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. Author manuscript; available in PMC: 2015 Sep 29.
Published in final edited form as: Oncogene. 2009 Dec 7;29(10):1486–1497. doi: 10.1038/onc.2009.443

Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms

L Massip 1, C Garand 1, A Labbé 1, È Perreault 1, RVN Turaga 1, VA Bohr 2, M Lebel 1
PMCID: PMC4586175  NIHMSID: NIHMS627880  PMID: 19966859

Abstract

Werner’s syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCδ and PKCβII in the membrane fraction of cells. In contrast, different DNA-damaging treatments known to activate PKCs did not induce RACK1/PKCs association in cells. Overall, our results indicate that a depletion of the WRN protein in normal fibroblasts causes the activation of several PKCs through translocation and association of RACK1 with such kinases.

Keywords: Werner syndrome, mass spectrometry, RACK1, PKC activation

Introduction

Werner’s syndrome (WS) is an autosomal recessive disorder that displays many clinical symptoms of normal aging at an early age. From their second decade of life onwards, patients with WS develop pathologies that prematurely resemble many traits of normal aging, such as cardiovascular diseases, osteoporosis or type II diabetes mellitus (reviewed in Epstein et al., 1966). Death generally occurs in the fourth decade of life from heart demise or cancer. Fibroblasts isolated from WS patients characteristically senesce prematurely in culture (Faragher et al., 1993) and display increased chromosomal aberrations (Melcher et al., 2000). The protein defective in WS (WRN) is a RecQ family 3′–5′ DNA helicase that also possesses a 3′–5′ exonuclease activity and is involved in DNA recombination, transcription, repair and telomere maintenance (reviewed in Cheng et al., 2007). A depletion of the WRN protein from cells renders them more sensitive to oxidative damage (Szekely et al., 2005). This is an important finding as the plasma from several WS patients and tissues from mice lacking a functional Wrn helicase domain exhibit a pro-oxidant state (Pagano et al., 2005; Massip et al., 2006). Oxidative damage that can lead to double-stranded DNA lesions throughout the genome, including telomeres, has been proposed to have a causative role in organismal aging (Beckman and Ames, 1998; Karanjawala et al., 2002; Parrinello et al., 2003).

Global expression analyses of mouse embryonic cells lacking a functional Wrn helicase domain has shown major changes in the expression of several kinases and phosphatases, at least at the mRNA level (Deschênes et al., 2005). In agreement with this observation, western blot analyses indicated increased levels of intracellular protein phosphorylation in such mutant cells. Furthermore, protein extracts from the liver and cardiac tissues from Wrn mutant mice indicated an increase in serine and tyrosine phosphorylation of several unknown proteins compared with wild-type animals. Several kinases exhibiting altered expression in this mouse model are known to be influenced by oxidative stress or are known to modify levels of cellular redox status (Deschênes et al., 2005). Human WS cells are also known to show increased stress signaling through the p38 mitogen-activated protein kinase (Davis et al., 2007; Davis and Kipling, 2008).

Several types of kinases can be activated upon cellular stress. These include ATM, protein kinase C (PKC), and p38 mitogen-activated protein kinase (reviewed in Kaneto et al., 2005; Nitti et al., 2008; Sedding, 2008). The exact mechanism by which these kinases are activated upon cellular stress is still elusive, but may require adaptor proteins. One such protein is the receptor for activated C-kinase 1 (RACK1). RACK1 is a conserved factor involved in a broad spectrum of mechanisms, such as cell proliferation, motility, apoptosis, signal transduction or protein translation/degradation. It functions as a docking platform for various molecules including protein kinases and phosphatases, thereby coordinating key signalization pathways (McCahill et al., 2002). Interestingly, RACK1 function is intimately connected to its subcellular localization and is related to typical age-related pathological processes, such as cancer, cardiac dysfunction or neurodegeneration (McCahill et al., 2002). RACK1 expression decreases with age in the rat brain and human leucocytes (Pascale et al., 1996; Corsini et al., 2005), whereas it increases in different kinds of carcinomas as compared with normal tissues (Berns et al., 2000; Egidy et al., 2008; Wang et al., 2008).

In this study, we used mass spectrometry analysis to identify proteins that copurify with a WRN protein containing a TAP tag (for tandem affinity purification). We identified RACK1 as one of the proteins interacting with this construct. RACK1 has no impact on the helicase activity of the WRN protein, but inhibits its exonuclease activity. Interestingly, depletion of the WRN protein causes relocation of a sub-population of RACK1 protein in the cytoplasm where it activates several PKC isoforms.

