Bioinformatics Analysis of Conserved Phosphorylation Sites in the DNA Damage Signaling Network
A total of 244 in vivo mapped phosphorylation sites on 33 human DDR-related proteins were manually collected from Phospho.ELM 
, Phosphosite 
, and a phospho-proteomic study of Polo Box substrates 
. The conservation level of these phosphorylation sites was measured by aligning predicted orthologues of these proteins in 11 species from the high-coverage vertebrate branch of Ensembl (release 46; 
) with the human seed sequences in which the sites had been mapped by mass spectrometry and other means. Genomes used in the analysis included Homo sapiens (human), Bos taurus (cow), Canis familiaris (dog), Danio rerio (zebrafish), Gallus gallus (chicken), Macaca mulatta (rhesus monkey), Mus musculus (house mouse), Ornithorhynchus anatinus (platypus), Rattus norvegicus (Norway rat), Tetraodon nigroviridis (fresh water pufferfish), and Xenopus tropicalis (western clawed frog).
Ortholog mapping was performed as follows: initially the human DDR sequences and phosphorylation sites were mapped to their corresponding Ensembl gene entries by sequence comparison. Next, genes orthologous to the DDR genes were retrieved from Ensembl. Orthologous relationships between human and other vertebrate genes in Ensembl were inferred from phylogenetic trees constructed from multiple sequence alignment of CDS sequences 
. A detailed description of Ensembl ortholog detection pipeline in release 46 is available at http://aug2007.archive.ensembl.org/info/data/compara/homology_method.html
. Finally, each DDR protein sequence and all spliced variants of its orthologous genes across the 11 species were aligned using multiple-sequence alignment program MAFFT (v6.240, E-INS-i option with default parameters) 
Cross-species conservation of the human phosphorylation sites was then computed by evaluating the average number of amino acid substitutions within a −5 to +5 residue window of the modified residue (S, T, or Y) across the 11 vertebrate genomes from the sequence alignments using Perl scripting. S→T and T→S transitions of the central phosphoresidue were permitted, but S/T→Y transitions were not. The conservation level for each phosphorylation site is reported as the average across the 11 genomes, as a percentage of conserved residues within the 11-mer window, if the corresponding S/T is conserved.
Information about which of the 244 in vivo mapped phosphorylation sites were phosphorylated by the specific kinases ATM/ATR, Cdk1/2, Chk1/2, and Plk1 was collected from Phospho.ELM 
and Phosphosite 
, along with whether phosphorylation at that site was known to create a binding site for the PBD of Plk1 
. In cases where multiple kinases are known to phosphorylate a single site, all of this information was retained and displayed. For sites where the upstream kinase was not experimentally known, we predicted the likely kinase responsible for phosphorylation at that site by computational analysis using the programs NetworKIN 
and NetPhorest 
Antibodies, Plasmids, and Reagents
Rabbit anti-53BP1 (304-A1) was from Novus Biologicals. Mouse anti-γ-H2AX (pS139, #05-636), rabbit anti-HistoneH3 pS10 (#06570), rabbit anti-Chk2 (#2662), rabbit anti-Chk2-pT68 (#2661), rabbit anti-53BP1-pS1778 (#2675), mouse anti-MPM2 (#05-368), and rabbit anti-Plk1 (#06-831) were purchased from Upstate. An additional rabbit anti-Chk2 antibody (#BL1432) was purchased from Bethyl Laboratories. Rabbit anti-Plk1 for immunoprecipitation was a kind gift from Dr. René Medema. Mouse anti-β-actin (A5441) was from Sigma. Mouse anti-Cyclin B1 (GNS1, sc-245), rabbit anti-GFP (sc-8334), and rabbit non-specific IgG (sc-2025) were from Santa Cruz Biotechnology. Mouse anti-GFP (clones 7.1 and 13.1) was from Roche. Rabbit anti-p-S380-53BP1 phospho-specific antibody was raised against peptide Pro-Phe-Iso-Val-Pro-Ser-pSer-Pro-Thr-Glu-Gln-Glu-Gly-Arg-Tyr and purified by Cell Signaling Technologies. Radiolabelled [32
P]-γ-ATP (3,000 Ci/mmol) was purchased from Amersham/GE Healthcare. Plk1 inhibitor (BI 2536) was synthesized following the procedure described by Munzert et al. 
. All other reagents and chemicals were from Sigma unless otherwise indicated.
