BCA protein assay reagent kit, SuperSignal West Dura Extended Duration substrate, and Pico chemiluminescent substrate were purchased from Pierce (Rockford, IL). Mouse anti-JNK1 antibody was from R&D Systems (Minneapolis, MN). Rabbit anti-Notch(Val1744) antibody, rabbit anti-SAPK/JNK MAPK antibody, mouse anti-phospho-SAPK/JNK antibody, and rabbit anti-JNK2 were purchased from Cell Signaling Technology (Beverly, MA). and rabbit anti-JNK3 was from Abgent (San Diego, CA). Rabbit anti-His probe (H-15) antibody, rabbit anti-GAPDH (FL-335) antibody, horseradish peroxidase (HRP)-conjugated anti-rabbit IgG, HRP-conjugated anti-rat IgG, and HRP-conjugated anti-mouse IgG were from Santa Cruz Biotechnology (Santa Cruz, CA), and Dual-Glo luciferase assay reagents, Steady-Glo luciferase assay reagents, and pRL-TK vector were from Promega (Madison, WI). Lipofectamine 2000 transfection reagent and DMEM were from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was from Biological Industries (Kibbutz Beit Haemek, Israel). Human Aβ40 colorimetric ELISA kit was from BioSource International (Camarillo, CA). TNF-α was from PeproTech Asia (Rehovot, Israel). HRP-conjugated anti-mouse IgG and ECL Western blotting detection reagents were from GE Healthcare (Waukesha, WI). FuGENE 6 transfection reagent, Expand long template PCR system, the Complete protease inhibitor cocktail, and the PCR nucleotide mixture were from Roche Applied Science (Indianapolis, IN). Mouse anti-phospho-Ser/Thr-Pro (MPM-2) antibody and active recombinant human JNK2 were from Upstate Biotechnology (Lake Placid, NY). Rat anti-presenilin-1 mAb, rabbit anti-APP C-terminal antibody, rabbit anti-presenilin-1-loop antibody, and mouse anti-Aβ protein (6E10) antibody were purchased from Chemicon (Temecula, CA). Rabbit anti-nicastrin was from Calbiochem (La Jolla, CA). The QuikChange site-directed mutagenesis kit was from Stratagene (La Jolla, CA). All other reagents were at least reagent grade and obtained from standard suppliers.
Cell Culture and Cell Lines
Human embryonic kidney cells (HEK293) and Chinese hamster ovary cells were maintained in DMEM supplemented with 10% FBS and 0.1 mg/ml penicillin and streptomycin. T-REx293 cells were purchased from Invitrogen and cultured in DMEM supplemented with 10% FBS and 5 μg/ml blasticidin. The generation of stably transfected cell lines, T16, T20, N7, and γ-30, was described previously (Kimberly et al., 2003
; Liao et al., 2004
; Bakshi et al., 2005
). HEK293-derived T16 cells were stably transfected with tetracycline-inducible Gal4/VP16-tagged full-length hAPP695 and a Gal4 promoter-driven firefly luciferase reporter gene. HEK293-derived T20 cells were stably transfected with tetracycline-inducible Gal4/VP16-tagged APP-C99 (a direct γ-secretase substrate) and a Gal4 promoter-driven firefly luciferase reporter gene. HEK293-derived N7 cells expressed mouse NΔE, a direct γ-secretase substrate. CHO-derived γ-30 cells overexpressed exogenous hPS1, hemagglutinin (HA)-tagged Aph1, and FLAG-tagged Pen-2. CHO-derived γNCT-36 cells overexpressed exogenous hPS1, His-tagged NCT, HA-tagged Aph1, and FLAG-tagged Pen-2. Cells were incubated in a humidified incubator at 37°C in 5% CO2
Transient Transfection of Mammalian Cells
Transfection of HEK293, T20, γ-30, and γNCT-36 cells was performed using FuGENE 6 transfection reagent or Lipofectamine 2000 transfection reagent as described by the manufacturers. Cells were plated onto six-well microplates or 10-cm plates and grown to ~60–80% confluency before transfection. On the day of transfection, the culture medium was replaced with fresh 2 ml/well or 10 ml/plate of DMEM supplemented with 10% FBS. DNA constructs (1 μg) and pRL-TK (0.1 μg) were diluted in 100 μl of serum-free DMEM and mixed with 3 μl of FuGENE 6 or Lipofectamine 2000, followed by incubation at room temperature for 15 min. Transfection mixtures were added dropwise into cell culture medium and incubated at 37°C for 24 h. Transfected cells were harvested by PBS containing 20 mM EDTA and lysed with 1× passive lysis buffer (PLB, Promega, Madison, WI) or CHAPSO lysis buffer (50 mM Tris-HCl, pH 8.0, 1% CHAPSO, 150 mM NaCl, 25 mM β-glycerophosphate, 1 mM Na3VO4, and the Complete protease inhibitor cocktail). Clarified lysates were subject to luciferase assay using the Dual-Glo luciferase assay reagent kit for the measurement of γ-secretase activity or affinity pulldown using HIS-Select Cobalt Affinity Gel (Sigma, St. Louis, MO) for the isolation of γ-secretase complexes. Protein content of lysates was determined by the BCA protein assay reagent kit.
