Physical and Functional Interaction of RACK1 and WRN in Genome Maintenance and Senescence
Figure S1 Examples of FACS analyses with GM08402 fibroblasts transfected with siControl RNA (on the left) and siWRN molecules (on the right).
Figure S2 Image showing overlay of the scans from the KinexTM antibody microarray using dye-labeled cell lysates from control siRNA transfected (in blue) and siWRN transfected normal human fibroblasts (GM08402) (in red). The 350 pan-specific and 258 phospho-site-specific antibodies were deposited in duplicate adjacent spots and the two samples were incubated on opposite sides of the same microarray (see for protocol details).
Figure S3 Phosphorylationof specific proteins in normal human GM08402 diploid fibroblasts and AG03141D fibroblasts derived from a WS patient. Detergent-solubilized lysates from control siRNA and siWRN transfected fibroblasts were subjected to KinetworksTM custom multi-sample screen (KCSS-1.0) analyses. The antibodies used in this study (from Kinexus Bioinformatics Corp.) were against phospho-threonine 232 of FOS, phospho-serines 21 of GSK3, IKK, phospho-serine 729 of PKC, PKC, and phospho-serine 910 of PKD.
Figure S4 Example of double-stranded breaks detected with an antibody against γ-H2AX in GM08402 diploid fibroblasts transfected with either scrambled control siRNA (siControl), siWRN, siRACK1, or siWRN+siRACK1 molecules (72 hours after transfection). DAPI staining is shown on the left.
Figure S5 Phosphorylation of PKCs and p38 MAPK in HT1080 cells. (a) Phosphorylation of PKC and PKCII in normal fibroblasts treated with either camptothecin (5 M for five hours), UV (five hours after irradiation with 40 J/m2 of UV), or H2O2 (0.5 mM for one hour) treatments. (b) Phosphorylation of p38 MAPK in HT1080 cells treated with either camptothecin (5 M for five hours) or H2O2 (0.5 mM for one hour) treatments. (c) Phosphorylation of p38 MAPK in HT1080 transfected with control siRNA or siWRN molecules (48 hours WRN knock down).
Supplemental materials and methods
Cell culture
The human fibrosarcoma HT1080 cell line, the normal human fibroblast cells (GM08402; passage 8 to 22), and the human Werner Syndrome fibroblast cells (AG03141D; passage 9 to 13) were all maintained in DMEM with 50 g/ml penicillin/streptomycin and 10% fetal bovine serum. The human osteosarcoma OsA-CL cell line was maintained in RPMI media supplemented with 50 g/ml penicillin/streptomycin and 10% fetal bovine serum. All chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON) unless otherwise stated. Stable pTAP-IRES-eGFP/puro, pTAP-WRN-IRES-eGFP/puro and pTAP-RACK1-IRES-eGFP/puro clones were obtained by transfecting HT1080 cells with a Nucleofector kit (Amaxa Biosystems, Gaithersburg, MD) and selecting with 0.5 g/ml puromycin. Cell fractionation was performed with the ProteoExtract® Subcellular Proteome Extraction Kit (BioVision Inc., Mountain View, CA).
Plasmids and siRNA molecules
The human WRN cDNA from a YFP-WRN construct (Bayntonet al., 2003) was first cloned into the InterplayTM Mammalian TAP system (Stratagene, La Jolla ,CA). This TAP sequence contains a calmodulin binding peptide followed by a streptavidin binding peptide. The whole TAP-WRN sequence was then cloned into a vector containing IRES-eGFP/puro sequences (Abbate et al., 2001) to generate the pTAPWRN-IRES-eGFP/puro vector. The GST-WRN chimeric constructs used in this study containing different fragments of the WRN cDNA was previously described (Von Kobbe et al., 2002).
The human RACK1 coding sequence was amplified from a HT1080 cells cDNA library with appropriate oligonucleotides for cloning into pGEX-2TK (GST-RACK1 construct) and into a new pTAP-IRES-eGFP/puro vector to generate the pTAP-RACK1-IRES-eGFP/puro construct. This final construct contains two FLAG sequences followed by two TEV cleavage sites and a streptavidin binding peptide cloned into the Eco47III of an IRES-eGFP/puro vector (Abbate et al., 2001).
