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Interaction of oncogenic papillomavirus E6 proteins with fibulin-1
BBRC Biochemical and Biophysical Research Communications 296 (2002) 962–969 www.academicpress.com
Interaction of oncogenic papillomavirus E6 proteins with fibulin-1 Minjie Du,1 Xueli Fan, Eva Hong, and Jason J. Chen* Department of Medicine, University of Massachusetts Medical School, LRB Room 323, 364 Plantation Street, Worcester, MA 01605-2324, USA Received 24 July 2002
Abstract Human papillomavirus (HPV) infection is the primary risk factor for the development of cervical cancer. The papillomavirus E6 gene is essential for virus-induced cellular transformation and the viral life cycle. Important insight into the mechanism of E6 function came from the discovery that cancer-related HPV E6 proteins promote the degradation of the tumor suppressor p53. However, mounting evidence indicates that interaction with p53 does not mediate all E6 activities. To explore the p53-independent functions of E6, we performed a yeast two-hybrid screen and identified fibulin-1 as an E6 binding protein. Fibulin-1 is a calciumbinding plasma and extracellular matrix protein that has been implicated in cellular transformation and tumor invasion. The interaction between E6 and fibulin-1 was demonstrated by both in vitro and in vivo assays. Fibulin-1 is associated specifically with cancer-related HPV E6s and the transforming bovine papillomavirus type 1 E6. Significantly, overexpression of fibulin-1 specifically inhibited E6-mediated transformation. These results suggest that fibulin-1 plays an important role in the biological activities of E6. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: HPV; Cancer; p53; Fibulin-1; Papillomavirus; E6; Invasion; Transformation
Papillomaviruses are small DNA viruses that infect various epithelial tissues, including the epidermis and the epithelial linings of the anogenital tract. A majority of the squamous cell abnormalities of the cervix that precede squamous cell carcinoma contain human papillomavirus (HPV) DNA [1]. HPVs that infect the anogenital tract can be classified as high risk and low risk on the basis of their clinical associations. The high-risk HPVs, such as types 16, 18, and 31, are strongly associated with the development of cervical carcinoma. More than 90% of cervical cancers contain HPV DNA. In contrast, infection with low risk HPVs rarely results in cancer (for review, see [2]). Among animal papillomaviruses, the bovine papillomavirus type 1 (BPV-1) had served as the prototype for studies of the molecular biology of the papillomaviruses before recent interest switched to the HPV studies. BPV-1 has the ability to
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Corresponding author. Fax: 1-508-856-6588. E-mail address: [email protected] (J.J. Chen). 1 Present address: Department of Molecular and Medical Genetics, University of Toronto, Toronto, Canada M5S 1A8.
replicate its genome and to induce cellular transformation in established murine cell lines. The transforming properties of high-risk HPVs primarily reside in two genes, E6 and E7. Antisense studies suggest that the continued expression of E6 and E7 is essential to maintain the transformed state of HPVpositive cells ([3] and references therein). The oncogenic activities of E6 have been reflected in many biological assays. These include immortalization of primary cells, transformation of established fibroblasts, resistance to terminal differentiation of human keratinocytes, modulation of apoptosis, tumorigenesis in animals, and abrogation of cell cycle check points (reviewed in [4]). A recent study has shown that E6 plays an essential role in HPV life cycle [5]. Human epithelial cells expressing HPV-16 E6 have been shown to induce invasiveness in an in vitro assay [6]. The papillomavirus E6 proteins are composed of approximately 150 amino acids. E6 proteins from different HPV types or among the animal and human papillomaviruses show moderate amino acid homology. A common feature of all E6 proteins is the presence of putative Cys–X–X–Cys motifs that are capable of binding zinc [7–9]. The E6 proteins are present at very
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low levels in cells [10]. E6 proteins have been detected in nuclear, non-nuclear membrane, and cytosolic fractions (reviewed in [4]). The ability of high-risk HPV E6 and E7 proteins to associate with the cellular tumor suppressors p53 and pRB, respectively, has been suggested as a mechanism by which the viral proteins induce tumors [11–14]. Association of E6 with p53 is mediated by the ubiquitin ligase E6-AP and leads to the degradation of p53 by the ubiquitination pathway [15,16]. E6 also has functions independent of inactivating p53 and has been shown to interact with multiple additional cellular proteins (reviewed in [4]). We identified a cellular protein E6BP that specifically interacts with E6s from cancer-related highrisk HPVs and BPV-1 [17]. Other proteins that directly bind E6s include the focal adhesion proteins, paxillin and zyxin, the interferon regulatory factor-3 (IRF-3), the c subunit of trans-Golgi network-specific clathrin adaptor (AP-1) complex, the transcriptional coactivator CBP/p300, the tyrosine kinase Tyk2, and the serine/ threonine kinase PKN [18–26]. In addition, several cellular proteins have been identified as the degradation target of E6. These include the human homolog of the Drosophila disk large tumor suppressor protein (hDlg), the oncoprotein Myc, human minichromosome maintenance 7 protein (hMCM7), a putative GAP protein E6TP1, PDZ domain-containing proteins MUPP1, hScrib and MAGI-1, thepro- apoptoticproteinBak, and the activation modulating factor Gps2 (or AMF-1) [27–37]. Although steady progress has been made in the identification of E6 interacting proteins, the biological relevance of these proteins to E6 remains to be established. Cellular proteins involved in E6-mediated resistance to terminal differentiation, maintaining viral episomes during viral life cycle, and induction of invasion have not been identified. Here, we describe the identification and characterization of fibulin-1 as a putative E6 interacting protein that may play an important role in E6-mediated cellular transformation and invasion.
Materials and methods Plasmids and retroviruses. DNA encoding truncated human fibulin1D from amino acids 262–566 was cloned as an EcoRI–XhoI fragment into pGEX4T-3 to express GST-fibulin in Escherichia coli. BPV-1 E6 was cloned as a BamHI–SalI fragment into BamHI–XhoI sites of pGEX3X to encode GST-BPV E6. A 2.3 kb EcoRI fragment containing the entire coding sequence of human fibulin-1D from pPuro-fibulin-1D [38] was inserted into the EcoRI site of pSG5 (Stratagene) to make pSG5fibulin-1D. The FLAGtagged HPV-16 E6 in pSG5 (pSG5-HPV16 E6-FLAG) was obtained from Dr. Qingshen Gao and Dr. Vimla Band [33]. pSGFLAGBE6 was constructed by amplifying BPV-1 E6 with PCR and cloning into pSG5. The PCR sense primer contains a FLAG tag between the first and second amino acids of BPV-1 E6.
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Plasmids encoding E6s from BPV-1, HPV-6, 11, 16, 18, and 31 for in vitro synthesis were described in [17]. Plasmid encoding HPV-8 E6 in the Bluescribe M13þ vector (pBS; Vector Cloning System) was provided by Iftner and co-workers [39]. Plasmid encoding HPV-16 E6 mutant with C-terminal amino acid 124–151 deletion in pSP65 vector was described in [40]. Plasmid encoding HPV-16 E7 in pSP65 vector was provided by Dr. Karen Vousden. The retrovirus vector pBabe Puro is a Moloney murine leukemia virus-based vector containing a puromycin resistance gene [41]. Retroviruses containing BPV-1 E6, HPV-16 E6, and HPV-16 E7 have been described [42–44]. Retroviruses containing the Harvey murine sarcoma virus v-ras and BPV-1 E5 were provided by Dr. Douglas Lowy and Dr. Daniel DiMaio, respectively. Cell culture and transformation assays. C127 is a non-transformed clonal line derived from the mammary tissue of an RIII mouse [45]. To establish stable cell lines overexpressing fibulin-1, plasmid encoding fibulin-1D in pSG5 was transfected into C127 cells using FuGENE 6 transfection reagent (Boehringer Mannheim) and NIH 3T3 cells by calcium phosphate-mediated transfection, respectively. All transfections were performed by cotransfection of the vector ptTA-hygro (provided by Dr. Dick Mosser) that confers hygromycin resistance. After hygromycin selection, individual colonies were collected and examined for fibulin-1 expression by Western blot using the mouse monoclonal anti-fibulin-1 IgG 3A11 [38]. All subsequent experiments were performed using fibulin-1 overexpressing cells within five passages. For focus formation studies, C127-derived cells were infected with amphotrophic retroviruses. Two days after infection, cells were split 1:3. Upon confluence of cells, medium was switched from Dulbecco’s minimum essential medium (DMEM) containing 10% fetal calf serum (FCS) to 5% FCS. Cells were fed with fresh medium every 3 days. Three weeks after infection, cells were stained with GIEMSA stain solution (Ricca Chemical, Texas) and foci were counted. For the colony formation assay, NIH 3T3-derived cells were infected with retroviruses produced in PA317 cells. Two days after infection, cells were pooled, seeded into DMEM containing 0.4% Noble agar and 10% fetal calf serum at a density of 5 104 /60-mm dish, and overlaid to DMEM containing 0.6% Noble agar and 10% fetal calf serum. Cells were fed with fresh 0.4% agar medium weekly. Colony formation was scored in 3–4 weeks under a microscope. Colonies composed of more than 16 cells were scored as positive. Protein preparation and interaction experiments. GST fusion proteins were expressed in E. coli strain DH5a. After induction with IPTG, cells were harvested, re-suspended in 50 ml low salt association buffer (LSAB, 100 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1% NP-40, and 1 mM phenylmethylsulfonyl fluoride), and lysed by sonication. SDS and DTT were added to the lysate immediately after sonication for a final concentration of 0.03% and 1 mM, respectively. After centrifugation at 10,000g for 10 min, supernatant was collected and mixed with glutathione–Sepharose beads (Pharmacia). After incubation, the beads were collected by centrifugation at 1000g, washed three times with 20 volumes of LSAB, and stored at 4 °C. In vitro-translated E6 proteins and fibulin-1 were prepared by using the rabbit reticulocyte lysate translation system (Promega) and 35 S-labeled cysteine and methionine, respectively (ICN Biomedicals, Irvine, CA). For in vitro binding, 5–30 ll glutathione–Sepharose containing 2.5 lg GST fusion proteins was combined with 2–20 ll 35 S-labeled in vitro-translated proteins in lysis buffer (100 mM NaCl, 100 mM Tris– HCl (pH 8.0), 0.5% Nonidet P-40, 0.1% dried milk, 2 mM DTT, and 1 mM PMSF) in a total volume of 250 ll. The mixtures were subjected to rotary shaking for 2–3 h at 4 °C. The mixtures were washed extensively with lysis buffer (1% Nonidet P-40), boiled in SDS loading buffer, and electrophoresed on SDS–polyacrylamide gels. Gels were dried and scanned by Molecular Imager (BioRad). The in vitro degradation assay was described previously [43]. Briefly, full-length fibulin-1D was translated in vitro, radiolabeled, and mixed in reaction mixtures containing E6 and rabbit reticulocyte
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lysate. Samples were incubated at 30 °C for 3 h. The remaining fibulin1 was quantified by SDS–PAGE and BioRad Molecular Imager. For in vivo association, COS-7 cells were transfected by electroporation with plasmids encoding FLAG-tagged E6 and full-length fibulin-1D in pSG5. Forty-eight hours after transfection, cell extracts were prepared in lysis buffer (containing 0.25% NP-40) and fibulin-1 was precipitated with specific monoclonal antibody. After extensive washing, the precipitates were fractionated by SDS–PAGE and immunoblotted with anti-fibulin-1 and anti-FLAG antibodies, respectively. The signals were detected by using the SuperSignal Chemiluminescent reagent (Pierce, IL). Yeast two-hybrid screen. A modified yeast two-hybrid system from Dr. Stanley Hollenberg was used to screen a random-primed mouse embryo cDNA library [46]. This system provides a nutritional selection for histidine prototrophs, followed by a selection for b-galactosidase activity. The bait hybrid used in the screen is a BPV-1 E6 mutant (R42W) fused in-frame with LexA DNA binding protein (LexABE6R42W). Yeast strain L40 containing the bait plasmid was transformed with the library DNA. The transformants were first screened for the ability to grow in the absence of histidine. Histidine prototrophs were then tested for b-galactosidase activity.
