Materials, antibodies, and vectors
We used the following reagents: anti–human Met, anti-Shp2, anti-Src, antiactin, GST-Shp2, and GST-SH2-Grb2 (Santa Cruz Biotechnology, Inc.); anti-β4 integrin (BD Biosciences and Chemicon); anti-Gab1, anti-Shc, anti-PI3K, antiphosphotyrosine, and anti-GST (Upstate Biotechnology); anti-Grb2 (Transduction Laboratories); antiactive and total ERK/MAPK (Promega); antiactive Src (nonphospho-Tyr529; Biosource International); PP2 (Calbiochem); wild-type Shp2 and catalytically inactive C/S Shp2 in pJ3H vector (obtained from B.G. Neel, Harvard Medical School, Boston, MA); and wild-type Src and kinase-dead K/R Src in pCMV5 vector (obtained from J. Brugge, Harvard Medical School). The β4ΔShp2
construct containing phenylalanine mutations of Y1257, Y1440, and Y1494 was generated by PCR amplification of the BssHII–NotI fragment of a β4 template already containing the Y1257F and Y1494F substitutions (obtained from L.M. Shaw, Harvard Medical School). To create the β4 siRNA expression vector, oligonucleotides used by Chung et al. (2004)
were annealed and ligated into pSUPER between the BglII and HindIII sites. BamHI- and XhoI-digested inserts were then subcloned into the pRLL5 lentiviral vector. A scrambled β4 oligonucleotide was used as a control. The constructs encoding for wild-type Met, kinase-inactive Met, wild-type β4, β4-1440F, and β4Δextra
have been described previously (Trusolino et al., 2001
). The β4 cDNA used in this study corresponds to PubMed accession no. AAC51632
and matches with the sequence originally cloned by Suzuki and Naitoh (1990)
Cell culture, transfection, and viral infection
COS-7, MDA-MB-435, and MDA-MB-231 cells were cultured in DME supplemented with 10% FBS (Invitrogen). The expression of exogenous proteins was obtained with LipofectAMINE- or LipofectAMINE 2000 (Invitrogen)–mediated transfection according to the manufacturer's protocol or with retroviral or lentiviral infection. Viral hybrid vectors were produced by the transient transfection of 293T cells. Viral supernatants were filtered through a 0.22-μm filter, and infections were performed in the presence of 4 μg/ml polybrene (Sigma-Aldrich).
For immunoprecipitations, 5 × 106 cells were lysed for 20 min at 4°C with 1 ml of a buffer containing 50 mM Hepes, pH 7.4, 5 mM EDTA, 2 mM EGTA, 150 mM NaCl, 10% glycerol, and 1% Triton X-100 in the presence of protease and phosphatase inhibitors. For β4–Shp2 coimmunoprecipitations in FG2 cells, 1% Brij58 was used instead of Triton X-100. Extracts were clarified at 12,000 rpm for 15 min, normalized with the BCA Protein Assay Reagent kit (Pierce Chemical Co.), and incubated with different mAbs for 2 h at 4°C. Immune complexes were collected with either protein G– or protein A–Sepharose, washed in lysis buffer in the presence of 1 M LiCl, and eluted. Total cellular proteins were extracted by solubilizing the cells in boiling SDS buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1% SDS). Extracts were electrophoresed on SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Hybond; GE Healthcare). Nitrocellulose-bound antibodies were detected by the ECL system (GE Healthcare). Unless otherwise indicated, cells were stimulated with 50 ng/ml HGF for 30 min. PP2 was generally used at a 10-μM concentration and applied to cells for 30 min. In experiments aimed at analyzing the association of Gab1 with signal transducers and the HGF-dependent activation of ERKs, mock cells and cells expressing wild-type β4 or β4ΔShp2 were transiently transfected with a Gab1 cDNA.
For far-Western analysis, membranes were blocked for 1 h in TBS, 0.1% Tween-20, 5% BSA, and 3 μm glutathione and were incubated for 3 h with 1 μg/ml GST fusion protein previously adsorbed to 3 μM glutathione in 0.5% BSA. Filters were subsequently decorated with anti-GST antibody.
Src kinase assays were performed on Src immunoprecipitates using a commercial kit (Upstate Biotechnology) based on the phosphorylation of a specific substrate peptide (KVEKIGEGTYGVVYK) using the transfer of the γ-phosphate of γ-[32P]ATP by Src. The phosphorylated substrate was then separated from the residual γ-[32P]ATP using phosphocellulose paper and quantified with a scintillation counter.
Soft agar assay
3,000 cells were resuspended in complete medium containing 0.5% Seaplaque agar. Cells were seeded in 24-well plates containing a 1% agar underlay and supplemented twice a week with complete medium. In some experiments, MDA-MB-435–β4 cells were treated twice a week with 5 μM PP2 or 10 μM PD98059. Colonies were stained by the incorporation of tetrazolium salts 2 (for MDA-MB-435) or 3 wk (for MDA-MB-231) after seeding. Colonies were coded and scored in a blinded fashion by a second observer. Colony numbers were obtained using a phase-contrast light microscope (DMIL; Leica) fitted with a 32-grid eyepiece at a total magnification of 20×. Images were captured with ImageReady software (Adobe) using a microscope (DMIL; Leica) and a 20 × 0.30 objective (Leica) equipped with a digital camera (DFC320; Leica).
Statistical and densitometric analysis
Results are means ± SEM. Comparisons were made using the two-tailed t test. P-values <0.05 were considered to be statistically significant. Blot images were captured using a molecular imager (ChemiDoc XRS; Bio-Rad Laboratories). Densitometric analysis was performed with analysis software (Quantity One 1-D; Bio-Rad Laboratories) installed on the imager. Images were arranged and labeled using Illustrator (Adobe).