scholarly journals Ras and Calcium Signaling Pathways Converge at Raf1 via the Shoc2 Scaffold Protein

2010 ◽  
Vol 21 (6) ◽  
pp. 1088-1096 ◽  
Author(s):  
Sayaka Yoshiki ◽  
Rie Matsunaga-Udagawa ◽  
Kazuhiro Aoki ◽  
Yuji Kamioka ◽  
Etsuko Kiyokawa ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Scaffold Protein ◽  
Ras Signaling ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Ras Activation ◽  
Camp Analogue

Situated downstream of Ras is a key signaling molecule, Raf1. Increase in Ca2+ concentration has been shown to modulate the Ras-dependent activation of Raf1; however, the mechanism underlying this effect remains elusive. Here, to characterize the role of Ca2+ in Ras signaling to Raf1, we used a synthetic guanine nucleotide exchange factor (GEF) for Ras, eGRF. In HeLa cells expressing eGRF, Ras was activated by the cAMP analogue 007 as efficiently as by epidermal growth factor (EGF), whereas the activation of Raf1, MEK, and ERK by 007 was about half of that by EGF. Using a biosensor based on fluorescence resonance energy transfer, it was found that activation of Raf1 at the plasma membrane required not only Ras activation but also an increase in Ca2+ concentration or inhibition of calmodulin. Furthermore, the Ca2+-dependent activation of Raf1 was found to be abrogated by knockdown of Shoc2, a scaffold protein that binds both Ras and Raf1. These observations indicated that the Shoc2 scaffold protein modulates Ras-dependent Raf1 activation in a Ca2+- and calmodulin-dependent manner.

scholarly journals Direct Spatial Control of Epac1 by Cyclic AMP

2009 ◽  
Vol 29 (10) ◽  
pp. 2521-2531 ◽  
Author(s):  
Bas Ponsioen ◽  
Martijn Gloerich ◽  
Laila Ritsma ◽  
Holger Rehmann ◽  
Johannes L. Bos ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Adhesion ◽  
Cyclic Amp ◽  
Conformational Change ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cell Junction ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

ABSTRACT Epac1 is a guanine nucleotide exchange factor (GEF) for the small G protein Rap and is directly activated by cyclic AMP (cAMP). Upon cAMP binding, Epac1 undergoes a conformational change that allows the interaction of its GEF domain with Rap, resulting in Rap activation and subsequent downstream effects, including integrin-mediated cell adhesion and cell-cell junction formation. Here, we report that cAMP also induces the translocation of Epac1 toward the plasma membrane. Combining high-resolution confocal fluorescence microscopy with total internal reflection fluorescence and fluorescent resonance energy transfer assays, we observed that Epac1 translocation is a rapid and reversible process. This dynamic redistribution of Epac1 requires both the cAMP-induced conformational change as well as the DEP domain. In line with its translocation, Epac1 activation induces Rap activation predominantly at the plasma membrane. We further show that the translocation of Epac1 enhances its ability to induce Rap-mediated cell adhesion. Thus, the regulation of Epac1-Rap signaling by cAMP includes both the release of Epac1 from autoinhibition and its recruitment to the plasma membrane.

Abstract P470: Disrupted HRAS/GRB2 Signaling In Induced-cardiomyocytes Derived From Patients With Noonan Syndrome Carrying SOS1 Gene Mutation

