Recombinant human PDGF (rHu PDGF-BB) was purchased from Intergen Company. The anti-Gal4 DNA binding domain (DBD) monoclonal antibody (RK5C1), the anti-N-terminal c-Fos polyclonal antibody (sc-52), the anti-phospho-ERK mouse monoclonal antibody (sc-7383), and the anti-total ERK2 rabbit polyclonal antibody (C-14) were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). The anti-C-terminal c-Fos polyclonal antibody (ab2) was from Geneka Biotechnology Inc. (Montreal, Quebec, Canada). The anti-phospho threonine-proline monoclonal antibody was from Cell Signaling Technology (Beverly, Mass.). The anti-tag monoclonal antibodies (for hemagglutinin [HA], AU5, and six-His) were from Covance (Berkeley, Calif.). The MEK inhibitor U0126 was obtained from Promega Corp. (Madison, Wis.). The p38 and MEK inhibitors SB203580 hydrochloride and PD98059 were purchased from Calbiochem-Novabiochem Corp. (La Jolla, Calif.). The JNK inhibitor SP600125 [anthra(1,9-cd)pyrazol-6(2H)-one], was from Biomol (Plymouth Meeting, Pa.). Protein phosphatase-2A (PP2A) and recombinant ERK2 (active) were purchased from Upstate Biotechnology (Lake Placid, N.Y.). [32P]ATP was obtained from New England Nuclear (Chicago, Ill.). All other reagents were of analytical grade.
The full-length cDNA from the murine c-fos
gene, containing amino acids (aa) 1 to 380, was PCR amplified and cloned as an Eco
I fragment into pCEFL-AU5, a modified pcDNAIII expression vector containing the elongation factor 1 (EF-1) promoter driving the expression of an in-frame N-terminal AU5 tag (34
). The expression vectors for the constitutively active MAPK kinases (MAPKKs; pCEV29-MEKEE, pCEFL-MEKK1, and pCEFL-MEK3EE), the HA-tagged MAPKs (ERK2, JNK1, and p38α), c-Jun (pCEFL-c-Jun) and a dominant negative MEK1 with alanine replacements of Ser-218 and -222 (pcDNAIII-MEKAA) have been previously described (10
). The B-chain of the human PDGF gene (c-sis
) was cloned into the pcDNAIII expression vector. The pAP-1 luciferase reporter plasmid was obtained from Stratagene. This construct has been designed by inserting seven tandem repeats of the AP-1 response element from the polyoma enhancer (TGA C TAA) into the pA3 vector (17
). To obtain the Gal4-c-FosTAD fusion protein, the C-terminal domain of mouse c-Fos (aa 209 to 380) (42
) was PCR amplified from its full-length cDNA and cloned in a pcDNAIII vector encoding the DBD of the yeast transcription factor Gal4 (pcDNAIII-GBDEX). A similar construct was made by fusing the full-length c-Fos cDNA to the same expression vector (Gal4-c-FosFL). A Gal4-driven luciferase reporter, pGal4-Luc (34
), was designed by inserting six tandem copies of Gal4-responsive elements and a minimum TATA initiator in place of the simian virus 40 (SV40) promoter in pGL3 (Promega).
Individual point mutations of pCEFL-AU5-c-Fos and Gal4-c-FosTAD on Thr-232, Thr-331, Thr-325, and Ser-374 were generated by site-directed mutagenesis (QuikChange site-directed mutagenesis kit; Stratagene). To obtain a pCEFL-AU5-c-Fos clone containing multiple alanine replacements on Thr-232, Thr-325, Thr-331, and Ser-374 (c-Fos-m), we used the QuikChange multisite-directed mutagenesis kit (Stratagene). The sequences of the primers used for mutagenesis will be provided upon request. Add-back mutants including a single potential phospho-acceptor site were generated by reintroducing each original residue on c-Fos-m, which was used as a template for mutagenesis. BamHI/NotI PCR fragments from c-FosTAD-m and the different mutants were also cloned in pcDNAIII-GBDEX to obtain expression plasmids for the corresponding Gal4-c-FosTAD fusion proteins.
To obtain the polyhistidine-tagged (six-His) c-Fos TAD constructs, the TADs from c-Fos wild type and the different c-Fos mutants were PCR amplified and cloned as BamHI/NotI fragments in a modified pRSET-A bacterial expression vector (Invitrogen Corp.) in which the HindIII recognition sequence at the polylinker was replaced by a NotI site.
