scholarly journals Phosphorylation of Ser111 in Rab8a modulates Rabin8 dependent activation by perturbation of side chain interaction networks

2019 ◽  
Author(s):  
Danial Pourjafar-Dehkordi ◽  
Sophie Vieweg ◽  
Aymelt Itzen ◽  
Martin Zacharias
Keyword(s):  
Intracellular Transport ◽  
Intramolecular Interaction ◽  
Salt Bridge ◽  
Small Gtpases ◽  
Side Chain ◽  
Rab Proteins ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Energy Simulations

AbstractGTPases are key-players in cellular signaling processes. Phosphorylation of Rab proteins, which belong to the Ras superfamily of small GTPases regulating intracellular transport, has recently been implicated in the pathogenesis of Parkinson Disease (PD). For Rab8a, it was shown that serine 111 phosphorylation (pS111) is dependent on the protein kinase PINK1, and that mimicking the phosphorylation at S111 by a serine/glutamate substitution (S111E) impaired Rab8a activation by its cognate nucleotide exchange factor (GEF) Rabin8. Here, we performed comparative Molecular Dynamics and free energy simulations on Rab8a and Rab8a:Rabin8 complexes to elucidate the molecular details on how pS111 and S111E may influence the interaction with Rabin8. The simulations indicate that S111E and pS111 establish an intramolecular interaction with arginine 79 (R79). In the complex, this interaction persists, and therefore perturbs a favorable intermolecular salt-bridge contact between R79 in Rab8a and the acidic aspartate 187 (D187) in Rabin8. Binding free analysis reveals that S111E and pS111, as well as the mutation R79A, in Rab8a drastically reduce the binding affinity to Rabin8. Combining the R79A mutation with S111E or pS111, respectively, nearly diminishes Rab8a-Rabin8 binding. In vitro experiments confirm our computational results showing that the nucleotide exchange rates of the respective Rab8a mutants are decreased by >80% in the presence of Rabin8 compared to wild type. In addition to specific insights into how S111 phosphorylation of Rab8a can influence GEF-mediated activation, the simulations demonstrate how side chain modifications in general can allosterically influence the network of surface side chain interactions between binding partners.

scholarly journals Identification of Regulators for Ypt1 GTPase Nucleotide Cycling

1998 ◽  
Vol 9 (10) ◽  
pp. 2819-2837 ◽  
Author(s):  
Sara Jones ◽  
Celeste J. Richardson ◽  
Robert J. Litt ◽  
Nava Segev
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Rab Proteins ◽  
Nucleotide Exchange ◽  
Gtp Hydrolysis ◽  
Golgi Transport ◽  
Transport Assay

Small GTPases of the Ypt/Rab family are involved in the regulation of vesicular transport. Cycling between the GDP- and GTP-bound forms and the accessory proteins that regulate this cycling are thought to be crucial for Ypt/Rab function. Guanine nucleotide exchange factors (GEFs) stimulate both GDP loss and GTP uptake, and GTPase-activating proteins (GAPs) stimulate GTP hydrolysis. Little is known about GEFs and GAPs for Ypt/Rab proteins. In this article we report the identification and initial characterization of two factors that regulate nucleotide cycling by Ypt1p, which is essential for the first two steps of the yeast secretory pathway. The Ypt1p-GEF stimulates GDP release and GTP uptake at least 10-fold and is specific for Ypt1p. Partially purified Ypt1p-GEF can rescue the inhibition caused by the dominant-negative Ypt1p-D124N mutant of in vitro endoplasmic reticulum-to-Golgi transport. This mutant probably blocks transport by inhibiting the GEF, suggesting that we have identified the physiological GEF for Ypt1p. The Ypt1p-GAP stimulates GTP hydrolysis by Ypt1p up to 54-fold, has a higher affinity for the GTP-bound form of Ypt1p than for the GDP-bound form, and is specific to a subgroup of exocytic Ypt proteins. The Ypt1p-GAP activity is not affected by deletion of two genes that encode known Ypt GAPs, GYP7and GYP1, nor is it influenced by mutations inSEC18, SEC17, or SEC22, genes whose products are involved in vesicle fusion. The GEF and GAP activities for Ypt1p localize to particulate cellular fractions. However, contrary to the predictions of current models, the GEF activity localizes to the fraction that functions as the acceptor in an endoplasmic reticulum-to-Golgi transport assay, whereas the GAP activity cofractionates with markers for the donor. On the basis of our current and previous results, we propose a new model for the role of Ypt/Rab nucleotide cycling and the factors that regulate this process.

