Rabbit anti-phosphotyrosine polyclonal and mouse anti-Src (clone GD11) monoclonal antibodies were obtained from Upstate Cell Signalling (Charlottesville, VA). Rabbit anti-ERK1 and MEK2 polyclonal antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-GFP polyclonal and mouse anti-MEK1 monoclonal antibodies were obtained from Invitrogen (Carlsbad, CA). Rabbit anti-L1 polyclonal antibody was a gift from Carl Lagenaur (University of Pittsburgh, Pittsburgh, PA). Mouse anti-myc monoclonal antibody (mAb) was obtained from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). Horseradish peroxidase (HRP)-conjugated sheep anti-mouse and donkey anti-rabbit antibodies were obtained from Amersham Biosciences (Piscataway, NJ). Donkey anti-mouse antibody conjugated to indocarbocyanine Cy3 and donkey anti-rabbit antibody conjugated to indodicarbocyanince Cy5 were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Human embryonic kidney (HEK)-293 and rat pheochromocytoma (PC)12 cells were obtained from American Type Culture Collection (Manassas, VA). BAPTA-AM, genistein, 5-iodotubercidin, ionomycin, LY294002, mastoparan, PD98059, protein kinase A (PKA), and protein kinase C (PKC) peptides, PP1, PP2, SB202190, and U0126, were obtained from BioMol Research Laboratories (Plymouth Meeting, PA). Ac-Y-EEIE, epidermal growth factor (EGF), erbstatin analog, NGF, and PAO were obtained from Sigma-Aldrich (St. Louis, MO). The codon-humanized pRluc and GFP2 vectors were obtained from PerkinElmer Life Sciences (Boston, MA). The small interference RNAs (siRNAs) for Abl, ERK1/2, MAPK14, MEK1, MEK2, and Src were obtained from Dharmacon (SMARTpool siGENOME; Chicago, IL).
Neurite Outgrowth Assays
Neurite outgrowth experiments were performed as described (Gil et al., 2003
) with slight modification. A 1-cm-diameter circle in a 35-mm Petri dish (Becton Dickinson, Franklin Lakes, NJ) was coated with poly-L-lysine (5 μg/ml in phosphate-buffered saline [PBS]); Chemicon, Temecula, CA) for 1 h at room temperature. After several washes with PBS, the coated area was dried under the hood. Aliquots of 1 μl of Ng-CAM (50 μg/ml; Gil et al., 2003
) or laminin (30 μg/ml, Becton Dickinson) were spotted on the coated area. Dishes were incubated for 1 h at room temperature, washed several times with PBS, and then blocked with 1% (wt/vol) bovine serum albumin. Cerebellar cells were prepared from P2-P4 mouse and plated on the prepared dishes in BME/B27/glucose/glutamine/Pen-Strep at a cell density of 3 × 105
cells/ml. Peptides and U0126 were diluted in dimethyl sulfoxide (10 mg/ml for peptides, 13 mM for U0126) and further diluted in media (final concentration 10 μg/ml peptide; 10 μM U0126) added to the cultures when cells were plated. Cultures were incubated for 2 d and fixed with 4% paraformaldehyde in 0.12 M phosphate buffer. Images were collected through CCD camera connected to a Zeiss Axiovert 100 inverted microscope (Thornwood, NY) and analyzed with NIH image.
Bioluminescence Resonance Energy Transfer (BRET) Construct Design
Bioluminescence resonance energy transfer (BRET) constructs were designed using vectors encoding Renilla luciferase and GFP2 (Sapphire GFP; Biosignal, PerkinElmer Life Sciences). Coding regions from each individual vector were copied by PCR with additional restriction sites, permitting their ligation into a single, concatenated coding region (GFP2:Rluc) between NotI and XhoI sites in a pcDNA3.1 Hygro (+) eukaryotic expression vector (Invitrogen). This chimeric construct (CHIM) encodes unique BsrGI and AscI sites in the intervening sequence. To create the reporter constructs from the CHIM construct, complimentary oligonucleotides derived from the L1-CAM coding region were synthesized (Sigma Genosys) with the addition of a 5′ overhang designed to generate a sticky end complimentary to the BsrGI and AscI sites. The addition of the reporter insert resulted in the deletion of two amino acids (SG) at the interface between GFP2 and Rluc found in the CHIM construct. Before ligation into the CHIM construct, oligonucleotide pairs were mixed in equimolar concentrations, heated to 94°C (4 min), and allowed to cool slowly to room temperature, permitting the annealing of the complementary regions.