Fig. 1. Schematic representation of fibulin-1 and truncated proteins used in this study. The three major structural elements found in fibulin1 are indicated as domains I, II, and III. Domain I contains three anaphylatoxin-like motifs. Domain II consists of nine EGF-like repeats, of which eight contain consensus sequence for calcium binding. Domain III is type specific sequence. The one shown in this figure is specific to fibulin-1D. The two-hybrid isolate ranges from EGF-like repeats 5 to 8. A truncated fibulin-1 containing EGF-like repeats 3–9 was expressed as a GST fusion for in vitro association assays.
Results Identification of fibulin-1 as an E6 interacting protein To identify cellular proteins that interact with E6, we used the yeast two-hybrid system. The bait hybrid was a BPV-1 E6 mutant (R42W) fused in-frame with LexA DNA binding protein. R42W is substantially reduced for transcriptional activation, yet competent for interaction with E6-AP in yeast [47]. BPV-1 E6 transforms cells but does not degrade p53. A modified version of the two-hybrid system provided by Dr. Stanley Hollenberg was used to screen a random-primed mouse embryo cDNA library [46]. A mouse library was chosen because BPV-1 E6 transforms the murine C127 cells. Approximately 1:6 106 transformants were screened and 140 histidine prototrophs were obtained. Of these histidine prototrophs, 76 were subsequently shown to be b-galactosidase positive. To test for specificity and reproducibility of the initial two-hybrid hits, a series of experiments were performed. First, the bait plasmid from yeast was removed. This was achieved by growing yeast in non-selective medium for the bait plasmid (on tryptophan containing plates), followed by transferring to low adenine plates. The bait plasmid contains the Ade2 gene and keeps the colonies formed by the L40 yeast white on low adenine plates. After the bait plasmid is lost, the yeast produces a red by-product that identifies these colonies. The resulting yeast was plated on X-gal plate to ascertain if the library DNA could produce b-galactosidase signal in the absence of a bait plasmid. Clones that scored positive were eliminated. Four cDNA clones encoding potential E6 interacting proteins were obtained. Sequence analysis of one clone revealed a 141-amino acid open reading frame encoding
a polypeptide with several EGF-like repeats (Fig. 1). This sequence represents a truncated version of fibulin-1 from amino acids 343–483. Fibulin-1 is a member of a growing family of cysteine-rich proteins with four variants named A, B, C, and D [48]. Based on its primary amino acid sequence, fibulin-1 can be divided into three parts. The N-terminal of fibulin-1 contains three cysteine-rich motifs similar to anaphylatoxins. The central core sequence of fibulin-1 contains nine EGF-like repeats [48]. The C-terminal region is unique to each variant. Fibulin-1 has been detected in the plasma, extracellular matrix (ECM), and blood [49]. Fibulin-1 associates with different ECM components such as fibronectin, laminin, and collagen IV [48,50,51]. The biological function of fibulin-1 remains to be established. Expression of fibulin-1 inhibits motility of human ovarian- and breast-cancer cells induced by fibronectin [52,53]. Elevated expression of fibulin-1 suppressed anchorage-independent growth, matrigel invasion, and tumor formation in human fibrosarcoma-derived cells [38]. These results suggest a role for fibulin-1 in tumor formation and invasion. Of particular interest to the papillomavirus-associated lesions, fibulin-1 has been detected at the dermal/epidermal border [54]. In vitro interaction of E6 and fibulin-1 The interaction of fibulin-1 and E6 was investigated using an in vitro binding experiment. cDNA encoding the central core sequence of human fibulin-1 was cloned into the pGEX plasmid (Fig. 1). Fibulin-1 was synthesized and purified from E. coli as a GST fusion and tested for association with 35 S-labeled, in vitro-translated BPV-1 E6 and HPV-16 E6 proteins. The HPV-16 E7 was included as a control. Our results demonstrated
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In reciprocal experiments, the truncated fibulin-1 from the original yeast two-hybrid screen was synthesized, 35 Slabeled by in vitro transcription, and translation and tested for association with GST-E6 fusion proteins. Our results indicated that fibulin-1 bound BPV-1 E6 and HPV-16 E6, but not HPV-6 E6 (data not shown). We also tested whether fibulin-1 can be degraded by E6 in vitro. Both fibulin-1 and E6 were prepared and 35 S-labeled by in vitro translation. They were incubated in a degradation assay in the presence of rabbit reticulocyte lysate. While the control p53 protein was efficiently degraded by HPV-16 E6, the level of fibulin-1 remained unchanged when incubated either with HPV16 E6 or BPV-1 E6 (data not shown). These results suggest that fibulin-1 is not a degradation target for E6, at least in vitro. In vivo interaction of fibulin-1 with E6
Fig. 2. In vitro interaction of E6 and fibulin-1. Glutathione–Sepharose beads containing GST-fibulin-1 fusion protein were combined with 35 S-labeled in vitro-translated E6 proteins in lysis buffer. The bound products were separated by SDS–PAGE and analyzed by Molecular Imager (BioRad). Input was directly loaded into the well and represents 10% of the [35 S]cysteine-labeled E6 proteins used in each binding reaction. (A) Fibulin-1 binds to BPV-1 E6 and HPV-16 E6. (B) Specific association of fibulin-1 with E6s from high-risk HPVs. The E6 proteins from different HPVs are indicated above each lane. HPV-16 E6M, a truncated form of E6 with C-terminal amino acid 124–151 deletion.
that fibulin-1 efficiently binds BPV-1 E6 and HPV-16 E6 (Fig. 2A). In contrast, fibulin-1 did not bind HPV-16 E7. We next investigated the ability of E6s from other HPV types to bind fibulin-1. GST-fibulin prepared from E. coli was tested for association with 35 S-labeled, in vitro-translated E6 proteins from multiple HPV types. HPV-16 E6 was included as a positive control. As shown in Fig. 2B, in addition to HPV-16 E6, fibulin-1 also bound efficiently to E6s from other high-risk HPVs such as types 18 and 31. In contrast, E6 from low-risk HPV-6 and HPV-11 or cutaneous HPV-8 did not bind fibulin-1. Thus, there is a correlation between biological risk for cervical cancer and the ability to bind fibulin-1. In addition, fibulin-1 did not bind the truncated form of E6 with C-terminal amino acid 124–151 deletion that was defective for immortalization of human mammary epithelial cells [40]. These results indicate that the association of fibulin-1 with oncogenic E6s is specific.
Since the level of neither fibulin-1 nor E6 is high in cells, it is not practical to detect their in vivo association with endogenous proteins. To assess the in vivo interaction of E6 and fibulin-1, a transient transfection assay was employed. Briefly, COS-7 cells were transfected by electroporation with plasmids encoding E6 and full-length human fibulin-1D in pSG5. To facilitate detection, E6 was expressed as epitope FLAG-tagged proteins. Epitope-tagged E6 was shown to be functionally equivalent to the wild-type E6 [55]. Cell extracts were prepared and fibulin-1 was precipitated with specific monoclonal antibody. The presence of E6 in fibulin1 precipitates was detected by Western blot. Our results demonstrated that both HPV-16 E6 and BPV-1 E6 could form a complex with fibulin-1 in vivo (Fig. 3).
Fig. 3. In vivo association of E6 and fibulin-1. COS-7 cells were transfected with plasmids encoding FLAG-tagged E6 (pSGFLAGBE6 and pSG5-HPV16 E6-FLAG) and full-length human fibulin-1D in pSG5 (pSG5fibulin-1D) by electroporation. Following transfection and incubation, cell extracts were prepared and fibulin-1 was precipitated with specific antibody. After extensive washing, the precipitates were fractionated by SDS–PAGE and immunoblotted with antiFLAG antibody. Upper panel, cell extract directly blotted by antifibulin-1 antibody. Lower panel, fibulin-1 precipitates blotted by antiFLAG antibody. The positions of fibulin-1 and E6 proteins are indicated by arrows.
