Articles
A Non-radioactive, Colorimetric ELISA For MAP Kinase Activity
Tue, 04/15/2003 - 9:59am
Marithea Smit and Jay Leng
The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily conserved signaling pathway that controls fundamental cellular processes such as proliferation, differentiation, survival and apoptosis in all eukaryotes. For example, the ability of growth factors to promote proliferation depends on the activation of receptor tyrosine kinases, which recruit the Ras family small G proteins and lead to the sequential activation of Raf (MAPK kinase kinase), MEK (MAPK kinase) and MAPK/extracellular signal-regulated kinase (ERK).(1, 2, 3, 4)
Phosphorylation analyses are straightforward
For years radioactive kinase assays, utilizing 32 P-γ-ATP as phosphate donor to the acceptor substrate, have been employed to assess kinase activity. The generation of phospho-specific antibodies in recent years has advanced the development of non-radioactive kinase activity detection methods with comparable sensitivity as radioactive methods.(5) These new reagents make phosphorylation analyses considerably more straightforward and less labor-intensive than what is required by radioactive kinase assay protocols.
Here we describe a non-radioactive, colorimetric MAPK activity assay in a convenient 96-well enzyme-linked immunosorbent assay (ELISA) format. This assay utilizes a biotinylated myelin basic protein (MBP) substrate that contains multiple phosphorylation sites and can be phosphorylated by a wide range of kinases, for example, phosphorylation on Thr97 residue by MAP kinases (MAPKs/ERKs). (Figure 1.) Following quenching of the enzymatic reaction with an inhibitor, both the phosphorylated and non-phosphorylated substrates are immobilized by binding to a streptavidin (SA)-coated plate. The fraction of phosphorylated substrate is visualized using a monoclonal anti-phospho-MBP antibody, secondary antibody conjugated to horseradish peroxidase (HRP) and an ensuing chromogenic substrate reaction. The enzymatic activity of ERK1/2 is therefore directly proportional to the optical density at 450 nm.
The kinase reaction
The kinase reactions were conducted in a 50 μl volume containing ERK sample and 200 ng biotinylated-MBP substrate in 50 mM Tris-HCl, pH 7.4, 30 mM NaCl, 15 mM MgCl2, 1 mM ATP, 2 mM DTT and 0.2 mg/ml BSA. Following incubation of 30 minutes at 30 C, the kinase reactions were terminated by adding 10 μl of 120 mM EDTA. 50 μl of each reaction mixture was then transferred to a SA-coated 96-well plate and incubated for 30 minutes at 37 C. The wells were then washed four times, thoroughly, with 1× PBS, 0.05% Tween 20 (wash buffer). To reduce background, SA-coated plates containing captured substrate, were blocked for 30 minutes at 37 C with 3% BSA in wash buffer. Following blocking of non-specific binding, 100 μl of a mouse anti-phospho-MBP at 0.5 μ was added to each well and incubated for 45 minutes at room temperature. Upon completion of the primary antibody incubation, the reaction wells were washed as above and an HRP-conjugated secondary antibody solution was added and incubated for 30 minutes at room temperature. The secondary antibody incubation was followed by a washing step as described before, after which TMB substrate (BioFX Laboratories, Owings Mills, Maryland) was added and incubated for five minutes. The HRP-TMB reaction was terminated upon addition of an equal volume of 0.5 N HCl. The optical density of each well was read on a standard microplate reader using 450 nm as primary wavelength. It was essential that a negative control (a sample without MAPK but all other components added) was utilized to present a value for any basal of mean absorbance. Background reading for negative control was subtracted from the reading of the kinase reaction sample prior to calculating the MAPK activity. Figure 2A presents the activity dose curve of purified recombinant ERK2 (New England Biolabs, MA) when activity was determined in the method described above.
