RESUMO
The primary role of Notch is to specify cellular identities, whereby the cells respond to amazingly small changes in Notch signalling activity. Hence, dosage of Notch components is crucial to regulation. Central to Notch signal transduction are CSL proteins: together with respective cofactors, they mediate the activation or the silencing of Notch target genes. CSL proteins are extremely similar amongst species regarding sequence and structure. We noticed that the fly homologue suppressor of hairless (Su(H)) is stabilised in transcription complexes. Using specific transgenic fly lines and HeLa RBPJKO cells we provide evidence that Su(H) is subjected to proteasomal degradation with a half-life of about two hours if not protected by binding to co-repressor hairless or co-activator Notch. Moreover, Su(H) stability is controlled by MAPK-dependent phosphorylation, matching earlier data for RBPJ in human cells. The homologous murine and human RBPJ proteins, however, are largely resistant to degradation in our system. Mutating presumptive protein contact sites, however, sensitised RBPJ for proteolysis. Overall, our data highlight the similarities in the regulation of CSL protein stability across species and imply that turnover of CSL proteins may be a conserved means of regulating Notch signalling output directly at the level of transcription.
Assuntos
Proteínas de Drosophila , Humanos , Animais , Camundongos , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas Correpressoras/metabolismo , Receptores Notch/metabolismo , Fosforilação , Proteínas Repressoras/metabolismo , Ligação ProteicaRESUMO
Cell fate choices during metazoan development are driven by the highly conserved Notch signalling pathway. Notch receptor activation results in release of the Notch intracellular domain (NICD) that acts as transcriptional co-activator of the DNA-binding protein CSL. In the absence of signal, a repressor complex consisting of CSL bound to co-repressors silences Notch target genes. The Drosophila repressor complex contains the fly CSL orthologue Suppressor of Hairless [Su(H)] and Hairless (H). The Su(H)-H crystal structure revealed a large conformational change within Su(H) upon H binding, precluding interactions with NICD. Based on the structure, several sites in Su(H) and H were determined to specifically engage in complex formation. In particular, three mutations in Su(H) were identified that affect interactions with the repressor H but not the activator NICD. To analyse the effects these mutants have on normal fly development, we introduced these mutations into the native Su(H) locus by genome engineering. We show that the three H-binding deficient Su(H) alleles behave similarly. As these mutants lack the ability to form the repressor complex, Notch signalling activity is strongly increased in homozygotes, comparable to a complete loss of H activity. Unexpectedly, we find that the abundance of the three mutant Su(H) protein variants is altered, as is that of wild type Su(H) protein in the absence of H protein. In the presence of NICD, however, Su(H) mutant protein persists. Apparently, Su(H) protein levels depend on the interactions with H as well as with NICD. Based on these results, we propose that in vivo levels of Su(H) protein are stabilised by interactions with transcription-regulator complexes.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/genética , Mutação , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Alelos , Animais , Sítios de Ligação , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Ligação Proteica , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
During development of higher animals, the Notch signalling pathway governs cell type specification by mediating appropriate gene expression responses. In the absence of signalling, Notch target genes are silenced by repressor complexes. In the model organism Drosophila melanogaster, the repressor complex includes the transcription factor Suppressor of Hairless [Su(H)] and Hairless (H) plus general co-repressors. Recent crystal structure analysis of the Drosophila Notch repressor revealed details of the Su(H)-H complex. They were confirmed by mutational analyses of either protein; however, only Su(H) mutants have been further studied in vivo. Here, we analyse three H variants predicted to affect Su(H) binding. To this end, amino acid replacements Phenylalanine 237, Leucines 245 and 247, as well as Tryptophan 258 to Alanine were introduced into the H protein. A cell-based reporter assay indicates substantial loss of Su(H) binding to the respective mutant proteins HFA, HLLAA and HWA. For in vivo analysis, UAS-lines HFA, HLLAA and HWA were generated to allow spatially restricted overexpression. In these assays, all three mutants resembled the HLD control, shown before to lack Su(H) binding, indicating a strong reduction of H activity. For example, the H variants were impaired in wing margin formation, but unexpectedly induced ectopic wing venation. Concurrent overexpression with Su(H), however, suggests that all mutant H protein isoforms are still able to bind Su(H) in vivo. We conclude that a weakening of the cohesion in the H-Su(H) repressor complex is sufficient for disrupting its in vivo functionality.
