This Sample Unit contains the full text of a Unit from Current Protocols in Cell Biology, including expert commentary sections with critical information designed to ensure the success of your experiments.

UNIT 14.3

Detection of MAP Kinase Signaling

Transmission of extracellular signals to their intracellular targets is mediated by a network of interacting proteins that relay biochemical messages, thereby controlling many cellular processes. Several related intracellular signaling pathways, collectively known as mitogen-activated protein kinase (MAPK) signaling cascades, have been elucidated in the past decade (Seger and Krebs, 1995). Transmission of signals via these cascades is usually initiated by activation of a small G protein (e.g., Ras) and followed by sequential stimulation of several sets of cytosolic protein kinases. Four distinct MAPK cascades, ERK (extracellular signal-related protein kinase), JNK (c-Jun NH₂-terminal kinase), SPK (stress-related protein kinase), and BMK (big MAPK), have been elucidated to date ( Figure 14.3.1) . Each is named after the subgroup of its MAPK components and is composed of up to five levels: MAP4K (MAPK kinase kinase kinase), MAP3K (MAPK kinase kinase), MAPKK (MAPK kinase), MAPK, and MAPKAPK (MAPK-activated protein kinase) (Figure 14.3.1). One or more components in each of these levels phosphorylates and activates components in the next level, until a downstream component phosphorylates a target regulatory molecule. These cascades can cooperate in transmitting signals from most extracellular stimuli and can thus determine a cell's fate in response to the ever-changing environment. For a detailed description of the MAPK cascades, see Background Information.

Since the majority of MAPK cascade components are kinases, the methods used to detect MAPK cascade activation involve determination of protein kinase activities. Immunoprecipitation of desired protein kinases followed by phosphorylation of specific substrates is a convenient and effective way to determine protein kinase activity (see Basic Protocol 1; Fig. 14.3.2). The activity assay is performed while the enzyme is still bound to the beads for immunoprecipitation, and the amount of incorporated phosphate is monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. An alternative method involves affinity purification with a specific substrate of the examined kinase, followed by phosphorylation; this assay is described for JNK (see Basic Protocol 3; (Fig. 14.3.2). When the identity of the kinase is not known, or there are no reagents available for its determination, an in-gel kinase assay should be used (see Basic Protocol 2; Fig. 14.3.2). The common procedure in which antibodies directed against the active form of MAPK cascade components are used is described in UNIT 14.2.

CAUTION: Investigators should wear gloves for all procedures involving radioactivity and should be careful not to contaminate themselves and their clothing. When working with ³²P, investigators should frequently check themselves and the working area for radioactivity using a hand-held monitor. Any radioactive contamination should be cleaned up using appropriate procedures. Radioactive waste should be placed in appropriately designated areas for disposal. Follow the guidelines provided by your local radiation safety advisor (also see APPENDIX 1D).

BASIC PROTOCOL 1

DETERMINATION OF MAP KINASE (ERK) ACTIVITY BY IMMUNOPRECIPITATION

Materials

Six 6-cm tissue culture plates of EGF-stimulated Rat1 cells (UNIT 14.2, Basic Protocol 1, steps 1 to 4)

PBS (APPENDIX 2A), ice cold

Homogenization buffer (UNIT 14.2), ice cold

Kinase buffer (UNIT 14.2), ice cold

Coomassie protein assay reagent (Pierce)

Protein standards: 5, 10, 20, 50, 100, and 200 µg/ml BSA in homogenization buffer

Protein A-Sepharose beads, swollen and packed (UNIT 14.2, Basic Protocol 2, steps 1 to 2)

Antibody for immunoprecipitation: anti-ERK C-terminal antibody (e.g., Sigma)

0.5 M LiCl solution, ice cold

RadioImmunoPrecipitation Assay (RIPA) buffer (UNIT 14.2), ice cold

3× reaction mixture (RM×3; see recipe)

2 mg/ml myelin basic protein (MBP)

4× SDS-PAGE sample buffer (UNIT 14.2)

15% SDS-polyacrylamide gel (UNIT 6.1)

Prestained protein markers, 16 to 175 kDa (New England Biolabs)

Running buffer: 25 mM Tris·Cl (pH 8.3)/188 mM glycine/0.1% SDS

Staining solution: 40% (v/v) methanol/7% (v/v) acetic acid/25 g/liter Coomassie brilliant blue R-250

Destaining solution: 15% (v/v) isopropanol/7% (v/v) acetic acid in water

X-ray film (not needed if phosphoimager is used)

1.5-ml microcentrifuge tubes, prechilled to 4°C (four sets of six, each labeled 1 to 6)

Stopwatch

1-ml pipet tips, prechilled to 4°C

Microcentrifuge, 4°C

96-well flat-bottomed microtiter plates

Microtiter plate reader, 595 nm wavelength

End-over-end rotator

Shields for radioactive work

Tube heater/shaker (e.g., Eppendorf Thermomixer) or water bath, 30°C

Flat container for washing gel

Whatman 3MM filter paper, cut larger than the gel

Film cassette for either X-ray film or phosphoimager

X-ray film developer or phosphoimager

Additional reagents and equipment for preparation of EGF-stimulated Rat1 cells (UNIT 14.2, Basic Protocol 1), SDS-PAGE (UNIT 6.1), and autoradiography (UNIT 6.3)

Prepare cytosolic extracts

1. Add 350 µl ice-cold homogenization buffer to each plate of EGF-stimulated Rat1 cells on ice (see also UNIT 14.2), tilt the plate gently, and scrape the cells into the buffer using a plastic scraper or rubber policeman. Using prechilled pipet tips, transfer the cells and buffer to labeled, prechilled 1.5-ml microcentrifuge tubes.

