RESUMO
Ras-related protein 1 (Rap1) is a major convergence point of the platelet-signaling pathways that result in talin-1 binding to the integrin ß cytoplasmic domain and consequent integrin activation, platelet aggregation, and effective hemostasis. The nature of the connection between Rap1 and talin-1 in integrin activation is an important remaining gap in our understanding of this process. Previous work identified a low-affinity Rap1-binding site in the talin-1 F0 domain that makes a small contribution to integrin activation in platelets. We recently identified an additional Rap1-binding site in the talin-1 F1 domain that makes a greater contribution than F0 in model systems. Here we generated mice bearing point mutations, which block Rap1 binding without affecting talin-1 expression, in either the talin-1 F1 domain (R118E) alone, which were viable, or in both the F0 and F1 domains (R35E,R118E), which were embryonic lethal. Loss of the Rap1-talin-1 F1 interaction in platelets markedly decreases talin-1-mediated activation of platelet ß1- and ß3-integrins. Integrin activation and platelet aggregation in mice whose platelets express only talin-1(R35E, R118E) are even more impaired, resembling the defect seen in platelets lacking both Rap1a and Rap1b. Although Rap1 is important in thrombopoiesis, platelet secretion, and surface exposure of phosphatidylserine, loss of the Rap1-talin-1 interaction in talin-1(R35E, R118E) platelets had little effect on these processes. These findings show that talin-1 is the principal direct effector of Rap1 GTPases that regulates platelet integrin activation in hemostasis.
Assuntos
Integrina beta1/metabolismo , Integrina beta3/metabolismo , Mutação Puntual , Talina/fisiologia , Trombopoese , Proteínas rap de Ligação ao GTP/fisiologia , Proteínas rap1 de Ligação ao GTP/fisiologia , Animais , Feminino , Integrina beta1/genética , Integrina beta3/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ativação Plaquetária , Agregação Plaquetária , Domínios Proteicos , Transdução de SinaisRESUMO
Androgen receptor (AR) splice variants (ARVs) are implicated in development of castration-resistant prostate cancer (CRPC). Upregulation of ARVs often correlates with persistent AR activity after androgen deprivation therapy (ADT). However, the genomic and epigenomic characteristics of ARV-dependent cistrome and the disease relevance of ARV-mediated transcriptome remain elusive. Through integrated chromatin immunoprecipitation coupled sequencing (ChIP-seq) and RNA sequencing (RNA-seq) analysis, we identified ARV-preferential-binding sites (ARV-PBS) and a set of genes preferentially transactivated by ARVs in CRPC cells. ARVs preferentially bind to enhancers located in nucleosome-depleted regions harboring the full AR-response element (AREfull), while full-length AR (ARFL)-PBS are enhancers resided in closed chromatin regions containing the composite FOXA1-nnnn-AREhalf motif. ARV-PBS exclusively overlapped with AR binding sites in castration-resistant (CR) tumors in patients and ARV-preferentially activated genes were up-regulated in abiraterone-resistant patient specimens. Expression of ARV-PBS target genes, such as oncogene RAP2A and cell cycle gene E2F7, were significantly associated with castration resistance, poor survival and tumor progression. We uncover distinct genomic and epigenomic features of ARV-PBS, highlighting that ARVs are useful tools to depict AR-regulated oncogenic genome and epigenome landscapes in prostate cancer. Our data also suggest that the ARV-preferentially activated transcriptional program could be targeted for effective treatment of CRPC.