Stealth small interference RNAs specific for human WRN and RACK1 mRNAs were purchased from Invitrogen Inc. (Burlington, ON). The sequences are (coding strand) 5'-UUAACCAGACUGUUAAGGCUCCAGG-3' for WRN and 5'-UAUCUCGAGAUCCAGAGACAAUCUG-3' for RACK1. Cells were transfected with siRNAs using Lipofectamine 2000 reagent (Invitrogen Inc. Burlington, ON) according to the manufacturer's instructions to knock down WRN and RACK1 protein levels. The siRNA against PKC was purchased from Santa Cruz Biotechnology (Santa Cruz, CA; cat. # sc-36253). Transfected cells were incubated at 37oC for 48 h. Transfection efficiency was determined with an Alexa-488 labeled control siRNA (Qiagen Inc, Mississauga, ON) as described previously (Turaga et al., 2007). The knock down efficiency was confirmed by Western blot analyses.
KinexTM antibody microarray screen andKinetworksTM custom multi-sample screen (KCSS-1.0) analyses
Proteins from normal human fibroblast cells (GM08402) transfected with control siRNA or siWRN molecules were subjected to a KinexTM antibody microarray screen at the Kinexus Bioinformatics Corp. (Vancouver, BC). This screen was performed in 2006. The screen uses antibodies to track the expression levels and phosphorylation states of 608 cell signaling proteins in duplicate (utilizing 258 phospho-site specific and 350 pan-specific antibodies). Details of the strategies and protocols can be found on their web site ( To confirm some of the data obtained with the antibody microarray screen, KinetworksTM custom multi-sample screen (KCSS-1.0) analyses on the following proteins were performed: phospho-FOS (threonine 232), PKC, phospho-PKC (serine 729), phospho-GSK3(serine 21), IKK, and phospho-serine 910 of PKD. Briefly, detergent-solubilized extracts (350 g of protein) from control siRNA and siWRN transfectedGM08402 cells were subjected to Western analyses as described on the Kinexus Bioinformatics Corp. website ( These screens use panels of highly validated commercial phosphosite-specific antibodies and 20-lane multi-channel blotters. The intensity of the ECL signals for the target protein bands on the Kinetworks immunoblots were quantified with a FluorS Max Imager and Quantity One software.
Immunoprecipitations and Western blots
For immunoprecipitations, cells were lysed in RIPA buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate]. Cell lysates were incubated overnight at 4°C with the appropriate antibodies and Protein A/G sepharose beads. Beads were washed and eluted proteins analyzed by SDS-PAGE. Proteins from the gels were transferred onto Amersham Hybond-P membranes (GE Healthcare Limited, Piscataway, NJ). Membranes were blocked two hours at room temperature in PBS containing 5% milk/0.1% Tween, washed in PBS-Tween (0.1%), and incubated overnight with the primary antibodies in PBS containing 5% milk overnight at 4°C. Blots were washed the next day in PBS-Tween and incubated two hours at room temperature with horseradish peroxidase-conjugated secondary antibody in PBS containing 5% milk. Blots were washed with PBS-Tween and proteins were revealed with chemiluminescence reagents (ECL Plus; GE Healthcare Limited, Piscataway, NJ). Polyclonal antibodies against the C-terminus and the N-terminus portion of human WRN were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Novus Biologicals (Littleton, CO), respectively. The monoclonal and polyclonal antibodies against RACK1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The other antibodies used in this report are the anti-PKC polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), the anti-phospho-PKC (threonine 507) polyclonal antibody (Cell Signaling Technology, Inc. Danvers, MA), the anti-PKC polyclonal antibody (Upstate Biotechnology, Lake Placid, NY), the anti-phospho-PKC (serine 729) polyclonal antibody (BioSource International Inc., Camarillo, CA), the anti-PKCII polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), the anti-phospho-PKCII (serine 641) polyclonal antibody (Cell Signaling Technology, Inc. Danvers, MA), the anti-PKD polyclonal antibody (Cell Signaling Technology, Inc. Danvers, MA), the anti-p38 MAPK polyclonal antibody and the anti-phospho-p38 (threonine180/tyrosine182) polyclonal antibody (Cell Signaling Technology, Inc. Danvers, MA), and the anti--actin monoclonal antibody (Sigma-Aldrich, St-Louis, MI). Horseradish coupled secondary antibodies were from GE Healthcare Limited (Piscataway, NJ).