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In the transient transfection experiment, the level of steady-state fibulin-1 was not affected by the expression of BPV-1 or HPV-16 E6. To explore this further, the endogenous fibulin-1 protein levels in several stable cell lines expressing E6 were examined by Western blot. Cells examined include C127 expressing BPV-1 E6, human keratinocytes containing HPV-16 E6, and HMEC immortalized by HPV-16 E6. Our results demonstrated that the level of fibulin-1 in these cells remains unchanged in the presence of E6 (data not shown). These data are consistent with our in vitro data and suggest that fibulin-1 is not a degradation target for E6. However, we cannot rule out the possibility that under some special physiological conditions, E6 may target fibulin-1 for degradation. Ectopic expression of fibulin-1 specifically inhibits E6 transformation To study the functional interaction of E6 and fibulin1, stable cell lines that overexpress fibulin-1 were established. Mouse C127 and NIH 3T3 cells were
transfected with full-length human fibulin-1D to make FB-C127 and FB-3T3 cells, respectively. Among seven independent clones of C127 derivatives tested, three were found to express high levels of fibulin-1. While fibulin-1 in the vector-transfected cells (pSG5–C127) was almost undetectable, strong bands are evident in the three clones (Fig. 4A). Similarly, three out of six NIH 3T3-derived clones were found to overexpress fibulin-1 (Fig. 4B). BPV-1 E6 induces the focus formation in C127 cells while HPV-16 E6 stimulates the anchorageindependent growth of NIH 3T3 cells in soft agar (reviewed in [4]). Both BPV-1 E5 and the cellular oncogene v-ras efficiently transform C127 cells but through seemingly different mechanisms than BPV-1 E6. BPV-1 E5 and HPV-16 E7 also induce anchorage-independent growth in NIH 3T3. FB-C127 and FB-3T3 cells were accordingly subjected to transformation by these oncogenes. As shown in Fig. 4, overexpression of fibulin-1 significantly reduced E6-induced transformation. In contrast, transformation by BPV-1 E5, HPV-16 E7, or oncogene v-ras was not affected. Moreover, the level of fibulin-1 correlated with its ability to inhibit E6-medi-
Fig. 4. Overexpression of fibulin-1 specifically inhibits E6 transformation. C127 and NIH 3T3 cells were transfected with full-length fibulin-1D gene under the control of SV40 promoter. The cells were plated in hygromycin B containing media. Multiple colonies were picked, expanded, and tested for the expression of fibulin-1 by Western blot and transformation by oncogenes. (A) Three clones (FB-C127-4, -6, and -7) along with vector control (pSG5-C127) of C127-derivatives were shown for fibulin-1 expression by Western blot (Top). Retroviruses expressing BPV-1 E5, E6, and v-ras were used to infect the cells for focus formation. Numbers in the middle panel represent foci of an average of two independent experiments for each clone. Representative plates are shown at the bottom. (B) Two clones (FB-3T3-1 and -2) along with vector control (pSG5-3T3) of NIH 3T3-derivatives were shown for fibulin-1 expression by Western blot (Top). Retroviruses expressing HPV-16 E6, E7, and BPV-1 E5 were used to infect the cells for growth on soft agar. Numbers in the middle panel represent colonies (>16 cells) of an average of three independent experiments, each done in triplicate. Representative plates are shown at the bottom (final magnification, 40).
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ated transformation. In FB-C127-6 and -7 cells that express higher levels of fibulin-1, more than 60% of E6induced focus formation were inhibited. In contrast, in FB-C127-4 cells that express less fibulin-1, modest inhibition (45%) of E6 transformation was observed (Fig. 4A). Similarly, FB-3T3-2 cells express more fibulin-1 than FB-3T3-1 cells and fewer E6-induced colonies were formed in FB-3T3-2 cells than in FB-3T3-1. Interestingly, although the number of colonies formed by BPV1 E6 was significantly reduced in FB-C127 cells, the size of E6-induced colonies in these cells appears to be increased in some experiments. The reason for the size increase is not known. Ideally, cell lines expressing E6binding defective fibulin-1 mutants should be used as controls. Since such mutants have not been identified, we used vector-transfected cells as controls. Apparently, vector control has its limitations and caution should be taken when interpreting results obtained by using these cells.
Discussion Over the past several years, more than a dozen putative E6 interacting proteins were identified. The biological significance of interaction between E6 and these cellular proteins remains to be established. In the present study, we describe the identification and characterization of fibulin-1 as a novel E6 binding protein. Fibulin-1 is a calcium-binding protein that has been implicated in a role in cellular transformation and tumor invasion. The interaction between E6 and fibulin-1 was demonstrated both in vitro and in vivo. Fibulin-1 is associated specifically with cancer-related HPV E6s and the transforming BPV-1 E6. Significantly, overexpression of fibulin-1 specifically inhibited both BPV-1 and HPV-16 E6-mediated transformation. This is probably the first example that ectopic expression of a cellular protein leads to inhibition of E6 transformation. These results suggest that fibulin-1 plays an important role in E6-mediated oncogenic activities. Fibulin-1 has been detected in cell membrane and ECM. One may wonder where fibulin-1 and E6 could possibly interact in vivo. Although there is no evidence that E6 localizes in the ECM, E6 has been detected in the cytoplasm and non-nuclear membrane fractions. In particular, we have observed peri-nuclear localization and Tong and Howley have demonstrated Golgi localization of E6 [17,19]. Newly synthesized membrane and ECM proteins must traverse the endoplasmic reticulum (ER) and then the Golgi complex en route to the cell surface. We speculate that E6 and fibulin-1 associate in the ER or Golgi. Alternatively, cytoplasmic E6 may bind to the cytoplasmic portion of the membrane-associated fibulin-1. Our data presented in this report support their in vivo interaction.
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Given the fact that its overexpression inhibits E6 transformation, fibulin-1 appears to be a tumor suppressor. This notion is consistent with the report that elevated expression of fibulin-1-suppressed anchorageindependent growth and tumor formation in human fibrosarcoma-derived cells. In addition, tumor-derived cell lines including the HPV containing cervical carcinoma HeLa cells showed a greatly reduced expression of fibulin-1 [38]. The expression of fibulin-1 has been shown to correlate with sites of active cellular migration in the embryo [56]. It has therefore been thought to play a role in the regulation of cell migration. Fibulin-1 inhibits motility of human ovarian- and breast-cancer cells induced by fibronectin [52]. A crucial event in the multistep process of malignant development is tumor invasion of surrounding tissues and metastasis to remote sites. This process requires tumor cells to detach from ECM and to migrate to other sites [52]. It is believed that increased cell motility facilitates invasion and metastasis of malignant tumors. It is well known that the motility of cells is regulated by ECM proteins [57]. HPV has been implicated a role in metastasis [58]. A recent study demonstrated that HPV-16 E6 acts at the malignant progression stage but weakly at the promotion stage [59]. Tumors arising in HPV-16 E6 transgenic mice were mostly invasive [60]. Cellular proteins involved in E6 invasion have not been identified. We hypothesize that fibulin-1 inhibits cell migration and invasion, while E6 inactivates fibulin-1 to promote cell migration and invasion. Interestingly, it was reported that estrogens increase the expression of fibulin-1 in ovarian cancer cells [61–63]. These results indicate that the role of fibulin-1 in cancer development is complicated. Although HPV-16, -18, and -31 are all considered high-risk types in terms of tumor progression, their pathogenic potential is different. While HPV-16 and -31 are primarily found in squamous cell carcinomas with HPV-16 as the predominant HPV type, HPV-18 is the predominant type in adenocarcinoma and in the small cell undifferentiated carcinoma of the cervix (reviewed in [64]). In vitro association experiments demonstrated that E6BP bound E6 from HPV-16 and HPV-31 more efficiently than HPV-18 E6 [17]. In contrast, hDlg bound HPV-18 E6 more efficiently than HPV-16 E6 [28]. Interestingly, fibulin-1 binds E6s from HPV-16, -18, and -31 with similar efficiency. These data are consistent with the potential role of fibulin-1 in tumor migration and/or invasion, which may not be confined to a specific carcinoma type. The two-hybrid isolate of fibulin-1 encodes amino acids 343–483. This region is present in all variants of fibulin-1. Sequences responsible for self-association, calcium-binding, and fibronectin-binding of fibulin-1 have been localized to amino acids 356–440 [65], a domain within the E6-binding region. This raises the possibility that binding of E6 to fibulin-1 may disrupt these
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functions. Interestingly, the binding region of E6BP has also been mapped to a calcium-binding domain [66]. Calcium ions and calcium-binding proteins are involved in cell signaling and cell differentiation. E6 may exert its inhibitory effect on epithelial cell differentiation through interaction with calcium-binding proteins. The E6binding domain of E6BP is conserved among several E6 binding proteins such as E6-AP, paxillin, and IRF-3. Fibulin-1 does not contain this consensus E6-binding motif. The E6-binding domain of fibulin-1 may therefore represent a new class of E6 interacting motifs. Additional studies are needed to address these possibilities.
Acknowledgments We thank Elliot J. Androphy for advice throughout the course of this work and critical reading of the manuscript; members of our laboratory for helpful suggestions; Virginia Baker for critical reading of the manuscript; Stanley Hollenberg for the yeast two-hybrid system; Scott Argraves and Waleed Twal for antibody; Daniel DiMaio, Denise Galloway, and Douglas Lowy for retroviruses; and Vimla Band, Michele Battle, Qingshen Gao, Thomas Iftner, Justin McCormick, Dick Mosser, Scott Vande Pol, and Karen Vousden for plasmids. MD was supported in part by the Dermatology Foundation Postdoctoral Fellowship. JJC was supported in part by the Massachusetts Breast Cancer Research Grant and the Dermatology Foundation Research Grant.