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 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 p21-Activated Kinase 1 Plays a Critical Role in Cellular Activation by Nef

2000 ◽  
Vol 20 (7) ◽  
pp. 2619-2627 ◽  
Author(s):  
Oliver T. Fackler ◽  
Xiaobin Lu ◽  
Jeffrey A. Frost ◽  
Matthias Geyer ◽  
Bing Jiang ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Critical Role ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Inhibitory Peptide ◽  
Nucleotide Exchange Factor ◽  
P21 Activated Kinase ◽  
Cytoskeletal Rearrangements

ABSTRACT The activation of Nef-associated kinase (NAK) by Nef from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. This induction occurs via the guanine nucleotide exchange factor Vav and the small GTPases Rac1 and Cdc42. In this study, we identified NAK as p21-activated kinase 1 (PAK1). PAK1 bound to Nef in vitro and in vivo. Moreover, the induction of cytoskeletal rearrangements such as the formation of trichopodia, the activation of Jun N-terminal kinase, and the increase of viral production were blocked by an inhibitory peptide that targets the kinase activity of PAK1 (PAK1 83-149). These results identify NAK as PAK1 and emphasize the central role its kinase activity plays in cytoskeletal rearrangements and cellular signaling by Nef.

Identification and characterisation of novel Mss4-binding Rab GTPases

2011 ◽  
Vol 392 (3) ◽  
Author(s):  
Viktor Wixler ◽  
Ludmilla Wixler ◽  
Anika Altenfeld ◽  
Stephan Ludwig ◽  
Roger S. Goody ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mutational Analysis ◽  
Binding Capacity ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Binding Studies ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Abstract The Mss4 (mammalian suppressor of yeast Sec4) is an evolutionarily highly conserved protein and is expressed in all mammalian tissues. Although its precise biological function is still elusive, it has been shown to associate with a subset of secretory Rab proteins (Rab1b, Rab3a, Rab8a, Rab10) and to possess a rather low guanine nucleotide exchange factor (GEF) activity towards them in vitro (Rab1, Rab3a and Rab8a). By screening a human placenta cDNA library with Mss4 as bait, we identified several Rab GTPases (Rab12, Rab13 and Rab18) as novel Mss4-binding Rab proteins. Only exocytic but no endocytic Rab GTPases were found in our search. The binding of Mss4 to Rab proteins was confirmed by direct yeast two-hybrid interaction, by co-immunoprecipitation from lysates of mammalian cells, by immunofluorescence colocalisation as well as by direct in vitro binding studies. Analysis of Mss4 catalytic activity towards different Rab substrates confirmed that it is a somewhat inefficient GEF. These data, together with our mutational analysis of Mss4-Rab binding capacity, support the already proposed idea that Mss4 functions rather as a chaperone for exocytic Rab GTPases than as a GEF.

scholarly journals The DHR1 Domain of DOCK180 Binds to SNX5 and Regulates Cation-independent Mannose 6-phosphate Receptor Transport

2008 ◽  
Vol 19 (9) ◽  
pp. 3823-3835 ◽  
Author(s):  
Shigeo Hara ◽  
Etsuko Kiyokawa ◽  
Shun-ichiro Iemura ◽  
Tohru Natsume ◽  
Thomas Wassmer ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Retrograde Transport ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Sorting Nexins ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Mannose 6 Phosphate

DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.

scholarly journals Hijacking Components of the Cellular Secretory Pathway for Replication of Poliovirus RNA

2006 ◽  
Vol 81 (2) ◽  
pp. 558-567 ◽  
Author(s):  
George A. Belov ◽  
Nihal Altan-Bonnet ◽  
Gennadiy Kovtunovych ◽  
Catherine L. Jackson ◽  
Jennifer Lippincott-Schwartz ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Brefeldin A ◽  
Rna Replication ◽  
Bound State ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Virus Growth ◽  
Guanine Nucleotide Exchange ◽  
Membrane Reorganization

ABSTRACT Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesicle-like structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase ∼4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.

scholarly journals The nucleoporin RanBP2 tethers the cAMP effector Epac1 and inhibits its catalytic activity

2011 ◽  
Vol 193 (6) ◽  
pp. 1009-1020 ◽  
Author(s):  
Martijn Gloerich ◽  
Marjolein J. Vliem ◽  
Esther Prummel ◽  
Lars A.T. Meijer ◽  
Marije G.A. Rensen ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Negative Regulator ◽  
Camp Signaling ◽  
Nuclear Pore ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Wide Range