Transfection of Small Interfering RNAs Targeting JNK1, JNK2, and JNK3
Small interfering RNA (siRNA) duplex oligoribonucleotides against human JNK1 (si-JNK1), JNK2 (si-JNK2), and JNK3 (si-JNK3) were chemically synthesized by Invitrogen. The target sequences for si-JNK1 were 5′-GGA GCU CAA GGA AUA GUA U-3′ (sense strand) and 5′-AUA CUA UUC CUU GAG CUC C-3′ (antisense strand); for si-JNK2, 5′-GCU CUG CGU CAC CCA UAC A-3′ (sense strand) and 5′-UGU AUG GGU GAC GCA GAG C-3′ (antisense strand); and for si-JNK3, 5′-GGG AUU UAA AAC CAA GUA A-3′ (sense strand) and 5′-UUA CUU GGU UUU AAA UCC C-3′ (antisense strand). The human GAPDH siRNAs and nonspecific scrambled siRNAs were from Ambion (Austin, TX). Gene-targeting siRNA oligos (100 pmol; for individual knockdown of JNK1, JNK2, or JNK3) were mixed with nonspecific scramble siRNAs (100 pmol) and transfected into T20 cells by using the Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. Additionally, two gene-targeting siRNA oligos (100 pmol each; for combinatorial knockdown of JNK1+JNK2, JNK1+JNK3, or JNK2+JNK3) were mixed and transfected into T20 cells. T20 cells (2.5 × 104/well) were plated in 12-well microplates and transfected with siRNAs for 24 h, followed by an additional incubation with culture medium containing 1 μg/ml tetracycline in the presence or absence of TNF-α (50 ng/ml) at 37°C for 9 h. Clarified lysates of transfected cells were prepared and analyzed by Steady-Glo luciferase assay reagents to determine γ-secretase activity, and conditioned media were analyzed by the colorimetric Aβ40 ELISA kit to measure secreted Aβ40. Protein contents of cell lysates were determined by BCA protein assay reagent kit. A nonspecific oligonucleotide (Silencer Negative Control 1, catalogue no. AM4611, Ambion) that is not homologous to any known genes was used as a negative control to rule out nonspecific cellular events caused by the introduction of the siRNAs into cells. The transcript levels of JNKs in transfected cells were quantitated by real-time PCR with isoform-specific primer pairs to determine the knockdown efficiency of respective siRNAs.
Real-Time PCR to Quantitate the RNA Transcripts of JNK Isoforms
Total RNA of transfected cells was prepared by using TRIzol (Invitrogen) and was used to generate the first strand cDNA by SuperScript III First-strand cDNA Synthesis Kit (Invitrogen). Equivalent amounts of cDNA were used in quantitative PCR on DNA Engine Opticon Real-Time PCR Detection System (Bio-Rad, Hercules, CA) with following isoform-specific primer pairs: For JNK1, forward primer: 5′-GTTTGCCACAAAATCCTC-3′, reverse primer: 5′-CTGAGAGCCATTGATCAC-3′; for JNK2, forward primer: 5′-TGAAACTTGCCCACCCTT-3′, reverse primer: 5′-CCTTGGAATATCACACAACCTTTC-3′; and for JNK3, forward primer: 5′-CCAACATTGGATGTGAAAATTGCCT-3′, reverse primer: 5′-GGTACGCTCTCTTGGCATGT-3′ (Katagiri et al., 2006
). The levels of human GAPDH transcripts (forward primer: 5′-GTGGTCTCCTCTGACTTCAAC-3′ and reverse primer: 5′-TCTCTTCCTCTTGTGCTCTTG-3′) were determined as an internal control. Cycling conditions were 95°C for 3 min, followed by 40 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. For quantitation, the transcript levels of JNK isoforms were individually normalized to those of GAPDH. The data were expressed as the average (±SD) of triplicate measurements from three independent experiments.
