tumor suppressor protein.

FIGURE 2.

FIGURE 2.

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Role of DDR kinase ATM in IR-induced PALB2 phosphorylation. A, schematic illustration of PALB2 protein, its domain organization, and the conservation of Ser-157 and Ser-376 across different species; B, chemical inhibition of ATM suppressed the IR-induced mobility shift of PALB2 proteins. Cells pretreated with KU55933 (10 μm, 2 h) or not were subjected to IR (10 Gy). Cells were lysed 4 h after and Western blotting experiments were performed using the indicated antibodies. * denotes nonspecific bands; C, validation of rabbit polyclonal antibodies against phosphorylated PALB2 at Ser-157 or Ser-376. Cells expressing the FLAG epitope-tagged WT PALB2, its S59A, S157A, or S376 mutants were irradiated (10 Gy), lysed, and FLAG-PALB2 proteins were immunoprecipitated (IP) under denaturing conditions using M2-agarose beads (Sigma). Immunoprecipitates were separated by SDS-PAGE and Western blotting experiments were performed to verify specificity of anti-PALB2 Ser(P)-157 and Ser(P)-376 antibodies; D, endogenous PALB2 proteins are phosphorylated in response to IR treatment. 293T cells were either irradiated (10 Gy) or left untreated. 4 h post-IR cells were lysed and PALB2 proteins were precipitated using protein A-agarose conjugated with anti-PALB2 antibodies; E, ATM inhibition suppressed DNA damage-induced PALB2 phosphorylation. 293T cells pre-treated with ATM inhibitor KU55933 (10 μm, 2 h) or dimethyl sulfoxide were irradiated or left untreated. 1 h post-IR whole cell extracts were prepared, separated by SDS-PAGE, and Western blotting experiments were performed; F, PALB2 is phosphorylated at Ser-157 and Ser-376 in response to different types of DNA damage. Cells expressing FLAG-tagged PALB2 were pre-treated with ATM inhibitor (KU55933) for 3 h before they were challenged with various DNA damaging agents, including IR (10 Gy), ultraviolet light (UV; 100 J/m2), or cisplatin (CIS; 33 μm). Cells were subsequently lysed and whole cell extracts (WCE) or immunoprecipitated PALB2 proteins were separated by SDS-PAGE for Western blotting analysis using indicated antibodies; G, requirement of ATM and ATR in PALB2 phosphorylation. Cell pre-treated with ATM- and/or ATR-targeting siRNAs were irradiated (10 Gy), lysed 1 h post-IR, and FLAG-PALB2 proteins were immunoprecipitated under denaturing conditions to evaluate the phosphorylation status at Ser-157 and Ser-376 using the indicated antibodies; H, siRNA-treated cells obtained from G were subjected to cell cycle profile analysis by propidium iodide staining; I, DNA-PK is not required for IR-induced PALB2 phosphorylation. Cells pre-treated with ATM inhibitor (10 μm), DNA-PK inhibitor (10 μm), or both for 2 h were irradiated (10 Gy). Phosphorylation status of PALB2 at Ser-157 and Ser-376 were evaluated 1 h post-IR by Western blotting.

The DDR Kinase ATM Is Required for IR-induced PALB2 Phosphorylation

Consistent with a master regulatory role of ATM in the DDR, ATM inhibition suppressed much of the IR-induced mobility shift of PALB2 proteins (Fig. 2B). To dissect how PALB2 phosphorylation at Ser-157 and Ser-376 are regulated, we raised antibodies against Ser(P)-157 and Ser(P)-376 in rabbits, and verified that the antibodies are specific for PALB2 Ser(P)-157 and Ser(P)-376, respectively (Fig. 2C). Using these antibodies we confirmed that PALB2 is phosphorylated at Ser-157 and Ser-376 in response to IR-inflicted genotoxic stress (Fig. 2D).

