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BIRB796 Inhibits All p38 MAPK Isoforms in Vitro and in Vivo*

  • Yvonne Kuma
    Affiliations
    Medical Research Council Protein Phosphorylation Unit, Dundee DD1 5EH, Scotland, United Kingdom
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  • Guadalupe Sabio
    Affiliations
    Medical Research Council Protein Phosphorylation Unit, Dundee DD1 5EH, Scotland, United Kingdom
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  • Jenny Bain
    Affiliations
    Division of Signal Transduction Therapy, Dundee DD1 5EH, Scotland, United Kingdom
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  • Natalia Shpiro
    Affiliations
    School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom
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  • Rodolfo Márquez
    Affiliations
    School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom
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  • Ana Cuenda
    Correspondence
    To whom correspondence should be addressed: MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, Scotland, UK. Tel.: 44-1382-344241l Fax: 44-1382-223778;
    Affiliations
    Medical Research Council Protein Phosphorylation Unit, Dundee DD1 5EH, Scotland, United Kingdom
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  • Author Footnotes
    * This work was supported by the U. K. Medical Research Council, The Royal Society, AstraZeneca, Boehringer Ingelheim, GlaxoSmith-Kline, Merck and Co., Merck KGaA, and Pfizer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:March 08, 2005DOI:https://doi.org/10.1074/jbc.M414221200
      The compound BIRB796 inhibits the stress-activated protein kinases p38α and p38β and is undergoing clinical trials for the treatment of inflammatory diseases. Here we report that BIRB796 also inhibits the activity and the activation of SAPK3/p38γ. This occurs at higher concentrations of BIRB796 than those that inhibit p38α and p38β and at lower concentrations than those that inhibit the activation of JNK isoforms. We also show that at these concentrations, BIRB796 blocks the stress-induced phosphorylation of the scaffold protein SAP97, further establishing that this is a physiological substrate of SAPK3/p38γ. Our results demonstrate that BIRB796, in combination with SB203580, a compound that inhibits p38α and p38β, but not the other p38 isoforms, can be used to identify physiological substrates of SAPK3/p38γ as well as those of p38α and p38β.
      The stress-activated protein kinase (SAPK)
      The abbreviations used are: SAPK, stress-activated protein kinase; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; MKK, MAPK kinase; MAPKAP, MAPK-activated protein; MAPKAP-K, MAPKAP-kinase; HEK, human embryonic kidney; EGF, epidermal growth factor.
      1The abbreviations used are: SAPK, stress-activated protein kinase; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; MKK, MAPK kinase; MAPKAP, MAPK-activated protein; MAPKAP-K, MAPKAP-kinase; HEK, human embryonic kidney; EGF, epidermal growth factor.
      p38 isoforms are mitogen-activated protein kinase (MAPK) family members that are activated by changes in the cellular environment, such as alterations in the concentration of nutrients, cytokines, cell-damaging agents, and changes in osmolarity of the surrounding medium (
      • Cohen P.
      ). They comprise p38α, p38β, SAPK3/p38γ (also known as ERK6), and SAPK4/p38δ. Each p38 isoform may have different biological functions and different physiological substrates, but they all phosphorylate substrates containing the minimal consensus sequence Ser/Thr-Pro. A major challenge of current research in this field is to identify the downstream physiological substrates and processes that each p38 MAPK regulates in the cell, as well as determining which “upstream” components regulate their activities. One of the most successful aids to the identification of physiological substrates has been the use of small cell-permeable compounds that are specific inhibitors of particular protein kinases. These compounds enter cells within minutes and act rapidly to suppress the activity of a particular kinase so that indirect effects caused, for example, by changes in gene expression or protein activity, a potential risk when cells deficient in a particular kinase are used, are excluded. Moreover, the use of protein kinase inhibitors avoids the need for transfection-based approaches, which have the potential to give misleading results since the fidelity of signaling can break down when components are overexpressed.
      Identification of physiological substrates for p38α and p38β has been greatly facilitated by the availability of specific inhibitors of these enzymes, such as the cell-permeant pyridinyl imidazole SB203580 and related compounds (
      • Cuenda A.
      • Rouse J.
      • Doza Y.N.
      • Meier R.
      • Cohen P.
      • Gallagher T.F.
      • Young P.R.
      • Lee J.C.
      ,
      • Davies S.P.
      • Reddy H.
      • Caivano M.
      • Cohen P.
      ). Substrates for p38α and p38β include other protein kinases, as well as several transcription factors and metabolic protein (
      • Kyriakis J.M.
      • Avruch J.
      ,
      • Kuma Y.
      • Campbell D.G.
      • Cuenda A.
      ). However, little is known about the physiological substrates for SAPK3/p38γ and SAPK4/p38δ as they are not inhibited by SB203580 (
      • Goedert M.
      • Cuenda A.
      • Craxton M.
      • Jakes R.
      • Cohen P.
      ,
      • Eyers P.A.
      • Craxton M.
      • Morrice N.
      • Cohen P.
      • Goedert M.
      ), and so far there are not any commercially available inhibitors for these kinases. Nevertheless, we have recently demonstrated that the synapse-associated proteins SAP90 and SAP97 are physiological substrates of SAPK3/p38γ by using a cell-permeant peptide that blocks the interaction between SAPK3/p38γ and these PDZ domain-containing proteins (
      • Sabio G.
      • Reuver S.
      • Feijoo C.
      • Hasegawa M.
      • Thomas G.M.
      • Centeno F.
      • Kuhlendahl S.
      • Leal-Ortiz S.
      • Goedert M.
      • Garner C.
      • Cuenda A.
      ,
      • Sabio G.
      • Arthur J.S.C.
      • Kuma Y.
      • Peggie M.
      • Carr J.
      • Murray-Tait V.
      • Centeno F.
      • Goedert M.
      • Morrice A.
      • Cuenda A.
      ). Moreover, by using small interfering RNA technology, we have also shown that, after cellular stress, the microtubule-associated protein Tau is an in vivo substrate of SAPK4/p38δ in neuroblastoma cells (
      • Feijoo C.
      • Campbell D.G.
      • Jakes R.
      • Goedert M.
      • Cuenda A.
      ).
      Recently, a new class of p38 inhibitors has been described. These are diaryl urea compounds, which bear little structural similarity to the other class of well characterized p38 inhibitors, the pyridinyl-imidazoles. The compound BIRB796 is one of the most potent compounds of this series and binds to p38α with both slow association and dissociation rates (
      • Pargellis C.
      • Tong L.
      • Churchill L.
      • Cirillo P.F.
      • Gilmore T.
      • Graham A.G.
      • Grob P.M.
      • Hickey E.R.
      • Moss N.
      • Pav S.
      • Regan J.
      ). BIRB796 inhibits p38α by a novel mechanism, indirectly competing with the binding of ATP. Structure determination revealed that, prior to binding, the kinase undergoes a reorganization of the activation loop exposing a critical binding domain and yielding a structure incompatible with ATP binding (
      • Pargellis C.