The pEGFP-m53BP1 expressing murine GFP-Tagged 53BP1 was kindly provided by Dr. Yasuhisa Adachi. The Nhe1-Apa1 fragment of pEGFP-m53BP1 was cloned in the retroviral plasmid pLNCX2 (Clontech) containing a synthetic linker to generate pLNCX2-GFP-m53BP1. PCR-based mutagenesis was used to create pLNCX2-GFP-m53BP1-317A, m53BP1-330A, m53BP1-376A, m53BP1-922A, m53BP1-1103A, and m53BP1-1620A. All plasmid constructs were verified by automated sequencing. pLNCX2-GFP-m53BP1Δ196–439 was created by a nested PCR on two m53BP1 PCR fragments surrounding the deletion. The resulting 53BP1 fragment containing the deletion was used to replace wt-m53BP1 in pLNCX2-GFP-m53BP1. Full-length human Chk2 was cloned from pGEX6P2-Chk2 and subcloned into the Nhe1-EcoR1 sites of pIRES2-GFP (Clontech). Serine/Threonine to Alanine mutations at positions 164, 168, 205, and 210 were obtained by side directed mutagenesis and validated using automated sequencing. Full-length FLAG-tagged Chk2 was a kind gift from Dr. Domenico Delia. VSV-G pseudotyped retroviruses were prepared according to standard techniques. In brief, HEK293T packaging cells were transfected with the pLNCX-2 and the packaging plasmids pMDg/p and pMDg in a 4
1 ratio. Virus-containing supernatant was harvested at 24 and 48 h after transfection, filtered through a 0.45 µM syringe filter, and used to infect U2OS osteosarcoma target cells. A plasmid encoding the PBD of Plk1 (aa. 326–603) fused to GST was described previously 
U2OS osteosarcoma cells were maintained in Dulbecco's Modified Eagle medium, supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. To obtain mitotic cell populations, cells were incubated with paclitaxel (1 µg/ml) or nocodazole (250 ng/ml, Sigma). Where indicated, cells were harvested by mitotic shake-off. Where indicated, DNA damage was induced using a gamma-cell 40 irradiator equipped with a 137Cesium source for indicated doses. Alternatively, cells were incubated with doxorubicin (0.5 µM) for 1 h.
Human breast cancer cell line MCF7 or human osteosarcoma U2OS cells were retrovirally infected with control pRetrosuper or pRetrosuper-53BP1 (53BP1-targeting sequence #1, 5′-GATACTGCCTCATCACAGT-3′
; 53BP1-targeting sequence #2 5′-GAACGAGGAGACGGTAATA-3′
) for three consecutive 12 h periods 
. Infected cells were selected with 2 µg/ml puromycin. pRS-53BP1-infected MCF7 cells were subsequently treated with 4 µM nutlin-3 to select for cells with a functional 53BP1 knockdown 
. The statistical analysis of colony numbers, S-phase content, and phospho-HistoneH3 content in control-infected or pRS-53BP1-infected MCF-7 cells was done using the unpaired t
test. Two-tailed p
values were calculated using GraphPath software.
The Plk1 kinase domain (residues 38–346) was made as a His6-tagged construct in Escherichia coli (E. coli) Rosetta cells (Novagen) and purified by Ni-NTA chromatography followed by gel filtration on a Superose-12 column. Recombinant full-length GST-Chk2 and a GST-Chk2 FHA domain (amino acids 1–219) fusion were expressed and purified from E. coli. In brief, full-length Chk2 was cloned into pGEX-6P1 (GE Healthcare) and transformed into BL21 (DE3) cells. Cells were grown at 37°C to an OD600 of 0.6 and the culture temperature was reduced to 18°C for 30 min before a final concentration of 0.3 mM IPTG was added for overnight expression. Cells were pelleted and washed with MTPBS (16 mM Na2HPO4, 4 mM NaH2PO4, 150 mM NaCl, pH 7.3) and lysed by sonication in the same buffer with the addition of benzonase. The lysate was clarified by centrifugation, and the GST-Chk2 fusion protein was captured on glutathione 4B resin. After washing with 30 column volumes of phosphate-buffered saline (PBS), Chk2 was cleaved off the GST tag on the resin with 3C protease at 4°C overnight. The eluted full-length Chk2 was further purified by anion exchange on a Resource Q column (GE Healthcare) equilibrated with 20 mM Tris pH 8.0, 50 mM NaCl, 0.5 mM TCEP, and developed with 20 mM Tris pH8, 1 M NaCl, and 0.5 mM TCEP. Peak fractions containing full-length Chk2 were pooled and further purified with a Superdex S200 gel filtration column (GE Healthcare).
