RESUMEN
The tumour suppressor, Lethal (2) giant larvae [Lgl; also known as L(2)gl], is an evolutionarily conserved protein that was discovered in the vinegar fly Drosophila, where its depletion results in tissue overgrowth and loss of cell polarity. Lgl links cell polarity and tissue growth through regulation of the Notch and the Hippo signalling pathways. Lgl regulates the Notch pathway by inhibiting V-ATPase activity via Vap33. How Lgl regulates the Hippo pathway was unclear. In this current study, we show that V-ATPase activity inhibits the Hippo pathway, whereas Vap33 acts to activate Hippo signalling. Vap33 physically and genetically interacts with the actin cytoskeletal regulators RtGEF (Pix) and Git, which also bind to the Hippo protein (Hpo) and are involved in the activation of the Hippo pathway. Additionally, we show that the ADP ribosylation factor Arf79F (Arf1), which is a Hpo interactor, is involved in the inhibition of the Hippo pathway. Altogether, our data suggest that Lgl acts via Vap33 to activate the Hippo pathway by a dual mechanism: (1) through interaction with RtGEF, Git and Arf79F, and (2) through interaction and inhibition of the V-ATPase, thereby controlling epithelial tissue growth.
Asunto(s)
Proteínas de Drosophila , Neoplasias , Animales , Adenosina Trifosfatasas/metabolismo , Polaridad Celular , Drosophila/metabolismo , Drosophila melanogaster , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Vía de Señalización Hippo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Capicua (Cic) proteins are conserved HMG-box transcriptional repressors that control receptor tyrosine kinase (RTK) signaling responses and are implicated in human neurological syndromes and cancer. While Cic is known to exist as short (Cic-S) and long (Cic-L) isoforms with identical HMG-box and associated core regions but distinct N termini, most previous studies have focused on Cic-S, leaving the function of Cic-L unexplored. Here we show that Cic-L acts in two capacities during Drosophila oogenesis: 1) as a canonical sensor of RTK signaling in somatic follicle cells, and 2) as a regulator of postmitotic growth in germline nurse cells. In these latter cells, Cic-L behaves as a temporal signal that terminates endoreplicative growth before they dump their contents into the oocyte. We show that Cic-L is necessary and sufficient for nurse cell endoreplication arrest and induces both stabilization of CycE and down-regulation of Myc. Surprisingly, this function depends mainly on the Cic-L-specific N-terminal module, which is capable of acting independently of the Cic HMG-box-containing core. Mirroring these observations, basal metazoans possess truncated Cic-like proteins composed only of Cic-L N-terminal sequences, suggesting that this module plays unique, ancient roles unrelated to the canonical function of Cic.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Proteínas HMGB , Oogénesis , Proteínas Represoras , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Drosophila melanogaster/fisiología , Proteínas HMGB/genética , Proteínas HMGB/fisiología , Oogénesis/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/fisiologíaRESUMEN
Arrestins control signaling via the G protein coupled receptors (GPCRs), serving as both signal terminators and transducers. Previous studies identified several structural elements in arrestins that contribute to their functions as GPCR regulators. However, the importance of these elements in vivo is unclear, and the developmental roles of arrestins are not well understood. We carried out an in vivo structure-function analysis of Kurtz (Krz), the single ortholog of mammalian ß-arrestins in the Drosophila genome. A combination of Krz mutations affecting the GPCR-phosphosensing and receptor core-binding ("finger loop") functions (Krz-KKVL/A) resulted in a complete loss of Krz activity during development. Endosome recruitment and bioluminescence resonance energy transfer (BRET) assays revealed that the KKVL/A mutations abolished the GPCR-binding ability of Krz. We found that the isolated "finger loop" mutation (Krz-VL/A), while having a negligible effect on GPCR internalization, severely affected Krz function, suggesting that tight receptor interactions are necessary for proper termination of signaling in vivo. Genetic analysis as well as live imaging demonstrated that mutations in Krz led to hyperactivity of the GPCR Mist (also known as Mthl1), which is activated by its ligand Folded gastrulation (Fog) and is responsible for cellular contractility and epithelial morphogenesis. Krz mutations affected two developmental events that are under the control of Fog-Mist signaling: gastrulation and morphogenesis of the wing. Overall, our data reveal the functional importance in vivo of direct ß-arrestin/GPCR binding, which is mediated by the recognition of the phosphorylated receptor tail and receptor core interaction. These Krz-GPCR interactions are critical for setting the correct level of Fog-Mist signaling during epithelial morphogenesis.
