Transcriptional regulation of a Ras nucleotide-exchange factor gene by extracellular signals in fission yeast

1994 ◽  
Vol 107 (12) ◽  
pp. 3635-3642 ◽  
Author(s):  
D.A. Hughes ◽  
N. Yabana ◽  
M. Yamamoto
Keyword(s):  
Feedback Loop ◽  
Nitrogen Starvation ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Exchange Factor ◽  
Physiological Signal ◽  
Extracellular Signals ◽  
Mating Pheromones ◽  
Nucleotide Exchange Factor ◽  
Pheromone Signalling

The ste6 gene of Schizosaccharomyces pombe encodes a putative GDP-GTP exchange factor for the ras1 gene product. Genetic analysis of the ste6 and ras1 genes has shown that they are required for mating and for the response to mating pheromones. In this study we show that expression of the ste6-encoded mRNA is induced by nitrogen starvation, the physiological signal that triggers mating and sexual differentiation. Exposure to mating pheromones enhances the induction of ste6 expression upon nitrogen starvation. Pheromone-induced expression requires not only the function of components of the pheromone-signalling pathway, but also ras1 function. Furthermore, mutants in which the Ras1 protein is activated have higher basal and induced levels of ste6 gene expression than wild-type cells. These observations indicate the existence of a positive-feedback loop through which Ras1 stimulates the expression of its own activator. Since Ste6 is likely to promote the exchange of guanine nucleotides on Ras1 protein, our results suggest an important role for GDP-GTP exchange in the regulation of Ras1 activity during the mating process in S. pombe.

scholarly journals Requirement of nucleotide exchange factor for Ypt1 GTPase mediated protein transport.

1995 ◽  
Vol 130 (5) ◽  
pp. 1051-1061 ◽  
Author(s):  
S Jones ◽  
R J Litt ◽  
C J Richardson ◽  
N Segev
Keyword(s):  
Essential Role ◽  
Protein Transport ◽  
Vesicular Transport ◽  
Mutant Protein ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Mutant Proteins ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor

Small GTPases of the rab family are involved in the regulation of vesicular transport. It is believed that cycling between the GTP- and GDP-bound forms, and accessory factors regulating this cycling are crucial for rab function. However, an essential role for rab nucleotide exchange factors has not yet been demonstrated. In this report we show the requirement of nucleotide exchange factor activity for Ypt1 GTPase mediated protein transport. The Ypt1 protein, a member of the rab family, plays a role in targeting vesicles to the acceptor compartment and is essential for the first two steps of the yeast secretory pathway. We use two YPT1 dominant mutations that contain alterations in a highly conserved GTP-binding domain, N121I and D124N. YPT1-D124N is a novel mutation that encodes a protein with nucleotide specificity modified from guanine to xanthine. This provides a tool for the study of an individual rab GTPase in crude extracts: a xanthosine triphosphate (XTP)-dependent conditional dominant mutation. Both mutations confer growth inhibition and a block in protein secretion when expressed in vivo. The purified mutant proteins do not bind either GDP or GTP. Moreover, they completely inhibit the ability of the exchange factor to stimulate nucleotide exchange for wild type Ypt1 protein, and are potent inhibitors of ER to Golgi transport in vitro at the vesicle targeting step. The inhibitory effects of the Ypt1-D124N mutant protein on both nucleotide exchange activity and protein transport in vitro can be relieved by XTP, indicating that it is the nucleotide-free form of the mutant protein that is inhibitory. These results suggest that the dominant mutant proteins inhibit protein transport by sequestering the exchange factor from the wild type Ypt1 protein, and that this factor has an essential role in vesicular transport.

scholarly journals Multifaceted Roles of the Ras Guanine-Nucleotide Exchange Factor ChRgf in Development, Pathogenesis, and Stress Responses of Colletotrichum higginsianum

2017 ◽  
Vol 107 (4) ◽  
pp. 433-443 ◽  
Author(s):  
Qiongnan Gu ◽  
Meijuan Chen ◽  
Junbin Huang ◽  
Yangdou Wei ◽  
Tom Hsiang ◽  
...  
Keyword(s):  
Stress Responses ◽  
Vegetative Growth ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Colletotrichum Higginsianum ◽  
Nucleotide Exchange Factor

