Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response
2012; Springer Nature; Volume: 31; Issue: 10 Linguagem: Inglês
10.1038/emboj.2012.86
ISSN1460-2075
AutoresDong-Hyun Lee, Aaron A. Goodarzi, Guillaume Adelmant, Yunfeng Pan, Penelope A. Jeggo, Jarrod A. Marto, Dipanjan Chowdhury,
Tópico(s)Ubiquitin and proteasome pathways
ResumoArticle10 April 2012free access Source Data Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response Dong-Hyun Lee Dong-Hyun Lee Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Aaron A Goodarzi Aaron A Goodarzi Genome Damage and Stability Centre, University of Sussex, East Sussex, UK Search for more papers by this author Guillaume O Adelmant Guillaume O Adelmant Department of Cancer Biology and Blais Proteomics Center, Dana Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Yunfeng Pan Yunfeng Pan Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Penelope A Jeggo Penelope A Jeggo Genome Damage and Stability Centre, University of Sussex, East Sussex, UK Search for more papers by this author Jarrod A Marto Jarrod A Marto Department of Cancer Biology and Blais Proteomics Center, Dana Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Dipanjan Chowdhury Corresponding Author Dipanjan Chowdhury Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Dong-Hyun Lee Dong-Hyun Lee Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Aaron A Goodarzi Aaron A Goodarzi Genome Damage and Stability Centre, University of Sussex, East Sussex, UK Search for more papers by this author Guillaume O Adelmant Guillaume O Adelmant Department of Cancer Biology and Blais Proteomics Center, Dana Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Yunfeng Pan Yunfeng Pan Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Penelope A Jeggo Penelope A Jeggo Genome Damage and Stability Centre, University of Sussex, East Sussex, UK Search for more papers by this author Jarrod A Marto Jarrod A Marto Department of Cancer Biology and Blais Proteomics Center, Dana Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Dipanjan Chowdhury Corresponding Author Dipanjan Chowdhury Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Dong-Hyun Lee1, Aaron A Goodarzi2, Guillaume O Adelmant3, Yunfeng Pan1, Penelope A Jeggo2, Jarrod A Marto3 and Dipanjan Chowdhury 1 1Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA 2Genome Damage and Stability Centre, University of Sussex, East Sussex, UK 3Department of Cancer Biology and Blais Proteomics Center, Dana Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA *Corresponding author. Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, 450 Brookline Ave, JF517 Boston, MA 02115, USA. Tel.:+1 617 582 8639; Fax:+1 617 582 8213; E-mail: [email protected] The EMBO Journal (2012)31:2403-2415https://doi.org/10.1038/emboj.2012.86 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Protein phosphatase PP4C has been implicated in the DNA damage response (DDR), but its substrates in DDR remain largely unknown. We devised a novel proteomic strategy for systematic identification of proteins dephosphorylated by PP4C and identified KRAB-domain-associated protein 1 (KAP-1) as a substrate. Ionizing radiation leads to phosphorylation of KAP-1 at S824 (via ATM) and at S473 (via CHK2). A PP4C/R3β complex interacts with KAP-1 and silencing this complex leads to persistence of phospho-S824 and phospho-S473. We identify a new role for KAP-1 in DDR by showing that phosphorylation of S473 impacts the G2/M checkpoint. Depletion of PP4R3β or expression of the phosphomimetic KAP-1 S473 mutant (S473D) leads to a prolonged G2/M checkpoint. Phosphorylation of S824 is necessary for repair of heterochromatic DNA lesions and similar to cells expressing phosphomimetic KAP-1 S824 mutant (S824D), or PP4R3β-silenced cells, display prolonged relaxation of chromatin with release of chromatin remodelling protein CHD3. Our results define a new role for PP4-mediated dephosphorylation in the DDR, including the regulation of a previously undescribed function of KAP-1 in checkpoint response. Introduction Genotoxic conditions