Proteomic Identification of Lynchpin Urokinase ... - ACS Publications

Jan 18, 2007 - Differential Proteome Expression Associated with Urokinase Plasminogen Activator Receptor (uPAR) Suppression in Malignant Epithelial ...
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Proteomic Identification of Lynchpin Urokinase Plasminogen Activator Receptor Protein Interactions Associated with Epithelial Cancer Malignancy Rohit G. Saldanha,† Mark P. Molloy,† Khalil Bdeir,‡ Douglas B. Cines,‡ Xiaomin Song,† Pauliina M. Uitto,† Paul H. Weinreb,§ Shelia M. Violette,§ and Mark S. Baker*,† Australian Proteome Analysis Facility Ltd and Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, 2109, NSW Australia, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6100, and Biogen Idec, Inc., Cambridge, Massachusetts 02142 Received October 4, 2006

Urokinase plasminogen activator (uPA) and its high affinity receptor (uPAR) play crucial proteolytic and non-proteolytic roles in cancer metastasis. In addition to promoting plasmin-mediated degradation of extracellular matrix barriers, cell surface engagement of uPA through uPAR binding results in the activation of a suite of diverse cellular signal transduction pathways. Because uPAR is bound to the plasma membrane through a glycosyl-phosphatidylinositol anchor, these signalling sequelae are thought to occur through the formation of multi-protein cell surface complexes involving uPAR. To further characterize uPAR-driven protein complexes, we co-immunoprecipitated uPAR from the human ovarian cancer cell line, OVCA 429, and employed sensitive proteomic methods to identify the uPARassociated proteins. Using this strategy, we identified several known, as well as numerous novel, uPAR associating proteins, including the epithelial restricted integrin, Rvβ6. Reverse immunoprecipitation using anti-β6 integrin subunit monoclonal antibodies confirmed the co-purification of this protein with uPAR. Inhibition of uPAR and/or β6 integrin subunit using neutralizing antibodies resulted in the inhibition of uPA-mediated ERK 1/2 phosphorylation and subsequent cell proliferation. These data suggest that the association of β6 integrin (and possibly other lynchpin cancer regulatory proteins) with uPAR may be crucial in co-transmitting uPA signals that induce cell proliferation. Our findings support the notion that uPAR behaves as a lynchpin in promoting tumorigenesis by forming functionally active multiprotein complexes. Keywords: uPA • uPAR • ovarian cancer • OVCA 429 • Rvβ6 integrin • metastasis • immunoprecipitation • ERK 1/2 • proliferation

Introduction Degradation of the extracellular matrix (ECM) is a hallmark of cancer metastasis and is an important prerequisite of tumor progression. Of the several protease enzymes that are implicated in matrix degradation, the urokinase plasminogen activation system is a prototypical cascade whose activity is closely associated with tissue remodelling and invasive phenotypes of several cancers.1,2 The zymogen plasminogen is ubiquitous in plasma (2 µM) and is known to exert multi-functional activity once activated due to the broad specificity of the terminal protease, plasmin.3 Plasminogen activation is brought about by two principal activators: tissue- and urokinase-plasminogen * To whom correspondence should be addressed. Professor Mark S. Baker, Australian Proteome Analysis Facility Ltd, Building F7B4, Macquarie University 2109, Australia. Telephone, +61 2 9850 6209; Fax, +61 2 9850 6200; E-mail, [email protected]. † Macquarie University. ‡ University of Pennsylvania School of Medicine. § Biogen Idec, Inc..

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Journal of Proteome Research 2007, 6, 1016-1028

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activators (tPA, uPA). Whereas the functions of tPA are predominantly in maintaining hemeostasis (i.e., fibrinolysis) and in neurobiology, uPA-activated plasminogen activation predominantly mediates the physiological and pathological tissue remodelling involving cells.2,4 The expression of a high-specificity glycosyl-phosphatidylinositol (GPI) linked uPA receptor (uPAR) on the surface of many cell types plays a major role in focusing plasmin activity both temporally and spatially, whereas surface co-localization of plasminogen on prolific plasminogen receptors promotes its activation in a positive feedback loop. Cell culture studies show that the receptor ligation of pro-uPA, combined with the binding of plasminogen to the C-terminal lysine residues of a range of plasma membrane proteins strongly enhances enzyme activity due to the reciprocal activation of both pro-enzymes.5 In fact, receptor bound uPA is at least 200-fold more efficient in activating plasminogen compared to free-circulating uPA. Thus, it comes as no surprise that uPA and uPAR have been shown to possess 10.1021/pr060518n CCC: $37.00

 2007 American Chemical Society

research articles

Identification of Lynchpin uPAR-Protein Interactions

greater utility as indicators of metastatic potential in cancer than plasmin activity per se.6 However, it is becoming increasingly evident that the function of the uPA-uPAR system is far more diverse than plasminogen activation alone, and its non-proteolytic involvement in several other cellular processes (e.g., proliferation, adhesion, migration, and chemotaxis) has gained considerable attention.7,8 However, uPAR is a GPI-linked receptor that lacks any transmembrane or cytoplasmic domain, and hence, taken alone, it is incapable of initiating intracellular signalling. However, most findings to date indicate (albeit in an indirect manner) that uPAR is capable of transmitting intracellular signals, most likely by interfacing with the extracellular domains of other transmembrane proteins on the cell surface.9-12 This has resulted in a shift of focus of the uPA-uPAR system toward a new paradigmsone that focuses on the ability of uPA and its receptor to mediate diverse cellular responses, both physiologically and pathologically in a non-proteolytic manner.13 It is assumed that the engagement of uPAR by uPA induces a change in receptor conformation, promoting its lateral interaction with other membrane proteins present in close proximity, possibly in discrete membrane “pools”. This is based on the observation that binding of uPA to uPAR induces adhesion, migration, proliferation, the induction of signalling events, and gene activation ultimately resulting in cellular differentiation.14,15 Some of the recently identified co-receptors include various integrins (both subunits or dimers), the Gprotein coupled receptor FPRL-1, the EGF-receptor (EGFR), the mannose-6-phosphate receptor, the family of low-density lipoprotein receptor-related proteins, p130, and others.8 The interactions between uPAR and these identified proteins are likely to be dynamic and can change depending on the functional state of the cell, the affinity of a given interaction, and the presence/absence of particular proteins on the plasma membrane. Fluorescence resonance emission transmission (FRET) studies in neutrophils demonstrate uPAR co-localization with the CR3 integrin in the resting state and its subsequent relocation to the advancing lamellipodia during migration, with the integrins translocating to the uropod.16 Similarly in this regard, the GPI-anchor of uPAR affords it relatively high flexibility and movement, allowing the receptor to interact dynamically with other proteins on the plasma membrane. Like other GPI-anchored proteins, uPAR has been shown to congregate in “lipid rafts” (islands rich in glycosphingolipids, cholesterol, and signalling components), which provides it with the infrastructure for its association with other co-receptors and downstream signalling intermediates.17 Several other groups have also shown uPAR co-localization in the plasma membrane invaginations known as “caveolae”, which contain signalling complexes together with the cholesterol-binding protein caveolin.10 Wei and co-workers have shown that uPAR binds and stabilizes caveolin/β1 integrin complexes, a disruption of which results in the abrogation of Src and focal adhesion kinase activities intracellularly.10 In this report, we have employed proteomic techniques to profile uPAR-protein interactions that may be of significance in a highly invasive ovarian cancer cell model. By coupling coimmunoprecipitation techniques using highly specific monoclonal antibodies with mass spectrometric identification techniques, we have identified a number of well-known interacting partners of the urokinase receptor, as well as many novel uPAR protein binding partners. Specifically, we identified that uPAR interacts with the integrin β6 subunit and this interaction is

shown to mediate cell proliferation and MAPK signal transduction in ovarian cancer cells.

