| ABSTRACT |
Protein Kinase cascades regulate many aspects of cellular biochemistry and physiology. Determining the direct substrates of protein kinases is important in understanding how these signaling enzymes exert their effects on cellular processes. A recent development in this area takes advantage of the similarity in the ATP-binding domains of protein kinases. Conserved residues in the ATP-binding site contain large side chains that come into close contact with the N6 position of bound ATP. Mutation of one or more of these large residues to alanine or glycine generates a "pocket" in the ATP-binding site that allows the mutant kinase, but not the wild-type kinase or other cellular kinases, to utilize analogs of ATP with bulky substituents synthesized onto the N6 position. Kinase reactions performed with a mutated kinase and radiolabeled ATP analogs in a cellular lysate allow specific labeling of direct substrates of the mutant kinase, which can later be identified by mass spectrometry or other means.
Once pocket mutations have been generated in the kinase, it is necessary to screen ATP analogs for their compatibility with the kinase mutant. This step requires an active form of the wild-type and mutant kinase, either as recombinant activated protein, or as protein that has been immunoprecipitated from transfected, stimulated cells. In addition, a known in vitro substrate for the kinase is also required. Screening the mutant kinases with ATP analogs is best performed in a two-step process. The first step involves assaying the ability of an ATP analog to inhibit the incorporation of radioactive phosphate from normal [y-32P]ATP into a known substrate in an in vitro kinase reaction. This method allows a large number of ATP analogs to be screened easily, without the need for creating numerous radiolabeled ATP analogs, saving time and bypassing the need for unnecessary radioactive work. Moreover, quantitative competition studies can be performed to identify analog-mutant pairs with high affinity.
It is important to keep in mind that there are two reasons an ATP analog can inhibit incorporation of labeled phosphate into substrate. The first is that the ATP analog is a good ATP source for use by the kinase and competes with the [y-32P]ATP as a substrate. The second is that the ATP analog is small enough to interact with the ATP-binding site of the kinase but is unable to be used by the kinase as an ATP source and is therefore simply blocking [y-32P]ATP from the ATP-binding site. To distinguish between these, it is necessary to directly test the ability of the mutant kinase to phosphorylate a substrate with analog ATP. Doing this requires either radiolabeled ATP analogs or, preferably, a phospho-specific antibody to the phosphorylation site on the known substrate. In our experiments, we used a commercially available phospho-specific antibody to the ERK2 substrate Elk1. A phospho-specific antibody allows a large number of ATP analogs to be screened without their having to be made radioactive. In this assay, active wild-type or mutant kinase is mixed with ATP analog and substrate in an in vitro kinase reaction. The reaction is then analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and either stained with Ponceau S and counted by Cerenkov counting (if [y-32P]ATP analog is used) or immunoblotted with a phosphospecific antibody (if available) to determine the extent of substrate phosphorylation. The kinase/ATP analog pair that provides the best phosphorylation of the substrate is then used for future experiments. Only those analogs that cannot be used by the wild-type kinase and other cellular kinases are chosen. |
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| MATERIALS |
| Buffers, Solutions, and Reagents |
- M2 Lysis buffer (cold) [II], freshly prepared
- 50 mM Tris-base (pH 7.4)
- 150 mM NaCl
- 10% glycerol
- 1% Triton X-100
- 0.5 mM EDTA
- 0.5 mM EGTA
- 50 mM NaF
- 40 mM β-glycerophosphate
- 5 mM tetrasodium pyrophosphate
- 0.1 mM sodium vanadate
- 10 µg/ml aprotinin
- 5 µg/ml leupeptin
- 2 mM phenylmethylsulfonyl fluoride (PMSF), fresh
- Acetic acid (1%)
- 10x Kinase buffer
- 250 mM HEPES (pH 7.4)
- 100 mM magnesium acetate
- 10 mM dithiothreitol (DTT)
- Kinase reaction buffer A (for the analog inhibition assay)
- 1x kinase buffer
- 10 µM ATP
- 100 µM ATP analog
- 10 µCi/reaction [y-32P]ATP
- 1-5 µg of a known substrate/reaction
- Kinase reaction buffer B (for the substrate phosphorylation assay)
- 1x kinase buffer
- 100 µM ATP analog
- 1-5 µg of a known substrate/reaction
- 10 µCi/reaction [y-32P]ATP analog (required only if a phospho-specific antibody to the substrate is not available)
- 1x PBS (cold)[I]
- 25.6 g of Na2HPO4 ˙7H2O
- 80 g of NaCl
- 2 g of KCl
- 2 g of KH2PO4
- Bring to 1 liter with distilled, deionized water; autoclave for 40 min at 121°C.
