scholarly journals Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1

2003 ◽  
Vol 160 (1) ◽  
pp. 17-23 ◽  
Author(s):  
Metello Innocenti ◽  
Emanuela Frittoli ◽  
Isabella Ponzanelli ◽  
John R. Falck ◽  
Saskia M. Brachmann ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Physical Interaction ◽  
Nucleotide Exchange ◽  
Downstream Target ◽  
Signaling Complex ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Phosphoinositide 3 Kinase

Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8–Abi1–Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Here, we show that Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8–Abi1–Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.

scholarly journals Mechanisms through which Sos-1 coordinates the activation of Ras and Rac

2002 ◽  
Vol 156 (1) ◽  
pp. 125-136 ◽  
Author(s):  
Metello Innocenti ◽  
Pierluigi Tenca ◽  
Emanuela Frittoli ◽  
Mario Faretta ◽  
Arianna Tocchetti ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Adaptor Protein ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Son Of Sevenless ◽  
Sequential Activation

Signaling from receptor tyrosine kinases (RTKs)* requires the sequential activation of the small GTPases Ras and Rac. Son of sevenless (Sos-1), a bifunctional guanine nucleotide exchange factor (GEF), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with Eps8 and E3b1–Abi-1, a RTK substrate and an adaptor protein, respectively. A mechanistic understanding of how Sos-1 coordinates Ras and Rac activity is, however, still missing. Here, we demonstrate that (a) Sos-1, E3b1, and Eps8 assemble into a tricomplex in vivo under physiological conditions; (b) Grb2 and E3b1 bind through their SH3 domains to the same binding site on Sos-1, thus determining the formation of either a Sos-1–Grb2 (S/G) or a Sos-1–E3b1–Eps8 (S/E/E8) complex, endowed with Ras- and Rac-specific GEF activities, respectively; (c) the Sos-1–Grb2 complex is disrupted upon RTKs activation, whereas the S/E/E8 complex is not; and (d) in keeping with the previous result, the activation of Ras by growth factors is short-lived, whereas the activation of Rac is sustained. Thus, the involvement of Sos-1 at two distinct and differentially regulated steps of the signaling cascade allows for coordinated activation of Ras and Rac and different duration of their signaling within the cell.

scholarly journals A conserved and regulated mechanism drives endosomal Rab transition

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Lars Langemeyer ◽  
Ann-Christin Borchers ◽  
Eric Herrmann ◽  
Nadia Füllbrunn ◽  
Yaping Han ◽  
...  
Keyword(s):  
Underlying Mechanism ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Molecular Determinants ◽  
Key Factor ◽  
Membrane Bound ◽  
Endosome Maturation

Endosomes and lysosomes harbor Rab5 and Rab7 on their surface as key proteins involved in their identity, biogenesis, and fusion. Rab activation requires a guanine nucleotide exchange factor (GEF), which is Mon1-Ccz1 for Rab7. During endosome maturation, Rab5 is replaced by Rab7, though the underlying mechanism remains poorly understood. Here, we identify the molecular determinants for Rab conversion in vivo and in vitro, and reconstitute Rab7 activation with yeast and metazoan proteins. We show (i) that Mon1-Ccz1 is an effector of Rab5, (ii) that membrane-bound Rab5 is the key factor to directly promote Mon1-Ccz1 dependent Rab7 activation and Rab7-dependent membrane fusion, and (iii) that this process is regulated in yeast by the casein kinase Yck3, which phosphorylates Mon1 and blocks Rab5 binding. Our study thus uncovers the minimal feed-forward machinery of the endosomal Rab cascade and a novel regulatory mechanism controlling this pathway.

Abstract 303: HDL Mimetic Peptide 4F Rescues Pre-existing Pulmonary Hypertension via MicroRNA 193-3p

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Salil Sharma ◽  
Soban Umar ◽  
Gabe Wong ◽  
Denise Mai ◽  
Mohamad Navab ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Intratracheal Instillation ◽  
Nucleotide Exchange ◽  
Mimetic Peptide ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Human Pulmonary Artery ◽  
Proliferation In Vitro