      • Tong L.
      • Churchill L.
      • Cirillo P.F.
      • Gilmore T.
      • Graham A.G.
      • Grob P.M.
      • Hickey E.R.
      • Moss N.
      • Pav S.
      • Regan J.
      ). BIRB796 demonstrated efficacy in an endotoxin (lipopolysaccharide)-stimulated mouse model of tumor necrosis factor-α production and in a mouse model of established collagen-induced arthritis (
      • Pargellis C.
      • Tong L.
      • Churchill L.
      • Cirillo P.F.
      • Gilmore T.
      • Graham A.G.
      • Grob P.M.
      • Hickey E.R.
      • Moss N.
      • Pav S.
      • Regan J.
      ). BIRB796 also displayed anti-inflammatory effects in a trial of human endotoxemia and has recently been in phase IIb/III clinical trials for the treatment of rheumatoid arthritis (
      • Pargellis C.
      • Tong L.
      • Churchill L.
      • Cirillo P.F.
      • Gilmore T.
      • Graham A.G.
      • Grob P.M.
      • Hickey E.R.
      • Moss N.
      • Pav S.
      • Regan J.
      ,
      • Branger J.
      • van den Blink B.
      • Weijer S.
      • Madwed J.
      • Bos C.L.
      • Gupta A.
      • Yong C.L.
      • Polmar S.H.
      • Olszyna D.P.
      • Hack C.E.
      • van Deventer S.J.
      • Peppelenbosch M.P.
      • van der Poll T.
      ,
      • Branger J.
      • van den Blink B.
      • Weijer S.
      • Gupta A.
      • van Deventer S.J.
      • Hack C.E.
      • Peppelenbosch M.P.
      • van der Poll T.
      ,
      • van den Blink B.
      • Branger J.
      • Weijer S.
      • Gupta A.
      • van Deventer S.J.
      • Peppelenbosch M.P.
      • van der Poll T.
      ).
      Here we show that in addition to p38α, other MAPK family members activated by cellular stress, such as p38β, SAPK3/p38γ, and SAPK4/p38δ, are also inhibited by BIRB796. We also show that in a cell-based assay, the use of different concentrations of BIRB796, in combination with other well characterized inhibitors of p38α/β, such as SB203580, can be a useful tool for the identification of new substrates of SAPK3/p38γ and thus for the elucidation of its physiological role.

      MATERIALS AND METHODS

      Protein Kinase Inhibitors—The compound 1-5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl]urea (BIRB796) was synthesized in four linear steps in percentage of overall yield starting from commercially available 4,4-dimethyl-3-oxopentanenitrile following the general procedure of Regan et al. (
      • Regan J.
      • Breitfelder S.
      • Cirillo P.
      • Gilmore T.
      • Graham A.G.
      • Hickey E.
      • Klaus B.
      • Madwed J.
      • Moriak M.
      • Moss N.
      • Pargellis C.
      • Pav S.
      • Proto A.
      • Swinamer A.
      • Tong L.
      • Torcellini C.
      ). SB 203580 was obtained from Calbiochem, and PD 184352 was made by custom synthesis.
      Antibodies—Phospho-specific antibodies that recognize SAP97 phosphorylated at Ser158 (antibody Phos-Ser158), Thr209 (antibody Phos-Thr209), Ser431 (antibody Phos-Ser431), or Ser442 (antibody Phos-Ser442) were raised against the peptides VSHSHIpSPIK (residues 152–161), NTDSLEpTPTYVNG (residues 203–215), DNHVpSPSSYLGQTP ASPARYSPISK, and DNHVSPSSYLGQTPApSPARYSPISK (residues 427–451) of rat SAP97 (
      • Sabio G.
      • Arthur J.S.C.
      • Kuma Y.
      • Peggie M.
      • Carr J.
      • Murray-Tait V.
      • Centeno F.
      • Goedert M.
      • Morrice A.
      • Cuenda A.
      ). An antibody that recognizes both phosphorylated and non-phosphorylated SAP97 was generated by injecting sheep at Diagnostics Scotland (Pennicuik, UK) with glutathione S-transferase-tagged SAP97 (
      • Sabio G.
      • Arthur J.S.C.
      • Kuma Y.
      • Peggie M.
      • Carr J.
      • Murray-Tait V.
      • Centeno F.
      • Goedert M.
      • Morrice A.
      • Cuenda A.
      ).
      Anti-p38β for immunoprecipitation and anti-p38α, anti-SAPK3, anti-MAPKAP-K2, anti-RSK2, and anti-ERK5 antibodies were obtained from the Division of Signal Transduction Therapy (Dundee, UK). These antibodies were affinity-purified as described (
      • Cuenda A.
      • Cohen P.
      • Buee-Scherrer V.
      • Goedert M.
      ) and used for immunoprecipitation and immunoblotting. A mouse anti-p38β2 antibody used for immunoblotting was purchased from Zymed Laboratories Inc. (San Francisco, CA). Antibodies that recognize p38α phosphorylated at Thr180 and Tyr182 (these antibodies also recognize phosphorylated p38β, SAPK3/p38γ, and SAPK4/p38δ), ERK1/ERK2, phospho-ERK1/ERK2 (Thr202/Tyr204), JNK1/JNK2, phospho-JNK1/JNK2 (Thr183/Tyr185), c-Jun, and c-Jun phosphorylated at Ser63 were obtained from Cell Signaling Technologies (Hitchin, UK).
      Protein Expression—All protein kinases were of human origin and were expressed either as glutathione S-transferase fusion protein, as maltose-binding protein in Escherichia coli, or as hexahistidine (His6)-tagged protein in insect Sf9 or Sf21 cells and purified as described previously (
      • Davies S.P.
      • Reddy H.
      • Caivano M.
      • Cohen P.
      ,
      • Bain J.
      • McLauchlan H.
      • Elliott M.
      • Cohen P.
      ).
      Activation of Protein Kinases—JNK1–3 were activated with MKK4; p38α, p38β, SAPK3/p38γ, and SAPK4/p38δ were activated with MKK6; protein kinase B, serum and glucocorticoid-inducible kinase, and S6K1 were activated with PDK1; ERK2 was activated with MKK1; MAPKAP-K1a, MAPKAP-K2, PRAK, and MSK1 were activated with ERK2; MKK1 was activated with c-Raf; and MKK6 and MKK4 were activated with MEKK-1 as described previously (
      • Davies S.P.
      • Reddy H.
      • Caivano M.
      • Cohen P.
      ,
      • Bain J.
      • McLauchlan H.
      • Elliott M.
      • Cohen P.
      ).
