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Elizabeth Sztul - One of the best experts on this subject based on the ideXlab platform.
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The Guanine Nucleotide Exchange Factor GBF1 Participates in Rotavirus Replication.
2019Co-Authors: Jose L Martinez, Elizabeth Sztul, Eunjoo Lee, Francesca Arnoldi, Elisabeth M. Schraner, Catherine Eichwald, Daniela Silva-ayala, Oscar R. Burrone, Susana López, Carlos F AriasAbstract:Cellular and viral Factors participate in the replication cycle of rotavirus. We report that the guanine Nucleotide Exchange Factor GBF1, which activates the small GTPase Arf1 to induce COPI transport processes, is required for rotavirus replication since knocking down GBF1 expression by RNA interference or inhibiting its activity by treatment with brefeldin A (BFA) or Golgicide A (GCA) significantly reduces the yield of infectious viral progeny. This reduction in virus yield was related to a block in virus assembly, since in the presence of either BFA or GCA, the assembly of infectious mature triple-layered virions was significantly prevented and only double-layered particles were detected. We report that the catalytic activity of GBF1, but not the activation of Arf1, is essential for the assembly of the outer capsid of rotavirus. We show that both BFA and GCA, as well as interfering with the synthesis of GBF1, alter the electrophoretic mobility of glycoproteins VP7 and NSP4 and block the trimerization of the virus surface protein VP7, a step required for its incorporation into virus particles. Although a posttranslational modification of VP7 (other than glycosylation) could be related to the lack of trimerization, we found that NSP4 might also be involved in this process, since knocking down its expression reduces VP7 trimerization. In support, recombinant VP7 protein overexpressed in transfected cells formed trimers only when cotransfected with NSP4.IMPORTANCE Rotavirus, a member of the family Reoviridae, is the major cause of severe diarrhea in children and young animals worldwide. Despite significant advances in the characterization of the biology of this virus, the mechanisms involved in morphogenesis of the virus particle are still poorly understood. In this work, we show that the guanine Nucleotide Exchange Factor GBF1, relevant for COPI/Arf1-mediated cellular vesicular transport, participates in the replication cycle of the virus, influencing the correct processing of viral glycoproteins VP7 and NSP4 and the assembly of the virus surface proteins VP7 and VP4.
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highly conserved motifs within the large sec7 arf guanine Nucleotide Exchange Factor gbf1 target it to the golgi and are critical for gbf1 activity
2018Co-Authors: Cristian A Pocognoni, George A Belov, Ekaterina G Viktorova, Eunjoo Lee, J T Wright, Justyna M Meissner, Garrett Sager, Elizabeth SztulAbstract:Cellular life requires the activation of the ADP-ribosylation Factors (ARFs) by Golgi brefeldin A-resistant Factor 1 (GBF1), a guanine Nucleotide Exchange Factor (GEF) with a highly conserved catal...
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dissecting the role of the arf guanine Nucleotide Exchange Factor gbf1 in golgi biogenesis and protein trafficking
2007Co-Authors: Tomasz Szul, Svetlana A Shestopal, Robert Grabski, Susan A Lyons, Yuichi Morohashi, Martin Lowe, Elizabeth SztulAbstract:COPI recruitment to membranes appears to be essential for the biogenesis of the Golgi and for secretory trafficking. Preventing COPI recruitment by expressing inactive forms of the ADP-ribosylation Factor (ARF) or the ARF-activating guanine Nucleotide Exchange Factor GBF1, or by treating cells with brefeldin A (BFA), causes the collapse of the Golgi into the endoplasmic reticulum (ER) and arrests trafficking of soluble and transmembrane proteins at the ER. Here, we assess COPI function in Golgi biogenesis and protein trafficking by preventing COPI recruitment to membranes by removing GBF1. We report that siRNA-mediated depletion of GBF1 causes COPI dispersal but does not lead to collapse of the Golgi. Instead, it causes extensive tubulation of the cis-Golgi. The Golgi-derived tubules target to peripheral ER-Golgi intermediate compartment (ERGIC) sites and create dynamic continuities between the ERGIC and the cis-Golgi compartment. COPI dispersal in GBF1-depleted cells causes dramatic inhibition of the trafficking of transmembrane proteins. Unexpectedly, soluble proteins continue to be secreted from GBF1-depleted cells. Our findings suggest that a secretory pathway capable of trafficking soluble proteins can be maintained in cells in which COPI recruitment is compromised by GBF1 depletion. However, the trafficking of transmembrane proteins through the existing pathway requires GBF1-mediated ARF activation and COPI recruitment.
