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Heidi C E Welch - One of the best experts on this subject based on the ideXlab platform.

  • norbin stimulates the catalytic activity and plasma membrane localization of the guanine nucleotide exchange factor p Rex1
    Journal of Biological Chemistry, 2016
    Co-Authors: Mark A Barber, David Oxley, Kirsti Hornigold, Martin J Baker, Judit Toth, Heidi C E Welch
    Abstract:

    P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates the small G protein (GTPase) Rac1 to control Rac1-dependent cytoskeletal dynamics, and thus cell morphology. Three mechanisms of P-Rex1 regulation are currently known: (i) binding of the phosphoinositide second messenger PIP3, (ii) binding of the Gβγ subunits of heterotrimeric G proteins, and (iii) phosphorylation of various serine residues. Using recombinant P-Rex1 protein to search for new binding partners, we isolated the G-protein-coupled receptor (GPCR)-adaptor protein Norbin (Neurochondrin, NCDN) from mouse brain fractions. Coimmunoprecipitation confirmed the interaction between overexpressed P-Rex1 and Norbin in COS-7 cells, as well as between endogenous P-Rex1 and Norbin in HEK-293 cells. Binding assays with purified recombinant proteins showed that their interaction is direct, and mutational analysis revealed that the pleckstrin homology domain of P-Rex1 is required. Rac-GEF activity assays with purified recombinant proteins showed that direct interaction with Norbin increases the basal, PIP3- and Gβγ-stimulated Rac-GEF activity of P-Rex1. Pak-CRIB pulldown assays demonstrated that Norbin promotes the P-Rex1-mediated activation of endogenous Rac1 upon stimulation of HEK-293 cells with lysophosphatidic acid. Finally, immunofluorescence microscopy and subcellular fractionation showed that coexpression of P-Rex1 and Norbin induces a robust translocation of both proteins from the cytosol to the plasma membrane, as well as promoting cell spreading, lamellipodia formation, and membrane ruffling, cell morphologies generated by active Rac1. In summary, we have identified a novel mechanism of P-Rex1 regulation through the GPCR-adaptor protein Norbin, a direct P-Rex1 interacting protein that promotes the Rac-GEF activity and membrane localization of P-Rex1.

  • a rac1 independent role for p Rex1 in melanoblasts
    Journal of Investigative Dermatology, 2015
    Co-Authors: Colin R Lindsay, Heidi C E Welch, Ang Li, William J Faller, Brad Ozanne, Laura M Machesky, Owen J Sansom
    Abstract:

