Preparation of Recombinant Proteins
The Plasmid pT7-UbcH5c and pET28a-PTEN-HA (C-terminal HA-tagged) were transformed into E. Coli strain BL21(DE3). The recombinant proteins were expressed and purified by Ni-NTA affinity chromatography according to standard procedures. Recombinant PTEN was further purified using a 1-ml HiTrap SP column (Amersham), and dialyzed against Buffer A (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT). The purified proteins were stored at −80°C in aliquots. The plasmid pGEX4T1-PTEN-HA was used to express recombinant GST-PTEN in BL21 strain according to standard procedures. For purification with glutathione-Sepharose, after the recombinant protein was bound to the column equilibrated with Buffer A, the column was washed with 10 x column volume of Buffer A supplemented with 500 mM NaCl. After washing again with Buffer A, the recombinant protein was eluted with Buffer A containing 15 mM reduced glutathione. The eluant was further purified with a 1-ml HiTrap Q column (Amersham). The purified GST-PTEN-HA was dialyzed against Buffer A, stored at −80°C in small aliquots.
In vitro Ubiquitination of PTEN
The reaction was carried out at 30°C for 1 hour in 15 μl reaction buffer (40 mM Tris-HCl, pH 7.5, 2 mM DTT, 5 mM MgCl2) containing the following components: 10 μg of ubiquitin (Boston Biochem, # U-100), 20 nM human E1 (Boston Biochem, # E302), 500 nM of recombinant UbcH5c, 2 μM ubiquitin aldehyde (Boston Biochem, #U-201), 5 mM ATP or ATPγS, and 150 ng of GST-PTEN. HeLa S-100 or different chromatographic fractions were added as E3 source as indicated in individual experiments. The reaction was terminated by adding 0.8 ml Pull-down Buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1% Triton X-100, 0.02% BSA, and 5 mM β-Mercaptoethanol). After addition of 8 μl of Glutathione Sepharose-4B, the samples were rotated at room temperature for 40 minutes. The beads were washed with 1 ml of Pull-down Buffer for 5 times. The proteins bound to beads were released by boiling in 30 μl of 2 x SDS-PAGE sample buffer for 5 minutes. The samples were then resolved by 8% SDS-PAGE followed by immunoblot analysis using a monoclonal anti-ubiquitin antibody (BD Biosciences, #550944). In the reactions using C-terminal HA-tagged recombinant PTEN instead of GST-PTEN as substrate, all the components were the same as described above except that 50 ng of recombinant PTEN-HA was added to replace GST-PTEN. After ubiquitination reaction, the samples were directly boiled with SDS-PAGE loading buffer and analyzed by immunoblotting against HA-tag (Babco, HA.11).
Purification and Identification of PTEN E3 Activity from HeLa S-100
All purification steps were carried out at 4°C, and chromatography was performed with an Amersham FPLC system. HeLa cell S-100 (HS100) in Buffer A (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT) supplemented with protease inhibitors was prepared from large-scale cell culture purchased from National Cell Culture Center. For the purification, 315 ml of HS100 (~1.5 g total protein) was applied to a Q-Sepharose column (60-ml bed volume) equilibrated with Buffer A. After washing the column with Buffer A containing 0.15 M NaCl, the PTEN ubiquitin ligase activity was eluted into 120 ml of Buffer A containing 0.35 M NaCl (480 mg protein). The activity was then subject to 20%–50% (saturation) ammonium sulfate precipitation. The protein pellet, which contained the activity, was dissolved in 40 ml Buffer A (199 mg protein) and then ammonium sulfate concentration was adjusted to 15%. The activity was subject to two runs through a 5-ml HiTrap Phenyl-Sepharose HP column (Amersham) equilibrated with 15% ammonium sulfate in Buffer A. After washing the column with 10% ammonium sulfate, the activity was eluted with a 10%–0% gradient of ammonium sulfate in 80 ml Buffer A, followed by 20 ml Buffer A. Fractions of 4 ml were collected, dialyzed, and assayed for activity. The fractions containing the activity peak were pooled (3.85 mg protein) and further fractionated with a 2-ml Hydroxyapatite (Bio-Rad) column by using a 3–200 mM phosphate gradient in 30 ml Buffer B (20 mM HEPES, pH 7.5, 10 mM KCl, 1 mM DTT). Fractions of 2 ml were collected, dialyzed, and assayed for activity. Then the activity fractions were combined (1.07 mg protein) and fractionated with a 1-ml HiTrap Heparin-Sepharose HP column. The activity was eluted by a gradient of 0–300 mM NaCl in 20 ml Buffer A. Fractions of 1 ml were collected, dialyzed, and assayed for activity. The active fractions were combined and underwent multiple runs through a 25-ml Superdex 200 gel-filtration column in Buffer A plus 50 mM NaCl. The activity fractions were combined and resolved by a Mono Q column with a 150–350 mM NaCl gradient in 20 ml Buffer A. Fractions of 1 ml were collected. After dialysis, activity assay were performed using 2 μl of each fraction, and SDS-PAGE followed by silver-staining was performed using 15 μl of each fraction. A single protein band correlating to the PTEN ubiquitin ligase activity was identified. The whole Mono Q fractions containing the purified activity were concentrated, resolved by SDS-PAGE, and stained with Coomassie blue. The protein band was subject to protein identity determination by performing mass spectrometry analysis (MALDI-TOF-MS/MS). The activity was identified as human NEDD4-1.