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dissection of membrane dynamics of the arf guanine Nucleotide Exchange Factor gbf1
2005Co-Authors: Tomasz Szul, Rafael Garciamata, Elizabeth Brandon, Svetlana A Shestopal, Cecilia Alvarez, Elizabeth SztulAbstract:ADP-ribosylation Factor (ARF)-facilitated recruitment of COP I to membranes is required for secretory traffic. The guanine Nucleotide Exchange Factor GBF1 activates ARF and regulates ARF/COP I dynamics at the endoplasmic reticulum (ER)–Golgi interface. Like ARF and coatomer, GBF1 peripherally associates with membranes. ADP-ribosylation Factor and coatomer have been shown to rapidly cycle between membranes and cytosol, but the membrane dynamics of GBF1 are unknown. Here, we used fluorescence recovery after photobleaching to characterize the behavior of GFP-tagged GBF1. We report that GBF1 rapidly cycles between membranes and the cytosol (t1/2 is approximately 17 ± 1 seconds). GBF1 cycles faster than GFP-tagged ARF, suggesting that in each round of association/dissociation, GBF1 catalyzes a single event of ARF activation, and that the activated ARF remains on membrane after GBF1 dissociation. Using three different approaches [expression of an inactive (E794K) GBF1 mutant, expression of the ARF1 (T31N) mutant with decreased affinity for GTP and Brefeldin A treatment], we show that GBF1 is stabilized on membranes when in a complex with ARF–GDP. GBF1 dissociation from ARF and membranes is triggered by its catalytic activity, i.e. the displacement of GDP and the subsequent binding of GTP to ARF. Our findings imply that continuous cycles of recruitment and dissociation of GBF1 to membranes are required for sustained ARF activation and COP I recruitment that underlies ER-Golgi traffic.
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the membrane tethering protein p115 interacts with gbf1 an arf guanine Nucleotide Exchange Factor
2003Co-Authors: Rafael Garciamata, Elizabeth SztulAbstract:The membrane‐transport Factor p115 interacts with diverse components of the membrane‐transport machinery. It binds two Golgi matrix proteins, a Rab GTPase, and various members of the soluble N ‐ethylmaleimide‐sensitive Factor attachment protein receptor (SNARE) family. Here, we describe a novel interaction between p115 and Golgi‐specific brefeldin‐A‐resistant Factor 1 (GBF1), a guanine‐Nucleotide Exchange Factor for ADP ribosylation Factor (ARF). GBF1 was identified in a yeast two‐hybrid screen, using full‐length p115 as bait. The interaction was confirmed biochemically, using in vitro and in vivo assays. The interacting domains were mapped to the proline‐rich region of GBF1 and the head region of p115. These proteins colocalize extensively in the Golgi and in peripheral vesicular tubular clusters. Mutagenesis analysis indicates that the interaction is not required for targeting GBF1 or p115 to membranes. Expression of the p115‐binding (pro‐rich) region of GBF1 leads to Golgi disruption, indicating that the interaction between p115 and GBF1 is functionally relevant.
Ian P Whitehead - One of the best experts on this subject based on the ideXlab platform.
