RESUMEN
p38 mitogen-activated protein kinase (MAPK) (p38/p38-alpha/CSBP2/RK) has been implicated in the regulation of many proinflammatory pathways. Because of this, it has received much attention as a potential drug target for controlling diseases such as rheumatoid arthritis, endotoxic shock, inflammatory bowel disease, osteoporosis, and many others. A number of small molecule inhibitors of this kinase have been described, and in this paper we have used surface plasmon resonance to directly measure and quantitate their binding to p38. Despite the relatively low molecular mass (approximately 400 Da) of these inhibitors, specific binding can be observed. For the two most potent inhibitors studied, SB 203580 and RWJ 67657, dissociation constants, K(d)'s, of 22 and 10 nm, respectively, were obtained. These values closely match the IC(5)0 values observed in a cell-based TNF alpha release assay implying that p38 plays a major role in TNF alpha release. The association and dissociation rates for the binding of these inhibitors to p38 have also been quantitated. SB 203580 and RWJ 67657 have very similar association rates of around 8 x 10(5) m(-1) x s(-1), and the differences in affinity are determined by different dissociation rates. The weaker binding compounds have dissociation rates similar to SB 203580, but the association rates vary by an order of magnitude or more. The direct measurement of compounds binding to p38 may help in understanding the difference between potency and efficacy for these inhibitors. This in turn may yield clues on how to develop better inhibitors.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Drosophila , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Unión Proteica , Resonancia por Plasmón de Superficie , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Tumor necrosis factor-alpha (TNF-alpha), a cytokine secreted by activated monocytes/macrophages and T lymphocytes, has been implicated in several disease states, including rheumatoid arthritis, inflammatory bowel disease, septic shock, and osteoporosis. Monocyte/macrophage production of TNF-alpha is dependent on the mitogen-activated protein kinase p38. RWJ 67657 (4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol -2-yl]-3-butyn-1-ol) inhibited the release of TNF-alpha by lipopolysaccharide (a monocyte stimulus)-treated human peripheral blood mononuclear cells with an IC(50) of 3 nM, as well as the release of TNF-alpha from peripheral blood mononuclear cells treated with the superantigen staphylococcal enterotoxin B (a T cell stimulus), with an IC(50) value of 13 nM. This compound was approximately 10-fold more potent than the literature standard p38 kinase inhibitor SB 203580 in all p38 dependent in vitro systems tested. RWJ 67657 inhibited the enzymatic activity of recombinant p38alpha and beta, but not gamma or delta, in vitro and had no significant activity against a variety of other enzymes. In contrast, SB 203580 significantly inhibited the tyrosine kinases p56 lck and c-src (IC(50) = 5 microM). RWJ 67657 did not inhibit T cell production of interleukin-2 or interferon-gamma and did not inhibit T cell proliferation in response to mitogens. RWJ 67657 inhibited TNF-alpha production in lipopolysaccharide-injected mice (87% inhibition at 50 mg/kg) and in rats (91% inhibition at 25 mg/kg) after oral administration. Based on these favorable biological properties, RWJ 67657 may have use as a treatment for inflammatory diseases.
