The preparation of plasmids expressing catalytically active PMR60, catalytically inactive PMR60°, Y650F mutant, and PMR60° deletions were described previously (Yang et al., 2004
; Yang and Schoenberg, 2004
). The mutations P262,265A and P270,273A were generated by site-directed mutagenesis of the proline residues at positions 262, 265, 270, and 273 using the QuikChange site-directed mutagenesis kit (Stratagene). Sense primer 5′- GTGCCAAAGAGCCTGCTTGCTTTGCCCTGAAGATTCCAC-3′ and antisense primer 5′-GTGGAATCTTCAGGGCAAAGCAAGCAGGCTCTTTGGCAC-3′ were used to generate P262,265A. Sense primer 5′-CCCTGAAGATTCCAGCAAATGACGCTCGAATTAGTAATCAGAG-3′ and antisense primer 5′-CTCTGATTACTAATTCGAGCGTCATTTGCTGGAATCTTCAGGG-3′ were used to generate P270,273A. The empty vector pUSEamp and plasmids encoding wild-type (WT-Src), kinase-inactive (KI-Src), dominant-negative (DN-Src), or constitutively-active (CA-Src) forms were from Upstate Biotechnology. The plasmid pET-32b-PMR was constructed by inserting the entire PMR60 sequence into pET32b between NcoI and NotI sites.
Monoclonal antibody to the c-Myc epitope tag (9E10), myc antibody-coupled beads, antibodies to GFP (B2), c-Src (SRC2 and B-12) and ribosomal protein S6 (E13) were purchased from Santa Cruz Biotechnology. PY20 was purchased from BD Biosciences, and horseradish peroxidase-coupled rabbit anti-mouse IgG and goat anti-rabbit IgG were purchased from Santa Cruz.
Cos-1 cells were cultured in Dulbecco’s modified Eagle’s medium plus 10% fetal bovine serum and 2 mM glutamine. U2OS cells were cultured in McCoy’s 5A medium plus 10% fetal bovine serum. For most experiments, 2.5 × 106 cells in log phase growth were transfected with 10 μg (total) of plasmid DNA by using LipofectAMINE (Invitrogen) following the manufacturer’s protocol. Unless otherwise indicated cells were collected 40 hr after transfection. To study EGF activation of c-Src Cos-1 cells were serum starved for 16 hr prior to adding 100 ng/ml EGF. To inhibit c-Src in these cells 10 μM PP3 or PP2 was added to the culture 30 min before addition of EGF.
Preparation of cell extracts
Cytoplasmic extracts were prepared as described previously (Yang and Schoenberg, 2004
). Briefly, cells were washed twice with ice-cold phosphate-buffered saline, then suspended in cell lysis buffer (10 mM HEPES-KOH, pH 7.5, 10 mM KCl, 5 mM MgCl2
, 50 mM NaF, 0.5% Nonidet P-40 (v/v), 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 25 μl/ml protease inhibitor mixture (Sigma), 10 μl/ml phosphatase inhibitors (Sigma), and 10 μl/ml RNaseOUT (Invitrogen)). After incubation for 15 min on ice the cells were homogenized with 30 strokes of a Dounce homogenizer (A pestle), and the homogenate was centrifuged for 15 min at 2,000 × g. Polysomes and mRNP complexes were separated on discontinuous sucrose gradients containing 1 ml each of 10% and 35% (w/v) sucrose in the buffers described above (Schoenberg and Cunningham, 1999
). Postmitochondrial extract was fractionated by sedimentation for 1 hr at 147,000 × g in a Sorvall Discovery M120 SE ultracentrifuge using a S55S-1123 rotor. Polysomes were collected from the bottom of the tube and mRNP complexes were collected at the interface between the 10% and 35% sucrose layers.
