DNA damage modulates nucleolar interaction of the Werner protein with the AAA ATPase p97/VCP

Abstract

We report a novel nucleolar interaction between the AAA ATPase p97/VCP and the Werner protein (WRNp), a member of the RecQ helicase family. p97/VCP mediates several important cellular functions in eucaryotic cells, including membrane fusion of the endoplasmic reticulum and Golgi and ubiquitin-dependent protein degradation. Mutations in the WRN gene cause Werner syndrome, a genetic disorder characterized by premature onset of aging symptoms, a higher incidence of cancer, and a high susceptibility to DNA damage caused by topoisomerase inhibitors. We observed that both WRNp and valosin-containing protein (VCP) were present in the nucleoplasm and in nucleolar foci in mammalian cells and that WRNp and p97/VCP physically interacted in the nucleoli. Importantly, the nucleolar WRNp/VCP complex was dissociated by treatment with camptothecin, an inhibitor of topoisomerase I, whereas other WRNp-associated protein complexes, such as WRNp/Ku 80, were not dissociated by this drug. Because WRN syndrome cells are sensitive to topoisomerase inhibitors, these observations suggest that the VCP/WRNp interaction plays an important role in WRN biology. We propose a novel role for VCP in the DNA damage response pathway through modulation of WRNp availability.

Full text

Molecular Biology of the Cell
Vol. 14, 4221–4229, October 2003
DNA Damage Modulates Nucleolar Interaction of the
Werner Protein with the AAA ATPase p97/VCP
Juneth Joaquin Partridge,* Joseph Onofrio Lopreiato, Jr.,
†
Martin Latterich,
‡
and Fred Eliezer Indig*
§㥋
*Laboratory of Cell Biology and
†
Laboratory of Molecular Pharmacology, Center for Cancer Research,
National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892;
‡
The Salk
Institute, La Jolla, California 92037; and
§
Laboratory of Molecular Gerontology, National Institute on
Aging, National Institutes of Health, Baltimore Maryland 21224
Submitted February 25, 2003; Revised May 27, 2003; Accepted May 27, 2003
Monitoring Editor: Pamela Silver
We report a novel nucleolar interaction between the AAA ATPase p97/VCP and the Werner protein (WRNp), a member
of the RecQ helicase family. p97/VCP mediates several important cellular functions in eucaryotic cells, including
membrane fusion of the endoplasmic reticulum and Golgi and ubiquitin-dependent protein degradation. Mutations in
the WRN gene cause Werner syndrome, a genetic disorder characterized by premature onset of aging symptoms, a higher
incidence of cancer, and a high susceptibility to DNA damage caused by topoisomerase inhibitors. We observed that both
WRNp and valosin-containing protein (VCP) were present in the nucleoplasm and in nucleolar foci in mammalian cells
and that WRNp and p97/VCP physically interacted in the nucleoli. Importantly, the nucleolar WRNp/VCP complex was
dissociated by treatment with camptothecin, an inhibitor of topoisomerase I, whereas other WRNp-associated protein
complexes, such as WRNp/Ku 80, were not dissociated by this drug. Because WRN syndrome cells are sensitive to
topoisomerase inhibitors, these observations suggest that the VCP/WRNp interaction plays an important role in WRN
biology. We propose a novel role for VCP in the DNA damage response pathway through modulation of WRNp
availability.
INTRODUCTION
The ATPases associated with diverse cellular activities
(AAA) proteins are a common family of Mg
2⫹
-dependent
ATPases that contain one or two conserved ATP-binding
domains (Ogura and Wilkinson, 2001). These domains, or
AAA cassettes (Patel and Latterich, 1998), consist of a con-
served sequence of 230-amino acid residues that include the
Walker A and B motifs. Another sequence-conserved do-
main, the second region of homology, is found in part of the
AAA cassette (Confalonieri and Duguet, 1995) but is not
present in the closely related AAA⫹ family (Neuwald et al.,
1999) of proteins. Cdc48p, p97, and valosin-containing pro-
tein (VCP) are 92- to 97-kDa orthologous members of a
subfamily of AAA ATPases originally defined in yeast, Xe-
nopus, and mammals, respectively (Moir et al., 1982; Peters et
al., 1990; Frohlich et al., 1991). This subfamily has a known
propensity to oligomerize and form homohexamers (Peters
et al., 1992) that are a part of multiprotein complexes (Ogura
and Wilkinson, 2001).