 

 

Special consideration should be given to the composition of homogenization buffer. The authors2 recommend using b-glycerophosphate, which serves both as a buffer and as a general phosphatase inhibitor, rather than Tris or HEPES. Sodium orthovanadate is used to inhibit tyrosine phosphatases, and the mixture of pepstatin-A, aprotinin, leupeptin, and benzamidine is used to inhibit proteinases. Ice-cold homogenization buffer blocks most of the phosphatase and proteinase activities in cell extracts.

2. Disrupt the cells by sonication (two 7-sec, 50-W pulses per 0.5-ml sample) on ice.

 

 

Sonication allows extraction of proteins from the cytosolic and nuclear fractions of the cells but not from the membrane fraction; therefore, the extract is called a cytosolic extract. Cellular extraction with nonionic detergents, which extract proteins from the membrane, cytosolic, and some nuclear fractions of the cell, makes determination of the protein concentration somewhat difficult but is often used (see UNIT 14.2). Extraction with RIPA buffer or by freeze-thawing can be used for some kinases, but these methods are less effective (see Critical Parameters).

3. Microcentrifuge the cytosolic extracts 15 min at 14,000 × g, 4°C. Transfer supernatants to fresh, prechilled, and prelabeled tubes. Take 5- to 10-µl aliquots of these extracts for protein determination. Store the remainder of each cytosolic extract on ice until needed.

 

 

Determine the protein concentration of each sample at this stage so that identical amounts of proteins from the different samples can be compared and the relative amount of protein kinases in each sample determined accurately. Comparing samples based on cell number, rather than protein concentration, can result in differences of up to 20% in the amount of protein. Such differences can cause even larger ones when phosphorylation is assessed immunologically.

Do not leave samples on ice >45 to 90 min, to avoid unnecessary exposure to phosphatases and proteinases (see Critical Parameters).

Determine protein concentration

4. Add 10 µl of each cytosolic extract to 190 µl kinase buffer in labeled 1.5-ml microcentrifuge tubes.

 

 

Dilutions of >1:20 are usually necessary to ensure that the sample protein concentrations are in the linear range of the protein determination assay. This dilution is not always necessary with some Coomassie protein assay reagents that have extended ranges.

5. Transfer 10 µl of each of the protein standards into two wells each of a 96-well, flat-bottomed microtiter plate.

 

 

Protein standards should be prepared in the same buffer as was used for the cell extraction.

6. Transfer 10 µl of each of the diluted cytosolic extracts into two wells of the same microtiter plate. Add 200 µl Coomassie protein assay reagent to all wells.
7. Place the microtiter plate in a microtiter plate reader and measure the absorbance of the samples at 595 nm (A₅₉₅). Calculate the protein concentrations of the cytosolic extracts by comparing the absorbance of the sample with the standard curve.
8. Based on the calculated protein concentrations, transfer a volume of each cytosolic extract containing 100 to 500 µg protein into a fresh, prechilled 1.5-ml microcentrifuge tube.

 

 

Good results are often obtained with 50 to 500 µg protein; the authors2 recommend using 300 µg. Since equal amounts of antibodies are used in each of the immunoprecipitation reactions, the protein concentration can vary slightly. To avoid inaccuracy, however, equal amounts of protein should be used in each of the samples to be immunoprecipitated.

Prepare antibody-conjugated protein A-Sepharose beads

9. Place 180 µl swollen, packed protein A-Sepharose beads in a 1.5-ml microcentrifuge tube. Add 320 µl PBS and 25 µl of the antibody to be conjugated, diluted according to the manufacturer's instructions. Rotate the mixture for 1 hr at room temperature on an end-over-end rotator to allow the antibody to bind to the protein A.

 

 

This step can be done at 4°C for 16 hr. Ideally, this step should be performed either before or simultaneously with the preparation of cytosolic extracts so that the immunoprecipitation step can proceed without delay.

Anti-C-terminal antibodies are generally used for the determination of kinase activity, because their binding to the kinase does not interfere with its kinase activity. Usually 1 to 5 µg per reaction is sufficient. Polyclonal antibodies are available from a number of suppliers (e.g., Transduction Laboratories, Sigma, Santa Cruz Biotechnology, Upstate Biotechnology, and Zymed Laboratories).

These volumes are calculated for ten reactions (usually 10 to 20 µl beads is used per reaction), but because of the density of the beads they will probably only be sufficient for eight reactions. The amounts can be scaled up as long as the proportions are maintained.

For easy handling of the resin, cut the ends of the pipet tips to enlarge their openings.

10. Resuspend beads in 1 ml ice-cold PBS and microcentrifuge 1 min at 14,000 × g, 4°C. Remove supernatant and add 1 ml ice-cold homogenization buffer. Repeat centrifugation three more times with homogenization buffer, ending with a final addition of an equal volume of ice-cold homogenization buffer.

 

 

Antibody-conjugated beads can be stored in homogenization buffer for <3 days at 4°C.

Immunoprecipitate kinase

11. Add 30 µl antibody-conjugated bead suspension (15 µl beads and 15 µl homogenization buffer) to a 300-µl sample of cytosolic extract containing 50 to 500 µg total protein in prechilled 1.5-ml microcentrifuge tubes. Rotate on end-over-end rotator for 2 hr at 4°C.

