The Detection of MAPK Signaling

Yoav Shaul1, Rony Seger1

1 Department of Biological Regulation, The Weizmann Institute of Science, Rehovot
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 18.12
DOI:  10.1002/0471142727.mb1812s73
Online Posting Date:  February, 2006
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Abstract

Mitogen‐activated protein kinase (MAPK) cascades are central pathways that participate in the intracellular transmission of extracellular signals. Each of the MAPK signaling cascades seems to consist of three to five tiers of protein kinases that sequentially activate each other by phosphorylation. Since the majority of MAPK cascade components are kinases, the methods used to detect their activation involve determining phosphorylation state and protein kinase activities. The primary method describes the use of immunoblotting with specific anti‐phospho antibody to detect activation of MAPK components. Alternative methods described are immunoprecipitation of desired protein kinases followed by phosphorylation of specific substrates and the use of an in‐gel kinase assay. These methods have proven useful in the study of the MAPK signaling cascades.

Keywords: MAPK; ERK; JNK; p38MAPK; BMK; protein kinases; antibodies

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Immunodetection of MAPK Activation using Anti‐Phospho MAPK Antibodies
  • Basic Protocol 2: Determination of MAPK (ERK) Activity by Immunoprecipitation
  • Basic Protocol 3: In‐Gel Kinase Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Immunodetection of MAPK Activation using Anti‐Phospho MAPK Antibodies

  Materials
  • Rat1 cells (ATCC #CRL‐2210)
  • DMEM containing 10% heat‐inactivated FBS (see appendix 22 and appendix 3F)
  • Starvation medium: DMEM containing 0.1% (v/v) heat‐inactivated FBS (see appendix 22 and appendix 3F)
  • 50 µg/ml epidermal growth factor (EGF) in EGF buffer (0.5 mg/ml BSA in PBS)
  • EGF buffer: 0.5 mg/ml bovine serum albumin (BSA) in phosphate‐buffered saline (PBS; appendix 22)
  • Phosphate‐buffered saline (PBS; appendix 22), ice‐cold
  • Buffer A (see recipe), ice‐cold
  • Buffer H (see recipe), ice‐cold
  • Protein standards: 5, 10, 20, 50, 100, and 200 µg/ml BSA in Buffer H (see recipe for buffer H)
  • Bradford protein assay reagent (Pierce or Bio‐Rad)
  • 4× sample buffer for SDS‐PAGE (see recipe)
  • 1.5 M Tris⋅Cl, pH 8.8 ( appendix 22)
  • 30% acrylamide/0.8% bisacrylamide (Table 97.80.4711)
  • 10% (w/v) ammonium persulfate (prepare fresh)
  • Tetramethylethelendiamine (TEMED)
  • 0.5 M Tris⋅Cl, pH 6.8 ( appendix 22)
  • Running buffer (see recipe)
  • Prestained protein markers
  • Transfer buffer (see recipe)
  • Blocking solution: 2% (w/v) bovine serum albumin (BSA) in TBST (see recipe for TBST)
  • Primary antibodies: monoclonal mouse anti‐phospho ERK antibody (Table 18.12.1) and polyclonal rabbit ati‐general ERK antibody (C‐terminus)
  • Tris‐buffered saline with Tween 20 (TBST; see recipe)
  • Secondary antibodies: alkaline phosphatase (AP)–coupled goat anti‐mouse antibody and horseradish peroxidase (HRP)–conjugated goat anti‐rabbit antibody
  • 6‐cm tissue culture dishes
  • 1‐ml pipet tips, precooled
  • 1.5‐ml microcentrifuge tubes precooled; four sets of six (each labeled 1 to 6)
  • Plastic (or rubber) policeman
  • Probe sonicator (e.g., Branson)
  • 96‐well flat‐bottom microtiter plate
  • Microtiter plate reader capable of reading at 595 nm
  • 95°C or boiling water bath
  • Gel‐casting apparatus: 7 × 10–cm glass plates, 1.5‐mm spacers, and 1.5‐mm comb with 10 teeth (also see unit 10.2)
  • Gel electrophoresis apparatus and power supply (also see unit 10.2)
  • 0.45‐µm nitrocellulose membrane (e.g., Schleicher & Schuell or Millipore), cut to gel size
  • Whatman 3MM paper (two sheets cut to gel size)
  • Transfer apparatus and power supply (also see unit 10.8)
  • Additional reagents and equipment for cell culture ( appendix 3F), SDS‐PAGE (unit 10.2), and visualization of immunoblotted proteins (units 10.8)
NOTE: All reagents and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) require altered levels of CO 2 to maintain pH 7.4.