    TO THE EDITOR Given the recent discovery of RAC1-activating mutations in melanoma, and our finding that PIP3-dependent Rac-exchanger 1 (PRex1) is overexpressed and drives metastasis in this cancer, an important question is to establish whether the functions of P-Rex1 are mediated specifically by Rac alone (Lindsay et al., 2011; Berger et al., 2012). Here we describe a Rac1-independent in vivo role for P-Rex1 through identification and characterization of a mouse coat color phenotype. P-Rex1 is a guanine-nucleotide exchange factor (GEF) for Rac, whose primary cell function is induction of actin-mediated membrane ruffling and lamellipodia formation at the leading edge of cell migration (Welch et al., 2002; Hill et al., 2005; Barber et al., 2007). To investigate this question we decided to examine the role of Rac1 and P-Rex1 in melanoblast development. Previously, we reported a “white belly” phenotype of mice with PRex1 deletion (Lindsay et al., 2011). Impaired melanoblast migration was mostly responsible for this phenotype, with melanoblasts lacking at the most distal points of migration (belly and paws). Constitutive deletion of Rac1 is embryonically lethal, but a coat color defect of mice with melanocyte-specific RAC1 abrogation (Tyr::Cre Rac1fl/fl) has also been described; these mice have a larger belly spot on their ventral side, suggesting that alternative Rho-GTPases can be activated to enable melanoblast migration to the perimeter of the Tyr::Cre Racfl/fl white belly (Sugihara et al., 1998; Li et al., 2011). A role for Rac1 in proliferation was also observed, as there was a marked reduction of melanoblast numbers in this phenotype. In line with these previous studies, and because mice with melanocyte-specific RAC1 abrogation require euthanization shortly after birth because of neurological problems, we used the same embryonic melanoblast reporter models to assess the downstream effects of P-Rex1 in vivo (Mackenzie et al., 1997; Mort et al., 2010; Li et al., 2011). Melanocyte-specific reporter mouse strains employed were Tyr::Cre Z/EG, which drives green fluorescent protein expression in the melanoblast lineage, and DCT::β-galactosidase (otherwise referred to as DCT-lacZ). First, we hypothesized that, if the effects of P-Rex1 were mediated exclusively via Rac1, double mutant Tyr::Cre Rac1fl/fl; P-Rex1−/− mice would exhibit the same coat color phenotype as Tyr::Cre Racfl/fl mice alone. However, Tyr::Cre Racfl/fl; P-Rex1−/− mice display a dramatic alteration in coat color phenotype from Tyr::Cre Racfl/fl mice (n=7; Figure 1a). The ventral and dorsal coats of these mice are almost entirely white, with hypo-pigmented limbs and tail. Graying pigmented areas were only observed in the head coat. We concluded from this experiment that P-Rex1 and Rac1 together constitute fundamental signaling components of the mouse coat color phenotype, with minimal rescue of melanoblast development conferred by other GEFs or Rho-GTPases. It was also clear that P-Rex1 must be able to exert phenotypic effects other than via Rac1. Figure 1 P-Rex1 and Rac1 are fundamental components of a mouse coat color phenotype. (a) Ventral coats of P-Rex1−/−, Tyr::Cre Rac1fl/fl and P-Rex1−/−;Tyr::Cre Rac1fl/flmice. Final photomicrograph shows dorsal and head coat of Tyr::Cre ... To explore the Rac1-independent effects of P-Rex1 further, we crossed Tyr::Cre Racfl/fl; P-Rex1−/− mice with mice carrying the melanoblast reporter DCT-lacZ transgene (methods detailed in Lindsay et al., 2011). Relative to Tyr::Cre Racfl/flmice or P-Rex1−/− embryos alone, Tyr::Cre Racfl/fl; P-Rex1−/− embryos at E15.5 displayed a substantial reduction in melanoblast numbers across their entire body (Figure 1b and c). To assess whether the cause of this reduction in melanoblast numbers could be accounted for by decreased proliferation ± increased cell death, we next treated our previously described primary immortalized Tyr::CrER2 INK4a−/− Racfl/fl melanocyte cell line with short interfering RNA to P-Rex1 (methods detailed in Li et al., 2012). The use of this model system also allowed us to delete Rac1 function when these cells were treated with 4-hydroxytamoxifen (OHT). We first used western blotting to confirm that efficient P-Rex1 knockdown and OHT-induced Rac deletion were achieved (Figure 1d). There was no increase in cleaved caspase-3 evident in the absence of P-Rex1 and/or Rac, suggesting that the reduced cell numbers observed in Tyr::Cre Racfl/fl; P-Rex1−/− embryos were not accounted for by increased cell death (Figure 1d). Growth curves and anti-BrdU immunofluorescence of the same cell lines confirmed that there was a reduced proliferation in P-Rex1-depleted cells, both in the presence and absence of OHT (Figure 1e; Supplementary Figure S1a and b online). Taken together, these results suggest, in addition to our previously reported effects of Rac1 deletion on mouse coat color, that the loss of a Rac1-independent proliferative effect of P-Rex1 also contributes to the coat color phenotype observed in Tyr::Cre Racfl/fl; P-Rex1−/− mice (Li et al., 2011). In line with our previous characterization of the P-Rex1 knockout phenotype alone, we next decided to delineate whether the coat color phenotype of Tyr::Cre Racfl/fl; P-Rex1−/− mice could also be a consequence of reduced melanoblast migration (Lindsay et al., 2011). To assess this, mice with the Z/EG double reporter transgene were crossed with Tyr::Cre Racfl/fl; P-Rex1−/−mice, driving green fluorescent protein expression in the melanoblast lineage (methods detailed in Lindsay et al., 2011; Figure 2a). Live imaging of melanoblasts was performed using E15.5 embryo skin from each genotype (Figure 2b; Supplementary Movies S1–3 online). Consistent with our previous work, significant reductions in migration speed were observed between wild-type, P-Rex1−/− and Tyr::Cre Racfl/fl melanoblasts (Figure 2c and d). However, there was no significant difference in speed between Tyr::Cre Racfl/fl; P-Rex1−/− and Tyr::Cre Racfl/fl melanoblasts alone, suggesting that there was no change in migratory characteristics to account for the Tyr::Cre Racfl/fl; P-Rex1−/− phenotype (Figure 2c and d). These results were matched by similar differences between the same genotypes when Euclidean distance was measured (Figure 2e), as well as no observable change in cell morphology evident in Tyr::Cre Racfl/fl; P-Rex1−/− compared with Tyr::Cre Racfl/fl melanoblasts alone (Supplementary Figure S1c online; methods detailed in Helmy and Azim, 2012). Finally, no cell death was seen in our melanoblast time-lapse movies of any genotype, again suggesting that P-Rex1 contributes to coat color phenotype by promoting cell proliferation using a Rac1-independent mechanism (Li et al., 2011; Supplementary Movies S1–3 online; methods detailed in Lindsay et al., 2011). Figure 2 P-Rex1 has no additional effect on migration compared with Rac1 alone. All experiments show embryo skin explants at E15.5 (a) Combined Z-stack confocal images of Z/EG melanoblasts from wild-type (control), P-Rex1−/−, Tyr::Cre Rac1fl/fl ... To conclude, we have elucidated a proliferative role of P-Rex1 when Rac1 is deleted. As E15.5 migratory characteristics are not altered in the Tyr::Cre Racfl/fl; P-Rex1−/− double mutant embryos, this suggests that the role of P-Rex1 in migration is almost exclusively mediated via Rac1. Here we focused on E15.5 embryos, a useful time point to observe the late migratory effects observed with previously described Prex and Rac phenotypes (Li et al., 2011; Lindsay et al., 2011). With a greater number of embryos, we would have performed further embryo time-point analyses at E13.5 to ensure there was no earlier melanoblast migratory deficit that could contribute to this phenotype, although even this experiment could not completely exclude such a possibility. One potential Rho-GTPase, RhoG, is a likely candidate for P-Rex1 interaction: it is the most structurally similar Rho-GTPase to Rac and has been shown to cooperate with Rac for induction of cell transformation (Roux et al., 1997). Moreover, there are distinct regulatory and functional similarities between P-Rex1 and Vav proteins, which have been characterized as the predominant GEFs required for RhoG activation (Samson et al., 2010; Lawson et al., 2011). Further studies are underway to investigate the phenotypes of these and other potential Rho-GTPases in melanoblast migration and melanomagenesis. Given that there is now considerable effort to generate Rac1 inhibitors, our data would suggest functions for proteins upstream of Rac1 that may become further therapeutic targets in melanoma. All experiments were conducted and approved in accordance with institutional and UK guidelines, and all animal studies were performed in accordance with local regulatory guidelines.