 

 

Conjugating the antibodies to protein A-Sepharose beads prior to adding them to the cytosolic extracts minimizes the time the samples are incubated with the antibodies, minimizing exposure of the kinases to phosphatases and proteinases in the extracts. This procedure also ensures that only antibodies recognized by protein A will be used for the immunoprecipitation. If polyclonal antibodies are added to the cytosolic extracts, antibodies that are not recognized by protein A can bind to the desired antigen but will not be precipitated when protein A-Sepharose beads are added, and will reduce the efficiency of immunoprecipitation.

12. Microcentrifuge protein A-Sepharose bead/cytosolic extract mixture 1 min at 14,000 × g, 4°C. Discard supernatant, add 1 ml ice-cold RIPA buffer, and centrifuge as before. Repeat centrifugation twice with 1 ml ice-cold 0.5 M LiCl and twice with 1 ml ice-cold kinase buffer.

 

 

These stringent washes remove most of the protein kinases that can nonspecifically interact with the protein A-Sepharose beads.

Perform phosphorylation reaction

13. After the last wash, completely remove kinase buffer from the conjugated beads by removing supernatant, microcentrifuging 1 min at 14,000 × g, 4°C, without adding more buffer, and gently removing residual buffer from above beads.

 
14. Resuspend beads in 15 µl water. Prepare the laboratory bench for working with small amounts of radioactivity.

 

 

Determination of enzymatic activity is not always accurate when enzymes are bound to beads. The kinase(s) of interest can be released from the beads at step 14 or 15 by the addition of excess immunizing peptide, allowing the phosphorylation reaction (steps 15 to 17) to be performed without the interference of the beads. The activity can then be measured as described below (steps 18 to 24) or by paper assay. If by paper assay, terminate the phosphorylation reaction by spotting 20 µl of each cytosolic extract on a phosphocellulose paper square (Whatman P81) and washing immediately with 150 mM phosphoric acid. Measure phosphate incorporation using scintillation cocktail and counter.

15. Add 10 µl RM×3 to each tube.

 

 

The most important components of RM×3 are the Mg²⁺ and [g-³²P]ATP, which are essential for the phosphorylation reaction. The use of 100 µM ATP with ~4000 cpm/pmol [g-³²P]ATP provides a good linear range and reproducible results. When the enzymatic activity of the kinases is low, which makes detection of phosphorylation difficult, the concentration of unlabeled ATP should be reduced to 10 to 20 µM and the amount of [g-³²P]ATP increased to 50,000 cpm/pmol. Addition of [g-³²P]ATP alone is not recommended because this will result in a nanomolar concentration of ATP, which is considerably below the K_M for ATP and may lead to nonspecific phosphorylation.

The b-glycerophosphate in the reaction mixture serves as a buffer, but can also inhibit residual phosphatases that may have nonspecifically bound to the beads. BSA serves as a carrier protein; it can be eliminated if purity is required. EGTA chelates Ca²⁺, which may interfere with some kinase activities, DTT keeps the proteins reduced, and sodium orthovanadate inhibits tyrosine phosphatases.

16. Add 5 µl of 2 mg/ml MBP to each tube and place the mixture in a 30°C Thermomixer or water bath.

 

 

MBP is probably not a physiological substrate for any MAPK, but it is a good general substrate for many kinases, including ERKs, in vitro. Substrates should be well phosphorylated by the desired kinases to allow accurate detection of the phosphorylation reaction.

17. Incubate 20 min at 30°C with either constant or frequent shaking.

 

 

If a Thermomixer is not available, a water bath or other heating device can be used.

18. Add 10 µl of 4× SDS-PAGE sample buffer to each tube to stop the phosphorylation reaction.

 
19. Boil for 5 min. Centrifuge for 1 min at 14,000 × g, room temperature.

Electrophorese the phosphorylation reaction products

20. Prepare a 15% separating gel with a 3% stacking gel (UNIT 6.1).

 
21. Load samples and prestained protein markers on the gel.

 

 

Load prestained markers into the first or second lane of the gel so that the first lane can be located on the dried gel and the molecular weights of the detected proteins determined.

22. Run the gel at 150 V, constant voltage, until the bromphenol blue dye is 0.5 cm from the bottom of the gel (~1 hr).
23. Place the gel in a flat container, add 50 ml staining solution, and let stand for 20 min at room temperature. Remove staining solution and add 50 ml destaining solution for 30 min. Repeat destaining three times with 50 ml destaining solution for 30 min each time.

 

 

This extensive destaining removes excess free [g-³²P]ATP which would affect the background radioactivity levels. Extensive destaining is not necessary if the phosphorylation of the desired proteins is very high. Alternatively, the proteins can be transferred to a nitrocellulose membrane (UNIT 14.2, Basic Protocol 1, steps 17 to 23) and then exposed as below.

24. Place the gel on Whatman 3MM filter paper, cover with plastic wrap, and dry the gel in a gel dryer for 1.5 hr at 80°C. Expose the gel in a phosphoimager or on X-ray film (UNIT 6.3).

 

 

Bands should appear at 16 to 21 kDa, which is the molecular weight of the four MBP isoforms.