Pulmonary hypertension (PH) is a chronic lung disease associated with severe vascular disorders leading to right ventricular(RV) failure. An HDL mimetic peptide, 4F, has been shown to be effective for the treatment of atherosclerosis and a number of inflammatory disorders. Here, we explored whether 4F can rescue advanced PH by controlling the expression of specific microRNAs (miRs). PH was induced in rats by a single injection of monocrotaline (MCT, 60mg/kg, s.c .) or by placing mice in hypoxia chamber(O2≤10%) for 21 days. MCT-rats or hypoxic mice received 4F therapy (50mg/kg/day, s.c .,days 21-30 in MCT model and days 14-21 in hypoxia model). We performed microRNA microarray analysis (non-affymetrix) in lung tissues of CTRL, PH and 4F-rescued groups. OE of miR193 was achieved by intratracheal instillation of 20nM dose at days 16, 21 and 26 in MCT model or at days 14 and 18 in hypoxia model. 4F therapy starting after the establishment of PH in both MCT and hypoxia models improved significantly RV pressure (RVP) and RV hypertrophy index (RVP=46±3 vs RVP=74±1 mmHg in PH; RV/LV+IVS=0.38±0.02 vs RV/(LV+IVS)=0.68±0.05 in PH, p<0.05 vs PH and in hypoxia model RVP=22±3.8 vs. 36.91±5.74 in PH and 20.93±2.52mmHg in ctrl, p<0.05 vs PH ). Microarray and qPCR showed downregulation of miR-193 by ~3 fold in MCT model. 4F therapy normalized miR-193 to ctrl levels. MiR-193 OE in both MCT-rats and hypoxic-mice rescued PH (RVP=38±5.5mmHg, RV/LV+IVS=0.37±0.034 in MCT-rats and RVP=25.48±0.88mmHg in hypoxic-mice). Lung sections showed increased arteriolar muscularization and ox-LDL deposition in the PH group, prevented by miR-193 therapy. In vivo, OE of miR-193 suppressed transcription of in-silico targets ALOX5, a lipoxygenase; IGF1R, insulin-like growth factor 1 receptor and ARHGEF12, a Rho guanine nucleotide exchange factor and decreased human pulmonary artery smooth muscle cell (HPASMC) proliferation in vitro in the presence of serum or 12-HETE by >2 folds whereas miR-193 KD increased proliferation. In conclusion, 4F rescues pre-existing severe PH by targeting genes associated with HETEs and HODEs production, inflammation and growth via inducing miR-193.

scholarly journals Coordinated regulation of AP2 uncoating from clathrin-coated vesicles by rab5 and hRME-6

2008 ◽  
Vol 183 (3) ◽  
pp. 499-511 ◽  
Author(s):  
Sophia Semerdjieva ◽  
Barry Shortt ◽  
Emma Maxwell ◽  
Sukhdeep Singh ◽  
Paul Fonarev ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Coated Vesicles ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Steady State Levels ◽  
Endocytic Vesicles ◽  
Interfering Rna

Here we investigate the role of rab5 and its cognate exchange factors rabex-5 and hRME-6 in the regulation of AP2 uncoating from endocytic clathrin-coated vesicles (CCVs). In vitro, we show that the rate of AP2 uncoating from CCVs is dependent on the level of functional rab5. In vivo, overexpression of dominant-negative rab5S34N, or small interfering RNA (siRNA)–mediated depletion of hRME-6, but not rabex-5, resulted in increased steady-state levels of AP2 associated with endocytic vesicles, which is consistent with reduced uncoating efficiency. hRME-6 guanine nucleotide exchange factor activity requires hRME-6 binding to α-adaptin ear, which displaces the ear-associated μ2 kinase AAK1. siRNA-mediated depletion of hRME-6 increases phospho-μ2 levels, and expression of a phosphomimetic μ2 mutant increases levels of endocytic vesicle-associated AP2. Depletion of hRME-6 or rab5S35N expression also increases the levels of phosphoinositide 4,5-bisphosphate (PtdIns(4,5)P2) associated with endocytic vesicles. These data are consistent with a model in which hRME-6 and rab5 regulate AP2 uncoating in vivo by coordinately regulating μ2 dephosphorylation and PtdIns(4,5)P2 levels in CCVs.

scholarly journals The eps8 Family of Proteins Links Growth Factor Stimulation to Actin Reorganization Generating Functional Redundancy in the Ras/Rac Pathway

2004 ◽  
Vol 15 (1) ◽  
pp. 91-98 ◽  
Author(s):  
Nina Offenhäuser ◽  
Alessandro Borgonovo ◽  
Andrea Disanza ◽  
Pascale Romano ◽  
Isabella Ponzanelli ◽  
...  
Keyword(s):  
Growth Factor ◽  
Functional Redundancy ◽  
Nucleotide Exchange ◽  
Lipid Kinase ◽  
Actin Remodeling ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Phosphoinositide 3 Kinase ◽  
Related Proteins