Cdc48p/p97/VCP is involved in two major and distinct
cellular pathways: homotypic membrane fusion of endo-
plasmic reticulum (ER) and Golgi fragments and ubiquitin-
dependent protein degradation. This versatility is achieved
through different sets of adaptor proteins (Patel and Latter-
ich, 1998). In ER membrane fusion, Cdc48p/p97/VCP is
complexed with Ufe1p and Shp1p (Latterich et al., 1995;
Patel et al., 1998; Lin et al., 2001) or with their vertebrate
orthologs p47 and syntaxin 5 in Golgi membrane fusion
(Acharya et al., 1995; Rabouille et al., 1995). It is likely that the
role of Cdc48p in ER membrane fusion is to remove a fusion
inhibitor, thus permitting membrane fusion to occur (Lin et
al., 2001). Cdc48p/p97/VCP also participates in the degra-
dation of ubiquitinated proteins in yeast via Ufd1p and
Npl4p and in mammals (Ghislain et al., 1996; Dai et al., 1998;
Meyer et al., 2000). Overall, the emerging hypothesis is that
Cdc48p and orthologs such as VCP may serve as an “un-
windase” or molecular motor to unfold proteins and extract
them from protein complexes or membranes, with pathway
specificity being conferred by adapter proteins (Patel and
Latterich, 1998). It is known that Cdc48p is required for
nuclear division, because cdc48 mutants arrest in the cell
cycle with an undivided nucleus (Latterich and Schekman,
1994; Latterich et al., 1995). Cdc48p has a nuclear localization
signal (NLS) located in the N-terminal domain that is essen-
tial for its nuclear localization (Madeo et al., 1998). It is
unclear whether VCP is also nuclear and whether it has a
functional NLS (Madeo et al., 1997; Muller et al., 1999).
Herein, we demonstrate that VCP has both nuclear and
nucleolar localization and embark on a study to determine
the role of nucleolar VCP.
Article published online ahead of print. Mol. Biol. Cell 10.1091/
mbc.E03–02–0111. Article and publication date are available at
www.molbiolcell.org/cgi/doi/10.1091/mbc.E03–02–0111.
㛳
Corresponding author. E-mail address: indigfr@grc.nia.nih.gov.
Abbreviations used: AAA, ATPases associated with diverse cel-
lular activities; CPT, camptothecin; DAPI, 4⬘,6-diamidino-2-phe-
nylindole, dihydrochloride; ER, endoplasmic reticulum; GFP,
green fluorescent protein; IP, immunoprecipitate; VCP, valosin-
containing protein; WRNp, Werner helicase protein; WS,
Werner Syndrome.
© 2003 by The American Society for Cell Biology 4221

Werner syndrome (WS) is a rare autosomal recessive ge-
netic disorder characterized by premature onset of aging
symptoms and higher incidence of cancer (Shen and Loeb,
2001). The WRN gene product is a 160-kDa protein homol-
ogous to the Escherichia coli RecQ DNA helicase (Yu et al.,
1996). Recombinant human Werner helicase (WRNp) exhib-
its RNA and DNA unwinding activities (Gray et al., 1997;
Suzuki et al., 1997) as well as exonuclease activity (Huang et
al., 1998; Suzuki et al., 1999). WRNp has an NLS near the C
terminus of the protein and has been described in both the
nucleoplasm and nucleolus. Recently, a nucleolar localiza-
tion signal has been determined for WRNp (von Kobbe and
Bohr, 2002). The regulation of WRNp nuclear dynamics is
unclear and differs between growing and resting cells and
also between mouse and human cells (Suzuki et al., 2001,
and references therein). The WRN protein forms functional
complexes with several cellular proteins, some of which
facilitate its helicase activity, such as replication protein A
(RPA) (Shen et al., 1998; Brosh et al., 1999) and telomere-
repeat binding factor 2 (TRF2) (Opresko et al., 2002). How-
ever, none of these interacting proteins explain the regula-
tion of WRNp nuclear localization.
In this study, we demonstrate that VCP is found in mam-
malian nuclei and report a novel nucleolar protein complex
between members of two protein families, the Werner pro-
tein of the RecQ Helicase family, and the AAA ATPase
p97/VCP. We further show that this complex is disrupted
by a drug to which WS cells show high sensitivity, the
topoisomerase I inhibitor camptothecin (CPT). These obser-
vations propose a novel role for members of the AAA pro-
tein family in regulating the availability of enzymes in-
volved in nucleic acid metabolism through physical
interaction.
MATERIALS AND METHODS
Proteins, Antibodies, and Cells
Rabbit anti-VCP antibody 5860 and chicken anti-VCP antibody 1469 (Aves,
Tigard, OR) were raised against purified bovine liver VCP (Indig and Latter-
ich, unpublished data). Briefly, liver homogenate was prepared in the pres-
ence of ATP and clarified by freezing ⫺80°C overnight, followed by pelleting
the precipitate by centrifugation. The clarified supernatant was loaded on
DEAE-Sepharose FF (Amersham Biosciences, Piscataway, NJ) and eluted with
a60–500 mM KCl gradient. VCP-containing fractions were detected by Coo-
massie-stained gels, pooled, and precipitated with ammonium sulfate at 40%
saturation. After dialysis, VCP was purified using a 5–30% sucrose gradient
and fractions collected were snap frozen and stored at ⫺80°C.
The antibodies 5860 and 1469 were compared with known mouse anti-p97
antibodies (see below) and found to be highly specific and selective, reacting
only with VCP from human, monkey, and hamster cells. Preimmune sera did
not detect VCP (Figure 4) and was negative in immunofluorescence (our
unpublished data). Antibodies 5860 and 1469 did not cross-react with other
AAA ATPases, including nuclear VCP-Like protein, a 110-kDa AAA protein
with unknown function (Germain-Lee et al., 1997).
