RESUMO
Extracellular vesicles (EVs) transfer complex biologic material between cells. However, the role of this process in vivo is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow (BM) niche elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells in vivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. The extracellular vesicles contained processed tRNAs (tiRNAs) known to modulate protein translation. 5'-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and, when transferred to granulocyte-monocyte progenitors, increased protein translation, cell proliferation, and myeloid differentiation. Upregulating EV transfer improved hematopoietic recovery from genotoxic injury and survival from fungal sepsis. Therefore, EV-mediated tiRNA transfer provides a stress-modulated signaling axis in the BM niche distinct from conventional cytokine-driven stress responses.
Assuntos
Vesículas Extracelulares , Células-Tronco Hematopoéticas , Medula Óssea , Células da Medula Óssea , HematopoeseRESUMO
Post-transcriptional mechanisms have the potential to influence complex changes in gene expression, yet their role in cell fate transitions remains largely unexplored. Here, we show that suppression of the RNA helicase DDX6 endows human and mouse primed embryonic stem cells (ESCs) with a differentiation-resistant, "hyper-pluripotent" state, which readily reprograms to a naive state resembling the preimplantation embryo. We further demonstrate that DDX6 plays a key role in adult progenitors where it controls the balance between self-renewal and differentiation in a context-dependent manner. Mechanistically, DDX6 mediates the translational suppression of target mRNAs in P-bodies. Upon loss of DDX6 activity, P-bodies dissolve and release mRNAs encoding fate-instructive transcription and chromatin factors that re-enter the ribosome pool. Increased translation of these targets impacts cell fate by rewiring the enhancer, heterochromatin, and DNA methylation landscapes of undifferentiated cell types. Collectively, our data establish a link between P-body homeostasis, chromatin organization, and stem cell potency.
Assuntos
Diferenciação Celular/genética , Plasticidade Celular/genética , RNA Helicases DEAD-box/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Linhagem Celular , Montagem e Desmontagem da Cromatina/genética , RNA Helicases DEAD-box/genética , Metilação de DNA , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/genética , Ontologia Genética , Homeostase/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/enzimologia , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Homeobox Nanog/metabolismo , Organoides/citologia , Organoides/diagnóstico por imagem , Organoides/metabolismo , Biossíntese de Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/metabolismo , RNA-Seq , Ribonucleoproteínas/genética , Ribossomos/metabolismoRESUMO
Development and differentiation are associated with profound changes to histone modifications, yet their in vivo function remains incompletely understood. Here, we generated mouse models expressing inducible histone H3 lysine-to-methionine (K-to-M) mutants, which globally inhibit methylation at specific sites. Mice expressing H3K36M developed severe anaemia with arrested erythropoiesis, a marked haematopoietic stem cell defect, and rapid lethality. By contrast, mice expressing H3K9M survived up to a year and showed expansion of multipotent progenitors, aberrant lymphopoiesis and thrombocytosis. Additionally, some H3K9M mice succumbed to aggressive T cell leukaemia/lymphoma, while H3K36M mice exhibited differentiation defects in testis and intestine. Mechanistically, induction of either mutant reduced corresponding histone trimethylation patterns genome-wide and altered chromatin accessibility as well as gene expression landscapes. Strikingly, discontinuation of transgene expression largely restored differentiation programmes. Our work shows that individual chromatin modifications are required at several specific stages of differentiation and introduces powerful tools to interrogate their roles in vivo.
