Chinese hamster ovary (CHO) cells, glioma CRL-1620 cells (glioma 1620), NIH 3T3, and MB-MDA-231 breast cancer cells (MDA-231) were obtained from American Type Culture Collection (Manassas, VA). All cells were cultured in DMEM with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% l-glutamine, and 1% nonessential amino acids (all from Invitrogen, Carlsbad, CA).
Antibodies and Immunohistochemistry
Rabbit polyclonal anti-PEA-15 (3099) was raised against a synthetic peptide containing the C-terminal 14 amino acids (EEEIIKLAPPPKKA) of PEA-15 as described previously (Ramos et al., 2000
). Anti-PEA-15 (4513) was raised against a glutathione S
-transferase-PEA-15 fusion protein (GST-PEA-15). Anti-PEA-15 (4513) was absorbed with GST-agarose, adjusted to pH 8.0 in 100 mM Tris, and bound to immobilized GST-PEA-15. Affinity-purified anti-PEA-15 (4513) was eluted using 100 mM glycine (pH 3.0) and neutralized with 1 M Tris (pH 8.0). Anti-PEA-15 phospho-S116 (p-PEA-15), which recognizes PEA-15 when it is phosphorylated at Ser116
, was produced at BioSource International (Camarillo, CA). Antiserum was generated using a chemically synthesized phosphorylated peptide (IRQP[pS]EEEIIKL) coupled to keyhole limpet hemocyanin and injected into specific pathogen-free rabbits. The resulting phosphorylation site-specific antibody was purified using both negative and positive peptide affinity purification. The rabbit antibody against lamins A/C was a gift from Dr. Larry Gerace (The Scripps Research Institute, La Jolla, CA). Antibodies against Influenza hemagglutinin (HA) tag and Rho GDI were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-FLAG antibody was obtained from Sigma-Aldrich (St. Louis, MO). Unless otherwise indicated, all antibodies were used at a 1:1000 dilution. Anti-rabbit and anti-mouse horseradish peroxidase (HRP)-conjugated antibodies (BioSource International) and SuperSignal Pico Chemiluminescent Substrate (Pierce Chemical, Rockford, IL) were used for Western blot detection.
Immunohistochemistry was performed on Formalin-fixed/paraffin-embedded breast tissue sections by immunoperoxidase staining. Paraffin-embedded sections were deparaffinized and then microwaved for 10 min in citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). After cooling, endogenous peroxidase was blocked with 0.3% H2O2 in phosphate-buffered saline (PBS) (1.7 mM KH2PO4, 5.2 mM Na2HPO4 150 mM NaCl, pH 7.4) for 10 min. Further blocking was done in 1% bovine serum albumin (BSA) (Sigma-Aldrich) in PBS for 20 min. Primary antibodies were incubated on the slides overnight at 4°C. Affinity purified anti-PEA-15 (4513) was used at 0.1 μg/μl, and anti-p-PEA-15 was used at a 1:25 dilution. Equivalent amounts of control rabbit IgG and 1% BSA were used as a control. After washing in PBS, goat anti-rabbit HRP secondary antibodies (1:500) were added to the slides for 30 min at room temperature. Staining was then developed with aminoethylcarbazole chromagen. Slides were counterstained with Mayer's hematoxylin. Dilutions and staining conditions were validated on fixed MB-MDA-231 and CRL 1620 cell lines before performing immunohistochemistry on breast tissue.
PEA-15 cDNA expression constructs used in this work have been described previously (Chou et al., 2003
). PEA-15 cDNA was expressed from two vectors, pCDNA3.1(+) (Invitrogen) for eukaryotic expression and pGEX2T (Amersham Biosciences UK, Little Chalfont, Buckinghamshire, United Kingdom) for in vitro protein production from bacterial cells. The PEA-15 mutant L123R was initially described in Hill et al
). All PEA-15 constructs in pCDNA3 included a C-terminal HA tag. Additional PEA-15 mutants S104D, S116D, S104A, and S116A, were generated with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) by using wild-type pGEX2T-PEA-15 or pCDNA3-PEA-15 as the template. All plasmid constructs were verified by DNA sequencing. The pEGFP-C1 vector was obtained from BD Biosciences Clontech (Palo Alto, CA).
