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

摘要

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 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) 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. 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 (1Cohen P. Trends Cell Biol. 1997; 7: 353-361Abstract Full Text PDF PubMed Scopus (515) Google Scholar). 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 (2Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1977) Google Scholar, 3Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3937) Google Scholar). Substrates for p38α and p38β include other protein kinases, as well as several transcription factors and metabolic protein (4Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Crossref PubMed Scopus (2877) Google Scholar, 5Kuma Y. Campbell D.G. Cuenda A. Biochem. J. 2004; 379: 133-139Crossref PubMed Scopus (50) Google Scholar). However, little is known about the physiological substrates for SAPK3/p38γ and SAPK4/p38δ as they are not inhibited by SB203580 (6Goedert M. Cuenda A. Craxton M. Jakes R. Cohen P. EMBO J. 1997; 16: 3563-3571Crossref PubMed Scopus (356) Google Scholar, 7Eyers P.A. Craxton M. Morrice N. Cohen P. Goedert M. Chem. Biol. 1998; 5: 321-328Abstract Full Text PDF PubMed Scopus (278) Google Scholar), 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 (8Sabio 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: 19-30Crossref PubMed Scopus (74) Google Scholar, 9Sabio 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: 1134-1145Crossref PubMed Scopus (194) Google Scholar). 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 (10Feijoo C. Campbell D.G. Jakes R. Goedert M. Cuenda A. J. Cell Sci. 2004; 118: 397-408Crossref Scopus (106) Google Scholar). 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 (11Pargellis 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: 268-272Crossref PubMed Scopus (794) Google Scholar). 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 (11Pargellis 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: 268-272Crossref PubMed Scopus (794) Google Scholar). 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 (11Pargellis 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: 268-272Crossref PubMed Scopus (794) Google Scholar). 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 (11Pargellis 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: 268-272Crossref PubMed Scopus (794) Google Scholar, 12Branger 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: 4070-4077Crossref PubMed Scopus (229) Google Scholar, 13Branger 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: 4446-4448Crossref PubMed Scopus (72) Google Scholar, 14van 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: 37-41Crossref PubMed Scopus (17) Google Scholar). 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. 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. (15Regan 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: 2994-3008Crossref PubMed Scopus (358) Google Scholar). 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 and (residues of SAP97 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: 1134-1145Crossref PubMed Scopus (194) Google Scholar). that both phosphorylated and SAP97 was by at with SAP97 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: 1134-1145Crossref PubMed Scopus (194) Google Scholar). for and and antibodies were obtained from the of These antibodies were as A. Cohen P. V. Goedert M. EMBO J. 1997; 16: PubMed Scopus Google and used for and A mouse used for was from that recognize p38α phosphorylated at and antibodies also recognize phosphorylated p38β, SAPK3/p38γ, and and c-Jun phosphorylated at were obtained from Cell Protein protein kinases were of human and were as as protein in or as protein in or cells and as S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3937) Google Scholar, J. H. M. Cohen P. Biochem. J. 2003; PubMed Scopus Google Scholar). of Protein were activated with p38α, p38β, SAPK3/p38γ, and SAPK4/p38δ were activated with protein kinase and and were activated with was activated with and were activated with was activated with and and were activated with as S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3937) Google Scholar, J. H. M. Cohen P. Biochem. J. 2003; PubMed Scopus Google Scholar). Protein Kinase protein kinase were linear with to were for at in using p38 MAPK isoforms and or with a in a for at in using The concentrations of and ATP in the were and were with were by of each by in were by the addition of of were four in to in or and and for protein kinases were as S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3937) Google Scholar, J. H. M. Cohen P. Biochem. J. 2003; PubMed Scopus Google Scholar). and were from using specific antibodies and as S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3937) Google Scholar, J. H. M. Cohen P. Biochem. J. 2003; PubMed Scopus Google Scholar, Cuenda A. Cohen P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, C.E. Morrice N. A. M. Cohen P. Biochem. 