RESUMEN
Canonical Wnt signalling plays important roles in early embryogenesis, such as axis formation due to its activation and head formation due to its inhibition. ß-catenin protein stability is a key factor in canonical Wnt signalling. Several E3 ubiquitin ligases contribute to ß-catenin degradation through the ubiquitin/proteasome system. We characterised an E3 ubiquitin ligase gene, Xenopus laevis macrophage erythroblast attacher (maea), during early development. maea transcripts were ubiquitously detected in early embryos. The expression levels of the Wnt target genes nodal homolog 3, gene 1 (nodal3.1), and siamois homeodomain 1 (sia1), which were induced by injection with ß-catenin mRNA, were reduced by maea.S mRNA co-injection. maea.S overexpression at the anterior dorsal region enlarged head structures, whereas Maea knockdown interfered with head formation in Xenopus embryos. Maea.S decreased and ubiquitinated ß-catenin protein. ß-catenin-4KRs protein, which mutated the four lysine (K) residues known as ubiquitinated sites to arginine (R) residues, was also ubiquitinated and degraded by Maea.S. These findings suggest that Maea contributes to ß-catenin degradation by ubiquitination of unknown lysine residues in early Xenopus development.
Asunto(s)
Proteínas del Citoesqueleto , Embrión no Mamífero , Proteínas de Xenopus , Xenopus laevis , beta Catenina , Animales , beta Catenina/genética , Regulación del Desarrollo de la Expresión Génica , Lisina/genética , Lisina/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Xenopus laevis/crecimiento & desarrollo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Proteínas del Citoesqueleto/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismoRESUMEN
We characterized Xenopus laevis C-C motif chemokine ligand 19.L (ccl19.L) and C-C motif chemokine ligand 21.L (ccl21.L) during early Xenopus embryogenesis. The temporal and spatial expression patterns of ccl19.L and ccl21.L tended to show an inverse correlation, except that the expression level was higher in the dorsal side at the gastrula stage. For example, even at the dorsal sector of the gastrulae, ccl19.L was expressed in the axial region and ccl21.L was expressed in the paraxial region. Dorsal overexpression of ccl19.L and ccl21.L and knockdown of Ccl19.L and Ccl21.L inhibited gastrulation, but their functions were different in cell behaviors during morphogenesis. Observation of Keller sandwich explants revealed that overexpression of both ccl19.L and ccl21.L and knockdown of Ccl21.L inhibited the convergent extension movements, while knockdown of Ccl19.L did not. ccl19.L-overexpressing explants attracted cells at a distance and ccl21.L-overexpressing explants attracted neighboring cells. Ventral overexpression of ccl19.L and ccl21.L induced secondary axis-like structures and chrd.1 expression at the ventral side. Upregulation of chrd.1 was induced by ligand mRNAs through ccr7.S. Knockdown of Ccl19.L and Ccl21.L inhibited gastrulation and downregulated chrd.1 expression at the dorsal side. The collective findings indicate that ccl19.L and ccl21.L might play important roles in morphogenesis and dorsal-ventral patterning during early embryogenesis in Xenopus.
Asunto(s)
Quimiocinas , Animales , Xenopus laevis/metabolismo , Ligandos , Receptores CCR7/metabolismo , Movimiento Celular , Quimiocinas/metabolismo , Diferenciación CelularRESUMEN
Chemokines play important roles in early embryogenesis, including morphogenesis and cell differentiation, before the immune system is established. We characterized Xenopus laevis CC-type chemokine receptor 7 S (ccr7.S) to clarify its role during early development. ccr7 transcripts were detected ubiquitously in early embryos. Dorsal overexpression of ccr7.S inhibited gastrulation, and ccr7.S mRNA-injected embryos had short axes and widely opened neural folds. Because the Keller sandwich explants of the injected embryos elongated well, ccr7.S might affect cell migration, but not convergent extension movements. Ventral ccr7.S overexpression induced secondary axes and chrd.1 upregulation in gastrula-stage embryos. Animal cap assays showed increased expression of neural and cement gland marker genes at later stages. Ccr7.S knockdown reduced chrd.1 expression and inhibited gastrulation at the dorsal side. Our findings suggest that ccr7.S plays important roles in morphogenetic movement and cell differentiation.
