scholarly journals Identification and characterization of Ral-binding protein 1, a potential downstream target of Ral GTPases.

1995 ◽  
Vol 15 (8) ◽  
pp. 4578-4584 ◽  
Author(s):  
S B Cantor ◽  
T Urano ◽  
L A Feig
Keyword(s):  
Mammalian Cells ◽  
Rho Gtpase ◽  
Binding Protein ◽  
Protein Domain ◽  
Nucleotide Exchange ◽  
Gtpase Activating Protein ◽  
Downstream Target ◽  
Nucleotide Exchange Factor ◽  
Ral Gtpases ◽  
Bud Site

Ral proteins constitute a distinct family of Ras-related GTPases. Although similar to Ras in amino acid sequence, Ral proteins are activated by a unique nucleotide exchange factor and inactivated by a distinct GTPase-activating protein. Unlike Ras, they fail to promote transformed foci when activated versions are expressed in cells. To identify downstream targets that might mediate a Ral-specific function, we used a Saccharomyces cerevisiae-based interaction assay to clone a novel cDNA that encodes a Ral-binding protein (RalBP1). RalBP1 binds specifically to the active GTP-bound form of RalA and not to a mutant Ral with a point mutation in its putative effector domain. In addition to a Ral-binding domain, RalBP1 also contains a Rho-GTPase-activating protein domain that interacts preferentially with Rho family member CDC42. Since CDC42 has been implicated in bud site selection in S. cerevisiae and filopodium formation in mammalian cells, Ral may function to modulate the actin cytoskeleton through its interactions with RalBP1.

scholarly journals GM130-dependent Control of Cdc42 Activity at the Golgi Regulates Centrosome Organization

2009 ◽  
Vol 20 (4) ◽  
pp. 1192-1200 ◽  
Author(s):  
Andrew Kodani ◽  
Irene Kristensen ◽  
Lan Huang ◽  
Christine Sütterlin
Keyword(s):  
Golgi Apparatus ◽  
Mammalian Cells ◽  
Rho Gtpase ◽  
Nucleotide Exchange ◽  
Interacting Protein ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Golgi Protein ◽  
And Function

The physical proximity of the Golgi apparatus and the centrosome is a unique feature of mammalian cells whose functional significance is only poorly understood. Here, we demonstrate that the previously described regulation of centrosome organization and function by the Golgi protein, GM130, involves a Golgi-associated complex consisting of GM130, the Rho GTPase, Cdc42, and its guanine nucleotide exchange factor, Tuba. We identified Tuba as a novel GM130-interacting protein and showed that this association controls Tuba-mediated activation of Cdc42 at the Golgi apparatus. Blocking either Tuba or Cdc42 activity reproduced the GM130 depletion phenotype of aberrant, nonfunctional centrosomes. Expression of constitutively active Cdc42 bypassed the requirement for GM130 in centrosome regulation, indicating that Cdc42 functions downstream of GM130. Our studies demonstrate that Cdc42 has a novel role in controlling centrosome organization in unstimulated cells in addition to its known function as a regulator of centrosome reorientation in stimulated cells. This first description of a regulatory pathway between the Golgi apparatus and the interphase centrosome that complements the known role of Golgi proteins in controlling spindle formation during mitosis and may provide an explanation for the pericentriolar position of the mammalian Golgi apparatus during interphase.

scholarly journals The Identification of cDNAs That Affect the Mitosis-to-Interphase Transition in Schizosaccharomyces pombe, Including sbp1, Which Encodes a spi1p-GTP–Binding Protein

Genetics ◽  
1998 ◽  
Vol 148 (2) ◽  
pp. 645-656
Author(s):  
Xiangwei He ◽  
Naoyuki Hayashi ◽  
Nathan G Walcott ◽  
Yoshiaki Azuma ◽  
Thomas E Patterson ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Genetic Interaction ◽  
Binding Protein ◽  
Medial Septum ◽  
Regulatory Proteins ◽  
Nucleotide Exchange ◽  
Gtpase Activating Protein ◽  
Guanine Nucleotide Exchange ◽  
System A ◽  
Nucleotide Exchange Factor