Assuntos
Androstenos/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/genética , Receptores Androgênicos/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/metabolismo , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Epigênese Genética , Genômica , Humanos , Masculino , Camundongos SCID , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Isoformas de Proteínas/metabolismo , Splicing de RNA , Receptores Androgênicos/genética , Proteínas rap de Ligação ao GTP/fisiologiaRESUMO
Rap has different biological functions on intracellular signaling pathways, such as regulating cell polarity, cell proliferation, cell differentiation, cell adhesion and cell movement. Furthermore, at tissue and organ level, Rap controls the establishment of neural polarity, synaptic growth, synaptic plasticity, neuronal migration and so on. Rap belongs to Ras family which contains two subtypes, Rap1 and Rap2. By binding GTP or GDP Rap transform between active or inactive state, and plays an important role as a molecular switch. Moreover, in the signal pathway of tumor, Rap inhibits cell transformation induced by the oncogene Ras, therefore inhibits the proliferation, invasion and migration of certain cancer cells by interacting with its downstream target molecules. In this review, we summarized the biological functions of Rap and discussed It's significance in cancer therapy and drug treatment of neurological diseases.
Assuntos
Proteínas rap de Ligação ao GTP/fisiologia , Adesão Celular , Movimento Celular , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças do Sistema Nervoso/metabolismo , Plasticidade Neuronal , Transdução de SinaisRESUMO
A single microRNA (miRNA) can regulate expression of multiple proteins, and expression of an individual protein may be controlled by numerous miRNAs. This regulatory pattern strongly suggests that synergistic effects of miRNAs play critical roles in regulating biological processes. miR-9 and miR-124, two of the most abundant miRNAs in the mammalian nervous system, have important functions in neuronal development. In this study, we identified the small GTP-binding protein Rap2a as a common target of both miR-9 and miR-124. miR-9 and miR-124 together, but neither miRNA alone, strongly suppressed Rap2a, thereby promoting neuronal differentiation of neural stem cells (NSCs) and dendritic branching of differentiated neurons. Rap2a also diminished the dendritic complexity of mature neurons by decreasing the levels of pAKT and pGSK3ß. Our results reveal a novel pathway in which miR-9 and miR-124 synergistically repress expression of Rap2a to sustain homeostatic dendritic complexity during neuronal development and maturation.
Assuntos
Regulação da Expressão Gênica/genética , MicroRNAs/genética , Neurogênese/genética , Proteínas rap de Ligação ao GTP/antagonistas & inibidores , Regiões 3' não Traduzidas/genética , Animais , Dendritos/ultraestrutura , Glicogênio Sintase Quinase 3 beta/fisiologia , Células HEK293 , Homeostase , Humanos , Camundongos , Células-Tronco Neurais/citologia , Neurônios/ultraestrutura , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Proteínas rap de Ligação ao GTP/genética , Proteínas rap de Ligação ao GTP/fisiologiaRESUMO
Rap2, a member of the GTP-binding proteins, is widely upregulated in many types of tumors. The specific effectors of Rap2 can affect multiple cancer-associated cellular processes, including cytoskeleton reorganization, proliferation, migration, and inflammation. However, the functional role of Rap2 in tumorigenesis and the interplay between different effectors remain to be fully elucidated. A more thorough understanding of the cancer-associated signaling networks of Rap2 is expected to facilitate drug discovery targeting Rap2 for cancer therapy. The present review mainly focused on recent studies on the functional and physical interactions between Rap2 and its effectors. We also speculated on the relevance of these pathways to tumorigenesis.
Assuntos
Proteínas rap de Ligação ao GTP/fisiologia , HumanosRESUMO
Neutrophils are the first line of cellular defense in response to infections and inflammatory injuries. However, neutrophil activation and accumulation into tissues trigger tissue damage due to release of a plethora of toxic oxidants and proteases, a cause of acute lung injury (ALI). Despite its clinical importance, the molecular regulation of neutrophil migration is poorly understood. The small GTPase Rap1b is generally viewed as a positive regulator of immune cell functions by controlling bidirectional integrin signaling. However, we found that Rap1b-deficient mice exhibited enhanced neutrophil recruitment to inflamed lungs and enhanced susceptibility to endotoxin shock. Unexpectedly, Rap1b deficiency promoted the transcellular route of diapedesis through endothelial cell. Increased transcellular migration of Rap1b-deficient neutrophils in vitro was selectively mediated by enhanced PI3K-Akt activation and invadopodia-like protrusions. Akt inhibition in vivo suppressed excessive Rap1b-deficient neutrophil migration and associated endotoxin shock. The inhibitory action of Rap1b on PI3K signaling may be mediated by activation of phosphatase SHP-1. Thus, this study reveals an unexpected role for Rap1b as a key suppressor of neutrophil migration and lung inflammation.
