scholarly journals The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation

2012 ◽  
Vol 23 (15) ◽  
pp. 2831-2844 ◽  
Author(s):  
Jos van Rijssel ◽  
Jeffrey Kroon ◽  
Mark Hoogenboezem ◽  
Floris P. J. van Alphen ◽  
Renske J. de Jong ◽  
...  
Keyword(s):  
Structure Formation ◽  
Apical Membrane ◽  
Transendothelial Migration ◽  
Dependent Manner ◽  
Membrane Protrusion ◽  
Nucleotide Exchange ◽  
Leukocyte Transmigration ◽  
Guanine Nucleotide Exchange ◽  
Leukocyte Transendothelial Migration ◽  
Nucleotide Exchange Factor

Leukocyte transendothelial migration involves the active participation of the endothelium through the formation of apical membrane protrusions that embrace adherent leukocytes, termed docking structures. Using live-cell imaging, we find that prior to transmigration, endothelial docking structures form around 80% of all neutrophils. Previously we showed that endothelial RhoG and SGEF control leukocyte transmigration. In this study, our data reveal that both full-length Trio and the first DH-PH (TrioD1) domain of Trio, which can activate Rac1 and RhoG, interact with ICAM-1 and are recruited to leukocyte adhesion sites. Moreover, upon clustering of ICAM-1, the Rho-guanine nucleotide exchange factor Trio activates Rac1, prior to activating RhoG, in a filamin-dependent manner. We further show that docking structure formation is initiated by ICAM-1 clustering into ring-like structures, which is followed by apical membrane protrusion. Interestingly, we find that Rac1 is required for ICAM-1 clustering, whereas RhoG controls membrane protrusion formation. Finally, silencing endothelial Trio expression or reducing TrioD1 activity without affecting SGEF impairs both docking structure formation and leukocyte transmigration. We conclude that Trio promotes leukocyte transendothelial migration by inducing endothelial docking structure formation in a filamin-dependent manner through the activation of Rac1 and RhoG.

scholarly journals The RhoA Guanine Nucleotide Exchange Factor, LARG, Mediates ICAM-1–Dependent Mechanotransduction in Endothelial Cells To Stimulate Transendothelial Migration

2014 ◽  
Vol 192 (7) ◽  
pp. 3390-3398 ◽  
Author(s):  
Elizabeth C. Lessey-Morillon ◽  
Lukas D. Osborne ◽  
Elizabeth Monaghan-Benson ◽  
Christophe Guilluy ◽  
E. Timothy O’Brien ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Guanine Nucleotide Exchange Factor ◽  
Transendothelial Migration ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

scholarly journals Par3 integrates Tiam1 and phosphatidylinositol 3-kinase signaling to change apical membrane identity

2017 ◽  
Vol 28 (2) ◽  
pp. 252-260 ◽  
Author(s):  
Travis R. Ruch ◽  
David M. Bryant ◽  
Keith E. Mostov ◽  
Joanne N. Engel
Keyword(s):  
Apical Membrane ◽  
Protein Localization ◽  
Apical Surface ◽  
Nucleotide Exchange ◽  
Phosphatidylinositol 3 Kinase ◽  
Signaling Mechanism ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Chemically Induced Dimerization ◽  
Phosphatidylinositol 3

Pathogens can alter epithelial polarity by recruiting polarity proteins to the apical membrane, but how a change in protein localization is linked to polarity disruption is not clear. In this study, we used chemically induced dimerization to rapidly relocalize proteins from the cytosol to the apical surface. We demonstrate that forced apical localization of Par3, which is normally restricted to tight junctions, is sufficient to alter apical membrane identity through its interactions with phosphatidylinositol 3-kinase (PI3K) and the Rac1 guanine nucleotide exchange factor Tiam1. We further show that PI3K activity is required upstream of Rac1, and that simultaneously targeting PI3K and Tiam1 to the apical membrane has a synergistic effect on membrane remodeling. Thus, Par3 coordinates the action of PI3K and Tiam1 to define membrane identity, revealing a signaling mechanism that can be exploited by human mucosal pathogens.

scholarly journals In Vitro Formation of Recycling Vesicles from Endosomes Requires Adaptor Protein-1/Clathrin and Is Regulated by Rab4 and the Connector Rabaptin-5

2004 ◽  
Vol 15 (11) ◽  
pp. 4990-5000 ◽  
Author(s):  
Adriana Pagano ◽  
Pascal Crottet ◽  
Cristina Prescianotto-Baschong ◽  
Martin Spiess
Keyword(s):  
Brefeldin A ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Vesicle Formation ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Vitro Assay ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and γ-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5.

scholarly journals G1/S Cyclin-dependent Kinase Regulates Small GTPase Rho1p through Phosphorylation of RhoGEF Tus1p inSaccharomyces cerevisiae

2008 ◽  
Vol 19 (4) ◽  
pp. 1763-1771 ◽  
Author(s):  
Keiko Kono ◽  
Satoru Nogami ◽  
Mitsuhiro Abe ◽  
Masafumi Nishizawa ◽  
Shinichi Morishita ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Guanine Nucleotide ◽  
Cyclin Dependent Kinase ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Temporal And Spatial ◽  
Cdk Phosphorylation

Rho1p is an essential small GTPase that plays a key role in the morphogenesis of Saccharomyces cerevisiae. We show here that the activation of Rho1p is regulated by a cyclin-dependent kinase (CDK). Rho1p is activated at the G1/S transition at the incipient-bud sites by the Cln2p (G1 cyclin) and Cdc28p (CDK) complex, in a process mediated by Tus1p, a guanine nucleotide exchange factor for Rho1p. Tus1p interacts physically with Cln2p/Cdc28p and is phosphorylated in a Cln2p/Cdc28p-dependent manner. CDK phosphorylation consensus sites in Tus1p are required for both Cln2p-dependent activation of Rho1p and polarized organization of the actin cytoskeleton. We propose that Cln2p/Cdc28p-dependent phosphorylation of Tus1p is required for appropriate temporal and spatial activation of Rho1p at the G1/S transition.

