CEACAM1 functionally interacts with filamin A and exerts a dual role in the regulation of cell migration

Esther Klaile1, Mario M. Müller1, Christoph Kannicht2, Bernhard B. Singer1 and Lothar Lucka1,*

1 Institut für Biochemie und Molekularbiologie, Charité, Universitätsmedizin Berlin, Campus Benjamin Franklin, 14195 Berlin, Germany
2 Octapharma, Molecular Biochemistry Berlin, Arnimallee 22, 14195 Berlin, Germany

* Author for correspondence (e-mail: lothar.lucka{at}charite.de)

Accepted 4 August 2005


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 Materials and Methods
 Results
 Discussion
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The carcinoembryonic antigen-related cell adhesion molecule CEACAM1 (CD66a) and the scaffolding protein filamin A have both been implicated in tumor cell migration. In the present study we identified filamin A as a novel binding partner for the CEACAM1-L cytoplasmic domain in a yeast two-hybrid screen. Direct binding was shown by surface plasmon resonance analysis and by affinity precipitation assays. The association was shown for human and rodent CEACAM1-L in endogenous CEACAM1-L expressing cells. To address functional aspects of the interaction, we used a well-established melanoma cell system. We found in different migration studies that the interaction of CEACAM1-L and filamin A drastically reduced migration and cell scattering, whereas each of these proteins when expressed alone, acted promigratory. CEACAM1-L binding to filamin A reduced the interaction of the latter with RalA, a member of the Ras-family of GTPases. Furthermore, co-expression of CEACAM1-L and filamin A led to a reduced focal adhesion turnover. Independent of the presence of filamin A, the expression of CEACAM1-L led to an increased phosphorylation of focal adhesions and to altered cytoskeletal rearrangements during monolayer wound healing assays. Together, our data demonstrate a novel mechanism for how CEACAM1-L regulates cell migration via its interaction with filamin A.

Key words: CEACAM1, Filamin A, RalA, Migration, Melanoma


    Introduction
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 Introduction
 Materials and Methods
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 Discussion
 References
 
Primary melanomas progress to malignancy via discrete but overlapping stages including dysplasia, radial growth phase, invasive vertical growth phase and metastasis. Alterations in cell-cell and cell-matrix interactions are associated with these stages of tumor progression. Changes in the expression or function of adhesion molecules, such as integrins, CD44, Mel-CAM, ICAM-1 and cadherins, have been reported in the progression of primary melanomas (Bogenrieder and Herlyn, 2002Go; Li et al., 2002Go), and are the main causes for altered adhesion and migration properties of melanocytes leading to invasiveness and metastatic spread.

Recently, CEACAM1 (CD66a), a member of the carcinoembryonic antigen (CEA) family of cell adhesion molecules, was reported to be upregulated in many melanomas, where its expression is associated with unfavorable prognosis and development of metastasis (Brummer et al., 2001Go; Thies et al., 2002Go). By interaction with ß3 integrins, CEACAM1-L was shown to enhance the migratory and invasory potential of melanoma cells (Ebrahimnejad et al., 2004Go). In non-pathologic conditions, it is expressed on the surface of epithelial, endothelial and hematopoietic cells (for review see Obrink, 1997Go). Alternative splicing events generate two major isoforms, CEACAM1-L and CEACAM1-S, which differ in the length of the cytoplasmic tail (Beauchemin et al., 1999Go). Both CEACAM1 isoforms mediate homophilic intercellular adhesion (Ocklind and Obrink, 1982Go) and are used as a receptor by pathogens (Dveksler et al., 1993Go; Virji et al., 1996Go). CEACAM1 triggers various cellular functions, such as proliferation, differentiation, morphogenesis and apoptosis (Busch et al., 2002Go; Huang et al., 1999Go; Kammerer et al., 1998Go; Kirshner et al., 2003Go; Muller et al., 2005Go). The ability to affect cellular signaling pathways is caused by interaction with a variety of cytoplasmic binding partners, such as kinases (Brummer et al., 1995Go; Skubitz et al., 1995Go) and phosphatases (Huber et al., 1999Go). Furthermore, it is linked to the actin cytoskeleton and associated proteins, for example paxillin, tropomyosin and talin (Da Silva-Azevedo and Reutter, 1999Go; Ebrahimnejad et al., 2000Go; Muller et al., 2005Go; Sadekova et al., 2000Go; Schumann et al., 2001Go).

In the present study, we report for the first time an interaction of the cytoplasmic domain of CEACAM1-L with filamin A (FLNa). FLNa mediates at least three functions that are crucial for cell shape modulation and motility (for review see Stossel et al., 2001Go; van der Flier and Sonnenberg, 2001Go). (1) FLNa homodimers crosslink filamentous actin to three-dimensional, orthogonal networks. (2) It anchors actin filaments to cell-extracellular matrix adhesion sites by binding to ß-integrin subunits. (3) FLNa provides a scaffold for small GTPases of the Ras and Rho families. FLNa has been implicated in migration by Cunningham et al. (Cunningham et al., 1992Go), who provided evidence that FLNa expression enhances migration of M2 melanoma cells in a concentration-dependent manner. Later, the small GTPase RalA was found to interact with FLNa in Cdc42-induced filopod production (Ohta et al., 1999Go). Ral proteins function in various cell biological processes, including vesicle trafficking, regulation of cell morphology, migration, transcription and oncogenesis (for review, see Feig, 2003Go). Here, we describe FLNa as a binding partner for human and rodent CEACAM1-L and we present a novel mechanism for how CEACAM1 regulates cell migration via the interaction with FLNa.


    Materials and Methods
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Antibodies, cell lines, vectors and materials
Monoclonal anti-rat CEACAM1 antibody mAb Be9.2 was described previously (Becker et al., 1986Go). Further monoclonal antibodies used were: anti-human CEACAM1/5 (4/3/17, Genovac, Freiburg, Germany); anti-CEACAM1/3/4/5/6/7 (IH4Fc, Immunotools, Friesoythe, Germany); anti-ß1 integrin (#0790), anti-CEACAM8 (80H3) (Immunotech, Marseille, France); anti-ß3 integrin (F11), anti-phosphotyrosine (PY99) (Santa Cruz, CA); anti-paxillin (#349), anti-ß1 integrin (#18); anti-FAK (#77), anti-N-cadherin (#32), (BD/Transduction Laboratories, CA); anti-Vinculin (hVIN-1); anti-ß-actin (AC-15) (Sigma, Deisenhofen, Germany); anti-P-glycoprotein (#C219, Calbiochem, Schwalbach, Germany). Polyclonal antibodies used were: rabbit-anti-human CEA, goat-anti-mouse Cy-2-conjugate and goat-anti-rabbit Rhodamine Red-X-conjugate (Dako, Hamburg, Germany). Control IgG from rabbit preimmune serum was purified with protein A-Sepharose. Anti-E-cadherin mAb (HECD1) was a gift from Ottmar Huber (Berlin, Germany).