      Protein Kinase Assays—All protein kinase assays were linear with respect to time. Assays were either performed manually for 10 min at 30 °C in 50-μl incubations using 150 nm p38 MAPK isoforms and 10 μm [γ-32P]ATP or performed with a Biomek 2000 laboratory automation workstation in a 96-well format (Beckman Instruments) for 40 min at ambient temperature in 25-μl incubations using [γ-33P]ATP. The concentrations of magnesium acetate and ATP in the assays were 10 mm and 10 μm, respectively, unless stated otherwise. Assays were initiated with MgATP. Manual assays were terminated by spotting aliquots of each incubation onto phosphocellulose paper followed by immersion in 75 mm phosphoric acid. Robotic assays were stopped by the addition of 5 μl of 0.5 m orthophosphoric acid. Aliquots were then spotted onto P81 filtermats, washed four times in 75 mm phosphoric acid to remove ATP, washed once in acetone (manual assays) or methanol (robotic assays), and then dried and counted for radioactivity. All protein kinases were assayed as described previously (
      • Davies S.P.
      • Reddy H.
      • Caivano M.
      • Cohen P.
      ,
      • Bain J.
      • McLauchlan H.
      • Elliott M.
      • Cohen P.
      ). MAPKAP-K2 and RSK2 were immunoprecipitated from cell lysates using specific antibodies and assayed as described (
      • Davies S.P.
      • Reddy H.
      • Caivano M.
      • Cohen P.
      ,
      • Bain J.
      • McLauchlan H.
      • Elliott M.
      • Cohen P.
      ,
      • Alessi D.R.
      • Cuenda A.
      • Cohen P.
      • Dudley D.T.
      • Saltiel A.R.
      ,
      • Haydon C.E.
      • Watt P.W.
      • Morrice N.
      • Knebel A.
      • Gaestel M.
      • Cohen P.
      ).
      Cell Culture, Transfection, and Lysis—Human embryonic kidney (HEK) 293 and HeLa cells were cultured in Dulbecco's modified Eagle's medium at 37 °C, supplemented with 10% fetal calf serum, 50 units of penicillin/ml, 50 μg/ml streptomycin (Invitrogen), and 2 mm glutamine (BioWhittaker). Mouse embryonic fibroblasts were cultured as described previously (
      • Sabio G.
      • Arthur J.S.C.
      • Kuma Y.
      • Peggie M.
      • Carr J.
      • Murray-Tait V.
      • Centeno F.
      • Goedert M.
      • Morrice A.
      • Cuenda A.
      ), and C2C12 myoblasts were cultured as described in Ref.
      • Cuenda A.
      • Cohen P.
      .
      Cells were exposed to 0.5 m sorbitol for 30 min or 100 ng/ml EGF for 10 min and then lysed in buffer A (50 mm Tris-HCl, pH 7.5, 1 mm EGTA, 1 mm EDTA, 1 mm sodium orthovanadate, 10 mm sodium fluoride, 50 mm sodium β-glycerophosphate, 5 mm pyrophosphate, 0.27 m sucrose, 0.1 mm phenylmethylsulfonyl fluoride, 1% (v/v) Triton X-100) plus 0.1% (v/v) 2-mercaptoethanol and Complete proteinase inhibitor mixture from Roche Applied Science. Lysates were centrifuged at 18,000 × g for 5 min at 4 °C, and the supernatants were removed, quick-frozen in liquid nitrogen, and stored at –20 °C until use. When required, cells were preincubated for 1 h without or with 10 μm SB 203580 or 10 μm PD 184352 or with different concentrations of BIRB796 for the times indicated in the figures.
      Immunoprecipitation from Cell Lysates—MAPKAP-K2 and SAP97 were immunoprecipitated from 0.1 to 1 mg of HEK293 cells, whereas RSK2 was immunoprecipitated from 0.1 mg of HeLa cell extract. Extracts were incubated with 3 and 5 μg of the specific antibodies coupled to protein G-Sepharose, respectively (Amersham Biosciences). After incubation for 2 h at 4 °C, the captured proteins were centrifuged at 13,000 × g, the supernatant was discarded, and the beads were washed once in buffer A containing 0.5 m NaCl and then washed twice in buffer A alone. Samples were denatured, electrophoresed in precast polyacrylamide gels (Invitrogen), and then immunoblotted. Quantification of protein phosphorylation and total protein after immunoblotting was carried out using the Odyssey™ infrared imaging system (LI-COR Biosciences).

      RESULTS

      BIRB796 Inhibits All Four SAPK/p38s Isoforms in Vitro— There are about 500 protein kinases encoded in the human genome, most of which belong to the same superfamily. It is therefore difficult to develop compounds that inhibit one particular protein kinase without inhibiting several related enzymes. Establishing the specificity of any particular inhibitor is a critical issue. We have previously examined the specificity of more than 40 commercially available compounds against a large panel of protein kinases and found that most inhibitors target more than one kinase (
      • Davies S.P.
      • Reddy H.
      • Caivano M.
      • Cohen P.
      ,
      • Bain J.
      • McLauchlan H.
      • Elliott M.
      • Cohen P.
      ). There is therefore a danger that in cell-based assays, the observed effects do not result from inhibition of the kinase of interest but rather from the inhibition of another protein kinase. As the selectivity of BIRB796 against only 11 different kinases was reported previously (
      • Pargellis C.
      • Tong L.
      • Churchill L.
      • Cirillo P.F.
      • Gilmore T.
      • Graham A.G.
      • Grob P.M.
      • Hickey E.R.
      • Moss N.
      • Pav S.
      • Regan J.
      ), we decided to extend the specificity of this compound to a larger panel of protein kinases. We found that, at a concentration of 10 μm, BIRB796 inhibited the activity of p38α, Lck, and JNK2 as described previously (
      • Pargellis C.
      • Tong L.
      • Churchill L.
      • Cirillo P.F.
      • Gilmore T.
      • Graham A.G.
      • Grob P.M.
      • Hickey E.R.
      • Moss N.
      • Pav S.
      • Regan J.
      ). However, it also inhibited the activity of the other three p38 MAPK isoforms p38β, SAPK3/p38γ, and SAPK4/p38δ. It had little effect on the other protein kinases in the panel (Table I).
      Table IInhibition of protein kinases by BIRB796 inhibitor The inhibitor concentrations used are shown in parentheses. Results are presented as activity relative to that in control incubations where inhibitor was omitted (means of duplicate determinations). The time of pre-incubation of BIRB796 with the kinase was 5 min. ATP concentration was 5 μm in SAPK3/p38γ, SAPK4/p38δ, protein kinase B (PKB), GSK3β, CDK2 and MKK1 assays; 20 μm in JNK1 to 3, PRAK, ROCK-II, p38β, CDK2/cyclinA, CHK1, MSK1, CSK, S6K1, cAMP-dependent protein kinase (PKA), CK1, MAPKAP-K2, serum and glucocorticoid-inducible kinase (SGK), protein kinase C-α (PKCα), and PDK1 assays; and 50 μm for the rest of the kinases listed in the table.
