scholarly journals Direct Spatial Control of Epac1 by Cyclic AMP

2009 ◽  
Vol 29 (10) ◽  
pp. 2521-2531 ◽  
Author(s):  
Bas Ponsioen ◽  
Martijn Gloerich ◽  
Laila Ritsma ◽  
Holger Rehmann ◽  
Johannes L. Bos ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Adhesion ◽  
Cyclic Amp ◽  
Conformational Change ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cell Junction ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

ABSTRACT Epac1 is a guanine nucleotide exchange factor (GEF) for the small G protein Rap and is directly activated by cyclic AMP (cAMP). Upon cAMP binding, Epac1 undergoes a conformational change that allows the interaction of its GEF domain with Rap, resulting in Rap activation and subsequent downstream effects, including integrin-mediated cell adhesion and cell-cell junction formation. Here, we report that cAMP also induces the translocation of Epac1 toward the plasma membrane. Combining high-resolution confocal fluorescence microscopy with total internal reflection fluorescence and fluorescent resonance energy transfer assays, we observed that Epac1 translocation is a rapid and reversible process. This dynamic redistribution of Epac1 requires both the cAMP-induced conformational change as well as the DEP domain. In line with its translocation, Epac1 activation induces Rap activation predominantly at the plasma membrane. We further show that the translocation of Epac1 enhances its ability to induce Rap-mediated cell adhesion. Thus, the regulation of Epac1-Rap signaling by cAMP includes both the release of Epac1 from autoinhibition and its recruitment to the plasma membrane.

scholarly journals Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins

2010 ◽  
Vol 30 (22) ◽  
pp. 5421-5431 ◽  
Author(s):  
Martijn Gloerich ◽  
Bas Ponsioen ◽  
Marjolein J. Vliem ◽  
Zhongchun Zhang ◽  
Jun Zhao ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Adhesion ◽  
Cyclic Amp ◽  
Cell Junction ◽  
Nucleotide Exchange ◽  
Erm Proteins ◽  
Spatial Regulation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Interfering Rna

ABSTRACT Epac1 is a guanine nucleotide exchange factor for the small G protein Rap and is involved in membrane-localized processes such as integrin-mediated cell adhesion and cell-cell junction formation. Cyclic AMP (cAMP) directly activates Epac1 by release of autoinhibition and in addition induces its translocation to the plasma membrane. Here, we show an additional mechanism of Epac1 recruitment, mediated by activated ezrin-radixin-moesin (ERM) proteins. Epac1 directly binds with its N-terminal 49 amino acids to ERM proteins in their open conformation. Receptor-induced activation of ERM proteins results in increased binding of Epac1 and consequently the clustered localization of Epac1 at the plasma membrane. Deletion of the N terminus of Epac1, as well as disruption of the Epac1-ERM interaction by an interfering radixin mutant or small interfering RNA (siRNA)-mediated depletion of the ERM proteins, impairs Epac1-mediated cell adhesion. We conclude that ERM proteins are involved in the spatial regulation of Epac1 and cooperate with cAMP- and Rap-mediated signaling to regulate adhesion to the extracellular matrix.

scholarly journals The RAP1 Guanine Nucleotide Exchange Factor Epac2 Couples Cyclic AMP and Ras Signals at the Plasma Membrane

2005 ◽  
Vol 281 (5) ◽  
pp. 2506-2514 ◽  
Author(s):  
Yu Li ◽  
Sirisha Asuri ◽  
John F. Rebhun ◽  
Ariel F. Castro ◽  
Nivanka C. Paranavitana ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Regulation of RalA GTPase by phosphatidylinositol 3-kinase as visualized by FRET probes

2006 ◽  
Vol 34 (5) ◽  
pp. 851-854 ◽  
Author(s):  
H. Yoshizaki ◽  
K. Aoki ◽  
T. Nakamura ◽  
M. Matsuda
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Subsequent Activation ◽  
Phosphoinositide 3 Kinase