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the rho specific guanine Nucleotide Exchange Factor dbs regulates breast cancer cell migration
2009Co-Authors: Zhuoming Liu, Homer Adams, Ian P WhiteheadAbstract:Dbs is a Rho-specific guanine Nucleotide Exchange Factor (RhoGEF) that regulates neurotrophin-3-induced cell migration in Schwann cells. Here we report that Dbs regulates cell motility in tumor-derived, human breast epithelial cells through activation of Cdc42 and Rac1. Cdc42 and Rac1 are activated in T47D cells that stably express onco- or proto-Dbs, and activation is dependent upon growth of the cells on collagen I. Transient suppression of expression of Cdc42 or Rac1 by small interfering RNAs attenuates Dbs-enhanced motility. Both onco- and proto-Dbs-enhanced motility correlates with an increase in tyrosine phosphorylation of focal adhesion kinase on Tyr-397 and p130(Cas) on Tyr-410 and an increase in the abundance of the Crk.p130(Cas) complex. Suppression of expression of Cdc42 or its effector, Ack1, reduces tyrosine phosphorylation of focal adhesion kinase and p130(Cas) and disrupts the Crk.p130(Cas) complex. We further determined that suppression of expression of Cdc42, Ack1, p130(Cas), or Crk reduces Rac1 activation and cell motility in Dbs-expressing cells to a level comparable with that in vector cells. Therefore, a cascade of activation of Cdc42 and Rac1 by Dbs through the Cdc42 effector Ack1 and the Crk.p130(Cas) complex is established. Suppression of the expression of endogenous Dbs reduces cell motility in both T47D cells and MDA-MB-231 cells, which correlates with the down-regulation of Cdc42 activity. This suggests that Dbs activates Cdc42 in these two human breast cancer cell lines and that the normal function of Dbs may be required to support cell movement.
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transformation by the rho specific guanine Nucleotide Exchange Factor dbs requires rock i mediated phosphorylation of myosin light chain
2006Co-Authors: Zhuoming Liu, Elena V Kostenko, Gwendolyn M Mahon, Oyenike O Olabisi, Ian P WhiteheadAbstract:Abstract Dbs was identified in a cDNA-based expression screen for sequences that can cause malignant growth when expressed in murine fibroblasts. In previous studies we have shown that Dbs is a Rho-specific guanine Nucleotide Exchange Factor that can activate RhoA and/or Cdc42 in a cell-specific manner. In this current study we have used a combination of genetic and pharmacological approaches to examine the relative contributions of RhoA·PRK and RhoA·ROCK signaling to Dbs transformation. Our analysis indicates that ROCK is activated in Dbs-transformed cells and that Dbs transformation is dependent upon ROCK I activity. In contrast, there appears to be no requirement for PRK activation in Dbs transformation. Dbs transformation is also associated with increased phosphorylation of myosin light chain and stress fiber formation, both of which occur in a ROCK-dependent manner. Suppression of myosin light chain expression by small interfering RNAs impairs Dbs focus formation, thus establishing a direct link between actinomyosin contraction and Rho-specific guanine Nucleotide Exchange Factor transformation.
Gary M Bokoch - One of the best experts on this subject based on the ideXlab platform.
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p rex1 is a primary rac2 guanine Nucleotide Exchange Factor in mouse neutrophils
2005Co-Authors: Xuemei Dong, Zhicheng Mo, Zhong Li, Gary M Bokoch, Dianqing WuAbstract:Summary Leukocyte chemoattractants regulate many leukocyte functions, including leukocyte chemotaxis, via the Rho family of small GTPases that include RhoA, Cdc42, and Rac [1–4]. Previous work has revealed mechanisms by which chemoattractants regulate RhoA and Cdc42 in mouse neutrophils [5–8], but the mechanisms for regulation of Rac remain unclear even though Rac is important for neutrophil functions [9–13]. Here, we characterized P-Rex1, a Gβγ and PIP 3 -regulated guanine Nucleotide Exchange Factor that was initially identified as a Rac activator in response to chemoattractants [14], for its roles in the regulation of Rac activity and neutrophil functions. We generated a mouse line in which the P-Rex1 gene is disrupted and found that P-Rex1 deficiency did not significantly affect Rac1 activation but diminished Rac2 activation in response to a chemoattractant fMLP in mouse neutrophils. This preference for Rac2 may partially result from the apparent higher affinity of P-Rex1 for Rac2 than for Rac1 because P-Rex1 was more readily immunoprecipitated with Rac2 S17N than Rac1 S17N . In addition, P-Rex1 deficiency significantly attenuated fMLP-induced F actin formation and superoxide production without affecting LPS- or PMA-induced production. Furthermore, P-Rex1 deficiency caused a chemotactic defect that is primarily attributed to a reduction in the migration rate rather than directionality.