Calculations of Fluorescence Resonance Energy Transfer Efficiency
The relationship between Förster resonance energy transfer (FRET) efficiency (E) and donor-acceptor separation (r) is described by the equation E = R06
), where R0
is the Förster distance at which transfer efficiency is 50% (Lakowicz, 1999
). Changes in r resulting from a 24% change in E were calculated using Δr/R0
= [(1/0.76E) − 1]1/6
− [(1/E) − 1]1/6
Near-confluent cultures of HEK-293 cells were harvested with trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA; Invitrogen) and resuspended to a density of 2.5 × 105 cells/ml. Aliquots (200 μl) of cell suspensions were added to white 96-well culture plates (CulturPlate; PerkinElmer Life Sciences) and incubated for 12 h at 37°C. HEK-293 cells were transfected with either 0.1 μg of DNA/well or 100 nM of siRNA/well using lipofectamine reagents (Lipofectamine Plus and Lipofectamine; Invitrogen) according to the manufacturer's instructions. After incubation of plates for either 48 h (DNA) or 72 h (siRNA) at 37°C the cells were washed once with warm DMEM without phenol red (Invitrogen), supplemented with 25 mM HEPES (Invitrogen). Transfected HEK-293 cells were treated with EGF for 15 min and inhibitors for 1 h (PD98059 and U0126) or 4 h (genistein). To each well, 10 μl of DeepBlueC substrate (final concentration of 5 μM; PerkinElmer Life Sciences) diluted in Dulbecco's PBS containing 0.1% (wt/vol) CaCl2, 0.1% (wt/vol) D-glucose, 0.1% (wt/vol) MgCl2, and 10 μg/ml aprotinin was added. The plates were immediately counted using the Fusion Universal Microplate Analyzer (PerkinElmer Life Sciences). Bioluminescence resulting from Rluc emission was counted at 410 nm using a 370–450-nm band pass filter, and the energy transferred to GFP2 was counted at 515 nm using a 500–530-nm bandpass filter. The efficiency of energy transfer between Rluc and GFP2 is determined by dividing acceptor emission intensity (GFP2) by donor emission intensity (Rluc). The resulting values reflect the proximity of GFP2 to Rluc and are referred to as the BRET ratio. Results from BRET assays were normalized against values obtained from untreated cells transfected with the L1-BRET construct.
Western Blots and Immunoprecipitation
Near-confluent cultures of HEK-293 cells, stably transfected with either L1-FIGQY or CHIM constructs, or ND7 cells were harvested with trypsin-EDTA and resuspended to a density of 6 × 105 cells/ml. Aliquots (5 ml) of cell suspensions were added to 100-mm cell culture dishes (Corning Life Sciences, Corning, NY) and incubated for 12 h at 37°C. Stably transfected HEK-293 cells were treated with genistein for 4 h at 37°C and ND7 cells for 1 h with 100 μM PD98059 and 15 min with 100 ng/ml NGF. Plates were washed with 5 ml of ice-cold PBS, and then cells were lysed with modified RIPA buffer (1% (wt/wt) IGEPAL CA-630, 1% (wt/vol) sodium deoxycholate, 0.1% (wt/vol) SDS, 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.2, 2 mM EDTA, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 10 mM benzamidine, 10 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin at 4°C for 20 min and centrifuged at 15,000 × g for 15 min at 4°C. The protein concentrations of the supernatants were determined by using the BCA protein assay (Pierce Chemical, Rockford, IL). The cell lysates were precleared with immobilized protein A (Pierce Chemical) for 3 h at 4°C. Immunoprecipitates were carried out with a rabbit anti-GFP or a rabbit anti-L1 polyclonal antibody and immobilized protein A beads overnight at 4°C. Alternatively, lysates were immunoprecipitated using agarose beads directly conjugated with rabbit anti-GFP (Vector Laboratories, Burlingame, CA). Beads were washed and resuspended in Laemmli buffer, analyzed by SDS-PAGE, and transferred to nitrocellulose membrane. The membrane was blocked, washed, and then incubated with 1 μg/ml anti-phosphotyrosine antibody overnight at 4°C. The blot was then incubated with HRP-conjugated goat anti-rabbit antibody at a dilution of 1:5000 and then developed using the enhanced chemiluminescence system (SuperSignal West Pico chemiluminescent substrate; Pierce Chemical). Membranes were stripped using 0.2 M glycine-HCl (pH 2.5) and reprobed with 0.5 μg/ml anti-GFP antibody for 2 h at room temperature or 2 μg/ml anti-L1 antibody overnight at 4°C. Densitometry of immunoblot films was carried out using a transilluminated flat-bed scanner (Umax Powerlook 1100; Dallas, TX), calibrated using a series of neutral density filters scanned under identical conditions and analyzed using NIH Image J (National Institutes of Health, Bethesda, MD). Measurements were normalized to loading controls for each lane.
HEK-293 cells were transfected with cDNA encoding an amino-terminal myc-epitope–tagged full-length wild-type L1-CAM and a carboxy-terminal GFP-tagged ankyrin B constructs using lipofectamine reagents. Transiently transfected HEK-293 cells were treated for 1 h with 100 μM PD98059 and 100 ng/ml with EGF. For immunolocalization, cells were fixed for 10 min using 1% (wt/vol) paraformaldehyde in 60 mM Pipes, 25 mM HEPES, 10 mM EGTA, and 2 mM MgCl2
. Staining was performed as described previously (Felsenfeld et al., 1999
). Briefly, ankyrin B was detected by indirect immunofluorescence using a rabbit anti-GFP polyclonal antibody and a donkey anti-rabbit antibody conjugated to indodicarbocyanince Cy5. L1-CAM was detected by indirect immunofluorescence using a mouse anti-myc mAb and a donkey anti-mouse antibody conjugated to indocarbocyanine Cy3. Confocal micrographs were collected on an Olympus microscope (Melville, NY) using a 60× objective at a plane intersecting cell–cell junctions.
Images were analyzed using NIH ImageJ. Densitometry was performed using a 5-pixel-wide line scan normal to the interface between two L1-CAM–positive cells. Signal maximum for ankyrin staining at the junction between cells was determined at the position of the maximal L1-CAM staining to ensure that we were quantifying membrane rather than juxtamembrane staining. Minima were determined from the regions of the line overlapping the cytoplasm of either of the two cells. Membrane localization index was determined using the equation index = max/(max − min) as described (Gil et al., 2003