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First published online December 31, 2008
doi: 10.1242/10.1242/jcs.036467

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EphA1 interacts with integrin-linked kinase and regulates cell morphology and motility

Tohru Yamazaki^*, Junko Masuda^*, Tsutomu Omori, Ryosuke Usui, Hitomi Akiyama and Yoshiro Maru

Department of Pharmacology, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan

View larger version (60K): [in this window] [in a new window]   Fig. 1. Yeast two-hybrid analysis for the interaction between EphA1 and ILK. (A) Schematic representation of the EphA1 and ILK constructs used in the yeast two-hybrid analysis. Bait constructs were the GAL4 DNA-binding domain fused to: the cytoplasmic region of EphA1 (CP), kinase-dead EphA1 (CP/KD), juxtamembrane-region deletion EphA1 (CP/JM), EphA1 with the C-terminal sterile motif (SAM) deletion (CP/SAM) and the SAM domain alone (SAM). Preys were the GAL4-activation domain fused to: full-length ILK (ILK), the N-terminal ankyrin-repeats (ANK), the C-terminal PH- and kinase domain (CAT), ILK with the ankyrin1 deletion (ANK1), ILK with the ankyrin1-2 deletion (ANK1-2) and ILK with the ankyrin1-3 deletion (ANK1-3). (B) Interactions between EphA1 and ILK in the yeast two-hybrid analysis. The left panel shows growth of transformed yeasts in synthetic dropout medium (SD-2; –Leu, –Trp). The middle panel indicates interactions between various EphA1 and ILK constructs (shown in the right panel) in SD-4 (–Leu, –Trp, –His, –Ade) in a series of dilution (dilution increases from left to right). Representative colonies from five independent experiments are shown.

View larger version (34K): [in this window] [in a new window]   Fig. 2. Interaction between EphA1 and ILK in mammalian cells. (A) Cells expressing GFP, WT (wild-type)-EphA1–GFP or KD (kinase dead)-EphA1–GFP were transfected with ILK-myc. 24 hours after transfection, cells were serum-starved for 12 hours and stimulated with ephrin-A1–Fc (1 µg/ml) for 10 minutes. Cell lysates were immunoprecipitated (IP) with anti-myc antibody followed by immunoblot (IB) analysis with the indicated antibodies. (B) HEK293 cells expressing WT-EphA1–GFP or SAM-EphA1–GFP were transfected with ILK-myc and treated as in A. Whole cell lysate (WCL; lane 1) and immunoprecipitates by control IgG (lane 2), anti()-GFP (lanes 3-6), anti-myc (lane 7) and no antibody (Ab) (lane 8) were subjected to anti-EphA1 and anti-ILK immunoblotting. (C) EBC-1 cells were serum-starved overnight and stimulated with ephrin-A1–Fc (1 µg/ml) for 10 minutes. Cell lysates were immunoprecipitated with anti-ILK antibody followed by immunoblot analysis with anti-EphA1 and anti-ILK antibody.

