RESUMEN
C3G is a Rap1 GEF that plays a pivotal role in platelet-mediated processes such as angiogenesis, tumor growth, and metastasis by modulating the platelet secretome. Here, we explore the mechanisms through which C3G governs platelet secretion. For this, we utilized animal models featuring either overexpression or deletion of C3G in platelets, as well as PC12 cell clones expressing C3G mutants. We found that C3G specifically regulates α-granule secretion via PKCδ, but it does not affect δ-granules or lysosomes. C3G activated RalA through a GEF-dependent mechanism, facilitating vesicle docking, while interfering with the formation of the trans-SNARE complex, thereby restricting vesicle fusion. Furthermore, C3G promotes the formation of lamellipodia during platelet spreading on specific substrates by enhancing actin polymerization via Src and Rac1-Arp2/3 pathways, but not Rap1. Consequently, C3G deletion in platelets favored kiss-and-run exocytosis. C3G also controlled granule secretion in PC12 cells, including pore formation. Additionally, C3G-deficient platelets exhibited reduced phosphatidylserine exposure, resulting in decreased thrombin generation, which along with defective actin polymerization and spreading, led to impaired clot retraction. In summary, platelet C3G plays a dual role by facilitating platelet spreading and clot retraction through the promotion of outside-in signaling while concurrently downregulating α-granule secretion by restricting granule fusion.
Asunto(s)
Actinas , Plaquetas , Retracción del Coagulo , Factor 2 Liberador de Guanina Nucleótido , Animales , Actinas/metabolismo , Plaquetas/metabolismo , Exocitosis/fisiología , Hemostasis , Factor 2 Liberador de Guanina Nucleótido/metabolismoRESUMEN
BACKGROUND: C3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. C3G is activated by tyrosine phosphorylation and interaction with Crk adaptor proteins, whose expression is elevated in multiple human cancers. However, the molecular details of C3G activation and the interplay between phosphorylation and Crk interaction are poorly understood. METHODS: We combined biochemical, biophysical, and cell biology approaches to elucidate the mechanisms of C3G activation. Binding of Crk adaptor proteins to four proline-rich motifs (P1 to P4) in C3G was characterized in vitro using isothermal titration calorimetry and sedimentation velocity, and in Jurkat and HEK293T cells by affinity pull-down assays. The nucleotide exchange activity of C3G over Rap1 was measured using nucleotide-dissociation kinetic assays. Jurkat cells were also used to analyze C3G translocation to the plasma membrane and the C3G-dependent activation of Rap1 upon ligation of T cell receptors. RESULTS: CrkL interacts through its SH3N domain with sites P1 and P2 of inactive C3G in vitro and in Jurkat and HEK293T cells, and these sites are necessary to recruit C3G to the plasma membrane. However, direct stimulation of the GEF activity requires binding of Crk proteins to the P3 and P4 sites. P3 is occluded in resting C3G and is essential for activation, while P4 contributes secondarily towards complete stimulation. Tyrosine phosphorylation of C3G alone causes marginal activation. Instead, phosphorylation primes C3G lowering the concentration of Crk proteins required for activation and increasing the maximum activity. Unexpectedly, optimal activation also requires the interaction of CrkL-SH2 domain with phosphorylated C3G. CONCLUSION: Our study revealed that phosphorylation of C3G by Src and Crk-binding form a two-factor mechanism that ensures tight control of C3G activation. Additionally, the simultaneous SH2 and SH3N interaction of CrkL with C3G, required for the activation, reveals a novel adaptor-independent function of Crk proteins relevant to understanding their role in physiological signaling and their deregulation in diseases. Video abstract.