Assuntos
Quimiotaxia de Leucócito/fisiologia , Infiltração de Neutrófilos/fisiologia , Neutrófilos/fisiologia , Proteínas rap de Ligação ao GTP/fisiologia , Actinas/metabolismo , Animais , Antígeno CD11b/metabolismo , Quimiotaxia de Leucócito/imunologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Doenças do Sistema Imunitário/imunologia , Doenças do Sistema Imunitário/fisiopatologia , Transtornos Leucocíticos/imunologia , Transtornos Leucocíticos/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Migração Transendotelial e Transepitelial/imunologia , Migração Transendotelial e Transepitelial/fisiologia , Proteínas rap de Ligação ao GTP/deficiência , Proteínas rap de Ligação ao GTP/genéticaRESUMO
Lymphocyte recruitment is regulated by signaling modules based on the activity of Rho and Rap small guanosine triphosphatases that control integrin activation by chemokines. We show that Janus kinase (JAK) protein tyrosine kinases control chemokine-induced LFA-1- and VLA-4-mediated adhesion as well as human T lymphocyte homing to secondary lymphoid organs. JAK2 and JAK3 isoforms, but not JAK1, mediate CXCL12-induced LFA-1 triggering to a high affinity state. Signal transduction analysis showed that chemokine-induced activation of the Rho module of LFA-1 affinity triggering is dependent on JAK activity, with VAV1 mediating Rho activation by JAKs in a Gαi-independent manner. Furthermore, activation of Rap1A by chemokines is also dependent on JAK2 and JAK3 activity. Importantly, activation of Rap1A by JAKs is mediated by RhoA and PLD1, thus establishing Rap1A as a downstream effector of the Rho module. Thus, JAK tyrosine kinases control integrin activation and dependent lymphocyte trafficking by bridging chemokine receptors to the concurrent and hierarchical activation of the Rho and Rap modules of integrin activation.
Assuntos
Integrinas/fisiologia , Janus Quinases/fisiologia , Linfócitos T/fisiologia , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Adesão Celular , Quimiocina CXCL12/metabolismo , Humanos , Integrina alfa4beta1/metabolismo , Integrina alfa4beta1/fisiologia , Integrinas/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno-1 Associado à Função Linfocitária/fisiologia , Fosfolipase D/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteínas Proto-Oncogênicas c-vav/fisiologia , Transdução de Sinais , Linfócitos T/metabolismo , Proteínas rap de Ligação ao GTP/fisiologia , Proteínas rho de Ligação ao GTP/fisiologiaRESUMO
Cell-matrix adhesion strongly influences developmental signaling. Resulting impacts on cell migration and tissue morphogenesis are well characterized. However, the in vivo impact of adhesion on fate induction remains ambiguous. Here, we employ the invertebrate chordate Ciona intestinalis to delineate an essential in vivo role for matrix adhesion in heart progenitor induction. In Ciona pre-cardiac founder cells, invasion of the underlying epidermis promotes localized induction of the heart progenitor lineage. We found that these epidermal invasions are associated with matrix adhesion along the pre-cardiac cell/epidermal boundary. Through targeted manipulations of RAP GTPase activity, we were able to manipulate pre-cardiac cell-matrix adhesion. Targeted disruption of pre-cardiac cell-matrix adhesion blocked heart progenitor induction. Conversely, increased matrix adhesion generated expanded induction. We were also able to selectively restore cell-matrix adhesion and heart progenitor induction through targeted expression of Ci-Integrin ß2. These results indicate that matrix adhesion functions as a necessary and sufficient extrinsic cue for regional heart progenitor induction. Furthermore, time-lapse imaging suggests that cytokinesis acts as an intrinsic temporal regulator of heart progenitor adhesion and induction. Our findings highlight a potentially conserved role for matrix adhesion in early steps of vertebrate heart progenitor specification.
