GTP hydrolysis mechanisms in ras p21 and in the ras-GAP complex studied by fluorescence measurements on tryptophan mutants

Antonny, Bruno; Chardin, Pierre Teilhard de; Roux, Michel J.; Chabre, Marc

doi:10.1021/bi00098a002

Cited by 48 publications

(38 citation statements)

References 29 publications

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“…This step is the potential location of GAP activation. The value of kcat obtained here is 5-10-fold lower than other estimates Antonny et al, 1991) and so may reflect the possibility that Pi release becomes rate limiting at very high GAP activation.…”

Section: Discussioncontrasting

confidence: 71%

Interaction of GTPase-activating protein with p21ras, measured using a continuous assay for inorganic phosphate release

Webb¹,

Hunter²

1992

Biochemical Journal

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The mechanism of GTPase-activating protein (GAP) activation of p2Iras GTP hydrolysis has been investigated by measuring the kinetics of release of Pi during the hydrolysis. The measurement uses a continuous spectroscopic assay forPi, based on a guanosine analogue, 2-amino-6-mercapto-7-methylpurine ribonucleoside, as substrate for purine nucleoside phosphorylase [Webb, M. R. (1992) Proc. Natl. Acad. Sci. U.S. A. 89, 4884-4887]. This phosphorolysis gives an absorbance increase at 360 nm, so that when the reaction is coupled to GTP hydrolysis, the change in absorbance gives the total amount of Pi released from the p2Iras. The rate of the absorbance increase gives the GTPase activity. This provides a non-radioactive method of determining p21r48 concentration and GAP activity. It was used to determine the interaction of GAP with wild-type p2lra8 and two mutants , all in the GTP ( Asp-12 mutant binds to GAP with the same affinity as the wild-type protein. A novel GTPase activity was characterized whereby the EDTA-induced nucleotide release and GAP-activated cleavage of bound GTP leads to steady-state turnover of GTP hydrolysis. An assay for GAP is described based on this activity.

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Section: Discussioncontrasting

confidence: 71%

Interaction of GTPase-activating protein with p21ras, measured using a continuous assay for inorganic phosphate release

Webb¹,

Hunter²

1992

Biochemical Journal

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“…In vitro ADP-ribosylation of Rac1 was performed with 10 g of purified Rac1 (12) loaded with 10 M GTP␥S, according to the procedure of Antonny et al (1). For ADP-ribosylation reactions, 1 g of Rac1-GTP␥S was incubated for 30 to 60 min at 25°C in 200 mM Tris acetate (pH 6.0) containing the indicated amount of purified His-tagged ExoS, 10 mM NAD, 1 mM MgCl 2 , and a source of the eukaryotic ExoS ADPRT cofactor-either 10 l of wheat germ extract or 1 M 14-3-3 protein (Upstate Biotechnology, Lake Placid, NY)-in a final reaction volume of 50 l.…”

Section: Methodsmentioning

confidence: 99%

Examination of the Coordinate Effects ofPseudomonas aeruginosaExoS on Rac1

et al. 2005

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Exoenzyme S (ExoS) is a bifunctional toxin directly translocated into eukaryotic cells by the Pseudomonas aeruginosa type III secretory (TTS) process. The amino-terminal GTPase-activating (GAP) activity and the carboxy-terminal ADP-ribosyltransferase (ADPRT) activity of ExoS have been found to target but exert opposite effects on the same low-molecular-weight G protein, Rac1. ExoS ADP-ribosylation of Rac1 is cell line dependent. In HT-29 human epithelial cells, where Rac1 is ADP-ribosylated by TTS-ExoS, Rac1 was activated and relocalized to the membrane fraction. Arg66 and Arg68 within the GTPase-binding region of Rac1 were identified as preferred sites of ExoS ADP-ribosylation. The modification of these residues by ExoS would be predicted to interfere with Rac1 inactivation and explain the increase in active Rac1 caused by ExoS ADPRT activity. Using ExoS-GAP and ADPRT mutants to examine the coordinate effects of the two domains on Rac1 function, limited effects of ExoS-GAP on Rac1 inactivation were evident in HT-29 cells. In J774A.1 macrophages, where Rac1 was not ADP-ribosylated, ExoS caused a decrease in the levels of active Rac1, and this decrease was linked to ExoS-GAP. Using immunofluorescence staining of Rac1 to understand the cellular basis for the targeting of ExoS ADPRT activity to Rac1, an inverse relationship was observed between Rac1 plasma membrane localization and Rac1 ADP-ribosylation. The results obtained from these studies have allowed the development of a model to explain the differential targeting and coordinate effects of ExoS GAP and ADPRT activity on Rac1 within the host cell.Pseudomonas aeruginosa is a ubiquitous, environmentally beneficial bacterium that can adapt to become a highly virulent opportunistic pathogen in compromised individuals. The versatility and pathogenicity of P. aeruginosa are multifactorial and relate to its ability to respond to its environment by the regulated production of a variety of cell-associated and extracellular products. The establishment of infection begins with the adherence of P. aeruginosa to host cells through type IV pili or nonpilus adherence mechanisms (42, 50, 52). After colonization, the organism ensures its survival in the host through the secretion of virulence factors, including exotoxin A (24), hemolysins (40), elastases LasA and LasB (2,20,21), and pigments (53). In addition to secreted virulence factors, P. aeruginosa is able to directly affect eukaryotic cell function through the contact-dependent translocation of effector proteins by the type III secretion system (59). Four P. aeruginosa-type III secretory (TTS) effector proteins have been identified, ExoS, ExoT, ExoU, and ExoY, each affecting eukaryotic cell function differently. Notably, the TTS system is found in both clinical and environmental P. aeruginosa isolates, suggesting an essential role of TTS in P. aeruginosa overall growth and survival (8,9,49). The integral relationship between P. aeruginosa TTS effectors and eukaryotic cell function is also evident in the requirement of eu...