Protein purifications
Recombinant His-tagged WRN protein was purified using an insect cell expression system, as described previously (Oren et al., 1999). Briefly, recombinant full length human WRN was over-expressed in insect cells and purified by chromatography, using DEAE-Sepharose, Q-Sepharose and Ni-NTA columns. Human GST-RACK1 and GST-WRN fragments were purified from BL21 E. Coli strain using standard procedures. Briefly, bacteria were induced with 0.1 mM isopropyl--D-thiogalactopyranoside for 6 h at 30°C. Bacteria were harvested,resuspended in NETN buffer (0.5% NP-40, 20 mM Tris-HCl pH 8.0, 100 mM NaCl, and 1 mM EDTA), and sonicated. After centrifugation at12,000 g for 10 min, the supernatant was incubatedwith glutathione-sepharose beads overnight at 4°C. Beads were washed five times with NETN and GST fusion proteins wereused for in vitro affinity binding assays as described previously (Gaudreault et al., 2004). GST without RACK1 expressed and treated in the same way was used as a negative control in each experiment. For WRN enzymatic assays, RACK1 was released from the beads by cleavage with biotinylated thrombin (Novagen, Madison, WI) for 2 hours at room temperature in thrombin cleavage buffer (20 mM Tris-HCl pH 8.4, 150 mM NaCl, 2.5 mM CaCl2). Beads were spun down and the supernatant was kept for the next step. Thrombin was captured by incubation with streptavidine agarose (Novagen, Madison, WI) for two hours on a rocking platform at room temperature. Agarose beads were spun down and RACK1 protein from the supernatant was concentrated onto Centricon-30 filters (Amicon, Bedford, MA) and used the same day for enzymatic assays. Protein concentration was determined using the Bradford assay. The purity of recombinant proteins was verified by SDS-PAGE, Coomassie staining, and Western blotting.
Mapping of RACK1 interaction to WRN protein was performed as follows. Briefly, cell lysates (RIPA buffer) were incubated with different GST-WRN fusion fragments coated beads overnight at 4°C. After extensive washing with lysis buffer, bound proteins were released by boiling in SDS sample buffer and analyzed by Western blotting with an anti-RACK1 antibody.
Helicase and Exonuclease assays
Exonuclease and helicase assays were performed exactly as described before for human WRN protein (Kamath-Loeb et al., 1998; Shen et al., 1998). The DNA substrate used for both assays is a forked DNA structure formed by the oligonucleotide 5'-TTTTTTTTTTTTTTTTTAGGGTTAGGGCATGCACTAC-3' and the partial complementary oligonucleotide 5'-GTAGTGCATGCCCTAACCCTAATTTTT TTTTTTTTTT-3'. These oligonucleotides were annealed to form a 22-bp forked duplex as described (Gaudreault et al., 2004). One strand was labeled with T4 polynucleotide kinase and [-32P] ATP and annealed to its complementary strand. This DNA substrate was incubated with the recombinant proteins as indicated in the figure legends for 30 min at 37 °C in the reaction buffer (40 mM Tris-HCl, pH 7.5, 4 mM MgCl2, 5 mM DTT, 2 mM ATP, and 0.1 mg/ml bovine serum albumin). Four l of loading buffer were added to the reaction (40% glycerol, 50 mM sodium EDTA, 2% SDS, and 1% bromophenol blue, pH 8.0) and the DNA was analyzed on native 12% polyacrylamide gel (in TBE buffer). For the exonuclease assays, radioactive DNA substrates were incubated with the indicated purified proteins in the same reaction buffer as indicated for the helicase reaction and the cleaved DNA products were boiled 10 min and separated on a denaturing gel (14% polyacrylamide, 8 M urea in TBE) for autoradiography.
