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‣ RGS4 and GAIP are GTPase-activating proteins for Gqα and block activation of phospholipase Cβ by γ-thio-GTP-Gqα

Hepler, John R.; Berman, David M.; Gilman, Alfred G.; Kozasa, Tohru
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 21/01/1997 Português
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RGS proteins constitute a newly appreciated and large group of negative regulators of G protein signaling. Four members of the RGS family act as GTPase-activating proteins (GAPs) with apparent specificity for members of the Giα subfamily of G protein subunits. We demonstrate here that two RGS proteins, RGS4 and GAIP, also act as GAPs for Gqα, the Gα protein responsible for activation of phospholipase Cβ. Furthermore, these RGS proteins block activation of phospholipase Cβ by guanosine 5′-(3-O-thio)triphosphate-Gqα. GAP activity does not explain this effect, which apparently results from occlusion of the binding site on Gα for effector. Inhibitory effects of RGS proteins on G protein-mediated signaling pathways can be demonstrated by simple mixture of RGS4 or GAIP with plasma membranes.

‣ Arabidopsis RopGAPs Are a Novel Family of Rho GTPase-Activating Proteins that Require the Cdc42/Rac-Interactive Binding Motif for Rop-Specific GTPase Stimulation1

Wu, Guang; Li, Hai; Yang, Zhenbiao
Fonte: American Society of Plant Physiologists Publicador: American Society of Plant Physiologists
Tipo: Artigo de Revista Científica
Publicado em /12/2000 Português
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The plant-specific Rop subfamily of Rho GTPases, most closely related to the mammalian Cdc42 and Rac GTPases, plays an important role in the regulation of calcium-dependent pollen tube growth, H2O2-mediated cell death, and many other processes in plants. In a search for Rop interactors using the two-hybrid method, we identified a family of Rho GTPase-activating proteins (GAP) from Arabidopsis, termed RopGAPs. In addition to a GAP catalytic domain, RopGAPs contain a Cdc42/Rac-interactive binding (CRIB) motif known to allow Cdc42/Rac effector proteins to bind activated Cdc42/Rac. This novel combination of a GAP domain with a CRIB motif is widespread in higher plants and is unique to the regulation of the Rop GTPase. A critical role for CRIB in the regulation of in vitro RopGAP activity was demonstrated using point and deletion mutations. Both types of mutants have drastically reduced capacities to stimulate the intrinsic Rop GTPase activity and to bind Rop. Furthermore, RopGAPs preferentially stimulate the GTPase activity of Rop, but not Cdc42 in a CRIB-dependent manner. In vitro binding assays show that the RopGAP CRIB domain interacts with GTP- and GDP-bound forms of Rop, as well as the transitional state of Rop mimicked by aluminum fluoride. The CRIB domain also promotes the association of the GAP domain with the GDP-bound Rop...

‣ Activity of Specific Lipid-regulated ADP Ribosylation Factor-GTPase–activating Proteins Is Required for Sec14p-dependent Golgi Secretory Function in Yeast

Yanagisawa, Lora L.; Marchena, Jennifer; Xie, Zhigang; Li, Xinmin; Poon, Pak P.; Singer, Richard A.; Johnston, Gerald C.; Randazzo, Paul A.; Bankaitis, Vytas A.
Fonte: The American Society for Cell Biology Publicador: The American Society for Cell Biology
Tipo: Artigo de Revista Científica
Publicado em /07/2002 Português
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Yeast phosphatidylinositol transfer protein (Sec14p) coordinates lipid metabolism with protein-trafficking events. This essential Sec14p requirement for Golgi function is bypassed by mutations in any one of seven genes that control phosphatidylcholine or phosphoinositide metabolism. In addition to these “bypass Sec14p” mutations, Sec14p-independent Golgi function requires phospholipase D activity. The identities of lipids that mediate Sec14p-dependent Golgi function, and the identity of the proteins that respond to Sec14p-mediated regulation of lipid metabolism, remain elusive. We now report genetic evidence to suggest that two ADP ribosylation factor-GTPase–activating proteins (ARFGAPs), Gcs1p and Age2p, may represent these lipid-responsive elements, and that Gcs1p/Age2p act downstream of Sec14p and phospholipase D in both Sec14p-dependent and Sec14p-independent pathways for yeast Golgi function. In support, biochemical data indicate that Gcs1p and Age2p ARFGAP activities are both modulated by lipids implicated in regulation of Sec14p pathway function. These results suggest ARFGAPs are stimulatory factors required for regulation of Golgi function by the Sec14p pathway, and that Sec14p-mediated regulation of lipid metabolism interfaces with the activity of proteins involved in control of the ARF cycle.

