Methods And Materials Report Example

Immunoprecipitation of Activated MAP Kinase

Abstract
MAP Kinases are protein kinases which have many roles in different biological processes. MAP Kinase ERK1/2 can be phosphorylated by another protein kinase at two amino acid residues and is involved in phosphorylating other target proteins. The following laboratory experiment examines the phosphorylation of MAP Kinase ERK1/2 from rat muscle cells under stimulating (in the presence of insulin) and inhibitory (in the presence of PD98059) conditions. Phosphorylated forms of MAP Kinase were detected by immunoprecipitation using an antibody that is specific for phosphotyrosine, followed by immunoblot analysis. The results of this experiment are discussed.

Introduction

MAP Kinases, also known as Mitogen-activated protein kinases, are protein kinases found in eukaryotes that are involved in the cellular response to different external stimuli and for regulating a variety of cell functions. MAP Kinase ERKs, or extracellular signal-regulated kinases, are involved in a signaling cascade that plays a role in several cellular processes including cell cycle progression, proliferation and differentiation. MAP Kinase ERK is in turn activated by kinases that are located upstream on the signaling pathway [1, 2]. The phosphorylation of MAP Kinase ERK via its tyrosine or threonine residue results in its translocation to the nucleus where it phosphorylates a number of nuclear targets. Insulin can act as a stimulating hormone in this cascade by enabling the insulin receptor to become phosphorylated, which in turn phosphorylates other proteins downstream on the pathway, including MAP Kinase ERK 1/2. Inhibitors such as PD98059 act to block this stimulation by directly binding to the phosphotyrosine of MAP Kinase and preventing it from becoming phosphorylated.
Immunoprecipitation is a technique used to precipitate a particular antigen such as a protein out of a solution. Antibodies that are bound to a solid such as a bead are used to bind to the target protein and pull it out of solution. This is a way to purify a specific protein away from many others and concentrate it. Antibodies that are bound to the antigen can then be caught by a second protein such as protein A, or by another secondary antibody [3, 4].

In the following experiment, lab rat skeletal muscle cells treated with combinations of the

stimulating hormone (insulin) and the inhibitor (PD98059) are examined for MAP kinase (ERK1/2) activity. MAP kinase ERK1/2 can be activated by the dual phosphorylation of amino acid residues Tyrosine and Threonine by an upstream kinase and thus its activity can be measured by determining the degree of phosphorylation. Anti-phosphotyrosine antibodies were used to immunoprecipitate the MAP Kinase. The immuoprecipitates were examined further by immunoblot analysis. Using this technique, the effects of insulin and inhibitor on MAP Kinase ERK1/2 phosphorylation could be determined. It was expected that stronger bands corresponding to MAP Kinase ERK1/2 should be detected on the immunoblot since insulin stimulates MAP Kinase phosphorylation, and weaker bands would be detected in the presence of the inhibitor.

