scholarly journals Conundrum, an ARHGAP18 orthologue, regulates RhoA and proliferation through interactions with Moesin

2013 ◽  
Vol 24 (9) ◽  
pp. 1420-1433 ◽  
Author(s):  
Amanda L. Neisch ◽  
Etienne Formstecher ◽  
Richard G. Fehon
Keyword(s):  
Growth Control ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Cell Cortex ◽  
Nucleotide Exchange ◽  
Filamentous Actin ◽  
Erm Proteins ◽  
Promote Cell Proliferation ◽  
Rhoa Activity ◽  
Guanine Nucleotide Exchange

RhoA, a small GTPase, regulates epithelial integrity and morphogenesis by controlling filamentous actin assembly and actomyosin contractility. Another important cytoskeletal regulator, Moesin (Moe), an ezrin, radixin, and moesin (ERM) protein, has the ability to bind to and organize cortical F-actin, as well as the ability to regulate RhoA activity. ERM proteins have previously been shown to interact with both RhoGEF (guanine nucleotide exchange factors) and RhoGAP (GTPase activating proteins), proteins that control the activation state of RhoA, but the functions of these interactions remain unclear. We demonstrate that Moe interacts with an unusual RhoGAP, Conundrum (Conu), and recruits it to the cell cortex to negatively regulate RhoA activity. In addition, we show that cortically localized Conu can promote cell proliferation and that this function requires RhoGAP activity. Surprisingly, Conu's ability to promote growth also appears dependent on increased Rac activity. Our results reveal a molecular mechanism by which ERM proteins control RhoA activity and suggest a novel linkage between the small GTPases RhoA and Rac in growth control.

scholarly journals Spatiotemporal dynamics of GEF-H1 activation controlled by microtubule- and Src-mediated pathways

2019 ◽  
Vol 218 (9) ◽  
pp. 3077-3097 ◽  
Author(s):  
Mihai L. Azoitei ◽  
Jungsik Noh ◽  
Daniel J. Marston ◽  
Philippe Roudot ◽  
Christopher B. Marshall ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factors ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Nucleotide Exchange ◽  
Rhoa Activity ◽  
Src Signaling ◽  
Cell Functions ◽  
Simultaneous Imaging ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

Rho family GTPases are activated with precise spatiotemporal control by guanine nucleotide exchange factors (GEFs). Guanine exchange factor H1 (GEF-H1), a RhoA activator, is thought to act as an integrator of microtubule (MT) and actin dynamics in diverse cell functions. Here we identify a GEF-H1 autoinhibitory sequence and exploit it to produce an activation biosensor to quantitatively probe the relationship between GEF-H1 conformational change, RhoA activity, and edge motion in migrating cells with micrometer- and second-scale resolution. Simultaneous imaging of MT dynamics and GEF-H1 activity revealed that autoinhibited GEF-H1 is localized to MTs, while MT depolymerization subadjacent to the cell cortex promotes GEF-H1 activation in an ~5-µm-wide peripheral band. GEF-H1 is further regulated by Src phosphorylation, activating GEF-H1 in a narrower band ~0–2 µm from the cell edge, in coordination with cell protrusions. This indicates a synergistic intersection between MT dynamics and Src signaling in RhoA activation through GEF-H1.

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 Divergent Mechanisms Activating RAS and Small GTPases Through Post-translational Modification

2021 ◽  
Vol 8 ◽  
Author(s):  
Natsuki Osaka ◽  
Yoshihisa Hirota ◽  
Doshun Ito ◽  
Yoshiki Ikeda ◽  
Ryo Kamata ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Guanine Nucleotide Exchange ◽  
Ras Superfamily ◽  
Switch Regions

