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doi: 10.1242/10.1242/jcs.00166

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Assembly of the PINCH-ILK-CH-ILKBP complex precedes and is essential for localization of each component to cell-matrix adhesion sites

¹ Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261, USA
² Structural Biology Program, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA

View larger version (36K): [in a new window]   Fig. 1. The Q40A point mutation within the LIM1 domain abolishes the ILK binding and the localization of PINCH to cell-ECM adhesion sites. (A) Three-dimensional structure of the PINCH LIM1 domain. Amino acid residues that comprise the PINCH binding site are labeled. (B,C) Complex formation with ILK. Lysates of C2C12 cells expressing GFP-tagged wild-type or mutant (Q40A) form of PINCH were mixed with rabbit anti-GPF antibodies. The GFP-PINCH (lane 3) and GFP-Q40A (lane 4) immunoprecipitates were analyzed by western blotting with HRP-conjugated anti-GFP antibodies (B) or mouse monoclonal anti-ILK antibody 65.1 and HRP-conjugated anti-mouse IgG antibodies (C). Lanes 1-2 were loaded with cell lysates (13 µg/lane) as indicated in the figure. (D-G) Subcellular localization. C2C12 cells transfected with expression vectors encoding GFP-Q40A (D,E) or GFP-PINCH (F,G) were plated on fibronectin-coated coverslips and stained with a mouse monoclonal anti-paxillin antibody (as a marker of focal adhesions) and a Rhodamine RedTX-conjugated anti-mouse IgG antibody. GFP-Q40A, GFP-PINCH and paxillin were visualized under a fluorescence microscope equipped with GFP (D,F) and rhodamine (E,G) filters. The experiments were performed three times and similar results were obtained. Bar, 10 µm.

View larger version (52K): [in a new window]   Fig. 2. The D31A point mutation disrupts the interaction with PINCH and impairs ILK localization to focal adhesions. (A-C) PINCH binding. Lysates of C2C12 cells expressing GFP-tagged wild-type or mutant (D31A) form of ILK were mixed with rabbit anti-GPF antibodies. The GFP-D31A (lane 3) and GFP-ILK (lane 4) immunoprecipitates were analyzed by western blotting with HRP-conjugated anti-GFP antibodies (A), rabbit anti-PINCH antibodies and HRP-conjugated anti-rabbit IgG antibodies (B), or mouse monoclonal anti-CH-ILKBP antibody 3B5 and HRP-conjugated anti-mouse IgG antibodies (C). Lanes 1 and 2 were loaded with cell lysates (10 µg/lane) as indicated in the figure. (D,G) Subcellular localization. C2C12 cells transfected with expression vectors encoding GFP-D31A (D,E) or GFP-ILK (F,G) were plated on fibronectin-coated coverslips and stained with a mouse monoclonal anti-paxillin antibody and a Rhodamine RedTX-conjugated anti-mouse IgG antibody. GFP-D31A, GFP-ILK and paxillin were visualized under a fluorescence microscope equipped with GFP (D,F) and rhodamine (E,G) filters. The experiments were performed twice and similar results were obtained. Bar, 10 µm.

View larger version (42K): [in a new window]   Fig. 3. The PH-like motif is not required for the ILK complex formation and localization to focal adhesions. (A-C) ILK complex formation. Lysates of C2C12 cells expressing GFP-ILK, GFP-tagged ILK mutant in which residues 180-190 within the PH-like motif were deleted (dPH) or GFP alone were mixed with rabbit anti-GPF antibodies. The GFP-ILK (lane 4), GFP-dPH (lane 5) and GFP (lane 6) immunoprecipitates were analyzed by western blotting with HRP-conjugated anti-GFP antibodies (A), rabbit anti-PINCH antibodies and HRP-conjugated anti-rabbit IgG antibodies (B), or mouse monoclonal anti-CH-ILKBP antibody 3B5 and HRP-conjugated anti-mouse IgG antibodies (C). Lanes 1-3 were loaded with cell lysates (15 µg/lane) as indicated in the figure. (D,E) Subcellular localization. C2C12 cells expressing GFP-dPH were plated on fibronectin-coated coverslips and stained with a mouse monoclonal anti-paxillin antibody and a Rhodamine RedTX-conjugated anti-mouse IgG antibody. GFP-dPH and paxillin were visualized under a fluorescence microscope equipped with GFP (D) and rhodamine (E) filters. The experiments were performed twice and similar results were obtained. Bar, 10 µm.