Basic Protocol 2: Determination of MAPK (ERK) Activity by Immunoprecipitation

  Materials
  • Rat1 cells (ATCC #CRL‐2210)
  • DMEM containing 10% heat‐inactivated FBS (see appendix 22 and appendix 3F)
  • Starvation medium: DMEM containing 0.1% (v/v) heat‐inactivated FBS (see appendix 22 and appendix 3F)
  • 50 µg/ml epidermal growth factor (EGF) in EGF buffer
  • EGF buffer: 0.5 mg/ml bovine serum albumin (BSA) in phosphate buffered saline (PBS; appendix 22)
  • Phosphate‐buffered saline (PBS; appendix 22), ice‐cold
  • Buffer A (see recipe), ice‐cold
  • Buffer H (see recipe), ice‐cold
  • Protein standards: 5, 10, 20, 50, 100 and 200 µg/ml BSA in Buffer H
  • Bradford protein assay reagent (Pierce or Bio‐Rad)
  • Protein A–Sepharose beads (Amersham Biosciences; 10 to 20 µl of packed beads per reaction)
  • Antibody for immunoprecipitation (e.g., anti‐ERK C‐terminus antibody, 1 to 5 µg per reaction according to the supplier's instructions)
  • RIPA buffer (see recipe), ice‐cold
  • Lithium chloride solution: 0.5 M LiCl/0.1 M Tris⋅Cl, pH 8.0 (see appendix 22 for Tris⋅Cl), ice‐cold
  • RM×3 (see recipe)
  • 2 mg/ml myelin basic protein (MBP), or other appropriate phosphorylation substrate at appropriate (usually lower) concentration
  • 4× sample buffer for SDS‐PAGE (see recipe)
  • 7 × 10–cm 15% SDS‐PAGE gel with stacking gel (unit 10.2; also see protocol 1)
  • Prestained protein markers
  • Staining solution (see recipe)
  • Destaining solution (see recipe)
  • 6‐cm tissue culture dishes
  • 1‐ml pipet tips, precooled
  • 1.5‐ml microcentrifuge tubes precooled; four sets of six (each labeled 1 to 6)
  • Plastic (or rubber) policeman
  • Probe sonicator (e.g., Branson)
  • 96‐well flat‐bottom microtiter plate
  • Microtiter plate reader capable of reading at 595 nm
  • End‐over‐end rotator
  • 30°C Thermomixer (Eppendorf) or water bath
  • Boiling water bath
  • Flat container for staining/destaining gel
  • Additional reagents and equipment for cell culture ( appendix 3F), SDS‐PAGE (unit 10.2), and autoradiography or phosphor imaging ( appendix 3A)
NOTE: All reagents and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) require altered levels of CO 2 to maintain pH 7.4.

Basic Protocol 3: In‐Gel Kinase Assay

  Materials
  • Rat1 cells (ATCC #CRL‐2210)
  • DMEM containing 10% heat‐inactivated FBS (see appendix 22 and appendix 3F)
  • Starvation medium: DMEM containing 0.1% (v/v) heat‐inactivated FBS (see appendix 22 and appendix 3F)
  • 50 µg/ml epidermal growth factor (EGF) in EGF buffer
  • EGF buffer: 0.5 mg/ml bovine serum albumin (BSA) in phosphate buffered saline (PBS; appendix 22)
  • Phosphate‐buffered saline (PBS; appendix 22), ice‐cold
  • Buffer A (see recipe), ice‐cold
  • Buffer H (see recipe) containing 1% (v/v) Triton X‐100, ice‐cold
  • Buffer H (see recipe), ice‐cold
  • Protein standards: 5, 10, 20, 50, 100 and 200 µg/ml BSA in Buffer H (see recipe for buffer H)
  • Bradford protein assay reagent (Pierce or Bio‐Rad)
  • 4× sample buffer for SDS‐PAGE (see recipe)
  • 1.5 M Tris⋅Cl, pH 8.8 ( appendix 22)
  • 30% acrylamide/0.8% bisacrylamide (Table 18.12.1)
  • 10% (w/v) ammonium persulfate (prepare fresh)
  • Tetramethylethelendiamine (TEMED)
  • 2 mg/ml myelin basic protein (MBP)
  • 0.5 M Tris⋅Cl, pH 6.8 ( appendix 22)
  • Running buffer (see recipe)
  • 20% (v/v) isopropanol/50 mM HEPES, pH 7.6
  • Renaturation buffer: 50 mM HEPES, pH 7.6 containing 5 mM 2‐mercaptoethanol
  • Renaturation buffer containing 6 M urea
  • Renaturation buffer containing 0.05% (v/v) Tween 20
  • In‐gel kinase buffer (see recipe)
  • In‐gel kinase reaction solution (see recipe)
  • 5% (w/v) trichloroacetic acid (TCA)/1% (w/v) sodium pyrophosphate
  • 6‐cm tissue culture dishes
  • 1‐ml pipet tips, precooled
  • 1.5‐ml microcentrifuge tubes precooled; four sets of six (each labeled 1 to 6)
  • Plastic (or rubber) policeman
  • 96‐well flat‐bottom microtiter plate
  • Microtiter plate reader capable of reading at 595 nm
  • Gel‐casting apparatus: 7 × 10–cm glass plates, 1.5‐mm spacers, and 1.5‐mm comb with 10 teeth (also see unit 10.2)
  • Gel electrophoresis apparatus and power supply (also see unit 10.2)
  • Flat containers for washing gel
  • 30°C water bath with proper shielding for radioactive work
  • Additional reagents and equipment for cell culture ( appendix 3F), SDS‐PAGE (unit 10.2), and autoradiography ( appendix 3A)
NOTE: All reagents and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) require altered levels of CO 2 to maintain pH 7.4.
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Literature Cited

Literature Cited
   Abe, J., Kusuhara, M., Ulevitch, R.J., Berk, B.C., and Lee, J.D. 1996. Big mitogen‐activated protein kinase 1 (BMK1) is a redox‐sensitive kinase. J. Biol. Chem. 271:16586‐16590.
   Abe, M.K., Kuo, W.L., Hershenson, M.B., and Rosner, M.R. 1999. Extracellular signal‐regulated kinase 7 (ERK7), a novel ERK with a C‐terminal domain that regulates its activity, its cellular localization, and cell growth. Mol. Cell Biol. 19:1301‐1312.
   Abe, M.K., Kahle, K.T., Saelzler, M.P., Orth, K., Dixon, J.E., and Rosner, M.R. 2001. ERK7 is an autoactivated member of the MAP kinase family. J. Biol. Chem. 276:21272‐21279.
   Ahn, N.G., Weiel, J.E., Chan, C.P., and Krebs, E.G. 1990. Identification of multiple epidermal growth factor‐stimulated protein serine/threonine kinases from Swiss 3T3 cells. J. Biol. Chem. 265:11487‐11494.