‣ GTPase-Activating Proteins for Cdc42

Smith, Gregory R.; Givan, Scott A.; Cullen, Paul; Sprague Jr., George F.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /06/2002 Português
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68.805%
The Rho-type GTPase, Cdc42, has been implicated in a variety of functions in the yeast life cycle, including septin organization for cytokinesis, pheromone response, and haploid invasive growth. A group of proteins called GTPase-activating proteins (GAPs) catalyze the hydrolysis of GTP to GDP, thereby inactivating Cdc42. At the time this study began, there was one known GAP, Bem3, and one putative GAP, Rga1, for Cdc42. We identified another putative GAP for Cdc42 and named it Rga2 (Rho GTPase-activating protein 2). We confirmed by genetic and biochemical criteria that Rga1, Rga2, and Bem3 act as GAPs for Cdc42. A detailed characterization of Rga1, Rga2, and Bem3 suggested that they regulate different subsets of Cdc42 function. In particular, deletion of the individual GAPs conferred different phenotypes. For example, deletion of RGA1, but not RGA2 or BEM3, caused hyperinvasive growth. Furthermore, overproduction or loss of Rga1 and Rga2, but not Bem3, affected the two-hybrid interaction of Cdc42 with Ste20, a p21-activated kinase (PAK) kinase required for haploid invasive growth. These results suggest Rga1, and possibly Rga2, facilitate the interaction of Cdc42 with Ste20 to mediate signaling in the haploid invasive growth pathway. Deletion of BEM3 resulted in cells with severe morphological defects not observed in rga1Δ or rga2Δ strains. These data suggest that Bem3 and...

‣ GGAPs, a New Family of Bifunctional GTP-Binding and GTPase-Activating Proteins

Xia, Chunzhi; Ma, Wenbin; Stafford, Lewis Joe; Liu, Chengyu; Gong, Liming; Martin, James F.; Liu, Mingyao
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2003 Português
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G proteins are molecular switches that control a wide variety of physiological functions, including neurotransmission, transcriptional activation, cell migration, cell growth. and proliferation. The ability of GTPases to participate in signaling events is determined by the ratio of GTP-bound to GDP-bound forms in the cell. All known GTPases exist in an inactive (GDP-bound) and an active (GTP-bound) conformation, which are catalyzed by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs), respectively. In this study, we identified and characterized a new family of bifunctional GTP-binding and GTPase-activating proteins, named GGAP. GGAPs contain an N-terminal Ras homology domain, called the G domain, followed by a pleckstrin homology (PH) domain, a C-terminal GAP domain, and a tandem ankyrin (ANK) repeat domain. Expression analysis indicates that this new family of proteins has distinct cell localization, tissue distribution, and even message sizes. GTPase assays demonstrate that GGAPs have high GTPase activity through direct intramolecular interaction of the N-terminal G domain and the C-terminal GAP domain. In the absence of the GAP domain, the N-terminal G domain has very low activity, suggesting a new model of GGAP protein regulation via intramolecular interaction like the multidomain protein kinases. Overexpression of GGAPs leads to changes in cell morphology and activation of gene transcription.

