Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A variety of systems use nucleoside triphosphate hydrolysis to control or provide energy for biological processes, mediated through protein-protein interactions. The nature of this coupling may vary, but often there is a degree of similarity. In this paper, two systems are compared: actomyosin in muscle and p21ras in a signal transduction pathway as yet undefined. The mechanism of the nucleotide triphosphate hydrolysis and the consequent changes in the protein-nucleotide complex have been investigated, to understand how the coupling to biological function is achieved. The basal nucleoside triphosphatase mechanisms are compared and the roles of proteins that activate the hydrolysis, actin and GAP, are discussed. The cleavage process was probed by stereochemical techniques to determine the basic mechanism, of either a phosphorylated enzyme intermediate or direct displacement of nucleoside diphosphate by water. Phosphate-water oxygen exchange probes were used to investigate nucleoside triphosphate and inorganic phosphate release steps. A new method of probing the kinetics of inorganic phosphate release directly has been developed. In muscle, this process seems likely to be related directly to force generation. In the GAP-ras system, measurement of phosphate release is allowing the mechanism of the GAP-p21ras interaction to be probed.
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PMID:The role of nucleoside triphosphate hydrolysis in transducing systems: p21ras and muscle. 135 Dec 91

Ras-GAP (GTPase activating protein) is a regulatory protein that stimulates the intrinsic guanosine triphosphatase (GTPase) activity of the proto-oncogene product p21ras. A domain of the neurofibromatosis gene product (NF1) that has sequence similarity to the catalytic domain of Ras-GAP and to yeast IRA gene products also has a specific stimulatory activity toward p21ras GTPase. Arachidonic acid and phosphatidic acid inactivate GAP, but no agents have been identified that stimulate GAP and thereby switch p21ras off. With the use of recombinant Ha-c-Ras and Ras-GAP, NF1, and GAP catalytic domains, it was found that prostaglandins PGF2 alpha and PGA2 stimulated Ras-GAP and that prostacyclin PGI2 inhibited Ras-GAP. The stimulatory effect of PGF2 alpha was saturable and structure-specific and competed with the inhibitory effect of arachidonic acid. Arachidonic acid also inhibited the catalytic activity of NF1, but prostaglandins were not stimulatory. These results suggest a mechanism for the allosteric control of Ras function through the modulation of arachidonate metabolism.
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PMID:Regulation of Ras-GAP and the neurofibromatosis-1 gene product by eicosanoids. 190 23

Phospholipase C gamma 1 (PLC gamma 1) and p21ras guanosine triphosphatase (GTPase) activating protein (GAP) bind to and are phosphorylated by activated growth factor receptors. Both PLC gamma 1 and GAP contain two adjacent copies of the noncatalytic Src homology 2 (SH2) domain. The SH2 domains of PLC gamma 1 synthesized individually in bacteria formed high affinity complexes with the epidermal growth factor (EGF)- or platelet derived growth factor (PDGF)-receptors in cell lysates, and bound synergistically to activated receptors when expressed together as one bacterial protein. In vitro complex formation was dependent on prior growth factor stimulation and was competed by intracellular PLC gamma 1. Similar results were obtained for binding of GAP SH2 domains to the PDGF-receptor. The isolated SH2 domains of other signaling proteins, such as p60src and Crk, also bound activated PDGF-receptors in vitro. SH2 domains, therefore, provide a common mechanism by which enzymatically diverse regulatory proteins can physically associate with the same activated receptors and thereby couple growth factor stimulation to intracellular signal transduction pathways.
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PMID:Binding of SH2 domains of phospholipase C gamma 1, GAP, and Src to activated growth factor receptors. 217 44

