RESUMEN
Chemical tools to monitor drug-target engagement of endogenously expressed protein kinases are highly desirable for preclinical target validation in drug discovery. Here, we describe a chemical genetics strategy to selectively study target engagement of endogenous kinases. By substituting a serine residue into cysteine at the DFG-1 position in the ATP-binding pocket, we sensitize the non-receptor tyrosine kinase FES towards covalent labeling by a complementary fluorescent chemical probe. This mutation is introduced in the endogenous FES gene of HL-60 cells using CRISPR/Cas9 gene editing. Leveraging the temporal and acute control offered by our strategy, we show that FES activity is dispensable for differentiation of HL-60 cells towards macrophages. Instead, FES plays a key role in neutrophil phagocytosis via SYK kinase activation. This chemical genetics strategy holds promise as a target validation method for kinases.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes , Proteínas Proto-Oncogénicas c-fes , Transportadoras de Casetes de Unión a ATP/química , Sistemas CRISPR-Cas , Diferenciación Celular , Línea Celular , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Edición Génica , Humanos , Macrófagos/metabolismo , Mutación , Neutrófilos , Fagocitosis , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-fes/química , Proteínas Proto-Oncogénicas c-fes/genética , Proteínas Proto-Oncogénicas c-fes/metabolismo , Transducción de Señal , Quinasa Syk/metabolismoAsunto(s)
Química Bioinorgánica/historia , Biología de Sistemas/historia , Espectroscopía de Resonancia por Spin del Electrón , Historia del Siglo XX , Historia del Siglo XXI , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Espectroscopía de Resonancia Magnética , Proteínas Proto-Oncogénicas c-fes/química , Proteínas Proto-Oncogénicas c-fes/metabolismoRESUMEN
FES and FES-related (FER) comprise a unique subfamily of protein-tyrosine kinases (PTKs) that signal downstream of several classes of receptors involved in regulating hematopoietic cell development, survival, migration, and inflammatory mediator release. Activated alleles of FES are potent inducers of myeloid differentiation, however FES-deficient mice have only subtle differences in hematopoiesis. This may reflect overlapping function of other kinases such as FER. Studies of FES- and FER-deficient mice have revealed more prominent roles in regulating the activation of mature innate immune cells, including macrophages and mast cells. Recently, new insights into regulation of FES/FER kinases has emerged with the characterization of their N-terminal phospholipid-binding and membrane targeting FER/CIP4 homology-Bin/Amphyphysin/Rvs (F-BAR) and F-BAR extension (FX) domains. The F-BAR/FX domains regulate subcellular localization and FES/FER kinase activation. FES kinase activity is also enhanced upon ligand binding to its SH2 domain, which may lead to further phosphorylation of the same ligand, or other ligand-associated proteins. In mast cells, SH2 ligands of FES/FER include KIT receptor PTK, and the high affinity IgE receptor (FceRI) that trigger rapid activation of FES/FER and signaling to regulators of the actin cytoskeleton and membrane trafficking. Recently, FES/FER have also been implicated in growth and survival signaling in leukemias driven by oncogenic KIT and FLT3 receptors. With further definition of their roles in immune cells and their progenitors, FES/FER may emerge as relevant therapeutic targets in inflammatory diseases and leukemias.