      Protein kinaseBIRB796BIRB796
      1 μm10 μm
      MKK171 ± 075 ± 12
      ERK279 ± 1272 ± 3
      JNK1α1106 ± 099 ± 1
      JNK2α260 ± 1250 ± 8
      JNK3α1100 ± 591 ± 6
      p38α3 ± 13 ± 1
      p38β7 ± 03 ± 0
      SAPK3/p38γ29 ± 63 ± 2
      SAPK4/p38δ47 ± 811 ± 0
      RSK159 ± 152 ± 8
      MAPKAP-K293 ± 690 ± 12
      MSK198 ± 283 ± 9
      PRAK71 ± 665 ± 12
      PKA88 ± 279 ± 2
      PKCα80 ± 370 ± 12
      PDK189 ± 189 ± 0
      PKB100 ± 8102 ± 5
      SGK94 ± 1393 ± 3
      S6K1119 ± 12108 ± 1
      GSK3β112 ± 5107 ± 7
      ROCK-II80 ± 378 ± 1
      AMPK
      AMPK, AMP-activated kinase
      89 ± 993 ± 9
      CHK196 ± 4106 ± 9
      CK285 ± 184 ± 4
      PHK
      PHK, phosphorylase kinase
      115 ± 0102 ± 10
      Lck42 ± 518 ± 0
      CSK
      CSK, C-terminal Src kinase
      101 ± 1293 ± 2
      CDK2/cyclinA102 ± 1593 ± 1
      CK187 ± 278 ± 2
      DYRK1a82 ± 979 ± 0
      NEK695 ± 993 ± 10
      a AMPK, AMP-activated kinase
      b PHK, phosphorylase kinase
      c CSK, C-terminal Src kinase
      Time-dependent Inhibition of p38 MAPKs by BIRB796 —It has been shown that BIRB796 inhibits p38α activity in a time-dependent manner due to its slow binding behavior (
      • Pargellis C.
      • Tong L.
      • Churchill L.
      • Cirillo P.F.
      • Gilmore T.
      • Graham A.G.
      • Grob P.M.
      • Hickey E.R.
      • Moss N.
      • Pav S.
      • Regan J.
      ). To examine whether or not BIRB976 affects the activity of other p38 isoforms similarly, we monitored the apparent inhibitory potency of the compound as a function of the time of preincubation with the kinase (Fig. 1A). We observed firstly that BIRB796 blocked the individual p38 MAPK activities at different potencies in vitro. Thus, this compound inhibited p38α more potently than p38β and inhibited p38β more potently than SAPK3/p38γ, whereas SAPK4/p38δ was inhibited the least (Fig. 1, A and B). Secondly, the apparent IC50 value for all p38 MAPK isoforms decreased as the time of preincubation with the inhibitor increased (Fig. 1, A and B). These results are consistent with the slow binding kinetics of this compound to the p38 MAPK isoforms.
      Figure thumbnail gr1
      Fig. 1Time-dependent inhibition of all four p38 MAPK isoforms by BIRB796. All four activated recombinant human p38 MAPKs (150 nm) were incubated at room temperature in the presence of the indicated concentrations of BIRB796 for 0 (open circles), 30 (gray circles), or 90 min (closed circles) before being assayed for 10 min using mielin basic protein as substrate as described under “Materials and Methods.”
      BIRB796 Is a Potent Inhibitor of p38 Isoforms in Cells—To test whether or not BIRB796 could inhibit each p38 MAPK in cells, we first investigated the effect that this compound had on the activation of one known physiological substrate of p38α, namely the protein kinase MAPKAP-K2. We incubated HEK293 cells with different concentrations of BIRB796 for 30 min or 2 h prior to stimulation with sorbitol (an osmotic shock) and examined the activation of MAPKAP-K2 by measuring its activity (Fig. 2A). MAPKAP-K2 activation was inhibited in these cells in a time-dependent manner with an apparent IC50 of 30 nm after 30 min or 8 nm after 2 h of preincubation with BIRB796 (Fig. 2A). MAPKAP-K2 activation was also blocked by preincubation of the cells with the p38 MAPK inhibitor SB203580 as described previously (
      • Cuenda A.
      • Rouse J.
      • Doza Y.N.
      • Meier R.
      • Cohen P.
      • Gallagher T.F.
      • Young P.R.
      • Lee J.C.
      ) (Fig. 2A).
      Figure thumbnail gr2
      Fig. 2Effect of BIRB796 on the activity and activation of p38α and p38β in cells. A, BIRB796 inhibits the activation of MAPKAP-K2. HEK293 cells were preincubated for 2 h (gray bars) or 30 min (black bars) in the presence of the indicated concentrations of BIRB796 or preincubated for 1 h in the presence of 10 μm SB203580. Cells were stimulated for 30 min with 0.5 m sorbitol in the continued presence or absence of the inhibitors and lysed. MAPKAP-K2 was immunoprecipitated from 0.2 mg of lysate protein and assayed as described under “Materials and Methods.” B, BIRB796 inhibits the activation of p38α. Lysates (50 μg) from HEK293 treated as in panel A were immunoblotted using either an antibody that recognizes phosphorylated p38α (Phos-p38α) or an antibody that recognizes both phosphorylated and unphosphorylated p38α. C, BIRB796 inhibits the activation of p38β. C2C12 myoblasts were treated as in panel A, and p38β was immunoprecipitated from 3 mg of cell lysate and immunoblotted with either the p38α phospho-specific antibody, which also recognizes p38β, or an antibody that recognizes both phosphorylated and unphosphorylated p38β.
      We also examined the phosphorylation of p38α and p38β under the same conditions and observed that the phosphorylation of these two kinases was also blocked by pretreatment of the cells with the compound (Fig. 2, B and C). These results suggest that the binding of BIRB796 to these p38 MAPKs is also impairing their phosphorylation by the upstream kinases MKK6/MKK3 and/or enhancing their dephosphorylation. On the other hand, preincubation of cells with the SB203580 did not prevent p38α phosphorylation by this agonist (Fig. 2B), although it blocked p38β phosphorylation, as shown previously (
      • Kuma Y.
      • Campbell D.G.
      • Cuenda A.
      ) (Fig. 2C).
      To test whether BIRB796 could also inhibit the phosphorylation, and therefore activation, of SAPK3/p38γ and SAPK4/p38δ, cells were preincubated with different concentrations of BIRB796 for 30 min or 2 h before stimulation with sorbitol. The phosphorylation of SAPK3/p38γ was blocked by this compound (Fig. 3A) with an apparent IC50 of 90 nm or 35 nm after 30 min or 2 h preincubation, respectively (Fig. 3B), which are 3 and 4-fold higher than the IC50 for blockade of MAPKAP-K2 activation. Since the expression level of SAPK4/p38δ in HEK293 cells is very low and this isoform is not activated in these cells after sorbitol treatment (
      • Sabio G.
      • Arthur J.S.C.
