Cell culture and treatment
HEK293 cells in passages 38–48 were cultured at 300 mOsm/kg in Eagle’s minimal essential medium (American Type Culture Collection, Manassas, VA) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT) as previously described (Irarrazabal et al.
). Osmolality of the control medium was 300 mOsm/kg, and at experiment-specific time points, medium was replaced with ones at 300 mOsm/kg, 200 mOsm/kg (NaCl added to NaCl-free medium; Biofluids, Rockville, MD), or 500 mOsm/kg (NaCl added). CDK5 inhibitors roscovitine and CDK1/5 inhibitor were from EMD Chemicals (Gibbstown, NJ). Inhibitors were added to medium bathing HEK293 cells 60 min before changing the osmolality and were added in any subsequent changes of medium. All cells were cultured at 37°C in 5% CO2
and were studied while subconfluent. Anti-TonEBP/OREBP, anti–aldose reductase, anti-BRG1, anti-CDK5 and anti–CDK5-phospho-Y15 antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA) and anti-tubulin from Rockland (Gilbertsville, PA). Anti-V5 was from AbD Serotec (Raleigh, NC). The rabbit anti–TonEBP/OREBP-phospho-T135 and –phospho-S134 were produced by PhosphoSolutions (Aurora, CO). HEK293 cells stably expressing TonEBP/OREBP-1–547-V5-His were previously described (Chen et al.
Plasmids and transfection
Human TonEBP/OREBP cDNA clone KIAA0827 was a gift from Takahiro Nagase (Kazusa DNA Research Institute, Chiba, Japan). Sequences coding for amino acids 1–547 or 1–1531 of KIAA0827 were cloned into expression vector pcDNA6V5-His (Invitrogen, Carlsbad, CA) to generate 1–547- or 1–1531-V5-His, as previously described (Irarrazabal et al.
; Zhang et al.
). Mutants S120A, S134A, T135A, and S155A of 1–1531-V5 were prepared by site-directed mutagenesis (QuikChange, Stratagene, La Jolla, CA). All constructs were generated using standard cloning procedures and verified by restriction enzyme digestion and DNA sequencing. HEK293 cells were transfected with Lipofectamine 2000 (Invitrogen), according to the supplier’s instructions. CDK5 WT or kinase dead dominant negative (KD) were kindly provided by Harish C. Pant (Laboratory of Neurochemistry, NINDS, Bethesda, MD). The ORE-X luciferase reporter construct contains two copies of human ORE-X element (Ferraris et al.
) in a plasmid with a minimal IL2 promoter (hTonE-GL3, a gift from S. N. Ho, University of California at San Diego, La Jolla, CA) (Trama et al.
). The binary GAL4/TAD reporter system was previously described (Ferraris et al.
After washing, cells were collected in buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, protease inhibitor cocktail tablet (Roche, Indianapolis, IN), and phosphatase cocktail inhibitor I and II (Sigma-Aldrich, St. Louis, MO). Cell extracts were precleared with mouse IgG plus Protein A/G PLUS-Agarose (Santa Cruz Biotechnologies) at 4ºC for 1 h and then incubated with mouse anti-V5 antibody or anti-CDK5 antibody plus Protein A/G PLUS-Agarose at 4ºC overnight. The agarose beads were washed gently once with the lysis buffer. Proteins were eluted with the SDS loading buffer, boiled for 5 min, then subjected to Western analysis or kept at 4°C for kinase assay.
