RESUMEN
Citizen science is a productive approach to include non-scientists in research efforts that impact particular issues or communities. In most cases, scientists at advanced career stages design high-quality, exciting projects that enable citizen contribution, a crowdsourcing process that drives discovery forward and engages communities. The challenges of having citizens design their own research with no or limited training and providing access to laboratory tools, reagents, and supplies have limited citizen science efforts. This leaves the incredible life experiences and immersion of citizens in communities that experience health disparities out of the research equation, thus hampering efforts to address community health needs with a full picture of the challenges that must be addressed. Here, we present a robust and reproducible approach that engages participants from Grade 5 through adult in research focused on defining how diet impacts disease signaling. We leverage the powerful genetics, cell biology, and biochemistry of Drosophila oogenesis to define how nutrients impact phenotypes associated with genetic mutants that are implicated in cancer and diabetes. Participants lead the project design and execution, flipping the top-down hierarchy of the prevailing scientific culture to co-create research projects and infuse the research with cultural and community relevance.
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Drosophila , Salud Pública , Animales , InvestigaciónRESUMEN
Casein kinase 2 (CK2) is an evolutionarily conserved serine/threonine kinase implicated in a wide range of cellular functions and known to be dysregulated in various diseases such as cancer. Compared to most other kinases, CK2 exhibits several unusual properties, including dual co-substrate specificity and a high degree of promiscuity with hundreds of substrates described to date. Most paradoxical, however, is its apparent constitutive activity: no definitive mode of catalytic regulation has thus far been identified. Here we demonstrate that copper enhances the enzymatic activity of CK2 both in vitro and in vivo. We show that copper binds directly to CK2, and we identify specific residues in the catalytic subunit of the enzyme that are critical for copper-binding. We further demonstrate that increased levels of intracellular copper result in enhanced CK2 kinase activity, while decreased copper import results in reduced CK2 activity. Taken together, these findings establish CK2 as a copper-regulated kinase and indicate that copper is a key modulator of CK2-dependent signaling pathways.
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Developing male germ cells are extremely sensitive to heat stress; consequently, anatomic and physiologic adaptations have evolved to maintain proper thermoregulation during mammalian spermatogenesis. At the cellular level, increased expression and activity of HSP70 family members occur in response to heat stress in order to refold partially denatured proteins and restore function. In addition, several kinase-mediated signaling pathways are activated in the testis upon hyperthermia. The p38 MAP kinase (MAPK) pathway plays an important role in mitigating heat stress, and recent findings have implicated the downstream p38 substrate, MAPKAP kinase 2 (MK2), in this process. However, the precise function that this kinase plays in spermatogenesis is not completely understood. Using a proteomics-based screen, we identified and subsequently validated that the testis-enriched HSP70 family member, HspA1L, is a novel substrate of MK2. We demonstrate that MK2 phosphorylates HspA1L solely on Ser241, a residue within the N-terminal nucleotide-binding domain of the enzyme. This phosphorylation event enhances the chaperone activity of HspA1L in vitro and renders male germ cells more resistant to heat stress-induced apoptosis. Taken together, these findings illustrate a novel stress-induced signaling cascade that promotes the chaperone activity of HspA1L with implications for understanding male reproductive biology.
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Proteínas HSP70 de Choque Térmico/fisiología , Respuesta al Choque Térmico/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Espermatogénesis/fisiología , Espermatozoides/metabolismo , Animales , Apoptosis/fisiología , Línea Celular , Masculino , Espermatozoides/citologíaRESUMEN
The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.
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Quinasa de la Caseína II/metabolismo , Pruebas de Enzimas/métodos , Animales , Línea Celular Tumoral , Drosophila melanogaster/metabolismo , Humanos , Fosforilación , Especificidad por SustratoRESUMEN
Lipid metabolism plays a critical role in female reproduction. During oogenesis, maturing oocytes accumulate high levels of neutral lipids that are essential for both energy production and the synthesis of other lipid molecules. Metabolic pathways within the ovary are partially regulated by protein kinases that link metabolic status to oocyte development. Although the functions of several kinases in this process are well established, the roles that many other kinases play in coordinating metabolic state with female germ cell development are unknown. Here, we demonstrate that the catalytic activity of casein kinase 2 (CK2) is essential for Drosophila oogenesis. Using an unbiased biochemical screen that leveraged an unusual catalytic property of the kinase, we identified a novel CK2 substrate in the Drosophila ovary, the lipid droplet-associated protein Jabba. We show that Jabba is essential for modulating ovarian lipid metabolism and for regulating female fertility in the fly. Our findings shed light on a CK2-dependent signaling pathway governing lipid metabolism in the ovary and provide insight into the long-recognized but poorly understood association between energy metabolism and female reproduction.