Abstract Perturbations of the spi1p GTPase system in fission yeast, caused by mutation or overexpression of several regulatory proteins, result in a unique terminal phenotype that includes condensed chromosomes, a wide medial septum, and a fragmented nuclear envelope. To identify potential regulators or targets of the spi1p GTPase system, a screen for cDNAs whose overexpression results in this terminal phenotype was conducted, and seven clones that represent three genes, named med1, med2, and med3 (mitotic exit defect), were identified. Their genetic interaction with the spi1p GTPase system was established by showing that the spi1p guanine nucleotide exchange factor mutant pim1-d1ts was hypersensitive to their overexpression. med1 encodes a homologue of the human Ran-binding protein, RanBP1, and has been renamed sbp1 (spi1-binding protein). sbp1p binds to spi1p-GTP and costimulates the GTPase-activating protein (GAP)-catalyzed GTPase activity. Cells in which sbp1p is depleted or overproduced phenocopy cells in which the balance between spi1p-GTP and spi1p-GDP is perturbed by other means. Therefore, sbp1p mediates and/or regulates the essential functions of the spi1p GTPase system. med2 and med3 encode novel fission yeast proteins that, based on our phenotypic analyses, are likely to identify additional regulators or effectors of the spi1p GTPase system.

scholarly journals XMog1, a nuclear Ran-binding protein in Xenopus, is a functional homologue of Schizosaccharomyces pombe Mog1p that co-operates with RanBP1 to control generation of Ran-GTP

2001 ◽  
Vol 114 (16) ◽  
pp. 3013-3023 ◽  
Author(s):  
Francisco J. Nicolás ◽  
William J. Moore ◽  
Chuanmao Zhang ◽  
Paul R. Clarke
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Nucleocytoplasmic Transport ◽  
Small Gtpase ◽  
Growth Defect ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Ran Gtp ◽  
Nuclear Envelope Formation

Ran is a multifunctional small GTPase of the Ras superfamily that plays roles in nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. By screening a Xenopus oocyte cDNA library for Ran-GTP-binding proteins using the two-hybrid system of co-expression in yeast, we identified XMog1, a 20.4 kDa polypeptide related to Mog1p in Saccharomyces cerevisiae and similar gene products in Schizosaccharomyces pombe, Arabidopsis and mammals. We show that cDNAs encoding XMog1 and S. cerevisiae Mog1p rescue the growth defect of S. pombe cells lacking mog1, demonstrating conservation of their functions. In Xenopus somatic cells and transfected mammalian cells, XMog1 is localised to the nucleus. XMog1 alone does not stimulate Ran GTPase activity or nucleotide exchange, but causes nucleotide release from Ran-GTP and forms a complex with nucleotide-free Ran. However, in combination with Ran-binding protein 1 (RanBP1), XMog1 promotes the release of GDP and the selective binding of GTP to Ran. XMog1 and RanBP1 also promote selective GTP loading onto Ran catalysed by the nuclear guanine nucleotide exchange factor, RCC1. We propose that Mog1-related proteins, together with RanBP1, facilitate the generation of Ran-GTP from Ran-GDP in the nucleus.

scholarly journals Activation of Cdc42 GTPase upon CRY2-Induced Cortical Recruitment Is Antagonized by GAPs in Fission Yeast

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2089 ◽  
Author(s):  
Iker Lamas ◽  
Nathalie Weber ◽  
Sophie G. Martin
Keyword(s):  
Fission Yeast ◽  
Positive Feedback ◽  
Antagonistic Activity ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Gtpase Activating Protein ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Cell Architecture

The small GTPase Cdc42 is critical for cell polarization in eukaryotic cells. In rod-shaped fission yeast Schizosaccharomyces pombe cells, active GTP-bound Cdc42 promotes polarized growth at cell poles, while inactive Cdc42-GDP localizes ubiquitously also along cell sides. Zones of Cdc42 activity are maintained by positive feedback amplification involving the formation of a complex between Cdc42-GTP, the scaffold Scd2, and the guanine nucleotide exchange factor (GEF) Scd1, which promotes the activation of more Cdc42. Here, we use the CRY2-CIB1 optogenetic system to recruit and cluster a cytosolic Cdc42 variant at the plasma membrane and show that this leads to its moderate activation also on cell sides. Surprisingly, Scd2, which binds Cdc42-GTP, is still recruited to CRY2-Cdc42 clusters at cell sides in individual deletion of the GEFs Scd1 or Gef1. We show that activated Cdc42 clusters at cell sides are able to recruit Scd1, dependent on the scaffold Scd2. However, Cdc42 activity is not amplified by positive feedback and does not lead to morphogenetic changes, due to antagonistic activity of the GTPase activating protein Rga4. Thus, the cell architecture is robust to moderate activation of Cdc42 at cell sides.

scholarly journals The BNIP-2 and Cdc42GAP Homology (BCH) Domain of p50RhoGAP/Cdc42GAP Sequesters RhoA from Inactivation by the Adjacent GTPase-activating Protein Domain