   Ahn, N.G., Seger, R., Bratlien, R.L., Diltz, C.D., Tonks, N.K., and Krebs, E.G. 1991. Multiple components in an epidermal growth factor‐stimulated protein kinase cascade: In vitro activation of myelin basic protein/microtubule‐associated protein‐2 kinase. J. Biol. Chem. 266:4220‐4227.
   Blank, J.L., Gerwins, P., Elliott, E.M., Sather, S., and Johnson, G.L. 1996. Molecular cloning of mitogen‐activated protein/ERK kinase kinases (MEKK) 2 and 3: Regulation of sequential phosphorylation pathways involving mitogen‐activated protein kinase and c‐Jun kinase. J. Biol. Chem. 271:5361‐5368.
   Boulton, T.G., Yancopoulos, G.D., Gregory, J.S., Slaughter, C., Moomaw, C., Hsu, J., and Cobb, M.H. 1990. An insulin‐stimulated protein kinase similar to yeast kinases involved in cell cycle control. Science 249:64‐67.
   Boulton, T.G., Nye, S.H., Robbins, D.J., Ip, N.Y., Radziejewska, E., Morgenbesser, S.D., DePinho, R.A., Panayotatos, N., Cobb, M.H., and Yancopoulos, G.D. 1991. ERK's: A family of protein‐serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 65:663‐675.
   Brushia, R.J. and Walsh, D.A. 1999. Phosphorylase kinase: The complexity of its regulation is reflected in the complexity of its structure. Front. Biosci. 4:D618‐D641.
   Cameron, S.J., Malik, S., Akaike, M., Lerner‐Marmarosh, N., Yan, C., Lee, J.D., Abe, J., and Yang, J. 2003. Regulation of epidermal growth factor‐induced connexin 43 gap junction communication by big mitogen–activated protein kinase1/ERK5 but not ERK1/2 kinase activation. J. Biol. Chem. 278:18682‐18688.
   Canagarajah, B.J., Khokhlatchev, A., Cobb, M.H., and Goldsmith, E.J. 1997. Activation mechanism of the MAP kinase ERK2 by dual phosphorylation. Cell 90:859‐869.
   Chadee, D.N. and Kyriakis, J.M. 2004. MLK3 is required for mitogen activation of B‐Raf, ERK and cell proliferation. Nat. Cell Biol. 6:770‐776.
   Chao, T.H., Hayashi, M., Tapping, R.I., Kato, Y., and Lee, J.D. 1999. MEKK3 directly regulates MEK5 activity as part of the big mitogen‐activated protein kinase 1 (BMK1) signaling pathway. J. Biol. Chem. 274:36035‐36038.
   Chayama, K., Papst, P.J., Garrington, T.P., Pratt, J.C., Ishizuka, T., Webb, S., Ganiatsas, S., Zon, L.I., Sun, W., Johnson, G.L., and Gelfand, E.W. 2001. Role of MEKK2‐MEK5 in the regulation of TNF‐alpha gene expression and MEKK2‐MKK7 in the activation of c‐Jun N‐terminal kinase in mast cells. Proc. Natl. Acad. Sci. U.S.A. 98:4599‐4604.
   Chen, Z., Hutchison, M., and Cobb, M.H. 1999. Isolation of the protein kinase TAO2 and identification of its mitogen‐activated protein kinase/extracellular signal‐regulated kinase kinase binding domain. J. Biol. Chem. 274:28803‐28807.
   Chen, Z., Gibson, T.B., Robinson, F., Silvestro, L., Pearson, G., Xu, B., Wright, A., Vanderbilt, C., and Cobb, M.H. 2001. MAP kinases. Chem. Rev. 101:2449‐2476.
   Cheng, M., Zhen, E., Robinson, M.J., Ebert, D., Goldsmith, E., and Cobb, M.H. 1996. Characterization of a protein kinase that phosphorylates serine 189 of the mitogen‐activated protein kinase homolog ERK3. J. Biol. Chem. 271:12057‐12062.
   Chow, C.W., Rincon, M., Cavanagh, J., Dickens, M., and Davis, R.J. 1997. Nuclear accumulation of NFAT4 opposed by the JNK signal transduction pathway. Science 278:1638‐1641.
   Chuderland, D. and Seger, R. 2005. Protein‐protein interactions in the regulation of the extracellular signal‐regulated kinase (ERK). Mol. Biotechnol. 29:47‐64.
   Cohen, P., Alessi, D.R., and Cross, D.A. 1997. PDK1, one of the missing links in insulin signal transduction? FEBS Lett. 410:3‐10.
   Crespo, P., Schuebel, K.F., Ostrom, A.A., Gutkind, A.S., and Bustel, X.P. 1997. Phosphotyrosine‐dependent activation of Rac‐1 GDP/GTP exchange by the vav proto‐oncogene product. Nature 385:169‐172.
   Cuenda, A., Cohen, P., Buee‐Scherrer, V., and Goedert, M. 1997. Activation of stress‐activated protein kinase‐3 (SAPK3) by cytokines and cellular stresses is mediated via SAPKK3 (MKK6): Comparison of the specificities of SAPK3 and SAPK2 (RK/p38). EMBO J. 16:295‐305.
   Dan, I., Watanabe, N.M., Kobayashi, T., Yamashita‐Suzuki, K., Fukagaya, Y., Kajikawa, E., Kimura, W.K., Nakashima, T.M., Matsumoto, K., Ninomiya‐Tsuji, J., and Kusumi, A. 2000. Molecular cloning of MINK, a novel member of mammalian GCK family kinases, which is up‐regulated during postnatal mouse cerebral development. FEBS Lett. 469:19‐23.