Asunto(s)
Factor 2 Liberador de Guanina Nucleótido , Proteínas Nucleares , Humanos , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Células HEK293 , Proteínas Nucleares/metabolismo , Nucleótidos/metabolismo , Proteínas Proto-Oncogénicas c-crk/metabolismo , Dominios Homologos src , Tirosina/metabolismoRESUMEN
Crkl is a protein involved in the onset of several cancer pathologies that exerts its function only through its protein-protein interaction domains, a SH2 domain and two SH3 domains. SH3 domains are small protein interaction modules that mediate the binding and recognition of proline-rich sequences. One of the main physiological interactors of Crkl is C3G (also known as RAPGEF1), an interaction with key implications in regulating cellular growth and differentiation, cell morphogenesis and adhesion processes. Thus, understanding the interaction between Crkl and C3G is fundamental to gaining information about the molecular determinants of the several cancer pathologies in which these proteins are involved. In this paper, through a combination of fast kinetics at different experimental conditions and site-directed mutagenesis, we characterize the binding reaction between the N-SH3 domain of Crkl and a peptide mimicking a specific portion of C3G. Our results show a clear effect of pH on the stability of the complex, due to the protonation of negatively charged residues in the binding pocket of N-SH3. Our results are discussed under the light of previous work on SH3 domains.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Mutagénesis Sitio-Dirigida/métodos , Proteínas Adaptadoras Transductoras de Señales/genética , Sitios de Unión , Adhesión Celular , Diferenciación Celular , Proliferación Celular , Factor 2 Liberador de Guanina Nucleótido/química , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos , Electricidad EstáticaRESUMEN
C3G (RAPGEF1) is a guanine nucleotide exchange factor (GEF) for GTPases from the Ras superfamily, mainly Rap1, although it also acts through GEF-independent mechanisms. C3G regulates several cellular functions. It is expressed at relatively high levels in specific brain areas, playing important roles during embryonic development. Recent studies have uncovered different roles for C3G in cancer that are likely to depend on cell context, tumour type, and stage. However, its role in brain tumours remained unknown until very recently. We found that C3G expression is downregulated in GBM, which promotes the acquisition of a more mesenchymal phenotype, enhancing migration and invasion, but not proliferation. ERKs hyperactivation, likely induced by FGFR1, is responsible for this pro-invasive effect detected in C3G silenced cells. Other RTKs (Receptor Tyrosine Kinases) are also dysregulated and could also contribute to C3G effects. However, it remains undetermined whether Rap1 is a mediator of C3G actions in GBM. Various Rap1 isoforms can promote proliferation and invasion in GBM cells, while C3G inhibits migration/invasion. Therefore, other RapGEFs could play a major role regulating Rap1 activity in these tumours. Based on the information available, C3G could represent a new biomarker for GBM diagnosis, prognosis, and personalised treatment of patients in combination with other GBM molecular markers. The quantification of C3G levels in circulating tumour cells (CTCs) in the cerebrospinal liquid and/or circulating fluids might be a useful tool to improve GBM patient treatment and survival.
Asunto(s)
Glioblastoma/metabolismo , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Animales , Glioblastoma/genética , Factor 2 Liberador de Guanina Nucleótido/genética , Humanos , Células Neoplásicas Circulantes/metabolismo , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
RAPGEF1 is a guanine nucleotide exchange factor responsible for transmitting extracellular signals to the Ras family of GTPase located at the inside of membrane. Here, we report for the first time a homozygous mutation of RAPGEF1 in a consanguineous family with two siblings affected by neuropsychiatric disorder. To confirm the correlation of the mutation and the phenotype, we utilized in silico analysis and established a zebrafish model. Survival rate was reduced in the rapgef1a-knockdown model, and the zebrafish showed global morphological abnormalities, particularly of brain and blood vessels. Co-application of human RAPGEF1 wildtype mRNA effectively rescued the abnormal phenotype, while that of RAPGEF1 mRNA carrying the human mutation did not. This work is the first report of a human Mendelian disease associated with RAPGEF1 and the first report of a zebrafish model built for this gene. The phenotype of zebrafish model provides further evidence that defective RAPGEF1 may lead to global developmental delay in human patients.
