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Johannes L. Bos - One of the best experts on this subject based on the ideXlab platform.
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Rap1 and Rap2 Antagonistically Control Endothelial Barrier Resistance
PloS one, 2013Co-Authors: Willem-jan Pannekoek, Johannes L. Bos, Jelena R. Linnemann, Patricia Brouwer, Holger RehmannAbstract:Rap1 and Rap2 are closely related proteins of the Ras family of small G-proteins. Rap1 is well known to regulate cell-cell adhesion. Here, we have analysed the effect of Rap-mediated signalling on endothelial permeability using electrical impedance measurements of HUVEC monolayers and subsequent determination of the barrier resistance, which is a measure for the ease with which ions can pass cell junctions. In line with its well-established effect on cell-cell junctions, depletion of Rap1 decreases, whereas activation of Rap1 increases barrier resistance. Despite its high sequence homology with Rap1, depletion of Rap2 has an opposite, enhancing, effect on barrier resistance. This effect can be mimicked by depletion of the Rap2 specific activator RasGEF1C and the Rap2 effector MAP4K4, establishing Rap2 signalling as an independent pathway controlling barrier resistance. As simultaneous depletion or activation of both Rap1 and Rap2 results in a barrier resistance comparable to control cells, Rap1 and Rap2 control barrier resistance in a reciprocal manner. This Rap1-antagonizing effect of Rap2 is established independent of junctional actin formation. These data establish that endothelial barrier resistance is determined by the combined antagonistic actions of Rap1 and Rap2.
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Rap1: a key regulator in cell-cell junction formation.
Journal of cell science, 2007Co-Authors: Matthijs R H Kooistra, Nadia Dubé, Johannes L. BosAbstract:Rap1 is a Ras-like small GTPase that is activated by many extracellular stimuli and strongly implicated in the control of integrin-mediated cell adhesion. Recent evidence indicates that Rap1 also plays a key role in formation of cadherin-based cell-cell junctions. Indeed, inhibition of Rap1 generates immature adherens junctions, whereas activation of Rap1 tightens cell-cell junctions. Interestingly, Rap1 guanine nucleotide exchange factors, such as C3G and PDZ-GEF, are directly linked to E-cadherin or to other junction proteins. Furthermore, several junction proteins, such as afadin/AF6 and proteins controlling the actin cytoskeleton, function as effectors of Rap1. These findings point to a role of Rap1 in spatial and temporal control of cell-cell junction formation.
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The role of Rap1 in integrin-mediated cell adhesion.
Biochemical Society Transactions, 2003Co-Authors: Johannes L. Bos, Jorrit Martijn Enserink, K. De Bruyn, B. Kuiperij, S. Rangarajan, Holger Rehmann, Jurgen Riedl, J. De Rooij, F. Van Mansfeld, Fried J. T. ZwartkruisAbstract:Rap1 is a member of the Ras-like small GTPases. Originally the protein was identified in a genome-wide screen for suppressors of Ras transformation, but the mechanism of this reversion remained elusive. We have investigated the signalling function of Rap1. We observed that Rap1 is activated by a large variety of stimuli, including growth factors, neurotransmitters and cytokines. Common second messengers like cAMP, diacylglycerol and calcium are mediators of this activation. These messengers activate guanine nucleotide exchange factors (GEFs), the most notable of which is Epac (exchange protein directly activated by cAMP). However, the downstream effectors of Rap1 are less clear. Although direct connections of Rap1 with the serine/threonine kinases Raf1 and B-raf have been reported, we were unable to find functional evidence for an interaction of endogenous Rap1 signalling with the Raf/extracellular-signal-regulated kinase (ERK) pathway. Instead we observe a clear connection of Rap1 with inside-out signalling to integrins. Indeed, introduction of a constitutively active Rap1 as well as Epac induces integrin-mediated cell adhesion, whereas inhibition of Rap1 signalling by the introduction of Rap1GAP (GTPase-activating protein) inhibits inside-out activation of integrins. More importantly, activation of a G s -protein-coupled receptor results in integrin-mediated cell adhesion, by a pathway involving Epac and Rap1. From these results, we conclude that one of the functions of receptor-induced Rap1 activation is inside-out regulation of integrins.