   Dan, I., Watanabe, N.M., and Kusumi, A. 2001. The Ste20 group kinases as regulators of MAP kinase cascades. Trends Cell Biol. 11:220‐230.
   Dan, C., Nath, N., Liberto, M., and Minden, A. 2002. PAK5, a new brain‐specific kinase, promotes neurite outgrowth in N1E‐115 cells. Mol. Cell Biol. 22:567‐577.
   Dashti, S., Efimova, R.T., and Eckert, R.L. 2001. MEK7‐dependent activation of p38 MAP kinase in keratinocytes. J. Biol. Chem. 276:8059‐8063.
   Davis, R.J. 2000. Signal transduction by the JNK group of MAP kinases. Cell 103:239‐252.
   Deak, M., Clifton, A.D., Lucocq, L.M., and Alessi, D.R. 1998. Mitogen‐ and stress‐activated protein kinase‐1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB. EMBO J. 17:4426‐4441.
   Derijard, B., Hibi, M., Wu, I.H., Barrett, T., Su, B., Deng, T., Karin, M., and 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 76:1025‐1027.
   Derijard, B., Raingeaud, J., Barrett, T., Wu, I.H., Han, J., Ulevitch, R.J., and Davis, R.J. 1995. Independent human MAP‐kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267:682‐685 [published erratum appears in Science 269:17].
   Diener, K., Wang, X.S., Chen, C., Meyer, C.F., Keesler, G., Zukowski, M., Tan, T.H., and Yao, Z. 1997. Activation of the c‐Jun N‐terminal kinase pathway by a novel protein kinase related to human germinal center kinase. Proc. Natl. Acad. Sci. U.S.A. 94:9687‐9692.
   Ducret, C., Maira, S.M., Lutz, Y., and Wasylyk, B. 2000. The ternary complex factor Net contains two distinct elements that mediate different responses to MAP kinase signalling cascades. Oncogene 19:5063‐5072.
   English, J.M., Pearson, G., Baer, R., and Cobb, M.H. 1998. Identification of substrates and regulators of the mitogen‐activated protein kinase ERK5 using chimeric protein kinases. J. Biol. Chem. 273:3854‐3860.
   English, J.M., Pearson, G., Hockenberry, T., Shivakumar, L., White, M.A., and Cobb, M.H. 1999. Contribution of the ERK5/MEK5 pathway to Ras/Raf signaling and growth control. J. Biol. Chem. 274:31588‐31592.
   Fan, G., Merritt, S.E., Kortenjann, M., Shaw, P.E., and Holzman, L.B. 1996. Dual leucine zipper‐bearing kinase (DLK) activates p46SAPK and p38mapk but not ERK2. J. Biol. Chem. 271:24788‐24793.
   Freshney, N.W., Rawlinson, L., Guesdon, F., Jones, E., Cowley, S., Hsuan, J., and Saklatvala, J. 1994. Interleukin‐1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27. Cell 78:1039‐1049.
   Fu, C.A., Shen, M., Huang, B.C., Lasaga, J., Payan, D.G., and Luo, Y. 1999. TNIK, a novel member of the germinal center kinase family that activates the c‐Jun N‐terminal kinase pathway and regulates the cytoskeleton. J. Biol. Chem. 274:30729‐30737.
   Fukunaga, R. and Hunter, T. 1997. MNK1, a new MAP kinase‐activated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates. EMBO J. 16:1921‐1933.
   Gabay, L., Seger, R., and Shilo, B.Z. 1997. In situ activation pattern of Drosophila EGF receptor pathway during development. Science 277:1103‐1106.
   Gallo, K.A. and Johnson, G.L. 2002. Mixed‐lineage kinase control of JNK and p38 MAPK pathways. Nat. Rev. Mol. Cell Biol. 3:663‐672.
   Gerwins, P., Blank, J.L., and Johnson, G.L. 1997. Cloning of a novel mitogen‐activated protein kinase kinase kinase, MEKK4, that selectively regulates the c‐Jun amino terminal kinase pathway. J. Biol. Chem. 272:8288‐8295.
   Gille, H., Sharrocks, A.D., and Shaw, P.E. 1992. Phosphorylation of transcription factor p62TCF by MAP kinase stimulates ternary complex formation at c‐fos promoter. Nature 358:414‐417.
   Goedert, M., Cuenda, A., Craxton, M., Jakes, R., and Cohen, P. 1997. Activation of the novel stress‐activated protein kinase SAPK4 by cytokines and cellular stresses is mediated by SKK3 (MKK6): Comparison of its substrate specificity with that of other SAP kinases. EMBO J. 16:3563‐3571.
   Gotoh, I., Adachi, M., and Nishida, E. 2001. Identification and characterization of a novel MAP kinase kinase kinase, MLTK. J. Biol. Chem. 276:4276‐4286.
   Gotoh, Y. and Nishida, E. 1995. Activation mechanism and function of the MAP kinase cascade. Mol. Reprod. Dev. 42:486‐492.
   Graves, J.D., Gotoh, Y., Draves, K.E., Ambrose, D., Han, D.K., Wright, M., Chernoff, J., Clark, E.A., and Krebs, E.G., 1998. Caspase‐mediated activation and induction of apoptosis by the mammalian Ste20‐like kinase Mst1. EMBO J. 17:2224‐2234.
   Gupta, S., Campbell, D., Derijard, B., and Davis, R.J. 1995. Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science 267:389‐393.
   Hagemann, C. and Blank, J.L. 2001. The ups and downs of MEK kinase interactions. Cell Signal 13:863‐875.
   Hagemann, C. and Rapp, U.R. 1999. Isotype‐specific functions of Raf kinases. Exp. Cell Res. 253:34‐46.
   Han, J., Lee, J.D., Bibbs, L., and Ulevitch, R.J. 1994. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265: 808‐811.
