Prepared by Swathi Arur
Protein Kinase (stock solutions of 1-10 mg/ml pure kinases) – for these assays, I use purified ERK2 kinase (NEB). For each enzyme, it is important to determine the optimal buffer, ionic strength, and pH for activity. If these conditions have not been established, the protocol listed below can be used as a starting point.
Substrate (stock solution of 10 mM) – Substrates typically contain one Serine /Threonine in a phosphorylation site motif. As a control I use the Myelin Basic Protein (obtained from Sigma), which was used by NEB to caliberate the ERK2 kinase.
In addition, the substrates should have a net positive charge to facilitate binding to phosphocellulose filters used in the assay. For quantitative binding to the phosphocellulose paper, it is recommended to have at least 2 basic residues and a free amino terminus. If a phosphorylation site motif is not known, a general tyrosine kinase substrate can be used. For example, “Myelin Basic Protein” (is a non-specific substrate for many MAPKs). For initial reactions, a substrate concentration of 0.7-1.5 mM should be used. To determine the kinetic parameters for phosphorylation of the synthetic peptide, a range of peptide concentrations is required
10X Kinase Buffer – contains 5 mg/mL BSA (to prevent kinase adsorption to the assay tube), 150 mM Tris-Cl (pH 7.5).
ATP/MgCl2 (purchased from Upstate Biotech- Magnesium/ATP Cocktail # 20-113) – a stock solution of 1-5 mM is convenient. Note that most MAPKs have Km values for ATP in the range 10-150 uM, so for kinetic experiments it is important to use saturating concentrations of ATP to arrive at values of Km and Vmax for the peptides.
[gamma-32P]ATP – 10 mCi/mL.
ERK2 Kinase Assays
a) AUTORADIOGRAPHY ASSAY:
- A standard kinase assay is carried out in a volume of 25 ul:
- 2.5 ul of 10X kinase buffer
- 5 ul of 1.0 mM MgCl2 /ATP (0.2 mM final concentration)
- [gamma-32P]ATP (100-500 cpm/pmol)
- 3 ul of 10 mM substrate (1.2 mM final concentration)
- 1 U of the ERK2 kinase
- H2O to 25 ul
1. Before the experiments, prepare a cocktail containing enough buffer, ATP, and [gamma-32P]ATP to complete the assays. For assays at different substrate concentrations, the substrate should be diluted and added separately to each tube. After dispensing the cocktail into 1.5 ml microcentrifuge tubes, place the tubes in a water bath at 30 degrees C for 30 minutes. Reactions should be initiated by the addition of kinase and allowed to proceed at 30 degrees C.
2. After the desired time, terminate the reactions by adding 2 X SDS sample buffer and run out on a gel.
3. Dry the gel on a Whatman 3.0 paper, and expose the dry gel to an autoradiogram and keep the cassette at -70Cfor 2 hrs. (for longer exposures make sure the film is dry again before putting a new autoradiogram onto it).
b) KINETIC ANALYSIS:
1. Conduct the kinase assay as above, this time use different concentrations of the substrates starting from 50nM to 1mM. Stop the reaction after 30 minutes by adding 45 ul ice-cold 10% trichloroacetic acid (TCA) to each reaction. Vortex the reactions.
3. Spin for 2 minutes in microcentrifuge (10K rpm).
4. Spot 35 ul of the supernatants onto 2.1-cm diameter Whatman P81 cellulose phosphate filter circles.
6. Wash the P81 filter circles three times with 500 ml cold 0.5% phosphoric acid (5-10 minutes per wash). The progress of the washing steps can be followed by removing the P81 filter circle for a blank reaction and checking it with a Geiger counter.
7. Wash once with 200 ml acetone at room temperature for 5 minutes.
8. Allow the filter circles to dry at room temperature.
9. Put filter circles into scintillation vials and measure 32P incorporation by counting the pads dry in a scintillation counter. The specific activity of ATP in a kinase reaction (e.g., in cpm/pmol) can be determined by spotting a small sample (2-5 ul) of the reaction onto a P81 filter circle and counting directly (no washing). Counts per minute obtained in the kinase reaction (minus blank) are then divided by the specific activity to determine the moles of phosphate transferred in the reaction.
Kinetics of Substrate Phosphorylation
The kinetic parameters for phosphorylation of a substrate by a MAPK can be determined using a variation on the protocol above.
1. Carry out a reaction at a high concentration of substrate to establish that the protein is a substrate.
2. Vary the enzyme concentration in the assay. The rate of substrate phosphorylation should be proportional to the enzyme concentration under the conditions of the assay. This experiment is also used to determine the amount of enzyme needed for the kinetic studies.
To determine rates, a time course of substrate phosphorylation should be carried out. In this case, prepare a larger enzyme reaction (we use 150 ul). At the desired time points, withdraw 25 ul aliquots and transfer them to microcentrifuge tubes containing 45 ul of ice-cold 10% TCA, and analyze the reactions as described above. Phosphorylation of the substrate should be linear with time, and for measurement of kinetic constants the initial rates of reaction (5%) should be used.
3. Vary the substrate concentration in the assay. Use a plot of velocity vs. peptide concentration to get an initial estimate of the value of Km. A wide range of substrate concentrations (e.g., 20 uM to 2 mM) should be used in this initial measurement.
4. To determine Km (substrate) and Vmax , vary the substrate concentration and measure the rate of phosphate transfer. A good range of substrate concentrations are the following multiples of Km: 0.125 x Km, 0.25 x Km, 0.5 x Km, 1.0 x Km, 2.0 x Km, 4.0 x Km, 8.0 x Km. The reactions should be carried out in triplicate for best results.
5. Kinetic constants are determined by weighted non-linear least-squares fit to the hyperbolic velocity vs. [substrate] plots using iterative programs such as NFIT (Island Products, Galveston, TX).
CALCULATION of RAR:
RAR: Relative Acceptor Ratio = Vmax / Km defined as an overall measure of the ability of a protein to function as a substrate.
I normalize the RAR of a given substrate with Myelin Basic Protein, i.e. calculate the value of RAR for each substrate and then divide it by the value of RAR of Myelin Basic Protein (thus setting MBP at 1.0).