BASIC PROTOCOL 2

IN-GEL KINASE ASSAY

Materials

Cell lysates from serum-starved, EGF-stimulated Rat1 cells (UNIT 14.2, Basic Protocol 1, steps 1 to 8) or cytosolic extracts (see Basic Protocol 1, steps 1 to 3)

4× SDS-PAGE sample buffer (UNIT 14.2)

2 mg/ml myelin basic protein (MBP)

1.5 M Tris·Cl, pH 8.8

30% acrylamide/0.8% bisacrylamide (UNIT 6.1)

10% ammonium persulfate

Stacking gel (UNIT 6.1)

TEMED

20% isopropanol/50 mM HEPES, pH 7.6

Renaturation buffer: 50 mM HEPES (pH 7.6)/5 mM 2-mercaptoethanol

Renaturation buffer/0.05% Tween 20

6 M urea in kinase buffer (UNIT 14.2)

In-gel kinase buffer: 20 mM HEPES (pH 7.6)/20 mM MgCl₂

In-gel kinase buffer/2 mM DTT/20 µM ATP/100 µCi [g-³²P]ATP

5% trichloroacetic acid (TCA)/1% sodium pyrophosphate (NaPPi)

Water bath, 30°C, with proper shielding for radioactive work

Additional reagents and equipment for SDS-PAGE (UNIT 6.1) and autoradiography (UNIT 6.3)

Procedure

1. Determine protein concentration of cell lysates (UNIT 14.2, Basic Protocol 1, steps 9 to 12) or cytosolic extracts (see Basic Protocol 1, steps 4 to 8). Based on the calculated protein concentrations, transfer a volume of each cell lysate containing 30 to 80 µg protein to a fresh 1.5-ml microcentrifuge tube.

 
2. Add ¼ vol of 4× SDS-PAGE sample buffer to each tube and mix thoroughly. Keep at 4°C.

 

 

IMPORTANT NOTE: Do not boil the samples.

3. Prepare 8 ml of 12% polyacrylamide gel (separating gel) containing MBP by mixing the following:

 

 

0.7 ml H₂O
2.0 ml MBP
2.0 ml of 1.5 M Tris·Cl, pH 8.8
3.2 ml 30% acrylamide/0.8% bisacrylamide
100 µl of 10% ammonium persulfate
6 µl TEMED.

After the separating gel polymerizes, add a 3% stacking gel (UNIT 6.1).

4. Load samples and prestained markers onto gel and run at 100 V until the dye front has reached 0.5 cm from the bottom of the gel (~90 min).

 

 

The gel should not be heated above 30°C; therefore, the voltage used for electrophoresis should be <100 V.

5. Remove the gel from the apparatus, cut off the stacking gel, and carefully place the gel in a flat container. Wash twice with 100 ml of 20% isopropanol/50 mM HEPES, pH 7.6, for 30 min each at room temperature. Repeat washing twice with 100 ml renaturation buffer for 30 min each at room temperature and twice with 100 ml of 6 M urea in kinase buffer for 15 min each at room temperature.

 

 

The second wash with 20% isopropanol/50 mM HEPES, pH 7.6, can be done overnight at 4°C.

6. Place the gel in 4°C cold room, remove 50 ml of washing solution, add 50 ml renaturation buffer/0.05% Tween 20, and shake for 15 min. Repeat twice, removing 50 ml of the washing solution, adding 50 ml renaturation buffer/0.05% Tween 20, and shaking for 15 min each time. Wash the gel three times with 100 ml renaturation buffer/0.05% Tween 20 for 15 min each time. Shake the gel overnight in the cold room.
7. Remove washing buffer and incubate the gel in 30 ml in-gel kinase buffer for 30 min at 30°C. Remove the buffer, add 20 ml in-gel kinase buffer/2 mM DTT/20 µM ATP/100 µCi [g-³²P]ATP, and incubate in 30°C water bath for 2 hr.

 

 

The amount of radioactive material is very high at this stage and the reaction should be performed with proper shielding. Make sure that the gel is straight in the flat container. Unequal distribution of the phosphorylation buffer can interfere with the phosphorylation reaction.

8. Wash the gel carefully four times with 5% TCA/1% NaPPi for 15 min each at room temperature. If the gel is still very radioactive, continue washing overnight.

 
9. Dry the gel (see Basic Protocol 1, step 30) and subject to autoradiography.

 

 

Bands should appear where kinases phosphorylated the MBP copolymerized in the gel (see Figure 14.3.2).

BASIC PROTOCOL 3

JNK ASSAY

Materials

Cytosolic extracts from serum-starved, EGF-stimulated Rat1 cells (see Basic Protocol 1, steps 1 to 3)

10× binding buffer (see recipe)

GST-Jun beads: GST-Jun(1-91) bound to glutathione-conjugated beads (see Support Protocol)

HB1B buffer (see recipe), ice cold

JNK kinase buffer (see recipe), ice cold and room temperature

10× ATP mix: 20 mM unlabeled ATP/2 µCi [g-³²P]ATP, freshly prepared

4× SDS-PAGE sample buffer (UNIT 14.2)

12% SDS-polyacrylamide gel (UNIT 6.1)

1.5-ml microcentrifuge tubes, prechilled

Microcentrifuge, 4°C

Tube heater/shaker (e.g., Eppendorf Thermomixer) or water bath, 30°C

Additional materials and equipment for SDS-PAGE (UNIT 6.1)

Procedure

1. Determine the protein concentration of cytosolic extracts (see Basic Protocol 1, steps 4 to 8).

 
2. Place 150 µl cytosolic extract or cell lysate (50 to 500 µg protein), 30 µl of 10× binding buffer, 100 µl water, and 20 µl GST-Jun beads in prechilled 1.5-ml microcentrifuge tubes.