The anti-VCP monoclonal antibody (mAb) MARA-1 (Schulte et al., 1994)
was a kind gift of Robbie Schulte and Bart Sefton (The Salk Institute, La Jolla,
CA). Anti-p97 mAb 58.13.3 was purchased from Research Diagnostics
(Flanders, NJ). Rabbit anti-WRN1 was purchased from Novus (Littleton, CO)
and mouse anti-WRNp mAb and SW13 cell lysate were from BD Transduc-
tion Laboratories (San Diego, CA). Rabbit anti-WRN H-300 and rabbit anti-
MPP2 polyclonals were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Rabbit anti-Ku 80 was purchased from Chemicon International
(Temecula, CA) and mouse anti-Ku 80 from NeoMarkers (Fremont, CA).
Rabbit anti-BRCA1 was purchased from Lab Vision (Fremont, CA) and mouse
anti-BRCA1 clone antibody-1 was from Oncogene Science (Cambridge, MA).
Horseradish peroxidase- and Cy3-conjugated secondary mAbs were pur-
chased from Jackson Immunoresearch Laboratories (West Grove, PA). Alexa
488-conjugated secondary mAbs and the DNA stain 4⬘,6-diamidino-2-phe-
nylindole, dihydrochloride (DAPI) were purchased from Molecular Probes
(Eugene, OR).
The monkey kidney cell line CV-1, the human glioma cell line MO59-K, the
human mesothelioma cell line NCI-H226, and the human lymphoblastoid
lines K562 and Jurkat were obtained from the American Type Culture Col-
lection (Manassas, VA).
Protease inhibitors leupeptin, phenylmethylsulfonyl fluoride, E
64
, chymo
-
statin, and pepstatin A were purchased from Calbiochem (San Diego, CA).
RNase A was from Worthington Biochemicals (Lakewood, NJ).
Plasmid Construction
The murine VCP cDNA clone was a kind gift from Dr. Ron Trible (National
Institutes of Health, Bethesda, MD). The following restriction fragments were
cloned into the pEGFP-c1 vector (BD Biosciences Clontech, Palo Alto, CA), by
using standard molecular biology techniques (Sambrook et al., 1989): the
whole VCP gene (Swissprot Q01853), VCP⌬C (VCP without C-terminal resi-
dues 639–806), and VCP⌬N (VCP without N-terminal residues 1–141).
Cell Culture, Transfection, and Camptothecin Treatment
Cells were maintained in DMEM (Quality Biological, Gaithersburg, MD)
supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan,
UT), l-glutamine, and antibiotics (Quality Biological), by using standard cell
culture technique (Indig et al., 1997). Cells were plated onto wells of a six-well
plate, ⬃100,000 cells/well. After overnight growth, subconfluent cells were
transfected with 0.5
␮
g of DNA by using LipofectAMINE Plus (Invitrogen,
Carlsbad, CA) according to manufacturer’s instructions.
Subconfluent cells propagated for nuclear extraction were treated with
camptothecin (CPT; Calbiochem) at 10
␮
M for 1 or 4 h. Control cells were left
untreated. The cells were trypsinized and harvested, washed once with
phosphate-buffered saline (PBS), and the cell pellet was either snap frozen
and stored at ⫺80°C, or immediately extracted as below.
Nuclear Extraction
The nuclear extraction procedure was adapted from (Abmayr and Workman,
2001). Hypotonic (10 mM KCl, 10 mM HEPES, pH 7.9, at 4°C, 1.5 mM MgCl
2
),
low salt (0.2 M KCl, 20 mM HEPES, pH 7.9, at 4°C, 25% glycerol, 1.5 mM
MgCl
2
, 0.2 mM EDTA), and high salt (similar to low salt buffer except with 1.2
M KCl) buffers were prepared. The following protease inhibitor cocktail was
added to hypotonic and low salt buffers immediately before use (final con-
centration): 2 mM phenylmethylsulfonyl fluoride, 0.5 mM leupeptin, 0.2 mM
chymostatin, 20
␮
M pepstatin A, 0.2 mM E64. All procedures were at 4°C.
Cell pellets were washed in 5 packed cell volumes of hypotonic buffer plus
inhibitors, centrifuged at ⬃1800 ⫻ g for 5 min, and then resuspended in 3
packed cell volumes of hypotonic buffer, and cells were allowed to swell on
ice for 10 min. More hypotonic buffer was added until cell density was 2 ⫻ 10
7
cells/ml. The cells were then lysed on ice by using 10 –20 strokes of a glass
Dounce homogenizer (type B pestle). Lysis (⬎90% of cells) was verified by
staining a sample with trypan blue and observing under a light microscope.
The lysate was centrifuged at 3300 ⫻ g, and this cytoplasmic extract was
transferred to new tubes and saved for later immunoprecipitation. The re-
maining nuclear pellet was resuspended in 2 packed nuclear volumes of low
salt buffer plus inhibitors. High salt buffer, half the new volume of nuclei in
low salt buffer, was added dropwise while gently vortexing. The suspension
was incubated at 4°C with continuous gentle mixing for 2 h and then centri-
fuged at ⬃18,000 ⫻ g for 40 min. The nuclear extract was transferred to fresh
tubes for immunoprecipitation or storage at ⫺80°C.
SDS-PAGE, Immunoblotting, and Immunoprecipitation
Nuclear or cytoplasmic extracts of equal protein content (determined by BCA
protein assay; Pierce Chemical, Rockford, IL) were immunoprecipitated as
described by Indig et al., 1997. Precipitated proteins were separated on poly-
acrylamide gels, immunoblotted, and chemiluminescence detected as de-
scribed previously (Indig et al., 1997).
