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1.
Science ; 381(6663): 1217-1225, 2023 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-37708276

RESUMEN

The mitogen-activated protein kinase (MAPK) p38α is a central component of signaling in inflammation and the immune response and is, therefore, an important drug target. Little is known about the molecular mechanism of its activation by double phosphorylation from MAPK kinases (MAP2Ks), because of the challenge of trapping a transient and dynamic heterokinase complex. We applied a multidisciplinary approach to generate a structural model of p38α in complex with its MAP2K, MKK6, and to understand the activation mechanism. Integrating cryo-electron microscopy with molecular dynamics simulations, hydrogen-deuterium exchange mass spectrometry, and experiments in cells, we demonstrate a dynamic, multistep phosphorylation mechanism, identify catalytically relevant interactions, and show that MAP2K-disordered amino termini determine pathway specificity. Our work captures a fundamental step of cell signaling: a kinase phosphorylating its downstream target kinase.


Asunto(s)
MAP Quinasa Quinasa 2 , MAP Quinasa Quinasa 6 , Proteína Quinasa 14 Activada por Mitógenos , Microscopía por Crioelectrón , Activación Enzimática , MAP Quinasa Quinasa 2/química , MAP Quinasa Quinasa 6/química , Proteína Quinasa 14 Activada por Mitógenos/química , Fosforilación , Especificidad por Sustrato , Conformación Proteica
2.
Protein Sci ; 30(4): 908-913, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33554397

RESUMEN

Mitogen-activated protein kinase (MAPK; p38, ERK, and JNK) cascades are evolutionarily conserved signaling pathways that regulate the cellular response to a variety of extracellular stimuli, such as growth factors and interleukins. The MAPK p38 is activated by its specific upstream MAPK kinases, MKK6 and MKK3. However, a comprehensive molecular understanding of how these cognate upstream kinases bind and activate p38 is still missing. Here, we combine NMR spectroscopy and isothermal titration calorimetry to define the binding interface between full-length MKK6 and p38. It was shown that p38 engages MKK6 not only via its hydrophobic docking groove, but also influences helix αF, a secondary structural element that plays a key role in organizing the kinase core. It was also shown that, unlike MAPK phosphatases, the p38 conserved docking (CD) site is much less affected by MKK6 binding. Finally, it was demonstrated that these interactions with p38 are conserved independent of the MKK6 activation state. Together, the results revealed differences between specificity markers of p38 regulation by upstream kinases, which do not effectively engage the CD site, and downstream phosphatases, which require the CD site for productive binding.


Asunto(s)
MAP Quinasa Quinasa 6/química , Proteínas Quinasas p38 Activadas por Mitógenos/química , Sitios de Unión , Activación Enzimática , Humanos , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/metabolismo , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica en Hélice alfa , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
3.
Fish Shellfish Immunol ; 92: 500-507, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31247318

RESUMEN

Mitogen-activated protein kinase 6 (MKK6) is one of the major important central regulatory proteins response to environmental and physiological stimuli. In this study, a novel MKK6, EcMKK6, was isolated from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. The open reading frame (ORF) of EcMKK6 is 1077 bp encoding 358 amino acids. EcMKK6 contains a serine/threonine protein kinase (S_TKc) domain, a tyrosine kinase catalytic domain, a conserved dual phosphorylation site in the SVAKT motif and a conserved DVD domain. By in situ hybridization (ISH) with Digoxigenin-labeled probe, EcMKK6 mainly located at the cytoplasm of cells, and a little appears in the nucleus. EcMKK6 mRNA can be detected in all eleven tissues examined, but the expression level is different in these tissues. After challenge with Vibrio alginolyticus and Singapore grouper iridovirus (SGIV), the transcription level of EcMKK6 was apparently up-regulated in the tissues examined. The data demonstrated that the sequence and the characters of EcMKK6 were conserved, EcMKK6 showed tissue-specific expression profiles in healthy grouper, and the expression was significantly varied after pathogen infection, indicating that EcMKK6 may play important roles in E. coioides during pathogen-caused inflammation.