Asunto(s)
Arrestinas/fisiología , Proteínas de Drosophila/fisiología , Drosophila/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Secuencias de Aminoácidos , Animales , Arrestinas/química , Regulación hacia Abajo , Drosophila/embriología , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Femenino , Gastrulación , Masculino , Modelos Moleculares , Conformación Proteica , Transducción de Señal , Relación Estructura-Actividad , Alas de Animales/embriologíaRESUMEN
The transcriptional repressor Capicua (Cic) controls tissue patterning and restricts organ growth, and has been recently implicated in several cancers. Cic has emerged as a primary sensor of signaling downstream of the receptor tyrosine kinase (RTK)/extracellular signal-regulated kinase (ERK) pathway, but how Cic activity is regulated in different cellular contexts remains poorly understood. We found that the kinase Minibrain (Mnb, ortholog of mammalian DYRK1A), acting through the adaptor protein Wings apart (Wap), physically interacts with and phosphorylates the Cic protein. Mnb and Wap inhibit Cic function by limiting its transcriptional repressor activity. Down-regulation of Cic by Mnb/Wap is necessary for promoting the growth of multiple organs, including the wings, eyes, and the brain, and for proper tissue patterning in the wing. We have thus uncovered a previously unknown mechanism of down-regulation of Cic activity by Mnb and Wap, which operates independently from the ERK-mediated control of Cic. Therefore, Cic functions as an integrator of upstream signals that are essential for tissue patterning and organ growth. Finally, because DYRK1A and CIC exhibit, respectively, prooncogenic vs. tumor suppressor activities in human oligodendroglioma, our results raise the possibility that DYRK1A may also down-regulate CIC in human cells.
Asunto(s)
Tipificación del Cuerpo/genética , Proteínas de Drosophila/genética , Drosophila/genética , Proteínas HMGB/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Proteínas Represoras/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Drosophila/crecimiento & desarrollo , Proteínas de Drosophila/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Proteínas HMGB/biosíntesis , Humanos , Neoplasias/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Represoras/biosíntesis , Alas de Animales/crecimiento & desarrollo , Quinasas DyrKRESUMEN
ß-Arrestins have been implicated in the regulation of multiple signalling pathways. However, their role in organism development is not well understood. In this study, we report a new in vivo function of the Drosophila ß-arrestin Kurtz (Krz) in the regulation of two distinct developmental signalling modules: MAPK ERK and NF-κB, which transmit signals from the activated receptor tyrosine kinases (RTKs) and the Toll receptor, respectively. Analysis of the expression of effectors and target genes of Toll and the RTK Torso in krz maternal mutants reveals that Krz limits the activity of both pathways in the early embryo. Protein interaction studies suggest a previously uncharacterized mechanism for ERK inhibition: Krz can directly bind and sequester an inactive form of ERK, thus preventing its activation by the upstream kinase, MEK. A simultaneous dysregulation of different signalling systems in krz mutants results in an abnormal patterning of the embryo and severe developmental defects. Our findings uncover a new in vivo function of ß-arrestins and present a new mechanism of ERK inhibition by the Drosophila ß-arrestin Krz.