Microscopy and Immunofluorescence
Cells were grown on coverslips overnight and fixed in 3.7% formaldehyde
followed by permeabilization with 0.2% Triton X-100 and then incubated with
appropriate antibodies and mounted on slides as described previously (Indig
et al., 1997). The following changes and additions were made to the procedure.
After incubation with secondary antibodies, coverslips were washed five
times in PBS, pH 8.5. When coverslips were stained with DAPI, they were
washed three times and then stained with 1
␮
M DAPI (in PBS), and coverslips
were incubated for 20 min at 37°C. RNase A (20
␮
g/ml) was also added to this
step. Coverslips were washed three more times in PBS, pH 8.5, before mount-
ing.
Fixed cells were studied with an Axioplan 2 microscope (Carl Zeiss, Thorn-
wood, NY) and images were captured with a Orca 2 charged-coupled device
camera (Hamamatsu, Bridgewater, NJ). Images were processed and decon-
volved using Openlab software (Improvision, Lexington, MA). Confocal mi-
croscopy was done with a TCS SP system (Leica Microsystems, Deerfield, IL),
and images were transferred into Photoshop (Adobe Systems, Palo Alto, CA).
Live cells were studied with an IX70 inverted microscope (Olympus, Melville,
J.J. Partridge et al.
Molecular Biology of the Cell4222

NY), and images were captured through a Cool Snap Fx charge-coupled
device and processed with IPLab (BioVision, Exton, PA) software.
RESULTS
VCP Is Localized to the Nucleus and Nucleolus of
Mammalian Cells
We examined the subcellular distribution of VCP in mam-
malian cells by indirect immunofluorescence. As shown in
Figure 1, antibodies against VCP stained both the cytoplasm
and the nucleus with a punctate pattern in simian and
human cells (antibody Rb5860 in simian CV1 cells, Figure
1A; antibody chicken 1469 in human NCI-H226, Figure 1B).
The cytoplasmic signal was primarily from the ER, where
VCP colocalized with the ER-specific stain 3,3⬘-dihexyl-ox-
acarbocyanine iodide (our unpublished data). Two patterns
of VCP nuclear fluorescence emerged: a punctate pattern
throughout the nucleus and several foci of intense fluores-
cent signal. These intensely staining foci were found to
correspond to nucleoli, as demonstrated by colocalization of
VCP to nucleolar markers such as fibrillarin (Figure 1B) and
nucleolin (our unpublished data). In contrast, antibodies
against VCP did not colocalize with antibodies against other
abundant nuclear proteins such as the M-phase phospho-
protein 2 (MPP2; Figure 1B). We used confocal microscopy
to verify that the VCP signal was not merely superimposed
on the nuclear area but intercalated within the nuclear DNA-
staining regions (Figure 1A, xz and xy sections). Similar
immunofluorescent patterns were observed with other hu-
man cell lines, including M059K (Figures 3 and 6), M059J,
K562, U87MG, and Jurkat and in Chinese hamster ovary
cells (our unpublished data). Identical patterns were ob-
served with several anti-VCP antibodies: two murine mono-
clonals (anti-p97, MARA-1; our unpublished data) and with
two polyclonal anti-VCP antibodies (rabbit 5860, Figure 1A;
chicken 1469, Figure 1B).
VCP Has an N-Terminal Domain Nuclear Localization
Signal
In contrast to its yeast ortholog Cdc48p, VCP did not possess
a known NLS and was thought not to be a nuclear protein
(Madeo et al., 1997, 1998). Because VCP clearly localized to
the nucleus (Figure 1), it was important to determine the
location of a putative NLS within the VCP sequence. Toward
this goal, a series of VCP mutants fused to the green fluo-
rescent protein (GFP) gene were cloned (Figure 2). The full-
length VCP-GFP expressed throughout the cell, in an ER-like
network in the cytoplasm and fluoresced intensely in the
nuclear region (Figure 2, second row). Deletion of the C-
terminal domain (⌬C-VCP-GFP) resulted in a diffuse nuclear
and cytoplasmic signal (Figure 2, forth row). Deletion of the
N-terminal domain (⌬N-VCP-GFP) prevented nuclear ex-
pression of the fusion protein and only an extranuclear
Figure 1. VCP is present in the nucleus and nucleolus of mammalian cells. Cells were grown on glass coverslips overnight and then fixed
in 3.7% formaldehyde and permeabilized with Triton X-100 as described in MATERIALS AND METHODS. (A) Confocal section of CV-1 cells
stained with Rb 5860 anti-VCP (1:500) and visualized with Cy3-conjugated secondary antibody (red), and with the DNA stain DAPI (blue).
xz and yz are confocal cross sections through the right-hand cell as marked. 630⫻; bar, 10
␮
m. (B) NCI-H226 cells were stained with chicken
1469 anti-VCP (1:200, red) and mouse anti-MPP2 (1:500, green) or mouse anti-fibrillarin (1:200, green). Images were merged to view
colocalization (yellow). 400⫻; bar, 10
␮
m.
VCP/Werner Nucleolar Complex
Vol. 14, October 2003 4223

cytoplasmic localization was visible (Figure 2, third row).