Assuntos
Polaridade Celular/fisiologia , Junções Célula-Matriz/fisiologia , Ciona intestinalis/embriologia , Indução Embrionária , Coração/embriologia , Células-Tronco/fisiologia , Animais , Animais Geneticamente Modificados , Adesão Celular/genética , Adesão Celular/fisiologia , Polaridade Celular/genética , Junções Célula-Matriz/genética , Junções Célula-Matriz/metabolismo , Cordados/embriologia , Cordados/genética , Cordados/metabolismo , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , Embrião não Mamífero , Indução Embrionária/genética , Indução Embrionária/fisiologia , Invertebrados/embriologia , Invertebrados/genética , Invertebrados/metabolismo , Modelos Biológicos , Células-Tronco/metabolismo , Proteínas rap de Ligação ao GTP/genética , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas rap de Ligação ao GTP/fisiologiaRESUMO
RUN domain is present in several proteins related to the functions of Rap and Rab family GTPases. Accumulating evidence supports the hypothesis that RUN domain-containing proteins act as a component of vesicle traffic and might be responsible for an interaction with a filamentous network linked to actin cytoskeleton or microtubules. That is to say, on one hand, RUN domains associate with Rab or Rap family proteins, on the other hand, they also might interact with motor proteins such as kinesin or myosin via intervention molecules. In this review, we summarize the background and current status of RUN domain research with an emphasis on the interaction between RUN domain and motor proteins with respect to the vesicle traffic on filamentous network.
Assuntos
Proteínas Motores Moleculares/metabolismo , Proteínas rab de Ligação ao GTP/fisiologia , Proteínas rap de Ligação ao GTP/fisiologia , Transporte Biológico , Estrutura Terciária de Proteína , Proteínas rab de Ligação ao GTP/química , Proteínas rap de Ligação ao GTP/químicaRESUMO
Misshapen/NIKs (Nck-interacting kinases)-related kinase (MINK) and closely related TRAF2/Nck-interacting kinase (TNIK) are proteins that specifically bind to activated Rap2 and are thus hypothesized to relay its downstream signal transduction. Activated Rap2 has been found to stimulate dendritic pruning, reduce synaptic density and cause removal of synaptic AMPA receptors (AMPA-Rs) (Zhu et al., 2005; Fu et al., 2007). Here we report that MINK and TNIK are postsynaptically enriched proteins whose clustering within dendrites is bidirectionally regulated by the activation state of Rap2. Expression of MINK and TNIK in neurons is required for normal dendritic arborization and surface expression of AMPA receptors. Overexpression of a truncated MINK mutant unable to interact with Rap2 leads to reduced dendritic branching and this MINK-mediated effect on neuronal morphology is dependent upon Rap2 activation. While similarly truncated TNIK also reduces neuronal complexity, its effect does not require Rap2 activity. Furthermore, Rap2-mediated removal of surface AMPA-Rs from spines is entirely abrogated by coexpression of MINK, but not TNIK. Thus, although both MINK and TNIK bind GTP-bound Rap2, these kinases employ distinct mechanisms to modulate Rap2-mediated signaling. MINK appears to antagonize Rap2 signal transduction by binding to activated Rap2. We suggest that MINK interaction with Rap2 plays a critical role in maintaining the morphological integrity of dendrites and synaptic transmission.