leading to double-stranded DNA breaks (DSBs) or replication stress are hazardous by virtue of their ability to stimulate both genomic instability and cellular transformation. To prevent such detrimental consequences, organisms have evolved a complex response to genotoxic stress, generally initiated by the phosphatidylinositol-3 (PI3) kinase-like family of protein kinases. A vast network of ∼700 proteins, including DNA repair and replication proteins, are phosphorylated by these kinases in response to DSBs or replication stress (Matsuoka et al, 2007). The phosphorylated proteins include factors involved in DNA replication and repair, apoptosis and/or cell cycle progression. The functional consequences of phosphorylation have been studied for only a small subset of these factors, and in many cases these modifications impact upon the DNA damage response (DDR). There is increasing evidence that protein phosphatases play an important role in the DSB-induced signalling cascade (Lee and Chowdhury, 2011). Two recent studies investigating the dynamics of phosphorylation, following induction of DSBs, show that over one-third of the captured phospho-peptides were dephosphorylated within minutes of DNA damage (Bennetzen et al, 2010; Bensimon et al, 2010). These data suggest that phosphatases not only play a role in counteracting the DSB-induced phosphorylation later in the damage response but also play a primary role in initiating the repair process. Interestingly, this suggestion is consistent with our recent study showing that PP4 dephosphorylates the essential replication protein A (RPA) on the RPA2 subunit, immediately after DNA damage and that this dephosphorylation event is critical for efficient repair of DSBs (Lee et al, 2010). In recent years, we and others have identified a role for the PP2A-like phosphatases (PP2AC, PP4C and PP6C) in the DNA damage response DDR (Chowdhury et al, 2005, 2008; Nakada et al, 2008; Mi et al, 2009; Douglas et al, 2010). The catalytic components of these enzymes are typically contained in dimeric or trimeric complexes with tissue/cell type-specific regulatory subunits conferring substrate specificity (Virshup, 2000; Shi, 2009). The catalytic subunit PP4C is significantly overexpressed in malignant lung and breast tissue, and PP4 deficiency impacts repair of DNA replication-mediated damage (Chowdhury et al, 2008; Wang et al, 2008). The role of PP4C in the DDR is broadly conserved in budding yeast, and its homolog, Pph3, impacts DNA repair and the cellular checkpoint response (Hastie et al, 2006; Keogh et al, 2006; O'Neill et al, 2007; Kim et al, 2010). Several putative PP4C-containing complexes have been identified in mammalian cells but their biological functions remain unclear (Gingras et al, 2005; Chen et al, 2008), and so far there are only a few bonafide substrates of PP4 (Zhang et al, 2005; Cha et al, 2008; Toyo-oka et al, 2008; Falk et al, 2010; Zhang and Durocher, 2010). Genetic deletion of the PP4 catalytic subunit PP4C in mice results in early embryonic lethality, underlining its importance in development and maintaining cell health (Shui et al, 2007). Therefore, systematic identification of PP4 substrates is necessary to elucidate its role in the DDR, and during development. Due to the lack of consensus targeting motifs, identifying substrates of Ser/Thr phosphatases has been a major challenge(Lee and Chowdhury, 2011). In recent times, interaction-based approaches, that is, tandem affinity purification/mass spectrometry, have been the only successful comprehensive strategy enabling the identification of substrates for Ser/Thr phosphatases (Wakula et al, 2003; Gingras et al, 2005; Arroyo et al, 2008). Here, we devised a proteomic method to identify proteins de-phosphorylated by PP4 based on the rationale that phosphoproteins enriched in the absence of a phosphatase are putative substrates. Quantitative phosphoproteomics in the context of PP4 depletion revealed that KRAB-domain-associated protein 1 (KAP-1) is a putative substrate of PP4. KAP-1 (also known as TRIM28, KRIP-1 and TIF1β) is a