Asunto(s)
Imidazoles/farmacología , Macrófagos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Monocitos/metabolismo , Proteínas Quinasas/metabolismo , Piridinas/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Antígenos/inmunología , División Celular/efectos de los fármacos , Perros , Relación Dosis-Respuesta a Droga , Enterotoxinas/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratas , Ratas Endogámicas Lew , Staphylococcus/fisiología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
T cell proliferation and cytokine production usually require stimulation via both the TCR/CD3 complex and the CD28 costimulatory receptor. Using purified human CD4+ peripheral blood T cells, we show that CD28 stimulation alone activates p38 alpha mitogen-activated protein kinase (p38 alpha). Cell proliferation induced by CD28 stimulation alone, a response attributed to CD4+CD45RO+ memory T cells, was blocked by the highly specific p38 inhibitors SB 203580 (IC50 = 10-80 nM) and RWJ 67657 (IC50 = 0.5-4 nM). In contrast, proliferation induced by anti-CD3 plus anti-CD28 mAbs was not blocked. Inhibitors of p38 also blocked CD4+ T cell production of IL-4 (SB 203580 IC50 = 20-100 nM), but not IL-2, in response to CD3 and CD28 stimulation. IL-5, TNF-alpha, and IFN-gamma production were also inhibited, but to a lesser degree than IL-4. IL-4 production was attributed to CD4+CD45RO+ T cells, and its induction was suppressed by p38 inhibitors at the mRNA level. In polarized Th1 and Th2 cell lines, SB 203580 strongly inhibited IL-4 production by Th2 cells (IC50 = 10-80 nM), but only partially inhibited IFN-gamma and IL-2 production by Th1 cells (<50% inhibition at 1 microM). In both Th1 and Th2 cells, CD28 signaling activated p38 alpha and was required for cytokine production. These results show that p38 alpha plays an important role in some, but not all, CD28-dependent cellular responses. Its preferential involvement in IL-4 production by CD4+CD45RO+ T cells and Th2 effector cells suggests that p38 alpha may be important in the generation of Th2-type responses in humans.
Asunto(s)
Antígenos CD28/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Interleucina-4/biosíntesis , Antígenos Comunes de Leucocito/análisis , Proteínas Quinasas Activadas por Mitógenos , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/enzimología , Células Th2/enzimología , Adulto , Linfocitos T CD4-Positivos/enzimología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Activación Enzimática/inmunología , Inhibidores Enzimáticos/farmacología , Humanos , Imidazoles/farmacología , Interleucina-2/biosíntesis , Interleucina-4/antagonistas & inhibidores , Interleucina-4/genética , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Piridinas/farmacología , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Células Th2/inmunología , Células Th2/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Interleukin (IL)-18 is functionally similar to IL-12 in mediating T helper cell type 1 (Th1) response and natural killer (NK) cell activity but is related to IL-1 in protein structure and signaling, including recruitment of IL-1 receptor-associated kinase (IRAK) to the receptor and activation of c-Jun NH2-terminal kinase (JNK) and nuclear factor (NF)-kappaB. The role of IRAK in IL-18-induced responses was studied in IRAK-deficient mice. Significant defects in JNK induction and partial impairment in NF-kappaB activation were found in IRAK-deficient Th1 cells, resulting in a dramatic decrease in interferon (IFN)-gamma mRNA expression. In vivo Th1 response to Propionibacterium acnes and lipopolysaccharide in IFN-gamma production and induction of NK cytotoxicity by IL-18 were severely impaired in IRAK-deficient mice. IFN-gamma production by activated NK cells in an acute murine cytomegalovirus infection was significantly reduced despite normal induction of NK cytotoxicity. These results demonstrate that IRAK plays an important role in IL-18-induced signaling and function.