  • p Rex1 directly activates rhog to regulate gpcr driven rac signalling and actin polarity in neutrophils
    Journal of Cell Science, 2014
    Co-Authors: George Damoulakis, Heidi C E Welch, Karen E Anderson, Len R Stephens, Laure Gambardella, Kent L Rossman, Campbell D Lawson, Yoshinori Fukui, Phillip T Hawkins
    Abstract:

    G-protein-coupled receptors (GPCRs) regulate the organisation of the actin cytoskeleton by activating the Rac subfamily of small GTPases. The guanine-nucleotide-exchange factor (GEF) P-Rex1 is engaged downstream of GPCRs and phosphoinositide 3-kinase (PI3K) in many cell types, and promotes tumorigenic signalling and metastasis in breast cancer and melanoma, respectively. Although P-Rex1-dependent functions have been attributed to its GEF activity towards Rac1, we show that P-Rex1 also acts as a GEF for the Rac-related GTPase RhoG, both in vitro and in GPCR-stimulated primary mouse neutrophils. Furthermore, loss of either P-Rex1 or RhoG caused equivalent reductions in GPCR-driven Rac activation and Rac-dependent NADPH oxidase activity, suggesting they both function upstream of Rac in this system. Loss of RhoG also impaired GPCR-driven recruitment of the Rac GEF DOCK2, and Factin, to the leading edge of migrating neutrophils. Taken together, our results reveal a new signalling hierarchy in which P-Rex1, acting as a GEF for RhoG, regulates Rac-dependent functions indirectly through RhoG-dependent recruitment of DOCK2. These findings thus have broad implications for our understanding of GPCR signalling to Rho GTPases and the actin cytoskeleton.

  • p Rex1 cooperates with pdgfrβ to drive cellular migration in 3d microenvironments
    PLOS ONE, 2013
    Co-Authors: Andrew D Campbell, Heidi C E Welch, Samuel Lawn, Lynn Mcgarry, Bradford W Ozanne, Jim C Norman
    Abstract:

    Expression of the Rac-guanine nucleotide exchange factor (RacGEF), P-Rex1 is a key determinant of progression to metastasis in a number of human cancers. In accordance with this proposed role in cancer cell invasion and metastasis, we find that ectopic expression of P-Rex1 in an immortalised human fibroblast cell line is sufficient to drive multiple migratory and invasive phenotypes. The invasive phenotype is greatly enhanced by the presence of a gradient of serum or platelet-derived growth factor, and is dependent upon the expression of functional PDGF receptor β. Consistently, the invasiveness of WM852 melanoma cells, which endogenously express P-Rex1 and PDGFRβ, is opposed by siRNA of either of these proteins. Furthermore, the current model of P-Rex1 activation is advanced through demonstration of P-Rex1 and PDGFRβ as components of the same macromolecular complex. These data suggest that P-Rex1 has an influence on physiological migratory processes, such as invasion of cancer cells, both through effects upon classical Rac1-driven motility and a novel association with RTK signalling complexes.

  • the guanine nucleotide exchange factor p Rex1 is activated by protein phosphatase 1α
    Biochemical Journal, 2012
    Co-Authors: Mark A Barber, Sylvia Thelen, Marcus Thelen, Annick Hendrickx, Monique Beullens, Hugo Ceulemans, David Oxley, Mathieu Bollen, Heidi C E Welch
    Abstract:

    : P-Rex1 is a GEF (guanine-nucleotide-exchange factor) for the small G-protein Rac that is activated by PIP3 (phosphatidylinositol 3,4,5-trisphosphate) and Gβγ subunits and inhibited by PKA (protein kinase A). In the present study we show that PP1α (protein phosphatase 1α) binds P-Rex1 through an RVxF-type docking motif. PP1α activates P-Rex1 directly in vitro, both independently of and additively to PIP3 and Gβγ. PP1α also substantially activates P-Rex1 in vivo, both in basal and PDGF (platelet-derived growth factor)- or LPA (lysophosphatidic acid)-stimulated cells. The phosphatase activity of PP1α is required for P-Rex1 activation. PP1β, a close homologue of PP1α, is also able to activate P-Rex1, but less effectively. PP1α stimulates P-Rex1-mediated Rac-dependent changes in endothelial cell morphology. MS analysis of wild-type P-Rex1 and a PP1α-binding-deficient mutant revealed that endogenous PP1α dephosphorylates P-Rex1 on at least three residues, Ser834, Ser1001 and Ser1165. Site-directed mutagenesis of Ser1165 to alanine caused activation of P-Rex1 to a similar degree as did PP1α, confirming Ser1165 as a dephosphorylation site important in regulating P-Rex1 Rac-GEF activity. In summary, we have identified a novel mechanism for direct activation of P-Rex1 through PP1α-dependent dephosphorylation.