Results

Short-term depletion of the WRN protein activates several PKC isoforms in normal human diploid fibroblasts

We first determined which kinases were activated in WRN-depleted cells. Cells derived from WS patients exhibit aneuploidy and chromosomal rearrangements (Melcher et al., 2000). Such alterations might affect the gene expression of kinases in a manner only indirectly related to the principal WS or age-related defects. In addition, WRN mutant cells may have undergone an adaptation process in vitro. To avoid these problems, we used short-term small interfering RNA (siRNA)-based inhibition of WRN to test the direct consequences of WRN protein loss on kinase activities in normal fibroblasts. Normal human diploid fibroblasts (GM08402 at passage 8) were transfected with a siRNA specific for WRN mRNA (referred to as siWRN hereafter). Scrambled siRNA was used for control transfections. Transfections were performed with Lipofectamine 2000 (Invitrogen Inc., Burlington, ON, Canada) on 50% confluent cell culture accordingly to the manufacturer’s instructions. Under these conditions, transfected cells only went through one population doubling. Although siWRN-transfected cells exhibited DNA damage during the first 48 h of WRN protein knock down (Turaga et al., 2007), this strategy permitted us to avoid the accumulation of fixed mutations that would occur only after several rounds of DNA replication. Transfection efficiency determined with an Alexa-488-labeled control siRNA was more than 95% (Turaga et al., 2007). Forty-eight hours after transfection, proteins were extracted for antibody microarray analyses. Two independent transfection experiments (control siRNA and siWRN) were conducted in duplicate to extract proteins. To assess the efficiency of the siWRN-mediated knockdown, we examined WRN protein levels in transfected cells. As indicated in Figure 1a, the most efficient knock down of WRN was achieved with HSS111385 siRNA (Invitrogen Inc.) and corresponded to ~80% decrease compared with control siRNA transfection. Fluorescence-activated cell sorting analyses were performed to determine the percentage of cycling cells 48 h after transfection with control siRNA and HSS111385 siWRN (at the time of protein extraction). As indicated in Figure 1b, siWRN-transfected cells showed an increase in the number of cells in the G1 phase, from 71 to 77%, and a decrease in the number of cells in the S phase, from 28 to 18%, compared with siRNA control-transfected fibroblasts. Examples of fluorescence-activated cell sorting analyses are provided in Supplementary Figure S1.

Figure 1.

Figure 1

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Impact of WRN protein depletion on GM08402 diploid fibroblasts cell cycle and phospho-proteins after transfection with scrambled siRNA and WRN-specific siRNA. (a) WRN protein detection with an anti-WRN antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in GM08402 cells 48 h after siRNA molecule transfection. The β-actin protein present in the lysate was used as control. (b) Percentage of GM08402 cells (at passage 8) in each phase of the cell cycle 48 h after transfection with control siRNA and siWRN molecules determined by FACS analysis. Experiments were performed in duplicate. (c) Detergent-solubilized lysates from control siRNA- and siWRN-transfected fibroblasts were subjected to Kinetworks custom multi-sample screen (KCSS-1.0) analyses (http://www.Kinexus.ca). Antibodies used in this study (from Kinexus Bioinformatics Corp.) were against phosphothreonine 232 of FOS, phospho-serines 21 of GSK3α/β, IKKβ, phosphoserine 729 of PKCε, PKCλ/ι and phospho-serine 910 of PKD. WRN, protein defective in patients with Werner’s syndrome; siRNA, small interfering RNA; FACS, fluorescence-activated cell sorting; PKC, protein kinase C.

Proteins from transfected cells were subjected to a Kinex antibody microarray screen at the Kinexus Bioinformatics Corp. (Vancouver, BC, Canada). The screen used antibodies to track expression levels and phosphorylation states of 608 cell-signaling proteins in duplicate (using 258 phospho-site-specific and 350 pan-specific antibodies). An image of the scans of the antibody microarray is shown in Supplementary Figure S2. Raw data obtained from the scans are shown in Supplementary Table 1. A list of proteins (some were phosphorylated) showing a two-fold difference between cells transfected with a control-scrambled siRNA and siWRN molecules is shown in Table 1. Every scan measurement that was flagged by the Kinexus service or showed an error range >35% was excluded. As indicated in Table 1, the expression level of one tyrosine phosphatase (PTP1D), two tyrosine kinases (FGFR2 and phospho-PDGFRα) and four PKC isoforms (phospho-PKCβII, phospho-PKCδ, phospho-PKCε and PKCλ/ι) were changed in fibroblasts transfected with siWRN compared with control siRNA molecules. In addition, siWRN transfection induced phosphorylation of FOS at threonine 232 and hypo-phosphorylation of the pRb tumor suppressor. The hypo-phosphorylation of pRb is consistent with the observed accumulation of cells in the G1 phase of the cell cycle 48 h after siWRN transfection. Follow-up studies conducted using the Kinetworks immunoblot services (Vancouver, BC, Canada) confirmed the changes observed with the antibody screen for phospho-FOS, phospho-PKCε and PKCλ/ι (Figure 1c). The phosphorylation of GSK3α/β, IKKβ and PKD was also examined and considered as the negative control (on the basis of the antibody array). These results indicate that the activity of several PKC isoforms is affected by short-term WRN depletion in normal fibroblasts. The same proteins were compared using normal fibroblasts (GM08402) and fibroblasts derived from aWS patient (AG03141D). Supplementary Figure S3 shows an increase in phospho-FOS, phospho-PKCε and PKCλ/ι in WS cells compared with normal fibroblasts. There was a small change in the phosphorylation of GSK3α/β and IKKβ. However, there was a marked decrease in PKD expression in WS cells. These results indicate that WRN-depleted cells behaved similarly to WS cells in these assays.

