Johannes L Bos

University Medical Center Utrecht, Utrecht, Utrecht, Netherlands

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Publications (146)1465.37 Total impact

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    ABSTRACT: The small GTPase Rap1 controls the actin cytoskeleton by regulating Rho GTPase signaling. We have recently established that the Rap1 effectors Radil and Rasip1, together with the Rho GTPase activating protein ArhGAP29, mediate Rap1-induced inhibition of Rho signaling in the processes of epithelial cell spreading and endothelial barrier function. Here we show that Rap1 induces the independent translocations of Rasip1 and a Radil-ArhGAP29 complex to the plasma membrane. This results in the formation of a multimeric protein complex required for Rap1-induced inhibition of Rho signaling and increased endothelial barrier function. Together with the previously reported spatiotemporal control of the Rap guanine nucleotide exchange factor Epac1, these findings elucidate a signaling pathway for spatiotemporal control of Rho signaling, that operates by successive protein translocations to and complex formation at the plasma membrane. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    Molecular and Cellular Biology 05/2015; DOI:10.1128/MCB.01453-14 · 5.04 Impact Factor
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    ABSTRACT: The second messenger cAMP is known to augment glucose-induced insulin secretion. However, its downstream targets in pancreatic β-cells have not been unequivocally determined. Therefore, we designed cAMP analogues by a structure-guided approach that act as Epac2-selective agonists both in vitro and in vivo. These analogues activate Epac2 about two orders of magnitude more potently than cAMP. The high potency arises from increased affinity as well as increased maximal activation. Crystallographic studies demonstrate that this is due to unique interactions. At least one of the Epac2-specific agonists, Sp-8-BnT-cAMPS (S-220), enhances glucose-induced insulin secretion in human pancreatic cells. Selective targeting of Epac2 is thus proven possible and may be an option in diabetes treatment.
    PLoS Biology 01/2015; 13(1):e1002038. DOI:10.1371/journal.pbio.1002038 · 11.77 Impact Factor
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    Lucas J M Bruurs · Johannes L Bos
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    ABSTRACT: Brush border formation during polarity establishment of intestinal epithelial cells is uniquely governed by the Rap2A GTPase, despite expression of the other highly similar Rap2 isoforms (Rap2B and Rap2C). We investigated the mechanisms of this remarkable specificity and found that Rap2C is spatially segregated from Rap2A signaling as it is not enriched at the apical membrane after polarization. In contrast, both Rap2A and Rap2B are similarly located at Rab11 positive apical recycling endosomes and inside the brush border. However, although Rap2B localizes similarly it is not equally activated as Rap2A during brush border formation. We reveal that the C-terminal hypervariable region allows selective activation of Rap2A, yet this selectivity does not originate from the known differential lipid modifications of this region. In conclusion, we demonstrate that Rap2 specificity during brush border formation is determined by two distinct mechanisms involving segregated localization and selective activation.
    PLoS ONE 09/2014; 9(9):e106687. DOI:10.1371/journal.pone.0106687 · 3.23 Impact Factor
  • European Journal of Cancer 07/2014; 50:S171-S172. DOI:10.1016/S0959-8049(14)50628-4 · 4.82 Impact Factor
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    ABSTRACT: PDZGEF is a guanine nucleotide exchange factor for the small G protein Rap. It was recently found that PDZGEF contributes to establishment of intestinal epithelial polarity downstream of the kinase Lkb1. By binding to phosphatidic acid enriched at the apical membrane, PDZGEF locally activates Rap2a resulting in induction of brush border formation via a pathway that includes the polarity players TNIK, Mst4 and Ezrin. Here we show that the PDZ domain of PDZGEF is essential and sufficient for targeting PDZGEF to the apical membrane of polarized intestinal epithelial cells. Inhibition of PLD and consequently production of phosphatidic acid inhibitis targeting of PDZGEF to the plasma membrane. Furthermore, localization requires specific positively charged residues within the PDZ domain. We conclude that local accumulation of PDZGEF at the apical membrane during establishment of epithelial polarity is mediated by electrostatic interactions between positively charged side chains in the PDZ domain and negatively charged phosphatidic acid.
    PLoS ONE 05/2014; 9(5):e98253. DOI:10.1371/journal.pone.0098253 · 3.23 Impact Factor