The infection process of Colletotrichum higginsianum, which causes a disease of crucifers, involves several key steps: conidial germination, appressorial formation, appressorial penetration, and invasive growth in host tissues. In this study, the ChRgf gene encoding a Ras guanine-nucleotide exchange factor protein was identified by screening T-DNA insertion mutants generated from Agrobacterium tumefaciens-mediated transformation that were unable to cause disease on the host Arabidopsis thaliana. Targeted gene deletion of ChRgf resulted in a null mutant (ΔChrgf-42) with defects in vegetative growth, hyphal morphology, and conidiation, and poor surface attachment and low germination on hydrophobic surfaces; however, there were no apparent differences in appressorial turgor pressure between the wild type and the mutant. The conidia of the mutant were unable to geminate on attached Arabidopsis leaves and did not cause any disease symptoms. Intracellular cyclic adenosine monophosphate levels in the ΔChrgf mutant were lower than that of the wild type. Our results suggest that ChRgf is a key regulator in response to salt and osmotic stresses in C. higginsianum, and indicate that it is involved in fungal pathogenicity. This gene seems to act as an important modulator upstream of several distinct signaling pathways that are involved in regulating vegetative growth, conidiation, infection-related structure development, and stress responses of C. higginsianum.

scholarly journals Oligomerization of the Sec7 domain Arf guanine nucleotide exchange factor GBF1 is dispensable for Golgi localization and function but regulates degradation

2016 ◽  
Vol 310 (6) ◽  
pp. C456-C469 ◽  
Author(s):  
Jay M. Bhatt ◽  
Ekaterina G. Viktorova ◽  
Theodore Busby ◽  
Paulina Wyrozumska ◽  
Laura E. Newman ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Sec7 Domain ◽  
And Function ◽  
In Cells

Members of the large Sec7 domain-containing Arf guanine nucleotide exchange factor (GEF) family have been shown to dimerize through their NH2-terminal dimerization and cyclophilin binding (DCB) and homology upstream of Sec7 (HUS) domains. However, the importance of dimerization in GEF localization and function has not been assessed. We generated a GBF1 mutant (91/130) in which two residues required for oligomerization (K91 and E130 within the DCB domain) were replaced with A and assessed the effects of these mutations on GBF1 localization and cellular functions. We show that 91/130 is compromised in oligomerization but that it targets to the Golgi in a manner indistinguishable from wild-type GBF1 and that it rapidly exchanges between the cytosolic and membrane-bound pools. The 91/130 mutant appears active as it integrates within the functional network at the Golgi, supports Arf activation and COPI recruitment, and sustains Golgi homeostasis and cargo secretion when provided as a sole copy of functional GBF1 in cells. In addition, like wild-type GBF1, the 91/130 mutant supports poliovirus RNA replication, a process requiring GBF1 but believed to be independent of GBF1 catalytic activity. However, oligomerization appears to stabilize GBF1 in cells, and the 91/130 mutant is degraded faster than the wild-type GBF1. Our data support a model in which oligomerization is not a key regulator of GBF1 activity but impacts its function by regulating the cellular levels of GBF1.

scholarly journals Oncogenic RAS isoforms show a hierarchical requirement for the guanine nucleotide exchange factor SOS2 to mediate cell transformation

2018 ◽  
Vol 11 (546) ◽  
pp. eaar8371 ◽  
Author(s):  
Erin Sheffels ◽  
Nancy E. Sealover ◽  
Chenyue Wang ◽  
Do Hyung Kim ◽  
Isabella A. Vazirani ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Cellular Transformation ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Ras Family ◽  
Ras Isoforms

About a third of tumors have activating mutations in HRAS, NRAS, or KRAS, genes encoding guanosine triphosphatases (GTPases) of the RAS family. In these tumors, wild-type RAS cooperates with mutant RAS to promote downstream effector activation and cell proliferation and transformation, suggesting that upstream activators of wild-type RAS are important modulators of mutant RAS-driven oncogenesis. The guanine nucleotide exchange factor (GEF) SOS1 mediates KRAS-driven proliferation, but little is understood about the role of SOS2. We found that RAS family members have a hierarchical requirement for the expression and activity of SOS2 to drive cellular transformation. In mouse embryonic fibroblasts (MEFs), SOS2 critically mediated mutant KRAS-driven, but not HRAS-driven, transformation. Sos2 deletion reduced epidermal growth factor (EGF)–dependent activation of wild-type HRAS and phosphorylation of the kinase AKT in cells expressing mutant RAS isoforms. Assays using pharmacological inhibitors revealed a hierarchical requirement for signaling by phosphoinositide 3-kinase (PI3K) in promoting RAS-driven cellular transformation that mirrored the requirement for SOS2. KRAS-driven transformation required the GEF activity of SOS2 and was restored in Sos2−/− MEFs by expression of constitutively activated PI3K. Finally, CRISPR/Cas9-mediated deletion of SOS2 reduced EGF-stimulated AKT phosphorylation and synergized with MEK inhibition to revert the transformed phenotype of human KRAS mutant pancreatic and lung tumor cells. These results indicate that SOS2-dependent PI3K signaling mediates mutant KRAS-driven transformation, revealing therapeutic targets in KRAS-driven cancers. Our data also reveal the importance of three-dimensional culture systems in investigating the mediators of mutant KRAS.