   Han, J., Lee, J.D., Jiang, Y., Li, Z., Feng, L., and Ulevitch, R.J. 1996. Characterization of the structure and function of a novel MAP kinase kinase (MKK6). J. Biol. Chem. 271:2886‐2891.
   Han, J., Jiang, Y., Li, Z., Kravchenko, V.V., and Ulevitch, R.J. 1997. Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature 386:296‐299.
   Hayashi, M., Tapping, R.I., Chao, T.H., Lo, J.F., King, C.C., Yang, Y., and Lee, J.D. 2001. BMK1 mediates growth factor‐induced cell proliferation through direct cellular activation of serum and glucocorticoid‐inducible kinase. J. Biol. Chem. 276:8631‐8634.
   Herskowitz, I. 1995. MAP kinase pathways in yeast: For mating and more. Cell 80:187‐197.
   Hibi, M., Lin, A., Smeal, T., Minden, A., and Karin, M. 1993. Identification of an oncoprotein‐ and UV‐responsive protein kinase that binds and potentiates the c‐Jun activation domain. Genes Dev. 7:2135‐2148.
   Hirai, S., Katoh, M., Terada, M., Kyriakis, J.M., Zon, L.I., Rana, A., Avruch, J., and Ohno, S. 1997. MST/MLK2, a member of the mixed lineage kinase family, directly phosphorylates and activates SEK1, an activator of c‐Jun N‐terminal kinase/stress‐activated protein kinase. J. Biol. Chem. 272:15167‐15173.
   Huang, C., Ma, W.Y., Maxiner, A., Sun, Y., and Dong, Z. 1999. p38 kinase mediates UV‐induced phosphorylation of p53 protein at serine 389. J. Biol. Chem. 274:12229‐12235.
   Hutchison, M., Berman, K.S., and Cobb, M.H. 1998. Isolation of TAO1, a protein kinase that activates MEKs in stress‐ activated protein kinase cascades. J. Biol. Chem. 273:28625‐28632.
   Ichijo, H., Nishida, E., Irie, K., ten Dijke, P., Saitoh, M., Moriguchi, T., Takagi, M., Matsumoto, K., Miyazono, K., and Gotoh, Y. 1997. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275:90‐94.
   Janknecht, R., Monte, D., Baert, J.L., and de Launoit, Y. 1996. The ETS‐related transcription factor ERM is a nuclear target of signaling cascades involving MAPK and PKA. Oncogene 13:1745‐1754.
   Johnston, A.M., Naselli, G., Gonez, L.J., Martin, R.M., Harrison, L.C., and DeAizpurua, H.J. 2000. SPAK, a STE20/SPS1‐related kinase that activates the p38 pathway. Oncogene 19:4290‐4297.
   Kallunki, T., Deng, T., Hibi, M., and Karin, M. 1996. c‐Jun can recruit JNK to phosphorylate dimerization partners via specific docking interactions. Cell 87:929‐939.
   Kamakura, S., Moriguchi, T., and Nishida, E. 1999. Activation of the protein kinase ERK5/BMK1 by receptor tyrosine kinases: Identification and characterization of a signaling pathway to the nucleus. J. Biol. Chem. 274:26563‐26571.
   Kasler, H. Victoria, G.J., Duramad, O., and Winoto, A. 2000. ERK5 is a novel type of mitogen‐activated protein kinase containing a transcriptional activation domain. Mol. Cell Biol. 20:8382‐8389.
   Kato, Y., Kravchenko, V.V., Tapping, R.I., Han, J., Ulevitch, R.J., and Lee, J.D. 1997. BMK1/ERK5 regulates serum‐induced early gene expression through transcription factor MEF2C. EMBO J. 16:7054‐7066.
   Kato, Y., Tapping, R.I., Huang, S., Watson, M.H., Ulevitch, R.J., and Lee, J.D. 1998. Bmk1/Erk5 is required for cell proliferation induced by epidermal growth factor. Nature 395:713‐716.
   Kiefer, F., Tibbles, L.A., Anafi, M., Janssen, A., Zanke, B.W., Lassam, N., Pawson, T., Woodgett, J.R., and Iscove, N.N. 1996. HPK1, a hematopoietic protein kinase activating the SAPK/JNK pathway. EMBO J. 15:7013‐7025.
   Kolesnick, R. and Xing, H.R. 2004. Inflammatory bowel disease reveals the kinase activity of KSR1. J. Clin. Invest. 114:1233‐1237.
   Kyriakis, J.M. and Avruch, J. 1996. Sounding the alarm: Protein kinase cascades activated by stress and inflamation. J. Biol. Chem. 271:24313‐24316.
   Kyriakis, J.M., App, H., Zhang, F.X., Banerjee, P., Brautigan, D.L., Rapp, U.R., and Avruch, J. 1992. Raf‐1 activates MAP kinase‐kinase. Nature 358:417‐421.
   Kyriakis, J.M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E.A., Ahmad, M.F., Avruch, J., and Woodgett, J.R. 1994. The stress‐activated protein kinase subfamily of c‐Jun kinases. Nature 369:156‐160.
   Lange‐Carter, C.A., Pleiman, C.M., Gardner, A.M., Blumer, K.J., and Johnson, G.L. 1993. A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf. Science 260:315‐317.
   Lee, J.C., Laydon, J.T., McDonnell, P.C., Gallagher, T.F., Kumar, S., Green, D., McNulty, D., Blumenthal, M.J., Heys, J.R., and Landvatter, S.W., et al. 1994. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372:739‐746.
   Leung, T., Manser, E., Tan, L., and Lim, L. 1995. A novel serine/threonine kinase binding the Ras‐related RhoA GTPase which translocates the kinase to peripheral membranes. J. Biol. Chem. 270:29051‐29054.
