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Fibroblast Growth Factors and their receptors (FGFRs) comprise a cell signaling module that can stimulate signaling by Ras and the kinases Raf, MEK, and ERK to regulate animal development and homeostatic functions. In Caenorhabditis elegans, the sole FGFR ortholog EGL-15 acts with the GRB2 ortholog SEM-5 to promote chemoattraction and migration by the sex myoblasts (SMs) and fluid homeostasis by the hypodermis (Hyp7). Cell-specific differences in EGL-15 signaling were suggested by the phenotypes caused by egl-15(n1457), an allele that removes a region of its C-terminal domain (CTD) known to bind SEM-5. To determine how mutations altered EGL-15 activity in the SMs and Hyp7, we used the kinase reporter ERK-KTR to measure activation of the ERK ortholog MPK-1. Consequences of egl-15(n1457) were cell-specific, resulting in loss of MPK-1 activity in the SMs and elevated activity in Hyp7. Previous studies of Hyp7 showed that loss of the CLR-1 phosphatase causes a fluid homeostasis defect termed "Clear" that is suppressed by reduction of EGL-15 signaling, a phenotype termed "Suppressor of Clear" (Soc). To identify mechanisms that permit EGL-15 signaling in Hyp7, we conducted a genetic screen for Soc mutants in the clr-1; egl-15(n1457) genotype. We report the identification of SOC-3, a protein with putative SEM-5-binding motifs and PH and PTB domains similar to DOK and IRS proteins. In combination with the egl-15(n1457) mutation, loss of either soc-3, the GAB1 ortholog soc-1, or the SHP2 ortholog ptp-2, reduced MPK-1 activation. We generated alleles of soc-3 to test the requirement for the SEM-5-binding motifs, finding that residue Tyr356 is required for function. We propose that EGL-15-mediated SM chemoattraction relies solely on the direct interaction between SEM-5 and the EGL-15 CTD. In Hyp7, EGL-15 signaling uses two mechanisms: the direct SEM-5 binding mechanism; and an alternative, CTD-independent mechanism involving SOC-3, SOC-1, and PTP-2. This work demonstrates that FGF signaling uses distinct, tissue-specific mechanisms in development, and identifies SOC-3 as a potential adaptor that facilitates Ras pathway activation by FGFR.
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Raf protein kinases act as Ras-GTP sensing components of the ERK signal transduction pathway in animal cells, influencing cell proliferation, differentiation, and survival. In humans, somatic and germline mutations in the genes BRAF and RAF1 are associated with malignancies and developmental disorders. Recent studies shed light on the structure of activated Raf, a heterotetramer consisting of Raf and 14-3-3 dimers, and raised the possibility that a Raf C-terminal distal tail segment (DTS) regulates activation. We investigated the role of the DTS using the Caenorhabditis elegans, which has a single Raf ortholog termed lin-45 . We discovered that truncations removing the DTS strongly enhanced lin-45(S312A) , a weak gain-of-function allele equivalent to RAF1 mutations found in patients with Noonan Syndrome. We generated mutations to test three elements of the LIN-45 DTS, which we termed the active site binding sequence (ASBS), the KTP motif, and the aromatic cluster. In the context of lin-45(S312A), mutation of either the ASBS, KTP motif, or aromatic cluster enhanced activity. We used AlphaFold to predict DTS protein interactions for LIN-45, fly Raf, and human BRAF, within the activated heterotetramer complex. We propose distinct functions for the LIN-45 DTS elements: i) the ASBS binds the kinase active site as an inhibitor, ii) phosphorylation of the KTP motif modulates DTS-kinase domain interaction, and iii) the aromatic cluster anchors the DTS in an inhibitory conformation. This work establishes that the Raf/LIN-45 DTS negatively regulates signaling in C. elegans and provides a model for its function in other Raf proteins.