Propidium iodide staining of nuclei verified that there was
no nuclear GFP expression when cells were transfected with
⌬N-VCP-GFP (our unpublished data).
VCP and WRNp Colocalize in the Nucleolus of Human
Glioma Cells and Coimmunoprecipitate in Nuclear
Extracts
To further characterize VCP function in the nucleus, we
initiated a search for possible VCP interacting proteins, fo-
cusing on proteins that showed similar punctated nuclear
and possible nucleolar distribution. VCP was not found in
promyelocytic leukemia (PML) nuclear bodies and did not
colocalize with proteins associated with DNA replication,
such as proliferating cell nuclear antigen, or with cell cycle-
specific proteins such as MPP2 (for example, Figure 1B). In
contrast, WRNp showed a staining pattern similar to VCP
throughout the nucleus with intense nucleolar signal that
colocalized with the VCP signal (Figure 3). To verify that the
pattern similarity was derived from colocalization and not
superimposition of different nucleolar layers, we analyzed
deconvolved, high-resolution nuclear images from 0.1-
␮
m
optical sections. As shown in Figure 3, the deconvolution
data indicated that VCP (green) and WRNp (red) colocalized
primarily in nucleolar foci (yellow, Figure 3, merge; also see
Figure 6, nontreated [NT]). This colocalization extended to
⬃1.0
␮
m in depth and was prominent in the DAPI-negative
areas (i.e., that contained RNA before RNase A treatment) of
the nucleolus (Figure 3).
We next examined whether VCP and WRNp formed a
stable complex in mammalian cells, as suggested by the
colocalization data. To that end, we used immunoblotting to
detect VCP in anti-WRNp immunoprecipitates of nuclear
extracts. As shown in Figure 4, the chicken anti-VCP anti-
body (Ch
␣
VCP) detected an ⬃100-kDa protein in purified
p97 (lane 1). In control experiments, VCP was not detected
in nuclear extracts immunoprecipitated by purified rabbit
IgG (lane 7) or chicken IgY (not shown), nor did VCP react
with preimmune chicken serum or purified IgY (lanes 2 and
3). Similarly, the monoclonal anti-VCP antibody (Figure 4,
lanes 4 – 6) identified the same 100-kDa protein in purified
p97 and in lysates from two cell lines, MO59K and SW13. An
identically migrating protein was detected by the anti-VCP
chicken antibody in immunoprecipitates with anti-WRNp
from Jurkat and MO59K cells (Figure 4, lanes 10 and 11).
Reciprocally, WRNp was detected in anti-VCP immunopre-
cipitates from both rabbit and chicken anti-VCP (Figure 4,
lanes 12 and 13). Coimmunoprecipitation was apparent in
several cell lines as well, such as MO59K, K562, and Jurkat,
by using various antibodies against VCP and WRNp (Figure
4, lanes 8 –13). These observations confirmed that VCP and
WRNp are present in the same protein complex, as indicated
by the indirect immunofluorescence studies.
Figure 2. The N-terminal domain of VCP is crucial for nuclear
localization. The murine VCP gene was fused to the pEGFP vector
as described in MATERIALS AND METHODS, and several domain-
deleted fusion proteins were constructed as described in text. Hu-
man glioma MO59K cells were transfected using the cationic lipid
method. After 48 h, live cells were examined under an Olympus
IX70 and images captured and merged with IPLab. Right, merged
image of GFP fluorescence (middle) with phase contrast (left). 400⫻,
bar, 5
␮
m.
Figure 3. VCP and WRNp colocalize in the nucleolus. MO59K cells
were processed for indirect immunofluorescence as described above
and stained with anti-WRNp mAb (1:100, red), chicken 1469 anti-
VCP (1:250, green), and the DNA stain DAPI (blue). By using
Openlab deconvolution software, 0.1-
␮
m Z-sections were obtained
on an Axioplan 2 microscope (Carl Zeiss) and deconvolved. Merge
is the merged image of the three fluorescence channels examined of
the same deconvolved Z-section. Two representative cells from
different experiments are shown; a nucleolar area of each cell was
enlarged (3⫻, bottom). 630⫻; bar, 5
␮
M.
J.J. Partridge et al.
Molecular Biology of the Cell4224

The Topoisomerase I Inhibitor CPT modulates the VCP–
WRNp Interaction
Camptothecin is known to have a specific effect on cells with
mutated WRN function, causing chromosomal damage, cell-
cycle arrest and rapid cell death (Poot et al., 1992, Pichierri et
al., 2000). To examine whether the WRNp/VCP interaction
was affected by DNA repair pathways, MO59K cells were
treated with 10
␮
M CPT for 1 or 4 h before immunoprecipi-
tation analysis or visualization of the WRN and VCP pro-
teins by indirect immunofluorescence. As shown in Figure 5,
a 4-h treatment with CPT led to a marked reduction in
WRNp detected in anti-VCP immunoprecipitates (Figure
5A). We also observed a reciprocal reduction in VCP de-
tected in anti-WRNp immonoprecipitates in nuclear extracts
from cells treated for 4 h with CPT (Figure 5, A and B,
compare4htountreated cells or cells treated for only 1 h).