Generation of a Stably Transfected Cell Line γNCT-36
The parental cell line γ-30 and nicastrin expression vector were gifts kindly provided by Dr. Michael Wolfe (Brigham and Women's Hospital and Harvard Medical School, Boston, MA), and the generation of CHO-derived γ-30 cells was previously described (Kimberly et al., 2003
). On the day of transfection, γ-30 cells on a 10-cm dish with ~50% confluency were transfected with 5 μg of pcDNA6-Nicastrin-V5-His (NCT-His) by FuGENE 6 transfection reagent according to the manufacturer's instructions. Transfected cells were cultured in DMEM supplemented with 10% FBS, 250 μg/ml hygromycin, 250 μg/ml zeocin, 2.5 μg/ml puromycin, 200 μg/ml geneticin (G418), and 5 μg/ml blasticidin (DMEM-HZPGB), and single colonies resistant to antibiotic selection were isolated individually using cloning cylinders. Each of the independent cell lines was screened for the overexpression of His-tagged NCT. Individually isolated cell lines were plated into 12-well microplates in DMEM-HZPGB for 24 h. Cells were lysed in CHAPSO lysis buffer, and His-tagged NCT along with other γ-secretase components was pulled down by HIS-Selected Cobalt Affinity Gel (Sigma). The affinity-purified proteins were resolved by SDS-PAGE and analyzed by Western blotting using rabbit anti-His antibody and rabbit anti-NCT antibody. The optimal cell line was the one exhibiting the highest amount of mature NCT. Based on this criterion, clone γNCT-36 was selected for future experiments ().
Figure 1. Generation of a cell line stably overexpressing His-tagged NCT. Cells of the parental clone γ-30 that overexpress human PS1, FLAG-tagged Pen-2, HA-tagged Aph1α2, and APP were transfected with pcDNA6-NCT-V5-His (His-NCT). Transfected cells (more ...)
TNF-α–elicited Phosphorylation of NCT and PSI-1
On the day of the experiment, γNCT-36 cells (107 cells/plates) in 10-cm culture dishes were preincubated with DMEM containing 0.5% FBS at 37°C for 3 h, followed by the addition of TNF-α to a final concentration of 50 ng/ml and incubation at 37°C for various intervals. For the treatment with JNK inhibitor, γNCT-36 cells were pretreated with SP600125 (10 μM) or DMSO (1%) for 30 min, followed by the addition of TNF-α (50 ng/ml) and incubation at 37°C for 1.5 h. Cells were harvested and lysed in a CHAPSO (1%)-containing buffer. Intact γ-secretase complexes derived from clarified lysates of treated and untreated γNCT-36 cells were purified by HIS-Selected Cobalt Affinity Gel (Sigma) through their affinity with His-tagged NCT. Affinity-purified proteins were resolved by SDS-PAGE and analyzed by Western blotting with anti-phospho-serine/threonine antibody to determine the phosphorylation of immature and mature NCT (~130 and 180 kDa) and full-length PS1 (~50 kDa). The same blots were stripped and reprobed with anti-NCT and anti-PS1-NTF antibodies, respectively, to determine the levels of total NCT and PS1.
Cell-based γ-Secretase Assays
The quantitative measurement of γ-secretase activity using T20 cells has been previously described (Liao et al., 2004
). To examine TNF-α–elicited effects on γ-secretase, T20 cells stably transfected with C99-GV and Gal4-Luc were trypsinized and plated onto six-well microplates at 5 × 105
cells/well with 1 ml/well serum-free DMEM. After incubation at 37°C overnight, cells were treated with 50 ng/ml TNF-α in DMEM containing 1 μg/ml tetracycline and incubated at 37°C for various intervals as specified. Cells incubated with serum-free DMEM containing 1 μg/ml tetracycline alone were used to define the basal level of γ-secretase activity at each time point. To terminate the reactions, cells were harvested with PBS containing 20 mM EDTA and dissolved in 100 μl of 1× PLB. After the removal of cell debris by centrifugation, the activity of expressed luciferase reporter gene in clarified lysates was determined by mixing 20 μl of lysates and 20 μl of Steady-Glo luciferase assay reagent in a 96-well LumiNunc microplate (Nunc, Naperville, IL). Luminescence in individual microwells was determined by a VictorLight microplate luminometer (PerkinElmer, Waltham, MA) and subsequently normalized by the protein content of the corresponding lysates. The protein concentrations of clarified lysates were determined by the BCA protein assay kit as described in manufacturer's instructions. The normalized luciferase signal emitted by T20 cells in serum-free DMEM without tetracycline was referred to as onefold of activation. For treatments with chemical compounds, such as compound E, DAPT, MAP kinase inhibitors, compounds were added into the medium to a final concentration of 10 μM or as specified, and the reactions were incubated at 37°C for various intervals as specified.