M2 and A7 cell lines (Cunningham et al., 1992Go) were kindly provided by Brigitte M. Jockusch and Wolfgang Ziegler (Braunschweig, Germany). Rat brain endothelial (RBE) cells transfected with rat CEACAM1-L were described previously (Muller et al., 2005Go). pCDNA1 vector containing the human CEACAM1-4L cDNA, kindly provided by Björn Öbrink (Stockholm, Sweden), was originally a gift from Thomas R. Barnett (Connecticut). pGEX-4T vector was from Amersham Biosciences (Upsala, Sweden). Restriction and DNA-modifying enzymes were from Fermentas (St Leon-Roth, Germany) or Invitrogen (Karlsruhe, Germany). All other reagents were from Sigma Aldrich (Deisenhofen, Germany) unless otherwise stated. All cDNA constructs used were verified by sequencing (Thermo Sequenase Primer Cycle Sequencing Kit, Amersham Biosciences, Uppsala, Sweden).

Yeast two-hybrid analysis
The yeast two-hybrid MATCHMAKER 3 GAL-4 based system (BD Biosciences Clontech, CA, USA) was used. The cytoplasmic domain of rat CEACAM1-L (aa 447-519, CC1-cyto) (Budt et al., 2002Go) was cloned into the BamHI/PstI sites of pGBKT7. Yeast strain AH109 was sequentially transformed with pGBKT7-CC1-cyto and the rat liver MATCHMAKER cDNA library. Yeast plasmid was extracted using the YEASTMAKER yeast plasmid isolation kit (BD Biosciences Clontech, CA). Specificity of reporter gene activation was confirmed by transformation of prey plasmid alone and by one-on-one transformations of prey plasmid pACT2-FLNa together with pGBKT7-DNA-BD, pGBKT7-53, pGBKT7-Lam (negative controls) or bait plasmid into yeast strains AH109 and CG1945. Clones were tested for growth on selection medium lacking leucine, tryptophan and histidine, or were grown on selection medium lacking leucine and tryptophan for 72 hours and tested for ß-GAL reporter gene activity in filter-lift assays and {alpha}-GAL reporter gene activity in liquid assays (substrate: p-nitrophenyl {alpha}-D-galactopyranoside).

Generation and purification of recombinant proteins
The cDNA encoding aa 2460-2647 of rat FLNa was cloned into the EcoRI and XhoI sites of the pGEX-4T plasmid, creating a GST-FLNa fusion protein. GST-FLNa (47 kDa) and GST (26 kDa) were expressed in E. coli BL21-star (Invitrogen, Karlsruhe, Germany) and purified using glutathione-Sepharose (Amersham Biosciences, Uppsala, Sweden). The generation, expression and purification of the His-tagged cytoplasmic domain of rat CEACAM1-L (aa 447-519, CC1-cyto-His) was done as described (Budt et al., 2002Go). The purified His-tagged cytoplasmic domain of the {alpha}3 integrin subunit was kindly provided by Diana Mutz (Berlin, Germany)

Surface plasmon resonance analysis
The His-tagged cytoplasmic domain of CEACAM1 (CC1-cyto-His) and of the {alpha}3 integrin subunit ({alpha}3-cyto-His, background control) were immobilized on a CM5 sensorchip (BIAcore AB, Uppsala, Sweden) using the Amine Coupling Kit (BIAcore AB, Uppsala, Sweden). For immobilization, 40 µl CC1-cyto-His or 80 µl {alpha}3-cyto-His (each 200 µg/ml in 10 mM sodium acetate, pH 2.7) were applied. All coupling reactions were carried out using a flow rate of 5 µl/minute. Biomolecular interaction analyses were carried out in HBS Buffer (150 mM NaCl, 0.05% (v/v) Tween 20, 3.4 mM EDTA, 10 mM HEPES, pH 7.4) at a flow rate of 10 µl/minute using the BIAcore® 2000 (BIAcore AB, Uppsala, Sweden). Between sample injections the surface was regenerated with 5 µl of 15 mM HCl.

Immunoprecipitation, binding assays and western blotting
For tyrosine phosphorylation, adherent cells were scraped into phospho-lysis buffer (50 mM Tris pH 7.2, 150 mM NaCl, 1% Triton X-100, 0.1% SDS) containing phosphatase inhibitors (1 mM NaVO3, 10 mM Na4P2O6, 100 mM NaF), 1 mM PMSF and a protease inhibitor cocktail (AEBSF, E-64, betastatin, leupeptin, aprotinin, Sigma). In all other cases cells were lysed in HBS (10 mM Hepes, pH 7.2, 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2,1.7 mM KCl2) containing 1% Triton X-100, 1 mM PMSF and protease inhibitor cocktail. For experiments shown in Fig. 2D, 2E (left panel) and Fig. 8A, 1 mg/ml DNaseI and 10 µM cytochalasin D were added. Rat liver was homogenized in PBS, cleared by centrifugation (5000 g), mixed with 5 volumes of HBS (see above) and lysed using a dounce tissue homogenizer. For immunoprecipitations, equal amounts of cleared lysates were incubated with the appropriate protein G-Sepharose-bound antibodies or Ig-controls, washed and subjected to SDS-PAGE (rat CEACAM1) or Tricin-SDS-PAGE (human CEACAM1 and p-Tyr). For affinity binding assays, 10 µg GST or GST-FLNa bound to glutathione-Sepharose were incubated with 5 µg CC1-cyto-His or 5 µg control-His, or RBE cell lysate for 2 hours at 4°C, washed and subjected to SDS-PAGE. Western blotting and subsequent protein detection on nitrocellulose filters by the horseradish peroxidase/ECL-method (Amersham Biosciences) were performed as described previously (Lucka et al., 1999Go). Chemiluminescence and quantification was done using the Fujifilm LAS-1000 digital system. Images were imported into Adobe Photoshop program for compilation.



