| ABSTRACT |
| Ideally, one would like to be able to directly phosphorylate substrates in an intact cell. This could potentially be performed by introducing ATP analog into live or permeabilized cells. However, cells are impermeable to ATP, and the addition of labeled ATP analog to digitonin-permeabilized cells results in the hydrolysis of the analog ATP within 1 min (Chaudhary et al. 2002). Therefore, we have chosen to apply this technique to cell lysates or fractions. Here we describe two approaches to detect direct substrates of a protein kinase. The first approach involves identification of kinase-associated substrates by immunoprecipitating a tagged form of the mutant kinase from transfected COS-1 cells and performing a kinase reaction by the addition of [γ-32P]ATP analog. The second approach involves the addition of recombinant mutant kinase and [γ-32P]ATP analog to cell lysates. We have used these techniques for the phosphorylation of ERK2 substrates; however, this methodology can be applied to other protein kinases. |
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| MATERIALS (for Approaches I and II, as Indicated) |
| Buffers, Solutions, and Reagents |
Recombinant kinase (II) [γ-32P]ATP analog (I+II) Sepharose CL-6B beads (I) Lipofectamine (I) Agonist (I), e.g., epidermal growth factor, serum, platelet-derived growth factor FLAG M2 agarose beads (I) SDS, 20% (I) M2 lysis buffer (II) (see Protocol 1) PBS, room temperature (I) and ice cold (I+II) (see Protocol 1) Serum-free medium (I, e.g., Dulbecco's modified Eagle medium) FLAG peptide (5 mg/ml in hypotonic lysis buffer) (I) Kinase buffer containing benzamidine, 10 mm and 100 mm NaCl (II) 2x Laemmli sample buffer (I) Hypotonic lysis buffer (I) 20 mm HEPES (pH 7.4) 2 mm EGTA 2 mm MgCl2
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| Cells |
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| Plasmids |
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| Special Equipment |
Tissue culture dishes (I+II) Hypodermic needle, 1 1/4'', 27 gauge (I) Miniprep Columns (I) Dialysis cassette, 10,000 molecular-weight cutoff (II) SDS-polyacrylamide gel (I) Rotator, set up in a cold room (4°C) (I) Boiling water bath, preset to 100°C (I) Incubator, preset to 37°C, 5% CO2 (I+II) Incubator, preset to 30°C (I+II) Microfuge cooled to 4°C (I+II)
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METHOD
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Approach I: Phosphorylation of Kinase-associated Substrates
It is important to standardize the procedure in small-scale reactions prior to scaling-up the procedure to identify novel substrates.
Plate 1 x 106 COS-1 cells onto 100-mm tissue culture dishes 1 day prior to transfection. Incubate the cells at 37°C in 5% CO2. Transfect the cells with 6 μg of plasmid DNA carrying epitope-tagged versions of the wild-type or mutant forms of the kinase of interest, and 24 μl of Lipofectamine, according to the manufacturer's instructions.
24-72 hr posttransfection, wash the cells twice with room-temperature PBS and serum-starve them in serum-free medium for 4-12 hr. Then stimulate the cells with agonist for 5-10 min.
Depending on which kinase is being studied, different starvation times and stimulants can be used.
Wash the cells twice with cold PBS on ice. Add hypotonic lysis buffer (0.5 ml/100-mm dish).
Hypotonic lysis buffer is utilized to preserve ERK2 interactions with associated proteins. For identifying interacting substrates that form stable associations, M2 lysis buffer (see Protocol 1) or another buffer with increasing concentrations of detergent or salts can be used (Harlow and Lane 1999).
Scrape the cells together and transfer them to a 1.5-ml microcentrifuge tube. Do not vortex. Lyse the cells by centrifuging at 15,000g in a microcentrifuge for 20 min at 4°C.
Transfer the supernatants (~1 mg of protein) into fresh tubes. Keep small aliquots aside (20 μg) to check the expression levels of the expressed proteins.
Wash the appropriate quantity of Sepharose CL-6B beads (30 μl/sample) with hypotonic buffer and mix with FLAG-M2 agarose beads (10 μl/sample), or beads conjugated to another antibody to the chosen epitope tag (~1 μg of antibody per 1 mg of protein lysate).