 

 

The amount of GST-Jun beads can vary according to the amount of the protein conjugated to the beads. Amounts of 2 to 4 µg protein per 20 µl beads usually give good results. The truncated form of Jun (residues 1 to 91) is recommended for these experiments, but similar results can be obtained with the full-length or 1-to-74 constructs.

3. Incubate 1 hr at 4°C with constant shaking. Microcentrifuge 2 min at 14,000 × g, 4°C, and discard the supernatant.

 

 

The JNKs bind to the GST-Jun on the beads during this incubation.

4. Resuspend pelleted beads in 1 ml ice-cold HB1B buffer and microcentrifuge for 2 min at 14,000 × g, 4°C. Discard supernatants and repeat centrifugation twice with 1 ml ice-cold HB1B buffer each time and once with 1 ml ice-cold JNK kinase buffer.

 

 

Remove the supernatant completely after each centrifugation.

5. Add 30 µl JNK kinase buffer at room temperature to the pelleted beads and place the tubes in 30°C Thermomixer or water bath. Add 3 µl of 10× ATP mix. Close the tubes and incubate 20 min with shaking.

 

 

Take proper precautions when working with radioactive material.

6. Add 11 µl of 4× SDS-PAGE sample buffer. Boil the samples 5 min.

 
7. Prepare 12% SDS-polyacrylamide gel with a 3% stacking gel (UNIT 6.1).

 
8. Load samples and prestained protein markers onto the gel. Run the gel at 150 V, constant voltage, until the bromphenol blue reaches 0.5 cm from the bottom of the gel (~1 hr).
9. Stain, dry, and analyze the gel (see Basic Protocol 1, steps 23 and 24).

 

 

A band should be detected at 46 kDa (the molecular weight of the truncated GST-Jun). In many cases, an additional band is observed at 30 kDa, which represents a degradation product of GST-Jun and is a good indication of JNK activity as well.

SUPPORT PROTOCOL

PREPARATION OF GST-JUN-GLUTATHIONE BEADS

Materials

Bacteria transformed with GST-Jun(1-91)-expressing plasmid (plasmid is available from several investigators; alternatively, similar proteins can be obtained from, e.g., Calbiochem)

LB medium with appropriate antibiotics (LB medium; APPENDIX 2A)

100 mM isopropyl-b-D-thioglactopyranoside (IPTG; store in aliquots <1 year at -20°C)

PBS/protease inhibitors (see recipe), ice cold

Washed glutathione beads (see recipe)

PBS/protease inhibitors/20% (v/v) glycerol, ice cold

End-over-end rotator

1. Grow bacteria transformed with GST-Jun-expressing plasmid at 30°C in 4 liters LB medium until optical density at 600 nm (OD₆₀₀) is 0.6 (~3 to 4 hr.
2. Add IPTG to 0.4 nM final and grow bacteria an additional 4 hr at 30°C.
3. Centrifuge 10 min at 6000 × g, 4°C.
4. Resuspend pellet in 80 ml ice-cold PBS/protease inhibitors, sonicate, and centrifuge 10 min at 15,000 × g, 4°C. Transfer supernatant to a fresh tube and repeat centrifugation.
5. Discard pellet, add 3 ml washed glutathione beads to supernatant, and rotate end over end 2 hr at 20 rpm, 4°C.
6. Wash beads with ice-cold PBS three times. Add 8 ml PBS/protease inhibitors/20% glycerol.

 

 

Beads can be stored 1 to 2 days at 4°C or <4 months at -20°C in ice-cold PBS/protease inhibitors/20% glycerol before being used in Basic Protocol 3.

REAGENTS AND SOLUTIONS

Binding buffer, 10×

1.5 M NaCl

220 mM HEPES, pH 7.7

20 mM MgCl₂

0.75% (v/v) Triton X-100

200 mM b-glycerophosphate

1 mM EDTA

1 mM sodium orthovanadate

1 mM phenylmethylsulfonyl fluoride (PMSF; APPENDIX 2A)

10 µg/ml aprotinin

10 µg/ml leupeptin

2 µg/ml pepstatin A

Store 2 months at 4°C

 

 

This buffer is derived from Hibi et al. (1993).

Glutathione beads, washed

Add 50 ml ice-cold PBS to 3 ml glutathione beads, centrifuge 5 min at 2000 × g, 4°C, and discard supernatant. Repeat centrifugation three times, adding 50 ml ice-cold PBS each time.

HB1B buffer

20 mM HEPES, pH 7.7

50 mM NaCl

0.1 mM EDTA

25 mM MgCl₂

0.05% (v/v) Triton X-100

Store 2 months at 4°C

JNK kinase buffer

20 mM HEPES, pH 7.7

20 mM MgCl₂

20 mM b-glycerophosphate

0.1 mM sodium orthovanadate

2 mM dithiothreitol (DTT; APPENDIX 2A), added fresh

Store without DTT for 2 months at 4°C

PBS/protease inhibitors

PBS containing:

10 µg/ml leupeptin (from 10 mg/ml stock solution)

10 µg/ml aprotinin (from 2 mg/ml stock solution)

2 µg/ml pepstatin A (from 10 mg/ml stock solution)

1 mM phenylmethylsulfonyl fluoride (PMSF; APPENDIX 2A)

Prepare fresh

 

 

Leupeptin and aprotinin are prepared in water; pepstatin is prepared in ethanol or methanol. All stock solutions are stored <1 year at -20°C.