Asunto(s)
Hematopoyesis/fisiología , Leucemia/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-fes/metabolismo , Animales , Hematopoyesis/genética , Humanos , Leucemia/genética , Masculino , Mastocitos/metabolismo , Ratones , Modelos Moleculares , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/inmunología , Proteínas Proto-Oncogénicas c-fes/química , Proteínas Proto-Oncogénicas c-fes/genética , Proteínas Proto-Oncogénicas c-fes/inmunología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Transducción de Señal , Testículo/metabolismoRESUMEN
Cell differentiation is compromised in acute leukemias. We report that mammalian target of rapamycin (mTOR) and S6 kinase (S6K) are highly expressed in the undifferentiated promyelomonocytic leukemic HL-60 cell line, whereas PLD2 expression is minimal. The expression ratio of PLD2 to mTOR (or to S6K) is gradually inverted upon in vitro induction of differentiation toward the neutrophilic phenotype. We present three ways that profoundly affect the kinetics of differentiation as follows: (i) simultaneous overexpression of mTOR (or S6K), (ii) silencing of mTOR via dsRNA-mediated interference or inhibition with rapamycin, and (iii) PLD2 overexpression. The last two methods shortened the time required for differentiation. By determining how PLD2 participates in cell differentiation, we found that PLD2 interacts with and activates the oncogene Fes/Fps, a protein-tyrosine kinase known to be involved in myeloid cell development. Fes activity is elevated with PLD2 overexpression, phosphatidic acid or phosphatidylinositol bisphosphate. Co-immunoprecipitation indicates a close PLD2-Fes physical interaction that is negated by a Fes-R483K mutant that incapacitates its Src homology 2 domain. All these suggest for the first time the following mechanism: mTOR/S6K down-regulationâPLD2 overexpressionâPLD2/Fes associationâphosphatidic acid-led activation of Fes kinaseâgranulocytic differentiation. Differentiation shortening could have a clinical impact on reducing the time of return to normalcy of the white cell counts after chemotherapy in patients with acute promyelocytic leukemia.
Asunto(s)
Diferenciación Celular , Leucemia Mieloide/patología , Fosfolipasa D/metabolismo , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Silenciador del Gen , Células HL-60 , Humanos , Cinética , Datos de Secuencia Molecular , Fosfolipasa D/genética , Proteínas Proto-Oncogénicas c-fes/química , Proteínas Proto-Oncogénicas c-fes/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Quinasas S6 Ribosómicas/genética , Proteínas Quinasas S6 Ribosómicas/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/deficiencia , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Dominios Homologos srcRESUMEN
A tendência atual do mercado cosmético é desenvolver produtos que contenham insumos de origem vegetal. O objetivo deste trabalho foi a aplicação da Tecnologia da Química Verde na síntese da rutina visando o aumento da estabilidade dessa em formulações cosméticas com sua eficácia antioxidante e fotoprotetora. Realizou-se a síntese química por meio da introdução de grupos carboxilatos às hidroxilas do dissacarídeo na molécula de rutina, gerando como produto final o succinato de rutina. Este derivado e/ou a rutina foram incorporados em 74 formulações-teste e, selecionadas 12 (sistemas emulsionados O/A), após serem submetidas à Avaliação Preliminar de Estabilidade (APE) e ao Teste de Estabilidade Acelerada (TEA), sob variações de temperatura e umidade. Utilizou-se agentes emolientes e silicones para facilitar a solubilização e/ou dispersão dos filtros químicos e físicos. A segunda etapa deste trabalho foi a avaliação da segurança do succinato de rutina, tendo como padrão a rutina, por meio do método alternativo de toxicidade in vitro, o XTT. Após o screening das concentrações ensaiadas, as que apresentaram menor nível de morte celular foram respectivamente, 0,1% ou 1 mg/mL (rutina) e 0,4% ou 4 mg/mL (succinato de rutina). Segundo os resultados do TEA, as formulações contendo succinato de rutina associada ou não aos filtros solares em ambas as bases cosméticas (A - Crodafos®CES + Uniox®C e B - Hostacerin®SAF) foram selecionadas para a continuidade do Teste de Estabilidade Normal (TEN). Neste teste, as emulsões fotoprotetoras foram avaliadas frente aos parâmetros: propriedades organolépticas (aspecto, cor e odor), aspectos físico-químicos (medição de pH e de viscosidade) e funcionais (atividade antirradicalar e eficácia fotoprotetora in vitro). Os resultados apresentados pela formulação MS (succinato de rutina associado aos filtros químicos e físico) foram: homogeneidade, a não modificação de cor e odor em temperatura ambiente, a não alterações significativas...