Tandem affinity purification and mass spectrometry analyses
The TAP-WRN protein was purified from a stable HT1080 clone expressing this protein construct with a TAP purification kit (Stratagene, LaJolla, CA) as described by the manufacturer. HT1080 cells containing an empty TAP vector were used as a control. Eluted proteins were analyzed by SDS-PAGE and lanes corresponding to control TAP and TAP-WRN expressing cells were cut into small gel slices. Gel slices were sent to the Proteomics platform of the QuebecGenomicCenter (Quebec City, Qc) for spetrometry analyses and protein identifications. Briefly, gel slices were disposed into 96-well plates and in-gel trypsin digestion was performed on a MassPrepTM liquid handling station (Waters, Mississauga, ON) according to the manufacturer's specifications. Peptide extracts were dried out using a SpeedVacTM. Peptide extracts were separated by online reversed-phase nanoscale capillary LC and analyzed by electrospray MS (ES MS/MS). The experiments were performed on a Thermo Surveyor MS pump connected to a LTQ linear ion trap mass spectrometer (Thermo Electron, San Jose, CA) equipped with a nanoelectrospray ion source (Thermo Electron, San Jose, CA). Peptide separation took place within a PicoFrit column BioBasic C18, 10 cm x 0.075 mm internal diameter (New Objective, Woburn, MA) with a linear gradient from 2% to 50% solvent B (acetonitrile, 0.1% formic acid) in 30 min, at 200 nl/min. Mass spectra were acquired using data-dependent acquisition mode (Xcalibure software, version 2.0). Each full-scan mass spectrum (400–2000 m/z) was followed by collision-induced dissociation of the seven most intense ions. The dynamic exclusion function was enabled (30 s exclusion), and the relative collisional fragmentation energy was set to 35%.
All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.2.0). Mascot was set up to search against human Uniref_100 protein database assuming a digestion with trypsin. Fragment and parent ion mass tolerance were, respectively, of 0.5 Da and 2.0 Da. Iodoacetamide derivative of cysteine was specified as a fixed modification. Deamidation of asparagines and glutamine, acetylation of lysine and arginine and oxidation of methionine were specified as variable modifications. Two missed cleavages were allowed. Scaffold (version 01_07_00; Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at >80.0% probability as specified by the Peptide Prophet algorithm (Keller et al., 2002) and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Using these stringent identification parameters, the rate of false positive identifications is <1%.
Indirect Immunolabeling and Confocal Microscopy
Cells were grown on glass coverslips for 24 hours, fixed at room temperature for 20 min in 4% paraformaldehyde, permeabilized with 0.15% Triton X-100 for 10 min, blocked with 3% BSA for 1 hour and incubated overnight at 4°C with the primary antibodies indicated in the figure legends in BSA 1%. The rabbit polyclonal antibodies against RACK1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), the mouse IgG antibody against WRN was purchased from Transduction Laboratories (Lexington, KY), and the mouse monoclonal antibody against PKC was purchased from BD Transduction LaboratoriesTM (Mississauga, ON). Alexa-488 and -594 coupled secondary antibodies (GE Healthcare Limited,Piscataway, NJ) were used for detection after one hour incubation in BSA 1% at room temperature. After washing, coverslips were mounted on glass slides and viewed on a Nikon inverted diaphot confocal microscope. Images were captured with a BioRad MRC1024 confocal microscope in appropriate channels and imageswere then overlaid and analyzed with Metamorph imaging system (Universal Imaging Corp., Downingtown, PA). Immunofluorescence experiments wererepeated at least three times. Approximately 50 cells were analyzedfor each treatment, and representative photographs areshown.
FACS analyses
The normal fibroblast primary cell strain (GM08402) were transfected using the Lipofectamine 2000 reagent (Invitrogen Inc. Burlington, ON). Forty-eight hours later, cells were fixed in 50% ethanol overnight at 4°C. Cells were then washed in PBS and incubated for 30 min at 37°C in a buffer containing propidium iodide and RNAses. Cell cycle analyses were performed on a Beckman-Coulter Epics Elite ESP (Cambridge, MA) flow activated cell sorter. Data were analyzed with the MultiCycle software (Phoenix Flow System, San Diego, CA).
Measurements of reactive oxygen species
Measurements of intracellular ROS with the dye 2'-7' dichlorofluorescein diacetate (Sigma-Aldrich Canada Ltd., Oakville, ON) were performed as described (Deschênes et al., 2005). Fluorescence measurements were performed with a Fluoroskan Ascent fluorescence spectrophotometer (Thermo Electron Inc., Milford, MA). The excitation and emission wavelengths used were 485 nm and 527 nm, respectively. The final result was expressed as units of fluorescence per g of protein. Protein concentrations were measured using the Bradford assay.
References for supplementary materials and methods
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