Table 1.

List of phospho-proteins exhibiting a greater than a two-fold change in normal human GM08402 fibroblasts transfected with small interference RNA against the WRN mRNA

Protein Phosphorylation site % CFCa % Error
FGFR2 Pan-specificb 99.7 29.27
FOS Threonine 232 155.7 16.96
Paxillin 1 Tyrosine 118 184.0 16.89
PDGFRα Tyrosine 742 97.0 9.33
PKCβII Threonine 641 162.4 34.51
PKCδ Threonine 507 138.9 33.94
PKCε Serine 729 309.9 21.86
PKCλ/ι Pan-specific 222.9 3.36
PTP1D Pan-specific −99.1 17.50
Rb Serine 612 −96.4 20.99
Rb Serines 807 + 871 −169.2 26.09
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Abbreviation: WRN, protein defective in patients with Werner’s syndrome.

a

% (percentage) change from control siRNA-transfected cells.

b

The antibody also recognizes the unphosphorylated protein.

Identification of RACK1 as a cellular partner of the WRN protein

To identify new WRN protein partners that may affect the activity of PKCs, WRN cDNA was cloned in frame with a TAP tag containing a calmodulin- and a streptavidin-binding peptide for TAP. As the expression of the TAP-WRN construct was toxic to normal human fibroblasts (GM08402), we transfected the fibrosarcoma cell line HT1080. Several stable viable clones were obtained. Copurification of proteins was achieved on exponentially growing HT1080 cells expressing either the TAP alone or the TAP-WRN construct. Unbound proteins at each step of the chromatography procedure were removed by extensive washing, thus obtaining proteins stringently bound to the TAP-WRN construct in cell lysates. Bound proteins were identified by liquid chromatography tandem mass spectrometry. Proteins identified from both HT1080 TAP and TAP-WRN expressing cells were considered artifacts, and were removed from the final list of potentialWRN interacting proteins. Table 2 lists 12 potential proteins interacting with the TAP-WRN construct in HT1080 cells. The first three proteins identified with 100% confidence included the Ku heterodimer (XRCC5/XRCC6 or Ku80/70) and the T-complex protein 1 subunit-γ (a member of the chaperonine family). Thus, the liquid chromatography tandem mass spectrometry analysis confirmed the WRN–Ku complex interaction in cells (Cooper et al., 2000; Li and Comai, 2000). The interaction with the chaperonine may be due to misfolding of a number of TAP-WRN molecules in cells. The fourth protein identified with 99% confidence was the Guanine nucleotide-binding protein (G protein), β-polypeptide 2-like 1 variant also known as RACK1. The other proteins listed in Table 2 were identified with <64% confidence and were not pursued further in this study.

Table 2.

List of TAP-WRN-binding proteins in the HT1080 fibrosarcoma cell line

MS/MS identified proteins Accession no. MW (kDA) % Confidence (no. of peptides found)
Werner’s syndrome protein Q14191 163 100 (49)
XRCC5 protein (Ku80) Q4VBQ5 83 100 (4)
T-complex protein 1 subunit-γ P49368 61 100 (2)
Ku 70 protein P12956 70 100 (2)
RACK1 protein Q53HU2 35 99 (2)
Hypothetical protein DDX18 Q4ZG72 62 64 (1)
Hypothetical protein SAP130 Q53T46 78 64 (1)
Leu-rich repeat-containing protein 45 Q96CN5 76 62 (1)
Protein FLJ25373 Q96LM3 46 58 (1)
WD40 protein Q6UXN9 35 55 (1)
ASCC3 protein Q4G1A0 84 53 (1)
ATPase family, AAA domain containing 3A Q5SV24 64 53 (1)
Protein DKFZp686I1974 Q6AW91 50 51 (1)
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Abbreviations: MS, mass spectrometry; MW, molecular weight; RACK1, receptor for activated C-kinase 1; TAP, tandem affinity purification; WRN, protein defective in patients with Werner’s syndrome.

RACK1 physically associates with WRN

To confirm the binding of RACK1 to the TAP-WRN construct and to exclude the possibility that this association could result from independent binding of these proteins to nucleic acid, TAP purification was repeated on cell extracts pretreated with benzonase and analyzed by western blotting. Figure 2a shows that RACK1 was still detected in the TAP-WRN precipitate, but not in precipitates from cells expressing TAP alone, suggesting that nucleic acids are not required for the WRN/RACK1 association. Coimmunoprecipitation experiments were then conducted on endogenous WRN proteins in HT1080 cells to detect endogenous RACK1. As indicated in Figure 2b, an antibody against the N-terminus portion of the WRN protein coimmunoprecipitated RACK1, but an antibody against the C-terminus region of the WRN protein did not. Neither of the anti-RACK1 antibodies tested immunoprecipitated RACK1, preventing us from performing the reverse coimmunoprecipitation experiments.