The current cosmetic market trend is to develop products containing vegetables raw materials. This work proposed to use the Technology of Green Chemical to increase the rutin stability in cosmetic formulas as regards of its antioxidant and photoprotective properties. The chemical synthesis was realized by the introduction of carboxylate groups on sugar moiety of rutin producing in rutin succinate. This derivative and/or rutin were incorporated into 74 test formulas. After the undergoing to preliminary and accelerated stabilities under different temperature and humidity conditions were selected 12 formulas (O/W emulsions). Emollient agents and silicones were used to improve the solubility and/or dispersion of the chemical and physical filters. The second stage of this work was to evaluate the safety of rutin succinate, rutin used as an internal standard, using the alternative method of in vitro toxicity, the XTT. After the screening of tested concentrations, the concentrations of the samples with the lowest level of cell death were 0.1% or 1 mg/mL (rutin) and 0.4% or 4 mg/mL (rutin succinate), respectively. According to results obtained in accelerated stability testing, the formulations containing rutin succinate in combination or not with UV filters in both O/W emulsions (A - Crodafos®CES + Uniox®C and B - Hostacerin®SAF) were selected for the long term stability test. In this test the sunscreens were evaluated in the following parameters: the organoleptic properties (appearance, color and odor), physico-chemical aspects (pH value and viscosity) and functional (antiradicalar activity and in vitro photoprotection efficacy). The results presented by the MS formula (rutin succinate associated with physical filter and chemical filters) were: uniformity, stability of color and odor at room temperature and showed no significant difference, as well stability in: pH and SPF (Sun Protection Factor) values, hysteresis area, antiradicalar activity. These results were...
Asunto(s)
Ácido Succínico/análisis , Ácido Succínico/efectos de la radiación , Ácido Succínico/síntesis química , Técnicas In Vitro , Factores Protectores , Rutina/análisis , Rutina/efectos de la radiación , Rutina/síntesis química , Química Farmacéutica , Tecnología Química Verde , Proteínas Proto-Oncogénicas c-fes/farmacocinética , Proteínas Proto-Oncogénicas c-fes/químicaRESUMEN
Non receptor protein tyrosine kinases are targets in the treatment of a number of diseases. This review focuses on the role of Fes tyrosine kinase and on the design of inhibitors of this protein. Fes and its homologously related protein Fer are the only two members of a distinct class of non receptor tyrosine kinases and they seem to play a role in cytoskeletal rearrangements and inside-out signaling associated with receptor-ligand, cell-matrix and cell-cell interactions. The knowledge of the three dimensional structure of this protein, in fact, has informed drug design, while at the same time it has helped to shed some light on the molecular mechanism at the basis of kinase activation and functions.
Asunto(s)
Antineoplásicos/química , Diseño de Fármacos , Neoplasias/enzimología , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas c-fes/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fes/metabolismo , Animales , Antineoplásicos/farmacología , Humanos , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-fes/químicaRESUMEN
The c-Fes protein-tyrosine kinase is the normal cellular ortholog of several avian and feline retroviral oncoproteins. Unlike its transforming viral counterparts, c-Fes tyrosine kinase activity is tightly regulated in vivo through a mechanism involving coiled-coil oligomerization domains and other unique structural features found in its long N-terminal region. This review is focused on the regulatory features and structural biology of c-Fes, which has been implicated in normal cellular growth regulation, the innate immune response, and tumorigenesis.
Asunto(s)
Proteínas Proto-Oncogénicas c-fes/genética , Proteínas Proto-Oncogénicas c-fes/metabolismo , Animales , Anticuerpos , Células de la Médula Ósea/enzimología , Transformación Celular Neoplásica , Transformación Celular Viral , Proteínas de Fusión gag-onc/inmunología , Humanos , Oncogenes , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-fes/químicaRESUMEN
Gefitinib inhibits epidermal growth factor-independent angiogenesis, but the molecular mechanism underlying this inhibition has yet to be defined. Here we show that gefitinib dose-dependently inhibited chemotaxis of endothelial cells toward fibroblast growth factor-2 (FGF-2), but not toward vascular endothelial growth factor-A (VEGF-A). Gefitinib inhibited lamellipodium formation by endothelial cells induced by FGF-2, but not by VEGF-A. Gefitinib at 10 microM did not inhibit autophosphorylation of FGF receptor 1 or VEGF receptor 2. A non-receptor protein tyrosine kinase, Fes, has two coiled-coil domains (CCDs) in its N-terminal region. Fes is activated by trans-autophosphorylation through CCD functions. An inactivating mutation in the second CCD abolished FGF-2 activation of Fes, indicating involvement of this CCD in FGF-2-induced Fes activation. Gefitinib-treatment decreased both CCD-independent and FGF-2- or VEGF-A-promoted Fes activity with a maximal decrease at 1 microM. The same results were observed in cells stably expressing kinase-inactive Fes; a dominant negative effect was observed in cells treated with FGF-2, but not with VEGF-A. Taken together, these results indicate that FGF-2 activates Fes via the second CCD, leading to lamellipodium formation and chemotaxis by endothelial cells, and gefitinib may act through Fes as an inhibitor of FGF-2-driven angiogenesis.