The ATM kinase orchestrates cellular responses to IR-induced DNA double-strand breaks (DSBs) by regulating the phosphorylation status of hundreds of DDR proteins (33). Accordingly, we first examined whether PALB2 phosphorylation at Ser-157 and Ser-376 required the master DDR kinase. To this end, we chemically inhibited ATM activity using the ATM-specific inhibitor KU55933, and found that phosphorylation of KAP1, an established ATM substrate (34), was efficiently suppressed (Fig. 2E). Under these experimental conditions we found that ATM activity was required for phosphorylation of PALB2 at Ser-157, and to a large extent Ser-376, in response to IR (Fig. 2E). Phosphorylation of PALB2 was also induced in cells challenged with ultraviolet light (UV) and the interstrand cross-linker cisplatin in an ATM-dependent manner (Fig. 2F).

To corroborate a role of ATM in PALB2 phosphorylation, we inactivated ATM by use of the RNAi approach. We also tested whether the ATR kinase, which plays predominant roles in replicative stress responses (35), may be important in the IR-induced PALB2 phosphorylation. Consistently, pre-treatment of cells with ATM targeting siRNAs inhibited the IR-induced PALB2 phosphorylation (Fig. 2G), without affecting cell distribution at different cell cycle phases (Fig. 2H). Intriguingly, whereas both DDR kinases appear to promote PALB2 phosphorylation at Ser-157, phosphorylation of PALB2 Ser-376 seems to be strictly ATM-dependent.

To more rigorously examine the specific requirement of the ATM kinase in PALB2 phosphorylation at Ser-157 and Ser-376, we also investigated whether the phosphatidylinositol 3-kinase-related kinase DNA-PK, which is key in DSB repair via non-homologous end-joining (36), may be important. In stark contrast to that of ATM, inactivation of DNA-PK activity did not affect PALB2 phosphorylation (Fig. 2I), illustrating a highly specific role of ATM in regulating PALB2 Ser-157 and Ser-376 phosphorylation.

Role of BRCA1 in ATM-dependent PALB2 Phosphorylation

Previous studies implicated BRCA1 in serving as a molecular scaffold to promote phosphorylation and activation of a number of ATM substrates, including p53, NBS1, CHK1, and CHK2 (5). Because BRCA1 directly interacts with PALB2 (20–22), we next tested whether BRCA1 may also be required for PALB2 phosphorylation. siRNA-mediated depletion of BRCA1 reversed the IR-induced mobility shift of PALB2 proteins on SDS-PAGE (Fig. 3, A and B), and led to marked suppression of DNA damage-induced phosphorylation of PALB2 at Ser-157, with only minor influence on the phosphorylation status of Ser-376 (Fig. 3C).

FIGURE 3.

FIGURE 3.

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The BRCA1-PALB2-BRCA2 complex integrity is important for PALB2 phosphorylation in response to IR. A, cell cycle distribution of cells treated with BRCA1-targeting (siBRCA1) or control (siCTR) siRNAs; B, requirement of BRCA1 in IR-induced mobility shift of PALB2. HeLa cells pre-treated with BRCA1 or control siRNAs were irradiated (10 Gy). 4 h post-IR whole cell extracts were prepared, separated by SDS-PAGE, and Western blotting experiments were performed using the indicated antibodies; C, PALB2 Ser-157 phosphorylation, and to a lesser extent, Ser-376 phosphorylation requires BRCA1. Phosphorylation status of PALB2 Ser-157 and Ser-376 was examined after BRCA1 depletion essentially as in B except that FLAG-PALB2 expressing cells were used; D, the BRCA1-PALB2 complex promotes full-blown PALB2 phosphorylation upon IR challenge. HeLa cells stably expressing the BRCA1-binding defective PALB2 mutant (ΔN42) were irradiated (10 Gy). 1 h post-IR cells were lysed and PALB2 phosphorylation at Ser-157 and Ser-376 was examined using the indicated antibodies essentially as in C. The PALB2 S157A/S376A (S/A) mutant was used as negative control.

