scholarly journals Activation of Cellular Arf GTPases by Poliovirus Protein 3CD Correlates with Virus Replication

2007 ◽  
Vol 81 (17) ◽  
pp. 9259-9267 ◽  
Author(s):  
George A. Belov ◽  
Courtney Habbersett ◽  
David Franco ◽  
Ellie Ehrenfeld

ABSTRACT We have previously shown that synthesis of poliovirus protein 3CD in uninfected HeLa cell extracts induces an increased association with membranes of the cellular Arf GTPases, which are key players in cellular membrane traffic. Arfs cycle between an inactive, cytoplasmic, GDP-bound form and an active, membrane-associated, GTP-bound form. 3CD promotes binding of Arf to membranes by initiating recruitment to membranes of guanine nucleotide exchange factors (GEFs), BIG1 and BIG2. GEFs activate Arf by replacing GDP with GTP. In poliovirus-infected cells, there is a dramatic redistribution of cellular Arf pools that coincides with the reorganization of membranes used to form viral RNA replication complexes. Here we demonstrate that Arf translocation in vitro can be induced by purified recombinant 3CD protein; thus, concurrent translation of viral RNA is not required. Coexpression of 3C and 3D proteins was not sufficient to target Arf to membranes. 3CD expressed in HeLa cells was retained after treatment of the cells with digitonin, indicating that it may interact with a membrane-bound host factor. A F441S mutant of 3CD was shown previously to have lost Arf translocation activity and was also defective in attracting the corresponding GEFs to membranes. A series of other mutations were introduced at 3CD residue F441. Mutations that retained Arf translocation activity of 3CD also supported efficient growth of virus, regardless of their effects on 3D polymerase elongation activity. Those that abrogated Arf activation by 3CD generated quasi-infectious RNAs that produced some plaques from which revertants that always restored the Arf activation property of 3CD were rescued.

2006 ◽  
Vol 26 (13) ◽  
pp. 4830-4842 ◽  
Author(s):  
Sonja G. Hunter ◽  
Guanglei Zhuang ◽  
Dana Brantley-Sieders ◽  
Wojciech Swat ◽  
Christopher W. Cowan ◽  
...  

ABSTRACT Angiogenesis, the process by which new blood vessels are formed from preexisting vasculature, is critical for vascular remodeling during development and contributes to the pathogenesis of diseases such as cancer. Prior studies from our laboratory demonstrate that the EphA2 receptor tyrosine kinase is a key regulator of angiogenesis in vivo. The EphA receptor-mediated angiogenic response is dependent on activation of Rho family GTPase Rac1 and is regulated by phosphatidylinositol 3-kinase. Here we report the identification of Vav2 and Vav3 as guanine nucleotide exchange factors (GEFs) that link the EphA2 receptor to Rho family GTPase activation and angiogenesis. Ephrin-A1 stimulation recruits the binding of Vav proteins to the activated EphA2 receptor. The induced association of EphA receptor and Vav proteins modulates the activity of Vav GEFs, leading to activation of Rac1 GTPase. Overexpression of either Vav2 or Vav3 in primary microvascular endothelial cells promotes Rac1 activation, cell migration, and assembly in response to ephrin-A1 stimulation. Conversely, loss of Vav2 and Vav3 GEFs inhibits Rac1 activation and ephrin-A1-induced angiogenic responses both in vitro and in vivo. In addition, embryonic fibroblasts derived from Vav2−/− Vav3−/− mice fail to spread on an ephrin-A1-coated surface and exhibit a significant decrease in the formation of ephrin-A1-induced lamellipodia and filopodia. These findings suggest that Vav GEFs serve as a molecular link between EphA2 receptors and the actin cytoskeleton and provide an important mechanism for EphA2-mediated angiogenesis.