Experimental Procedures Antibodies and Reagents. Monoclonal antibodies against uPAR (clone R3 and R4) were kindly donated by Dr. Neils Behrendt (Finsen Laboratory, Copenhagen).18 Monoclonal antibodies against the β6 subunit of the Rvβ6 integrin (6.3G9 and 6.2A1) were from Biogen Idec, Cambridge, MA., U.S.A., as previously described.19 Recombinant single chain uPA (scuPA) was from Dr. Douglas Cines (U. Penn, Philadelphia, PA, U.S.A.) whereas recombinant Rvβ6 (extracellular domain) was a kind donation from Dr. Dean Sheppard (UCSF, U.S.A.).20,21 IgG1 isotype control antibodies were purchased from Chemicon (Temecula, CA, U.S.A.). Goat anti-mouse and anti-rabbit horse radish peroxidase conjugated secondary antibodies were purchased from Cell Signalling Technology (Danvers, MA, U.S.A.). Streptavidin-HRP was purchased from Molecular probes (Eugene, OR, U.S.A.). One-dimension SDS-PAGE gels (10%) were purchased from Invitrogen, Australia. All cell culture media used in these experiments were purchased from Gibco, Invitrogen, Australia. Cell Culture. The epithelial ovarian cancer cell line OVCA 429 was kindly provided by Dr. Robert Bast (MD Andersen Cancer Research Centre, Houston, USA). The cells were cultured in DMEM media supplemented with 10% heat inactivated FBS, 100 µg/mL penicillin, 100 µg/mL streptomycin, 10 mM HEPES, and 6 mM L-glutamine. Cultures were maintained at 37 °C in the presence of 5% CO2. Co-immunoprecipitation. For each co-immunoprecipitation experiment, OCVA 429 cells (3 × 107 cells) were harvested and lysed in 3 mL of Triton cell lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, pH 7.0), supplemented with cocktail protease inhibitors (Roche Applied Sciences), EDTA; 100 µM and sodium orthovanadate; 200 µM as per published procedures.22 After incubating on ice for 10 min, the cells were sonicated for 60 s (15-s bursts), centrifuged at 15.8 × g for 10 min at 4 °C, and the clarified supernatant recovered. Total protein concentrations were determined using the Fluoroprofile protein quantitation assay kit (Sigma, Australia). Equal quantities of the cell lysates (2 mg/mL) were precleared with protein-G Sepharose (50 µg) and streptavidinagarose (50 µg) for 2 h prior to incubation with 50 µg (1 µg/µL) of monoclonal antibodies to either uPAR (clone R4), β6 (clone 6.3G9), or an IgG1 isotype control antibody at 4 °C overnight. Immunocomplexes were precipitated with protein-G sepharose beads and extensively washed with Triton wash buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100, pH 7.0) to remove nonspecific binding proteins. The immunocomplex components were eluted off the beads in two fractions to facilitate the identification of components both by mass spectrometry and Western blotting. The beads were initially incubated in a low pH glycine buffer (100 mM glycine, 150 µL, pH 2.5) for 15 min, centrifuged, and supernatants collected in a 1/10 volume of 0.5 M NH4HCO3, pH 8 (fraction 1). Subsequently, the beads were boiled in 4 × SDS sample buffer for 15 min to remove any residual, high-affinity bound proteins (fraction 2). The fractions were analyzed by one-dimensional SDS-PAGE under non-reducing conditions for mass spectrometric analysis or transferred to nitrocellulose membranes (Bio-Rad, Australia) for immunodetection. SDS-PAGE Analysis of Protein Extracts. Co-immunoprecipitated samples (10 µg) were mixed with NuPAGE LDS sample Journal of Proteome Research • Vol. 6, No. 3, 2007 1017

research articles buffer (4 µL) in the absence of reducing agent, loaded onto 10% NuPAGE Bis-Tris PAGE gels (Invitrogen, Carlsbard, CA), and electrophoresis performed as per the manufacturer’s instructions. Gels were stained using Sypro Ruby (Molecular Probes, Eugene, OR) and visualized using a Typhoon laser scanner (GE Healthcare, Uppsala, Sweden). The gel was then counter stained with 0.25% Coomassie Brilliant Blue G-250 prior to excision of bands for mass spectrometric analysis. For Western blotting, the blots were probed with biotinylated monoclonal antibodies (1:1000 dilution) overnight and specific proteins detected by horseradish peroxidase conjugated streptavidin (1:2000 dilution; Molecular Probes, Eugene, OR). Visualization was performed using enhanced chemiluminescence (ECL Plus, GE Health care). Protein Identification by Mass Spectrometry and Database Searching. Protein bands detected after co-immunoprecipitation were excised for mass spectrometric analysis. After reduction and alkylation, the gel plugs were digested overnight at 37 °C using trypsin (150 ng, 25 mM NH4HCO3, pH 7.8). The resultant tryptic peptides were analyzed by nanoLC-MS/MS using a QSTAR XL mass spectrometer (Applied Biosystems, Foster City, CA) operated in an information dependent acquisition mode (IDA). Briefly, the peptide digest (15 µL) was injected onto a Michrom Peptide Captrap (Michrom; Auburn, CA) for preconcentration and desalted with 0.1% formic acid at a flow rate of 10 µL/min. The precolumn was then switched into line with the analytical column containing C18 RP silica (150 µm × 100 mm, Protocol C18, 3 micron, SGE, Australia). Peptides were eluted from the column using a linear solvent gradient from H2O:CH3CN (95:5, + 0.1% formic acid) to H2O:CH3CN (50: 50, + 0.1% formic acid) at 500 nL/ min over a 40-min period. The eluted peptides were subjected to positive-ion nanoflow electrospray. Under the IDA mode, TOF/MS survey scans were acquired (m/z 400-2000, 1.0 s), with the four most abundant multiply charged ions (counts > 25) in the survey scan sequentially being subjected to MS/MS analysis. MS/MS spectra were accumulated for 1 s (m/z 50-2000). Peak lists for each LCMS/MS analysis was generated using a mascot script plug-in (mascot.dll; ABI/MDX Sciex analyst) to the Analyst QS software package (version 1.4). Specific criteria used in the generation of the Mascot generic files (.mgf) included the analysis of +2, +3, and +4 precursor ions and the exclusion of MS/MS spectra with less than 10 peaks. All MS/MS peaks below 1% of the overall intensity were removed, the spectra centroided and deisotoped prior to submission for database searching to the Mascot search program (version 1.8). All database searches were performed against the forward and reversed NCBInr database (May 2006) focusing on the Homo sapiens taxonomy subset. The reversed database was generated using a Perl script program that was developed in house. The following parameters were selected for the database searches: selection of trypsin as the cleavage enzyme with a maximum of 1 missed cleavage allowed, detection of monoisotopic masses, peptide precursor mass tolerance of ( 0.6 Da, and the MS/MS product ion tolerance of ( 0.8 Da. Carboxyamidomethylation of cysteine and oxidation of methionine were chosen as the variable modifications. Proteins whose MS/MS data satisfied the following threshold criteria were reported as positive protein hits in the Mascot searches: inclusion of only bold red peptides in protein searches to match protein hits, an ion score cutoff of 40 with a p value of 0.05 to remove ambiguous, low-scoring peptides. 1018