Asunto(s)
Antineoplásicos/farmacología , Quimiotaxis/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Neovascularización Patológica/metabolismo , Proteínas Proto-Oncogénicas c-fes/metabolismo , Quinazolinas/farmacología , Western Blotting , Regulación hacia Abajo , Células Endoteliales/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Gefitinib , Humanos , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-fes/química , Proteínas Proto-Oncogénicas c-fes/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The c-fes proto-oncogene encodes a unique nonreceptor protein-tyrosine kinase (c-Fes) that contributes to the differentiation of myeloid hematopoietic, vascular endothelial, and some neuronal cell types. Although originally identified as the normal cellular homologue of the oncoproteins encoded by avian and feline transforming retroviruses, c-Fes has recently been implicated as a tumor suppressor in breast and colonic epithelial cells. Structurally, c-Fes consists of a unique N-terminal region harboring an FCH domain, two coiled-coil motifs, a central SH2 domain, and a C-terminal kinase domain. In living cells, c-Fes kinase activity is tightly regulated by a mechanism that remains unclear. Previous studies have established that c-Fes forms high molecular weight oligomers in vitro, suggesting that the dual coiled-coil motifs may regulate the interconversion of inactive monomeric and active oligomeric states. Here we show for the first time that c-Fes forms oligomers in live cells independently of its activation status using a YFP bimolecular fluorescence complementation assay. We also demonstrate that both N-terminal coiled-coil regions are essential for c-Fes oligomerization in transfected COS-7 cells as well as HCT 116 colorectal cancer and K-562 myeloid leukemia cell lines. Together, these data provide the first evidence that c-Fes, unlike c-Src, c-Abl, and other nonreceptor tyrosine kinases, is constitutively oligomeric in both its repressed and active states. This finding suggests that conformational changes, rather than oligomerization, may govern its kinase activity in vivo.
Asunto(s)
Prueba de Complementación Genética , Proteínas Proto-Oncogénicas c-fes/biosíntesis , Proteínas Proto-Oncogénicas c-fes/genética , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Células COS , Chlorocebus aethiops , Activación Enzimática/genética , Silenciador del Gen , Células HCT116 , Humanos , Immunoblotting , Células K562 , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Microscopía Fluorescente , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-fes/química , Transducción de Señal/genéticaRESUMEN
The flow of molecular information through normal and oncogenic signaling pathways frequently depends on protein phosphorylation, mediated by specific kinases, and the selective binding of the resulting phosphorylation sites to interaction domains present on downstream targets. This physical and functional interplay of catalytic and interaction domains can be clearly seen in cytoplasmic tyrosine kinases such as Src, Abl, Fes, and ZAP-70. Although the kinase and SH2 domains of these proteins possess similar intrinsic properties of phosphorylating tyrosine residues or binding phosphotyrosine sites, they also undergo intramolecular interactions when linked together, in a fashion that varies from protein to protein. These cooperative interactions can have diverse effects on substrate recognition and kinase activity, and provide a variety of mechanisms to link the stimulation of catalytic activity to substrate recognition. Taken together, these data have suggested how protein kinases, and the signaling pathways in which they are embedded, can evolve complex properties through the stepwise linkage of domains within single polypeptides or multi-protein assemblies.