transcriptional corepressor, which recruits several components of the gene silencing machinery, including heterochromatin protein 1 (HP1) and the chromodomain-helicase-DNA-binding protein 3 (CHD3), to specific genomic loci (Lechner et al, 2000; Schultz et al, 2001, 2002). KAP1 and HP1β interaction is compromised by phosphorylation of KAP1 at S473 (Chang et al, 2008), and this modification occurs in the mitotic phase of the cell cycle (Beausoleil et al, 2004; Chang et al, 2008). In response to DSBs there is rapid, but transient, ATM-mediated phosphorylation of KAP-1 at serine 824 (S824) both at DNA repair foci and throughout the nucleus (Ziv et al, 2006; Goodarzi et al, 2008; Noon et al, 2010). Whereas pan-nuclear pS824-KAP1 dissipates rapidly, pS824-KAP1 foci can persist for longer times. Phosphorylation of KAP-1 at S824 specifically impacts repair of DSBs in heterochromatin (Goodarzi et al, 2008, 2010, 2011; Noon et al, 2010). The justification for pursuing a PP4 substrate would be provided by any data showing that removal of the phosphorylated form of the protein is necessary for restoration to a 'normal', pre-phosphorylation state. Interestingly, constitutive expression of phosphomimetic (S824D) KAP-1 has a distinct cellular phenotype to cells expressing wild-type (WT) KAP-1, with de-repression of several stress-response genes (Li et al, 2007, 2010) and global chromatin relaxation (Ziv et al, 2006). The short half-life of DSB-induced phospho-S824 KAP-1 and the functional consequences of expressing constitutively phosphorylated (S824D) KAP-1 suggested that regulated de-phosphorylation of KAP-1 may be necessary for restoring the 'normal' cellular state following DNA damage. Therefore, we investigated the link between PP4C and KAP-1. Biochemical and cytological studies confirmed that PP4C dephosphorylates KAP-1 at S824 and regulates its role in chromatin compaction and gene expression. Furthermore, we observe that CHK2-mediated phosphorylation of another KAP-1 residue, S473, plays a role in enforcing the G2/M checkpoint after ionizing radiation (IR). A PP4C/R3β complex dephosphorylates KAP-1 at S473 to facilitate cell cycle progress. Results Differential phosphoproteomics in PP4C-silenced cells In an effort to broadly profile potential PP4C substrates, we utilized the proteomic strategy outlined in Figure 1A. Duplicate samples of nuclear proteins isolated from cells treated with control (scrambled siRNA) and siRNA targeting PPC4 were digested with trypsin and the resulting peptides were encoded with isobaric iTRAQ tags (Ross et al, 2004). Phosphopeptides were purified with iron-NTA IMAC (Ficarro et al, 2009a) and analyzed by LC-MS/MS (Ficarro et al, 2009b). Data from replicate samples were processed within our multiplierz software framework (Askenazi et al, 2009) and used to derive a 95% acceptance region (Zhang et al, 2010) for iTRAQ intensity ratios (Figure 1B). This approach allowed us to capture experimental variance at the level of enzymatic digestion. Based on this analysis, we identified significant hyperphosphorylation (P-value ?0.05; 197 peptides) in the context of PP4C depletion (Figure 1B). Interestingly, several of the corresponding proteins have well-established roles in the DDR, including KAP-1, CHD4 and 53BP1. In order to validate our phosphoproteomics approach and further explore the potential links between the DDR and subsequent phosphatase activity, we utilized a pan-phosphoserine antibody to immunoprecipate phosphoproteins from PP4C-silenced cells subjected to DNA damage. The immunoprecipitate was probed for KAP-1, 53BP1 and CHD4, along with RPA2, a known PP4 substrate, as a control. All these proteins showed a distinct enrichment in the immunoprecipitates from PP4C-silenced cells exposed to IR (Figure 1C). The enrichment in undamaged cells was detectable but moderate. To verify these results, we examined substrate phosphorylation using the mobility shift of phosphorylated proteins. To enhance the analysis, we used a phosphate-binding metal complex (phos–tag), which enhances the mobility shift of phosphorylated