Browse
Submit Data
Databases
API
Help

Dataset Information

24 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Identification of guanine nucleotide exchange factors that increase Cdc42 activity in primary human endothelial cells.

ABSTRACT: The Rho GTPase family is involved in actin dynamics and regulates the barrier function of the endothelium. One of the main barrier-promoting Rho GTPases is Cdc42, also known as cell division control protein 42 homolog. Currently, regulation of Cdc42-based signalling networks in endothelial cells (ECs) lack molecular details. To examine these, we focused on a subset of 15 Rho guanine nucleotide exchange factors (GEFs), which are expressed in the endothelium. By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1. A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs. Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1. Additionally, we generated truncated GEF constructs that comprise only the catalytic dbl homology (DH) domain or together with the adjacent pleckstrin homology domain (DHPH). The DH domain by itself did not activate Cdc42, whereas the DHPH domain of ITSN1, ITSN2 and PLEKHG1 showed activity towards Cdc42. Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42, which will be of great value for the field of vascular biology.

SUBMITTER: Reinhard NR

PROVIDER: S-EPMC7939571 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Identification of Rac guanine nucleotide exchange factors promoting Lgl1 phosphorylation in glioblastoma

Project description:The protein Lgl has key roles in the regulation of cell polarity. We have shown that Lgl is inactivated by hyperphosphorylation in glioblastoma as a consequence of PTEN loss and aberrant activation of the PI 3-kinase pathway; this contributes to glioblastoma pathogenesis both by promoting invasion and repressing glioblastoma cell differentiation. Lgl is phosphorylated by atypical protein kinase C in a complex with Par6 and either activated Cdc42 or activated Rac. Here we have investigated the role of specific Rac guanine nucleotide exchange factors in Lgl hyperphosphorylation in glioblastoma. We used CRISPR/Cas9 to knockout PREX1, a PI 3-kinase pathway-responsive Rac guanine nucleotide exchange factor that is overexpressed in glioblastoma. Knockout of PREX1 in patient-derived glioblastoma cells resulted in a reduction in Lgl phosphorylation and this could be reversed by re-expressing PREX1. PREX1 knockout cells showed reduced motility and altered phenotype suggestive of partial neuronal differentiation; consistent with this, RNA-seq analyses of these cells identified sets of PREX1-regulated genes with roles in promoting cell motility and repressing neuronal differentiation. Knockout of PREX1 in glioblastoma cells derived from a second patient did not affect Lgl phosphorylation. These cells overexpressed a short isoform of the Rac guanine nucleotide exchange factor TIAM1; knockdown of TIAM1 in PREX1-knockout cells from this patient reduced Lgl phosphorylation. These data show that PREX1 links aberrant PI 3-kinase to Lgl phosphorylation in glioblastoma, but that TIAM1 can also promote Lgl phosphorylation in a subset of patients. While this shows redundant mechanisms for Lgl phosphorylation, PREX1 appears to have a non-redundant role in glioblastoma cell motility, as this was impaired in PREX1 knockout cells from both patients.

2021-08-23 | GSE162783 | GEO

Identification of Rac guanine nucleotide exchange factors promoting Lgl1 phosphorylation in glioblastoma.

Project description:The protein Lgl1 is a key regulator of cell polarity. We previously showed that Lgl1 is inactivated by hyperphosphorylation in glioblastoma as a consequence of PTEN tumour suppressor loss and aberrant activation of the PI 3-kinase pathway; this contributes to glioblastoma pathogenesis both by promoting invasion and repressing glioblastoma cell differentiation. Lgl1 is phosphorylated by atypical protein kinase C that has been activated by binding to a complex of the scaffolding protein Par6 and active, GTP-bound Rac. The specific Rac guanine nucleotide exchange factors that generate active Rac to promote Lgl1 hyperphosphorylation in glioblastoma are unknown. We used CRISPR/Cas9 to knockout PREX1, a PI 3-kinase pathway-responsive Rac guanine nucleotide exchange factor, in patient-derived glioblastoma cells. Knockout cells had reduced Lgl1 phosphorylation, which was reversed by re-expressing PREX1. They also had reduced motility and an altered phenotype suggestive of partial neuronal differentiation; consistent with this, RNA-seq analyses identified sets of PREX1-regulated genes associated with cell motility and neuronal differentiation. PREX1 knockout in glioblastoma cells from a second patient did not affect Lgl1 phosphorylation. This was due to overexpression of a short isoform of the Rac guanine nucleotide exchange factor TIAM1; knockdown of TIAM1 in these PREX1 knockout cells reduced Lgl1 phosphorylation. These data show that PREX1 links aberrant PI 3-kinase signaling to Lgl1 phosphorylation in glioblastoma, but that TIAM1 is also to fill this role in a subset of patients. This redundancy between PREX1 and TIAM1 is only partial, as motility was impaired in PREX1 knockout cells from both patients.

| S-EPMC8551657 | biostudies-literature

Intermediates in the guanine nucleotide exchange reaction of Rab8 protein catalyzed by guanine nucleotide exchange factors Rabin8 and GRAB.