      • Kuma Y.
      • Peggie M.
      • Carr J.
      • Murray-Tait V.
      • Centeno F.
      • Goedert M.
      • Morrice A.
      • Cuenda A.
      ), the effect of BIRB796 on the activation of this kinase was studied in mouse embryonic fibroblasts. Surprisingly, SAPK4/p38δ phosphorylation after osmotic shock was enhanced at low doses of the inhibitor and blocked at higher concentrations of BIRB796 (Fig. 4). The apparent IC50 for SAPK4/p38δ was ∼20-fold higher than the IC50 for SAPK3/p38γ. Phosphorylation of SAPK4/p38δ after stimulation was also enhanced when cells were preincubated with the p38α/p38β inhibitor SB203580 (Fig. 4). One possible explanation for this result is that p38α/p38β negatively regulates SAPK4/p38δ activation by an unknown mechanism. Alternatively the binding of BIRB796 and SB203580 to p38α/p38β may allow MKK6 and/or MKK3 to activate SAPK4/p38δ preferentially.
      Figure thumbnail gr3
      Fig. 3Effect of BIRB796 on the activation of SAPK3/p38γ. A, HEK293 cells were preincubated with different concentrations of BIRB796 or 10 μm SB203580 as indicated in before being exposed to 0.5 m sorbitol for 30 min. Cell lysates (50 μg) were immunoblotted with either the p38α phospho-specific antibody (Phos-p38α), which also recognizes phosphorylated SAPK3/p38γ (Phos-SAPK3), or an antibody that recognizes both phosphorylated and unphosphorylated SAPK3/p38γ (SAPK3). B, the intensity of the bands in the immunoblots from panel A was quantified as described under “Materials and Methods,” and the percentage of inhibition of SAPK3/p38γ phosphorylation was calculated. Results in panel B are shown as the mean ± S.E. for duplicate determinations from two experiments.
      Figure thumbnail gr4
      Fig. 4Effect of BIRB796 on the activation of SAPK4/p38δ in mouse embryonic fibroblasts. Mouse embryonic fibroblasts were preincubated for 2 h with different concentrations of BIRB796 or 10 μm SB203580 as indicated before being exposed to 0.5 m sorbitol for 30 min. Endogenous SAPK4/p38δ was immunoprecipitated from 2 mg of cell lysate, and the pellets were immunoblotted using the p38α phospho-specific antibody that also recognizes SAPK4/p38δ (Phos-SAPK4) or using an antibody that recognizes both phosphorylated and unphosphorylated SAPK4/p38δ (SAPK4).
      BIRB796 Blocks the Phosphorylation of the SAPK3/p38γ Substrate SAP97 in Cells—The scaffold protein SAP97 is the mammalian homologue of the Drosophila discs large tumor suppressor and a physiological substrate of SAPK3/p38γ. SAP97 becomes phosphorylated in HEK293 cells at four major residues (Ser158, Thr209, Ser431, and Ser442) in response to osmotic stress (
      • Sabio G.
      • Arthur J.S.C.
      • Kuma Y.
      • Peggie M.
      • Carr J.
      • Murray-Tait V.
      • Centeno F.
      • Goedert M.
      • Morrice A.
      • Cuenda A.
      ) (Fig. 5A). The phosphorylation of all four residues was greatly reduced when cells were pretreated with 1 μm BIRB796 before stimulation but not by pretreatment with 10 μm SB203580 (Fig. 5A). The apparent IC50 for inhibition of Ser158 phosphorylation was 150 nm or 60 nm after 30 min or 2 h preincubation, respectively (Fig. 5, B and C), similar to the IC50 for inhibition of SAPK3/p38γ.
      Figure thumbnail gr5
      Fig. 5Effect of BIRB796 on SAP97 phosphorylation. A, phosphorylation of SAP97 in HEK293 cells after osmotic shock. Cells were incubated for 2 h with or without 10 μm SB203580 or 1 μm BIRB796 and then exposed for 20 min to 0.5 m sorbitol. Endogenous SAP97 was immunoprecipitated from 0.5 to 5 mg of cell lysate, and the pellets were immunoblotted using an antibody that recognizes SAP97 phosphorylated at Ser158 (Phos-Ser158), Thr209 (Phos-Thr209), Ser431 (Phos-Ser431), Ser442 (Phos-Ser442), and an antibody that recognizes unphosphorylated and phosphorylated SAP97 equally well (SAP97). B, HEK293 cells were preincubated with different concentrations of BIRB796 or 10 μm SB203580 as indicated before being exposed to 0.5 m sorbitol for 30 min. SAP97 was immunoprecipitated from 1 mg of cell lysate and immunoblotted using either anti-Phos-Ser158 or the antibody that recognizes unphosphorylated and phosphorylated SAP97. C, the intensity of the bands in immunoblots from panel B was quantified as described under “Materials and Methods,” and the percentage of inhibition of SAP97 phosphorylation was calculated. Results in panel C are shown as the mean ± S.E. for duplicate determinations from two experiments.
      BIRB796 Inhibits JNK Activation in Vivo—We also examined the effect of the inhibitor BIRB796 on the activation of the c-Jun N-terminal kinase (JNK), which is also a MAPK family members activated by stress. Exposure of cells to osmotic shock caused an increase in the phosphorylation of both alternative spliced isoforms (46 and 54 kDa) of JNK1 and JNK2, which was completely impaired when cells were preincubated with 10 μm BIRB796 (Fig. 6A). Inhibition of p54(JNK1/2) phosphorylation was more sensitive to BIRB796 than p46(JNK1/2) phosphorylation (Fig. 6A). Thus, after a 2-h preincubation with the inhibitor, the apparent IC50 for p46(JNK1/2) was 1 μm, and for p54(JNK1/2), it was 350 nm, whereas the apparent IC50 for p54(JNK1/2) was higher than 1 μm when cells were incubated for 30 min with BIRB796 prior to stimulation. These results suggest that BIRB796 inhibits JNK by a mechanism similar to the p38 MAPK isoforms.
      Figure thumbnail gr6
      Fig. 6Effect of BIRB796 on the activation of JNK1/2 and the phosphorylation of c-Jun. HEK293 cells were preincubated with different concentrations of BIRB796 or 10 μm SB203580 as indicated in before being exposed to 0.5 m sorbitol for 30 min. A, cell lysates (100 μg) were immunoblotted with either the JNK1/2 phospho-specific antibody (Phos-JNK1/2) or an antibody that recognizes both phosphorylated and unphosphorylated JNK1/2 (JNK1/2). B, c-Jun from 0.2 mg of cell lysate was immunoblotted using an antibody that recognizes c-Jun phosphorylated at Ser63 (Phos-C-Jun) and an antibody that recognizes unphosphorylated and phosphorylated protein equally well (C-Jun). The intensity of the bands in immunoblots from panels A and B was quantified as described under “Materials and Methods,” and the percentage of inhibition of JNK1/JNK2 and c-Jun phosphorylation was calculated. Results are shown as the mean ± S.E. for duplicate determinations from two experiments.