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These threshold criteria were selected based on the determination of the false discovery rates (FDR) for each MS data set.23,24 The FDR was calculated by determining the number of peptides matched against the reversed database as a percentage of peptides identified against the forward database.24 Cell Proliferation Assays. OVCA 429 cells (5000 cells/well) were seeded into 96-well plates (CELLSTAR; Greiner, Frickenhausen, Germany) and cultured for 18 h in serum containing DMEM. After washing with PBS, the monoadherent cells were subjected to a glycine acid shock buffer wash (100 µL) for 3 min at 4 °C to elute receptor bound uPA,25 washed in serumfree media, and then serum starved for 3 h at 37 °C in the presence of 5% CO2. The cells were subsequently treated with neutralizing uPAR-specific antibodies (Clone R3, 50 µg/mL, 100 µL), neutralizing β6-specific antibodies (6.3G9, 10 µg/mL, 100 µL), a mixture of both (R3, 100 µg/mL, 50 µL + 6.3G9, 20 µg/ mL, 50 µL) or an irrelevant, isotype-matched IgG1 control antibody (60 µg/mL, 100 µL) for 2 h prior to stimulation with single chain uPA (scuPA, 10 nM) at 37 °C for 48 h. Cell proliferation was measured using the WST-1 cell proliferation reagent (Roche Applied Sciences, Germany), which provides a colorimetric index of cell viability. In each experiment, basal cell proliferation was determined by assessing the difference in cell viability at time points 0 and after 48 h under serumstarved conditions. The effects of uPA induced cell proliferation was expressed as a the relative increase in the percentage of absorbance compared to the serum-starved controls at 48 h and the effects of neutralizing antibodies on cell growth were expressed as a percentage decrease in absorbance relative to the positive control (10 nM scuPA stimulation after 48 h). uPAR and Rvβ6 Neutralization Antibody Effects on ERK Phosphorylation. To determine whether the inhibitory effects of the neutralizing antibodies on cell proliferation are mediated via the MAPK signalling pathway we proceeded to examine their effects on the ERK 1/2 phosphorylation. OVCA 429 cells (3.5 × 104 cells/well) were seeded in 6-well culture (CELLSTAR; Greiner, Frickenhausen, Germany). The adherent cells were acid shock buffer washed as described earlier to elute endogenous, uPAR-bound uPA. After washing with serum-free media, the cells were pretreated with neutralizing uPAR antibodies (Clone R3, 50 µg /mL, 1 mL), neutralizing β6 antibodies (Clone 6.3G9, 10 µg/ mL, 1 mL), a mixture of both (anti-uPAR, R3; 50 µg/ mL + anti-β6, 6.3G9; 10 µg/ mL, 1 mL), or a nonspecific isotype control IgG1 antibody (60 µg /mL, 1 mL, R&D Systems) for 12 h under serum-free conditions. Basal ERK phosphorylation levels were recorded under serum-starved conditions, whereas the positive control comprised of recording ERK1/2 phosphorylation secondary to stimulation with DMEM containing 10% FBS. uPA mediated ERK phosphorylation was performed by stimulating the adherent cells with 10 nM scuPA for 15 min in the absence/presence of neutralizing antibodies following which the cells were extensively washed in sterile PBS. Cell extracts were prepared in ice-cold Triton cell lysis buffer (150 µL/well) and sonicated on ice for 60 s (4 × 15 s bursts). The extracts were clarified by centrifugation at 15.8 × g for 10 min at 4 °C, and the protein content was determined by Fluoroprofile protein quantitation assay (Sigma). Equal amounts of proteins (5 µg) were Western blotted and probed for phosphorylated ERK1/2 (pThr202/Tyr204, Cell signalling technology). The blots were stripped using standard stripping buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 788 µL of β-mercaptoethanol) at 50 °C for 30 min, washed, reblocked, and

Identification of Lynchpin uPAR-Protein Interactions

Figure 1. SDS-PAGE analysis of uPAR-protein complexes. Proteins were immunoprecpitated from the total cell lysates of OVCA 429 using mouse monoclonal anti-uPAR antibodies (Clone R4) or a mouse monoclonal IgG1 control antibody and resolved on parallel 10% SDS-polyacrylamide gels. The lanes are labeled as follows: A and B correspond to fractions F1 and F2, respectively, from the anti-uPAR co-immunoprecipitation experiment whereas C and D correspond to fractions F1* and F2*, respectively, from the mouse IgG1 control co-immunoprecipitation experiment. Protein bands labeled 1-12 corresponding to visualized bands from lanes A and B were excised for LC-MS/MS analysis to identify uPAR-interacting proteins. Corresponding MW matched bands from the IgG1 control experiment (lanes C and D) are labeled 1*-12* and were analyzed by LC-MS/MS to identify truenegative proteins and exclude false-positive proteins.

subsequently probed with mouse anti-human β-actin (Sigma) to confirm equal protein loading onto the gels.

Results Proteomic Analysis of uPAR Co-immunoprecipitates. Our previous studies have clearly shown that the expression of uPA and uPAR are consistently up-regulated in malignant ovarian carcinomas.26 Additionally, we and others have also demonstrated, using ovarian cancer cell lines as in vitro models, that overexpression of these cell surface proteins strongly correlate with proteolytic degradation of ECM proteins as well as enhanced ERK 1/2 phosphorylation signals.27 On the basis of these previously described characteristics, we affinity purified uPAR-protein complexes from the cell lysates of a malignant ovarian cancer cell line, OVCA 429 and identified the components using proteomic techniques. We chose to use a monoclonal antibody toward uPAR (Clone R4) that does not interfere with uPA-uPAR interactions in order to optimize the immunoprecipitation procedure. One-dimensional SDS-PAGE analysis of the uPAR and IgG1 immunoprecipitated complexes revealed the presence of several protein bands in the Mr ≈ 20220 kDa (Figure 1) including those corresponding to immunoglobulin heavy and light chains (45 and 20 kDa, respectively). Importantly, several faint bands at ∼50-100 kDa, which varied significantly in their intensity between the anti-uPAR (Figure 1 A,B) and IgG1 negative control (Figure 1 C,D) pull-down experiments, were also observed. Visualized protein bands from the uPAR co-immunoprecipitation experiments (Figure 1, No: 1-12) and the corresponding