Cyclic adenosine monophosphate (cAMP) is a second messenger that relays a wide range of hormone responses. In this paper, we demonstrate that the nuclear pore component RanBP2 acts as a negative regulator of cAMP signaling through Epac1, a cAMP-regulated guanine nucleotide exchange factor for Rap. We show that Epac1 directly interacts with the zinc fingers (ZNFs) of RanBP2, tethering Epac1 to the nuclear pore complex (NPC). RanBP2 inhibits the catalytic activity of Epac1 in vitro by binding to its catalytic CDC25 homology domain. Accordingly, cellular depletion of RanBP2 releases Epac1 from the NPC and enhances cAMP-induced Rap activation and cell adhesion. Epac1 also is released upon phosphorylation of the ZNFs of RanBP2, demonstrating that the interaction can be regulated by posttranslational modification. These results reveal a novel mechanism of Epac1 regulation and elucidate an unexpected link between the NPC and cAMP signaling.

scholarly journals The Guanine Nucleotide Exchange Factor Tiam1 Affects Neuronal Morphology; Opposing Roles for the Small GTPases Rac and Rho

1997 ◽  
Vol 139 (3) ◽  
pp. 797-807 ◽  
Author(s):  
Frank N. van Leeuwen ◽  
Hendrie E.T. Kain ◽  
Rob A. van der Kammen ◽  
Frits Michiels ◽  
Onno W. Kranenburg ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Small Gtpases ◽  
Neuronal Morphology ◽  
Laminin Receptor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Neurite Formation

The invasion-inducing T-lymphoma invasion and metastasis 1 (Tiam1) protein functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. Differentiation-dependent expression of Tiam1 in the developing brain suggests a role for this GEF and its effector Rac1 in the control of neuronal morphology. Here we show that overexpression of Tiam1 induces cell spreading and affects neurite outgrowth in N1E-115 neuroblastoma cells. These effects are Rac-dependent and strongly promoted by laminin. Overexpression of Tiam1 recruits the α6β1 integrin, a laminin receptor, to specific adhesive contacts at the cell periphery, which are different from focal contacts. Cells overexpressing Tiam1 no longer respond to lysophosphatidic acid– induced neurite retraction and cell rounding, processes mediated by Rho, suggesting that Tiam1-induced activation of Rac antagonizes Rho signaling. This inhibition can be overcome by coexpression of constitutively active RhoA, which may indicate that regulation occurs at the level of Rho or upstream. Conversely, neurite formation induced by Tiam1 or Rac1 is further promoted by inactivating Rho. These results demonstrate that Rac- and Rho-mediated pathways oppose each other during neurite formation and that a balance between these pathways determines neuronal morphology. Furthermore, our data underscore the potential role of Tiam1 as a specific regulator of Rac during neurite formation and illustrate the importance of reciprocal interactions between the cytoskeleton and the extracellular matrix during this process.

Human p63RhoGEF, a novel RhoA-specific guanine nucleotide exchange factor, is localized in cardiac sarcomere

2002 ◽  
Vol 115 (3) ◽  
pp. 629-640 ◽  
Author(s):  
Michel Souchet ◽  
Elodie Portales-Casamar ◽  
David Mazurais ◽  
Susanne Schmidt ◽  
Isabelle Léger ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Small Gtpases ◽  
Fiber Formation ◽  
Pleckstrin Homology Domain ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Intact Cells ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

The Rho small GTPases are crucial proteins involved in regulation of signal transduction cascades from extracellular stimuli to cell nucleus and cytoskeleton. It has been reported that these GTPases are directly associated with cardiovascular disorders. In this context, we have searched for novel modulators of Rho GTPases, and here we describe p63RhoGEF a new Db1-like guanine nucleotide exchange factor (GEF). P63RhoGEF encodes a 63 kDa protein containing a Db1 homology domain in tandem with a pleckstrin homology domain and is most closely related to the second Rho GEF domain of Trio. Northern blot and in situ analysis have shown that p63RhoGEF is mainly expressed in heart and brain. In vitro guanine nucleotide exchange assays have shown that p63RhoGEF specifically acts on RhoA. Accordingly, p63RhoGEF expression induces RhoA-dependent stress fiber formation in fibroblasts and in H9C2 cardiac myoblasts. Moreover, we show that p63RhoGEF activation of RhoA in intact cells is dependent on the presence of the PH domain. Using a specific anti-p63RhoGEF antibody, we have detected the p63RhoGEF protein by immunocytochemistry in human heart and brain tissue sections. Confocal microscopy shows that p63RhoGEF is located in the sarcomeric I-band mainly constituted of cardiac sarcomeric actin. Together, these results show that p63RhoGEF is a RhoA-specific GEF that may play a key role in actin cytoskeleton reorganization in different tissues, especially in heart cellular morphology.

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