2020 ◽  
Author(s):  
Stefanie Lübke ◽  
Carina Braukmann ◽  
Karl-Heinz Rexer ◽  
Lubjinka Cigoja ◽  
Susanne F. Önel

AbstractGuanine nucleotide exchange factors (GEF) of the BRAG subfamily activate small Arf GTPases, which are pivotal regulators of intracellular membrane traffic and actin dynamics. Here, we demonstrate a novel interaction between the Abl-interactor (Abi) and the BRAG family member Schizo. We mapped the SH3 domain of Abi to interact with the N-terminal region of Schizo. This region is additionally involved in the binding of the cytodomain of the cell adhesion molecule N-cadherin. In schizo loss of function mutants, we detected increased amounts of N-cadherin. In contrast, the expression of the GEF (Sec7) and the membrane-binding (pleckstrin homology) domains decreased amounts of N-cadherin, indicating a crucial role of the Sec7-PH module in regulating N-cadherin levels. Unlike other Sec7 GEFs, where the catalytic Sec7 domain is autoinhibited, the Sec7 and PH domain of BRAG2 are constitutively accessible, raising the question how GEF activity is controlled in a spatial and temporal manner. Our genetic analyzes demonstrate that the nature of the Abi Schizo interaction is to antagonize Schizo function and to restore wild-type amounts of N-cadherin.


2003 ◽  
Vol 77 (21) ◽  
pp. 11408-11416 ◽  
Author(s):  
Mark H. Fogg ◽  
Natalya L. Teterina ◽  
Ellie Ehrenfeld

ABSTRACT Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation.


1998 ◽  
Vol 18 (12) ◽  
pp. 7444-7454 ◽  
Author(s):  
Gwo-Jen Day ◽  
Raymond D. Mosteller ◽  
Daniel Broek

ABSTRACT The Ras-related GTPases are small, 20- to 25-kDa proteins which cycle between an inactive GDP-bound form and an active GTP-bound state. The Ras superfamily includes the Ras, Rho, Ran, Arf, and Rab/YPT1 families, each of which controls distinct cellular functions. The crystal structures of Ras, Rac, Arf, and Ran reveal a nearly superimposible structure surrounding the GTP-binding pocket, and it is generally presumed that the Rab/YPT1 family shares this core structure. The Ras, Rac, Ran, Arf, and Rab/YPT1 families are activated by interaction with family-specific guanine nucleotide exchange factors (GEFs). The structural determinants of GTPases required for interaction with family-specific GEFs have begun to emerge. We sought to determine the sites on YPT1 which interact with GEFs. We found that mutations of YPT1 at position 42, 43, or 49 (effector loop; switch I), position 69, 71, 73, or 75 (switch II), and position 107, 109, or 115 (alpha-helix 3–loop 7 [α3-L7]) are intragenic suppressors of dominant interfering YPT1 mutant N22 (YPT1-N22), suggesting these mutations prevent YPT1-N22 from binding to and sequestering an endogenous GEF. Mutations at these positions prevent interaction with the DSS4 GEF in vitro. Mutations in the switch II and α3-L7 regions do not prevent downstream signaling in yeast when combined with a GTPase-defective (activating) mutation. Together, these results show that the YPT1 GTPase interacts with GEFs in a manner reminiscent of that for Ras and Arf in that these GTPases use divergent sequences corresponding to the switch I and II regions and α3-L7 of Ras to interact with family-specific GEFs. This finding suggests that GTPases of the Ras superfamily each may share common features of GEF-mediated guanine nucleotide exchange even though the GEFs for each of the Ras subfamilies appear evolutionarily unrelated.


Blood ◽  
2010 ◽  
Vol 116 (16) ◽  
pp. 2921-2931 ◽  
Author(s):  
Ande Satyanarayana ◽  
Kristbjorn Orri Gudmundsson ◽  
Xiu Chen ◽  
Vincenzo Coppola ◽  
Lino Tessarollo ◽  
...  