Asunto(s)
Factor 2 Liberador de Guanina Nucleótido/genética , Mutación , Pez Cebra/embriología , Pez Cebra/genética , Animales , Modelos Animales de Enfermedad , Embrión no Mamífero/anomalías , Embrión no Mamífero/irrigación sanguínea , Femenino , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Semivida , Humanos , Masculino , Trastornos del Humor/genética , Neuronas Motoras/patología , Linaje , Fenotipo , Proteínas de Pez Cebra/genéticaRESUMEN
Glioblastoma (GBM) is the most aggressive tumor from the central nervous system (CNS). The current lack of efficient therapies makes essential to find new treatment strategies. C3G, a guanine nucleotide exchange factor for some Ras proteins, plays a dual role in cancer, but its function in GBM remains unknown. Database analyses revealed a reduced C3G mRNA expression in GBM patient samples. C3G protein levels were also decreased in a panel of human GBM cell lines as compared to astrocytes. Based on this, we characterized C3G function in GBM using in vitro and in vivo human GBM models. We report here that C3G downregulation promoted the acquisition of a more mesenchymal phenotype that enhanced the migratory and invasive capacity of GBM cells. This facilitates foci formation in anchorage-dependent and -independent growth assays and the generation of larger tumors in xenografts and chick chorioallantoic membrane (CAM) assays, but with a lower cell density, as proliferation was reduced. Mechanistically, C3G knock-down impairs EGFR signaling by reducing cell surface EGFR through recycling inhibition, while upregulating the activation of several other receptor tyrosine kinases (RTKs) that might promote invasion. In particular, FGF2, likely acting through FGFR1, promoted invasion of C3G-silenced GBM cells. Moreover, ERKs mediate this invasiveness, both in response to FGF2- and serum-induced chemoattraction. In conclusion, our data show the distinct dependency of GBM tumors on C3G for EGF/EGFR signaling versus other RTKs, suggesting that assessing C3G levels may discriminate GBM patient responders to different RTK inhibition protocols. Hence, patients with a low C3G expression might not respond to EGFR inhibitors.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Movimiento Celular/fisiología , Glioblastoma/metabolismo , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Regulación hacia Abajo , Receptores ErbB/metabolismo , Glioblastoma/patología , Humanos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The expression of Ras-specific guanine nucleotide-releasing factor 2 (RasGRF2) in lung adenocarcinomas was examined using immunohistochemistry in relation to clinicopathological characteristics and prognosis. In comparison to low expression, high expression of RasGRF2 was more closely associated with poor prognosis. Interestingly, expression of phosphorylated epithelial cell transforming 2 (pECT2), which - like RasGRF2 - is also a guanine-nucleotide exchange factor, was also associated with prognosis, and patients with high expression of both RasGRF2 and pECT2 had a much poorer outcome than those who were negative for both.
Asunto(s)
Adenocarcinoma del Pulmón/patología , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Pronóstico , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Femenino , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Factores de Intercambio de Guanina Nucleótido ras/metabolismoRESUMEN
GSK3ß, a ubiquitously expressed Ser/Thr kinase, regulates cell metabolism, proliferation and differentiation. Its activity is spatially and temporally regulated dependent on external stimuli and interacting partners, and its deregulation is associated with various human disorders. In this study, we identify C3G (RapGEF1), a protein essential for mammalian embryonic development as an interacting partner and substrate of GSK3ß. In vivo and in vitro interaction assays demonstrated that GSK3ß and Akt are present in complex with C3G. Molecular modelling and mutational analysis identified a domain in C3G that aids interaction with GSK3ß, and overlaps with its nuclear export sequence. GSK3ß phosphorylates C3G on primed as well as unprimed sites, and regulates its subcellular localization. Over-expression of C3G resulted in activation of Akt and inactivation of GSK3ß. Huntingtin aggregate formation, dependent on GSK3ß inhibition, was enhanced upon C3G overexpression. Stable clones of C2C12 cells generated by CRISPR/Cas9 mediated knockdown of C3G, that cannot differentiate, show reduced Akt activity and S9-GSK3ß phosphorylation compared to wild type cells. Co-expression of catalytically active GSK3ß inhibited C3G induced myocyte differentiation. C3G mutant defective for GSK3ß phosphorylation, does not alter S9-GSK3ß phosphorylation and, is compromised for inducing myocyte differentiation. Our results show complex formation and reciprocal regulation between GSK3ß and C3G. We have identified a novel function of C3G as a negative regulator of GSK3ß, a property important for its ability to induce myogenic differentiation.