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Proteínas Portadoras/metabolismo , Quinasa de la Caseína II/metabolismo , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Metabolismo de los Lípidos , Oogénesis , Ovario/metabolismo , Células 3T3-L1 , Animales , Animales Modificados Genéticamente , Proteínas Portadoras/química , Proteínas Portadoras/genética , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/química , Quinasa de la Caseína II/genética , Cruzamientos Genéticos , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Femenino , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Microscopía Fluorescente , Ovario/citología , Ovario/enzimología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Interferencia de ARN , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por SustratoRESUMEN
Developing male germ cells are exquisitely sensitive to environmental insults such as heat and oxidative stress. An additional characteristic of these cells is their unique dependence on RNA-binding proteins for regulating posttranscriptional gene expression and translational control. Here we provide a mechanistic link unifying these two features. We show that the germ cell-specific RNA-binding protein deleted in azoospermia-like (Dazl) is phosphorylated by MAPKAP kinase 2 (MK2), a stress-induced protein kinase activated downstream of p38 MAPK. We demonstrate that phosphorylation of Dazl by MK2 on an evolutionarily conserved serine residue inhibits its interaction with poly(A)-binding protein, resulting in reduced translation of Dazl-regulated target RNAs. We further show that transgenic expression of wild-type human Dazl but not a phosphomimetic form in the Drosophila male germline can restore fertility to flies deficient in boule, the Drosophila orthologue of human Dazl. These results illuminate a novel role for MK2 in spermatogenesis, expand the repertoire of RNA-binding proteins phosphorylated by this kinase, and suggest that signaling by the p38-MK2 pathway is a negative regulator of spermatogenesis via phosphorylation of Dazl.
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Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Animales , Drosophila/metabolismo , Expresión Génica , Células Germinativas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , ARN/metabolismo , Proteínas con Motivos de Reconocimiento de ARN , Espermatogénesis/genética , Espermatogénesis/fisiología , Testículo/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The enzyme CTP synthase (CTPS) dynamically assembles into macromolecular filaments in bacteria, yeast, Drosophila, and mammalian cells, but the role of this morphological reorganization in regulating CTPS activity is controversial. During Drosophila oogenesis, CTPS filaments are transiently apparent in ovarian germline cells during a period of intense genomic endoreplication and stockpiling of ribosomal RNA. Here, we demonstrate that CTPS filaments are catalytically active and that their assembly is regulated by the non-receptor tyrosine kinase DAck, the Drosophila homologue of mammalian Ack1 (activated cdc42-associated kinase 1), which we find also localizes to CTPS filaments. Egg chambers from flies deficient in DAck or lacking DAck catalytic activity exhibit disrupted CTPS filament architecture and morphological defects that correlate with reduced fertility. Furthermore, ovaries from these flies exhibit reduced levels of total RNA, suggesting that DAck may regulate CTP synthase activity. These findings highlight an unexpected function for DAck and provide insight into a novel pathway for the developmental control of an essential metabolic pathway governing nucleotide biosynthesis.
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Ligasas de Carbono-Nitrógeno/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Oogénesis , Proteínas Tirosina Quinasas/metabolismo , Animales , Ligasas de Carbono-Nitrógeno/genética , Drosophila/genética , Drosophila/fisiología , Proteínas de Drosophila/genética , Femenino , Ovario/metabolismo , Transporte de Proteínas , Proteínas Tirosina Quinasas/genética , ARN/metabolismoRESUMEN
A healthy diet improves adult stem cell function and delays diseases such as cancer, heart disease, and neurodegeneration. Defining molecular mechanisms by which nutrients dictate stem cell behavior is a key step toward understanding the role of diet in tissue homeostasis. In this paper, we elucidate the mechanism by which dietary cholesterol controls epithelial follicle stem cell (FSC) proliferation in the fly ovary. In nutrient-restricted flies, the transmembrane protein Boi sequesters Hedgehog (Hh) ligand at the surface of Hh-producing cells within the ovary, limiting FSC proliferation. Upon feeding, dietary cholesterol stimulates S6 kinase-mediated phosphorylation of the Boi cytoplasmic domain, triggering Hh release and FSC proliferation. This mechanism enables a rapid, tissue-specific response to nutritional changes, tailoring stem cell divisions and egg production to environmental conditions sufficient for progeny survival. If conserved in other systems, this mechanism will likely have important implications for studies on molecular control of stem cell function, in which the benefits of low calorie and low cholesterol diets are beginning to emerge.