Asunto(s)
Arrestinas/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Inhibidores Enzimáticos/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Transducción de Señal/fisiología , Receptores Toll-Like/metabolismo , Animales , Arrestinas/farmacología , Western Blotting , Células Cultivadas , Drosophila/metabolismo , Proteínas de Drosophila/farmacología , Inhibidores Enzimáticos/farmacología , Técnicas de Inactivación de Genes , Inmunoprecipitación , Hibridación Fluorescente in Situ , Mutación/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The human dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) is implicated in the pathology of Down syndrome, microcephaly, and cancer, however the exact mechanism through which it functions is unknown. Here, we have studied the role of the Drosophila ortholog of DYRK1A, Minibrain (Mnb), in brain development. The neuroblasts (neural stem cells) that eventually give rise to differentiated neurons in the adult brain are formed from a specialized tissue in the larval optic lobe called the neuroepithelium, in a tightly regulated process. Molecular marker analysis of mnb mutants revealed alterations in the neuroepithelium and neuroblast regions of developing larval brains. Using affinity purification-mass spectrometry (AP-MS), we identified the novel Mnb binding partners Ral interacting protein (Rlip) and RALBP1 associated Eps domain containing (Reps). Rlip and Reps physically and genetically interact with Mnb, and the three proteins may form a ternary complex. Mnb phosphorylates Reps, and human DYRK1A binds to the Reps orthologs REPS1 and REPS2. Furthermore, Mnb engages the small GTPase Ras-like protein A (Rala) to regulate brain and wing development. This work uncovers a previously unrecognized early role of Mnb in the neuroepithelium and defines the functions of the Mnb/Reps/Rlip/Rala signaling network in brain development. Significance statement: The kinase Minibrain(Mnb)/DYRK1A regulates the development of the brain and other tissues across many organisms. Here we show the critical importance of Mnb within the developing neuroepithelium. Advancing our understanding of Mnb function, we identified novel protein interactors of Mnb, Reps and Rlip, which function together with Mnb to regulate growth in Drosophila melanogaster . We also identify and characterize a role for the small GTPase Rala in Mnb-regulated growth and nervous system development. This work reveals an early role of Mnb in brain development and identifies a new Mnb/Reps/Rlip/Rala signaling axis.
RESUMEN
The canonical function of the Hippo signaling pathway is the regulation of organ growth. How this pathway controls cell-fate determination is less well understood. Here, we identify a function of the Hippo pathway in cell-fate decisions in the developing Drosophila eye, exerted through the interaction of Yorkie (Yki) with the transcriptional regulator Bonus (Bon), an ortholog of mammalian transcriptional intermediary factor 1/tripartite motif (TIF1/TRIM) family proteins. Instead of controlling tissue growth, Yki and Bon promote epidermal and antennal fates at the expense of the eye fate. Proteomic, transcriptomic, and genetic analyses reveal that Yki and Bon control these cell-fate decisions by recruiting transcriptional and post-transcriptional co-regulators and by repressing Notch target genes and activating epidermal differentiation genes. Our work expands the range of functions and regulatory mechanisms under Hippo pathway control.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Diferenciación Celular , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Vía de Señalización Hippo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mamíferos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica , Transducción de Señal , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
The Germinal Center Kinase III (GckIII) pathway is a Hippo-like kinase module defined by sequential activation of Ste20 kinases Thousand and One (Tao) and GckIII, followed by nuclear dbf2-related (NDR) kinase Tricornered (Trc). We previously uncovered a role for the GckIII pathway in Drosophila melanogaster tracheal (respiratory) tube morphology. The trachea form a network of branched epithelial tubes essential for oxygen transport, and are structurally analogous to branched tubular organs in vertebrates, such as the vascular system. In the absence of GckIII pathway function, aberrant dilations form in tracheal tubes characterized by mislocalized junctional and apical proteins, suggesting that the pathway is important in maintaining tube integrity in development. Here, we observed a genetic interaction between trc and Cerebral cavernous malformations 3 (Ccm3), the Drosophila ortholog of a human vascular disease gene, supporting our hypothesis that the GckIII pathway functions downstream of Ccm3 in trachea, and potentially in the vertebrate cerebral vasculature. However, how GckIII pathway signaling is regulated and the mechanisms that underpin its function in tracheal development are unknown. We undertook biochemical and genetic approaches to identify proteins that interact with Trc, the most downstream GckIII pathway kinase. We found that known GckIII and NDR scaffold proteins are likely to control GckIII pathway signaling in tracheal development, consistent with their conserved roles in Hippo-like modules. Furthermore, we show genetic interactions between trc and multiple enzymes in glycolysis and oxidative phosphorylation, suggesting a potential function of the GckIII pathway in integrating cellular energy requirements with maintenance of tube integrity.
