# The Valosin-containing Protein (VCP) Is a Target of Akt Signaling Required for Cell Survival

Franck Vandermoere, Ikram El Yazidi-Belkoura, Christian Slomianny, Yohann

Demont, Gabriel Bidaux, Eric Adriaenssens, Jérôme Lemoine, Hubert

Hondermarck

► To cite this version:

Franck Vandermoere, Ikram El Yazidi-Belkoura, Christian Slomianny, Yohann Demont, Gabriel Bidaux, et al.. The Valosin-containing Protein (VCP) Is a Target of Akt Signaling Required for Cell Survival. Journal of Biological Chemistry, 2006, 281 (20), pp.14307-14313. 10.1074/jbc.M510003200. hal-03060251

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# THE VALOSIN-CONTAINING PROTEIN (VCP) IS A TARGET OF AKT SIGNALING REQUIRED FOR CELL SURVIVAL

Franck Vandermoere¹, Ikram El Yazidi- Belkoura¹, Christian Slomianny², Yohann Demont¹,

Gabriel Bidaux², Eric Adriaenssens¹, Jérôme Lemoine³, Hubert Hondermarck¹*

¹ERI-8 INSERM, EA-1033, “Growth factor signaling in breast cancer. Functional proteomics”,

²EMI-0228 INSERM and ³UMR-8576 CNRS. IFR-118,

University of Sciences and Technologies Lille, France.

Running title: VCP is a target of Akt signaling

*Address correspondence to Hubert Hondermarck, ERI-8 INSERM, EA-1033, batiment SN3, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq, France; tel +33 (0)3 20 43 40 97, fax +33 (0)3 20 47 40 38 ; hubert.hondermarck@univ-lille1.fr

The serine/threonine kinase Akt is a key mediator of cell survival and growth, but its precise mechanism of action and more specifically the nature of its signaling partners largely remains to be elucidated. Here we show, using a proteomics-based approach, that the valosin-containing protein (VCP), a member of the AAA (ATPases associated with a variety of cellular activities) family, is a target of Akt signaling. SDS-PAGE of Akt co-immunoprecipitated proteins obtained from MCF-7 breast cancer cells revealed the increase of a 97 kDa band under Akt activation. Mass spectrometry analysis allowed the identification of VCP and we have shown a serine/threonine phosphorylation on a Akt consensus site upon activation by growth factors. Site-directed mutagenesis identified Ser351, Ser745, and Ser747 as Akt phosphorylation sites on VCP. Confocal microscopy indicated a co-localisation between Akt and VCP upon Akt stimulation. Interestingly, siRNA against VCP induced an inhibition of the growth factor-induced activation of NF-kB and a potent pro-apoptotic effect. Together, these data identify VCP as an essential target of Akt signaling.

The valosin-containing protein (VCP) belongs to the AAA (ATPases associated with various cellular activities) family, whose members are characterized by having highly conserved ATPase domain(s) with high sequence similarities and ring structures consisting of homo-oligomers (1,2). VCP, also known as VAT in archaebacteria, CDC48p in yeast, TER94 in Drosophila and p97 in Xenopus, is one of the most evolutionarily conserved proteins that is ubiquitous and abundant in cells, accounting for

more than 1% of total cellular proteins. Like other AAA proteins, VCP has been shown to be involved in a wide variety of ATP-dependent cellular processes such as ubiquitin-mediated proteolysis, DNA repair, membrane fusion and dynamic of subcellular compartments, gene expression and cell growth. Admittedly, it acts as a molecular chaperone that unfolds or unwinds proteins, although the detailed mechanisms of VCP function remain to be determined, as well as its regulation mechanisms in both physiological and pathological conditions.

During the course of studying the molecular partners of the serine/threonine kinase Akt, a key regulator of cell survival that is activated by growth factors, we detected VCP in Akt co-immunoprecipitated material. The interaction between Akt and VCP was further studied and from the functional point of view, we showed that disruption of VCP using siRNA impaired the cell survival signaling mediated by Akt. We propose that VCP is an important player in the Akt-mediated signaling of cell survival.