We also took advantage of a BRCA1-binding defective PALB2 mutant that was previously characterized (ΔN42) (21, 24, 25), and examined how IR treatment affects PALB2 phosphorylation at Ser-157 and Ser-376 when its ability to interact with BRCA1 was compromised. Consistent with a critical role of the BRCA1-PALB2 complex formation in the DDR, defects in BRCA1-binding hampered PALB2 phosphorylation at Ser-157 (Fig. 3D). Interestingly, the PALB2 ΔN42 mutant partially supported IR-induced phosphorylation at Ser-376. Together, these data suggest that BRCA1, via its interaction with PALB2, promoted PALB2 phosphorylation in response to IR-induced DNA damage.

Phosphorylation of PALB2 Is Dispensable for HR Repair

Given the established role of PALB2 in HR repair, we first explored the functional significance of PALB2 phosphorylation at Ser-157 and Ser-376 using the Maria Jasin gene conversion assay (Fig. 4A). Accordingly, depletion of PALB2 resulted in substantial reduction in rates of DNA repair events (Fig. 4B). We generated siRNA-resistant versions of wild type PALB2, its phospho-dead mutant S157A/S376A (S/A) and phospho-mimicking mutants S157D/S376D (S/D) and S157E/S376E (S/E), and reconstituted these expression constructs into PALB2-depleted cells to evaluate how manipulating the phosphorylation status of PALB2 affects HR repair. We included the PALB2 L21A mutant, which we and others have shown to be HR deficient (19, 21), as a negative control (Fig. 4B). Notably, introduction of various PALB2 phospho-mutants restored HR repair to similar extents as did wild type PALB2 (Fig. 4B). Because all PALB2 phospho-mutants tested migrated to DSBs (Fig. 4C), restored IR-induced RAD51 foci formation upon depletion of endogenous PALB2 (Fig. 4D), retained their ability to interact with BRCA1, BRCA2, and RAD51 (Fig. 5, A–C), and were indistinguishable in their stability in the cycloheximide pulse-chase assay (Fig. 5D), we concluded that PALB2 phosphorylation at Ser-157 and Ser-376 do not play major roles in DSB repair via HR, at least in the context of gene conversion.

FIGURE 4.

FIGURE 4.

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PALB2 phosphorylation is dispensable for HR repair. A, schematic illustration showing the Maria Jasin gene conversion assay; B, no marked difference in homologous recombination-based repair ability was observed in PALB2 phospho-mutant expressing cells. The Maria Jasin gene conversion assay was performed by introducing the I-SceI expression plasmid together with either siRNA-resistant PALB2 or its phospho-mutants into PALB2-depleted U2OS-DR-GFP cells. Percentage of GFP-positive cells was measured using a BD Canto II analyzer and corresponds to the relative homologous recombination efficiency in each cell line. Results represent mean ± S.D. of at least three independent experiments. PALB2 L21A, BRCA1-binding defective PALB2; *, p < 0.05 compared with control siRNA (siCTR)-treated cells; C, PALB2 phosphorylation does not affect its double-strand break localization. Wild type PALB2 or its various phospho-mutants were transfected into HeLa cells. Cells were treated with 10 Gy IR and processed 6 h after for immunofluorescence staining experiment. Overlays of HA (PALB2) and γH2AX signals were determined by the ImageJ software. Quantification is shown on the right; D, PALB2 phospho-mutants supported IR-induced RAD51 foci formation. PALB2 siRNA-treated cells were evaluated for their ability to promote RAD51 foci in response to IR treatment (left). siRNA-resistant versions of PALB2 and its phospho-mutants were examined for their ability to support RAD51 IRIFs (right). Nuclei were counterstained with DAPI.

FIGURE 5.

FIGURE 5.

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PALB2 phosphorylation is dispensable for its interaction with BRCA1, BRCA2, and RAD51. A–C, PALB2 phosphorylation does not affect its interaction with BRCA1, BRCA2, or RAD51. Co-immunoprecipitation (IP) experiments were performed using lysates derived from cells co-transfected with various PALB2 expression plasmids and BRCA1, BRCA2, or RAD51 constructs. ΔC32, BRCA2-binding defective PALB2 mutant. D, phosphorylation of PALB2 does not affect protein stability. PALB2-expressing cells were treated with cycloheximide (CHX) and irradiated immediately. Cells were collected at the indicated time points post treatment for Western blotting analysis of protein expression. S/A, S157A/S376A; S/D, S157D/S376D; S/E, S157E/S376E.