References [1] M.H. Schiffman, H.M. Bauer, R.N. Hoover, A.G. Glass, D.M. Cadell, B.B. Rush, D.R. Scott, M.E. Sherman, R.J. Kurman, S. Wacholder, et al., J. Natl. Cancer Inst. 85 (1993) 958–964. [2] H. zur Hausen, J. Natl. Cancer Inst. 92 (2000) 690–698. [3] L.M. Alvarez-Salas, A.E. Cullinan, A. Siwkowski, A. Hampel, J.A. DiPaolo, Proc. Natl. Acad. Sci. USA 95 (1998) 1189–1194. [4] L. Rapp, J.J. Chen, Biochim. Biophys. Acta 1378 (1998) F1–F19. [5] J.T. Thomas, W.G. Hubert, M.N. Ruesch, L.A. Laimins, Proc. Natl. Acad. Sci. USA 96 (1999) 8449–8454. [6] D.V. Mythily, S. Krishna, V. Tergaonkar, J. Gen. Virol. 80 (1999) 1707–1713. [7] M.S. Barbosa, R. Schlegel, Oncogene 4 (1989) 1529–1532. [8] S.R. Grossman, L.A. Laimins, Oncogene 4 (1989) 1089–1093. [9] T. Kanda, S. Wantanabe, S. Zanma, H. Sato, A. Furuno, K. Yoshiike, Virology 185 (1991) 536–543. [10] E.J. Androphy, N.L. Hubbert, J.T. Schiller, D.R. Lowy, EMBO J. 6 (1987) 989–992. [11] B.A. Werness, A.J. Levine, P.M. Howley, Science 248 (1990) 76–79. [12] N. Dyson, P.M. Howley, K. Munger, E. Harlow, Science 243 (1989) 934–937. [13] K. Munger, B.A. Werness, N. Dyson, W.C. Phelps, E. Harlow, P.M. Howley, EMBO J. 8 (1989) 4099–4105. [14] J.R. Gage, C. Meyers, F.O. Wettstein, J. Virol. 64 (1990) 723–730. [15] J.M. Huibregtse, M. Scheffner, P.M. Howley, EMBO J. 10 (1991) 4129–4135. [16] M. Scheffner, J.M. Huibregtse, R.D. Vierstra, P.M. Howley, Cell 75 (1993) 495–505. [17] J.J. Chen, C.E. Reid, V. Band, E.J. Androphy, Science 269 (1995) 529–531.
[18] X. Tong, P.M. Howley, Proc. Natl. Acad. Sci. USA 94 (1997) 4412–4417. [19] X. Tong, R. Salgia, J.-L. Li, J.D. Griffin, P.M. Howley, J. Virol. 72 (1998) 476–482. [20] L.V. Ronco, A.Y. Karpova, M. Vidal, P.M. Howley, Genes Dev. 12 (1998) 2061–2072. [21] D. Patel, S.M. Huang, L.A. Baglia, D.J. McCance, EMBO J. 18 (1999) 5061–5072. [22] H. Zimmermann, R. Degenkolbe, H.U. Bernard, M.J. O’Connor, J. Virol. 73 (1999) 6209–6219. [23] S. Li, S. Labrecque, M.C. Gauzzi, A.R. Cuddihy, A.H. Wong, S. Pellegrini, G.J. Matlashewski, A.E. Koromilas, Oncogene 18 (1999) 5727–5737. [24] Q. Gao, A. Kumar, S. Srinivasan, L. Singh, H. Mukai, Y. Ono, D.E. Wazer, V. Band, J. Biol. Chem. 275 (2000) 14824– 14830. [25] H. Zimmermann, C.H. Koh, R. Degenkolbe, M.J. O’Connor, A. Muller, G. Steger, J.J. Chen, Y. Liu, E. Androphy, H.U. Bernard, J. Gen. Virol. 81 (2000) 2617–2623. [26] Y.Y. Degenhardt, S. Silverstein, J. Virol. 75 (2001) 11791–11802. [27] S.S. Lee, R.S. Weiss, R.T. Javier, Proc. Natl. Acad. Sci. USA 94 (1997) 6670–6675. [28] T. Kiyono, A. Hiraiwa, M. Fujita, Y. Hayashi, T. Akiyama, M. Ishibashi, Proc. Natl. Acad. Sci. USA 94 (1997) 11612–11616. [29] S. Gross-Mesilaty, E. Reinstein, B. Bercovich, K.E. Tobias, A.L. Schwartz, C. Kahana, A. Ciechanover, Proc. Natl. Acad. Sci. USA 95 (1998) 8058–8063. [30] I. Kukimoto, S. Aihara, K. Yoshiike, T. Kanda, Biochem. Biophys. Res. Commun. 249 (1998) 258–262. [31] M. Thomas, L. Banks, Oncogene 17 (1998) 2943–2954. [32] C. Kuhne, L. Banks, J. Biol. Chem. 273 (1998) 34302–34309. [33] Q. Gao, S. Srinivasan, S.N. Boyer, D.E. Wazer, V. Band, Mol. Cell. Biol. 19 (1999) 733–744. [34] Y.Y. Degenhardt, S.J. Silverstein, J. Virol. 75 (2001) 151–160. [35] S.S. Lee, B. Glaunsinger, F. Mantovani, L. Banks, R.T. Javier, J. Virol. 74 (2000) 9680–9693. [36] S. Nakagawa, J.M. Huibregtse, Mol. Cell. Biol. 20 (2000) 8244– 8253. [37] B.A. Glaunsinger, S.S. Lee, M. Thomas, L. Banks, R. Javier, Oncogene 19 (2000) 5270–5280. [38] J. Qing, V.M. Maher, H. Tran, W.S. Argraves, R.W. Dunstan, J.J. McCormick, Oncogene 15 (1997) 2159–2168. [39] A. Schmitt, J.B. Harry, B. Rapp, F.O. Wettstein, T. Iftner, J. Virol. 68 (1994) 7051–7059. [40] S. Dalal, Q. Gao, E.J. Androphy, V. Band, J. Virol. 70 (1996) 683–688. [41] J.P. Morgenstern, H. Land, Nucleic Acids Res. 18 (1990) 3587– 3596. [42] J.J. Chen, Y. Hong, E.J. Androphy, Virology 236 (1997) 30–36. [43] Y. Liu, J.J. Chen, Q. Gao, S. Dalal, Y. Hong, C.P. Mansur, V. Band, E.J. Androphy, J. Virol. 73 (1999) 7297–7307. [44] C.L. Halbert, G.W. Demers, D.A. Galloway, J. Virol. 65 (1991) 473–478. [45] D.R. Lowy, E. Rands, E.M. Scolnick, J. Virol. 26 (1978) 291–298. [46] S.M. Hollenberg, R. Sternglanz, P.F. Cheng, H. Weintraub, Mol. Cell. Biol. 15 (1995) 3813–3822. [47] R. Ned, S. Allen, S. Vande Pol, J. Virol. 71 (1997) 4866–4870. [48] T.C. Pan, M. Kluge, R.Z. Zhang, U. Mayer, R. Timpl, M.L. Chu, Eur. J. Biochem. 215 (1993) 733–740. [49] W.S. Argraves, H. Tran, W.H. Burgess, K. Dickerson, J. Cell Biol. 111 (1990) 3155–3164. [50] W.S. Argraves, K. Dickerson, W.H. Burgess, E. Ruoslahti, Cell 58 (1989) 623–629. [51] K. Balbona, H. Tran, S. Godyna, K.C. Ingham, D.K. Strickland, W.S. Argraves, J. Biol. Chem. 267 (1992) 20120–20125. [52] Y. Hayashido, A. Lucas, C. Rougeot, S. Godyna, W.S. Argraves, H. Rochefort, Int. J. Cancer 75 (1998) 654–658.
M. Du et al. / Biochemical and Biophysical Research Communications 296 (2002) 962–969 [53] W.O. Twal, A. Czirok, B. Hegedus, C. Knaak, M.R. Chintalapudi, H. Okagawa, Y. Sugi, W.S. Argraves, J. Cell Sci. 114 (2001) 4587–4598. [54] A. Aspberg, S. Adam, G. Kostka, R. Timpl, D. Heinegard, J. Biol. Chem. 274 (1999) 20444–20449. [55] L. Sherman, R. Schlegel, J. Virol. 70 (1996) 3269–3279. [56] S.G. Spence, W.S. Argraves, L. Walters, J.E. Hungerford, C.D. Little, Dev. Biol. 151 (1992) 473–484. [57] S.K. Akiyama, K. Olden, K.M. Yamada, Cancer Metastasis Rev. 14 (1995) 173–189. [58] J. Czegledy, C. Iosif, O. Forslund, R. Willen, B.G. Hansson, J. Med. Virol. 54 (1998) 183–185. [59] S. Song, A. Liem, J.A. Miller, P.F. Lambert, Virology 267 (2000) 141–150. [60] S. Song, G.A. Gulliver, P.F. Lambert, Proc. Natl. Acad. Sci. USA 95 (1998) 2290–2295.
969
[61] G.M. Clinton, C. Rougeot, J. Derancourt, P. Roger, A. Defrenne, S. Godyna, W.S. Argraves, H. Rochefort, Proc. Natl. Acad. Sci. USA 93 (1996) 316–320. [62] P. Roger, P. Pujol, A. Lucas, P. Baldet, H. Rochefort, Am. J. Pathol. 153 (1998) 1579–1588. [63] F. Moll, D. Katsaros, G. Lazennec, N. Hellio, P. Roger, P.L. Giacalone, D. Chalbos, T. Maudelonde, H. Rochefort, P. Pujol, Oncogene 21 (2002) 1097–1107. [64] K.V. Shah, P.M. Howley, in: B.N. Fields, D.M. Knipe, P.M. Howley (Eds.), Fields Virology, vol. 2, third ed., LippincottRaven, Philadelphia, 1996, pp. 2077–2109. [65] H. Tran, W.J. VanDusen, W.S. Argraves, J. Biol. Chem. 272 (1997) 22600–22606. [66] J.J. Chen, Y. Hong, E. Rustamzadeh, J.D. Baleja, E.J. Androphy, J. Biol. Chem. 273 (1998) 13537–13544.