Asunto(s)
Desarrollo Embrionario , Gástrula , Animales , Diferenciación Celular/genética , Desarrollo Embrionario/genética , Gástrula/metabolismo , Morfogénesis/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genéticaRESUMEN
We describe a method of generating three-notochord explants to analyze the cell movements of convergent extension (CE) during Xenopus laevis gastrulation and neurulation. This method uses standard microsurgical techniques under a fluorescence stereomicroscope to combine notochordal sectors of gastrulae, side by side (lateral surfaces apposed) into a single explant. Three-notochord explants cultured on bovine serum albumin (BSA)-coated glass converged mediolaterally and extended in the anterior-posterior direction. The individual notochordal cells showed the mediolaterally oriented, bipolar tractional motility and the resulting mediolaterally oriented cell intercalation characteristic of CE, thereby reproducing both the in vivo tissue and the cell movements in an explant. Image analysis of three-notochord explants reveals the effects of overexpressions or knockdowns of genes, of manipulation of the extracellular matrix, and of exposure to chemical reagents on morphogenesis during gastrulation and neurulation, compared with control explants. Moreover, since three-notochord explants provide two zones of cell intercalation between notochords, individual cell behaviors between notochords of different characteristics and experimental treatments can be observed at the same time.
Asunto(s)
Gástrula , Notocorda , Animales , Movimiento Celular , Morfogénesis , Xenopus laevisRESUMEN
Cell signaling pathways, such as Wnt, Hedgehog (Hh), Notch, and Hippo, are essential for embryogenesis, organogenesis, and tissue homeostasis. In this study, we analyzed 415 genes involved in these pathways in the allotetraploid frog, Xenopus laevis. Most genes are retained in two subgenomes called L and S (193 homeologous gene pairs and 29 singletons). This conservation rate of homeologs is much higher than that of all genes in the X. laevis genome (86.9% vs 60.2%). Among singletons, 24 genes are retained in the L subgenome, a rate similar to the average for all genes (82.8% vs 74.6%). In addition, as general components of signal transduction, we also analyzed 32 heparan sulfate proteoglycan (HSPG)-related genes and eight TLE/Groucho transcriptional corepressors-related genes. In these gene sets, all homeologous pairs have been retained. Transcriptome analysis using RNA-seq data from developmental stages and adult tissues demonstrated that most homeologous pairs of signaling components have variable expression patterns, in contrast to the conservative expression profiles of homeologs for transcription factors. Our results indicate that homeologous gene pairs for cell signaling regulation have tended to become subfunctionalized after allotetraploidization. Diversification of signaling pathways by subfunctionalization of homeologs may enhance environmental adaptability. These results provide insights into the evolution of signaling pathways after polyploidization.
Asunto(s)
Perfilación de la Expresión Génica , Proteínas Hedgehog/genética , Receptores Notch/genética , Transducción de Señal/genética , Proteínas Wnt/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Animales , Receptores Frizzled/biosíntesis , Receptores Frizzled/genética , Expresión Génica , Genoma , Proteínas Hedgehog/biosíntesis , Anotación de Secuencia Molecular , Receptores Notch/biosíntesis , Fracciones Subcelulares/metabolismo , Sintenía , Tetraploidía , Transcriptoma , Proteínas Wnt/biosíntesis , Vía de Señalización Wnt/genética , Proteínas de Xenopus/biosíntesisRESUMEN
Several chemokine molecules control cell movements during early morphogenesis. However, it is unclear whether chemokine molecules affect cell fate. Here, we identified and characterized the CXC-type chemokine ligand in Xenopus laevis, Xenopus CXCLh (XCXCLh), during early embryogenesis. XCXCLh is expressed in the dorsal vegetal region at the gastrula stage. Both overexpression and knockdown of XCXCLh in the dorsal region inhibited gastrulation. XCXCLh contributed to the attraction of mesendodermal cells and accelerated the reassembly of scratched culture cells. Also, XCXCLh contributed to early endodermal induction. Overexpression of VegTmRNA or high concentrations of calcium ions induced XCXCLh expression. XCXCLh may play roles in both cell movements and differentiation during early Xenopus embryogenesis.