Project description:Small G-proteins of the Ras superfamily control the temporal and spatial coordination of intracellular signaling networks by acting as molecular on/off switches. Guanine nucleotide exchange factors (GEFs) regulate the activation of these G-proteins through catalytic replacement of GDP by GTP. During nucleotide exchange, three distinct substrate·enzyme complexes occur: a ternary complex with GDP at the start of the reaction (G-protein·GEF·GDP), an intermediary nucleotide-free binary complex (G-protein·GEF), and a ternary GTP complex after productive G-protein activation (G-protein·GEF·GTP). Here, we show structural snapshots of the full nucleotide exchange reaction sequence together with the G-protein substrates and products using Rabin8/GRAB (GEF) and Rab8 (G-protein) as a model system. Together with a thorough enzymatic characterization, our data provide a detailed view into the mechanism of Rabin8/GRAB-mediated nucleotide exchange.

| S-EPMC3820881 | biostudies-literature

The Cdc42 guanine nucleotide exchange factor FGD1 regulates osteogenesis in human mesenchymal stem cells.

Project description:Loss of function mutations in FGD1 result in faciogenital dysplasia, an X-linked human developmental disorder that adversely affects the formation of multiple skeletal structures. FGD1 encodes a guanine nucleotide exchange factor that specifically activates Cdc42, a Rho family small GTPase that regulates a variety of cellular behaviors. We have found that FGD1 is expressed in human mesenchymal stem cells (hMSCs) isolated from adult bone marrow. hMSCs are multipotent cells that can differentiate into many cell types, including fibroblasts, osteoblasts, adipocytes, and chondrocytes, and are thought to play a role in maintaining musculoskeletal tissues throughout life. We demonstrate an active role of FGD1 in osteogenic differentiation of hMSCs. During osteogenic differentiation of hMSCs in culture, we observed up-regulation of both FGD1 expression and Cdc42 activity. Activating FGD1/Cdc42 signaling by overexpression of either FGD1 or constitutively active Cdc42 promoted hMSC osteogenesis, while inhibiting Cdc42 signaling by either dominant negative mutants of FGD1 or Cdc42 suppressed osteogenesis. These results demonstrate an important role for FGD1/Cdc42 signaling in hMSC osteogenesis and suggest that the defects in bone remodeling in faciogenital dysplasia may persist throughout adult life and serve as a potential pathway that may be targeted for enhancing bone regeneration.

| S-EPMC3070587 | biostudies-literature

A crystallographic view of interactions between Dbs and Cdc42: PH domain-assisted guanine nucleotide exchange.

Project description:Dbl-related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. While it is known that the DH domain is the principal catalytic subunit, recent biochemical data indicate that for some Dbl-family proteins, such as Dbs and Trio, PH domains may cooperate with their associated DH domains in promoting guanine nucleotide exchange of Rho GTPases. In order to gain an understanding of the involvement of these PH domains in guanine nucleotide exchange, we have determined the crystal structure of a DH/PH fragment from Dbs in complex with Cdc42. The complex features the PH domain in a unique conformation distinct from the PH domains in the related structures of Sos1 and Tiam1.Rac1. Consequently, the Dbs PH domain participates with the DH domain in binding Cdc42, primarily through a set of interactions involving switch 2 of the GTPase. Comparative sequence analysis suggests that a subset of Dbl-family proteins will utilize their PH domains similarly to Dbs.

| S-EPMC125919 | biostudies-literature

Bacterial guanine nucleotide exchange factors SopE-like and WxxxE effectors.

Project description:Subversion of Rho family small GTPases, which control actin dynamics, is a common infection strategy used by bacterial pathogens. In particular, Salmonella enterica serovar Typhimurium, Shigella flexneri, enteropathogenic Escherichia coli (EPEC), and enterohemorrhagic Escherichia coli (EHEC) translocate type III secretion system (T3SS) effector proteins to modulate the Rho GTPases RhoA, Cdc42, and Rac1, which trigger formation of stress fibers, filopodia, and lamellipodia/ruffles, respectively. The Salmonella effector SopE is a guanine nucleotide exchange factor (GEF) that activates Rac1 and Cdc42, which induce "the trigger mechanism of cell entry." Based on a conserved Trp-xxx-Glu motif, the T3SS effector proteins IpgB1 and IpgB2 of Shigella, SifA and SifB of Salmonella, and Map of EPEC and EHEC were grouped together into a WxxxE family; recent studies identified the T3SS EPEC and EHEC effectors EspM and EspT as new family members. Recent structural and functional studies have shown that representatives of the WxxxE effectors share with SopE a 3-D fold and GEF activity. In this minireview, we summarize contemporary findings related to the SopE and WxxxE GEFs in the context of their role in subverting general host cell signaling pathways and infection.

| S-EPMC2849395 | biostudies-literature

Chlamydial entry involves TARP binding of guanine nucleotide exchange factors.