      The phosphorylation of one physiological substrate of JNK, the transcription factor c-Jun, was also examined (Fig. 6B). After exposure of the cells to osmotic shock in the presence of increasing concentrations of BIRB796, c-Jun was immunoblotted using a phospho-specific antibody, which specifically recognizes Ser63. Phosphorylation of c-Jun was completely blocked at 10 μm BIRB796 with an apparent IC50 of 1 μm after 2 h of preincubation with the inhibitor (Fig. 6B), similar to the IC50 for inhibition of p46(JNK1/2) activation under these conditions (Fig. 6A). Taken together, these results show that BIRB796 blocks JNK1/2 activation and activity in HEK293 cells but at higher concentrations than those needed to block p38α, p38β, or SAPK3/p38γ.
      BIRB796 Does Not Inhibit ERK1/2 or ERK5 Activation in Vivo—To test further the specificity of BIRB796, we examined its effect on the activation and activity of other MAPK family members, ERK1/2 and ERK5. These experiments were carried out in HeLa cells in which the activation of the ERK1/2 and ERK5 pathways by EGF is well characterized (
      • Mody N.
      • Leitch J.
      • Armstrong C.
      • Dixon J.
      • Cohen P.
      ).
      Phosphorylation of ERK1/ERK2 and activation of RSK2, one of their in vivo substrates, were not prevented by preincubation for up to 2 h of the cells with even 10 μm BIRB796, whereas preincubation with the classical MAPK pathway inhibitor PD184352 completely blocked ERK1/ERK2 phosphorylation and RSK2 activation (Fig. 7, A and B). These results indicated that BIRB796 does not inhibit the activation and activity of ERK1/ERK2.
      Figure thumbnail gr7
      Fig. 7BIRB796 has no effect on ERK1/2 or ERK5 activation. A, HeLa cells were preincubated either for 2 h in the presence of the indicated concentrations of BIRB796 or for 1 h in the presence of 10 μm PD184352 and then for 10 min with 100 ng/ml EGF in the continued presence of the inhibitors. Lysates (50 μg) were immunoblotted using either an antibody that recognizes phosphorylated ERK1/2 (Phos-ERK1/2) or an antibody that recognizes both phosphorylated and unphosphorylated ERK1/2 (ERK1/2). B, RSK2 was immunoprecipitated from 0.1 mg of protein lysate from cells treated as in panel A and assayed as described under “Materials and Methods.” C, lysates (50 μg) from cells treated as in panel A were immunoblotted using anti-ERK5 antibody that recognizes both phosphorylated and unphosphorylated form of the kinase (Phos-ERK5) to detect the band-shift caused by phosphorylation.
      The phosphorylation of ERK5 could be detected by a small decrease in its electrophoretic mobility using an antibody that recognizes the phosphorylated and the dephosphorylated forms of the protein equally well (
      • Mody N.
      • Leitch J.
      • Armstrong C.
      • Dixon J.
      • Cohen P.
      ). Preincubation of HeLa cells with PD184352 inhibited the EGF-induced phosphorylation of ERK5, as expected from earlier studies (
      • Mody N.
      • Leitch J.
      • Armstrong C.
      • Dixon J.
      • Cohen P.
      ,
      • Kamakura S.
      • Moriguchi T.
      • Nishida E.
      ) (Fig. 7C). However, increasing concentrations of BIRB796 did not affect the mobility of the kinase caused by its phosphorylation and therefore did not block the activation of ERK5 induced by EGF (Fig. 7C).
      BIRB796 Inhibits the Phosphorylation of All Four SAPK/p38s Isoforms in Vitro—To examine whether or not the binding of BIRB796 to p38 MAPKs or JNKs also impaired their phosphorylation by the upstream kinases MKK6 or MKK4, respectively, and/or accelerated their dephosphorylation, we first studied the in vitro phosphorylation of each p38 MAPK isoform by MKK6 and the phosphorylation of the JNK2α2 isoform by MKK4. We found that BIRB796 blocked the individual p38 MAPK and the JNK2α2 phosphorylation at the same potency (Fig. 8A). On the other hand, pretreatment of the cells with the phosphatase inhibitor vanadate, to prevent MAPK dephosphorylation, did not cause any effect on the inhibition of the phosphorylation of SAPK3/p38γ, p38α, or JNK1/2 by BIRB796 after sorbitol stimulation (Fig. 8B). As a control, we checked that preincubation of cells with vanadate increased the phosphorylation of ERK1/2, indicating that the phosphatase inhibitor was working (Fig. 8B). All of these results suggest that the binding of BIRB796 to the p38 MAPKs or JNK1/2 is impairing their phosphorylation by the upstream kinase MKK6 or MKK4 rather than enhancing their dephosphorylation.
      Figure thumbnail gr8
      Fig. 8Effect of BIRB796 on the phosphorylation of p38 MAPKs and JNKs. A, the rate of phosphorylation, of each p38 isoform and JNK2α2, relative to control in the absence of BIRB796 (100%). MKK6 and MKK4 were activated in vitro (see “Materials and Methods”). The phosphorylation of each p38 was studied at MKK6 concentration of 10 units/ml for p38α, 100 units/ml for p38β, 50 units/ml for SAPK3/p38γ, or 20 units/ml for SAPK4/p38δ, and the phosphorylation of JNK2α2 was studied at an MKK4 concentration of 10 units/ml. All four recombinant human p38 MAPKs or JNK2α2 (1 μg) were incubated at room temperature with MKK6 or MKK4, respectively, in the presence of the indicated concentration of BIRB796 for 30 min before starting the reaction with Mg-[γ32P]ATP for 10 min as described under “Materials and Methods.” Results are shown as the mean ± S.E. for duplicate determinations from two experiments. B, HEK293 cells were preincubated for 2 h with different concentrations of BIRB796 or 10 μm SB203580 as indicated and for 1 h in the presence or absence of 10 mm orthovanadate before being exposed to 0.5 m sorbitol for 30 min. Cell lysates (50 μg) were immunoblotted with either the p38α phospho-specific antibody, which also recognizes phosphorylated SAPK3/p38γ (Phos-p38α), or an antibody that recognizes both phosphorylated and unphosphorylated p38α (p38α). Cell lysates (100 μg) were immunoblotted with either the JNK1/2 phospho-specific antibody (Phos-JNK1/2) or an antibody that recognizes both phosphorylated and unphosphorylated JNK1/2 (JNK1/2). As control, lysates (50 μg) from cells incubated for 1 h with or without 10 mm orthovanadate were immunoblotted using either an antibody that recognizes phosphorylated ERK1/2 (Phos-ERK1/2) or an antibody that recognizes both phosphorylated and unphosphorylated ERK1/2 (ERK1/2).

