Plasmids and cDNAs
pBI and pBI/EGFP were purchased from BD Biosciences (San Jose, CA). The cDNAs of Rab5:wild type (WT), Rab5:Q79L, Rab5:S34N, and Rab5:N133I were generated by polymerase chain reaction (PCR) with the previously made pGEX or pH2J1 constructs as templates (Liang et al., 2000
; Li and Liang, 2001
) and subcloned into the MluI restriction site of pBI and pBI/EGFP. Rat RabGAP5 cDNA was purchased from Invitrogen (Carlsbad, CA). The TrkA, RN-tre, and TSC2 cDNAs were kindly provided by Brian Rudkin (Laboratoire de Biologie Moleculaire et Cellulaire, Lyon, France), P. Paolo Di Fiore (European Institute of Oncology, Milan, Italy), and Kun-Liang Guan (Department of Biological Chemistry, University of Michigan, Ann Arbor, MI), respectively. We subcloned the TrkA cDNA into the MluI site of pBI. The RabGAP5 and RN-tre cDNAs were subcloned in pcDNA3 and pBI/TrkA vectors, either with or without the Myc tag.
The affinity-purified rabbit anti-RabGAP5 antibody was kindly provided by Francis Barr's laboratory (Max-Planck Institute of Biochemistry, Martinsried, Germany). Monoclonal antibodies for actin, FLAG, and Myc were purchased from Sigma-Aldrich (St. Louis, MO), whereas the anti-Rab5 monoclonal antibody (mAb), anti-hemagglutinin (HA) mAb, and anti-TrkA rabbit antiserum were from BD Biosciences, Santa Cruz Biotechnology (Santa Cruz, CA), and Upstate Biotechnology (Lake Placid, NY), respectively. The anti-pTrkA rabbit antiserum was from Cell Signaling Technology (Beverly, MA).
Cell Culture and Transfection
Tet-Off PC12 cells (BD Biosciences) were grown in 35-mm culture dishes in DMEM (Invitrogen) supplemented with 10% heat-inactivated horse serum (Invitrogen), 5% heat-inactivated fetal bovine serum (FBS; Invitrogen), 20 U/ml penicillin/streptomycin (Invitrogen), 1 mM l-glutamine (Invitrogen), and 200 μg/ml Geneticin (G-418; Invitrogen). Cells were incubated at 37°C in a humidified incubator with 10% CO2. For transfection, cells were seeded at a density of 2 × 105 cells/dish, grown to 70–80% confluence, and transfected with the indicated plasmids by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.
Neurite Outgrowth Assay
After cotransfection with pBI/EGFP, cells were allowed to recover in full growth medium and to express the recombinant proteins for 24 h. The growth medium was then replaced with a medium containing only 0.5% horse serum (no FBS) and 50 ng/ml NGF. The medium was incubated at 37°C for 6 d, with replenishment of NGF every 2 d until the sixth day. The NGF concentration was 50 ng/ml unless indicated otherwise. On different days as indicated, cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 30 min, and neurite outgrowth was observed with either an inverted fluorescence microscope (transiently transfected cells) or a phase-contrast microscope (cloned cell lines). We used a Nikon Diaphot 300 microscope for both purposes. The images were captured by a digital camera, stored in a connected computer, and analyzed with the Nikon ACT-1 software. Differentiated cells were defined as those containing at least one neurite twice as long as the cell body diameter. The percentage of differentiated cells in each case was determined from transfected cells (i.e., cells expressing green fluorescent protein). Standard error of the mean (SEM) was calculated from three to five independent experiments.
Confocal Fluorescence Microscopy
We used a Leica confocal laser scanning microscope with Ar-488 and Kr-568 laser excitation in the Flow and Image laboratory on campus and followed a procedure described previously (Li and Liang, 2001
). Briefly Tet-Off PC12 cells were grown on coverslips coated with collagen IV and transfected with pBI and/or pcDNA3 constructs expressing various enhanced green fluorescent protein (EGFP)-Rab5:S34N, EGFP-Rab5:Q79L, red fluorescent protein (RFP)-Rab5:Q79L, TrkA, or TrkA-EGFP as indicated. RFP represents ds-Red monomer from BD Biosciences. At 24 h posttransfection, the cells were treated with 50 ng/ml NGF for the indicated times, and then they were processed for immunofluorescence microscopy. Cells were rinsed three times with phosphate-buffered saline (PBS) and fixed for 20 min with 4% paraformaldehyde (wt/vol in PBS) at room temperature, followed by permeabilization with 0.1% Triton X-100 (in PBS) for 5 min. The cells were then stained with the anti-pTrkA antibody that specifically recognizes the cytoplasmic domain of activated TrkA and a secondary antibody (goat anti-rabbit IgG conjugated with Alexa568; Invitrogen). The coverslips were then mounted in PBS on glass slides and viewed with the Leica confocal microscope.