Expression of recombinant PMR60
The plasmid pET32b-PMR was transformed into E. coli BL21(DE3). Cells were grown to an optical density (O.D.)600 of ~1.2 and induced with 0.5 mM IPTG for 4 hr at 37°C. Cells were lyzed by sonication in the buffer containing 1 X PBS, 5 mM EDTA, 5 mM DTT, 1 mg/ml lysozyme and 1 mM PMSF. After centrifugation, the pellets were extensively washed with 1 X PBS, 5 mM EDTA, 5 mM DTT, 2 M Urea, 2% Triton X-100, then dissolved in 20 mM NaH2PO4, 500 mM NaCl, 6 M guanidine-HCl, 2 mM beta-mercaptoethanol, pH8.0. The clarified lysate was loaded onto HiTrap chelating HP column (Amersham Bioscience). After extensive washing with 20 mM NaH2PO4, 500 mM NaCl, 10 mM imidazole, pH7.5, bound proteins were eluted with a linear gradient of 10 mM to 500 mM imidazole. The purified proteins were renatured in 20 mM Tris-HCl, pH 8.5, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, 9.6 mM NaCl, 0.4 mM KCl, and further purified by ion exchange chromatography on a Fast-flow DEAE-Sepharose column.
Immunoprecipitation and Western blot analysis
For immunoprecipitation with myc antibody cell lysates were incubated with monoclonal antibody-coupled beads on a rocking platform for 3 hr at 4°C. For immunoprecipitation with rabbit anti-Src antibody cell lysates were incubated with antibody for 3 hr at 4°C followed by 20 μl of protein A-agarose (Santa Cruz Biotechnolgy) and incubation for 1 hr on a rocking platform at 4°C. The beads were washed four times with IPP150 buffer (10 mM Tris-HCl, pH7.5, 150 mM NaCl, 0.1% NP-40) and suspended in SDS-PAGE sample buffer. For Western blot analysis, the immunoprecipitates were separated on a 10% SDS-PAGE and electroblotted onto Immobilon-P membrane (Millipore). The membrane was blocked for 1 hr at room temperature in 5% nonfat dry milk in TBST buffer (20 mM Tris-HCl, pH7.5, 150 mM NaCl, 0.1% NP-40), then incubated with the primary antibody for 4 hr at room temperature, washed and incubated with horseradish peroxidase-conjugated secondary antibody for 1 hr. Blots were developed with SuperSignal west pico chemiluminescent substrate (Pierce).
In vitro kinase assays
HPLC purified N-terminal biotin-labeled peptides containing the tyrosine phosphorylation site of PMR60 (ARDGDRFFYEQP) without or with phosphotyrosine at position 9 (corresponding to Y650) were obtained from American Peptide Company, Inc. (Sunnydale, CA). 6 units of purified recombinant c-Src (Upstate Biotechnology, Inc) was incubated with 250 μM of each synthetic peptide or different amounts of recombinant PMR60 dissolved in 50 μl of 25 mM Tris-HCl, pH 7.5, 30 mM MgCl2, 12.5 mM MnCl2, 1 mM EGTA, 60 μM sodium orthovanadate, 1 mM dithiothreitol). Reactions were initiated by adding of 1 μl of γ-[32P]ATP (3,000 Ci/mmol, PerkinElmer) and incubated for 30 min at 30ºC. The reaction was stopped by the addition of SDS sample buffer, and phosphorylated samples separated by SDS-PAGE were visualized by PhosphorImager.
Ribonuclease protection assay
8 × 105
cells were transiently transfected with plasmids expressing albumin and luciferase mRNA and co-transfected with empty vector (pcDNA3), or plasmid expressing catalytically active myc-PMR60, plus vector pUSEamp or plamids encoding CA- or DN -Src. Total RNA was isolated with TRIzol reagent (Invitrogen). The antisense albumin riboprobe was prepared with the MAXIscipt in vitro
Transcription Kit (Ambion) using T7 promoter from a pcRII-Topo plasmid containing exons 14 and 15 of albumin cDNA. The antisense firefly luciferase riboprobe was synthesized with T3 promoter from a pBluescript(SK) plasmid containing the first 153 nucleotides of firefly luciferase cDNA. Ribonuclease protection assay was done as described previously (Yang and Schoenberg, 2004
) with 5 μg of total RNA hybridized to 600 pg of each riboprobe using the Ribonuclease Protection Assay III Kit (Ambion). Protected probe was separated on a denaturing 6% polyacrylamide-urea gel and quantified by PhosphorImager analysis.