Cell Recovery Correlated with Reversal of PALB2 Phosphorylation

The observation that PALB2 mediates the DNA damage checkpoint control prompted us to investigate the kinetics of PALB2 phosphorylation in the context of cell arrest and recovery in response to DNA damage (13). We first examined whether PALB2 phosphorylation at Ser-157 and Ser-376 are specific to interphase cells, as aberrant activation of the DDR during mitosis appears to promote chromosome instability (37–39). To this end we harvested mitotic cells by use of the microtubule-depolymerizing drug nocodazole, and compared the phosphorylation status of PALB2 in an asynchronized cell population in response to 10 Gy IR (Fig. 6A). Remarkably, PALB2 phosphorylation was limited to asychronized cells, strongly supporting the idea that mechanisms have evolved to promote PALB2 phosphorylation in response to IR strictly in interphase cells (Fig. 6, B and C).

FIGURE 6.

FIGURE 6.

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PALB2 phosphorylation is coupled to DDR activation and deactivation. A, PALB2 Ser-157 and Ser-376 are specifically phosphorylated during interphase. Mitotic cells were obtained after nocodazole treatment for 12 h by the shake-off method. Mitotic cells and asynchronous cells were irradiated (10 Gy) separately. 1 h post-IR, cells were lysed under denaturing conditions. Immunoprecipitation (IP) was performed using either anti-PALB2 antibodies (B) or anti-FLAG-agarose beads (C). Phosphorylation of PALB2 at Ser-157 and Ser-376 was examined using the indicated antibodies; D and E, IR-induced PALB2 phosphorylation at Ser-157 and Ser-376 are reversed as cells transit into mitosis. Cells were harvested according to the experimental procedures depicted in D. Whole cell extracts (WCE) and immunoprecipitated PALB2 proteins were prepared for Western blotting analysis of PALB2 phosphorylation using the indicated antibodies (E). N, non-mitotic cells; M, mitotic cells.

Given the temporal regulation of PALB2 phosphorylation we hypothesized that the interphase-specific phosphorylation of PALB2 may be coupled to DDR activation and deactivation. To address this possibility, we took advantage of the DNA damage-regulated G2/M checkpoint, which would allow us to efficiently separate cells that are undergoing DNA repair (G2-phase) from cells that have recovered from the checkpoint (mitotic cells). To this end we synchronized cells at the G1/S boundary by use of the double thymidine block protocol. Cells were subsequently released into S phase before they were irradiated with a recoverable dose of IR (3 Gy; Fig. 6D). We then collected cells that have entered mitosis (indicative of cells that have recovered from the G2/M checkpoint), and analyzed their PALB2 phosphorylation status in parallel to those that were non-mitotic (cells that remained arrested at the G2/M border). Remarkably, we found that PALB2 is phosphorylated at Ser-157 and Ser-376 only in cells that are arrested at the G2/M checkpoint (Fig. 6E). It is noteworthy to mention that mitotic PALB2 proteins, as compared with their interphase counterparts, displayed mobility shifts compatible with mitotic phosphorylation (Fig. 6, B, C, and E).

Dysregulated PALB2 Phosphorylation Perturbs DDRs

Given the coupling of cell arrest and recovery with the status of PALB2 phosphorylation, we explored whether PALB2, and its ATM-dependent phosphorylation at Ser-157 and Ser-376, may be important in promoting DDRs. We first determined the kinetics of DDRs in cells depleted of PALB2 by monitoring ATM autophosphorylation and KAP1 phosphorylation (KAP1-pS824), both of which are established and robust DSB respondents (34, 40). In line with important roles of PALB2 in DDRs, cells pre-treated with PALB2-targeting siRNAs exhibited sustained DDR activation, as judged by the delay in resolution of phosphorylation of ATM and KAP1 (Fig. 7A). Consistent with important roles of PALB2 phosphorylation in driving DNA damage responses, cells expressing PALB2 S157A/S376A (S/A) resulted in prolonged DDR activation as compared with its wild type counterpart (Fig. 7B).

