Identification of Kinase-substrate Pairs Using High Throughput Screening

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

The overall goal of this procedure is to identify kinases that phosphorylate a substrate of interest using high throughput screening methods. This is accomplished by first transecting cells with plasmids expressing kinases FU to glutathione as transferase or GST. The second step is to perform A GST kinase pulldown.

Next, the samples are loaded into gels, which are then run and stained with kumasi brilliant blue dye. The final step is to dry the gels and expose them to auto radiography film. Ultimately by developing and interpreting the resulting films, one is able to identify kinase substrate pairs as each lane is representative of a distinct kinase assay.

Existing methods for identifying a kinase for a known phosphorylated substrate include using bioinformatic approaches to search for a consensus site in the substrate, detecting complexes between kinase and substrates using biochemical techniques and a trial and error approach, searching for conte consensus substrates on the basis of their known biological function. These approaches are time consuming and do not always meet with success. Our approach permits rapid identification of kinase substrate pairs on the basis of functional outcomes.

When we first had the idea for this method, we were concerned that there would be insufficient specificity in vitro. As it turns out, the substrate specificity is excellent and we often find that only family member kinases are able to phosphorylate a given substrate in the screen. This is particularly evident when we multiplex using multiple substrates in each screen, demonstrating the procedure will be Courtney, a technician from my laboratory Begin the procedure with preparation of reagents, plates, and cells as described in the text protocol.

If plates containing kinase plasmids have been frozen, thaw at room temperature and centrifuge at 1900 times G for three minutes to collect any moisture at the bottom of the wells. Mix 8.6 milliliters of reduced serum medium with 312.7 microliters of lipid-based transfection reagent, and allow the mixture to sit for five minutes to each well. Of the 96 well plate containing the kinase plasmids at 10 microliters of the reduced serum medium.

Using an automated liquid dispenser fitted with a small volume cassette, then add 10 microliters of the reduced serum medium transfection reagent mix per well, using an automated liquid dispenser fitted with a small volume cassette and allow the plate to sit for 20 to 45 minutes. Next, resus suspend 2 93 T cells at 0.75 million cells per milliliter in 80 milliliters of complete delcos modified equals medium or DMEM at 100 microliters of the cell suspension per well. Using an automated liquid dispenser fitted with a standard volume cassette, check the wells under a microscope for uniform cell distribution before returning the plate to a 37 degree Celsius incubator for 24 hours.

To begin the GST kinase pull down experiment. Make a four millimolar per vanadate solution by mixing 60 microliters of 0.2 molar sodium vanadate with 540 microliters of water in a second tube mixed 2.7 microliters of 30%peroxide and 1.4 milliliters of phosphate buffered saline or PBS. Add the two solutions together and let the mixture sit for 15 minutes before use.

Using a multi-channel pipette, dispense two microliters of 0.25 molar calcium chloride in each well, followed by 2.5 microliters of the proven date solution. Incubate each plate at 37 degrees Celsius for 10 minutes, and then place on ice, keeping the plates on ice. Remove the medium from each well, using a vacuum immediately at 50 microliters per well of ice cold lysis buffer.

Using an automated liquid dispenser fitted with the standard cassette, let the plate sit for 30 minutes on ice to lice. After spinning the plates at 1900 times G for three minutes at four degrees Celsius, scrape the cells from each well using a multi-channel pipette and transfer all contents to appropriately labeled vbo. 96 well plates spin the plates at 1900 times G for 10 minutes at four degrees Celsius during the spin fill glutathione coated plates with 100 microliters per well of ice cold lysis buffer.

As a rinse, keep the plates on ice. Following centrifugation of the bottom plates, invert the glutathione plates over a sink to shake out the lysis buffer and blot on a paper towel. Transfer the lysis buffer from the V bottom plates to the glutathione plates by tilting the plate and using a multi-channel pipette, being careful to not disturb the pellet on the bottom.

Then cover the plates and leave on ice for a minimum of two hours. To bind close to the end of the two hour binding step, prepare a radioactivity workstation ensuring the necessary safety precautions are in place for radioactive work. Set the hybridization oven to 30 degrees Celsius.