Asunto(s)
Lubina/genética , Lubina/inmunología , Enfermedades de los Peces/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/inmunología , Secuencia de Aminoácidos , Animales , Infecciones por Virus ADN/inmunología , Infecciones por Virus ADN/veterinaria , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Perfilación de la Expresión Génica/veterinaria , MAP Quinasa Quinasa 6/química , Filogenia , Ranavirus/fisiología , Alineación de Secuencia/veterinaria , Vibriosis/inmunología , Vibriosis/veterinaria , Vibrio alginolyticus/fisiología
4.
Cancer Lett ; 442: 126-136, 2019 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-30391783

RESUMEN

Gossypetin as a hexahydroxylated flavonoid found in many flowers and Hibiscus. It exerts various pharmacological activities, including antioxidant, antibacterial and anticancer activities. However, the anticancer capacity of gossypetin has not been fully elucidated. In this study, gossypetin was found to inhibit anchorage-dependent and -independent growth of esophageal cancer cells. To identify the molecular target(s) of gossypetin, various signaling protein kinases were screened and results indicate that gossypetin strongly attenuates the MKK3/6-p38 signaling pathway by directly inhibiting MKK3 and MKK6 protein kinase activity in vitro. Mechanistic investigations showed that arginine-61 in MKK6 is critical for binding with gossypetin. Additionally, the inhibition of cell growth by gossypetin is dependent on the expression of MKK3 and MKK6. Gossypetin caused G2 phase cell cycle arrest and induced intrinsic apoptosis by activating caspases 3 and 7 and increasing the expression of BAX and cytochrome c. Notably, gossypetin suppressed patient-derived esophageal xenograft tumor growth in an in vivo mouse model. Our findings suggest that gossypetin is an MKK3 and MKK6 inhibitor that could be useful for preventing or treating esophageal cancer.


Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias Esofágicas/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Flavonoides/farmacología , MAP Quinasa Quinasa 3/antagonistas & inhibidores , MAP Quinasa Quinasa 6/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Sitios de Unión , Línea Celular Tumoral , Neoplasias Esofágicas/enzimología , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/enzimología , Carcinoma de Células Escamosas de Esófago/patología , Femenino , Flavonoides/química , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa 6/química , MAP Quinasa Quinasa 6/metabolismo , Ratones SCID , Simulación del Acoplamiento Molecular , Conformación Proteica , Inhibidores de Proteínas Quinasas/química , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
5.
Biosci Rep ; 38(6)2018 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-30442872

RESUMEN

Mitogen-activated protein kinase kinases (MKKs) are important components of the MAPK signaling pathways, which play a key role in responding to stress and inflammatory stimuli. Here, a new MKK gene, AccMKK6, was identified and functionally analyzed in Apis cerana cerana Real-time quantitative PCR (qPCR) and Western blot analysis demonstrated that the AccMKK6 expression level was up-regulated by several environmental stresses. Moreover, the knockdown of AccMKK6 by RNA interference technology altered the expression levels of some antioxidant genes. In addition, the knockdown of AccMKK6 resulted in increased malonyldialdehyde (MDA) concentration and decreased antioxidant-related enzymes activity in honeybees. To explore the MAPK signaling pathways involved in AccMKK6, we identified the transcription factor kayak in A. cerana cerana We analyzed the interactions of AccMKK6, Accp38b, and Acckayak using the yeast two-hybrid system. AccMKK6 and Acckayak showed similar expression profiles after several stress treatments. In addition, the expression level of Acckayak was significantly increased when AccMKK6 was silenced. Therefore, we speculate that AccMKK6 may be involved in the MAPK cascades, which play a crucial role in counteracting oxidative stress caused by external stimuli.


Asunto(s)
Abejas/metabolismo , Proteínas de Insectos/metabolismo , MAP Quinasa Quinasa 6/metabolismo , Sistema de Señalización de MAP Quinasas , Estrés Oxidativo , Mapas de Interacción de Proteínas , Secuencia de Aminoácidos , Animales , Abejas/química , Abejas/genética , Proteínas de Insectos/química , Proteínas de Insectos/genética , MAP Quinasa Quinasa 6/química , MAP Quinasa Quinasa 6/genética , Filogenia , Interferencia de ARN , Alineación de Secuencia
6.
Proc Natl Acad Sci U S A ; 114(11): E2096-E2105, 2017 03 14.
Artículo en Inglés | MEDLINE | ID: mdl-28242696