FIGURE 7.

FIGURE 7.

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ATM-dependent PALB2 phosphorylation promotes DDRs. A, cells pre-treated with control siRNAs (siCTR) and PALB2-targeting siRNAs (siPALB2) were irradiated. Whole cell extracts were prepared at the indicated time points for Western blotting analysis using antibodies against phosphorylated ATM (ATM-pS1981) and KAP1 (KAP1-pS824); B, siRNA-resistant versions of PALB2 or its S157A/S376A (S/A) mutant were expressed in cells to evaluate ATM and KAP phosphorylation events. Phosphorylation status of ATM and KAP1 were evaluated over a time course that spanned 24 h; C–E, U2OS cells transfected with either control (siCTR) or PALB2-targeting siRNAs (siPALB2) were irradiated and the number of 53BP1 (C) or γH2AX (E) foci positive cells were counted (n = 200) at the indicated time points. For reconstitution experiments siRNA-resistant versions of PALB2 constructs were transfected into cells treated with PALB2-specific siRNAs. Immunofluorescence staining experiments were performed using anti-53BP1 or anti-γH2AX antibodies. Results represent mean ± S.D. of three independent experiments. F and G, 53BP1 and γH2AX IRIF were similarly evaluated in HeLa cells. p < 0.05 versus control siCTR-treated cells.

The tumor suppressor 53BP1 is key in DSB repair, and sustained accumulation of 53BP1 at DNA damage foci is indicative of aberrant DDRs (41). To complement the above analysis of the DDR, we also monitored 53BP1 accumulation at, and its time-dependent resolution from, DSBs. Consistently, PALB2 inactivation did not affect the IR-induced foci formation (IRIF) of 53BP1, but resulted in a substantial delay in the clearance of 53BP1 from DSBs (Fig. 7, C and D). To further probe the functional significance of PALB2 phosphorylation in the DDR, we reconstituted PALB2 knockdown cells with siRNA-resistant PALB2 expression constructs (wild type and S157A/S376A), and quantified 53BP1 IRIF over a time course that spanned 24 h. Intriguingly, whereas wild type PALB2 promoted 53BP1 IRIF clearance, cells expressing the PALB2 phospho-mutant exhibited defects in resolving 53BP1 accumulation from DSBs at late time points (Fig. 7, C and D). Similar requirements of PALB2 and its phosphorylation at Ser-157 and Ser-376 were observed for clearance of γH2AX IRIF during cell recovery from IR-induced DNA damage (Fig. 7E). More importantly, we also excluded cell type-specific effects and showed that PALB2 phosphorylation is equally important in cell recovery from IR-induced DNA damage in HeLa cells (Fig. 7, F and G). Together, our data uncover a pivotal role of dynamic phosphorylation of PALB2 in cellular responses and recovery to DNA damage.

Discussion

The Fanconi anemia protein PALB2 safeguards genome integrity by participating in multiple DDR mechanisms, including HR repair and cell cycle checkpoint control. However, exactly how this multifunctional tumor suppressor operates within the context of the DDR network is not clear. In this study, we systematically analyzed the phosphorylation profile of the Fanconi anemia protein PALB2 and explored the functional significance of its DNA damage-induced modifications. Consistent with versatility of post-translational modification systems in orchestrating DDRs, we found that PALB2 phosphorylation is dynamically regulated, and is important in cellular responses to genotoxic stress.