The experiments were carried out according to the instructions in the lab manual [5]. On Day 1, rat muscle cells will be made quiescent by serum starving for 5 hours prior to the beginning of the experiment. Media was aspirated away and replaced with 1.5 mL of starvation medium per well. Insulin was diluted to 10 -5M and added to the cell culture medium at a 1:100 dilution to a final concentration of 100 mM. The inhibitor PD98059 was provided at a concentration of 2.5 mM and diluted to 1:100 a final concentration of 25 μM. Buffer B was prepared by adding 5 µL of 1M Na2VO4 and protease inhibitor cocktail to 5 mL Buffer A (50 mM HEPES, pH 7.6, 150 mM NaCl, 10% glycerol (vol/vol), 1% Triton X-100 (vol/vol), 30 mM sodium pyrophosphate, 10 mM NaF and 1 mM EDTA). This will result in a final concentration of 1 mM Na2VO4, 10 μM E-64, 1 μM pepstatin A, 1 μM leupeptin and 0.2 mM PMSF. A plate of L6 muscle cells was used and each well was labeled as follows: Well #1 Control, Well #2 Insulin, Well #3 PD98059, and Well #4 PD98059 + Insulin. The inhibitor PD98059 was added to Wells 3 and 4 and an equal volume of Buffer B was added to Wells 1 and 2. The wells were incubated for 30 minutes in an incubator at 37 oC. Insulin was added to wells 2 and 4, and an equal volume of Buffer B was added to the other two wells. All wells were incubated for 10 minutes at 37 oC. After stimulation with insulin, the medium was aspirated off and the cells were gently washed with 1 X PBS. PBS was aspirated and 0.75 mL Buffer B was added. The cells were scraped off the wells with a blue pipette tip and transferred into pre-labeled eppendorf tubes. 1 µg goat anti-phosphotyrosine antibody was added to each tube. Tubes were capped, sealed with parafilm and incubated with rotation overnight at 4 oC.
The following day, a 25 µL slurry of protein A-sepharose beads was added to each sample. Samples were capped, sealed with parafilm and incubated with rotation for 1 hour. Samples were washed by centrifugation for 2 minutes to collect the beads, supernatant was withdrawn and discarded and 0.75 ml PBS containing 1 mM Na2VO4 and protease inhibitor cocktail was added to resuspend each bead pellet. This washing step was repeated, then 35 µL of 2X Laemmli sample buffer was added to each pellet and the samples were boiled for 5 minutes [6]. Samples were stored at -20 oC overnight. The next day, a 12% SDA-polyacrylamide gel was prepared with a stacking gel and samples were thawed and centrifuged for 2 minutes. Twenty-five µL of the supernatant of each sample was loaded onto the gel. A molecular weight marker (Blue Prestained Protein Standard, Broad Range (11-190 kDa)New England Biolabs) was included as a standard [7]. The gel was run at 200V until the dye front came off the bottom of the gel; proteins were then transferred to a PVDF membrane by electrotransfer at 100V for 90 minutes. The PVDF membrane was incubated in blocking buffer C containing 5% non-fat milk for 1 hour at room temperature with gentle rocking, then incubated overnight at 4 oC with primary antibody to ERK (p42/44 MAPK) at a dilution of 1:1000 in the same blocking buffer.
The following day, the membrane was washed four times for 10 minutes each in Buffer C with rocking at room temperature, then secondary antibody was added to the membrane for 1 hour at room temperature. The membrane was washed in buffer C another four times, then rinsed with distilled water. Signal was detected by mixing equal volumes from each container of ECL solution and coating the membrane in this solution for 1 minute. The membrane was placed in a page protector and taken to a dark room where the membrane was exposed to film and developed.

Results

The polyacrylamide gel was stained with Coomassie blue after electrotransfer in order to determine whether any protein remained and had not been transferred onto the PVDF membrane. While no higher molecular weight protein bands could be detected on the majority of the gel, weak lower molecular weight protein bands could be observed (below 20 kDa), indicating that the transfer was not complete (data not shown). It is possible that the transfer required a higher voltage or a longer time period, or perhaps the samples were overloaded and there was too much lower molecular weight protein to transfer onto the membrane. The molecular weight marker was used to determine the size of the bands on the Coomassie stained gel as well as on the membrane. The results of the immunoprecipitation are depicted in Figure 1, and the molecular weight markers are illustrated on the left hand side of the figure. The arrow points to the band that represents MAP Kinase ERK1/2.
Figure 1. Immunoblot of MAP Kinase ERK1/2 Expression in Rat Muscle Cells.
Samples from each well was loaded onto an SDS-PAGE gel, run at 200V until the dye front came off the bottom, then electrotransferred onto PVDF membrane at 100V for 90 minutes. Primary antibodies specific to p42/44 MAPK and secondary anti-rat antibodies conjugated to ECL were used to detect signal by chemiluminescence. Molecular weight marker is shown on the left. Band representing MAP Kinase is indicated by an arrow.