Abstract RapGEF2 is one of many guanine nucleotide exchange factors (GEFs) that specifically activate Rap1. Here, we generated RapGEF2 conditional knockout mice and studied its role in embryogenesis and fetal as well as adult hematopoietic stem cell (HSC) regulation. RapGEF2 deficiency led to embryonic lethality at ∼ E11.5 due to severe yolk sac vascular defects. However, a similar number of Flk1+ cells were present in RapGEF2+/+ and RapGEF2−/− yolk sacs indicating that the bipotential early progenitors were in fact generated in the absence of RapGEF2. Further analysis of yolk sacs and embryos revealed a significant reduction of CD41 expressing cells in RapGEF2−/− genotype, suggesting a defect in the maintenance of definitive hematopoiesis. RapGEF2−/− cells displayed defects in proliferation and migration, and the in vitro colony formation ability of hematopoietic progenitors was also impaired. At the molecular level, Rap1 activation was impaired in RapGEF2−/− cells that in turn lead to defective B-raf/ERK signaling. Scl/Gata transcription factor expression was significantly reduced, indicating that the defects observed in RapGEF2−/− cells could be mediated through Scl/Gata deregulation. Inducible deletion of RapGEF2 during late embryogenesis in RapGEF2cko/ckoERcre mice leads to defective fetal liver erythropoiesis. Conversely, inducible deletion in the adult bone marrow, or specific deletion in B cells, T cells, HSCs, and endothelial cells has no impact on hematopoiesis.


Blood ◽  
2011 ◽  
Vol 117 (21) ◽  
pp. 5744-5750 ◽  
Author(s):  
Kan Chen ◽  
Wei Li ◽  
Jennifer Major ◽  
Shaik Ohidar Rahaman ◽  
Maria Febbraio ◽  
...  

AbstractPlatelet hyperactivity associated with hyperlipidemia contributes to development of a pro-thrombotic state. We previously showed that oxidized LDL (oxLDL) formed in the setting of hyperlipidemia and atherosclerosis initiated a CD36-mediated signaling cascade leading to platelet hyperactivity. We now show that the guanine nucleotide exchange factors Vav1 and Vav3 were tyrosine phosphorylated in platelets exposed to oxLDL. Pharmacologic inhibition of src family kinases abolished Vav1 phosphorylation by oxLDL in vitro. Coimmunoprecipitations revealed the tyrosine phosphorylated form of src kinase Fyn was associated with Vav1 in platelets exposed to oxLDL. Using a platelet aggregation assay, we demonstrated that Vav1 deficiency, Fyn deficiency, or Vav1/Vav3 deficiency protected mice from diet-induced platelet hyperactivity. Furthermore, flow cytometric analysis revealed that Vav1/Vav3 deficiency significantly inhibited oxLDL-mediated integrin αIIbβIII activation of platelets costimulated with ADP. Finally, we showed with an in vivo carotid artery thrombosis model that genetic deletion of Vav1 and Vav3 together may prevent the development of occlusive thrombi in mice fed a high-fat diet. These findings implicate Vav proteins in oxLDL-mediated platelet activation and suggest that Vav family member(s) may act as critical modulators linking a prothrombotic state and hyperlipidemia.


2015 ◽  
Vol 26 (2) ◽  
pp. 238-255 ◽  
Author(s):  
Ning Wang ◽  
Mo Wang ◽  
Yi-Hua Zhu ◽  
Timothy W. Grosel ◽  
Daokun Sun ◽  
...  