The GST-Chk2 FHA domain cloned into pGex-4T1 was transfected into BL21(DE3) cells, grown to an OD600 of 0.8, and induced with 1 mM IPTG at 37°C for 6 h. Cells were lysed in PBS containing 1 mM DTT and a mixture of protease inhibitors and disrupted by sonication. Benzonase (Novagen) was added at room temperature for 30 min and the lysate cleared via centrifugation. Roughly 500 µL of PBS-equilibrated GSH beads were added to the lysate and incubated at 4°C with rocking overnight. Non-bound material was aspirated off followed by 4×10 mL washes with PBS containing 0.2% NP-40 and 1 mM DTT, and the GST-Chk2 FHA domain eluted off the beads by incubation in 2.5 mL of elution buffer (20 mM HEPES pH 7.2, 40 mM glutathione, and 1 mM DTT; EB+G) at 4°C overnight. The purified GST-Chk2 FHA domain was dialyzed against elution buffer lacking 40 mM glutathione (EB) using a Slide-A-Lyzer (Pierce) dialysis cassette with a molecular weight cut-off of 6–8 kDa at 4°C with three buffer exchanges. Purity was assessed by SDS-PAGE and the protein concentration determined by absorbance at A280 using an extinction coefficient of 71,780 M−1 cm−1.
Chk2 FHA Domain-Phospho-Peptide Binding Assay
Streptavidin beads (Pierce, 75 pmol/µL gel) were incubated with a 5-fold molar excess (relative to binding capacity) of a biotinylated phosphothreonine-oriented peptide library (B-4pT5; biotin-Gly-AHA-Gly-AHA-Met-Ala-X-X-X-X-pThr-X-X-X-X-X-Ala-Tyr-Lys-Lys-Lys-NH2, where X indicates a equimolar degenerate mixture of all amino acids except Cys, and pThr denotes phosphothreonine) in 50 mM Tris pH 7.5, 150 mM NaCl, 0.5% NP-40, and 1 mM EDTA for 30 min at 4°C. Beads were washed five times with the same buffer to remove unbound peptides and then 20 µL of the bead-immobilized library was incubated with 10 µg of GST-Chk2 FHA domain prior to or following in vitro phosphorylation of the FHA domain by Plk1 kinase in the presence of [32P]-γATP. After a 60 min incubation, the beads were washed five times with 50 mM Tris pH 7.5, 150 mM NaCl, 0.5% NP-40, and 1 mM EDTA. Bead-bound protein was released by addition of SDS sample buffer with heating to 95°C and resolved by SDS-PAGE on 10% denaturing gels. Gels were analyzed by autoradiography using a phosphorscreen and a Typhoon variable mode imager (GE Healthcare, or transferred to PVDF membrane and immunoblotted using an HRP-conjugated anti-GST antibody to visualize binding of the GST-FHA domain).
U2OS cells were seeded on glass cover slips and treated as indicated. After treatment, cells were fixed in 3.8% formaldehyde in PBS for 15 min at room temperature. Subsequently, cells were permeabilized with 0.1% TritonX100 in PBS for 5 min. After extensive washing, cells were blocked and stained in PBS-0.05% Tween20 and mounted on slides. Images were acquired on a Zeiss Axioplan-2 inverted microscope, equipped with a Hamamatsu Orca-ER digital camera using OpenLab software.
Immunoprecipitation, In Vitro Phosphorylation, and Kinase Assays
After the indicated treatments, U2OS cells were lysed in lysis buffer (1% TritonX-100, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 50 mM beta-glycerophosphate, 10 mM sodium pyrophosphate, 30 mM NaF, 1 mM benzamidine, 2 mM EGTA, 100 µM NaVO4, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml microcystin-LR) for 15 min at 4°C and cleared by high speed centrifugation. Protein concentrations were measured using the bicinchoninic acid assay (Pierce). 53BP1 was immunoprecipitated from 500 µg of clarified cell lysate using 3 µg of anti-53BP1 antibody and 50 µls of Protein-A-conjugated agarose beads (50% slurry) for 16 h. Immunoprecipitations were extensively washed and analyzed by SDS-Page and Western blotting. Alternatively, immunoprecipitations were subjected to in vitro phosphorylation by resuspension in kinase buffer (50 mM Tris-HCL pH7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, 2 mM dithiothreitol, 0.01% BRIJ35, and 150 mM NaCl2), followed by addition of 25 µM unlabelled ATP, 10 µCi of [32P]-γ-ATP, and recombinant Cyclin A-Cdk2, Cyclin B-Cdk1, or Plk1 for 30 min. Kinase reactions were analyzed by SDS-page and autoradiography.