Asunto(s)
Interleucina-18/farmacología , Células Asesinas Naturales/inmunología , Proteínas Quinasas Activadas por Mitógenos , Proteínas Quinasas/deficiencia , Transducción de Señal/fisiología , Células TH1/inmunología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/biosíntesis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Quimera , Cruzamientos Genéticos , Infecciones por Citomegalovirus/inmunología , Citotoxicidad Inmunológica/efectos de los fármacos , Depresión Química , Activación Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Interferón gamma/biosíntesis , Interferón gamma/genética , Quinasas Asociadas a Receptores de Interleucina-1 , Subunidad alfa del Receptor de Interleucina-18 , Interleucina-4/biosíntesis , Interleucina-4/genética , Proteínas Quinasas JNK Activadas por Mitógenos , Lipopolisacáridos/inmunología , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Propionibacterium acnes/inmunología , Proteínas Quinasas/genética , Proteínas Quinasas/fisiología , Receptores de Interleucina/fisiología , Receptores de Interleucina-18 , Células TH1/citología , Células Th2/citología , Células Th2/inmunologíaRESUMEN
p38 mitogen-activated protein kinase (MAPK) (p38) is involved in various cellular responses, including LPS stimulation of monocytes, resulting in production of proinflammatory cytokines such as TNF-alpha. However, the function of p38 during antigenic stimulation of T cells is largely unknown. Stimulation of the human Th cell clone HA-1.70 with either the superantigen staphylococcal enterotoxin B (SEB) or with a specific antigenic peptide resulted in p38 activation and the release of TNF-alpha. MAPK-activated protein kinase-2 (MAPKAPK-2), an in vivo substrate for p38, was also activated by T cell signaling. SB 203580, a selective inhibitor of p38, blocked p38 and MAPKAPK-2 activation in the T cell clone but did not completely inhibit TNF-alpha release. PD 098059, a selective inhibitor of MAPK kinase 1 (MEK1), blocked activation of extracellular signal-regulated kinase (ERK) and partially blocked TNF-alpha production by the clone. In human peripheral T cells, p38 was not activated by SEB, but rather by CD28 cross-linking, whereas in the human leukemic T cell line Jurkat, p38 was activated by CD3 and CD28 cross-linking in an additive fashion. TNF-alpha production by peripheral T cells in response to SEB and anti-CD28 mAb correlated more closely with ERK activity than with p38 activity. Therefore, various forms of T cell stimulation can activate the p38 pathway depending on the cells examined. Furthermore, unlike LPS-stimulated monocytes, TNF-alpha production by T cells is only partially p38-dependent.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Lipopolisacáridos/inmunología , Activación de Linfocitos , Proteínas Quinasas Activadas por Mitógenos , Monocitos/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Regulación hacia Arriba/inmunología , Anticuerpos Monoclonales/metabolismo , Antígenos CD28/sangre , Antígenos CD28/inmunología , Antígenos CD28/fisiología , Calcineurina/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Células Clonales/enzimología , Células Clonales/inmunología , Células Clonales/metabolismo , Enterotoxinas/farmacología , Activación Enzimática/inmunología , Antígenos de Histocompatibilidad Clase II/farmacología , Humanos , Células Jurkat , Activación de Macrófagos , Monocitos/metabolismo , Staphylococcus aureus/inmunología , Superantígenos/farmacología , Linfocitos T/enzimología , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosAsunto(s)
Aminopiridinas/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Proteínas Quinasas Activadas por Mitógenos , Pirroles/farmacología , Animales , Artritis Experimental/patología , Humanos , Técnicas In Vitro , Interleucina-1/antagonistas & inhibidores , Lipopolisacáridos , Masculino , Ratones , Ratones Endogámicos BALB C , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Ratas , Ratas Endogámicas Lew , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Lamina-associated polypeptide (LAP) 2 of the inner nuclear membrane (now LAP2beta) and LAP2alpha are related proteins produced by alternative splicing, and contain a common 187 amino acid N-terminal domain. We show here that, unlike LAP2beta, LAP2alpha behaved like a nuclear non-membrane protein in subcellular fractionation studies and was localized throughout the nuclear interior in interphase cells. It