## EXPERIMENTAL PROCEDURES

*Materials*- Cell culture reagents were purchased from Biowhittaker (France). Recombinant human fibroblast growth factor-2 (FGF-2) was from R&D systems (France). Wortmannin (PI3K inhibitor) was from Calbiochem (France). C2 ceramide analogue (N-acetyl-D-sphingosine), 5-Fluorouracil, camptothecin, etoposide, Hoechst 33258, G250 brilliant blue, DMSO and peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G antibody were from Sigma (France). Dynabeads M-280 tosylactivated were from Dynal Biotech (France). Constitutively
active Akt and Wild-type Akt pUSE encoding
plasmids were from Upstate (NY).

*Cell culture, NF-κB activation, and induction of apoptosis*- Breast cancer cell lines MCF-7, T-47D, BT-20 were obtained from the American Type Culture Collection and routinely grown as monolayers as previously described (3). Apoptosis of MCF-7 cells was induced by treatment with ceramide analogue C2 (10 µM for 24 h), previously described as a pro-apoptotic agent for human breast cancer cells (4). The determination of both the NF-κB activation (luciferase reporter assay) and the proportion of cells in apoptosis (Hoechst staining) were performed as described (3). For Apoptosis induction by chemotherapeutic agents, MCF-7 breast cancer cells were treated 3h with 5-Fluorouracil (100 µM), camptothecin (0,1 nM) or etoposide (10 µM) and replaced in new culture medium. After 24 h, cells were fixed with cold methanol (-20°C) for 10 min and washed twice with phosphate-buffered saline (PBS) before staining with 1µg/mL Hoechst 33258 and apoptosis quantification.

*Immunoprecipitation of Akt and interacting proteins*- MCF-7 cells were rinsed by phosphate-buffered saline (PBS) pH 7.5, serum-starved for 3h in minimum essential medium (MEM-Earle's salts) and treated for 24 h by 10 µM C2 ceramide in fresh minimal essential medium. Proteins were extracted from cells after 10 min of 5 ng/mL FGF-2 stimulation in 100 µM pervanadate. To inhibit PI3K/Akt, cells were pretreated for 3 h by 100 nM wortmannin before FGF-2 stimulation. Then, cells were rinsed with PBS pH 7.5 and scratched in lysis buffer (150 mM NaCl, 50 mM Tris pH 7.5, 0.1% SDS, 1% Nonidet P-40, 100 µM sodium orthovanadate). After centrifugation (10, 000 g, 5 min), proteins were quantified using Bradford's method (Bio-Rad). Akt and interacting proteins were co-immunoprecipitated from equal quantities of proteins using antibodies covalently bound to magnetic beads, according to Dynal biotech protocole using 2.5 µg of anti-Akt for 500 µg of total proteins. Immunoprecipitated material was rinsed three times by PBS pH 7.5 before a 5 min boiling in Laemmli buffer 1X.

*Gradient gel electrophoresis*- Akt
immunoprecipitated material was solubilized in
SDS solution (0.3% SDS and 1% β-

mercaptoethanol) before SDS-PAGE migration
and colloidal blue staining (G250 brilliant blue).

*Protein identification by mass spectrometry*- Protein identification was performed by Matrix Assisted Laser Desorption Ionisation-Time Of Flight (MALDI-TOF) and tandem mass spectrometry (MS-MS). The protein band of interest was trypsin digested as previously described (5). MALDI-TOF and MS-MS analysis of the trypsin digests were performed on a Voyager reflector instrument (Applied biosystems) and a Q-STAR (Perseptive biosystems) in positive ion mode. For MALDI-TOF, mass measurements were made after peak smoothing and internal calibration using the average mass of the two autolysis trypsin fragment ions at m/z 842.510 and 2211.105. Protein sequence database searching was performed with MS-Fit (http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm) and Mascot (http://www.matrixscience.com/).

*Western blotting*- Co-immunoprecipitated proteins were separated by 10% SDS-PAGE and Western blotted with anti-VCP (1/2000), anti-Akt and anti-phospho-(Ser/Thr) Akt substrate antibodies in experimental conditions described in (3).

*Confocal microscopy*- After Akt activation using FGF-2, cells were successively rinsed with PBS pH 7.5, fixed 20 min with paraformaldehyde 4%, permeabilized for 20 min at room temperature with PBS pH 7.5; 0.05% saponine; 50 mM ammonium chloride and blocked for 30 min with PBS pH 7.5; 0.05% saponine; 2% bovine serumalbumine. Then, cells were incubated for 2h at room temperature in a blocking solution containing 1/50 rabbit phospho-Akt antibody (cell Signaling, UK) and 1/50 mouse VCP antibody (Abcam, UK), rinsed with PBS pH 7.5, incubated for 1h at 37°C in a blocking solution containing 1/4000 alexa fluor 546 goat anti-rabbit IgG and 1/3000 alexa fluor 488 goat anti-mouse IgG. Cells were rinsed with PBS and mounted under mowiol.