Cloning of Full-Length NEDD4-1
HeLa cell genomic DNA was prepared as described in Molecular Cloning manual book. Using it as template, nest PCR were performed to obtain DNA sequence containing the N-terminal fragment of NEDD4-1, a GC-rich sequence (First round PCR used primers AATATTAAAAAGCATGATTATTCC and TCGTCCTCCAGGAGCCCGA. The second round PCR used primers ATAACCTTATTCCACAGGAGG and ACACCTCC ACCGCGCAAGT). The C-terminal fragment of NEDD4-1 was amplified by PCR from clone KIAA0093 using primers CTCCACAGTTGCCTGCCCT and GCCCTCGAGCTA ATCAACTCCATCAAAGCC (3’-XhoI). The two PCR products overlap with each other for 66 bp. Then by mixing the N-terminal and C-terminal PCR fragments as template and using Primer 5’-EcoRI (CCGGAATTCTATGGCACAAAGCTTACGATTGC) and Primer 3’-XhoI, the full-length NEDD4-1 was amplified by PCR and cloned into pCDNA3.1 using EcoRI and XhoI sites. For NEDD4-1-HA construct, a HA-tag was inserted at the C-terminus by PCR. All constructs were verified by DNA sequencing.
NEDD4-1 RNAi Constructs
The shRNA constructs against human NEDD-1 were generated using pSuperRetro vector according to manufacturer’s procedure (OligoEngine). The NEDD4-1 DNA sequences used in the RNAi constructs are: 5’TGGCGATTTGTAAACCGAA3’ (iN4A); 5’TGCAGAA CAGGCTGAGGAA3’ (iN4B); and ATGAAACTTCGCCGAGCAA (iN4C).
Transfection and in vivo PTEN-Ubiquitination Assay
Cell culture transfection was performed using polyethylenimine (PEI) reagent, or, where indicated, electroporation approach (Amaxa) for high transfection efficiency (>65%). For in vivo PTEN-ubiquitination assay, PEI transfection was performed when 293T cells in 10-cm plates reached ~ 50% confluence. Plasmids encoding for PTEN, NEDD4-1, and HA- or His-tagged ubiquitin were used in transfection as indicated in individual experiments. Twenty four hours after transfection, cells were treated with or without 25 μM proteasome inhibitor MG132 (CalBiochem) for 6 hours. The cells were washed with PBS, pelleted, and lysed in 0.1 ml of PBS plus 1% NP-40, 25 μM MG132, 2 μM ubiquitin aldehyde, and protease inhibitor cocktail. The lysates were centrifuged to obtain cytosolic proteins. Subsequently, we follow a published protocol (Treier et al., 1994
) with minor modifications. Briefly, individual samples were diluted with 1 ml of Guanidine Buffer (6 M guanidine, 0.1 M sodium phosphate, pH 8.0, 0.2% Titon-X100, 10 mM imidazole) before addition of 20 μl TALON Cobalt transition metal beads (BD Biosciences) beads pre-washed with Guanidine Buffer (GB). After rotating at 4°C for 3 hour, the beads were washed twice with 1ml of GB followed by two washes with 1 ml of GB/TI, which composes 1:3 (vol) of GB vs. buffer TI (20 mM Tris-HCl, pH 6.8, 0.2% Titon-X100, 20 mM imidazole, 0.2% SDS). Then the beads were washed twice with TI followed by two washes with TI plus 1 M NaCl. After another wash with TI, the supernatant was completely removed with thin tips. The proteins were released from the beads by boiling in 40 μl of 2x SDS-PAGE sample buffer plus 200 mM imidazole for 10 min. Twenty-five μl of the samples were subjected to immunoblot against antibodies as specified in individual experiments.