proteins during resolution by SDS–PAGE (Kinoshita et al, 2009). Control and PP4C-silenced cell lysates were analyzed using phos–tag. Consistent with Figure 1C, there was a detectable increase in the phosphorylated form of KAP-1, 53BP1 and CHD4 in the absence of PP4C (Figure 1D). Collectively, these findings verify the potential importance of our screening method and show that several DDR proteins are putative substrates of PP4C. Next, we focused on substantiating direct dephosphorylation and in vivo significance for a single substrate. Figure 1.PP4C influences the phosphorylation status of multiple DDR proteins. (A) Schematic for phosphoproteomics-based identification of putative PP4C substrates. (B) Identification of regulated phosphorylation in response to PP4C depletion. The geometric mean of iTRAQ spectral peak height ratios for replicate samples was plotted as a function of the sum of the geometric means of all iTRAQ spectral peak heights. Maximum approximate conditional likelihood (MACL) was used to determine an intensity-based variance function from which the 95% acceptance region was calculated (grey curve and Supplementary Table 1). Several DDR proteins, including KAP1, CHD4 and TP53BP1, exhibited hyperphosphorylation in response to deplection of PP4C. (C) Validation of the PP4 targets. (Left panel) HeLa S3 cells transfected with PP4C or scrambled siRNAs were exposed to IR, lysed after 2 h and immunoprecipitated (IP) using a pan-phosphoSer antibody and probed for DDR proteins as indicated. The relative band intensities are provided below each immunoblot. (Right panel) Proteins identified as putative PP4 substrates were confirmed using Phos–tag. HeLa S3 cells were transfected with PP4C siRNA and irradiated with 10-Gy IR. Lysates were subjected to SDS–PAGE containing 20 μM Phos–tag and immunobloted with indicated antibodies. Lysates treated with λ protein phosphatase (λPP) served as control for the Phos–tag-induced mobility shift. Download figure Download PowerPoint KAP-1 is a substrate of a PP4/R3β complex To investigate whether a PP4 complex impacts the phosphorylation of KAP-1 at S824, we silenced the regulatory subunits of PP4, and evaluated basal levels of phosphorylated KAP-1 using a phospho-S824-specific antibody. Although detectable by mass spectrometry, the basal pS824-KAP-1 level in cells without exogenous DNA damage is not visible by immunoblotting using 20–100 μg of cell lysate (Figure 2A). We used 200 μg of cell lysate to observe a pS824-KAP-1 signal in PP4-deficient cells. The increase in p S824-KAP-1 obtained by silencing PP4C was also observed by silencing PP4R3β (Figure 2A). Knocking down the other subunits, PP4R1, PP4R2 or PP4R3α, had a relatively modest effect on pS824-KAP-1 (Supplementary Figure 1). In response to IR there is a rapid increase in pS824-KAP-1, which peaks within 1 h, and significantly drops by 3 h. In the absence of either PP4C or PP4R3β there is a significantly higher amount of pS824-KAP-1 in cells 3 h after IR (Figure 2B). Similar results were obtained using other DNA damaging agents such as bleomycin and doxorubicin (Supplementary Figure 2). Furthermore, there is also persistence of focal pS824-KAP-1 in PP4R3β-silenced primary human fibroblasts most evident between 4–16 h post IR (Figure 2C). Figure 2.KAP-1 phosphorylation on S824 is regulated by a PP4C–R3β complex. (A) In unperturbed cells, phosphorylation of KAP-1 on S824 is elevated in PP4-depleted cells. HeLa cells transfected with siRNAs against indicated PP4 subunits were harvested after 72 h and immunoblotting was performed with the indicated antibodies. (B) HeLa cells transfected with siRNAs against PP4C or PP4R3β were irradiated and harvested at the indicated times and p-KAP-1 was assessed by immunoblot using phospho-KAP-1 antibody (phosphoSerine 824). The kinetics of pS824-KAP-1 formation was monitored after irradiation by loading 20 μg of cell lysate per lane. (C) PP4R3β depletion attenuates pS824-KAP-1 turnover after IR. Primary human fibroblasts were transfected with control or PP4R3β siRNAs. After 72 h, cells