Wash the mixture of beads twice in hypotonic lysis buffer and then add them to the lysates prepared in step 5 (40 μl per sample) for immunoprecipitation. Incubate the beads in lysate for 1-2 hr at 4°C with constant rotation.
Wash the immunoprecipitates three times with hypotonic lysis buffer (1 ml per wash for each sample). After the final wash, aspirate the few remaining drops of buffer using a 1 1/4 inch, 27 gauge needle.
Perform kinase reactions in a total volume of 40 ml containing kinase buffer and 10 μCi [γ-32P]ATP analog for 3-15 min at 30°C.
We find that shorter kinase reaction times are preferable, but the time of incubation should be optimized for each kinase and cell type. We use pilot reactions with labeled analog to identify conditions for which the reaction can be scaled up. Scale-up reactions are performed with unlabeled ATP analog and tracer amounts of labeled ATP analog on the immunoprecipitate. Eventually, reactions should be scaled up to give silver or Coomassie blue stainable amounts of the protein of interest to allow identification by mass spectrometry.
Pool labeled and unlabeled (when scaling up) kinase reactions and add 80 μl of FLAG peptide (5 mg/ml in hypotonic lysis buffer). Elute FLAG ERK2-QG and its associated proteins by incubating on ice for 15 min followed by incubation at 30°C for 15 min, with occasional vortexing.
Resolve the samples in 1x Laemmli sample buffer on a 1-dimensional SDS-polyacrylamide gel.
To perform isoelectric focusing prior to SDS-polyacrylamide electrophoresis, add 20% SDS to the samples to a final concentration of 2% and boil the samples for 4 min. Remove Sepharose CL-6B and M2 agarose beads by loading the reaction into a miniprep column placed in a microcentrifuge tube and centrifuging for 30 sec at 15,000g.
Approach II: Phosphorylation of Substrates in Cell Lysates with Exogenous Kinase
This approach uses recombinant protein kinase to phosphorylate substrates in a cell lysate
Plate cells and incubate at 37°C and 5% CO2 until the cells are 80% confluent. Wash the cells twice on ice with cold PBS. Drain the dishes well. Add 0.5 ml of fresh M2 lysis buffer per 100-mm dish and scrape the cells into microcentrifuge tubes on ice. Keep the samples on ice for 15 min, vortexing occasionally.
Centrifuge the samples at 15,000g in a microcentrifuge at 4°C for 15 min. Transfer the supernatant to a fresh tube on ice.
Pool the lysates and dialyze them against kinase buffer (without ATP or DTT) containing 10 mm benzamidine and 100 mm NaCl. Dialyze twice for 2 hr each in 1 liter of buffer using a 10,000 MWCO Dialysis Cassette.
This will get rid of cellular ATP. Perform a protein assay on the lysate. Dialyzed lysates can be stored at -70°C until use
Add purified, active recombinant mutant kinase to 100 μg of cell lysate.
We use 10-100 ng of activated ERK2, but this must be determined empirically according to the specific activity of the kinase of interest.
Add 10 μCi of radiolabeled ATP analog. Mix and incubate at 30°C for 3-15 min.
We find that short reaction times (3 min) are often preferable. This is most likely due to depletion of the analog over time and the ability of phosphatases in the reaction to dephosphorylate substrates.
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REFERENCES
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Chaudhary A., Brugge J.S., and Cooper J.A. 2002. Direct phosphorylation of focal adhesion kinase by c-src: Evidence using a modified nucleotide pocket kinase and ATP analog. Biochem. Biophys. Res. Commun. 294: 293-300.
Harlow E. H. and Lane D.L. 1999. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. |
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Anyone using the procedures in this protocol does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in this protocol and has no liability in connection with the use of these materials. Materials used in this protocol may be considered hazardous and should be used with caution. For a full listing of cautions regarding these material, please consult:
Protein: Protein Interactions, Second Edition: A Molecular Cloning Manual, edited by Erica A. Golemis and Peter D. Adams, ? 2005 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. 457-461. |
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