Reaction mixture, 3× (RM×3)

75 mM b-glycerophosphate, pH 7.3

30 mM MgCl₂

0.9% (w/v) BSA

3 mM DTT (APPENDIX 2A)

3 mM EGTA

0.3 mM sodium orthovanadate

100 µM [g-³²P]ATP (~4000 cpm/pmol)

Store without [g-³²P]ATP for 1 year at -20°C

COMMENTARY

Background Information

The MAPK cascades

The activation of each of these cascades seems to be initiated by a small GTP-binding protein that transmits the signal to protein kinases, commonly referred to as MAPK kinase kinase kinases (MAP4Ks). Then the signal is transmitted downstream through the cascade by enzymes at the next levels that are referred to as MAP3K (MAPK kinase kinase), MAPKK (MAPK kinase), MAPK, and MAPK-activated protein kinases (MAPKAPKs). It should be noted that in some of the cascades, the signals seem to be transmitted from the small GTP-binding protein directly to the MAP3K level of the cascade. The existence of four to five levels in each MAPK cascade allows for signal amplification, determination of specificity, and tight regulation of the ransmitted signal.

The four distinct MAPK cascades that are currently known are named according to the subgroup of their MAPK components: the ERK (extracellular signal-related protein kinase), JNK (c-Jun NH₂-terminal kinase), SPK (stress-related protein kinase or p38MAPK), and BMK (big MAPK or ERK 5) cascades. These MAPK cascades cooperate to transmit signals to their intracellular targets, thereby initiating cellular processes such as proliferation, differentiation, development, stress response, and apoptosis. In this section, the various MAPK cascades will be briefly reviewed.

The ERK cascade, also known as the p42, p44 MAPK cascade, was the first MAPK cascade elucidated (Seger and Krebs, 1995). This cascade is initiated by the small G protein Ras, which upon stimulation causes membrane translocation and activation of the protein serine/threonine kinase Raf1. Once activated, Raf1 continues transmission of the signal by phosphorylating two regulatory serine residues located in the activation loop of MEK, causing its full activation. Other kinases that can also activate MEK are A-Raf, B-Raf, Mos TPL2, and MEKK2 (Seger and Krebs, 1995; Salmeron et al., 1996; Wang et al., 1996), which all seem to phosphorylate the same regulatory residues of MEK. Activated MEK is a dual-specificity protein kinase that appears to be the only kinase capable of specifically phosphorylating and activating the next kinase in this cascade, which is ERK.

ERK activation requires phosphorylation of two regulatory residues, threonine and tyrosine, that reside in a TEY phosphorylation motif (Payne et al., 1991; Canagarajah et al., 1997). Phosphorylation of threonine and tyrosine residues is essential for the activation of all MAPKs. In the other cascades, however, the identity of the middle amino acid in the TXY motif of the MAPK varies, which probably determines the specificity of the signal.

ERK appears to be an important regulatory molecule. ERK,which by itself can phosphorylate regulatory targets in the cytosol such as phospholipase A₂ (PLA₂; Lin et al., 1993), can translocate into and phosphorylate substrates in the nucleus such as ELK1 (Chen et al., 1992; Marais et al., 1993) and can ransmit the signal to the MAPKAPK level. The main MAPKAPK of the ERK cascade is RSK, which can also translocate to the nucleus upon activation and phosphorylate a set of nuclear substrates different from those phosphorylated by ERK. MNK is another MAPKAPK that is activated also by the SPK cascade (Fukunaga and Hunter, 1997; Waskiewicz et al., 1997). Activation of the ERK cascade was initially implicated in the transmission and control of mitogenic signals; this cascade is now known to be important for differentiation, development, stress response, learning and memory, and morphology determination.

ERKs are activated primarily by mitogenic signals, whereas other MAPK cascades are activated mainly by cellular stress, such as heat shock, ischemia, UV irradiation, and cytokines (Woodgett et al., 1996) and are referred to as stress-activated protein kinase (SAPK) cascades (Kyriakis and Avruch, 1996). The SPK (p38 MAPK) cascade consists of MAPKs that contain a glycine residue in their TXY activation motif (TGY; Han et al., 1994).

Many kinases in the MAPKK, MAP3K, and MAP4K levels have been implicated in the SPK cascade (Figure 14.3.1), but their individual roles are not yet known. GCK1 and HPK1 (Kiefer et al., 1996; Pombo et al., 1995), and probably also PAK1 (Zhang et al., 1995), may belong to the MAP4K level of SPK, although it is not clear whether they are involved in the activation of SAPKs. Twelve distinct kinases have already been implicated in the MAP3K level of this cascade: MEKK1 to 5, MTK1, MLK2, MLK3, ASK1, TPL2, DLK, and TAK1 (Yamaguchi et al., 1995; Blank et al., 1996; Fan et al., 1996; Salmeron et al., 1996; Wang et al., 1996; Deacon and Blank, 1997; Gerwins et al., 1997; Takekawa et al., 1997; Wang et al., 1997). At the MAPKK level, SKK3, SKK6 (MEK6), SKK2 (MKK3), and SKK1 (MKK4, SEK1, JNKK1) seem to play major roles in the activation of all SPKs (Kyriakis and Avruch, 1996).