RESUMEN

Apoptosis signal-regulating kinases (ASK1-3) are apical kinases of the p38 and JNK MAP kinase pathways. They are activated by diverse stress stimuli, including reactive oxygen species, cytokines, and osmotic stress; however, a molecular understanding of how ASK proteins are controlled remains obscure. Here, we report a biochemical analysis of the ASK1 kinase domain in conjunction with its N-terminal thioredoxin-binding domain, along with a central regulatory region that links the two. We show that in solution the central regulatory region mediates a compact arrangement of the kinase and thioredoxin-binding domains and the central regulatory region actively primes MKK6, a key ASK1 substrate, for phosphorylation. The crystal structure of the central regulatory region reveals an unusually compact tetratricopeptide repeat (TPR) region capped by a cryptic pleckstrin homology domain. Biochemical assays show that both a conserved surface on the pleckstrin homology domain and an intact TPR region are required for ASK1 activity. We propose a model in which the central regulatory region promotes ASK1 activity via its pleckstrin homology domain but also facilitates ASK1 autoinhibition by bringing the thioredoxin-binding and kinase domains into close proximity. Such an architecture provides a mechanism for control of ASK-type kinases by diverse activators and inhibitors and demonstrates an unexpected level of autoregulatory scaffolding in mammalian stress-activated MAP kinase signaling.


Asunto(s)
MAP Quinasa Quinasa Quinasa 5/química , MAP Quinasa Quinasa Quinasa 5/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Humanos , MAP Quinasa Quinasa 6/química , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/metabolismo , MAP Quinasa Quinasa Quinasa 5/genética , Modelos Biológicos , Modelos Moleculares , Mutación , Fosforilación , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Relación Estructura-Actividad , Especificidad por Sustrato
7.
Int J Mol Sci ; 17(11)2016 Nov 22.
Artículo en Inglés | MEDLINE | ID: mdl-27879664

RESUMEN

Chloranthalactone B (CTB), a lindenane-type sesquiterpenoid, was obtained from the Chinese medicinal herb Sarcandra glabra, which is frequently used as a remedy for inflammatory diseases. However, the anti-inflammatory mechanisms of CTB have not been fully elucidated. In this study, we investigated the molecular mechanisms underlying these effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. CTB strongly inhibited the production of nitric oxide and pro-inflammatory mediators such as prostaglandin E2, tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 in RAW264.7 cells stimulated with LPS. A reverse-transcription polymerase chain reaction assay and Western blot further confirmed that CTB inhibited the expression of inducible nitric oxide synthase, cyclooxygenase-2, TNF-α, and IL-1ß at the transcriptional level, and decreased the luciferase activities of activator protein (AP)-1 reporter promoters. These data suggest that inhibition occurred at the transcriptional level. In addition, CTB blocked the activation of p38 mitogen-activated protein kinase (MAPK) but not c-Jun N-terminal kinase or extracellular signal-regulated kinase 1/2. Furthermore, CTB suppressed the phosphorylation of MKK3/6 by targeting the binding sites via formation of hydrogen bonds. Our findings clearly show that CTB inhibits the production of inflammatory mediators by inhibiting the AP-1 and p38 MAPK pathways. Therefore, CTB could potentially be used as an anti-inflammatory agent.


Asunto(s)
Antiinflamatorios/farmacología , Lactonas/farmacología , Lipopolisacáridos/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Sesquiterpenos/farmacología , Factor de Transcripción AP-1/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Línea Celular , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Dinoprostona/antagonistas & inhibidores , Dinoprostona/biosíntesis , Regulación de la Expresión Génica , Inflamación/prevención & control , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lipopolisacáridos/farmacología , MAP Quinasa Quinasa 3/antagonistas & inhibidores , MAP Quinasa Quinasa 3/química , MAP Quinasa Quinasa 3/genética , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa 6/antagonistas & inhibidores , MAP Quinasa Quinasa 6/química , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Moleculares , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Transducción de Señal , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Transcripción Genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
8.
J Biol Chem ; 290(44): 26661-74, 2015 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-26370088