Cycloheximide chase experiments
To examine the effect of NEDD4-1 overexpression on PTEN degradation, three 10-cm plates of HeLa cells were co-transfected with 2μg/plate of PTEN plasmid and 6μg/plate of NEDD4-1 plasmid or control plasmid. To examine the effect of NEDD4-1 RNAi on PTEN degradation, three 10-cm plates of HeLa cells were co-transfected with 0.8 μg/plate of PTEN plasmid and 3.2 μg/plate of RNAi construct against human NEDD4-1 (iN4A) or control plasmid. Ten hours after transfection, the cells from the triplicate plates were pooled together and re-plated into four 6-cm plates to minimize the variation in transfection among the plates. Twelve hours later after re-plating, 50 μg/ml of cycloheximide was added to the 6-cm plates, and the cycloheximide treatment was terminated at 0, 3, 6, and 9 hours time points as indicated. The cell lysates were made, and protein concentration was determined. Subsequently, twenty μg of total protein from each sample, except 60 μg of total protein from the RNAi vector control samples, was analyzed by immunoblotting as indicated.
Soft-Agar Colony Formation Assay
Retroviruses were generated using pBabe-puro vector alone, pBabe-puro-NEDD4-1, and pBabe-puro-Ras. Primary MEFs (passage 3) were plated in 10-cm plates (2 x 105/plate). After overnight, they were infected by three rounds of infection (4 hours each round) with indicated retroviruses. Forty-eight hours after infection, cells were allowed to grow for three days in medium containing 2 μg/ml puromycin. After recovering from puromycin selection for 12 hours in puromycin-free medium, viral-infected cells were trypsinized and plated for soft-agar colony formation assay. For each assay, 5 x 105 viral-infected cells were plated with soft-agar medium to individual wells in 6-well plates. The plates were incubated for two to three weeks, and phase-contrast microscopic pictures were taken for each sample using a digital camera coupled to a microscope to show colony formation. Quantification and standard deviation were obtained from results of three independent experiments.
Human Cancer Tissues and Microarray Analysis
Tumor tissues and clinical data were obtained through an IRB-approved protocol at Memorial Sloan-Kettering Cancer Center. Tumors were microdissected to ensure that at least 70% of cells present were tumor. RNA was extracted using Trizol (Invitrogen, Carlsbad CA) followed by cleanup with Rneasy columns (Qiagen, Valencia CA). Double stranded cDNA was produced using a ds cDNA synthesis kit (Invitrogen) followed by production of labelled RNA probe using an in vitro transcription kit (Enzo Diagnostics, Farmingdale NY). Hybridization, washing and imaging of Affymetrix U133 Plus v. 2.0 microarrays (Affymetrix, Santa Clara CA) were performed according to manufacturer's protocols. Microarray data were normalized, background subtracted and log transformed using the robust multi-array average (RMA) method (Irizarry et al., 2003
) within bioconductor for R (Gentleman et al., 2004
Immunohistochemistry of mouse prostates
IHC was carried out with the automated staining processor (Ventana Medical Systems, Arizona) for mouse prostates as previously described (Trotman et al., 2003
) using the following antibodies: rabbit anti-phospho-Akt (Ser473-IHC specific, Cell signalling # 9277), rabbit anti-PTEN (Ab-2, NeoMarkers), anti-Nedd4 (Upstate, # 07-049). For correlation of PTEN and NEDD4-1 status in Ptenhy/−
mice, single prostate glands on a total of 9 adjacent sections from 2 animals (6-month old) were classified for NEDD4-1 and PTEN high or low staining. A total of 16 glands were scored, yielding an inverse correlation for PTEN and NEDD4-1 level as following: no gland with PTEN high/NEDD4-1 high or PTEN low/NEDD4-1 low; 11 glands with PTEN high/NEDD4-1 low (normal glands); 5 glands with PTEN low/NEDD4-1 high (tumor glands, p-Akt-positive). The statistical significance of this inverse correlation was calculated using a chi-square analysis (P < 0.001).
DU-145 cells and PC3 cells were infected with iN4A or iLacZ pSuper Retrovirus, selected with 2 μg/ml puromycin for 4 days, and allowed to recover in the absence of puromycin. Subsequently, 106 DU-145 or PC3 tumor cells, bearing NEDD4-1 RNAi or LacZ RNAi retroviruses as indicated, were suspended in 100 μl 10% FCS and 100 μl Matrigel (BD Biosciences), and subcutaneously injected into the right flank of 6-week-old athymic nude mice (NCRNU-M, Taconic Farms Inc.) in triplicates. Every 3 days tumor size was measured with a caliper, and volumes were calculated as L × W2 × 0.52, where L is the longest and W is the shortest diameter.