Arthur M Mercurio - One of the best experts on this subject based on the ideXlab platform.

  • p Rex1 promotes resistance to vegf vegfr targeted therapy in prostate cancer
    Cell Reports, 2016
    Co-Authors: Hira Lal Goel, Bryan M Pursell, Leonard D Shultz, Dale L Greiner, Rolf A Brekken, Craig Vander W Kooi, Arthur M Mercurio
    Abstract:

    Summary Autocrine VEGF signaling is critical for sustaining prostate and other cancer stem cells (CSCs), and it is a potential therapeutic target, but we observed that CSCs isolated from prostate tumors are resistant to anti-VEGF (bevacizumab) and anti-VEGFR (sunitinib) therapy. Intriguingly, resistance is mediated by VEGF/neuropilin signaling, which is not inhibited by bevacizumab and sunitinib, and it involves the induction of P-Rex1, a Rac GEF, and consequent Rac1-mediated ERK activation. This induction of P-Rex1 is dependent on Myc. CSCs isolated from the PTEN pc−/− transgenic model of prostate cancer exhibit Rac1-dependent resistance to bevacizumab. Rac1 inhibition or P-Rex1 downregulation increases the sensitivity of prostate tumors to bevacizumab. These data reveal that prostate tumors harbor cells with stem cell properties that are resistant to inhibitors of VEGF/VEGFR signaling. Combining the use of available VEGF/VEGFR-targeted therapies with P-Rex1 or Rac1 inhibition should improve the efficacy of these therapies significantly.

  • P-Rex1 Promotes Resistance to VEGF/VEGFR-Targeted Therapy in Prostate Cancer
    Cell Reports, 2016
    Co-Authors: Hira Lal Goel, Bryan M Pursell, Leonard D Shultz, Dale L Greiner, Rolf A Brekken, Craig Vander W Kooi, Arthur M Mercurio
    Abstract:

    Summary Autocrine VEGF signaling is critical for sustaining prostate and other cancer stem cells (CSCs), and it is a potential therapeutic target, but we observed that CSCs isolated from prostate tumors are resistant to anti-VEGF (bevacizumab) and anti-VEGFR (sunitinib) therapy. Intriguingly, resistance is mediated by VEGF/neuropilin signaling, which is not inhibited by bevacizumab and sunitinib, and it involves the induction of P-Rex1, a Rac GEF, and consequent Rac1-mediated ERK activation. This induction of P-Rex1 is dependent on Myc. CSCs isolated from the PTEN pc−/− transgenic model of prostate cancer exhibit Rac1-dependent resistance to bevacizumab. Rac1 inhibition or P-Rex1 downregulation increases the sensitivity of prostate tumors to bevacizumab. These data reveal that prostate tumors harbor cells with stem cell properties that are resistant to inhibitors of VEGF/VEGFR signaling. Combining the use of available VEGF/VEGFR-targeted therapies with P-Rex1 or Rac1 inhibition should improve the efficacy of these therapies significantly.

Lorraine J Gudas - One of the best experts on this subject based on the ideXlab platform.

  • Rex1 zfp42 null mice show impaired testicular function abnormal testis morphology and aberrant gene expression
    Developmental Biology, 2011
    Co-Authors: Naira C Rezende, Ailan Lu, Sebastien Monette, Willie Mark, Lorraine J Gudas
    Abstract:

    Abstract Rex1 (Zfp42), GeneID 132625, is a gene whose expression is closely associated with pluripotency/multipotency in both mouse and human embryonic stem cells. To study the function of the murine Rex1 gene in vivo, we have used cre/lox technology to create Rex1(floxed) mice and mice deficient in Rex1 gene function. Rex1−/−males are characterized by an age-associated decrease in sperm counts, abnormal sperm morphology, and mild testicular atrophy. We characterized global patterns of gene expression in primary germ cells by microarray and identified the growth hormone responsive gene, GRTP1, as a transcript present at a 4.5 fold higher level in wild type (WT) compared to Rex1−/− mice. We analyzed immature germ cell (Dazl), proliferating (PCNA), and Sertoli cell populations, and quantitated levels of apoptosis in Rex1−/− as compared to WT testes. We evaluated the expression of proteins previously reported to correlate with Rex1 expression, such as STAT3, phospho-STAT3, p38, and phospho-p38 in the testis. We report a distinct cellular localization of total STAT3 protein in Rex1−/− affected testes. Our data suggest that loss of Rex1 leads to impaired testicular function.

