The overall goal of this in vitro kinase assay is to identify phosphorylation sites in a protein of interest that are specific for the kinase cyclin-dependent kinase 1. The identification of CDK1 specific phosphorylation sites is important as they provide mechanistic insights into how CDK1 controls the cell cycle. Cell cycle regulation is critical for phase full chromosome segregation and defects lead to chromosomal aberrations and cancer.
The main advantage of this technique is that is relies on purified proteins therefore it can be applied to any model organism and yields reliable results, especially when combined with functional studies in cells. This method is important as the known number of CDK1 targets is still low, despite the fact that CDK1 phosphorylates an estimated eight to 13%of the proteum. Though this method identifies CDK1 specific phosphorylation sites it can be modified to identify phosphorylation sites for other kinases provided that the purified kinase is available.
Generally individuals new to this method will struggle because the conditions for the kinase assay have to be optimized for each protein target to obtain high phosphorylation efficiency. Preferably 100%Use IGPS 3.0 software and visit the web server to predict the CDK1 specific phosphorylation sites in the CENP-F target protein sequence. Check the phosphorylation sites against the CDK1 consensus sequence.
Start the protein expression by preparing a pre-culture. Following the manufacturer's instructions, transform one microliter of the expression plasmid into 50 microliters of E.coli competent cells. After adding the plasmid, incubate the cells on ice for 30 minutes.
Then incubate the cells for 45 seconds at 42 degrees Celsius. After the incubation place the cells on ice for one minute then add 400 microliters of LB to the cells, then incubate the culture for one hour at 37 degrees Celsius while shaking. Then plate 200 microliters of the culture on LB agar plates, supplemented with the antibiotics of interest, depending upon the construct.
Next incubate the plates at 37 degrees Celsius for 12 to 20 hours without shaking. Then pick a colony from the LB agar plate and inoculate the colony in 50 milliliters of LB medium supplemented with the desired antibiotic combination. Incubate the culture at 37 degrees Celsius while shaking at 160 to 180 revolutions per minute for 12 to 20 hours.
For each liter of LB medium add the antibiotics and 10 milliliters of 40%weight by volume of sterile filtered glucose solution. To this medium add 20 milliliters of the densely grown pre-culture. Then incubate the culture at 37 degrees Celsius while shaking at 160 to 180 revolutions per minute.
Check the absorbance of the culture at 600 nanometers in regular intervals. Once the absorbance reaches 0.5 to 0.6, induce protein expression by adding 0.2 millimolar IPTG. Then incubate the culture at 37 degrees Celsius for three hours while shaking.
After three hours harvest the cells by centrifugation at 4, 100 times G for 15 minutes at four degrees Celsius, using a swing out rotor. After the centrifugation, expel the supernatant. Resuspend the cell pellet in 20 milliliters of GST binding buffer for CENP-F and his six binding buffer for karyopherin alpha for each liter of culture.
Then store the cells at minus 80 degrees Celsius. To purify CENP-F, first thaw the cells with CENP-F construct. Then adjust the volume to 20 milliliters with a GST binding buffer.
Once the volume is adjusted add all the reductants and the protease inhibitors one after another to prepare the cell suspension for subsequent lysis. Next transfer the cell suspension in a glass beaker and immerse the beaker in an ice water bath. Then sonicate the culture.
After sonication add 250 micromolar PMSF to the lysate, then inspect the lysate, which should be transparent at this stage. Then centrifuge the lysate at 12, 000 to 40, 000 times G for 25 minutes at four degrees Celsius. Next prepare the column by adding two milliliters of glutathione agarose one to one slurry to a disposable chromatography column.
To equilibrate the column wash it first with 25 milliliters of ultra pure water and then 25 milliliters of GST binding buffer. Once the centrifugation is over, decant the supernatant then filter the supernatant through a 0.2 micrometer pour size. Next pour the lysate on the plugged column for the binding step.
Cap the column and incubate it at four degrees Celsius for 30 minutes while gently nutating and avoid foaming. After 30 minutes transfer the column on a rack and let the resin settle. Then collect the flow through and wash the column twice with 25 milliliters of GST binding buffer.
Once the washing is complete, again plug the column. Then add 400 microliters of GST binding buffer and 250 microliters of PS protease stock with activity of 1000 units per milligram to the column. Again cap the column and swirl the column gently to resuspend the resin in the buffer, then incubate the column at four degrees Celsius for 16 to 20 hours.