‣ The Role of Cdc42p GTPase-activating Proteins in Assembly of the Septin Ring in Yeast

Caviston, Juliane P.; Longtine, Mark; Pringle, John R.; Bi, Erfei
Fonte: The American Society for Cell Biology Publicador: The American Society for Cell Biology
Tipo: Artigo de Revista Científica
Publicado em /10/2003 Português
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The septins are a conserved family of GTP-binding, filament-forming proteins. In the yeast Saccharomyces cerevisiae, the septins form a ring at the mother-bud neck that appears to function primarily by serving as a scaffold for the recruitment of other proteins to the neck, where they participate in cytokinesis and a variety of other processes. Formation of the septin ring depends on the Rho-type GTPase Cdc42p but appears to be independent of the actin cytoskeleton. In this study, we investigated further the mechanisms of septin-ring formation. Fluorescence-recovery-after-photobleaching (FRAP) experiments indicated that the initial septin structure at the presumptive bud site is labile (exchanges subunits freely) but that it is converted into a stable ring as the bud emerges. Mutants carrying the cdc42V36G allele or lacking two or all three of the known Cdc42p GTPase-activating proteins (GAPs: Bem3p, Rga1p, and Rga2p) could recruit the septins to the cell cortex but were blocked or delayed in forming a normal septin ring and had accompanying morphogenetic defects. These phenotypes were dramatically enhanced in mutants that were also defective in Cla4p or Gin4p, two protein kinases previously shown to be important for normal septin-ring formation. The Cdc42p GAPs colocalized with the septins both early and late in the cell cycle...

‣ Inhibition by phospholipids of the interaction between R-ras, rho, and their GTPase-activating proteins.

Tsai, M H; Hall, A; Stacey, D W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1989 Português
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Certain lipids were found to inhibit the interaction between rho and R-ras proteins and their respective GTPase-activating proteins (GAP). Inhibitory lipids were similar for each protein but differed significantly from those previously found to inhibit the interaction between ras protein and GAP activity. These data raise the possibility that ras and related proteins are controlled biologically by interactions between lipids and GAP molecules.

‣ Characterization of membrane-localized and cytosolic Rac-GTPase-activating proteins in human neutrophil granulocytes: contribution to the regulation of NADPH oxidase.

Geiszt, M; Dagher, M C; Molnár, G; Havasi, A; Faure, J; Paclet, M H; Morel, F; Ligeti, E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/05/2001 Português
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We have investigated the intracellular localization and molecular identity of Rac-GTPase-activating proteins (Rac-GAPs) in human neutrophils. Immunoblot analysis detected the presence of both p190RhoGAP and Bcr mainly in the cytosol. An overlay assay performed with [gamma-(32)P]GTP-bound Rac revealed dominant GAP activity related to a 50 kDa protein both in the membrane and cytosol. This activity could be identified by Western blotting and immunoprecipitation with specific antibody directed against the GAP domain of p50RhoGAP. Using a semirecombinant or fully purified cell-free activation assay of the Rac-activated enzyme NADPH oxidase, we demonstrated the regulatory effect of both the membrane-localized and soluble GAPs. We suggest that in neutrophil granulocytes Rac-GAPs have redundant function and represent suitable targets for both the up-regulation and down-regulation of the NADPH oxidase.

‣ GAP1 Family Members Constitute Bifunctional Ras and Rap GTPase-activating Proteins*

