Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.1.25 (triphosphatase)
 1,529 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the present study we show that expression of the neural PKC-substrate B-50 (growth-associated protein [GAP-43]) in Rat-1 fibroblasts induced the formation of filopodial extensions during spreading. This morphological change was accompanied by an enhanced formation of peripheral actin filaments and by accumulation of vinculin immunoreactivity in filopodial focal adhesions, colocalizing with B-50. In time lapse experiments, the B-50-induced filopodial extensions were shown to stay in close contact with the substratum and appeared remarkably stable, resulting in a delayed lamellar spreading of the fibroblasts. The morphogenetic effects of the B-50 protein were entirely dependent on the integrity of the two N-terminal cysteines involved in membrane association (C3C4), but were not significantly affected by mutations of the PKC-phosphorylation site (S41) or deletion of the C terminus (177-226). Cotransfection of B-50 with dominant negative Cdc42 or Rac did not prevent B-50-induced formation of filopodial cells, whereas this process could be completely blocked by cotransfection with dominant negative Rho or Clostridium botulinum C3-transferase. Conversely, constitutively active Rho induced a similar filopodial phenotype as B-50. We therefore propose that the induction of surface extensions by B-50 in spreading Rat-1 fibroblasts depends on Rho-guanosine triphosphatase function.
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PMID:B-50/GAP-43-induced formation of filopodia depends on Rho-GTPase. 961 74

Members of the regulators of G protein signaling (RGS) family stimulate the intrinsic guanosine triphosphatase (GTPase) activity of the alpha subunits of certain heterotrimeric guanine nucleotide-binding proteins (G proteins). The guanine nucleotide exchange factor (GEF) for Rho, p115 RhoGEF, has an amino-terminal region with similarity to RGS proteins. Recombinant p115 RhoGEF and a fusion protein containing the amino terminus of p115 had specific activity as GTPase activating proteins toward the alpha subunits of the G proteins G12 and G13, but not toward members of the Gs, Gi, or Gq subfamilies of Galpha proteins. This GEF may act as an intermediary in the regulation of Rho proteins by G13 and G12.
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PMID:p115 RhoGEF, a GTPase activating protein for Galpha12 and Galpha13. 966 63

Signaling pathways that link extracellular factors to activation of the monomeric guanosine triphosphatase (GTPase) Rho control cytoskeletal rearrangements and cell growth. Heterotrimeric guanine nucleotide-binding proteins (G proteins) participate in several of these pathways, although their mechanisms are unclear. The GTPase activities of two G protein alpha subunits, Galpha12 and Galpha13, are stimulated by the Rho guanine nucleotide exchange factor p115 RhoGEF. Activated Galpha13 bound tightly to p115 RhoGEF and stimulated its capacity to catalyze nucleotide exchange on Rho. In contrast, activated Galpha12 inhibited stimulation by Galpha13. Thus, p115 RhoGEF can directly link heterotrimeric G protein alpha subunits to regulation of Rho.
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PMID:Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13. 966 63

Scrape loading Clostridium botulinum C3 exoenzyme into primary peripheral blood human T lymphocytes (PB T cells) efficiently adenosine diphosphate (ADP)-ribosylates and thus inactivates the guanosine triphosphatase (GTPase) Rho. Basal adhesion of PB T cells to the beta1 integrin substrate fibronectin (Fn) was not inhibited by inactivation of Rho, nor was upregulation of adhesion using phorbol myristate acetate (PMA; 10 ng/ml) or Mn++ (1 mM) affected. Whereas untreated PB T cells adherent to Fn remain spherical, C3-treated PB T cells extend F-actin-containing pseudopodia. Inactivation of Rho delayed the kinetics of PMA-dependent PB T cell homotypic aggregation, a process involving integrin alphaLbeta2. Although C3 treatment of PB T cells did not prevent adhesion to the beta1 integrin substrate Fn, it did inhibit beta1 integrin/CD3-mediated costimulation of proliferation. Analysis of intracellular cytokine production at the single cell level demonstrated that ADP-ribosylation of Rho inhibited beta1 integrin/ CD3 and CD28/CD3 costimulation of IL-2 production within 6 h of activation. Strikingly, IL-2 production induced by PMA and ionomycin was unaffected by C3 treatment. Thus, the GTPase Rho is a novel regulator of T lymphocyte cytoarchitecture, and functional Rho is required for very early events regulating costimulation of IL-2 production in PB T cells.
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PMID:Adenosine diphosphate (ADP)-ribosylation of the guanosine triphosphatase (GTPase) rho in resting peripheral blood human T lymphocytes results in pseudopodial extension and the inhibition of T cell activation. 976

