RESUMEN
The precise regulation of both the magnitude and the duration of Janus kinase (JAK) catalytic activity is essential for the cytokine orchestration of many biological processes, and the dysregulation of JAK activity has pathological implications. Immunosuppressive disease states, such as X-linked severe combined immunodeficiency, arise from inappropriate JAK inhibition. In contrast, a limited number of cancers, primarily leukemias, result from constitutive or enhanced activation of JAK activity. JAKs are no longer implicated only in classic cytokine receptor-mediated signaling pathways, but are now also known to integrate indirectly into other receptor-mediated signal transduction processes. Therefore, an increasing number of therapeutic applications exist for biological-response modifiers that can restore aberrant JAK activity to normal levels. Exciting breakthroughs in both physiological and pharmacological methods of selective inhibition of cytokine-JAK-signal transducers and activators of transcription pathways have recently emerged in the form of suppressors of cytokine signaling (also known as cytokine-inducible SH2 protein, JAK-binding protein, or STAT-induced STAT inhibitor) proteins and novel dimethoxyquinazoline derivatives, respectively. The basis of these and other mechanisms of negative regulation of JAK activity, including the suppression of jak expression levels caused by tumor- or pathogen-derived agents, the complex interactions of JAKs with phosphatases, and the redox regulation of JAK catalytic activity, is the focus of this review.
Asunto(s)
Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Animales , Transformación Celular Neoplásica , Citocinas/metabolismo , Activación Enzimática , Humanos , Tolerancia Inmunológica , Janus Quinasa 1 , Janus Quinasa 2 , Janus Quinasa 3 , Neoplasias/enzimología , Oxidación-Reducción , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Proteínas/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The Ku heterodimeric protein (Ku80/Ku70) is an essential component of the double-strand break DNA repair pathway in mammalian cells. We have recently defined a central region within Ku80 that is required for heterodimerization with Ku70. We now identified a core region within Ku80 (amino acids 210 to 531) that is necessary for binding of Ku to DNA ends. Interaction with Ku70 and DNA end binding are important for Ku80 function in vivo, since Ku80 mutants lacking DNA end binding activity were unable to restore radiation resistance in Ku80 deficient fibroblast cell lines. However, Ku80 mutants were identified that retained DNA end binding activity but were unable to restore radiation survival, thus pointing to additional functional properties of Ku80. An N-terminal deletional mutant of Ku80 was able to suppress wild type Ku80 function for radiation survival in several cell lines, thus demonstrating dominant negative function.
Asunto(s)
Antígenos Nucleares , Supervivencia Celular , ADN Helicasas , Reparación del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Radiación Ionizante , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Dimerización , Embrión de Mamíferos , Fibroblastos , Humanos , Autoantígeno Ku , Ratones , Mutación , Proteínas Nucleares/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Relación Estructura-Actividad , TransfecciónRESUMEN
Here we report the expression of a fork head domain protein in human T helper cells. We cloned and characterized a fork head cDNA from human T helper cell mRNA using differential display RT-PCR. The cDNA contains a 546-nucleotide (nt) open reading frame (ORF) that codes for the carboxyl-terminal 180 amino acids (aa) of the recently identified fkhrl1 gene. This ORF does not contain the characteristic DNA-binding domain found in members of the forkhead protein family. In-vitro transcription/translation of this cDNA expressed a protein of approximately 20 kDa. We have generated antibodies that specifically immunoprecipitated the in-vitro-translated 20-kDa protein. This antibody also recognizes in human T lymphocytes a 70-kDa protein corresponding in size to that predicted for the fkhrl1 gene product. The mRNA levels for fkhrl1 is elevated in T helper-induced lymphocytes in comparison to PHA-stimulated T lymphocytes. Further characterization of FKHRL1 and its related family members should shed light on the transcriptional mechanisms of this fork head gene subfamily and their role in T helper cell differentiation and regulation of cell growth.