Kupzig, Sabine; Deaconescu, Delia; Bouyoucef, Dalila; Walker, Simon A.; Liu, Qing; Polte, Christian L.; Daumke, Oliver; Ishizaki, Toshimasa; Lockyer, Peter J.; Wittinghofer, Alfred; Cullen, Peter J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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GAP1IP4BP is a member of the GAP1 family of Ras GTPase-activating proteins (Ras GAPs) that includes GAP1m, CAPRI, and RASAL. Composed of a central Ras GAP domain, surrounded by amino-terminal C2 domains and a carboxyl-terminal pleckstrin homology/Bruton’s tyrosine kinase domain, GAP1IP4BP has previously been shown to possess an unexpected GAP activity on the Ras-related protein Rap, besides the predicted Ras GAP activity (Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P., and Irvine, R. F. (1995) Nature 376, 527–530). Here we have shown that GAP1IP4BP is indeed an efficient Ras/Rap GAP, having Kms of 213 and 42 μM and estimated kcats of 48 and 16 s−1 for Ras and Rap, respectively. For this dual activity, regions outside the Ras GAP domain are required, as the isolated domain (residues 291–569) retains a pronounced Ras GAP activity yet has very low activity toward Rap. Interestingly, mutagenesis of the Ras GAP argi-nine finger, and surrounding residues important in Ras binding, inhibit both Ras and Rap GAP activity of GAP1IP4BP. Although the precise details by which GAP1IP4BP can function as a Rap GAP remain to be determined, these data are consistent with Rap associating with GAP1IP4BP through the Ras-binding site within the Ras GAP domain. Finally...

‣ The Rho GTPase-activating proteins RGA-3 and RGA-4 are required to set the initial size of PAR domains in Caenorhabditis elegans one-cell embryos

Schonegg, Stephanie; Constantinescu, Alexandru T.; Hoege, Carsten; Hyman, Anthony A.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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Caenorhabditis elegans embryos establish cortical domains of PAR proteins of reproducible size before asymmetric cell division. The ways in which the size of these domains is set remain unknown. Here we identify the GTPase-activating proteins (GAPs) RGA-3 and RGA-4, which regulate the activity of the small GTPase RHO-1. rga-3/4(RNAi) embryos have a hypercontractile cortex, and the initial relative size of their anterior and posterior PAR domains is altered. Thus, RHO-1 activity appears to control the level of cortical contractility and concomitantly the size of cortical domains. These data support the idea that in C. elegans embryos the initial size of the PAR domains is set by regulating the contractile activity of the acto-myosin cytoskeleton through the activity of RHO-1. RGA-3/4 have functions different from CYK-4, the other known GAP required for the first cell division, showing that different GAPs cooperate to control the activity of the acto-myosin cytoskeleton in the first cell division of C. elegans embryos.

‣ ROLE OF CHIMAERINS, A GROUP OF Rac-SPECIFIC GTPase ACTIVATING PROTEINS, IN T-CELL RECEPTOR SIGNALING

Caloca, María José; Delgado, Pilar; Alarcón, Balbino; Bustelo, Xosé R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Chimaerins are GTPase-activating proteins that inactivate the GTP-hydrolase Rac1 in a diacylglycerol-dependent manner. To date, the study of chimaerins has been done mostly in neuronal cells. Here, we show that α2- and β2-chimaerin are expressed at different levels in T-cells and that they participate in T-cell receptor signaling. In agreement with this, we have observed that α2- and β2-chimaerins translocate to the T-cell/B-cell immune synapse and, using both gain- and loss-of-function approaches, demonstrated that their catalytic activity is important for the inhibition of the T-cell receptor- and Vav1-dependent stimulation of the transcriptional factor NF-AT. Mutagenesis-based approaches have revealed the molecular determinants that contribute to the biological program of chimaerins during T-cell responses. Unexpectedly, we have found that the translocation of chimaerins to the T-cell/B-cell immune synapse does not rely on the canonical binding of diacylglycerol to the C1 region of these GTPase-activating proteins. Taken together, these results identify chimaerins as candidates for the downmodulation of Rac1 in T-lymphocytes and, in addition, uncover a novel regulatory mechanism that mediates their activation in T-cells.

‣ Arf GTPase-activating proteins and their potential role in cell migration and invasion

Campa, Fanny; Randazzo, Paul A
Fonte: Landes Bioscience Publicador: Landes Bioscience
Tipo: Artigo de Revista Científica
Publicado em //2008 Português
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Cell migration is central to normal physiology in embryogenesis, the inflammatory response and wound healing. In addition, the acquisition of a motile and invasive phenotype is an important step in the development of tumors and metastasis. Arf GTPase-activating proteins (GAPs) are nonredundant regulators of specialized membrane surfaces implicated in cell migration. Part of Arf GAP function is mediated by regulating the ADP ribosylation factor (Arf) family GTP-binding proteins. However, Arf GAPs can also function independently of their GAP enzymatic activity, in some cases working as Arf effectors. In this commentary, we discuss examples of Arf GAPs that function either as regulators of Arfs or independently of the GTPase activity to regulate membrane structures that mediate cell adhesion and movement.