were irradiated, fixed at the indicated times and immunostained for pS824-KAP1 (red), γH2AX (green) and DAPI (blue). Right Panel: The average pS824-KAP-1 signal intensity per nucleus was quantified using ImageJ software. Data represent average and s.d. of three independent experiments. (D) PP4C/PP4R3β interacts with KAP-1. HeLa S3 cells stably expressing empty vector (FH-Ctrl), FH-tagged KAP-1 (top and bottom panel), and PP4C or PP4R3β (middle panel) were subjected to immunoprecipitation using anti-FLAG beads at indicated time points before or after IR. PP4R3β or control siRNAs were transfected in cells 72 h prior to immunoprecipitation (right panel, bottom). The immunoprecipitate was probed with antibodies against endogenous KAP-1, PP4C and PP4R3β as indicated. Figure source data can be found with the Supplementary data. Download figure Download PowerPoint R3β mediates the interaction of KAP-1 and PP4C We reasoned that a PP4C/R3β complex-mediated dephosphorylation of KAP-1 would require interaction of these proteins. We analyzed the association of these proteins by reciprocal immunoprecipitation/immunoblot assays using lysates from HeLa cells expressing FLAG and HA (FH)-tagged KAP-1, PP4C or PP4R3β. KAP-1 associates with PP4C and PP4R3β, and the interaction is enhanced by DNA damage (Figure 2D, upper and middle panel). To examine whether PP4C and PP4R3β independently associate with KAP-1, we silenced PP4R3β and observed that the interaction of PP4C with KAP-1 is dramatically reduced in the absence of PP4R3β (Figure 2D, lower panel). Together, these results suggest that a PP4C/R3β complex interacts with KAP-1 and regulates its phosphorylation status. Impact of PP4C/R3β on pS824-KAP-1 is not due to ectopic activation of ATM KAP-1 is a bonafide ATM substrate, and moderate (0–20 Gy) doses of IR do not induce the formation of pS824-KAP1 in ATM-deficient cells (Ziv et al, 2006; Noon et al, 2010). However, ectopic activation of ATM can potentially cause enhanced pS824-KAP-1 levels. PP4 dephosphorylates multiple proteins (H2AX and RPA2) (Chowdhury et al, 2008; Lee et al, 2010) involved in the DDR and it is feasible that PP4 deficiency indirectly activates ATM-induced DNA damage signalling, which in turn leads to the persistence of pS824-KAP-1. In that scenario, continued ATM activity would be necessary for maintaining the pS824-KAP-1 signal in PP4-deficient cells. To evaluate this possibility, we inhibited ATM immediately after IR-induced formation of pS824-KAP-1. The ATM inhibitor (ATMi) has an immediate impact on ATM activity (Shibata et al, 2011), and pS824-KAP-1 levels diminish rapidly after treatment with ATMi (Figure 3A). Depletion of PP4C, or PP4R3β, causes persistence of pS824-KAP-1 several hours after IR even in the presence of ATMi (Figure 3B). A recent study suggested that PP1β de-phosphorylated DNA damage-induced pS824-KAP-1, and PP1α de-phosphorylated basal pS824-KAP-1 (Li et al, 2010). Relative to control cells, there was a small increase in pS824-KAP-1 in PP1α- and PP1β-silenced cells, in the presence of ATMi (Figure 3B). This would suggest that the impact of PP1 on cellular levels of pS824-KAP-1 may be primarily due to inappropriate activation of ATM. Figure 3.Hyperphosphorylation of S824-KAP-1 in PP4-silenced cells is not due to ectopic activation of ATM. (A) pS824-KAP-1 is lost rapidly in the absence of ongoing ATM activity. (Upper panel) 1BR3 primary fibroblasts were irradiated with 3-Gy IR and, 0.5 h later, were incubated with either DMSO or 10 μM of ATMi and harvested at the indicated times. Cells were fixed and immunostained for pS824-KAP-1 (red), γH2AX (green) and DAPI (blue). (Lowerpanel) GM02188 lymphoblastoid cells were irradiated with 10-Gy IR and, 20 min later, were incubated with DMSO or ATMi. Cells were harvested at the indicated times, processed and immunoblotted for pS824-KAP-1 and total KAP-1 (top panel). Immunoblot signal was quantified and plotted (lower panel). The half-lives of signal decay were calculated. (B) PP4C/PP4R3β depletion attenuates pS824-KAP-1 turnover after IR independent of ATM