SDS-PAGE and Western Blot Analysis
Clarified cell lysates containing equivalent amounts of proteins were mixed with equal volumes of 2× SDS sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 2% dithiothreitol, and 5% β-mercaptoethanol) and boiled at 100°C for 5 min. Denatured proteins were resolved in Tris glycine polyacrylamide gels (10 or 12%) and transferred electrophoretically to polyvinylidene difluoride membranes (Pall Corporation, East Hills, NY). Unbound areas on the membranes were blocked by a blocking buffer [3% BSA, 0.1% Tween-20 in TBS (TBST)] at room temperature for 1 h, followed by a brief rinse with TBST. Blocked membranes were probed with appropriate dilutions of primary antibody in the blocking buffer at room temperature for 1 h. After extensive washes with TBST to remove unbound primary antibody, washed membranes were incubated with appropriate HRP-conjugated secondary antibody in TBST at room temperature for 1 h. After the removal of unbound secondary antibody, antibody-reacted proteins were visualized by chemiluminescence using the SuperSignal West Femto or Dura reagents. Images were captured by and processed with ChemiGenius2 (Syngene, Frederick, MD). The antibodies used and their dilutions were as follows: anti-phospho-JNK and anti-JNK (Cell Signaling Technology, Beverly, MA), 1:1000; anti-phospho-Ser/Thr (MPM-2), 1:4000; anti-PS1-NTF (N-19), 1:4000; anti-nicastrin, 1:4000; anti-Aβ (6E10), 1:1000; anti-APP-CTF, 1:4000; anti-GAPDH, 1:4000; HRP-conjugated anti-mouse IgG (Amersham Biosciences, Piscataway, NJ) 1:10,000; and HRP-conjugated anti-rabbit IgG (Santa Cruz), 1:1000.
Site-directed Mutagenesis of Human PS1
The expression vector encoding the full-length human wild-type PS1 (pcDNA3.1-PS1) was kindly provided by Dr. Michael Wolfe (Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Kimberly et al., 2003
). To generate a human PS1 mutant (PS1-S319AT320A) that lacks a putative phosphorylation site, adjacent serine and threonine (S319
) in PS1 predicted to be susceptible to phosphorylation (http://www.cbs.dtu.dk/services/NetPhos/
; the algorithm of this analysis can be found in Blom et al., 1999
) were both replaced with alanines using the QuikChange site-directed mutagenesis kit according to the manufacturer's instructions. Two complementary oligonucleotides containing the desired mutations, flanked by unmodified nucleotide sequences, were synthesized. The paired primers for the generation of mutant PS1-S319AT320A were S319AT320A-forward (5′-AAG TAT AAT GCA GAA GC
CA GAA AGG GAG TCA CAA-3′) and S319AT320A-reverse (5′-AGC TCG TGA CTC AGG TGC
GCG ATG AGG CCC TAG-3′). The underlined nucleotides denote the base changes made to incorporate the desired missense mutations. The mutagenized sequences were confirmed by DNA sequencing.
Microsomal Preparation of γ-Secretase
The microsomal preparation and solubilization of γ-secretase from γ-30 and γNCT-36 cells were performed as previously described (Fraering et al., 2004
). A total of 108
γNCT-36 cells was harvested and resuspended in 10 ml of MES buffer (50 mM MES, pH 6.0, 150 mM NaCl, 5 mM MgCl2
, 5 mM CaCl2
, and the Complete protease inhibitor cocktail). The cells were lysed by being processed through a French Press (Thermo Fisher Scientific, Waltham, MA) once at 1500 psi. Nuclei and cellular debris were removed by centrifugation at 1500 g
for 10 min, and the postnuclear supernatant was saved and centrifuged at 100,000× g
for 1 h at 4°C to pellet the total membranes (to yield the membrane preparation). Membrane pellets were resuspended in 2 ml of ice-cold MES buffer (0.1 M NaHCO3
, pH 11.3) and were then repelleted at 100,000× g
for 1 h at 4°C to obtain the total microsomal membranes.