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
VINOTH SIGAMANI ◽  
Narasimman Gurusamy ◽  
SHEEJA RAJASINGH ◽  
Rajasingh Johnson
Keyword(s):  
Gene Mutation ◽  
Noonan Syndrome ◽  
Guanine Nucleotide Exchange Factor ◽  
Signaling Molecules ◽  
Ras Signaling ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Background: Noonan syndrome (NS), a dominant autosomal genetic disorder that prevents normal development, and exhibits cardiac defects, which is estimated to appear in 50% to 90% of patients. Son of sevenless homolog 1 (SOS1) gene mutation has been identified as a major gene causing NS and attributes to the development of cardiomyopathy and congenital heart defects. SOS1 is a guanine nucleotide exchange factor for RAS and is known to interact with growth factor receptor-bound protein 2 (GRB2). Recently, we have generated induced pluripotent stem cells (iPSCs)-derived cardiomyocytes (iCMCs) from cardiac fibroblasts obtained from a NS patient carrying SOS1 gene variant 1654A>G. Hypothesis: Since NS is known to have aberrant RAS-MAPK signaling, we hypothesize that iCMCs derived from NS patient (NS-iCMCs) may have atypical RAS signaling leading to the development of cardiomyopathy. Methods and Results: We have compared the normal skin fibroblast-derived iPSCs (N-iPSCs) and N-iCMCs with NS patient-derived induced NS-iPSCs and NS-iCMCs. Our qRT-PCR results showed that the mRNA expressions of signaling molecules HRAS, GRB2 and SOS1 were significantly decreased in NS-iCMCs compared with N-iCMCs (Figure A), and further confirmed through the protein expression by Western immunoblotting (Figure B). These results were in association with a significantly decreased mRNA and protein expressions of cardiac transcription factor GATA4, and structural proteins alpha sarcomeric actinin-2 (ACTN2), cardiac troponin T (TNNT2) and tropomyosin alpha-1 (TPM1) in NS-iCMCs compared with N-iCMCs. Further studies are underway to explore the difference in the guanine nucleotide exchange factor (GEF) activity and ERK activation between NS-iCMCs and N-iCMCs. Conclusion: Our current findings clearly indicate that the SOS1-associated signaling molecules HRAS and GRB2 were disrupted in NS-iCMCs, which may result in the development of cardiomyopathy in NS patients.

scholarly journals In Vitro Formation of Recycling Vesicles from Endosomes Requires Adaptor Protein-1/Clathrin and Is Regulated by Rab4 and the Connector Rabaptin-5

2004 ◽  
Vol 15 (11) ◽  
pp. 4990-5000 ◽  
Author(s):  
Adriana Pagano ◽  
Pascal Crottet ◽  
Cristina Prescianotto-Baschong ◽  
Martin Spiess
Keyword(s):  
Brefeldin A ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Vesicle Formation ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Vitro Assay ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and γ-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5.

scholarly journals Ccpg1, a Novel Scaffold Protein That Regulates the Activity of the Rho Guanine Nucleotide Exchange Factor Dbs

2006 ◽  
Vol 26 (23) ◽  
pp. 8964-8975 ◽  
Author(s):  
Elena V. Kostenko ◽  
Oyenike O. Olabisi ◽  
Sutapa Sahay ◽  
Pedro L. Rodriguez ◽  
Ian P. Whitehead
Keyword(s):  
Family Member ◽  
Guanine Nucleotide Exchange Factor ◽  
Scaffold Protein ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Novel Scaffold ◽  
Exchange Activity

ABSTRACT Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) with in vitro exchange activity specific for RhoA and Cdc42. Like many RhoGEF family members, the in vivo exchange activity of Dbs is restricted in a cell-specific manner. Here we report the characterization of a novel scaffold protein (designated cell cycle progression protein 1 [Ccpg1]) that interacts with Dbs and modulates its in vivo exchange specificity. When coexpressed in mammalian cells, Ccpg1 binds to the Dbl homology/pleckstrin homology domain tandem motif of Dbs and inhibits its exchange activity toward RhoA, but not Cdc42. Expression of Ccpg1 correlates with the ability of Dbs to activate endogenous RhoA in cultured cells, and suppression of endogenous Ccpg1 expression potentiates Dbs exchange activity toward RhoA. The isolated Dbs binding domain of Ccpg1 is not sufficient to suppress Dbs exchange activity on RhoA, thus suggesting a regulatory interaction. Ccpg1 mediates recruitment of endogenous Src kinase into Dbs-containing complexes and interacts with the Rho family member Cdc42. Collectively, our studies suggest that Ccpg1 represents a new class of regulatory scaffold protein that can function as both an assembly platform for Rho protein signaling complexes and a regulatory protein which can restrict the substrate utilization of a promiscuous RhoGEF family member.

scholarly journals Activation of Rac and Cdc42 Video Imaged by Fluorescent Resonance Energy Transfer-Based Single-Molecule Probes in the Membrane of Living Cells