- Ponceau S solution
- 0.5% (w/v) in 1% acetic acid
- 2x Laemmli sample buffer
- 100 mM Tris (pH 6.8)
- 2% SDS
- 20% glycerol
- 4% β-mercaptoethanol (added fresh)
- Dulbecco's modified Eagle medium (DMEM)
- Fetal bovine serum (FBS)
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| Cells and Antibodies |
- COS-1 African green monkey kidney epithelial cells (ATCC)
- Antibodies to the epitope tag on the protein kinase of interest
- A phospho-specific antibody to a known substrate of your kinase (if available)
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| Gels |
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| Special Equipment |
- Microcentrifuge with cooling facility
- Transfer tank for western blotting
- 30°C water bath
- Scintillation counter
- Heating block, preset to 100°C (I)
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| Additional Reagents |
- [y-32P]ATP (6000 Ci/mmole)
- Agonist (I); e.g., epidermal growth factor, serum, platelet-derived growth factor Lipofectamine
- M2 agarose (for FLAG-tagged protein kinases)
- Protein A- or protein G-conjugated agarose or Sepharose CL-6 beads (for preparation of antibody, see step 7)
- BCA protein assay kit
- Hypodermic needle 1", 27 gauge
- Syringe, 1 cc
- Nitrocellulose membrane
- Vacuum flask
- Shaker, preset to 4°C
- Incubator, preset to 37°C [I]
- 60-mm tissue culture dishes [I]
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| Plasmids |
- Expression plasmid of choice
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METHOD
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Cellular Transfection and Immunoprecipitation
Before proceeding with the experiments outlined below, all kinase pocket mutants should be characterized for their expression, localization, specific activity, and substrate specificity. The results should be compared with those obtained from the wild-type kinase to ensure that the mutation does not significantly alter these characteristics.
- Grow COS-1 cells in DMEM supplemented with 10% FBS. Plate cells the day before transfection at 4 x 105 cells per dish in 60-mm tissue culture dishes. Incubate at 37°C and 5% CO2 overnight.
- Transfect cells with 2 µg of expression plasmid using Lipofectamine, according to the manufacturer's specifications.
We have found that a 1:4 ratio of DNA to Lipofectamine is optimal for transfection of COS-1 cells.
- Incubate the cells for 24-72 hours at 37°C and 5% CO2. The rest of step 3 and step 4 are optional and can be varied depending on the kinase of interest. For mitogen-activated kinases, serum-starve transfected cells by washing twice with room-temperature phosphate-buffered saline (PBS) and placing in serum-free media for 4-12 hr prior to harvest.
- Stimulate the cells for 10 min before harvest with an agonist that activates the kinase of interest. Harvest the cells at 24-72 hr posttransfection.
- Wash the cells twice in cold PBS on ice. Drain the dishes well. Add 0.5 ml of M2 lysis buffer and scrape each dish of cells into microcentrifuge tubes. Vortex. Let the cells lyse on ice for 15 min, vortexing occasionally. Centrifuge at 15,000g and 4°C in a microcentrifuge for 15 min. Transfer the supernatant to a fresh tube.
- Perform a protein assay on the cell lysates using a BCA assay kit from Pierce. We typically obtain 0.5 mg of protein per 6-cm dish.