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Fig. 2. FLNa directly binds to CEACAM1-L. (A) Affinity precipitation of purified CC1-cyto-His or His-tagged control protein by purified GST or GST-FLNa (aa 2460-2647) bound to glutathione-Sepharose. Associated CC1-cyto-His or control-His was detected by mAb anti-Xpress. (B) SPR binding kinetics of GST-FLNa to immobilized CC1-cyto-His. Representative overlaid sensograms illustrating the real-time binding of GST-FLNa at various concentrations (42, 84, 126 and 168 nM from bottom to top) to CC1-cyto-His immobilized on a CM5 chip with substracted background are shown. The inset shows the primary sensogramms of the flow cells with immobilized CC1-cyto-His and immobilized control protein (background) after injection of 168 nM GST-FLNa. RU, relative units. (C) Affinity precipitation of CEACAM1-L from lysates of CEACAM1-L-transfected rat brain endothelial cells (RBE) by purified GST or GST-FLNa bound to glutathione-Sepharose. Associated CEACAM1-L was detected by rat CEACAM1 specific mAb Be9.2. (D) RBE cell lysate and rat liver lysate were immunoprecipitated with mAb Be9.2 or an Ig-control. CEACAM1 was detected with mAb Be9.2. Co-precipitated FLNa was detected by mAb FLMN01. (E) Cell lysates from human colon carcinoma HT29 cells or freshly prepared and PMA-treated human granulocytes were immunoprecipitated with human CEACAM1 reactive mAb 4/3/17 or Ig-control. CEACAM1 was detected with mAb 4/3/17. Coprecipitated FLNa was detected with mAb FLMN01.

 


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Fig. 8. Wounding and adhesion enhance binding of CEACAM1 to FLNa thereby disturbing the interaction of FLNa with the small GTPase RalA. (A) A7-CC1 cells were left untreated, wounded with a multichannel pipette, allowed to re-adhere to laminin-1, or kept in suspension for 1 hour prior to lysis. Lysates were immunoprecipitated with anti-CEACAM1 mAb 4/3/17. Blots of precipitations and of lysates were developed for co-precipitated FLNa with mAb FLMN01 and quantified. Mean amounts of FLNa co-precipitated by CEACAM1 are shown in the diagram with their standard deviations. (n=3) (B,C) Ral activation assay. A7 and A7-CC1 cells were treated as described above and precipitated with Sepharose-bound Ral-GTP binding domain. Blots of precipitations and of lysates were developed for RalA (B) and for FLNa (C) and quantified. Mean amounts of Ral-GTP and of co-precipitated FLNa are shown in the diagrams with their standard deviations (n=3).

 
RalA activation and affinity binding assay
The detection of Ral-GTP was performed using the GST-fused Ral-binding domain of Ral-binding protein 1 (RalBP1) immobilized with glutathione-agarose beads (Upstate, NY). Cells were lysed in HBS (20 mM HEPES pH 7.2, 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 1 mM PMSF and protease inhibitor cocktail), precleared with 50 µl glutathione-Sepharose and precipitated using 10 µg RalBP1 fusion protein. Samples were washed and subjected to western blot analysis.

Cell culture
M2 and A7 cells were transfected with pCDNA1 vector containing human CEACAM1-4L cDNA (Barnett et al., 1989Go), using lipofectamine (Invitrogen, Karlsruhe, Germany), selected by G418-resistance and cloned by limiting dilution. M2, A7 and derived cell lines were grown in MEM medium containing Earles salts, 2% FCS and 8% newborn calf serum. Rat brain endothelial (RBE) cells and HT29 cells were cultured in RPMI medium/10% FCS. Human granulocytes were prepared as reported (Singer et al., 2002Go). Prior to lysis, the granulocytes were incubated with 10 ng/ml PMA in RPMI medium/10% FCS and were allowed to adhere on cell culture plates for 1 hour.

Characterization of M2, A7, M2-CC1 and A7-CC1 cells
For flow cytometric analysis, cells were incubated sequentially with primary antibody (10 µg/ml) and FITC-conjugated goat anti-mouse F(ab')2 (Jackson ImmunoResearch Europe, Cambridgeshire, UK) in PBS at 4°C for 1 hour. Background fluorescence was determined using an unspecific Ig-control instead of primary antibodies. Flow cytometry was performed with a FACScan instrument and analyzed by CellQuest software (BD Biosciences, CA). RNA isolations using RNazol (Peqlab, Erlangen, Germany) and reverse transcriptions using SuperscriptIII (Invitrogen, Karlsruhe, Germany), were performed according to the manufacturer's guidelines. Polymerase chain reaction was performed using CEACAM1 primers with the following sequences: CCAGCAGGAGACACCATGG and AGGTGAGAGGCCATTTTCTTG. Products after 35 PCR-cycles were analyzed by agarose gel electrophoresis.

Wound healing, migration and invasion studies
Wound healing assays: monolayers were wounded 24 hours after plating by scratching with a pipette tip. Debris was removed by washing. Images were taken at 20x magnification (Nicon Ph1 DL; NA 0.4) with a Nicon TMS microscope equipped with a Nicon F-601 camera. Distances between wound edges were measured at five sites/image (n=3). Alternatively, wounded monolayers were subjected to time-lapse studies for 3 hours at 10x magnification using a Zeiss Axiovert 200 microscope equipped with the AxioCam digital system. Transwell migration assays: after coating the outer side of the membrane of Boyden Transwell migration chambers (diameter 6.5 mm, pore size 8 µm; Costar Corp., Cambridge, MA) with 5 mg/l gelatin/PBS for 1 hour at RT, the filters were placed into the lower chamber containing M2 conditioned medium. 1x105 cells were allowed to migrate for 2 hours. Non-migrated cells on the upper membrane were removed with a cotton swab. Migrated cells were fixed in 4% PFA/0.025% saponin/PBS for 30 minutes, stained with crystal violet and counted (n=3 in duplicate, 5 unit fields/chamber were counted). Cell invasion assays were performed using BD Biocoat Matrigel Invasion Chambers (8 µm pore size; BD Labware, Bedford, MA) according to the manufacturer's guidelines. 1x105 cells per well were allowed to migrate towards M2 conditioned medium for 24 hours. Cells were fixed, stained and counted as described for Transwell migration assays.



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Fig. 1 . Identification of FLNa as a CEACAM1-L-interacting protein by yeast two-hybrid analysis. AH109 yeast cells were co-transfected with pACT2-FLNa and pGBKT7-CC1-cyto or negative control pGBKT7-Lam, and tested for ß-GAL reporter gene activity in filter-lift assays (A) and {alpha}-GAL reporter gene activity in liquid assays (B). (C) Schematic representation of FLNa and the partial FLNa cDNA clone found in the yeast two-hybrid screen (ABD, actin binding domain; 1-24, filamin repeats; H, hinge regions).