The MAPK level components of the SPK cascade are p38MAPK (also known as RK, Hog, SAPK2a, and CSBP; Han et al., 1994), SAPK2b, SAPK3, and SAPK4 (Kyriakis et al., 1994; Cuenda et al., 1997; Goedert et al., 1997). Once these SPKs are activated, they either transmit the signal to the MAPKAPK level components MAPKAPK 2 and 3 (Stokoe et al., 1992; McLaughlin et al., 1996) and MNK, or phosphorylate regulatory molecules such as PLA₂ (Kramer et al., 1996) and the transcription factors ATF2, ELK1 (Karin, 1995), CHOP (Wang and Ron, 1996), and MEF2C (Han et al., 1997).

JNKs (also called SAPK1), which comprise a third MAPK subgroup, are also SAPKs. These enzymes are not closely related to the above SPKs, however, mainly because they contain TPY rather than TGY residues in their activation motif. Like the other MAPK cascades, the JNK cascade is triggered by the small GTPases (Crespo et al., 1997) Rac and CDC42. The signals are then transmitted via MAP4K (?) and MAP3K components that are largely shared with the SPK cascades. Since the SPK and JNK cascades are not always simultaneously activated, the signals must be separately regulated to allow separate cascades; the mode of this regulation is unknown as yet.

At the MAPKK level, the JNKs can be activated by at least three dual-specificity enzymes (JNKKs): SEK1 (SKK1, MKK4; Yan et al., 1994), MKK7 (Holland et al., 1997; Tournier et al., 1997), and JNKK2 (which may be an MKK7 isoform; Lu et al., 1997). All three JNKKs seem to be able to activate the components at the MAPK level, JNK1 to 3, SPKs which have molecular weights 46, 54, and 52 kDa, respectively. No enzymes at the MAPK-APK level and no cytosolic targets have been identified for JNKs, but these enzymes appear to be major regulators of nuclear processes, in particular transcription. Shortly after activation, JNKs translocate to the nucleus where they physically associate with and activate their target transcription factors (e.g., c-Jun, ATF, Elk, etc.).

The BMKs comprise another MAPK subgroup (Zhou et al., 1995; Abe et al., 1996) with molecular weights of ~110 kDa. BMK1 (also known as ERK5) and MEK5 are the only known components of this MAPK cascade. Like ERKs, BMKs contain a TEY phosphorylation motif, but they seem to be involved primarily in stress processes, and therefore may belong to the stress-activated subgroup of MAPKs. The C termini of BMKs are unique and appear to be activated by several extracellular stimuli, including osmotic and oxidative stresses.

Kinase cascades other than the MAPK cascades are also activated in response to mitogenic stimulation. These include the NIK-IKK1/2 (DiDonato et al., 1997; Regnier et al., 1997) and PI3K-PDK-AKT-GSK3 (Cohen et al., 1997) Rho-dependent pathways (Leung et al., 1995) and the phosphokinase A (PKA)-phosphorylase kinase pathway (Campbell et al., 1995). Because of their distinct characteristics, these pathways are usually not considered to be genuine MAPK cascades, although they are involved in transmission of many extracellular signals.

All the pathways mentioned are apparently activated to some extent by distinct extracellular agents and, as a result of their action in an elaborate network, determine the outcome of each stimulation. The full dimensions of this network, the mode of regulation of its components, and the mechanisms by which these cascades determine cell fates in response to various stimuli have yet to be fully elucidated.

Analysis of cascade enzymes

Most components of the MAPK cascades belong to the large family of protein kinases, which consists of more than 2000 distinct members (Hunter, 1994). For studying protein kinases in general and MAPK components in particular, specific detection of the activity of the desired protein kinase is essential. The activity of a particular protein kinase can be singled out from a multitude of related activities that might mask its activity in two main ways. One method uses a specific substrate that is recognized only by the d desired protein kinase. This method is good for detecting kinases like MEK, which seems to specifically and selectively phosphorylate its downstream component, ERK. The other, and more common, method is to isolate the protein kinase and then use a general substrate as an indicator of its activity. This method has been used successfully in studies of MAPK cascades.

In one of the first methods used for the systematic detection of protein kinases involved in growth factor signaling, protein kinases were isolated using Mono Q fast protein liquid chromatography (FPLC; Ahn et al., 1990). This method involves stimulating tissue culture cells, fractionating the cytosolic extracts of these cells on a Mono Q column (Pharmacia Biotech), and examining the resulting fractions for protein kinase activity. Since fractionation with Mono Q columns is extremely reproducible, kinases that are activated upon stimulation can be detected by comparing the elution profiles of kinases from activated and unactivated cells. The advantages of this method are (1) the ability to identify novel kinases and measure their activity, (2) the ability to detect the overall activity of many protein kinases, and (3) its good linear range, which allows determination of the ratio between the activities of distinct protein kinases at a given time. The main disadvantage of this method is that separation of various protein kinases is not always complete. In addition, this is a very laborious method and it is difficult to examine more then one sample per day.

Another method that the authors2 have found useful in detecting novel protein kinases is the in-gel kinase assay (see Basic Protocol 2; Kameshita and Fujisawa, 1989). This technique involves copolymerization of a given substrate in an SDS-polyacrylamide gel, electrophoresis of the samples of interest on the copolymerized gel, and in-gel phosphorylation in the presence of [g-³²P]ATP. The advantage of this method is that it reveals the molecular weight of the kinases with the desired specificity, which helps to identify the enzymes of interest. Also, several samples can be examined simultaneously. The main disadvantages are that (1) not all protein kinases can be renatured in the SDS gel, (2) each in-gel assay takes ~3 days, and (3) there is a narrow linear range of protein kinase activities, which can interfere with detection of the increase in induction of protein kinases upon stimulation.