- Prepare the antibody for the immunoprecipitation (Harlow and Lane 1999). We use M2 agarose for FLAG-tagged ERKs. If the antibody to the epitope tag on the protein is not preconjugated, prebind the primary antibody for your epitope tag to protein A- or protein G-conjugated agarose or Sepharose CL-6 beads. If necessary, use a secondary antibody to link the primary antibody to the beads (Harlow and Lane 1999). The amount of antibody to use will be antibody- and protein-specific. For the small-scale immunoprecipitations described in step 8, 0.1 µg of antibody per immunoprecipitation should be sufficient. Batch prebinding can be performed with enough antibody and conjugated beads for all of your reactions. Prebinding should be performed in 1 ml of M2 cell lysis buffer for 1 hr at 4°C with constant agitation. Additional unconjugated Sepharose CL-6 beads can be included in the prebinding to increase the bead volume for each kinase reaction. We find that a total of 30 µl of bead volume is optimal for each reaction. After prebinding, centrifuge the antibody mixture at 15,000g in a microfuge for 30 sec. Aspirate the supernatant and wash the beads twice in M2 lysis buffer to remove unbound antibody. Aspirate the wash buffer. Resuspend the beads in an equal volume of M2 lysis buffer to make a 1:1 bead:buffer slurry.
- Aliquot 50 µg of cellular protein (as determined in step 6) into fresh microcentrifuge tubes, using one tube for each reaction (use more or less protein, depending on the abundance and activity of your kinase). We typically perform kinase reactions in duplicate to control for variability in the assay. Add M2 lysis buffer to a total volume of 750 µl.
- Aliquot the preconjugated antibody (60 µl of 1:1 bead:buffer slurry from step 7) into each of the tubes containing cell lysate. Incubate at 4°C for 2 hr with gentle rocking.
- Centrifuge the samples at 15,000g at 4°C for 30 sec. Aspirate the supernatant. Wash the beads three times with 1 ml of cold M2 lysis buffer, followed by two washes with 1x kinase buffer. Aspirate the final wash buffer down to the beads. Keep the samples on ice.
- Just before starting the kinase reaction, aspirate the residual kinase buffer with a 27 gauge needle attached to a 1-cc syringe and a vacuum flask. Keep the beveled side of the needle facing the wall of the tube to prevent aspirating any beads. Use this immunoprecipitated protein for the analog inhibition assay and the substrate phosphorylation assay.
- Analog inhibition assay and substrate phosphorylation assay: 2x 50- µg aliquots of cellular protein are required for the analog inhibition assay and a further 2x 50 µg are required for substrate phosphorylation.
- Add 40 µl of kinase reaction mixture to each tube on ice. (Note: Kinase reaction mixture A is for analog inhibition assays. Kinase reaction mixture B is for the substrate phosphorylation assay.) Mix gently by flicking the tube with your finger.
- Incubate samples in a 30°C water bath for 10 min. Place the samples on ice and immediately add 40 µl of 2x Laemmli sample buffer to stop the reaction. Vortex the samples.
- Heat the samples for 4 min in a boiling water bath or heating block. Centrifuge the samples at 15,000g at room temperature for 30 sec.
- Load the supernatant on an SDS-polyacrylamide gel and electrophorese.
- Transfer the gel to nitrocellulose membrane.
- Place the membrane in a glass dish. Stain the membrane for 5 min with Ponceau S solution. Remove the stain and destain the membrane twice, for 5 min each, with 1% acetic acid.
- If [y-32P]ATP or [y-32P]ATP analog is used, cut the substrate bands out of the gel and count them individually by dry Cerenkov counting on a scintillation counter. If a phospho-specific antibody to the substrate is available, leave the blot intact and immunoblot with the antibody (Harlow and Lane 1999).
- For the analog inhibition assay, calculate the percentage of labeled phosphorylation from each reaction. The cpm from duplicate assays should be averaged, and the value obtained from the kinase reactions that did not contain ATP analog should be set to 100%. Then compare the average counts from the other kinase reactions to those of the control reaction and express the ratio as a percentage.
- For the substrate phosphorylation assay, use the mutant kinase/ATP analog pair that gives the highest phosphorylation, either through immunoblotting or radioactive measurements, for future studies.
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REFERENCES
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| Harlow E. H. and Lane D.L. 1999. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. |