   Lin, J.L., Chen, H.C., Fang, H.I., Robinson, D., Kung, H.J., and Shih, H.M. 2001. MST4, a new Ste20‐related kinase that mediates cell growth and transformation via modulating ERK pathway. Oncogene 20:6559‐6569.
   Lin, L.L., Wartmann, M., Lin, A.Y., Knopf, J.L., Seth, A., and Davis, R.J. 1993. cPLA2 is phosphorylated and activated by MAP kinase. Cell 72:269‐278.
   MacGillivray, M.K., Cruz, T.F., and McCulloch, C.A. 2000. The recruitment of the interleukin‐1 (IL‐1) receptor‐associated kinase (IRAK) into focal adhesion complexes is required for IL‐1beta‐induced ERK activation. J. Biol. Chem. 275:23509‐23515.
   Malinin, N.L., Boldin, M.P., Kovalenko, A.V., and Wallach, D. 1997. MAP3K‐related kinase involved in NF‐kappaB induction by TNF, CD95 and IL‐1. Nature 385:540‐544.
   Manning, G., Whyte, D.B., Martinez, R., Hunter, T., and Sudarsanam, S. 2002. The protein kinase complement of the human genome. Science 298:1912‐1934.
   Marshall, C.J. 1995. Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal‐regulated kinase activation. Cell 80:179‐185.
   McLaughlin, M.M., Kumar, S., McDonnell, P.C., Van Horn, S., Lee, J.C., Livi, G.P., and Young, P.R. 1996. Identification of mitogen‐activated protein (MAP) kinase‐activated protein kinase‐3, a novel substrate of CSBP p38 MAP kinase. J. Biol. Chem. 271:8488‐8492.
   Milne, D.M., Campbell, D.G., Caudwell, F.B., and Meek, D.W. 1994. Phosphorylation of the tumor suppressor protein p53 by mitogen‐activated protein kinases. J. Biol. Chem. 269:9253‐9260.
   Milne, D.M., Campbell, L.E., Campbell, D.G., and Meek, D.W. 1995. p53 is phosphorylated in vitro and in vivo by an ultraviolet radiation‐induced protein kinase characteristic of the c‐Jun kinase, JNK1. J. Biol. Chem. 270:5511‐5518.
   Moore, T.M., Garg, R., Johnson, C., Coptcoat, M.J., Ridley, A.J., and Morris, J.D. 2000. PSK, a novel STE20‐like kinase derived from prostatic carcinoma that activates the c‐Jun N‐terminal kinase mitogen‐activated protein kinase pathway and regulates actin cytoskeletal organization. J. Biol. Chem. 275:4311‐4322.
   Moriguchi, T., Kuroyanagi, N., Yamaguchi, K., Gotoh, Y., Irie, K., Kano, T., Shirakabe, K., Muro, Y., Shibuya, H., Matsumoto, K., Nishida, E., and Hagiwara, M. 1996. A novel kinase cascade mediated by mitogen‐activated protein kinase kinase 6 and MKK3. J. Biol. Chem. 271:13675‐13679.
   Morrison, D.K. and Davis, R.J. 2003. Regulation of MAP kinase signaling modules by scaffold proteins in mammals. Annu. Rev. Cell Dev. Biol. 19:91‐118.
   Morton, S., Davis, R.J., McLaren, A., and Cohen, P. 2003. A reinvestigation of the multisite phosphorylation of the transcription factor c‐Jun. EMBO J. 22:3876‐3886.
   Murphy, L.O., Smith, S., Chen, R.H., Fingar, D.C., and Blenis, J. 2002. Molecular interpretation of ERK signal duration by immediate early gene products. Nat. Cell Biol. 4:556‐564.
   Nakano, K., Yamauchi, J., Nakagawa, K., Itoh, H., and Kitamura, N. 2000. NESK, a member of the germinal center kinase family that activates the c‐Jun N‐terminal kinase pathway and is expressed during the late stages of embryogenesis. J. Biol. Chem. 275:20533‐20539.
   Ni, H., Wang, X.S., Diener, K., and Yao, Z. 1998. MAPKAPK5, a novel mitogen‐activated protein kinase (MAPK)‐activated protein kinase, is a substrate of the extracellular‐regulated kinase (ERK) and p38 kinase. Biochem. Biophys. Res. Commun. 243:492‐496.
   Noguchi, K., Kitanaka, C., Yamana, H., Kokubu, A., Mochizuki, T., and Kuchino, Y. 1999. Regulation of c‐Myc through phosphorylation at Ser‐62 and Ser‐71 by c‐Jun N‐terminal kinase. J. Biol. Chem. 274:32580‐32587.
   O'Hagan, R.C., Tozer, R.G., Symons, M., McCormick, F., and Hassell, J.A. 1996. The activity of the Ets transcription factor PEA3 is regulated by two distinct MAPK cascades. Oncogene 13:1323‐1333.
   Ono, K. and Han, J. 2000. The p38 signal transduction pathway: Activation and function. Cell Signal 12:1‐13.
   Palmer, A., Gavin, A.C., and Nebreda, A.R. 1998. A link between MAP kinase and p34(cdc2)/cyclin B during oocyte maturation: p90(rsk) phosphorylates and inactivates the p34(cdc2) inhibitory kinase Myt1. EMBO J. 17:5037‐5047.
   Payne, D.M., Rossomando, A.J., Martino, P., Erickson, A.K., Her, J.‐H., Shabanowitz, J., Hunt, D.F., Weber, M.J., and Sturgill, T.W. 1991. Identification of the regulatory phosphorylation sites in pp42/mitogen activated protein kinase (MAP kinase). EMBO J. 10:885‐892.