Adducin is a membrane skeletal protein that binds to actin filaments (F-actin) and thereby promotes the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such as ruffling membranes. Rho-associated kinase (Rho- kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates alpha-adducin and thereby enhances the F-actin-binding activity of alpha-adducin in vitro. Here we identified the sites of phosphorylation of alpha-adducin by Rho-kinase as Thr445 and Thr480. We prepared antibody that specifically recognized alpha-adducin phosphorylated at Thr445, and found by use of this antibody that Rho-kinase phosphorylated alpha-adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated alpha-adducin accumulated in the membrane ruffling area of Madin-Darby canine kidney (MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF). The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-kinase, or alpha-adducinT445A,T480A (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migration in NRK49F cells. alpha-AdducinT445D,T480D (substitution of Thr445 and Thr480 by Asp), but not alpha-adducinT445A,T480A, counteracted the inhibitory effect of the dominant negative Rho-kinase on the TPA-induced membrane ruffling in MDCK cells. Taken together, these results indicate that Rho-kinase phosphorylates alpha-adducin downstream of Rho in vivo, and that the phosphorylation of adducin by Rho-kinase plays a crucial role in the regulation of membrane ruffling and cell motility.
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PMID:Phosphorylation of adducin by Rho-kinase plays a crucial role in cell motility. 1020 29

Proliferation, differentiation, and survival of erythroid progenitor cells are mainly regulated by stem cell factor (SCF) and erythropoietin (Epo). Using normal human progenitors, we analyzed the role of Ca2+-sensitive protein kinase C (PKC) subtypes and of G-protein-coupled receptor ligands on growth factor-dependent DNA synthesis. We show that stimulation of DNA synthesis by the two growth factors requires activation of PKCalpha. Inhibitors of Ca2+-activated PKC subtypes blocked the growth factor-induced 3H-thymidine incorporation. SCF and Epo caused no significant translocation of PKCalpha into the membrane, but treatment of intact cells with either of the two cytokines resulted in enhanced activity of immunoprecipitated cytosolic PKCalpha. Stimulation of PKC with the phorbol ester PMA mimicked the cytokine effect on DNA synthesis. Epo-, SCF-, and PMA-induced thymidine incorporation was potently inhibited by thrombin (half-maximal inhibition with 0.1 U/mL). This effect was mediated via the G-protein-coupled thrombin receptor and the Rho guanosine triphosphatase. Adenosine diphosphate caused a modest Ca2+-dependent stimulation of DNA synthesis in the absence of cytokines and specifically enhanced the effect of SCF. Cyclic 3', 5'-adenosine monophosphate exerted a selective inhibitory effect on Epo-stimulated thymidine incorporation. Our results define PKCalpha as major intermediate effector of cytokine signaling and suggest a role for thrombin in controlling erythroid progenitor proliferation.
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PMID:Erythropoietin- and stem cell factor-induced DNA synthesis in normal human erythroid progenitor cells requires activation of protein kinase Calpha and is strongly inhibited by thrombin. 1038 4