Assuntos
Epigênese Genética , Histonas/metabolismo , Leucemia de Células T/genética , Lisina/metabolismo , Metionina/metabolismo , Teratoma/genética , Animais , Transplante de Medula Óssea , Linhagem da Célula/genética , Modelos Animais de Doenças , Doxiciclina/farmacologia , Células Eritroides/metabolismo , Células Eritroides/patologia , Feminino , Granulócitos/metabolismo , Granulócitos/patologia , Histonas/genética , Leucemia de Células T/induzido quimicamente , Leucemia de Células T/metabolismo , Leucemia de Células T/patologia , Masculino , Metilação , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/patologia , Mutação , Transdução de Sinais , Análise de Sobrevida , Linfócitos T/metabolismo , Linfócitos T/patologia , Teratoma/induzido quimicamente , Teratoma/metabolismo , Teratoma/patologiaRESUMO
The microglial receptors CD33 and TREM2 have been associated with risk for Alzheimer's disease (AD). Here, we investigated crosstalk between CD33 and TREM2. We showed that knockout of CD33 attenuated amyloid beta (Aß) pathology and improved cognition in 5xFAD mice, both of which were abrogated by additional TREM2 knockout. Knocking out TREM2 in 5xFAD mice exacerbated Aß pathology and neurodegeneration but reduced Iba1+ cell numbers, all of which could not be rescued by additional CD33 knockout. RNA-seq profiling of microglia revealed that genes related to phagocytosis and signaling (IL-6, IL-8, acute phase response) are upregulated in 5xFAD;CD33-/- and downregulated in 5xFAD;TREM2-/- mice. Differential gene expression in 5xFAD;CD33-/- microglia depended on the presence of TREM2, suggesting TREM2 acts downstream of CD33. Crosstalk between CD33 and TREM2 includes regulation of the IL-1ß/IL-1RN axis and a gene set in the "receptor activity chemokine" cluster. Our results should facilitate AD therapeutics targeting these receptors.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Cognição , Glicoproteínas de Membrana/genética , Microglia/metabolismo , Placa Amiloide/patologia , Receptores Imunológicos/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Reação de Fase Aguda/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Camundongos , Camundongos Knockout , Microglia/patologia , Fagocitose/genéticaRESUMO
The zinc finger CCCTC-binding protein (CTCF) carries out many functions in the cell. Although previous studies sought to explain CTCF multivalency based on sequence composition of binding sites, few examined how CTCF post-translational modification (PTM) could contribute to function. Here, we performed CTCF mass spectrometry, identified a novel phosphorylation site at Serine 224 (Ser224-P), and demonstrate that phosphorylation is carried out by Polo-like kinase 1 (PLK1). CTCF Ser224-P is chromatin-associated, mapping to at least a subset of known CTCF sites. CTCF Ser224-P accumulates during the G2/M transition of the cell cycle and is enriched at pericentric regions. The phospho-obviation mutant, S224A, appeared normal. However, the phospho-mimic mutant, S224E, is detrimental to mouse embryonic stem cell colonies. While ploidy and chromatin architecture appear unaffected, S224E mutants differentially express hundreds of genes, including p53 and p21. We have thus identified a new CTCF PTM and provided evidence of biological function.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fase G2 , Mitose , Fosfosserina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Fator de Ligação a CCCTC/química , Caseína Quinase II/metabolismo , Proliferação de Células , Cromatina , Sequência Conservada , DNA/metabolismo , Análise Mutacional de DNA , Humanos , Interfase , Proteínas de Membrana/metabolismo , Camundongos , Mutação/genética , Fosforilação , Ploidias , Ligação Proteica , RNA/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Quinase 1 Polo-LikeRESUMO
Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple cell types.
Assuntos
Reprogramação Celular , Fibroblastos/citologia , Músculo Esquelético/citologia , Células-Tronco/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Camundongos , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Distrofia Muscular Animal/patologia , Proteína MyoD/metabolismo , Fator de Transcrição PAX7/metabolismo , Regeneração/efeitos dos fármacos , Células Satélites de Músculo Esquelético/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Nicho de Células-Tronco/efeitos dos fármacos , Transplante de Células-Tronco , Células-Tronco/efeitos dos fármacos , TransgenesRESUMO
Polycomb repressive complex 2 (PRC2-EZH2) methylates histone H3 at lysine 27 (H3K27) and is required to maintain gene repression during development. Misregulation of PRC2 is linked to a range of neoplastic malignancies, which is believed to involve methylation of H3K27. However, the full spectrum of non-histone substrates of PRC2 that might also contribute to PRC2 function is not known. We characterized the target recognition specificity of the PRC2 active site and used the resultant data to screen for uncharacterized potential targets. The RNA polymerase II (Pol II) transcription elongation factor, Elongin A (EloA), is methylated by PRC2 in vivo. Mutation of the methylated EloA residue decreased repression of a subset of PRC2 target genes as measured by both steady-state and nascent RNA levels and perturbed embryonic stem cell differentiation. We propose that PRC2 modulates transcription of a subset of low expression target genes in part via methylation of EloA.