View larger version (44K): [in this window] [in a new window]   Fig. 3. Functional expression of EphA1 in HEK293 cells. (A) The expression vector alone (pCMV-Tag4, Mock) or containing cDNA encoding WT- or KD (V638R)-EphA1–FLAG was stably transfected into HEK293 (293) cells. Expression levels of EphA1 and EphA2 in HEK293 Mock cells (lane 1), HEK293 WT-EphA1–FLAG cells (lane 2), K562 cells (lane 3) and EBC-1 cells (lane 4) are shown. Lysates from the indicated cells were analyzed by anti-EphA1, anti-EphA2 and anti-actin immunoblotting. (B) Mock, WT-EphA1–FLAG (WT) and KD-EphA1–FLAG (KD) cells were serum starved for 12 hours and stimulated with the indicated concentrations of ephrin-A1–Fc for 10 minutes, then lysed. EphA1 receptors were immunoprecipitated (IP) with anti-FLAG antibody and analyzed by immunoblotting (IB) with PY-20 (PY) and M2 (FLAG) antibody. (C) Immobilized ephrin-A1–Fc mediates adhesion of WT- and KD-EphA1–FLAG cells but not Mock cells. Cells were plated onto plastic culture plates or fibronectin (FN)-coated (1 µg/ml) 96-well plates with the indicated dosage of immobilized ephrin-A1–Fc (0-5 µg/ml) for 30 minutes. Adherent cells were fixed and stained with crystal violet. Dyes were extracted and measured at A590. (D) Mock, WT-EphA–FLAG and KD-EphA–FLAG cells were treated as in B (ephrin-A1–Fc at 1 µg/ml). Anti-FLAG immunoprecipitates were subjected to anti-FAK (upper) and anti-FLAG (lower) immunoblotting. (E) Mock, WT-EphA–FLAG and KD-EphA–FLAG cells were replated on FN-coated dishes with or without immobilized ephrin-A1–Fc (1 µg/ml) for 5 or 15 minutes. Then, cells were lysed and immunoprecipitated with anti-FAK antibodies for immunoblot analysis. Phosphorylated and total FAK in suspension (Sus) and in adhered cells were detected using anti-phosphotyrosine (PY) antibody followed by anti-FAK (FAK) antibody.

View larger version (33K): [in this window] [in a new window]   Fig. 4. EphA1 inhibits cell spreading in a kinase-activity-dependent manner. (A) Mock, WT-EphA1–FLAG (WT), KD-EphA1–FLAG (KD) and SAM-EphA1–FLAG (SAM) HEK293 cells (see Figs 1 and 2) and EBC-1 cells were plated onto FN-coated (1 µg/ml) coverslips with (right) or without (left) immobilized ephrin-A1–Fc (1 µg/ml) for 30 minutes and stained with rhodamine-conjugated phalloidin. (B) Quantification of spreading in the cells shown in A. (C) GFP images of HEK293 WT-EphA1–GFP cells stimulated by control Fc or ephrin-A1–Fc. (D) HEK293 (293), Rat1 and MCF7 cells expressing GFP or WT-EphA1–GFP were subjected to spreading assays as in A, and stained by phalloidin and DAPI. Only cells after ephrin-A1–Fc stimulation are shown. Scale bars: 10 µm.

View larger version (23K): [in this window] [in a new window]   Fig. 5. EphA1 is required for the ephrin-A1–Fc-induced spreading defect in endothelial cells. (A) NP31 ATF3-Tet cells were deprived of tetracycline [Tet (–)] for 12 hours to induce EphA1 expression. Cells stimulated by ephrin-A1–Fc (right panels) with (lower four panels) or without (top four panels) anti()-EphA1 siRNAs at 80 nM were analyzed by spreading assays. In the Tet system, GFP is co-induced in cells deprived of tetracycline, which enables recognition of EphA1-expressing cells (green) merged with phalloidin (red) to give yellow color. DAPI is blue. Scale bar: 10 µm. (B) Quantification of A.

View larger version (33K): [in this window] [in a new window]   Fig. 6. ROCK inhibitor (Y-27632) attenuates the inhibitory effect of EphA1 on cell spreading and migration. (A) Activities of Rho-family GTPases in HEK293 Mock, WT-EphA1–FLAG (WT) and KD-EphA1–FLAG (KD) cells were measured by immunoblot analysis as described in the Materials and Methods. (B) Quantification of A with means ± s.d. from three independent experiments. (C) The same set of cells as shown in A were plated onto FN-coated (1 µg/ml) coverslips with immobilized ephrin-A1–Fc (1 µg/ml) in serum-reduced medium containing DMSO (vehicle; middle column) or Y-27632 (10 µM; right column) for 30 minutes. Cells were fixed and stained with rhodamine-labeled phalloidin. Scale bar: 10 µm. (D) Quantification of the spreading of the cells shown in C. (E) Modified Boyden-chamber migration analysis of the same set of cells was performed as described in the Materials and Methods. Values show means ± s.d. from nine randomly selected high-power fields.