After the incubation period is over add four milliliters of GST binding buffer to elute the CENP-F fragment from the column that has been proteolytically cleaved. Next to elute the GST tag again plug the column. Then add four milliliters of GST elution buffer containing glutathione to the column.
Next incubate the column for 10 minutes to elute the bound GST tag. After 10 minutes collect the eluate. Then perform SDS polyacrylamide gel electrophoresis to analyze the PS protease and glutathione elution fractions.
Next to concentrate the protein sample, pipette the PS protease eluate into the upper compartment of a centrifugal filter unit with a three kilodalton molecular weight cutoff. Then centrifuge the eluate at 4, 100 times G in 10 to 15 minute increments at four degrees Celsius in a swing out rotor, to concentrate the sample. After each increment mix the protein sample in the concentration filter by gently pipetting up and down to avoid formation of a concentration gradient.
To purify CENP-F by gel filtration, attach a gel filtration column to a fast protein liquid chromatography system. Next equilibrate the column with one column volume of gel filtration buffer. Next centrifuge the CENP-F fragment in a microcentrifuge tube at 21, 700 times G for 20 minutes at four degrees Celsius.
After the centrifugation is over, inject the sample on the column, then elute the column with gel filtration buffer and collect 0.6 milliliter fractions of the CENP-F fragment. Next analyze the gel filtration profile and run an SDS-PAGE of the peak fractions. Pool fractions that contain pure CENP-F.
Then concentrate the purified CENP-F fragment to 3.3 milligrams per milliliter. To determine the protein concentration dilute the CENP-F fragment in a one to 100 ratio with ultra pure water. Next record the spectrum from 220 to 300 nanometer wavelength in the specrophotometer.
Next prepare aliquots of 50 microliters of the purified CENP-F in 0.5 milliliter microtubes, then flash freeze the tubes in liquid nitrogen and store at minus 80 degrees Celsius. For the kinase assay mix the CENP-F fragment with ATP and CDK1 cyclin B along with other buffers in a 0.5 milliliter microtube. Then prepare a second sample without CDK1 cyclin B as a negative control.
Then incubate the samples in a water bath at 30 degrees Celsius for one to 16 hours. For the successful identification of the phosphorylation sites by mass spectrometry, it is critical that the phosphorylation efficiency is as high as possible, preferably 100%To increase the phosphorylation efficiency add more kinase and increase the incubation time to 16 hours. Then add equal volumes of six molar guanidine hydrochloride solution to the remaining kinase assay reaction and the negative control and perform mass spectrometry to identify the phosphorylation site.
To obtain high quality mass spectrometry data it's also important that the purified protein sample is highly pure and homogenous. First SDS-PAGE analysis is done which shows a clear upward shift of the band on the gel for the phosphorylated CENP-F compared to the negative control without CDK1, indicating that the cNLS of CENP-F is a CDK1 substrate. Next mass spectrometry is done to analyze the number and efficiency of the CENP-F phosphorylation sites.
The ESI ion trap mass spectrum of the intact in vitro phosphorylated CENP-F shows that there are five peaks, indicating four phosphorylation sites and the fifth peak is the unphosphorylated protein. Next this size exclusion elution profile shows that the elution volume of the karyopherin alpha peak shifts to a higher mass on adding the wild type CENP-F, which is not observed on adding the CENP-F mutant. This indicates that the phosphomimetic version S3048D of CENP-F does weaken the interaction of the CENP-F cNLS with karyopherin alpha.
Finally SDS-PAGE analysis done shows that the wild type CENP-F fragment with intact cNLS coelutes with karyopherin alpha, indicating strong interaction. However the S3048D mutant of CENP-F and karyopherin alpha elute in separate peaks. While attempting this procedure it's important to remember to check the obtained phosphorylation sites against the CDK1 consensus phosphorylation sequence and to verify that the identified substrate localizes in the same cellular compartment as CDK1.
Following this procedure functional verification should be performed in the cells or animal models in order to confirm that the identified phosphorylation sites have a physiological function. A number of tools are available for functional verification such as specific CDK1 inhibitors and phosphomimetic mutations. Due to the large number of CDK1 substrates in the proteum, many human CDK1 substrates remain to be discovered and the in vitro kinase assay will be an important tool to identify, map and verify these sites.
Identification of these phosphorylation sites enables mechanistic studies of how CDK1 controls the cell cycle. After watching this video you should have a good understanding of how to identify CDK1 specific phosphorylation sites and target proteins by an in vitro kinase assay.