co-fractionated with LAP2beta in nuclear lamina/matrix-enriched fractions upon extraction of nuclei with detergent, salt and nucleases. During metaphase LAP2alpha dissociated from chromosomes and became concentrated around the spindle poles. Furthermore, LAP2alpha was mitotically phosphorylated, and phosphorylation correlated with increased LAP2alpha solubility upon extraction of cells in physiological buffers. LAP2alpha relocated to distinct sites around chromosomes at early stages of nuclear reassembly and intermediarily co-localized with peripheral lamin B and intranuclear lamin A structures at telophase. During in vitro nuclear assembly LAP2alpha was dephosphorylated and assembled into insoluble chromatin-associated structures, and recombinant LAP2alpha was found to interact with chromosomes in vitro. Some LAP2alpha may also associate with membranes prior to chromatin attachment. Altogether the data suggest a role of LAP2alpha in post-mitotic nuclear assembly and in the dynamic structural organization of the nucleus.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN , Interfase , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Animales , Células CHO , Núcleo Celular/ultraestructura , Cricetinae , Detergentes , Células HeLa , Humanos , Microscopía Inmunoelectrónica , Mitosis , Fosforilación , Sales (Química)RESUMEN
Interleukin (IL)-1 is a proinflammatory cytokine with pleiotropic effects in inflammation. IL-1 binding to its receptor triggers a cascade of signaling events, including activation of the stress-activated mitogen-activated protein (MAP) kinases, c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase, as well as transcription factor nuclear factor kappaB (NF-kappaB). IL-1 signaling results in cellular responses through induction of inflammatory gene products such as IL-6. One of the earliest events in IL-1 signaling is the rapid interaction of IL-1 receptor-associated kinases, IRAK and IRAK-2, with the receptor complex. The relative roles of IRAK and IRAK-2 in IL-1 signaling pathways and subsequent cellular responses have not been previously determined. To evaluate the importance of IRAK in IL-1 signaling, IRAK-deficient mouse fibroblast cells were prepared and studied. Here we report that IL-1-mediated activation of JNK, p38, and NF-kappaB were all reduced in embryonic fibroblasts deficient in IRAK expression. In addition, IL-6 production in response to IL-1 was also dramatically reduced in IRAK-deficient embryonic fibroblasts and in skin fibroblasts prepared from IRAK-deficient mice. Our results demonstrate that IRAK plays an essential proximal role in coordinating multiple IL-1 signaling pathways for optimal induction of cellular responses.
Asunto(s)
Interleucina-1/metabolismo , Interleucina-6/biosíntesis , Proteínas Quinasas Activadas por Mitógenos , Proteínas Quinasas/metabolismo , Receptores de Interleucina-1/metabolismo , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/citología , Quinasas Asociadas a Receptores de Interleucina-1 , Proteínas Quinasas JNK Activadas por Mitógenos , Masculino , Ratones , Mutación , FN-kappa B/metabolismo , Proteínas Quinasas/genética , Transducción de Señal , Piel/citología , Cromosoma X , Proteínas Quinasas p38 Activadas por MitógenosAsunto(s)
Proteínas Portadoras/aislamiento & purificación , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Choque Térmico/aislamiento & purificación , Isomerasa de Peptidilprolil/metabolismo , Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Calcineurina/metabolismo , Proteínas Portadoras/análisis , Ciclosporina/metabolismo , Proteínas de Unión al ADN/análisis , Inhibidores Enzimáticos/farmacología , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico/análisis , Inmunosupresores/farmacología , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Isomerasa de Peptidilprolil/aislamiento & purificación , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosforilación , Polienos/metabolismo , Unión Proteica/fisiología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Sirolimus , Tacrolimus/análogos & derivados , Tacrolimus/farmacología , Proteínas de Unión a TacrolimusRESUMEN
The MAP kinase p38 plays a key role in the biosynthesis of the inflammatory cytokines TNF-alpha and IL-1. We have developed a novel series of potent p38 inhibitors that could lead to new methods of treatment for inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease.