Assuntos
Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Mutação , Receptores Notch/metabolismo , Fatores de Transcrição/genética , Animais , Olho Composto de Artrópodes/crescimento & desenvolvimento , Olho Composto de Artrópodes/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Ligação Proteica , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismoRESUMO
Notch is a conserved signaling pathway that specifies cell fates in metazoans. Receptor-ligand interactions induce changes in gene expression, which is regulated by the transcription factor CBF1/Su(H)/Lag-1 (CSL). CSL interacts with coregulators to repress and activate transcription from Notch target genes. While the molecular details of the activator complex are relatively well understood, the structure-function of CSL-mediated repressor complexes is poorly defined. In Drosophila, the antagonist Hairless directly binds Su(H) (the fly CSL ortholog) to repress transcription from Notch targets. Here, we determine the X-ray structure of the Su(H)-Hairless complex bound to DNA. Hairless binding produces a large conformational change in Su(H) by interacting with residues in the hydrophobic core of Su(H), illustrating the structural plasticity of CSL molecules to interact with different binding partners. Based on the structure, we designed mutants in Hairless and Su(H) that affect binding, but do not affect formation of the activator complex. These mutants were validated in vitro by isothermal titration calorimetry and yeast two- and three-hybrid assays. Moreover, these mutants allowed us to solely characterize the repressor function of Su(H) in vivo.
Assuntos
Proteínas de Drosophila/química , Drosophila melanogaster , Proteínas Repressoras/química , Fatores de Transcrição/química , Animais , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , DNA/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Termodinâmica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: In Drosophila, the development of the fly eye involves the activity of several, interconnected pathways that first define the presumptive eye field within the eye anlagen, followed by establishment of the dorso-ventral boundary, and the regulation of growth and apoptosis. In Lobe (L) mutant flies, parts of the eye or even the complete eye are absent because the eye field has not been properly defined. Manifold genetic interactions indicate that L influences the activity of several signalling pathways, resulting in a conversion of eye tissue into epidermis, and in the induction of apoptosis. As information on the molecular nature of the L mutation is lacking, the underlying molecular mechanisms are still an enigma. RESULTS: We have identified Protein Kinase D (PKD) as a strong modifier of the L mutant phenotype. PKD belongs to the PKC/CAMK class of Ser/Thr kinases that have been involved in diverse cellular processes including stress resistance and growth. Despite the many roles of PKD, Drosophila PKD null mutants are without apparent phenotype apart from sensitivity to oxidative stress. Here we report an involvement of PKD in eye development in the sensitized genetic background of Lobe. Absence of PKD strongly enhanced the dominant eye defects of heterozygous L 2 flies, and decreased their viability. Moreover, eye-specific overexpression of an activated isoform of PKD considerably ameliorated the dominant L 2 phenotype. This genetic interaction was not allele specific but similarly seen with three additional, weaker L alleles (L 1 , L 5 , L G ), demonstrating its specificity. CONCLUSIONS: We propose that PKD-mediated phosphorylation is involved in underlying processes causing the L phenotype, i.e. in the regulation of growth, the epidermal transformation of eye tissue and apoptosis, respectively.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Epistasia Genética , Proteínas do Olho/genética , Olho/embriologia , Olho/metabolismo , Mutação , Proteína Quinase C/genética , Animais , Olho/ultraestrutura , Estudos de Associação Genética , Organogênese/genética , FenótipoRESUMO
BACKGROUND: DNA damage generally results in the activation of ATM/ATR kinases and the downstream checkpoint kinases Chk1/Chk2. In Drosophila melanogaster, the ATR homologue meiotic 41 (mei-41) is pivotal to DNA damage repair and cell cycle checkpoint signalling. Although various mei-41 mutant alleles have been analyzed in the past, no gain-of-function allele is yet available. To fill this gap, we have generated transgenic flies allowing temporal and tissue-specific induction of mei-41. RESULTS: Overexpression of mei-41 in wing and eye anlagen affects proliferation and a G2/M checkpoint even in the absence of genomic stress. Similar consequences were observed following the overexpression of the downstream kinase Grapes (Grp) but not of Loki (Lok), encoding the respective Drosophila Chk1 and Chk2 homologues, in agreement with their previously reported activities. Moreover, we show that irradiation induced cell cycle arrest was prolonged in the presence of ectopic mei-41 expression. Similar to irradiation stress, mei-41 triggered the occurrence of a slower migrating form of Grp, implying specific phosphorylation of Grp in response to either signal. Using a p53R-GFP biosensor, we further show that overexpression of mei-41 was sufficient to elicit a robust p53 activation in vivo. CONCLUSION: We conclude that overexpression of the Drosophila ATR homologue mei-41 elicits an effectual DNA damage response irrespective of irradiation.