Figure 2.

Figure 2

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Interaction of RACK1 with the WRN protein. (a) Coprecipitation of RACK1 with the TAP-WRN protein in HT1080 fibrosarcoma cells. Proteins from TAP and TAP-WRN expressing cells were eluted from the streptavidin beads and analyzed by SDS–PAGE with antibodies against WRN and RACK1 proteins. (b) Coimmunoprecipitation of human WRN protein with RACK1. Approximately 2 mg of proteins from human HT1080 cells were immunoprecipitated with antibodies against the N- or C-terminus region of the human WRN protein. Control antibodies were of the same IgG species. Immunoprecipitates were analyzed by western blotting with the anti-WRN antibody (WRN; top panel) and an antibody against RACK1. Proteins were revealed with an ECL kit. The anti-WRN (N-ter) antibody is from Novus Biologicals (Littleton, CO, USA). The anti-WRN (C-ter) and anti-RACK1 antibodies are from Santa Cruz Biotechnology. The ‘input’ lane corresponds to 20 μg of total cell lysate. (c) Binding of purified WRN protein to GST-RACK1 affinity Sepharose beads as revealed by immunoblots with an anti-WRN antibody. GST Sepharose beads were used as a negative control. The experiment is presented in duplicate. (d) Interaction of RACK1 with different domains of WRN in whole-cell extract. Immunoblot against RACK1 protein bound to different GST-WRN affinity Sepahrose beads. Human HT1080 whole-cell extracts were incubated with 50 μg of the GST-WRN fragments or GST-linked glutathione-Sepharose beads overnight. Proteins bound to the affinity beads were analyzed by SDS–PAGE with antibodies against RACK1. (e) Schematic representation of different WRN fragments that were used in the WRN affinity chromatography experiments. Each domain of the WRN protein is indicated on the full WRN protein figure. The amino-acid residues of the WRN fragments used in this study are indicated on the top of each construct. Binding of RACK1 is indicated on the right by the ‘+’ sign. The ‘−’ sign indicates no binding detected. RACK1, receptor for activated C-kinase 1; TAP, tandem affinity purification; WRN, protein defective in patients with Werner’s syndrome.

To determine whether the WRN/RACK1 association was direct, purified His-WRN protein was incubated with purified GST-RACK1 immobilized on glutathione-Sepharose beads (GE-Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). GST containing beads were used as control. As shown in Figure 2c, WRN bound GST-RACK1 but not GST, suggesting that this complex is formed through a protein–protein interaction. We next mapped the region of the WRN protein required for RACK1 binding. Different GST-WRN fragment constructs were immobilized on glutathione-Sepharose resins and incubated with HT1080 cell lysates. Bound proteins were then probed with an anti-RACK1 antibody. As shown in Figures 2d and e, RACK1 bound strongly to amino-acid residues 949–1092 of the WRN protein. RACK1 also bound weakly to residues 239–499 and very weakly to residues 499–946 of the WRN protein. These results indicate that RACK1 interacts mainly with the RQC domain of the WRN protein and weakly to the acidic repeats in the N-terminus region of WRN. It does not interact with residues 1072–1236 at the C-terminus region of WRN.

RACK1 inhibits the exonuclease activity of WRN protein

We next investigated whether RACK1 could affect the exonuclease and DNA helicase enzymatic activities of WRN in vitro. WRN and RACK1 proteins were thus purified in vitro, mixed, and the exonuclease/helicase activity of this complex was assayed on a forked DNA duplex. No DNA binding or helicase/exonuclease activity was observed for purified RACK1 alone. RACK1 had no impact on WRN helicase activity. In contrast, a strong inhibition of the WRN exonuclease activity was noted at all tested RACK1 concentrations, suggesting a specific inhibitory function for this interaction (Figure 3a).

Figure 3.

Figure 3

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The impact of purified RACK1 on DNA helicase and exonuclease activities of purified WRN protein, and the impact of RACK1 depletion on DNA damage in cells. (a) The indicated concentration of purified human RACK1 protein was incubated with 3nM of purified WRN protein, and the indicated radioactive DNA substrate under standard conditions for helicase activity for 30 min at 37 °C. Reactions were stopped in the appropriate dye buffer, and DNA products were run on a 12% native polyacrylamide gel. The double- and single-stranded DNA structures are depicted on the left of the autoradiogram. The 5′-labeled strand of the duplex is represented by an asterisk (*). The triangle represents heat-denatured DNA. (b) The indicated concentrations of purified human RACK1 and WRN proteins were incubated with a radioactive DNA-forked structure under the same buffer conditions as for the helicase assay for 30 min at 37 °C. Reactions were stopped in the appropriate dye buffer, heat denatured, and the DNA substrates were analyzed on a 12% denaturing gel. (c) The percentage of cells displaying foci of DNA damage detected by the anti-γ-H2AX antibody. The percentage of cells exhibiting 0–10 foci, 11–20 foci or >20 foci is depicted for each transfection experiment (150 cells per transfection were analyzed). The resulting contingency table is displayed as a histogram. (d) Extent of DNA breaks quantified by the alkaline comet assay. The tails of broken DNA from 100 cells were measured to obtain the mean tail length (in μm). All experiments were repeated twice (unpaired Student’s t-test: *P=0.049; **P<0.001. Bars represent s.e.m.). RACK1, receptor for activated C-kinase 1; WRN, protein defective in patients with Werner’s syndrome.