Asunto(s)
Antiinflamatorios/síntesis química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Proteínas Quinasas Activadas por Mitógenos , Antiinflamatorios/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Imidazoles/farmacología , Péptidos y Proteínas de Señalización Intracelular , Lipopolisacáridos/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridinas/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Early T-cell receptor mediated signal transduction involves the activation of several tyrosine protein kinases. One of these tyrosine kinases, p56lck, is expressed primarily in T-cells and Natural Killer (NK) cells and has been shown to be critical for their proliferative and effector functions. Indandiones have been identified as a potent and selective chemical class that inhibits p56lck.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Proteína Tirosina Quinasa CSK , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Indanos/química , Indanos/farmacología , Células Asesinas Naturales/enzimología , Modelos Químicos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Transducción de Señal , Linfocitos T/enzimología , Familia-src QuinasasRESUMEN
Thymopoietins (TMPOs, previously abbreviated TPs) alpha (75 kDa), beta (51 kDa), and gamma (39 kDa) are related nuclear proteins expressed in many or all tissues. TMPO alpha is present diffusely throughout the nucleus, while TMPOs beta and gamma are localized to the nuclear membrane. Here we report the cloning and analysis of a single TMPO gene encoding TMPOs alpha, beta, and gamma, which are produced by alternative mRNA splicing, as previously inferred from cDNA sequences. The eight exons of the TMPO gene are spread over approximately 35 kb. Exon 4, which is spliced into TMPO alpha mRNA, contains sequences that encode a putative basic nuclear localization motif. Exon 8, which is spliced into TMPO beta and gamma mRNAs, encodes a hydrophobic putative membrane-spanning domain that is thought to target TMPOs beta and gamma to the nuclear membrane. TMPO beta appears to be the human homologue of the recently described rat protein LAP2 (lamina-associated polypeptide 2), which is thought to play an important role in the regulation of nuclear architecture by binding lamin B1 and chromosomes in a manner regulated by phosphorylation during mitosis (K. Furukawa and L. Gerace, La Jolla, pers. comm., 22 Nov. 1994). The human TMPO gene maps to chromosome band 12q22.
Asunto(s)
Proteínas de Unión al ADN , Genes , Proteínas de la Membrana/genética , Proteínas Nucleares/genética , Ratas/genética , Timopoyetinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromosomas Humanos Par 12 , ADN Complementario/genética , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Empalme del ARN , Especificidad de la EspecieRESUMEN
Thymopoietin (TP) was originally isolated as a 5-kDa 49-aa protein from bovine thymus in studies of the effects of thymic extracts on neuromuscular transmission and was subsequently observed to affect T-cell differentiation and function. We now report the isolation of cDNA clones for three alternatively spliced mRNAs that encode three distinct human T-cell TPs. Proteins encoded by these mRNAs, which we have named TP alpha (75 kDa), TP beta (51 kDa), and TP gamma (39 kDa), contain identical N-terminal regions, including sequences nearly identical to that of the originally isolated 49-aa protein, but divergent C-terminal regions. TP mRNAs are expressed in many tissues, most abundantly in adult thymus and fetal liver of the tissues so far examined. Distinct structural domains and functional motifs in TPs alpha, beta, and gamma suggest that the proteins have unique functions and may be directed to distinct subcellular compartments.
Asunto(s)
Empalme Alternativo , ARN Mensajero/biosíntesis , Timopoyetinas/biosíntesis , Timo/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Línea Celular , Clonación Molecular , ADN Complementario/aislamiento & purificación , ADN Complementario/metabolismo , Escherichia coli , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Poli A/aislamiento & purificación , Poli A/metabolismo , ARN/aislamiento & purificación , ARN/metabolismo , ARN Mensajero/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Linfocitos T , Timopoyetinas/genética , Timopoyetinas/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
In addition to their clinical utility in tissue transplantation the immunosuppressive agents FK-506 (Prograf) and rapamycin, have proven to be valuable tools for gaining insight into the biochemistry of T-cell activation. The findings that the protein phosphatase calcineurin and cell cycle control are key elements in T-cell activation and proliferation are the direct result of investigations into the mechanism of action of FK-506 and rapamycin and provide potentially novel therapeutic targets.