Discussion

Insulin plays a key role in activating a variety of cellular pathways. It does so by binding to inulin receptor substrate 1 (IRS1). Phosphorylation of a tyrosine residue in the insulin receptor enables IRS-1 to bind to these receptors through its phosphotyrosine binding domains. As a result, tyrosine residues located on IRS-1 become phosphorylated by insulin receptors, and IRS-1 is then able to activate several signaling pathways, including the MAP kinase ERK1/2 pathway [8, 9]. This leads to signaling cascades that result in proliferation, differentiation and cell cycle progression.
In this lab experiment, protein from rat muscle cells was immunoprecipitated with anti phosphotyrosine antibodies that were conjugated to beads. This enabled all phosphorylated forms of MAP kinase ERK1/2 to be targeted. The MAPK antibody complexes were brought out of solution through the addition of protein A sepharose beads. Protein A binds to immunoglobulins, and the Sepharose beads enable the complex to be precipitated [4]. The samples were then separated according to molecular weight on a polyacrylamide gel and then electrotransferred onto a membrane, where the primary MAP Kinase antibodies were applied. Positive signal was detected using the secondary goat anti-rabbit HRP antibody, which could then be detected using the ECL chemiluminescence system.
The bands detected on the immunoblot were compared with a molecular weight standard, as illustrated in Figure 1. A band with a molecular weight of approximately 42 kDa was observed in the control lane A as well as in the other lanes. One larger band of approximately 100 kDa in size, as well as smaller bands of 25 kDa or less were also observed on the immunoblot. It is possible that these extra bands are artifacts, and represent cross -reactivity between the antibodies used in this experiment and other proteins found in rat muscle cells. It is also possible that the large band represents dimer aggregates of MAP Kinase (since it is approximately twice its size) and the smaller bands represent degradation products of the Kinase. The fact that all of these bands are fainter in the sample that had been incubated with inhibitor alone would suggest the latter, rather than the former.
The anti-phosphotyrosine immunoprecipitates detected on the gel illustrate the effect of both insulin and an inhibitor on MAP Kinase phosphorylation. Lane A contains a control sample in which only buffer was added to the rat cells. The band representing MAP Kinase could be clearly detected. In lane B, the sample was incubated in the presence of insulin. A stronger band representing MAP Kinase was observed, indicating that more of the phosphorylated form of the protein was present and that the MAP Kinase had been activated. The fact that there was a higher level of phosphorylated MAP Kinase in the presence of insulin means that more of this protein would be immunoprecipitated out of solution with the anti-phosphotyrosine, and would result in a stronger band on the immunoblot. In the presence of insulin + inhibitor in lane C, however, only weak bands were detected, suggesting that the extent of phosphorylation of the tyrosine taking place on the Map Kinase was much lower than in the control sample. This inhibitor is known to bind to the active, unphosphorylated form of MAP Kinase and prevent its phosphorylation, even in the presence of glucose [10, 11]. When inhibitor alone was provided to the sample as shown in lane D, the level of expression of phosphotyrosine on the MAP Kinase resembled the control sample in lane 1. This was a surprise, as we expected the inhibitor to block phosphorylation of the MAP kinase. An explanation or this unexpected result is that no inhibitor was added to the tube, or that the inhibitor had somehow become inactivated during the experiment. The level of phosphorylated MAP Kinase in lane 4 appeared to be greater than the control in lane 1, suggesting that MAP Kinase had been stimulated, rather than repressed. It is possible that the total amounts of protein loaded in each sample are incorrect,and that would explain the difference in intensities between lanes 1 and 4. However, the bands observed on the Coomassie stained protein gel does not support this possibility (data not shown).
In conclusion, the immunoprecipitation technique works well to identify different states of phosphorylation of the MAP kinase in the presence of both stimulants and inhibitors. Improvements could include a repeat of the experiment and running a longer gel to further distinguish the different bands. It would be interesting to compare these results with a Western blot that did not include immunoprecipitated samples, but rather entire protein samples. The bands could also be quantitated by densitometric analysis, to provide a more numerical result.

References

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