2010 ◽  
Vol 21 (18) ◽  
pp. 3232-3246 ◽  
Author(s):  
Yi Ting Zhou ◽  
Li Li Chew ◽  
Sheng-cai Lin ◽  
Boon Chuan Low
Keyword(s):  
Rho Gtpases ◽  
Rho Gtpase ◽  
Intramolecular Interaction ◽  
Complete Loss ◽  
Protein Domain ◽  
Binding Motif ◽  
Gtpase Activating Protein ◽  
New Paradigm ◽  
Cell Rounding ◽  
In Cis

The BNIP-2 and Cdc42GAP homology (BCH) domain is a novel regulator for Rho GTPases, but its impact on p50-Rho GTPase-activating protein (p50RhoGAP or Cdc42GAP) in cells remains elusive. Here we show that deletion of the BCH domain from p50RhoGAP enhanced its GAP activity and caused drastic cell rounding. Introducing constitutively active RhoA or inactivating GAP domain blocked such effect, whereas replacing the BCH domain with endosome-targeting SNX3 excluded requirement of endosomal localization in regulating the GAP activity. Substitution with homologous BCH domain from Schizosaccharomyces pombe, which does not bind mammalian RhoA, also led to complete loss of suppression. Interestingly, the p50RhoGAP BCH domain only targeted RhoA, but not Cdc42 or Rac1, and it was unable to distinguish between GDP and the GTP-bound form of RhoA. Further mutagenesis revealed a RhoA-binding motif (residues 85-120), which when deleted, significantly reduced BCH inhibition on GAP-mediated cell rounding, whereas its full suppression also required an intramolecular interaction motif (residues 169-197). Therefore, BCH domain serves as a local modulator in cis to sequester RhoA from inactivation by the adjacent GAP domain, adding to a new paradigm for regulating p50RhoGAP signaling.

scholarly journals ErbB2 directly activates the exchange factor Dock7 to promote Schwann cell migration

2008 ◽  
Vol 181 (2) ◽  
pp. 351-365 ◽  
Author(s):  
Junji Yamauchi ◽  
Yuki Miyamoto ◽  
Jonah R. Chan ◽  
Akito Tanoue
Keyword(s):  
Cell Migration ◽  
Schwann Cell ◽  
Rho Gtpase ◽  
Morphological Changes ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Schwann Cell Migration ◽  
Subsequent Activation

The cellular events that precede myelination in the peripheral nervous system require rapid and dynamic morphological changes in the Schwann cell. These events are thought to be mainly controlled by axonal signals. But how signals on the axons are coordinately organized and transduced to promote proliferation, migration, radial sorting, and myelination is unknown. We describe that the axonal signal neuregulin-1 (NRG1) controls Schwann cell migration via activation of the atypical Dock180-related guanine nucleotide exchange factor (GEF) Dock7 and subsequent activation of the Rho guanine triphosphatases (GTPases) Rac1 and Cdc42 and the downstream c-Jun N-terminal kinase. We show that the NRG1 receptor ErbB2 directly binds and activates Dock7 by phosphorylating Tyr-1118. Dock7 knockdown, or expression of Dock7 harboring the Tyr-1118–to–Phe mutation in Schwann cells, attenuates the effects of NRG1. Thus, Dock7 functions as an intracellular substrate for ErbB2 to promote Schwann cell migration. This provides an unanticipated mechanism through which ligand-dependent tyrosine phosphorylation can trigger the activation of Rho GTPase-GEFs of the Dock180 family.

scholarly journals Structure of the C-terminal guanine nucleotide exchange factor module of Trio in an autoinhibited conformation reveals its oncogenic potential

2019 ◽  
Vol 12 (569) ◽  
pp. eaav2449 ◽  
Author(s):  
Sumit J. Bandekar ◽  
Nadia Arang ◽  
Ena S. Tully ◽  
Brittany A. Tang ◽  
Brenna L. Barton ◽  
...  
Keyword(s):  
Rho Gtpase ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Independent Manner ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Factor Module

