RESUMO
Nearly all cell surface receptors utilize one or more of the mitogen-activated protein kinase cascades in their repertoire of signal transduction mechanisms. Recent advances in the study of such cascades include the cloning of genes encoding novel members of the cascades, further definition of the roles of the cascades in responses to extracellular signals, and examination of cross-talk between different cascades.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , MAP Quinase Quinase Quinase 1 , Quinases de Proteína Quinase Ativadas por Mitógeno , Animais , Humanos , MAP Quinase Quinase 1 , Mamíferos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-raf , Transdução de SinaisRESUMO
A family of protein kinases, known alternatively as microtubule-associated protein-2/myelin basic protein kinases or extracellular signal-regulated kinases, is activated by numerous hormones, growth factors and other extracellular stimuli. At least two members of this family function as intermediate kinases in protein phosphorylation cascades. Their mechanisms of activation may involve autophosphorylation, which occurs on both threonine and tyrosine residues.
Assuntos
Proteínas Quinases/metabolismo , Transdução de Sinais , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Espaço Extracelular , Quinase 3 da Glicogênio Sintase , Substâncias de Crescimento/metabolismo , Hormônios/metabolismo , Modelos Biológicos , Fosforilação , Proteínas Quinases S6 Ribossômicas , Treonina/metabolismo , Tirosina/metabolismoRESUMO
A hormonally defined medium was used to isolate a homogeneous epithelioid cell population from canine kidney. Monolayers of these cells form domes, an indication of active ion transport, and this process is inhibited by ouabain. This technique allows the isolation of primary cultures of renal epithelial cells, free of fibroblasts, for the characterization of biochemical and physiological properties related to renal function.
Assuntos
Rim/citologia , Animais , Transporte Biológico Ativo , Adesão Celular , Células Cultivadas , Meios de Cultura , Cães , Epitélio/metabolismo , Feminino , Masculino , Sódio/metabolismoRESUMO
A protein kinase characterized by its ability to phosphorylate microtubule-associated protein-2 (MAP2), is thought to be an early intermediate in an insulin-stimulated phosphorylation cascade and in a variety of other mammalian cell responses to extracellular signals. A complementary DNA that encodes this protein serine-threonine kinase has been cloned, and the protein designated extracellular signal-regulated kinase 1 (ERK1). ERK1 has striking similarity to two protein kinases, KSS1 and FUS3, from yeast. The yeast kinases function in an antagonistic manner to regulate the cell cycle in response to mating factors. Thus, ERK1 and the two yeast kinases constitute a family of evolutionarily conserved enzymes involved in regulating the response of eukaryotic cells to extracellular signals.
Assuntos
Ciclo Celular/fisiologia , Sistema Nervoso Central/enzimologia , DNA/genética , Proteínas Quinases Ativadas por Mitógeno , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Linhagem Celular , Fibroblastos/enzimologia , Humanos , Insulina/farmacologia , Proteína Quinase 3 Ativada por Mitógeno , Dados de Sequência Molecular , Fosforilação , Proteínas Quinases/genética , Ratos , Receptor de Insulina/metabolismo , Leveduras/enzimologiaRESUMO
The stimulus-dependent nuclear localization of the extracellular-signal- regulated kinases ERK1 and ERK2 is required for many of their actions, including induction of neurites in PC12 cells and transformation of fibroblasts. Phosphorylation of ERK2 causes it to form dimers, and the most flexible portions of the ERK2 molecule provide the surfaces for dimerization. It is thought that dimerization promotes nuclear localization of ERK2 by its effects on import, export or retention in cytoplasmic and nuclear compartments. Dimerization might also influence substrate interactions.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Núcleo Celular/metabolismo , Dimerização , Humanos , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Fosforilação , Conformação Proteica , Transdução de SinaisRESUMO
Mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs including embryogenesis, proliferation, differentiation and apoptosis based on cues derived from the cell surface and the metabolic state and environment of the cell. In mammals, there are more than a dozen MAPK genes. The best known are the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK(1-3)) and p38(alpha, beta, gamma and delta) families. ERK3, ERK5 and ERK7 are other MAPKs that have distinct regulation and functions. MAPK cascades consist of a core of three protein kinases. Despite the apparently simple architecture of this pathway, these enzymes are capable of responding to a bewildering number of stimuli to produce exquisitely specific cellular outcomes. These responses depend on the kinetics of their activation and inactivation, the subcellular localization of the kinases, the complexes in which they act, and the availability of substrates. Fine-tuning of cascade activity can occur through modulatory inputs to cascade component from the primary kinases to the scaffolding accessory proteins. Here, we describe some of the properties of the three major MAPK pathways and discuss how these properties govern pathway regulation and activity.