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Numerous factors have been implicated in the cell-cell interactions that lead to elimination of cells via cell competition, a context-dependent process of cell selection in somatic tissues that is based on comparisons of cellular fitness. Here, we use a series of genetic tests in Drosophila to explore the relative contribution of the pleiotropic cytokine tumor necrosis factor α (TNFα) in Myc-mediated cell competition (also known as Myc supercompetition or Myc cell competition). We find that the sole Drosophila TNF, Eiger (Egr), its receptor Grindelwald (Grnd/TNF receptor), and the adaptor proteins Traf4 and Traf6 are required to eliminate wild-type "loser" cells during Myc cell competition. Although typically the interaction between Egr and Grnd leads to cell death by activating the intracellular Jun N-terminal kinase (JNK) stress signaling pathway, our experiments reveal that many components of canonical JNK signaling are dispensable for cell death in Myc cell competition, including the JNKKK Tak1, the JNKK Hemipterous and the JNK Basket. Our results suggest that Egr/Grnd signaling participates in Myc cell competition but functions in a role that is largely independent of the JNK signaling pathway.
Assuntos
Proteínas de Drosophila , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Competição entre as Células/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Transdução de Sinais , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Proteínas de Ligação a DNA , Proteínas de Membrana , Fatores de TranscriçãoRESUMO
FBXW7 is a critical regulator of cell cycle, cell signaling, and development. A highly conserved F-box protein and component of the SKP1-Cullin-F-box (SCF) complex, FBXW7 functions as a recognition subunit within a Cullin-RING E3 ubiquitin ligase responsible for ubiquitinating substrate proteins and targeting them for proteasome-mediated degradation. In human cells, FBXW7 promotes degradation of a large number of substrate proteins, including many that impact disease, such as NOTCH1, Cyclin E, MYC, and BRAF. A central focus for investigation has been to understand the molecular mechanisms that allow the exquisite substrate specificity exhibited by FBXW7. Recent work has produced a clearer understanding of how FBXW7 physically interacts with both high-affinity and low-affinity substrates. We review new findings that provide insights into the consequences of "hotspot" missense mutations of FBXW7 that are found in human cancers. Finally, we discuss how the FBXW7-substrate interaction, and the kinases responsible for substrate phosphorylation, contribute to patterned protein degradation in C. elegans development.
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Caenorhabditis elegans , Proteínas F-Box , Humanos , Animais , Proteína 7 com Repetições F-Box-WD/genética , Proteínas Culina , Proteínas F-Box/genética , Ciclo CelularRESUMO
Signaling by the kinase cascade composed of Raf, MEK, and ERK is critical for animal development and is often inappropriately activated in human malignancies. We sought to identify factors that control signaling mediated by the Caenorhabditis elegans Raf ortholog LIN-45. A genetic screen showed that the degradation of LIN-45 required the E3/E4 ubiquitin ligase UFD-2. Both UFD-2 and its partner, the ATP-dependent segregase CDC-48, were required for the developmental regulation of LIN-45 protein abundance. We showed that UFD-2 acted in the same pathway as the E3 ubiquitin ligase SCFSEL-10 to decrease LIN-45 abundance in cells in which Raf-MEK-ERK signaling was most highly active. UFD-2 also reduced the protein abundance of activated LIN-45 carrying a mutation equivalent to the cancer-associated BRAF(V600E) variant. Our structure-function studies showed that the disruption of LIN-45 domains that mediate protein-protein interactions, including the conserved cysteine-rich domain and 14-3-3 binding motifs, were required for UFD-2-independent degradation of LIN-45. We propose a model in which UFD-2 and CDC-48 act downstream of SCFSEL-10 to remove LIN-45 from its protein interaction partners and facilitate proteasomal targeting and degradation. These findings imply that UFD-2 and CDC-48 may be important for Raf degradation during normal and oncogenic Ras and MAPK signaling in mammalian cells.