The C-terminal guanine nucleotide exchange factor (GEF) module of Trio (TrioC) transfers signals from the Gαq/11subfamily of heterotrimeric G proteins to the small guanosine triphosphatase (GTPase) RhoA, enabling Gαq/11-coupled G protein–coupled receptors (GPCRs) to control downstream events, such as cell motility and gene transcription. This conserved signal transduction axis is crucial for tumor growth in uveal melanoma. Previous studies indicate that the GEF activity of the TrioC module is autoinhibited, with release of autoinhibition upon Gαq/11binding. Here, we determined the crystal structure of TrioC in its basal state and found that the pleckstrin homology (PH) domain interacts with the Dbl homology (DH) domain in a manner that occludes the Rho GTPase binding site, thereby suggesting the molecular basis of TrioC autoinhibition. Biochemical and biophysical assays revealed that disruption of the autoinhibited conformation destabilized and activated the TrioC module in vitro. Last, mutations in the DH-PH interface found in patients with cancer activated TrioC and, in the context of full-length Trio, led to increased abundance of guanosine triphosphate–bound RhoA (RhoA·GTP) in human cells. These mutations increase mitogenic signaling through the RhoA axis and, therefore, may represent cancer drivers operating in a Gαq/11-independent manner.

scholarly journals Novel Functions of Ect2 in Polar Lamellipodia Formation and Polarity Maintenance during “Contractile Ring-Independent” Cytokinesis in Adherent Cells

2008 ◽  
Vol 19 (1) ◽  
pp. 8-16 ◽  
Author(s):  
Masamitsu Kanada ◽  
Akira Nagasaki ◽  
Taro Q.P. Uyeda
Keyword(s):  
Mammalian Cells ◽  
Polar Regions ◽  
Nucleotide Exchange ◽  
Contractile Ring ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Ht1080 Cells ◽  
Lamellipodia Formation

Some mammalian cells are able to divide via both the classic contractile ring-dependent method (cytokinesis A) and a contractile ring-independent, adhesion-dependent method (cytokinesis B). Cytokinesis A is triggered by RhoA, which, in HeLa cells, is activated by the guanine nucleotide-exchange factor Ect2 localized at the central spindle and equatorial cortex. Here, we show that in HT1080 cells undergoing cytokinesis A, Ect2 does not localize in the equatorial cortex, though RhoA accumulates there. Moreover, Ect2 depletion resulted in only modest multinucleation of HT1080 cells, enabling us to establish cell lines in which Ect2 was constitutively depleted. Thus, RhoA is activated via an Ect2-independent pathway during cytokinesis A in HT1080 cells. During cytokinesis B, Ect2-depleted cells showed narrower accumulation of RhoA at the equatorial cortex, accompanied by compromised pole-to-equator polarity, formation of ectopic lamellipodia in regions where RhoA normally would be distributed, and delayed formation of polar lamellipodia. Furthermore, C3 exoenzyme inhibited equatorial RhoA activation and polar lamellipodia formation. Conversely, expression of dominant active Ect2 in interphase HT1080 cells enhanced RhoA activity and suppressed lamellipodia formation. These results suggest that equatorial Ect2 locally suppresses lamellipodia formation via RhoA activation, which indirectly contributes to restricting lamellipodia formation to polar regions during cytokinesis B.

scholarly journals Identification and characterization of EBP, a novel EEN binding protein that inhibits Ras signaling and is recruited into the nucleus by the MLL-EEN fusion protein

Blood ◽  
2004 ◽  
Vol 103 (4) ◽  
pp. 1445-1453 ◽  
Author(s):  
Judy Wai Ping Yam ◽  
Dong-Yan Jin ◽  
Chi Wai So ◽  
Li Chong Chan
Keyword(s):  
Fusion Protein ◽  
Binding Protein ◽  
Chromosomal Translocation ◽  
Direct Interaction ◽  
Cellular Transformation ◽  
Sh3 Domain ◽  
Ras Signaling ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Src Homology

Abstract The chimeric MLL-EEN fusion protein is created as a result of chromosomal translocation t(11;19)(q23;p13). EEN, an Src homology 3 (SH3) domain–containing protein in the endophilin family, has been implicated in endocytosis, although little is known about its role in leukemogenesis mediated by the MLL-EEN fusion protein. In this study, we have identified and characterized EBP, a novel EEN binding protein that interacts with the SH3 domain of EEN through a proline-rich motif PPERP. EBP is a ubiquitous protein that is normally expressed in the cytoplasm but is recruited to the nucleus by MLL-EEN with a punctate localization pattern characteristic of the MLL chimeric proteins. EBP interacts simultaneously with EEN and Sos, a guanine-nucleotide exchange factor for Ras. Coexpressoin of EBP with EEN leads to suppression of Ras-induced cellular transformation and Ras-mediated activation of Elk-1. Taken together, our findings suggest a new mechanism for MLL-EEN–mediated leukemogenesis in which MLL-EEN interferes with the Ras-suppressing activities of EBP through direct interaction.

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