Depletion of RACK1 in normal cells induces DNA damage

As RACK1 affects the exonuclease activity of WRN in vitro, we next examined its impact on overall DNA damage in normal diploid fibroblasts (GM08402). Normal fibroblasts were transfected with a scrambled control siRNA, siRNA against RACK1 (siRACK1), siWRN, or both siRACK1 and siWRN molecules. Forty-eight hours later, cells were processed for immunofluorescence analysis with an antibody against γ-H2AX, which marks double-stranded DNA breaks (Rogakou et al., 1998). The number of nuclear foci stained by anti-γ-H2AX was calculated on the basis of 150 transfected cells. Supplementary Figure S4 shows an example of transfected cells stained with anti-γ-H2AX and DAPI. As indicated in Figure 3c, the number of fibroblasts with >20 nuclear foci (double-stranded breaks) was 50% higher in cells transfected with siRACK1 or siWRN molecules. The number of cells with >20 nuclear foci was increased twofold in cells transfected with both siRACK1 and siWRN molecules compared with control siRNA-transfected cells.

We also quantified the extent of double- and single-stranded breaks by alkaline comet assay in these transfected cells. As indicated in Figure 3d, the mean tail length of damaged DNA in siRACK1-transfected cells was comparable with that in siWRN-transfected fibroblasts. When both siRACK1 and siWRN molecules were transfected, the mean tail length increased above those levels observed in the siRACK1-transfected cells (Figure 3d). Overall, these results indicate that a depletion of RACK1 protein in fibroblasts induced DNA damage in normal diploid fibroblasts. However, there was no synergistic effect when both RACK1 and WRN proteins were depleted in fibroblasts.

WRN protein colocalizes with nuclear

RACK1 in normal and tumor cell lines

We next examined the localization of RACK1 and WRN proteins by immunofluorescence in HT1080 cells. RACK1 is localized in both the cytoplasm and the nucleus of HT1080 cells (Figure 4, top row). Only a partial colocalization was observed between RACK1 and WRN in the cell nucleus. We also examined the localization of RACK1 and WRN proteins in normal diploid fibroblasts (GM08402) and several cancer cell lines. Figure 4 shows that the WRN protein is mainly localized to the nucleoli of normal diploid fibroblasts as described earlier (Marciniak et al., 1998), with some in the nucleoplasm. Interestingly, a fraction of nucleoplasmic WRN colocalized with nuclear RACK1. There was no or very little RACK1 in the nucleoli (Figure 4, bottom row).

Figure 4.

Figure 4

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Nuclear RACK1 colocalizes with nucleoplasmic WRN protein in normal and tumor cells. Immuno-localization of RACK1 and WRN proteins in HT1080 fibrosarcoma cells is shown in the top row. The bottom row shows immuno-localization of RACK1 and WRN proteins in normal GM08402 fibroblasts. Anti-mouse Alexa-594-labeled and anti-rabbit Alexa-488-conjugated secondary antibodies were used to visualize WRN and RACK1 by confocal microscopy at 568 and 488 nm, respectively. Images depict representative cells at ×600. In the merged images, a yellow color appears where RACK1 (red) and WRN (green) fluorescence signals coincide. RACK1, receptor for activated C-kinase 1; WRN, protein defective in patients with Werner’s syndrome.

We then examined the impact of WRN depletion on RACK1 proteins in HT1080 cells. As shown in Figure 5a, the total cellular RACK1 levels did not change upon WRN depletion in HT1080 cells (Figure 5a). However, immunofluorescence analyses with anti-RACK1 antibodies indicated a partial loss of RACK1 proteins in the nuclei of siWRN-transfected HT1080. The same result was obtained with normal GM08402 fibroblasts transfected with siWRN molecules (Figure 5b). RACK1 is an adaptor protein which relocalizes to membrane fractions upon activation of different signal transduction pathways (Battaini et al., 1997; Grosso et al., 2008). We thus examined levels of RACK1 protein in the membrane fractions of transfected cells. Fractionation of transfected cells into nuclear, cytosolic and membrane fractions were performed, and the amount of RACK1 was quantified by western blotting. As indicated in Figure 5c (left panels), there was an increase in RACK1 association with the membrane fraction in siWRN-transfected HT1080 cells compared with control siRNA-transfected cells. Concomitantly, we detected a decrease in nuclear RACK1 in siWRN-transfected cells compared with control siRNA-transfected cells (Figure 5c, right panels). No change was observed in levels of cytoplasmic β-tubulin or nuclear HNRPK splicing factor in all transfected cells.

Figure 5.