‣ Tuberous Sclerosis Tumor Suppressor Complex-like Complexes Act as GTPase-activating Proteins for Ral GTPases*

Shirakawa, Ryutaro; Fukai, Shuya; Kawato, Mitsunori; Higashi, Tomohito; Kondo, Hirokazu; Ikeda, Tomoyuki; Nakayama, Ei; Okawa, Katsuya; Nureki, Osamu; Kimura, Takeshi; Kita, Toru; Horiuchi, Hisanori
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
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The small GTPases RalA and RalB are multifunctional proteins regulating a variety of cellular processes. Like other GTPases, the activity of Ral is regulated by the opposing effects of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Although several RalGEFs have been identified and characterized, the molecular identity of RalGAP remains unknown. Here, we report the first molecular identification of RalGAPs, which we have named RalGAP1 and RalGAP2. They are large heterodimeric complexes, each consisting of a catalytic α1 or α2 subunit and a common β subunit. These RalGAP complexes share structural and catalytic similarities with the tuberous sclerosis tumor suppressor complex, which acts as a GAP for Rheb. In vitro GTPase assays revealed that recombinant RalGAP1 accelerates the GTP hydrolysis rate of RalA by 280,000-fold. Heterodimerization was required for this GAP activity. In PC12 cells, knockdown of the β subunit led to sustained Ral activation upon epidermal growth factor stimulation, indicating that the RalGAPs identified here are critical for efficient termination of Ral activation induced by extracellular stimuli. Our identification of RalGAPs will enable further understanding of Ral signaling in many biological and pathological processes.

‣ Analysis of Rab GTPase-Activating Proteins Indicates that Rab1a/b and Rab43 Are Important for Herpes Simplex Virus 1 Secondary Envelopment ▿

Zenner, Helen L.; Yoshimura, Shin-ichiro; Barr, Francis A.; Crump, Colin M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2011 Português
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Assembly of herpes simplex virus 1 (HSV-1) occurs in the cytoplasm, where the capsid and tegument bud into host cell membranes. It is at this point that the viral glycoproteins are incorporated into the virion, as they are located at the assembly site. We investigated the role of the Rab GTPases in coordinating the assembly process by overexpressing 37 human Rab GTPase-activating proteins (GAPs) and assessing infectious titers. Rab GTPases are key cellular regulators of membrane trafficking events that, by their membrane association and binding of effector proteins, ensure the appropriate fusion of membranes. We identified that TBC1D20 and RN-tre and their partner Rabs, Rab1a/b and Rab43, respectively, are important for virion assembly. In the absence of Rab1a/b, the viral glycoproteins are unable to traffic from the endoplasmic reticulum to the assembly compartment, and thus unenveloped particles build up in the cytoplasm. The defect resulting from Rab43 depletion is somewhat more complex, but it appears that the fragmentation and dispersal of the trans-Golgi network and associated membranes render these compartments unable to support secondary envelopment.

‣ Watch the GAP: Emerging Roles for IQ Motif-Containing GTPase-Activating Proteins IQGAPs in Hepatocellular Carcinoma