RESUMEN

MAPKs bind to many of their upstream regulators and downstream substrates via a short docking motif (the D-site) on their binding partner. MAPKs that are in different families (e.g. ERK, JNK, and p38) can bind selectively to D-sites in their authentic substrates and regulators while discriminating against D-sites in other pathways. Here we demonstrate that the short hydrophobic region at the distal end of the D-site plays a critical role in determining the high selectivity of JNK MAPKs for docking sites in their cognate MAPK kinases. Changing just 1 or 2 key hydrophobic residues in this submotif is sufficient to turn a weak JNK-binding D-site into a strong one, or vice versa. These specificity-determining differences are also found in the D-sites of the ETS family transcription factors Elk-1 and Net. Moreover, swapping two hydrophobic residues between these D-sites switches the relative efficiency of Elk-1 and Net as substrates for ERK versus JNK, as predicted. These results provide new insights into docking specificity and suggest that this specificity can evolve rapidly by changes to just 1 or 2 amino acids.


Asunto(s)
Factor de Transcripción Activador 2/química , Proteínas Quinasas JNK Activadas por Mitógenos/química , MAP Quinasa Quinasa 4/química , MAP Quinasa Quinasa 6/química , Proteína Quinasa 1 Activada por Mitógenos/química , Factor de Transcripción Activador 2/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Regulación de la Expresión Génica , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Isoenzimas/química , Isoenzimas/genética , Proteínas Quinasas JNK Activadas por Mitógenos/genética , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 6/genética , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Alineación de Secuencia , Transducción de Señal
9.
Biochemistry ; 52(33): 5645-55, 2013 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-23848432

RESUMEN

A robust, high-throughput method has been developed to screen one-bead-one-compound peptide libraries to systematically profile the sequence specificity of protein kinases. Its ability to provide individual sequences of the preferred substrates permits the identification of sequence contextual effects and nonpermissive residues. Application of the library method to kinases Pim1, MKK6, and Csk revealed that Pim1 and Csk are highly active toward peptide substrates and recognize specific sequence motifs, whereas MKK6 has little activity or sequence selectivity against peptide substrates. Pim1 recognizes peptide substrates of the consensus RXR(H/R)X(S/T); it accepts essentially any amino acid at the S/T-2 and S/T+1 positions, but strongly disfavors acidic residues (Asp or Glu) at the S/T-2 position and a proline residue at the S/T+1 position. The selected Csk substrates show strong sequence covariance and fall into two classes with the consensus sequences of (D/E)EPIYϕXϕ and (D/E)(E/D)S(E/D/I)YϕXϕ (where X is any amino acid and ϕ is a hydrophobic amino acid). Database searches and in vitro kinase assays identified phosphatase PTP-PEST as a Pim1 substrate and phosphatase SHP-1 as a potential Csk substrate. Our results demonstrate that the sequence specificity of protein kinases is defined not only by favorable interactions between permissive residue(s) on the substrate and their cognate binding site(s) on the kinase but also by repulsive interactions between the kinase and nonpermissive residue(s).


Asunto(s)
Biblioteca de Péptidos , Péptidos/metabolismo , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Western Blotting , Proteína Tirosina Quinasa CSK , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Proteínas Fúngicas , MAP Quinasa Quinasa 6/química , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/metabolismo , Proteínas Quinasas Activadas por Mitógenos/química , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Estructura Molecular , Péptidos/química , Péptidos/genética , Unión Proteica , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 12/química , Proteína Tirosina Fosfatasa no Receptora Tipo 12/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/química , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Rodaminas/química , Rodaminas/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Familia-src Quinasas/química , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismo
10.
J Biol Chem ; 288(32): 23322-30, 2013 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-23744074

