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

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.

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Trio amino-terminal guanine nucleotide exchange factor domain expression promotes actin cytoskeleton reorganization, cell migration and anchorage-independent cell growth

Journal of Cell Science ◽

10.1242/jcs.112.12.1825 ◽

1999 ◽

Vol 112 (12) ◽

pp. 1825-1834 ◽

Cited By ~ 3

Author(s):

K. Seipel ◽

Q.G. Medley ◽

N.L. Kedersha ◽

X.A. Zhang ◽

S.P. O'Brien ◽

...

Keyword(s):

Cell Migration ◽

Cell Growth ◽

Actin Cytoskeleton ◽

Guanine Nucleotide Exchange Factor ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Exchange Factor ◽

Amino Terminal ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor

Rho family GTPases regulate diverse cellular processes, including extracellular signal-mediated actin cytoskeleton reorganization and cell growth. The functions of GTPases are positively regulated by guanine nucleotide exchange factors, which promote the exchange of GDP for GTP. Trio is a complex protein possessing two guanine nucleotide exchange factor domains, each with adjacent pleckstrin homology and SH3 domains, a protein serine/threonine kinase domain with an adjacent immunoglobulin-like domain and multiple spectrin-like domains. To assess the functional role of the two Trio guanine nucleotide exchange factor domains, NIH 3T3 cell lines stably expressing the individual guanine nucleotide exchange factor domains were established and characterized. Expression of the amino-terminal guanine nucleotide exchange factor domain results in prominent membrane ruffling, whereas cells expressing the carboxy-terminal guanine nucleotide exchange factor domain have lamellae that terminate in miniruffles. Moreover, cells expressing the amino-terminal guanine nucleotide exchange factor domain display more rapid cell spreading, haptotactic cell migration and anchorage-independent growth, suggesting that Trio regulates both cell motility and cell growth. Expression of full-length Trio in COS cells also alters actin cytoskeleton organization, as well as the distribution of focal contact sites. These findings support a role for Trio as a multifunctional protein that integrates and amplifies signals involved in coordinating actin remodeling, which is necessary for cell migration and growth.

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

Phytopathology ◽

10.1094/phyto-03-16-0137-r ◽

2017 ◽

Vol 107 (4) ◽

pp. 433-443 ◽

Cited By ~ 8

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.

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

AJP Cell Physiology ◽

10.1152/ajpcell.00185.2015 ◽

2016 ◽

Vol 310 (6) ◽

pp. C456-C469 ◽

Cited By ~ 12

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.

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

Science Signaling ◽

10.1126/scisignal.aar8371 ◽

2018 ◽

Vol 11 (546) ◽

pp. eaar8371 ◽

Cited By ~ 7

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.

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