Asunto(s)
Quimiocinas/metabolismo , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/fisiología , Proteínas de Xenopus/metabolismo , Animales , Quimiocinas/genética , Endodermo/metabolismo , Gastrulación/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine of key importance for controlling embryogenesis and tissue homeostasis. How TGF-beta signals are attenuated and terminated is not well understood. Here, we show that TMEPAI, a direct target gene of TGF-beta signaling, antagonizes TGF-beta signaling by interfering with TGF-beta type I receptor (TbetaRI)-induced R-Smad phosphorylation. TMEPAI can directly interact with R-Smads via a Smad interaction motif. TMEPAI competes with Smad anchor for receptor activation for R-Smad binding, thereby sequestering R-Smads from TbetaRI kinase activation. In mammalian cells, ectopic expression of TMEPAI inhibited TGF-beta-dependent regulation of plasminogen activator inhibitor-1, JunB, cyclin-dependent kinase inhibitors, and c-myc expression, whereas specific knockdown of TMEPAI expression prolonged duration of TGF-beta-induced Smad2 and Smad3 phosphorylation and concomitantly potentiated cellular responsiveness to TGF-beta. Consistently, TMEPAI inhibits activin-mediated mesoderm formation in Xenopus embryos. Therefore, TMEPAI participates in a negative feedback loop to control the duration and intensity of TGF-beta/Smad signaling.
Asunto(s)
Proteínas de la Membrana/fisiología , Transducción de Señal , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Activinas/metabolismo , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Mesodermo/crecimiento & desarrollo , Ratones , Modelos Biológicos , Células 3T3 NIH , ARN Mensajero/metabolismo , XenopusRESUMEN
In the canonical Wnt signaling pathway, the translocation of ß-catenin is important for the activation of target genes in the nucleus. However, the molecular mechanisms underlying its nuclear localization remain unclear. In the present study, we found IQGAP1 to be a regulator of ß-catenin function via importin-ß5. In Xenopus embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of ß-catenin and expression of Wnt target genes during early embryogenesis. Depletion of endogenous importin-ß5 associated with IQGAP1 also reduced expression of Wnt target genes and the nuclear localization of IQGAP1 and ß-catenin. Moreover, a small GTPase, Ran1, contributes to the nuclear translocation of ß-catenin and the activation of Wnt target genes. These results suggest that IQGAP1 functions as a regulator of translocation of ß-catenin in the canonical Wnt signaling pathway.
Asunto(s)
Núcleo Celular/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , beta Carioferinas/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Dishevelled , Embrión no Mamífero/metabolismo , Células HEK293 , Humanos , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosfoproteínas/metabolismo , Unión Proteica , Xenopus , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Proteínas Activadoras de ras GTPasa/genéticaRESUMEN
With no lysine (K) (WNK) kinase family is conserved among many species and regulates SPAK/OSR1 and ion cotransporters. WNK is also involved in developmental and cellular processes, but the molecular mechanisms underlying its regulation in these processes remain unknown. In this study, we found that WNK4 is involved in fibroblast growth factor (FGF) signaling during Xenopus development. In Xenopus embryos, depletion of WNK4 by antisense morpholino oligonucleotides (MOs) results in a severe defect in anterior development and impaired expression of endogenous anterior markers. Defects in head formation or expression of anterior marker genes caused by suppression of endogenous WNK4 expression could be rescued by expression of wild-type WNK4, but not mutant WNK4 lacking its kinase activity. It is notable that morphants of Xenopus WNK4 inhibited the expression of anterior marker genes and the target genes induced by FGF signaling. Moreover, knockdown of Wnk4 significantly reduced the phosphorylation level of Osr1 induced by FGF. These results provide the first evidence that FGF signaling regulates WNK4 function required for anterior formation in Xenopus development.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas de Xenopus/metabolismo , Xenopus/embriología , Xenopus/metabolismo , Animales , Oligonucleótidos Antisentido , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas de Xenopus/antagonistas & inhibidoresRESUMEN
A transcriptional corepressor, Xenopus furry (Xfurry), is expressed in the chordamesodermal region and induces secondary dorsal axes when overexpressed on the ventral side of the embryo. The N-terminal furry domain functions as a repressor, and the C-terminal leucine zipper (LZ) motifs /coiled-coil structure, found only in vertebrate homologs, contributes to the nuclear localization. The engrailed repressor (enR)+LZ repressor construct, which has properties similar to Xfurry, induced several chordamesodermal genes. In contrast, an antisense morpholino oligonucleotide, Xfurry-MO, and the activating construct, herpes simplex virus protein (VP16)+LZ, had effects opposite those of Xfurry overexpression. Because blocking protein synthesis with cycloheximide superinduced several Xfurry transcriptional targets, and because expression of enR+LZ induced such genes under cycloheximide treatment, we analyzed the role of an Xfurry transcriptional target, microRNA miR-15. Cycloheximide reduced the expression of primary miR-15 (pri-miR-15), whereas miR-15 reduced the expression of genes superinduced by cycloheximide treatment. These results show that Xfurry regulates chordamesodermal genes by contributing to repression of pretranscriptional gene silencing by miR-15.