View larger version (114K): [in a new window]   Fig. 4. Disruption of the interaction with CH-ILKBP inhibits the localization of ILK to focal adhesions. (A-C) Co-immunoprecipitation. The immunoprecipitates were prepared from C2C12 cells expressing GFP-ILK or GFP-tagged ILK mutant in which residues 180-215 were deleted (D215) with anti-GFP antibodies as described in Fig. 3. The GFP-ILK (lane 1) and GFP-D215 (lane 2) immunoprecipitates were analyzed by western blotting with HRP-conjugated anti-GFP antibodies (A), mouse monoclonal anti-CH-ILKBP antibody 3B5 and HRP-conjugated anti-mouse IgG antibodies (B), or rabbit anti-PINCH antibodies and HRP-conjugated anti-rabbit IgG antibodies (C). The sample loaded on lane 3 was prepared as those of lanes 1 and 2 except that the cell lysates were omitted (to show bands derived from rabbit IgG, which were strong when HRP-conjugated anti-rabbit IgG antibodies were used, such as in panel C). (D,E) Subcellular localization. C2C12 cells expressing GFP-D215 were plated on fibronectin-coated coverslips and stained with a mouse monoclonal anti-paxillin antibody and a Rhodamine RedTX-conjugated anti-mouse IgG antibody. GFP-D215 and paxillin were visualized under a fluorescence microscope equipped with GFP (D) and rhodamine (E) filters. The experiments were performed twice and similar results were obtained. Bar, 10 µm.

View larger version (36K): [in a new window]   Fig. 5. The formation of the PINCH-ILK-CH-ILKBP complex precedes cell adhesion and spreading. CH-ILKBP was immunoprecipitated from C2C12 cells that were maintained in suspension (lane 1) or had adhered and spread on fibronectin for 0.5 hour (lane 2), 1 hour (lane 3) or 20 hours (lane 4) with monoclonal anti-CH-ILKBP antibody 1D4. The immunoprecipitates were analyzed by western blotting with mouse monoclonal anti-CH-ILKBP antibody 3B5 (A), mouse monoclonal anti-ILK antibody 65.1 (B) and rabbit anti-PINCH antibodies (C), respectively. Lane 5 was loaded with a sample that was prepared as those of lanes 1 and 2 except that the cell lysates were omitted. Note that PINCH and ILK formed a complex with CH-ILKBP before cells were in contact with fibronectin (lane 1). The experiments were performed three times and similar results were obtained.

View larger version (43K): [in a new window]   Fig. 6. Protein kinase C regulates the formation of the PINCH-ILK-CH-ILKBP complex. (A-C) CH-ILKBP was immunoprecipitated from lysate of human mesangial cells that were treated with calphostin C (1 µM) for 50 minutes (lane 2), or that of untreated human mesangial cells as a control (lane 1) with monoclonal anti-CH-ILKBP antibody 1D4. The immunoprecipitates were analyzed by western blotting with mouse monoclonal anti-CH-ILKBP antibody 3B5 (A), mouse monoclonal anti-ILK antibody 65.1 (B) and rabbit polyclonal anti-PINCH antibodies (C), respectively. The sample loaded on lane 3 was prepared as those of lanes 1 and 2, except that the cell lysates were omitted (to show mouse IgG bands, which were apparent in A and B). Note that treatment of cells with protein kinase C inhibitor calphostin C significantly reduced the amounts of ILK and PINCH associated with CH-ILKBP (compare lanes 1 and 2). Similar results have been obtained with other cell types including human IMR-90 fibroblasts and mouse C2C12 myoblasts (not shown in the figure). (D-F) Human 293 embryonal kidney cells expressing FLAG-ILK was incubated in normal medium (lane 1) or medium containing 0.2 µM calphostin C (lane 2) or 0.4 µM cytochalasin D for 60 minutes. FLAG-ILK was immunoprecipitated from the cell lysates with monoclonal anti-FLAG antibody M2 as described in Materials and Methods. The immunoprecipitates were analyzed by western blotting with mouse monoclonal anti-FLAG antibody M2 (D), mouse monoclonal anti-CH-ILKBP antibody 3B5 (E) and rabbit polyclonal anti-PINCH antibodies (F), respectively. Note that calphostin C (lane 2), but not cytochalasin D (lane 3), inhibited the interactions of FLAG-ILK with CH-ILKBP (E) and PINCH (F). The experiments were performed four times and similar results were obtained.

View larger version (54K): [in a new window]   Fig. 7. The F438A point mutation does not inhibit the interactions with PINCH and CH-ILKBP but impairs the localization of ILK to focal adhesions. (A-C) ILK complex formation. Lysates of C2C12 cells expressing GFP, GFP-ILK or GFP-ILK F438A point mutant were mixed with rabbit anti-GPF antibodies. The GFP (lane 4), GFP-ILK (lane 5), GFP-F438A (lane 6) immunoprecipitates were analyzed by western blotting with HRP-conjugated anti-GFP antibodies (A), mouse monoclonal anti-CH-ILKBP antibody 3B5 and HRP-conjugated anti-mouse IgG antibodies (B), or rabbit anti-PINCH antibodies and HRP-conjugated anti-rabbit IgG antibodies (C). Lanes 1-3 were loaded with cell lysates (10 µg/lane) as indicated in the figure. (D,E) Subcellular localization. C2C12 cells expressing GFP-F438A were plated on fibronectin coated coverslips and stained with a mouse monoclonal anti-paxillin antibody and a Rhodamine RedTX-conjugated anti-mouse IgG antibody. GFP-F438A and paxillin were visualized under a fluorescence microscope equipped with GFP (D) and rhodamine (E) filters. The data shown are representative of three experiments. Bar, 10 µm.

View larger version (30K): [in a new window]   Fig. 8. A model of the assembly and focal adhesion localization of the PINCH-ILK-CH-ILKBP complex. See text for further details.