The Mono Q fractionation and in-gel kinase assay methods are mainly used to identify or characterize novel protein kinases. The resolution of these two methods is not always adequate, however, and more specific and convenient methods are recommended for the characterization of a given protein kinase. Such specific methods often require the isolation of the protein kinase of interest, although a specific activator or substrate can sometimes be used (as is the case with PKA or MEK). In studies of MAPKs, the desired protein kinases are often isolated by immunoprecipitation with specific antibodies directed to the C-terminal domain of the kinase or by immunoblot analysis with antibodies to the activated kinase (see UNIT 14.2). Antibodies can also be used to detect slower migration on SDS-polyacrylamide gel electrophoresis (PAGE) that occurs upon phosphorylation of regulatory residues of some MAPKs. This gel shift does not always correlate with enzymatic activity, however, as was shown for ERK and for Raf1. Methods for affinity purification that do not involve antibodies can sometime be used to isolate given protein kinases (see Basic Protocol 3). Although affinity techniques (including immunoprecipitation) are often used, it should be noted that the attachment to a solid support that occurs in this method can interfere with the accurate detection of the kinase activity.

Critical Parameters

Another consideration for successful detection of phosphoproteins is minimization of protein degradation and dephosphorylation. During extraction, most cellular organelles break, exposing phosphoproteins to phosphatases and proteinases (also see UNIT 14.1). To minimize the effects of these enzymes, specific inhibitors of phosphatases and proteinases can be added to the extraction buffers and extraction can be performed at low temperature. Phosphatases are usually efficient enzymes, however, and extractions should be performed as fast as possible even if these precautions are taken.

The success of the immunoprecipitation protocol also depends on the quality and specificity of the antibodies used. The antibodies employed should recognize only the desired protein kinase, not isoforms or irrelevant enzymes. The antibodies should also not interfere with the enzymatic activity of the enzymes tested. In addition, the amount of protein in the different samples and the dilution of antibodies should be optimized to avoid nonspecific recognition of excess proteins. Stringent washing of the immunoprecipitates is necessary to reduce nonspecific precipitation of contaminating kinases. Washing may not completely prevent coimmunoprecipitation of protein kinases other than those desired, however, and these might interfere with the phosphorylation reaction. In this case, it may be necessary to use a specific substrate or direct assaying methods (e.g., in-gel kinase assay).

Other parameters that should be considered for accurate comparison of protein kinases are: (1) starvation of the cells before activation may interfere with activation of the desired protein kinase or may cause activation of some SAPKs; (2) the optimal length of stimulation may vary from cell to cell and from one protein kinase to the other; therefore, appropriate time points for each kinase should be determined; and (3) for accurate comparison of the activities of protein kinases, detection should be performed in the linear range of the phosphorylation reaction. Thus, the amount of protein used for immunoprecipitation, the concentration of antibodies, the length of the phosphorylation reaction, and the exposure to X-ray film or to the phosphoimager should be optimized in order to reach linearity. If necessary, a standard curve with the protein kinases of interest can be made, and serial dilutions of the cytosolic extracts or a time course of the phosphorylation can be used to ensure one is working in a linear range.

Anticipated Results

Time Considerations

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Schematic representation of mitogen-activated protein kinase (MAPK) cascades. The extracellular signal-related protein kinase (ERK) cascade is represented by light gray shading, the c-Jun NH₂-terminal (JNK) cascade by dark gray shading, the stress-related protein kinase (SPK) cascade by white, and the big MAPK (BMK) cascade by stippled shading. Components that are shared by more than one cascade are indicated by combinations of shading. The connections between components from different levels are shown by arrows; the specifics of these interactions have yet to be defined. Abbreviations: MAP4K, MAPK kinase kinase kinase; MAP3K, MAPK kinase kinase; MAPKK, MAPK kinase; MAPKAPK, MAPK-activated protein kinase.

Detection of mitogen-activated protein kinase (MAPK) activity by the methods described in this unit. (A) Detection of extracellular signal-related protein kinase (ERK) activity by immunoprecipitation and myelin basic protein (MBP) phosphorylation. NIH-3T3 cells were grown to subconfluency in 6-cm plates and then starved as described in UNIT 14.2, Basic Protocol 1. Cells were then stimulated with 100 µM sodium orthovanadate and 200 µM hydrogen peroxide (VOOH) for 15 min, or 50 ng/ml epidermal growth factor (EGF) for 5 min, or left untreated (basal). Cytosolic extracts were prepared by sonication and the resulting proteins (300 µg) were incubated with either 30 µl anti-ERK C-terminal antibody (Santa Cruz iotechnology)-conjugated protein A-Sepharose beads (+) or with unconjugated protein A-Sepharose beads (-). The phosphorylation reaction on MBP was performed as described in Basic Protocol 1. (B) Detection of protein kinase activity by the in-gel kinase assay. MCF7 cells overexpressing the ErbB-2 receptor were stimulated with 50 ng/ml EGF for the indicated times. The in-gel kinase assay was p performed as described in Basic Protocol 2. ERK1 and ERK2 bands are indicated. The identity of other bands was not determined. (C) Detection of c-Jun NH2-terminal protein kinase (JNK) activity by an affinity assay. aT3-1 cells were treated with 100 nM D-Trp gonadotropin-releasing hormone for the indicated times. The JNK assay was performed as described in Basic Protocol 3. GST-cJun and a degradation product (p30) are indicated. Abbreviation: GST-Jun, glutathione-S-transferase-conjugated cJun.