Sos-1, a guanine nucleotide exchange factor (GEF), eps8 and Abi1, two signaling proteins, and the lipid kinase phosphoinositide 3-kinase (PI3-K), assemble in a multimolecular complex required for Rac activation leading to actin cytoskeletal remodeling. Consistently, eps8 –/– fibroblasts fail to form membrane ruffles in response to growth factor stimulation. Surprisingly, eps8 null mice are healthy, fertile, and display no overt phenotype, suggesting the existence of functional redundancy within this pathway. Here, we describe the identification and characterization of a family of eps8-related proteins, comprising three novel gene products, named eps8L1, eps8L2, and eps8L3. Eps8Ls display collinear topology and 27–42% identity to eps8. Similarly to eps8, eps8Ls interact with Abi1 and Sos-1; however, only eps8L1 and eps8L2 activate the Rac-GEF activity of Sos-1, and bind to actin in vivo. Consistently, eps8L1 and eps8L2, but not eps8L3, localize to PDGF-induced, F-actin–rich ruffles and restore receptor tyrosine kinase (RTK)-mediated actin remodeling when expressed in eps8 –/– fibroblasts. Thus, the eps8Ls define a novel family of proteins responsible for functional redundancy in the RTK-activated signaling pathway leading to actin remodeling. Finally, the patterns of expression of eps8 and eps8L2 in mice are remarkably overlapping, thus providing a likely explanation for the lack of overt phenotype in eps8 null mice.

scholarly journals The Rho-Guanine Nucleotide Exchange Factor Domain of Obscurin Activates RhoA Signaling in Skeletal Muscle

2009 ◽  
Vol 20 (17) ◽  
pp. 3905-3917 ◽  
Author(s):  
Diana L. Ford-Speelman ◽  
Joseph A. Roche ◽  
Amber L. Bowman ◽  
Robert J. Bloch
Keyword(s):  
Skeletal Muscle ◽  
Guanine Nucleotide Exchange Factor ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Obscurin is a large (∼800-kDa), modular protein of striated muscle that concentrates around the M-bands and Z-disks of each sarcomere, where it is well positioned to sense contractile activity. Obscurin contains several signaling domains, including a rho-guanine nucleotide exchange factor (rhoGEF) domain and tandem pleckstrin homology domain, consistent with a role in rho signaling in muscle. We investigated the ability of obscurin's rhoGEF domain to interact with and activate small GTPases. Using a combination of in vitro and in vivo approaches, we found that the rhoGEF domain of obscurin binds selectively to rhoA, and that rhoA colocalizes with obscurin at the M-band in skeletal muscle. Other small GTPases, including rac1 and cdc42, neither associate with the rhoGEF domain of obscurin nor concentrate at the level of the M-bands. Furthermore, overexpression of the rhoGEF domain of obscurin in adult skeletal muscle selectively increases rhoA expression and activity in this tissue. Overexpression of obscurin's rhoGEF domain and its effects on rhoA alter the expression of rho kinase and citron kinase, both of which can be activated by rhoA in other tissues. Injuries to rodent hindlimb muscles caused by large-strain lengthening contractions increases rhoA activity and displaces it from the M-bands to Z-disks, similar to the effects of overexpression of obscurin's rhoGEF domain. Our results suggest that obscurin's rhoGEF domain signals at least in part by inducing rhoA expression and activation, and altering the expression of downstream kinases in vitro and in vivo.

scholarly journals Ras recruits mitotic exit regulator Lte1 to the bud cortex in budding yeast

2003 ◽  
Vol 161 (5) ◽  
pp. 889-897 ◽  
Author(s):  
Satoshi Yoshida ◽  
Ryuji Ichihashi ◽  
Akio Toh-e
Keyword(s):  
Small Gtpase ◽  
Mitotic Exit ◽  
Effector Protein ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Downstream Effector ◽  
Nucleotide Exchange Factor ◽  
Family Protein

ACdc25 family protein Lte1 (low temperature essential) is essential for mitotic exit at a lowered temperature and has been presumed to be a guanine nucleotide exchange factor (GEF) for a small GTPase Tem1, which is a key regulator of mitotic exit. We found that Lte1 physically associates with Ras2-GTP both in vivo and in vitro and that the Cdc25 homology domain (CHD) of Lte1 is essential for the interaction with Ras2. Furthermore, we found that the proper localization of Lte1 to the bud cortex is dependent on active Ras and that the overexpression of a derivative of Lte1 without the CHD suppresses defects in mitotic exit of a Δlte1 mutant and a Δras1 Δras2 mutant. These results suggest that Lte1 is a downstream effector protein of Ras in mitotic exit and that the Ras GEF domain of Lte1 is not essential for mitotic exit but required for its localization.