  • transcriptional regulation of Rex1 zfp42 in normal prostate epithelial cells and prostate cancer cells
    Journal of Cellular Physiology, 2010
    Co-Authors: Ailan Lu, Lorraine J Gudas
    Abstract:

    Rex1 (zfp42) was identified by our laboratory because of its reduced expression in F9 teratocarcinoma stem cells after retinoic acid (RA) treatment. The Rex1 (Zfp42) gene is currently widely used as a marker of embryonic stem cells. We compared the transcriptional regulation of the human Rex1 gene in NTera-2 (NT-2) human teratocarcinoma, normal human prostate epithelial cells (PrEC), and prostate cancer cells (PC-3) by promoter/luciferase analyses. Oct4, Sox2, Nanog, and Dax1 transcripts are expressed at higher levels in NT-2 and PrEC cells than in PC-3 cells. Co-transfection analyses showed that YY1 and Rex1 are positive regulators of hRex1 transcription in NT-2 and PrEC cells, whereas Nanog is not. Serial deletion constructs of the hRex1 promoter were created and analyzed, by which we identified a potential negative regulatory site that is located between -1kb to -0.4 kb of the hRex1 promoter. We also delineated regions of the hRex1 promoter between -0.4 kb and the TSS that, when mutated, reduced transcriptional activation; these are putative Rex1 binding sites. Mutation of a putative Rex1 binding site in electrophoretic mobility shift assays (EMSA) resulted in reduced protein binding. Taken together, our results indicate that hRex1 binds to the hRex1 promoter region at -298 bp and positively regulates hRex1 transcription, but that this regulation is lost in PC-3 human prostate cancer cells. This lack of positive transcriptional regulation by the hRex1 protein may be responsible for the lack of Rex1 expression in PC-3 prostate cancer cells.

  • analysis of Rex1 zfp42 function in embryonic stem cell differentiation
    Developmental Dynamics, 2009
    Co-Authors: Kymora B Scotland, Siming Chen, Renia Sylvester, Lorraine J Gudas
    Abstract:

    Rex1 (zfp42) is a zinc finger protein expressed primarily in undifferentiated stem cells, both in the embryo and the adult. Upon all-trans retinoic acid induced differentiation of murine embryonic stem (ES) cells, Rex1 mRNA levels decrease several fold. To characterize the function(s) of Rex1 more extensively, we generated Rex1 double knockout ES cell lines. The disruption of the Rex1 gene enhanced the expression of ectoderm, mesoderm, and endoderm markers as compared to wild-type (Wt) cells. We propose that Rex1 acts to reduce retinoic acid induced differentiation in ES cells. We performed microarray analyses on Wt and Rex1−/− cells cultured in the presence or absence of LIF to identify potential Rex1 targets. We also evaluated gene expression in a Wt line that overexpresses Rex1 and in a Rex1−/− line in which Rex1 expression was restored. These data, taken together, suggest that Rex1 influences differentiation, cell cycle regulation, and cancer progression. Developmental Dynamics 238:1863–1877, 2009. © 2009 Wiley-Liss, Inc.

  • transcriptional activation of the suppressor of cytokine signaling 3 socs 3 gene via stat3 is increased in f9 Rex1 zfp 42 knockout teratocarcinoma stem cells relative to wild type cells
    Journal of Molecular Biology, 2008
    Co-Authors: Juliana Xu, Renia Sylvester, Siming Chen, Ann P Tighe, Lorraine J Gudas
    Abstract:

    Abstract Rex1 (Zfp42), first identified as a gene that is transcriptionally repressed by retinoic acid (RA), encodes a zinc finger transcription factor expressed at high levels in F9 teratocarcinoma stem cells, embryonic stem cells, and other stem cells. Loss of both alleles of Rex1 by homologous recombination alters the RA-induced differentiation of F9 cells, a model of pluripotent embryonic stem cells. We identified Suppressor of Cytokine Signaling-3 ( SOCS-3 ) as a gene that exhibits greatly increased transcriptional activation in RA, cAMP, and theophylline (RACT)-treated F9 Rex1 −/− cells (∼ 25-fold) as compared to wild-type (WT) cells (∼ 2.5-fold). By promoter deletion, mutation, and transient transfection analyses, we have shown that this transcriptional increase is mediated by the STAT3 DNA-binding elements located between − 99 to − 60 in the SOCS-3 promoter. Overexpression of STAT3 dominant-negative mutants greatly diminishes this SOCS-3 transcriptional increase in F9 Rex1 −/− cells. This increase in SOCS-3 transcription is associated with a four- to fivefold higher level of tyrosine-phosphorylated STAT3 in the RACT-treated F9 Rex1 −/− cells as compared to WT. Dominant-negative Src tyrosine kinase, Jak2, and protein kinase A partially reduce the transcriptional activation of the SOCS 3 gene in RACT-treated F9 Rex1 null cells. In contrast, parathyroid hormone peptide enhances the effect of RA in F9 Rex1 −/− cells, but not in F9 WT. Thus, Rex1, which is highly expressed in stem cells, inhibits signaling via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, thereby modulating the differentiation of F9 cells.

Jose Vazquezprado - One of the best experts on this subject based on the ideXlab platform.

  • camp dependent activation of the rac guanine exchange factor p Rex1 by type i protein kinase a pka regulatory subunits
    Journal of Biological Chemistry, 2019
    Co-Authors: Sendi Rafael Adamegarcia, Guadalupe Reyescruz, Silvio J Gutkind, Rodolfo Daniel Cervantesvillagrana, Lennis Beatriz Ordunacastillo, Susan S Taylor, Jose Vazquezprado
    Abstract:

    : Regulatory subunits of protein kinase A (PKA) inhibit its kinase subunits. Intriguingly, their potential as cAMP-dependent signal transducers remains uncharacterized. We recently reported that type I PKA regulatory subunits (RIα) interact with phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-Rex1), a chemotactic Rac guanine exchange factor (RacGEF). Because P-Rex1 is known to be phosphorylated and inhibited by PKA, its interaction with RIα suggests that PKA regulatory and catalytic subunits may fine-tune P-Rex1 activity or those of its target pools. Here, we tested whether RIα acts as a cAMP-dependent factor promoting P-Rex1-mediated Rac activation and cell migration. We observed that Gs-coupled EP2 receptors indeed promote endothelial cell migration via RIα-activated P-Rex1. Expression of the P-Rex1-PDZ1 domain prevented RIα/P-Rex1 interaction, P-Rex1 activation, and EP2-dependent cell migration, and P-Rex1 silencing abrogated RIα-dependent Rac activation. RIα-specific cAMP analogs activated P-Rex1, but lost this activity in RIα-knockdown cells, and cAMP pulldown assays revealed that P-Rex1 preferentially interacts with free RIα. Moreover, purified RIα directly activated P-Rex1 in vitro We also found that the RIα CNB-B domain is critical for the interaction with P-Rex1, which was increased in RIα mutants, such as the acrodysostosis-associated mutant, that activate P-Rex1 at basal cAMP levels. RIα and Cα PKA subunits targeted distinct P-Rex1 molecules, indicated by an absence of phosphorylation in the active fraction of P-Rex1. This was in contrast to the inactive fraction in which phosphorylated P-Rex1 was present, suggesting co-existence of dual stimulatory and inhibitory effects. We conclude that PKA's regulatory subunits are cAMP-dependent signal transducers.

  • gβγ signaling to the chemotactic effector p Rex1 and mammalian cell migration is directly regulated by gαq and gα13 proteins
    Journal of Biological Chemistry, 2019
    Co-Authors: Rodolfo Daniel Cervantesvillagrana, Guadalupe Reyescruz, Silvio J Gutkind, Sendi Rafael Adamegarcia, Irving Garciajimenez, Victor Manuel Coloraparicio, Yarely Mabell Beltrannavarro, Gabriele M Konig, Evi Kostenis, Jose Vazquezprado
    Abstract:

    : G protein-coupled receptors stimulate Rho guanine nucleotide exchange factors that promote mammalian cell migration. Rac and Rho GTPases exert opposing effects on cell morphology and are stimulated downstream of Gβγ and Gα12/13 or Gαq, respectively. These Gα subunits might in turn favor Rho pathways by preventing Gβγ signaling to Rac. Here, we investigated whether Gβγ signaling to phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-Rex1), a key Gβγ chemotactic effector, is directly controlled by Rho-activating Gα subunits. We show that pharmacological inhibition of Gαq makes P-Rex1 activation by Gq/Gi-coupled lysophosphatidic acid receptors more effective. Moreover, chemogenetic control of Gi and Gq by designer receptors exclusively activated by designer drugs (DREADDs) confirmed that Gi differentially activates P-Rex1. GTPase-deficient GαqQL and Gα13QL variants formed stable complexes with Gβγ, impairing its interaction with P-Rex1. The N-terminal regions of these variants were essential for stable interaction with Gβγ. Pulldown assays revealed that chimeric Gα13-i2QL interacts with Gβγ unlike to Gαi2-13QL, the reciprocal chimera, which similarly to Gαi2QL could not interact with Gβγ. Moreover, Gβγ was part of tetrameric Gβγ-GαqQL-RGS2 and Gβγ-Gα13-i2QL-RGS4 complexes, whereas Gα13QL dissociated from Gβγ to interact with the PDZ-RhoGEF-RGS domain. Consistent with an integrated response, Gβγ and AKT kinase were associated with active SDF-1/CXCL12-stimulated P-Rex1. This pathway was inhibited by GαqQL and Gα13QL, which also prevented CXCR4-dependent cell migration. We conclude that a coordinated mechanism prioritizes Gαq- and Gα13-mediated signaling to Rho over a Gβγ-dependent Rac pathway, attributed to heterotrimeric Gi proteins.

  • protein kinase a pka type i interacts with p Rex1 a rac guanine nucleotide exchange factor effect on pka localization and p Rex1 signaling
    Journal of Biological Chemistry, 2016
    Co-Authors: Lydia Chavezvargas, Guadalupe Reyescruz, Sendi Rafael Adamegarcia, Rodolfo Daniel Cervantesvillagrana, Susan S Taylor, Alejandro Castillokauil, Jessica G H Bruystens, Shigetomo Fukuhara, Naoki Mochizuki, Jose Vazquezprado
    Abstract:

    Abstract Morphology of migrating cells is regulated by Rho GTPases and fine-tuned by protein interactions and phosphorylation. PKA affects cell migration potentially through spatiotemporal interactions with regulators of Rho GTPases. Here we show that the endogenous regulatory (R) subunit of type I PKA interacts with P-Rex1, a Rac guanine nucleotide exchange factor that integrates chemotactic signals. Type I PKA holoenzyme interacts with P-Rex1 PDZ domains via the CNB B domain of RIα, which when expressed by itself facilitates endothelial cell migration. P-Rex1 activation localizes PKA to the cell periphery, whereas stimulation of PKA phosphorylates P-Rex1 and prevents its activation in cells responding to SDF-1 (stromal cell-derived factor 1). The P-Rex1 DEP1 domain is phosphorylated at Ser-436, which inhibits the DH-PH catalytic cassette by direct interaction. In addition, the P-Rex1 C terminus is indirectly targeted by PKA, promoting inhibitory interactions independently of the DEP1-PDZ2 region. A P-Rex1 S436A mutant construct shows increased RacGEF activity and prevents the inhibitory effect of forskolin on sphingosine 1-phosphate-dependent endothelial cell migration. Altogether, these results support the idea that P-Rex1 contributes to the spatiotemporal localization of type I PKA, which tightly regulates this guanine exchange factor by a multistep mechanism, initiated by interaction with the PDZ domains of P-Rex1 followed by direct phosphorylation at the first DEP domain and putatively indirect regulation of the C terminus, thus promoting inhibitory intramolecular interactions. This reciprocal regulation between PKA and P-Rex1 might represent a key node of integration by which chemotactic signaling is fine-tuned by PKA.