Asunto(s)
Neoplasias/metabolismo , Transducción de Señal/fisiología , Familia-src Quinasas/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Evolución Molecular , Modelos Moleculares , Fosforilación , Conformación Proteica , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Proto-Oncogénicas c-fes/química , Proteínas Proto-Oncogénicas c-fes/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Proteína Tirosina Quinasa ZAP-70/química , Proteína Tirosina Quinasa ZAP-70/metabolismo , Dominios Homologos src , Familia-src Quinasas/químicaRESUMEN
This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcepsilonRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcepsilonRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcepsilonRI beta chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcepsilonRI signaling and potential regulation the actin reorganization in mast cells.
Asunto(s)
Mastocitos/metabolismo , Dominios y Motivos de Interacción de Proteínas/fisiología , Proteínas Proto-Oncogénicas c-fes/metabolismo , Receptores de IgE/metabolismo , Dominios Homologos src/fisiología , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Proteínas Sanguíneas/metabolismo , Degranulación de la Célula/fisiología , Citoplasma/metabolismo , Humanos , Microscopía Fluorescente , Microtúbulos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fosfatidilinositoles/metabolismo , Fosforilación , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Proteínas Proto-Oncogénicas c-fes/química , Proteínas Proto-Oncogénicas c-fes/genética , Dominios Homologos src/genéticaRESUMEN
The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.
Asunto(s)
Proteínas Proto-Oncogénicas c-abl/química , Proteínas Proto-Oncogénicas c-fes/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Activación Enzimática , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Proto-Oncogénicas c-fes/metabolismoRESUMEN
The intracellular behavior of human FCHO1 protein was investigated by live-cell imaging microscopy. The fluorescence intensity of green fluorescent protein (GFP)-FCHO1 fluctuated periodically in a perinuclear region approximately every 100 s, reminding us of the periodic fluctuations of clathrin reported in our recent work. The periodicity of FCHO1 was temporally correlated with that of clathrin, suggesting that FCHO1 is involved in clathrin-coated vesicle formation.
Asunto(s)
Vesículas Cubiertas por Clatrina/metabolismo , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/fisiología , Proteínas/fisiología , Línea Celular , Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/química , Genes Reporteros , Aparato de Golgi/metabolismo , Humanos , Proteínas de la Membrana , Proteínas Asociadas a Microtúbulos/química , Antígenos de Histocompatibilidad Menor , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-fes/química , Proteínas Proto-Oncogénicas c-fes/fisiología , Homología de Secuencia de AminoácidoRESUMEN
Protein Tyrosine Kinases (PTKs) are important molecules in intra- and inter-cellular communication, playing a major role in signal transduction processes. We have previously identified and characterized the molecular structure of a new PTK in Schistosoma mansoni, SmFes. SmFes exhibits the characteristic features of Fes/Fps protein tyrosine kinase subfamily of which it is the first member described in helminths. Herein, we show that genes orthologous to SmFes are also present in other Schistosoma species and the transcript is detected in Schistosoma japonicum. The SmFes protein was detected at all the main life-cycle stages and was most abundant in cercariae and newly-transformed schistosomula. However, no protein was detected in schistosomula maintained in vitro for 7 days. By immunolocalization assays we showed that SmFes is particularly concentrated at the terebratorium of miracidia and tegument of cercaria and schistosomula skin-stage. These findings suggest that SmFes may play a role in signal transduction pathways involved in larval transformation after penetration into intermediate and definitive hosts.