scholarly journals PtdIns(3,4,5)P3-dependent Rac Exchanger 1 (PREX1) Rac-Guanine Nucleotide Exchange Factor (GEF) Activity Promotes Breast Cancer Cell Proliferation and Tumor Growth via Activation of Extracellular Signal-regulated Kinase 1/2 (ERK1/2) Signaling

2016 ◽  
Vol 291 (33) ◽  
pp. 17258-17270 ◽  
Author(s):  
Heng-Jia Liu ◽  
Lisa M. Ooms ◽  
Nuthasuda Srijakotre ◽  
Joey Man ◽  
Jessica Vieusseux ◽  
...  
Keyword(s):  
Cell Growth ◽  
Tumor Growth ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Breast Cancers ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

PtdIns(3,4,5)P3-dependent Rac exchanger 1 (PREX1) is a Rac-guanine nucleotide exchange factor (GEF) overexpressed in a significant proportion of human breast cancers that integrates signals from upstream ErbB2/3 and CXCR4 membrane surface receptors. However, the PREX1 domains that facilitate its oncogenic activity and downstream signaling are not completely understood. We identify that ERK1/2 MAPK acts downstream of PREX1 and contributes to PREX1-mediated anchorage-independent cell growth. PREX1 overexpression increased but its shRNA knockdown decreased ERK1/2 phosphorylation in response to EGF/IGF-1 stimulation, resulting in induction of the cell cycle regulators cyclin D1 and p21WAF1/CIP1. PREX1-mediated ERK1/2 phosphorylation, anchorage-independent cell growth, and cell migration were suppressed by inhibition of MEK1/2/ERK1/2 signaling. PREX1 overexpression reduced staurosporine-induced apoptosis whereas its shRNA knockdown promoted apoptosis in response to staurosporine or the anti-estrogen drug tamoxifen. Expression of wild-type but not GEF-inactive PREX1 increased anchorage-independent cell growth. In addition, mouse xenograft studies revealed that expression of wild-type but not GEF-dead PREX1 resulted in the formation of larger tumors that displayed increased phosphorylation of ERK1/2 but not AKT. The impaired anchorage-independent cell growth, apoptosis, and ERK1/2 signaling observed in stablePREX1knockdown cells was restored by expression of wild-type but not GEF-dead-PREX1. Therefore, PREX1-Rac-GEF activity is critical for PREX1-dependent anchorage-independent cell growth and xenograft tumor growth and may represent a possible therapeutic target for breast cancers that exhibit PREX1 overexpression.

scholarly journals Sil1, a nucleotide exchange factor for BiP, is not required for antibody assembly or secretion

2015 ◽  
Vol 26 (3) ◽  
pp. 420-429 ◽  
Author(s):  
Viraj P. Ichhaporia ◽  
Tyler Sanford ◽  
Jenny Howes ◽  
Tony N. Marion ◽  
Linda M. Hendershot
Keyword(s):  
B Cells ◽  
Antibody Production ◽  
Autosomal Recessive ◽  
Ex Vivo ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor ◽  
Endoplasmic Reticulum Chaperone

Sil1 is a nucleotide exchange factor for the endoplasmic reticulum chaperone BiP, and mutations in this gene lead to Marinesco–Sjögren syndrome (MSS), a debilitating autosomal recessive disease characterized by multisystem defects. A mouse model for MSS was previously produced by disrupting Sil1 using gene-trap methodology. The resulting Sil1Gt mouse phenocopies several pathologies associated with MSS, although its ability to assemble and secrete antibodies, the best-characterized substrate of BiP, has not been investigated. In vivo antigen-specific immunizations and ex vivo LPS stimulation of splenic B cells revealed that the Sil1Gt mouse was indistinguishable from wild-type age-matched controls in terms of both the kinetics and magnitude of antigen-specific antibody responses. There was no significant accumulation of BiP-associated Ig assembly intermediates or evidence that another molecular chaperone system was used for antibody production in the LPS-stimulated splenic B cells from Sil1Gt mice. ER chaperones were expressed at the same level in Sil1WT and Sil1Gt mice, indicating that there was no evident compensation for the disruption of Sil1. Finally, these results were confirmed and extended in three human EBV-transformed lymphoblastoid cell lines from individuals with MSS, leading us to conclude that the BiP cofactor Sil1 is dispensable for antibody production.

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