To measure kinase activity in cell lysate samples, immune complex kinase assays are often performed. In these assays, the kinase in the cell lysate is captured by an antibody, immunoprecipitated and then subjected to kinase activity reactions in the presence of 32P-γ-TP. The activity of the kinase in the reaction is analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and autoradiography. In an attempt to determine ERK activity from cell lysates, and to show the activation of ERK in vivo by its upstream kinase, MEK, we transiently transfected COS-7 cells with wild-type ERK2 along with a dominant active MEK1 using Lipofectamine 2000 (Invitrogen, Carlsbad, California). Following the transfection, the cell lysate samples were prepared as follows: cell media was removed and cells washed once with ice-cold 1× PBS. Pre-chilled detergent lysis buffer was added at 1 ml per each 100 mm cell culture dish. After 10 minutes incubation on ice, cells were harvested with a rubber policeman and the cell lysate collected by centrifugation at 12,000 ×g for 10 minutes. For immunoprecipitation of MAPK, clear lysates (0.5-2 mg of total protein) were incubated with an anti-ERK2 antibody (Santa Cruz Biotechnology, Santa Cruz, California) for 1-12 hours at 4 C on a shaking platform. Following the incubation the mixture was added to 50-100 μl of protein A Sepharose® beads (RepliGen, Waltham, Massachusetts), pre-washed and equilibrated in lysis buffer, and incubated for an additional 1-4 hours at 4 C on a shaking platform. The beads were collected by centrifugation at 2000 ×g at 4 C and supernatants carefully removed by aspiration. The bead pellets were washed three times with lysis buffer followed by two more washes with the kinase assay buffer (50 mM Tris-HCl, pH7.4; 5 mM MgCl2; 2 mM DTT). Following the removal of the supernatant the precipitates, containing the MAPK-antibody complex, were used directly in the kinase activity assay as described above. Figure 2B reveals that the Erk2 expression level for both transfected samples was similar as determined by an anti-ERK2 immunoblot. The activation of MAPKs occurs through phosphorylation of Thr202 and Tyr204 by a MAP kinase kinase (MAPKK or MEK).(1) In transfected COS-7 cells, the activation of ERK2 by a dominant active MEK1 is clearly shown in an immunoblot using anti-phospho-ERK (Cell Signaling, Technology, Beverly, Massachusetts. (Figure 2B.) Figure 2C illustrates the immunoprecipitation of ERK2 in the cytosolic fraction by an anti-ERK2 antibody and activity determination as described in the assay protocol. As a result, when MBP was used as substrate, the activation of ERK2 by MEK induced its activity with an approximate three-fold increase.
Summary
In conclusion, this non-radioactive MAPK (ERK1/2) activity assay provides a simple, convenient, and specific method for measuring ERK1/2 activity. Rapid analyses of purified as well as endogenous MAPK activity, in vitro high throughout screening of inhibitors can be performed with this assay. It is highly specific for serine/threonine kinases and does not cross-react with tyrosine kinases. The uniqueness of this assay lies with the unbound biotinylated substrate that allows determination of endogenous MAPK activity levels following immunoprecipitation. Therefore, we strongly believe that many researchers in the signal transduction field will favor this assay, due to its simplicity and high throughput format.
About the authors
Marithea Smit and Jay Leng work at the Beckman Institute For Biomedical Research in Temecula, California. Address correspondence to Dr. Jay Leng, Beckman Institute For Biomedical Research, 28835 Single Oak Drive, Temecula, CA 92590, USA. e-mail: jleng@beckmaninstitute.org. More information is also available from: Chemicon International, Temecula, CA. 800-437-7500; chemicon.org
The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily conserved signaling pathway that controls fundamental cellular processes such as proliferation, differentiation, survival and apoptosis in all eukaryotes. For example, the ability of growth factors to promote proliferation depends on the activation of receptor tyrosine kinases, which recruit the Ras family small G proteins and lead to the sequential activation of Raf (MAPK kinase kinase), MEK (MAPK kinase) and MAPK/extracellular signal-regulated kinase (ERK).(1, 2, 3, 4)
Phosphorylation analyses are straightforward
For years radioactive kinase assays, utilizing 32 P-γ-ATP as phosphate donor to the acceptor substrate, have been employed to assess kinase activity. The generation of phospho-specific antibodies in recent years has advanced the development of non-radioactive kinase activity detection methods with comparable sensitivity as radioactive methods.(5) These new reagents make phosphorylation analyses considerably more straightforward and less labor-intensive than what is required by radioactive kinase assay protocols.
Here we describe a non-radioactive, colorimetric MAPK activity assay in a convenient 96-well enzyme-linked immunosorbent assay (ELISA) format. This assay utilizes a biotinylated myelin basic protein (MBP) substrate that contains multiple phosphorylation sites and can be phosphorylated by a wide range of kinases, for example, phosphorylation on Thr97 residue by MAP kinases (MAPKs/ERKs). (Figure 1.) Following quenching of the enzymatic reaction with an inhibitor, both the phosphorylated and non-phosphorylated substrates are immobilized by binding to a streptavidin (SA)-coated plate. The fraction of phosphorylated substrate is visualized using a monoclonal anti-phospho-MBP antibody, secondary antibody conjugated to horseradish peroxidase (HRP) and an ensuing chromogenic substrate reaction. The enzymatic activity of ERK1/2 is therefore directly proportional to the optical density at 450 nm.