Asunto(s)
Silenciador del Gen , MicroARNs/genética , Proteínas Represoras/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Transporte Activo de Núcleo Celular , Animales , Cicloheximida/farmacología , Regulación de la Expresión Génica , Notocorda , Proteínas de Xenopus/fisiologíaRESUMEN
We cloned the gene for the CXC-type chemokine ligand, Xenopus CXCLC (XCXCLC), the transcripts of which were detected at the dorsal midline during the gastrula and neurula stages. XCXCLC overexpression resulted in the attraction of nearby mesodermal cells, and the excess of chemoattractant interfered with convergent and extension movements. The direction of the deep neural plate cells around the notoplate was also controlled by XCXCLC. Fluorescence signals for XCXCLC + enhanced green fluorescent protein derivatives accumulated around the notochord region. These results indicate that XCXCLC attracts adjacent cells to the midline region, so as to ensure accurate lateral-medial directional tissue convergence during gastrulation and neurulation.
Asunto(s)
Movimiento Celular , Quimiocinas CXC/metabolismo , Gastrulación , Morfogénesis , Neurulación , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Animales , Diferenciación Celular , Quimiocinas CXC/genética , Proteínas Fluorescentes Verdes , Placa Neural/crecimiento & desarrollo , ARN Mensajero/genética , Receptores de Quimiocina/metabolismo , Transducción de Señal , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
ß-Catenin is an important component of the Wnt signalling pathway. As dysregulation or mutation of this pathway causes many diseases, including cancer, the ß-Catenin level is carefully regulated by the destruction complex in the Wnt signalling pathway. However, the mechanisms underlying the regulation of ß-Catenin ubiquitination and degradation remain unclear. Here, we find that WNK (With No Lysine [K]) kinase is a potential regulator of the Wnt signalling pathway. We show that WNK protects the interaction between ß-Catenin and the Glucose-Induced degradation Deficient (GID) complex, which includes an E3 ubiquitin ligase targeting ß-Catenin, and that WNK regulates the ß-Catenin level. Furthermore, we show that WNK inhibitors induced ß-Catenin degradation and that one of these inhibitors suppressed xenograft tumour development in mice. These results suggest that WNK is a previously unrecognized regulator of ß-Catenin and a therapeutic target of cancer.
Asunto(s)
Proteína Quinasa Deficiente en Lisina WNK 1 , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo , Animales , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitinación/fisiología , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismoRESUMEN
The TATA box-binding protein (TBP) is an essential component of transcription initiation complexes of all three eukaryotic RNA polymerases. Recent knockdown studies revealed that basic transcription factors are essential not only for gene transcription but also for regulating specific gene expression. However, the mechanism of and the effect by regulation of TBP expression are unknown during early embryogenesis. Here we show that the alteration of concentration of each TBP-type gene affected mutually one another's expression, suggesting that an optimal ratio of concentrations of TBP-type genes induce expression of specific genes.
Asunto(s)
Notocorda/embriología , Proteína de Unión a TATA-Box/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Xenopus laevis/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , ADN Complementario/genética , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Proteínas Fluorescentes Verdes/genéticaRESUMEN
The noncanonical wnt/planar cell polarity (PCP) pathway [1] regulates the mediolaterally (planarly) polarized cell protrusive activity and intercalation that drives the convergent extension movements of vertebrate gastrulation [2], yet the underlying mechanism is unknown. We report that perturbing expression of Xenopus PCP genes, Strabismus (Xstbm), Frizzled (Xfz7), and Prickle (Xpk), disrupts radially polarized fibronectin fibril assembly on mesodermal tissue surfaces, mediolaterally polarized motility, and intercalation. Polarized motility is restored in Xpk-perturbed explants but not in Xstbm- or Xfz7-perturbed explants cultured on fibronectin surfaces. The PCP complex, including Xpk, first regulates polarized surface assembly of the fibronectin matrix, which is necessary for mediolaterally polarized motility, and then, without Xpk, has an additional and necessary function in polarizing motility. These results show that the PCP complex regulates several cell polarities (radial, planar) and several processes (matrix deposition, motility), by indirect and direct mechanisms, and acts in several modes, either with all or a subset of its components, during vertebrate morphogenesis.
