Our work focuses on the development of tools that regulate the activity of tyrosine phosphatases. We've applied the rapamycin regulated system to several phosphatases and shown that it imparts specific and temporal regulation of our protein of interest. We've aimed to determine the morphological changes induced by the activation of SHIP-2 phosphatase.
The RapR tool has allowed us to determine transient effects of phosphatase activation in cells, and we've determined complex specific downstream effects of a phosphatase involved in cell spreading and migration. Additionally, we've determined domain-driven behaviors of the same phosphatase. This has contributed to our understanding of phosphatase mechanisms in regulating cell morph photodynamics and downstream signaling.
Our RapR tool offers the ability to specifically and temporally regulate a phosphatase of interest. Once inserted into the phosphatase, the iFKBP domain acts as an allosteric switch that, once bound to rapamycin, restores activity to the phosphatase, acting as an on switch. We can also use the rapamycin-regulated system to reconstruct signaling complexes.
Our findings using the RapR tool have opened the door to determining the effects of SHIP-2s interactome on its ability to regulate cell activity. We found that SHIP-2s in SH2 domain binding is crucial in cell retraction. Using the RapR tool, we can further investigate what binding partners may be responsible for this regulation.
To begin, obtain HEK-293T cells. Inflate 800, 000 cells per well in a six-well tissue culture plate in supplemented DMEM media. Incubate the plate overnight at 37 degrees Celsius with 5%carbon dioxide to achieve 60 to 80%cofluency.
Next, mix one microgram of each plasma DNA with 200 microliters of serum-free DMEM and six microliters of transfection reagent. After 15 minutes of incubation, dispense 100 microliters of transfection mixture per well of cells. Incubate overnight in a 37 degrees Celsius and 5%carbon dioxide incubator.
On day three, add an appropriate amount of either rapamycin or ethanol separately to the cells, and incubate them for one hour in a 37 degrees Celsius and 5%carbon dioxide incubator. Then place the plate on ice, and gently wash the cells with three milliliters of cold PBS. After removing the PBS, add 300 microliters of lysis buffer containing either rapamycin or ethanol, and scrape the cells with a cell scraper.
Transfer the sample to a 1.5-milliliter tube and microcentrifuge it for 10 minutes at 1, 000 g at four degrees Celsius. To check for expression levels, aliquot 20 microliters of the supernatant into a new 1.5-milliliter tube. Incubate the sample with 20 microliters of 2x Laemmli buffer containing 5%beta-mercaptoethanol at 95 to 100 degrees Celsius for five minutes.
To begin, culture and prepare the HEK-293T cells expressing RapR-Shp2 for immunoprecipitation. For preparing the Sepharose, add resuspended protein G Sepharose into a 1.5-milliliter tube. After adding the lysis buffer, microcentrifuge the tube for one minute at 2, 000 g, and carefully remove the lysis buffer.
Then resuspend the Sepharose in 380 microliters of lysis buffer by inverting the tube a few times. Incubate the Sepharose with BSA and the antibody on a rotator at four degrees Celsius for 1.5 hours. After washing the Sepharose, as demonstrated earlier, use a cut tip to carefully resuspend it in the lysis buffer.
Add the supernatant from the prepared HEK-293T cells expressing RapR-Shp2 to 50 microliters of the suspended Sepharose in a new 1.5-milliliter tube, and incubate the tube on a rotator at four degrees Celsius for 1.5 to two hours. To begin, obtain the HEK-293T cells expressing RapR-Shp2, and perform immunoprecipitation with protein G Sepharose. After immunoprecipitation, wash the Sepharose two times each with 0.5 milliliters of lysis buffer, followed by 0.5 milliliters of wash buffer.
Remove as much buffer as possible after the final spin. Then add 40 microliters of the phospho-paxillin Imidazole Buffer mixture to each sample with or without one micromolar rapamycin. Flick the tube gently, and immediately place it in the heating shaker for 40 minutes at 32 degrees Celsius.
Set the heating shaker to 1, 000 revolutions per minute to ensure full agitation of the sample. To stop the reaction, add 40 microliters of 2x Laemmli Buffer to each sample, and incubate them at 95 to 100 degrees for five minutes. After the samples cool down, load 15 microliters of each sample onto a 4 to 15%gradient SDS polyacrylamide gel.
Run a standard Western blot protocol using PVDF membranes. Finally, blot using Anti-FLAG antibody to detect the RapR-Shp2 construct and anti-phospho-paxillin to detect phosphorylated paxillin in the samples. Active RapR-Shp2 showed no phospho-paxillin, similar to the constitutively active Shp2, indicating retained full activity.
Inactive RapR-Shp2 and Dominant Negative Shp2 showed similar phospho-paxillin levels, indicating no activity when inactive.