Interaction of oncogenic papillomavirus E6 proteins with fibulin-1 Minjie Du,1 Xueli Fan, Eva Hong, and Jason J. Chen* Department of Medicine, University of Massachusetts Medical School, LRB Room 323, 364 Plantation Street, Worcester, MA 01605-2324, USA Received 24 July 2002
Abstract Human papillomavirus (HPV) infection is the primary risk factor for the development of cervical cancer. The papillomavirus E6 gene is essential for virus-induced cellular transformation and the viral life cycle. Important insight into the mechanism of E6 function came from the discovery that cancer-related HPV E6 proteins promote the degradation of the tumor suppressor p53. However, mounting evidence indicates that interaction with p53 does not mediate all E6 activities. To explore the p53-independent functions of E6, we performed a yeast two-hybrid screen and identified fibulin-1 as an E6 binding protein. Fibulin-1 is a calciumbinding plasma and extracellular matrix protein that has been implicated in cellular transformation and tumor invasion. The interaction between E6 and fibulin-1 was demonstrated by both in vitro and in vivo assays. Fibulin-1 is associated specifically with cancer-related HPV E6s and the transforming bovine papillomavirus type 1 E6. Significantly, overexpression of fibulin-1 specifically inhibited E6-mediated transformation. These results suggest that fibulin-1 plays an important role in the biological activities of E6. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: HPV; Cancer; p53; Fibulin-1; Papillomavirus; E6; Invasion; Transformation
Papillomaviruses are small DNA viruses that infect various epithelial tissues, including the epidermis and the epithelial linings of the anogenital tract. A majority of the squamous cell abnormalities of the cervix that precede squamous cell carcinoma contain human papillomavirus (HPV) DNA [1]. HPVs that infect the anogenital tract can be classified as high risk and low risk on the basis of their clinical associations. The high-risk HPVs, such as types 16, 18, and 31, are strongly associated with the development of cervical carcinoma. More than 90% of cervical cancers contain HPV DNA. In contrast, infection with low risk HPVs rarely results in cancer (for review, see [2]). Among animal papillomaviruses, the bovine papillomavirus type 1 (BPV-1) had served as the prototype for studies of the molecular biology of the papillomaviruses before recent interest switched to the HPV studies. BPV-1 has the ability to
*
Corresponding author. Fax: 1-508-856-6588. E-mail address: [email protected] (J.J. Chen). 1 Present address: Department of Molecular and Medical Genetics, University of Toronto, Toronto, Canada M5S 1A8.
replicate its genome and to induce cellular transformation in established murine cell lines. The transforming properties of high-risk HPVs primarily reside in two genes, E6 and E7. Antisense studies suggest that the continued expression of E6 and E7 is essential to maintain the transformed state of HPVpositive cells ([3] and references therein). The oncogenic activities of E6 have been reflected in many biological assays. These include immortalization of primary cells, transformation of established fibroblasts, resistance to terminal differentiation of human keratinocytes, modulation of apoptosis, tumorigenesis in animals, and abrogation of cell cycle check points (reviewed in [4]). A recent study has shown that E6 plays an essential role in HPV life cycle [5]. Human epithelial cells expressing HPV-16 E6 have been shown to induce invasiveness in an in vitro assay [6]. The papillomavirus E6 proteins are composed of approximately 150 amino acids. E6 proteins from different HPV types or among the animal and human papillomaviruses show moderate amino acid homology. A common feature of all E6 proteins is the presence of putative Cys–X–X–Cys motifs that are capable of binding zinc [7–9]. The E6 proteins are present at very
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low levels in cells [10]. E6 proteins have been detected in nuclear, non-nuclear membrane, and cytosolic fractions (reviewed in [4]). The ability of high-risk HPV E6 and E7 proteins to associate with the cellular tumor suppressors p53 and pRB, respectively, has been suggested as a mechanism by which the viral proteins induce tumors [11–14]. Association of E6 with p53 is mediated by the ubiquitin ligase E6-AP and leads to the degradation of p53 by the ubiquitination pathway [15,16]. E6 also has functions independent of inactivating p53 and has been shown to interact with multiple additional cellular proteins (reviewed in [4]). We identified a cellular protein E6BP that specifically interacts with E6s from cancer-related highrisk HPVs and BPV-1 [17]. Other proteins that directly bind E6s include the focal adhesion proteins, paxillin and zyxin, the interferon regulatory factor-3 (IRF-3), the c subunit of trans-Golgi network-specific clathrin adaptor (AP-1) complex, the transcriptional coactivator CBP/p300, the tyrosine kinase Tyk2, and the serine/ threonine kinase PKN [18–26]. In addition, several cellular proteins have been identified as the degradation target of E6. These include the human homolog of the Drosophila disk large tumor suppressor protein (hDlg), the oncoprotein Myc, human minichromosome maintenance 7 protein (hMCM7), a putative GAP protein E6TP1, PDZ domain-containing proteins MUPP1, hScrib and MAGI-1, thepro- apoptoticproteinBak, and the activation modulating factor Gps2 (or AMF-1) [27–37]. Although steady progress has been made in the identification of E6 interacting proteins, the biological relevance of these proteins to E6 remains to be established. Cellular proteins involved in E6-mediated resistance to terminal differentiation, maintaining viral episomes during viral life cycle, and induction of invasion have not been identified. Here, we describe the identification and characterization of fibulin-1 as a putative E6 interacting protein that may play an important role in E6-mediated cellular transformation and invasion.
Materials and methods Plasmids and retroviruses. DNA encoding truncated human fibulin1D from amino acids 262–566 was cloned as an EcoRI–XhoI fragment into pGEX4T-3 to express GST-fibulin in Escherichia coli. BPV-1 E6 was cloned as a BamHI–SalI fragment into BamHI–XhoI sites of pGEX3X to encode GST-BPV E6. A 2.3 kb EcoRI fragment containing the entire coding sequence of human fibulin-1D from pPuro-fibulin-1D [38] was inserted into the EcoRI site of pSG5 (Stratagene) to make pSG5fibulin-1D. The FLAGtagged HPV-16 E6 in pSG5 (pSG5-HPV16 E6-FLAG) was obtained from Dr. Qingshen Gao and Dr. Vimla Band [33]. pSGFLAGBE6 was constructed by amplifying BPV-1 E6 with PCR and cloning into pSG5. The PCR sense primer contains a FLAG tag between the first and second amino acids of BPV-1 E6.
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Plasmids encoding E6s from BPV-1, HPV-6, 11, 16, 18, and 31 for in vitro synthesis were described in [17]. Plasmid encoding HPV-8 E6 in the Bluescribe M13þ vector (pBS; Vector Cloning System) was provided by Iftner and co-workers [39]. Plasmid encoding HPV-16 E6 mutant with C-terminal amino acid 124–151 deletion in pSP65 vector was described in [40]. Plasmid encoding HPV-16 E7 in pSP65 vector was provided by Dr. Karen Vousden. The retrovirus vector pBabe Puro is a Moloney murine leukemia virus-based vector containing a puromycin resistance gene [41]. Retroviruses containing BPV-1 E6, HPV-16 E6, and HPV-16 E7 have been described [42–44]. Retroviruses containing the Harvey murine sarcoma virus v-ras and BPV-1 E5 were provided by Dr. Douglas Lowy and Dr. Daniel DiMaio, respectively. Cell culture and transformation assays. C127 is a non-transformed clonal line derived from the mammary tissue of an RIII mouse [45]. To establish stable cell lines overexpressing fibulin-1, plasmid encoding fibulin-1D in pSG5 was transfected into C127 cells using FuGENE 6 transfection reagent (Boehringer Mannheim) and NIH 3T3 cells by calcium phosphate-mediated transfection, respectively. All transfections were performed by cotransfection of the vector ptTA-hygro (provided by Dr. Dick Mosser) that confers hygromycin resistance. After hygromycin selection, individual colonies were collected and examined for fibulin-1 expression by Western blot using the mouse monoclonal anti-fibulin-1 IgG 3A11 [38]. All subsequent experiments were performed using fibulin-1 overexpressing cells within five passages. For focus formation studies, C127-derived cells were infected with amphotrophic retroviruses. Two days after infection, cells were split 1:3. Upon confluence of cells, medium was switched from Dulbecco’s minimum essential medium (DMEM) containing 10% fetal calf serum (FCS) to 5% FCS. Cells were fed with fresh medium every 3 days. Three weeks after infection, cells were stained with GIEMSA stain solution (Ricca Chemical, Texas) and foci were counted. For the colony formation assay, NIH 3T3-derived cells were infected with retroviruses produced in PA317 cells. Two days after infection, cells were pooled, seeded into DMEM containing 0.4% Noble agar and 10% fetal calf serum at a density of 5 104 /60-mm dish, and overlaid to DMEM containing 0.6% Noble agar and 10% fetal calf serum. Cells were fed with fresh 0.4% agar medium weekly. Colony formation was scored in 3–4 weeks under a microscope. Colonies composed of more than 16 cells were scored as positive. Protein preparation and interaction experiments. GST fusion proteins were expressed in E. coli strain DH5a. After induction with IPTG, cells were harvested, re-suspended in 50 ml low salt association buffer (LSAB, 100 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1% NP-40, and 1 mM phenylmethylsulfonyl fluoride), and lysed by sonication. SDS and DTT were added to the lysate immediately after sonication for a final concentration of 0.03% and 1 mM, respectively. After centrifugation at 10,000g for 10 min, supernatant was collected and mixed with glutathione–Sepharose beads (Pharmacia). After incubation, the beads were collected by centrifugation at 1000g, washed three times with 20 volumes of LSAB, and stored at 4 °C. In vitro-translated E6 proteins and fibulin-1 were prepared by using the rabbit reticulocyte lysate translation system (Promega) and 35 S-labeled cysteine and methionine, respectively (ICN Biomedicals, Irvine, CA). For in vitro binding, 5–30 ll glutathione–Sepharose containing 2.5 lg GST fusion proteins was combined with 2–20 ll 35 S-labeled in vitro-translated proteins in lysis buffer (100 mM NaCl, 100 mM Tris– HCl (pH 8.0), 0.5% Nonidet P-40, 0.1% dried milk, 2 mM DTT, and 1 mM PMSF) in a total volume of 250 ll. The mixtures were subjected to rotary shaking for 2–3 h at 4 °C. The mixtures were washed extensively with lysis buffer (1% Nonidet P-40), boiled in SDS loading buffer, and electrophoresed on SDS–polyacrylamide gels. Gels were dried and scanned by Molecular Imager (BioRad). The in vitro degradation assay was described previously [43]. Briefly, full-length fibulin-1D was translated in vitro, radiolabeled, and mixed in reaction mixtures containing E6 and rabbit reticulocyte
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lysate. Samples were incubated at 30 °C for 3 h. The remaining fibulin1 was quantified by SDS–PAGE and BioRad Molecular Imager. For in vivo association, COS-7 cells were transfected by electroporation with plasmids encoding FLAG-tagged E6 and full-length fibulin-1D in pSG5. Forty-eight hours after transfection, cell extracts were prepared in lysis buffer (containing 0.25% NP-40) and fibulin-1 was precipitated with specific monoclonal antibody. After extensive washing, the precipitates were fractionated by SDS–PAGE and immunoblotted with anti-fibulin-1 and anti-FLAG antibodies, respectively. The signals were detected by using the SuperSignal Chemiluminescent reagent (Pierce, IL). Yeast two-hybrid screen. A modified yeast two-hybrid system from Dr. Stanley Hollenberg was used to screen a random-primed mouse embryo cDNA library [46]. This system provides a nutritional selection for histidine prototrophs, followed by a selection for b-galactosidase activity. The bait hybrid used in the screen is a BPV-1 E6 mutant (R42W) fused in-frame with LexA DNA binding protein (LexABE6R42W). Yeast strain L40 containing the bait plasmid was transformed with the library DNA. The transformants were first screened for the ability to grow in the absence of histidine. Histidine prototrophs were then tested for b-galactosidase activity.