Assuntos
Diferenciação Celular , Metilação de DNA , Elonguina/metabolismo , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Histonas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Transcrição Gênica , Células 3T3-L1 , Animais , Elonguina/genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Camundongos , Mutação , Complexo Repressor Polycomb 2/genética , TransfecçãoRESUMO
Cell transplantation into immunodeficient mice has revolutionized our understanding of regeneration, stem cell self-renewal, and cancer; yet models for direct imaging of engrafted cells has been limited. Here, we characterize zebrafish with mutations in recombination activating gene 2 (rag2), DNA-dependent protein kinase (prkdc), and janus kinase 3 (jak3). Histology, RNA sequencing, and single-cell transcriptional profiling of blood showed that rag2 hypomorphic mutant zebrafish lack T cells, whereas prkdc deficiency results in loss of mature T and B cells and jak3 in T and putative Natural Killer cells. Although all mutant lines engraft fluorescently labeled normal and malignant cells, only the prkdc mutant fish reproduced as homozygotes and also survived injury after cell transplantation. Engraftment into optically clear casper, prkdc-mutant zebrafish facilitated dynamic live cell imaging of muscle regeneration, repopulation of muscle stem cells within their endogenous niche, and muscle fiber fusion at single-cell resolution. Serial imaging approaches also uncovered stochasticity in fluorescently labeled leukemia regrowth after competitive cell transplantation into prkdc mutant fish, providing refined models to assess clonal dominance and progression in the zebrafish. Our experiments provide an optimized and facile transplantation model, the casper, prkdc mutant zebrafish, for efficient engraftment and direct visualization of fluorescently labeled normal and malignant cells at single-cell resolution.
Assuntos
Proteína Quinase Ativada por DNA/deficiência , Imageamento Tridimensional/métodos , Transplante de Neoplasias , Fenômenos Ópticos , Análise de Célula Única/métodos , Peixe-Zebra/metabolismo , Anemia/patologia , Animais , Sequência de Bases , Células Clonais , Proteína Quinase Ativada por DNA/metabolismo , Modelos Animais de Doenças , Raios gama , Homozigoto , Humanos , Hospedeiro Imunocomprometido/efeitos da radiação , Proteínas Luminescentes/metabolismo , Células Musculares/patologia , Células Musculares/efeitos da radiação , Mutação/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Regeneração/efeitos da radiação , Transplante Homólogo , Recombinação V(D)J/genética , Proteínas de Peixe-Zebra/metabolismo , Proteína Vermelha FluorescenteRESUMO
Parathyroid hormone (PTH) activates receptors on osteocytes to orchestrate bone formation and resorption. Here we show that PTH inhibition of SOST (sclerostin), a WNT antagonist, requires HDAC4 and HDAC5, whereas PTH stimulation of RANKL, a stimulator of bone resorption, requires CRTC2. Salt inducible kinases (SIKs) control subcellular localization of HDAC4/5 and CRTC2. PTH regulates both HDAC4/5 and CRTC2 localization via phosphorylation and inhibition of SIK2. Like PTH, new small molecule SIK inhibitors cause decreased phosphorylation and increased nuclear translocation of HDAC4/5 and CRTC2. SIK inhibition mimics many of the effects of PTH in osteocytes as assessed by RNA-seq in cultured osteocytes and following in vivo administration. Once daily treatment with the small molecule SIK inhibitor YKL-05-099 increases bone formation and bone mass. Therefore, a major arm of PTH signalling in osteocytes involves SIK inhibition, and small molecule SIK inhibitors may be applied therapeutically to mimic skeletal effects of PTH.
