Identification of Rac guanine nucleotide exchange factors promoting Lgl1 phosphorylation in glioblastoma
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA683013 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13208284_1.fastq.gz | Fastqsanger.gz | |||
| SRR13208284_2.fastq.gz | Fastqsanger.gz | |||
| SRR13208285_1.fastq.gz | Fastqsanger.gz | |||
| SRR13208285_2.fastq.gz | Fastqsanger.gz | |||
| SRR13208286_1.fastq.gz | Fastqsanger.gz |
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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
Project description:Extracellular superoxide dismutase (SOD3), which dismutases hydrogen peroxide to superoxide anion at cell membranes, mimics RAS oncogene action inducing primary cell immortalization at sustained low-level expression while high expression activates cancer barrier signaling through p53-p21 growth arrest pathway. We have previously demonstrated that the growth regulation of SOD3 occurs at the level of RAS and is mediated through non-transcriptional and transcriptional routes. Therefore, in the current work we assayed the growth suppressive mechanisms of SOD3 by characterizing the main signal transduction routes from the cell membrane into the nucleus. Based on our data robust over-expression of SOD3 in anaplastic thyroid cancer 8505c cells increased EGFR, RYK, ALK, FLT3, and EPHA10 tyrosine kinase receptor phosphorylation with consequent downstream SRC, FYN, YES, HCK, and LYN kinase activation. However, RAS pull-down experiment suggested lack of mitogen pathway stimulation that was confirmed by MEK1/2 and ERK1/2 Western blot. Interestingly, mRNA expression analysis indicated that SOD3 regulated in a dose dependent manner the expression of selected guanine nucleotide exchange factors (Rho GEF16, Ral GEF RGL1), GTPase activating proteins (ArfGAP ADAP2, Ras GAP RASAL1, RGS4), and Rho guanine nucleotide disassociation inhibitors (Rho GDI 2) therefore controlling the signal transduction through RAS GTPases to downstream signal transduction pathways. Our current data suggests a SOD3-induced activation of growth signal transduction is controlled in a dose dependent manner through GEF, GAP, and GDI.
2014-12-31 | GSE56546 | GEO
Project description:Rho Guanine Nucleotide Exchange Factor ARHGEF10 is a putative tumor suppressor in pancreatic ductal adenocarcinoma
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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.
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Project description:Abscisic acid (ABA) plays essential roles in plant development and responses to environmental stress. ABA induces subcellular translocation and degradation of the guanine nucleotide exchange factor RopGEF1 thus facilitating ABA core signal transduction. However, the underlying mechanisms for ABA-triggered RopGEF1 trafficking/degradation remain unknown. Studies have revealed that RopGEFs associate with receptor-like kinases to convey developmental signals to small ROP GTPases. However, how the activities of RopGEFs are modulated is not well understood. Type 2C protein phosphatases stabilize the RopGEF1 protein, indicating that phosphorylation may trigger RopGEF1 trafficking and degradation. We have screened inhibitors followed by several protein kinase mutants and find that quadruple mutant plants in the Arabidopsis calcium-dependent protein kinases (CPKs) cpk3/4/6/11 disrupt ABA-induced trafficking and degradation of RopGEF1. Moreover, cpk3/4/6/11 partially impairs ABA inhibition of germination. Several CPKs interact with RopGEF1. CPK4 binds to and phosphorylates RopGEF1 and promotes the degradation of RopGEF1. CPK-mediated phosphorylation of RopGEF1 at specific N-terminal serine residues causes the degradation of RopGEF1 and mutation of these sites also compromises the RopGEF1 over-expression phenotype in root hair development in Arabidopsis. Our findings establish the physiological and molecular functions and relevance of calcium-dependent protein kinases in regulation of RopGEF1 and illuminate physiological roles of a CPK-GEF-ROP module in abscisic acid signaling and plant development. We further discuss that CPK-dependent RopGEF degradation during abiotic stress could provide a mechanism for down-regulation of RopGEF-dependent growth responses.
2019-11-12 | PXD009421 | Pride
Project description:Abscisic acid (ABA) plays essential roles in plant development and responses to environmental stress. ABA induces subcellular translocation and degradation of the guanine nucleotide exchange factor RopGEF1 thus facilitating ABA core signal transduction. However, the underlying mechanisms for ABA-triggered RopGEF1 trafficking/degradation remain unknown. Studies have revealed that RopGEFs associate with receptor-like kinases to convey developmental signals to small ROP GTPases. However, how the activities of RopGEFs are modulated is not well understood. Type 2C protein phosphatases stabilize the RopGEF1 protein, indicating that phosphorylation may trigger RopGEF1 trafficking and degradation. We have screened inhibitors followed by several protein kinase mutants and find that quadruple mutant plants in the Arabidopsis calcium-dependent protein kinases (CPKs) cpk3/4/6/11 disrupt ABA-induced trafficking and degradation of RopGEF1. Moreover, cpk3/4/6/11 partially impairs ABA inhibition of germination. Several CPKs interact with RopGEF1. CPK4 binds to and phosphorylates RopGEF1 and promotes the degradation of RopGEF1. CPK-mediated phosphorylation of RopGEF1 at specific N-terminal serine residues causes the degradation of RopGEF1 and mutation of these sites also compromises the RopGEF1 over-expression phenotype in root hair development in Arabidopsis. Our findings establish the physiological and molecular functions and relevance of calcium-dependent protein kinases in regulation of RopGEF1 and illuminate physiological roles of a CPK-GEF-ROP module in abscisic acid signaling and plant development. We further discuss that CPK-dependent RopGEF degradation during abiotic stress could provide a mechanism for down-regulation of RopGEF-dependent growth responses.