2002 ◽  
Vol 22 (18) ◽  
pp. 6582-6591 ◽  
Author(s):  
Reina E. Itoh ◽  
Kazuo Kurokawa ◽  
Yusuke Ohba ◽  
Hisayoshi Yoshizaki ◽  
Naoki Mochizuki ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Leading Edge ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Gtpase Activating Proteins

ABSTRACT Rho family G proteins, including Rac and Cdc42, regulate a variety of cellular functions such as morphology, motility, and gene expression. We developed fluorescent resonance energy transfer-based probes which monitored the local balance between the activities of guanine nucleotide exchange factors and GTPase-activating proteins for Rac1 and Cdc42 at the membrane. These probes, named Raichu-Rac and Raichu-Cdc42, consisted of a Cdc42- and Rac-binding domain of Pak, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras. With these probes, we video imaged the Rac and Cdc42 activities. In motile HT1080 cells, activities of both Rac and Cdc42 gradually increased toward the leading edge and decreased rapidly when cells changed direction. Under a higher magnification, we observed that Rac activity was highest immediately behind the leading edge, whereas Cdc42 activity was most prominent at the tip of the leading edge. Raichu-Rac and Raichu-Cdc42 were also applied to a rapid and simple assay for the analysis of putative guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) in living cells. Among six putative GEFs and GAPs, we identified KIAA0362/DBS as a GEF for Rac and Cdc42, KIAA1256 as a GEF for Cdc42, KIAA0053 as a GAP for Rac and Cdc42, and KIAA1204 as a GAP for Cdc42. In conclusion, use of these single-molecule probes to determine Rac and Cdc42 activity will accelerate the analysis of the spatiotemporal regulation of Rac and Cdc42 in a living cell.

scholarly journals Hormonal regulation of phospholipase Cepsilon through distinct and overlapping pathways involving G12 and Ras family G-proteins

2004 ◽  
Vol 378 (1) ◽  
pp. 129-139 ◽  
Author(s):  
Grant G. KELLEY ◽  
Sarah E. REKS ◽  
Alan V. SMRCKA
Keyword(s):  
G Proteins ◽  
Hormonal Regulation ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Independent Manner ◽  
Hormonal Stimulation ◽  
Nucleotide Exchange Factor ◽  
Ras Activation ◽  
Ras Family ◽  
Sphingosine 1 Phosphate

PLC∊ (phospholipase C∊) is a novel PLC that has a CDC25 guanine nucleotide exchange factor domain and two RA (Ras-association) domains of which the second (RA2) is critical for Ras activation of the enzyme. In the present studies, we examined hormonal stimulation to elucidate receptor-mediated pathways that functionally regulate PLC∊. We demonstrate that EGF (epidermal growth factor), a receptor tyrosine kinase agonist, and LPA (lysophosphatidic acid), S1P (sphingosine 1-phosphate) and thrombin, GPCR (G-protein-coupled receptor) agonists, stimulate PLC∊ overexpressed in COS-7 cells. EGF stimulated PLC∊ in an RA2-dependent manner through Ras and Rap. In contrast, LPA, S1P and thrombin stimulated PLC∊ by both RA2-independent and -dependent mechanisms. To determine the G-proteins that mediate the effects of these GPCR agonists, we co-expressed constitutively active G-proteins with PLC∊ and found that Gα12, Gα13, Rho, Rac and Ral stimulate PLC∊ in an RA2-independent manner; whereas TC21, Rap1A, Rap2A and Rap2B stimulate PLC∊ in an RA2-dependent manner similar to H-Ras. Of these G-proteins, we show that Gα12/Gα13 and Rap partly mediate the effects of LPA, S1P and thrombin to stimulate PLC∊. In addition, the stimulation by LPA and S1P is also partly sensitive to pertussis toxin. These studies demonstrate diverse hormonal regulation of PLC∊ by distinct and overlapping pathways.