Asunto(s)
Glucógeno Sintasa Quinasa 3 beta/metabolismo , Factor 2 Liberador de Guanina Nucleótido/química , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Mutación , Mioblastos/citología , Animales , Células COS , Diferenciación Celular , Línea Celular , Chlorocebus aethiops , Citoplasma/metabolismo , Regulación de la Expresión Génica , Factor 2 Liberador de Guanina Nucleótido/genética , Células HEK293 , Humanos , Ratones , Desarrollo de Músculos , Mioblastos/metabolismo , FosforilaciónRESUMEN
Mice lacking C3G (RapGEF1), a ubiquitously expressed protein essential for neuronal differentiation, show multiple defects in brain development. Function of C3G in neurogenesis is poorly defined. Here, we identify brain specific expression of a novel C3G isoform in mice and humans. This isoform has an insert in the Crk-binding region, generating a polypeptide of 175 kDa, unlike the previously known 140 kDa form expressed in all other tissues. In the adult mouse brain, C3G expression is seen in neurons, but was not detectable in GFAP-positive cells. C3G levels were high in the CA3 region of hippocampus and in mitral cells of olfactory bulb. Neural progenitor cells positive for Doublecortin and Nestin, show expression of C3G. During development, C3G is expressed in precursor cells prior to their differentiation into mature neurons or astrocytes. The 175 kDa as well as 140 kDa forms are seen in embryonic mouse brain, while only the 175 kDa variant is seen in post-natal brain. Human cerebral organoids generated from induced pluripotent stem cells predominantly expressed the 140 kDa polypeptides, and the 175 kDa isoform appeared upon maturation. This study describes developmental regulation and neuronal expression of a brain specific isoform of C3G, a molecule essential for normal development of the mammalian brain.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Expresión Génica , Factor 2 Liberador de Guanina Nucleótido/genética , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Animales , Encéfalo/embriología , Hipocampo/metabolismo , Humanos , Ratones , Bulbo Olfatorio/metabolismo , Organoides/metabolismo , Péptidos/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
OBJECTIVE: To investigate the functions of eIF3b in chronic myelogenous leukemia (CML). METHODS: The expression of eIF3b was inhibited by transfecting aspecifically designed shRNA into the CML cell lines of TK-6 and K562. The CCK8 assay was conducted to determine cell viability, and flow cytometry was used to examine the change in the cell cycle and cell apoptosis. RNAsequencing was applied to screen the candidate targets of eIF3b to identify the underlying mechanisms of eIF3b.An in vivo tumour xenograft mouse model was established by injecting shRNA transfected cells into the NCG mice. The tumour size and body weight of mice were monitored every other day. The mice were sacrificed 2 weeks after the tumour cell injection. The expression of eIF3b and target genes in the tumour tissues were determined by immunohistochemical staining and Western blotting. RESULTS: The group with inhibited expression of eIF3b led to about 50% lower cell viability compared with that of the control group (P < 0.05). Flow cytometry suggested that the percentage of increase in apoptotic cells was eight times higher than those in control group for TK-6 and K562 cells (P < 0.05). However, the difference between the cell amounts in the S phase for the experiment and control groups was not significant. After RNAsequencing and further validation via qPCR, C3G was screened as the potential target of eIF3b involved in the cell proliferation and apoptosis of CML cell lines. Subsequent in vivo analysis proved that the inhibition of eIF3b suppressed tumour formation and decreased C3G expression, thereby indicating that C3G was the potential target of eIF3b. CONCLUSION: eIF3b is correlated with the cell proliferation and cell apoptosis of CML. Moreover, eIF3b regulation most probably occurs via regulating the expression of C3G.
Asunto(s)
Apoptosis/fisiología , Proliferación Celular/fisiología , Factores Eucarióticos de Iniciación/metabolismo , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Animales , Xenoinjertos , Humanos , Células K562 , RatonesRESUMEN
Sildenafil, a phosphodiesterase-5 inhibitor is FDA approved drug against erectile dysfunction. It is currently undergoing many clinical trials, alone or in combinations against different diseases. Treatment of neural progenitor cells with sildenafil is known to regulate their basal cGMP levels and enhance neurogenesis and differentiation. cGMP as well as cAMP are known to play a central role in the maintenance, repair and remodelling of the nervous system. In the present study, we report the neurodifferentiation property of sildenafil in neuroblastoma cancer cell line IMR-32. Sildenafil was found to induce the formation of neurite outgrowths that were found expressing neuronal markers, such as NeuN, NF-H and ßIII tubulin. IS00384, a recently discovered PDE5 inhibitor by our laboratory, was also found to induce neurodifferentiation of IMR-32 cells. The effect of IS00384 on differentiation was even more profound than sildenafil. Both the compounds were found to elevate and activate the Guanine nucleotide exchange factor C3G, which is a regulator of differentiation in IMR-32 cells. They were also found to elevate the levels of cGMP and activate the AMPK-ACC and PI3K-Akt signalling pathways. These pathways are known to play important role in cytoskeletal rearrangements necessary for differentiation. This study highlights the role of phosphodiesterases-5 in neurodifferentiation and use of sildenafil and IS00384 as small molecule tools to study the process of cellular differentiation.