To verify the specificity of the CPT effect on the VCP/
WRNp interaction, we examined the effect of this drug on
the abundance of these two proteins as well as on other
multiprotein complexes that were reported to contain VCP
or WRNp (Figure 5, C–F). As shown in Figure 5C, the WRNp
signal did not diminish in immunoprecipitates with anti-
WRNp antibodies (lane 1 versus 3), nor in total nuclear
extracts (lane 7 versus 9) from cells treated with CPT. Sim-
ilarly, we did not observe a decrease of total nuclear VCP
after 4 h CPT treatment in most experiments (Figure 5D).
Moreover, in contrast to the effect of CPT on the VCP/
WRNp complex, CPT effected neither the nuclear abundance
of the Ku-80 protein that is known to interact with WRNp
(Cooper et al., 2000; Li and Comai, 2000), nor the ability of
Ku and WRNp antibodies to immunoprecipitate these pro-
teins (Figure 5E). Finally, we have also investigated complex
formation between BRCA1 and VCP (Zhang et al., 2000a).
We have confirmed this interaction, because BRCA1 was
detected in VCP immunoprecipitates (IPs) from both nuclear
and cytoplasmic extracts (Figure 5F). This complex was not
disrupted by CPT, and BRCA1 was not recruited to the
WRNp complex after CPT treatment (Figure 5C, lanes 4– 6).
BRCA1 was not detected in WRNp IPs, nor was WRNp
found in BRCA1 IPs (Figure 5, C and F), indicating that the
VCP/BRCA1 and VCP/WRNp were separate and distinct
nuclear protein complexes.
We used indirect immunofluorescence to determine
whether CPT dissociates the VCP/WRNp complex in the
nucleolus. Figure 6 shows enlarged deconvolved images of
representative nuclei from MO59K cells that were fixed with
formaldehyde after CPT treatment, and stained with anti-
bodies against VCP and WRNp. The NT nucleolus showed
a similar fluorescence pattern for both anti-WRNp (green)
and anti-VCP (red), with intense colocalization (merge, yel-
low) in the DAPI-negative central region of the nucleolus.
After1hofCPTtreatment, the nucleolar colocalization was
diminished (Figure 6, 1 h). After4hofCPTtreatment, very
little colocalization remained in the central nucleolar area
and both WRNp and VCP showed a different nucleolar
staining pattern (Figure 6, 4 h). These data suggest that CPT
treatment specifically disrupts the nucleolar complex
formed between the VCP and the WRN proteins.
DISCUSSION
We have shown that VCP localizes to mammalian nuclei,
where it physically associated with the Werner helicase in
the nucleolus. This conclusion was based on the specific and
reciprocal coimmunoprecipitation of these nucleolar pro-
teins and precise colocalization by indirect immunofluores-
cence in deconvolved thin sections. Furthermore, treatment
of cells with camptothecin causes the abrogation of the
VCP–Werner complex. The dissociation of VCP and WRNp
can be followed in high-resolution deconvolved fluorescent
images of the nucleolus. Our data further suggest that VCP
and WRNp both participate in other multiprotein complexes
that are not disrupted by CPT.
We demonstrated that the AAA ATPase p97/VCP, known
for its roles in homotypic membrane fusion and ubiquitin-
directed proteolysis, was abundant in the nucleus. VCP/p97
showed a robust intranuclear signal, especially in nucleoli
and was immunoprecipitated from nuclear extracts. Several
AAA⫹ (but not AAA) proteins are present in the nucleus
and participate in the nucleic acid metabolic pathways, such
as DNA replication and damage repair (Ogura and Wilkin-
son, 2001) and ribosome biogenesis (Brown, 2001). We ob-
served that nuclear VCP did not localize to PML bodies or
replication factories, indicating that VCP probably does not
Figure 4. VCP and WRNp reciprocally coimmunopre-
cipitate. Nuclear extracts were immunoprecipitated and
immunoblotted as described in MATERIALS AND
METHODS. Equal amounts of total protein were immu-
noprecipitated with 20
␮
l of rabbit anti-Werner helicase
(Rb
␣
W) and either the chicken (1469) or rabbit (5860)
anti-VCP polyclonal antibodies. Rabbit IgG was used as
a negative control. Immunoprecipitated proteins (40
␮
l/
lane) were electrophoresed on a 7.5% polyacrylamide
gel and then immunoblotted to polyvinylidene difluo-
ride membranes. Aftera1hincubation with primary
antibodies and appropriate horseradish peroxidase-con-
jugated secondary antibodies, proteins were visualized
by enhanced chemiluminescence. The results are pre-
sented as a composite image of Jurkat (lanes 7–10),
MO59K (lanes 11 and 12) or K562 (lane 13) precipitates,
or total cell lysates of MO56K (lane 5) or SW13 (lane 6)
cells. Lanes 1– 4 contain purified bovine liver VCP, 0.5
␮
g, immunoblotted with chicken anti-VCP 1469 (1:2000, lane 1), or anti-VCP mAb (lane 4). As a control, 2.0
␮
g of purified VCP was
immunoblotted with 10
␮
g/ml preimmune chicken serum (preimmune 1:200, lane 2) or 10
␮
g/ml purified chicken IgY (IgY, lane 3). The
expected position of VCP and WRNp are indicated on the right and molecular masses in kilodaltons are in the middle. In some cell lysates
(example, lane 5), an ⬃60-kDa protein, apparently a VCP fragment, is detected by anti-VCP antibody. VCP multimers are occasionally
detected, for example, in purified VCP (lane 1).