NIH 3T3 mouse fibroblasts were routinely cultured in serum-supplemented medium composed of Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Inc.), 10% calf serum, and penicillin-streptomycin-amphotericin B (Life Technologies, Inc.). HEK-293T cells were grown in DMEM containing 10% fetal bovine serum (FBS) and the above antimicrobial mixture.
Preparation of nuclear extracts.
Cells were collected after overnight starvation followed by induction with PDGF (20 ng/ml) or serum with or without previous treatment with MAPK inhibitors. The monolayers were washed with Tris-buffered saline and lysed with buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), and 0.5% NP-40. Cell lysates were centrifuged, and the resulting nuclei (pellet) were disrupted with extraction buffer (20 mM HEPES [pH 7.9], 0.5 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT). Cell debris were separated by low-speed centrifugation. Nuclei aliquots (2 to 5 μg of protein/μl) were stored at −70°C until use. All steps were performed at 4°C.
NIH 3T3 and HEK-293T cells were plated in complete medium and allowed to grow overnight to 70 to 80% confluence in six-well plates unless otherwise noted. The cells were transfected for 3 h in serum-free DMEM containing up to 2 μg of total plasmid DNA together with the Lipofectamine Plus reagent (Life Technologies, Inc.) according to the protocol suggested by the manufacturer.
Luciferase reporter assays.
Cells were transfected with different expression vectors together with 0.1 μg of each luciferase reporter and 0.01 μg of pRL-null (a plasmid encoding the luciferase gene from Renilla reniformis), which served as an internal control for transfection efficiency. The total amount of transfected DNA was normalized by adjusting it with pcDNAIII-β-gal, an expression vector for the enzyme β-galactosidase. Cells were lysed in passive lysis buffer (Promega) 24 h posttransfection. Cell lysates (50 μl/well) were transferred to a 96-well luminometer plate, and firefly and Renilla luciferase activities were assayed using the Dual-Luciferase reporter system (Promega). Light emission was quantified using a Microliter plate luminometer as specified by the manufacturer (DINEX Tech).
NIH 3T3 cells were grown up to 10 to 20% confluence in 10-cm plates and transfected following the calcium-phosphate precipitation technique. The day after transfection, cells were washed and incubated in DMEM supplemented with 5% calf serum. The cultures were maintained in the same medium, with medium changes every 3 days, until the appearance of foci from transformed cells was evident (usually 15 to 25 days after transfection).
Purification of GST and six-His fusion proteins.
Escherichia coli BL-21 Lys cells (Promega Corp.) were transformed with the vectors pGEX-4T3 and pRSET-A encoding glutathione acetyl transferase (GST) and polyhistidine (six-His) fusion proteins, respectively. Bacteria were grown in Luria-Bertani medium until the optical density reached 0.5. Protein synthesis was stimulated by the addition of 1 mM isopropyl-β-thiogalactopyranoside, and bacteria were allowed to grow for 3 h. The cells were collected by centrifugation (3,000 × g, 30 min) and resuspended in buffer containing phosphate-buffered saline (PBS), 1% Triton X-100, 1 mM EDTA, 2 μg of aprotinin/ml, 2 μg of leupeptin/ml, and 1 mM PMSF. The cell suspension was sonicated, and cellular debris was removed by centrifugation. The supernatant was incubated (1 h, 4°C) with 300 μl of glutathione-Sepharose 4B (Amersham-Pharmacia Biotech). The beads were pelleted by centrifugation, washed three times with PBS, 1% Triton X-100, 2 μg of aprotinin/ml, 2 μg of leupeptin/ml, 1 mM PMSF and then twice with PBS, 2 μg of aprotinin/ml, 2 μg of leupeptin/ml, 1 mM PMSF. GST fusion proteins were eluted with glutathione buffer (10 mM glutathione in 50 mM Tris [pH 8.0], 2 μg of aprotinin/ml, 2 μg of leupeptin/ml, 1 mM PMSF).
Six-His-tagged proteins were isolated using nickel-nitrilotriacetic acid magnetic agarose beads (Qiagen) following the manufacturer's protocol for bacterial expression and purification under native conditions.
In vitro kinase assays.