Asunto(s)
Polaridad Celular/genética , Matriz Extracelular/metabolismo , Gástrula/metabolismo , Regulación del Desarrollo de la Expresión Génica , Transducción de Señal/fisiología , Xenopus/embriología , Animales , Extensiones de la Superficie Celular/metabolismo , Fibronectinas/metabolismo , Colorantes Fluorescentes , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Compuestos Orgánicos , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Xenopus/metabolismoRESUMEN
Glucose transporters (GLUTs) are transmembrane proteins that play an essential role in sugar uptake and energy supply. Thirteen GLUT genes have been described and GLUT1 is the most abundantly expressed member of the family in animal tissues. Deficiencies in human GLUT1 are associated with many diseases, such as metabolic abnormalities, congenital brain defects and oncogenesis. It was suggested recently that Xenopus GLUT1 (xGLUT1) is upregulated by Activin/Nodal signaling, although the developmental role of xGLUT1 remains unclear. Here, we investigated the expression pattern and function of xGLUT1 during Xenopus development. Whole-mount in situ hybridization analysis showed expression of xGLUT1 in the mesodermal region of Xenopus embryos, especially in the dorsal blastopore lip at the gastrula stage. From the neurula stage, it was expressed in the neural plate, eye field, cement gland and somites. Loss-of-function analyses using morpholino antisense oligonucleotides against xGLUT1 (xGLUT1MO) caused microcephaly and axis elongation error. This elongation defect of activin-treated animal caps occurred without downregulation of early mesodermal markers. Moreover, dorsal-marginal explant analysis revealed that cell movement was suppressed in dorsal marginal zones injected with xGLUT1MO. These findings implicate xGLUT1 as an important player during gastrulation cell movement in Xenopus.
Asunto(s)
Movimiento Celular , Gástrula/fisiología , Transportador de Glucosa de Tipo 1/fisiología , Proteínas de Xenopus/fisiología , Xenopus laevis/embriología , Xenopus laevis/fisiología , Animales , Clonación Molecular , ADN Complementario , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Hibridación in Situ , Microinyecciones , Microscopía por Video , Modelos Biológicos , Mutación , Oligonucleótidos Antisentido/farmacología , Técnicas de Cultivo de Órganos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMEN
The molecular events driving specification of the kidney have been well characterized. However, how the initial kidney field size is established, patterned, and proportioned is not well characterized. Lhx1 is a transcription factor expressed in pronephric progenitors and is required for specification of the kidney, but few Lhx1 interacting proteins or downstream targets have been identified. By tandem-affinity purification, we isolated FRY like transcriptional coactivator (Fryl), one of two paralogous genes, fryl and furry (fry), have been described in vertebrates. Both proteins were found to interact with the Ldb1-Lhx1 complex, but our studies focused on Lhx1/Fry functional roles, as they are expressed in overlapping domains. We found that Xenopus embryos depleted of fry exhibit loss of pronephric mesoderm, phenocopying the Lhx1-depleted animals. In addition, we demonstrated a synergism between Fry and Lhx1, identified candidate microRNAs regulated by the pair, and confirmed these microRNA clusters influence specification of the kidney. Therefore, our data shows that a constitutively-active Ldb1-Lhx1 complex interacts with a broadly expressed microRNA repressor, Fry, to establish the kidney field.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Riñón/embriología , Riñón/metabolismo , Proteínas con Homeodominio LIM/metabolismo , MicroARNs/genética , Organogénesis/genética , Factores de Transcripción/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Tipificación del Cuerpo/genética , Línea Celular , Cromatografía Liquida , Orden Génico , Vectores Genéticos/genética , Complejos Multiproteicos/metabolismo , Unión Proteica , Proteínas Represoras/metabolismo , Espectrometría de Masas en Tándem , Xenopus laevisRESUMEN
The stability of ß-catenin is very important for canonical Wnt signaling. A protein complex including Axin/APC/GSK3ß phosphorylates ß-catenin to be degraded by ubiquitination with ß-TrCP. In the recent study, we isolated WDR26, a protein that binds to Axin. Here, we found that WDR26 is a negative regulator of the canonical Wnt signaling pathway, and that WDR26 affected ß-catenin levels. In addition, WDR26/Axin binding is involved in the ubiquitination of ß-catenin. These results suggest that WDR26 plays a negative role in ß-catenin degradation in the Wnt signaling pathway.
