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 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

Most members of the guanosine triphosphatase (GTPase) superfamily hydrolyze guanosine triphosphate (GTP) quite slowly unless stimulated by a GTPase activating protein or GAP. The alpha subunits (G alpha) of the heterotrimeric G proteins hydrolyze GTP much more rapidly and contain an approximately 120-residue insert not found in other GTPases. Interactions between a G alpha insert domain and a G alpha GTP-binding core domain, both expressed as recombinant proteins, show that the insert acts biochemically as a GAP. The results suggest a general mechanism for GAP-dependent hydrolysis of GTP by other GTPases.
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PMID:Separate GTP binding and GTPase activating domains of a G alpha subunit. 826 82

Na,K-ATPase is an integral membrane protein complex involved in the regulation of intracellular Na+ and K+ ion levels. The number of accessible Na,K-ATPase complexes on the surface of human platelets before and after activation by adenosine 5'-diphosphate (ADP) was quantitated using ouabain, a highly specific inhibitor of this enzyme. Studies with [3H]ouabain revealed an increase in the number of accessible ouabain binding sites from approximately 55 per resting platelet to approximately 625 per ADP-activated platelet. Ultrastructural localization of this ion transport complex was also studied in resting human platelets or following activation by ADP. Localization was performed using a novel colloidal gold affinity probe which employed ouabain (ouabain-GAP) as a ligand specific for the Na,K-ATPase. Qualitatively, the number of surface-associated ouabain-GAP labels per platelet was observed to increase following activation by ADP. Fracture-labeling studies of frozen preparations of resting and freshly activated platelets suggested ouabain binding sites were associated with intracellular platelet structures during the early stages of activation. Together these results suggest an increase in the amount of ouabain binding sites at the surface of human platelets as an event of activation and that at least some of these additional sites may originate from intracellular sites.
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PMID:Appearance of additional ouabain binding sites on platelet surfaces following activation. 839 97

Unlike the alpha subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins, Ras-related GTP-binding proteins have hitherto been considered not to bind or become activated by tetrafluoroaluminate (AIF4-). However, the product of the proto-oncogene ras in its guanosine diphosphate (GDP)-bound form interacted with AIF4 - in the presence of stoichiometric amounts of either of the guanosine triphosphatase (GTPase)-activating proteins (GAPs) p120GAP and neurofibromin. Neither oncogenic Ras nor a GAP mutant without catalytic activity produced such a complex. Together with the finding that the Ras-binding domain of the protein kinase c-Raf, whose binding site on Ras overlaps that of the GAPs, did not induce formation of such a complex, this result suggests that GAP and neurofibromin stabilize the transition state of the GTPase reaction of Ras.
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PMID:Formation of a transition-state analog of the Ras GTPase reaction by Ras-GDP, tetrafluoroaluminate, and GTPase-activating proteins. 865 79

The aim of this study was to estimate the anticataract action of vitamin E using an in vitro methylprednisolone (MP)-induced cataract model. The same severity of early cortical cataract was induced in lenses isolated from male Wistar rats aged 6 weeks by incubation with MP (1.5 mg/ml) in TC-199 medium. The cataractous lenses showed slight increases in lipid peroxide (LPO) content and Na+/K+ ratio and slight decreases in reduced glutathione (GSH) content and glyceraldehyde-3-phosphate dehydrogenase (GAP-DH), a sensitive index of oxidative stress, and Na+,K(+)-ATPase activities. When the cataractous lenses were further incubated in TC-199 medium with and without vitamin E (250 micrograms/ml) for 48 h, the progression of cataract was prevented in the vitamin E-treated lenses, but not in the vitamin E-untreated lenses. The vitamin E-untreated lenses showed a decrease in vitamin E content and an increase in water content in addition to further increases in LPO content and Na+/K+ ratio and further decreases in GSH content and GAP-DH and Na+,K(+)-ATPase activities. In contrast, the changes of these components and enzymes except for GSH were attenuated in the vitamin E-treated lenses. From these results, it can be estimated that vitamin E prevents in vitro cataractogenesis in rat lenses treated with MP by protecting the lenses against oxidative damage and loss of membrane function.
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PMID:Anticataract action of vitamin E: its estimation using an in vitro steroid cataract model. 888 85

The three-dimensional structure of the complex between human H-Ras bound to guanosine diphosphate and the guanosine triphosphatase (GTPase)-activating domain of the human GTPase-activating protein p120GAP (GAP-334) in the presence of aluminum fluoride was solved at a resolution of 2.5 angstroms. The structure shows the partly hydrophilic and partly hydrophobic nature of the communication between the two molecules, which explains the sensitivity of the interaction toward both salts and lipids. An arginine side chain (arginine-789) of GAP-334 is supplied into the active site of Ras to neutralize developing charges in the transition state. The switch II region of Ras is stabilized by GAP-334, thus allowing glutamine-61 of Ras, mutation of which activates the oncogenic potential, to participate in catalysis. The structural arrangement in the active site is consistent with a mostly associative mechanism of phosphoryl transfer and provides an explanation for the activation of Ras by glycine-12 and glutamine-61 mutations. Glycine-12 in the transition state mimic is within van der Waals distance of both arginine-789 of GAP-334 and glutamine-61 of Ras, and even its mutation to alanine would disturb the arrangements of residues in the transition state.
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PMID:The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. 951 63