RESUMEN

The MAP kinase cascades, composed of a MAP3K, a MAP2K, and a MAPK, control switch responses to extracellular stimuli and stress in eukaryotes. The most important feature of these modules is thought to be the two double phosphorylation reactions catalyzed by MAP3Ks and MAP2Ks. We addressed whether the reactions are sequential or random in the p38 MAP kinase module. Mass spectrometry was used to track the phosphorylation of the MAP2K MEK6 by two MAP3Ks, TAO2 and ASK1, and the subsequent phosphorylation of p38α by MEK6/S*T* (where S (Ser) and T (Thr) are the two phosphorylation sites and * denotes phosphorylation). Both double phosphorylation reactions are precisely ordered. MEK6 is phosphorylated first on Thr-211 and then on Ser-207 by both MAP3Ks. This is the first demonstration of a precise reaction order for a MAP2K. p38α is phosphorylated first on Tyr-182 and then on Thr-180, the same reaction order observed previously in ERK2. Thus, intermediates were MEK6/ST* and p38α/TY*. Similarly, the phosphorylation of the p38α transcription factor substrate ATF2 occurs in a precise sequence. Progress curves for the appearance of intermediates were fit to kinetic models. The models confirmed the reaction order, revealed processivity in the phosphorylation of MEK6 by ASK1, and suggested that the order of phosphorylation is dictated by both binding and catalysis rates.


Asunto(s)
MAP Quinasa Quinasa 6/química , MAP Quinasa Quinasa Quinasa 5/química , Quinasas Quinasa Quinasa PAM/química , Proteína Quinasa 14 Activada por Mitógenos/química , Modelos Químicos , Proteínas Quinasas/química , Factor de Transcripción Activador 2/química , Factor de Transcripción Activador 2/genética , Factor de Transcripción Activador 2/metabolismo , Animales , Humanos , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/metabolismo , MAP Quinasa Quinasa Quinasa 5/genética , MAP Quinasa Quinasa Quinasa 5/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 14 Activada por Mitógenos/genética , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Modelos Biológicos , Fosforilación/fisiología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Ratas
11.
J Biochem ; 151(5): 541-9, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22383536

RESUMEN

Mitogen-activated protein kinase kinase 6 (MAP2K6) plays a crucial role in the p38 MAP kinase signal cascade that regulates various stress-induced responses and is associated with pathological conditions. The crystal structure of human non-phosphorylated MAP2K6 (npMAP2K6) complexed with an ATP analogue was determined at 2.6 Å resolution and represents an auto-inhibition state of MAP2K6. Three characteristics of short α-helices configured in the activation loop region, termed activation helices (AH1, AH2 and AH3), are important in controlling the auto-inhibition mechanism. AH1 displaces the αC-helix, a component essential for forming the active configuration, away from the active site. AH1 and AH2 were found to enclose the γ-phosphate, the leaving group of ATP. A comparison with the related enzymes, MAP2K1 and MAP2K4 reveals that MAP2K6 has the unique auto-inhibition mechanism mediated by the three activation helices.


Asunto(s)
MAP Quinasa Quinasa 6/antagonistas & inhibidores , MAP Quinasa Quinasa 6/química , Cristalografía por Rayos X , Humanos , MAP Quinasa Quinasa 6/metabolismo , Modelos Moleculares , Conformación Proteica , Relación Estructura-Actividad
12.
Biochem J ; 443(1): 95-102, 2012 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-22248149

RESUMEN

MAPK (mitogen-activated protein kinase) pathways have been implicated in stress signalling in plants. In the present study, we performed yeast two-hybrid screening to identify partner MAPKs for OsMKK (Oryza sativa MAPK kinase) 6, a rice MAPK kinase, and revealed specific interactions of OsMKK6 with OsMPK3 and OsMPK6. OsMPK3 and OsMPK6 each co-immunoprecipitated OsMKK6, and both were directly phosphorylated by OsMKK6 in vitro. An MBP (myelin basic protein) kinase assay of the immunoprecipitation complex indicated that OsMPK3 and OsMPK6 were activated in response to a moderately low temperature (12°C), but not a severely low temperature (4°C) in rice seedlings. A constitutively active form of OsMKK6, OsMKK6DD, showed elevated phosphorylation activity against OsMPK3 and OsMPK6 in vitro. OsMPK3, but not OsMPK6, was constitutively activated in transgenic plants overexpressing OsMKK6DD, indicating that OsMPK3 is an in vivo target of OsMKK6. Enhanced chilling tolerance was observed in the transgenic plants overexpressing OsMKK6DD. Taken together, our data suggest that OsMKK6 and OsMPK3 constitute a moderately low-temperature signalling pathway and regulate cold stress tolerance in rice.