Assuntos
Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/genética , Dano ao DNA , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Animais Geneticamente Modificados , Divisão Celular , Drosophila melanogaster/efeitos da radiação , Fase G2RESUMO
In multicellular organisms, growth and proliferation is adjusted to nutritional conditions by a complex signaling network. The Insulin receptor/target of rapamycin (InR/TOR) signaling cascade plays a pivotal role in nutrient dependent growth regulation in Drosophila and mammals alike. Here we identify Cyclin G (CycG) as a regulator of growth and metabolism in Drosophila. CycG mutants have a reduced body size and weight and show signs of starvation accompanied by a disturbed fat metabolism. InR/TOR signaling activity is impaired in cycG mutants, combined with a reduced phosphorylation status of the kinase Akt1 and the downstream factors S6-kinase and eukaryotic translation initiation factor 4E binding protein (4E-BP). Moreover, the expression and accumulation of Drosophila insulin like peptides (dILPs) is disturbed in cycG mutant brains. Using a reporter assay, we show that the activity of one of the first effectors of InR signaling, Phosphoinositide 3-kinase (PI3K92E), is unaffected in cycG mutants. However, the metabolic defects and weight loss in cycG mutants were rescued by overexpression of Akt1 specifically in the fat body and by mutants in widerborst (wdb), the B'-subunit of the phosphatase PP2A, known to downregulate Akt1 by dephosphorylation. Together, our data suggest that CycG acts at the level of Akt1 to regulate growth and metabolism via PP2A in Drosophila.
Assuntos
Ciclina G/fisiologia , Drosophila melanogaster/metabolismo , Animais , Peso Corporal , Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Corpo Adiposo/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Metabolismo dos Lipídeos , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
The Notch signaling pathway is highly conserved in all animal metazoa: upon Notch receptor activation, transcription of Notch target genes is turned on by an activator complex that centers on the transcription factor CSL. In the absence of signal, CSL assembles transcriptional repression complexes that display remarkable evolutionary diversity. The major antagonist of Notch signaling in insects named Hairless was originally identified in Drosophila melanogaster. It binds to the Drosophila CSL homologue Suppressor of Hairless [Su(H)] and recruits the two general co-repressors, Groucho and C-terminal binding protein. Whereas the majority of Notch signaling components is conserved between insects and vertebrates, Hairless is found only in insects. Here, we present the analysis of the Hairless gene from Daphnia pulex and, hence, for the first time from a crustacean. Daphnia and Drosophila Hairless protein sequences are highly diverged. Known functional domains, however, the Su(H), Groucho and the C-terminal binding protein interactions domains, are well conserved. Moreover, direct binding of the Daphnia Hairless protein and the respective Drosophila interaction partners was detected, demonstrating the conservation at the molecular level. In addition, interaction between Daphnia Hairless and Drosophila Su(H) was demonstrated in vivo, as co-overexpression of the respective genes during Drosophila development resulted in the expected downregulation of Notch activity in the fly. Structural models show that the Hairless-Su(H) repressor complexes from Daphnia and Drosophila are almost indistinguishable from one another. Amino acid residues in direct contact within the Hairless-Su(H) complex are at absolutely identical positions in the two homologues.