Asunto(s)
Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Diferenciación Celular , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Proteínas de Unión al ADN/análisis , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead , Regulación del Desarrollo de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Pruebas de Precipitina , Biosíntesis de Proteínas , ARN Mensajero/análisis , ARN Mensajero/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Linfocitos T Colaboradores-Inductores/química , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/metabolismo , Factores de Transcripción/análisisRESUMEN
Cytokines, hormones and hematopoietic growth factors transduce biological signals across the cell membrane via a highly conserved family of single membrane-spanning receptors. The intracellular signal transducing machinery responsible for mediating these responses has remained largely unknown. However, recent identification of a homologous class of tyrosine kinases, Janus Kinases (JAKs), and a related family of transcription factors, signal transducers and activators of transcription (STATs), has shed new light on the molecular mechanisms responsible for mediating hematopoietin signaling and immune response. Current research efforts within the field of cytokine signaling have now shifted to understanding how these molecules are activated by hematopoietic receptors, positively and negatively regulated by kinases and phosphatases, and how they impact on gene transcription to ultimately coordinate cell homeostasis, proliferation and differentiation. This article will review some of our results identifying the involvement of JAKs, STATs, and secondary effector molecules activated following engagement of hematopoietic receptors for IL-2, IL-4, and prolactin. Here, we provide evidence for the ingenious ability of cytokine receptors to selectively recruit and activate these proteins among a repertoire of possible alternative biochemical messengers as a means to affect unique and general cell responses.
Asunto(s)
Citocinas/fisiología , Factores de Crecimiento de Célula Hematopoyética/fisiología , Transducción de Señal/fisiología , Animales , Proteínas de Unión al ADN/fisiología , Humanos , Interleucina-2/fisiología , Interleucina-4/fisiología , Modelos Biológicos , Prolactina/fisiología , Proteínas Tirosina Quinasas/fisiología , Receptores de Citocinas/fisiología , Transactivadores/fisiologíaRESUMEN
The activation of Janus kinases (JAKs) is crucial for propagation of the proliferative response initiated by many cytokines. The proliferation of various cell lines, particularly those of hematopoietic origin, is also modulated by mediators of oxidative stress such as nitric oxide and thiol redox reagents. Herein we demonstrate that nitric oxide and other thiol oxidants can inhibit the autokinase activity of rat JAK2 in vitro, presumably through oxidation of crucial dithiols to disulfides within JAK2. The reduced form of JAK2 is the most active form, and the oxidized JAK2 form is inactive. Nitric oxide pretreatment of quiescent Ba/F3 cells also inhibits the interleukin 3-triggered in vivo activation of JAK2, a phenomenon that correlates with inhibited proliferation. Furthermore, we observed that the autokinase activity of JAK3 responds in a similar fashion to thiol redox reagents in vitro and to nitric oxide donors in vivo. We suggest that the thiol redox regulation of JAKs may partially explain the generally immunosuppressive effects of nitric oxide and of other thiol oxidants.
Asunto(s)
Óxido Nítrico/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Reactivos de Sulfhidrilo/farmacología , Animales , Baculoviridae , Células Cultivadas , Ditiotreitol/farmacología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Yodobenzoatos/farmacología , Janus Quinasa 1 , Janus Quinasa 2 , Janus Quinasa 3 , Ratones , Oxidación-Reducción , Ratas , SpodopteraRESUMEN
The Janus family of protein-tyrosine kinases has long been known to function in signal transduction pathways initiated by a host of cytokines. A brief overview of the role of Janus kinases (Jaks) in both cytokine and noncytokine signaling pathways highlights the broad physiologic importance of this kinase family. New insights into the structural and mechanistic regulatory aspects of Janus kinases are rapidly emerging. Recent mutational analyses allow the dissection of Jaks into three distinct structural domains governing receptor affiliation, autoregulation, and catalysis. A fourth domain determining substrate specificity is as yet poorly defined and is, therefore, discussed in the context of known substrates and inhibitors, a collection of molecules that have been expanded recently to include Stam and Jab. The proposed mechanism of the interconversion of Janus kinases from inactive to fully active enzymes involves three states of enzymatic activity. Additional layers of regulation can be independently superimposed on this multistate model, providing a simplified description of the behavior of Janus kinases under normal and pathologic circumstances.