In Vitro Kinase Assay
JNK-dependent phosphorylation of γ-secretase components was carried out as described previously (Wei et al., 1999
). In brief, the total microsomal membranes prepared from 108
γNCT-36 cells were resuspended in 0.6 ml of solubilization buffer (1% digitonin, 25 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MgCl2
, 5 mM CaCl2
, and the Complete protease inhibitor cocktail) and incubated at 4°C for 1 h, followed by centrifugation at 16,100×g
for 10 min at 4°C to remove insoluble materials. γ-Secretase complexes in the solubilized supernatant was isolated by HIS-Select Cobalt Affinity Gel (Sigma, 150 μl of 50% suspension) through the affinity with His-tagged NCT and eluted by the elution buffer (50 mM sodium phosphate, pH 8.0, 0.3 M sodium chloride, 250 mM imidazole). Eluates of equivalent volume containing the affinity-precipitated γ-secretase complexes were then incubated with 0.2 μg of active JNK2 (Upstate) and 200 μM ATP in kinase reaction buffer in the presence or absence of SP600125 (20 μM) for 1.5 h at 30°C. Kinase reactions were terminated by the addition of SDS sample buffer. Proteins were resolved by 10–12% SDS-PAGE, and phosphorylated γ-secretase components were detected by Western blotting with anti-phospho-Ser/Thr antibodies. Duplicate blots were probed with anti-JNK2, anti-PS1, and anti-NCT antibodies for the identification of phosphorylated proteins.
To assess the JNK phosphorylation motif of PS1, synthetic peptide containing either the wild-type Ser319-Thr320 motif (ST; NH2-KYNAESTERESQ-COOH) or an altered motif substituting both Ser319 and Thr320 residues with alanines (AA; NH2-KYNAEAAERESQ-COOH) were synthesized by Genesis Biotech (Taipei, Taiwan) and were used as competitors of in vitro JNK phosphorylation of PS1 as described above. Boldface letters denote the critical amino acids essential for JNK phosphorylation in wild-type PS1 and those substituted by alanines in mutant peptide.
Cell-Free γ-Secretase Assay
To determine the microsomal γ-secretase activity in response to JNK phosphorylation, we used a robust in vitro cell-free assay to assess the microsomal membrane-associated γ-secretase activity (McLendon et al., 2000
; Qin et al., 2003
; Weggen et al., 2003
). Briefly, the total microsomal membranes derived from γNCT-36 or T20 cells were resuspended in 0.5 ml of kinase reaction buffer (25 mM Tris, pH 7.5, 5 mM β-glycerolphosphate, 2 mM 2-mercaptoethanol, 0.1 mM Na3
, 10 mM MgCl2
, Complete protease inhibitor cocktail, and 0.02 mM EDTA). An aliquot of resuspended membrane (38 μl) was incubated with 0.2 μg of active JNK2 (Upstate) and 200 μM ATP (to a final volume of 40 μl) for 2 h at 37 or 4°C. Isolated γ-secretase has been previously shown to exhibit a pH optimum at pH 7.5 and be able to cleave solubilized APP-CTFs in cell-free conditions (Esler et al., 2002
; Fraering et al., 2004
). Reactions lacking JNK2 were included as controls. Kinase reactions were terminated by the addition of SDS sample buffer and boiling at 100°C for 10 min. Proteins were resolved by tricine (16.5%) SDS-PAGE, and the levels of C99, C83, and AICD were visualized by Western blotting using anti-Aβ1–17 (clone 6E10, for C99) and anti-APP C-terminus (for C83 and AICD) antibodies, respectively.
Quantitative Densitometry and Statistical Analysis
Quantitative analysis of Western blots was conducted with the TotalLab v2.01 program by determining the relative density of the immunoreactive bands after acquisition of the blot image with ChemiGenius2 (Syngene). Phosphorylation levels were defined as the ratio of phospho-protein to total protein. Results were expressed as the mean (±SD) of triplicate measurements from a representative experiment. Statistical analyses were done by a one-tailed Student's t test, and p < 0.05 was considered significant.