Assuntos
Osso e Ossos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Regulação da Expressão Gênica , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/genética , Glicoproteínas/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Knockout , Osteócitos/citologia , Osteócitos/metabolismo , Osteogênese/genética , Hormônio Paratireóideo/metabolismo , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Ligante RANK/antagonistas & inibidores , Ligante RANK/genética , Ligante RANK/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
ZAP70 [zeta-chain (TCR)-associated protein kinase, 70-kDa], is required for T cell activation. ZAP70 deficiencies in humans and null mutations in mice lead to severe combined immune deficiency. Here, we describe a zap70 loss-of-function mutation in zebrafish (zap70 y442 ) that was created using transcription activator-like effector nucleases (TALENs). In contrast to what has been reported for morphant zebrafish, zap70 y442 homozygous mutant zebrafish displayed normal development of blood and lymphatic vasculature. Hematopoietic cell development was also largely unaffected in mutant larvae. However, mutant fish had reduced lck:GFP + thymic T cells by 5 days postfertilization that persisted into adult stages. Morphological analysis, RNA sequencing, and single-cell gene expression profiling of whole kidney marrow cells of adult fish revealed complete loss of mature T cells in zap70 y442 mutant animals. T cell immune deficiency was confirmed through transplantation of unmatched normal and malignant donor cells into zap70 y442 mutant zebrafish, with T cell loss being sufficient for robust allogeneic cell engraftment. zap70 mutant zebrafish show remarkable conservation of immune cell dysfunction as found in mice and humans and will serve as a valuable model to study zap70 immune deficiency.
RESUMO
Sox2 expression marks gastric stem and progenitor cells, raising important questions regarding the genes regulated by Sox2 and the role of Sox2 itself during stomach homeostasis and disease. By using ChIP-seq analysis, we have found that the majority of Sox2 targets in gastric epithelial cells are tissue specific and related to functions such as endoderm development, Wnt signaling, and gastric cancer. Unexpectedly, we found that Sox2 itself is dispensable for gastric stem cell and epithelial self-renewal, yet Sox2(+) cells are highly susceptible to tumorigenesis in an Apc/Wnt-driven mouse model. Moreover, Sox2 loss enhances, rather than impairs, tumor formation in Apc-deficient gastric cells in vivo and in vitro by inducing Tcf/Lef-dependent transcription and upregulating intestinal metaplasia-associated genes, providing a mechanistic basis for the observed phenotype. Together, these data identify Sox2 as a context-dependent tumor suppressor protein that is dispensable for normal tissue regeneration but restrains stomach adenoma formation through modulation of Wnt-responsive and intestinal genes.
Assuntos
Adenoma/genética , Proteína da Polipose Adenomatosa do Colo/genética , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição SOXB1/genética , Neoplasias Gástricas/genética , Adenoma/metabolismo , Adenoma/patologia , Proteína da Polipose Adenomatosa do Colo/deficiência , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Camundongos Transgênicos , Fatores de Transcrição SOXB1/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Via de Sinalização WntRESUMO
Activating mutations in the phosphoinositide 3-kinase (PI3K) signaling pathway are frequently identified in cancer. To identify pathways that support PI3K oncogenesis, we performed a genome-wide RNAi screen in isogenic cell lines harboring wild-type or mutant PIK3CA to search for PI3K synthetic-lethal (SL) genes. A combined analysis of these results with a meta-analysis of two other large-scale RNAi screening data sets in PI3K mutant cancer cell lines converged on ribosomal protein translation and proteasomal protein degradation as critical nononcogene dependencies for PI3K-driven tumors. Genetic or pharmacologic inhibition of either pathway alone, but not together, selectively killed PI3K mutant tumor cells in an mTOR-dependent manner. The expression of ribosomal and proteasomal components was significantly up-regulated in primary human colorectal tumors harboring PI3K pathway activation. Importantly, a PI3K SL gene signature containing the top hits of the SL genes identified in our meta-analysis robustly predicted overall patient survival in colorectal cancer, especially among patients with tumors with an activated PI3K pathway. These results suggest that disruption of protein turnover homeostasis via ribosome or proteasome inhibition may be a novel treatment strategy for PI3K mutant human tumors.