Asunto(s)
Inmunosupresores/farmacología , Polienos/farmacología , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Tacrolimus/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Humanos , Activación de Linfocitos/efectos de los fármacos , SirolimusRESUMEN
The immunosuppressant rapamycin (RAP) is a potent inhibitor of the entry of interleukin (IL)-2-stimulated T cells into S-phase. Earlier results indicated that RAP treatment arrested the growth of the murine IL-2-dependent T cell line CTLL-2 in late G1-phase. To explore further the interactions of RAP with the cell cycle control machinery in T cells, we examined the effects of RAP treatment on the activation of the cyclin-dependent kinases p34cdc2 and p33cdk2 in G1-phase CTLL-2 cells. Stimulation of factor-deprived cells with IL-2 led to the assembly of high molecular weight complexes containing active p34cdc2 and p33cdk2. The appearance of these complexes was explained, at least in part, by the association of both cyclin-dependent kinases with IL-2-induced cyclin A. RAP treatment profoundly inhibited both cyclin A expression and the appearance of active cyclin A-cyclin-dependent kinase complexes in IL-2-stimulated, late G1-phase CTLL-2 cells. Although p34cdc2 activation was largely dependent on association with cyclin A, a significant proportion of the active p33cdk2 pool was complexed with cyclin E. In contrast to cyclin A, the IL-2-induced accumulation of cyclin E in G1-phase cells was only partially suppressed by RAP, and cyclin E-p33cdk2 complexes were readily detected in drug-treated cells. These cyclin E-cyclin-dependent kinase complexes were nonetheless devoid of histone H1 kinase activity. The inhibitory effects of RAP on the activation of cyclin E- and cyclin A-associated cyclin-dependent kinases suggest that one or both events participate in the regulation of T cell entry into S-phase.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes , Inmunosupresores/farmacología , Interleucina-2/farmacología , Polienos/farmacología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Linfocitos T/efectos de los fármacos , Secuencia de Aminoácidos , Northern Blotting , División Celular/efectos de los fármacos , Línea Celular , Cromatografía en Gel , Quinasa 2 Dependiente de la Ciclina , Ciclinas/biosíntesis , Ciclinas/aislamiento & purificación , Sondas de ADN , Replicación del ADN/efectos de los fármacos , Activación Enzimática , Humanos , Cinética , Datos de Secuencia Molecular , Oligopéptidos/inmunología , Protamina Quinasa/metabolismo , Proteínas Quinasas/aislamiento & purificación , Proteínas Recombinantes/farmacología , Sirolimus , Linfocitos T/enzimologíaRESUMEN
The macrolide rapamycin (RAP) is a potent inhibitor of interleukin-2 (IL-2)-induced T-cell proliferation. Current models suggest that RAP, when complexed to its intracellular receptor, FK506-binding protein, interferes with an IL-2 receptor-coupled signaling pathway required for cell-cycle progression from G1- to S-phase. Here we show that RAP treatment inhibits the growth of an IL-2-dependent cytotoxic T-cell line, CTLL-2, in late G1-phase, just prior to entry of the cells into S-phase. In contrast, RAP-treated CTLL-2 cells retained the ability to respond to IL-2 with enhanced cytolytic activity, indicating that RAP was not a general suppressant of cellular responsiveness to IL-2. Subsequent studies revealed that IL-2 stimulation triggered a delayed activation of the p34cdc2 kinase, the timing of which correlated with the G1- to S-phase transition. The IL-2-dependent increase in p34cdc2 kinase activity was blocked by RAP. The RAP sensitivity of the p34cdc2 activation mechanism implicates this signaling pathway in the control of S-phase commitment in IL-2-stimulated T-cells.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Citotoxicidad Inmunológica/efectos de los fármacos , Inmunosupresores/farmacología , Polienos/farmacología , Linfocitos T Citotóxicos/efectos de los fármacos , Animales , Proteína Quinasa CDC2/antagonistas & inhibidores , División Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Fase G1/efectos de los fármacos , Interleucina-2/farmacología , Cinética , Ratones , Fase S/efectos de los fármacos , Sirolimus , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/enzimología , Timidina/metabolismoRESUMEN