  • Sarah V Consonni · Madelon M Maurice · Johannes L Bos
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    ABSTRACT: The Dishevelled, EGL-10 and pleckstrin (DEP) domain is a globular protein domain that is present in about ten human protein families with well-defined structural features. A picture is emerging that DEP domains mainly function in the spatial and temporal control of diverse signal transduction events by recruiting proteins to the plasma membrane. DEP domains can interact with various partners at the membrane, including phospholipids and membrane receptors, and their binding is subject to regulation.
    Nature Reviews Molecular Cell Biology 04/2014; DOI:10.1038/nrm3791 · 36.46 Impact Factor
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    ABSTRACT: Epithelial cell migration is crucial for the development and regeneration of epithelial tissues. Aberrant regulation of epithelial cell migration has a major role in pathological processes such as the development of cancer metastasis and tissue fibrosis. Here, we report that in response to factors that promote cell motility, the Rap guanine exchange factor RAPGEF2 is rapidly phosphorylated by I-kappa-B-kinase-β and casein kinase-1α and consequently degraded by the proteasome via the SCF(βTrCP) ubiquitin ligase. Failure to degrade RAPGEF2 in epithelial cells results in sustained activity of Rap1 and inhibition of cell migration induced by HGF, a potent metastatic factor. Furthermore, expression of a degradation-resistant RAPGEF2 mutant greatly suppresses dissemination and metastasis of human breast cancer cells. These findings reveal a molecular mechanism regulating migration and invasion of epithelial cells and establish a key direct link between IKKβ and cell motility controlled by Rap-integrin signaling.
    Developmental Cell 11/2013; 27(5). DOI:10.1016/j.devcel.2013.10.023 · 10.37 Impact Factor
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    ABSTRACT: Previously, exchange protein directly activated by cAMP 2 (Epac2) and PKA were known to play a role in glucose-stimulated insulin secretion (GSIS) by pancreatic β cells. The present study shows that Epac1 mRNA is also expressed by β cells. Therefore, we generated mice and embryonic stem (ES) cells with deletion of the Epac1 gene to define its role in β-cell biology and metabolism. The homozygous Epac1-knockout (Epac1(-/-)) mice developed impaired glucose tolerance and GSIS with deranged islet cytoarchitecture, which was confirmed by isolated islets from adult Epac1(-/-) mice. Moreover, Epac1(-/-) mice developed more severe hyperglycemia with increased β-cell apoptosis and insulitis after multiple low-dose streptozotocin (MLDS; 40 mg/kg) treatment than Epac1(+/+) mice. Interestingly, Epac1(-/-) mice also showed metabolic defects, including increased respiratory exchange ratio (RER) and plasma triglyceride (TG), and more severe diet-induced obesity with insulin resistance, which may contributed to β-cell dysfunction. However, islets differentiated from Epac1(-/-) ES cells showed insulin secretion defect, reduced Glut2 and PDX-1 expression, and abolished GLP-1-stimulated PCNA induction, suggesting a role of Epac1 in β-cell function. The current study provides in vitro and in vivo evidence that Epac1 has an important role in GSIS of β cells and phenotype resembling metabolic syndrome. Kai, A. K. L., Lam, A. K. M., Chen, Y., Tai, A. C. P., Zhang, X., Lai, A. K. W., Yeung, P. K. K., Tam, S., Wang, J., Lam, K. S., Vanhoutte, P. M., Bos, J. L., Chung, S. S. M., Xu, A., Chung, S. K. Exchange protein activated by cAMP 1 (Epac1)-deficient mice develop β-cell dysfunction and metabolic syndrome.
    The FASEB Journal 06/2013; 27(10). DOI:10.1096/fj.13-230433 · 5.48 Impact Factor
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    ABSTRACT: Rap1 is a small GTPase regulating cell-cell adhesion, cell-matrix adhesion, and actin rearrangements, all processes dynamically coordinated during cell spreading and endothelial barrier function. Here, we identify the adaptor protein ras-interacting protein 1 (Rasip1) as a Rap1-effector involved in cell spreading and endothelial barrier function. Using Förster resonance energy transfer, we show that Rasip1 interacts with active Rap1 in a cellular context. Rasip1 mediates Rap1-induced cell spreading through its interaction partner Rho GTPase-activating protein 29 (ArhGAP29), a GTPase activating protein for Rho proteins. Accordingly, the Rap1-Rasip1 complex induces cell spreading by inhibiting Rho signaling. The Rasip1-ArhGAP29 pathway also functions in Rap1-mediated regulation of endothelial junctions, which controls endothelial barrier function. In this process, Rasip1 cooperates with its close relative ras-association and dilute domain-containing protein (Radil) to inhibit Rho-mediated stress fiber formation and induces junctional tightening. These results reveal an effector pathway for Rap1 in the modulation of Rho signaling and actin dynamics, through which Rap1 modulates endothelial barrier function.