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“…As summarized in Table I , we investigated its effects on the intrinsic tryptophan fluorescence of Rab5; the latter provides a sensitive probe for local conformational changes in GTP-binding proteins (41)(42)(43)(44)(45). We previously characterized guanine nucleotide-dependent fluorescence changes for Rab5 (39).…”

Section: Influence Of Mg 2ϩ On the Structure And Function Of Rab5mentioning

confidence: 99%

Influence of Mg2+ on the Structure and Function of Rab5

Pan

Sanford

Wessling‐Resnick

1996

Journal of Biological Chemistry

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Rab proteins are a family of Ras-like small molecular weight GTPases that are localized to distinct subcellular compartments (1, 2) and believed to regulate specific steps of intracellular membrane trafficking (3-6). The functional cycle of Rab proteins involves the delivery of the GDP-bound forms to the target membrane by a GDP dissociation inhibitor (GDI) 1 (7-9), the exchange of GDP for GTP at membrane surface catalyzed by a guanine nucleotide exchange factor (GEF) (8, 9) and the retrieval of the GDP-bound forms from the membrane by GDI after GTP hydrolysis and membrane fusion (7). Localized on plasma membrane, clathrin-coated vesicles, and early endosomes (2), Rab5 has been shown to play an important role in early events of endocytosis (4, 5), although the exact mechanism of its function remains to be determined.It is known that Mg 2ϩ is essential for GTPase function and structure. Crystallographic studies of several GTP-binding proteins reveal a single Mg 2ϩ in the guanine nucleotide binding pocket, coordinating between the protein and guanine nucleotide in both GDP-and GTP analog-bound conformations (10 -15). Effects of Mg 2ϩ on guanine nucleotide binding, GTPase activity, and the structural integrity of GTP-binding proteins have been widely documented (16 -32). A key observation is that Mg 2ϩ inhibits GDP release from Ras-like GTP-binding proteins and therefore prevents binding of . However, the exact mechanism for this inhibitory effect and its physiological significance remains unknown.Important functional roles of the N-terminal domains of several Ras-like GTP-binding proteins also have been noted in studies of guanine nucleotide exchange (33-38). Myristoylation at the N terminus of ARF enhances its rate of GDP release (27), and N-terminal truncation of ARF results in loss of function by reducing its affinity for GDP and permitting GDP/GTP exchange in the absence of phospholipids (33). Moreover, deletion of the N terminus enables isolation of ARF in a nucleotide-free form (38). Finally, deletion of the N-terminal domain of Rab5 results in a loss of function (34 -37) and interferes with the protein's post-translational processing (37). These observations suggest that N-terminal domains of Ras-like GTP-binding proteins may participate in the regulation of guanine nucleotide exchange and represent crucial structural domains necessary for the function of the proteins.We have investigated mechanisms through which Mg 2ϩ and the N-terminal domain of Rab5 participate in its regulation of GDP release. . While the structure and function of Rab5 , a C-terminal truncation mutant, is influenced by Mg 2ϩ in the same fashion as Rab5WT , an N-and C-terminal truncation mutant, Rab5 , is resistant to the cation's effects. Thus, inhibition of GDP release by Mg 2ϩ appears to be exerted via chemical constraints due to the cation's coordination between GDP and Rab5, as well as conformational restraints involving the protein's N-terminal domain that are induced by Mg 2ϩ coordination with Ser 34 of Rab5. Based on the correl...

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GTP hydrolysis mechanisms in ras p21 and in the ras-GAP complex studied by fluorescence measurements on tryptophan mutants

Cited by 48 publications

References 29 publications

Interaction of GTPase-activating protein with p21ras, measured using a continuous assay for inorganic phosphate release

Interaction of GTPase-activating protein with p21ras, measured using a continuous assay for inorganic phosphate release

Examination of the Coordinate Effects ofPseudomonas aeruginosaExoS on Rac1

Influence of Mg2+ on the Structure and Function of Rab5

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