The crystal structure at 2.7 A resolution of the normal human c-H-ras oncogene protein lacking a flexible carboxyl-terminal 18 residue reveals that the protein consists of a six-stranded beta sheet, four alpha helices, and nine connecting loops. Four loops are involved in interactions with bound guanosine diphosphate: one with the phosphates, another with the ribose, and two with the guanine base. Most of the transforming proteins (in vivo and in vitro) have single amino acid substitutions at one of a few key positions in three of these four loops plus one additional loop. The biological functions of the remaining five loops and other exposed regions are at present unknown. However, one loop corresponds to the binding site for a neutralizing monoclonal antibody and another to a putative "effector region"; mutations in the latter region do not alter guanine nucleotide binding or guanosine triphosphatase activity but they do reduce the transforming activity of activated proteins. The data provide a structural basis for understanding the known biochemical properties of normal as well as activated ras oncogene proteins and indicate additional regions in the molecule that may possibly participate in other cellular functions.
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PMID:Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21. 244 79

Bacterially synthesized c-Ha-ras protein (Ras) was incubated with guanosine triphosphatase (GTPase) activating (GA) protein in the presence of various phospholipids. The stimulation of Ras GTPase activity by GA protein was inhibited in some cases. Among the lipids most active in blocking GA protein activity were lipids that show altered metabolism during mitogenic stimulation. These included phosphatidic acid (containing arachidonic acid), phosphatidylinositol phosphates, and arachidonic acid. Other lipids, including phosphatidic acid with long, saturated side chains, diacylglycerols, and many other common phospholipids, were unable to alter GA protein activity. The interaction of lipids with GA protein might be important in the regulation of Ras activity during mitogenic stimulation.
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PMID:The effect of GTPase activating protein upon ras is inhibited by mitogenically responsive lipids. 253 92

A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras as well as with N-ras proteins. To identify the region of ras p21 with which GAP interacts, 21 H-ras mutant proteins were purified and tested for their ability to undergo stimulation of GTPase activity by GAP. Mutations in nonessential regions of H-ras p21 as well as mutations in its carboxyl-terminal domain (residues 165-185) and purine binding region (residues 117 and 119) did not decrease the ability of the protein to respond to GAP. In addition, an antibody against the carboxyl-terminal domain did not block GAP activity, supporting the conclusion that GAP does not interact with this region. Transforming mutations at positions 12, 59, and 61 (the phosphoryl binding region) abolished GTPase stimulation by GAP. Point mutations in the putative effector region of ras p21 (amino acids 35, 36, and 38) were also insensitive to GAP. However, a point mutation at position 39, shown previously not to impair effector function, did not alter GAP-p21 interaction. These results indicate that GAP interaction may be essential for ras p21 biological activity and that it may be a ras effector protein.
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PMID:Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. 283 17

Human choriocarcinoma cell lines, preserving many biological properties of normal trophoblasts, are good models for investigating of trophoblastic cell biology. Most of these cells express various oncogenes, which might have essential roles for biological characteristics of choriocarcinoma cells. Of these oncogenes, ras gene family has been known to play the key roles in cell growth, transformation and differentiation. In order to investigate the roles of ras genes on various unique characters of trophoblasts, the author transfected the viral H- or K-ras oncogene into a human choriocarcinoma cell line, CC1, and established choriocarcinoma cell lines acquired up-regulated activity of ras genes. v-ras-expressing clones exhibited almost the same growth capacities as control clones, but only v-H-ras clones formed the many fluid-filled hemispherical "domes" in a cell layer. Microscopic observation of these domes clarified that the accumulation of fluid between cell layer and the surface of culture dish has resulted in dome formation, which is characteristic of the polarized epithelial cells and represents an in vitro morphologic expression of vectorial transport function. Since these clones exhibited higher Na(+)-K(+)-ATPase activity than other clones and dome formation was inhibited with the treatment of ouabain, a specific inhibitor of Na(+)-K(+)-ATPase, dome formation may be due to the augmentation of the vectorial fluid transport driven by Na(+)-K(+)-ATPase. In addition, the expression of human chorionic gonadotropin (hCG) beta was examined to investigate the effects of ras genes on peptide hormone production which is one of the differentiated functions of trophoblasts. v-K-ras-expressing clones secreted significantly more hCG than controls, while v-H-ras did not seem to affect hCG-producing activity. These results obtained in this study indicate that H-ras gene may facilitate the vectorial transcellular fluid transport from maternal site to fetus, while K-ras gene is associated with some endocrine functions such as hCG production in trophoblasts.
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PMID:[Roles of ras genes on biological properties of human choriocarcinoma cells]. 759 Jun 7