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Sequential regulation of the small GTPase Rap1 in human platelets.
Molecular and cellular biology, 2000Co-Authors: Barbara Franke, Miranda Van Triest, Kim M. T. De Bruijn, Gijsbert Van Willigen, H. Karel Nieuwenhuis, Claude Negrier, Jan-willem N. Akkerman, Johannes L. BosAbstract:Rap1, a small GTPase of the Ras family, is ubiquitously expressed and particularly abundant in platelets. Previously we have shown that Rap1 is rapidly activated after stimulation of human platelets with alpha-thrombin. For this activation, a phospholipase C-mediated increase in intracellular calcium is necessary and sufficient. Here we show that thrombin induces a second phase of Rap1 activation, which is mediated by protein kinase C (PKC). Indeed, the PKC activator phorbol 12-myristate 13-acetate induced Rap1 activation, whereas the PKC-inhibitor bisindolylmaleimide inhibited the second, but not the first, phase of Rap1 activation. Activation of the integrin alpha(IIb)beta(3), a downstream target of PKC, with monoclonal antibody LIBS-6 also induced Rap1 activation. However, studies with alpha(IIb)beta(3)-deficient platelets from patients with Glanzmann's thrombasthenia type 1 show that alpha(IIb)beta(3) is not essential for Rap1 activation. Interestingly, induction of platelet aggregation by thrombin resulted in the inhibition of Rap1 activation. This downregulation correlated with the translocation of Rap1 to the Triton X-100-insoluble, cytoskeletal fraction. We conclude that in platelets, alpha-thrombin induces Rap1 activation first by a calcium-mediated pathway independently of PKC and then by a second activation phase mediated by PKC and, in part, integrin alpha(IIb)beta(3). Inactivation of Rap1 is mediated by an aggregation-dependent process that correlates with the translocation of Rap1 to the cytoskeletal fraction.
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Activation of the Ras-related GTPase Rap1 by thymocyte TCR engagement and during selection
European journal of immunology, 2000Co-Authors: Derk Amsen, Johannes L. Bos, Ada M. Kruisbeek, Kris A. ReedquistAbstract:Signals mediated by activation of the small GTPase Ras play an essential role both in thymocyte development and in TCR-mediated activation of mature T cells. Given the critical requirement of Ras signaling pathways in thymocyte development, and recent indications that Rap1 may negatively regulate Ras-dependent signaling pathways, we examined the possible involvement of Rap1 in thymocyte TCR signaling. We find that Rap1 and proposed regulators of Rap1 (the proto-oncogene product Cbl, Crk family adaptor proteins, and the Rap1 guanine nucleotide exchange factor C3G) are expressed at equivalent levels in both double-negative and double-positive murine thymocytes. Rap1 was transiently activated following TCR stimulation of both total thymocytes and purified double-positive thymocytes, and this activation correlated with tyrosine phosphorylation of Cbl and Cbl association with CrkL. TCR-dependent Rap1 activation was enhanced by co-stimulation through CD28 and could be mimicked by treatment of thymocytes with phorbol ester and calcium. In contrast to mature peripheral T lymphocytes, Rap1 stimulation by CD3 ligation in thymocytes did not require intracellular calcium mobilization. Intriguingly, we found a clear elevation of activated Rap1 in thymocytes undergoing positive selection, suggesting a functional role for Rap1 in thymocyte development and selection.
Eric R Kandel - One of the best experts on this subject based on the ideXlab platform.
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Rap1 couples cAMP signaling to a distinct pool of p42/44MAPK regulating excitability, synaptic plasticity, learning, and memory.