scholarly journals Arf3p GTPase is a key regulator of Bud2p activation for invasive growth in Saccharomyces cerevisiae

2013 ◽  
Vol 24 (15) ◽  
pp. 2328-2339 ◽  
Author(s):  
Jia-Wei Hsu ◽  
Fang-Jen S. Lee
Keyword(s):  
Bound State ◽  
Invasive Growth ◽  
Nucleotide Exchange ◽  
General Applicability ◽  
Glucose Depletion ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Adp Ribosylation Factor

The regulation and signaling pathways involved in the invasive growth of yeast have been studied extensively because of their general applicability to fungal pathogenesis. Bud2p, which functions as a GTPase-activating protein (GAP) for Bud1p/Rsr1p, is required for appropriate budding patterns and filamentous growth. The regulatory mechanisms leading to Bud2p activation, however, are poorly understood. In this study, we report that ADP-ribosylation factor 3p (Arf3p) acts as a regulator of Bud2p activation during invasive growth. Arf3p binds directly to the N-terminal region of Bud2p and promotes its GAP activity both in vitro and in vivo. Genetic analysis shows that deletion of BUD1 suppresses the defect of invasive growth in arf3Δ or bud2Δ cells. Lack of Arf3p, like that of Bud2p, causes the intracellular accumulation of Bud1p-GTP. The Arf3p–Bud2p interaction is important for invasive growth and facilitates the Bud2p–Bud1p association in vivo. Finally, we show that under glucose depletion–induced invasion conditions in yeast, more Arf3p is activated to the GTP-bound state, and the activation is independent of Arf3p guanine nucleotide-exchange factor Yel1p. Thus we demonstrate that a novel spatial activation of Arf3p plays a role in regulating Bud2p activation during glucose depletion–induced invasive growth.

scholarly journals Overexpression of Translation Elongation Factor 1A Affects the Organization and Function of the Actin Cytoskeleton in Yeast

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1425-1436
Author(s):  
Raj Munshi ◽  
Kimberly A Kandl ◽  
Anne Carr-Schmid ◽  
Johanna L Whitacre ◽  
Alison E M Adams ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Elongation Factor ◽  
Nucleotide Exchange ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
And Function

Abstract The translation elongation factor 1 complex (eEF1) plays a central role in protein synthesis, delivering aminoacyl-tRNAs to the elongating ribosome. The eEF1A subunit, a classic G-protein, also performs roles aside from protein synthesis. The overexpression of either eEF1A or eEF1Bα, the catalytic subunit of the guanine nucleotide exchange factor, in Saccharomyces cerevisiae results in effects on cell growth. Here we demonstrate that overexpression of either factor does not affect the levels of the other subunit or the rate or accuracy of protein synthesis. Instead, the major effects in vivo appear to be at the level of cell morphology and budding. eEF1A overexpression results in dosage-dependent reduced budding and altered actin distribution and cellular morphology. In addition, the effects of excess eEF1A in actin mutant strains show synthetic growth defects, establishing a genetic connection between the two proteins. As the ability of eEF1A to bind and bundle actin is conserved in yeast, these results link the established ability of eEF1A to bind and bundle actin in vitro with nontranslational roles for the protein in vivo.

scholarly journals Premature Platelet Activation and Resistance to P2Y12 Inhibitors in Rasa3 Mutant Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 91-91
Author(s):  
Wolfgang Bergmeier ◽  
David S Paul ◽  
Lucia Stefanini ◽  
Raymond F. Robledo ◽  
E. Ricky Chan ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
P2y12 Inhibitors ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Abstract The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the guanine nucleotide exchange factor CalDAG-GEFI and an unknown regulator operating downstream of the ADP receptor, P2Y12, the target of antithrombotic therapy. Here we provide evidence that the GTPase-activating protein, RASA3, is a critical inhibitor of platelet activation and the missing link in the P2Y12/RAP1 signaling pathway. Genetic inactivation of Rasa3 led to premature activation and markedly reduced lifespan of circulating platelets in mice (t1/2=14 hrs vs. 55 hrs in controls). The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Importantly, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Thus, constitutively active RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling. At sites of vascular injury, P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation. Our findings provide critical mechanistic insights for the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders. Disclosures No relevant conflicts of interest to declare.

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