Figure 5

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The impact of WRN depletion on RACK1 in HT1080 and GM08402 cells. (a) Expression levels of total WRN and RACK1 proteins in control siRNA- and siWRN-transfected HT1080 cells. The β-actin protein is used as loading control. (b) RACK1 protein localization detected by immunofluorescence in HT1080 and GM08402 cells transfected with control siRNA or siWRN molecules. (c) Expression levels of RACK1 proteins in the membrane fraction of control siRNA- and siWRN-transfected HT1080 cells (panels on the left). TRAP1 protein was used as loading control for the membrane fraction (including mitochondrial membranes). β-Tubulin was used as a control for cytosolic fractions. Expression levels of RACK1 proteins in the nuclear and cytoplasmic fractions of control siRNA- and siWRN-transfected HT1080 cells (panels on the right). β-Tubulin was used as a control for the cytoplasmic fraction. Heterogeneous nucleoriboprotein K (HNRPK) was used as a control for the nuclear fraction. RACK1, receptor for activated C-kinase 1; WRN, protein defective in patients with Werner’s syndrome; siRNA, small interfering RNA.

RACK1 interacts with several PKC isoforms upon WRN depletion

RACK1 interacts with several PKC isoforms upon different physiological conditions. These interactions are considered important for PKC activation and phosphorylation of specific substrates in cells. We thus examined the association of RACK1 with three different PKCs in WRN-depleted HT1080 cells. To look at RACK1–PKC interactions, each PKC isoform was coprecipitated with a TAP-RACK1 construct. TAP-RACK1 and TAP expressing HT1080 cells were transfected with control siRNA or siWRN, and the TAP construct was purified along with their complex. The eluted TAP–RACK1 complex was then analyzed by western blotting with antibodies against PKCβII, PKCδ and PKCε. As indicated in Figure 6a, PKCβII and PKCδ isoforms were associated with RACK1 only in WRN-depleted cells. In contrast, PKCε did not associate with TAP-RACK1 under the same conditions. These results indicate that loss of WRN causes a subpopulation of RACK1 proteins to bind to PKCβII and PKCδ. Noticeably, Table 1 shows that WRN depletion in normal diploid fibroblasts also increases the phosphorylation of PKCβII at its threonine 641 and PKCδ at its threonine 507. Such phosphorylation is required for the activation of PKCβII (Edwards et al., 1999) and PKCδ (Parekh et al., 1999) in cells, respectively.We next determined whether phosphorylated PKCβII and PKCδ (activated PKCs) were still bound to TAP-RACK1 in HT1080 cells. As shown in Figure 6b, both phosphorylated PKCβII (at its threonine 642) and PKCδ (at its threonine 507) were bound to TAP-RACK1 in WRN-depleted cells.

Figure 6.

Figure 6

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The impact of WRN protein depletion on RACK1/PKC associations. (a) Coprecipitation of three different protein kinase C with the TAP-RACK1 protein in HT1080 fibrosarcoma cells. Proteins from TAP and TAP-RACK1 expressing cells were eluted from the streptavidin beads and analyzed by SDS–PAGE with antibodies against PKCδ, PKCε and PKCβII. (b) Coprecipitation of two phosphorylated PKC with the TAP-RACK1 protein in HT1080 fibrosarcoma cells. Proteins from TAP and TAP-RACK1 expressing cells were eluted from the streptavidin beads and analyzed by SDS–PAGE with antibodies against phospho-threonine 507 of PKCδ and phospho-threonine 641 of PKCβII. TAP-RACK1 is shown in the top panel with an anti-RACK1 antibody. RACK1, receptor for activated C-kinase 1; PKC, protein kinase C; WRN, protein defective in patients with Werner’s syndrome; TAP, tandem affinity purification.

A knock down of the WRN protein is known to cause oxidative stress and DNA damage (Das et al., 2007; Turaga et al., 2007). We thus observed the interaction of PKCs with TAP-RACK1 at different time points after camptothecin (5 μM), ultraviolet (40 J/m2) or H2O2 (0.5mM) treatments. No increase in RACK1/WRN or RACK1/TAP-WRN association was detected under these conditions (data not shown), although increased phosphorylation of PKCβII (at its threonine 642) and PKCδ (at its threonine 507) was detected after ultraviolet or peroxide treatments (Supplementary Figure S5a). Camptothecin and peroxide treatments increased p38 mitogen-activated protein kinase phosphorylation in HT1080 cells, indicating that such treatments induced cellular stress (Supplementary Figure S5b). However, a depletion of the WRN protein did not increase p38 phosphorylation in HT1080 (Supplementary Figure S5c), confirming the antibody microarray screen (Supplementary Table 1).

RACK1 modulates PKCδ and PKCβII phosphorylation in WRN-depleted cells

We determined the impact of knocking down RACK1 protein levels on PKC phosphorylation in WRN-depleted normal fibroblasts. GM08402 fibroblasts were transfected with either control siRNA, siWRN or both siWRN and siRACK1 molecules. As indicated in Figure 7, depletion of the WRN protein increased the phosphorylation of PKCδ, PKCβII and PKCε. Knocking down RACK1 in WRN-depleted cells significantly decreased the phosphorylation of PKCβII (at its threonine 642) and PKCδ (at its threonine 507). However, it had no impact on the phosphorylation of PKCε (at its serine 729) induced by depletion of the WRN protein (Figure 7, bottom panels). These results suggest that depletion of WRN activates PKCε independently of RACK1.