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Proteínas de Caenorhabditis elegans , Ubiquitina-Proteína Ligases , Animais , Humanos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular , Mamíferos , Quinases de Proteína Quinase Ativadas por Mitógeno , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , UbiquitinasRESUMO
Activation of a canonical EGFR-Ras-Raf-ERK cascade initiates patterning of multipotent vulval precursor cells (VPCs) of Caenorhabditis elegans We have previously shown that this pathway includes a negative-feedback component in which MPK-1/ERK activity targets the upstream kinase LIN-45/Raf for degradation by the SEL-10/FBXW7 E3 ubiquitin ligase. This regulation requires a Cdc4 phosphodegron (CPD) in LIN-45 that is conserved in BRAF. Here, we identify and characterize the minimal degron that encompasses the CPD and is sufficient for SEL-10-mediated, MPK-1-dependent protein degradation. A targeted screen of conserved protein kinase-encoding genes yielded gsk-3 (an ortholog of human GSK3B) and cdk-2 (a CDK2-related kinase) as required for LIN-45 degron-mediated turnover. Genetic analysis revealed that LIN-45 degradation is blocked at the second larval stage due to cell cycle quiescence, and that relief of this block during the third larval stage relies on activation of CDKs. Additionally, activation of MPK-1 provides spatial pattern to LIN-45 degradation but does not bypass the requirement for gsk-3 and cdk-2 This analysis supports a model whereby MPK-1/ERK, GSK-3/GSK3 and CDK-2/CDK2, along with SEL-10/FBXW7, constitute a regulatory network that exerts spatial and temporal control of LIN-45/Raf degradation during VPC patterning.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Quinase 3 da Glicogênio Sintase/genética , Vulva/crescimento & desenvolvimento , Quinases raf/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Retroalimentação Fisiológica , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Fosfotransferases/genética , Proteólise , Transdução de Sinais/genética , Ubiquitina-Proteína Ligases , Vulva/metabolismoRESUMO
The process of apoptosis in epithelia involves activation of caspases, delamination of cells, and degradation of cellular components. Corpses and cellular debris are then rapidly cleared from the tissue by phagocytic blood cells. In studies of the Drosophila TNF, Eiger (Egr) and cell death in wing imaginal discs, the epithelial primordia of fly wings, we noticed that dying cells appeared to transiently accumulate in egr3 mutant wing discs, raising the possibility that their phagocytic engulfment by hemocytes was impaired. Further investigation revealed that lymph glands and circulating hemocytes from egr3 mutant larvae were completely devoid of NimC1 staining, a marker of phagocytic hemocytes. Genome sequencing uncovered mutations in the NimC1 coding region that are predicted to truncate the NimC1 protein before its transmembrane domain, and provide an explanation for the lack of NimC staining. The work that we report here demonstrates the presence of these NimC1 mutations in the widely used egr3 mutant, its sister allele, egr1 , and its parental strain, Regg1GS9830 As the egr3 and egr1 alleles have been used in numerous studies of immunity and cell death, it may be advisable to re-evaluate their associated phenotypes.
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Proteínas de Drosophila , Drosophila , Animais , Drosophila/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Mutação , Receptores Imunológicos , Fator de Necrose Tumoral alfaRESUMO
Kinase translocation reporters (KTRs) are genetically encoded fluorescent activity sensors that convert kinase activity into a nucleocytoplasmic shuttling equilibrium for visualizing single-cell signaling dynamics. Here, we adapt the first-generation KTR for extracellular signal-regulated kinase (ERK) to allow easy implementation in vivo. This sensor, "ERK-nKTR," allows quantitative and qualitative assessment of ERK activity by analysis of individual nuclei and faithfully reports ERK activity during development and neural function in diverse cell contexts in Caenorhabditis elegans. Analysis of ERK activity over time in the vulval precursor cells, a well-characterized paradigm of epidermal growth factor receptor (EGFR)-Ras-ERK signaling, has identified dynamic features not evident from analysis of developmental endpoints alone, including pulsatile frequency-modulated signaling associated with proximity to the EGF source. The toolkit described here will facilitate studies of ERK signaling in other C. elegans contexts, and the design features will enable implementation of this technology in other multicellular organisms.