Asunto(s)
Proteínas de Drosophila , Proteínas Serina-Treonina Quinasas , Animales , Humanos , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Quinasas del Centro Germinal/genética , Quinasas del Centro Germinal/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de SeñalRESUMEN
Signalling activity of the Notch receptor, which plays a fundamental role in metazoan cell fate determination, is controlled at multiple levels. We uncovered a Notch signal-controlling mechanism that depends on the ability of the non-visual beta-arrestin, Kurtz (Krz), to influence the degradation and, consequently, the function of the Notch receptor. We identified Krz as a binding partner of a known Notch-pathway modulator, Deltex (Dx), and demonstrated the existence of a trimeric Notch-Dx-Krz protein complex. This complex mediates the degradation of the Notch receptor through a ubiquitination-dependent pathway. Our results establish a novel mode of regulation of Notch signalling and define a new function for non-visual beta-arrestins.
Asunto(s)
Arrestinas/fisiología , Proteínas de Drosophila/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Animales , Arrestinas/genética , Arrestinas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Femenino , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos , Mutación/fisiología , Técnicas del Sistema de Dos Híbridos , Ubiquitina/metabolismo , Alas de Animales/crecimiento & desarrollo , beta-ArrestinasRESUMEN
Melanin is a widely distributed phenolic pigment that is biosynthesized from tyrosine and its hydroxylated product, dopa, in all animals. However, recent studies reveal a significant deviation from this paradigm, as insects appear to use dopamine rather than dopa as the major precursor of melanin. This observation calls for a reconsideration of the insect melanogenic pathway. While phenoloxidases and laccases can oxidize dopamine for dopaminechrome production, the fate of dopaminechrome remains undetermined. Dopachrome decarboxylase/tautomerase, encoded by yellow-f/f2 of Drosophila melanogaster, can convert dopaminechrome into 5,6-dihydroxyindole, but the same enzyme from other organisms does not act on dopaminechrome, suggesting the existence of a specific dopaminechrome tautomerase (DPT). We now report the identification of this novel enzyme that biosynthesizes 5,6-dihydroxyindole from dopaminechrome in Drosophila. Dopaminechrome tautomerase acted on both dopaminechrome and N-methyl dopaminechrome but not on dopachrome or other aminochromes tested. Our biochemical and molecular studies reveal that this enzyme is encoded by the yellow-h gene, a member of the yellow gene family, and advance our understanding of the physiological functions of this gene family. Identification and characterization of DPT clarifies the precursor for melanin biosynthetic pathways and proves the existence of an independent melanogenic pathway in insects that utilizes dopamine as the primary precursor.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Oxidorreductasas Intramoleculares , Melaninas , Animales , Animales Modificados Genéticamente , Línea Celular , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Indoles/metabolismo , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Melaninas/biosíntesis , MutaciónRESUMEN
The extracellular signal-regulated kinase (ERK) pathway is an essential component of developmental signaling in metazoans. Previous models of pathway activation suggested that dissociation of activated dually phosphorylated ERK (dpERK) from MAPK/ERK kinase (MEK), a kinase that phosphorylates ERK, and other cytoplasmic anchors, is sufficient for allowing ERK interactions with its substrates. Here, we provide evidence for an additional step controlling ERK's access to substrates. Specifically, we demonstrate that interaction of ERK with its substrate Capicua (Cic) is controlled at the level of ERK phosphorylation, whereby Cic binds to dpERK much stronger than to unphosphorylated ERK, both in vitro and in vivo. Mathematical modeling suggests that the differential affinity of Cic for dpERK versus ERK is required for both down-regulation of Cic and stabilizing phosphorylated ERK. Preferential association of Cic with dpERK serves two functions: it prevents unproductive competition of Cic with unphosphorylated ERK and contributes to efficient signal propagation. We propose that high-affinity substrate binding increases the specificity and efficiency of signal transduction through the ERK pathway.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas HMGB/genética , Sistema de Señalización de MAP Quinasas/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteínas Represoras/genética , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Proteínas HMGB/metabolismo , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Fosforilación , Unión Proteica , Ratas , Proteínas Represoras/metabolismo , Transducción de SeñalRESUMEN