Small GTPases, which are binary switches regulating various signal transduction cascades, function not only to relay signals but also to integrate them from multiple signalling branches. For example, RalA activity is regulated by at least three signalling cascades involving Ras, Rac or PI3K (phosphoinositide 3-kinase). To untangle such complicated regulatory mechanisms, we have been developing probes for GTPases, kinases and phosphatidylinositols based on the principle of FRET (fluorescence resonance energy transfer). We demonstrated previously that, upon EGF (epidermal growth factor) stimulation, Ras activity increases diffusely in the plasma membrane, whereas RalA activity increases predominantly in lamellipodial protrusions. Here, we show that the level of PtdIns(3,4,5)P3 is increased diffusely in the plasma membrane, whereas, in the central region, the level of PtdIns(3,4)P2 is increased more in the nascent lamellipodia than in the plasma membrane. The distribution and time course of Akt activation are similar to those of increased PtdIns(3,4)P2 levels. These observations suggest that the increase in PtdIns(3,4)P2 and the subsequent activation of Akt may be responsible for the localized activation of RalA. Thus the signals from Ras and PI3K converge at the level of Ral GEFs (guanine nucleotide-exchange factors), and this convergence restricts the area of RalA activation.

scholarly journals Ras and Calcium Signaling Pathways Converge at Raf1 via the Shoc2 Scaffold Protein

2010 ◽  
Vol 21 (6) ◽  
pp. 1088-1096 ◽  
Author(s):  
Sayaka Yoshiki ◽  
Rie Matsunaga-Udagawa ◽  
Kazuhiro Aoki ◽  
Yuji Kamioka ◽  
Etsuko Kiyokawa ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Scaffold Protein ◽  
Ras Signaling ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Ras Activation ◽  
Camp Analogue

Situated downstream of Ras is a key signaling molecule, Raf1. Increase in Ca2+ concentration has been shown to modulate the Ras-dependent activation of Raf1; however, the mechanism underlying this effect remains elusive. Here, to characterize the role of Ca2+ in Ras signaling to Raf1, we used a synthetic guanine nucleotide exchange factor (GEF) for Ras, eGRF. In HeLa cells expressing eGRF, Ras was activated by the cAMP analogue 007 as efficiently as by epidermal growth factor (EGF), whereas the activation of Raf1, MEK, and ERK by 007 was about half of that by EGF. Using a biosensor based on fluorescence resonance energy transfer, it was found that activation of Raf1 at the plasma membrane required not only Ras activation but also an increase in Ca2+ concentration or inhibition of calmodulin. Furthermore, the Ca2+-dependent activation of Raf1 was found to be abrogated by knockdown of Shoc2, a scaffold protein that binds both Ras and Raf1. These observations indicated that the Shoc2 scaffold protein modulates Ras-dependent Raf1 activation in a Ca2+- and calmodulin-dependent manner.

scholarly journals The Parkinson's disease–associated kinase LRRK2 regulates genes required for cell adhesion, polarization, and chemotaxis in activated murine macrophages

2020 ◽  
Vol 295 (31) ◽  
pp. 10857-10867 ◽  
Author(s):  
Daniel R. Levy ◽  
Atul Udgata ◽  
Panagiotis Tourlomousis ◽  
Martyn F. Symmons ◽  
Lee J. Hopkins ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Crohn’S Disease ◽  
Cell Adhesion ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Leucine-rich repeat kinase 2 (LRRK2) encodes a complex protein that includes kinase and GTPase domains. Genome-wide association studies have identified dominant LRRK2 alleles that predispose their carriers to late-onset idiotypic Parkinson's disease (PD) and also to autoimmune disorders such as Crohn's disease. Considerable evidence indicates that PD initiation and progression involve activation of innate immune functions in microglia, which are brain-resident macrophages. Here we asked whether LRRK2 modifies inflammatory signaling and how this modification might contribute to PD and Crohn's disease. We used RNA-Seq–based high-resolution transcriptomics to compare gene expression in activated primary macrophages derived from WT and Lrrk2 knockout mice. Remarkably, expression of a single gene, Rap guanine nucleotide exchange factor 3 (Rapgef3), was strongly up-regulated in the absence of LRRK2 and down-regulated in its presence. We observed similar regulation of Rapgef3 expression in cells treated with a highly specific inhibitor of LRRK2 protein kinase activity. Rapgef3 encodes an exchange protein, activated by cAMP 1 (EPAC-1), a guanine nucleotide exchange factor that activates the small GTPase Rap-1. Rap-1 mediates cell adhesion, polarization, and directional motility, and our results indicate that LRRK2 modulates chemotaxis of microglia and macrophages. Dominant PD-associated LRRK2 alleles may suppress EPAC-1 activity, further restricting motility and preventing efficient migration of microglia to sites of neuronal damage. Functional analysis in vivo in a subclinical infection model also indicated that Lrrk2 subtly modifies the inflammatory response. These results indicate that LRRK2 modulates the expression of genes involved in murine immune cell chemotaxis.