Neuron, 2003Co-Authors: Alexei Morozov, Isabel A. Muzzio, Rusiko Bourtchouladze, Niels Van-strien, Kyle A.b. Lapidus, Deqi Yin, Danny G. Winder, J. Paige Adams, J. David Sweatt, Eric R KandelAbstract:Learning-induced synaptic plasticity commonly involves the interaction between cAMP and p42/44MAPK. To investigate the role of Rap1 as a potential signaling molecule coupling cAMP and p42/44MAPK, we expressed an interfering Rap1 mutant (iRap1) in the mouse forebrain. This expression selectively decreased basal phosphorylation of a membrane-associated pool of p42/44MAPK, impaired cAMP-dependent LTP in the hippocampal Schaffer collateral pathway induced by either forskolin or theta frequency stimulation, decreased complex spike firing, and reduced the p42/44MAPK-mediated phosphorylation of the A-type potassium channel Kv4.2. These changes correlated with impaired spatial memory and context discrimination. These results indicate that Rap1 couples cAMP signaling to a selective membrane-associated pool of p42/44MAPK to control excitability of pyramidal cells, the early and late phases of LTP, and the storage of spatial memory.
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Rap1 couples camp signaling to a distinct pool of p42 44mapk regulating excitability synaptic plasticity learning and memory
Neuron, 2003Co-Authors: Alexei Morozov, Isabel A. Muzzio, Rusiko Bourtchouladze, Kyle A.b. Lapidus, Deqi Yin, Danny G. Winder, Niels Vanstrien, Paige J Adams, David J Sweatt, Eric R KandelAbstract:Learning-induced synaptic plasticity commonly involves the interaction between cAMP and p42/44MAPK. To investigate the role of Rap1 as a potential signaling molecule coupling cAMP and p42/44MAPK, we expressed an interfering Rap1 mutant (iRap1) in the mouse forebrain. This expression selectively decreased basal phosphorylation of a membrane-associated pool of p42/44MAPK, impaired cAMP-dependent LTP in the hippocampal Schaffer collateral pathway induced by either forskolin or theta frequency stimulation, decreased complex spike firing, and reduced the p42/44MAPK-mediated phosphorylation of the A-type potassium channel Kv4.2. These changes correlated with impaired spatial memory and context discrimination. These results indicate that Rap1 couples cAMP signaling to a selective membrane-associated pool of p42/44MAPK to control excitability of pyramidal cells, the early and late phases of LTP, and the storage of spatial memory.
Alexei Morozov - One of the best experts on this subject based on the ideXlab platform.
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Rap1 couples cAMP signaling to a distinct pool of p42/44MAPK regulating excitability, synaptic plasticity, learning, and memory.
Neuron, 2003Co-Authors: Alexei Morozov, Isabel A. Muzzio, Rusiko Bourtchouladze, Niels Van-strien, Kyle A.b. Lapidus, Deqi Yin, Danny G. Winder, J. Paige Adams, J. David Sweatt, Eric R KandelAbstract:Learning-induced synaptic plasticity commonly involves the interaction between cAMP and p42/44MAPK. To investigate the role of Rap1 as a potential signaling molecule coupling cAMP and p42/44MAPK, we expressed an interfering Rap1 mutant (iRap1) in the mouse forebrain. This expression selectively decreased basal phosphorylation of a membrane-associated pool of p42/44MAPK, impaired cAMP-dependent LTP in the hippocampal Schaffer collateral pathway induced by either forskolin or theta frequency stimulation, decreased complex spike firing, and reduced the p42/44MAPK-mediated phosphorylation of the A-type potassium channel Kv4.2. These changes correlated with impaired spatial memory and context discrimination. These results indicate that Rap1 couples cAMP signaling to a selective membrane-associated pool of p42/44MAPK to control excitability of pyramidal cells, the early and late phases of LTP, and the storage of spatial memory.