Regulation of RalA GTPase by phosphatidylinositol 3-kinase as visualized by FRET probes

2006 ◽  
Vol 34 (5) ◽  
pp. 851-854 ◽  
Author(s):  
H. Yoshizaki ◽  
K. Aoki ◽  
T. Nakamura ◽  
M. Matsuda
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Subsequent Activation ◽  
Phosphoinositide 3 Kinase

Small GTPases, which are binary switches regulating various signal transduction cascades, function not only to relay signals but also to integrate them from multiple signalling branches. For example, RalA activity is regulated by at least three signalling cascades involving Ras, Rac or PI3K (phosphoinositide 3-kinase). To untangle such complicated regulatory mechanisms, we have been developing probes for GTPases, kinases and phosphatidylinositols based on the principle of FRET (fluorescence resonance energy transfer). We demonstrated previously that, upon EGF (epidermal growth factor) stimulation, Ras activity increases diffusely in the plasma membrane, whereas RalA activity increases predominantly in lamellipodial protrusions. Here, we show that the level of PtdIns(3,4,5)P3 is increased diffusely in the plasma membrane, whereas, in the central region, the level of PtdIns(3,4)P2 is increased more in the nascent lamellipodia than in the plasma membrane. The distribution and time course of Akt activation are similar to those of increased PtdIns(3,4)P2 levels. These observations suggest that the increase in PtdIns(3,4)P2 and the subsequent activation of Akt may be responsible for the localized activation of RalA. Thus the signals from Ras and PI3K converge at the level of Ral GEFs (guanine nucleotide-exchange factors), and this convergence restricts the area of RalA activation.

The cAMP Sensor Epac2 Is a Direct Target of Antidiabetic Sulfonylurea Drugs

Science ◽  
2009 ◽  
Vol 325 (5940) ◽  
pp. 607-610 ◽  
Author(s):  
Chang-Liang Zhang ◽  
Megumi Katoh ◽  
Tadao Shibasaki ◽  
Kohtaro Minami ◽  
Yasuhiro Sunaga ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Gut Hormones ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Promising Target ◽  
Guanosine Triphosphatase

Epac2, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rap1, is activated by adenosine 3′,5′-monophosphate. Fluorescence resonance energy transfer and binding experiments revealed that sulfonylureas, widely used antidiabetic drugs, interact directly with Epac2. Sulfonylureas activated Rap1 specifically through Epac2. Sulfonylurea-stimulated insulin secretion was reduced both in vitro and in vivo in mice lacking Epac2, and the glucose-lowering effect of the sulfonylurea tolbutamide was decreased in these mice. Epac2 thus contributes to the effect of sulfonylureas to promote insulin secretion. Because Epac2 is also required for the action of incretins, gut hormones crucial for potentiating insulin secretion, it may be a promising target for antidiabetic drug development.

scholarly journals Integration of Transforming Growth Factor β and RAS Signaling Silences a RAB5 Guanine Nucleotide Exchange Factor and Enhances Growth Factor-Directed Cell Migration

2007 ◽  
Vol 28 (5) ◽  
pp. 1573-1583 ◽  
Author(s):  
Hailiang Hu ◽  
Marc Milstein ◽  
Joanne M. Bliss ◽  
Minh Thai ◽  
Gautam Malhotra ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Guanine Nucleotide Exchange Factor ◽  
Transforming Growth Factor Β ◽  
Ras Signaling ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

ABSTRACT Transforming growth factor β (TGF-β) receptor (TβR) signaling contributes to normal development as well as tumorigenesis. Here we report that RIN1, a RAB5 guanine nucleotide exchange factor (GEF) and down regulator of receptor tyrosine kinases (RTKs), promotes TβR signaling through enhanced endocytosis. TβR activation induces SNAI1 (Snail), a transcription repressor that reduces RIN1 expression, providing a negative feedback mechanism to control TβR trafficking and downstream signaling. Persistent RAS signaling disrupts this equilibrium by stabilizing SNAI1 protein, resulting in strong silencing of RIN1 and stabilization of RTKs. TGF-β-induced RIN1 silencing in breast cancer cells prolonged sensitivity to hepatocyte growth factor, a ligand for the MET-type RTK, and enhanced growth factor-directed cell motility. We conclude that in some tumor cells TβR and RAS signals are integrated through the silencing of RIN1, leading to a reduction in RAB5-mediated endocytosis. These findings shed new light on the basis for distinct interpretations of TGF-β signaling by normal versus transformed cells.

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