scholarly journals Modulation of Rho Guanine Exchange Factor Lfc Activity by Protein Kinase A-Mediated Phosphorylation

2009 ◽  
Vol 29 (21) ◽  
pp. 5963-5973 ◽  
Author(s):  
David Meiri ◽  
Melissa A. Greeve ◽  
Andrea Brunet ◽  
Dina Finan ◽  
Clark D. Wells ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Dynein Motor ◽  
Nucleotide Exchange Factor ◽  
Kinase A ◽  
Exchange Activity

ABSTRACT Lfc is a guanine nucleotide exchange factor (GEF) for Rho that demonstrates an unusual ability to associate with microtubules. While several phosphorylated residues have been detected in the Lfc polypeptide, the mechanism(s) by which phosphorylation regulates the exchange activity of Lfc remains unclear. We confirm that Lfc is a phosphorylated protein and demonstrate that 14-3-3 interacts directly and in a phosphorylation-dependent manner with Lfc. We identify AKAP121 as an Lfc-binding protein and show that Lfc is phosphorylated in an AKAP-dependent manner by protein kinase A (PKA). Forskolin treatment induced 14-3-3 binding to Lfc and suppressed the exchange activity of wild-type Lfc on RhoA. Importantly, a mutant of Lfc that is unable to associate with 14-3-3 proteins was resistant to inhibition by forskolin. Tctex-1, a dynein motor light chain, binds to Lfc in a competitive manner with 14-3-3.

scholarly journals Coordinated RhoA signaling at the leading edge and uropod is required for T cell transendothelial migration

2010 ◽  
Vol 190 (4) ◽  
pp. 553-563 ◽  
Author(s):  
Sarah J. Heasman ◽  
Leo M. Carlin ◽  
Susan Cox ◽  
Tony Ng ◽  
Anne J. Ridley
Keyword(s):  
T Cells ◽  
T Cell ◽  
Rho Gtpases ◽  
Leading Edge ◽  
Transendothelial Migration ◽  
Nucleotide Exchange ◽  
Rhoa Activity ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Rhoa Signaling

Transendothelial migration (TEM) is a tightly regulated process whereby leukocytes migrate from the vasculature into tissues. Rho guanosine triphosphatases (GTPases) are implicated in TEM, but the contributions of individual Rho family members are not known. In this study, we use an RNA interference screen to identify which Rho GTPases affect T cell TEM and demonstrate that RhoA is critical for this process. RhoA depletion leads to loss of migratory polarity; cells lack both leading edge and uropod structures and, instead, have stable narrow protrusions with delocalized protrusions and contractions. By imaging a RhoA activity biosensor in transmigrating T cells, we find that RhoA is locally and dynamically activated at the leading edge, where its activation precedes both extension and retraction events, and in the uropod, where it is associated with ROCK-mediated contraction. The Rho guanine nucleotide exchange factor (GEF) GEF-H1 contributes to uropod contraction but does not affect the leading edge. Our data indicate that RhoA activity is dynamically regulated at the front and back of T cells to coordinate TEM.

scholarly journals SAC-1 ensures epithelial endocytic recycling by restricting ARF-6 activity

2018 ◽  
Vol 217 (6) ◽  
pp. 2121-2139 ◽  
Author(s):  
Dan Chen ◽  
Chao Yang ◽  
Sha Liu ◽  
Weijian Hang ◽  
Xianghong Wang ◽  
...  
Keyword(s):  
Negative Regulator ◽  
Dependent Manner ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Endocytic Recycling ◽  
Concentration Dependent Manner ◽  
Guanosine Diphosphate ◽  
Guanine Nucleotide Exchange ◽  
Phosphoinositide Phosphatase ◽  
Nucleotide Exchange Factor

Arf6/ARF-6 is a crucial regulator of the endosomal phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) pool in endocytic recycling. To further characterize ARF-6 regulation, we performed an ARF-6 interactor screen in Caenorhabditis elegans and identified SAC-1, the homologue of the phosphoinositide phosphatase Sac1p in yeast, as a novel ARF-6 partner. In the absence of ARF-6, basolateral endosomes show a loss of SAC-1 staining in epithelial cells. Steady-state cargo distribution assays revealed that loss of SAC-1 specifically affected apical secretory delivery and basolateral recycling. PI(4,5)P2 levels and the endosomal labeling of the ARF-6 effector UNC-16 were significantly elevated in sac-1 mutants, suggesting that SAC-1 functions as a negative regulator of ARF-6. Further analyses revealed an interaction between SAC-1 and the ARF-6-GEF BRIS-1. This interaction outcompeted ARF-6(guanosine diphosphate [GDP]) for binding to BRIS-1 in a concentration-dependent manner. Consequently, loss of SAC-1 promotes the intracellular overlap between ARF-6 and BRIS-1. BRIS-1 knockdown resulted in a significant reduction in PI(4,5)P2 levels in SAC-1-depleted cells. Interestingly, the action of SAC-1 in sequestering BRIS-1 is independent of SAC-1’s catalytic activity. Our results suggest that the interaction of SAC-1 with ARF-6 curbs ARF-6 activity by limiting the access of ARF-6(GDP) to its guanine nucleotide exchange factor, BRIS-1.

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.

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