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 Activation of Rac and Cdc42 Video Imaged by Fluorescent Resonance Energy Transfer-Based Single-Molecule Probes in the Membrane of Living Cells

2002 ◽  
Vol 22 (18) ◽  
pp. 6582-6591 ◽  
Author(s):  
Reina E. Itoh ◽  
Kazuo Kurokawa ◽  
Yusuke Ohba ◽  
Hisayoshi Yoshizaki ◽  
Naoki Mochizuki ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Leading Edge ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Gtpase Activating Proteins

ABSTRACT Rho family G proteins, including Rac and Cdc42, regulate a variety of cellular functions such as morphology, motility, and gene expression. We developed fluorescent resonance energy transfer-based probes which monitored the local balance between the activities of guanine nucleotide exchange factors and GTPase-activating proteins for Rac1 and Cdc42 at the membrane. These probes, named Raichu-Rac and Raichu-Cdc42, consisted of a Cdc42- and Rac-binding domain of Pak, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras. With these probes, we video imaged the Rac and Cdc42 activities. In motile HT1080 cells, activities of both Rac and Cdc42 gradually increased toward the leading edge and decreased rapidly when cells changed direction. Under a higher magnification, we observed that Rac activity was highest immediately behind the leading edge, whereas Cdc42 activity was most prominent at the tip of the leading edge. Raichu-Rac and Raichu-Cdc42 were also applied to a rapid and simple assay for the analysis of putative guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) in living cells. Among six putative GEFs and GAPs, we identified KIAA0362/DBS as a GEF for Rac and Cdc42, KIAA1256 as a GEF for Cdc42, KIAA0053 as a GAP for Rac and Cdc42, and KIAA1204 as a GAP for Cdc42. In conclusion, use of these single-molecule probes to determine Rac and Cdc42 activity will accelerate the analysis of the spatiotemporal regulation of Rac and Cdc42 in a living cell.

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 DRhoGEF2 and Diaphanous Regulate Contractile Force during Segmental Groove Morphogenesis in the Drosophila Embryo

2008 ◽  
Vol 19 (5) ◽  
pp. 1883-1892 ◽  
Author(s):  
Shai Mulinari ◽  
Mojgan Padash Barmchi ◽  
Udo Häcker
Keyword(s):  
Cell Shape ◽  
Cell Formation ◽  
Small Gtpases ◽  
Cell Junctions ◽  
Drosophila Embryo ◽  
Cell Cortex ◽  
Nucleotide Exchange ◽  
Apical Constriction ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

Morphogenesis of the Drosophila embryo is associated with dynamic rearrangement of the actin cytoskeleton mediated by small GTPases of the Rho family. These GTPases act as molecular switches that are activated by guanine nucleotide exchange factors. One of these factors, DRhoGEF2, plays an important role in the constriction of actin filaments during pole cell formation, blastoderm cellularization, and invagination of the germ layers. Here, we show that DRhoGEF2 is equally important during morphogenesis of segmental grooves, which become apparent as tissue infoldings during mid-embryogenesis. Examination of DRhoGEF2-mutant embryos indicates a role for DRhoGEF2 in the control of cell shape changes during segmental groove morphogenesis. Overexpression of DRhoGEF2 in the ectoderm recruits myosin II to the cell cortex and induces cell contraction. At groove regression, DRhoGEF2 is enriched in cells posterior to the groove that undergo apical constriction, indicating that groove regression is an active process. We further show that the Formin Diaphanous is required for groove formation and strengthens cell junctions in the epidermis. Morphological analysis suggests that Dia regulates cell shape in a way distinct from DRhoGEF2. We propose that DRhoGEF2 acts through Rho1 to regulate acto-myosin constriction but not Diaphanous-mediated F-actin nucleation during segmental groove morphogenesis.

scholarly journals Function of the N-terminus of zizimin1: autoinhibition and membrane targeting

2007 ◽  
Vol 409 (2) ◽  
pp. 525-533 ◽  
Author(s):  
Nahum Meller ◽  
M. Jody Westbrook ◽  
John D. Shannon ◽  
Chittibabu Guda ◽  
Martin A. Schwartz
Keyword(s):  
Limited Proteolysis ◽  
Small Gtpases ◽  
Guanine Nucleotide Exchange Factors ◽  
Pleckstrin Homology Domain ◽  
Membrane Targeting ◽  
Nucleotide Exchange ◽  
Rho Proteins ◽  
Cellular Functions ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