Rho GTPases, activated by Rho guanine nucleotide exchange factors (GEFs), are conserved molecular switches for signal transductions that regulate diverse cellular processes, including cell polarization and cytokinesis. The fission yeast Schizosaccharomyces pombe has six Rho GTPases (Cdc42 and Rho1–Rho5) and seven Rho GEFs (Scd1, Rgf1–Rgf3, and Gef1–Gef3). The GEFs for Rho2–Rho5 have not been unequivocally assigned. In particular, Gef3, the smallest Rho GEF, was barely studied. Here we show that Gef3 colocalizes with septins at the cell equator. Gef3 physically interacts with septins and anillin Mid2 and depends on them to localize. Gef3 coprecipitates with GDP-bound Rho4 in vitro and accelerates nucleotide exchange of Rho4, suggesting that Gef3 is a GEF for Rho4. Consistently, Gef3 and Rho4 are in the same genetic pathways to regulate septum formation and/or cell separation. In gef3∆ cells, the localizations of two potential Rho4 effectors—glucanases Eng1 and Agn1—are abnormal, and active Rho4 level is reduced, indicating that Gef3 is involved in Rho4 activation in vivo. Moreover, overexpression of active Rho4 or Eng1 rescues the septation defects of mutants containing gef3∆. Together our data support that Gef3 interacts with the septin complex and activates Rho4 GTPase as a Rho GEF for septation in fission yeast.


2022 ◽  
Vol 8 ◽  
Author(s):  
Mengqi Li ◽  
Qingzheng Jiao ◽  
Wenqiang Xin ◽  
Shulin Niu ◽  
Mingming Liu ◽  
...  

Atherosclerosis is a leading cause of cardiovascular disease, and atherosclerotic cardiovascular disease accounts for one-third of global deaths. However, the mechanism of atherosclerosis is not fully understood. It is well-known that the Rho GTPase family, especially Rho A, plays a vital role in the development and progression of arteriosclerosis. Rho guanine nucleotide exchange factors (Rho GEFs), which act upstream of Rho GTPases, are also involved in the atheromatous pathological process. Despite some research on the role of Rho GEFS in the regulation of atherosclerosis, the number of studies is small relative to studies on the essential function of Rho GEFs. Some studies have preliminarily revealed Rho GEF regulation of atherosclerosis by experiments in vivo and in vitro. Herein, we review the advances in research on the relationship and interaction between Rho GEFs and atheroma to provide a potential reference for further study of atherosclerosis.


2001 ◽  
Vol 276 (50) ◽  
pp. 47530-47541 ◽  
Author(s):  
Yuan Gao ◽  
Jingchuan Xing ◽  
Michel Streuli ◽  
Thomas L. Leto ◽  
Yi Zheng

Signaling specificity of Rho GTPase pathways is achieved in part by selective interaction between members of the Dbl family guanine nucleotide exchange factors (GEFs) and their Rho GTPase substrates. For example, Trio, GEF-H1, and Tiam1 are a subset of GEFs that specifically activate Rac1 but not the closely related Cdc42. The Rac1 specificity of these GEFs appears to be governed by Rac1-GEF binding interaction. To understand the detailed mechanism underlying the GEF specificity issue, we have analyzed a panel of chimeras made between Rac1 and Cdc42 and examined a series of point mutants of Rac1 made at the switch I, switch II, and β2/β3regions for their ability to interact with and to be activated by the GEFs. The results reveal that Rac1 residues of both the switch I and switch II regions are involved in GEF docking and GEF-mediated nucleotide disruption, because mutation of Asp38, Asn39, Gln61, Tyr64, or Arg66/Leu67into Ala results in the loss of GEF binding, whereas mutation at Tyr32, Asp65, or Leu70/Ser71leads to the loss of GEF catalysis while retaining the binding capability. The region between amino acids 53–72 of Rac1 is required for specific recognition and activation by the GEFs, and Trp56in β3appears to be the critical determinant. Introduction of Trp56to Cdc42 renders it fully responsive to the Rac-specific GEFin vitroand in cells. Further, a polypeptide derived from the β3region of Rac1 including the Trp56residue serves as a specific inhibitor for Rac1 interaction with the GEFs. Taken together, these results indicate that Trp56is the necessary and sufficient determinant of Rac1 for discrimination by the subset of Rac1-specific GEFs and suggest that a compound mimicking Trp56action could be explored as an interfering reagent specifically targeting Rac1 activation.


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