 
Immunofluorescence
Cells were grown on tissue culture-treated eight-well chamber slides. Cells were fixed for 20 minutes in 4% PFA/PBS at RT and permeabilized with 0.025% saponin or 0.1% Triton X-100 or were fixed for 2 minutes with methanol at -20°C. Slides were blocked with 1% BSA/PBS for 1 hour, incubated with the appropriate primary antibodies (10 µg/ml) for 2 hours, washed and incubated with the appropriate CY2- or Rhodamine Red-X-conjugated secondary antibodies (1:100) and in some cases with TRITC-phalloidin (1 ng/ml) for 1 hour. Samples were mounted in Elvanol (70% PBS, 25% glycerol, 5% Polyviol, 0.5% ß-mercaptoethanol, 1 mg/ml phenylendiamine). Indirect immunofluorescence was visualized at 63x magnification (Zeiss plan-Neofluar; NA 1.25) or 40x magnification (Zeiss plan-Neofluar; NA 0.90) using a Zeiss Axiovert 200 microscope equipped with the AxioCam digital system. In some cases, cells were examined at 63x magnification (Zeiss Plan-Apochromat; NA 1.4) on a confocal laser-beam microscope (Zeiss Axiovert 100 microscope/Micro Systems LSM). Images were imported into Adobe Photoshop for compilation.


    Results
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 Materials and Methods
 Results
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 References
 
Identification of FLNa as a novel CEACAM1-L interacting protein by the yeast two-hybrid technique
A yeast two-hybrid screen against a rat liver cDNA library, using the cytoplasmic domain of rat CEACAM1-L (CC1-cyto) as bait, identified FLNa as a putative CEACAM1-L interacting protein. Other isolated clones encoded either false positives or will be described elsewhere. The specificity of the interaction was verified by one-on-one transformation into yeast strain AH109. Only FLNa/CC1-cyto double transformants tested positive in ß-galactosidase filter-lift assays and {alpha}-galactosidase liquid assays (Fig. 1A,B). Similar results were obtained with yeast strain CG1945 (data not shown). The interacting clone encoded the carboxy-terminal 187 aa of rat FLNa (aa 2460-2647) (Fig. 1C). The sequence comprises 59 aa of filamin-repeat 23, the second hinge region and the complete filamin-repeat 24.

Direct binding of FLNa to CEACAM1-L
Direct binding of FLNa to CEACAM1-L was shown by two in vitro binding assays. In affinity precipitation studies, purified GST-FLNa (aa 2460-2647) bound to glutathione-Sepharose was able to precipitate the purified His-tagged cytoplasmic domain of CEACAM1 (CC1-cyto-His) (Fig. 2A). The specificity of this interaction was confirmed with glutathione-Sepharose-bound GST as well as with a His-tagged control protein. These results were further confirmed by surface plasmon resonance (SPR)-based binding measurements. SPR analyses revealed a concentration-dependent binding of GST-FLNa to CC1-cyto-His, further supporting a direct interaction of both molecules (Fig. 2B). Additionally, GST-FLNa but not GST alone precipitated CEACAM1-L from lysates of CEACAM1-L-transfected rat brain endothelial (RBE) cells (Fig. 2C). To confirm the interaction in vivo, co-immunoprecipitation experiments were performed using anti-rat CEACAM1 mAb Be9.2 or an Ig isotype control. FLNa was co-immunoprecipitated with CEACAM1-L in CEACAM1-L-transfected RBE cells and in rat liver lysates (Fig. 2D). To analyze the interaction in other species, we tested the association of FLNa and CEACAM1 in human cells expressing both proteins endogenously. FLNa was co-immunoprecipitated by human CEACAM1 reactive mAb 4/3/17 from HT29 colon carcinoma cell lysates and from lysates of freshly prepared adherent human granulocytes (Fig. 2E). In all experiments, the amount of co-precipitated FLNa corresponded to 0.5-1.5% of the total FLNa content. This is well in accordance with data on other FLNa interacting proteins (except for actin), that only bind to a small fraction of FLNa (Marti et al., 1997Go; Ohta et al., 1999Go).

CEACAM1 expression in M2 and A7 melanoma cells
To investigate functional aspects of the FLNa-CEACAM1-L interaction, we used the human FLNa deficient melanoma cell line M2 and the derived, full length FLNa-rescued cell line A7 (Cunningham et al., 1992Go). In both cell lines no endogenous CEACAM1 expression could be detected, either by flow cytometric analysis or by western blot or RT-PCR analysis (Fig. 3A-C). Flow cytometric analysis and western blot also revealed the absence of other CEA-family molecules, namely transmembrane CEACAM3 and CEACAM4 as well as GPI anchored CEACAM5, CEACAM6, CEACAM7 and CEACAM8 (data not shown). M2 and A7 cells were transfected with human CEACAM1-4L cDNA, resulting in the cell lines M2-CC1 and A7-CC1, respectively. Stable M2-CC1 and A7-CC1 clones chosen for further investigations showed quantitatively similar CEACAM1-L cell surface and overall expression levels in FACS analysis (Fig. 3A) and western blot (Fig. 3B), where CEACAM1-L migrated as a diffuse band of approximately 130 kDa. The A7-CC1 clones used in this study and the parental A7 cells expressed similar amounts of FLNa (Fig. 3B). The experiments on migration and invasion described below were performed with at least two independent CEACAM1-L-expressing clones with similar results (data not shown). Mock transfectants exhibited the same morphology and behavior as the parental M2 and A7 cells (data not shown).



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Fig. 3. CEACAM1-L transfection of human M2 and A7 melanoma cells. M2 and A7 cells were stably transfected with human CEACAM1-4L cDNA and cloned by limiting dilution, resulting in the cell lines M2-CC1 and A7-CC1. (A) FACS analysis of M2, A7, M2-CC1 and A7-CC1 cells using human CEACAM1 reactive mAb 4/3/17 (bold lines) or an Ig-control (gray area). (B) Western blot of equal amounts of M2, A7, M2-CC1 and A7-CC1 cell lysates, developed with FLNa-specific mAb FLMN01 and human CEACAM1 reactive mAb 4/3/17. (C) RT-PCR analysis of M2, A7, M2-CC1 and A7-CC1 cells using CEACAM1-specific primers. (D) Cell lysates of A7-CC1 cells were immunoprecipitated with human CEACAM1 reactive mAb 4/3/17 or an Ig-control. CEACAM1 was detected with mAb 4/3/17. Co-immunoprecipitated FLNa was detected with mAb FLMN01. A7-CC1 cells were grown sparsely (E) or were allowed to adhere for 1 hour to laminin-1 (F) and double-stained for CEACAM1 (mAb 4/3/17, red) and FLNa (mAb FLMN01, green) and visualized by confocal microscopy. Co-localization is shown in the merged images (yellow). The white lines in the merged images show the courses of the Z-scans. Bars, 5 µm.

