Mitogen-activated protein (MAP) kinases are serine/threonine kinases that are activated by phosphorylation and are involved in the cellular response to various physiologic stimuli and stress conditions. Because MAP kinases play an important role in cellular functioning, a screening assay to determine the phosphorylation of MAP kinase upon various conditions was desirable. Therefore, we have developed a cellular enzyme-linked immunosorbent assay (Cell-ELISA), in which the phosphorylated forms of p42MAPK and p44MAPK are detected. We show that in this Cell-ELISA, MAP kinase becomes phosphorylated in a dose- and time-dependent manner under proliferative or stress conditions. This dose- and time-dependent phosphorylation agrees with observations using classical gel-electrophoresis and Western blotting techniques. Furthermore, we show that our assay is applicable to different cell types and that serum-starvation is not required for detection of an increase in MAP kinase phosphorylation. From these experiments, it is concluded that the Cell-ELISA is a reliable and fast method for quantitative detection of the phosphorylation, and thus the activation, of MAP kinase. This assay is applicable for a large-scale screening of the effectivity of biological or chemical compounds that modulate the cellular response to physiologic stimuli or stress through phosphorylation and activation of MAP kinase.
References
1.
Pelech, S.L., Sanghera, S.J. (1992). Mitogen-activated protein kinases: Versatile transducers for cell signaling. Trends Biochem. Sci.17:233-238.
2.
Sturgill, T.W., Wu, J. (1991). Recent progress in characterization of protein kinase cascades for phosphorylation of ribosomal protein S6. Biochim. Biophys. Acta.1092:350-357.
3.
Bittorf, T., Jaster, R., Brock, J. (1994). Rapid activation of the MAP kinase pathway in hematopoietic cells by erythropoietin, granulocyte-macrophage colony-stimulating factor and interleukin-3. Cell. Signal. 6:305-311.
4.
Boulton, T.G., Nye, S.H., Robbins, D.J., Ip, N.Y., Radziejewska, E., Morgenbesser, S.D., DePinho, A., Panayotatos, N., Cobb, M.H., Yancopoulos, G.D. (1991). ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell. 65:663-675.
5.
Lee, J.C., Laydon, J.T., McDonnell, P.C., Gallagher, T.F., Kumar, S., Green, D., McNulty, D., Blumenthal, M.J., Heys, J.R., Landvatter, S.W., Strickler, J.E., McLaughlin, E.E., Siemens, I.R., Fisher, R.M., Livi, G.P., White, J.R., Adams, J.L., Young, P.R. (1994). A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature. 372:739-746.
6.
Guyton, K.Z., Liu, Y., Gorospe, M., Xu, Q., Holbrook, N.J. (1996). Activation of mitogen-activated protein kinase by H202. Role in cell survival following oxidant injury. J. Biol. Chem.271:4138-4142.
7.
Rossomando, A.J., Payne, D.M., Weber, M.J., Sturgill, T.W. (1989). Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase. Proc. Natl. Acad. Sci. USA.86:6940-6943.
8.
Stevenson, M.A., Pollock, S.S., Coleman, C.N., Calderwood, S.K. (1994). X-irradiation, phorbol esters, and H202 stimulate mitogen-activated protein kinase activity in NIH-3T3 cells through the formation of reactive oxygen intermediates. Cancer Res.54:12-15.
9.
Meloche, S., Seuwen, K., Pages, G., Pouyssegur, J. (1992). Biphasic and synergistic activation of p44mapk (ERKI) by growth factors: correlation between late phase activation and mitogenicity. Mol. Endocrinol.6:845-854.
10.
Pages, G., Lenormand, P., L'Allemain, G., Chambard, J-C., Meloche, S., Pouyssegur, J. (1993). Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc. Natl. Acad. Sci. U S A.90:8319-8323.
11.
Cowley, S., Paterson, H., Kemp, P., Marshall, C.J. (1994). Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell. 77:841-852.
12.
