Yeast Two-Hybrid Library Screening
cDNAs corresponding to full-length human ILK were amplified from a human bone marrow cDNA library (CLONTECH Laboratories, Inc.) by PCR. These fragments were subcloned into pAS2-1, and the resultant ILK–pAS2-1 was used to screen human bone marrow and fetal liver cDNA libraries (CLONTECH Laboratories, Inc.) in Y190(a) yeast strain. Cotransformants of the bait and library plasmids were grown for 3–7 d at 30°C on minimum essential plates lacking histidine, tryptophan, leucine, and uracil and containing 35 mM 3-aminotriazole (Sigma-Aldrich). Positive colonies were further screened for β-galactosidase activity according to the manufacturer's instructions (CLONTECH Laboratories, Inc.). Y187(a) yeast strain was also used only for β-galactosidase assay to verify the two-hybrid interactions. Approximately 1 × 106 and 4 × 106 clones were screened, respectively, and seven independent clones positive for growth on plates lacking histidine and β-galactosidase activity were identified. Sequencing analysis revealed that five of these clones contained cDNA inserts encoding a 570-bp overlapping sequence. Subsequent backscreening against phage cDNA libraries from NEC and Jurkat cell lines to obtain the corresponding full cDNA resulted in the identification of several affixin cDNAs with the same ORF but containing different 5′ or 3′ noncoding regions.
Northern Blot Analysis
To analyze the tissue distribution of mRNA expression, multiple tissue Northern blot membrane (CLONTECH Laboratories, Inc.) was probed with a 32P-labeled human affixin cDNA probe, corresponding to amino acid residues 206–481 of the l-affixin prepared using a random-primed DNA labeling kit (Amersham Pharmacia Biotech). The hybridization was performed according to the manufacturer's instructions (CLONTECH Laboratories, Inc.), and an x-ray film was exposed at −80°C for 10 d with an intensifying screen.
Affixin and ILK Mutations
Affixin and ILK deletion mutants were generated by PCR using appropriate primers. Point mutations in ILK mutants (E359K, K220M, K220A) were introduced using a QuickChange site-directed mutagenesis kit (Stratagene). The fidelities of the amplified sequences were all verified by DNA sequencing.
CHO-K1 cells were maintained at 37°C in a humidified atmosphere of 5% CO2 in F-12 medium containing 10% FCS (Cell Culture Technologies, Inc.), 100 U/ml penicillin, and 100 μg/ml streptomycin. COS-7 cells were cultured under the same conditions as those for CHO-K1, except for the use of DME instead of F-12 medium. cDNA transfection was performed by either electroporation for the immunoprecipitation assay or lipofection using a Fugene6 transfection reagent (Roche) for immunofluorescence analysis. When performing the replating assay, CHO-K1 cells were transfected with the appropriate expression plasmids, harvested 48 h later by incubating in 0.05% trypsin in PBS containing 0.02% (wt/wt) EDTA, washed two times with PBS, and replated on fibronectin-coated coverslips.
The antibodies used in this study were anti-Flag and antivinculin monoclonal antibodies (Sigma-Aldrich), anti-FAK monoclonal antibodies (Transduction Laboratories), anti-ILK monoclonal antibody (Upstate Biotechnology), anti–α-actinin monoclonal antibody (provided by Yukiko Hayashi, National Institute of Neuroscience, NCNP, Japan), anti-T7 monoclonal antibody (Novagen), and FITC-phalloidin and rhodamine-phalloidin (Molecular Probes). Antiaffixin antibodies were generated in rabbits using glutathione S-transferase (GST)–ss-affixin as an antigen and affinity purified with the antigen before use.