Assuntos
Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/genética , Animais , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/fisiopatologia , Genômica , Células HCT116 , Células HEK293 , Humanos , Camundongos , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Ribossomos/genéticaRESUMO
Tumors exhibit numerous recurrent hemizygous focal deletions that contain no known tumor suppressors and are poorly understood. To investigate whether these regions contribute to tumorigenesis, we searched genetically for genes with cancer-relevant properties within these hemizygous deletions. We identified STOP and GO genes, which negatively and positively regulate proliferation, respectively. STOP genes include many known tumor suppressors, whereas GO genes are enriched for essential genes. Analysis of their chromosomal distribution revealed that recurring deletions preferentially overrepresent STOP genes and underrepresent GO genes. We propose a hypothesis called the cancer gene island model, whereby gene islands encompassing high densities of STOP genes and low densities of GO genes are hemizygously deleted to maximize proliferative fitness through cumulative haploinsufficiencies. Because hundreds to thousands of genes are hemizygously deleted per tumor, this mechanism may help to drive tumorigenesis across many cancer types.
Assuntos
Proliferação de Células , Transformação Celular Neoplásica , Genes Neoplásicos , Haploinsuficiência , Neoplasias/genética , Neoplasias/patologia , Deleção de Sequência , Linhagem Celular , Linhagem Celular Tumoral , Mapeamento Cromossômico , Genes Essenciais , Genes Recessivos , Genes Supressores de Tumor , Hemizigoto , Humanos , Modelos Genéticos , OncogenesRESUMO
Intracellular reactive oxygen species are generated as a by-product of normal metabolic processes and can both damage cellular constituents and function as important signalling species. This signalling often involves changes in the thiol redox balance. As an antioxidant, glutathione serves in maintaining the reduced state of cellular protein thiol groups. The paper by Cross and Templeton appearing in this issue of the Biochemical Journal describes a mechanism by which glutathionylation plays a key role in the regulation of the kinase activity of MEKK1 [MAP (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase kinase; MAP3K] in response to oxidative stresses. This type of post-translational-modification glutathionylation may represent a general mechanism by which protein kinase function can be regulated.
Assuntos
Glutationa/química , Proteínas Quinases/química , Proteínas Quinases/fisiologiaRESUMO
Neuronal precursor cells have the capacity to engage the Raf-MEK-ERK signal module to drive either of two distinctly different regulatory programs, proliferation and differentiation. This is, at least in part, a consequence of stimulus-specific shaping of the kinase cascade response. For example, the mitogen EGF induces a transient ERK activation, whereas the neurotrophin NGF induces prolonged ERK activation. Here we define a novel component of the regulatory machinery contributing to the selective integration of MAP kinase signaling with discrete biological responses. We show that the scaffold/adaptor protein CNK2/MAGUIN-1 is required for NGF- but not EGF-induced ERK activation. In addition, CNK2 makes a separate, essential contribution to the coupling of NGF signaling to membrane/cytoskeletal remodeling. We propose that CNK2 integrates multiple regulatory pathways that must function in concert to drive an appropriate biological response to external stimuli.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Expressão Gênica/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Western Blotting , Proteínas de Transporte/fisiologia , Primers do DNA , Ativação Enzimática , Células HeLa , Humanos , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana/fisiologia , Proteínas dos Microfilamentos , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Neuritos/metabolismo , Células PC12 , Plasmídeos/genética , Testes de Precipitina , RNA Interferente Pequeno/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/metabolismoRESUMO
Genetic analysis of Ras signaling has unveiled the participation of non-enzymatic accessory proteins in signal transmission. These proteins, KSR, CNK, and Sur-8, can interact with multiple core components of the Ras/MAP kinase cascade and may contribute to the structural organization of this cascade. However, the precise biochemical nature of the contribution of these proteins to Ras signaling is currently unknown. Here we show directly that CNK and KSR are required for stimulus dependent Raf kinase activation. CNK is required for membrane recruitment of Raf, while KSR is likely required to couple Raf to upstream kinases. These results demonstrate that CNK and KSR are integral components of the cellular machinery mediating Raf activation.