Immortalized human prostate epithelial PWR-1E cells (a gift from Dr. S.C. Chauhan, University of South Dakota), PC-3 human prostate cancer cells, SKBR-3 human breast cancer cells and HEK293 human embryonic kidney cells were maintained according to ATCC’s recommendation.
Antibodies and reagents
Primary antibodies targeting the following antigens were used: goat anti-human RARRES1 (Cat# AF4255, R&D Systems, Minneapolis, MN), anti-tyrosinated tubulin and anti-Eg5 (Abcam, Cambridge, MA), anti-E-cadherin (BD-Transduction Laboratories, San Jose, CA), anti-Cyclin D1 (EMD-Calbiochem, Gibbstown, NJ), anti-HA (Millipore, Temecula, CA), anti-histone-H4 (Cell Signaling, Danvers, MA), anti-detyrosinated tubulin (AbD Serotec, Oxford, England), and anti-AGBL2, and anti-pan-cadherin (Sigma-Aldrich, St. Louis, MO). The following RNAis (Dharmacon, Lafayette, CO) were used: for RARRES1 knockdown, GUACACGGCUCAUCGAGAA and AAAGAGGGAUGUAAAGUUC. For AGBL2 knock-down: GCACACUUCUACCCAUAUA and UGGACAAGAUGUAGAUUUA.
siRNA, expression constructs and transfection
Variant and full-length human RARRES1 isoforms were directionally cloned into the BglII and HindIII sites in the pEYFP-N1 vector (Clontech). N-RARRES1 (+121 - +897) and full-length RARRES1 (+1 - +897) were cloned into the pGlue vector as codon optimized versions by Genscript (Piscataway, NJ). AGBL2 (cat#M-012937-00) and non-targeting control siRNAs (cat#D-001210-01-20) were from Dharmacon (Lafayette, CO). Plasmid DNA and siRNA constructs were either introduced to cells by electroporation mediated by an Amaxa nucleoporator (Amaxa-Lonza, Gaithersburg, MD), or by Fugene 6 transfection reagent (Roche, Indianapolis, IN).
Detection of AGBL family members by qPCR
Total RNA was extracted from indicated cell lines with Trizol reagent (Invitrogen) and isolated with an RNeasy purification kit (Qiagen). Single-stranded cDNA was prepared from 400 ng of RNA by TaqMan reverse transcription reagents (Applied Biosystems) following the manufacturer’s guidelines. Real-time PCR was then performed and monitored on a 7900 HT system (Applied Biosystems) using TaqMan universal PCR master mix and the following inventoried primer/probe sets: GAPDH–Hs99999905_m1, AGBL1/Nna1–Hs00328701_m1, AGBL2–Hs00417079_m1, AGBL3–Hs00227489_m1, AGBL4–Hs00262179_m1, and AGBL5–Hs00222447_m1 (Applied Biosystems). Plotted ΔCt values were determined by subtracting control (GAPDH) cycle threshold values from each target cycle threshold. Where targets did not return a threshold value after 40 cycles of PCR, the transcript was determined to be absent.
RARRES1 (full-length) forward 5’-CAACAAGAGGATTACCTGCTTTACAAG-3’ and reverse 5’-GAGCAGAGTTCAGTGTGCATG-3’ primers (generating a 630 base pair amplicon). For β-actin; forward 5’-CCACTGGCATCGTGATGGAC-3’ and reverse 5’-GCGGATGTCCACGTCACACT-3’ primers (generating a 350 base-pair amplicon). Thermal cycling for RARRES1 was done according to the following profile: 30 minutes at 50°C, 15 minutes at 95°C for RT reaction followed by PCR cycling of, 30 seconds at 94°C, 45 seconds at 52°C, one minute at 72°C, for indicated number of cycles with a subsequent final elongation at 72°C for 10 minutes.
Whole cell lysates of untreated PWR-1E cells were made using RIPA buffer followed by digestion with the glycosidase PNGase F, or no enzyme (NEB, Ipswich, MA) according to the manufacturer’s instructions.