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/fisiologiaRESUMO
As detected by coimmunoprecipitation from PC12 cells, NGF induces rapid association between ERK1 (a growth factor-activated serine/threonine protein kinase) and gp140prototrk NGF receptors. In contrast, no such association is found with the closely related ERK2. Anti-trk immunocomplexes generated from NGF-treated cells also contain protein kinase activity that shares many properties with soluble ERK1. The association of both ERK1 protein and ERK-like kinase activity with gp140prototrk is maximal by 5 min of NGF treatment, persists for approximately 1 hr, and subsequently declines by 18 hr. Treatment with either basic fibroblast growth factor, epidermal growth factor, or orthovanadate also leads to association of ERK1 with gp140prototrk without tyrosine phosphorylation of the latter. The interaction between ERK1 and gp140prototrk may prove relevant to the NGF mechanism.
Assuntos
Proteínas Quinases Ativadas por Mitógeno , Fatores de Crescimento Neural/farmacologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Vanadatos/farmacologia , Animais , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Cinética , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Peso Molecular , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Células PC12 , Proteínas Serina-Treonina Quinases/isolamento & purificação , Receptor trkARESUMO
Mitogen-activated protein (MAP) kinases comprise a family of ubiquitous proline-directed, protein-serine/threonine kinases, which participate in signal transduction pathways that control intracellular events including acute responses to hormones and major developmental changes in organisms. MAP kinases lie in protein kinase cascades. This review discusses the regulation and functions of mammalian MAP kinases. Nonenzymatic mechanisms that impact MAP kinase functions and findings from gene disruption studies are highlighted. Particular emphasis is on ERK1/2.
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Humanos , Proteínas Quinases Ativadas por Mitógeno/química , FosforilaçãoRESUMO
Although the function of the p42/p44 mitogen-activated protein (MAP) kinase pathway in long-term potentiation at hippocampal CA3-CA1 synapses has been well described, relatively little is known about the importance of the p38 MAP kinase pathway in synaptic plasticity. Here we show that the p38 MAP kinase pathway, a parallel signaling cascade activated by distinct upstream kinases, mediates the induction of metabotropic glutamate receptor-dependent long-term depression at CA3-CA1 synapses. Thus, two parallel MAP kinase pathways contribute to opposing forms of long-term plasticity at a central synapse.
Assuntos
Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Animais , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Sinapses/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
BACKGROUND: Mitogen-activated protein (MAP) kinases are ubiquitous components of many signal transduction pathways. Constitutively active variants have been isolated for every component of the extracellular-signal-regulated kinase 1 (ERK1) and ERK2 MAP kinase pathway except for the ERK itself. RESULTS: To create an activated ERK2 variant, we fused ERK2 to the low activity form of its upstream regulator, the MAP kinase kinase MEK1. The ERK2 in this fusion protein was active in the absence of extracellular signals. Expression of the fusion protein in mammalian cells did not activate endogenous ERK1 or ERK2. It was sufficient, however, to induce activation of the transcription factors Elk-1 and AP-1, neurite extension in PC12 cells in the absence of nerve growth factor, and foci of morphologically and growth-transformed NIH3T3 cells, if the fusion protein was localized to the nucleus. A cytoplasmic fusion protein was without effect. CONCLUSIONS: Activation of ERK2 is sufficient to cause several transcriptional and phenotypic responses in mammalian cells. Nuclear localization of activated ERK2 is required to induce these events.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Núcleo Celular/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Neuritos/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transcrição Gênica , Células 3T3 , Sequência de Aminoácidos , Animais , Linhagem Celular , Linhagem Celular Transformada , Núcleo Celular/enzimologia , Códon , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Luciferases/biossíntese , MAP Quinase Quinase 1 , Camundongos , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Fatores de Crescimento Neural/fisiologia , Células PC12 , Fenótipo , Mutação Puntual , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