Rho family small GTPases are critical regulators of multiple cellular functions. Dbl-homology-domain-containing proteins are the classical GEFs (guanine nucleotide exchange factors) responsible for activation of Rho proteins. Zizimin1 is a Cdc42-specific GEF that belongs to a second family of mammalian Rho-GEFs, CZH [CDM (Ced-5/DOCK180/Myoblast city)-zizimin homology] proteins, which possess a novel type of GEF domain. CZH proteins can be divided into a subfamily related to DOCK 180 and a subfamily related to zizimin1. The two groups share two conserved regions named the CZH1 (or DHR1) domain and the CZH2 (DHR2 or DOCKER) domains, the latter exhibiting GEF activity. We now show that limited proteolysis of zizimin1 suggests the existence of structural domains that do not correspond to those identified on the basis of homologies. We demonstrate that the N-terminal half binds to the GEF domain through three distinct areas, including the CZH1, to inhibit the interaction with Cdc42. The N-terminal PH (pleckstrin homology) domain binds phosphoinositides and mediates zizimin1 membrane targeting. These results define two novel functions for the N-terminal region of zizimin1.

scholarly journals The RhoGEF Trio: A Protein with a Wide Range of Functions in the Vascular Endothelium

2021 ◽  
Vol 22 (18) ◽  
pp. 10168
Author(s):  
Lanette Kempers ◽  
Amber J. M. Driessen ◽  
Jos van Rijssel ◽  
Martijn A. Nolte ◽  
Jaap D. van Buul
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Function ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Endothelial Cell Function ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Wide Range ◽  
Range Of Functions

Many cellular processes are controlled by small GTPases, which can be activated by guanine nucleotide exchange factors (GEFs). The RhoGEF Trio contains two GEF domains that differentially activate the small GTPases such as Rac1/RhoG and RhoA. These small RhoGTPases are mainly involved in the remodeling of the actin cytoskeleton. In the endothelium, they regulate junctional stabilization and play a crucial role in angiogenesis and endothelial barrier integrity. Multiple extracellular signals originating from different vascular processes can influence the activity of Trio and thereby the regulation of the forementioned small GTPases and actin cytoskeleton. This review elucidates how various signals regulate Trio in a distinct manner, resulting in different functional outcomes that are crucial for endothelial cell function in response to inflammation.

scholarly journals Autoactivation of small GTPases by the GEF–effector positive feedback modules

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 1676 ◽  
Author(s):  
Andrew B. Goryachev ◽  
Marcin Leda
Keyword(s):  
Cell Biology ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Gtpase Activating Proteins ◽  
Recent Developments ◽  
Functional Consequences ◽  
Exciting Area

Small GTPases are organizers of a plethora of cellular processes. The time and place of their activation are tightly controlled by the localization and activation of their regulators, guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Remarkably, in some systems, the upstream regulators of GTPases are also found downstream of their activity. Resulting feedback loops can generate complex spatiotemporal dynamics of GTPases with important functional consequences. Here we discuss the concept of positive autoregulation of small GTPases by the GEF–effector feedback modules and survey recent developments in this exciting area of cell biology.

scholarly journals Rab35–GEFs, DENND1A and folliculin differentially regulate podocalyxin trafficking in two- and three-dimensional epithelial cell cultures

2020 ◽  
Vol 295 (11) ◽  
pp. 3652-3663
Author(s):  
Riko Kinoshita ◽  
Yuta Homma ◽  
Mitsunori Fukuda
Keyword(s):  
Cell Cultures ◽  
Three Dimensional ◽  
Coiled Coil ◽  
Culture Conditions ◽  
Small Gtpases ◽  
Asymmetric Structure ◽  
Inositol Polyphosphate ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

