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 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

Ras plays an essential role in lymphocyte development and function. However, in vivo consequence(s) of regulation of Ras activity by guanosine triphosphatase (GTPase)-activating proteins (GAPs) on lymphocyte development and function are not known. In this study we demonstrate that neurofibromin, the protein encoded by the NF1 tumor suppressor gene functions as a GAP for Ras in T cells. Loss of Nf1 in T cells results in enhanced Ras activation, which is associated with thymic and splenic hyperplasia, and an increase in the absolute number of immature and mature T-cell subsets compared with control mice. Interestingly, in spite of a profound T-cell expansion and higher thymidine incorporation in unstimulated Nf1-deficient T cells, T-cell receptor and interleukin-2 receptor-mediated proliferation of thymocytes and mature T cells was substantially reduced compared with control mice. Collectively, these results identify neurofibromin as a GAP for Ras in T cells for maintaining immune homeostasis in vivo.
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PMID:Lymphoproliferative defects in mice lacking the expression of neurofibromin: functional and biochemical consequences of Nf1 deficiency in T-cell development and function. 1239 9

Our studies have shown that RLIP76 (RALBP1), a 76 kDa Ral-binding, Rho/Rac-GAP and Ral effector protein, is a novel multispecific transporter of xenobiotics as well as GS-Es. Like previously characterized ABC transporters, it mediates ATP-dependent transport of structurally unrelated amphiphilic xenobiotics and displays inherent ATPase activity, which is stimulated by its substrate allocrites. It does not have significant sequence homology with ABC transporters and differs from the ABC transporters in several other important aspects, including (i) lack of any close homologs in humans, (ii) lack of a classical Walker domain, (iii) integral membrane association without clearly defined transmembrane domains and (iv) its role as a direct link to Ras/Ral/Rho and EGF-R signaling through its multifunctional nature, including GAP activity, regulation of exocytosis as well as clathrin-coated pit-mediated receptor endocytosis. Its multifunctional nature derives from the presence of multiple motifs, including a Rho/Rac GAP domain, a Ral effector domain binding motif, 2 distinct ATP-binding domains, a H(+)-ATPase domain, PKC and tyrosine kinase phosphorylation sites and the ability to undergo fragmentation into multiple smaller peptides which participate as components of macromolecular functional complexes. One of the physiologic functions of RLIP76 is regulation of intracellular concentration of the electrophilic intermediates of oxidative lipid metabolism by mediating efflux of GS-E formed from oxidative degradation of arachidonic acid, including leukotrienes and the 4HNE-GSH conjugate. RLIP76-mediated transport of amphiphilic chemotherapeutic agents such as anthracyclines and vinca alkaloids as well as GS-E produced during oxidative metabolism places this multifunctional protein in a central role as a resistance mechanism for preventing apoptosis caused by chemotherapeutic agents and a variety of external/internal stressors, including oxidative stress, heat shock and radiation.
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PMID:Transport of glutathione conjugates and chemotherapeutic drugs by RLIP76 (RALBP1): a novel link between G-protein and tyrosine kinase signaling and drug resistance. 1286 21

We previously identified a guanosine triphosphatase (GTPase)-activating protein (GAP) male germ cell Rac GAP (MgcRacGAP) that enhanced interleukin-6 (IL-6)-induced macrophage differentiation of murine M1 leukemia cells. Later, MgcRacGAP was found to play crucial roles in cell division. However, how MgcRacGAP enhanced IL-6-induced differentiation remained elusive. Here we show that MgcRacGAP enhances IL-6-induced differentiation through enhancement of signal transducer and activator of transcription-3 (STAT3) activation. MgcRacGAP, Rac, and STAT3 formed a complex in IL-6-stimulated M1 cells, where MgcRacGAP interacted with Rac1 and STAT3 through its cysteine-rich domain and GAP domain. In reporter assays, the wild-type MgcRacGAP enhanced transcriptional activation of STAT3 while a GAP-domain deletion mutant (DeltaGAP) did not significantly enhance it, suggesting that the GAP domain was required for enhancement of STAT3-dependent transcription. Intriguingly, M1 cells expressing DeltaGAP had no effect on the differentiation signal of IL-6, while forced expression of MgcRacGAP rendered M1 cells hyperresponsive to the IL-6-induced differentiation. Moreover, knockdown of MgcRacGAP by RNA interference profoundly suppressed STAT3 activation, implicating MgcRacGAP in the STAT3-dependent transcription. All together, our data not only reveal an important role for MgcRacGAP in STAT3 activation, but also demonstrate that MgcRacGAP regulates IL-6-induced cellular differentiation in which STAT3 plays a pivotal role.
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PMID:A GTPase-activating protein binds STAT3 and is required for IL-6-induced STAT3 activation and for differentiation of a leukemic cell line. 1528 13