Three-pool (membrane, cytoplasmic and nuclear/cell debris fractions) cell fractionation was carried out as previously described (9
Cell Lysis and Tandem Affinity Purification (TAP)
A stable clone expressing a level of exogenous pGlue RARRES1close to the endogenous level of this protein was grown in 5 dishes (150 mm each). At 90% confluency, medium was discarded and each dish was lysed in 0.5 mL of a lysis buffer composed of 10% glycerol, 50 mM Hepes-NaOH pH 8.0, 100 mM NaCl, 2 mM EDTA, 0.1% NP-40, 2 mM DTT, 10 mM NaF, 5 nM calyculin A, 50 mM β-glycerolphosphate, and 1 × Complete MiniTM
protease inhibitor (Roche, Indianapolis, IN). Lysates were harvested by scraping and two freeze-thaw cycles were performed to improve protein recovery. Lysates were then centrifuged at 15,000 × g for 15 minutes, supernatant were recovered and incubated for 4 hours with 100 μL of streptavidin beads (Streptavidin SepharoseTM
High Performance, GE Healthcare) pre-washed 3 times with the lysis buffer. The slurry was then centrifuged at 1,500 × g for 2 minutes and the precipitate containing the streptavidin beads was recovered and washed 3 times with lysis buffer and 2 times with the TEV buffer supplied with the AcTEVTM
Protease kit (Cat no. 12575-015, Invitrogen, Carlsbad, CA). Streptavidin beads were then incubated with 200 units of TEV protease in 150μL of TEV buffer overnight at 4°C. The slurry was then centrifuged at 1,500 × g for 2 minutes and the supernatant was recovered. The precipitated beads were washed twice with 200 μL of TEV buffer, centrifuged, and supernatants were pooled. The final pool volume was diluted 1:1 (v/v) wih calmodulin-binding buffer composed of 10 mM β-mercaptoethanol, 10 mM Hepes-NaOH pH8.0, 150 mM NaCl, 1 mM MgOAc, 1 mM imidazole, 0.1% NP-40, and 2mM CaCl2
. The mixture was then incubated for 90 minutes at 4°C with 100 μL of Calmodulin beads (Calmodulin SepharoseTM
4B, GE Healthcare) pre-washed 3 times with Calmodulin-binding buffer. The slurry was then centrifuged at 1,500 × g for 2 minutes, the supernatant was discarded and the precipitated beads were washed twice with a Calmodulin- rinsing buffer composed of 50 mM Ammonium Bicarbonate pH 8.0, 75 mM NaCl, 1 mM MgOAc, 1 mM imidazole, and 2 mM CaCl2
. Each wash was followed by centrifugation and supernatant shedding. 150 μL of a calmodulin-elution buffer composed of 50 mM Ammonium bicarbonate pH 8.0 and 25 mM EGTA was then added to the calmodulin resin. The slurry was then vortex mixed, centrifuged at 1,500 × g, and the supernatant was collected. This elution step was repeated twice and supernatants were pooled together (Figure S1
Trypsin Digestion and Tandem Mass Spectrometry Analysis (LC-MS/MS)
The pull-down sample was vacuum-dried and reconstituted with 20 μL of a buffer composed of 500 mM triethylammonium bicarbonate pH 8.5. The protein sample was then denatured by adding 1 μL of a 2% SDS solution followed by the addition of 2 μL of a reducing reagent composed of 50 mM TRIS-(2-carboxyethyl)phosphine (TCEP). The mixture was then incubated at 60°C for 1 hour followed by the addition of 1 μL of a cysteine blocking reagent composed of 200 mM methyl methane-thiosulfonate (MMTS) in isopropanol. Trypsin digestion was then performed by adding 10 μL of 1 μg/μL trypsin solution in 80 mM CaCl2
. Samples were incubated overnight at 37°C then vacuum dried and reconstituted with 10 μL of a 2% acetonitrile and 0.1% formic acid in distilled deionized water as described previously (10
). NanoLC/MS/MS was performed using a Q-Star Elite (Applied Biosystems, Foster City, CA) equipped with a nanoAcquity UPLC system (Waters, Milford, MA). Sample separations were performed using a 1.7 μm nanoAcquity BEH130 C18 (100 μm × 100 mm) at a flow rate of 400 nL/min. Tryptic digests were eluted using the following gradient: 100% of Solvent A (97.9% water, 2%Acetonitrile, 0.1% Formic acid (v/v/v)) for 1 hour; then from 100% solvent A to 100% solvent B (2% water, 97.8%Acetonitrile, 0.1% Formic acid (v/v/v)) in 2 hours; A 100% solvent B flow was maintained for 1 hour followed by a return to 100% of solvent A flow in 15 min. Mass spectrometer settings were as follows: Ion spray voltage 2300 V, interface heater temperature 220 °C, cone voltage 20 V, and collision energy 8 V.