We conducted a mutation analysis of the most conserved region of the neurofibromatosis type 1 (NF1) gene, the guanine triphosphatase (GTPase) activating protein (GAP)-related domain (NF1 GRD), to which the function of tumour suppressor is attributed. Sixty primary neuroectodermal tumours were analysed. The rationale for the study was based on the likelihood of finding structural alterations resulting in loss of function of this region in tumours of neuroepithelial tissues, where the activity of neurofibromin seems to be crucial in regulating the mechanisms of signal transduction and cell transformation mediated by p21 ras. Following analysis of the whole NF1 GRD sequence, no mutations were identified in the tumours analysed. We conclude that the loss of NF1 gene tumour suppressor function, that might lead or contribute to the development of malignancies in neuroectodermal tissues, is not due to structural abnormalities of the region of the gene which interacts with p21 ras.
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PMID:The guanine triphosphatase (GTPase) activating protein (GAP)-related domain of the neurofibromatosis type 1 gene is not mutated in neural crest-derived sporadic tumours. 971 12

A prolonged ouabain blockade of the Na(+),K(+)-ATPase detaches cells from each other and from the substrate. This suggests the existence of a link between pump (P) and attachment (A). In the present work, we report that MDCK-W cells treated with ouabain increase tyrosine phosphorylation and content of active MAP kinase, redistribute molecules involved in cell attachment (occludin, ZO-1, desmoplakin, cytokeratin, alpha-actinin, vinculin and actin), and detach. Genistein and UO126, inhibitors of protein tyrosine kinase and of MAP kinase kinase, respectively, block this detachment. The content of P190(Rho-GAP), a GTPase activating protein of the Rho small G-protein subfamily, is increased by ouabain, suggesting that both the Rho/Rac and MAPK pathways are involved. Another clone of MDCK cells whose Na(+),K(+)-ATPase has a negligible affinity for the drug, show none of the effects described for MDCK-W and remain attached. Ma104 cells, a line that has a high affinity for ouabain and stops pumping, fail to modify phosphorylation, as well as the pattern of distribution of attaching molecules, and remain in the monolayer. Taken together, these results suggest that there is a mechanism (P-->A) that transduces a blockade of the pump in a detachment of the cell from neighbors and substrate, in which Ma104 cells are faulty.
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PMID:Relationship between Na(+),K(+)-ATPase and cell attachment. 1056 41

The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein alpha subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single G alpha(i/o)-Loco (GoLoco) motif predicted to represent a second G alpha interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with G alpha(i1), G alpha(i2), and G alpha(i3) in their GDP-bound forms. In GTP gamma S binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by G alpha(i1). Both regions also stabilize G alpha(i1) in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF(4)(-). Our results indicate that both RGS12 and RGS14 harbor two distinctly different G alpha interaction sites: a previously recognized N-terminal RGS box possessing G alpha(i/o) GAP activity and a C-terminal GoLoco region exhibiting G alpha(i) GDI activity. The presence of two, independent G alpha interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple G alpha GAP activity.
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PMID:RGS12 and RGS14 GoLoco motifs are G alpha(i) interaction sites with guanine nucleotide dissociation inhibitor Activity. 1138 33

Coculture with stromal cells tends to maintain normal hematopoietic progenitors and their leukemic counterparts in an undifferentiated, proliferative state. An example of this effect is seen with megakaryocytic differentiation, wherein stromal contact renders many cell types refractory to potent induction stimuli. This inhibitory effect of stroma on megakaryocytic differentiation correlates with a blockade within hematopoietic cells of protein kinase C-epsilon (PKC-epsilon) up-regulation and of extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase activation, both of which have been implicated in promoting megakaryocytic differentiation. In this study K562DeltaRafER.5 cells, expressing an estradiol-responsive mutant of the protein kinase Raf-1, were used to determine the relevance and stage of ERK/MAPK pathway blockade by stromal contact. Activation of DeltaRafER by estradiol overrode stromal blockade of megakaryocytic differentiation, implicating the proximal stage of the ERK/MAPK pathway as a relevant control point. Because stromal contact blocked delayed but not early ERK activation, the small guanosine triphosphatase (GTPase) Rap1 was considered as a candidate inhibitory target. Activation assays confirmed that Rap1 underwent sustained activation as a result of megakaryocytic induction, as previously described. As with ERK activation, stromal contact selectively blocked delayed but not early Rap1 activation, having no effect on Ras activation. Enforced expression of either wild-type Rap1 or the GTPase (GAP) resistant mutant Rap1 V12 failed to override stromal inhibition, suggesting that the inhibitory mechanism does not involve GAP up-regulation but rather may target upstream guanine nucleotide exchange factor (GEF) complexes. Accordingly, coimmunoprecipitation demonstrated stromally induced alterations in a protein complex associated with c-Cbl, a scaffolding factor for Rap1-GEF complexes.
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PMID:Stromal inhibition of megakaryocytic differentiation is associated with blockade of sustained Rap1 activation. 1239 69


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