*VCP RNA interference*- VCP siRNA was designed and validated as described by Wójcik *et al.* (6). Corresponding sequences are Forward 5'-TAAGACGCTGCTGGCCAA-3' and Reverse 5'-CATCGCGGAGTTCTGATTTT-3'. The siRNAs were synthesized using the Silencer® siRNA construction kit (Ambion, Austin, TX) according to manufacturer's instructions and
transfection was performed as previously
described (7).

Site-directed mutagenesis of VCP- CDNA of VCP was obtained by PCR on MCF-7 cells cDNA bank using following primers that incorporatd Knp1 site and Not1 site at respectively 5' and 3' ends of the cDNA of VCP: « Vcp-dir+KpnI » 5'-ATCGGTACCGCTCGTAGCCGTTACC-3', « Vcp-rev+Not1 » 5'-ATGCGGCCGCTGAACTGTTCAGACTGGA G-3'. PCR product was inserted and in topoA vector and amplified in E. coli bacteria. Then double digestion by Kpn1 and Not1 was used to insert VCP cDNA in CMVTNT vector that allowed expression in mammalian cells. VCP serine 352 mutation to alanine S351A, S745A, S747A, and a triple mutant were carried out using PCR with mutant primers. The primers used to generate the mutants were as follows: S351A:5'GACCCAACGCCATTGACCC3', S745A:5'GCCCCGACGTGCTGTCAGCG-3', S747A:5'CGACGTTCTGTCGACGATAATGA C-3', S745AS747A:5'CGACGTGCTGTCGC CGATAATG-3'. Cloning and mutation were confirmed by DNA sequencing. Expression of pCMVTNT-VCP, pCMVTNT-VCPS351A, pCMVTNT-VCPS745A, pCMVTNT-VCPS747A, and pCMVTNT-VCPS745AS747A plasmids was carried out by transfection in MCF-7 cells.

RESULTS

VCP is co-immunoprecipitated with activated Akt. The protocole used for identification of VCP in Akt co-immunoprecipitates is summarized in Fig. 1A. Immunoprecipitates obtained with Akt antibodies were resolved on SDS-polyacrylamide gel. Two experimental conditions were compared. In the first, Akt was not activated and in the second, Akt was activated by cell stimulation with FGF-2. Silver staining or Coomassie blue staining of the gel revealed the appearance of a 97 kDa band in the Akt stimulated condition (Fig. 1B). The equiloading was assessed by detection of Akt (60kDa) and IgG (70 kDa). The 97 kDa band was cut out and trypsin digested before mass spectrometry analysis. Mass spectra from MALDI-TOF (Fig. 1C) and MS-MS (Fig. 1D) allowed the establishment of a peptide mass fingerprint and aminoacid sequence respectively, thereby leading to the identification of VCP.

From the peptide fingerprint, a total of 31
experimental masses matched theoretical masses
from VCP, leading to a protein sequence
covering of 23% and this identification was
reinforced by the sequencing of 12 consecutive
amino acids (Table 1).

**The interaction between Akt and VCP is dependent on Akt activation and leads to VCP phosphorylation.** The interaction between Akt and VCP was further confirmed by confocal microscopy showing a cellular co-localization of VCP and activated Akt (pAkt) upon growth factor stimulation (Fig. 2A). Importantly, reverse co-immunoprecipitation, with anti-VCP antibody, followed by anti-Akt Western-blotting led to the same result (Fig. 2B), hence confirming the interaction between Akt and VCP upon fibroblast growth factor-2 stimulation. The interaction was further confirmed in different breast cancer cell types and under stimulation by insulin-like growth factor 1 and epidermal growth factor (Fig. 2C). Moreover, the interaction between VCP and Akt was abolished by treatment of the cells with the PI3K/Akt pharmacological inhibitor wortmannin (Fig. 2B), thus indicating the requirement of Akt activation for interaction with VCP. Using anti-phospho-Akt substrate (PAS) antibody, that recognizes phosphorylated serine or threonine in a consensus site for Akt, we have been able to show that Akt activation resulted in VCP phosphorylation (Fig. 2B). This Western-blotting experiment showed that VCP contained a serine or a threonine phosphorylated, upon growth factor stimulation, on a site corresponding to Akt phosphorylation consensus. Interestingly, treatment by wortmannin resulted in a decrease in VCP phosphorylation, confirming that VCP is a target of Akt signaling.