Sample preparation for mass spectrometry
HEK 293 cells stably transfected with TonEBP/OREBP-1–547-V5-His were grown in 15-cm dishes. Osmolality was increased to 500 mOsm/kg by adding NaCl for 2 h, and then nuclear extracts were prepared with NE-PER (Pierce, Rockford, IL), according to the supplier’s instructions, with added protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail I and II (Sigma-Aldrich). Extracts were precleared for 1 h with rabbit IgG, biotin conjugated (Santa Cruz Biotechnology), on Dynabeads (Invitrogen) for 1 h. Precleared supernatants were incubated overnight with rabbit anti-V5, biotin conjugated (ICL Lab, Newberg, OR), on Dynabeads (Invitrogen). Beads were washed three times with buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1% Triton X-100 and three times with phosphate-buffered saline (PBS) containing 1% Triton X-100. Both buffers included phosphatase and protease inhibitor cocktails. The beads were resuspended in 6 M guanidine-HCl/50mM NH4HCO3 to denature the proteins and elute them from the beads. Protein samples were then reduced with 100 mM dithiothreitol (DTT) for 1 h at 56°C and alkylated using 100 mM iodoacetamide for 1 h at room temperature in the dark. The sample buffer was exchanged to 50 mM NH4HCO3 (for trypsin digestion) or to 100 mM Tris-HCl, 10 mM CaCl2, pH 7.6 (for endoproteinase Arg-C digestion). Amicon Ultra Centrifugal Filter Devices (Millipore, Billerica, MA) were used for buffer exchange. Proteins were digested with trypsin (Promega, Madison, WI) or endoproteinase Arg-C (Roche Applied Science, Mannheim, Germany) in a ratio of 1:50 wt/wt overnight at 37°C. Peptide samples were desalted using a 1-ml hydrophilic-lipophilic–balanced cartridge (Oasis, Milford, MA), followed by volume reduction in vacuo. Samples were resuspended in 50 μl of 5% acetic acid, pH 2.5–3.0 and then loaded onto an IMAC column (Pierce) for phosphopeptide enrichment. Peptides were incubated with this Ga3+ resin for 20 min agitating gently every 5 min and then washed and eluted according to the supplier’s protocol. Samples were then dried in vacuo, resuspended in 1% formic acid, and desalted with C18 Ziptips (Millipore) before analysis by MS/MS.
Phosphopeptides identification and validation
IMAC eluates and flow-through fractions were both analyzed on an Agilent 1100 nanoflow system (Agilent Technologies, Palo Alto, CA) LC connected to a Finnigan LTQ mass spectrometer (Thermo Electron, San Jose, CA) equipped with a nanoelectrospray ion source. MS spectra were analyzed with BIOWORKS software (Thermo Electron), using the SEQUEST search algorithm for peptide identification. Peak masses were searched against the most current version of the Human RefSeq Database (National Center for Biotechnology Information) and its reversed complement with the following parameters: fixed carbamidomethylation of Cys; variable phosphorylation of Ser, Thr, and Tyr when searching MS2
; and use of a preliminary exclusion filter to remove poor-quality spectra. The filter removed any spectrum with the following values for the charge state of the peptide and the associated Xcorr score: +1 ≤ 1.5; +2 ≤ 2.0; and +3 ≤ 2.5. Xcorr takes into account the number of peaks matched between actual and theoretical spectra and is directly proportional to spectral quality. We used Ascore, a probability-based score (Beausoleil et al.
), to estimate the probability of correct phosphorylation site localization.
Beads containing CDK5 immunoprecipitated from cell extracts were suspended in Kinase Buffer (Cell Signaling Technology, Danvers, MA) and then incubated with 1 μg TonEBP/OREBP-T135 peptide (QQHPSTPKRH) in assay buffer supplemented with 200 μM ATP (Cell Signaling Technology) at 30°C for 30 min. Supernatants were analyzed by dot blot for phosphorylated TonEBP/OREBP-T135. The relative amounts of CDK5 present on the beads were measured by Western analysis after decanting the supernatant. For in vitro kinase assay, the following recombinant kinases were purchased from Cell Signaling Technology: ERK1, JNK1, PKAc, PKC, p38, CDK1, and CDK5; 100 ng of recombinant kinase was incubated in kinase buffer containing 200 μM ATP and 1 μg TonEBP-T135 peptide in 30 μl final volume for 30 min at 30°C and analyzed by dot blot for phosphorylated TonEBP/OREBP-T135.
Antibodies against TonEBP/OREBP–phospho-S134 and -T135 were prepared by PhosphoSolutions. To verify their specificity, dilutions of the peptide containing TonEBP/OREBP amino acids 130–139 (QQHPSTPKRH), unphosphorylated or phosphorylated at S134, T135, or both, in 50 μl PBS were spotted on nitrocellulose membranes with a manifold suction device (Bio-Rad Laboratories, Hercules, CA). Nonspecific binding was blocked by incubating membranes at room temperature for 1 h with Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE) diluted 1:1 with PBS. Membranes were incubated with rabbit anti–TonEBP/OREBP phospho-S134 or anti–TonEBP/OREBP phospho-T135 at 4°C overnight. After washing with 0.1% Tween-20 in PBS, blots were incubated with Alexa Fluor 680 goat anti–rabbit IgG (Molecular Probes, Carlsbad, CA) for 1 h in the dark. Blots were visualized and quantified using an Odyssey Infrared Imager (LI-COR Biosciences).