Within the leaf of an angiosperm, the vascular system is constructed in a complex network pattern called venation. The formation of this vein pattern has been widely studied as a paradigm of tissue pattern formation in plants. To elucidate the molecular mechanism controlling the vein patterning process, we previously isolated Arabidopsis mutants van1 to van7, which show a discontinuous vein pattern. Here we report the phenotypic analysis of the van3 mutant in relation to auxin signaling and polar transport, and the molecular characterization of the VAN3 gene and protein. Double mutant analyses with pin1, emb30-7/gn and mp, and physiological analyses using the auxin-inducible marker DR5::GUS and an auxin transport inhibitor indicated that VAN3 may be involved in auxin signal transduction, but not in polar auxin transport. Positional cloning identified VAN3 as a gene that encodes an adenosine diphosphate (ADP)-ribosylation factor-guanosine triphosphatase (GTPase) activating protein (ARF-GAP). It resembles animal ACAPs and contains four domains: a BAR (BIN/amphiphysin/RVS) domain, a pleckstrin homology (PH) domain, an ARF-GAP domain and an ankyrin (ANK)-repeat domain. Recombinant VAN3 protein showed GTPase-activating activity and a specific affinity for phosphatidylinositols. This protein can self-associate through the N-terminal BAR domain in the yeast two-hybrid system. Subcellular localization analysis by double staining for Venus-tagged VAN3 and several green-fluorescent-protein-tagged intracellular markers indicated that VAN3 is located in a subpopulation of the trans-Golgi network (TGN). Our results indicate that the expression of this gene is induced by auxin and positively regulated by VAN3 itself, and that a specific ACAP type of ARF-GAP functions in vein pattern formation by regulating auxin signaling via a TGN-mediated vesicle transport system.
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PMID:VAN3 ARF-GAP-mediated vesicle transport is involved in leaf vascular network formation. 1574 78

The brain-specific synaptic guanosine triphosphatase (GTPase)-activating protein (SynGAP) is important in synaptic plasticity. It shows dual specificity for the small guanine nucleotide-binding proteins Rap and Ras. Here, we show that RapGAP activity of SynGAP requires its C2 domain. In contrast to the isolated GAP domain, which does not show any detectable RapGAP activity, a fragment comprising the C2 and GAP domains (C2-GAP) stimulates the intrinsic GTPase reaction of Rap by approximately 1 x 10(4). The C2-GAP crystal structure, complemented by modelling and biochemical analyses, favours a concerted movement of the C2 domain towards the switch II region of Rap to assist in GTPase stimulation. Our data support a catalytic mechanism similar to that of canonical RasGAPs and distinct from the canonical RapGAPs. SynGAP presents the first example, to our knowledge, of a GAP that uses a second domain for catalytic activity, thus pointing to a new function of C2 domains.
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PMID:The C2 domain of SynGAP is essential for stimulation of the Rap GTPase reaction. 1832 56

p190-A and -B Rho GAPs (guanosine triphosphatase activating proteins) are the only cytoplasmatic proteins containing FF domains. In p190-A Rho GAP, the region containing the FF domains has been implicated in binding to the transcription factor TFII-I. Moreover, phosphorylation of Tyr308 within the first FF domain inhibits this interaction. Because the structural determinants governing this mechanism remain unknown, we sought to solve the structure of the first FF domain of p190-A Rho GAP (RhoGAPFF1) and to study the potential impact of phosphorylation on the structure. We found that RhoGAPFF1 does not fold with the typical (alpha1-alpha2-3(10)-alpha 3) arrangement of other FF domains. Instead, the NMR data obtained at 285 K show an alpha1-alpha2-alpha 3-alpha 4 topology. In addition, we observed that specific contacts between residues in the first loop and the fourth helix are indispensable for the correct folding and stability of this domain. The structure also revealed that Tyr308 contributes to the domain hydrophobic core. Furthermore, the residues that compose the target motif of the platelet-derived growth factor receptor alpha kinase form part of the alpha 3 helix. We observed that the phosphorylation reaction requires a previous step including domain unfolding, a process that occurs at 310 K. In the absence of phosphorylation, the temperature-dependent RhoGAPFF1 folding/unfolding process is reversible. However, phosphorylation causes an irreversible destabilization of the RhoGAPFF1 structure, which probably accounts for the inhibitory effect that it exerts on the TFII-I interaction. Our results link the ability of a protein domain to be phosphorylated with conformational changes in its three-dimensional structure.
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PMID:NMR structural studies on human p190-A RhoGAPFF1 revealed that domain phosphorylation by the PDGF-receptor alpha requires its previous unfolding. 1939 45

Beta1-containing adhesions at the plasma membrane function as dynamic complexes to provide bidirectional communication between the cell and its environment, yet commonly are used by pathogens to gain host cell entry. Recently, the cholesterol-lowering drug simvastatin was found to inhibit host invasion through beta1-containing adhesion complexes. To better understand the regulatory mechanisms controlling adhesion formation and uptake and the use of these complexes by Staphylococcus aureus, the primary etiologic agent in sepsis, bacteremia and endocarditis, we investigated the mechanism of inhibition by simvastatin. In response to simvastatin, adhesion complexes diminished as well as beta1 trafficking to the plasma membrane required to initiate adhesion formation. Simvastatin stimulated CDC42 activation and coupling to p85, a small-guanosine triphosphatase (GTPase) activating protein (GAP), yet sequestered CDC42 coupled to p85 within the cytosol. Loss of p85 GAP activity through use of genetic strategies decreased host cell invasion as well as beta1 trafficking. From these findings, we propose a mechanism whereby p85 GAP activity localized within membrane compartments facilitates beta1 trafficking. By sequestering p85 within the cytosol, simvastatin restricts the availability and uptake of the receptor used by pathogenic strains to gain host cell entry.
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PMID:GTPase activating protein function of p85 facilitates uptake and recycling of the beta1 integrin. 1991 8


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