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P-Rex1 Is a Primary Rac2 Guanine Nucleotide Exchange Factor in Mouse Neutrophils
2005Co-Authors: Xuemei Dong, Gary M Bokoch, Caiying GuoAbstract:Leukocyte chemoattractants regulate many leukocyte functions, including leukocyte chemotaxis, via the Rho family of small GTPases that include RhoA, Cdc42, and Rac. Previous work has revealed mechanisms by which chemoattractants regulate RhoA and Cdc42 in mouse neutrophils, but the mechanisms for regulation of Rac remain unclear even though Rac is important for neutrophil functions. Here, we characterized P-Rex1, a Gbetagamma and PIP(3)-regulated guanine Nucleotide Exchange Factor that was initially identified as a Rac activator in response to chemoattractants, for its roles in the regulation of Rac activity and neutrophil functions. We generated a mouse line in which the P-Rex1 gene is disrupted and found that P-Rex1 deficiency did not significantly affect Rac1 activation but diminished Rac2 activation in response to a chemoattractant fMLP in mouse neutrophils. This preference for Rac2 may partially result from the apparent higher affinity of P-Rex1 for Rac2 than for Rac1 because P-Rex1 was more readily immunoprecipitated with Rac2(S17N) than Rac1(S17N). In addition, P-Rex1 deficiency significantly attenuated fMLP-induced F actin formation and superoxide production without affecting LPS- or PMA-induced production. Furthermore, P-Rex1 deficiency caused a chemotactic defect that is primarily attributed to a reduction in the migration rate rather than directionality.
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Nucleotide Exchange Factor gef h1 mediates cross talk between microtubules and the actin cytoskeleton
2002Co-Authors: Mira Krendel, Frank Zenke, Gary M BokochAbstract:Nucleotide Exchange Factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton
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Nucleotide Exchange Factor gef h1 mediates cross talk between microtubules and the actin cytoskeleton
2002Co-Authors: Mira Krendel, Frank Zenke, Gary M BokochAbstract:Regulation of the actin cytoskeleton by microtubules is mediated by the Rho family GTPases. However, the molecular mechanisms that link microtubule dynamics to Rho GTPases have not, as yet, been identified. Here we show that the Rho guanine Nucleotide Exchange Factor (GEF)-H1 is regulated by an interaction with microtubules. GEF-H1 mutants that are deficient in microtubule binding have higher activity levels than microtubule-bound forms. These mutants also induce Rho-dependent changes in cell morphology and actin organization. Furthermore, drug-induced microtubule depolymerization induces changes in cell morphology and gene expression that are similar to the changes induced by the expression of active forms of GEF-H1. Furthermore, these effects are inhibited by dominant-negative versions of GEF-H1. Thus, GEF-H1 links changes in microtubule integrity to Rho-dependent regulation of the actin cytoskeleton.
Yong Xiong - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of a guanine Nucleotide Exchange Factor encoded by the scrub typhus pathogen orientia tsutsugamushi
2020Co-Authors: Christopher Lim, Jason M Berk, Alyssa Blaise, Josie Bircher, Anthony J Koleske, Mark Hochstrasser, Yong XiongAbstract:Rho family GTPases regulate an array of cellular processes and are often modulated by pathogens to promote infection. Here, we identify a cryptic guanine Nucleotide Exchange Factor (GEF) domain in the OtDUB protein encoded by the pathogenic bacterium Orientia tsutsugamushi A proteomics-based OtDUB interaction screen identified numerous potential host interactors, including the Rho GTPases Rac1 and Cdc42. We discovered a domain in OtDUB with Rac1/Cdc42 GEF activity (OtDUBGEF), with higher activity toward Rac1 in vitro. While this GEF bears no obvious sequence similarity to known GEFs, crystal structures of OtDUBGEF alone (3.0 A) and complexed with Rac1 (1.7 A) reveal striking convergent evolution, with a unique topology, on a V-shaped bacterial GEF fold shared with other bacterial GEF domains. Structure-guided mutational analyses identified residues critical for activity and a mechanism for Nucleotide displacement. Ectopic expression of OtDUB activates Rac1 preferentially in cells, and expression of the OtDUBGEF alone alters cell morphology. Cumulatively, this work reveals a bacterial GEF within the multifunctional OtDUB that co-opts host Rac1 signaling to induce changes in cytoskeletal structure.