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Técnicas Biossensoriais/métodos , Caenorhabditis elegans/citologia , Caenorhabditis elegans/enzimologia , Linhagem da Célula , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Animais , Linhagem Celular , Movimento Celular , Núcleo Celular/metabolismo , Feminino , Genes Reporter , Células Germinativas/citologia , Mamíferos , Mutação/genética , Mioblastos/citologia , Neurônios/citologia , Fosforilação , Reprodutibilidade dos Testes , Células-Tronco/citologia , Células-Tronco/metabolismo , Frações Subcelulares/metabolismo , Vulva/citologiaRESUMO
In growing tissues, cell fitness disparities can provoke interactions that promote stronger cells at the expense of the weaker in a process called cell competition. The mechanistic definition of cell fitness is not understood, nor is it understood how fitness differences are recognized. Drosophila cells with extra Myc activity acquire "supercompetitor" status upon confrontation with wild-type (WT) cells, prompting the latter's elimination via apoptosis. Here we show that such confrontation enhances glycolytic flux in Myc cells and promotes their fitness and proliferation in a p53-dependent manner. Whereas p53 loss in noncompeting Myc cells is inconsequential, its loss impairs metabolism, reduces viability, and prevents the killing activity of Myc supercompetitor cells. We propose that p53 acts as a general sensor of competitive confrontation to enhance the fitness of the "winner" population. Our findings suggest that the initial confrontation between precancerous and WT cells could enhance cancer cell fitness and promote tumor progression.
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Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Drosophila/citologia , Proteínas de Drosophila/genética , Instabilidade Genômica , Glicólise , Fosforilação Oxidativa , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
The conserved E3 ubiquitin ligase component named SEL-10 in Caenorhabditis elegans and Fbw7 in mammals targets substrates for ubiquitin-mediated degradation through a high-affinity binding site called a Cdc4 phosphodegron (CPD). As many known substrates of Fbw7 are oncoproteins, the identification of new substrates may offer insight into cancer biology as well as aspects of proteome regulation. Here, we evaluated whether the presence of an evolutionarily conserved CPD would be a feasible complement to proteomics-based approaches for identifying new potential substrates. For functional assessments, we focused on LIN-45, a component of the signal transduction pathway underlying vulval induction and the ortholog of human Braf, an effector of Ras in numerous cancers. Our analysis demonstrates that LIN-45 behaves as a bona fide substrate of SEL-10, with mutation of the CPD or loss of sel-10 resulting in increased activity and protein stability in vivo. Furthermore, during vulval induction, the downstream kinase MPK-1/ERK is also required for LIN-45 protein degradation in a negative feedback loop, resulting in degradation of LIN-45 where ERK is highly active. As the CPD consensus sequence is conserved in human Braf, we propose that Fbw7 may also regulate Braf stability in some cell contexts. We discuss the implications of our findings for vulval development in C. elegans, the potential applicability to human Braf, and the value of a CPD-based predictive approach for human Fbw7 substrates.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Ciclo Celular/metabolismo , Retroalimentação Fisiológica , Proteínas Proto-Oncogênicas B-raf/metabolismo , Transdução de Sinais/genética , Quinases raf/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Regulação para Baixo , Humanos , Ligação Proteica , Proteínas Proto-Oncogênicas B-raf/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Vertebrados/genética , Quinases raf/genéticaRESUMO
The Myc transcription factor regulates fundamental processes in a cell's life: its growth, division, and survival. Myc is conserved throughout metazoan phyla, and its identification in the fruit fly, Drosophila melanogaster has led to new insights in Myc's physiological roles. In this review, we describe recent research on the biology of Myc and its family members in Drosophila, paying particular attention to its role in the control of growth during development.
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Animais Geneticamente Modificados , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Transdução de Sinais , Animais , Regulação da Expressão Gênica no Desenvolvimento , TransativadoresRESUMO
Experiments in both vertebrates and invertebrates have illustrated the competitive nature of growth and led to the idea that competition is a mechanism of regulating organ and tissue size. We have assessed competitive interactions between cells in a developing organ and examined their effect on its final size. We show that local expression of the Drosophila growth regulator dMyc, a homolog of the c-myc protooncogene, induces cell competition and leads to the death of nearby wild-type cells in developing wings. We demonstrate that cell competition is executed via induction of the proapoptotic gene hid and that both competition and hid function are required for the wing to reach an appropriate size when dMyc is expressed. Moreover, we provide evidence that reproducible wing size during normal development requires apoptosis. Modulating dmyc levels to create cell competition and hid-dependent cell death may be a mechanism used during normal development to control organ size.