VCP/Werner Nucleolar Complex
Vol. 14, October 2003 4225

participate in functions associated with these nuclear struc-
tures. Interestingly, a role in DNA repair for VCP has been
implied by a report associating VCP with the nuclear protein
BRCA1 (Zhang et al., 2000a). We confirmed this interaction
and demonstrated that the VCP/BRCA1 complex is distinct
from the VCP/WRNp complex, because WRNp was not
observed in VCP/BRCA1 complexes and vice versa. These
observations suggest that the nuclear role of VCP may in-
volve regulating the abundance of its partner proteins, such
as WRNp and BRCA1, modulating the availability of these
proteins to participate in metabolic processes in response to
DNA damage.
We found that similar to Cdc48p, the N-terminal do-
main of VCP was essential for nuclear localization. When
the N-terminal portion was deleted, the ⌬N-VCP-GFP
fusion protein was unable to enter the nucleus and re-
mained in the cytoplasm. When the C-terminal portion of
VCP was deleted, the ⌬C-VCP-GFP fusion protein was
expressed throughout the cell, but it was no longer con-
centrated in the nucleus. This indicated that although the
N-terminal portion of VCP was responsible for nuclear
localization, the C-terminal domain has a role in retaining
VCP in the nucleus, leading eventually to a high nuclear
concentration of VCP. In yeast, a Cdc48p/VCP fusion
protein showed that the C-terminal domain was function-
ally interchangeable between the orthologs, but not the
N-terminal domain. Cdc48p containing the VCP N-termi-
nal was unable to gain nuclear entry (Madeo et al., 1997).
Frohlich and coworkers identified a bipartite NLS (Rob-
bins et al., 1991) spanning residues 15–31 in Cdc48p that is
mostly absent from VCP. However, as they had noted, a
stretch of basic amino acid residues, KGKKRK, that re-
Figure 5. CPT treatment effects VCP and WRNp coimmunoprecipitates. MO59K cells were treated with 10
␮
MCPTfor1or4horwere
untreated controls (0 h). In all panels, the molecular mass in kilodaltons is indicated to the right or left. (A) Rabbit anti-WRNp and rabbit
anti-VCP precipitates were treated as described in Figure 4 and Western blotted with mouse anti-WRNp (lanes 1– 4) or chicken anti-VCP
(lanes 5 and 6) as described. (B) A single immunoblot from the same experiment of VCP (lane 3) and WRNp (lanes 4 – 6) IPs probed with
chicken anti-VCP. Lane 1, 0.5
␮
g of purified bovine liver VCP; lane 2, MO59K total cell lysate (40
␮
g). (C) Mouse anti-WRNp detects WRNp
in rabbit anti-WRNp precipitates (lanes 1–3) and in total nuclear extract (lanes 7–9), but not in rabbit anti-BRCA1 IPs (lanes 4–6). Cells were
treated with CPT and Western blotted as described in text. (D) Cells were treated as described above with CPT and extracts were
immunoblotted with chicken 1469 anti-VCP as described in text. Three different experiments are shown, one performed with K562 extracts
(lanes 1–6) and two with MO59K extracts (lanes 8–13 and 15–20). Lanes 7 and 14, 0.5
␮
g of purified bovine liver VCP. (E) WRNp was detected
in rabbit anti-Ku 80 precipitates (lanes 6–8) and Ku-80 was detected in rabbit anti-WRNp precipitates (lanes 2– 4) of nuclear extracts. Lane
1, 20
␮
l of SW13 total lysate; lane 5, 20
␮
l of MO59K total lysate. (F) BRCA1 detected in VCP, but not WRNp IPs. MO59K cells were separated
into cytoplasmic and nuclear extracts as described in MATERIALS AND METHODS and precipitated with rabbit anti-WRNp or Rb 5860
anti-VCP and probed for BRCA1.
J.J. Partridge et al.
Molecular Biology of the Cell4226

sembles the SV40 T-antigen NLS (Kalderon et al., 1984) is
identified at Cdc48p position 70–75 (Madeo et al., 1997).
This sequence is highly conserved in VCP (KGKKRR,
position 60 – 65) and can function as the VCP NLS. We
should bear in mind, however, that the N-terminal do-
main of Cdc48p/p97/VCP is also thought to be involved
in protein binding of adaptor molecules, such as p47
(Coles et al., 1999; Rouiller et al., 2000; Zhang et al., 2000b).
Therefore, we cannot rule out the less likely possibility
that the N-terminal deletion affected the interaction of
VCP with a cofactor essential for its nuclear entry.
