scholarly journals Nuclear Import of the Ran Exchange Factor, Rcc1, Is Mediated by at Least Two Distinct Mechanisms

2000 ◽  
Vol 149 (4) ◽  
pp. 835-850 ◽  
Author(s):  
Michael E. Nemergut ◽  
Ian G. Macara
Keyword(s):  
Nuclear Import ◽  
Nuclear Protein ◽  
Nucleotide Exchange ◽  
Ran Gtpase ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Interphase Cells ◽  
Importin Α

RCC1, the only known guanine-nucleotide exchange factor for the Ran GTPase, is an ∼45-kD nuclear protein that can bind chromatin. An important question concerns how RCC1 traverses the nuclear envelope. We now show that nuclear RCC1 is not exported readily in interphase cells and that the import of RCC1 into the nucleoplasm is extremely rapid. Import can proceed by at least two distinct mechanisms. The first is a classic import pathway mediated by basic residues within the NH2-terminal domain (NTD) of RCC1. This pathway is dependent upon both a preexisting Ran gradient and energy, and preferentially uses the importin-α3 isoform of importin-α. The second pathway is not mediated by the NTD of RCC1. This novel pathway does not require importin-α or importin-β or the addition of any other soluble factor in vitro; however, this pathway is saturable and sensitive only to a subset of inhibitors of classical import pathways. Furthermore, the nuclear import of RCC1 does not require a preexisting Ran gradient or energy. We speculate that this second import pathway evolved to ensure that RCC1 never accumulates in the cytoplasm.

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.

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 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 Crystal structure of a guanine nucleotide exchange factor encoded by the scrub typhus pathogenOrientia tsutsugamushi

2020 ◽  
Vol 117 (48) ◽  
pp. 30380-30390
Author(s):  
Christopher Lim ◽  
Jason M. Berk ◽  
Alyssa Blaise ◽  
Josie Bircher ◽  
Anthony J. Koleske ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Sequence Similarity ◽  
Ectopic Expression ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Rho family GTPases regulate an array of cellular processes and are often modulated by pathogens to promote infection. Here, we identify a cryptic guanine nucleotide exchange factor (GEF) domain in the OtDUB protein encoded by the pathogenic bacteriumOrientia tsutsugamushi. A proteomics-based OtDUB interaction screen identified numerous potential host interactors, including the Rho GTPases Rac1 and Cdc42. We discovered a domain in OtDUB with Rac1/Cdc42 GEF activity (OtDUBGEF), with higher activity toward Rac1 in vitro. While this GEF bears no obvious sequence similarity to known GEFs, crystal structures of OtDUBGEFalone (3.0 Å) and complexed with Rac1 (1.7 Å) reveal striking convergent evolution, with a unique topology, on a V-shaped bacterial GEF fold shared with other bacterial GEF domains. Structure-guided mutational analyses identified residues critical for activity and a mechanism for nucleotide displacement. Ectopic expression of OtDUB activates Rac1 preferentially in cells, and expression of the OtDUBGEFalone alters cell morphology. Cumulatively, this work reveals a bacterial GEF within the multifunctional OtDUB that co-opts host Rac1 signaling to induce changes in cytoskeletal structure.

scholarly journals The nucleoporin Nup50 activates the Ran guanyl-nucleotide exchange factor RCC1 to promote mitotic NPC assembly

2021 ◽  
Author(s):  
Guillaume Holzer ◽  
Paola De Magistris ◽  
Cathrin Gramminger ◽  
Ruchika Sachdev ◽  
Adriana Magalska ◽  
...  
Keyword(s):  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor ◽  
Egg Extracts ◽  
Importin Α ◽  
Pore Complexes ◽  
Xenopus Egg Extracts

During mitotic exit, thousands of nuclear pore complexes (NPCs) assemble concomitant with the nuclear envelope to build a transport-competent nucleus. We show here that Nup50 plays a crucial role in NPC assembly that is independent of its well-established function in nuclear transport. RNAi-mediated downregulation in cells or immunodepletion of the protein in Xenopus egg extracts interferes with NPC assembly. We define a conserved central region of 46 residues in Nup50 that is crucial for Nup153 and MEL28/ELYS binding, and NPC interaction. Surprisingly, neither NPC interaction nor binding of Nup50 to importin α, β, the GTPase Ran or chromatin is crucial for its function in the assembly process. Instead, we discovered that an N-terminal fragment of Nup50 can stimulate the Ran guanine exchange factor RCC1 and NPC assembly, indicating that Nup50 acts via the Ran system in mitotic NPC reformation. In support of this conclusion, Nup50 mutants defective in RCC1 binding and stimulation cannot replace the wild type protein in in vitro NPC assembly assays.

scholarly journals Lck regulates Vav activation of members of the Rho family of GTPases.

