RESUMEN
The Wilms tumor suppressor gene Wt1 encodes a zinc finger transcription factor that is essential for development of multiple organs including kidneys, gonads, spleen and heart. In mammals Wt1 comprises 10 exons with two characteristic splicing events: inclusion or skipping of exon 5 and alternative usage of two splice donor sites between exons 9 and 10. Most fish including zebrafish and medaka possess two wt1 paralogs, wt1a and wt1b, both lacking exon 5. Here we have characterized wt1 in guppy, platyfish and the short-lived African killifish Nothobranchius furzeri. All fish except zebrafish show alternative splicing of exon 4 of wt1a but not of wt1b with the wt1a(-exon 4) isoform being the predominant splice variant. With regard to function, Wt1a(+exon 4) showed less dimerization but stimulated transcription more effectively than the Wt1a(-exon 4) isoform. A specific knockdown of wt1a exon 4 in zebrafish was associated with anomalies in kidney development demonstrating a physiological function for Wt1a exon 4. Interestingly, alternative splicing of exon 4 seems to be an early evolutionary event as it is observed in the single wt1 gene of the sturgeon, a species that has not gone through teleost-specific genome duplication.
Asunto(s)
Empalme Alternativo/genética , Peces/anomalías , Riñón/anomalías , Pronefro/anomalías , Proteínas WT1/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Ciprinodontiformes/embriología , Ciprinodontiformes/genética , Peces/genética , Fundulidae/embriología , Fundulidae/genética , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Datos de Secuencia Molecular , Morfolinos/genética , Oryzias/embriología , Oryzias/genética , Poecilia/embriología , Poecilia/genética , Isoformas de Proteínas/genética , Multimerización de Proteína , Alineación de Secuencia , Análisis de Secuencia de ADN , Técnicas del Sistema de Dos Híbridos , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
Heterotrimeric G proteins in physiological and pathological processes have been extensively studied so far. However, little is known about mechanisms regulating the cellular content and compartmentalization of G proteins. Here, we show that the association of nucleoside diphosphate kinase B (NDPK B) with the G protein betagamma dimer (Gbetagamma) is required for G protein function in vivo. In zebrafish embryos, morpholino-mediated knockdown of zebrafish NDPK B, but not NDPK A, results in a severe decrease in cardiac contractility. The depletion of NDPK B is associated with a drastic reduction in Gbeta(1)gamma(2) dimer expression. Moreover, the protein levels of the adenylyl cyclase (AC)-regulating Galpha(s) and Galpha(i) subunits as well as the caveolae scaffold proteins caveolin-1 and -3 are strongly reduced. In addition, the knockdown of the zebrafish Gbeta(1) orthologs, Gbeta(1) and Gbeta(1like), causes a cardiac phenotype very similar to that of NDPK B morphants. The loss of Gbeta(1)/Gbeta(1like) is associated with a down-regulation in caveolins, AC-regulating Galpha-subunits, and most important, NDPK B. A comparison of embryonic fibroblasts from wild-type and NDPK A/B knockout mice demonstrate a similar reduction of G protein, caveolin-1 and basal cAMP content in mammalian cells that can be rescued by re-expression of human NDPK B. Thus, our results suggest a role for the interaction of NDPK B with Gbetagamma dimers and caveolins in regulating membranous G protein content and maintaining normal G protein function in vivo.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , Proteínas de Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/genética , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas de Unión al GTP Heterotriméricas/genética , Proteínas de Unión al GTP Heterotriméricas/fisiología , Humanos , Immunoblotting , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Contracción Miocárdica/genética , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Nucleósido Difosfato Quinasas NM23/genética , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
The Wilms' tumor suppressor gene wt1 encodes a zinc-finger transcription factor that plays an important role in the development of the mammalian genitourinary system. Mutations in WT1 in humans lead to anomalies of kidney and gonad development and cause Wilms' tumor, a pediatric kidney cancer. The inactivation of both wt1 alleles in mice gives rise to multiple organ defects, among them agenesis of kidney, spleen, and gonads. In zebrafish, an ortholog of wt1 has been described that is expressed in the pronephric field and is later restricted to the podocytes. Here, we report the existence of a second wt1 gene in zebrafish, which we have named wt1b (we named the initial gene wt1a). The overall sequence identity of the two Wt1 proteins is 70% and 92% between the zinc-finger regions, respectively. In contrast to wt1a, wt1b is expressed from the earliest stages of development onward, albeit at low levels. Both wt1a and wt1b are expressed in the intermediate mesoderm, with wt1b being restricted to a smaller area lying at the caudal end of the wt1a expression domain. In adult fish, high expression levels for both genes can be found in gonads, kidney, heart, spleen, and muscle.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Proteínas WT1/genética , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Embrión no Mamífero/química , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Humanos , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas WT1/química , Proteínas WT1/metabolismo , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrolloRESUMEN
Increasing evidence suggests that reversible phosphorylation of histidine residues in proteins is important for signaling cascades in eukaryotic cells. Recently, the first eukaryotic protein histidine phosphatase (PHP) was identified. The beta1-subunit of heterotrimeric G proteins (Gbeta) undergoes phosphorylation on His266 which is apparently involved in receptor-independent G protein activation. We studied whether phosphorylated Gbeta-subunits are substrates of PHP. Phosphorylated Gbetagamma dimers of the retinal G protein transducin and Gbeta in membrane preparations of H10 cells (neonatal rat cardiomyocytes) were dephosphorylated by PHP. Overexpression of PHP in H10 cells showed that PHP and Gbeta also interfere within cells. In membranes of cells overexpressing PHP, the amount of phosphorylated Gbeta was largely reduced. Both our in vitro and cell studies indicate that phosphorylated Gbeta-subunits of heterotrimeric G proteins are substrates of PHP. Therefore, PHP might play a role in the regulation of signal transduction via heterotrimeric G proteins.