research articles molecular weight matched bands from the IgG1 isotype control experiments (Figure 1, No: 1*-12*) were excised from the SDSPAGE gel, digested and the analyzed by LC-MS/MS. By imposing a stringent individual MS/MS ion cutoff score of >40 on the proteins identified from the MASCOT database searches, the false discovery rate of proteins identified due to poor ion spectra was reported to be less than 1% with the exception of a single dataset (corresponding to Band no. 8, Figure 1), which reported a FDR of 1.17% (Table 1). This overall, low falsepositive rate allowed high confidence of protein assignment in each band. Table 1 lists the proteins that were identified from each excised band of the uPAR and corresponding IgG1 negative control immunoprecipitation experiments. Proteins detected in both control and uPAR pull-downs were eliminated from further consideration. Of the proteins found specifically in the uPAR pull-downs, we identified 5 proteins that have previously been shown to either interact or associate with uPAR and/or influence uPA-uPAR mediated plasminogen activation functions. These include thrombospondin-1 (a matrix protein), R-enolase (a plasminogen receptor), and nucleolin.28-30 In addition, we identified 8 novel uPAR associated proteins, several of which have been independently reported as being relevant in the context of tumor invasion in various cancers. Noteworthy among these are Rvβ6 integrin, galectin-3, gelsolin, ezrin, and stratifin. The reported functions of each of the novel uPAR associated proteins identified in this experiment are elaborated upon in Table 2. Interestingly, we also observed differences in the observed and calculated molecular weights of some of the proteins identified in the 1D-SDS-PAGE gel bands (Table 1). For example, LC-MS/MS analysis of band 4 in the SDS-PAGE gel (Mr ≈ 35-40 kDa) identified both subunits of the Rvβ6 integrin whose calculated molecular weights were 115 and 97 kDa, respectively. The apparent lack of concordance between the calculated and observed molecular weight of proteins here might be relevant considering the close proximity of these proteins to cell surface proteolytic machinery and shall be expanded upon in the discussion. Rvβ6 Integrin Co-purifies With the Urokinase Receptor. Of particular note in the identification of novel uPAR interacting proteins was the detection of the epithelial restricted β6 integrin subunit in several of the gel bands from the uPAR immunoprecipitated complex. This is an interesting observation considering that the Rvβ6 integrin, which forms a heterodimer with the Rv integrin subunit, has previously been implicated as a critical player in the malignant transformation of epithelial cancer.31,32 Indeed, β6 integrin is not detected in normal epithelial tissues but frequently is up-regulated during inflammation, wound healing, and more recently, tumorigenesis in epithelial cells.33,34 To confirm the proteomic identifications, Western blotting of the uPAR immunocomplexes was carried out using anti-β6 mAb (Clone 6.2A1), which clearly showed the co-isolation of two reactive bands (Figure 2A) whose molecular masses corresponded with the mature and immature (incompletely glycosylated) forms of the β6 integrin subunit from OVCA 429 cell lysate.35 Not surprisingly and in agreement with our proteomic findings, immunoreactive β6 subunit was not detected in isotype control pull-down experiments (Figure 2B). Further validation of the potential interaction between uPAR and β6 integrin was performed by conducting reverse immunoprecipitation on the OVCA 429 cell lysate using an Rvβ6 integrin specific monoclonal antibody (Clone 6.3G9). The detection of uPAR in the β6 pull-downs but not in isotype control experiment (Figure 2C,D) clearly demonstrates a speJournal of Proteome Research • Vol. 6, No. 3, 2007 1019

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Table 1. Proteins Identified from the Immunoprecipitation of uPAR Complexesa

band #/MW

1

2

3

protein

Myosin-1 (Myosin heavy chain, nonmuscle IIb) Integrin beta-6 subunit Cytokeratin 9 Heat shock 70kDa protein 8 isoform 1 Keratin 10 BiP L-plastin polypeptide Alpha-tubulin Annexin A2, isoform 2 Gelsolin isoform b Thrombospondin-1 N-Terminal Domain Signal recognition particle 68 Ezrin (p81)

NCBI accession

gi|28317

calc MW (Da)

#peptides/sec coverage (%)

Mascot score

9/7

474

gi|9446402 gi435476

89244 62320

2/5 2/3

123 107

gi|5729877

71082

12/31

746

Y/5

gi|40354192 gi|1143492 gi|190030 gi|37492 gi|16306978 gi|4504165 gi|88191913

59020 72185 64352 50810 38822 86043 23668

14/24 10/19 5/12 4/11 2/9 2/4 1/10

701 651 308 248 122 100 74

Y/7 Y/1 N Y/4 N N N

gi|6690741

70712

1/3

68

N

gi|119717

69470

1/1

53

N

gi|67782365 gi|55665783 gi|338695 gi|303618 gi|9446402

51354 28890 50240 57065 892449

12/33 8/24 7/24 1/2 1/3

725 565 395 69 59

4

Cytokeratin 8 Enolase 1, (alpha) Annexin A2, isoform 2 67 kda laminin receptor Integrin alpha-V precursor Integrin beta-6 subunit

gi|181573 gi|13325278 gi|16306978 gi|250127 gi|4504763 gi|9446402

53529 47139 38822 32860 117062 89244

21/39 6/20 4/15 2/7 1/1 1/3

1139 364 255 125 61 51

5

Keratin 1 Keratin 10 Peroxiredoxin 6 Epithelial cell marker protein 1 Nucleolin Beta-tubulin

gi|11935049 gi|623409 gi|77744395 gi|62131678 gi|189306 gi|338695

661987 57384 25133 26658 76355 50240

12/26 12/31 3/19 2/9 2/3 1/4

746 686 170 122 99 52

Keratin 1 Hornerin Putative MAPK activating protein Dermcidin, precursor Cofilin Galectin -3 binding protein

gi|11935049 gi|28557150 gi|31455537

66198 48797 43037

24/40 2/7 2/7

1280 120 96

gi|16751921 gi|5031635 gi|5031863

11391 18719 66202

1/12 1/15 1/2

75 68 45

Nonmuscle myosin heavy chain IIb Plectin Integrin beta-6 subunit

gi|1346640

229824

38/24

2281

gi|1296662 gi|9446402

533408 89244

2/1 1/3

95 66

90 kDa heat shock protein Putative MAPK activating protein Aminopeptidase

gi|306891 gi|31455537

83584 43037

17/27 4/21

998 228

gi|1657268

99151

1/1

45

Cytokeratin 8 Phospholipase C-alpha Annexin A2, isoform 2

gi|181573 gi|303618 gi|16306978

53529 57065 38822

18/42 2/4 2/9

998 102 109

Cytokeratin 18 Eukaryotic translation initiation factor 4A1

gi|30311 gi|4503529

47305 50457

21/52 3/10

1280 155

Enolase 1, (alpha) Annexin A2, isoform 2 67 kda laminin receptor Interleukin enhancer binding factor 2, 45kD

gi13325278 gi|16306978 gi|250127 gi|13385872

47481 38822 32860 43263

8/24 5/20 2/10 2/13

482 310 144 102

Mutant beta-actin Putative MAPK activating protein

gi|28336 gi|31455537

42128 43037

1/3 1/2

65 61

7

8

9

10

11

12

detection in IgG1 Co-IP/ # peptides.

229824

Keratin 7 Tropomyosin 3 Beta-tubulin Phospholipase C-alpha Integrin beta-6 subunit

6

FDR (%)

Y/8 0.1

0.41

0.1

0.65

0.1

0.1

N Y/9

Y/6 Y/12 Y/1 N N N N Y/4 N N N Y/2 Y/7 Y/2 N N Y/4 Y/8 N N Y/1 N N Y/132

0.1

1.17

Y/48 N N N N

0.1

0.54

0.1

0.1

Y/17 N N Y/5 N N Y/9 N Y/1 Y/4 Y/3

a Proteins identified by LC-MS/MS analysis of uPAR immunocomplexes from OVCA 429 cell lysates (marked in bold). Proteins are grouped into the 1D SDS-PAGE band that they were identified from (Figure 1). False-positive proteins that were co-isolated and identified from the negative control IgG1 isotype antibody immunocomplexes, peptides identified, sequence coverage, and Mascot scores are also enumerated.