Guanosine triphosphatase activating protein (GAP) is an essential component of Ras signaling pathways. GAP functions in different cell types as a deactivator and a transmitter of cellular Ras signals. A domain (amino acids 275 to 351) encompassing the Src homology region 3 (SH3) of GAP was found to be essential for GAP signaling. A monoclonal antibody was used to block germinal vesicle breakdown (GVBD) induced by the oncogenic protein Ha-ras Lys12 in Xenopus oocytes. The monoclonal antibody, which was found to recognize the peptide containing amino acids 275 to 351 within the amino-terminal domain of GAP, did not modify the stimulation of the Ha-Ras-GTPase by GAP. Injection of peptides corresponding to amino acids 275 to 351 and 317 to 326 blocked GVBD induced by insulin or by Ha-Ras Lys12 but not that induced by progesterone. These findings confirm that GAP is an effector for Ras in Xenopus oocytes and that the SH3 domain is essential for signal transduction.
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PMID:Identification of the SH3 domain of GAP as an essential sequence for Ras-GAP-mediated signaling. 767 7

Guanosine triphosphatase activating protein (GAP) is an important modulator of p21ras (Ras)-dependent signal transduction in mammalian cells and in insulin-induced maturation of Xenopus oocytes. A synthetic octapeptide from the catalytic domain of GAP, residues 899-906 (F899VFLRLIC906), inhibited GAP-stimulated hydrolysis of GTP to GDP by Ras in an in vitro biochemical assay (IC50 = 12 microM). The peptide was assayed for its ability to block insulin- (Ras-dependent) and progesterone- (Ras-independent) induced maturation of stage VI Xenopus laevis oocytes, marked by germinal vesicle breakdown (GVBD). Microinjection of 50 pmol of the peptide inhibited insulin- but not progesterone-induced GVBD by 50%. A 7-residue peptide lacking F899, GAP(900-906)-NH2, failed to inhibit GAP-stimulated GTPase activity and did not block GVBD. Replacement of the cysteine residue at position 906 with methionine resulted in a peptide with prolonged inhibitory activity in the oocyte. Moreover, sequential replacement of specific L-amino acid residues with the corresponding D-amino acids produced a peptide with a two-fold increased half-life after injection into oocytes. None of the peptides tested affected progesterone induced GVBD, suggesting that the modifications did not result in loss of specificity. These studies show that (a) peptides that were able to inhibit GAP-stimulated Ras GTPase activity in vitro were also able to block Ras-dependent GVBD in oocytes, and (b) specific substitutions in these peptides can result in improved stability in oocytes.
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PMID:Ras-dependent maturation of Xenopus oocytes is blocked by modified peptides of GTPase activating protein (GAP). 778 68

We examined the differential effects of the H-ras oncogene and the K-ras oncogene on cisplatin sensitivity in murine NIH/3T3 cells transfected with these oncogenes. Although the NIH/3T3 cells transformed with H-ras oncogenes (EJ-NIH/3T3 and Ha8-21) showed an increased resistance to cisplatin compared to the parental NIH/3T3, the cell lines transformed with K-ras oncogenes (DT and 1,8DNP2-2-5) did not. Compared with NIH/3T3, the 2 H-ras transformants reduced both the accumulation of cisplatin and the Na+,K(+)-ATPase activity in the membrane fraction. On the other hand, we observed no significant difference in cellular accumulation of cisplatin or in Na+,K(+)-ATPase activity between parental NIH/3T3 and the K-ras transformants. Since these ras transformants did not affect the cellular metallothionein content, transcriptional level of DNA polymerase beta or activity of glutathione-S-transferase which is not associated with cisplatin sensitivity, these results suggest that cisplatin resistance is brought about by the H-ras oncogene, but not by K-ras, and that induction of cisplatin resistance by H-ras is mainly due to a reduction of cisplatin accumulation and an impairment of Na+,K(+)-ATPase activity in the membrane fraction.
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PMID:Differential Na+,K(+)-ATPase activity and cisplatin sensitivity between transformants induced by H-ras and those induced by K-ras. 807 52


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