**Identification of Akt phosphorylation sites on VCP.** In our study, we demonstrated that FGF-2 stimulates both the interaction between Akt and VCP and also the phosphorylation of VCP on a site recognized by PAS antibody. The Akt-phosphorylated consensus site is commonly defined as R\*X\*XX(S/T) (8,9). We performed bioinformatics search to identify putative Akt phosphorylation site on VCP using the Scansite software (http://scansite.mit.edu/motifscanner). The putative Akt phosphorylation sites on VCP are AATN<ins>RPN</ins>S<ins>351</ins>, AMRFARRS<ins>745</ins> and RFARRS<ins>RV</ins>S<ins>747</ins>. To test the involvement of S351, S745, and S747 we performed site-
directed mutagenesis and we generated pCMVTNT-VCP<sup>S351A</sup>, pCMVTNT-VCP<sup>S745A</sup>, and pCMVTNT-VCP<sup>S747A</sup> and pCMVTNT-VCP<sup>S351AS745AS747A</sup>. These VCP wild type and mutant cDNAs were transfected in MCF-7 cells in parallel with Akt constitutively activated (AktCA) and after immunoprecipitation, phosphorylation on Akt consensus site was detected with PAS antibody. The results (Fig. 2D) show that wild type VCP was phosphorylated on PAS consensus to a greater extent as compared to S351A, S745A and S748A mutants. The triple mutant S351A-S745A-S747A was phosphorylated to an even lesser extent as no phosphorylation was detected. These results indicate that S351, S745 and S747 are Akt phosphorylation sites on VCP.

**VCP is functionally involved in Akt antiapoptotic signaling and is required for cell survival.** The functional involvement of VCP in Akt signaling was shown by the treatment of MCF-7 cells with siRNA directed against Akt or VCP. Treatment by 100 nM siRNA directed against Akt or VCP resulted in a strong reduction of the pro-survival effect of FGF-2 for breast cancer cells (Fig. 3A). Similarly, transfection with the triple VCP mutant S351A-S745A-S747A resulted in a reduction of FGF-2 anti-apoptotic activity confirming the functional involvement of the detected phosphorylation site. Negative dominant Akt also resulted in the inhibition of FGF-2 survival effect, in contrast with constitutively activated Akt. It has been previously demonstrated that the Akt-mediated anti-antiapoptotic signaling in breast cancer cells requires the activation of the nuclear factor kappaB (NF-κB) (7). We have evaluated the impact of VCP on NF-κB activation induced by Akt. Treatment with 100 nM siRNA directed against Akt or VCP, as well as transfection with dominant negative Akt or VCP mutant S351A-S745A-S747A resulted in a strong inhibition of NF-κB activation by FGF-2 (Fig. 3B). The efficiency of cell treatment by siRNA directed against Akt and VCP was demonstrated by Western-blotting showing the disappearance of Akt and VCP with increasing concentrations of siRNA (Fig. 3C). Together, these data show that VCP is involved in the growth factor-stimulated antiapoptotic signaling mediated by Akt.

Interestingly, high concentrations of siRNA against VCP were shown to induce a potent apoptosis in breast cancer cells that was not rescued by overexpression of a dominant-active

Akt mutant (Fig. 4A). Cell treatment with increasing concentration of siRNA against VCP resulted in increase of breast cancer cell death, and it is worth noting that for identical concentrations, no apoptosis was induced by the control siRNA. For example, 50% of apoptosis was observed for 150 nM siRNA against VCP after a 48 h treatment, whereas no induction of cell death was detected with control siRNA. In conclusion, VCP depletion dramatically induced, by itself, a massive apoptosis in breast cancer cells, demonstrating the crucial role played by this protein in cell survival. Interestingly, siRNA directed against VCP were found to induce a decrease in the level of phospho-IκBα (Fig. 4B). This is consistent with the decrease of NF-κB activation that was also observed when cells were treated with siRNA against VCP (Fig. 3B). Moreover, we have shown that depletion in VCP resulted in a potentiation of the proapoptotic effect of various chemotherapeutic drugs (Fig. 4C). The proapototic effect of 5FU, camptothecine and etoposide obtained with MCF-7 cells was potentiated by siRNA directed against VCP (at the 100nM dose that has no apoptotic effect by itself). This demonstrates the significance of VCP under conditions of apoptosis induced by anti-cancer treatment drugs.