  • sphingosine 1 phosphate receptor s1p1 is regulated by direct interactions with p Rex1 a rac guanine nucleotide exchange factor
    Biochemical and Biophysical Research Communications, 2010
    Co-Authors: Benjamin Alejandro Ledezmasanchez, Alejandro Garciaregalado, Maria Luisa Guzmanhernandez, Jose Vazquezprado
    Abstract:

    Sphingosine-1-phosphate (S1P) receptors S1P 1 are emerging molecular targets for the treatment of cancer, vascular and immune diseases, due to their pivotal role in cell migration and survival of immune and endothelial cells. A therapeutic strategy to control S1P 1 function is based on agonists that promote changes on S1P 1 expression at the plasma membrane. Here, we explored the hypothesis that cell surface expression and function of S1P 1 are influenced by direct interactions with P-Rex1, a guanine nucleotide exchange factor for Rac. We demonstrate that P-Rex1-PDZ domains interact with S1P 1 -carboxyl terminal tail and full length receptor monomers and dimers. Endothelial cells transfected with P-Rex1-PDZ domains show an increased migratory response to S1P. S1P 1 trafficking to intracellular compartments is diminished by coexpression of P-Rex1. We conclude that S1P 1 signaling linked to cell migration is facilitated by a functional interaction with P-Rex1 via a mechanism that involves the maintenance of S1P 1 receptors at the cell membrane.

  • p Rex1 links mammalian target of rapamycin signaling to rac activation and cell migration
    Journal of Biological Chemistry, 2007
    Co-Authors: Ivette Hernandeznegrete, Jorge Carreteroortega, Hans Rosenfeldt, Ricardo Hernandezgarcia, Victor J Calderonsalinas, Guadalupe Reyescruz, Silvio J Gutkind, Jose Vazquezprado
    Abstract:

    Abstract Polarized cell migration results from the transduction of extra-cellular cues promoting the activation of Rho GTPases with the intervention of multidomain proteins, including guanine exchange factors. P-Rex1 and P-Rex2 are Rac GEFs connecting Gβγ and phosphatidylinositol 3-kinase signaling to Rac activation. Their complex architecture suggests their regulation by protein-protein interactions. Novel mechanisms of activation of Rho GTPases are associated with mammalian target of rapamycin (mTOR), a serine/threonine kinase known as a central regulator of cell growth and proliferation. Recently, two independent multiprotein complexes containing mTOR have been described. mTORC1 links to the classical rapamycin-sensitive pathways relevant for protein synthesis; mTORC2 links to the activation of Rho GTPases and cytoskeletal events via undefined mechanisms. Here we demonstrate that P-Rex1 and P-Rex2 establish, through their tandem DEP domains, interactions with mTOR, suggesting their potential as effectors in the signaling of mTOR to Rac activation and cell migration. This possibility was consistent with the effect of dominant-negative constructs and short hairpin RNA-mediated knockdown of P-Rex1, which decreased mTOR-dependent leucine-induced activation of Rac and cell migration. Rapamycin, a widely used inhibitor of mTOR signaling, did not inhibit Rac activity and cell migration induced by leucine, indicating that P-Rex1, which we found associated to both mTOR complexes, is only active when in the mTORC2 complex. mTORC2 has been described as the catalytic complex that phosphorylates AKT/PKB at Ser-473 and elicits activation of Rho GTPases and cytoskeletal reorganization. Thus, P-Rex1 links mTOR signaling to Rac activation and cell migration.

Christina Anne Mitchell - One of the best experts on this subject based on the ideXlab platform.

  • p Rex1 and p rex2 racgefs and cancer
    Biochemical Society Transactions, 2017
    Co-Authors: Nuthasuda Srijakotre, Lisa M Ooms, Christina M Lucato, Andrew M Ellisdon, Christina Anne Mitchell
    Abstract:

    Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger (P-Rex) proteins are RacGEFs that are synergistically activated by phosphatidylinositol 3,4,5-trisphosphate and Gβγ subunits of G-protein-coupled receptors. P-Rex1 and P-Rex2 share similar amino acid sequence homology, domain structure, and catalytic function. Recent evidence suggests that both P-Rex proteins may play oncogenic roles in human cancers. P-Rex1 and P-Rex2 are altered predominantly via overexpression and mutation, respectively, in various cancer types, including breast cancer, prostate cancer, and melanoma. This review compares the similarities and differences between P-Rex1 and P-Rex2 functions in human cancers in terms of cellular effects and signalling mechanisms. Emerging clinical data predict that changes in expression or mutation of P-Rex1 and P-Rex2 may lead to changes in tumour outcome, particularly in breast cancer and melanoma.