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Rap1 couples camp signaling to a distinct pool of p42 44mapk regulating excitability synaptic plasticity learning and memory
Neuron, 2003Co-Authors: Alexei Morozov, Isabel A. Muzzio, Rusiko Bourtchouladze, Kyle A.b. Lapidus, Deqi Yin, Danny G. Winder, Niels Vanstrien, Paige J Adams, David J Sweatt, Eric R KandelAbstract:Learning-induced synaptic plasticity commonly involves the interaction between cAMP and p42/44MAPK. To investigate the role of Rap1 as a potential signaling molecule coupling cAMP and p42/44MAPK, we expressed an interfering Rap1 mutant (iRap1) in the mouse forebrain. This expression selectively decreased basal phosphorylation of a membrane-associated pool of p42/44MAPK, impaired cAMP-dependent LTP in the hippocampal Schaffer collateral pathway induced by either forskolin or theta frequency stimulation, decreased complex spike firing, and reduced the p42/44MAPK-mediated phosphorylation of the A-type potassium channel Kv4.2. These changes correlated with impaired spatial memory and context discrimination. These results indicate that Rap1 couples cAMP signaling to a selective membrane-associated pool of p42/44MAPK to control excitability of pyramidal cells, the early and late phases of LTP, and the storage of spatial memory.
Maria A. Blasco - One of the best experts on this subject based on the ideXlab platform.
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Rap1 protects from obesity through its extratelomeric role regulating gene expression
Cell Reports, 2013Co-Authors: Paula Martínez, Gonzalo Gomezlopez, Fernando Garcia, Evi M Mercken, Sarah J Mitchell, Juana M Flores, Rafael De Cabo, Maria A. BlascoAbstract:Rap1 is part of shelterin, the protective complex at telomeres. Rap1 also binds along chromosome arms, where it is proposed to regulate gene expression. To investigate the nontelomeric roles of Rap1 in vivo, we generated a Rap1 whole-body knockout mouse. These mice show early onset of obesity, which is more severe in females than in males. Rap1-deficient mice show accumulation of abdominal fat, hepatic steatosis, and high-fasting plasma levels of insulin, glucose, cholesterol, and alanine aminotransferase. Gene expression analyses of liver and visceral white fat from Rap1-deficient mice before the onset of obesity show deregulation of metabolic programs, including fatty acid, glucose metabolism, and PPARα signaling. We identify Pparα and Pgc1α as key factors affected by Rap1 deletion in the liver. We show that Rap1 binds to Pparα and Pgc1α loci and modulates their transcription. These findings reveal a role for a telomere-binding protein in the regulation of metabolism.
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Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites
Nature Cell Biology, 2010Co-Authors: Paula Martínez, Maria Thanasoula, Ana R. Carlos, Gonzalo Gómez-lópez, Agueda M. Tejera, Stefan Schoeftner, Orlando Dominguez, David G. Pisano, Madalena Tarsounas, Maria A. BlascoAbstract:The shelterin complex binds and protects mammalian telomeres. The shelterin component, Rap1, binds to non-telomeric regions and has extra-telomeric functions in transcriptional gene regulation. Rap1 is a component of the shelterin complex at mammalian telomeres, but its in vivo role in telomere biology has remained largely unknown to date. Here we show that Rap1 deficiency is dispensable for telomere capping but leads to increased telomere recombination and fragility. We generated cells and mice deleted for Rap1; mice with Rap1 deletion in stratified epithelia were viable but had shorter telomeres and developed skin hyperpigmentation in adulthood. By performing chromatin immunoprecipitation coupled with ultrahigh-throughput sequencing, we found that Rap1 binds to both telomeres and to extratelomeric sites through the (TTAGGG)_2 consensus motif. Extratelomeric Rap1-binding sites were enriched at subtelomeric regions, in agreement with preferential deregulation of subtelomeric genes in Rap1 -deficient cells. More than 70% of extratelomeric Rap1-binding sites were in the vicinity of genes, and 31% of the genes deregulated in Rap1 -null cells contained Rap1-binding sites, suggesting a role for Rap1 in transcriptional control. These findings place a telomere protein at the interface between telomere function and transcriptional regulation.