Fig. 1. Schematic representation of fibulin-1 and truncated proteins used in this study. The three major structural elements found in fibulin1 are indicated as domains I, II, and III. Domain I contains three anaphylatoxin-like motifs. Domain II consists of nine EGF-like repeats, of which eight contain consensus sequence for calcium binding. Domain III is type specific sequence. The one shown in this figure is specific to fibulin-1D. The two-hybrid isolate ranges from EGF-like repeats 5 to 8. A truncated fibulin-1 containing EGF-like repeats 3–9 was expressed as a GST fusion for in vitro association assays.
Results Identification of fibulin-1 as an E6 interacting protein To identify cellular proteins that interact with E6, we used the yeast two-hybrid system. The bait hybrid was a BPV-1 E6 mutant (R42W) fused in-frame with LexA DNA binding protein. R42W is substantially reduced for transcriptional activation, yet competent for interaction with E6-AP in yeast [47]. BPV-1 E6 transforms cells but does not degrade p53. A modified version of the two-hybrid system provided by Dr. Stanley Hollenberg was used to screen a random-primed mouse embryo cDNA library [46]. A mouse library was chosen because BPV-1 E6 transforms the murine C127 cells. Approximately 1:6 106 transformants were screened and 140 histidine prototrophs were obtained. Of these histidine prototrophs, 76 were subsequently shown to be b-galactosidase positive. To test for specificity and reproducibility of the initial two-hybrid hits, a series of experiments were performed. First, the bait plasmid from yeast was removed. This was achieved by growing yeast in non-selective medium for the bait plasmid (on tryptophan containing plates), followed by transferring to low adenine plates. The bait plasmid contains the Ade2 gene and keeps the colonies formed by the L40 yeast white on low adenine plates. After the bait plasmid is lost, the yeast produces a red by-product that identifies these colonies. The resulting yeast was plated on X-gal plate to ascertain if the library DNA could produce b-galactosidase signal in the absence of a bait plasmid. Clones that scored positive were eliminated. Four cDNA clones encoding potential E6 interacting proteins were obtained. Sequence analysis of one clone revealed a 141-amino acid open reading frame encoding
a polypeptide with several EGF-like repeats (Fig. 1). This sequence represents a truncated version of fibulin-1 from amino acids 343–483. Fibulin-1 is a member of a growing family of cysteine-rich proteins with four variants named A, B, C, and D [48]. Based on its primary amino acid sequence, fibulin-1 can be divided into three parts. The N-terminal of fibulin-1 contains three cysteine-rich motifs similar to anaphylatoxins. The central core sequence of fibulin-1 contains nine EGF-like repeats [48]. The C-terminal region is unique to each variant. Fibulin-1 has been detected in the plasma, extracellular matrix (ECM), and blood [49]. Fibulin-1 associates with different ECM components such as fibronectin, laminin, and collagen IV [48,50,51]. The biological function of fibulin-1 remains to be established. Expression of fibulin-1 inhibits motility of human ovarian- and breast-cancer cells induced by fibronectin [52,53]. Elevated expression of fibulin-1 suppressed anchorage-independent growth, matrigel invasion, and tumor formation in human fibrosarcoma-derived cells [38]. These results suggest a role for fibulin-1 in tumor formation and invasion. Of particular interest to the papillomavirus-associated lesions, fibulin-1 has been detected at the dermal/epidermal border [54]. In vitro interaction of E6 and fibulin-1 The interaction of fibulin-1 and E6 was investigated using an in vitro binding experiment. cDNA encoding the central core sequence of human fibulin-1 was cloned into the pGEX plasmid (Fig. 1). Fibulin-1 was synthesized and purified from E. coli as a GST fusion and tested for association with 35 S-labeled, in vitro-translated BPV-1 E6 and HPV-16 E6 proteins. The HPV-16 E7 was included as a control. Our results demonstrated
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In reciprocal experiments, the truncated fibulin-1 from the original yeast two-hybrid screen was synthesized, 35 Slabeled by in vitro transcription, and translation and tested for association with GST-E6 fusion proteins. Our results indicated that fibulin-1 bound BPV-1 E6 and HPV-16 E6, but not HPV-6 E6 (data not shown). We also tested whether fibulin-1 can be degraded by E6 in vitro. Both fibulin-1 and E6 were prepared and 35 S-labeled by in vitro translation. They were incubated in a degradation assay in the presence of rabbit reticulocyte lysate. While the control p53 protein was efficiently degraded by HPV-16 E6, the level of fibulin-1 remained unchanged when incubated either with HPV16 E6 or BPV-1 E6 (data not shown). These results suggest that fibulin-1 is not a degradation target for E6, at least in vitro. In vivo interaction of fibulin-1 with E6
Fig. 2. In vitro interaction of E6 and fibulin-1. Glutathione–Sepharose beads containing GST-fibulin-1 fusion protein were combined with 35 S-labeled in vitro-translated E6 proteins in lysis buffer. The bound products were separated by SDS–PAGE and analyzed by Molecular Imager (BioRad). Input was directly loaded into the well and represents 10% of the [35 S]cysteine-labeled E6 proteins used in each binding reaction. (A) Fibulin-1 binds to BPV-1 E6 and HPV-16 E6. (B) Specific association of fibulin-1 with E6s from high-risk HPVs. The E6 proteins from different HPVs are indicated above each lane. HPV-16 E6M, a truncated form of E6 with C-terminal amino acid 124–151 deletion.
that fibulin-1 efficiently binds BPV-1 E6 and HPV-16 E6 (Fig. 2A). In contrast, fibulin-1 did not bind HPV-16 E7. We next investigated the ability of E6s from other HPV types to bind fibulin-1. GST-fibulin prepared from E. coli was tested for association with 35 S-labeled, in vitro-translated E6 proteins from multiple HPV types. HPV-16 E6 was included as a positive control. As shown in Fig. 2B, in addition to HPV-16 E6, fibulin-1 also bound efficiently to E6s from other high-risk HPVs such as types 18 and 31. In contrast, E6 from low-risk HPV-6 and HPV-11 or cutaneous HPV-8 did not bind fibulin-1. Thus, there is a correlation between biological risk for cervical cancer and the ability to bind fibulin-1. In addition, fibulin-1 did not bind the truncated form of E6 with C-terminal amino acid 124–151 deletion that was defective for immortalization of human mammary epithelial cells [40]. These results indicate that the association of fibulin-1 with oncogenic E6s is specific.
Since the level of neither fibulin-1 nor E6 is high in cells, it is not practical to detect their in vivo association with endogenous proteins. To assess the in vivo interaction of E6 and fibulin-1, a transient transfection assay was employed. Briefly, COS-7 cells were transfected by electroporation with plasmids encoding E6 and full-length human fibulin-1D in pSG5. To facilitate detection, E6 was expressed as epitope FLAG-tagged proteins. Epitope-tagged E6 was shown to be functionally equivalent to the wild-type E6 [55]. Cell extracts were prepared and fibulin-1 was precipitated with specific monoclonal antibody. The presence of E6 in fibulin1 precipitates was detected by Western blot. Our results demonstrated that both HPV-16 E6 and BPV-1 E6 could form a complex with fibulin-1 in vivo (Fig. 3).
Fig. 3. In vivo association of E6 and fibulin-1. COS-7 cells were transfected with plasmids encoding FLAG-tagged E6 (pSGFLAGBE6 and pSG5-HPV16 E6-FLAG) and full-length human fibulin-1D in pSG5 (pSG5fibulin-1D) by electroporation. Following transfection and incubation, cell extracts were prepared and fibulin-1 was precipitated with specific antibody. After extensive washing, the precipitates were fractionated by SDS–PAGE and immunoblotted with antiFLAG antibody. Upper panel, cell extract directly blotted by antifibulin-1 antibody. Lower panel, fibulin-1 precipitates blotted by antiFLAG antibody. The positions of fibulin-1 and E6 proteins are indicated by arrows.