activity. HeLa cells were transfected with siRNAs. After 72 h, cells were irradiated with 10-Gy IR. At 0.5 h post IR, DMSO or ATMi was added. Cells were harvested at the indicated times and immunoblotting was performed and band intensities quantified using the Odyssey Infrared Imaging System. The intensity of pS824-KAP-1 was normalized relative to total KAP-1 and represented graphically. (C) PP4R3β depletion attenuates pS824-KAP-1 turnover after IR independent of ATM activity. Primary human fibroblasts were transfected with siRNAs. After 72 h, cells were irradiated with 3-Gy IR. At 0.5 h post IR, DMSO or ATMi was added. Cells were fixed at the indicated times and immunostained for pS824-KAP1 (red), γH2AX (green) and DAPI (not shown). The data were quantified as in Figure 2C. (D) PP4R3β regulates pS824-KAP-1 at late-repairing IRIF. Primary human fibroblasts were transfected with control or PP4R3β siRNA. After 72 h, cells were irradiated with 8-Gy IR. After 24 h, ATMi was added (t=0 min) and cells were fixed at the indicated times up to 120 min. Fixed cells were then immunostained for pS824-KAP-1 (red), γH2AX (green) and DAPI (blue). The average pS824KAP-1 signal intensity (from (A)) overlapping with γH2AX foci (i.e., 'at IRIF') was quantified using ImageJ software. Data represent average and s.d. of three independent experiments. The approximate half-life (t1/2) of pS824-KAP-1 signal following the addition of ATMi is indicated. Data represent average and s.d. of three independent experiments. Figure source data can be found with the Supplementary data. Download figure Download PowerPoint Cytological detection of pS824-KAP-1 shows a very distinct localization pattern (Goodarzi et al, 2010). Shortly after DNA damage, pS824-KAP-1 is detected throughout the nucleus, and the pS824-KAP-1 foci at DSBs are not distinctly visible. After several hours, pan-nuclear pS824-KAP-1 dissipates, and intense pS824-KAP-1 foci at late-repairing IR-induced foci (IRIF) are observable at sites marked by γ-H2AX foci and heterochromatic markers (Noon et al, 2010). Addition of ATMi shortly after IR leads to a rapid decrease in nuclear pS824-KAP1 in control cells. In agreement with the immunoblots shown in Figure 3B, PP4R3β-silenced cells show persistence of nuclear pS824-KAP-1 even in the presence of ATMi (Figure 3C). Importantly, pS824-KAP1 at late-repairing foci is critical for HC-DSB repair whereas pan-nuclear pS824-KAP1 appears to be dispensable (Noon et al, 2010). To study the impact of PP4R3β depletion on late pS824-KAP-1 foci, ATMi was added 24 h after a higher dose of IR (8 Gy). Under these conditions only late-repairing IRIF with pS824-KAP-1 foci are detectable. Consistent with previous results, pS824-KAP1 foci at late-repairing IRIF disappeared rapidly following the addition of an ATM inhibitor in control cells, while persisting selectively in the PP4R3β-depleted cells (Figure 3D). In all these experiments, the depletion of PP4R3β was observed only in cells transfected with the PP4R3β siRNA. These results strongly suggest that the PP4C–R3β complex directly dephosphorylates KAP-1, and that the persistence of pS824-KAP1 in PP4-depleted cells is not due to prolonged activation of ATM. Functional impact of PP4-mediated dephosphorylation of SS824-KAP-1 So far, two major functional consequences of expressing the phosphomimetic (S824D) KAP-1 mutant have been reported; one is the global de-condensation of chromatin, which is reflected by increased susceptibility to micrococcal nuclease (MNase) (Ziv et al, 2006) and the second is the ectopic de-repression of specific stress-response genes (Li et al, 2007, 2010). The molecular details of pS824-KAP-1-induced chromatin relaxation in the context of DSB repair has been recently elucidated. KAP-1 phosphorylation triggers the release of the nucleosome remodeler, CHD3, from DSB sites allowing localized de-condensation of chromatin (Goodarzi et al, 2011). We hypothesize that PP4 deficiency is functionally equivalent to expressing the phosphomimetic (S824D) KAP-1 mutant. To test this idea, we compared the expression