In Vitro Protein Production
BL21 competent bacteria were transfected with pGEX2T-PEA-15 plasmids (wild-type or mutant) and induced to express protein with 1 mM isopropyl β-d-thiogalactoside for 2–3 h. The bacteria were lysed in a PBS buffer containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 2 mM dithiothreitol (DTT), 5 μg/ml aprotinin, and 0.5 mM leupeptin. Wild-type and mutant GST-PEA-15 were then enriched from total bacterial lysate by binding to glutathione-Sepharose 4B beads (Amersham Biosciences UK). Bead-bound GST-PEA-15 was washed with PBS and eluted using 20 mM glutathione. After dialysis against PBS, purified GST-PEA-15 was stored at –70°C.
In Vitro Phosphorylation and ERK Binding
NIH 3T3 cells were lysed in a kinase active buffer (1% NP-40, 25 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM NaF, 1 mM EGTA, 2 mM DTT, 3 mM MgCl2, 2 mM CaCl2, 1 mM NaVO4, EDTA-free protease inhibitor cocktail [Roche Diagnostics, Mannheim, Germany]) to which 1 mM ATP with 0.022% [γ-32P]ATP was added. One hundred micrograms of total cell lysate in 100 μl was then incubated with Sepharose-immobilized GST-PEA-15 for 2 h at 37°C in the presence or absence of lipid activator [50 μg/ml l-α-phosphatidyl-l-serine sodium salt (Sigma-Aldrich) and 50 μM phorbol 12-myristate 13-acetate (PMA; EMD Biosciences, La Jolla, CA) in 25 mM Tris, pH 7.4. In some cases, PKC or CamKII were inhibited by addition of 1 μM bisindoylmaleimide I (Bis) or 10 μM KN-62 (EMD Biosciences), respectively.
At the end of the incubation, beads were sedimented by centrifugation at 22,000 × g and washed three times with PBS. The bead bound PEA-15 was solubilized by digestion of the immobilized GST-PEA-15 fusion protein with thrombin (100 U/ml) in a buffer containing 100 mM Tris, pH 8.0, 150 mM NaCl, 2.5 mM CaCl2. Eluted proteins were resolved by SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. Incorporation of 32P was assayed by autoradiography. The blots were stained with antibodies reactive with ERK1/2 (Santa Cruz Biotechnology) to detect bound ERK1/2 and antibodies against PEA-15 to assay loading of the affinity matrix.
CHO cells were cotransfected with 0.5 μg of pCDNA3-FLAG-ERK2 and either 1.5 μg of pCDNA3-HA-PEA-15, pCDNA3-HA-PEA-15 mutants, or empty vector. Transient transfections were carried out using Lipofectamine and Plus reagents as per manufacturer's protocol (Invitrogen). Cells were harvested 24 h posttransfection and scraped into 1 ml of lysis buffer (20 mM HEPES, pH 7.4, 2 mM EGTA, 2 mM MgCl2, 2 mM NaVO4, protease inhibitors cocktail). Total cell lysates were homogenized by 20 serial passages through a 27-gauge needle and then centrifuged at 22,000 × g for 10 min. The supernatant was precleared with 20 μl of protein G-Sepharose beads (~50% slurry; Amersham Biosciences UK) at 4°C for 30 min. For each condition, 2 μg of anti-HA antibody and 20 μl of protein G-Sepharose beads were added to 500 μg of cleared lysate, and incubated for 2 h at 4°C. Immunoprecipitates were washed three times in lysis buffer and solubilized in 5× SDS-PAGE sample buffer [10% (wt/vol) SDS, 250 mM Tris, pH 6.8, 500 mM DTT, 50% glycerol, 1 mg/ml bromphenol blue]. Western blotting with anti-FLAG was used to assay ERK coimmunoprecipitation and with anti-HA to detect immunopre-cipitated PEA-15. Separately, 25 μg of total cell lysate was immunoblotted for FLAG-ERK and HA-PEA-15 to verify comparable expression in all samples.