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Identification of Lynchpin uPAR-Protein Interactions

Table 2. Unique uPAR Binding Proteins Isolated from uPAR Co-immunoprecipitation Experiments of OVCA 429 Cell Lysatesa protein

Integrin beta-6 subunit

Gelsolin isoform b

NCBI accession

gi|9446402

gi|4504165

MS/MS sequence

ion scores

K.DSGNILQLIISAYEELR.S

40

Epithelial restricted integrin.

K.LVQNNVLLIFAVTQEQVH LYENYAK.L

86

Fibronectin receptor.

R.EVQGFESATFLGYFK.S

58

R.DPDQTDGLGLSYLSSHI ANVER.V

42

Thrombospondin-1 N-Terminal Domain

gi|88191913

NRIPESGGDNSVFDIFELTG AAR.K

74

L-plastin

gi|303618

K.LSPEELLLR.W

47 84

Ezrin (p81)

gi|119717

K.MINLSVPDTIDER.T + Oxidation (M) R.YTLNILEEIGGGQK.V K.FSLVGIGGQDLNEGNR.T R.VNHLYSDLSDALVIF QLYEK.I K.IGFPWSEIR.N

53

phospholipase C-alpha

gi|303618

R.LAPEYEAAATR.L

69

K.YGVSGYPTLK.I

52

R.ELSDFISYLQR.E

50

polypeptide

enolase 1, (alpha)

67 kda laminin receptor

integrin alpha-V precursor

gi13325278

gi|250127

gi|4504763

putative functions

R.GNPTVEVDLFTSK.G

52 67 58

105

R.AAVPSGASTGIYEAL ELR.D K.DATNVGDEGGFAPNIL ENKEGLELLK.T R.YISPDQLADLYK.S

70

K.AGYTDKVVIGMDVAA SEFFR.S R.IGAEVYHNLK.N K.TIAPALVSK.K K.VVIGMDVAASEFFR.S R.AIVAIENPADVSVISSR.N

51 48 47 43 88

K.FAAATGATPIAGR.F

55

K.VDLAVLAAVEIR.G

61

64 56

Activation of latent TGF-β. Potential marker of EMT. Actin regulatory protein associated with cell growth and motility. Decreased expression in malignant cancers (e.g., breast, ovaries, colon, prostrate, etc). Loss of expression indicative of poor prognosis and malignancy. Regulates angiogenesis and myofibroblast growth and migration. Activates latent TGF-β on cell surface. Inhibits MMP activity. Increased expression in several cancers, including ovarian cancer. Enhances proliferatory and invasive potential in colon cancer. Capable of modulating integrin functions.

Membrane cytoskeletal protein of the ERM family. Regulates cell adhesion, motility and survival. Increased expression reported in metastatic pancreatic, colorectal and breast cancer cells. Involved in tyrosine kinase mediated signal transduction. Cleaves uPAR GPI anchor Known to influence growth-factor mediated cell motility and cancer cell invasiveness. Activates cofilin, profilin and actin mediated cytoskeleton changes. Glycolytic enzyme

reference

33, 37, 75, 84

85-87

88-90

91-93

94-98

99-101

29, 102

Binds plasminogen when expressed on the surface of cells. Enhances activation to plasmin by presenting lysine cleavage sites. Prevents plasmin inactivation by anti-plasmin.

Cell surface laminin receptor. Increased expressions reported in breast, ovarian, non-small cell lung and prostrate cancers and strongly correlate with malignant potential. Proposed to increase invasion by inducing conformational changes in laminin for proteolytic degradation. Promotes adhesion to ECM protein Vitronectin and laminin. Promotes MAPK activation. Increased expression associated with increased uPAR expression and cell migration/invasion.

103, 104

105-107

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Table 2. (Continued) protein

epithelial cell marker protein 1 (stratifin)

NCBI accession

gi|62131678

MS/MS sequence

ion scores

K.DSTLIMQLLR.D

66

K.VAGMDVELTVEER.N

56

Nucleolin

gi|189306

K.NDLAVVDVR.I K.VTQDELKEVFEDAA EIR.L

42 57

cofilin

gi|5031635

R.YALYDATYETK.E

68

Galectin-3 binding protein

gi|5031863

R.SDLAVPSELALLK.A

45

putative functions

reference

Influences cell-cycle progression, apoptosis and mitogenic signalling. Epithelial restricted protein. Tumor suppressive; inactivation leads to immortalization.

66, 108

Putative marker of tumor angiogenesis. Binds to basic FGF, midkine (13 KDa cytokine). Acts as a shuttle protein from cell surface to nucleus. Directly linked to HPV-18 induced cervical carcinogenesis. Influences integrin mediated actin reorganization. Promotes continued extension of leading edge of cells. Influences motility and migration based on integrin-ECM interactions. Known ligand of cell surface glycoprotein, galectin-3 Promotes homotypic cell adhesion by interacting with integrins. Increased expression associated with malignant cell transformation

28, 61

56, 59, 109, 110

62, 63, 65

a This table is a filtered list of Table 1 and includes unique peptides identified by MS/MS analysis (only bold red peptides), respective ion scores, and their putative functions.

cific interaction of uPAR with the epithelial restricted integrin subunit β6 in OVCA 429 cells. Further studies are currently being conducted to identify whether a similar interaction as such exists in other malignant ovarian cancer cell lines as well as “map” the precise epitope sites of interactions between the two proteins. uPAR and β6 Integrin Association Co-ordinately Regulate Cell Proliferation. The enhanced expression of both uPA and uPAR is associated with an increase in tumor cell growth and invasive capabilities.36 In addition, in vitro and in vivo studies by Agrez et al. have demonstrated that the enhanced expression of the Rvβ6 integrin in colon cancer cells promotes tumor cell growth and extracellular matrix degradation via direct interactions with the MAP kinase signalling pathway.37,38 As exogenous scuPA is known to promote ovarian cancer cell proliferation through uPAR engagement,39 we sought to determine whether this could co-ordinately be regulated by the interaction between uPAR and the β6 integrin subunit. Elution of cell surface, receptor bound uPA by glycine acid shock buffer washing and subsequent stimulation of the cells with 10 nM scuPA for 48 h, resulted in a 47 ( 14% increase in cell proliferation relative to the unstimulated, serum-starved OVCA 429 cells (10 nM uPA vs S/S 48 h, Figure 3). Pretreatment of the cells with uPAR neutralizing antibodies (Clone R3, 50 µg/mL, which interferes with uPA-uPAR binding) reduced uPA driven proliferation by approximately 55 ( 12% compared to the scuPA stimulated cells (anti-uPAR vs 10 nM uPA, Figure 3) confirming that uPA-mediated cell growth is dependent on uPAR ligation. Interestingly, pretreatment of the cell monolayers with β6 function-blocking antibodies (Clone 6.3G9, 10 µg/ mL which inhibits the integrins ability to bind fibronectin and/ or activate latent TGF-β1) also inhibited uPA-stimulated proliferation at a similar level (58 ( 9% Anti-β6 vs 10 nM uPA) while preincubation with an irrelevant IgG1 isotype control antibody had no statistically significant effect (determined using the student t test, p < 0.01) on proliferation compared to uPA 1022