2002; PubMed Scopus Google Scholar). Cell and embryonic and cells were in medium at with of and embryonic were as 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: 1134-1145Crossref PubMed Scopus (194) Google Scholar), and were as in A. Cohen P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google were to for or for and in A and from were at for at and the were in and at cells were for or with SB 203580 or PD 184352 or with different concentrations of BIRB796 for the in the from Cell and SAP97 were from to of was from of were with and of the specific antibodies to protein for at the proteins were at the was and the were in A containing and in A were in and of protein phosphorylation and protein after was using the BIRB796 in are about protein kinases in the human most of which to the is to compounds that inhibit one particular protein kinase several related the of any particular is a critical We have the of than commercially available compounds against a of protein kinases and that most inhibitors than one kinase S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3937) Google Scholar, J. H. M. Cohen P. Biochem. J. 2003; PubMed Scopus Google Scholar). is a that in cell-based the effects not from of the kinase of but from the of protein the of BIRB796 against different kinases was (11Pargellis 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: 268-272Crossref PubMed Scopus (794) Google Scholar), we to the of this compound to a of protein kinases. We that, at a concentration of BIRB796 inhibited the activity of p38α, and as (11Pargellis 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: 268-272Crossref PubMed Scopus (794) Google Scholar). However, also inhibited the activity of the other p38 MAPK isoforms p38β, SAPK3/p38γ, and SAPK4/p38δ. little the other protein kinases in the of protein kinases by BIRB796 The concentrations used are shown in are as activity to that in was of The of of BIRB796 with the kinase was ATP concentration was in SAPK3/p38γ, SAPK4/p38δ, protein kinase and in to p38β, protein kinase and kinase protein kinase and and for the of the kinases in the kinase in a new of p38 by BIRB796 has been shown that BIRB796 inhibits p38α activity in a to its slow binding (11Pargellis 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: 268-272Crossref PubMed Scopus (794) Google Scholar). or not the activity of other p38 isoforms we the of the compound as a of the of with the kinase We that BIRB796 the p38 MAPK at different in this compound inhibited p38α than p38β and inhibited p38β than SAPK3/p38γ, SAPK4/p38δ was inhibited the A and the for all p38 MAPK isoforms as the of with the A and These results are with the slow binding of this compound to the p38 MAPK isoforms. BIRB796 a of p38 in or not BIRB796 inhibit each p38 MAPK in we the that this compound the activation of one known physiological substrate of p38α, the protein kinase We cells with different concentrations of BIRB796 for or prior to with and the activation of by its activity activation was inhibited in these cells in a with an of after or after of with BIRB796 activation was also by of the cells with the p38 MAPK SB203580 as (2Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1977) Google We also the phosphorylation of p38α and p38β the and that the phosphorylation of these kinases was also by of the cells with the compound and These results that the binding of BIRB796 to these p38 is also their phosphorylation by the kinases their the other of cells with the SB203580 not p38α phosphorylation by this p38β as shown Y. Campbell D.G. Cuenda A. Biochem. J. 2004; 379: 133-139Crossref PubMed Scopus (50) Google BIRB796 also inhibit the and of SAPK3/p38γ and SAPK4/p38δ, cells were with different concentrations of BIRB796 for or with The phosphorylation of SAPK3/p38γ was by this compound with an of or after or which are and higher than the for of the expression of SAPK4/p38δ in cells is and this isoform is not activated in these cells after treatment 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: 1134-1145Crossref PubMed Scopus (194) Google Scholar), the of BIRB796 the activation of this kinase was in mouse embryonic SAPK4/p38δ phosphorylation after was at of the and at higher concentrations of BIRB796 The for SAPK4/p38δ was higher than the for SAPK3/p38γ. of SAPK4/p38δ after was also when cells were with the SB203580 One for this is that regulates SAPK4/p38δ activation by an the binding of BIRB796 and SB203580 to may to SAPK4/p38δ of BIRB796 the activation of SAPK4/p38δ in mouse embryonic embryonic were for with different concentrations of BIRB796 or SB203580 as to for SAPK4/p38δ was from of and the were using the p38α that also SAPK4/p38δ or using an that both phosphorylated and SAPK4/p38δ BIRB796 the of the SAPK3/p38γ SAP97 in scaffold protein SAP97 is the of the tumor and a physiological substrate of SAPK3/p38γ. SAP97 phosphorylated in cells at four major and in to 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: 1134-1145Crossref PubMed Scopus (194) Google The phosphorylation of all four was greatly when cells were with BIRB796 but not by with SB203580 The for of Ser158 phosphorylation was or after or and to the for of SAPK3/p38γ. BIRB796 JNK in also the of the BIRB796 the activation of the c-Jun N-terminal kinase which is also a MAPK family members activated by of cells to an in the phosphorylation of both isoforms and of and which was when cells were with BIRB796 of phosphorylation was to BIRB796 than phosphorylation after a with the the for was and for was the for was higher than when cells were for with BIRB796 prior to These results that BIRB796 inhibits JNK by a to the p38 MAPK isoforms. The phosphorylation of one physiological substrate of JNK, the transcription was also of the cells to in the of concentrations of BIRB796, c-Jun was using a which of c-Jun was at BIRB796 with an of after of with the to the for of activation these these results show that BIRB796 blocks activation and activity in cells but at higher concentrations than those to p38α, p38β, or SAPK3/p38γ. BIRB796 or in further the of BIRB796, we its the activation and activity of other MAPK family and These were in cells in which the activation of the and by is well characterized N. J. C. J. Cohen P. FEBS Lett. 2001; PubMed Scopus Google Scholar). of and activation of one of their in vivo substrates, were not by for to of the cells with BIRB796, with the MAPK phosphorylation and activation A and These results that BIRB796 not inhibit the activation and activity of The phosphorylation of be by a small in its using an that the phosphorylated and the of the protein well N. J. C. J. Cohen P. FEBS Lett. 2001; PubMed Scopus Google Scholar). of cells with inhibited the phosphorylation of as from N. J. C. J. Cohen P. FEBS Lett. 2001; PubMed Scopus Google Scholar, S. T. