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First published online 15 November 2005
doi: 10.1242/jcs.02660

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

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

View larger version (23K): [in a new window]   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 -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).

View larger version (29K): [in a new window]   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.

View larger version (37K): [in a new window]   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.

View larger version (18K): [in a new window]   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.

View larger version (52K): [in a new window]   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.

View larger version (66K): [in a new window]   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.

View larger version (89K): [in a new window]   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).

View larger version (28K): [in a new window]   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).