scholarly journals Essential Role of Vav Family Guanine Nucleotide Exchange Factors in EphA Receptor-Mediated Angiogenesis

2006 ◽  
Vol 26 (13) ◽  
pp. 4830-4842 ◽  
Author(s):  
Sonja G. Hunter ◽  
Guanglei Zhuang ◽  
Dana Brantley-Sieders ◽  
Wojciech Swat ◽  
Christopher W. Cowan ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Rac1 Gtpase ◽  
Rho Family ◽  
Epha Receptor

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.

Identification of residues of the H-ras protein critical for functional interaction with guanine nucleotide exchange factors

1994 ◽  
Vol 14 (2) ◽  
pp. 1104-1112
Author(s):  
R D Mosteller ◽  
J Han ◽  
D Broek
Keyword(s):  
Random Mutagenesis ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Ras Proteins ◽  
Ras Protein ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

Ras proteins are activated in vivo by guanine nucleotide exchange factors encoded by genes homologous to the CDC25 gene of Saccharomyces cerevisiae. We have taken a combined genetic and biochemical approach to probe the sites on Ras proteins important for interaction with such exchange factors and to further probe the mechanism of CDC25-catalyzed GDP-GTP exchange. Random mutagenesis coupled with genetic selection in S. cerevisiae was used to generate second-site mutations within human H-ras-ala15 which could suppress the ability of the Ala-15 substitution to block CDC25 function. We transferred these second-site suppressor mutations to normal H-ras and oncogenic H-rasVal-12 to test whether they induced a general loss of function or whether they selectively affected CDC25 interaction. Four highly selective mutations were discovered, and they affected the surface-located amino acid residues 62, 63, 67, and 69. Two lines of evidence suggested that these residues may be involved in binding to CDC25: (i) using the yeast two-hybrid system, we demonstrated that these mutants cannot bind CDC25 under conditions where the wild-type H-Ras protein can; (ii) we demonstrated that the binding to H-Ras of monoclonal antibody Y13-259, whose epitope has been mapped to residues 63, 65, 66, 67, 70, and 73, is blocked by the mouse sos1 and yeast CDC25 gene products. We also present evidence that the mechanism by which CDC25 catalyzes exchange is more involved than simply catalyzing the release of bound nucleotide and passively allowing nucleotides to rebind. Most critically, a complex of Ras and CDC25 protein, unlike free Fas protein, possesses significantly greater affinity for GTP than for GDP. Furthermore, the Ras CDC25 complex is more readily dissociated into free subunits by GTP than it is by GDP. Both of these results suggest a function for CDC25 in promoting the selective exchange of GTP for GDP.

scholarly journals Distinct Subclasses of Small GTPases Interact with Guanine Nucleotide Exchange Factors in a Similar Manner

1998 ◽  
Vol 18 (12) ◽  
pp. 7444-7454 ◽  
Author(s):  
Gwo-Jen Day ◽  
Raymond D. Mosteller ◽  
Daniel Broek
Keyword(s):  
Alpha Helix ◽  
Small Gtpases ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Structural Determinants ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Ras Superfamily

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.

scholarly journals Identification of residues of the H-ras protein critical for functional interaction with guanine nucleotide exchange factors.

1994 ◽  
Vol 14 (2) ◽  
pp. 1104-1112 ◽  
Author(s):  
R D Mosteller ◽  
J Han ◽  
D Broek
Keyword(s):  
Random Mutagenesis ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Ras Proteins ◽  
Ras Protein ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

Ras proteins are activated in vivo by guanine nucleotide exchange factors encoded by genes homologous to the CDC25 gene of Saccharomyces cerevisiae. We have taken a combined genetic and biochemical approach to probe the sites on Ras proteins important for interaction with such exchange factors and to further probe the mechanism of CDC25-catalyzed GDP-GTP exchange. Random mutagenesis coupled with genetic selection in S. cerevisiae was used to generate second-site mutations within human H-ras-ala15 which could suppress the ability of the Ala-15 substitution to block CDC25 function. We transferred these second-site suppressor mutations to normal H-ras and oncogenic H-rasVal-12 to test whether they induced a general loss of function or whether they selectively affected CDC25 interaction. Four highly selective mutations were discovered, and they affected the surface-located amino acid residues 62, 63, 67, and 69. Two lines of evidence suggested that these residues may be involved in binding to CDC25: (i) using the yeast two-hybrid system, we demonstrated that these mutants cannot bind CDC25 under conditions where the wild-type H-Ras protein can; (ii) we demonstrated that the binding to H-Ras of monoclonal antibody Y13-259, whose epitope has been mapped to residues 63, 65, 66, 67, 70, and 73, is blocked by the mouse sos1 and yeast CDC25 gene products. We also present evidence that the mechanism by which CDC25 catalyzes exchange is more involved than simply catalyzing the release of bound nucleotide and passively allowing nucleotides to rebind. Most critically, a complex of Ras and CDC25 protein, unlike free Fas protein, possesses significantly greater affinity for GTP than for GDP. Furthermore, the Ras CDC25 complex is more readily dissociated into free subunits by GTP than it is by GDP. Both of these results suggest a function for CDC25 in promoting the selective exchange of GTP for GDP.