The VCP protein was associated with WRNp as seen by
immunofluorescence and reciprocal coimmunoprecipita-
tion. The Werner helicase has been shown to interact with
multiple protein complexes: p53 (Blander et al., 1999; Spillare
et al., 1999), Ku 80/76 (Cooper et al., 2000; Li and Comai,
2000), topoisomerase I (Lebel et al., 1999), and FEN1 (Brosh
et al., 2001), among others. The VCP/WRNp complex de-
scribed herein was dissociated by treatment of cells with the
DNA-damaging agent camptothecin, whereas other WRN-
associated complexes, such as WRNp/Ku, were not. CPT is
a DNA topoisomerase I inhibitor that blocks topoisomerase
I kinase activity (Rossi et al., 1996) and causes double-strand
DNA breaks (Pommier et al., 1998; Pourquier and Pommier,
2001). Cells and cell lines derived from Werner Syndrome
patients were shown to be sensitive to the genotoxins camp-
tothecin and 4-nitroquinoline-1-oxide (Poot et al., 1992; Og-
burn et al., 1997; Poot et al., 1999). We observed that other
genotoxic agents, such as hydroxyurea and bleomycin, did
not dissociate the VCP/WRNp complex as did CPT (Indig,
unpublished data). In contrast, CPT had no effect on the
interaction between WRNp and Ku 80 and on intranuclear
levels of immunoprecipitated WRNp. Similarly, topoisom-
erase I was shown to dissociate from nucleoli after treatment
with the CPT derivative topotecan, whereas hydroxyurea
had no effect (Danks et al., 1996). In another WRNp-associ-
ated complex, the induction of p53 by CPT was reduced in
WS cells (Blander et al., 2000). These molecular data are
consistent with the observation that CPT has a specific effect
on the survival of WS cells (Shen and Loeb, 2001) compared
with other DNA-damaging agents, such as UV irradiation,
hydroxyurea, bleomycin, and alkylating agents (Shen and
Loeb, 2000).
We localized the VCP/WRNp interaction to the nucleolus.
For many years, the nucleolus was largely considered to be
a ribosome factory (Schwarzacher and Wachtler, 1993). With
the localization to the nucleolus of proteins that are not
involved in ribosome genesis, it is apparent that the nucle-
olus has other roles in the cell (Andersen et al., 2002). An
example of novel nucleolar function is seen in the phospha-
tase Cdc14p, which is sequestered to the nucleolus by its
inhibitor, Cfi1p/Net1p, and released into the nucleoplasm
during anaphase (Visintin et al., 1999; Stegmeier et al., 2002).
The Werner helicase has been localized to the nucleolus
(Gray et al., 1998; Marciniak et al., 1998) and in fact possesses
a nucleolar targeting sequence (von Kobbe and Bohr, 2002).
However, the role of WRNp in the nucleolus is unclear.
After treatment with CPT, WRNp translocated to intranu-
clear repair foci that included the repair proteins Rad50 and
RPA (Sakamoto et al., 2001). WRNp also translocated from
the nucleolus to the nucleoplasm after treatment with the
genotoxic agent 4-nitroquinoline-1-oxide (Gray et al., 1998)
and serum starvation (Suzuki et al., 2001). It has been pro-
posed that tyrosine phosphorylation, either by direct mod-
ification of WRNp or of a putative “WRN-nucleolar carrier”
may modulate the nucleolar trafficking of WRNp (Gray et al.,
1998). Interestingly, VCP is known to be tyrosine-phospho-
rylated at its C-terminal domain and that hydrogen peroxide
treatment greatly increases VCP tyrosine phosphorylation
(Schulte et al., 1994). It has been suggested that another
Figure 6. CPT treatment dissociates VCP
and WRNp in the nucleolus. MO59K cells
were treated with 10
␮
MCPTfor1or4hor
were NT and then processed for indirect
immunofluorescence as described in Figure
3. WRN (green) and VCP (red) are the dif-
ferent fluorescent channels of the same
0.1-
␮
m section examined. Merged is the re-
sult of coloring both channels and present-
ing them in the same image, with yellow
indicating colocalization. NT, 630⫻; 1 and
4 h, 1000⫻.
VCP/Werner Nucleolar Complex
Vol. 14, October 2003 4227

intranuclear structure, PML bodies, act as a nuclear depot of
proteins, that are released upon viral attack (Negorev et al.,
2001). Similarly, the sequestered WRNp is released upon
accumulation of damaged DNA in the cell. Our observation
that VCP and WRNp dissociate from each other and move
away from the nucleolus after CPT treatment supports this
suggestion. Together, we propose that VCP may play a role
in the response to DNA damage by modulating the nucleo-
lar trafficking of WRNp.
What role does the VCP ATPase play in Werner helicase
biology? The P97/VCP hexamer is known to undergo a
conformational change upon ATP binding that could be
translated into mechanical work (Rouiller et al., 2000), such
as protein complex dissociation. Other AAA proteins are
thought to function in a similar manner (Ogura and Wilkin-
son, 2001). When DNA damage has occurred (from CPT, for
example) that requires WRNp elsewhere (like in DNA repair
complexes), VCP binds ATP and dissociates the protein
complex, enabling WRNp to translocate from the nucleolus.
We propose that VCP recruits or sequesters WRNp to the
nucleolus, to be released as required through VCP action. It
is probable that VCP is involved in more than one nuclear
pathway and that the elucidation of the VCP nuclear inter-
actions should provide important insights into normal nu-
clear function and nuclear response to DNA damage.
ACKNOWLEDGMENTS
This article is dedicated to the memory of Professor Shmaryahu Blumberg,
who suddenly passed away in December 2001. We thank Drs. Michael Gottes-
man, Vilhelm Bohr, and Mirit I. Aladjem for critical reading of the manu-
script. This work was in part sponsored by grants GM-54729 from the Na-
tional Institutes of Health and RG 183/1999-M from the Human Frontier
Science Program, awarded to M.L.
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