Asunto(s)
MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa 6/metabolismo , Sistema de Señalización de MAP Quinasas , Oryza/fisiología , Proteínas de Plantas/metabolismo , Estrés Fisiológico , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Clima Frío , Frío , MAP Quinasa Quinasa 3/química , MAP Quinasa Quinasa 3/genética , MAP Quinasa Quinasa 6/química , MAP Quinasa Quinasa 6/genética , Mutagénesis Sitio-Dirigida , Oryza/enzimología , Fosforilación , Proteínas de Plantas/química , Proteínas de Plantas/genética , Unión Proteica
13.
Methods Mol Biol ; 661: 223-37, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20811986

RESUMEN

MAPK cascade components have been the subject of structural analysis, advancing our understanding of how these enzymes are activated and how they interact. A surprising finding has been that unique inactive conformers are adopted by many of these kinases. These inactive conformers are interesting and often require experimental phases to determine their crystal structures because molecular replacement techniques are not successful. Here, we describe the preparation of MAP2K MEK6 and MAP3K TAO2 substituted with selenomethionine (SeMet) for de novo phasing. TAO2 and SeMet TAO2 were expressed in insect cells.


Asunto(s)
MAP Quinasa Quinasa 6/química , MAP Quinasa Quinasa 6/metabolismo , Quinasas Quinasa Quinasa PAM/química , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas , Animales , Línea Celular , Cristalización , Escherichia coli/genética , Humanos , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/aislamiento & purificación , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/aislamiento & purificación , Ratones , Fosforilación , Ratas , Selenometionina/metabolismo
14.
Structure ; 17(1): 96-104, 2009 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-19141286

RESUMEN

MAP2Ks are dual-specificity protein kinases functioning at the center of three-tiered MAP kinase modules. The structure of the kinase domain of the MAP2K MEK6 with phosphorylation site mimetic aspartic acid mutations (MEK6/DeltaN/DD) has been solved at 2.3 angstroms resolution. The structure reveals an autoinhibited elongated ellipsoidal dimer. The enzyme adopts an inactive conformation, based upon structural queues, despite the phosphomimetic mutations. Gel filtration and small-angle X-ray scattering analysis confirm that the crystallographically observed ellipsoidal dimer is a feature of MEK6/DeltaN/DD and full-length unphosphorylated wild-type MEK6 in solution. The interface includes the phosphate binding ribbon of each subunit, part of the activation loop, and a rare "arginine stack" between symmetry-related arginine residues in the N-terminal lobe. The autoinhibited structure likely confers specificity on active MAP2Ks. The dimer may also serve the function in unphosphorylated MEK6 of preventing activation loop phosphorylation by inappropriate kinases.


Asunto(s)
MAP Quinasa Quinasa 1/química , MAP Quinasa Quinasa 6/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Dimerización , Activación Enzimática , Humanos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 6/antagonistas & inhibidores , MAP Quinasa Quinasa 6/metabolismo , Ratones , Modelos Moleculares , Imitación Molecular , Datos de Secuencia Molecular , Fosforilación , Conformación Proteica , Ratas , Dispersión de Radiación , Homología de Secuencia de Aminoácido , Especificidad por Sustrato
15.
Antioxid Redox Signal ; 9(11): 1803-13, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17854274

RESUMEN

Rac-dependent NADPH oxidases generate reactive oxygen species used in cell signaling and microbial killing or both. Whereas the mechanisms leading to NADPH oxidase activation are fairly well studied, the mechanisms that control downregulation of this enzyme complex remain unclear. We hypothesized that reactive oxygen species produced by NADPH oxidase may autoregulate the complex by inhibiting Rac activity. To this end, we searched for binding partners of Rac1 and identified a tyrosine-phosphorylated fragment of MKK6 that bound to Rac1 under redox-stress conditions. Constitutively active MKK6 interacted directly with Rac1 in vitro, and this interaction was enhanced when MKK6 was phosphorylated on tyrosine 219. Both Rac1 and Rac2 immunoprecipitated an MKK6 fragment under conditions that elevate cellular peroxide levels in 293 and RAW cells, respectively. Constitutively active and wild-type MKK6 enhanced Rac-GTPase activity in vitro, and their overexpression inhibited PMA-induced NADPH oxidase activation in RAW cells. In contrast, a Y219F mutant of MKK6 only partially enhanced Rac1 GTPase activity, and its overexpression did not alter PMA-induced NADPH oxidase activation in RAW cells. Last, MKK6 deficiency led to an increase in Rac1-GTP levels in brain tissue. Our findings suggest that MKK6 downregulates NADPH oxidase activity by enhancing Rac-GTPase activity.