Healthy mitochondrial networks are maintained via balanced integration of mitochondrial quality control processes (biogenesis, fusion, fission, and mitophagy). The purpose of this study was to investigate the effects of severe obesity and type 2 diabetes (T2D) on mitochondrial network morphology and expression of proteins regulating mitochondrial quality control processes in cultured human myotubes. Primary human skeletal muscle cells were isolated from biopsies from lean, severely obese nondiabetic individuals and severely obese type 2 diabetic individuals (n = 8-9/group) and were differentiated to myotubes. Mitochondrial network morphology was determined in live cells via confocal microscopy and protein markers of mitochondrial quality control were measured by immunoblotting. Myotubes from severely obese nondiabetic and type 2 diabetic humans exhibited fragmented mitochondrial networks (P < 0.05). Mitochondrial fission protein Drp1 (Ser616) phosphorylation was higher in myotubes from severely obese nondiabetic humans when compared with the lean controls (P < 0.05), while mitophagy protein Parkin expression was lower in myotubes from severely obese individuals with T2D in comparison to the other groups (P < 0.05). These data suggest that regulatory proteins in mitochondrial quality control processes, specifically mitochondrial fission protein Drp1 (Ser616) phosphorylation and mitophagy protein Parkin, are intrinsically dysregulated at cellular level in skeletal muscle from severely obese nondiabetic and type 2 diabetic humans, respectively. These differentially expressed mitochondrial quality control proteins may play a role in mitochondrial fragmentation evident in skeletal muscle from severely obese and type 2 diabetic humans. Novelty Mitochondrial network morphology and mitochondrial quality control proteins are intrinsically dysregulated in skeletal muscle cells from severely obese humans with or without T2D.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Proteínas Mitocondriales/metabolismo , Recambio Mitocondrial/fisiología , Fibras Musculares Esqueléticas/metabolismo , Obesidad Mórbida/metabolismo , Adulto , Femenino , Humanos , Biogénesis de OrganelosRESUMEN
In Drosophila, the same set of genes that are used for cuticle pigmentation and sclerotization are present in the nervous system and are responsible for neurotransmitter recycling. In this study, we carried out biochemical analysis to determine whether insects have the enzymatic machinery to make melanic component of neuromelanin. We focused our attention on two key enzymes of melanogenesis, namely phenoloxidase and dopachrome decarboxylase/tautomerase. Activity staining of the proteins isolated from the Drosophila larval brain tissue, separated by native polyacrylamide gel electrophoresis, indicated the presence of these two enzymes. Mass spectral sequence analysis of the band also supported this finding. To best of our knowledge, this is the first report on the presence of the enzymatic machinery to make melanin part of neuromelanin in any insect brain.
Asunto(s)
Drosophila melanogaster/enzimología , Melaninas/biosíntesis , Secuencia de Aminoácidos , Animales , Biomarcadores/metabolismo , Vías Biosintéticas , Encéfalo/metabolismo , Dopamina/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Melaninas/química , Modelos Biológicos , Monofenol Monooxigenasa/metabolismo , Neuronas/metabolismo , PigmentaciónRESUMEN
Epithelial cell polarity is linked to the control of tissue growth and tumorigenesis. The tumor suppressor and cell polarity protein lethal-2-giant larvae (Lgl) promotes Hippo signaling and inhibits Notch signaling to restrict tissue growth in Drosophila melanogaster Notch signaling is greater in lgl mutant tissue than in wild-type tissue because of increased acidification of endosomal vesicles, which promotes the proteolytic processing and activation of Notch by γ-secretase. We showed that the increased Notch signaling and tissue growth defects of lgl mutant tissue depended on endosomal vesicle acidification mediated by the vacuolar adenosine triphosphatase (V-ATPase). Lgl promoted the activity of the V-ATPase by interacting with Vap33 (VAMP-associated protein of 33 kDa). Vap33 physically and genetically interacted with Lgl and V-ATPase subunits and repressed V-ATPase-mediated endosomal vesicle acidification and Notch signaling. Vap33 overexpression reduced the abundance of the V-ATPase component Vha44, whereas Lgl knockdown reduced the binding of Vap33 to the V-ATPase component Vha68-3. Our data indicate that Lgl promotes the binding of Vap33 to the V-ATPase, thus inhibiting V-ATPase-mediated endosomal vesicle acidification and thereby reducing γ-secretase activity, Notch signaling, and tissue growth. Our findings implicate the deregulation of Vap33 and V-ATPase activity in polarity-impaired epithelial cancers.