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google However, concentrations of BIRB796 not the of the kinase by its phosphorylation and not the activation of by BIRB796 the of in or not the binding of BIRB796 to p38 or also their phosphorylation by the kinases or their we the in phosphorylation of each p38 MAPK isoform by and the phosphorylation of the isoform by We that BIRB796 the p38 MAPK and the phosphorylation at the the other of the cells with the to MAPK not any the of the phosphorylation of SAPK3/p38γ, p38α, or by BIRB796 after a we that of cells with the phosphorylation of that the was of these results that the binding of BIRB796 to the p38 or is their phosphorylation by the kinase or than their Recently, has been that the diaryl urea compound BIRB796 is a of the protein kinase p38α (11Pargellis 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: 268-272Crossref PubMed Scopus (794) Google Scholar). the of a particular is a critical as a for protein kinases are a class of enzymes, most of which to the and the of the family is within the T. PubMed Scopus Google Scholar), is to compounds that inhibit one particular protein kinase several related this we to the of the BIRB796 The in the that BIRB796 inhibits the activity in and the activation in the cell, of all p38 MAPK and isoforms. of p38α has shown that BIRB796 binds to a novel within the of the which is a by in the by the which to a structure incompatible with ATP binding (11Pargellis 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: 268-272Crossref PubMed Scopus (794) Google Scholar). Moreover, have demonstrated that this class of compound has slow binding with the for a (11Pargellis 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: 268-272Crossref PubMed Scopus (794) Google Scholar). Our in and in vivo show that BIRB796 inhibits all p38 MAPK and JNK isoforms in a that the association of this compound with each of these kinases is to the of the BIRB796 binding is to the in the that is in a between the major of the kinase domain (11Pargellis 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: 268-272Crossref PubMed Scopus (794) Google Scholar). the in this are all of these kinases, we have that BIRB796 is for p38α and p38β than for the SAPK3/p38γ and SAPK4/p38δ isoforms and other members of the MAPK This be to the that this diaryl urea also the containing (11Pargellis 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: 268-272Crossref PubMed Scopus (794) Google a to p38α and p38β. Moreover, we also show that BIRB796 the phosphorylation of p38 or by the kinase or but not their in Our results that the by the binding of the to the MAPK may the structure of both its phosphorylation and the for the the phosphorylation of p38 or We also show that of cells with BIRB796 prior to blocks phosphorylation of the physiological substrates of p38α and SAPK3/p38γ. BIRB796 the phosphorylation by of SAP97, which is a SAPK3/p38γ Recently, we have shown that the phosphorylation of different PDZ domain-containing proteins by SAPK3/p38γ, such as SAP97, is the interaction of the sequence of the kinase with the PDZ domain of these proteins (8Sabio 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: 19-30Crossref PubMed Scopus (74) Google Scholar, M. Cuenda A. Thomas G.M. V. Cohen P. Goedert M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). this we a cell-permeant peptide to identify different SAPK3/p38γ substrates in This peptide blocks the phosphorylation of PDZ domain-containing proteins by SAPK3/p38γ but not phosphorylation by other MAPK in by the association of the kinase with the PDZ domain of the protein substrate (8Sabio 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: 19-30Crossref PubMed Scopus (74) Google Scholar, 9Sabio 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: 1134-1145Crossref PubMed Scopus (194) Google Scholar). The peptide the of the SAPK3/p38γ to the domain of the human protein (8Sabio 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: 19-30Crossref PubMed Scopus (74) Google Scholar, 9Sabio 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: 1134-1145Crossref PubMed Scopus (194) Google Scholar, M. Y. L. S. M. M. 2002; PubMed Scopus Google Scholar). This peptide has been useful in the of PDZ domain-containing proteins as SAPK3/p38γ substrates such as (8Sabio 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: 19-30Crossref PubMed Scopus (74) Google or SAP97 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: 1134-1145Crossref PubMed Scopus (194) Google Scholar). However, the availability of new cell-permeant SAPK3/p38γ inhibitors be useful in to the physiological of this kinase and to other substrates that not PDZ The results in this show that is to the concentration of BIRB796 in the medium to inhibit particular stress-activated protein kinases in combination with the specific example, in cells to that p38 and BIRB796 at inhibits at also inhibits SAPK3/p38γ. physiological substrates for SAPK3/p38γ can be by the phosphorylation of which is by of cells with BIRB796 but not by higher concentrations than BIRB796 also blocks the JNK However, the concentration for may from to this is to the concentration of BIRB796 to suppress the activity of a particular MAPK by by in the phosphorylation of a substrate of the protein kinase We the protein production and at the of of by H. and J. for expression and of and and we L. for in the