Glutathione S-Transferase (GST) Pull-Down Assay
We cloned the cDNA of the Rab5-binding domain (R5BD, residues 739-862) of Rabaptin5 into the pGEX vector (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). The resulting construct was termed pGEX/Rabaptin-5(R5BD), which expressed the fusion protein GST-R5BD in the Escherichia coli strain DH5α upon isopropyl β-d-thiogalactoside induction. GST-R5BD was then affinity purified with glutathione-Sepharose 4B resin (GE Healthcare). Rab5 proteins (WT, Q79L, and S34N) were expressed in Tet-Off PC12 cells by transfection of corresponding pBI constructs and incubation at 37°C for 24 h. Cells were then treated with 50 ng/ml NGF for the indicated times (untreated cells served as controls), followed by washing with ice-cold PBS and lysis for 5 min in the lysis buffer, which contained 25 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 0.1% NP-40, 10% glycerol, 1 mM dithiothreitol, and protease inhibitor cocktail (Sigma-Aldrich). Lysates were clarified by centrifugation at 10,000g for 2 min at 4°C, and an aliquot (200 μl) of the supernatant was incubated with 20 μl of GST-R5BD bound to the glutathione-Sepharose 4B resin for 10 min at 4°C on a rotating mixer. The resin was subsequently rinsed with the lysis buffer, resuspended in SDS sample buffer, boiled for 3 min, and subjected to SDS-PAGE (15% gel), followed by immunoblot analysis with the anti-Rab5 mAb. The results were quantified by densitometry using Densitometer SI (GE Healthcare).
In Vivo GTPase-activating Protein (GAP) Assay
Cells were cotransfected with pBI/Rab5 and a pBI or pcDNA3 construct expressing one of the GAPs. At 24 h posttransfection, the Rab5-GTP level in the cell was determined by the GST pull-down assay described above.
Establishment of Stable PC12 Cell Lines
Tet-Off PC12 cells were cotransfected with pBI/FLAG-Rab5:Q79L (or pBI/FLAG-Rab5:S34N) and pTK-hyg at a 20:1 ratio by using the Lipofectamine 2000-mediated procedure as described above. The cells were then selected with 150 μg/ml hygromycin (BD Biosciences) in the presence of 1 μg/ml doxycycline (Dox; BD Biosciences). After 3 wk, hygromycin-resistant colonies began to grow. Individual colonies were isolated and transferred to 24-well plates in triplicates, with two samples maintained in the presence of Dox and one sample without Dox to induce the expression of cloned Rab5 proteins for 2 d. Recombinant Rab5 proteins containing the FLAG epitope were identified by immunoblot analysis with the anti-FLAG antibody, and clones with inducible recombinant protein expression were selected and scaled up for further assays.
Cell Growth Rate
Cells were seeded at a density of 1 × 105 cells/well in a six-well plate and incubated at 37°C. Cell numbers were counted each day up to 6 d with a hemacytometer (Hausser Scientific, Horsham, PA), after trypsinization and resuspension in the medium at each time point. The results were averaged from triplicate samples, and error bars represented SEM from three independent experiments.
Coimmunoprecipitation (coIP) Assay
Cells were transfected with pBI/TrkA constructs that coexpressed RabGAP5 or mutants (all contained Myc tag) via Lipofectamine 2000 as described above. Cells were allowed to recover in full growth medium and to express the recombinant proteins for 24 h at 37°C. Before cell lysis, cells were starved in serum-free medium for 24 h and then either treated or not treated with 50 ng/ml NGF for 5 min as indicated. Cells were then rinsed with ice-cold PBS, pH 7.4, and lysed on ice for 45 min in the lysis buffer, which contained 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 2 mM sodium orthovanadate, 2 mM para-nitrophenol phosphate, and protease inhibitor cocktail (Sigma- Aldrich). After clearing at 10,000 × g for 15 min, cell lysates were incubated with anti-Myc antibody-conjugated agarose beads (Sigma-Aldrich) for 4 h at 4°C. The beads were then washed four times with the lysis buffer and boiled for 3 min in SDS sample buffer, followed by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis with anti-TrkA, anti-pTrkA, and anti-Myc antibodies. The results were quantified by densitometry using Densitometer SI (GE Healthcare).
RNA Interference (RNAi) of RabGAP5
We used the pSUPER vector (Oligoengine) to express specific short hairpin RNAs (shRNAs) to knock down RabGAP5 expression in PC12 cells. The 19- or 21-mer oligonucleotides were designed, annealed, and then cloned into pSUPER according to the manufacturer's instructions. The targeting regions included four sequences in rat RabGAP5 and one sequence in its human counterpart as a control, including 5′-GCATCTGGGACCTGTTCTTCT-3′ (for rat shRNA1), 5′-GCCCTATTTGAACATGGATTG-3′ (for rat shRNA2), 5′-GGCAAAGAACATCAAACAA-3′ (for rat shRNA3), 5′-GGCCCTATTTGAACATGGA-3′ (for rat shRNA4), and 5′-GCAGAGCAACCAGAGTTCTAC-3′ (for human shRNA).
The effectiveness of RabGAP5 knockdown was confirmed by immunoblot analysis. Because of low transfection efficiency, the shRNA constructs were cotransfected with pBI/myc-RabGAP5, followed by immunoblot analysis 48 h later with the anti-myc mAb to examine the level of myc-RabGAP5 expression. For neurite outgrowth analysis, PC12 cells were transfected with either the pSUPER vector alone as a negative control or with each of the shRNA constructs. In this case, pBI/EGFP was cotransfected to identify the transfected cells by fluorescence microscopy, and neurite outgrowth was measured at 48 h posttransfection and for 5 days thereafter with the assay described above.