Schmidt, Valentina A.
Fonte: Hindawi Publishing Corporation Publicador: Hindawi Publishing Corporation
Tipo: Artigo de Revista Científica
Português
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IQ motif-containing GTPase-activating proteins IQGAP1 and IQGAP2 are highly homologous multidomain scaffolding proteins. Their major function consists of integration of Rho GTPase and Ca2+/calmodulin signals with cell adhesive and cytoskeletal reorganizational events. Recent studies showed that they play an important role in carcinogenesis. There is growing evidence that IQGAP2 is a novel tumor suppressor counteracting the effects of IQGAP1, an oncogene, in several cancers, especially in hepatocellular carcinoma (HCC). While HCC is highly prevalent and one of the deadliest cancers worldwide, the signaling pathways involved are not fully understood and treatment of advanced disease still represents an area of high unmet medical need. This paper compiles various findings from studies in mouse models, cell lines, and patient samples that support future development of IQGAPs into new therapeutic targets. It also discusses distinct features of IQGAP2 in an attempt to provide insight into the mechanism of the seemingly paradoxical opposing roles of the two very similar IQGAP proteins in carcinogenesis.

‣ Plant ERD2s self-interact and interact with GTPase-activating proteins and ADP-ribosylation factor 1

Xu, Guoyong; Liu, Yule
Fonte: Landes Bioscience Publicador: Landes Bioscience
Tipo: Artigo de Revista Científica
Publicado em 01/09/2012 Português
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ERD2s (ER luminal protein receptors)-mediated retrograde transport is one of the most substantial processes to maintain the endoplasmic reticulum (ER) homeostasis. It is completed by the recognition of the escaped ER luminal proteins, the gathering into COP I vesicle, and the fusion and releasing into the ER. ERD2s can recognize HDEL/KDEL motifs at the C-terminal of the escaped ER luminal proteins at the Golgi to initiate the retrograde transport. However, these mechanisms remain largely unknown in plants. We recently found that two Nicotiana benthamiana homologs, ERD2a and ERD2b, functioned as ER luminal protein receptors, were required for both HDEL/KDEL motifs-mediated ER retrieval and participated in cell death triggered by ER stress and nonhost pathogens. Here, we provide a set of new data that ERD2a/2b can form homo- or hetero-oligomerization and interact with both the ADP-ribosylation factor 1 (ARF1) and its potential GTPase-activating proteins (GAP) indicated by the firefly luciferase complementation imaging assay (LCI). These evidences further support the ER luminal protein receptor function of ERD2a/2b in plants and suggest their evolutionarily conserved mechanism during the retrograde trafficking. We also analyze the characteristics of ERD2s within a species and among different species.

‣ A RhoGEF and Rho family GTPase-activating protein complex links the contractile ring to cortical microtubules at the onset of cytokinesis

Somers, W.; Saint, R.
Fonte: Cell Press Publicador: Cell Press
Tipo: Artigo de Revista Científica
Publicado em //2003 Português
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78.74386%
The mechanism that positions the cytokinetic contractile ring is unknown, but derives from the spindle midzone. We show that an interaction between the Rho GTP exchange factor, Pebble, and the Rho family GTPase-activating protein, RacGAP50C, connects the contractile ring to cortical microtubules at the site of furrowing in D. melanogaster cells. Pebble regulates actomyosin organization, while RacGAP50C and its binding partner, the Pavarotti kinesin-like protein, regulate microtubule bundling. All three factors are required for cytokinesis. As furrowing begins, these proteins colocalize to a cortical equatorial ring. We propose that RacGAP50C-Pavarotti complexes travel on cortical microtubules to the cell equator, where they associate with the Pebble RhoGEF to position contractile ring formation and coordinate F-actin and microtubule remodeling during cytokinesis.

‣ Biochemical characterization of baculovirus-expressed rap1A/Krev-1 and its regulation by GTPase-activating proteins.