The immunoregulant FK-506 potently inhibits particular calcium-associated signal transduction events that occur early during T-lymphocyte activation and during IgE receptor-mediated exocytosis in mast cells. FK-506 binds to a growing family of receptors termed FK-506-binding proteins (FKBPs), the most abundant being a 12-kDa cytosolic receptor, FKBP12. To date, there is no formal evidence proving that FKBP12 is the sole receptor mediating the immunosuppressive effects or toxic side effects of FK-506. Using gel filtration chromatography as an assay for novel FK-506-binding proteins, we identified FK-506 binding activities in extracts prepared from calf brain and from JURKAT cells. Both of these new activities comigrated with apparent molecular masses of 110 kDa. However, further characterization of both binding activities revealed that the two are not identical. The 110-kDa activity observed in brain extracts appears to be the FKBP12.FK-506.calcineurin (CaN) complex previously reported (Liu, J., Farmer, J., Lane, W., Friedman, J., Weissman, I., and Schreiber, S. (1991) Cell 66, 807-815) while the 110 kDa activity observed in JURKAT cells is a novel FK-506-binding protein. Our characterization of the FKBP12.FK-506.CaN complex reveals a dependence upon calmodulin (CaM) for formation of the complex and demonstrates that the peptidyl-prolyl cis-trans isomerase (PPIase) activity of FKBP12 is not required for binding of FKBP12.FK-506 to CaN or for inhibition of CaN phosphatase activity. The novel FK-506-binding protein in JURKAT cells has been purified to homogeneity, migrates with an apparent mass of 51 kDa on denaturing gels, and has been termed FKBP51. Like FKBP12, FKBP51 has PPIase activity, but, unlike FKBP12.FK-506, FKBP51.FK-506 does not complex with or inhibit the phosphatase activity of, CaN. These results indicate that complex formation with CaN may not be a general property of the FKBPs. Peptide sequencing reveals that FKBP51 may be similar, if not identical, to hsp56, a component of non-transformed steroid receptors.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Choque Térmico/metabolismo , Tacrolimus/metabolismo , Isomerasas de Aminoácido/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/metabolismo , Calcineurina , Proteínas de Unión a Calmodulina/metabolismo , Proteínas Portadoras/aislamiento & purificación , Bovinos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Ciclosporinas/metabolismo , Electroforesis en Gel de Poliacrilamida , Proteínas de Choque Térmico/aislamiento & purificación , Datos de Secuencia Molecular , Peso Molecular , Oligodesoxirribonucleótidos , Isomerasa de Peptidilprolil , Fosfoproteínas Fosfatasas/metabolismo , Homología de Secuencia de Aminoácido , Proteínas de Unión a TacrolimusRESUMEN
Cyclosporin A and FK-506 are important therapeutic agents that have found widespread use in preventing graft rejection during tissue transplantation. Research efforts aimed at elucidating the molecular mechanism of action of these drugs have, in addition to defining their immunosuppressive functions, led to the identification of two new gene families whose products may function as components of several diverse signal transduction pathways. In the presence of the immunosuppressive drugs, some members of the receptor families interact with the Ca2+/calmodulin-dependent protein phosphatase 2B, also known as calcineurin. Inhibition of phosphatase activity may effect several downstream biochemical processes. In this way, cyclosporin A and FK-506 have proved to be useful probes of signaling events in both lymphocytic and other cell types.