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mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites
Nature Cell Biology, 2010Co-Authors: Paula Martínez, Maria Thanasoula, Ana R. Carlos, Agueda M. Tejera, Stefan Schoeftner, Orlando Dominguez, David G. Pisano, Madalena Tarsounas, Gonzalo Gomezlopez, Maria A. BlascoAbstract:Rap1 is a component of the shelterin complex at mammalian telomeres, but its in vivo role in telomere biology has remained largely unknown to date. Here we show that Rap1 deficiency is dispensable for telomere capping but leads to increased telomere recombination and fragility. We generated cells and mice deleted for Rap1; mice with Rap1 deletion in stratified epithelia were viable but had shorter telomeres and developed skin hyperpigmentation in adulthood. By performing chromatin immunoprecipitation coupled with ultrahigh-throughput sequencing, we found that Rap1 binds to both telomeres and to extratelomeric sites through the (TTAGGG)(2) consensus motif. Extratelomeric Rap1-binding sites were enriched at subtelomeric regions, in agreement with preferential deregulation of subtelomeric genes in Rap1-deficient cells. More than 70% of extratelomeric Rap1-binding sites were in the vicinity of genes, and 31% of the genes deregulated in Rap1-null cells contained Rap1-binding sites, suggesting a role for Rap1 in transcriptional control. These findings place a telomere protein at the interface between telomere function and transcriptional regulation.
Titia De Lange - One of the best experts on this subject based on the ideXlab platform.
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talen gene knockouts reveal no requirement for the conserved human shelterin protein Rap1 in telomere protection and length regulation
Cell Reports, 2014Co-Authors: Shaheen Kabir, Dirk Hockemeyer, Titia De LangeAbstract:The conserved protein Rap1 functions at telomeres in fungi, protozoa, and vertebrates. Like yeast Rap1, human Rap1 has been implicated in telomere length regulation and repression of nonhomologous end-joining (NHEJ) at telomeres. However, mouse telomeres lacking Rap1 do not succumb to NHEJ. To determine the functions of human Rap1, we generated several transcription activator-like effector nuclease (TALEN)-mediated human cell lines lacking Rap1. Loss of Rap1 did not affect the other components of shelterin, the modification of telomeric histones, the subnuclear position of telomeres, or the 3' telomeric overhang. Telomeres lacking Rap1 did not show a DNA damage response, NHEJ, or consistent changes in their length, indicating that Rap1 does not have an important function in protection or length regulation of human telomeres. As human Rap1, like its mouse and unicellular orthologs, affects gene expression, we propose that the conservation of Rap1 reflects its role in transcriptional regulation rather than a function at telomeres.
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identification of human Rap1 implications for telomere evolution
Cell, 2000Co-Authors: Stephanie Oestreich, Titia De LangeAbstract:It has been puzzling that mammalian telomeric proteins, including TRF1, TRF2, tankyrase, and TIN2 have no recognized orthologs in budding yeast. Here, we describe a human protein, hRap1, that is an ortholog of the yeast telomeric protein, scRap1p. hRap1 has three conserved sequence motifs in common with scRap1, is located at telomeres, and affects telomere length. However, while scRap1 binds telomeric DNA directly, hRap1 is recruited to telomeres by TRF2. Extending the comparison of telomeric proteins to fission yeast, we identify S. pombe Taz1 as a TRF ortholog, indicating that TRFs are conserved at eukaryotic telomeres. The data suggest that ancestral telomeres, like those of vertebrates, contained a TRF-like protein as well as Rap1. We propose that budding yeast preserved Rap1 at telomeres but lost the TRF component, possibly concomitant with a change in the telomeric repeat sequence.