scholarly journals Substrate binding by the yeast Hsp110 nucleotide exchange factor and molecular chaperone Sse1 is not obligate for its biological activities

2017 ◽  
Vol 28 (15) ◽  
pp. 2066-2075 ◽  
Author(s):  
Veronica M. Garcia ◽  
Nadinath B. Nillegoda ◽  
Bernd Bukau ◽  
Kevin A. Morano
Keyword(s):  
Molecular Chaperone ◽  
Biological Activities ◽  
Substrate Binding ◽  
Normal Growth ◽  
Cellular Protein ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor

The highly conserved heat shock protein 70 (Hsp70) is a ubiquitous molecular chaperone essential for maintaining cellular protein homeostasis. The related protein Hsp110 (Sse1/Sse2 in Saccharomyces cerevisiae) functions as a nucleotide exchange factor (NEF) to regulate the protein folding activity of Hsp70. Hsp110/Sse1 also can prevent protein aggregation in vitro via its substrate-binding domain (SBD), but the cellular roles of this “holdase” activity are poorly defined. We generated and characterized an Sse1 mutant that separates, for the first time, its nucleotide exchange and substrate-binding functions. Sse1sbd retains nucleotide-binding and nucleotide exchange activities while exhibiting severe deficiencies in chaperone holdase activity for unfolded polypeptides. In contrast, we observed no effect of the SBD mutation in reconstituted disaggregation or refolding reactions in vitro. In vivo, Sse1sbd successfully heterodimerized with the yeast cytosolic Hsp70s Ssa and Ssb and promoted normal growth, with the exception of sensitivity to prolonged heat but not other proteotoxic stress. Moreover, Sse1sbd was fully competent to support Hsp90-dependent signaling through heterologously expressed glucocorticoid receptor and degradation of a permanently misfolded protein, two previously defined roles for Sse1. We conclude that despite conservation among eukaryotic homologues, chaperone holdase activity is not an obligate function in the Hsp110 family.

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 Binding of ISRIB reveals a regulatory site in the nucleotide exchange factor eIF2B

Science ◽  
2018 ◽  
Vol 359 (6383) ◽  
pp. 1533-1536 ◽  
Author(s):  
Alisa F. Zyryanova ◽  
Félix Weis ◽  
Alexandre Faille ◽  
Akeel Abo Alard ◽  
Ana Crespillo-Casado ◽  
...  
Keyword(s):  
Cellular Response ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Nucleotide Exchange ◽  
Regulatory Subunits ◽  
Eukaryotic Translation ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor

The integrated stress response (ISR) is a conserved translational and transcriptional program affecting metabolism, memory, and immunity. The ISR is mediated by stress-induced phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, reverses the attenuation of eIF2B by phosphorylated eIF2α, protecting mice from neurodegeneration and traumatic brain injury. We describe a 4.1-angstrom-resolution cryo–electron microscopy structure of human eIF2B with an ISRIB molecule bound at the interface between the β and δ regulatory subunits. Mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB analogs in vivo and ISRIB binding in vitro. Our findings point to a site in eIF2B that can be exploited by ISRIB to regulate translation.

scholarly journals Gef1p, a New Guanine Nucleotide Exchange Factor for Cdc42p, Regulates Polarity inSchizosaccharomyces pombe

2003 ◽  
Vol 14 (1) ◽  
pp. 313-323 ◽  
Author(s):  
Pedro M. Coll ◽  
Yadira Trillo ◽  
Amagoia Ametzazurra ◽  
Pilar Perez
Keyword(s):  
Cell Morphology ◽  
Rho Gtpases ◽  
Genetic Interaction ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Schizosaccharomyces pombe cdc42+regulates cell morphology and polarization of the actin cytoskeleton. Scd1p/Ral1p is the only described guanine nucleotide exchange factor (GEF) for Cdc42p in S. pombe. We have identified a new GEF, named Gef1p, specifically regulating Cdc42p. Gef1p binds to inactive Cdc42p but not to other Rho GTPases in two-hybrid assays. Overexpression of gef1+increases specifically the GTP-bound Cdc42p, and Gef1p is capable of stimulating guanine nucleotide exchange of Cdc42p in vitro. Overexpression ofgef1+causes changes in cell morphology similar to those caused by overexpression of the constitutively active cdc42G12V allele. Gef1p localizes to the septum. gef1+deletion is viable but causes a mild cell elongation and defects in bipolar growth and septum formation, suggesting a role for Gef1p in the control of cell polarity and cytokinesis. The double mutant gef1Δ scd1Δ is not viable, indicating that they share an essential function as Cdc42p activators. However, both deletion and overexpression of either gef1+orscd1+causes different morphological phenotypes, which suggest different functions. Genetic evidence revealed a link between Gef1p and the signaling pathway of Shk1/Orb2p and Orb6p. In contrast, no genetic interaction between Gef1p and Shk2p-Mkh1p pathway was observed.