Asunto(s)
Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Animales , Historia del Siglo XX , Humanos , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/historia , Transducción de SeñalRESUMEN
Microinjection of expression plasmids encoding either JAK2 or hyperactive (Ndelta661)rJAK2 into serum-starved NIH 3T3 cells resulted in 20-30-fold induction of DNA synthesis. Control microinjections of buffer or parental pcDNA3 vector resulted in only 3-5-fold induction of DNA synthesis. Induction of DNA synthesis was blocked when plasmid encoding JAK2 was microinjected in the presence of the JAK2-selective inhibitor AG-490, whereas AG-490 did not block DNA synthesis induced by microinjected plasmid encoding (Ndelta661)rJAK2. The ability of JAK2 to initiate the G(o)/S cell cycle transition is comparable to that of other proto-oncogenes, and supports a mechanistic role for overexpressed Janus kinases in carcinogenesis.
Asunto(s)
ADN/biosíntesis , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Tirfostinos , Células 3T3 , Animales , Replicación del ADN/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Janus Quinasa 2 , Cinética , Ratones , Microinyecciones/métodos , Nitrilos/farmacología , Plásmidos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Transfección/métodosRESUMEN
Three forms of rat JAK2 (type 2 Janus tyrosine kinase) were produced via the baculovirus expression vector system. Recombinant baculoviruses encoded either the full-length rat jak2 cloned from the Nb2-SP cell line (rJAK2), a carboxyl-terminal deletion mutant lacking the putative catalytic domain (rJAK2(C delta 795)), or an amino-terminal deletion mutant containing the putative catalytic domain ((N delta 661)rJAK2). The proteins produced in infected Sf21 cells were assayed for phosphotyrosine content and autophosphorylating activity. Tyrosine phosphorylation of rJAK2 was not observed 1 day postinfection when rJAK2 was initially produced but was apparent 2 or more days postinfection when the rJAK2 level had significantly increased. Tyrosine phosphorylation of rJAK2(C delta 795) was not observed; further, coproduction of rJAK2(C delta 795) with rJAK2 blocked tyrosine phosphorylation of rJAK2, consistent with previously published results (Zhuang, H., Patel, S. V., He, T-C., Sonsteby, S. K., Niu, Z., and Wojchowski, D. M. (1994) J. Biol. Chem. 269, 21411-21414). Mutant (N delta 661)rJAK2 exhibited a robust tyrosine phosphorylation signal. A second 62-kDa tyrosine phosphoprotein co-immunoprecipitated with (N delta 661)rJAK2 but not with rJAK2 or rJAK2(C delta 795). Both rJAK2 and (N delta 661)rJAK2 incorporated phosphate under in vitro kinase assay conditions, but rJAK2(C delta 795) did not. A JAK2 oligomer with interacting catalytic sites and/or inhibitory sites would provide a simple model to describe these results.
Asunto(s)
Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Animales , Baculoviridae , Western Blotting , Clonación Molecular , Janus Quinasa 2 , Cinética , Mutagénesis , Fosfotirosina/análisis , Proteínas Tirosina Quinasas/análisis , Proteínas Tirosina Quinasas/biosíntesis , Ratas , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Transducción de Señal , Spodoptera , Factores de Tiempo , TransfecciónRESUMEN
A complete cDNA clone encoding the rat JAK2 protein tyrosine kinase was isolated from an Nb2-SP (rat pre-T lymphoma cell line) cDNA library. The nucleotide (nt) and deduced amino acid (aa) sequences for this clone were determined and an open reading frame of 3399 bp, encoding a protein of a deduced mass of 130 kDa, was found. The coding regions of the rat and murine Jak2 clones share 93.4% nt identity and 97.1% aa identity. Northern analysis demonstrated that the 5-kb mRNA is highly abundant in brain and spleen, less abundant in skeletal muscle and testis, and detectable in kidney, heart, lung and liver. Translation of the rat Jak2 mRNA in rabbit reticulocytes results in a protein which is specifically immunoprecipitated by antibodies (Ab) recognizing JAK2, but not by Ab recognizing JAK1.
