scholarly journals Ras Binding Triggers Ubiquitination of the Ras Exchange Factor Ras-GRF2

2001 ◽  
Vol 21 (6) ◽  
pp. 2107-2117 ◽  
Author(s):  
Carmen L. de Hoog ◽  
Jackie A. Koehler ◽  
Marni D. Goldstein ◽  
Paul Taylor ◽  
Daniel Figeys ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Small Gtpase ◽  
Dominant Negative ◽  
26S Proteasome ◽  
Guanine Nucleotide ◽  
Sequence Motifs ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange

ABSTRACT Ras is a small GTPase that is activated by upstream guanine nucleotide exchange factors, one of which is Ras-GRF2. GRF2 is a widely expressed protein with several recognizable sequence motifs, including a Ras exchanger motif (REM), a PEST region containing a destruction box (DB), and a Cdc25 domain. The Cdc25 domain possesses guanine nucleotide exchange factor activity and interacts with Ras. Herein we examine if the DB motif in GRF2 results in proteolysis via the ubiquitin pathway. Based on the solved structure of the REM and Cdc25 regions of the Son-of-sevenless (Sos) protein, the REM may stabilize the Cdc25 domain during Ras binding. The DB motif of GRF2 is situated between the REM and the Cdc25 domains, tempting speculation that it may be exposed to ubiquitination machinery upon Ras binding. GRF2 protein levels decrease dramatically upon activation of GRF2, and dominant-negative Ras induces degradation of GRF2, demonstrating that signaling downstream of Ras is not required for the destruction of GRF2 and that binding to Ras is important for degradation. GRF2 is ubiquitinated in vivo, and this can be detected using mass spectrometry. In the presence of proteasome inhibitors, Ras-GRF2 accumulates as a high-molecular-weight conjugate, suggesting that GRF2 is destroyed by the 26S proteasome. Deleting the DB reduces the ubiquitination of GRF2. GRF2 lacking the Cdc25 domain is not ubiquitinated, suggesting that a protein that cannot bind Ras cannot be properly targeted for destruction. Point mutations within the Cdc25 domain that eliminate Ras binding also eliminate ubiquitination, demonstrating that binding to Ras is necessary for ubiquitination of GRF2. We conclude that conformational changes induced by GTPase binding expose the DB and thereby target GRF2 for destruction.

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 Divergent Mechanisms Activating RAS and Small GTPases Through Post-translational Modification

2021 ◽  
Vol 8 ◽  
Author(s):  
Natsuki Osaka ◽  
Yoshihisa Hirota ◽  
Doshun Ito ◽  
Yoshiki Ikeda ◽  
Ryo Kamata ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Guanine Nucleotide Exchange ◽  
Ras Superfamily ◽  
Switch Regions

RAS is a founding member of the RAS superfamily of GTPases. These small 21 kDa proteins function as molecular switches to initialize signaling cascades involved in various cellular processes, including gene expression, cell growth, and differentiation. RAS is activated by GTP loading and deactivated upon GTP hydrolysis to GDP. Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) accelerate GTP loading and hydrolysis, respectively. These accessory proteins play a fundamental role in regulating activities of RAS superfamily small GTPase via a conserved guanine binding (G)-domain, which consists of five G motifs. The Switch regions lie within or proximal to the G2 and G3 motifs, and undergo dynamic conformational changes between the GDP-bound “OFF” state and GTP-bound “ON” state. They play an important role in the recognition of regulatory factors (GEFs and GAPs) and effectors. The G4 and G5 motifs are the focus of the present work and lie outside Switch regions. These motifs are responsible for the recognition of the guanine moiety in GTP and GDP, and contain residues that undergo post-translational modifications that underlie new mechanisms of RAS regulation. Post-translational modification within the G4 and G5 motifs activates RAS by populating the GTP-bound “ON” state, either through enhancement of intrinsic guanine nucleotide exchange or impairing GAP-mediated down-regulation. Here, we provide a comprehensive review of post-translational modifications in the RAS G4 and G5 motifs, and describe the role of these modifications in RAS activation as well as potential applications for cancer therapy.

scholarly journals Crystal and cryo-EM structures of the cytosolic G protein alpha chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1

2020 ◽  
Author(s):  
Levi J. McClelland ◽  
Kaiming Zhang ◽  
Tung-Chung Mou ◽  
Jake Johnston ◽  
Cindee Yates-Hansen ◽  
...  
Keyword(s):  
G Protein ◽  
Domain Architecture ◽  
Guanine Nucleotide Exchange Factor ◽  
Chaperone Activity ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα)1. Ric-8A is essential to life in multicellular eukaryotes by virtue of its chaperone activity that is required for Gα biogenesis and membrane localization2, 3. Ric-8A adopts an armadillo (ARM)/HEAT repeat domain architecture and is structurally unrelated to G Protein-Coupled Receptors (GPCR)4. Both GEF and chaperone activities are stimulated by Casein Kinase II phosphorylation5. The mechanisms by which Ric-8A catalyzes GDP release and GTP binding to Gα, or exerts chaperone activity are unknown. Here, we report the structure of the nanobody-stabilized complex of nucleotide-free Gαi1 (isoform 1 of Gα family i) and phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. We find that Ric-8A envelops the GTPase domain of Gα, disrupting all three switch regions that convey Gα nucleotide-binding and signaling activity, and displaces the C-terminal helix and helical domain of Gα. These cooperative interactions dismantle the GDP binding site and promote GDP release, while protecting structural elements of Gα that are dynamic in the nucleotide-free state. The structures also show how in vivo phosphorylation stabilizes Gα-binding elements of Ric-8A, thereby enhancing its GEF and chaperone activities.

scholarly journals Regulation of type V adenylate cyclase by Ric8a, a guanine nucleotide exchange factor

2007 ◽  
Vol 406 (3) ◽  
pp. 383-388 ◽  
Author(s):  
Shyi-Chyi Wang ◽  
Hsing-Lin Lai ◽  
Yi-Ting Chiu ◽  
Ren Ou ◽  
Chuen-Lin Huang ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Guanine Nucleotide Exchange Factor ◽  
Dominant Negative ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
N Terminus ◽  
Type V

In the present study, we demonstrate that AC5 (type V adenylate cyclase) interacts with Ric8a through directly interacting at its N-terminus. Ric8a was shown to be a GEF (guanine nucleotide exchange factor) for several α subunits of heterotrimeric GTP binding proteins (Gα proteins) in vitro. Selective Gα targets of Ric8a have not yet been revealed in vivo. An interaction between AC5 and Ric8a was verified by pull-down assays, co-immunoprecipitation analyses, and co-localization in the brain. Expression of Ric8a selectively suppressed AC5 activity. Treating cells with pertussis toxin or expressing a dominant negative Gαi mutant abolished the suppressive effect of Ric8a, suggesting that interaction between the N-terminus of AC5 and a GEF (Ric8a) provides a novel pathway to fine-tune AC5 activity via a Gαi-mediated pathway.

Sign in / Sign up

Export Citation Format

Share Document