Asunto(s)
Neuroblastoma/metabolismo , Neurogénesis/efectos de los fármacos , Inhibidores de Fosfodiesterasa 5/farmacología , Citrato de Sildenafil/farmacología , Quinasas de la Proteína-Quinasa Activada por el AMP , Antígenos Nucleares/metabolismo , Línea Celular Tumoral , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Humanos , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma/enzimología , Proteínas de Neurofilamentos/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Citrato de Sildenafil/química , Tubulina (Proteína)/metabolismoRESUMEN
BACKGROUND: Megakaryopoiesis allows platelet formation, which is necessary for coagulation, also playing an important role in different pathologies. However, this process remains to be fully characterized. C3G, an activator of Rap1 GTPases, is involved in platelet activation and regulates several differentiation processes. METHODS: We evaluated C3G function in megakaryopoiesis using transgenic mouse models where C3G and C3GΔCat (mutant lacking the GEF domain) transgenes are expressed exclusively in megakaryocytes and platelets. In addition, we used different clones of K562, HEL and DAMI cell lines with overexpression or silencing of C3G or GATA-1. RESULTS: We found that C3G participates in the differentiation of immature hematopoietic cells to megakaryocytes. Accordingly, bone marrow cells from transgenic C3G, but not those from transgenic C3GΔCat mice, showed increased expression of the differentiation markers CD41 and CD61, upon thrombopoietin treatment. Furthermore, C3G overexpression increased the number of CD41+ megakaryocytes with high DNA content. These results are supported by data obtained in the different models of megakaryocytic cell lines. In addition, it was uncovered GATA-1 as a positive regulator of C3G expression. Moreover, C3G transgenic megakaryocytes from fresh bone marrow explants showed increased migration from the osteoblastic to the vascular niche and an enhanced ability to form proplatelets. Although the transgenic expression of C3G in platelets did not alter basal platelet counts, it did increase slightly those induced by TPO injection in vivo. Moreover, platelet C3G induced adipogenesis in the bone marrow under pathological conditions. CONCLUSIONS: All these data indicate that C3G plays a significant role in different steps of megakaryopoiesis, acting through a mechanism dependent on its GEF activity.
Asunto(s)
Plaquetas/citología , Diferenciación Celular , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Megacariocitos/citología , Adipogénesis , Línea Celular Tumoral , Humanos , Megacariocitos/metabolismo , PloidiasRESUMEN
C3G (Crk SH3 domain binding guanine nucleotide releasing factor) (Rap guanine nucleotide exchange factor 1), essential for mammalian embryonic development, is ubiquitously expressed and undergoes regulated nucleocytoplasmic exchange. Here we show that C3G localizes to SC35-positive nuclear speckles and regulates splicing activity. Reversible association of C3G with speckles was seen on inhibition of transcription and splicing. C3G shows partial colocalization with SC35 and is recruited to a chromatin and RNase-sensitive fraction of speckles. Its presence in speckles is dependent on intact cellular actin cytoskeleton and is lost on expression of the kinase Clk1. Rap1, a substrate of C3G, is also present in nuclear speckles, and inactivation of Rap signaling by expression of GFP-Rap1GAP alters speckle morphology and number. Enhanced association of C3G with speckles is seen on glycogen synthase kinase 3 beta inhibition or differentiation of C2C12 cells to myotubes. CRISPR/Cas9-mediated knockdown of C3G resulted in altered splicing activity of an artificial gene as well as endogenous CD44. C3G knockout clones of C2C12 as well as MDA-MB-231 cells showed reduced protein levels of several splicing factors compared with control cells. Our results identify C3G and Rap1 as novel components of nuclear speckles and a role for C3G in regulating cellular RNA splicing activity.
