SMN point mutations E134K, Y272C, and G279V were introduced into a N-terminal myc-epitope-tagged expression plasmid, pCS2-MT-SMN, by site-directed mutagenesis using a QuikChange site-directed mutagenesis kit (Stratagene). A short hairpin RNA (shRNA)-resistant form of myc-SMNΔ7 was generated by introducing silent mutations in the shRNA-targeting region (G863A, C867T, A870T, and T873C) of myc-tagged SMNΔ7 cDNA. Cells were transfected with FUGENE 6 (Roche, Indianapolis, IN) according to the manufacturer's protocol.
Protein extraction and semiquantitative Western blotting.
Cells were lysed in 0.1% Nonidet P-40-0.5% sodium deoxycholate and sonicated for 10 s. For experiments studying SMN ubiquitination, the cell lysates were denatured with 1% sodium dodecyl sulfate (SDS) and then renatured with 4.5% Triton X-100 prior to immunoprecipitation with anti-SMN antibody (BD Transduction Laboratories, San Diego, CA). In addition, cell lysis buffer was supplemented with 2.5 μM ubiquitin aldehyde (Boston Biochem, Cambridge, MA) to inhibit deubiquitinating enzymes and 10 μM MG132 (Biomol International, LP, Plymouth Meeting, PA) to block proteasome degradation. Total protein concentrations were determined by using a BCA protein assay kit (Pierce, Rockford, IL) according to the manufacturer's protocol. Protein lysates were separated on 12 or 15% Tris-glycine acrylamide gels and transferred to membranes. The membranes were probed with mouse anti-SMN antibody at 1:3,000 (clone 8; BD Transduction Laboratories), mouse anti-β-actin antibody at 1:10,000 (Sigma-Aldrich, St. Louis, MO), anti-Gemin 3 antibody at 1:1,000 (12H12; Sigma-Aldrich), anti-Gemin 5 at 1:500 (Abcam, Inc., Cambridge, MA), anti-Gemin 6 at 1:500 (Abcam), anti-Gemin 2 antibody at 1:1,000 (2E17; Abcam), anti-myc at 1:1,000 (Sigma-Aldrich), and antiubiquitin at 1:1,000 (FK2; Biomol International). Quantitative immunoblotting was performed as suggested by the manufacturer (Li-Cor, Lincoln, NE). The membranes were scanned on an Odyssey infrared imaging system (Li-Cor), and the intensity of the protein bands were analyzed by using software provided by the manufacturer. SMN and actin were visualized with a peroxidase-linked goat anti-mouse immunoglobulin G (IgG) secondary antibody at 1:10,000 (Jackson Immuno Labs, Inc., West Grove, PA) and chemiluminescence detection (Perkin-Elmer Life Sciences, Oak Brook, IL). Densitometry of the resulting bands was done by using NIH Image software, and SMN protein amounts were corrected based on actin values.
Cell culture and drug treatment.
The human fibroblast cell line from a 3-year-old type I SMA patient with two copies of SMN2
(GM03813; Coriell Cell Repository, Camden, NJ) was maintained as previously described (39
). HEK293T cells were maintained in Dulbecco modified Eagle medium supplemented with 2 mM glutamine, 10% fetal bovine serum, and 1% penicillin-streptomycin (100 U/ml) in an atmosphere of 5% CO2
and 37°C. For drug treatments, cells were plated and treated 24 h later with 10 μM MG132 (Biomol International), 5 mM Calpeptin (Biomol International), 10 mM ammonium chloride, 1 μM myristoylated PKI, or vehicle for the designated times. The cells were then harvested for RNA or protein isolation and quantified as previously described (39
). The average value of untreated cells was normalized to 1.
shRNA knockdown of endogenous SMN.
HEK293T cells were successfully transfected with four predesigned SMN shRNA vectors or scrambled vector (SureSilencing; SuperArray, Frederick, MD) according to the manufacturer's instructions. Silencing of SMN expression was confirmed by reverse transcription-PCR analysis and immunoblotting. The shRNA plasmid (insert 5′-AAGGTGCTCACATTCCTTAAATT-3′) showed the highest level of SMN knockdown and was chosen for subsequent experiments.
Pulse-chase protein labeling.
