Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.
This method can help answer key questions in the cell-signaling field such as the importance of different protein domains in receptor enzyme interaction and enzymatic activation. The main advantage of this technique is that it allows concomitant testing of receptor enzyme interactions and the functional consequences of this interaction on the enzyme activity. For each plate, use 10 milliliters of DMEM supplemented with 10%FBS and 1%penicillin and streptomycin.
The day before transfection, seed human embryonic kidney 293-T cells into 12 10-centimeter plates using a hemocytometer to count the cells. Incubate at 37 degrees Celsius in 5%C02. Once the cells are 80 to 90%confluent, use a lipid-based transfection agent to transfect five of the plates.
Perform the transfection following the manufacturer's instructions for adherence cells in 10-centimeter plates. Then transfect an identical second set of five plates along with one additional plate serving as a nontransfected control for the measurement of expressed protein amount before the immunoprecipitation. Incubate the transfected plates in a tissue culture incubator at 37 degrees Celsius in 5%CO2 for 48 hours.
Use a fluorescent microscope to examine the cells for GFP expression and ensure the transfection has successfully occurred. Mix 15 microliters of 100-millimolar sodium orthovanadate with 50 microliters of 30%hydrogen peroxide to prepare a fresh pervanadate mixture. To phosphorylate the tagged versions of PD-1, remove the media from the PD-1-GFP transfected cells.
Add 10 milliliters of plain DMEM and 10 microliters of pervanadate to each plate. Incubate in the dark at room temperature for 15 minutes. After this, wash the cells three times in ice-cold PBS using five milliliters of PBS per wash.
While performing immunoprecipitation, all steps should be performed either on ice or at four degrees Celsius. Supplement the lysis buffer with protease inhibitors by dissolving one tablet of the inhibitors in 10 milliliters of buffer. Also add one millimolar of sodium orthovanadate to the buffer that will be used on the PD-1-GFP-transfected plates.
Add 500 microliters of ice-cold lysis buffer to the cells, making sure to add the lysis buffer containing the sodium orthovanadate to only the plates containing PD-1-GFP-transfected cells. Use a cell scraper to immediately remove and collect the cells from the plates. Transfer the lysates into 1.5-milliliter cold tubes and rotate them at 0.005 times gravity and at four degrees Celsius for 30 minutes.
To collect the post-nuclei supernatant from the lysates, spin them down at 10, 000 times gravity and four degrees Celsius for 10 minutes. Transfer the supernatants into new tubes and discard the pellet. Store the supernatants for the second set of six plates on ice for later WCL analysis.
To begin preparing anti-GFP beads, gently shake the bottle containing the beads before opening to prevent settling. Remove 40 microliters of the anti-GFP beads from the slurry per each condition. Centrifuge at 500 times gravity and four degrees Celsius for three minutes.
Remove the supernatant to wash the beads, making sure to minimize contact between the pipette and the beads to prevent loss. Resuspend the beads in 80 microliters of lysis buffer per sample. Add the washed beads directly to the cell lysate from the PD-1-GFP-expressing cells of the first set of four plates.
Rotate at 0.005 times gravity for 30 minutes at four degrees Celsius to immunoprecipitate the GFP-tagged proteins. Wash the beads three times using one milliliter of cold lysis buffer that does not contain orthovanadate for each wash. Then centrifuge at 2, 500 times gravity for 10 seconds.
Divide the lysate of the active SHP2 from the first set of plates into three equal portions and add a portion to each of the three tubes containing the washed PD-1-GFP-containing beads. Add 1/3 of the lysate volume from the nontransfected cells to the second tube of the wild-type, PD-1-GFP beads. Discard the remaining 2/3.
Incubate the beads for four hours at four degrees Celsius with gentle rotation at 0.005 times gravity. After this, wash the beads twice using one milliliter of cold lysis buffer for each wash. Add 80 microliters of lysis buffer to each sample, ensuring that the total volume in each tube is 100 microliters.
Using a cut pipette tip, pipette gently up and down to mix. Then transfer 50 microliters from each tube into two, fresh, 1.5 milliliter tubes. Wash the beads once with phosphatase wash buffer.
Then remove the supernatant completely. Add 100 microliters of assay buffer to the beads and incubate at 30 degrees Celsius for 30 minutes under gentle agitation. When the buffer turns yellow, terminate the reaction by adding 50 microliters of one molar solution of sodium hydroxide.
Centrifuge at 2, 500 times gravity for 10 seconds. After this, transfer the supernatant into two wells of half area in a 96-well plate. Read the absorbents at 405 nanometers and express the results as relative optical density over the control wild-type version of PD-1-GFP.
First spin down the beads at 2, 000 times gravity and four degrees Celsius for 30 seconds and remove the supernatant. Add 20 microliters of two times Laemmli buffer and boil at 95 degrees Celsius for five minutes. Using a BCA kit, measure the protein concentration of the input controls.
Transfer 30 microliters of the most-diluted sample to a new tube. Use lysis buffer to dilute the rest of the input controls to the same concentration as the most-diluted sample. Then transfer 30 microliters of each of the diluted input controls to a new tube.
Add equal volumes of two times Laemmli buffer to the lysates and boil them 95 degrees Celsius for five minutes. After this, spin down the beads at 2, 000 times gravity and four degrees Celsius for 30 seconds before performing Western Blot analysis as outlined in the text protocol. In this study, a combined co-immunoprecipitation and enzymatic activity assay is developed for the parallel assessment of receptor enzyme interactions and activation.
Unsurprisingly, SHP2 failed to bind to PD-1 when ITSM was mutated. Remarkably, the mutant version of the ITIM inhibited SHP2 binding only to a limited extent. Nevertheless, the SHP2-phosphatase activity assay reveals that ITIM and ITSM are equally indispensable for the enzymatic activity.
This reveals a two-step activation model in which SHP2 is folded into an autoinhibited conformation under resting conditions. Upon activation of PD-1, SHP2 is recruited to the phosphorylated ITSM. However, the ITIM must also be phosphorylated to unfold SHP2 into its active conformation.
After its development, this technique can pave the way for researchers in the field of cell signaling to explore the function of protein domains in receptor-enzyme interactions.