Assuntos
Proteínas de Artrópodes/metabolismo , Daphnia/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Daphnia/genética , Daphnia/crescimento & desenvolvimento , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Notch/antagonistas & inibidores , Proteínas Repressoras/química , Proteínas Repressoras/genética , Homologia de Sequência , Transdução de Sinais , Homologia Estrutural de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Cyclin G (CycG) belongs to the atypical cyclins, which have diverse cellular functions. The two mammalian CycG genes, CycG1 and CycG2, regulate the cell cycle in response to cell stress. Detailed analyses of the role of the single Drosophila cycG gene have been hampered by the lack of a mutant. We generated a null mutant in the Drosophila cycG gene that is female sterile and produces ventralised eggs. This phenotype is typical of the downregulation of epidermal growth factor receptor (EGFR) signalling during oogenesis. Ventralised eggs are also observed in mutants (for example, mutants of the spindle class) that are defective in meiotic DNA double-strand break repair. Double-strand breaks (DSBs) induce a meiotic checkpoint by activating Mei-41 kinase (the Drosophila ATR homologue), thereby indirectly causing dorsoventral patterning defects. We provide evidence for the role of CycG in meiotic checkpoint control. The increased incidence of DSBs in cycG mutant germaria may reflect inefficient DSB repair. Therefore, the downregulation of Mei-W68 (an endonuclease that induces meiotic DSBs), Mei-41, or Drosophila melanogaster Chk2 (a downstream kinase that initiates the meiotic checkpoint) rescues the cycG mutant eggshell phenotype. In vivo, CycG associates with Rad9 and BRCA2. These two proteins are components of the 9-1-1 complex, which is involved in sensing DSBs and in activating meiotic checkpoint control. Therefore, we propose that CycG has a role in an early step of meiotic recombination repair, thereby affecting EGFR-mediated patterning processes during oogenesis.
Assuntos
Ciclina G/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Meiose/genética , Reparo de DNA por Recombinação/genética , Animais , Padronização Corporal/genética , Ciclina G/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Imunoprecipitação , Masculino , Proteínas Mutantes/metabolismo , Mutação/genética , Oócitos/citologia , Oócitos/metabolismo , Ovário/citologia , Ovário/metabolismo , Oviposição/fisiologia , Óvulo/metabolismo , Ligação Proteica/genética , Reprodutibilidade dos TestesRESUMO
The Enhancer of split complex [E(spl)-C] comprises twelve genes of different classes. Seven genes encode proteins of with a basic-helix-loop-helix-orange (bHLH-O) domain that function as transcriptional repressors and serve as effectors of the Notch signalling pathway. They have been named E(spl)m8-, m7-, m5-, m3-, mß-, mγ- and mδ-HLH. Four genes, E(spl)m6-, m4-, m2- and mα-BFM are intermingled and encode Notch repressor proteins of the Bearded-family (BFM). The complex is split by a single gene of unrelated function, encoding a Kazal-type protease inhibitor (Kaz-m1). All members within a family, bHLH-O or BFM, are very similar in structure and in function. In an attempt to generate specific mutants, we have mobilised P-element constructs residing next to E(spl)m7-HLH and E(spl)mγ-HLH, respectively. The resulting deletions were mapped molecularly and by cytology. Two small deletions affected only E(spl)m7-HLH and E(spl)mδ. The deficient flies were viable without apparent phenotype. Larger deletions, generated also by X-ray mutagenesis, uncover most of the E(spl)-C. The phenotypes of homozygous deficient embryos were analysed to characterize the respective loss of Notch signalling activity.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Mapeamento Cromossômico , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Repressoras/genética , Deleção de Sequência , Animais , Genes de Insetos , Teste de Complementação Genética , Família Multigênica , Mutagênese , Fenótipo , Regiões Promotoras Genéticas , Receptores Notch/genética , Transdução de SinaisRESUMO
Cellular differentiation during eumetazoan development is based on highly conserved signalling pathways. Two of them, the Notch and the EGFR signalling pathways, are closely intertwined. We have identified two potential target sites of the Mitogen activated kinase (MAPK), the downstream effector kinase of EGFR, within Hairless (H), the major antagonist of Notch signalling in Drosophila. Assuming that phosphorylation of these sites modulates H activity, a direct influence of EGFR signalling on Notch pathway regulation might be possible. This hypothesis was tested by generating a phospho-deficient and a phospho-mimetic H isoform and by assaying for their biological activity. We first addressed the binding of known H interaction partners Su(H), Gro, CtBP and Pros26.4 which was similar between mutant and wild type H. Next we assayed eye, wing and bristle development which are strongly affected by the overexpression of H due to the inhibition of Notch signalling. Overexpression of the mutant constructs resulted in phenotypes similar to wildtype H overexpression, yet with subtle differences in phenotypic severity. However, large variations suggest that the mutated residues may be critical for the overall structure or stability of H. Albeit of minor impact, EGFR may fine tune Notch signalling via MAPK dependent phosphorylation of H.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Sistema de Sinalização das MAP Quinases , Fatores de Transcrição/genética , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Olho/crescimento & desenvolvimento , Fenótipo , Fosforilação , Mutação Puntual , Isoformas de Proteínas/genética , Asas de Animais/crescimento & desenvolvimentoRESUMO