Protein identification was performed using ProteinPilotTM
software with the following settings (11
): Sample type: Identification; Cysteine alkylation: MMTS; Digestion: Trypsin; Instrument: QSTAR ESI; Species: Homo sapiens; Min S/N Filter: 10; Precursor tolerance 75 ppm; Maximum missed cleavage: 1; and MS/MS fragment tolerance of 0.3 Da.
Western blotting was performed as described previously (12
). Cells were lysed with RIPA buffer, loaded onto 4–12% gradient polyacrylamide gel. Amounts of proteins loaded were: 1 μg of total cell lysates for tubulin immunoblots and 10μg for all other immunoblots. Gels were electrophoresed at 100V until the end of the separation. Proteins contained within the gel were then electroblotted onto a nitrocellulose membrane (50 V for 50 min). Western blot analyses were accomplished utilizing a 1 μg/mL dilution of primary antibody followed by incubation with a horseradish peroxidase-conjugated secondary antibody against the appropriate species. Visualization of the bands was then accomplished by the addition of a 1 to 1 ratio of Super Signal West Pico-Stable Peroxidase Solution and Luminol/Enhancer Solution (Pierce, Rockford, IL) and by developing the chemiluminescent signal in the dark using Kodak Scientific Imaging Film (Kodak cat. 1651496), Fixer and Replenisher/Developer and Replenisher (Kodak cat. 1901859) according to the manufacturer’s instructions.
Hek 293 Cells were lysed using RIPA buffer and centrifuged at 14,000 × g for 15 min. The supernatant was recovered and pre-cleared by adding 1μg of normal IgG premixed with 20 μL of A/G protein bead slurry. The mixture was incubated for 30 minutes at 4 °C and then centrifuged at 1,000 × g for 5 min. The supernatant was recovered and mixed with 10 μL of primary antibody and incubated for 1 hour at 4 °C. 20 μL of A/G bead slurry was then added and incubated at 4 °C for 1 hour. Samples were then centrifuged and supernatants were discarded. The precipitate was boiled for 3 minutes after adding 20 μL of SDS-PAGE sample buffer to release the complex from the beads. Western blotting was then performed as described above.
AGBL2 structure was predicted using human carboxypeptidase A1 (CPA1) (PDB: 1V77) as a template. The sequence identity of AGBL2 and CPA1 is 27%. The missing loops were built using the ‘loop model’ building option in the Modeller9v7. The model was refined further by molecular dynamics simulations followed by energy minimization using SANDER module of AMBER 10.0. The quality of the refined model was checked with PROCHECK. Docking of the ‘EEY’ peptide motif was carried out with the SurFlexDock. Molecular Dynamics (MD) simulations and energy minimization were performed using the AMBER10.0 package.
To assess the levels of detyrosinated tubulin in control versus AGBL2, RARRES1 knocked-down, and/or paclitaxel treated Hek293 cells, 50,000 cells were plated on cover slides (Fisher brand, microscope Cover Glass, #12-545-100 18CIR-1). After 24 hrs, cells were treated with paclitaxel or DMSO at a final concentration of 5 μM for 2 hours. HEK293 cells were then fixed in 3.7% paraformaldehyde/PBS for 10 minutes at room temperature followed by washing the cells 3 times with PBS. Post-permeabilization was performed by adding PBST (PBS/Tween 20) and incubating the cells for 5 mins at room temperature. Cells were then washed 3 times with PBS followed by the addition of the primary antibody that consisted of a rabbit polyclonal anti-detyrosinated tubulin (ABD Serotec, Oxford, England) with a final dilution of 1:250. Cells were then washed 3 times with PBS before adding the secondary antibody that consisted of an Alexa 488-conjugated anti-Rabbit IgG diluted 1:300 (Invitrogen, Carlsbad, CA). DAPI was also added at a 1:50 dilution for nuclei detection. Cells were washed 3 times with PBS and Images were obtained using a 60X oil lens on the Olympus FV 300 Confocal microscope. Consistent laser intensity or camera exposure levels for each fluorescent marker in each experiment were used. For image analysis and quantification, measurements were made using Metamorph Image analysis software ver. 7.0. Average intensity was calculated from integrated intensity and area for each selected area. Quantitation of fluorescence signals from five random fields for each treatment was performed. An example of an original image used for quantitation is included in the supplementary materials (Figure S2)