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crystal structure of a guanine Nucleotide Exchange Factor encoded by the scrub typhus pathogen orientia tsutsugamushi
2020Co-Authors: Christopher Lim, Jason M Berk, Alyssa Blaise, Josie Bircher, Anthony J Koleske, Mark Hochstrasser, Yong XiongAbstract:Rho GTPases regulate an array of cellular processes and are often modulated by pathogens to promote infection. Here, we identify a guanine Nucleotide Exchange Factor (GEF) domain in the OtDUB protein encoded by the pathogenic bacterium Orientia tsutsugamushi. A proteomics-based OtDUB interaction screen identified Rho GTPases Rac1 and Cdc42. We discovered a new domain in OtDUB with GEF activity toward Rac1 in vitro. While this GEF bears no sequence similarity to known GEFs, crystal structures of OtDUBGEF alone and complexed with Rac1 reveal striking convergent evolution, with a distinct topology, on a V-shaped bacterial GEF fold. Structure-guided mutational analyses identified residues critical for activity and a novel mechanism for Nucleotide displacement. Ectopic expression of OtDUB activates Rac1 in cells, and expression of the OtDUBGEF alters cell morphology. Cumulatively, this work reveals a novel bacterial GEF within the multifunctional OtDUB that co-opts host Rac1 signaling to evoke changes in cytoskeletal structure.
Zhuoming Liu - One of the best experts on this subject based on the ideXlab platform.
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the rho specific guanine Nucleotide Exchange Factor dbs regulates breast cancer cell migration
2009Co-Authors: Zhuoming Liu, Homer Adams, Ian P WhiteheadAbstract:Dbs is a Rho-specific guanine Nucleotide Exchange Factor (RhoGEF) that regulates neurotrophin-3-induced cell migration in Schwann cells. Here we report that Dbs regulates cell motility in tumor-derived, human breast epithelial cells through activation of Cdc42 and Rac1. Cdc42 and Rac1 are activated in T47D cells that stably express onco- or proto-Dbs, and activation is dependent upon growth of the cells on collagen I. Transient suppression of expression of Cdc42 or Rac1 by small interfering RNAs attenuates Dbs-enhanced motility. Both onco- and proto-Dbs-enhanced motility correlates with an increase in tyrosine phosphorylation of focal adhesion kinase on Tyr-397 and p130(Cas) on Tyr-410 and an increase in the abundance of the Crk.p130(Cas) complex. Suppression of expression of Cdc42 or its effector, Ack1, reduces tyrosine phosphorylation of focal adhesion kinase and p130(Cas) and disrupts the Crk.p130(Cas) complex. We further determined that suppression of expression of Cdc42, Ack1, p130(Cas), or Crk reduces Rac1 activation and cell motility in Dbs-expressing cells to a level comparable with that in vector cells. Therefore, a cascade of activation of Cdc42 and Rac1 by Dbs through the Cdc42 effector Ack1 and the Crk.p130(Cas) complex is established. Suppression of the expression of endogenous Dbs reduces cell motility in both T47D cells and MDA-MB-231 cells, which correlates with the down-regulation of Cdc42 activity. This suggests that Dbs activates Cdc42 in these two human breast cancer cell lines and that the normal function of Dbs may be required to support cell movement.
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transformation by the rho specific guanine Nucleotide Exchange Factor dbs requires rock i mediated phosphorylation of myosin light chain
2006Co-Authors: Zhuoming Liu, Elena V Kostenko, Gwendolyn M Mahon, Oyenike O Olabisi, Ian P WhiteheadAbstract:Abstract Dbs was identified in a cDNA-based expression screen for sequences that can cause malignant growth when expressed in murine fibroblasts. In previous studies we have shown that Dbs is a Rho-specific guanine Nucleotide Exchange Factor that can activate RhoA and/or Cdc42 in a cell-specific manner. In this current study we have used a combination of genetic and pharmacological approaches to examine the relative contributions of RhoA·PRK and RhoA·ROCK signaling to Dbs transformation. Our analysis indicates that ROCK is activated in Dbs-transformed cells and that Dbs transformation is dependent upon ROCK I activity. In contrast, there appears to be no requirement for PRK activation in Dbs transformation. Dbs transformation is also associated with increased phosphorylation of myosin light chain and stress fiber formation, both of which occur in a ROCK-dependent manner. Suppression of myosin light chain expression by small interfering RNAs impairs Dbs focus formation, thus establishing a direct link between actinomyosin contraction and Rho-specific guanine Nucleotide Exchange Factor transformation.