CHO cells were transfected with cDNA encoding wild-type PEA-15, phosphomimetic mutants, or empty vector in a 3:1 ratio to HA-tagged ERK2. Cells were allowed to recover from transfection by allowing growth in complete media for 24 h. Next, cells were maintained in serum-free media for an additional 24 h. Cells were stimulated for 3 h with 10% FBS and then harvested and suspended at 5 × 106 cells/ml in fractionation buffer (20 mM HEPES, pH 7.5, 1.5 mM MgCl2, 5 mM KCl, 1 mM DTT, 1 mM NaVO4, with Complete EDTA Free protease inhibitor cocktail [Roche Diagnostics]). After incubation on ice for 20 min, cells were homogenized by shearing through a 23-gauge needle. Twenty-five microliters of total cell lysate was saved, and the remaining sample was centrifuged at 3000 × g to sediment the nuclei. The supernatant was then spun at 20,800 × g for 30 min at 4°C to separate the sedimented membrane fraction from the soluble cytosolic fraction. The nuclear pellet was washed two times with fractionation buffer, resuspended in 250 μl, and loaded onto a 250-μl cushion formed by 1 M sucrose in fractionation buffer, and centrifuged at 2250 × g for 10 min at 4°C. The pellet was then extracted into 250 μl of fractionation buffer containing 1% NP-40. Total, cytosolic, and nuclear fractions were resolved by SDS-PAGE and analyzed by Western blotting.
Serum Response Element reporter assay
CHO cells were transfected with 1.0 μg of wild-type pCDNA3-HA-PEA-15, 0.33 μg of pSRE-Luc (BD Biosciences Clontech), and 0.33 μg of pRL-TK (Promega, Madison, WI). Cells were grown in complete medium for 24 h and then shifted to serum-free media for 24 h. Cells were then stimulated by addition 10% FBS for 3 h, harvested, and resuspended in 250 μl of passive lysis buffer (supplied with dual luciferase assay kit; Promega). Twenty microliters of total lysate was placed in one well of a 96-well plate, and 100 μl of luciferase assay reagent (dual-luciferase reporter assay kit; Promega) was added. Firefly luciferase activity was assayed by measuring light emission using a 96-well plate Lucy 2 Luminometer (Anthos Labtec Instruments, Salzburg, Austria) with 1-s integration. One hundred microliters Stop-and-glo reagent (dual-luciferase reporter assay kit; Promega) was added to stop firefly luciferase activity and assay Renilla luciferase activity to correct for transfection efficiency. All conditions were assayed in triplicate in each experiment, and each experiment was performed in triplicate.
CHO cells were transfected with 1.0 μg of pCDNA3-HA-PEA-15 and 0.33 μg of pEGFP-C1 (GFP) vector as a transfection reporter. Cells were grown in complete medium for 24 h and then shifted to serum-free media for 24 h. Bromodeoxyuridine (BrdU, 10 μM; BD Biosciences PharMingen, San Diego, CA) was added and 15 min later 10% FBS was added. Cells were harvested after 45 min, resuspended in 50 μl of ice-cold PBS, and fixed by addition of 1 ml of 1% formaldehyde in PBS for 2 min at room temperature. After washing one time with PBS, the cells were resuspended in 100 μl of PBS and incubated with 1 ml of cold 70% ethanol for 5 min on ice. The cells were then washed two times with PBS, resuspended in 50 μl of PBS, and incubated with 20 U of DNase I (Roche Diagnostics) for 30 min. To detect BrdU, 1 μl of anti-BrdU antibody (BD Biosciences PharMingen) was added for 30 min at room temperature. After one wash with PBS, 1 μl of phycoerythrin-conjugated F(ab′)2 goat anti-mouse IgG (BD Biosciences PharMingen) was added for 30 min at room temperature. Cells were analyzed by flow cytometry for BrdU incorporation by using green fluorescent protein (GFP) expression as a marker for transfection. The percentage of change in BrdU incorporation was calculated by comparing the geometric mean fluorescence intensity of the BrdU staining of each sample to serum-starved vector-transfected cells. Whole cell lysates were analyzed for transfected protein expression by Western blotting.
Analytical two-color flow cytometry was carried out as described previously (Chou et al., 2003
). Briefly, CHO cells were transfected with 0.1 μg of pEGFP as transfection marker and a combination of 1.5 μg of pCDNA1-Raf-CAAX with 1.5 μg of various pCDNA3-HA-PEA-15 constructs. Transient transfections were carried out using Lipofectamine and Plus reagents using the manufacturer's protocol. After 24 h in normal growth media, cells were harvested and analyzed for transfection efficiency (GFP) and integrin binding to 3Fn-(9-11). Integrin activation was quantified as an activation index (AI) as defined in Chou et al
). The percentage of reversal of suppression was calculated as described previously (Chou et al., 2003