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.

The guanine-nucleotide-exchange factor P-Rex1 is activated by protein phosphatase 1α

2012 ◽  
Vol 443 (1) ◽  
pp. 173-183 ◽  
Author(s):  
Mark A. Barber ◽  
Annick Hendrickx ◽  
Monique Beullens ◽  
Hugo Ceulemans ◽  
David Oxley ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Guanine Nucleotide Exchange Factor ◽  
Site Directed Mutagenesis ◽  
Similar Degree ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

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.

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 Thyrotropin Activates Guanosine 5′-Diphosphate/Guanosine 5′-Triphosphate Exchange on the Rate-Limiting Endocytic Catalyst, Rab5a, in Human Thyrocytes in Vivo and in Vitro

2007 ◽  
Vol 92 (7) ◽  
pp. 2803-2810 ◽  
Author(s):  
Marie-France van den Hove ◽  
Karine Croizet-Berger ◽  
Donatienne Tyteca ◽  
Charlotte Selvais ◽  
Philippe de Diesbach ◽  
...  
Keyword(s):  
Hormone Secretion ◽  
Thyroid Tissue ◽  
Whole Body ◽  
Nucleotide Exchange ◽  
Membrane Recruitment ◽  
Exchange Factor ◽  
Whole Body Metabolism ◽  
Rate Limiting

Abstract Context: We have previously reported that the TSH receptor/cAMP cascade enhances the coordinate expression of the rate-limiting endocytic catalysts, Rab5a and Rab7, which respectively promote thyroglobulin (Tg) internalization and transfer to lysosomes, thereby accelerating thyroid hormone secretion. Objective: We address whether TSH further controls Rab5a activity by promoting its GTP-bound state. Design: We compared Rab5a activation in seven pairs of hyperactive and corresponding quiescent thyroid tissues; TSH effect was reproduced on polarized cultures of normal human thyrocytes. Patients: We studied seven euthyroid patients bearing hyperactive autonomous adenomas; normal thyroid tissue for culture. Main Outcome Measurements: Rab5a GDP/GTP exchange factor activity [Rab5a-guanine nucleotide exchange factor (GEF)], expression of Rabex-5 (a Rab5a-GEF), and function of thyrocytes in vitro were the main outcome measures. Results: In autonomous adenomas, constitutive activation increased both total activity and sedimentability (membrane recruitment) of Rab5a-GEF, compared with perinodular tissues. Increased Rab5a-GEF activity correlated with increased expression of Rabex-5 and Rab5a, as well as with Tg store depletion. In polarized human thyrocyte monolayers, TSH did not affect total Rab5a-GEF activity after 2 h but promoted its membrane recruitment; after 4 d, TSH increased both Rab5a-GEF activity and Rabex-5 expression and recruitment onto membranes where Rabex-5 coimmunoprecipitated with Rabaptin-5 and Rab5a. Sedimentable Rab5a-GEF perfectly correlated with apical endocytosis and lysosomal transfer of 125I-Tg, and with basolateral secretion of 125I-derived hormones. Conclusion: This study provides the first clinical and experimental evidence that regulation of the activity of a rate-limiting endocytic catalyst finely tunes a tightly controlled cellular function that ultimately governs whole body metabolism.

Sign in / Sign up

Export Citation Format

Share Document