   Pearson, G., English, J.M., White, M.A., and Cobb, M.H. 2001. ERK5 and ERK2 cooperate to regulate NF‐kappaB and cell transformation. J. Biol. Chem. 276:7927‐7931.
   Peraldi, P., Frodin, M., Barnier, J.V., Calleja, V., Scimeca, J.C., Filloux, C., Calothy, G., and Van Obberghen, E. 1995. Regulation of the MAP kinase cascade in PC12 cells: B‐Raf activates MEK‐1 (MAP kinase or ERK kinase) and is inhibited by cAMP. FEBS Lett. 357:290‐296.
   Pombo, C.M., Kehrl, J.H., Sanchez, I., Katz, P., Avruch, J., Zon, L.I., Woodgett, J.R., Force, T., and Kyriakis, J.M. 1995. Activation of the SAPK pathway by the human STE20 homologue germinal centre kinase. Nature 377:750‐754.
   Posada, J., Yew, N., Ahn, N.G., Vande‐Woude, G.F., and Cooper, J.A. 1993. Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro. Mol. Cell. Biol. 13:2546‐2552.
   Raingeaud, J., Whitmarsh, A.J., Barrett, T., Derijard, B., and Davis, R.J. 1996. MKK3‐ and MKK6‐regulated gene expression is mediated by the p38 mitogen‐activated protein kinase signal transduction pathway. Mol. Cell Biol. 16:1247‐1255.
   Raman, M. and Cobb, M.H. 2003. MAP kinase modules: Many roads home. Curr. Biol. 13:R886‐R888.
   Rana, A., Gallo, K., Godowski, P., Hirai, S., Ohno, S., Zon, L., Kyriakis, J.M., and Avruch, J. 1996. The mixed lineage kinase SPRK phosphorylates and activates the stress‐activated protein kinase activator, SEK‐1. J. Biol. Chem. 271:19025‐19028.
   Raviv, Z., Kalie, E., and Seger, R. 2004. MEK5 and ERK5 are localized in the nuclei of resting as well as stimulated cells, while MEKK2 translocates from the cytosol to the nucleus upon stimulation. J. Cell Sci. 117:1773‐1784.
   Ray, L.B. and Sturgill, T.W. 1987. Characterization of insulin‐stimulated microtubule‐associated protein kinase: Rapid isolation and stabilization of a novel serine/threonine kinase from 3T3‐L1 cells. Proc. Natl. Acad. Sci. U.S.A. 84:1502‐1506.
   Robbins, D.J. and Cobb, M.H. 1992. Extracellular signal‐regulated kinases 2 autophosphorylates on a subset of peptides phosphorylated in intact cells in response to insulin and nerve growth factor: Analysis by peptide mapping. Mol. Biol. Cell 3:299‐308.
   Roux, P.P. and Blenis, J. 2004. ERK and p38 MAPK‐activated protein kinases: A family of protein kinases with diverse biological functions. Microbiol. Mol. Biol. Rev. 68:320‐344.
   Rubinfeld, H. and Seger, R. 2004. The ERK cascade as a prototype of MAPK signaling pathways. Methods Mol. Biol. 250:1‐28.
   Sabourin, L.A. and Rudnicki, M.A. 1999. Induction of apoptosis by SLK, a Ste20‐related kinase. Oncogene 18:7566‐7575.
   Sakuma, H., Ikeda, A., Oka, S., Kozutsumi, Y., Zanetta, J.P., and Kawasaki, T. 1997. Molecular cloning and functional expression of a cDNA encoding a new member of mixed lineage protein kinase from human brain. J. Biol. Chem. 272:28622‐28629.
   Salmeron, A., Ahmad, T.B., Carlile, G.W., Pappin, D., Narsimhan, R.P., and Ley, S.C. 1996. Activation of MEK‐1 and SEK‐1 by Tpl‐2 proto‐oncoprotein, a novel MAP kinase kinase kinase. EMBO J. 15:817‐826.
   Sanchez, I., Hughes, R.T., Mayer, B.J., Yee, K., Woodgett, J.R., Avruch, J., Kyriakis, J.M., and Zon, L.I. 1994. Role of SAPK/ERK kinase‐1 in the stress‐activated pathway regulating transcription factor c‐Jun. Nature 372:794‐798.
   Seger, R. and Krebs, E.G., 1995. The MAPK signaling cascade. FASEB J. 9:726‐735.
   Seger, R., Ahn, N.G., Posada, J., Munar, E.S., Jensen, A.M., Cooper, J.A., Cobb, M.H., and Krebs, E.G., 1992. Purification and characterization of MAP kinase activator(s) from epidermal growth factor stimulated A431 cells. J. Biol. Chem. 267:14373‐14381.
   Sgouras, D.N., Athanasiou, M.A., Beal, G.J. Jr., Fisher, R.J., Blair D.G., and Mavrothalassitis, G.J. 1995. ERF: An ETS domain protein with strong transcriptional repressor activity, can suppress ets‐associated tumorigenesis and is regulated by phosphorylation during cell cycle and mitogenic stimulation. EMBO J. 14:4781‐4793.
   Shi, C.S. and Kehrl, J.H. 1997. Activation of stress‐activated protein kinase/c‐Jun N‐terminal kinase, but not NF‐kappaB, by the tumor necrosis factor (TNF) receptor 1 through a TNF receptor–associated factor 2‐ and germinal center kinase related‐dependent pathway. J. Biol. Chem. 272:32102‐32107.
   Stokoe, D., Campbell, D.G., Nakielny, S., Hidaka, H., Leevers, S.J., Marshall, C., and Cohen, P. 1992. MAPKAP kinase‐2: A novel protein kinase activated by mitogen‐activated protein kinase. EMBO J. 11:3985‐3994.