Blood cells in Drosophila serve primarily innate immune responses. Various stressors influence blood cell homeostasis regarding both numbers and the proportion of blood cell types. The principle molecular mechanisms governing hematopoiesis are conserved amongst species and involve major signaling pathways like Notch, Toll, JNK, JAK/Stat or RTK. Albeit signaling pathways generally rely on the activity of protein kinases, their specific contribution to hematopoiesis remains understudied. Here, we assess the role of Serine/Threonine kinases with the potential to phosphorylate the transcription factor Su(H) in crystal cell homeostasis. Su(H) is central to Notch signal transduction, and its inhibition by phosphorylation impedes crystal cell formation. Overall, nearly twenty percent of all Drosophila Serine/Threonine kinases were studied in two assays, global and hemocyte-specific overexpression and downregulation, respectively. Unexpectedly, the majority of kinases influenced crystal cell numbers, albeit only a few were related to hematopoiesis so far. Four kinases appeared essential for crystal cell formation, whereas most kinases restrained crystal cell development. This group comprises all kinase classes, indicative of the complex regulatory network underlying blood cell homeostasis. The rather indiscriminative response we observed opens the possibility that blood cells measure their overall phospho-status as a proxy for stress-signals, and activate an adaptive immune response accordingly.
Assuntos
Proteínas de Drosophila , Proteínas Serina-Treonina Quinases , Animais , Proteínas Serina-Treonina Quinases/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Células Sanguíneas/metabolismo , Homeostase , Serina/metabolismo , Treonina/metabolismoRESUMO
Hairless (H) encodes the major antagonist in the Notch signaling pathway, which governs cellular differentiation of various tissues in Drosophila. By binding to the Notch signal transducer Suppressor of Hairless (Su(H)), H assembles repressor complexes onto Notch target genes. Using genome engineering, three new H alleles, HFA, HLLAA and HWA were generated and a phenotypic series was established by several parameters, reflecting the residual H-Su(H) binding capacity. Occasionally, homozygous HWA flies develop to adulthood. They were compared with the likewise semi-viable HNN allele affecting H-Su(H) nuclear entry. The H homozygotes were short-lived, sterile and flightless, yet showed largely normal expression of several mitochondrial genes. Typical for H mutants, both HWA and HNN homozygous alleles displayed strong defects in wing venation and mechano-sensory bristle development. Strikingly, however, HWA displayed only a loss of bristles, whereas bristle organs of HNN flies showed a complete shaft-to-socket transformation. Apparently, the impact of HWA is restricted to lateral inhibition, whereas that of HNN also affects the respective cell type specification. Notably, reduction in Su(H) gene dosage only suppressed the HNN bristle phenotype, but amplified that of HWA. We interpret these differences as to the role of H regarding Su(H) stability and availability.
Assuntos
Alelos , Proteínas de Drosophila , Drosophila melanogaster , Asas de Animais , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais/genéticaRESUMO
bHLH-O proteins are a subfamily of the basic-helix-loop-helix transcription factors characterized by an 'Orange' protein-protein interaction domain. Typical members are the Hairy/E(spl), or Hes, proteins, well studied in their ability, among others, to suppress neuronal differentiation in both invertebrates and vertebrates. Hes proteins are often effectors of Notch signalling. In vertebrates, another bHLH-O protein group, the Hey proteins, have also been shown to be Notch targets and to interact with Hes. We have studied the single Drosophila Hey orthologue. We show that it is primarily expressed in a subset of newly born neurons, which receive Notch signalling during their birth. Unlike in vertebrates, however, Hey is not expressed in precursor cells and does not block neuronal differentiation. It rather promotes one of two alternative fates that sibling neurons adopt at birth. Although in the majority of cases Hey is a Notch target, it is also expressed independently of Notch in some lineages, most notably the larval mushroom body. The availability of Hey as a Notch readout has allowed us to study Notch signalling during the genesis of secondary neurons in the larval central nervous system.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Neurogênese/fisiologia , Neurônios/metabolismo , Receptores Notch/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Drosophila/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Hibridização In Situ , Larva/citologia , Larva/crescimento & desenvolvimento , Larva/metabolismo , Neurogênese/genética , Neuroglia/metabolismo , Neurônios/citologiaRESUMO
By using mass spectrometry, we have identified Ser 402 as a new phosphorylation site within the catalytic domain of human slingshot 1 (SSH1). Phosphorylation at this site inhibits substrate binding and, thus, phosphatase activity in vitro, resulting in enrichment of phosphorylated cofilin in monolayer cell culture. We further demonstrate that protein kinase D (PKD) is upstream from Ser 402 phosphorylation. Accordingly, expression of active PKD in Drosophila phenotypically mimics the loss of SSH activity by inducing accumulation of phosphorylated cofilin and filamentous actin. We thus identify a universal mechanism by which PKD controls SSH1 phosphatase activity.