Figure 7.

Figure 7

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The impact of RACK1 on the phosphorylation of PKCδ, PKCβII and PKCε in WRN-depleted GM08402 cells. Immunoblot analyses with antibodies against WRN, RACK1, PKCδ, phospho-threonine 507 of PKCδ, total PKCδ, phosphothreonine 641 of PKCβII, total PKCβII, phospho-serine 729 of PKCε, and total PKCε 48 h after GM08402 cells were transfected with control-scrambled siRNA, and siWRN with or without siRACK1 molecules. RACK1, receptor for activated C-kinase 1; PKC, protein kinase C; WRN, protein defective in patients with Werner’s syndrome; siRNA, small interfering RNA.

Knock down of RACK1 and PKCδ diminishes induction of reactive oxygen species production in WRN-depleted normal fibroblasts

As WRN depletion increases reactive oxygen species (ROS) production in normal GM08402 fibroblasts, we examined the impact of knocking down RACK1 or PKCδ in such cells. As shown in Figures 8a and b, the efficiency of RACK1 and PKCδ knock down was approximately 64–42% in GM08402 fibroblasts. Depletion of WRN increased ROS formation by 34% (Figure 8c). ROS levels were decreased to near basal levels when both siWRN and siRACK1 molecules were transfected. The decrease was not significant (P-value of 0.065; Student’s unpaired t-test), but there was an obvious trend toward normalization. In contrast, siPKCδ molecules significantly decreased ROS production in WRN-depleted fibroblasts (P-value <0.05; Figure 8c).

Figure 8.

Figure 8

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The impact of RACK1 protein knock down on reactive oxygen species (ROS) production and senescence markers in WRN-depleted normal human GM08402 fibroblasts. (a) RACK1 protein levels in GM08402 fibroblasts 48 h after transfection with control-scrambled siRNA or siRACK1 molecules. β-actin is used as control. (b) PKCδ protein levels in GM08402 fibroblasts 48 h after transfection with control-scrambled siRNA or siPKCδ molecules. RACK1 protein level is used as a control. (c) Intracellular ROS levels in GM08402 cells transfected with control siRNA (siControl), siWRN, siWRN+siRACK1 or siWRN+siPKCδ molecules in combination. Experiments were performed in triplicates (unpaired Student’s t-test; *P<0.05). Bars represent s.e.m.

Discussion

In this study, we examined the impact of WRN depletion on the cellular phosphorylation of specific kinases in normal and cancer cells. Previous observations indicated that cells derived from mice with a deletion in the helicase domain of the murine Wrn gene exhibited changes in the phosphorylation status of several kinases, including PKCδ (Massip et al., 2009). The major kinases that exhibited expression and activity changes in short-termWRN-depleted normal fibroblasts were PKCβII, PKCδ, PKCε and PKCλ/ι. Such kinases can be activated by changes in either insulin or glucose levels and transport (for example, PKCβII and PKCλ/ι), shear stress (PKCβII), intracellular redox status (PKCβII and PKCδ), cellular morphology changes (PKCε), growth/differentiation factors (PKCβII and PKCε) or DNA damage (PKCδ) (Li and Xu, 2000; Ottaviani et al., 2001; Besson et al., 2002; Yoshida et al., 2003; Stawowy et al., 2005; Sajan et al., 2006; Min et al., 2009). In contrast to several PKCs, a knock down of the WRN protein (up to 72 h) was insufficient to induce phosphorylation and activation of p38 as described for fibroblasts derived from WS patients (Davis et al., 2007). Finally, phosphorylation of the ATM protein (and thus its activation) was not detected, although a 48 h knock down of the WRN protein was sufficient to cause double-stranded breaks (Turaga et al., 2007). This result is consistent with a recent report indicating that the WRN protein is required for ATM activation upon DNA damage, which causes replication fork collapse (Cheng et al., 2008).