Assuntos
Diferenciação Celular/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptores de AMPA/metabolismo , Transdução de Sinais/genética , Proteínas rap de Ligação ao GTP/fisiologia , Animais , Células Cultivadas , Dendritos/metabolismo , Dendritos/ultraestrutura , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Mutação/genética , Neurônios/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/fisiologia , Transmissão Sináptica/fisiologia , Proteínas rap de Ligação ao GTP/genética , Proteínas rap de Ligação ao GTP/metabolismoRESUMO
Signaling by the B cell receptor (BCR) promotes integrin-mediated adhesion and cytoskeletal reorganization. This results in B cell spreading, which enhances the ability of B cells to bind antigens and become activated. Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related cytoplasmic tyrosine kinases that regulate cell adhesion, cell morphology, and cell migration. In this report we show that BCR signaling and integrin signaling collaborate to induce the phosphorylation of Pyk2 and FAK on key tyrosine residues, a modification that increases the kinase activity of Pyk2 and FAK. Activation of the Rap GTPases is critical for BCR-induced integrin activation as well as for BCR- and integrin-induced reorganization of the actin cytoskeleton. We now show that Rap activation is essential for BCR-induced phosphorylation of Pyk2 and for integrin-induced phosphorylation of Pyk2 and FAK. Moreover Rap-dependent phosphorylation of Pyk2 and FAK required an intact actin cytoskeleton as well as actin dynamics, suggesting that Rap regulates Pyk2 and FAK via its effects on the actin cytoskeleton. Importantly B cell spreading induced by BCR/integrin co-stimulation or by integrin engagement was inhibited by short hairpin RNA-mediated knockdown of either Pyk2 or FAK expression and by treatment with PF-431396, a chemical inhibitor that blocks the kinase activities of both Pyk2 and FAK. Thus Pyk2 and FAK are downstream targets of the Rap GTPases that play a key role in regulating B cell morphology.
Assuntos
Receptor do Fator Ativador de Células B/fisiologia , Linfócitos B/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Integrinas/fisiologia , Proteínas rap de Ligação ao GTP/fisiologia , Actinas/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Matriz Extracelular/metabolismo , Imunofluorescência , Quinase 1 de Adesão Focal/genética , Quinase 2 de Adesão Focal/genética , Immunoblotting , Integrinas/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Fosforilação , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas rap de Ligação ao GTP/genéticaRESUMO
BACKGROUND: The platelet alpha2beta1 integrin functions as both an adhesion and signaling receptor upon exposure to collagen. Recent studies have indicated that alpha2beta1 function can be activated via inside-out signaling, similar to the prototypical platelet integrin alphaIIbbeta3. However, signaling molecules that regulate alpha2beta1 activation in platelets are not well defined. A strong candidate molecule is the small GTPase Rap1b, the dominant platelet isoform of Rap1, which regulates alphaIIbbeta3 activation. OBJECTIVES: We hypothesized that Rap1b positively regulates alpha2beta1 during agonist-induced platelet activation. METHODS: To test whether Rap1b activates alpha2beta1 downstream of glycoprotein (GP)VI or other platelet receptors, we stimulated platelets purified from Rap1b-/- or wild-type mice with diverse agonists and measured alpha2beta1 activation using fluorescein isothiocyanate-labeled monomeric collagen. We also examined the role of Rap1b in outside-in signaling pathways by analyzing adhesion and spreading of Rap1b-/- or wild-type platelets on monomeric, immobilized collagen. Finally, we monitored the activation status of related Rap GTPases to detect changes in signaling pathways potentially associated with Rap1b-mediated events. RESULTS: Rap1b-/- platelets displayed comparable ADP-induced or thrombin-induced alpha2beta1 activation as wild-type platelets, but reduced convulxin-dependent alpha2beta1 activation. Rap1b-/- platelets exhibited increased spreading on immobilized collagen but similar adhesion to immobilized collagen compared to wild-type platelets. Rap1b-/- platelets also showed Rap1a and Rap2 activation upon agonist stimulation, possibly revealing functional compensation among Rap family members. CONCLUSIONS: Rap1b is required for maximal GPVI-induced but not ADP-induced activation of alpha2beta1 in murine platelets.