We have previously shown that inhibition of phosphatidylinositol (PI) 3-kinase severely attenuates the activation of extracellular signal-regulated kinase (Erk) following engagement of integrin/fibronectin receptors and that Raf is the critical target of PI 3-kinase regulation [1]. To investigate how PI 3-kinase regulates Raf, we examined sites on Raf1 required for regulation by PI 3-kinase and explored the mechanisms involved in this regulation. Serine 338 (Ser338), which was critical for fibronectin stimulation of Raf1, was phosphorylated in a PI 3-kinase-dependent manner following engagement of fibronectin receptors. In addition, fibronectin activation of a Raf1 mutant containing a phospho-mimic mutation (S338D) was independent of PI 3-kinase. Furthermore, integrin-induced activation of the serine/threonine kinase Pak-1, which has been shown to phosphorylate Raf1 Ser338, was also dependent on PI 3-kinase activity and expression of a kinase-inactive Pak-1 mutant blocked phosphorylation of Raf1 Ser338. These results indicate that PI 3-kinase regulates phosphorylation of Raf1 Ser338 through the serine/threonine kinase Pak. Thus, phosphorylation of Raf1 Ser338 through PI 3-kinase and Pak provides a co-stimulatory signal which together with Ras leads to strong activation of Raf1 kinase activity by integrins.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Serina/metabolismo , Animais , Células COS , Integrinas/metabolismo , Mutagênese , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-raf/genética , Serina/genética , Quinases Ativadas por p21RESUMO
The adverse effects of lipopolysaccharide (LPS) are mediated primarily by tumor necrosis factor alpha (TNF-alpha). TNF-alpha production by LPS-stimulated macrophages is regulated at the levels of both transcription and translation. It has previously been shown that several mitogen-activated protein kinases (MAPKs) are activated in response to LPS. We set out to determine which MAPK signaling pathways are activated in our system and which MAPK pathways are required for TNF-alpha gene transcription or TNF-alpha mRNA translation. We confirm activation of the MAPK family members extracellular-signal-regulated kinases 1 and 2 (ERK1 and ERK2), p38, and Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), as well as activation of the immediate upstream MAPK activators MAPK/ERK kinases 1 and 4 (MEK1 and MEK4). We demonstrate that LPS also activates MEK2, MEK3, and MEK6. Furthermore, we demonstrate that dexamethasone, which inhibits the production of cytokines, including TNF-alpha, significantly inhibits LPS induction of JNK/SAPK activity but not that of p38, ERK1 and ERK2, or MEK3, MEK4, or MEK6. Dexamethasone also blocks the sorbitol but not anisomycin stimulation of JNK/SAPK activity. A kinase-defective mutant of SAPKbeta, SAPKbeta K-A, blocked translation of TNF-alpha, as determined by using a TNF-alpha translational reporting system. Finally, overexpression of wild-type SAPKbeta was able to overcome the dexamethasone-induced block of TNF-alpha translation. These data confirm that three MAPK family members and their upstream activators are stimulated by LPS and demonstrate that JNK/SAPK is required for LPS-induced translation of TNF-alpha mRNA. A novel mechanism by which dexamethasone inhibits translation of TNF-alpha is also revealed.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Lipopolissacarídeos/farmacologia , Proteínas Quinases Ativadas por Mitógeno , Fator de Necrose Tumoral alfa/biossíntese , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Genes Reporter , Proteínas Quinases JNK Ativadas por Mitógeno , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Modelos Biológicos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Alteration of the TAL1 gene is the most common genetic lesion found in T-cell acute lymphoblastic leukemia. TAL1 encodes phosphoproteins, pp42TAL1 and pp22TAL1, that represent phosphorylated versions of the full-length (residues 1 to 331) and truncated (residues 176 to 331) TAL1 gene products, respectively. Both proteins contain