Asunto(s)
Factor 2 Liberador de Guanina Nucleótido/metabolismo , Factor 2 Liberador de Guanina Nucleótido/fisiología , Empalme del ARN/fisiología , Animales , Diferenciación Celular , Línea Celular , Línea Celular Tumoral , Estructuras del Núcleo Celular/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Proteínas Nucleares , Unión Proteica , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas , Procesamiento Postranscripcional del ARN/fisiología , ARN Mensajero/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Factores de Empalme Serina-Arginina/fisiología , Complejo Shelterina , Transducción de Señal , Empalmosomas , Proteínas de Unión a Telómeros/metabolismoRESUMEN
Various parameters of neurogenesis were analyzed in parallel in the two neurogenic areas (the Dentate Gyrus[DG] and the Subventricular Zone[SVZ]/Rostral Migratory Stream[RMS]/Main Olfactory Bulb[MOB] neurogenic system) of adult WT and KO mouse strains for the Ras-GRF1/2 genes (Ras-GRF1-KO, Ras-GRF2-KO, Ras-GRF1/2-DKO). Significantly reduced numbers of doublecortin[DCX]-positive cells were specifically observed in the DG, but not the SVZ/RMS/MOB neurogenic region, of Ras-GRF2-KO and Ras-GRF1/2-DKO mice indicating that this novel Ras-GRF2-dependent phenotype is spatially restricted to a specific neurogenic area. Consistent with a role of CREB as mediator of Ras-GRF2 function in neurogenesis, the density of p-CREB-positive cells was also specifically reduced in all neurogenic regions of Ras-GRF2-KO and DKO mice. Similar levels of early neurogenic proliferation markers (Ki67, BrdU) were observed in all different Ras-GRF genotypes analyzed but significantly elevated levels of nestin-immunolabel, particularly of undifferentiated, highly ramified, A-type nestin-positive neurons were specifically detected in the DG but not the SVZ/RMS/MOB of Ras-GRF2-KO and DKO mice. Together with assays of other neurogenic markers (GFAP, Sox2, Tuj1, NeuN), these observations suggest that the deficit of DCX/p-CREB-positive cells in the DG of Ras-GRF2-depleted mice does not involve impaired neuronal proliferation but rather delayed transition from the stem cell stage to the differentiation stages of the neurogenic process. This model is also supported by functional analyses of DG-derived neurosphere cultures and transcriptional characterization of the neurogenic areas of mice of all relevant Ras-GRF genotypes suggesting that the neurogenic role of Ras-GRF2 is exerted in a cell-autonomous manner through a specific transcriptional program.
Asunto(s)
Diferenciación Celular/fisiología , Giro Dentado/citología , Células-Madre Neurales/citología , Neurogénesis/fisiología , ras-GRF1/metabolismo , Animales , Giro Dentado/metabolismo , Proteína Doblecortina , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NestinaRESUMEN
The adaptor protein CrkII regulates T cell adhesion by recruiting the guanine nucleotide exchange factor C3G, an activator of Rap1. Subsequently, Rap1 stimulates the integrin LFA-1, which leads to T cell adhesion and interaction with antigen-presenting cells (APCs). The adhesion of T cells to APCs is critical for their proper function and education. The interface between the T cell and the APC is known as the immunological synapse. It is characterized by the specific organization of proteins that can be divided into central supramolecular activation clusters (c-SMACs) and peripheral SMACs (p-SMACs). Through total internal reflection fluorescence (TIRF) microscopy and experiments with supported lipid bilayers, we determined that activated Rap1 was recruited to the immunological synapse and localized to the p-SMAC. C3G and the active (dephosphorylated) form of CrkII also localized to the same compartment. In contrast, inactive (phosphorylated) CrkII was confined to the c-SMAC. Activation of CrkII and its subsequent movement from the c-SMAC to the p-SMAC depended on the phosphatase SHP-1, which acted downstream of the T cell receptor. In the p-SMAC, CrkII recruited C3G, which led to Rap1 activation and LFA-1-mediated adhesion of T cells to APCs. Functionally, SHP-1 was necessary for both the adhesion and migration of T cells. Together, these data highlight a signaling pathway in which SHP-1 acts through CrkII to reshape the pattern of Rap1 activation in the immunological synapse.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Sinapsis Inmunológicas/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogénicas c-crk/metabolismo , Linfocitos T/inmunología , Inmunidad Adaptativa , Animales , Adhesión Celular , Proteínas Activadoras de GTPasa/genética , Factor 2 Liberador de Guanina Nucleótido/genética , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Células HEK293 , Humanos , Sinapsis Inmunológicas/metabolismo , Células Jurkat , Activación de Linfocitos/inmunología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Ratones , Ratones Transgénicos , Fosforilación , Cultivo Primario de Células , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Proteínas Proto-Oncogénicas c-crk/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Análisis de la Célula Individual , Linfocitos T/metabolismoRESUMEN
C3G (RapGEF1) is a ubiquitously expressed guanine nucleotide exchange factor that functions in signaling pathways regulating cell proliferation, apoptosis, and actin reorganization. It is essential for differentiation and early embryonic development in mice. Overexpressed C3G shows predominant cytoplasmic localization, but endogenous C3G is a component of nuclear fractions in a variety of cell types. Coexpression of importin-α and inhibition of nuclear export by leptomycin B resulted in predominant nuclear localization of C3G. Functional NLSs, NES, and GSK3-ß-dependent phosphorylation regulate its dynamic nuclear localization. C3G translocates to the nucleus in response to myogenic differentiation and sublethal dose of cisplatin. C3G is associated with chromatin and nuclear matrix fractions. Cells with C3G localized in the nucleus showed peripheralization of heterochromatin and reduced histone modifications associated with euchromatin. Short hairpin RNA-mediated depletion of C3G in epithelial cells resulted in reduced expression of CDK inhibitors and the histone demethylase KDM5A. Myoblast clones with CRISPR/Cas9-mediated knockout of C3G failed to show repression of histone marks and did not show up-regulation of myosin heavy chain and myotube formation when grown in differentiation medium. Our results document regulated nucleocytoplasmic exchange of C3G in response to physiological stimuli and provide insights into nuclear functions for C3G.