1997 ◽  
Vol 17 (3) ◽  
pp. 1346-1353 ◽  
Author(s):  
J Han ◽  
B Das ◽  
W Wei ◽  
L Van Aelst ◽  
R D Mosteller ◽  
...  
Keyword(s):  
Animal Cell ◽  
Guanine Nucleotide Exchange Factor ◽  
Biological Data ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Rho Family

Vav is a member of a family of oncogene proteins that share an approximately 250-amino-acid motif called a Dbl homology domain. Paradoxically, Dbl itself and other proteins containing a Dbl domain catalyze GTP-GDP exchange for Rho family proteins, whereas Vav has been reported to catalyze GTP-GDP exchange for Ras proteins. We present Saccharomyces cerevisiae genetic data, in vitro biochemical data, and animal cell biological data indicating that Vav is a guanine nucleotide exchange factor for Rho-related proteins, but in similar genetic and biochemical experiments we fail to find evidence that Vav is a guanine nucleotide exchange factor for Ras. Further, we present data indicating that the Lck kinase activates the guanine nucleotide exchange factor and transforming activity of Vav.

scholarly journals Crystal structure of a novel guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

2020 ◽  
Author(s):  
Christopher Lim ◽  
Jason M. Berk ◽  
Alyssa Blaise ◽  
Josie Bircher ◽  
Anthony J. Koleske ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Ectopic Expression ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Orientia Tsutsugamushi ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

AbstractRho family GTPases regulate an array of cellular processes and are often modulated by pathogens to promote infection. Here, we identify a cryptic guanine nucleotide exchange factor (GEF) domain in the OtDUB protein encoded by the pathogenic bacterium Orientia tsutsugamushi. A proteomics-based OtDUB interaction screen identified numerous potential host interactors, including the Rho-GTPases Rac1 and Cdc42. We discovered a new domain in OtDUB with Rac1/Cdc42 GEF activity (OtDUBGEF), with higher activity toward Rac1 in vitro. While this GEF bears no obvious sequence similarity to known GEFs, crystal structures of OtDUBGEF alone (3.0 Å) and complexed with Rac1 (1.7 Å) reveal striking convergent evolution, with a distinct topology, on a V-shaped bacterial GEF fold shared with other bacterial GEF domains. Structure-guided mutational analyses identified residues critical for activity and a novel mechanism for nucleotide displacement. Ectopic expression of OtDUB activates Rac1 preferentially in cells, and expression of the OtDUBGEF alone alters cell morphology. Cumulatively, this work reveals a novel bacterial GEF within the multifunctional OtDUB that co-opts host Rac1 signaling to evoke changes in cytoskeletal structure.

scholarly journals In Vivo Analysis of Importin α Proteins Reveals Cellular Proliferation Inhibition and Substrate Specificity

2004 ◽  
Vol 24 (23) ◽  
pp. 10246-10255 ◽  
Author(s):  
Christina Quensel ◽  
Beate Friedrich ◽  
Thomas Sommer ◽  
Enno Hartmann ◽  
Matthias Kohler
Keyword(s):  
Nuclear Import ◽  
Cellular Proliferation ◽  
Minor Effect ◽  
Nucleotide Exchange ◽  
Down Regulation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Importin Α ◽  
A Minor

ABSTRACT The “classical” nuclear import pathway depends on importins α and β. Humans have only one importin β, while six α importins have been described. Whether or not distinct α importins are essential for specific import pathways in living human cells is unclear. We used RNA interference technology to specifically down-regulate the expression of ubiquitously expressed human α importins in HeLa cells. Down-regulation of importins α3, α5, α7, and β strongly inhibited HeLa cell proliferation, while down-regulation of importins α1 and α4 had only a minor effect or no effect. Nucleoplasmin import was not prevented by down-regulation of any α importin, indicating that the importin α/β pathway was generally not affected. In contrast, importin α3 or α5 down-regulation specifically inhibited the nuclear import of the Ran guanine nucleotide exchange factor, RCC1. Coinjection of recombinant α importins and RCC1 into down-regulated cells demonstrated that these transport defects were specifically caused by the limited availability of importin α3 in both cases. Thus, importin α3 is the only α importin responsible for the classical nuclear import of RCC1 in living cells.

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