EphA3 Cloning and Mutagenesis
The full-length human EphA3 cDNA clone was purchased from Invitrogen (clone MGC:71556; GenBank accession number NP_005224.2) and subcloned into the pEGFP-IRES2 vector (Clontech, Mountain View, CA) between the EcoRI and BamHI restriction sites. The EphA3 mutants were generated using the QuikChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) following the manufacturer's instructions. Wild-type and mutant EphA3 alkaline phosphatase (AP) fusion proteins were generated by subcloning cDNAs encoding wild-type and mutant extracellular domains (amino acids 1–537) into the pAPtag-2 vector (GenHunter, Nashville, TN) between BglII and BspEI restriction sites, except for the G228R mutant which was cloned between the BamHI and BspEI restriction sites.
Cell Lines and Transfection
HEK 293T cells were grown in Dulbecco’s Modified Eagle Medium (Cellgro, Manassas, VA) supplemented with 10% fetal bovine serum, 1 mM L-glutamine, 1 mM sodium pyruvate and antibiotics. Lipofectamine Plus (Invitrogen, Carlsbad, CA) was used to transfect HEK 293T cells with wild-type and mutant EphA3 cDNA in the pEGFP-IRES2 vector, following the manufacturer’s instructions and using various amounts of DNA (1–8 µg) to obtain similar wild-type and mutant EphA3 protein levels (see and ).
Figure 2 Effects of mutations in the intracellular region of EphA3 on receptor tyrosine phosphorylation. (A) Representative immunoblots showing tyrosine phosphorylation (PTyr) and total levels of wild-type EphA3 and the indicated mutants expressed by transient (more ...)
Figure 3 Effects of mutations in the extracellular region of EphA3 on ephrin ligand binding. (A) Curves showing the binding of wild-type and mutant EphA3 AP to ephrin-A5 Fc immobilized in ELISA wells. For each EphA3 AP concentration, averages ± SEM from (more ...)
For immunoblotting, cells were lysed in modified RIPA buffer (50 mM TrisHCl, pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA) containing protease and phosphatase inhibitors. Cell lysates were separated by SDS-PAGE followed by immunoblotting with an anti-EphA3 antibody (sc-919, Santa Cruz Biotechnology, Santa Cruz, CA) or an anti-human Fc antibody (Jackson ImmunoResearch Laboratories, West Grove PA) followed by either an anti-rabbit or an anti-mouse secondary antibody conjugated to horseradish peroxidase (HRP) or with a phosphotyrosine antibody conjugated to HRP (610012, BD Biosciences, San Jose CA). Immunoblots were developed with HyGLO chemiluminescence HRP detection reagent (Denville Scientific, Metuchen NJ).
Production of EphA3 AP
Ten µg wild-type or mutant EphA3 AP plasmid DNA were transiently transfected into HEK 293T cells with Lipofectamine 2000 (Invitrogen, Carlsbad CA) according to the manufacturer’s instructions. Culture medium containing secreted EphA3 AP fusion proteins was concentrated using Amicon Ultra-Centrifugal filters (Millipore, Billerica MA). The concentration of the AP fusion proteins was estimated based on AP activity measurements (26
Quantification of EphA3-Ephrin-A5 Binding by Enzyme-Linked Immunosorbent Assay (ELISA)
Protein A-coated 96-well plates (Pierce, Rockford IL) were used to immobilize ephrin-A5 Fc (0.1 µg/mL) (R&D Systems, Minneapolis, MN) or Fc control (MP Biomedical, Solon OH) for 1 hour at room temperature in 3% BSA in Tris buffered saline with 0.01% Tween-20 (TBST). Various amounts of culture medium containing concentrated EphA3 AP fusion proteins diluted in TBST were subsequently added for 1 hour at room temperature. After 3 washes with TBST, binding was quantified using p-nitrophenyl phosphate (pNPP; Pierce Scientific, Rockford IL) as the substrate. Dissociation constants (KD) were calculated using nonlinear regression and GraphPad Prism (Graphpad Software, Inc, La Jolla CA).
Assessment of EphA3-Ephrin-A5 Binding by Pulldown Assay
Transfected cells were lysed in modified RIPA buffer and lysates were incubated with 1 µg of ephrin-A5 Fc immobilized on GammaBind Plus Sepharose beads (GE Healthcare, Piscataway NJ). Complexes were washed 3 times with RIPA buffer, boiled in sample buffer, separated by SDS-PAGE and probed by immunoblotting for EphA3.
In Vitro Kinase Assay
HEK 293T cells expressing wild-type or mutant EphA3 were lysed in RIPA buffer without phosphatase inhibitors. EphA3 was immunoprecipitated with 2.5 µg anti-EphA3 antibody (37-3200, Life Science Technologies-Invitrogen, Carlsbad CA) and immobilized on GammaBind Plus sepharose. Immunoprecipitates were then incubated with 25 mM Hepes pH 7.5, 10 mM MnCl2, 10 MgCl2, 1 mM Na orthovanadate, 0.1% Triton X-100 and 150 µM ATP with phosphatase inhibitors for 30 min at 30°C and reactions were analyzed by immunoblotting for phosphotyrosine and EphA3. Immunoblots were quantified using Image J.
To measure phosphorylation of enolase by EphA3, immunoprecipitates with 1.25 µg anti-EphA3 antibody (Santa Cruz, sc-919) were washed twice with HNTG buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol and 0.1% Triton X-100) and then twice with kinase reaction buffer (10 mM Hepes, pH 7.5, 25 mM MgCl2 and 10 mM MnCl2). Rabbit muscle enolase (Sigma) was acid denatured and 6.25 µg were added to each kinase reaction together with 5 µCi [γ-32P]-ATP. 32P incorporated into enolase and EphA3 after 30 min incubation at 30°C was quantified from scans obtained with a Storm phosphorimager using Image J.
Measurement of Ephrin-Binding Domain Folding
Protein-A plates were coated with 0.5 µg/mL anti-EphA3 antibody (sc-919, Santa Cruz Biotechnology) in 3% bovine serum albumin (BSA) in TBST to capture EphA3 from transiently transfected HEK 293T cells lysed in modified RIPA buffer. The amounts of cell lysates used were sufficient to saturate the binding sites of the coated anti-EphA3 antibody, as verified by comparing the results with those obtained with twice as much lysate (data not shown). This ensured that the same amount of EphA3 was immobilized in all the wells. EphA3 with a properly folded ephrin-binding domain was detected using 2 µg/mL IIIA4 anti-EphA3 antibody (from Millipore, Billerica MA or kind gift from A. Boyd) in 3% BSA in TBST, followed by a goat-anti-mouse AP antibody (AP124A, Millipore, Billerica MA) and binding was quantified using pNPP as the substrate.
Quantification of Cell Surface EphA3
HEK 293T cells expressing wild-type or mutant EphA3 were incubated with EZ-link SulfoNHS-LC-Biotin (Pierce-Thermo Scientific, Rockford IL) to biotinylate cell surface proteins. Lysates were captured using an aanti-EphA3 antibody recognizing an intracellular epitope (Santa Cruz; see previous section) and biotin labeling was detected using a streptavidin-HRP conjugate (Pierce-Thermo Scientific, Rockford IL). The amounts of cell lysates used were sufficient to saturate the binding sites of the coated anti-EphA3 antibody, as verified by comparing the results with those obtained with twice as much lysate (data not shown). This ensured that the same amount of EphA3 was immobilized in all the wells.