RAS is a founding member of the RAS superfamily of GTPases. These small 21 kDa proteins function as molecular switches to initialize signaling cascades involved in various cellular processes, including gene expression, cell growth, and differentiation. RAS is activated by GTP loading and deactivated upon GTP hydrolysis to GDP. Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) accelerate GTP loading and hydrolysis, respectively. These accessory proteins play a fundamental role in regulating activities of RAS superfamily small GTPase via a conserved guanine binding (G)-domain, which consists of five G motifs. The Switch regions lie within or proximal to the G2 and G3 motifs, and undergo dynamic conformational changes between the GDP-bound “OFF” state and GTP-bound “ON” state. They play an important role in the recognition of regulatory factors (GEFs and GAPs) and effectors. The G4 and G5 motifs are the focus of the present work and lie outside Switch regions. These motifs are responsible for the recognition of the guanine moiety in GTP and GDP, and contain residues that undergo post-translational modifications that underlie new mechanisms of RAS regulation. Post-translational modification within the G4 and G5 motifs activates RAS by populating the GTP-bound “ON” state, either through enhancement of intrinsic guanine nucleotide exchange or impairing GAP-mediated down-regulation. Here, we provide a comprehensive review of post-translational modifications in the RAS G4 and G5 motifs, and describe the role of these modifications in RAS activation as well as potential applications for cancer therapy.

scholarly journals The Small GTPases in Fungal Signaling Conservation and Function

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1039
Author(s):  
Mitzuko Dautt-Castro ◽  
Montserrat Rosendo-Vargas ◽  
Sergio Casas-Flores
Keyword(s):  
General Rule ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Plant Interactions ◽  
New Family ◽  
Ras Gtpases ◽  
Guanine Nucleotide Exchange ◽  
Small Proteins ◽  
And Function

Monomeric GTPases, which belong to the Ras superfamily, are small proteins involved in many biological processes. They are fine-tuned regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Several families have been identified in organisms from different kingdoms. Overall, the most studied families are Ras, Rho, Rab, Ran, Arf, and Miro. Recently, a new family named Big Ras GTPases was reported. As a general rule, the proteins of all families have five characteristic motifs (G1–G5), and some specific features for each family have been described. Here, we present an exhaustive analysis of these small GTPase families in fungi, using 56 different genomes belonging to different phyla. For this purpose, we used distinct approaches such as phylogenetics and sequences analysis. The main functions described for monomeric GTPases in fungi include morphogenesis, secondary metabolism, vesicle trafficking, and virulence, which are discussed here. Their participation during fungus–plant interactions is reviewed as well.

Characterization of the activation of small GTPases by their GEFs on membranes using artificial membrane tethering

2017 ◽  
Vol 474 (7) ◽  
pp. 1259-1272 ◽  
Author(s):  
François Peurois ◽  
Simon Veyron ◽  
Yann Ferrandez ◽  
Ilham Ladid ◽  
Sarah Benabdi ◽  
...  
Keyword(s):  
Small Gtpases ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Gtpase Activating Proteins ◽  
Membrane Tethering ◽  
Quantitative Understanding ◽  
Membrane Bound ◽  
Histidine Tag

Active, GTP-bound small GTPases need to be attached to membranes by post-translational lipid modifications in order to process and propagate information in cells. However, generating and manipulating lipidated GTPases has remained difficult, which has limited our quantitative understanding of their activation by guanine nucleotide exchange factors (GEFs) and their termination by GTPase-activating proteins. Here, we replaced the lipid modification by a histidine tag in 11 full-length, human small GTPases belonging to the Arf, Rho and Rab families, which allowed to tether them to nickel–lipid-containing membranes and characterize the kinetics of their activation by GEFs. Remarkably, this strategy uncovered large effects of membranes on the efficiency and/or specificity in all systems studied. Notably, it recapitulated the release of autoinhibition of Arf1, Arf3, Arf4, Arf5 and Arf6 GTPases by membranes and revealed that all isoforms are efficiently activated by two GEFs with different regulatory regimes, ARNO and Brag2. It demonstrated that membranes stimulate the GEF activity of Trio toward RhoG by ∼30 fold and Rac1 by ∼10 fold, and uncovered a previously unknown broader specificity toward RhoA and Cdc42 that was undetectable in solution. Finally, it demonstrated that the exceptional affinity of the bacterial RabGEF DrrA for the phosphoinositide PI(4)P delimits the activation of Rab1 to the immediate vicinity of the membrane-bound GEF. Our study thus validates the histidine-tag strategy as a potent and simple means to mimic small GTPase lipidation, which opens a variety of applications to uncover regulations brought about by membranes.