Quilliam, L A; Der, C J; Clark, R; O'Rourke, E C; Zhang, K; McCormick, F; Bokoch, G M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1990 Português
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Normal human rap1A and 35A rap1A (which encodes a protein with a Thr-35----Ala mutation) were cloned into a baculovirus transfer vector and expressed in Sf9 insect cells. The resulting proteins were purified, and their nucleotide binding, GTPase activities, and responsiveness to GTPase-activating proteins (GAPs) were characterized and compared with those of Rap1 purified from human neutrophils. Recombinant wild-type Rap1A bound GTP gamma S, GTP, and GDP with affinities similar to those observed for neutrophil Rap1 protein. The rate of exchange of GTP by Rap1 without Mg2+ was much slower than that by Ras. The basal GTPase activities by both recombinant proteins were lower than that observed with the neutrophil Rap1, but the GTPase activity of the neutrophil and wild-type recombinant Rap1 proteins could be stimulated to similar levels by Rap-GAP activity in neutrophil cytosol. In contrast to wild-type Rap1A, the GTPase activity of 35A Rap was unresponsive to Rap-GAP stimulation. Neither recombinant Rap1A nor neutrophil Rap1 protein GTPase activity could be stimulated by recombinant Ras-GAP at a concentration 25-fold higher than that required to hydrolyze 50% of H-Ras-bound GTP under similar conditions. These results suggest that the putative effector domains (amino acids 32 to 40) shared between Rap1 and Ras are functionally similar and interact with their respective GAPs. However...

‣ Identification of the catalytic domains and their functionally critical arginine residues of two yeast GTPase-activating proteins specific for Ypt/Rab transport GTPases.

Albert, S; Will, E; Gallwitz, D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/10/1999 Português
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Ypt/Rab proteins constitute the largest subfamily of the Ras superfamily of monomeric GTPases and are regulators of vesicular protein transport. Their slow intrinsic GTPase activity (10(-4)-10(-3) min(-1) at 30 degrees C) has to be accelerated to switch the active to the inactive conformation. We have identified the catalytic domain within the C-terminal halves of two yeast GTPase-activating proteins (GAPs), Gyp1p and Gyp7p, with specificity for Ypt/Rab GTPases. The catalytically active fragments of Gyp1p and Gyp7p were more active than the full-length proteins and accelerated the intrinsic GTP hydrolysis rates of their preferred substrates by factors of 4.5 x 10(4) and 7.8 x 10(5), respectively. The K(m) values for the Gyp1p and Gyp7p active fragments (143 and 42 microM, respectively) indicate that the affinities of those GAPs for their substrates are very low. The catalytic domains of Gyp1p and Gyp7p contain five invariant arginine residues; substitutions of only one of them (R343 in Gyp1p and R458 in the analogous position of Gyp7p) rendered the GAPs almost completely inactive. We suggest that Ypt/Rab-GAPs, like Ras- and Rho-GAPs, follow the same mode of action and provide a catalytic arginine ('arginine finger') in trans to accelerate the GTP hydrolysis rate of the transport GTPases.

‣ Arabidopsis thaliana ROP GTPase activating proteins: At the crossroad of cell division and cell expansion

Stöckle, Dorothee
Fonte: Universität Tübingen Publicador: Universität Tübingen
Tipo: Dissertation; info:eu-repo/semantics/doctoralThesis
Português
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Cell division is a fundamental process in every living organism. Therefore it is important to investigate this mechanism. Higher plants developed a separate kind of cell division since the cells are not motile unlike mammal or yeast cells. Land plants have to overcome the rigidity of the cellulosic cell wall. Plants developed specific cytoskeletal arrays, the preprophase band and the phragmoplast. The preprophase band predicts the insertion site of the future, partitioning new wall, the cell plate. The cell plate is formed by vesicle fusion, initiated in the center of the cell and expands towards the parental plasma membrane, where the preprophase band originally predicted the insertion sites. The cell plate formation is facilitated by the plant specific cytoskeletal array, the phragmoplast. Since the preprophase band disappears at the end of prophase it is important to maintain the positional information it provides. In the model plant Arabidopsis thaliana (A. thaliana) two members of the kinesin-12 class motor proteins, PHRAGMOPLAST ORIENTING KINESIN (POK) 1 and 2 preserve this information while localizing at the former position of the preprophase band throughout mitosis and cytokinesis. The absence of these two proteins results in mis-positioned cell walls indicating that POK1 and POK2 carry out an important function in cell wall positioning during cell division. For better understanding of POK function and its role in plant cell division...