Asunto(s)
Proteína Axina/metabolismo , Proteínas/metabolismo , Vía de Señalización Wnt , Proteínas Adaptadoras Transductoras de Señales , Animales , Células HEK293 , Humanos , Unión Proteica , Ubiquitinación , Xenopus , beta Catenina/metabolismoRESUMEN
Autophosphorylation of amino-acid residues is part of the folding process of various protein kinases. Conventional chemical screening of mature kinases has missed inhibitors that selectively interfere with the folding process. Here we report a cell-based assay that evaluates inhibition of a kinase at a transitional state during the folding process and identify a folding intermediate-selective inhibitor of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), which we refer to as FINDY. FINDY suppresses intramolecular autophosphorylation of Ser97 in DYRK1A in cultured cells, leading to its degradation, but does not inhibit substrate phosphorylation catalysed by the mature kinase. FINDY also suppresses Ser97 autophosphorylation of recombinant DYRK1A, suggesting direct inhibition, and shows high selectivity for DYRK1A over other DYRK family members. In addition, FINDY rescues DYRK1A-induced developmental malformations in Xenopus laevis embryos. Our study demonstrates that transitional folding intermediates of protein kinases can be targeted by small molecules, and paves the way for developing novel types of kinase inhibitors.
Asunto(s)
Bioensayo , Pliegue de Proteína/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Tiazoles/farmacología , Secuencia de Aminoácidos , Animales , Biomarcadores/metabolismo , Cantaridina/farmacología , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Toxinas Marinas , Datos de Secuencia Molecular , Ácido Ocadaico/farmacología , Oxazoles/farmacología , Fosforilación/efectos de los fármacos , Plásmidos/química , Plásmidos/metabolismo , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes , Alineación de Secuencia , Tiazoles/química , Transfección , Xenopus laevis/embriología , Quinasas DyrKRESUMEN
We have cloned Xenopus orthologues of the Drosophila planar cell polarity (PCP) gene Prickle. Xenopus Prickle (XPk) is expressed in tissues at the dorsal midline during gastrulation and early neurulation. XPk is later expressed in a segmental pattern in the presomitic mesoderm and then in recently formed somites. XPk is also expressed in the tailbud, pronephric duct, retina, and the otic vesicle. The complex expression pattern of XPk suggests that PCP signaling is used in a diverse array of developmental processes in vertebrate embryos.
Asunto(s)
Proteínas de Unión al ADN/genética , Xenopus/embriología , Xenopus/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Polaridad Celular/genética , Clonación Molecular , ADN/genética , Proteínas de Unión al ADN/química , Drosophila/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Genes de Insecto , Hibridación in Situ , Proteínas con Dominio LIM , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Chemokine molecules play important roles in the immune system. However, several chemokine molecules are expressed during early development before the immune system is established. Using reverse transcriptionpolymerase chain reaction (RT-PCR) and overexpression of chemokine molecules, we identified and characterized Xenopus laevis CXC-type chemokine ligands (XCXCL13L1, XCXCL13L2, XCXCLa, XCXCLb, XCXCLd, and XCXCLe) and receptors (XCXCR1/2, XCXCR3, XCXCR5, XCXCR6, and XCXCRa) during early development. The CXC-type ligands have low identity with genes for human CXC ligands (CXCL). With the exception of XCXCRa, the CXC receptors (CXCR) identified in the present study had high (ï¾40%65%) identity with human CXCR genes. Although the expression patterns for the CXCL and CXCR genes differed, transcript levels for all genes were very low during early embryogenesis. Overexpression of XCXCL13L1, XCXCL13L2, XCXCLa, XCXCR3, XCXCR6, and XCXCRa interfered with gastrulation and neural fold closure. The results of the present study suggest that several chemokine molecules are related to cell movements during early morphogenesis.