Traverse, S., Gomez, N., Paterson, H., Marshall, C., Cohen, P. (1992). Sustained activation of the mitogen-activated protein (MAP) kinase cascade may be required for differentiation of PC 12 cells. Comparison of the effects of nerve growth factor and epidermal growth factor. Biochem. J.288:351-355.
13.
Traverse, S., Seedorf, K., Paterson, H., Marshall, C.J., Cohen, P., Ullrich, A. (1994). EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor. Curr. Biol.4:694-701.
14.
Cantoni, O., Boscoboinik, D., Fiorani, M., Stauble, B., Azzi, A. (1996). The phosphorylation state of MAP-kinases modulates the cytotoxic responses of smooth muscle cells to hydrogen peroxide. FEBS Lett.389:285-288.
Derijard, B., Hibi, M., Wu, I-H., Barrett, T., Su, B., Deng, T., Karin, M., Davis, R.J. (1994). JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell. 76:1025-1037.
17.
Han, J., Lee, J-D., Bibbs, L., Ulevitch, R.J. (1994). A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science. 265:808-811.
18.
Kyriakis, J.M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E.A., Ahmad, M.F., Avruch, J., Woodgett, J.R. (1994). The stress-activated protein kinase subfamily of c-Jun kinases. Nature. 369:156-160.
19.
Davis, R.J. (1994). MAPKs: new JNK expands the group. Trends Biochem. Sci.19:470-473.
20.
Wu, J., Michel, H., Dent, P., Haystead, T., Hunt, D.F., Sturgill, T.W. (1993). Activation of MAP kinase by a dual specificity Tyr/1br kinase. Adv.-Second-Messenger-Phosphoprotein-Res.28:219-225.
21.
Wu, J., Harrison, J.K., Dent, P., Lynch, K.R., Weber, M.J., Sturgill, T.W. (1993). Identification and characterization of a new mammalian mitogen-activated protein kinase kinase, MKK2. Mol. Cell. Biol.13:4539-4548.
22.
Zhang, F., Strand, A., Robbins, D., Cobb, M.H., Goldsmith, J. (1994). Atomic structure of the MAP kinase ERK2 at 2.3 Angstrom resolution. Nature. 367:704-711.
23.
Zheng, C-F., Guan, K-L. (1993). Dephosphorylation and inactivation of the mitogen-activated protein kinase by a mitogen-induced Thr/Tyr protein phosphatase. J. Biol. Chem.268:16116-16119.
24.
Harary, I., Farley, B. (1963). In vitro studies of single rat heart cells. I. Growth and organization. Exp. Cell Res.29:451-465.
25.
Post, J.A., Langer, G.A., Op den Kamp, J.A.F., Verkleij, A.J. (1988). Phospholipid asymmetry in cardiac sarcolemma. Analysis of intact and "gas-dissected" membranes. Biochim. Biophys. Acta.943:256-266.
26.
Posada, J., Cooper, J.A. (1992). Requirements for phosphorylation of MAP kinase during meiosis in Xenopus oocytes. Science. 255:212-215.
27.
Chajry, N., Martin, P.M., Pages, G., Cochet, C., Afdel, K., Berthois, Y. (1994). Relationship between the MAP kinase activity and the dual effect of EGF on A431 cell proliferation. Biochem. Biophys. Res. Common.203:984-990.
28.
Muller, J.M., Cahill, M.A., Rupec, R.A., Baeuerle, P.A., Nordheim, A. (1997). Antioxidants as well as oxidants activate c-fos via Ras-dependent activation of extracellular-signal-regulated kinase 2 and Elk-l. Eur. J. Biochem.244:45-52.
29.
Aikawa, R., Komuro, I., Yamazaki, T., Zou, Y., Kudoh, S., Tanaka, M., Shiojima, I., Hiroi, Y., Yazaki, Y. (1997). Oxidative stress activates extracellular signal-regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats. J. Clin. Invest.100:1813-1821.