## DISCUSSION

The serine/threonine kinase Akt is increasingly appearing as a general mediator of cell survival signaling, with high potentialities as a drug target in various diseases characterized by a deregulation of the balance between cell apoptosis and cell growth, such as cancer (10). Akt is mainly, but not exclusively, activated through the phosphatidylinositol 3-kinase (PI3-K) pathway that is stimulated by a wide range of tyrosine kinase-growth factor receptors. Although Akt signaling remains incompletely known, it noticeably promotes cell survival by indirectly activating the pro-survival transcription factor NF-κB, through the stimulation of the inhibitor I-kappaB kinase (IKK). Akt activation of IKK results in the phosphorylation and subsequent degradation of IκB through the ubiquitin-mediated proteasome proteolysis, allowing NF-κB to translocate into the nucleus where it stimulates target gene expression (11). Due to its involvement in several pathologies such as cancer, the signaling initiated by Akt is the subject of increasing
attention and we indicate here the involvement
of VCP in the growth factor-induced
antiaoptotic signaling mediated by the Akt/NF-
b pathway. In contrast to classical approaches
to studying signal transduction (based on the use
of specific antibodies to identify signaling
partners), proteomic approaches have the
advantage of allowing the identification of
unexpected partners and this is of considerable
potential for defining new therapeutic targets in
pathologies such as breast cancer (12).
Interestingly, VCP has previously been shown to
associate directly with the alpha form of IκB
(IκBα) and is co-purified with the mammalian
26S proteasome involved in the ubiquitin-
dependent proteasome degradation pathway of
IκBα (13,14). In breast cancer cells, growth
factors such as fibroblast growth factor-2 (FGF-
2) and nerve growth factor (NGF) have been
shown to stimulate cell growth and survival
through the activation of a PI3-K/Akt/NFκB
mediated pathway (7,15,16). In this context, our
results indicate that the NF-κB activation and the
subsequent effects on cell survival are
functionally regulated through the recruitment
and phosphorylation of VCP by the Akt
pathway.

The Akt-phosphorylated consensus site is
commonly defined as R*X/RXX(S/T) (8,9). In fact,
the primary sequence of VCP does not contain
the precise consensus sequence predicted for an
Akt substrate: the arginine at position -5 is
lacking. However, VCP encodes a peptide
sequence that is consistent with a pattern of other
Akt-phosphorylated targets, where Arginine at
position -5 is not present as in insulin-response
element-binding protein 1 (KERCQ*S*1036) (17),
CREB LSR*RPS133Y* (18) and ATP-citrate lyase
(TPAPS*RTA*S*F) (19). The bioinformatic
analysis performed with scansite indicated three
potential Akt phosphorylation site in VCP and
we have used site-directed mutagenesis to
confirm their involvement. We demonstrated that
S351, S745 and S747 on VCP are
phosphorylated on a site recognized by PAS
antibody corresponding to Akt consensus site.
The phosphospecific Akt substrate antibody
(PAS) that we have used here was raised against
Akt R*X/RXX(pS/pT) consensus sequence, but
does not appear to strictly require arginine in the
-5 position for phosphoprotein recognition, as
was obtained for ATP-citrate lyase (19).
Interestingly it has very recently been shown, in
rat hippocampal tissue, a functional interation

between Akt and VCP that also leads to VCP
phosphorylation on the same corresponding
serine residues (20). Therefore, these three serine
residues appear to be generally crucial for VCP
regulation by Akt.