 
The interaction of FLNa and CEACAM1-L was confirmed in the A7-CC1 double transfectants by co-immunoprecipitation of FLNa by mAb 4/3/17 mAb (Fig. 3D). Confocal images of sparsely grown A7-CC1 cells showed a co-localization of FLNa and CEACAM1-L mainly at cell-cell contacts (Fig. 3E). When cells were allowed to adhere on laminin-1 for 1 hour, both proteins co-localized strongly in distinct regions of the dorsal plasma membrane of single cells (Fig. 3F). M2, A7, M2-CC1 or A7-CC1 cells showed no significant differences, either in proliferation, as determined in a BrdU incorporation approach, or in spontaneous apoptosis rates, as analyzed by annexin-V/propidium iodide staining (see supplementary material Fig. S1A).

Expression of FLNa and CEACAM1-L affects cell migration
To test for cell migration, we performed chemotactic Transwell assays with all four cell lines in response to a gradient of M2-conditioned medium. As described by Cunningham et al. (Cunningham et al., 1992Go), FLNa-expressing A7 cells migrated at a significantly higher rate through a porous membrane compared with slow migrating wild-type M2 cells (3.5-fold±0.9) (Fig. 4A). CEACAM1-L expression in M2 cells enhanced motility to the same level as FLNa expression (3.5-fold±0.4). By contrast, A7-CC1 cells expressing both, FLNa and CEACAM1-L, showed a 40% reduced motility compared with single transfected M2-CC1 or A7 cells (0.6-fold±0.2). Compared with wild-type M2 cells, A7-CC1 cells still showed an enhanced migration (1.9-fold±0.6).



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Fig. 4. Expression of FLNa and CEACAM1-L affects cell migration. (A) Cell migration of M2, A7, M2-CC1 and A7-CC1 cells was determined in a Transwell Boyden chamber assay. Mean migrations relative to M2 wild type cells in 3 independent experiments performed in triplicate are shown with their standard deviations. Student's t-test revealed (*1) P<0.005 compared with M2, and (*2) P<0.01 compared with A7 cells. (B) Cell invasion of M2, A7, M2-CC1 and A7-CC1 cells was determined using BD Biocoat Matrigel Invasion Chamber assays. Mean invasions relative to M2 wild-type cells in three independent experiments performed in triplicate are shown with their standard deviations. Student's t-test revealed (*3) P<0.006 compared with M2, and (*4) P<0.01 compared with A7 cells.

 
Transwell invasion assays were performed to test whether CEACAM1-L and FLNa expression alters the invasiveness of the M2 melanoma cells. The cells were allowed to penetrate a reconstituted basement membrane (Matrigel) in response to a gradient of M2 conditioned medium. FLNa-expressing A7 cells exhibited a strongly enhanced invasiveness compared with low invasive M2 cells (7.2-fold±2.3) (Fig. 4B). CEACAM1-L expression did not alter the invasive properties of wild-type M2 cells on the extracellular matrix gel (1.2-fold±0.6). Co-expression of CEACAM1-L and FLNa in A7-CC1 cells reduced their invasive capacity to 50% of that of parental A7 cells (0.5-fold±0.1). However, compared with low invasive wild-type M2 cells, the double transfectants still showed a higher invasiveness (3.6-fold±0.5).

Matrigel consists of a complex mixture of extracellular matrix proteins and adhesion to these proteins is mediated by integrins, which thereby control migration and invasion. We examined adhesive properties of the melanoma cells in binding kinetics and found that M2, A7, M2-CC1 and A7-CC1 cells adhered equally well on tissue culture plates coated with Matrigel, vitronectin, fibronectin, laminin-1, collagen I or collagen IV (see supplementary material Fig. S2). We found no significant differences in cell surface expression levels of tested integrin subunits ß1, {alpha}3, {alpha}6 and {alpha}v on all four cell lines (see supplementary material Table S1). Integrin subunit ß3 could not be detected on any of the four cell lines by FACS (see supplementary material Table S1) or western blot analysis (data not shown), indicating a different mechanism of CEACAM1-mediated cell migration enhancement from that reported by Ebrahimnejad et al. (Ebrahimnejad et al., 2004Go).

FLNa-CEACAM1-L interaction inhibits wound healing and cell scattering
In wound healing assays, confluent cells were wounded by scratching with a pipette tip and cells were allowed to migrate into the cleared area. Wild-type M2 cells covered 52% (±8%) of the wound area after 24 hours and 64% (±6%) after 48 hours (Fig. 5A,B). Wound closure of this slow migrating cell line was complete after 72 hours (data not shown). By contrast, FLNa-expressing A7 and CEACAM1-L-expressing M2-CC1 cells covered 66% (±11%) and 69% (±12%) of the wound area after 24 hours and showed nearly complete wound closure after 48 hours (A7=89%±6%; M2-CC1=92%±7%). Strikingly, the A7-CC1 double transfectants exhibited a strongly reduced migration during wound healing and closed only 16% (±7%) of the cleared area within 24 hours. After 48 hours, most of the wound area remained uncovered (wound closure=24%±10%) and A7-CC1 cells were not able to close the wound area even after 72 hours (data not shown). BrdU-incorporation assays ensured that these effects were not due to differences in proliferation at the wound edge (see supplementary material Fig. S1B) but represent major differences in migration.



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Fig. 5. FLNa-CEACAM1-L interaction inhibits wound healing. (A) Monolayers of M2, A7, M2-CC1 and A7-CC1 cells were scratched with a pipette tip and images were taken immediately, 24 and 48 hours after wounding. Bar, 100 µm. (B) Distances between wound edges after 0, 24 and 48 hours were measured. Mean wound closure after 24 and 48 hours in 4 independent experiments and standard deviation are shown. (C) Time-lapse analysis of M2, A7, M2-CC1 and A7-CC1 cells after wounding. Single cells detaching from the wound edge are labeled with an asterisk. Representative details of the supplementary Movies are shown. (D) Spontaneous cell scattering of M2, A7, M2-CC1 and A7-CC1 cells. Single cells were seeded sparsely and were allowed to grow for 10 days. Bar, 10 µm.