scholarly journals Vav guanine nucleotide exchange factors link hyperlipidemia and a prothrombotic state

Blood ◽  
2011 ◽  
Vol 117 (21) ◽  
pp. 5744-5750 ◽  
Author(s):  
Kan Chen ◽  
Wei Li ◽  
Jennifer Major ◽  
Shaik Ohidar Rahaman ◽  
Maria Febbraio ◽  
...  
Keyword(s):  
Oxidized Ldl ◽  
Flow Cytometric Analysis ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Prothrombotic State ◽  
Aggregation Assay ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

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.

scholarly journals ITAM Signaling by Vav Family Rho Guanine Nucleotide Exchange Factors Regulates Interstitial Transit Rates of Neutrophils In Vivo

PLoS ONE ◽  
2009 ◽  
Vol 4 (2) ◽  
pp. e4652 ◽  
Author(s):  
Daniel B. Graham ◽  
Bernd H. Zinselmeyer ◽  
Francesca Mascarenhas ◽  
Ryan Delgado ◽  
Mark J. Miller ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

scholarly journals The Emerging Role of Rho Guanine Nucleotide Exchange Factors in Cardiovascular Disorders: Insights Into Atherosclerosis: A Mini Review

2022 ◽  
Vol 8 ◽  
Author(s):  
Mengqi Li ◽  
Qingzheng Jiao ◽  
Wenqiang Xin ◽  
Shulin Niu ◽  
Mingming Liu ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Rho Gtpase ◽  
Guanine Nucleotide Exchange Factors ◽  
Atherosclerotic Cardiovascular Disease ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

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.

New insights into the Ras onco-protein and its interactions with the Raf-1-1 kinase

1996 ◽  
Vol 54 ◽  
pp. 878-879
Author(s):  
Sharon Campbell ◽  
Helen Mott ◽  
Sheng Zhong ◽  
Jonelle Drugan ◽  
John Carpenter
Keyword(s):  
Bound States ◽  
Direct Interaction ◽  
Nucleotide Binding ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Biochemical Effect ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

The Ras proteins are members of a large superfamily of nucleotide-binding proteins that cycle between active GTP- and inactive GDP-bound states. They are positively regulated by guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound state and negatively regulated by GTPase activating proteins (GAPs) that stimulate formation of the inactive GDP-complexed protein. Structural mutations that activate Ras oncogenic potential either impair GAP-stimulated GTPase activity or promote enhanced intrinsic nucleotide exchange. The net result of either biochemical effect is to favor elevated levels of Ras-GTP in vivo.We are investigating selected Ras variants containing substitutions at conserved sites thought to be important for guanine nucleotide binding, GTP hydrolysis, GTP/GDP interconversion and protein recognition, to better understand the role of these conserved amino acids in Ras-mediated signal transduction. Recent studies conducted at the U. of North Carolina will be presented, where we have characterized a novel activating mutation in Ras using multi-dimensional NMR spectroscopy and established a region involved in direct interaction with guanine nucleotide exchange factors.

scholarly journals Trp56of Rac1 Specifies Interaction with a Subset of Guanine Nucleotide Exchange Factors

2001 ◽  
Vol 276 (50) ◽  
pp. 47530-47541 ◽  
Author(s):  
Yuan Gao ◽  
Jingchuan Xing ◽  
Michel Streuli ◽  
Thomas L. Leto ◽  
Yi Zheng
Keyword(s):  
Rho Gtpase ◽  
Specific Inhibitor ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Binding Interaction ◽  
Critical Determinant ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

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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