Polarized epithelial cells have functionally distinct apical and basolateral membranes through which they communicate with external and internal bodily environments, respectively. The establishment and maintenance of this asymmetric structure depend on polarized trafficking of specific cargos, but the precise molecular mechanism is incompletely understood. We previously showed that Rab35, a member of the Rab family small GTPases, differentially regulates the trafficking of an apical cargo, podocalyxin (PODXL), in two-dimensional (2D) and three-dimensional (3D) Madin–Darby canine kidney (MDCK) II cell cultures through specific interactions with two distinct effectors, OCRL inositol polyphosphate-5-phosphatase (OCRL) and ArfGAP with coiled-coil, ankyrin repeat and pleckstrin homology domains 2 (ACAP2), respectively. However, whether the upstream regulators of Rab35 also differ depending on the culture conditions remains completely unknown. Here, we investigated four known guanine nucleotide exchange factors (GEFs) of Rab35, namely DENN domain–containing 1A (DENND1A), DENND1B, DENND1C, and folliculin (FLCN), and demonstrate that DENND1A and FLCN exhibit distinct requirements for Rab35-dependent PODXL trafficking under the two culture conditions. In 3D cell cultures, only DENDN1A-knockout cysts exhibited the inverted localization of PODXL similar to that of Rab35-knockout cysts. Moreover, the DENN domain, harboring GEF activity toward Rab35, was required for proper PODXL trafficking to the apical membrane. By contrast, FLCN-knockdown cells specifically accumulated PODXL in actin-rich structures similar to the Rab35-knockdown cells in 2D cell cultures. Our findings indicate that two distinct functional cascades of Rab35, the FLCN-Rab35-OCRL and the DENND1A-Rab35-ACAP2 axes, regulate PODXL trafficking in 2D and 3D MDCK II cell cultures, respectively.

scholarly journals Pleiotropic Roles of Calmodulin in the Regulation of KRas and Rac1 GTPases: Functional Diversity in Health and Disease

2020 ◽  
Vol 21 (10) ◽  
pp. 3680 ◽  
Author(s):  
Francesc Tebar ◽  
Albert Chavero ◽  
Neus Agell ◽  
Albert Lu ◽  
Carles Rentero ◽  
...  
Keyword(s):  
Biological Response ◽  
Direct Interaction ◽  
Small Gtpases ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Calmodulin Binding ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Cellular Events ◽  
And Migration

Calmodulin is a ubiquitous signalling protein that controls many biological processes due to its capacity to interact and/or regulate a large number of cellular proteins and pathways, mostly in a Ca2+-dependent manner. This complex interactome of calmodulin can have pleiotropic molecular consequences, which over the years has made it often difficult to clearly define the contribution of calmodulin in the signal output of specific pathways and overall biological response. Most relevant for this review, the ability of calmodulin to influence the spatiotemporal signalling of several small GTPases, in particular KRas and Rac1, can modulate fundamental biological outcomes such as proliferation and migration. First, direct interaction of calmodulin with these GTPases can alter their subcellular localization and activation state, induce post-translational modifications as well as their ability to interact with effectors. Second, through interaction with a set of calmodulin binding proteins (CaMBPs), calmodulin can control the capacity of several guanine nucleotide exchange factors (GEFs) to promote the switch of inactive KRas and Rac1 to an active conformation. Moreover, Rac1 is also an effector of KRas and both proteins are interconnected as highlighted by the requirement for Rac1 activation in KRas-driven tumourigenesis. In this review, we attempt to summarize the multiple layers how calmodulin can regulate KRas and Rac1 GTPases in a variety of cellular events, with biological consequences and potential for therapeutic opportunities in disease settings, such as cancer.

ARF small GTPases in the developmental function mediated by ARF regulators GNOM and VAN3

2022 ◽  
Author(s):  
Maciek Adamowski ◽  
Ivana Matijević ◽  
Jiří Friml
Keyword(s):  
Small Gtpases ◽  
Guanine Nucleotide Exchange Factors ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Gene Group ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Gtpase Activating Proteins ◽  
Developmental Patterning ◽  
Branching Angle

ARF small GTPases are molecular switches acting in intracellular trafficking. Their cycles of activity are controlled by regulators, ARF Guanine nucleotide Exchange Factors (ARF-GEFs) and ARF GTPase Activating Proteins (ARF-GAPs). The ARF-GEF GNOM (GN) and the ARF-GAP VAN3 share a prominent function in auxin-mediated developmental patterning, but the ARFs which they might control were not identified. We conducted a loss-of-function and localization-based screening of the ARF/ARF-LIKE gene family in Arabidopsis thaliana with the primary aim of identifying functional partners of GN and VAN3, while extending the limited understanding of this gene group as a whole. We identified a function of ARLA1 in branching angle control. Mutants lacking the variably localized ARLB1, ARFB1, ARFC1, ARFD1, and ARF3, even in high order combinations, do not exhibit any evident phenotypes. Loss of function arfa1 phenotypes support a major role of ARFA1 in growth and development overall, but patterning defects typical to gn loss of function are not found. ARFA1 are not localized at the plasma membrane, where GN and VAN3 carry out developmental patterning function according to current models. Taken together, putative ARF partners of GN and VAN3 in developmental patterning cannot be conclusively identified.

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