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stimulation of OVCA 429 (19 ( 17%, IgG1 control vs 10 nM uPA). To examine whether uPAR and Rvβ6 promoted uPAmediated cell proliferation via mutually independent pathways, we further measured proliferation under the combined blocking effects of both antibodies. Combining anti-uPAR and anti-β6 neutralizing antibodies did not demonstrate an additive inhibitory effect on uPA mediated cell proliferation (61 ( 12%, Figure 3), suggesting that the inhibitory effects are probably mediated via the same pathway. We also found that the inhibition of uPA driven cell proliferation was greater than that of the serum-starved controls indicating that the antibodies might act on the basal cell proliferation rate. However, pretreatment with neutralizing antibodies on unstimulated, nonacid washed OVCA 429 cells did not result in a reduction of cell growth below serum-starved control levels (unpublished observations). These observations were not surprising considering that OVCA 429 cells produce high levels of endogenous uPA.40,41 It is possible that preclearing and subsequent blocking of available urokinase receptors and/ or its lateral interacting partners (such as Rvβ6 integrin) could prevent both exogenously added and/or endogenous produced uPA from further propagating cell growth, effectively driving cell proliferation below that of the serum starved cells that still produce uPA albeit at basal levels. This data strongly suggests that β6 integrin supports the propagation of uPA initiated cellular proliferation either via an interaction with uPAR or with uPA, in an uPAR independent manner. Whatever the mechanism, it is clear that proliferation can be co-ordinately regulated by some interaction involving uPA:uPAR and the β6 integrin subunit axis. uPA Stimulated ERK Phosphorylation Can Be Abrogated by β6 Blocking Antibodies. Ligation of uPAR by uPA results in the phosphorylation of the ERK 1/2 in different ovarian, colon, and squamous cancer cell lines.12,22,42 Ossowski and others have previously shown that ERK phosphorylation after uPA engagement of uPAR occurred through R5β1 integrin interactions in

research articles

Identification of Lynchpin uPAR-Protein Interactions

an uPAR-β6 association. Incubation with an isotype matched (IgG1) antibody demonstrated a marginal decrease in ERK 1/2 phosphorylation secondary to scuPA stimulation (18 ( 10%) but was greater than serum-mediated ERK activation indicating a small degree of nonspecific inhibition attributable to antibody concentrations.

Discussion

Figure 2. Western blotting and reverse co-immunoprecipitation validation of OVCA 429 extracts. Precleared OVCA 429 cell lysates were subjected to immunoprecipitation using (A) monoclonal anti-uPAR (Clone R4), (B) isotype matched control IgG1 antibody, (C) monoclonal anti-β6 integrin subunit (Clone 6.3G9), or (D) isotype matched control IgG1 antibody. The immunoprecipitated fractions were western blotted and probed for the presence of β6 (A and B) using anti-β6 (Clone 6.2A1) or uPAR (C and D) using anti-uPAR (Clone R3). Lanes are designated as follows: (M) molecular weight markers, (OVCA 429) - OVCA 429 whole-cell lysate, and (IP) - immunoprecipitated fractions. Western blotting confirmed the presence of β6 integrin subunit in the total cell lysate and in the anti-uPAR immunoprecipitated fractions while none was detected in the IgG1 isotype control (A and B). Similarly, uPAR was detected in OVCA 429 whole-cell lysates as well as the anti-β6 immunoprecipitated fractions whereas no bands were detected in the IgG1 isotype control (C and D), confirming a physical association between uPAR and Rvβ6 integrin.

malignant Hep3 cancer cells.12 As uPAR lacks a transmembrane domain, in our studies we proceeded to examine whether ERK phosphorylation secondary to uPA stimulation in OVCA 429 cells is similarly influenced by potential uPAR-β6 interactions. Stimulation of serum-starved OVCA 429 cells with 10 nM scuPA increased ERK phosphorylation by 56 ( 3% compared to the baseline serum-starved cells (Figure 4A,B). Inhibiting uPA-uPAR binding by preincubation with anti-uPAR neutralizing antibodies reduced uPA induced ERK phosphorylation by 40 ( 6 % highlighting uPAR’s role in MAPK activation. Similarly, pretreatment with the β6 integrin function-blocking antibody (Clone 6.3G9) reduced uPA mediated ERK activation by about 45 ( 5% in the ovarian cancer cell line (Figure 4). These results serve to highlight the contention that the β6 integrin subunit does indeed contribute to uPA mediated ERK activation through some type of interaction with uPAR. Interestingly, the combined neutralization of both uPAR and β6 integrin resulted in a synergistic inhibitory effect on ERK1/2 phosphorylation (57.4 ( 2%, Figure 4 A&B) suggesting that physical cross-talk between uPAR and/or uPA with β6 integrin is an important part of the pathway of MAPK signalling by uPA in this cell model. However, considering that ERK activity is critical to cell growth, the lack of a similar synergistic effect on cell proliferation seems to suggest that persistent and sustained ERK activity might be the net effect of several pathways and not exclusively

The involvement of the urokinase plasminogen activator (uPA) and its receptor (uPAR) in the pathology of human cancers has been well documented.43-45 High expression levels of both these proteins have been reported in several human cancers including that of the breast, lung, bladder, prostrate, liver, pleura, pancreas, ovaries, and brain.45 Cancers with elevated uPA and/or uPAR levels are often associated with poor prognosis, lowered survival rates, and an increased preponderance toward evidence of cancer metastasis.44,46 The increased expression of uPA and/or uPAR in epithelial cancer was initially presumed to facilitate only the dysregulation of plasmin activity and the consequent degradation of extracellular matrix barriers, a characteristic feature of tumor invasion. However, increasing evidence now serves to suggest that the uPA-uPAR system functions in a more diverse way than simply plasminogen activation and recently its nonproteolytic contribution in several other cellular processes (e.g., proliferation, adhesion, migration, and chemotaxis) has gained increasing evidence.47,48 We hypothesize that ligand binding induces a change in uPAR conformation, enhancing its interactions with auxiliary ligands. This is based on the observation that uPA engagement of its receptor is critical in the induction of adhesion, migration, proliferation, the modulation of signalling systems that result in gene activation, and cellular differentiation in several cell types.8 Structural studies indicate that the three domains of uPAR are organized in an almost globular fashion generating a deep internal cavity into which uPA (via the growth factor domain) becomes buried, leaving the whole external surface of uPAR available for other protein:protein interactions.49,50 Some of the uPAR-protein interactions whose functions have been partially characterized include ECM proteins like laminin and vitronectin, several integrin subunits, G-protein coupled receptor FPRL1/LXAR4.8,51,52 Using sensitive proteomic detection methodologies, we have identified several proteins (Tables 1 and 2) from OVCA 429 cells that are associated with uPAR, likely influencing its expression levels or modulating its functions via direct or indirect means. Interestingly, a number of proteins detected here have previously been reported by others in independent studies to interact with uPAR, indirectly validating the methodology used to identify novel uPAR-protein interactions. For example, in our proteomic experiments, we identified thrombospondin-1 (TSP-1) as one of the proteins that co-immunoprecpitated with uPAR. This matrix protein has been shown to increase expression of uPA and uPAR as well as enhance the invasive phenotype of different cancers in a TGF-β dependent and independent mechanism.30,53-55 Cofilin, a cytoskeletal protein also identified in the pull-down experiment, is dependent on uPAR and Rvβ3 integrin for its activation (i.e., phosphorylation).56 Loss of uPAR or Rvβ3 expression in glioblastoma cancer cells causes cofilin dephosphorylation, resulting in the inhibition of intracellular signal transduction (pAkt, p38 MAPK and PI3kinase) and subsequent loss of cell migration and invasion properties.56-59 We identified nucleolin, a nucleolar protein that is known to shuttle between the plasma membrane surface and Journal of Proteome Research • Vol. 6, No. 3, 2007 1023