Assuntos
Integrina alfa2beta1/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Purinérgicos P2/metabolismo , Proteínas rap de Ligação ao GTP/fisiologia , Animais , Plaquetas/citologia , Plaquetas/metabolismo , Forma Celular , Colágeno , Camundongos , Camundongos Knockout , Adesividade Plaquetária , Transdução de Sinais , Proteínas rap de Ligação ao GTP/deficiênciaRESUMO
Intracranial hemorrhage (ICH) is a particularly severe form of stroke whose etiology remains poorly understood, with a highly variable appearance and onset of the disease (Felbor et al., 2006; Frizzell, 2005; Lucas et al., 2003). In humans, mutations in any one of three CCM genes causes an autosomal dominant genetic ICH disorder characterized by cerebral cavernous malformations (CCM). Recent evidence highlighting multiple interactions between the three CCM gene products and other proteins regulating endothelial junctional integrity suggests that minor deficits in these other proteins could potentially predispose to, or help to initiate, CCM, and that combinations of otherwise silent genetic deficits in both the CCM and interacting proteins might explain some of the variability in penetrance and expressivity of human ICH disorders. Here, we test this idea by combined knockdown of CCM pathway genes in zebrafish. Reducing the function of rap1b, which encodes a Ras GTPase effector protein for CCM1/Krit1, disrupts endothelial junctions in vivo and in vitro, showing it is a crucial player in the CCM pathway. Importantly, a minor reduction of Rap1b in combination with similar reductions in the products of other CCM pathway genes results in a high incidence of ICH. These findings support the idea that minor polygenic deficits in the CCM pathway can strongly synergize to initiate ICH.
Assuntos
Hemorragias Intracranianas/genética , Proteínas Associadas aos Microtúbulos/genética , Animais , Sequência de Bases , Adesão Celular/fisiologia , Células Cultivadas , Clonagem Molecular , Primers do DNA , Humanos , Imuno-Histoquímica , Junções Intercelulares , Microscopia Eletrônica de Transmissão , Peixe-Zebra , Proteínas rap de Ligação ao GTP/fisiologiaRESUMO
We demonstrate that lck promoter-driven conditional expression of transgenic SPA-1, a Rap GTPase-activation protein, causes a profound defect of alphabeta T-cell development at the CD4/CD8 double-negative (DN) stage due to enhanced cell death without affecting gammadelta T-cell development. The effect was specific to the DN stage, because CD4 promoter-driven SPA-1 expression hardly affected T-cell development. Rap1A17, a dominant-negative Rap mutant, interfered with the generation of double-positive (DP) cells from Rag2(-/-) fetal thymocytes in vitro in the presence of anti-CD3epsilon antibody and Notch ligand. Rap GTPases were activated in a DN cell line by the expression of self-oligomerizing CD3 (CD8:CD3epsilon chimera), which substituted autonomous pre-T-cell receptor (TCR) signal, inducing CD69 expression and CD25 down-regulation. Reciprocally, expression of C3G, a Rap guanine nucleotide exchange factor, in both normal and Rag2(-/-) DN cells markedly enhanced Notch-dependent generation and expansion of DP cells without additional anti-CD3epsilon antibody, thus bypassing pre-TCR. Defective alphabeta T-cell development in the conditional SPA-1-transgenic mice was restored completely by introducing a p53(-/-) mutation. These results suggest that endogenous Rap GTPases downstream of pre-TCR play an essential role in rescuing pre-T cells from the p53-mediated checkpoint response, thus allowing Notch-mediated expansion and differentiation.
