This protocol allows for the specific labeling and subsequent enrichment and identification of endogenous CK2 substrates from a complex biological sample such as a cell or tissue lysate. This method can be applied to any cell type or tissue. Thus facilitating, the study of CK2 in various biological contexts.
Demonstrating the procedure will be John Chojnowski, a graduate student from my laboratory. To begin, mechanically lyse the tissue sample or cultured cells as outlined in the text protocol to collect a total of 900 microliters of sample. Centrifuge at 17, 500 times gravity and at four degrees celsius for three minutes.
Then, transfer 270 microliters of the supernatant to each of three new 1.7 milliliter microcentrifuge tubes. Remove 40 microliters of the remaining supernatant to be used as an input control, and transfer it to a new tube. Place all of the samples on ice.
First, label each of the three sample tubes as shown here. To the Kinase Reaction tube, add 2.7 microliters of CK2 and 2.7 microliters of 2.5 millimolar GTPgammaS. Flick the tube to mix, and immediately place it on ice.
To the GTPgammaS only tube, add 2.7 microliters of 2.5 millimolar GTPgammaS, and 2.7 microliters of Lysis Buffer. Flick this tube to mix, and immediately place it on ice. To the PNBM Only tube add 5.4 microliters of Lysis Buffer.
Flick the tube to mix, and immediately place it on ice. Incubate all three tubes in a water bath at 30 degrees Celsius for one minute. After this, add 13.5 microliters of PNBM at 12 milligrams per milliliter to each tube.
Invert the tubes to mix the samples, and incubate the samples at room temperature for one hour. While the samples are incubating, label each of the columns for the sample to be loaded, as shown here. Prepare the columns by inverting each column several times to resuspend the Sephadex G-25 resin in the storage buffer.
Attach each column to a clamp stand, and let it sit undisturbed for approximately five minutes to let the resin settle. Next, place a tube below the bottom opening of each column. Remove the caps from both the top and the bottom of each column, to allow the storage buffer to drain into the tubes by gravity.
Once the storage buffer is depleted, add approximately 2.7 milliliters of Lysis Buffer to each column to equilibrate them, making sure to collect and discard the flow through. Repeat this process of adding Lysis Buffer and discarding the flow through, three times. To begin, load each sample into its respective column.
Collect and discard the flow through. Add 420 microliters of Lysis Buffer to each column to wash the samples. Let the Lysis Buffer filter through the column, and collect and discard the flow through.
Then, place tubes into position under each column for sample collection. Add 500 microliters of Lysis Buffer to each column to elute the samples. Collect the flow through, which now contains an enriched population of thiophosphorylated and alkylated CK2 substrates in the kinase reaction.
The GTPgammaS only reaction will contain substrates thiophosphorylated and alkylated by any endogenous CK2 present. The PNMB only reaction will contain background alkylated proteins. Remove 80 microliters from each Elution as an Elution Input Control sample.
Next, split each sample into two tubes that each contain 200 microliters. Label each of the tubes to be either anti-thiophosphate ester or immunoglobulin G, as shown here. Add 2.8 micrograms of anti-thiophosphate ester antibody to each of the anti-thiophosphate ester tubes.
Add 2.8 micrograms of Isotype Control antibody to each of the IGG tubes. Then place the tubes on a rotator at four degrees Celsius for two hours. During the last 15 minutes of the sample incubation, use a clean razor blade to cut the end off of a P200 pipette tip to increase the gauge size.
Immediately prior to use, briefly vortex the storage tube containing the agarose beads. Using the cut pipette tip, transfer 100 microliters of the beads slurry per immunoprecipitation to a new 1.7 milliliter microcentrifuge tube. It is important to vortex the beads before each transfer to prevent beads from settling.
Centrifuge the tubes at 17, 500 times gravity and at four degrees Celsius for one minute. Remove and discard the supernatant. Resuspend the beads by adding 200 microliters of Lysis Buffer and briefly vortexing to mix.
Repeat this process of centrifuging and washing the beads three times. After the final wash, place the beads on ice until ready to proceed. When the samples have finished incubating, centrifuge them at 17, 500 times gravity and at four degrees Celsius for three minutes.
Then, transfer 200 microliters of each sample to the tubes containing the washed beads. Place the tubes on a rotator at four degrees Celsius for one hour. Next, centrifuge the tubes at 17, 500 times gravity and at four degrees Celsius for one minute.
Remove 40 microliters from each supernatant to save as a depletion control. Remove and discard the remainder of the supernatant, being careful not to disturb the beads. Wash the samples by adding 200 microliters of Lysis Buffer to each and vortexing briefly.
Centrifuge at 17, 500 times gravity and at four degrees Celsius for one minute, discarding the supernatant. Repeat this wash and centrifugation process three times taking care to not disturb the beads. After this, add 50 microliters of 2X sample buffer to each sample containing beads.
For all other samples, which include the Input Control, the Elution Input Controls and the Depletion Controls, add 8 microliters of 6X Sample Buffer. Pipette each tube up and down to mix with the exception of tubes containing beads, and heat all samples at 95 degrees Celsius for five minutes before proceeding with SDS-PAGE. To assess if the immunoprecipitation was successful, run 25 to 30 microliters of each sample that was eluded from beads on a separate 12.5%polyacrylamide gel.
Stain each gel with Coomassie Blue to visualize enriched proteins from various stages of the experimental protocol. Using new, clean razor blades, carefully excise any unique bands present in the kinase reaction anti-thiophosphate ester IP Lane while making note of their approximate molecular weights. A new razor blade should be used for each unique band.
The underlying basis of this technique is this technique is the unusual ability of CK2 to use GTP for phosphoryl group transfer. Addition of exogenous CK2 holoenzyme along with the GTP analog, GTPgammaS, to a cell lysate, results in a thiophosphorylation of endogenous CK2 substrates. Subsequent treatment of the lysate with the alkylating reagent p-Nitrobenzyl mesylate generates thiophosphate ester moiety on these specific substrate proteins, that can then be immunoprecipitated using an anti-thiophosphate ester antibody and ultimately identified by Mass spectrometry.
In the representative positive results shown here, CK2 dependent, thiophosphorylation and subsequent alkylation were successful. As expected, an enhanced anti-thiophosphate ester signal by Western blotting is observed only in the lane containing the complete kinase reaction, and not in the GTPgammaS only and PNMB only treated samples. A Coomassie Blue stained gel of the immunoprecipitated and alluded proteins, also demonstrate a positive result.
As multiple unique bands are evident only in the anti-thiophosphate ester IP Lane, in which the lysate was incubated with exogenous CK2 and GTPgammaS. The marked band is then excised from the gel and submitted for protein identification by mass spectrometry. Following this procedure, putative CK2 substrates identified by a mass spectrometry must be validated using an additional approach, such as by CK2 dependent phosphorylation in a standard in vitro kinase assay.