Pulse-chase analysis was performed as previously described (34
). In brief, HEK293T cells were incubated in cysteine-methionine-free medium (Sigma-Aldrich) for 2 h or 12 h followed by incubation in cysteine-methionine-free medium containing 100 μCi of 35
S-labeled cysteine-methionine (GE Healthcare, Piscataway, NJ) for 1 h. After labeling, the cells were washed once with culture medium containing 10-fold excess of unlabeled methionine and cysteine (5 mM each) and then incubated further in the same medium. Cells were collected at the indicated time points and processed for immunoprecipitation with anti-SMN antibody (BD Transduction Laboratories), anti-Gemin 3 antibody (Sigma-Aldrich), anti-Gemin 5 (Abcam), and anti-Gemin 6 antibody (Abcam). Immunoprecipitations were carried out for 6 h at 4°C with antibodies bound to protein G-Sepharose (Sigma-Aldrich). After three washes with lysis buffer (100 mM NaCl, 10 mM Tris-HCl [pH 7.4], 2.5 mM MgCl2
, 0.1% NP-40, and protease [Roche]) and phosphatase inhibitor cocktails (Sigma-Aldrich), bound proteins were eluted by boiling in SDS-PAGE sample buffer. Immunocomplexes were separated on SDS-10% PAGE. Gels were dried and exposed to a phosphorimager screen, and the signal visualized with a Storm phosphorimager system (Molecular Dynamics, Piscataway, NJ). Densitometric analysis of protein bands was carried out using ImageQuant PhosphorImager software (Molecular Dynamics).
Ubiquitin proteasome degradation assays.
Purified glutathione S-transferase (GST)-SMN and GST-SMNΔ7 were iodinated with Na125I by using IodoBeads (Pierce) to a specific activity of 0.25 mCi/mg of protein. 125I-GST-SMN (100,000 cpm) was incubated at 30°C for 6 h in a total volume of 40 μl of reaction mixture containing 50 mM HEPES (pH 7.5); 4 mM ATP; 5 mM MgCl2; 40 mM KCl; 1 mM dithiothreitol (DTT); an energy-regenerating system (10 μg of creatine kinase/ml, 5 mM creatine phosphate, 4 mM GTP); 20 μg of ubiquitin conjugation enzyme fraction (Boston Biochem); 1 μg of ubiquitin aldehyde (Sigma-Aldrich); 4 μg of ubiquitin (Sigma-Aldrich); and 2.5 μg of GST, GST-SMN, or GST-SMNΔ7. Reactions were stopped by adding SDS sample buffer, boiled for 5 min, and resolved on 12% SDS-polyacrylamide gel electrophoresis (PAGE) gels. Phosphorimager analysis was used for quantification.
Equal amounts of ubiquitinated 125I-GST-SMN and 125I-GST-SMNΔ7 were incubated with 5 nM purified 26S proteasome (Biomol, Plymouth Meeting, PA) for 30 min at 37°C in the presence or absence of MG132. 125I-GST-SMN and 125I-GST-SMNΔ7 were precipitated at the designated times on ice with 100 μl of 10% trichloroacetic acid (TCA), and the amount of TCA-soluble protein fragments in the supernatant was quantified by using a gamma counter.
Sucrose gradient centrifugation.
Cell extracts were prepared in cell lysis buffer. For sucrose gradient centrifugation experiments, cell extracts were centrifuged on 10 ml of 10 to 30% sucrose gradients in lysis buffer for 4 h at 38,000 rpm in a Sorvall Discovery 90 ultracentrifuge at 4°C. The fractions were then pooled by threes. Immunoprecipitations were carried out as described above. For experiments involving incorporation of radioactive material, proteins were resolved by SDS-12% PAGE, and the gels were subsequently dried and exposed to a phosphorimager screen as described above.
All of the GST fusion proteins were expressed from the GST expression vector pGEX-5X (GE Healthcare) in Escherichia coli strain BL21(DE3)/pLysS and purified by using glutathione-Sepharose according to the manufacturer's protocol (GE Healthcare).
In vitro PKA phosphorylation.
Purified GST-SMN (1 μg) was incubated with the catalytic subunit of PKA (1 U) for 2 h at room temperature in a buffer (300 μl) containing 50 mM Tris (pH 7.5), 1 mM EDTA, 10 mM MgCl2, 200 mM NaCl, and 0.2 mM DTT in the presence of 300 μM ATP and [γ-32P]ATP. Phosphorylation was stopped by the addition of excess EDTA (15 mM), and samples were resolved by SDS-12% PAGE. Gels were dried and exposed to a phosphorimager screen as described above.
RNA extraction and quantification.
The cells were homogenized in 1 ml of TRIzol reagent (Invitrogen, Carlsbad, CA), and total RNA was isolated and converted to cDNA as previously described (15
). Primers to amplify human SMN, hGUS, and the 18S RNA were purchased from Applied Biosystems. Quantitative reverse transcription-PCRs (RT-PCRs) were run in triplicate using the ABI Prism 7900 sequence detector system (Applied Biosystems, Foster City, CA). The level of each transcript was quantified by the ddCt method using hGUS or 18S as an endogenous control. The average value of the untreated cells was normalized to 1.
Biochemical data were analyzed by using the GraphPad Prism software package (GraphPad Software, Inc., San Diego, CA) and compared statistically by either t tests or one-way analysis of variance followed by the Newman-Keuls multiple comparison post hoc test. To compare differences between the three groups, a nonparametric equality of medians test was performed, since the data were not normally distributed. If this was statistically significant, then a pairwise comparison between the two treatment groups was performed using a Mann-Whitney test.