Assuntos
Diferenciação Celular/genética , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Proteínas rap de Ligação ao GTP/fisiologia , Animais , Morte Celular/genética , Morte Celular/imunologia , Diferenciação Celular/imunologia , Proliferação de Células , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/fisiologia , Genes p53/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores Notch/fisiologia , Transdução de Sinais/fisiologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Timo/imunologia , Timo/metabolismo , Proteínas rap de Ligação ao GTP/genéticaRESUMO
The role of tubular injury in diabetic nephropathy is relatively unknown, despite that apoptosis of tubular epithelial cells is commonly observed in human renal biopsies. The GTPase Ras-proximate-1 (Rap1b) is upregulated in the hyperglycemic state and is known to increase B-Raf, an antiapoptotic effector protein. In this study, the effects of high glucose on renal tubular apoptosis and the potential ability for Rap1b to ameliorate these effects were investigated. In the kidneys of diabetic mice, apoptotic tubular cells and dysmorphic mitochondria were observed, Bcl-2 expression was decreased, and Bax expression was increased. Total Rap1b expression was slightly increased, but its associated GTPase activity was significantly decreased. In vitro, high extracellular glucose led to decreased Bcl-2 expression, reduced Rap1b GTPase activity, and increased levels of both Bax and GTPase activating protein in a proximal tubular cell line (HK-2). These changes were accompanied by increased DNA fragmentation, decreased high molecular weight mitochondrial DNA, altered mitochondrial morphology and function, disrupted Bcl-2-Bax and Bcl-2-Rap1b interactions, and reduced cell survival. Overexpression of Rap1b partially prevents these abnormalities. Furthermore, the BH4 domain of Bcl-2 was found to be required for successful protein-protein interaction between Bcl-2 and Rap1b. In summary, these data suggest that Rap1b ameliorates glucose-induced mitochondrial dysfunction in renal tubular cells.
Assuntos
Glucose/metabolismo , Mitocôndrias/metabolismo , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas rap de Ligação ao GTP/fisiologia , Animais , Apoptose , Linhagem Celular , Citocromos c/metabolismo , DNA Mitocondrial/metabolismo , Diabetes Mellitus Experimental/metabolismo , Marcação In Situ das Extremidades Cortadas , Túbulos Renais/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Within the Ras superfamily of GTPases, Rap1 and Rap2 are the closest homologs to Ras. In non-neural cells, Rap signaling can antagonize Ras signaling. In neurons, Rap also seems to oppose Ras in terms of synaptic function. Whereas Ras is critical for long-term potentiation (LTP), Rap1 has been shown to be required for long-term depression (LTD), and Rap2 has been implicated in depotentiation. Moreover, active Rap1 and Rap2 cause loss of surface AMPA receptors and reduced miniature EPSC amplitude and frequency in cultured neurons. The role of Rap signaling in vivo, however, remains poorly understood. To study the function of Rap2 in the brain and in behavior, we created transgenic mice expressing either constitutively active (Rap2V12) or dominant-negative (Rap2N17) mutants of Rap2 in postnatal forebrain. Multiple lines of Rap2N17 mice showed only weak expression of the transgenic protein, and no phenotype was observed. Rap2V12 mice displayed fewer and shorter dendritic spines in CA1 hippocampal neurons, and enhanced LTD at CA3-CA1 synapses. Behaviorally, Rap2V12 mice showed impaired spatial learning and defective extinction of contextual fear, which correlated with reduced basal phosphorylation of extracellular signal-regulated kinase (ERK) and blunted activation of ERK during fear extinction training. Our data support the idea that Rap2 opposes Ras-ERK signaling in the brain, thereby inhibiting dendritic spine development/maintenance, promoting synaptic depression rather than LTP, and impairing learning. The findings also implicate Rap2 signaling in fear extinction mechanisms, which are thought to be aberrant in anxiety disorders and posttraumatic stress disorder.