Asunto(s)
Eucromatina/fisiología , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Factor 2 Liberador de Guanina Nucleótido/fisiología , Código de Histonas/fisiología , Actinas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Diferenciación Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , Eucromatina/metabolismo , Ácidos Grasos Insaturados/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factor 2 Liberador de Guanina Nucleótido/genética , Ratones , Desarrollo de Músculos , Señales de Localización Nuclear , Fosforilación , Transducción de Señal , Regulación hacia ArribaRESUMEN
BACKGROUND: Vasodilator-Stimulated Phosphoprotein (VASP) is involved in the inhibition of agonist-induced platelet aggregation by cyclic nucleotides and the adhesion of platelets to the vascular wall. αIIbß3 is the main integrin responsible for platelet activation and Rap1b plays a key role in integrin signalling. We investigated whether VASP is involved in the regulation of Rap1b in platelets since VASP-null platelets exhibit augmented adhesion to endothelial cells in vivo. METHODS: Washed platelets from wild type and VASP-deficient mice were stimulated with thrombin, the purinergic receptors agonist ADP, or the thromboxane A2 receptor agonist U46619 and Rap1b activation was measured using the GST-RalGDS-RBD binding assay. Interaction of VASP and Crkl was investigated by co-immunoprecipitation, confocal microscopy, and pull-down assays using Crkl domains expressed as GST-fusion proteins. RESULTS: Surprisingly, we found that activation of Rap1b in response to thrombin, ADP, or U46619 was significantly reduced in platelets from VASP-null mice compared to platelets from wild type mice. However, inhibition of thrombin-induced activation of Rap1b by nitric oxide (NO) was similar in platelets from wild type and VASP-null mice indicating that the NO/cGMP/PKG pathway controls inhibition of Rap1b independently from VASP. To understand how VASP regulated Rap1b, we investigated association between VASP and the Crk-like protein (Crkl), an adapter protein which activates the Rap1b guanine nucleotide exchange factor C3G. We demonstrated the formation of a Crkl/VASP complex by showing that: 1) Crkl co-immunoprecipitated VASP from platelet lysates; 2) Crkl and VASP dynamically co-localized at actin-rich protrusions reminiscent of focal adhesions, filopodia, and lamellipodia upon platelet spreading on fibronectin; 3) recombinant VASP bound directly to the N-terminal SH3 domain of Crkl; 4) Protein Kinase A (PKA) -mediated VASP phosphorylation on Ser157 abrogated the binding of Crkl. CONCLUSIONS: We identified Crkl as a novel protein interacting with VASP in platelets. We propose that the C3G/Crkl/VASP complex plays a role in the regulation of Rap1b and this explains, at least in part, the reduced agonist-induced activation of Rap1b in VASP-null platelets. In addition, the fact that PKA-dependent VASP phosphorylation abrogated its interaction with Crkl may provide, at least in part, a rationale for the PKA-dependent inhibition of Rap1b and platelet aggregation.