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Figure 3. Effects of uPAR and/or β6 blockade on uPA driven cell proliferation. OVCA 429 cells (5000/well) were seeded in a 96-well plate and serum starved for 3 h. The cells were acid washed and pretreated with anti-uPAR (R4, 50 µg/mL), anti-β6 (6.3G9, 10 µg/mL), a combination of both (R4, 50 µg/mL + 6.3G9, 10 µg/mL), or IgG1 (60 µg/mL) as indicated for 2 h before stimulating with 10 nM scuPA for 48 h. Cell growth was determined by measuring cell viability under the indicated conditions and time points using the WST-1 cell proliferation assay. Basal cell proliferation was determined by measuring cell viability under serum-starved conditions at the start (S/S 0 h) and end (S/S 48 h) of the 48 h incubation period. Stimulation with 10 nM scuPA resulted in a 46.5 ( 14% increase in cell growth compared to the serum-starved controls at 48 h (S/S 48 h). The inhibitory effects of the neutralizing antibodies on uPA induced cell proliferation were expressed as a percent decrease in viable cells relative to uPA stimulation (10 nM uPA) after the 48-h incubation period. Bar graphs are representative of mean ( SD; n ) 9 and statistical significance determined using student t test, p < 0.01.

the nucleus, providing a mechanism for outside-in signalling in cells.60 Nucleolin has been shown to physically interact and complex with uPAR and casein kinase 2 (CK2), which results in the induction of uPA mediated proliferation in vascular smooth muscle cells.28,61 We also were able to expand on the repertoire of currently known uPAR associated proteins and identify several other new candidate proteins that appear to associate with the receptor in the ovarian cancer cell line, OVCA 429. Although the expression and/or functions of several of these identified proteins have been found to be dysregulated in malignant cancers, their association with uPAR has not previously been demonstrated. Table 2 enumerates these candidate proteins and briefly describes their reported role(s) in promoting tumorigenesis. A few examples include galectin-3 binding protein (LG3BP), a secreted glycoprotein that is a known ligand of Galectin-3 (Gal-3).62 Both gal-3 and its ligand have been found to be overexpressed in several cancers including that of the stomach, liver, colon, ovaries, and neuroglia cells.63 Galectin-3 is phosphorylated by CK-2, which in turn regulates MAPK activation, thereby controlling mitogenic properties.64,65 Another novel candidate identified was the epithelial cell marker protein, stratifin. It belongs to the family of 14-3-3σ acid dimeric proteins and plays an influential role in regulating multiple signalling pathways via its phosphoserine/threonine binding domains.66 Protein-protein interactions involving stratifin have been reported to influence cell cycle progression, MAPK activation, and the co-ordination of integrin signalling.66 Alterations in 14-3-3 σ expression have been associated with several cancers.67 We, along with others, have previously demonstrated that Rvβ6 integrin expression correlates with that of uPA and uPAR levels in ovarian cancer cell lines and is associated with the development of an invasive phenotype.27 However, here for the 1024

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first time we have been able to demonstrate using orthogonal immunospecific approaches combined with proteomics that uPAR and the β6 integrin co-purify each other suggesting a physical association. We extended this by partially characterizing the functional consequences of such an interaction. The β6 integrin subunit is unique in that it is expressed only in association with the Rv subunit and is restricted to epithelial cells of the skin and mucosal surfaces.68 Although low or absent in normal epithelial cells, expression is often up-regulated in specific growth emulating conditions such as inflammation, wound healing, fibrosis and more recently a variety of epithelial tumors such as malignant colonic, ovarian, pancreatic, ovarian, and oral carcinomas.33,37,69,70 On the basis of these observations, it has recently been proposed that the expression of the β6 integrin subunit is related to the development of an epithelial to mesenchymal transformation (EMT) in these cancers, enhancing their metastatic potential.32 uPAR as an Rvβ6 Integrin Ligand. The demonstration of a physical association between uPAR and the β6 integrin points to a novel pathway by which uPA mediates tumor growth in epithelial cancers. In almost all the epithelial cancer models examined, Rvβ6 has been reported to be concentrated at the invading margins of the epithelial cells. This conveniently colocalizes in the same area as concerted proteolytic activities in cancer cells.34,68,71,72 This prompts the question of whether expression of the β6 subunit exerts any influence on the expression and/or function of proteolytic machinery in malignant cancer cells or vice versa? There is indirect evidence to support the hypothesis that the increased expression of the integrin is strongly associated with enhanced proteolytic activity in cell culture studies.71,73,74 We have previously shown that ovarian cancer cell lines that constitutively express high levels of β6 integrin also have enhanced HMW-uPA, pro-MMP-2 and -9 expression, increase

Identification of Lynchpin uPAR-Protein Interactions

Figure 4. Effects of uPAR/Rvβ6 blockade on ERK 1/2 phosphorylation. (A) OVCA 429 cells were acid shock buffer washed and cultured in serum-free media for 12 h in 6-well culture plates in the presence/absence of uPAR and/or β6 specific-neutralizing antibodies as indicated. The cells were treated with 10 nM scuPA or media supplemented with 10% FBS as indicated for 15 min; cell extracts were obtained and subjected to immunoblot analysis to determine for phosphorylated ERK. The blots were subsequently stripped and re-probed for β-actin to confirm equal protein loading and normalization. (B) Semiquantitative densitometric analysis of data in 4A after normalization to β-actin levels. Bar graphs represent the percent increase/decrease in ERK phosphorylation relative to stimulation with serum containing media (FBS; control). The graph is representative of data from 4 independent experiments after normalization to β-actin levels and error bars indicate ( std. deviation. Serum starvation decreased ERK phosphorylation by 40% whereas scuPA stimulation resulted in a 56 ( 3% increase in phosphorylation compared to serumstarvation. Pretreatment with anti-uPAR (Clone R3, 50 µg/mL), anti-β6 (Clone 6.3G9, 10 µg/mL) or a combination of both antibodies (40 µg/mL anti-uPAR + 10 µg/mL anti- β6) resulted in a 40 ( 6, 45 ( 5, and 57.4 ( 2% decrease in ERK phosphorylation, respectively. Pretreatment with an isotype control IgG1 antibody (60 µg/mL) demonstrated a marginal decrease in phospho-ERK levels compared to uPA stimulation (18 ( 10%) but not FBS stimulated controls indicating a nonspecific antibody mediated inhibitory effect.