 
Time-lapse imaging revealed that in addition to the typical sheet-like migration that occurs during wound closure, single M2-CC1 cells began to separate from the wound edge directly after wounding (Fig. 5C) (see supplementary material Movie 3), indicating an enhanced cell scattering induced by the expression of CEACAM1. M2 and A7 cells showed this behavior to a lesser extend (Fig. 5C) (see supplementary material Movie 1 and 2) with complete detachment occurring first after 6-10 hours (data not shown). The double-transfected A7-CC1 cells showed no cell scattering at all (Fig. 5C) (see supplementary material Movie 4). Comparable results were obtained when single cells were seeded sparsely and were allowed to grow for 10 days. M2, A7 and M2-CC1 cells grew in colonies exhibiting a frayed border (Fig. 5D). In particular, M2-CC1 cells showed enhanced isolation of single cells. By contrast, A7-CC1 cells grew in tight colonies with smooth borders, exhibiting no cell scattering at all.

We tested all cell lines for differences in E-cadherin expression, since inhibition of cell scattering of epithelial cells is normally mediated by adherens junctions, which contain E-cadherin as a basic component. Confocal images of immunostainings of E-cadherin revealed a punctate, exclusively intracellular distribution in all four cell lines (see supplementary material Fig. S3B). As described for progressing melanoma, we found an increase in N-cadherin expression, which was localized in cell-cell contacts (see supplementary material Fig. S3A). Western blot analysis showed similar N-cadherin expression levels in all four cell lines (data not shown). According to these results, the differences in cell migration and cell scattering of the cell lines were not due to variations in E- or N-cadherin expression levels.

CEACAM1-L and FLNa are recruited to cell-cell contacts after wounding
When the cellular localization of CEACAM1-L was analyzed in freshly wounded monolayers of A7-CC1 and M2-CC1 cells, CEACAM1-L was distributed diffusely with a small portion localized occasionally in cell-cell contacts (data not shown). One hour after wounding, most of the CEACAM1-L was recruited to cell-cell contacts in both cell lines (Fig. 6A). After incubation for 18 hours, migrating M2-CC1 cells still exhibited an intense staining for CEACAM1-L in areas of cell-cell contact. A7-CC1 cells displayed a smooth wound edge after 18 hours and maintained an increased accumulation of CEACAM1-L in cell-cell contacts. The cellular distribution of FLNa during wound healing was not affected by CEACAM1-L transfection. While staining was diffuse in confluent A7 and A7-CC1 cells (data not shown), FLNa was recruited to cell-cell contacts 1 hour after wounding and located mainly to cortical regions of the leading edge in migrating cells after 18 hours (Fig. 6B).



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Fig. 6. CEACAM1-L and FLNa are recruited to cell-cell contacts after wounding. (A) Indirect immunofluorescence of CEACAM1 (mAb 4/3/17) in M2-CC1 and A7-CC1 cells. (B) Indirect immunofluorescence of FLNa (mAb FLMN01) in A7 and A7-CC1 cells. Monolayers were fixed at the indicated time points. Bars, 5 µm.

 

CEACAM1-L expression alters cytoskeletal rearrangements and tyrosine phosphorylation in M2 and A7 cells during wound healing
Phalloidin staining was used to analyze the influence of FLNa and CEACAM1-L expression on the actin cytoskeleton. Freshly wounded monolayers of all four cell lines showed a diffuse F-actin distribution (data not shown). Within 1 hour, all cell lines began to show a polarized morphology with a leading lamella stretched into the cleared area (Fig. 7A). Wild-type M2 cells and FLNa-expressing A7 cells formed stress fibers after 1 hour, and F-actin was assembled at cell-cell contacts and cortical regions. After 18 hours, no more stress fibers were visible. In contrast to the CEACAM1-L-negative cells, M2-CC1 and A7-CC1 cells exhibited a stronger F-actin assembly at cell-cell contacts and cortical regions and only a very weak stress fiber formation after 1 hour. After 18 hours, staining for F-actin in M2-CC1 cells was comparable to that in M2 cells. A7-CC1 cells again did not detach from the wound edge. They no longer showed a leading lamella extending into the wound, but maintained the strong F-actin accumulation in cell-cell contacts and cortical regions.



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Fig. 7. CEACAM1-L expression alters F-actin assembly and tyrosine phosphorylation in M2 and A7 cells during wound healing. Indirect immunofluorescence of F-actin (phalloidin, A) and phospho-tyrosine (p-Tyr, B) in M2, A7, M2-CC1 and A7-CC1 cells. Wounded monolayers were fixed at the indicated time points. Arrowheads indicate mature focal adhesions (B, right panel). Bars, 5 µm (C) Indirect immunofluorescence of vinculin in M2, A7, M2-CC1 and A7-CC1 cells. Monolayers were fixed 1 hour after wounding. Bar, 5 µm. (D,E) Phospho-tyrosine specific immunoprecipitations. Monolayers of M2, A7, M2-CC1 and A7-CC1 cells were either left untreated or were wounded with a multichannel pipette 1 hour prior to lysis. Equal amounts of lysates were immunoprecipitated with anti-p-Tyr mAb PY99. Blots were developed for FAK (D) and paxillin (E) (n=3).

 
M2 and A7 cells displayed a diffuse staining for phosphorylated tyrosine (p-Tyr) directly after wounding (data not shown). After 1 hour, dot-like structures at the cell periphery and in the cell body were stained by anti-p-Tyr mAb (Fig. 7B). The dot-like structures were identified as focal adhesions as described below. After 18 hours, M2 and A7 cells exhibited fewer but larger focal adhesions that stained for p-Tyr and were mainly located at the cell periphery. In contrast to the parental cell lines, both M2-CC1 and A7-CC1 cells showed punctate rather than diffuse p-Tyr staining patterns directly after wounding (data not shown). After 1 hour, both cell lines exhibited a very strong p-Tyr staining of dot-like focal adhesions and cell-cell contacts. The tyrosine phosphorylation was more intense and more organized compared with M2 or A7 cells. After 18 hours, migrating M2-CC1 cells stained strongly for p-Tyr at cell-cell contacts and formed a few large focal adhesions that were located at the cell periphery in tips of protrusions. However, the p-Tyr pattern of A7-CC1 cells after 18 hours resembled the pattern obtained after 1 hour. Focal adhesions were numerous and distributed all over the cell, although they concentrated at the cell periphery. Thus, M2, A7 and M2-CC1 cells showed a comparable long-term focal adhesion turnover during migration upon wounding, in spite of differences in F-actin assembly and tyrosine phosphorylation after 1 hour in M2-CC1 cells. However, A7-CC1 cells remained non-motile after overnight incubation and maintained the enhanced phosphorylation status of focal adhesions.