Asunto(s)
Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Secuencia Conservada , ADN Complementario/genética , Biblioteca de Genes , Janus Quinasa 2 , Linfoma/enzimología , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Proteínas Tirosina Quinasas/biosíntesis , Ratas , Análisis de Secuencia de ADN , Distribución Tisular , Transcripción Genética , Células Tumorales CultivadasRESUMEN
The calmodulin binding domain of the type I adenylyl cyclase has recently been identified as an amino acid sequence (residues 495-522) that contains 2 cysteine residues. Therefore, we examined the effect of several sulfhydryl reagents on the calmodulin sensitivity of the enzyme. Treatment of membranes containing the type I adenylyl cyclase with N-ethylmaleimide rapidly inhibited basal, calcium/calmodulin-stimulated, and forskolin-stimulated adenylyl cyclase activity. When the enzyme was treated with limiting amounts of o-iodosobenzoate, which oxidizes vicinal sulfhydryls to disulfides, stimulation by Ca2+ and calmodulin was eliminated at concentrations which did not affect basal adenylyl cyclase activity. Calmodulin stimulation of the enzyme was restored by treatment with dithiothreitol or glutathione which reduce disulfides to free thiols. NO and sodium nitroprusside also reversible inhibited calmodulin stimulation of the enzyme. We propose that the loss in calmodulin sensitivity caused by these reagents may be due to the oxidation one or more sets of vicinal thiols present in the enzyme.
Asunto(s)
Adenilil Ciclasas/química , Calcio/farmacología , Calmodulina/farmacología , Cisteína/química , Yodobenzoatos/química , Óxido Nítrico/química , Inhibidores de Adenilato Ciclasa , Adenilil Ciclasas/metabolismo , Animales , Calcio/antagonistas & inhibidores , Calmodulina/antagonistas & inhibidores , Bovinos , Células Cultivadas , Activación Enzimática , Etilmaleimida/farmacología , Cinética , Nitroprusiato/farmacología , Compuestos de Sulfhidrilo/químicaRESUMEN
A water-soluble, highly polar, heat-stable, small molecule has been isolated from cell-free extracts of the halotolerant green alga Dunaliella salina. This compound, soluble inhibitory factor (SIF), when added to in vivo light-activated, thylakoid membrane-bound preparations of the D. salina coupling factor 1 (CF1), causes a rapid inactivation of the ATPase activity. SIF must be in its oxidized form to inactivate the CF1 ATPase and probably functions by oxidizing the reduced form of the light-activated enzyme. SIF has been purified to homogeneity and characterized by UV-visible and IR absorption spectroscopy, 1H and 13C NMR spectroscopy, and mass spectrometry. SIF has five different kinds of nonexchangeable protons and seven different kinds of carbon atoms. Three of the carbon atoms and one proton are part of a heterocyclic (imidazole) ring. One carbon atom is a carbonyl (carboxylic acid). One carbon atom and three protons form a methyl group attached to the aromatic ring. One carbon atom and two protons are a methylene group, and one carbon atom (an alpha-amino carbon) is attached to a single proton. In addition, in its reduced form, SIF contains a thiol group attached to the heterocyclic ring. From high resolution mass spectrometry, the molecular weight of SIF was determined to be 401 (M + H+) and is consistent with the composition being C14H21N6O4S2. The UV absorption of SIF shows a large increase at 240 nm upon reduction. An effective difference extinction coefficient for this absorbance change has been calculated to be 6.84 meq/cm. A comparison of SIF with the oxidized form of ovothiol A (1-N-methyl-4-mercaptohistidine disulfide) shows the two compounds to be identical in all respects. In addition, ovothiol A disulfide is as effective as SIF in inhibiting the light-triggered, CF1 ATPase activity. It is concluded, therefore, that SIF and L-1-N-methyl-4-mercaptohistidine disulfide are identical.