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Proliferación Celular/efectos de los fármacos , Colesterol en la Dieta/farmacología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efectos de los fármacos , Proteínas Hedgehog/metabolismo , Folículo Ovárico/efectos de los fármacos , Células Madre/efectos de los fármacos , Animales , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/metabolismo , Femenino , Folículo Ovárico/citología , Folículo Ovárico/metabolismo , Ovario/citología , Ovario/efectos de los fármacos , Ovario/metabolismo , Fosforilación , Proteínas Quinasas S6 Ribosómicas/metabolismo , Células Madre/citología , Células Madre/metabolismoRESUMEN
Synaptic transmission is mediated by a complex set of molecular events that must be coordinated in time and space. While many proteins that function at the synapse have been identified, the signaling pathways regulating these molecules are poorly understood. Pak5 (p21-activated kinase 5) is a brain-specific isoform of the group II Pak kinases whose substrates and roles within the central nervous system are largely unknown. To gain insight into the physiological roles of Pak5, we engineered a Pak5 mutant to selectively radiolabel its substrates in murine brain extract. Using this approach, we identified two novel Pak5 substrates, Pacsin1 and Synaptojanin1, proteins that directly interact with one another to regulate synaptic vesicle endocytosis and recycling. Pacsin1 and Synaptojanin1 were phosphorylated by Pak5 and the other group II Paks in vitro, and Pak5 phosphorylation promoted Pacsin1-Synaptojanin1 binding both in vitro and in vivo. These results implicate Pak5 in Pacsin1- and Synaptojanin1-mediated synaptic vesicle trafficking and may partially account for the cognitive and behavioral deficits observed in group II Pak-deficient mice.
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Proteínas del Tejido Nervioso/metabolismo , Neuronas/enzimología , Neuropéptidos/metabolismo , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Vesículas Sinápticas/enzimología , Quinasas p21 Activadas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Transporte Biológico , Encéfalo/enzimología , Péptidos y Proteínas de Señalización Intracelular , Ratones , Modelos Biológicos , Fosforilación , Unión Proteica , Especificidad por SustratoRESUMEN
Phospholipid-enriched membranes such as the plasma membrane can serve as direct regulators of kinase signaling. Pak1 is involved in growth factor signaling at the plasma membrane, and its dysregulation is implicated in cancer. Pak1 adopts an autoinhibited conformation that is relieved upon binding to membrane-bound Rho GTPases Rac1 or Cdc42, but whether lipids also regulate Pak1 in vivo is unknown. We show here that phosphoinositides, particularly PIP(2), potentiate Rho-GTPase-mediated Pak1 activity. A positively charged region of Pak1 binds to phosphoinositide-containing membranes, and this interaction is essential for membrane recruitment and activation of Pak1 in response to extracellular signals. Our results highlight an active role for lipids as allosteric regulators of Pak1 and suggest that Pak1 is a "coincidence detector" whose activation depends on GTPases present in phosphoinositide-rich membranes. These findings expand the role of phosphoinositides in kinase signaling and suggest how altered phosphoinositide metabolism may upregulate Pak1 activity in cancer cells.