2019-11-12 | PXD009422 | Pride
Project description:The adaptor protein ASC contributes to innate immunity through the assembly of caspase-1-activating inflammasome complexes. We demonstrate that ASC plays an inflammasome-independent cell-intrinsic role in adaptive immune cells. Asc-/- mice displayed defective antigen presentation by dendritic cells and lymphocyte migration due to impaired Rac-mediated actin polymerization. Genome-wide analysis showed that ASC, but not Nlrp3 or caspase-1, controls mRNA stability and expression of DOCK2, a guanine nucleotide exchange factor that mediates Rac-dependent signaling in immune cells. DOCK2-deficient dendritic cells showed similar defective antigen uptake as Asc-/- cells. Ectopic expression of DOCK2 in ASC-deficient cells restored Rac-mediated actin polymerization, antigen uptake and chemotaxis. Thus, ASC shapes adaptive immunity independently of inflammasomes by modulating DOCK2-dependent Rac activation and F-actin polymerization in dendritic cells and lymphocytes. Three replicates of naïve WT and three replicates of Asc-/- bone marrow derived dendritic cells were analyzed on the Affymetrix HT MG-430 PM plate array.
2012-07-08 | E-GEOD-30769 | biostudies-arrayexpress
Project description:Extra-hematopoietic immunomodulatory role of the guanine exchange factor DOCK-2
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Project description:Guanine nucleotide exchange factors (GEFs) activate Rho GTPases by catalyzing the exchange of bound GDP for GTP, thereby resulting in downstream effector recognition. Two metazoan families of GEFs have been described: Dbl-GEF family members that share conserved Dbl homology (DH) and Pleckstrin homology (PH) domains and the more recently described Dock180 family members that share little sequence homology with the Dbl family and are characterized by conserved Dock homology regions 1 and 2 (DHR-1 and -2, respectively). While extensive characterization of the Dbl family has been performed, less is known about how Dock180 family members act as GEFs, with only a single X-ray structure having recently been reported for the Dock9-Cdc42 complex. To learn more about the mechanisms used by the founding member of the family, Dock180, to act as a Rac-specific GEF, we set out to identify and characterize its limit functional GEF domain. A C-terminal portion of the DHR-2 domain, composed of approximately 300 residues (designated as Dock180(DHR-2c)), is shown to be necessary and sufficient for robust Rac-specific GEF activity both in vitro and in vivo. We further show that Dock180(DHR-2c) binds to Rac in a manner distinct from that of Rac-GEFs of the Dbl family. Specifically, Ala(27) and Trp(56) of Rac appear to provide a bipartite binding site for the specific recognition of Dock180(DHR-2c), whereas for Dbl family Rac-GEFs, Trp(56) of Rac is the sole primary determinant of GEF specificity. On the basis of our findings, we are able to define the core of Dock180 responsible for its Rac-GEF activity as well as highlight key recognition sites that distinguish different Dock180 family members and determine their corresponding GTPase specificities.
| S-EPMC3048587 | biostudies-literature
Project description:The adaptor protein ASC contributes to innate immunity through the assembly of caspase-1-activating inflammasome complexes. We demonstrate that ASC plays an inflammasome-independent cell-intrinsic role in adaptive immune cells. Asc-/- mice displayed defective antigen presentation by dendritic cells and lymphocyte migration due to impaired Rac-mediated actin polymerization. Genome-wide analysis showed that ASC, but not Nlrp3 or caspase-1, controls mRNA stability and expression of DOCK2, a guanine nucleotide exchange factor that mediates Rac-dependent signaling in immune cells. DOCK2-deficient dendritic cells showed similar defective antigen uptake as Asc-/- cells. Ectopic expression of DOCK2 in ASC-deficient cells restored Rac-mediated actin polymerization, antigen uptake and chemotaxis. Thus, ASC shapes adaptive immunity independently of inflammasomes by modulating DOCK2-dependent Rac activation and F-actin polymerization in dendritic cells and lymphocytes.
2012-07-09 | GSE30769 | GEO