CEACAM1-L alters phosphorylation of focal adhesion kinase (FAK) and paxillin during wound healing
Dot-like structures stained by anti-p-Tyr antibody were identified as focal adhesions using anti-vinculin (Fig. 7C), anti-ß1 integrin and anti-paxillin (data not shown) antibodies, each revealing a dot-like pattern similar to that found in p-Tyr-stained cells. Several proteins become phosphorylated on tyrosine residues after integrin binding to extracellular matrix proteins, e.g. FAK and paxillin, which have been implicated in focal adhesion function and turnover (Crowe and Ohannessian, 2004Go; Ilic et al., 1995Go). To investigate the phosphorylation status of these proteins, p-Tyr-specific immunoprecipitations of untreated cells or of cells wounded with a multichannel pipette 1 hour prior to lysis were performed. Proteins were analyzed by SDS-PAGE and precipitated FAK and paxillin were quantified. Untreated cells of all four cell lines exhibited similar phosphorylation levels of both proteins (Fig. 7D,E). However, 1 hour after wounding, CEACAM1-L-expressing M2-CC1 and A7-CC1 cells showed a markedly stronger increase in tyrosine phosphorylation of FAK and paxillin than the parental M2 and A7 cells. Thus, in both CEACAM1-L expressing cell lines, at least FAK and paxillin are responsible for the observed stronger p-Tyr staining of focal adhesions.

Alteration of co-immunoprecipitation of FLNa by CEACAM1-L during wound healing and adhesion
Since FLNa is an actin cytoskeleton-associated protein, we tested whether the binding of FLNa to CEACAM1-L in A7-CC1 cells was altered under conditions that cause a reorganization of the actin cytoskeleton. Processes like migration and adhesion are characterized by an increase in F-actin assembly and cell-matrix adhesion nucleation. From untreated confluent cells, about 1% (±0.3%) of the total FLNa was co-precipitated (Fig. 8A). When cells were wounded 1 hour prior to lysis to induce migration, or allowed to adhere on laminin-1, the amount of co-precipitated FLNa was strongly increased to 2.6% (±0.4%) and 2.7% (±0.7%) of total FLNa. When cells lack a matrix for adherence, the normally well-organized actin cytoskeleton and its adhesion plaques disassemble. Accordingly, when cells were kept in suspension 1 hour prior to lysis, there was virtually no co-precipitation of FLNa (0.11%±0.13%). These findings indicate a correlation between the presence of cell-matrix adhesions and the strength of the FLNa-CEACAM1-L interaction.

CEACAM1 binding to FLNa interferes with FLNa-RalA interaction
The small GTPase RalA was reported to bind to the 24th filamin repeat of FLNa, thereby regulating migration (Ohta et al., 1999Go). The used melanoma cell lines express RalA at a similar level (data not shown). We performed pulldown assays with the agarose-bound Ral-binding domain of Ral-binding protein 1 (RalBP1), which exclusively binds to the GTP-bound form of Ral proteins and immunoblotted the samples with a RalA specific antibody. To investigate whether CEACAM1 has an impact on RalA activation, we tested A7 and A7-CC1 cells under the same conditions where FLNa co-precipitation by CEACAM1 was altered in A7-CC1 cells. Wounding as well as adhesion increased RalA activation from 5% RalA-GTP in untreated cells to about 10% RalA-GTP in both cell lines (Fig. 8B). Keeping the A7 and A7-CC1 cells in suspension mildly reduced the content of active RalA to approximately 2.5% of total RalA. Thus, both cell lines showed highly similar alterations in their RalA-GTP levels in response to wounding, adhesion and absence of adhesion. Since RalA was shown to bind to FLNa in a GTP-specific manner (Ohta et al., 1999Go), we tested for co-precipitated FLNa in the RalA-GTP pulldowns described in Fig. 8B. In untreated A7 cells and in A7 cells kept in suspension about 1% of total FLNa was co-precipitated (Fig. 8C). After wounding or during adhesion about 3% of total FLNa was co-precipitated, indicating a relation between the concentration of GTP-bound RalA and the amount of co-precipitated FLNa. In A7-CC1 double-transfectants, we found a strongly reduced amount of FLNa co-precipitated by RalA-GTP in untreated, wounded and adhering cells (0.3-0.5% of total FLNa), irrespective of the RalA activation levels. Only when A7-CC1 cells were kept in suspension, an amount of FLNa comparable to that found in A7 cells (approximately 1%) was co-precipitated. Thus, binding of CEACAM1 to FLNa interferes with the interaction of FLNa with RalA. Only when cells were kept in suspension and CEACAM1 did not bind to FLNa, RalA-GTP was able to bind and precipitate FLNa in double transfected A7-CC1 cells.


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Integrins, Ig-CAMs, cadherins, and other cell adhesion molecules, regulate a variety of signaling events including those involved in differentiation, migration and survival. The underlying mechanisms often involve the ability of cell adhesion molecules to initiate the formation of organized structures or scaffolds that permit the efficient flow of information in signaling pathways (for review, see Juliano, 2002Go). In the present study we describe the scaffold FLNa as a novel CEACAM1-L interacting protein. The CEACAM1-L-FLNa association was found in a yeast two-hybrid screen and verified by in vitro and in vivo binding assays. Single expression of CEACAM1-L or FLNa in M2 cells strongly enhanced migration by differential alterations on the molecular level, demonstrating the different modes of action of these proteins. Co-expression of FLNa and CEACAM1-L most profoundly prevented cell scattering, thereby drastically reducing cell migration. Our data indicate that an altered interaction of FLNa with the small GTPase RalA possibly plays a role in the mechanistic scenario leading to immobility.

FLNa links the peripheral actin cytoskeleton to integrins (Loo et al., 1998Go; Sharma et al., 1995Go) and is targeted by a variety of different signaling molecules, e.g. FLNa interacting protein (FILIP), ribosomal S6 kinase, protein kinase C{alpha}, and GTPases of the Rho- and Rac-families, which thereby implicate FLNa in cell shape modulation and integrin-dependent adhesion and motility (Nagano et al., 2002Go; Ohta et al., 1999Go; Tigges et al., 2003Go; Woo et al., 2004Go). GTPases have the extraordinary ability to remodel actin-based cytoskeletal structures. We observed that FLNa-transfected A7 cells and A7 cells cotransfected with CEACAM1-L (A7-CC1) show pronounced differences in the actin cytoskeleton architecture and lamellipodium formation during wound-induced migration. FLNa has been proposed to coordinate the integration of extracellular signals leading to Rho GTPase activation (Bellanger et al., 2000Go) and the binding sites of the GTPases Rac1, Cdc42, RhoA and RalA all lie within the filamin repeat 24 (Ohta et al., 1999Go). Interestingly, we found that this repeat interacts with CEACAM1-L. Therefore, we wondered whether the CEACAM1-L-FLNa interaction may influence GTPase signaling. Of all GTPases mentioned above only RalA binds to FLNa in a GTP-dependent manner. The expression of CEACAM1-L had no influence on the activation of RalA. However, the CEACAM1-L-FLNa co-expression inhibited RalA binding to FLNa significantly. The prevention of FLNa-RalA binding was specific since both CEACAM1-L and RalA seemed to target the same small FLNa sub-fraction comprising about 1-3% of total FLNa. Numerous studies have implicated Ral proteins in migration (Camonis and White, 2005Go; Feig, 2003Go). For example, expression of the activated form of RalA enhances the motility of skeletal myoblasts (Suzuki et al., 2000Go) while the expression of a dominant-negative variant of Ral inhibits migration in epithelial cells in Drosophila ovary (Lee et al., 1996Go). Moreover, the interaction of RalA with FLNa is necessary for Cdc42-induced filopod production as demonstrated in Swiss 3T3 cells and in the M2/A7 cell system (Ohta et al., 1999Go). From these data a mechanistic model emerges that comprises RalA and FLNa as effectors in CEACAM1-L mediated signaling regarding the (negative) regulation of migration. Apparently, CEACAM1-L binding to FLNa interfered with FLNa-GTPase interactions by competing for the binding sites in filamin repeat 24.