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In the transient transfection experiment, the level of steady-state fibulin-1 was not affected by the expression of BPV-1 or HPV-16 E6. To explore this further, the endogenous fibulin-1 protein levels in several stable cell lines expressing E6 were examined by Western blot. Cells examined include C127 expressing BPV-1 E6, human keratinocytes containing HPV-16 E6, and HMEC immortalized by HPV-16 E6. Our results demonstrated that the level of fibulin-1 in these cells remains unchanged in the presence of E6 (data not shown). These data are consistent with our in vitro data and suggest that fibulin-1 is not a degradation target for E6. However, we cannot rule out the possibility that under some special physiological conditions, E6 may target fibulin-1 for degradation. Ectopic expression of fibulin-1 specifically inhibits E6 transformation To study the functional interaction of E6 and fibulin1, stable cell lines that overexpress fibulin-1 were established. Mouse C127 and NIH 3T3 cells were
transfected with full-length human fibulin-1D to make FB-C127 and FB-3T3 cells, respectively. Among seven independent clones of C127 derivatives tested, three were found to express high levels of fibulin-1. While fibulin-1 in the vector-transfected cells (pSG5–C127) was almost undetectable, strong bands are evident in the three clones (Fig. 4A). Similarly, three out of six NIH 3T3-derived clones were found to overexpress fibulin-1 (Fig. 4B). BPV-1 E6 induces the focus formation in C127 cells while HPV-16 E6 stimulates the anchorageindependent growth of NIH 3T3 cells in soft agar (reviewed in [4]). Both BPV-1 E5 and the cellular oncogene v-ras efficiently transform C127 cells but through seemingly different mechanisms than BPV-1 E6. BPV-1 E5 and HPV-16 E7 also induce anchorage-independent growth in NIH 3T3. FB-C127 and FB-3T3 cells were accordingly subjected to transformation by these oncogenes. As shown in Fig. 4, overexpression of fibulin-1 significantly reduced E6-induced transformation. In contrast, transformation by BPV-1 E5, HPV-16 E7, or oncogene v-ras was not affected. Moreover, the level of fibulin-1 correlated with its ability to inhibit E6-medi-
Fig. 4. Overexpression of fibulin-1 specifically inhibits E6 transformation. C127 and NIH 3T3 cells were transfected with full-length fibulin-1D gene under the control of SV40 promoter. The cells were plated in hygromycin B containing media. Multiple colonies were picked, expanded, and tested for the expression of fibulin-1 by Western blot and transformation by oncogenes. (A) Three clones (FB-C127-4, -6, and -7) along with vector control (pSG5-C127) of C127-derivatives were shown for fibulin-1 expression by Western blot (Top). Retroviruses expressing BPV-1 E5, E6, and v-ras were used to infect the cells for focus formation. Numbers in the middle panel represent foci of an average of two independent experiments for each clone. Representative plates are shown at the bottom. (B) Two clones (FB-3T3-1 and -2) along with vector control (pSG5-3T3) of NIH 3T3-derivatives were shown for fibulin-1 expression by Western blot (Top). Retroviruses expressing HPV-16 E6, E7, and BPV-1 E5 were used to infect the cells for growth on soft agar. Numbers in the middle panel represent colonies (>16 cells) of an average of three independent experiments, each done in triplicate. Representative plates are shown at the bottom (final magnification, 40).
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ated transformation. In FB-C127-6 and -7 cells that express higher levels of fibulin-1, more than 60% of E6induced focus formation were inhibited. In contrast, in FB-C127-4 cells that express less fibulin-1, modest inhibition (45%) of E6 transformation was observed (Fig. 4A). Similarly, FB-3T3-2 cells express more fibulin-1 than FB-3T3-1 cells and fewer E6-induced colonies were formed in FB-3T3-2 cells than in FB-3T3-1. Interestingly, although the number of colonies formed by BPV1 E6 was significantly reduced in FB-C127 cells, the size of E6-induced colonies in these cells appears to be increased in some experiments. The reason for the size increase is not known. Ideally, cell lines expressing E6binding defective fibulin-1 mutants should be used as controls. Since such mutants have not been identified, we used vector-transfected cells as controls. Apparently, vector control has its limitations and caution should be taken when interpreting results obtained by using these cells.
Discussion Over the past several years, more than a dozen putative E6 interacting proteins were identified. The biological significance of interaction between E6 and these cellular proteins remains to be established. In the present study, we describe the identification and characterization of fibulin-1 as a novel E6 binding protein. Fibulin-1 is a calcium-binding protein that has been implicated in a role in cellular transformation and tumor invasion. The interaction between E6 and fibulin-1 was demonstrated both in vitro and in vivo. Fibulin-1 is associated specifically with cancer-related HPV E6s and the transforming BPV-1 E6. Significantly, overexpression of fibulin-1 specifically inhibited both BPV-1 and HPV-16 E6-mediated transformation. This is probably the first example that ectopic expression of a cellular protein leads to inhibition of E6 transformation. These results suggest that fibulin-1 plays an important role in E6-mediated oncogenic activities. Fibulin-1 has been detected in cell membrane and ECM. One may wonder where fibulin-1 and E6 could possibly interact in vivo. Although there is no evidence that E6 localizes in the ECM, E6 has been detected in the cytoplasm and non-nuclear membrane fractions. In particular, we have observed peri-nuclear localization and Tong and Howley have demonstrated Golgi localization of E6 [17,19]. Newly synthesized membrane and ECM proteins must traverse the endoplasmic reticulum (ER) and then the Golgi complex en route to the cell surface. We speculate that E6 and fibulin-1 associate in the ER or Golgi. Alternatively, cytoplasmic E6 may bind to the cytoplasmic portion of the membrane-associated fibulin-1. Our data presented in this report support their in vivo interaction.
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Given the fact that its overexpression inhibits E6 transformation, fibulin-1 appears to be a tumor suppressor. This notion is consistent with the report that elevated expression of fibulin-1-suppressed anchorageindependent growth and tumor formation in human fibrosarcoma-derived cells. In addition, tumor-derived cell lines including the HPV containing cervical carcinoma HeLa cells showed a greatly reduced expression of fibulin-1 [38]. The expression of fibulin-1 has been shown to correlate with sites of active cellular migration in the embryo [56]. It has therefore been thought to play a role in the regulation of cell migration. Fibulin-1 inhibits motility of human ovarian- and breast-cancer cells induced by fibronectin [52]. A crucial event in the multistep process of malignant development is tumor invasion of surrounding tissues and metastasis to remote sites. This process requires tumor cells to detach from ECM and to migrate to other sites [52]. It is believed that increased cell motility facilitates invasion and metastasis of malignant tumors. It is well known that the motility of cells is regulated by ECM proteins [57]. HPV has been implicated a role in metastasis [58]. A recent study demonstrated that HPV-16 E6 acts at the malignant progression stage but weakly at the promotion stage [59]. Tumors arising in HPV-16 E6 transgenic mice were mostly invasive [60]. Cellular proteins involved in E6 invasion have not been identified. We hypothesize that fibulin-1 inhibits cell migration and invasion, while E6 inactivates fibulin-1 to promote cell migration and invasion. Interestingly, it was reported that estrogens increase the expression of fibulin-1 in ovarian cancer cells [61–63]. These results indicate that the role of fibulin-1 in cancer development is complicated. Although HPV-16, -18, and -31 are all considered high-risk types in terms of tumor progression, their pathogenic potential is different. While HPV-16 and -31 are primarily found in squamous cell carcinomas with HPV-16 as the predominant HPV type, HPV-18 is the predominant type in adenocarcinoma and in the small cell undifferentiated carcinoma of the cervix (reviewed in [64]). In vitro association experiments demonstrated that E6BP bound E6 from HPV-16 and HPV-31 more efficiently than HPV-18 E6 [17]. In contrast, hDlg bound HPV-18 E6 more efficiently than HPV-16 E6 [28]. Interestingly, fibulin-1 binds E6s from HPV-16, -18, and -31 with similar efficiency. These data are consistent with the potential role of fibulin-1 in tumor migration and/or invasion, which may not be confined to a specific carcinoma type. The two-hybrid isolate of fibulin-1 encodes amino acids 343–483. This region is present in all variants of fibulin-1. Sequences responsible for self-association, calcium-binding, and fibronectin-binding of fibulin-1 have been localized to amino acids 356–440 [65], a domain within the E6-binding region. This raises the possibility that binding of E6 to fibulin-1 may disrupt these
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functions. Interestingly, the binding region of E6BP has also been mapped to a calcium-binding domain [66]. Calcium ions and calcium-binding proteins are involved in cell signaling and cell differentiation. E6 may exert its inhibitory effect on epithelial cell differentiation through interaction with calcium-binding proteins. The E6binding domain of E6BP is conserved among several E6 binding proteins such as E6-AP, paxillin, and IRF-3. Fibulin-1 does not contain this consensus E6-binding motif. The E6-binding domain of fibulin-1 may therefore represent a new class of E6 interacting motifs. Additional studies are needed to address these possibilities.
Acknowledgments We thank Elliot J. Androphy for advice throughout the course of this work and critical reading of the manuscript; members of our laboratory for helpful suggestions; Virginia Baker for critical reading of the manuscript; Stanley Hollenberg for the yeast two-hybrid system; Scott Argraves and Waleed Twal for antibody; Daniel DiMaio, Denise Galloway, and Douglas Lowy for retroviruses; and Vimla Band, Michele Battle, Qingshen Gao, Thomas Iftner, Justin McCormick, Dick Mosser, Scott Vande Pol, and Karen Vousden for plasmids. MD was supported in part by the Dermatology Foundation Postdoctoral Fellowship. JJC was supported in part by the Massachusetts Breast Cancer Research Grant and the Dermatology Foundation Research Grant.