VCP is known to interact with other adaptor
proteins, which are proposed as functionning in
various cellular pathways. The list of proteins
known to interact with VCP includes clathrin
(21), the p47 membrane fusion required for
organelle biogenesis (22), the VCP p97/p47
complex- interacting protein (VCPIP135) (23),
the small VCP-interacting protein (SVIP) (24)
and the breast/ovarian cancer susceptibility gene
product (BRCA1) (25). BRCA1 is of particular
interest in the context of breast cancer, as this
protein is one of the most prominent players
identified in breast tumorigenesis, with 50% of
familial breast tumors exhibiting BRCA1
mutations (26). BRCA1 presumably acts as a
DNA unwinding factor in DNA damage repair,
although VCP is predominantly found in the
cytoplasm and less abundantly in the nucleus.
However, VCP can be translocated to the
nucleus and *in vitro* studies have revealed that
VCP, via its N- terminal region, binds to amino
acid residues 303-625 in the BRCA1 protein
(25). Interestingly, the cell growth regulator
heregulin has been shown to induce
phosphorylation of BRCA1 through PI3-
Kinase/Akt in breast cancer cells (27), and
therefore our data suggest that VCP might
participate in the interaction between Akt and
BRCA1, leading to DNA damage-repair function
and an antiapoptotic effect under growth factor
stimulation. However, our study also suggests
that other known biological activities of VCP,
such as those related to intracellular trafficking,
ubiquitin mediated proteolysis and activation of
transcription (28) might be regulated by Akt
through the activation of VCP. In our study, a
potent induction of apoptosis was observed in
breast cancer cells treated with siRNA against
VCP, thereby demonstrating the crucial role
played by this protein in the survival of cancer
cells. VCP has been described as an important
regulator of protein ubiquitination and
interestingly it was reported before that VCP is
involved in the ubiquitin-proteasome-mediated
degradation of IκB (13). In our study, we
demonstrated that siRNA directed against VCP
induced a decrease in the level of phospho-IκB.
As the phosphorylation of IκB is the germane
event leading to the proteolytic proteasome-
mediated degradation of IκB responsible for
the release and nuclear translocation of NF-κB,
the decrease in phospho-IκB that we observe
with siRNA directed against VCP is consistent
with the concomitant inhibition of NF-κB and
provides a biochemical basis for the cell
survival activity of VCP. In addition, the
potential of VCP as a therapeutic target is
particularly well illustrated by the potentiation
of the proapototic effect of chemotherapeutic
drugs that we have obtained with anti-VCP

siRNA. Hence, VCP targeting appears as a
rational approach to sensitize breast cancer
cells to chemotherapy.

In conclusion, we identified VCP as an
essential new target in the Akt signaling
pathway that is necessary to its related
antiapoptotic effect and therefore, this protein
appears as a new player to be considered in
cancer cell survival, with a correlative
potential as a target for cancer therapy.

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## FOOTNOTES

This work is supported by grants from the Ligue Nationale Contre le Cancer, the Association pour la Recherche sur le Cancer (Contract 3305), the Fondation pour la Recherche Médicale (FRM comité du Nord), the Institut Universitaire de France, the Region Nord-pas-de-Calais and the Genopole of Lille. FV was a recipient of a fellowship from the French Ministry for Research and Education and the Ligue National Contre le Cancer.

## FIGURE LEGENDS

**Figure 1.** VCP is co-immunoprecipitated with activated Akt. A, Akt co-immunoprecipitated material was resolved on SDS-polyacrylamide gel. Two experimental conditions were compared: Akt activated by cell stimulation with FGF-2 for 15 min versus the control in which Akt was not activated. The 97 kDa protein band was analyzed by mass spectrometry (MALDI-TOF and MS/MS) after trypsin digestion. B, Silver staining of the SDS-PAGE gel revealed the appearance of a 97 kDa band in the Akt stimulated condition. The levels of both immunoprecipitated-Akt and IgG were identical under the two conditions. C, Peptide mass fingerprint of the trypsin-digested 97 kDa band obtained by MALDI-TOF analysis. D, ESI-MS-MS spectrum, corresponding to the 1328 Da peptide, allowing the determination of a 12 amino-acid sequence corresponding to VCP.