the basic helix-loop-helix motif, a DNA-binding and protein dimerization motif common to several known transcriptional regulatory factors. We now report that serine residue 122 (S122) is a major phosphorylation site of pp42TAL1 in leukemic cell lines and transfected COS1 cells. In vivo phosphorylation of S122 is induced by epidermal growth factor with a rapid time course that parallels activation of the ERK/MAP2 protein kinases. Moreover, S122 is readily phosphorylated in vitro by the extracellular signal-regulated protein kinase ERK1. These data suggest that TAL1 residue S122 serves as an in vivo substrate for ERK/MAP2 kinases such as ERK1. Therefore, S122 phosphorylation may provide a mechanism whereby the properties of TAL1 polypeptides can be modulated by extracellular stimuli.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Linhagem Celular Transformada , Chlorocebus aethiops , Fator de Crescimento Epidérmico/farmacologia , Humanos , Leucemia , Proteína Quinase 3 Ativada por Mitógeno , Dados de Sequência Molecular , Mapeamento de Peptídeos , Fosforilação , Ratos , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Células Tumorais CultivadasRESUMO
The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Meios de Cultivo Condicionados , Primers do DNA , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , MAP Quinase Quinase Quinases , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Tirosina Quinases/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Transfecção , Quinases Ativadas por p21RESUMO
The structures of four serine/threonine protein kinases have been determined recently. By comparing these structures with that of the cAMP-dependent protein kinase (cAPK), it is now possible to see how the activity of these regulatory enzymes is controlled. Low activity is maintained through the conformation of the phosphorylation lip, domain rotations, and binding of substrate analog inhibitors and autoinhibitory domains.
Assuntos
Proteínas Quinases/química , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Conformação ProteicaRESUMO
The phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) in response to insulin in Rat 1 HIRc B cells and in response to nerve growth factor (NGF) in PC12 cells has been examined. ERK1 and ERK2 are phosphorylated on serine in the absence of the stimuli and additionally on tyrosine and threonine residues after exposure to NGF and insulin. NGF stimulates tyrosine phosphorylation of ERK1 more rapidly than threonine phosphorylation. Two-dimensional phosphopeptide maps of both ERK1 and ERK2 phosphorylated in intact cells treated with NGF or with insulin display the same three predominant phosphopeptides that comigrate when digests of ERK1 and ERK2 are mixed. As many as five additional phosphopeptides are detected under certain conditions. Autophosphorylated recombinant ERK2 also contains the three tryptic phosphopeptides found in ERKs labeled in intact cells. These experiments demonstrate that ERK1 and ERK2 are phosphorylated on related sites in response to two distinct extracellular signals. The data also support the possibility that autophosphorylation may be involved in the activation of the ERKs.
Assuntos
Insulina/fisiologia , Proteínas Quinases Ativadas por Mitógeno , Fatores de Crescimento Neural/fisiologia , Fosfopeptídeos/metabolismo , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Dados de Sequência Molecular , Mapeamento de Peptídeos , FosforilaçãoRESUMO
Microtubule-associated proteins (MAP), such as tau, modulate the extent and rate of microtubule assembly and play an essential role in morphogenetic processes, such as axonal growth. We have examined the mechanism by which tau affects microtubule polymerization by examining the kinetics of microtubule assembly and disassembly through direct observation of microtubules using dark-field microscopy. Tau increases the rate of polymerization, decreases the rate of transit into the shrinking phase (catastrophe), and inhibits the rate of depolymerization. Tau strongly suppresses the catastrophe rate, and its ability to do so is independent of its ability to increase the elongation rate. Thus, tau generates a partially stable but still dynamic state in microtubules. This state is perturbed by phosphorylation by MAP2 kinase, which affects all three activities by lowering the affinity of tau for the microtubule lattice.