  • the phosphatidylinositol 3 4 5 trisphosphate dependent rac exchanger 1 ras related c3 botulinum toxin substrate 1 p Rex1 rac1 complex reveals the basis of rac1 activation in breast cancer cells
    Journal of Biological Chemistry, 2015
    Co-Authors: Christina M Lucato, Lisa M Ooms, Christina Anne Mitchell, Michelle L Halls, James C Whisstock, Andrew M Ellisdon
    Abstract:

    Abstract The P-Rex (phosphatidylinositol(3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion and metastasis in several human cancers. The family is unique among Rho GEFs as their activity is regulated by the synergistic binding of PIP3 and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 A crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1:Rac1 interface reveal a critical role of this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. Structural data indicate the PIP3/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP3 and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site.

  • The Phosphatidylinositol (3,4,5)-Trisphosphate-dependent Rac Exchanger 1·Ras-related C3 Botulinum Toxin Substrate 1 (P-Rex1·Rac1) Complex Reveals the Basis of Rac1 Activation in Breast Cancer Cells
    Journal of Biological Chemistry, 2015
    Co-Authors: Christina M Lucato, Lisa M Ooms, Christina Anne Mitchell, Michelle L Halls, James C Whisstock, Andrew M Ellisdon
    Abstract:

    Abstract The P-Rex (phosphatidylinositol(3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion and metastasis in several human cancers. The family is unique among Rho GEFs as their activity is regulated by the synergistic binding of PIP3 and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 A crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1:Rac1 interface reveal a critical role of this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. Structural data indicate the PIP3/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP3 and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site.

  • identification of p Rex1 as a novel rac1 guanine nucleotide exchange factor gef that promotes actin remodeling and glut4 protein trafficking in adipocytes
    Journal of Biological Chemistry, 2011
    Co-Authors: Demosthenes Balamatsias, Joanne E Waters, Rajendra Gurung, Anne Kong, Absorn Sriratana, Charles G Bailey, John E J Rasko, Tony Tiganis, Lance S Macaulay, Christina Anne Mitchell
    Abstract:

    Phosphoinositide 3-kinase (PI3K) signaling promotes the translocation of the glucose transporter, GLUT4, to the plasma membrane in insulin-sensitive tissues to facilitate glucose uptake. In adipocytes, insulin-stimulated reorganization of the actin cytoskeleton has been proposed to play a role in promoting GLUT4 translocation and glucose uptake, in a PI3K-dependent manner. However, the PI3K effectors that promote GLUT4 translocation via regulation of the actin cytoskeleton in adipocytes remain to be fully elucidated. Here we demonstrate that the PI3K-dependent Rac exchange factor, P-Rex1, enhances membrane ruffling in 3T3-L1 adipocytes and promotes GLUT4 trafficking to the plasma membrane at submaximal insulin concentrations. P-Rex1-facilitated GLUT4 trafficking requires a functional actin network and membrane ruffle formation and occurs in a PI3K- and Rac1-dependent manner. In contrast, expression of other Rho GTPases, such as Cdc42 or Rho, did not affect insulin-stimulated P-Rex1-mediated GLUT4 trafficking. P-Rex1 siRNA knockdown or expression of a P-Rex1 dominant negative mutant reduced but did not completely inhibit glucose uptake in response to insulin. Collectively, these studies identify a novel RacGEF in adipocytes as P-Rex1 that, at physiological insulin concentrations, functions as an insulin-dependent regulator of the actin cytoskeleton that contributes to GLUT4 trafficking to the plasma membrane.

  • p Rex1 a multidomain protein that regulates neurite differentiation
    Journal of Cell Science, 2008
    Co-Authors: Joanne E Waters, Lisa M Ooms, Megan Victoria Astle, Demosthenes Balamatsias, Rajendra Gurung, Christina Anne Mitchell
    Abstract:

    The Rac-GEF P-Rex1 promotes membrane ruffling and cell migration in response to Rac activation, but its role in neuritogenesis is unknown. Rac1 promotes neurite differentiation; Rac3, however, may play an opposing role. Here we report that in nerve growth factor (NGF)-differentiated rat PC12 cells, P-Rex1 localised to the distal tips of developing neurites and to the axonal shaft and growth cone of differentiating hippocampal neurons. P-Rex1 expression inhibited NGF-stimulated PC12 neurite differentiation and this was dependent on the Rac-GEF activity of P-Rex1. P-Rex1 inhibition of neurite outgrowth was rescued by low-dose cytochalasin D treatment, which prevents actin polymerisation. P-Rex1 activated Rac3 GTPase activity when coexpressed in PC12 cells. In the absence of NGF stimulation, targeted depletion of P-Rex1 in PC12 cells by RNA interference induced the spontaneous formation of β-tubulin-enriched projections. Following NGF stimulation, enhanced neurite differentiation, with neurite hyper-elongation correlating with decreased F-actin at the growth cone, was demonstrated in P-Rex1 knockdown cells. Interestingly, P-Rex1-depleted PC12 cells exhibited reduced Rac3 and Rac1 GTPase activity. This study has identified P-Rex1 as a Rac3-GEF in neuronal cells that localises to, and regulates, actin cytoskeletal dynamics at the axonal growth cone to in turn regulate neurite differentiation.