    Proceedings of the National Academy of Sciences 06/2013; 110(28). DOI:10.1073/pnas.1306595110 · 9.81 Impact Factor
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    ABSTRACT: The small GTPase Rap1 is required for proper cell-cell junction formation and also plays a key role in mediating cAMP-induced tightening of adherens junctions and subsequent increased barrier function of endothelial cells. To further study how Rap1 controls barrier function, we performed quantitative global phosphoproteomics in human umbilical vein endothelial cells (HUVECs) prior to and after Rap1 activation by the Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP-AM (007-AM). Tryptic digests were labeled using stable isotope dimethyl labeling, enriched with phosphopeptides by strong cation exchange (SCX), followed by titanium(iv) immobilized metal affinity chromatography (Ti-IMAC) and analyzed by high resolution mass spectrometry. We identified 19 859 unique phosphopeptides containing 17 278 unique phosphosites on 4594 phosphoproteins, providing the largest HUVEC phosphoproteome to date. Of all identified phosphosites, 220 (∼1%) were more than 1.5-fold up- or downregulated upon Rap activation, in two independent experiments. Compatible with the function of Rap1, these alterations were found predominantly in proteins regulating the actin cytoskeleton, cell-cell junctions and cell adhesion.
    Molecular BioSystems 03/2013; 9(4):732-49. DOI:10.1039/c3mb25524g · 3.18 Impact Factor
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    ABSTRACT: 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.
    PLoS ONE 02/2013; 8(2):e57903. DOI:10.1371/journal.pone.0057903 · 3.23 Impact Factor
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    Willem-Jan Pannekoek · Anneke Post · Johannes L Bos
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    ABSTRACT: The small G-protein Rap1 plays an important role in the regulation of endothelial barrier function, a process controlled largely by cell-cell adhesions and their connection to the actin cytoskeleton. During the various stages of barrier dynamics, different guanine nucleotide exchange factors (GEFs) control Rap1 activity, indicating that Rap1 integrates multiple input signals. Once activated, Rap1 induces numerous signaling cascades, together responsible for the increased endothelial barrier function. Most notably, Rap1 activation results in the inhibition of Rho to decrease radial stress fibers and the activation of Cdc42 to increase junctional actin. This implies that Rap regulates endothelial barrier function by dual control of cytoskeletal tension. The molecular details of the signaling pathways are becoming to be elucidated.
    Cell adhesion & migration 01/2013; 8(2). DOI:10.4161/cam.27352 · 3.40 Impact Factor
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    ABSTRACT: Epac proteins are activated by binding of the second messenger cAMP and then act as guanine nucleotide exchange factors for Rap proteins. The Epac proteins are involved in the regulation of cell adhesion and insulin secretion. Here we have determined the structure of Epac2 in complex with a cAMP analogue (Sp-cAMPS) and RAP1B by X-ray crystallography and single particle electron microscopy. The structure represents the cAMP activated state of the Epac2 protein with the RAP1B protein trapped in the course of the exchange reaction. Comparison with the inactive conformation reveals that cAMP binding causes conformational changes that allow the cyclic nucleotide binding domain to swing from a position blocking the Rap binding site towards a docking site at the Ras exchange motif domain.

Publication Stats

19k Citations
1,465.37 Total Impact Points

Institutions

  • 2001–2014
    • University Medical Center Utrecht
      • Department of Physiological Chemistry
      Utrecht, Utrecht, Netherlands
  • 2008
    • Hubrecht Institute
      Utrecht, Utrecht, Netherlands
  • 1992–2008
    • Universiteit Utrecht
      • • Bijvoet Institute for Biomolecular Research
      • • Department of Physiological Chemistry
      Utrecht, Utrecht, Netherlands
  • 1994–2005
    • Friedrich Miescher Institute for Biomedical Research
      Bâle, Basel-City, Switzerland
  • 2003
    • Max Planck Institute of Molecular Physiology
      Dortmund, North Rhine-Westphalia, Germany
    • University of Bergen
      Bergen, Hordaland, Norway
  • 1989–1990
    • Leiden University
      Leyden, South Holland, Netherlands
  • 1988
    • Johns Hopkins University
      • Department of Pathology
      Baltimore, MD, United States