RESUMO
p38 has been shown to be a critical enzyme in the pro-inflammatory cytokine pathway and is a member of the mitogen-activated protein (MAP) kinase family. While the details for p38 activation and subsequent signal transduction have begun to be elucidated, little is known about the kinetic mechanism for p38. In this study, we have determined the kinetic mechanism for p38 MAP kinase. Data from initial velocity patterns in the presence and absence of a dead-end inhibitor and two triarylimidazole p38 inhibitors were consistent with an ordered sequential mechanism for p38 with protein substrate, glutathione S-transferase-activating transcription factor 2 (GST-ATF2), binding before ATP. The ATP analog, adenylyl methylenediphosphonate (AMP-PCP), and two triarylimidazoles were competitive inhibitors versus ATP and uncompetitive inhibitors versus GST-ATF2. Equilibrium binding studies utilizing a tritiated ATP-competitive inhibitor were also consistent with this mechanism and suggest an inability of ATP to bind to p38 in the absence of protein substrate. Moreover, the Michaelis constant for GST-ATF2 was 12-fold greater than the dissociation constant, indicating that the binding of ATP affected the binding of GST-ATF2. An ordered sequential mechanism with protein substrate binding first is unique to p38 compared to cyclic AMP-dependent protein kinase (cAPK) and most tyrosine kinases and helps to explain the interaction between enzyme, substrates, and inhibitors.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Fatores de Transcrição/metabolismo , Fator 2 Ativador da Transcrição , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Clonagem Molecular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Inibidores Enzimáticos/farmacologia , Glutationa Transferase/genética , Humanos , Imidazóis/farmacologia , Cinética , Estrutura Molecular , Fosforilação , Ligação Proteica , Piridinas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/enzimologia , Fatores de Transcrição/genética , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Interleukin-1 beta (IL-1 beta) converting enzyme (ICE) processes the precursor of the cytokine IL-1 beta to a mature, biologically active form in monocytes and macrophages. To further understand the role of ICE in regulating IL-1 beta-mediated biological functions, we have isolated several genomic clones encoding the full-length murine ICE gene. Southern blot comparison of murine genomic DNA and the clones indicates that ICE is a compact, single-copy gene 8616 bp in size. We sequenced the entire gene as well as 1.0-kb segment upstream of the coding region and determined that the gene consists of 10 exons whose organization parallels the functional organization of the ICE proenzyme in that the prodomain and p20 and p10 subunits of ICE are encoded by three clusters of exons. Two initiation sites, 37 and 32 nucleotides upstream of the initiator methionine, were identified by primer extension analysis. The 5' region of the ICE gene lacks a TATA box, a CAAT box, and SP1 sites. However, the presence of a completely conserved 14-bp sequence spanning the transcription initiation site of both the murine and the human ICE genes suggests that this sequence plays a role in transcription.
Assuntos
Mapeamento Cromossômico , Precursores Enzimáticos/genética , Metaloendopeptidases/genética , Camundongos/genética , Células 3T3 , Animais , Sequência de Bases , Southern Blotting , Caspase 1 , Linhagem Celular , Clonagem Molecular , Cosmídeos , DNA/análise , DNA/genética , Éxons , Íntrons , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Mapeamento por Restrição , TATA BoxRESUMO
Interleukin-1 beta-converting enzyme (ICE) was purified from dialyzed cytoplasmic extracts of THP.1 human monocytic cells by a combination of DEAE-5PW and SP-5PW ion exchange and C4 reverse phase high performance liquid chromatography. Sequence information from tryptic and Asp.N peptides on the isolated 20-kDa (p20) and a 10-kDa (p10) proteins enabled the subsequent cloning of ICE (Thornberry, N. A., Bull, H. G., Calaycay, J. R., Chapman, K. T., Howard, A. D., Kostura, M. J., Miller, D. K., Molineaux, S. M., Weidner, J. R., Aunins, J., Elliston, K. O., Ayala, J. M., Casano, F. J., Chin, J., Ding, G. J.-F., Egger, L. A., Gaffney, E. P., Limjuco, G., Palyha, O. C., Raju, S. M., Rolando, A. M., Salley, J. P., Yamin, T.-T., Lee, T. D., Shively, J. E., MacCross, M., Mumford, R. A., Schmidt, J. A., and Tocci, M. J. (1992) Nature 356, 768-774) and localized the active site Cys. Immunoblots with ICE specific antibodies and NH2-terminal sequencing indicated that ICE active column fractions contained in addition to p20 and p10 an alternatively processed form of the p20 protein (p22) containing an extra 16 amino acids NH2-terminal to the p20. Furthermore, immunoblot analysis of the ion exchange column effluent showed that p20 and p22 were found together in three separate fractions distinguished by differences in p10: an intact p10 with complete ICE activity, a COOH-terminally truncated form of p10 with decreased ICE activity, and an absence of p10 with no ICE activity. These results indicate that the p10 protein is essential for ICE activity and that the ICE holoenzyme contains an intact p10 subunit paired with a p20 or p22 catalytic subunit.
Assuntos
Metaloendopeptidases/isolamento & purificação , Metaloendopeptidases/metabolismo , Sequência de Aminoácidos , Anticorpos , Caspase 1 , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Citoplasma/enzimologia , Eletroforese em Gel de Poliacrilamida , Endopeptidases , Humanos , Immunoblotting , Metionina/metabolismo , Dados de Sequência Molecular , Monócitos , Fragmentos de Peptídeos/isolamento & purificação , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Murine interleukin 1 beta (IL-1 beta) convertase (mICE) was identified in cytosolic extracts of peritoneal exudate cells (PECs) and macrophage cell lines. mICE cleaves both the human and mouse IL-1 beta precursors (pIL-1 beta) at sites 1 and 2 but fails to cleave a human pIL-1 beta (Asp116 to Ala) mutant at site 2, indicating that Asp is required to the left of the scissile bond. Ac-Tyr-Val-Ala-Asp-amino-4-methyl coumarin, patterned after site 2 of human pIL-1 beta, is a fluorogenic substrate for mICE, while the tetrapeptide aldehyde Ac-Tyr-Val-Ala-Asp-CHO is a potent inhibitor (Ki = 3 nM) that prevents generation and release of mature IL-1 beta by PECs (IC50 = 7 microM). Cloning of a full-length 1.4-kb cDNA shows that mICE is encoded as a 402-aa proenzyme (p45) that can be divided into a prodomain (Met1-Asp122), followed by a p20 subunit (Gly123-Asp296), a connecting peptide (Ser297-Asp314), and a p10 subunit (Gly315-His402). At the amino acid level, p45, p20, and p10 are 62%, 60%, and 81% identical with human IL-1 beta convertase (hICE). The active site Cys284 lies within a completely conserved stretch of 18 residues; however, Ser289 in hICE, which aligns with the catalytic region of serine and viral cysteinyl proteases, is absent from mICE. Expression in Escherichia coli of a truncated cDNA encoding Asn119-His402 generated active enzyme, which was autocatalytically processed at three internal Asp-Xaa bonds to generate a p20 subunit (Asn119-Asp296) complexed with either p11 (Ala309-His402) or p10. Recombinant mICE cleaves murine pIL-1 beta accurately at the Asp117-Val118 bond. The striking similarities of the human and murine enzymes will make it possible to assess the therapeutic potential of hICE inhibitors in murine models of disease.