   Strahl, T., Gille, H., and Shaw, P.E. 1996. Selective response of ternary complex factor Sap1a to different mitogen‐activated protein kinase subgroups. Proc. Natl. Acad. Sci. U.S.A. 93:11563‐11568.
   Sturgill, T.W., Ray, L.B., Erikson, E., and Maller, J.L. 1988. Insulin‐stimulated MAP‐2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature 334:715‐718.
   Su, Y.C., Han, J., Xu, S., Cobb, M., and Skolnik, E.Y. 1997. NIK is a new Ste20‐related kinase that binds NCK and MEKK1 and activates the SAPK/JNK cascade via a conserved regulatory domain. EMBO J. 16:1279‐1290.
   Sun, W., Wei, X., Kesavan, K., Garrington, T.P., Fan, R., Mei, J., Anderson, S.M., Gelfand, E.W., and Johnson, G.L. 2003. MEK kinase 2 and the adaptor protein Lad regulate extracellular signal‐regulated kinase 5 activation by epidermal growth factor via Src. Mol. Cell Biol. 23:2298‐2308.
   Terasawa, K., Okazaki, K., and Nishida, E. 2003. Regulation of c‐Fos and Fra‐1 by the MEK5‐ERK5 pathway. Genes Cells 8:263‐273.
   Tournier, C., Whitmarsh, A.J., Cavanagh, J., Barrett, T., and Davis, R.J. 1997. Mitogen‐activated protein kinase kinase 7 is an activator of the c‐Jun NH2‐terminal kinase. Proc. Natl. Acad. Sci. U.S.A 94:7337‐7342.
   Tung, R.M. and Blenis, J. 1997. A novel human SPS1/STE20 homologue, KHS, activates Jun N‐terminal kinase. Oncogene 14:653‐659.
   Varfolomeev, E.E. and Ashkenazi, A. 2004. Tumor necrosis factor: An apoptosis JuNKie? Cell 116:491‐497.
   Wang, X.S., Diener, K., Tan, T.H., and Yao, Z. 1998. MAPKKK6, a novel mitogen‐activated protein kinase kinase kinase, that associates with MAPKKK5. Biochem. Biophys. Res. Commun. 253:33‐37.
   Wang, X.Z. and Ron, D. 1996. Stress‐induced phosphorylation and activation of the transcription factor CHOP (GADD153) by p38 MAP Kinase. Science 272:1347‐1349.
   Waskiewicz, A.J., Flynn, A., Proud, C.G., and Cooper, J.A. 1997. Mitogen‐activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2. EMBO J. 16:1909‐1920.
   Whitmarsh, A.J., Shore, P., Sharrocks, A.D., and Davis, R.J. 1995. Integration of MAP kinase signal transduction pathways at the serum response element. Science 269:403‐407.
   Xu, Z., Maroney, A.C., Dobrzanski, P., Kukekov, N.V., and Greene, L.A. 2001. The MLK family mediates c‐Jun N‐terminal kinase activation in neuronal apoptosis. Mol. Cell Biol. 21:4713‐4724.
   Xu, B.E., Stippec, S., Lenertz, L., Lee, B.H., Zhang, W., Lee, Y.K., and Cobb, M.H. 2004. WNK1 activates ERK5 by an MEKK2/3‐dependent mechanism. J. Biol. Chem. 279:7826‐7831.
   Yan, M., Dai, T., Deak, J.C., Kyriakis, J.M., Zon, L.I., Woodgett, J.R., and Templeton, D.J. 1994. Activation of stress‐activated protein kinase by MEKK1 phosphorylation of its activator SEK1. Nature 372:798‐800.
   Yang, B.S., Hauser, C.A., Henkel, G., Colman, M.S., Van Beveren, C., Stacey, K.J., Hume, D.A., Maki, R.A., and Ostrowski, M.C. 1996. Ras‐mediated phosphorylation of a conserved threonine residue enhances the transactivation activities of c‐Ets1 and c‐Ets2. Mol. Cell Biol. 16:538‐547.
   Yang, C.C., Ornatsky, O.I., McDermott, J.C., Cruz, T.F., and Prody, C.A. 1998. Interaction of myocyte enhancer factor 2 (MEF2) with a mitogen‐activated protein kinase, ERK5/BMK1. Nucl. Acids Res. 26:4771‐4777.
   Yang, J.J. 2002. Mixed lineage kinase ZAK utilizing MKK7 and not MKK4 to activate the c‐Jun N‐terminal kinase and playing a role in the cell arrest. Biochem. Biophys. Res. Commun. 297:105‐110.
   Yao, Z. and Seger, R. 2004. The molecular mechanism of MAPK/ERK inactivation. Curr. Genomics 5:385‐393.
   Yao, Z., Zhou, G., Wang, X.S., Brown, A., Diener, K., Gan, H., and Tan, T.H. 1999. A novel human STE20‐related protein kinase, HGK, that specifically activates the c‐Jun N‐terminal kinase signaling pathway. J. Biol. Chem. 274:2118‐2125.
   Yung, Y., Dolginov, Y., Yao, Z., Rubinfeld, H., Michael, D., Hanoch, T., Roubini, E., Lando, Z., Zharhary, D., and Seger, R. 1997. Detection of ERK activation by a novel monoclonal antibody. FEBS Lett. 408:292‐296.
   Zhang, S., Han, J., Sells, M.A., Chernoff, J., Knaus, U.G., Ulevitch, R.J., and Bokoch, G.M. 1995. Rho family GTPases regulate p38 mitogen‐activated protein kinase through the downstream mediator Pak1. J. Biol. Chem. 270:23934‐23936.
   Zhou, G., Bao, Z.Q., and Dixon, J.E. 1995. Components of a new human protein kinase signal transduction pathway. J. Biol. Chem. 270:12665‐12669.
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