View larger version (35K): [in this window] [in a new window]   Fig. 7. EphA1 inhibits the kinase activity of ILK. (A) Anti-FLAG (upper) and anti-ILK (lower) immunoblotting of endogenous ILK immunoprecipitated with anti-ILK antibody from the same set of cells as shown in Fig. 3D. (B) Mock and WT-EphA1–FLAG (WT) cells were plated onto FN-coated (1 µg/ml) dishes with or without immobilized ephrin-A1–Fc (1 µg/ml) for 15 minutes. Wortmannin (WM; 100 nM) was used for 30 minutes prior to the re-spreading of cells. ILK was immunoprecipitated and subjected to ILK kinase assay as described in the Materials and Methods. (C) Normalized kinase activities against the total amount of immunoprecipitated ILK (arrowhead) were quantified. Data on the right show means ± s.d. of three independent experiments. (D) HEK293 cells were transfected with control non-silencing RNA (Cont) or ILK siRNA (40 or 80 nM). Immunoblot analysis was performed 2 days after transfection. Cells transfected with control siRNA or ILK siRNA (80 nM) were plated on FN-coated (1 µg/ml) coverslips for 30 minutes. Cells were then stained with rhodamine-conjugated phalloidin (red) and DAPI (blue). Scale bar: 10 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 8. An active form of ILK (S343D) blocks the inhibitory effect of EphA1. (A) HEK293 (293) WT-EphA1–GFP and SAM-EphA1–GFP cells were transfected with wild-type ILK-myc (myc), starved of serum for 12 hours and then stimulated with ephrin-A1–Fc or control Fc (1 µg/ml) for 15 minutes. Cells were fixed and immunostained by anti-myc antibody (red). DAPI is blue; GFP is green. Note the colocalization of EphA1-GFP and ILK-myc in the merged images even before ephrin-A1–Fc stimulation in WT-EphA1–GFP cells (see the area with an arrowhead in the inset). Scale bar: 20 µm. (B) The same set of cells as is shown in A, with additional transfection with ILK-S343D, was subjected to spreading assays. The number of spreading cells over that of myc-positive cells was calculated. Data show means ± s.d. of three independent experiments. (C) ILK and RhoA activities in HEK293 WT-EphA1–FLAG cells transfected with wild-type ILK-myc (WT) or ILK-S343D–myc (S343D) were measured by immunoblot analysis as described in the Materials and Methods. (D) HEK293 WT-EphA1–GFP cells transfected with vector alone (lanes 1, 2) or ILK-S343D–myc (lane 3) were subjected to cell migration assays as described in Fig. 6E in the absence (lane 1) or presence (lanes 2, 3) of ephrin-A1–Fc.

View larger version (34K): [in this window] [in a new window]   Fig. 9. A dominant-negative ILK rescues the EphA1-mediated spreading defect. (A) HEK293 cells were co-transfected with EphA1-FLAG and myc-tagged ILK, CAT or ANK (see Fig. 1), and anti-myc immunoprecipitates (IP) were subjected to anti-FLAG (upper) and anti-myc (lower) immunoblotting. Specific bands are indicated by arrows. (B) HEK293 (293) Mock and WT-EphA1–FLAG (EphA1) cells were transfected with GFP (control) or ANK-GFP, and their expression was confirmed by anti-GFP immunoblotting. (C) The same set of cells as is shown in B was subjected to spreading assays. (D) A model for the regulation of cell spreading by EphA1. Ephrin-A1 activates EphA1, which recruits ILK to the SAM domain of EphA1. ECM activates the FAK-PI3K-ILK cascade. EphA1 could activate FAK but also inhibit ILK downstream of FAK. ILK suppresses Rho-ROCK but its de-inhibition by EphA1 through ILK inhibition results in the spreading defect. The kinase activity of ILK is negatively regulated by EphA1 kinase (through a direct or indirect mechanism). The suppression of Rac1 and activation of the RhoA-ROCK pathway might result in the increase of cell contractility, and inhibition of cell spreading and migration.

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