**Figure 2.** Interaction between Akt and VCP is dependent on Akt activation. A, Confocal microscopy revealed the co-localization of VCP and Akt upon FGF-2 induced stimulation of Akt. Activated Akt appears in red and VCP in green. B, Reverse co-immunoprecipitation with anti-VCP was separated by SDS-PAGE before Western-blotting using anti-Akt. A band of 60 kDa, corresponding to Akt was found in Akt activated conditions, confirming the interaction between Akt and VCP. The PI3K/Akt pharmacological inhibitor wortmannin inhibited the interaction between Akt and VCP. In addition, immunoprecipitated VCP was resolved by SDS-PAGE before Western-blotting using PAS (phospho-Akt substrate) antibody. The 97 kDa band corresponding to VCP was found in Akt-activated conditions, confirming that interaction between Akt and VCP correlated with phosphorylation of VCP on an Akt consensus site. The PI3K/Akt pharmacological inhibitor wortmannin inhibited this phosphorylation. C, The same co-immunoprecipitation experiment was reproduced with stimulation of Akt by three different growth factors: fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF). D, MCF-7 cells were transfected with pCMVTNT plasmids encoding VCP wild type, VCP S351A, VCP S745A, or VCP S351AS745AS747A and cotransfected with empty vector or vector encoding constitutively active Akt (AktCA). Immunoprecipitated VCP was then resolved by SDS-PAGE before Western-blotting using PAS antibody.

**Figure 3.** VCP is functionally involved in Akt mediated antiapoptotic signaling. A, Quantification of apoptosis with Hoechst staining revealed that siRNA against VCP or Akt (100 nM) inhibited the antiapoptotic effect of FGF-2. B, Transfection of MCF-7 cells with siRNA directed against Akt or VCP or transfection with plasmid encoding a negative dominant Akt, resulted in a strong reduction of NF-κB activation by FGF-2. In contrast, transfection with plasmid encoding a constitutively active form of Akt, mimicked the stimulation of NF-κB induced by FGF-2. C, Western-blotting detection of Akt and VCP levels in cells treated by increasing concentrations of siRNA. The efficiency of siRNA was tested 48 h after cell transfection.