Assuntos
Fosfoproteínas Fosfatases/metabolismo , Serina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Ativação Enzimática/genética , Células HEK293 , Células HeLa , Humanos , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/genética , Fosforilação , Ligação Proteica , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
Cellular differentiation relies on the highly conserved Notch signaling pathway. Notch activity induces gene expression changes that are highly sensitive to chromatin landscape. We address Notch gene regulation using Drosophila as a model, focusing on the genetic and molecular interactions between the Notch antagonist Hairless and the histone chaperone Asf1. Earlier work implied that Asf1 promotes the silencing of Notch target genes via Hairless (H). Here, we generate a novel HΔCT allele by genome engineering. Phenotypically, HΔCT behaves as a Hairless gain of function allele in several developmental contexts, indicating that the conserved CT domain of H has an attenuator role under native biological contexts. Using several independent methods to assay protein-protein interactions, we define the sequences of the CT domain that are involved in Hairless-Asf1 binding. Based on previous models, where Asf1 promotes Notch repression via Hairless, a loss of Asf1 binding should reduce Hairless repressive activity. However, tissue-specific Asf1 overexpression phenotypes are increased, not rescued, in the HΔCT background. Counterintuitively, Hairless protein binding mitigates the repressive activity of Asf1 in the context of eye development. These findings highlight the complex connections of Notch repressors and chromatin modulators during Notch target-gene regulation and open the avenue for further investigations.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Proteínas Repressoras/genética , Proteínas de Drosophila/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Alelos , Receptores Notch/genética , Receptores Notch/metabolismo , Drosophila/genética , Cromatina/metabolismoRESUMO
In general, cyclins control the cell cycle. Not so the atypical cyclins, which are required for diverse cellular functions such as for genome stability or for the regulation of transcription and translation. The atypical Cyclin G (CycG) gene of Drosophila has been involved in the epigenetic regulation of abdominal segmentation, cell proliferation and growth, based on overexpression and RNAi studies, but detailed analyses were hampered by the lack of a cycG mutant. For further investigations, we subjected the cycG locus to a detailed molecular analysis. Moreover, we studied a cycG null mutant that we recently established. The mutant flies are homozygous viable, however, the mutant females are sterile and produce ventralized eggs. Here we show that this egg phenotype is primarily a consequence of a defective Epidermal Growth Factor Receptor (EGFR) signalling pathway. By using different read outs, we demonstrate that cycG loss is tantamount to lowered EGFR signalling. Inferred from epistasis experiments, we conclude that CycG promotes the Grk signal in the oocyte. Abnormal accumulation but regular secretion of the Grk protein suggests defects of Grk translation in cycG mutants rather than transcriptional regulation. Accordingly, protein accumulation of Vasa, which acts as an oocyte specific translational regulator of Grk in the oocyte is abnormal. We propose a role of cycG in processes that regulate translation of Grk and hence, influence EGFR-mediated patterning processes during oogenesis.