Our results indicate the fact that RACK1 is required for the activation of PKCδ and PKCβII in WRN-depleted cells. Most importantly, RACK1 was the fourth most abundant protein partner to the TAP-WRN construct in HT1080 cells. Other proteins included the Ku heterodimer, a known partner (Cooper et al., 2000; Li and Comai, 2000), which established the suitability of our TAP strategy to identify new WRN cellular partners. We also detected the ATPase AAA protein, another protein interaction reported previously (Indig et al., 2004). It is noteworthy that few TAP-WRN interacting proteins were detected in our screen, as we extensively washed the complexes at each step of the procedure to capture the most stringent WRN protein interactants. RACK1 binds mainly to the RECQ domain and more weakly to the acidic domain of WRN (between the exonuclease and helicase domains of WRN). It inhibits the exonuclease activity of WRN without affecting its helicase activity in vitro. Interestingly, RACK1 has the opposite effect on WRN exonuclease activity than the Ku complex (Cooper et al., 2000). Thus, RACK1 and Ku complex could specifically coregulate the exonuclease activity of WRN depending on the levels of DNA damage or cellular conditions. Notably, depletion of RACK1 causes DNA damage in cells. It is possible that in the absence of RACK1, there is a dysregulation of WRN exonuclease activities leading to increased DNA damage (single-stranded gaps or double-stranded breaks). RACK1 may also associate and regulate other DNA repair proteins. Experiments are currently underway to identify other DNA repair enzymes that interact with the TAP-RACK1 construct. Although the binding of RACK1 to the WRN protein is outside the exonuclease domain (and helicase domain), the reason for its inhibitory effect on the exonuclease of WRN remains unknown. It is noteworthy that there are other examples of WRN interacting partners that regulate its exonuclease activity, but bind to other regions of the WRN protein. For example, the main binding regions of the KU heterodimer lie outside the exonuclease domain but strongly stimulates the exonuclease of WRN (Karmakar et al., 2002). The full-length WRN protein has not yet been crystallized, and its structure has therefore not been determined. Such crystallography analysis will be an important advance in the visualization of the three-dimensional structure of the WRN protein and in the understanding of the regulation of the exonuclease. Finally, we have no evidence that RACK1 affects the nucleolar localization of WRN (data not shown) as it binds to a region of WRN containing the nucleolar localization sequence (von Kobbe and Bohr, 2002). It is noteworthy that several proteins other than RACK1 bind to this particular region 949–109 (for example, TRF2 and FEN1; Brosh et al., 2001; Opresko et al., 2002), and we have no indication that any of these might affect WRN nuclear localization, but it does remain an interesting question for further pursuit.

Depletion of WRN also induces oxidative stress in addition to DNA damage (Szekely et al., 2005; Turaga et al., 2007). The exact mechanism by which depletion of WRN impacts the redox status of cells is unclear. However, it is known that WRN affects (directly or indirectly) the expression of several genes involved in lipidogenesis or oxidative metabolism (Massip et al., 2009; Turaga et al., 2009). Such changes in the cellular redox status will affect PKC activities (Konishi et al., 1997; Ohmori et al., 1998) through phosphorylation by an unknown kinase. In this study, we detected phosphorylation of PKCbII and PKCd upon ultraviolet or peroxide treatments. However, we did not observe an association of these PKCs with the TAP-RACK1 construct in the lysate of treated cells. This suggests that the oxidative stress and DNA damage observed in WRN-depleted cells may not be the inducers of the RACK1/PKCs association. A depletion of the WRN protein in cells causes a displacement of a portion of RACK1 from the nucleus to the cytoplasm in both HT1080 and GM08402 cells, where it interacts with several PKCs. It is unknown whether RACK1 molecules that were interacting with the WRN protein are the exact molecules that translocated into the cytoplasm upon WRN depletion. However, we observed an increase in RACK1 association with PKCδ and PKCβII upon WRN depletion in HT1080 cells. RACK1 is well known to regulate the activity of PKCβII, PKCδ and PKCε (Pass et al., 2001; Besson et al., 2002; Osmanagic-Myers and Wiche, 2004; Grosso et al., 2008; Slager et al., 2008). An increase in PKCε phosphorylation was detected in siWRN-transfected cells, but we did not detect an association of TAP-RACK1/PKCε. Furthermore, siRACK1 did not inhibit PKCε phosphorylation in WRN-depleted cells. This suggests that PKCε phosphorylation is independent of RACK1 in WRN protein-depleted cells unlike PKCβII and PKCδ.

It is noteworthy that PKCδ is known to modulate ROS production from the mitochondria (Kohda and Gemba, 2005). As WS patients are known to exhibit a pro-oxidant state (Pagano et al., 2005), there is the interesting possibility that depletion of WRN activates PKCδ through RACK1 displacement from the nucleus to the cytoplasm, which in turn modulates ROS production from the mitochondria. Accordingly, we decreased the production of ROS in WRN-depleted cells by transfecting siPKCδ molecules. The siRACK molecules were less efficient in reducing ROS production in WRN-depleted cells maybe because of a partial knock down of RACK1 proteins in cells. Indeed, we detected a certain phosphorylation level of PKCδ in siRACK1-transfected cells. Thus, application of appropriate kinase inhibitors may well form the basis of antiaging therapies for individuals with WS as recently proposed (Davis et al., 2007).

Materials and methods

The ‘Materials and methods’ section can be found at the Journal’s web site as Supplementary Materials.

Supplementary Material

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Table S1

Acknowledgments

We are grateful toMrs N Roberge for the FACS analyses (Centre de Recherche en Cancérologie, Quebec City, Qc). This study was supported by the Canadian Institutes of Health Research to ML and in part by funds from the intramural Program of the National Institute on Aging, NIH to VAB.ML is a senior scholar from the Fonds de la Recherche en Santédu Québec.

Footnotes

Conflict of interest

The authors declare no conflict of interest.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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Table S1
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