Invert the glutathione plates over a sink to shake out the lysis buffer and blot on a paper towel. Rinse the wells three times with 100 microliters of lysis buffer without PMSF. Do not let wells sit dry.

Keep them in the rinse until ready to proceed. Next, prepare 55 milliliters of one X kinase buffer or one x kb as described in the text protocol. Add 50 microliters of one x kb to each well of the plate using an automated liquid dispenser fitted with a standard volume cassette.

Then prepare solution A by making a solution containing the substrate of interest and myelin basic protein or MVP as detailed in the text protocol one at a time. Invert the plates over a sink to remove the one XKB rinse plot on a paper towel and immediately add 30 microliters of solution A.Using an automated liquid dispenser fitted with a small volume cassette. Keep the plates on ice.

Next, prepare solution B in the radioactivity work area as described in the text protocol at 20 microliters of solution B per well. Using a repeater pipette, which aids in mixing due to ejection force cover and incubate the plate in a 30 degree Celsius hybridization oven for 30 minutes. After 30 minutes, transfer the plates back to ice.

Then add 50 microliters of two x sodium eccle sulfate or SDS lysis buffer to each well using a multichannel pipette. All work in this section should be performed in an area designated for radio activity. Turn on the hybridization oven and set it to 85 degrees Celsius.

Once the oven has reached temperature, transfer the plates to the oven and incubate for 10 minutes to denature the samples. Next load. 26 well precast gels with 15 microliters of each reaction.

Using a multi-channel pipette to fill several wells at once, care must be taken that all tips align with corresponding wells. Before adding the samples, run the gel at 150 volts. Do not let the blue line run off of the bottom of the gel as this contains the unincorporated A TP.Then dismantle the gels and remove the unincorporated a TP as it will darken the gel exposure on the films.

Place the gels in labeled containers and cover with kumasi stain for 15 minutes. Next, remove the kumasi stain. Briefly rinse the gels with water and add detain solution.

Detain the gels until the proteins are clearly visible. A band for MVP and a band for the substrate should be visible for each sample. To dry the gels, cut a large sheet of filter paper and place it on the dryer.

Wet a cellophane sheet in distilled water until it is smooth and wrinkle free and place it on top of the paper. Lay the gels on top of the cellophane sheet making a note of the order of the gels. Wet a second cellophane sheet and place on top of the gels.

Roll out all bubbles for a nice uniform surface. Close the flap, turn on the vacuum and drive the gels for three hours at 80 degrees Celsius. Once the gels are dry, expose them to double emulsion auto radiography film using a screen to intensify the signal.

Wrap the cassette with saran wrap or a plastic bag and seal with tape to keep out frost before storing the cassette at minus 80 degrees Celsius overnight. The following day, remove the cassette from the freezer and let it thaw at room temperature. Develop the film in a dark room using a film processor according to the manufacturer's instructions shown.

Here are representative results from a screen 180 kinases were screened using A GST tagged peptide substrate corresponding to amino acids 268 through 283 from kreb regulated transcriptional coactivator two or C RTC two, as well as the classic kinase assay substrate myelin basic protein or MBP only two. Kinases mark two and the highly related kinase, mark three phosphorylated. The CRTC two peptide MBP is included as an internal control in all assays as it contains many phosphoryl relatable residues and runs at 18 kilodaltons toward the bottom of the gel.

This allows for an interpretation of specificity. Some kinases will robustly phosphorylate a substrate and MVP. Of note here is that the wells containing GST alone always purify some endogenous kinase activity.

Thus, there is always background phosphorylation in the assay. While this does not rule out that the phosphorylation of the substrate is real, it does suggest that in the in vitro setting, the kinase may be less selective. It is particularly informative to include multiple substrates of differing molecular weights to draw conclusions regarding kinase substrate specificity.

As the screen is in vitro and additional levels of complexity occur in vivo, a candidate kinase must be validated in cells. For example, a candidate kinase may be have the capacity to phosphorylate a substrate in vitro it not be expressed in the same cell type or in the same sub cells of a compartment as the substrate. This is typically done using RNAi mediated silencing of the candidate.

It is also possible to perform a secondary screen using a non phosphoryl relatable mutant of the substrate to confirm specificity.