Assuntos
Tubulina (Proteína)/metabolismo , Proteínas tau/farmacologia , Animais , Bovinos , Técnicas In Vitro , Cinética , Substâncias Macromoleculares , Microscopia Eletrônica , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Polímeros/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestruturaRESUMO
BACKGROUND: The mitogen-activated protein (MAP) kinase, ERK2, is a tightly regulated enzyme in the ubiquitous Ras-activated protein kinase cascade. ERK2 is activated by phosphorylation at two sites, Y185 and T183, that lie in the phosphorylation lip at the mouth of the catalytic site. To ascertain the role of these two residues in securing the low-activity conformation of the enzymes we have carried out crystallographic analyses and assays of phosphorylation-site mutants of ERK2. RESULTS: The crystal structures of four mutants, T183E (threonine at residue 183 is replaced by glutamate), Y185E, Y185F and the double mutant T183E/Y185E, were determined. When T183 is replaced by glutamate, few conformational changes are observed. By contrast, when Y185 is replaced by glutamate, 19 residues become disordered, including the entire phosphorylation lip and an adjacent loop. The conservative substitution of phenylalanine for Y185 also induces relatively large conformational changes. A binding site for phosphotyrosine in the active enzyme is putatively identified on the basis of the high-resolution refinement of the structure of wild-type ERK2. CONCLUSIONS: The remarkable disorder observed throughout the phosphorylation lip when Y185 is mutated shows that the stability of the phosphorylation lip is rather low. Therefore, only modest amounts of binding energy will be required to dislodge the lip for phosphorylation, and it is likely that these residues will be involved in conformational changes associated with both with binding to kinases and phosphatases and with activation. Furthermore, the low-activity structure is specifically dependent on Y185, whereas there is no such dependency on T183. Both residues, however, participate in forming the active enzyme, contributing to its tight control.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cristalização , Cristalografia por Raios X , Ativação Enzimática , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Glicina/química , Glicina/metabolismo , Proteína Quinase 1 Ativada por Mitógeno , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Fosforilação , Fosfotreonina/química , Fosfotreonina/metabolismo , Fosfotirosina , Conformação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Relação Estrutura-Atividade , Tirosina/análogos & derivados , Tirosina/química , Tirosina/metabolismoRESUMO
BACKGROUND: The mitogen-activated protein (MAP) kinases are important signaling molecules that participate in diverse cellular events and are potential targets for intervention in inflammation, cancer, and other diseases. The MAP kinase p38 is responsive to environmental stresses and is involved in the production of cytokines during inflammation. In contrast, the activation of the MAP kinase ERK2 (extracellular-signal-regulated kinase 2) leads to cellular differentiation or proliferation. The anti-inflammatory agent pyridinylimidazole and its analogs (SB [SmithKline Beecham] compounds) are highly potent and selective inhibitors of p38, but not of the closely-related ERK2, or other serine/threonine kinases. Although these compounds are known to bind to the ATP-binding site, the origin of the inhibitory specificity toward p38 is not clear. RESULTS: We report the structural basis for the exceptional selectivity of these SB compounds for p38 over ERK2, as determined by comparative crystallography. In addition, structural data on the origin of olomoucine (a better inhibitor of ERK2) selectivity are presented. The crystal structures of four SB compounds in complex with p38 and of one SB compound and olomoucine in complex with ERK2 are presented here. The SB inhibitors bind in an extended pocket in the active site and are complementary to the open domain structure of the low-activity form of p38. The relatively closed domain structure of ERK2 is able to accommodate the smaller olomoucine. CONCLUSIONS: The unique kinase-inhibitor interactions observed in these complexes originate from amino-acid replacements in the active site and replacements distant from the active site that affect the size of the domain interface. This structural information should facilitate the design of better MAP-kinase inhibitors for the treatment of inflammation and other diseases.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Inibidores Enzimáticos/química , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno , Trifosfato de Adenosina/metabolismo , Domínio Catalítico/efeitos dos fármacos , Diferenciação Celular , Divisão Celular , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Humanos , Imidazóis/química , Imidazóis/farmacologia , Cinetina , Modelos Químicos , Modelos Moleculares , Conformação Proteica , Purinas/química , Purinas/farmacologia , Piridinas/química , Piridinas/farmacologia , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Alteration of the TAL1 gene is the most common genetic lesion found in patients with T cell acute lymphoblastic leukemia. TAL1 encodes a basic helix-loop-helix transcription factor that is phosphorylated on serine residue 122 by the mitogen-activated protein (MAP) kinase ERK1. Here we show that the amino-terminal sequences of TAL1 (residues 1-166) function in vivo as a transcriptional activation domain. Mutation of serine residue 122 reduces the potency of the transactivation domain by more than half. The data suggest that the amino-terminal transactivation domain of TAL1 is positively regulated by S122 phosphorylation and that the functional properties of TAL1 can be influenced by signal transduction pathways that involve the MAP kinases.