IP/kinase assays for Chk2 activity were performed as generally described 
using lysates from either interphase cells or from mitotic cells generated by treating U2OS cells with 0.25 µg/ml nocodazole for 16 h followed by harvesting of the mitotitc non-adherent cells by gentle shaking. In brief, Protein A microtiter strips (Pierce) were coated overnight with 1.0 µg of anti-Chk2 antibody (Bethyl) or non-specific rabbit IgG per well and washed three times with blocking buffer (1% bovine serum albumin in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Triton X-100). Cell lysates (100 µgs) were placed in each antibody-coated well, incubated for 3 h, then washed twice with wash buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl) and twice with kinase wash buffer (20 mM Tris-HCl (pH 7.5), 15 mM MgCl2
, 5 mM beta-glycerophosphate, 1 mM EGTA, 0.2 mM Na3
, 0.2 mM DTT). Kinase reactions were performed in a total volume of 60 µl containing 20 mM Tris-HCl (pH 7.5), 15 mM MgCl2
, 5 mM β-glycerophosphate, 1 mM EGTA, 0.2 mM Na3
, 0.2 mM DTT, 0.4 µM protein kinase A inhibitor, 4 µM protein kinase C inhibitor, 4 µM calmidazolium, 25 µM ATP, 10 µCi [32
P]-γ-ATP, and 10 µM of Chk2tide substrate. Reactions were incubated for 60 min at 37°C, then terminated by addition of 60 µl of 20 mM EDTA. Forty µl of the terminated reaction mixture was transferred to a phosphocellulose filter plate (Millipore, Bedford, MA) containing 100 µl, 75 mM H3
, and mixed. The reaction contents were vacuum-filtered and washed five times with 75 mM H3
and three times with 70% ethanol. Scintillation counting was performed using a Microbeta TRILUX luminescence counter.
In vitro phosphorylation of recombinant Chk2 or the Chk2 FHA domain by Plk1 was performed by incubating 3–10 µgs of the substrate proteins with Plk1 kinase domain in 50 mM Tris pH 7.5 containing 150 mM NaCl, 10 mM MgCl2, 100 µg/ml bovine serum albumen, 5 mM DTT, and 100–500 µM unlabelled ATP, in the presence or absence of 10–20 µCi [32P]-γ-ATP, for 60–120 min at 30°C. Kinase assays of recombinant Chk2 before or after Plk1 phosphorylation were performed in the above buffer containing 1 mM DTT, 20 µCi [32P]-γ-ATP, and 50 µM Chk2tide in a final reaction volume of 50 µl at 30°C for 60 min. Samples were quenched with an equal volume of 0.05% H3PO4, and 5 µl of the reaction spotted onto P81 paper, air dried, washed extensively with 0.05% H3PO4, and analyzed by scintillation counting.
Identification of Plk1 phosphorylation sites in the Chk2 FHA domain following in vitro phosphorylation was performed by separating the reaction products by SDS-PAGE. Gel slices containing Chk2 were excised, alkylated with iodoacetamide, and digested with trypsin. Peptides were resolved by nano-flow reversed phase liquid chromatography (Agilent 1100, Palo Alto, CA) and analyzed with a LTQ-Orbitrap equipped with a nanoelectrospray ionization source (Thermo, Bremen, Germany). Peptide and protein identification was analyzed using the Spectrum Mill MS Proteomics Workbench software (Agilent).
For the in vivo mobility shift analysis of Chk2, 293T cells were transfected with FLAG-tagged full-length hChk2. Twenty-four h after transfection, cells were treated with paclitaxel in combination with DMSO or in combination with Plk1 inhibitor for 8 h. Cell lysates were cleared by centrifugation and mixed with M2 FLAG resin for overnight immunoprecipitation. After washing, samples were analyzed by SDS-PAGE.
Cells were harvested with Trypsin/EDTA, washed with PBS, and subsequently fixed in ice-cold 70% ethanol for 4–16 h. After washing, cells were stained with anti-phospho-Histone H3 (1
200) or anti-phospho-γ-H2AX (1
100) in PBS-0.05% Tween20 and counterstained with Alexa647-conjugated secondary antibodies in PBS-0.05% Tween20. Cells were treated with Propidium Iodide/RNAse and analyzed on a Becton Dickinson FACScalibur using Cellquest software. A minimum of 10,000 events were counted.