The ATM kinase is pivotal in orchestrating the mammalian DDR network (33). Indeed, not only is PALB2 specifically phosphorylated at two ATM consensus sites in response to IR treatment (Fig. 2A), chemical inhibition of ATM activity or depletion of ATM by use of siRNAs compromised the DNA damage-induced PALB2 phosphorylation (Fig. 2), strongly implicating an unprecedented connection between the master DDR kinase and the tumor suppressor protein PALB2. In line with the overlaps between the ATM and ATR kinases, IR-induced PALB2 phosphorylation at Ser-157 also required the replicative stress response ATR kinase, raising the possibility that ATM- and ATR-dependent DDR signaling events may converge onto PALB2 for proper coupling of cell proliferation and DNA repair processes. Indeed, our observations that dysregulation of PALB2 phosphorylation resulted in sustained DSB accumulation of 53BP1 and γH2AX (Fig. 7) clearly illustrate the functional relevance of these phosphorylation events during cell recovery from IR-induced DNA damage. Mechanistically how phosphorylated PALB2 orchestrates cell recovery from DDRs will require further exploration, but given its wide repertoire of DDRs the dynamic regulation of PALB2 phosphorylation may represent a means via which to coordinate different genome integrity protection protocols.

PALB2 physically links BRCA1 and BRCA2 and plays an important role in HR repair (5). Interestingly, we found that BRCA1 promoted PALB2 phosphorylation at Ser-157, whereas phosphorylation of PALB2 Ser-376 was only marginally affected in the absence of BRCA1 (Fig. 3, B and C). This paralleled our observation where disrupting the BRCA1-PALB2 interaction similarly compromised IR-induced PALB2 phosphorylation at Ser-157, and to a lesser extent, Ser-376 (Fig. 3D). It is noteworthy to mention that the requirement of BRCA1 is reminiscent with the more important role of the ATR kinase in promoting PALB2 phosphorylation at Ser-157, and raises the possibility that the two ATM/ATR phosphoresidues may exhibit differential kinetics in response to different types of DNA damage. Future work will be needed to explore this possibility.

In search for disease relevance of PALB2 phosphorylation we also retrieved PALB2 mutations from the COSMIC (Catalogue of Somatic Mutations in Cancer) database. We identified three cancer-derived mutations on the PALB2 sequence that surrounded Ser-157 and Ser-376 (K152N, Q377E, and K379N) and found that the Q377E mutation abrogated PALB2 phosphorylation specifically at Ser-376 (Fig. 8), highlighting the possibility that dysregulated PALB2 phosphorylation may perturb DDRs and promote genome instability.

FIGURE 8.

FIGURE 8.

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Analysis of COSMIC-derived PALB2 mutations. PALB2 and its mutants were examined for their ability to support Ser-157 and Ser-376 phosphorylation in response to IR treatment. Irradiated cells were lysed 1 h after IR and lysates were subjected to immunoprecipitation (IP) experiments followed by protein separation by SDS-PAGE. Western blotting experiments were carried out essentially as described. WCE, whole cell extracts.

In summary, we have provided a systematic phosphorylation analysis of the Fanconi anemia protein PALB2, and have revealed important roles of PALB2 Ser-157 and Ser-376 in driving cellular responses to genotoxic stress. Our study thus provides a new scope to further decipher mechanistically how the ATM kinase may orchestrate PALB2-dependent DDRs.

Author Contributions

Y. G., S. M. H. S., and M. S. Y. H. designed the experiments, Y. G. and W. F. performed the experiments, all authors analyzed and interpreted the data, Y. G. and M. S. Y. H. wrote the manuscript.

Acknowledgments

We thank Dr. Bing Xia for communicating unpublished findings, Dr. Maria Jasin for the DR-GRP gene conversion reporter, Dr. Junjie Chen for continuous support, The Faculty Core Facility of The LKS Faculty of Medicine (University of Hong Kong) for technical assistance, and all members of the Huen Laboratory for discussion and support.

*

The work was supported in part by the Research Grants Council Hong Kong Project number HKU_786512M (to M. S. Y. H.) and NSFC Young Scientist Fund Project number 31301115 (to W. F.). The authors declare that they have no conflicts of interest with the contents of this article.

4
The abbreviations used are:
HR
homologous recombination
DDR
DNA damage response
IR
ionizing radiation
ATM
Ataxia telangiectasia mutated
ATR
ATM and Rad3-related
Gy
gray
DSB
double-strand breaks
IRIF
IR-induced foci formation.

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