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endosomas/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Ácidos/metabolismo , Animales , Proteínas Portadoras/genética , Polaridad Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Epitelio/crecimiento & desarrollo , Epitelio/metabolismo , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Femenino , Proteínas de la Membrana/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Supresoras de Tumor/genética , ATPasas de Translocación de Protón Vacuolares/genética , Vacuolas/metabolismoRESUMEN
Hippo-like pathways are ancient signaling modules first identified in yeasts. The best-defined metazoan module forms the core of the Hippo pathway, which regulates organ size and cell fate. Hippo-like kinase modules consist of a Sterile 20-like kinase, an NDR kinase, and non-catalytic protein scaffolds. In the Hippo pathway, the upstream kinase Hippo can be activated by another kinase, Tao-1. Here, we delineate a related Hippo-like signaling module that Tao-1 regulates to control tracheal morphogenesis in Drosophila melanogaster. Tao-1 activates the Sterile 20-like kinase GckIII by phosphorylating its activation loop, a mode of regulation that is conserved in humans. Tao-1 and GckIII act upstream of the NDR kinase Tricornered to ensure proper tube formation in trachea. Our study reveals that Tao-1 activates two related kinase modules to control both growth and morphogenesis. The Hippo-like signaling pathway we have delineated has a potential role in the human vascular disease cerebral cavernous malformation.
Asunto(s)
Morfogénesis , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Tráquea/embriología , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Quinasas del Centro Germinal , Células HEK293 , Humanos , Proteínas Serina-Treonina Quinasas/genética , Tráquea/metabolismoRESUMEN
BACKGROUND: The Myc oncoprotein is an important regulator of cellular growth in metazoan organisms. Its levels and activity are tightly controlled in vivo by a variety of mechanisms. In normal cells, Myc protein is rapidly degraded, but the mechanism of its degradation is not well understood. RESULTS: Here we present genetic and biochemical evidence that Archipelago (Ago), the F box component of an SCF-ubiquitin ligase and the Drosophila ortholog of a human tumor suppressor, negatively regulates the levels and activity of Drosophila Myc (dMyc) protein in vivo. Mutations in archipelago (ago) result in strongly elevated dMyc protein levels and increased tissue growth. Genetic interactions indicate that ago antagonizes dMyc function during development. Archipelago binds dMyc and regulates its stability, and the ability of Ago to bind dMyc in vitro correlates with its ability to inhibit dMyc accumulation in vivo. CONCLUSIONS: Our data indicate that archipelago is an important inhibitor of dMyc in developing tissues. Because archipelago can also regulate Cyclin E levels and Notch activity, these results indicate how a single F box protein can be responsible for the degradation of key components of multiple pathways that control growth and cell cycle progression.
Asunto(s)
Ciclo Celular/fisiología , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/metabolismo , Proteínas F-Box/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Northern Blotting , Pesos y Medidas Corporales , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas F-Box/genética , Proteínas F-Box/fisiología , Citometría de Flujo , Inmunohistoquímica , Datos de Secuencia Molecular , Mutación/genética , Pruebas de Precipitina , Interferencia de ARN , Alineación de Secuencia , Factores de Transcripción/metabolismo , Transfección , Alas de Animales/citologíaRESUMEN
Elucidation of biological functions of signaling proteins is facilitated by studying their protein-protein interaction networks. Affinity purification combined with mass spectrometry (AP-MS) has become a favorite method to study protein complexes. Here we describe a procedure for single-step purification of ERK (Rolled) and associated proteins from Drosophila cultured cells. The use of the streptavidin-binding peptide (SBP) tag allows for a highly efficient isolation of native ERK signaling complexes, which are suitable for subsequent analysis by mass spectrometry. Our analysis of the ERK interactome has identified both known and novel signaling components. This method can be easily adapted for SBP-based purification of protein complexes in any expression system.