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Activadores de Enzimas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Quinasas p21 Activadas/metabolismo , Secuencia de Aminoácidos , Animales , Extractos Celulares , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Activación Enzimática/efectos de los fármacos , Humanos , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Factor de Crecimiento Derivado de Plaquetas/farmacología , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Xenopus , Quinasas p21 Activadas/química , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Endocytosed proteins are either delivered to the lysosome to be degraded or are exported from the endosomal system and delivered to other organelles. Sorting of the Saccharomyces cerevisiae reductive iron transporter, composed of the Fet3 and Ftr1 proteins, in the endosomal system is regulated by available iron; in iron-starved cells, Fet3-Ftr1 is sorted by Snx3/Grd19 and retromer into a recycling pathway that delivers it back to the plasma membrane, but when starved cells are exposed to iron, Fet3-Ftr1 is targeted to the lysosome-like vacuole and is degraded. We report that iron-induced endocytosis of Fet3-Ftr1 is independent of Fet3-Ftr1 ubiquitylation, and after endocytosis, degradation of Fet3-Ftr1 is mediated by the multivesicular body (MVB) sorting pathway. In mutant cells lacking any component of the ESCRT protein-dependent MVB sorting machinery, the Rsp5 ubiquitin ligase, or in wild-type cells expressing Fet3-Ftr1 lacking cytosolic lysyl ubiquitin acceptor sites, Fet3-Ftr1 is constitutively sorted into the recycling pathway independent of iron status. In the presence and absence of iron, Fet3-Ftr1 transits an endosomal compartment where a subunit of the MVB sorting receptor (Vps27), Snx3/Grd19, and retromer proteins colocalize. We propose that this endosome is where Rsp5 ubiquitylates Fet3-Ftr1 and where the recycling and degradative pathways diverge.
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Proteínas Portadoras/metabolismo , Endosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Hierro/farmacología , Lisina/metabolismo , Modelos Biológicos , Mutación/genética , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Ubiquitinación/efectos de los fármacosRESUMEN
Amajor function of the endocytic system is the sorting of cargo to various organelles. Endocytic sorting of the yeast reductive iron transporter, which is composed of the Fet3 and Ftr1 proteins, is regulated by available iron. When iron is provided to iron-starved cells, Fet3p-Ftr1p is targeted to the lysosome-like vacuole and degraded. In contrast, when iron is not available, Fet3p-Ftr1p is maintained on the plasma membrane via an endocytic recycling pathway requiring the sorting nexin Grd19/Snx3p, the pentameric retromer complex, and the Ypt6p Golgi Rab GTPase module. A recycling signal in Ftr1p was identified and found to bind directly to Grd19/Snx3p. Retromer and Grd19/Snx3p partially colocalize to tubular endosomes, where they are physically associated. After export from the endosome, Fet3p-Ftr1p transits through the Golgi apparatus for resecretion. Thus, Grd19/Snx3p, functions as a cargo-specific adapter for the retromer complex, establishing a precedent for a mechanism by which sorting nexins expand the repertoire of retromer-dependent cargos.
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Proteínas Portadoras/fisiología , Endocitosis , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Membrana Celular/metabolismo , Ceruloplasmina/metabolismo , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes/análisis , Hierro/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/metabolismoRESUMEN
ADP-ribosylation factor (Arf) GTP-binding proteins are among the best-characterized members of the Ras superfamily of GTPases, with well-established functions in membrane-trafficking pathways. A recent watershed of genomic and structural information has identified a family of conserved related proteins: the Arf-like (Arl) GTPases. The best-characterized Arl protein, Arl2, regulates the folding of beta tubulin, and recent data suggest that Arl1 and Arf-related protein 1 (ARFRP1) are localized to the trans-Golgi network (TGN), where they function, in part, to regulate the tethering of endosome-derived transport vesicles. Other Arl proteins are localized to the cytosol, nucleus, cytoskeleton and mitochondria, which indicates that Arl proteins have diverse roles that are distinct from the known functions of traditional Arf GTPases.
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Factores de Ribosilacion-ADP/fisiología , GTP Fosfohidrolasas/fisiología , Proteínas de la Membrana/fisiología , Factores de Ribosilacion-ADP/metabolismo , Animales , GTP Fosfohidrolasas/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologíaRESUMEN
Myristoylation of ARF family GTPases is required for their association with Golgi and endosomal membranes, where they regulate protein sorting and the lipid composition of these organelles. The Golgi-localized ARF-like GTPase Arl3p/ARP lacks a myristoylation signal, indicating that its targeting mechanism is distinct from myristoylated ARFs. We demonstrate that acetylation of the N-terminal methionine of Arl3p requires the NatC N(alpha)-acetyltransferase and that this modification is required for its Golgi localization. Chemical crosslinking and fluorescence microscopy experiments demonstrate that localization of Arl3p also requires Sys1p, a Golgi-localized integral membrane protein, which may serve as a receptor for acetylated Arl3p.