The kinase reaction
The kinase reactions were conducted in a 50 μl volume containing ERK sample and 200 ng biotinylated-MBP substrate in 50 mM Tris-HCl, pH 7.4, 30 mM NaCl, 15 mM MgCl2, 1 mM ATP, 2 mM DTT and 0.2 mg/ml BSA. Following incubation of 30 minutes at 30 C, the kinase reactions were terminated by adding 10 μl of 120 mM EDTA. 50 μl of each reaction mixture was then transferred to a SA-coated 96-well plate and incubated for 30 minutes at 37 C. The wells were then washed four times, thoroughly, with 1× PBS, 0.05% Tween 20 (wash buffer). To reduce background, SA-coated plates containing captured substrate, were blocked for 30 minutes at 37 C with 3% BSA in wash buffer. Following blocking of non-specific binding, 100 μl of a mouse anti-phospho-MBP at 0.5 μ was added to each well and incubated for 45 minutes at room temperature. Upon completion of the primary antibody incubation, the reaction wells were washed as above and an HRP-conjugated secondary antibody solution was added and incubated for 30 minutes at room temperature. The secondary antibody incubation was followed by a washing step as described before, after which TMB substrate (BioFX Laboratories, Owings Mills, Maryland) was added and incubated for five minutes. The HRP-TMB reaction was terminated upon addition of an equal volume of 0.5 N HCl. The optical density of each well was read on a standard microplate reader using 450 nm as primary wavelength. It was essential that a negative control (a sample without MAPK but all other components added) was utilized to present a value for any basal of mean absorbance. Background reading for negative control was subtracted from the reading of the kinase reaction sample prior to calculating the MAPK activity. Figure 2A presents the activity dose curve of purified recombinant ERK2 (New England Biolabs, MA) when activity was determined in the method described above.
To measure kinase activity in cell lysate samples, immune complex kinase assays are often performed. In these assays, the kinase in the cell lysate is captured by an antibody, immunoprecipitated and then subjected to kinase activity reactions in the presence of 32P-γ-TP. The activity of the kinase in the reaction is analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and autoradiography. In an attempt to determine ERK activity from cell lysates, and to show the activation of ERK in vivo by its upstream kinase, MEK, we transiently transfected COS-7 cells with wild-type ERK2 along with a dominant active MEK1 using Lipofectamine 2000 (Invitrogen, Carlsbad, California). Following the transfection, the cell lysate samples were prepared as follows: cell media was removed and cells washed once with ice-cold 1× PBS. Pre-chilled detergent lysis buffer was added at 1 ml per each 100 mm cell culture dish. After 10 minutes incubation on ice, cells were harvested with a rubber policeman and the cell lysate collected by centrifugation at 12,000 ×g for 10 minutes. For immunoprecipitation of MAPK, clear lysates (0.5-2 mg of total protein) were incubated with an anti-ERK2 antibody (Santa Cruz Biotechnology, Santa Cruz, California) for 1-12 hours at 4 C on a shaking platform. Following the incubation the mixture was added to 50-100 μl of protein A Sepharose® beads (RepliGen, Waltham, Massachusetts), pre-washed and equilibrated in lysis buffer, and incubated for an additional 1-4 hours at 4 C on a shaking platform. The beads were collected by centrifugation at 2000 ×g at 4 C and supernatants carefully removed by aspiration. The bead pellets were washed three times with lysis buffer followed by two more washes with the kinase assay buffer (50 mM Tris-HCl, pH7.4; 5 mM MgCl2; 2 mM DTT). Following the removal of the supernatant the precipitates, containing the MAPK-antibody complex, were used directly in the kinase activity assay as described above. Figure 2B reveals that the Erk2 expression level for both transfected samples was similar as determined by an anti-ERK2 immunoblot. The activation of MAPKs occurs through phosphorylation of Thr202 and Tyr204 by a MAP kinase kinase (MAPKK or MEK).(1) In transfected COS-7 cells, the activation of ERK2 by a dominant active MEK1 is clearly shown in an immunoblot using anti-phospho-ERK (Cell Signaling, Technology, Beverly, Massachusetts. (Figure 2B.) Figure 2C illustrates the immunoprecipitation of ERK2 in the cytosolic fraction by an anti-ERK2 antibody and activity determination as described in the assay protocol. As a result, when MBP was used as substrate, the activation of ERK2 by MEK induced its activity with an approximate three-fold increase.
Summary
In conclusion, this non-radioactive MAPK (ERK1/2) activity assay provides a simple, convenient, and specific method for measuring ERK1/2 activity. Rapid analyses of purified as well as endogenous MAPK activity, in vitro high throughout screening of inhibitors can be performed with this assay. It is highly specific for serine/threonine kinases and does not cross-react with tyrosine kinases. The uniqueness of this assay lies with the unbound biotinylated substrate that allows determination of endogenous MAPK activity levels following immunoprecipitation. Therefore, we strongly believe that many researchers in the signal transduction field will favor this assay, due to its simplicity and high throughput format.
About the authors
Marithea Smit and Jay Leng work at the Beckman Institute For Biomedical Research in Temecula, California. Address correspondence to Dr. Jay Leng, Beckman Institute For Biomedical Research, 28835 Single Oak Drive, Temecula, CA 92590, USA. e-mail: jleng@beckmaninstitute.org. More information is also available from: Chemicon International, Temecula, CA. 800-437-7500; chemicon.org