The actin cytoskeleton undergoes extensive remodeling during cell morphogenesis and motility. The small guanosine triphosphatase Rho regulates such remodeling, but the underlying mechanisms of this regulation remain unclear. Cofilin exhibits actin-depolymerizing activity that is inhibited as a result of its phosphorylation by LIM-kinase. Cofilin was phosphorylated in N1E-115 neuroblastoma cells during lysophosphatidic acid-induced, Rho-mediated neurite retraction. This phosphorylation was sensitive to Y-27632, a specific inhibitor of the Rho-associated kinase ROCK. ROCK, which is a downstream effector of Rho, did not phosphorylate cofilin directly but phosphorylated LIM-kinase, which in turn was activated to phosphorylate cofilin. Overexpression of LIM-kinase in HeLa cells induced the formation of actin stress fibers in a Y-27632-sensitive manner. These results indicate that phosphorylation of LIM-kinase by ROCK and consequently increased phosphorylation of cofilin by LIM-kinase contribute to Rho-induced reorganization of the actin cytoskeleton.
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PMID:Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. 1043 59

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and cell surface growth are polarized, mediating bud emergence, bud growth, and cytokinesis. We have determined whether p21-activated kinase (PAK)-family kinases regulate cell and actin polarization at one or several points during the yeast cell cycle. Inactivation of the PAK homologues Ste20 and Cla4 at various points in the cell cycle resulted in loss of cell and actin cytoskeletal polarity, but not in depolymerization of F-actin. Loss of PAK function in G1 depolarized the cortical actin cytoskeleton and blocked bud emergence, but allowed isotropic growth and led to defects in septin assembly, indicating that PAKs are effectors of the Rho-guanosine triphosphatase Cdc42. PAK inactivation in S/G2 resulted in depolarized growth of the mother and bud and a loss of actin polarity. Loss of PAK function in mitosis caused a defect in cytokinesis and a failure to polarize the cortical actin cytoskeleton to the mother-bud neck. Cla4-green fluorescent protein localized to sites where the cortical actin cytoskeleton and cell surface growth are polarized, independently of an intact actin cytoskeleton. Thus, PAK family kinases are primary regulators of cell and actin cytoskeletal polarity throughout most or all of the yeast cell cycle. PAK-family kinases in higher organisms may have similar functions.
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PMID:PAK-family kinases regulate cell and actin polarization throughout the cell cycle of Saccharomyces cerevisiae. 1056 85

To investigate the role of RhoA on the intracellular membrane dynamics of lysosomes in rat hepatoma cells (MM1), we analyzed the localization of lysosomal aspartic proteinase cathepsin D by confocal immunofluorescence microscopy in the dominant active RhoA-transfected cells. Here we show that the transfection of the dominant active form of human small guanosine triphosphatase (GTPase) RhoA in MMI cells, a highly invasive cell line, causes the redistribution and spreading of small punctate structures stained for cathepsin D throughout the cytoplasm. We found that the microtubule organization was markedly different in the two cell lines: uniformly developed and polymerized microtubule filaments were seen in the mock transfectants; however, the dynamic organization of microtubules was less pronounced in the active RhoA transfectants. Furthermore, we found for the first time that a selective inhibitor of Rho-associated kinase (p160ROCK), Y-27632, impeded the subcellular spreading of cathepsin D staining and promoted reclustering of cathepsin D toward the perinuclear region in the active RhoA-transfected cells. To our knowledge, this is the first indication that the RhoA/ROCK-mediated signaling pathway is involved in the intracellular membrane dynamics of lysosomes by regulating the cytoskeletal microtubule organization as well as the actin cytoskeletons.
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PMID:Small guanosine triphosphatase Rho/Rho-associated kinase as a novel regulator of intracellular redistribution of lysosomes in invasive tumor cells. 1099 80

Rhodopsin is essential for photoreceptor morphogenesis; photoreceptors lacking rhodopsin degenerate in humans, mice, and Drosophila. Here we report that transgenic expression of a dominant-active Drosophila Rho guanosine triphosphatase, Drac1, rescued photoreceptor morphogenesis in rhodopsin-null mutants; expression of dominant-negative Drac1 resulted in a phenotype similar to that seen in rhodopsin-null mutants. Drac1 was localized in a specialization of the photoreceptor cortical actin cytoskeleton, which was lost in rhodopsin-null mutants. Thus, rhodopsin appears to organize the actin cytoskeleton through Drac1, contributing a structural support essential for photoreceptor morphogenesis.
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PMID:Rescue of photoreceptor degeneration in rhodopsin-null Drosophila mutants by activated Rac1. 1118 46


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