Assuntos
Padronização Corporal , Ciclina G/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Oócitos/crescimento & desenvolvimento , Animais , Cromossomos de Insetos/genética , Cromossomos de Insetos/metabolismo , Ciclina G/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Loci Gênicos , Mutação , Oócitos/citologia , Oócitos/metabolismo , Oogênese , Fenótipo , Biossíntese de Proteínas , Receptores de Peptídeos de Invertebrados/genética , Receptores de Peptídeos de Invertebrados/metabolismo , Transdução de Sinais , Transcrição Gênica , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismoRESUMO
Notch signaling is fundamental to the regulation of cellular differentiation, cell growth and cell death in mammals as well as in invertebrates like Drosophila. Upon activation, the Notch receptor is cleaved and the intracellular part ICN assembles an activator complex around Suppressor of Hairless [Su(H)] that activates Notch target genes. Hairless (H) is the major antagonist of the Notch signaling pathway in Drosophila. In the absence of Notch signal, H binds to Su(H) and recruits two general co-repressors, Groucho (Gro) and C-terminal Binding Protein (CtBP); this repression complex downregulates Notch target genes. Previously we have shown that Gro and CtBP are recruited simultaneously to H and that they act in concert during wing and embryonic development. However, Gro and CtBP are utilized context-dependently by other transcription factors. Hence differential co-repressor recruitment by the Su(H)-H repressor complex is likewise conceivable. Here, we investigated the requirement for the co-repressors Gro and CtBP in H mediated Notch repression during several phases of eye development. Whereas both co-repressors appear likewise important during the specification of photoreceptor cells, we find differential requirement for the regulation of proliferation and cell death, respectively. During the early proliferative phase, H preferentially recruits Gro to inhibit Notch mediated growth of the eye disc. Elimination of superfluous interommatidial pigment cells, which depends on a late Notch signal, is antagonized by H and predominantly CtBP. In summary, differential recruitment of the co-repressors Gro and CtBP by H in a context-dependent manner ensures fine tuning of Notch signaling activity during eye development.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Olho/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Olho/crescimento & desenvolvimento , Olho/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Varredura , Ligação Proteica , Receptores Notch/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The Notch signaling pathway is pivotal to cellular differentiation. Activation of this pathway involves proteolysis of the Notch receptor and the release of the biologically active Notch intracellular domain, acting as a transcriptional co-activator of Notch target genes. While the regulation of Notch signaling dynamics at the level of ligand-receptor interaction, endocytosis, and transcriptional regulation has been well studied, little is known about factors influencing Notch cleavage. We identified EP555 as a suppressor of the Notch antagonist Hairless (H). EP555 drives expression of CG32521 encoding membrane-bound proteins, which we accordingly rename membrane-bound Notch regulator (mnr). Within the signal-receiving cell, upregulation of Mnr stimulates Notch receptor activation, whereas a knockdown reduces it, without apparent influence on ligand-receptor interaction. We provide evidence that Mnr plays a role in γ-secretase-mediated intramembrane cleavage of the Notch receptor. As revealed by a fly-eye-based reporter system, γ-secretase activity is stimulated by the overexpression of Mnr, and is inhibited by its knockdown. We conclude that Mnr proteins support Notch signaling activity by fostering the cleavage of the Notch receptor. With Mnr, we identified a membrane-bound factor directly augmenting Notch intra-membrane processing, thereby acting as a positive regulator of Notch signaling activity.
Assuntos
Drosophila melanogaster , Receptores Notch , Secretases da Proteína Precursora do Amiloide , Animais , Transdução de SinaisRESUMO
The mammalian protein kinase D family is involved in manifold cellular processes including cell migration and motility. Recently it was shown that human PKD1 and PKD2 phosphorylate and thereby inhibit Slingshot 1 Like (SSH1L), a phosphatase which is central to the regulation of actin cytoskeletal dynamics. We noted before that the overexpression of a constitutively active form of Drosophila PKD (PKD-SE) affects the fly retina and the resultant phenotypes suggest underlying defects in the actin cytoskeleton. Drosophila Slingshot, however, does not possess the phosphorylation site known to be targeted in SSH1L by human PKD1. Here we show that Drosophila PKD, despite this lack of conservation, nevertheless negatively regulates Slingshot. Overexpression of the active PKD-SE protein causes cellular defects that are similar to those of slingshot mutants. These include aberrant bristle morphology and positioning of photoreceptor nuclei. Interestingly, the observed nuclear mispositioning is due to a disturbance of the cytoskeleton rather than the epithelial organization. In accordance, overexpression of PKD-SE results in an accumulation of filamentous actin. This enrichment is modified by changes in slingshot gene doses, in line with an antagonistic relationship between PKD and slingshot. We conclude that similar to mammals, Drosophila PKD is a negative regulator of Ssh, with the premise of a different target phosphorylation site in Ssh.