In addition to this, CEACAM1-L could alter spatial and temporal qualities of other signaling complexes hosted by FLNa. Migration depends on integrin-mediated anchorage to the extracellular matrix (McGary et al., 2002Go). Eighteen hours after wounding, M2-CC1 cells showed a normal focal adhesion complex formation, while co-expression of FLNa and CEACAM1-L resulted in maintenance of numerous focal adhesions. Since migration involves coordinated focal adhesion assembly and disassembly (Schlaepfer et al., 1999Go), the altered focal adhesion turnover is likely to be another reason for the observed reduction in cell migration. Integrin signaling events have been shown to be modulated by FLNa (Calderwood et al., 2001Go; Glogauer et al., 1998Go) and also by CEACAM1 in different cell types (Muller et al., 2005Go). We have demonstrated recently, that CEACAM1-L binds to talin and affects integrin-dependent signaling in endothelial cells. In melanoma cells, an interaction of CEACAM1-L and ß3 integrins enhanced cell migration and invasion depending on the presence of Tyr-488 within the cytoplasmic domain of CEACAM1-L (Brummer et al., 2001Go; Ebrahimnejad et al., 2004Go). However, our in vitro binding data with purified recombinant proteins from E. coli indicate a tyrosine phosphorylation-independent association of FLNa with CEACAM1-L. That finding agrees with the results for other cytoplasmic proteins like calmodulin, tropomyosin and G-actin (Edlund et al., 1996Go; Schumann et al., 2001Go). Due to the lack of ß3-integrin expression in the cell system used, our findings describe an additional mechanism of the CEACAM1-L-mediated impact on integrin-dependent cell migration and motility that can be altered by FLNa. Importantly, in our cell system the CEACAM1-L expression did not enhance invasion, but rather reduced FLNa induced invasiveness.

Yet, our data also shed a light on the postulated role of CEACAM1-L in tumor progression and metastasis. Cancer cells have to detach from neighboring cells and loosen their adhesions to the basement membrane. They acquire a migratory phenotype and degrade or remodel the extracellular matrix. We found CEACAM1-L translocating to cell-cell contacts in both FLNa-negative and -positive cells after monolayer wounding. However, we observed differential effects of the CEACAM1-L expression on the maintenance of cell-cell contacts. Co-expressed with FLNa, CEACAM1-L expression led to reinforced cell-cell adhesion, while CEACAM1-L-expressing, FLNa-negative M2-CC1 melanoma cells frequently detached from neighboring cells. This behavior of the M2-CC1 cells may reflect the postulated role of CEACAM1 in melanoma progression (Brummer et al., 2001Go; Ebrahimnejad et al., 2004Go; Thies et al., 2002Go). Cell scattering of M2-CC1 cells is not in conflict to the homophilic binding capacities of CEACAM1 (Ocklind and Obrink, 1982Go). Hunter et al. showed that the expression and upregulation of CEACAM1-L in rat NBT-II cells undergoing epithelial-mesenchymal transition does not lead to strengthened cell-cell adhesion and does not prevent these cells from scattering (Hunter et al., 1994Go). From our results we conclude that the observed differential scattering behaviors of M2-CC1 and A7-CC1 cells were not due to altered E-cadherin or N-cadherin expression levels. Possibly, the CEACAM1-FLNa interaction strengthens the anchorage of the cortical actin skeleton to the plasma membrane. Sundberg et al. demonstrated that MDCK-cells expressing CEACAM1-L show a decreased abundance of desmosomes that is accompanied by a disorganization of cytokeratin filaments (Sundberg et al., 2004Go), but a possible effect on migration was not studied. In in vitro assays, impairment of cell migration caused by CEACAM1-L in FLNa-expressing cells was strongly increased when cells were in contact with each other (wound healing assays) and less when the experiments were carried out with single cells (Transwell assays). These data indicate that the properties of CEACAM1-L also depend on the molecular background and the surrounding tissue, e.g. during the radial and vertical growth phase of a tumor versus metastasis. The influence of CEACAM1-L on RalA signaling via FLNa-binding extends the possibilities how CEACAM1-L could be implicated in oncogenesis. There is increasing evidence that the Ras effector RalA plays a substantial role in Ras-induced oncogenesis (Gonzalez-Garcia et al., 2005Go; Tchevkina et al., 2005Go). Further investigation of how CEACAM1 interferes with the RalA signaling pathway also could reveal an explanation for the contradictory potential of CEACAM1 to either suppress tumorigenic characteristics, e.g. in many coloncarcinoma (Neumaier et al., 1993Go; Nollau et al., 1997Go; Rosenberg et al., 1993Go) or to enhance oncogenic properties, e.g. in melanoma (Brummer et al., 2001Go; Ebrahimnejad et al., 2004Go; Thies et al., 2002Go).

In conclusion, the present study identifies CEACAM1-L as interaction partner of FLNa. Furthermore, the results provide new mechanistic insights into the roles of CEACAM1-L and FLNa during cell migration and clearly demonstrate a functional interconnection of both proteins. For the first time, we could show that in addition to its pro-migratory properties CEACAM1-L is also able to reduce migration.


    Acknowledgments
 
This work has been supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 366 `Zelluläre Signalerkennung und -umsetzung', and the Sonnenfeld-Stiftung. We acknowledge T. Scott, UK, for critically reading the manuscript.


    Footnotes
 
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/118/23/5513/DC1


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