HEK-293T cells were allowed to grow to up to 70 to 80% confluence in 6-cm plates and then transfected with expression vectors for HA-tagged kinases alone or in combination with the respective upstream activating molecules. The cells were incubated in serum-supplemented medium overnight after transfection (Lipofectamine Plus) and then serum starved for 6 h (ERK2) or 2 h (JNK and p38α) before lysis for kinase reaction. The plates were washed with iced-cold PBS and resuspended in lysis buffer (25 mM HEPES [pH 7.5], 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 20 mM β-glycerophosphate, 1 mM Na-vanadate, 1% NP-40, 1 mM PMSF, 20 μg of aprotinin/ml, and 20 μg of leupeptin/ml). Cleared lysates were subjected to immunoprecipitation using anti-HA monoclonal antibodies (1 h, 4°C) followed by incubation with protein G-Sepharose (Gamma-Bind G Sepharose; Amersham-Pharmacia Biotech). The beads were pelleted by centrifugation and washed three times with PBS, 1% NP-40, 2 mM Na-vanadate, followed by one wash with 100 mM Tris (pH 7.5), 0.5 M LiCl and one final wash with kinase reaction buffer (12.5 mM morpholinepropanesulfonic acid [MOPS] [pH 7.5], 12.5 mM glycerophosphate, 7.5 mM MgCl2, 0.5 mM EGTA, 0.5 mM sodium fluoride, 0.5 mM Na-vanadate). The reactions were initiated by the addition of 30 μl of reaction mixture (kinase buffer plus 10 μCi of [γ32P]ATP, 20 μM unlabeled ATP, 1 mM DTT, 0.5 to 1 μg of substrate) to the immunocomplexes. After 30 min at 30°C, the reactions were terminated by the addition of sodium dodecyl sulfate (SDS) sample buffer (400 mM Tris-HCl [pH 6.8], 10% SDS, 50% glycerol, 500 mM DTT, 2 μg of bromophenol blue/ml), followed by 5 min of boiling. ERK2 kinase activity was assayed using 0.5 μg of myelin basic protein (Sigma) as a positive control. JNK and p38α activities were assayed using 1 μg of purified GST-ATF2 (aa 1 to 96). Denatured samples were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide gels, and autoradiographs were taken from dried gels using X-Omat Kodak films. Cold kinase assays were performed identically except for the omission of radioactive ATP from the reaction mix. The samples were immunoblotted using anti-phospho-specific antibodies as described below.
Western blot analysis.
Cleared cell lysates were combined with SDS sample buffer, boiled for 5 min, and resolved by SDS-10% PAGE. Fractionated proteins were electrophoretically transferred to polyvinylidene fluoride membranes (Immobilon-P; Millipore). Nonspecific binding sites were blocked with 5% nonfat dried milk in PBS containing 0.05% Tween 20 (PBS-T) and incubated (1 h, room temperature) with the appropriate dilution of each primary antiserum or monoclonal antibody. The membranes were repeatedly washed with PBS-T prior to incubation with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies (ICN-Cappel). Immunoreactive protein bands were visualized by enhanced chemiluminescence detection (ECL Plus System; Amersham Biosciences).
Northern blot analysis.
NIH 3T3 cells were grown to 70% confluence in 10-cm plates and serum starved overnight. Cells were left untreated or treated with 20 ng of PDGF/ml for different times, in the absence or presence of U0126. Cells were washed with cold PBS, and total RNA was extracted by homogenization in TRIzol (GIBCO BRL) according to the manufacturer's specifications. For Northern blotting, 20 μg of total RNA was fractionated in 2% formaldehyde-agarose gels, transferred to nylon membranes, and hybridized with a 32P-labeled cDNA probe from the murine c-Fos TAD, which was prepared using the Prime-a-Gene labeling system (Promega). Accuracy in gel loading and transfer was confirmed by fluorescence under UV light upon ethidium bromide staining of the gels.
NIH 3T3 and HEK-293T cells overexpressing c-Fos proteins were rapidly washed with cold PBS, scraped, and collected by centrifugation. Cells were disrupted by freeze-thaw cycles and resuspended in a buffer containing 20 mM MOPS (pH 7.3), 150 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 20 μg of leupeptin/ml. Protein aliquots were incubated in the absence or presence of 0.5 U of PP2A (2 h, 30°C). The reactions were stopped by adding SDS sample buffer and processed for immunoblotting as described above.