Asunto(s)
Proteínas Proto-Oncogénicas c-fes/fisiología , Schistosoma mansoni/enzimología , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Biomphalaria , Western Blotting , Secuencia Conservada , Femenino , Regulación Enzimológica de la Expresión Génica , Interacciones Huésped-Parásitos/fisiología , Masculino , Ratones , Modelos Estructurales , Proteínas Proto-Oncogénicas c-fes/biosíntesis , Proteínas Proto-Oncogénicas c-fes/química , Proteínas Proto-Oncogénicas c-fes/genética , Schistosoma mansoni/genética , Schistosoma mansoni/fisiologíaRESUMEN
The proto-oncogene tyrosine protein kinase c-fps/fes encodes a structurally unique protein (Fes) of the nonreceptor protein-tyrosine kinase (PTK) family. Its expression has been demonstrated in myeloid haematopoietic cells, vascular endothelial cells and in neurons. In human-derived and murine-derived cell lines, the activated form of this kinase can induce cellular transformation; moreover, it has been shown that Fes is involved in the regulation of cell-cell and cell-matrix interactions mediated by adherens junctions and focal adhesions. The N-terminus of Fes contains the FCH (Fps/Fes/Fer/CIP4 homology) domain, which is unique to the Fes/Fer kinase family. It is followed by three coiled-coil domains and an SH2 (Src-homology 2) domain. The catalytic region (Fes-CR) is located at the C-terminus of the protein. The successful expression, purification and crystallization of the catalytic part of Fes (Fes-CR) are described.
Asunto(s)
Dominio Catalítico/fisiología , Cristalografía por Rayos X/métodos , Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas Proto-Oncogénicas c-fes/biosíntesis , Secuencia de Aminoácidos , Animales , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/aislamiento & purificación , Humanos , Ratones , Datos de Secuencia Molecular , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-fes/química , Proteínas Proto-Oncogénicas c-fes/aislamiento & purificaciónRESUMEN
The recently introduced fully automated protein NMR structure determination algorithm (FLYA) yields, without any human intervention, a three-dimensional (3D) protein structure starting from a set of two- and three-dimensional NMR spectra. This paper investigates the influence of reduced sets of experimental spectra on the quality of NMR structures obtained with FLYA. In a case study using the Src homology domain 2 from the human feline sarcoma oncogene Fes (Fes SH2), five reduced data sets selected from the full set of 13 three-dimensional spectra of the previously determined conventional structure were used to calculate the protein structure. Three reduced data sets utilized only CBCA(CO)NH and CBCANH for the backbone assignments and two data sets used only CBCA(CO)NH. All, some, or none of the five original side-chain assignment spectra were used. Results were compared with those of a FLYA calculation for the complete set of spectra and those of the conventionally determined structure. In four of the five cases tested, the three-dimensional structures deviated by less than 1.3 A in backbone RMSD from the conventionally determined Fes SH2 reference structure, showing that the FLYA algorithm is remarkably stable and accurate when used with reduced sets of input spectra.
Asunto(s)
Algoritmos , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas Proto-Oncogénicas c-fes/química , Dominios Homologos src , Humanos , Conformación ProteicaRESUMEN
The human c-fes locus encodes a non-receptor protein-tyrosine kinase implicated in myeloid, vascular endothelial, and neuronal cell differentiation. A recent analysis of the tyrosine kinome in colorectal cancer identified c-fes as one of only seven genes with consistent kinase domain mutations. Although four mutations were identified (M704V, R706Q, V743M, S759F), the consequences of these mutations on Fes kinase activity were not explored. To address this issue, Fes mutants with these substitutions were co-expressed with STAT3 in human 293T cells. Surprisingly, the M704V, R706Q, and V743M mutations substantially reduced Fes autophosphorylation and STAT3 Tyr-705 phosphorylation compared with wild-type Fes, whereas S759F had little effect. These mutations had a similar impact on Fes kinase activity in a yeast expression system, suggesting that they inhibit Fes by affecting kinase domain structure. We have also demonstrated for the first time that endogenous Fes is strongly expressed at the base of colonic crypts where it co-localizes with epithelial cells positive for the progenitor cell marker Musashi-1. In contrast to normal colonic epithelium, Fes expression was reduced or absent in colon tumor sections from most individuals. Fes protein levels were also low or absent in a panel of human colorectal cancer cell lines, including HT-29 and HCT 116 cells. Introduction of Fes into these lines with a recombinant retrovirus suppressed their growth in soft agar. Together, our findings strongly implicate the c-Fes protein-tyrosine kinase as a tumor suppressor rather than a dominant oncogene in colorectal cancer.