level of KAP-1 target genes, p21 and Gadd45α, in PP4R3β-silenced cells, and cells where the endogenous KAP-1 had been replaced with the S824D- or S824A-KAP-1 mutant. IR-induced expression of p21 and Gadd45α is significantly enhanced by the depletion of PP4R3β or by the expression of phosphomimetic (S824D) KAP-1 mutant (Figure 4A). Conversely in cells expressing the phosphonull (S824A) KAP-1 mutant, these genes are not responsive to DNA damage. We also conducted chromatin relaxation assays using partial MNase digestion in the same set of cells. The depletion of PP4R3β, or expression of the S824D-KAP-1 mutant, caused a constitutive increase in cellular chromatin accessibility (Figure 4B and C). Consistent with previous data (Ziv et al, 2006), there is no additional de-condensation of chromatin after IR in cells expressing the S824D-KAP-1 mutant (Figure 4C). Continued ATM activity is required for the pS824-KAP-1-mediated dispersal of CHD3 from late-repairing IRIF (Goodarzi et al, 2011). We assessed the level of CHD3 at late IRIFs in PP4R3β-silenced cells (Figure 4D). As anticipated based on the previously observed persistence of pS824-KAP-1 following PP4R3β depletion (Figure 3D), silencing PP4R3β impedes the relocalization of CHD3 to late-repairing DSBs following the addition of ATMi. Together, these results strongly suggest that PP4-mediated dephosphorylation of KAP-1 S824 has a significant impact on its cellular function, and is necessary for an optimal DDR. Figure 4.PP4-mediated dephosphorylation of KAP-1 on S824 impacts its role in the DNA damage response. (A) PP4-mediated regulation of KAP-1 impacts the expression of Gadd45α and p21. HeLa cells were transfected with PP4R3β siRNA, or endogenous KAP-1 was replaced with WT, mutant forms A (KAP1-S824A) or D (KAP1-S824D). After IR, cells were harvested and RNA purified at indicated times and quantitative real-time PCR (qRT–PCR) was performed. Data represent average and s.d. of four independent experiments. (B, C) PP4R3β depletion and KAP1 phosphomimetic mutant (S824D) prolong damage-induced chromatin relaxation. HeLa cells were transfected with control or PP4R3β siRNA, or endogenous KAP-1 was replaced with WT or KAP1-S824D mutant (D). Cells were irradiated and harvested at indicated times. Nuclei were purified, treated with micrococcal nuclease; DNA isolated and analyzed as described in experimental procedures. The relative intensity of each nucleosomic form (mono, bi, tri or poly) is expressed as the percentage of the total signal (for a given lane). Bar graphs represent average and s.d. of three independent experiments. The KAP-1 replacement has been shown in the immunoblot with endogenous and Myc-tagged KAP-1 indicated. (D) The 'return' of CHD3 to sites of slow-repairing DSBs following ATMi addition is significantly attenuated in PP4R3β-depleted cells. Primary human fibroblasts were transfected with either control or PP4R3β siRNA. After 72 h, cells were irradiated with 8-Gy IR. At 24 h post IR, cells were treated with ATMi and harvested 10, 30, 60 and 120 min later. To examine the retention of CHD3 at sites of late-repairing damage, cells were pre-extracted with PBS containing 0.1% (v/v) Triton × 100 for 30 s before being fixed and immunostained for CHD3, γH2AX and DAPI (left panel). The signal intensity of CHD3 at regions of γH2AX foci (as determined by computer analysis) was measured (∼200 foci per sample). The data represent the mean and s.d. of multiple experiments. siRNA-mediated knock-down efficiency was independently verified for each experiment and was >80% (of cells with good knock-down) (right panel). It shows representative images at 60 min time point after ATMi addition from quantified data. In the zoomed-in and three-dimensional plotted images, note the relative difference in red–green overlap (yellow signal), indicative of changes in CHD3 abundance at sites of ongoing DSB repair, following the addition of ATMi for 1 h. Download figure Download PowerPoint CHK2 phosphorylates KAP-1 at S473 in response to
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