      DISCUSSION

      Recently, it has been described that the diaryl urea compound BIRB796 is a highly selective inhibitor of the protein kinase p38α (
      • Pargellis C.
      • Tong L.
      • Churchill L.
      • Cirillo P.F.
      • Gilmore T.
      • Graham A.G.
      • Grob P.M.
      • Hickey E.R.
      • Moss N.
      • Pav S.
      • Regan J.
      ). To establish the specificity of a particular inhibitor is a critical issue as a means for therapeutic intervention. Since protein kinases are a large class of enzymes, most of which belong to the same family, and the degree of homology across the entire family is relatively high, especially within the catalytic core (
      • Hanks S.K.
      • Quinn A.M.
      • Hunter T.
      ), it is difficult to develop compounds that inhibit one particular protein kinase without inhibiting several related enzymes. For this reason, we decided to reexamine the specificity of the BIRB796 compound. The work described in the present study shows that BIRB796 inhibits the activity in vitro, and the activation in the cell, of all p38 MAPK and JNK1/2 isoforms.
      Structural analysis of p38α has shown that BIRB796 binds to a novel site within the ATP-binding pocket of the kinase, which is created a by conformational change in the enzyme induced by the inhibitor, which yields to a structure incompatible with ATP binding (
      • Pargellis C.
      • Tong L.
      • Churchill L.
      • Cirillo P.F.
      • Gilmore T.
      • Graham A.G.
      • Grob P.M.
      • Hickey E.R.
      • Moss N.
      • Pav S.
      • Regan J.
      ). Moreover, solution studies have demonstrated that this class of compound has slow binding kinetics, consistent with the requirement for a conformational change (
      • Pargellis C.
      • Tong L.
      • Churchill L.
      • Cirillo P.F.
      • Gilmore T.
      • Graham A.G.
      • Grob P.M.
      • Hickey E.R.
      • Moss N.
      • Pav S.
      • Regan J.
      ). Our in vitro and in vivo data show that BIRB796 inhibits all p38 MAPK and JNK isoforms in a time-dependent manner, suggesting that the association of this compound with each of these kinases is slow, probably due to the requirement of the previously described conformational change. BIRB796 binding is targeted to the Phe residue in the conserved DFG motif that is buried in a hydrophobic pocket between the two major lobes of the kinase domain (
      • Pargellis C.
      • Tong L.
      • Churchill L.
      • Cirillo P.F.
      • Gilmore T.
      • Graham A.G.
      • Grob P.M.
      • Hickey E.R.
      • Moss N.
      • Pav S.
      • Regan J.
      ). Although the residues in this pocket are highly conserved among all of these kinases, we have observed that BIRB796 is more selective for p38α and p38β than for the SAPK3/p38γ and SAPK4/p38δ isoforms and other members of the MAPK family. This could be due to the fact that this diaryl urea inhibitor also utilizes the hydrophobic pocket containing Thr106 (
      • Pargellis C.
      • Tong L.
      • Churchill L.
      • Cirillo P.F.
      • Gilmore T.
      • Graham A.G.
      • Grob P.M.
      • Hickey E.R.
      • Moss N.
      • Pav S.
      • Regan J.
      ) a residue unique to p38α and p38β.
      Moreover, we also show that BIRB796 impairs the phosphorylation of p38 MAPKs or JNKs by the upstream kinase MKK6 or MKK4 but does not affect their dephosphorylation in vivo. Our results suggest that the conformational change caused by the binding of the inhibitor to the MAPK may affect the structure of both its phosphorylation site and the docking site for the upstream activator, therefore impairing the phosphorylation of p38 MAPKs or JNKs.
      We also show that incubation of cells with BIRB796 prior to stimulation blocks phosphorylation of the physiological substrates of p38α and SAPK3/p38γ. In particular, BIRB796 impairs the phosphorylation induced by osmotic shock of SAP97, which is a SAPK3/p38γ substrate.
      Recently, we have shown that the phosphorylation of different PDZ domain-containing proteins by SAPK3/p38γ, such as SAP97, is dependent on the interaction of the C-terminal sequence -ETXL of the kinase with the PDZ domain of these proteins (
      • Sabio G.
      • Reuver S.
      • Feijoo C.
      • Hasegawa M.
      • Thomas G.M.
      • Centeno F.
      • Kuhlendahl S.
      • Leal-Ortiz S.
      • Goedert M.
      • Garner C.
      • Cuenda A.
      ,
      • Hasegawa M.
      • Cuenda A.
      • Spillantini M.G.
      • Thomas G.M.
      • Buee-Scherrer V.
      • Cohen P.
      • Goedert M.
      ). Exploiting this characteristic, we designed a cell-permeant peptide to identify different SAPK3/p38γ substrates in vivo. This peptide blocks the phosphorylation of PDZ domain-containing proteins by SAPK3/p38γ specifically, but not phosphorylation by other MAPK in intact cells, by preventing the association of the kinase with the PDZ domain of the protein substrate (
      • Sabio G.
      • Reuver S.
      • Feijoo C.
      • Hasegawa M.
      • Thomas G.M.
      • Centeno F.
      • Kuhlendahl S.
      • Leal-Ortiz S.
      • Goedert M.
      • Garner C.
      • Cuenda A.
      ,
      • Sabio G.
      • Arthur J.S.C.
      • Kuma Y.
      • Peggie M.
      • Carr J.
      • Murray-Tait V.
      • Centeno F.
      • Goedert M.
      • Morrice A.
      • Cuenda A.
      ). The peptide contains the last nine residues of the SAPK3/p38γ fused to the cell-membrane transduction domain of the human immunodeficiency virus-type I (HIV-1) Tat protein (
      • Sabio G.
      • Reuver S.
      • Feijoo C.
      • Hasegawa M.
      • Thomas G.M.
      • Centeno F.
      • Kuhlendahl S.
      • Leal-Ortiz S.
      • Goedert M.
      • Garner C.
      • Cuenda A.
      ,
      • Sabio G.
      • Arthur J.S.C.
      • Kuma Y.
      • Peggie M.
      • Carr J.
      • Murray-Tait V.
      • Centeno F.
      • Goedert M.
      • Morrice A.
      • Cuenda A.
      ,
      • Aarts M.
      • Liu Y.
      • Liu L.
      • Besshoh S.
      • Arundine M.
      • Gurd J.W.
      • Wang Y.T.
      • Salter M.W.
      • Tymianski M.
      ). This peptide has been very useful in the validation of PDZ domain-containing proteins as SAPK3/p38γ substrates such as SAP90/PSD95 (
      • Sabio G.
      • Reuver S.
      • Feijoo C.
      • Hasegawa M.
      • Thomas G.M.
      • Centeno F.
      • Kuhlendahl S.
      • Leal-Ortiz S.
      • Goedert M.
      • Garner C.
      • Cuenda A.
      ) or SAP97 (
      • Sabio G.
      • Arthur J.S.C.