References [1] M.H. Schiffman, H.M. Bauer, R.N. Hoover, A.G. Glass, D.M. Cadell, B.B. Rush, D.R. Scott, M.E. Sherman, R.J. Kurman, S. Wacholder, et al., J. Natl. Cancer Inst. 85 (1993) 958–964. [2] H. zur Hausen, J. Natl. Cancer Inst. 92 (2000) 690–698. [3] L.M. Alvarez-Salas, A.E. Cullinan, A. Siwkowski, A. Hampel, J.A. DiPaolo, Proc. Natl. Acad. Sci. USA 95 (1998) 1189–1194. [4] L. Rapp, J.J. Chen, Biochim. Biophys. Acta 1378 (1998) F1–F19. [5] J.T. Thomas, W.G. Hubert, M.N. Ruesch, L.A. Laimins, Proc. Natl. Acad. Sci. USA 96 (1999) 8449–8454. [6] D.V. Mythily, S. Krishna, V. Tergaonkar, J. Gen. Virol. 80 (1999) 1707–1713. [7] M.S. Barbosa, R. Schlegel, Oncogene 4 (1989) 1529–1532. [8] S.R. Grossman, L.A. Laimins, Oncogene 4 (1989) 1089–1093. [9] T. Kanda, S. Wantanabe, S. Zanma, H. Sato, A. Furuno, K. Yoshiike, Virology 185 (1991) 536–543. [10] E.J. Androphy, N.L. Hubbert, J.T. Schiller, D.R. Lowy, EMBO J. 6 (1987) 989–992. [11] B.A. Werness, A.J. Levine, P.M. Howley, Science 248 (1990) 76–79. [12] N. Dyson, P.M. Howley, K. Munger, E. Harlow, Science 243 (1989) 934–937. [13] K. Munger, B.A. Werness, N. Dyson, W.C. Phelps, E. Harlow, P.M. Howley, EMBO J. 8 (1989) 4099–4105. [14] J.R. Gage, C. Meyers, F.O. Wettstein, J. Virol. 64 (1990) 723–730. [15] J.M. Huibregtse, M. Scheffner, P.M. Howley, EMBO J. 10 (1991) 4129–4135. [16] M. Scheffner, J.M. Huibregtse, R.D. Vierstra, P.M. Howley, Cell 75 (1993) 495–505. [17] J.J. Chen, C.E. Reid, V. Band, E.J. Androphy, Science 269 (1995) 529–531.
[18] X. Tong, P.M. Howley, Proc. Natl. Acad. Sci. USA 94 (1997) 4412–4417. [19] X. Tong, R. Salgia, J.-L. Li, J.D. Griffin, P.M. Howley, J. Virol. 72 (1998) 476–482. [20] L.V. Ronco, A.Y. Karpova, M. Vidal, P.M. Howley, Genes Dev. 12 (1998) 2061–2072. [21] D. Patel, S.M. Huang, L.A. Baglia, D.J. McCance, EMBO J. 18 (1999) 5061–5072. [22] H. Zimmermann, R. Degenkolbe, H.U. Bernard, M.J. O’Connor, J. Virol. 73 (1999) 6209–6219. [23] S. Li, S. Labrecque, M.C. Gauzzi, A.R. Cuddihy, A.H. Wong, S. Pellegrini, G.J. Matlashewski, A.E. Koromilas, Oncogene 18 (1999) 5727–5737. [24] Q. Gao, A. Kumar, S. Srinivasan, L. Singh, H. Mukai, Y. Ono, D.E. Wazer, V. Band, J. Biol. Chem. 275 (2000) 14824– 14830. [25] H. Zimmermann, C.H. Koh, R. Degenkolbe, M.J. O’Connor, A. Muller, G. Steger, J.J. Chen, Y. Liu, E. Androphy, H.U. Bernard, J. Gen. Virol. 81 (2000) 2617–2623. [26] Y.Y. Degenhardt, S. Silverstein, J. Virol. 75 (2001) 11791–11802. [27] S.S. Lee, R.S. Weiss, R.T. Javier, Proc. Natl. Acad. Sci. USA 94 (1997) 6670–6675. [28] T. Kiyono, A. Hiraiwa, M. Fujita, Y. Hayashi, T. Akiyama, M. Ishibashi, Proc. Natl. Acad. Sci. USA 94 (1997) 11612–11616. [29] S. Gross-Mesilaty, E. Reinstein, B. Bercovich, K.E. Tobias, A.L. Schwartz, C. Kahana, A. Ciechanover, Proc. Natl. Acad. Sci. USA 95 (1998) 8058–8063. [30] I. Kukimoto, S. Aihara, K. Yoshiike, T. Kanda, Biochem. Biophys. Res. Commun. 249 (1998) 258–262. [31] M. Thomas, L. Banks, Oncogene 17 (1998) 2943–2954. [32] C. Kuhne, L. Banks, J. Biol. Chem. 273 (1998) 34302–34309. [33] Q. Gao, S. Srinivasan, S.N. Boyer, D.E. Wazer, V. Band, Mol. Cell. Biol. 19 (1999) 733–744. [34] Y.Y. Degenhardt, S.J. Silverstein, J. Virol. 75 (2001) 151–160. [35] S.S. Lee, B. Glaunsinger, F. Mantovani, L. Banks, R.T. Javier, J. Virol. 74 (2000) 9680–9693. [36] S. Nakagawa, J.M. Huibregtse, Mol. Cell. Biol. 20 (2000) 8244– 8253. [37] B.A. Glaunsinger, S.S. Lee, M. Thomas, L. Banks, R. Javier, Oncogene 19 (2000) 5270–5280. [38] J. Qing, V.M. Maher, H. Tran, W.S. Argraves, R.W. Dunstan, J.J. McCormick, Oncogene 15 (1997) 2159–2168. [39] A. Schmitt, J.B. Harry, B. Rapp, F.O. Wettstein, T. Iftner, J. Virol. 68 (1994) 7051–7059. [40] S. Dalal, Q. Gao, E.J. Androphy, V. Band, J. Virol. 70 (1996) 683–688. [41] J.P. Morgenstern, H. Land, Nucleic Acids Res. 18 (1990) 3587– 3596. [42] J.J. Chen, Y. Hong, E.J. Androphy, Virology 236 (1997) 30–36. [43] Y. Liu, J.J. Chen, Q. Gao, S. Dalal, Y. Hong, C.P. Mansur, V. Band, E.J. Androphy, J. Virol. 73 (1999) 7297–7307. [44] C.L. Halbert, G.W. Demers, D.A. Galloway, J. Virol. 65 (1991) 473–478. [45] D.R. Lowy, E. Rands, E.M. Scolnick, J. Virol. 26 (1978) 291–298. [46] S.M. Hollenberg, R. Sternglanz, P.F. Cheng, H. Weintraub, Mol. Cell. Biol. 15 (1995) 3813–3822. [47] R. Ned, S. Allen, S. Vande Pol, J. Virol. 71 (1997) 4866–4870. [48] T.C. Pan, M. Kluge, R.Z. Zhang, U. Mayer, R. Timpl, M.L. Chu, Eur. J. Biochem. 215 (1993) 733–740. [49] W.S. Argraves, H. Tran, W.H. Burgess, K. Dickerson, J. Cell Biol. 111 (1990) 3155–3164. [50] W.S. Argraves, K. Dickerson, W.H. Burgess, E. Ruoslahti, Cell 58 (1989) 623–629. [51] K. Balbona, H. Tran, S. Godyna, K.C. Ingham, D.K. Strickland, W.S. Argraves, J. Biol. Chem. 267 (1992) 20120–20125. [52] Y. Hayashido, A. Lucas, C. Rougeot, S. Godyna, W.S. Argraves, H. Rochefort, Int. J. Cancer 75 (1998) 654–658.
M. Du et al. / Biochemical and Biophysical Research Communications 296 (2002) 962–969 [53] W.O. Twal, A. Czirok, B. Hegedus, C. Knaak, M.R. Chintalapudi, H. Okagawa, Y. Sugi, W.S. Argraves, J. Cell Sci. 114 (2001) 4587–4598. [54] A. Aspberg, S. Adam, G. Kostka, R. Timpl, D. Heinegard, J. Biol. Chem. 274 (1999) 20444–20449. [55] L. Sherman, R. Schlegel, J. Virol. 70 (1996) 3269–3279. [56] S.G. Spence, W.S. Argraves, L. Walters, J.E. Hungerford, C.D. Little, Dev. Biol. 151 (1992) 473–484. [57] S.K. Akiyama, K. Olden, K.M. Yamada, Cancer Metastasis Rev. 14 (1995) 173–189. [58] J. Czegledy, C. Iosif, O. Forslund, R. Willen, B.G. Hansson, J. Med. Virol. 54 (1998) 183–185. [59] S. Song, A. Liem, J.A. Miller, P.F. Lambert, Virology 267 (2000) 141–150. [60] S. Song, G.A. Gulliver, P.F. Lambert, Proc. Natl. Acad. Sci. USA 95 (1998) 2290–2295.
969
[61] G.M. Clinton, C. Rougeot, J. Derancourt, P. Roger, A. Defrenne, S. Godyna, W.S. Argraves, H. Rochefort, Proc. Natl. Acad. Sci. USA 93 (1996) 316–320. [62] P. Roger, P. Pujol, A. Lucas, P. Baldet, H. Rochefort, Am. J. Pathol. 153 (1998) 1579–1588. [63] F. Moll, D. Katsaros, G. Lazennec, N. Hellio, P. Roger, P.L. Giacalone, D. Chalbos, T. Maudelonde, H. Rochefort, P. Pujol, Oncogene 21 (2002) 1097–1107. [64] K.V. Shah, P.M. Howley, in: B.N. Fields, D.M. Knipe, P.M. Howley (Eds.), Fields Virology, vol. 2, third ed., LippincottRaven, Philadelphia, 1996, pp. 2077–2109. [65] H. Tran, W.J. VanDusen, W.S. Argraves, J. Biol. Chem. 272 (1997) 22600–22606. [66] J.J. Chen, Y. Hong, E. Rustamzadeh, J.D. Baleja, E.J. Androphy, J. Biol. Chem. 273 (1998) 13537–13544.