Project description:Chlamydia trachomatis attachment to cells induces the secretion of the elementary body-associated protein TARP (Translocated Actin Recruiting Protein). TARP crosses the plasma membrane where it is immediately phosphorylated at tyrosine residues by unknown host kinases. The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment. We show that TARP participates directly in chlamydial invasion activating the Rac-dependent signaling cascade to recruit actin. TARP functions by binding two distinct Rac guanine nucleotide exchange factors (GEFs), Sos1 and Vav2, in a phosphotyrosine-dependent manner. The tyrosine phosphorylation profile of the sequence YEPISTENIYESI within TARP, as well as the transient activation of the phosphatidylinositol 3-kinase (PI3-K), appears to determine which GEF is utilized to activate Rac. The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate. Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways. Collectively, these data implicate TARP in signaling to the actin cytoskeleton remodeling machinery, demonstrating a mechanism by which C.trachomatis invades non-phagocytic cells.

| S-EPMC2279300 | biostudies-literature

Small-molecule inhibitors of P-Rex guanine-nucleotide exchange factors.

Project description:P-Rex1 and P-Rex2 are guanine-nucleotide exchange factors (GEFs) that activate Rac small GTPases in response to the stimulation of G protein-coupled receptors and phosphoinositide 3-kinase. P-Rex Rac-GEFs regulate the morphology, adhesion and migration of various cell types, as well as reactive oxygen species production and cell cycle progression. P-Rex Rac-GEFs also have pathogenic roles in the initiation, progression or metastasis of several types of cancer. With one exception, all P-Rex functions are known or assumed to be mediated through their catalytic Rac-GEF activity. Thus, inhibitors of P-Rex Rac-GEF activity would be valuable research tools. We have generated a panel of small-molecule P-Rex inhibitors that target the interface between the catalytic DH domain of P-Rex Rac-GEFs and Rac. Our best-characterized compound, P-Rex inhibitor 1 (PREX-in1), blocks the Rac-GEF activity of full-length P-Rex1 and P-Rex2, and of their isolated catalytic domains, in vitro at low-micromolar concentration, without affecting the activities of several other Rho-GEFs. PREX-in1 blocks the P-Rex1 dependent spreading of PDGF-stimulated endothelial cells and the production of reactive oxygen species in fMLP-stimulated mouse neutrophils. Structure-function analysis revealed critical structural elements of PREX-in1, allowing us to develop derivatives with increased efficacy, the best with an IC50 of 2 µM. In summary, we have developed PREX-in1 and derivative small-molecule compounds that will be useful laboratory research tools for the study of P-Rex function. These compounds may also be a good starting point for the future development of more sophisticated drug-like inhibitors aimed at targeting P-Rex Rac-GEFs in cancer.

| S-EPMC9645260 | biostudies-literature

The Cdc42 guanine nucleotide exchange factor FGD6 coordinates cell polarity and endosomal membrane recycling in osteoclasts.

Project description:The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation.

| S-EPMC4140270 | biostudies-literature

Activation of G Proteins by Guanine Nucleotide Exchange Factors Relies on GTPase Activity.

Project description:G proteins are an important family of signalling molecules controlled by guanine nucleotide exchange and GTPase activity in what is commonly called an 'activation/inactivation cycle'. The molecular mechanism by which guanine nucleotide exchange factors (GEFs) catalyse the activation of monomeric G proteins is well-established, however the complete reversibility of this mechanism is often overlooked. Here, we use a theoretical approach to prove that GEFs are unable to positively control G protein systems at steady-state in the absence of GTPase activity. Instead, positive regulation of G proteins must be seen as a product of the competition between guanine nucleotide exchange and GTPase activity--emphasising a central role for GTPase activity beyond merely signal termination. We conclude that a more accurate description of the regulation of G proteins via these processes is as a 'balance/imbalance' mechanism. This result has implications for the understanding of intracellular signalling processes, and for experimental strategies that rely on modulating G protein systems.

| S-EPMC4795700 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data