**Figure 4.** VCP is required for breast cancer cells survival. A, Dose-effect of siRNA against VCP on breast cancer cell apoptosis. MCF-7 cells were transfected with increasing amounts (0, 50, 100,
150, 200 nM) of siRNA directed against VCP (or control siRNA) and cotransfected with plasmid encoding Akt constitutively active (AktCA). After Hoescht staining, apoptotic cells were counted and results are expressed as the mean +/- SD of five independent experiments. B, Western blotting detection of phospho-IκBα in MCF-7 cells treated with increasing concentrations (same as before) siRNA directed against VCP. C, MCF-7 cells were transfected with 100 nM siRNA against VCP or with nontargeting siRNA and were treated with C2, 5-Fluorouracil (5FU), camptothecin (campto.) or etoposide (eto.) before serum deprivation. Percentage of apoptotic cells was determined after Hoescht staining.
<table><caption>**Table 1.** Identification of VCP, from MALDI-TOF obtained peptide masses. The amino acid sequence from the Swiss-Prot database of each matching peptide, the delta mass between measured and matched masses and the number of cleavages missed by trypsin are reported. The identification of VCP was comforted by the full MS/MS sequencing of the underlined peptide.</caption><thead><tr><th>MH+ submitted</th><th>MH+ matched</th><th>delta mass (Da)</th><th>Miss peptide sequence consistent with mass</th></tr></thead><tbody><tr><td>783.42</td><td>783.36</td><td>0.06</td><td>0 506FGMTPSK512 Oxidation (M)</td></tr><tr><td>797.40</td><td>797.40</td><td>0.00</td><td>1 360FGRFDR365</td></tr><tr><td>819.47</td><td>819.46</td><td>0.01</td><td>0 149GDIFLVR155</td></tr><tr><td>837.52</td><td>837.52</td><td>-0.01</td><td>0 232AIGVKPPR239</td></tr><tr><td>947.58</td><td>947.56</td><td>0.02</td><td>1 148KGDIFLVR155</td></tr><tr><td>951.50</td><td>951.47</td><td>0.03</td><td>1 288KAFEEAEK295</td></tr><tr><td>986.54</td><td>986.53</td><td>0.02</td><td>0 218EMVELPLR225</td></tr><tr><td>1033.54</td><td>1033.52</td><td>0.02</td><td>1 746SVSDNDIRK754</td></tr><tr><td>1034.46</td><td>1034.43</td><td>0.03</td><td>0 734DHFEEAMR741</td></tr><tr><td>1049.55</td><td>1049.54</td><td>0.01</td><td>0 669DVDLEFLAK677</td></tr><tr><td>1050.48</td><td>1050.42</td><td>0.06</td><td>0 734DHFEEAMR741 Oxidation (M)</td></tr><tr><td>1051.53</td><td>1051.52</td><td>0.01</td><td>0 46MDELQLFR53</td></tr><tr><td>1067.53</td><td>1067.51</td><td>0.01</td><td>0 46MDELQLFR53 Oxidation (M)</td></tr><tr><td>1075.55</td><td>1075.53</td><td>0.02</td><td>0 278LAGESESNLR287</td></tr><tr><td>1118.60</td><td>1118.57</td><td>0.02</td><td>1 701ESIESEIRR709</td></tr><tr><td>1172.68</td><td>1172.66</td><td>0.02</td><td>0 240GILLYGPPGTGK251</td></tr><tr><td>1190.58</td><td>1190.53</td><td>0.05</td><td>1 733RDHFEEAMR741</td></tr><tr><td>1203.65</td><td>1203.63</td><td>0.02</td><td>1 278LAGESESNLRK288</td></tr><tr><td>1206.59</td><td>1206.52</td><td>0.06</td><td>1 733RDHFEEAMR741 Oxidation (M)</td></tr><tr><td>1329.71</td><td>1329.68</td><td>0.03</td><td>0 454WALSQSNPSALR465</td></tr><tr><td>1629.84</td><td>1629.80</td><td>0.04</td><td>1 754KYEMFAQLQQSR766</td></tr><tr><td>1645.82</td><td>1645.79</td><td>0.03</td><td>1 754KYEMFAQLQQSR766 Oxidation (M)</td></tr><tr><td>1660.83</td><td>1660.82</td><td>0.01</td><td>1 363FDREVDIGIPDATGR377</td></tr><tr><td>1667.85</td><td>1667.94</td><td>-0.09</td><td>1 212QLAQIKEMVELPLR225</td></tr><tr><td>1695.87</td><td>1695.82</td><td>0.04</td><td>0 600VINQILTEMDGMSTK614 Oxidation (M)</td></tr><tr><td>1711.87</td><td>1711.82</td><td>0.05</td><td>0 600VINQILTEMDGMSTK614 2 Oxidation (M)</td></tr><tr><td>1823.95</td><td>1823.92</td><td>0.03</td><td>1 600VINQILTEMDGMSTKK615 Oxidation (M)</td></tr><tr><td>1839.93</td><td>1839.91</td><td>0.02</td><td>1 600VINQILTEMDGMSTKK615 2 Oxidation (M)</td></tr><tr><td>2156.00</td><td>2155.97</td><td>0.03</td><td>0 714QTNPSAMEVEEDDPVPEIR732</td></tr><tr><td>2312.10</td><td>2312.07</td><td>0.02</td><td>1 714QTNPSAMEVEEDDPVPEIRR733</td></tr><tr><td>2328.08</td><td>2328.07</td><td>0.01</td><td>1 714QTNPSAMEVEEDDPVPEIRR733 Oxidation (M)</td></tr></tbody></table>
A.

MCF-7 cells
+/- FGF-2
▼
Akt co-immunoprecipitation
▼
SDS-PAGE
▼
Trypsin digestion of
the 97 kDa regulated band
▼
Mass spectrometry
(MALDI-TOF and ESI-MS/MS)
and database searching

B.

<figure><img src="image_2.png" /><figcaption>Figure 1AB</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Figure 1C</figcaption></figure>

Vandermoere *et al.*
<figure><img src="image_4.png" /><figcaption>Figure 1D</figcaption></figure>

Vandermoere *et al.*
<figure><img src="image_5.png" /><figcaption>Figure 2A</figcaption></figure>

Vandermoere *et al.*
B.

<figure><img src="image_6.png" /></figure>

C.

<figure><img src="image_7.png" /></figure>

D.

<figure><img src="image_8.png" /><figcaption>Figure 2BCD</figcaption></figure>

Vandermoere *et al.*
<figure><img src="image_9.png" /><figcaption>B.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Figure 3AB</figcaption></figure>
C.

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>**Figure 3C**</figcaption></figure>
A.

<figure><img src="image_13.png" /></figure>

B.

<figure><img src="image_14.png" /><figcaption>Figure 4AB</figcaption></figure>

Vandermoere *et al.*
C.

<figure><img src="image_15.png" /><figcaption>Figure 4C</figcaption></figure>

Vandermoere *et al.*