Asunto(s)
Ciclosporina/farmacología , Tacrolimus/farmacología , Isomerasas de Aminoácido/fisiología , Animales , Calcineurina , Proteínas de Unión a Calmodulina/fisiología , Proteínas Portadoras/fisiología , Ciclosporina/toxicidad , Humanos , Activación de Linfocitos , Isomerasa de Peptidilprolil , Fosfoproteínas Fosfatasas/fisiología , Linfocitos T/inmunología , Tacrolimus/toxicidad , Proteínas de Unión a TacrolimusRESUMEN
FK-506 inhibits Ca(2+)-dependent transcription of lymphokine genes in T cells, and thereby acts as a powerful immunosuppressant. However, its potential therapeutic applications may be seriously limited by several side effects, including nephrotoxicity and neurotoxicity. At present, it is unclear whether these immunosuppressive and toxic effects result from interference with related biochemical processes. FK-506 is known to interact with FK-binding protein-12 (FKBP-12), an abundant cytosolic protein with cis-trans peptidyl-prolyl isomerase activity (PPIase) activity. Because rapamycin (RAP) similarly binds to FKBP-12, although it acts in a manner different from FK-506, by inhibiting T cell responses to lymphokines, such an interaction with FKBP-12 is not sufficient to mediate immunosuppression. Recently, it was found that the complex of FKBP-12 with FK-506, but not with RAP, inhibits the phosphatase activity of calcineurin. Here, we used L-685,818, the C18-hydroxy, C21-ethyl derivative of FK-506, to explore further the role of FKBP-12 in the immunosuppressive and toxic actions of FK-506. Although L-685,818 bound with high affinity to FKBP-12 and inhibited its PPIase activity, it did not suppress T cell activation, and, when complexed with FKBP-12, did not affect calcineurin phosphatase activity. However, L-685,818 was a potent antagonist of the immunosuppressive activity of both FK-506 and RAP. Moreover, L-685,818 did not induce any toxicity in dogs and rats or in a mouse model of acute FK-506 nephrotoxicity, but it blocked the effect of FK-506 in this model. Therefore, FK-506 toxicity involves the disruption of biochemical mechanisms related to those implicated in T cell activation. Like immunosuppression, this toxicity is not due to the inhibition of the PPIase activity of FKBP-12, but may be linked to the inhibition of the phosphatase activity of calcineurin by the drug FKBP-12 complex.
Asunto(s)
Inmunosupresores/farmacología , Polienos/antagonistas & inhibidores , Tacrolimus/análogos & derivados , Tacrolimus/toxicidad , Isomerasas de Aminoácido/antagonistas & inhibidores , Animales , Calcineurina , Proteínas de Unión a Calmodulina/antagonistas & inhibidores , Proteínas de Unión a Calmodulina/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Femenino , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Isomerasa de Peptidilprolil , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Sirolimus , Linfocitos T/inmunología , Tacrolimus/antagonistas & inhibidores , Tacrolimus/farmacología , Proteínas de Unión a TacrolimusRESUMEN
The immunosuppressants FK506 and rapamycin prevent T-cell activation and also inhibit the growth of certain strains of the yeast Saccharomyces cerevisiae. It has previously been shown that yeast contains a 12-kDa cytosolic FK506-binding protein (yFKBP-12), which also possesses peptidylprolyl cis-trans isomerase activity, and that fkb1 strains lacking yFKBP-12 are resistant to rapamycin and sensitive to FK506. The absence of yFKBP-12 permitted the detection and isolation of a second FK506- and rapamycin-binding protein, which is about 13 kDa in size (yFKBP-13) and membrane-associated. Purified yFKBP-13 binds FK506 with 15-fold lower affinity than yFKBP-12 and has peptidylprolyl cis-trans isomerase activity with a similar substrate profile. The sequence of the first 37 N-terminal amino acids was determined, and the yFKBP-13 gene (FKB2) was cloned and sequenced. A hydrophobic putative signal sequence precedes the N terminus of the mature protein. yFKBP-13 most closely resembles the membrane-associated human FKBP-13, which also possesses a signal peptide, whereas yFKBP-12 most closely resembles human FKBP-12. fkb2 and fkb1 fkb2 mutants are viable and unaltered in their sensitivity to FK506, suggesting that yeast possesses an additional target for this drug. Furthermore, fkb2 null mutations confer no change in rapamycin sensitivity. These findings show that yFKBP-13 and yFKBP-12 have distinct functions within the cell.