Asunto(s)
Plaquetas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al GTP rap/metabolismo , Animales , Plaquetas/efectos de los fármacos , Moléculas de Adhesión Celular/genética , Células Cultivadas , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Humanos , Ratones , Proteínas de Microfilamentos/genética , Fosfoproteínas/genética , Unión Proteica , Proteínas Proto-Oncogénicas c-crk/metabolismo , Agonistas Purinérgicos/farmacología , Trombina/farmacologíaRESUMEN
C3G, a Guanine nucleotide Exchange Factor (GEF) for Rap1 and R-Ras, has been shown to play important roles in development and cancer. Previous studies determined that C3G regulates cell death through down-regulation of p38α MAPK activity. Here, we found that C3G knock-down in MEFs and HCT116 cells promotes migration and invasion through Rap1-mediated p38α hyper-activation. These effects of C3G were inhibited by Rap1 knock-down or inactivation. The enhanced migration observed in C3G depleted HCT116 cells was associated with reduction in E-cadherin expression, internalization of ZO-1, actin cytoskeleton reorganization and decreased adhesion. We also found that matrix metalloproteases MMP2 and MMP9 are involved in the pro-invasive effect of C3G down-regulation. Additionally, our studies revealed that both C3G and p38α collaborate to promote growth of HCT116 cells in vitro and in vivo, possibly by enhancing cell survival. In fact, knocking-down C3G or p38α individually or together promoted cell death in vitro, although only the double C3G-p38α silencing was able to increase cell death within tumors. Notably, we found that the pro-tumorigenic function of C3G does not depend on p38α or Rap1 activation. Altogether, our studies uncover novel mechanisms by which C3G controls key aspects of tumorigenesis.
Asunto(s)
Carcinogénesis/metabolismo , Neoplasias Colorrectales/patología , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Animales , Movimiento Celular/fisiología , Neoplasias Colorrectales/metabolismo , Activación Enzimática/fisiología , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Células HCT116 , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Desnudos , Invasividad Neoplásica/patologíaRESUMEN
The establishment of a polarized morphology is essential for the development and function of neurons. During the development of the mammalian neocortex, neurons arise in the ventricular zone (VZ) from radial glia cells (RGCs) and leave the VZ to generate the cortical plate (CP). During their migration, newborn neurons first assume a multipolar morphology in the subventricular zone (SVZ) and lower intermediate zone (IZ). Subsequently, they undergo a multi-to-bipolar (MTB) transition to become bipolar in the upper IZ by developing a leading process and a trailing axon. The small GTPases Rap1A and Rap1B act as master regulators of neural cell polarity in the developing mouse neocortex. They are required for maintaining the polarity of RGCs and directing the MTB transition of multipolar neurons. Here we show that the Rap1 guanine nucleotide exchange factor (GEF) C3G (encoded by the Rapgef1 gene) is a crucial regulator of the MTB transition in vivo by conditionally inactivating the Rapgef1 gene in the developing mouse cortex at different time points during neuronal development. Inactivation of C3G results in defects in neuronal migration, axon formation and cortical lamination. Live cell imaging shows that C3G is required in cortical neurons for both the specification of an axon and the initiation of radial migration by forming a leading process.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Factor 2 Liberador de Guanina Nucleótido/genética , Neocórtex/metabolismo , Neurogénesis/genética , Neuronas/metabolismo , Animales , Polaridad Celular , Embrión de Mamíferos , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neocórtex/citología , Neocórtex/embriología , Neuronas/citología , Transducción de Señal , Imagen de Lapso de Tiempo , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
RapGEF1 (C3G) is a ubiquitously expressed protein that is essential for mammalian embryonic development. We have shown earlier that C3G regulates cytoskeletal dynamics and is required for neuronal differentiation. To determine if C3G plays a wider role in differentiation of multiple tissue types, we examined its role in skeletal muscle differentiation using the model system of C2C12 cells in culture. C3G protein is highly expressed in mouse skeletal muscle and its transcript and protein levels increase as C2C12 cells are induced to differentiate. Increase in C3G was predominantly seen in the nuclei of myotubes. Ectopic expression of C3G promoted myotube formation when cells were cultured in growth as well as differentiation medium and, enhanced MHC levels were associated with C3G expression. C3G induced differentiation required its catalytic and protein interaction domains and was dependent on the function of cellular R-Ras. Knockdown of cellular C3G using small hairpin RNA reduced expression of muscle specific markers and ß-catenin, resulting in impaired differentiation. Disabling C3G function also resulted in enhanced cell death suggesting that cellular C3G is required for cell survival. In cells grown in growth medium, over-expressed C3G increased Akt activity, and C3G knockdown reduced it. C3G expression also suppressed cyclin D1 levels, and induced p27 expression, molecules involved in regulating cell proliferation. Endogenous C3G localizes to focal adhesions in myotubes and C3G expressing cells show distinct stress fibers, elongation and parallel alignment. Expression of a dominant negative construct of C3G, disrupts actin cytoskeleton and formation of focal adhesions resulting in detachment of cells from the substratum and inhibition of differentiation. Our results provide evidence that C3G plays an important role in myogenic differentiation by coordinating cell cycle exit, actin dynamics and survival signaling.