Assuntos
Espinhas Dendríticas/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Medo/fisiologia , Depressão Sináptica de Longo Prazo/genética , Sistema de Sinalização das MAP Quinases , Aprendizagem em Labirinto/fisiologia , Proteínas rap de Ligação ao GTP/genética , Animais , Espinhas Dendríticas/patologia , Extinção Psicológica/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/genética , Medo/psicologia , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibição Neural/genética , Comportamento Espacial/fisiologia , Transmissão Sináptica/genética , Proteínas rap de Ligação ao GTP/fisiologiaRESUMO
Rap1 is a small GTPase that belongs to Ras superfamily. This ubiquitously expressed GTPase is a key regulator of integrin functions. Rap1 exists in two isoforms: Rap1a and Rap1b. Although Rap1 has been extensively studied, its isoform-specific functions in B cells have not been elucidated. In this study, using gene knockout mice, we show that Rap1b is the dominant isoform in B cells. Lack of Rap1b significantly reduced the absolute number of B220(+)IgM(-) pro/pre-B cells and B220(+)IgM(+) immature B cells in bone marrow. In vitro culture of bone marrow-derived Rap1b(-/-) pro/pre-B cells with IL-7 showed similar proliferation levels but reduced adhesion to stromal cell line compared with wild type. Rap1b(-/-) mice displayed reduced splenic marginal zone (MZ) B cells, and increased newly forming B cells, whereas the number of follicular B cells was normal. Functionally, Rap1b(-/-) mice showed reduced T-dependent but normal T-independent humoral responses. B cells from Rap1b(-/-) mice showed reduced migration to SDF-1, CXCL13 and in vivo homing to lymph nodes. MZ B cells showed reduced sphingosine-1-phosphate-induced migration and adhesion to ICAM-1. However, absence of Rap1b did not affect splenic B cell proliferation, BCR-mediated activation of Erk1/2, p38 MAPKs, and AKT. Thus, Rap1b is crucial for early B cell development, MZ B cell homeostasis and T-dependent humoral immunity.
Assuntos
Formação de Anticorpos , Linfócitos B/citologia , Linfócitos B/imunologia , Quimiotaxia de Leucócito , Proteínas rap de Ligação ao GTP/fisiologia , Animais , Medula Óssea , Adesão Celular , Proliferação de Células , Células Cultivadas , Linfonodos , Camundongos , Camundongos Knockout , Baço , Linfócitos T/imunologiaRESUMO
Chemotaxis, the directional movement towards a chemical compound, is an essential property of many cells and has been linked to the development and progression of many diseases. Eukaryotic chemotaxis is a complex process involving gradient sensing, cell polarity, remodelling of the cytoskeleton and signal relay. Recent studies in the model organism Dictyostelium discoideum have shown that chemotaxis does not depend on a single molecular mechanism, but rather depends on several interconnecting pathways. Surprisingly, small G-proteins appear to play essential roles in all these pathways. This review will summarize the role of small G-proteins in Dictyostelium, particularly highlighting the function of the Ras subfamily in chemotaxis.
Assuntos
Quimiotaxia , Dictyostelium/fisiologia , Proteínas rap de Ligação ao GTP/fisiologia , Proteínas ras/fisiologia , Animais , Dictyostelium/enzimologia , Dictyostelium/metabolismo , Transdução de SinaisRESUMO
B-cell development is orchestrated by complex signaling networks. Rap1 is a member of the Ras superfamily of small GTP-binding proteins and has 2 isoforms, Rap1a and Rap1b. Although Rap1 has been suggested to have an important role in a variety of cellular processes, no direct evidence demonstrates a role for Rap1 in B-cell biology. In this study, we found that Rap1b was the dominant isoform of Rap1 in B cells. We discovered that Rap1b deficiency in mice barely affected early development of B cells but markedly reduced marginal zone (MZ) B cells in the spleen and mature B cells in peripheral and mucosal lymph nodes. Rap1b-deficient B cells displayed normal survival and proliferation in vivo and in vitro. However, Rap1b-deficient B cells had impaired adhesion and reduced chemotaxis in vitro, and lessened homing to lymph nodes in vivo. Furthermore, we found that Rap1b deficiency had no marked effect on LPS-, BCR-, or SDF-1-induced activation of mitogen-activated protein kinases and AKT but clearly impaired SDF-1-mediated activation of Pyk-2, a key regulator of SDF-1-mediated B-cell migration. Thus, we have discovered a critical and distinct role of Rap1b in mature B-cell trafficking and development of MZ B cells.