SDS-PAGE and Immunoblot Analysis
For analysis of affixin expression in various rat tissues, each organ was excised from 10-wk-old Sprague Dawley rats deeply anesthetized with diethyl ether, washed with ice-cold PBS, and frozen immediately in liquid nitrogen. The tissue blocks were crushed using a Cryo-Press (Diatron) precooled in liquid nitrogen, and the resultant powder was suspended in 10 vol (vol/wt) of SDS sample buffer, homogenized with a Polytron homogenizer (Kinematica), and sonicated. 15-μg aliquots of these samples were loaded in each lane. Electrophoresis was carried out by one-dimensional SDS-PAGE (10 or 12% polyacrylamide). The separated proteins were transferred onto PVDF membranes, which were subsequently blocked with 5% skimmed milk. The membranes were treated with appropriate antibodies, and antibody reactions were visualized by a chemiluminescence ECL system (Amersham Pharmacia Biotech).
Cells cultured in 10-cm dishes were suspended in 200 μl lysis buffer containing 20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 μg/ml leupeptin, 1 mM PMSF, 1% Triton X-100, 0.1% deoxycholate, and 0.1% SDS. After a 30-min incubation on ice, the lysates were clarified by centrifugation at 14,000 rpm for 30 min. 15 μl of protein G–Sepharose (Amersham Pharmacia Biotech) conjugated with 2 μg of affinity-purified antiaffixin, anti-Flag antibodies, or control normal rabbit IgG were incubated with the cell lysates for 1 h at 4°C. After washing with lysis buffer, the immunocomplex was solubilized by adding SDS sample buffer to the resin.
CHO-K1 cells or those transfected with expression plasmids were cultured on fibronectin-coated coverslips for 48 h and, after washing with PBS, fixed with 1 or 2% formaldehyde in PBS for 15 min and then permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature. In some experiments, cells were fixed with 100% methanol. The cells were blocked with PBS containing 10% calf serum for 1 h at room temperature and then treated with appropriate primary antibodies for 45 min at 37°C in a moist chamber. After washing with PBS containing 0.05% Tween 20, the cells were incubated with secondary antibodies (Cy3-conjugated goat anti–rabbit [Amersham Pharmacia Biotech] and Alexa488-conjugated goat anti–mouse antibodies [Molecular Probes]) at 37°C for 45 min. After washing, samples were observed under a fluorescence microscope (BX50; Olympus) equipped with a cooled CCD camera (Photometrics). Human skeletal muscle sample was obtained by biopsy for diagnostic purpose with informed consent. Thin sections of 6-μm thickness were fixed for 10 min with 100% acetone at −20°C, blocked for 15 min at 37°C with 2% BSA and 0.5% goat serum in PBS, and then processed for immunofluorescence analysis. Confocal microscopic analysis was performed using a Bio-Rad Laboratories Radiance 2000 scan head mounted on a Nikon Eclipse E600 microscope.
Purification of Recombinant Affixin and Its Mutant from Escherichia Coli
GST–ss-affixin and GST–RP2 fusion proteins were induced in E. coli with isopropyl β-d-thio-galactopyranoside (Amersham Pharmacia Biotech), and the proteins were purified with glutathione–Sepharose 4B beads (Amersham Pharmacia Biotech). The GST linker sites of these fusion proteins were digested with PreScission™ protease (Amersham Pharmacia Biotech) according to the manufacturer's protocol, and the excised recombinant proteins eluted from the resin were dialyzed against the appropriate buffers before use.
In Vitro Kinase Assay
COS-7 cells transfected with expression vectors encoding Flag-tagged ILK or its mutants were lysed in 20 mM Hepes, pH 7.0, 150 mM NaCl, 1 mM EDTA, 10 μg/ml leupeptin, 1 mM PMSF, 1% Triton X-100, and 0.1% deoxycholate. Immunoprecipitates by anti-Flag antibody were extensively washed with lysis buffer and then kinase reaction buffer (50 mM Hepes, pH 7.0, 10 mM MnCl2, 10 mM MgCl2, 2 mM NaF, 1 mM Na3VO4) and subjected to protein kinase assays in 20 μl kinase reaction buffer containing 10 μCi [γ-32P]ATP and an appropriate substrate (myelin basic protein [MBP] or recombinant affixin). After incubation for 60 min at 30°C, the reaction mixture was resolved by 10% SDS-PAGE, and bands were visualized by a Bio-imaging analyzer system (BAS2000; Fuji).