scholarly journals Active Arf6 Recruits ARNO/Cytohesin GEFs to the PM by Binding Their PH Domains

2007 ◽  
Vol 18 (6) ◽  
pp. 2244-2253 ◽  
Author(s):  
Lee Ann Cohen ◽  
Akira Honda ◽  
Peter Varnai ◽  
Fraser D. Brown ◽  
Tamas Balla ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Family Member ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Biochemical Assays ◽  
Inositol Phospholipids ◽  
Sec7 Domain

ARNO is a soluble guanine nucleotide exchange factor (GEF) for the Arf family of GTPases. Although in biochemical assays ARNO prefers Arf1 over Arf6 as a substrate, its localization in cells at the plasma membrane (PM) suggests an interaction with Arf6. In this study, we found that ARNO activated Arf1 in HeLa and COS-7 cells resulting in the recruitment of Arf1 on to dynamic PM ruffles. By contrast, Arf6 was activated less by ARNO than EFA6, a canonical Arf6 GEF. Remarkably, Arf6 in its GTP-bound form recruited ARNO to the PM and the two proteins could be immunoprecipitated. ARNO binding to Arf6 was not mediated through the catalytic Sec7 domain, but via the pleckstrin homology (PH) domain. Active Arf6 also bound the PH domain of Grp1, another ARNO family member. This interaction was direct and required both inositol phospholipids and GTP. We propose a model of sequential Arf activation at the PM whereby Arf6-GTP recruits ARNO family GEFs for further activation of other Arf isoforms.

scholarly journals Identification of a Plasma Membrane-associated Guanine Nucleotide Exchange Factor for ARF6 in Chromaffin Cells

2000 ◽  
Vol 275 (21) ◽  
pp. 15637-15644 ◽  
Author(s):  
Anne-Sophie Caumont ◽  
Nicolas Vitale ◽  
Marc Gensse ◽  
Marie-Christine Galas ◽  
James E. Casanova ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Chromaffin Cells ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

EHD2 mediates trafficking from the plasma membrane by modulating Rac1 activity

2011 ◽  
Vol 439 (3) ◽  
pp. 433-445 ◽  
Author(s):  
Sigi Benjamin ◽  
Hilla Weidberg ◽  
Debora Rapaport ◽  
Olga Pekar ◽  
Marina Nudelman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rho Gtpases ◽  
Actin Polymerization ◽  
Inhibitory Effect ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
System A ◽  
Nucleotide Exchange Factor ◽  
Rac1 Activity ◽  
The Impact

EHDs [EH (Eps15 homology)-domain-containing proteins] participate in different stages of endocytosis. EHD2 is a plasma-membrane-associated EHD which regulates trafficking from the plasma membrane and recycling. EHD2 has a role in nucleotide-dependent membrane remodelling and its ATP-binding domain is involved in dimerization, which creates a membrane-binding region. Nucleotide binding is important for association of EHD2 with the plasma membrane, since a nucleotide-free mutant (EHD2 T72A) failed to associate. To elucidate the possible function of EHD2 during endocytic trafficking, we attempted to unravel proteins that interact with EHD2, using the yeast two-hybrid system. A novel interaction was found between EHD2 and Nek3 [NIMA (never in mitosis in Aspergillus nidulans)-related kinase 3], a serine/threonine kinase. EHD2 was also found in association with Vav1, a Nek3-regulated GEF (guanine-nucleotide-exchange factor) for Rho GTPases. Since Vav1 regulates Rac1 activity and promotes actin polymerization, the impact of overexpression of EHD2 on Rac1 activity was tested. The results indicated that wt (wild-type) EHD2, but not its P-loop mutants, reduced Rac1 activity. The inhibitory effect of EHD2 overexpression was partially rescued by co-expression of Rac1 as measured using a cholera toxin trafficking assay. The results of the present study strongly indicate that EHD2 regulates trafficking from the plasma membrane by controlling Rac1 activity.

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