Western analysis was performed as previously described (Irarrazabal et al.
). Briefly, cells were lysed with buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1% Triton X-100 (for whole-cell extracts) or with NE-PER (Pierce) for separate nuclear and cytoplasmic fractions, according to the supplier’s instructions. Protease inhibitor mixture (Roche Diagnostics, Alameda, CA) and phosphatase inhibitor cocktails I (Sigma-Aldrich) were included in the lysis buffers. Total protein concentration was measured in cell extracts by BCA assay (Pierce), and proteins were separated on 4–12% gradient Novex Bis-Tris or 3–8% gradient Tris-Acetate gels and transferred electrophoretically to nitrocellulose membranes (Invitrogen). Nonspecific binding was blocked by incubating membranes for 1 h at room temperature with Odyssey Blocking Buffer (LI-COR Biosciences) diluted 1:1 with PBS. Membranes were then incubated with mouse anti-V5 (Invitrogen) or rabbit anti–TonEBP/OREBP phospho-S134 or rabbit anti–TonEBP/OREBP phospho-T135 at 4°C overnight. After washing with 0.1% Tween-20 in PBS, blots were incubated with Alexa Fluor 680 goat anti–rabbit IgG or Alexa Fluor 780 or 800 goat anti–mouse IgG (Molecular Probes) for 1 h in the dark. Blots were visualized and quantified using an Odyssey Infrared Imager (LI-COR Biosciences).
Nuclear to cytoplasmic ratio
Nuclear to cytoplasmic ratio was determined as previously described (Ferraris and Burg, 2007
). Cytoplasmic and nuclear proteins were extracted separately by using NE-PER (Pierce). Specific proteins in each fraction were measured by Western analysis. The relative amounts of TonEBP/OREBP in the cytoplasmic and nuclear fractions and the nuclear/cytoplasmic ratio were calculated from the relative concentrations of the respective protein in each cytoplasmic or nuclear extract and the relative volumes of the extracts.
Immunofluorescence studies of HeLa cells
The cells were fixed for immunofluorescence with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 PBS, and then blocked with 1% bovine serum albumin. Primary antibodies were rabbit anti-TonEBP/OREBP and mouse anti-CDK5. The secondary antibodies were conjugated to Alexa 488 or 633 fluorophores (Invitrogen), respectively. Images were acquired using a Zeiss LSM 510 META microscope with a 40× NA = 1.3, oil immersion objective (Carl Zeiss MicroImaging, Thornwood, NY).
Reporter experiments were performed as previously described. In brief, for assay of the transcriptional activity of native TonEBP/OREBP, HEK293 cells expressing stably transfected ORE-X IL2 min GL3_Bsd or IL2 promoter only (control) reporters were used (Irarrazabal et al.
). For assay of TonEBP/OREBP transactivating activity, cells expressing the stably transfected GAL4 reporter (pFR-Luc) and GAL4dbd-548–1531, which contain the recombinant TonEBP/OREBP transactivation domain, were used (Ferraris et al.
). HEK293 cells were preincubated with CDK1/5 inhibitor for 1 h followed by 4 or 16 h at either 300 or 500 mOsm/kg (NaCl added).
Pathogen-free male Sprague-Dawley rats (Taconic Farms, Germantown, NY) or homozygous Brattleboro rats (Harlan Sprague-Dawley, Indianapolis, IN) weighing 150–200 g were used. All experiments were conducted in accord with animal protocol H-0110R1 approved by the Animal Care and Use Committee of the National Heart, Lung, and Blood Institute. Homogenates were prepared from the inner medullas, in Laemmli buffer (1.5% SDS, 10 mM Tris, pH 6.8), containing a protease/phosphatase inhibitor cocktail (Pierce). Following determination of total protein concentration (Pierce BCA, Rockland, IL), samples were heated to 60°C for 15 min and then stored in 6% glycerol plus 40 mM DTT for Western analysis.
Data were compared by analysis of variance (ANOVA), followed by a Student–Newman–Keuls posttest. Normalized data were transformed before ANOVA. Results are expressed as means ± standard error of the mean (SEM) (n = number of independent experiments). Differences were considered significant for P < 0.05.