plasmin mediated basement membrane degradation and display high pERK 1/2 activity.27,75 Our new data extends these findings by demonstrating that β6 integrin could mediate uPA dependent cell proliferation and intracellular signal transduction, (i.e., ERK 1/2) by interacting with uPAR in ovarian cancer cell lines (Figure 3). A number of in vivo studies support a role for β6 integrin in inducing an invasive phenotype in cancer cells, particularly in a nonproteolytic dependent fashion. De novo expression of the β6 subunit in the colon cancer cell lines enhances the ability of the transfected cells to form aggressive tumors in nude mice compared to their β6 deficient counterparts.37 Conversely, the down regulation of β6 in these cells results in the inhibition of tumor growth in vivo as well as a concomitant decrease in MAPK activity. In agreement with these previously reported

research articles findings, our ERK inhibition assays found that uPA-mediated ERK 1/2 phosphorylation was effectively inhibited when the β6 subunit, which is known to mediate latent TGF-β activation, was abrogated by an Rvβ6 neutralizing antibody (6.3G9). This is quite interesting considering that the β6 subunit comprises a unique 11 amino acid sequence within its cytoplasmic domain that is capable of binding phosphorylated ERK 1/2.38 Mutational deletion of this terminal sequence is known to abrogate ERK phosphorylation, decrease MMP-2 activity, and reduce tumor growth in vivo.74 Thus, the ability of uPA to mediate ERK 1/2 phosphorylation could be dependent on a physical interaction between its receptor, uPAR, and the β6 integrin subunit. However, we also observed a synergistic inhibition of ERK 1/2 phosphorylation when both uPAR and the β6 subunit were blocked simultaneously compared to uPAR or β6 blockage alone (Figure 3). Our interpretation of this observation is that ERK activation by uPA is not exclusive to β6 and can be mediated by other uPAR-protein interactions or by uPA itself interacting with other low-affinity receptors.76 We suspect that this synergistic inhibitory effect might be transient with alternative uPA/uPAR-protein interactions compensating for the deficit in ERK activity over a period of time. This would potentially explain why a similar synergistic effect was not observed in our cell proliferation assay 48 h after uPA stimulation. In any case, our findings support a novel function for the β6 integrin, one which allows it to behave as a transmitter of uPA mitogenic signalling in epithelial cancer cells. Through what pathway does the uPAR-β6 interaction potentially promulgate the mitogenic functions of uPAR? We suspect that the TGF-β signalling pathway might play a key role in mediating some of the pro-metastatic properties of uPA in ovarian cancer, on the basis of our results and those reported by others.37,38,77,78 For example, malignant ovarian cancers have been reported to express higher levels of TGF-β in addition to uPAR and Rvβ6 integrin compared to normal ovarian epithelial tissues.27,75,79 Because the β6 integrin is a potent activator of latent TGF-β, we propose that uPA-mediated cell-proliferation in OVCA 429 may be linked with the integrin interacting with the TGF-β signalling pathway. Although there is currently no direct evidence to demonstrate this, preliminary immunological crossover pull-down experiments (Saldahna and Baker, unpublished data) suggest that this is a plausible explanation considering uPAR and β6 physically contact each other and that the blocking β6 antibody (Clone 6.3G9) against the β6-LAPTGF-β interaction inhibits uPA mediated cell proliferation and MAPK activation. Another observation from the SDS-PAGE analysis of the coimmunoprecipitated fractions was the apparent differences between the experimentally observed and calculated molecular weight of some of the uPAR-associating proteins (Table 1). Discrepancies between the observed and predicted molecular weight of proteins is not uncommon, and recent reports indicate that the number of proteins varying in their observed molecular weights (as determined by 1D-SDS-PAGE and MS analysis) can range from 20 to 55%.80,81 There are several biologically relevant explanations that can account for these discrepancies. For example, the migration of a protein at a higher than expected molecular weight in SDS-PAGE can be attributed to post-translation modification (PTM) of the protein and/or oligomerization. Additionally, the migrations of proteins at lower than calculated molecular weight may occur due to Journal of Proteome Research • Vol. 6, No. 3, 2007 1025

research articles alternative splicing, proteolytic processing, and removal of signal peptides, precursor fragments, or PTM’s. In this study, we have identified several proteins that putatively associate with uPAR. Given that uPAR anchors proteolytic activity that generates plasmin (a protease with specificity identical to trypsin), it is highly plausible that proteins in close proximity to uPAR might be substrates for the proteolytic activity involved. Based on this conjecture, one explanation to account for the variation in predicted and observed molecular weight as determined by SDS-PAGE is the migration of partially processed proteolytic fragments of the proteins. For example, the β6 integrin subunit was detected in several SDS-PAGE bands ranging from ∼115-40 kDa (Figure 1A,B) despite its calculated molecular weight being ∼97 kDa. Calculation of the theoretical molecular weight of the three possible fragments of the integrin subunit (AA1-372, AA318788, and AA318-372) that include the peptides that were definitively identified by LC-MS/MS analysis (i.e., Lys318-Lys343 and Lys355-Arg372, Table 2) reveal a variable molecular weight of 41, 51, and 6.1 kDa, respectively. At least two of these fragments are consistent with our detection at the observed molecular weight region. Whether the generation of these protein fragments have any biologically relevant functions are currently unknown and is the subject of ongoing investigations. From these observations, one could broadly propose that uPA and uPAR overexpression sub-serve an important function in promoting malignancy, which is by forming dynamic, multiprotein complexes that are capable of generating new tumor promoting factors and/or dysregulating critical growth factor pathways. Our recent data serves to suggest that lynchpin proteins that are frequently up-regulated in malignant ovarian epithelial cancers such as uPA, uPAR, and β6 integrin promotes the formation of novel multi-protein complexes (forthwith referred to as the metastasome) that are otherwise absent or minimally present in normal ovarian epithelial cells. Other lynchpin protein interactions that implicate uPAR (as well as other integrins) in tumorigenesis have previously been reported in the literature. Ossowski et al. have reported that uPAR interactions with the R5β1 integrin are critical to modulating the pro-invasive functions of EGF in Hep3 carcinoma cells via the MAPK pathway.12,82,83 In this proposed model, our data suggests that the co-operative interaction between uPAR and Rvβ6 might influence how uPA and TGF-β support cell proliferation and promote tumorigenesis. The contribution of the other proteins co-isolated with uPAR in the metastasome are the subject of further investigations. Abbreviations: WST-1, (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate); uPA, urokinase plasminogen activator; uPAR, urokinase plasminogen activator receptor; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; GPI anchor, glycosylphosphatidylinositol anchor; FPRL-1, formyl peptide-receptor-like1; FRET, fluorescence resonance energy transfer; mAb, monoclonal antibody; scuPA, single chain uPA; E-64, L-transepoxysuccinyl-L-leucylamido(4-guanidino)butane; IDA, information dependent acquisition; µg, microgram; µL, microliter; TGFβ, transforming growth factor-beta; pAKT, activated Akt; pI3K, phosphoinositide3-kinase; CK-2, casein kinase-2; Gal-3, galectin-3; EMT, epithelial to mesenchymal transformation; RIPA, radio immunoprecipitation buffer.

Acknowledgment. We thank Dr Niels Behrendt and Dr. Gunilla Hoyer-Hansen from the Finsen Laboratory, Rigshos1026

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pitalet, Copenhagen, for the kind gift of the uPAR monoclonal antibodies (R3 and R4). We thank Dr. Dean Sheppard from the Lung Biology Centre, UCSF, U.S.A., for the kind gift of β6 monoclonal antibody (10D5), recombinant Rvβ6 heterodimer protein, and valuable intellectual discussions. We also thank Dr. Jamie Sherman and Mr. Brett Cooke for their assistance in bioinformatic based analyses. We acknowledge Dr. Donna Badgewell and Professor Robert Bast Jr. from the MD Anderson Cancer centre for their contributions of the ovarian cancer cell lines. This project was partially supported by a Macquarie University Research and Developmental grant (MURDG). The research was facilitated by access to the Australian Proteome Analysis Facility Ltd. established under the Australian Commonwealth Government Major National Research Facility Scheme.

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