      • Kuma Y.
      • Peggie M.
      • Carr J.
      • Murray-Tait V.
      • Centeno F.
      • Goedert M.
      • Morrice A.
      • Cuenda A.
      ). However, the availability of new cell-permeant SAPK3/p38γ inhibitors would be extremely useful in helping to elucidate the physiological role of this kinase and to find other possible substrates that do not contain PDZ domains.
      The results presented in this study show that it is possible to vary the concentration of BIRB796 in the culture medium to differentially inhibit particular stress-activated protein kinases in combination with the more specific p38α/β inhibitor SB203580. For example, in HEK293 cells exposed to osmotic shock that activates p38 MAPKs and JNKs, BIRB796 at 0.1 μm inhibits p38α/β specifically, whereas at 0.3 μm, it also inhibits SAPK3/p38γ. Thus, physiological substrates for SAPK3/p38γ can be identified by identifying proteins, the phosphorylation of which is completely blocked by preincubation of HEK293 cells with 0.3 μm BIRB796 but not by 10 μm SB203580. At higher concentrations than 1 μm, BIRB796 also substantially blocks the JNK pathway. However, the precise concentration needed for inhibition may vary from cell to cell. For this reason, it is important to examine the minimum concentration of BIRB796 required to suppress the activity of a particular MAPK by 80–90% by checking in parallel the phosphorylation of a validated substrate of the protein kinase studied.

      Acknowledgments

      We thank the protein production and antibody purification teams at the Division of Signal Transduction Therapy, University of Dundee, coordinated by Drs. H. McLauchlan and J. Hastie, for expression and purification of enzymes and antibodies, and we thank L. Brown for help in culturing the cells.

      References

        • Cohen P.
        Trends Cell Biol. 1997; 7: 353-361
        • Cuenda A.
        • Rouse J.
        • Doza Y.N.
        • Meier R.
        • Cohen P.
        • Gallagher T.F.
        • Young P.R.
        • Lee J.C.
        FEBS Lett. 1995; 364: 229-233
        • Davies S.P.
        • Reddy H.
        • Caivano M.
        • Cohen P.
        Biochem. J. 2000; 351: 95-105
        • Kyriakis J.M.
        • Avruch J.
        Physiol. Rev. 2001; 81: 807-869
        • Kuma Y.
        • Campbell D.G.
        • Cuenda A.
        Biochem. J. 2004; 379: 133-139
        • Goedert M.
        • Cuenda A.
        • Craxton M.
        • Jakes R.
        • Cohen P.
        EMBO J. 1997; 16: 3563-3571
        • Eyers P.A.
        • Craxton M.
        • Morrice N.
        • Cohen P.
        • Goedert M.
        Chem. Biol. 1998; 5: 321-328
        • Sabio G.
        • Reuver S.
        • Feijoo C.
        • Hasegawa M.
        • Thomas G.M.
        • Centeno F.
        • Kuhlendahl S.
        • Leal-Ortiz S.
        • Goedert M.
        • Garner C.
        • Cuenda A.
        Biochem. J. 2004; 380: 19-30
        • Sabio G.
        • Arthur J.S.C.
        • Kuma Y.
        • Peggie M.
        • Carr J.
        • Murray-Tait V.
        • Centeno F.
        • Goedert M.
        • Morrice A.
        • Cuenda A.
        EMBO J. 2005; 24: 1134-1145
        • Feijoo C.
        • Campbell D.G.
        • Jakes R.
        • Goedert M.
        • Cuenda A.
        J. Cell Sci. 2004; 118: 397-408
        • Pargellis C.
        • Tong L.
        • Churchill L.
        • Cirillo P.F.
        • Gilmore T.
        • Graham A.G.
        • Grob P.M.
        • Hickey E.R.
        • Moss N.
        • Pav S.
        • Regan J.
        Nat. Struct. Biol. 2002; 9: 268-272
        • Branger J.
        • van den Blink B.
        • Weijer S.
        • Madwed J.
        • Bos C.L.
        • Gupta A.
        • Yong C.L.
        • Polmar S.H.
        • Olszyna D.P.
        • Hack C.E.
        • van Deventer S.J.
        • Peppelenbosch M.P.
        • van der Poll T.
        J. Immunol. 2002; 168: 4070-4077
        • Branger J.
        • van den Blink B.
        • Weijer S.
        • Gupta A.
        • van Deventer S.J.
        • Hack C.E.
        • Peppelenbosch M.P.
        • van der Poll T.
        Blood. 2003; 101: 4446-4448
        • van den Blink B.
        • Branger J.
        • Weijer S.
        • Gupta A.
        • van Deventer S.J.
        • Peppelenbosch M.P.
        • van der Poll T.
        J. Clin. Immunol. 2004; 24: 37-41
        • Regan J.
        • Breitfelder S.
        • Cirillo P.
        • Gilmore T.
        • Graham A.G.
        • Hickey E.
        • Klaus B.
        • Madwed J.
        • Moriak M.
        • Moss N.
        • Pargellis C.
        • Pav S.
        • Proto A.
        • Swinamer A.
        • Tong L.
        • Torcellini C.
        J. Med. Chem. 2002; 45: 2994-3008
        • Cuenda A.
        • Cohen P.
        • Buee-Scherrer V.
        • Goedert M.
        EMBO J. 1997; 16: 295-305
        • Bain J.
        • McLauchlan H.
        • Elliott M.
        • Cohen P.
        Biochem. J. 2003; 371: 199-204
        • Alessi D.R.
        • Cuenda A.
        • Cohen P.
        • Dudley D.T.
        • Saltiel A.R.
        J. Biol. Chem. 1995; 270: 27489-27494
        • Haydon C.E.
        • Watt P.W.
        • Morrice N.
        • Knebel A.
        • Gaestel M.
        • Cohen P.
        Arch. Biochem. Biophys. 2002; 397: 224-231
        • Cuenda A.
        • Cohen P.
        J. Biol. Chem. 1999; 274: 4341-4346
        • Mody N.
        • Leitch J.
        • Armstrong C.
        • Dixon J.
        • Cohen P.
        FEBS Lett. 2001; 502: 21-24
        • Kamakura S.
        • Moriguchi T.
        • Nishida E.
        J. Biol. Chem. 1999; 274: 26563-26571
        • Hanks S.K.
        • Quinn A.M.
        • Hunter T.
        Science. 1988; 241: 42-52
        • Hasegawa M.
        • Cuenda A.
        • Spillantini M.G.
        • Thomas G.M.
        • Buee-Scherrer V.
        • Cohen P.
        • Goedert M.
        J. Biol. Chem. 1999; 274: 12626-12631
        • Aarts M.
        • Liu Y.
        • Liu L.
        • Besshoh S.
        • Arundine M.
        • Gurd J.W.
        • Wang Y.T.
        • Salter M.W.
        • Tymianski M.
        Science. 2002; 298: 846-850