Asunto(s)
Aminoácidos Sulfúricos/metabolismo , Chlorophyta/enzimología , Histidina/análogos & derivados , ATPasas de Translocación de Protón/metabolismo , Aminoácidos Sulfúricos/química , Aminoácidos Sulfúricos/aislamiento & purificación , Ditiotreitol/farmacología , Histidina/química , Histidina/aislamiento & purificación , Histidina/metabolismo , Cinética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Estructura Molecular , Rotación Óptica , Oxidación-Reducción , EspectrofotometríaRESUMEN
The chloroplast coupling factor 1 complex (CF1) contains an epsilon-subunit which inhibits the CF1 ATPase activity. Chloroform treatment of Chlamydomonas reinhardtii thylakoid membranes solubilizes only forms of the enzyme which apparently lack the delta-subunit. Four interrelated observations are described in this paper. (1) The dithiothreitol- (DTT) induced ATPase activation of CF1(-delta) and the DTT-induced formation of a physically resolvable CF1(-delta,epsilon) from the CF1(-delta) precursor are compared. The similar time-courses of these two phenomena suggest that the dissociation of the epsilon-subunit is an obligatory process in the DTT-induced ATPase activation of soluble CF1. (2) The reversible dissociation of the epsilon-subunit of the CF1 is demonstrated by the exchange of subunits between distinguishable oligomers. 35S-labelled chloroplast coupling factor 1 lacking the delta and epsilon subunits [CF1(-delta,epsilon)] was added to a solution of non-radioactive coupling factor 1 lacking only the delta subunit [CF1(-delta)]. After separation of the two enzyme forms, via high resolution anion-exchange chromatography, radioactivity was detected in the chromatographic fractions containing CF1(-delta). (3) epsilon-deficient CF1 can be resolved from DTT pretreated epsilon-containing CF1 for several days after the removal of DTT. On the other hand, brief incubation of the DTT pretreated epsilon-containing CF1 with low concentrations of o-iodosobenzoate results in chromatographs containing only the peak of epsilon-containing CF1. A simple explanation for this phenomenon is that reduction of CF1 with DTT increases the apparent dissociation constant for the epsilon-subunit to an estimated 3.5 x 10(-8) M (+/- 1.0 x 10(-8) M) from a value of less than or equal to 5 x 10(-11) M for the oxidized enzyme. (4) ATPase activity data show that oxidation of the epsilon-deficient enzyme does not completely inhibit its manifest activity, but oxidation of DTT pre-treated CF1 which contains the epsilon-subunit completely inhibits manifest activity. A simple model is proposed for the influence of the oxidation state of the soluble enzyme on the distribution of ATPase-inactive and ATPase-active subunit configurations.
Asunto(s)
Chlamydomonas/enzimología , Ditiotreitol/farmacología , ATPasas de Translocación de Protón/metabolismo , Adenosina Trifosfatasas/metabolismo , Chlamydomonas/efectos de los fármacos , Cromatografía , Yodobenzoatos/farmacología , Cinética , Conformación Proteica , SolubilidadRESUMEN
The sensitivity of the catalytic activities of the D. salina chloroplast coupling factor 1 (CF1) to chemical modification by N-ethylmaleimide has been investigated. When D. salina thylakoid membranes are treated with N-ethylmaleimide, both photophosphorylation and the inducible CF1 ATPase activity are partially (approx. 60%) inhibited. The inhibition of both activities does not require the presence of a proton-motive force, and the inhibition of photophosphorylation is directly related to the N-ethylmaleimide-covalent modification of CF1 as shown by the time-course for the inhibition and the maximal extent of inhibition. Treatment of the purified, latent, D. salina CF1 with low concentrations of N-ethylmaleimide also results in the partial (approx. 60%) inhibition of the inducible ATPase activity (I50 approximately 50 microM). The inhibition does not require the presence of the chemical modifier during the activation of the enzyme. N-ethylmaleimide-induced inhibition of the ATPase activity of either membrane-bound or solubilized CF1 is partially reversed by either prolonged incubation at low concentrations of N-ethylmaleimide or short incubation times at high concentrations of N-ethylmaleimide. The results are interpreted as indicating multiple binding sites on the D. salina CF1 that have different rates of reactivity with N-ethylmaleimide. Those sites (or site) that react rapidly with N-ethylmaleimide cause(s) an inhibition of both ATP synthase and ATPase activities, whereas those sites (or site) that react more slowly partially restore(s) the original ATPase activity. The effects of N-ethylmaleimide on the catalytic activity of D. salina CF1 are probably mediated by N-ethylmaleimide-induced conformational changes of the enzyme.