Asunto(s)
Regulación Enzimológica de la Expresión Génica , MAP Quinasa Quinasa 6/metabolismo , Superóxidos/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Adenoviridae/genética , Animales , Línea Celular , Células Cultivadas , Escherichia coli/genética , Glutatión Transferasa/metabolismo , Peróxido de Hidrógeno/farmacología , MAP Quinasa Quinasa 6/química , MAP Quinasa Quinasa 6/genética , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Transgénicos , Mutación , Oxidantes/farmacología , Fosforilación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Tirosina/metabolismo , Proteínas de Unión al GTP rac/análisis
16.
Science ; 312(5777): 1211-4, 2006 May 26.
Artículo en Inglés | MEDLINE | ID: mdl-16728640

RESUMEN

Yersinia species use a variety of type III effector proteins to target eukaryotic signaling systems. The effector YopJ inhibits mitogen-activated protein kinase (MAPK) and the nuclear factor kappaB (NFkappaB) signaling pathways used in innate immune response by preventing activation of the family of MAPK kinases (MAPKK). We show that YopJ acted as an acetyltransferase, using acetyl-coenzyme A (CoA) to modify the critical serine and threonine residues in the activation loop of MAPKK6 and thereby blocking phosphorylation. The acetylation on MAPKK6 directly competed with phosphorylation, preventing activation of the modified protein. This covalent modification may be used as a general regulatory mechanism in biological signaling.


Asunto(s)
Proteínas Bacterianas/metabolismo , Quinasa I-kappa B/metabolismo , MAP Quinasa Quinasa 6/metabolismo , Yersinia/metabolismo , Acetilcoenzima A/metabolismo , Acetilación , Acetiltransferasas/metabolismo , Dominio Catalítico , Línea Celular , Sistema Libre de Células , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , MAP Quinasa Quinasa 6/química , Sistema de Señalización de MAP Quinasas , FN-kappa B/metabolismo , Fosforilación , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Transducción de Señal , Treonina/metabolismo , Yersinia/patogenicidad
17.
Biochemistry ; 44(50): 16475-90, 2005 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-16342939

RESUMEN

Inhibition of p38alpha MAP kinase is a potential approach for the treatment of inflammatory disorders. MKK6-dependent phosphorylation on the activation loop of p38alpha increases its catalytic activity and affinity for ATP. An inhibitor, BIRB796, binds at a site used by the purine moiety of ATP and extends into a "selectivity pocket", which is not used by ATP. It displaces the Asp168-Phe169-Gly170 motif at the start of the activation loop, promoting a "DFG-out" conformation. Some other inhibitors bind only in the purine site, with p38alpha remaining in a "DFG-in" conformation. We now demonstrate that selectivity pocket compounds prevent MKK6-dependent activation of p38alpha in addition to inhibiting catalysis by activated p38alpha. Inhibitors using only the purine site do not prevent MKK6-dependent activation. We present kinetic analyses of seven inhibitors, whose crystal structures as complexes with p38alpha have been determined. This work includes four new crystal structures and a novel assay to measure K(d) for nonactivated p38alpha. Selectivity pocket compounds associate with p38alpha over 30-fold more slowly than purine site compounds, apparently due to low abundance of the DFG-out conformation. At concentrations that inhibit cellular production of an inflammatory cytokine, TNFalpha, selectivity pocket compounds decrease levels of phosphorylated p38alpha and beta. Stabilization of a DFG-out conformation appears to interfere with recognition of p38alpha as a substrate by MKK6. ATP competes less effectively for prevention of activation than for inhibition of catalysis. By binding to a different conformation of the enzyme, compounds that prevent activation offer an alternative approach to modulation of p38alpha.


Asunto(s)
MAP Quinasa Quinasa 6/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , MAP Quinasa Quinasa 6/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores
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