scholarly journals G-protein binding features and regulation of the RalGDS family member, RGL2

2008 ◽  
Vol 415 (1) ◽  
pp. 145-154 ◽  
Author(s):  
Elisa Ferro ◽  
David Magrini ◽  
Paolo Guazzi ◽  
Thomas H. Fischer ◽  
Sara Pistolesi ◽  
...  
Keyword(s):  
Protein Binding ◽  
G Protein ◽  
Phosphorylation Site ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Mouse Species ◽  
Human And Mouse

RGL2 [RalGDS (Ral guanine nucleotide dissociation stimulator)-like 2] is a member of the RalGDS family that we have previously isolated and characterized as a potential effector for Ras and the Ras analogue Rap1b. The protein shares 89% sequence identity with its mouse orthologue Rlf (RalGDS-like factor). In the present study we further characterized the G-protein-binding features of RGL2 and also demonstrated that RGL2 has guanine-nucleotide-exchange activity toward the small GTPase RalA. We found that RGL2/Rlf properties are well conserved between human and mouse species. Both RGL2 and Rlf have a putative PKA (protein kinase A) phosphorylation site at the C-terminal of the domain that regulates the interaction with small GTPases. We demonstrated that RGL2 is phosphorylated by PKA and phosphorylation reduces the ability of RGL2 to bind H-Ras. As RGL2 and Rlf are unique in the RalGDS family in having a PKA site in the Ras-binding domain, the results of the present study indicate that Ras may distinguish between the different RalGDS family members by their phosphorylation by PKA.

scholarly journals Activation of Cdc42 GTPase upon CRY2-Induced Cortical Recruitment Is Antagonized by GAPs in Fission Yeast

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2089 ◽  
Author(s):  
Iker Lamas ◽  
Nathalie Weber ◽  
Sophie G. Martin
Keyword(s):  
Fission Yeast ◽  
Positive Feedback ◽  
Antagonistic Activity ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Gtpase Activating Protein ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Cell Architecture

The small GTPase Cdc42 is critical for cell polarization in eukaryotic cells. In rod-shaped fission yeast Schizosaccharomyces pombe cells, active GTP-bound Cdc42 promotes polarized growth at cell poles, while inactive Cdc42-GDP localizes ubiquitously also along cell sides. Zones of Cdc42 activity are maintained by positive feedback amplification involving the formation of a complex between Cdc42-GTP, the scaffold Scd2, and the guanine nucleotide exchange factor (GEF) Scd1, which promotes the activation of more Cdc42. Here, we use the CRY2-CIB1 optogenetic system to recruit and cluster a cytosolic Cdc42 variant at the plasma membrane and show that this leads to its moderate activation also on cell sides. Surprisingly, Scd2, which binds Cdc42-GTP, is still recruited to CRY2-Cdc42 clusters at cell sides in individual deletion of the GEFs Scd1 or Gef1. We show that activated Cdc42 clusters at cell sides are able to recruit Scd1, dependent on the scaffold Scd2. However, Cdc42 activity is not amplified by positive feedback and does not lead to morphogenetic changes, due to antagonistic activity of the GTPase activating protein Rga4. Thus, the cell architecture is robust to moderate activation of Cdc42 at cell sides.

scholarly journals The DHR1 Domain of DOCK180 Binds to SNX5 and Regulates Cation-independent Mannose 6-phosphate Receptor Transport

2008 ◽  
Vol 19 (9) ◽  
pp. 3823-3835 ◽  
Author(s):  
Shigeo Hara ◽  
Etsuko Kiyokawa ◽  
Shun-ichiro Iemura ◽  
Tohru Natsume ◽  
Thomas Wassmer ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Retrograde Transport ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Sorting Nexins ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Mannose 6 Phosphate

DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.

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