Asunto(s)
Proteínas Portadoras , Cromatografía de Afinidad , Quinasas MAP Reguladas por Señal Extracelular/aislamiento & purificación , Complejos Multiproteicos/aislamiento & purificación , Animales , Línea Celular , Cromatografía de Afinidad/métodos , Drosophila , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica , Orden Génico , Vectores Genéticos/genética , Complejos Multiproteicos/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , TransfecciónRESUMEN
Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, organism development, and disease. It is often desirable to obtain PPI information using in vivo material, to gain the most natural and unbiased view of the interaction networks. The fruit fly Drosophila melanogaster is an excellent platform to study PPIs in vivo, and lends itself to straightforward approaches to isolating material for biochemical experiments. In particular, fruit fly embryos represent a convenient type of tissue to study PPIs, due to the ease of collecting animals at this developmental stage and the fact that the majority of proteins are expressed in embryogenesis, thus providing a relevant environment to reveal most PPIs. Here we present a protocol for collection of Drosophila embryos at medium scale (0.5-1 g), which is an ideal amount for a wide range of proteomic applications, including analysis of PPIs by affinity purification-mass spectrometry (AP-MS). We describe our designs for 1 L and 5 L cages for embryo collections that can be easily and inexpensively set up in any laboratory. We also provide a general protocol for embryo collection and protein extraction to generate lysates that can be directly used in downstream applications such as AP-MS. Our goal is to provide an accessible means for all researchers to carry out the analyses of PPIs in vivo.
Asunto(s)
Drosophila/embriología , Técnicas de Cultivo de Embriones/métodos , Espectrometría de Masas/métodos , Proteómica/métodos , Animales , Drosophila/metabolismoRESUMEN
The Hippo pathway is a conserved signaling cascade that modulates tissue growth. Although its core elements are well defined, factors modulating Hippo transcriptional outputs remain elusive. Here we show that components of the steroid-responsive ecdysone (Ec) pathway modulate Hippo transcriptional effects in imaginal disc cells. The Ec receptor coactivator Taiman (Tai) interacts with the Hippo transcriptional coactivator Yorkie (Yki) and promotes expression of canonical Yki-responsive genes. Tai enhances Yki-driven growth, while Tai loss, or a form of Tai unable to bind Yki, suppresses Yki-driven tissue growth. This growth suppression is not correlated with impaired induction of canonical Hippo-responsive genes but with suppression of a distinct pro-growth program of Yki-induced/Tai-dependent genes, including the germline stem cell factors nanos and piwi. These data reveal Hippo/Ec pathway crosstalk in the form a Yki-Tai complex that collaboratively induces germline genes as part of a transcriptional program that is normally repressed in developing somatic epithelia.
Asunto(s)
Proteínas Argonautas/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/genética , Receptores de Esteroides/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas Argonautas/biosíntesis , Células Cultivadas , Proteínas de Drosophila/biosíntesis , Drosophila melanogaster , Epitelio/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Interferente Pequeño , Proteínas de Unión al ARN/biosíntesis , Factor de Células Madre , Transactivadores/genética , Factores de Transcripción/genética , Transcripción Genética , Alas de Animales/embriología , Proteínas Señalizadoras YAPRESUMEN
The Salvador-Warts-Hippo (Hippo) pathway is a conserved regulator of organ size and is deregulated in human cancers. In epithelial tissues, the Hippo pathway is regulated by fundamental cell biological properties, such as polarity and adhesion, and coordinates these with tissue growth. Despite its importance in disease, development, and regeneration, the complete set of proteins that regulate Hippo signaling remain undefined. To address this, we used proteomics to identify proteins that bind to the Hippo (Hpo) kinase. Prominent among these were PAK-interacting exchange factor (known as Pix or RtGEF) and G-protein-coupled receptor kinase-interacting protein (Git). Pix is a conserved Rho-type guanine nucleotide exchange factor (Rho-GEF) homologous to Beta-PIX and Alpha-PIX in mammals. Git is the single Drosophila melanogaster homolog of the mammalian GIT1 and GIT2 proteins, which were originally identified in the search for molecules that interact with G-protein-coupled receptor kinases. Pix and Git form an oligomeric scaffold to facilitate sterile 20-like kinase activation and have also been linked to GTPase regulation. We show that Pix and Git regulate Hippo-pathway-dependent tissue growth in D. melanogaster and that they do this in parallel to the known upstream regulator Fat cadherin. Pix and Git influence activity of the Hpo kinase by acting as a scaffold complex, rather than enzymes, and promote Hpo dimerization and autophosphorylation of Hpo's activation loop. Therefore, we provide important new insights into an ancient signaling network that controls the growth of metazoan tissues.