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Query: EC:3.4.11.18 (MAP)
 7,412 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The rapid modulation of ligand-binding affinity ("activation") is a central property of the integrin family of cell adhesion receptors. The Ras family of small GTP-binding proteins and their downstream effectors are key players in regulating integrin activation. H-Ras can suppress integrin activation in fibroblasts via its downstream effector kinase, Raf-1. In contrast, to H-Ras, a closely related small GTP-binding protein R-Ras has the opposite activity, and promotes integrin activation. To gain insight into the regulation of integrin activation by Ras GTPases, we created a series of H-Ras/R-Ras chimeras. We found that a 35-amino acid stretch of H-Ras was required for full suppressive activity. Furthermore, the suppressive chimeras were weak activators of the ERK1/2 MAP kinase pathway, suggesting that the suppression of integrin activation may be independent of the activation of the bulk of ERK MAP kinase. Additional data demonstrating that the ability of H-Ras or Raf-1 to suppress integrin activation was unaffected by inhibition of bulk ERK1/2 MAP kinase activation supported this hypothesis. Thus, the suppression of integrin activation is a Raf kinase induced regulatory event that can be mediated independently of bulk activation of the ERK MAP-kinase pathway.
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PMID:Suppression of integrin activation by activated Ras or Raf does not correlate with bulk activation of ERK MAP kinase. 1213 66

Some strains of Escherichia coli related to acute cystitis or colitis produce a toxin named cytotoxic necrotizing factor 1 (CNF-1). CNF-1 mediates its effects on epithelial cells or phagocytes via the permanent activation of small GTP-binding proteins, caused by the toxin-induced deamidation of Glu(63) of p21 Rho. The behavior of peripheral blood T lymphocytes during the acute phase of bacterial colitis has been poorly investigated. Our study was conducted to test whether (i) peripheral blood T lymphocytes can be activated by CNF-1 and (ii) CNF-1-activated T lymphocytes are cytotoxic against intestinal epithelial cells. Activation of T lymphocytes by CNF-1 was assessed by electrophoresis, flow cytometry, confocal microscopy, and electron microscopy studies. Assays for migration and adherence of CNF-1-treated T lymphocytes were performed in Transwell chambers with T84 intestinal epithelial cells grown on polycarbonate semipermeable filters. CNF-1 induced a decrease in the electrophoretic mobility of the GTP-binding protein Rho in treated T lymphocytes. CNF-1 provoked an increase in the content of actin stress fibers and pseudopodia in T lymphocytes. Several adherence molecules were clustered into cytoplasmic projections in CNF-1-treated T lymphocytes and adherence of such lymphocytes on the basolateral pole of T84 was increased, resulting in cytotoxicity toward epithelial cells. Such enhanced adherence in response to CNF-1 was dependent on p42-44(MAP) kinase activation of T lymphocytes. Taken together, these results suggest that CNF-1, by acting on T lymphocytes, may increase in an important fashion the virulence of certain strains of E. coli against the intestinal epithelia.
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PMID:Rho GTPase is activated by cytotoxic necrotizing factor 1 in peripheral blood T lymphocytes: potential cytotoxicity for intestinal epithelial cells. 1259 28

Thrombin activates human platelets through three different membrane receptors, the protease-activated receptors PAR-1 and PAR-4 and the glycoprotein Ib (GPIb)-IX-V complex. We investigated the contribution of these three receptors to thrombin-induced activation of the small GTPase Rap1B. We found that, similarly to thrombin, selective stimulation of either PAR-1 or PAR-4 by specific activating peptides caused accumulation of GTP-bound Rap1B in a dose-dependent manner. By contrast, in PAR-1- and PAR-4-desensitized platelets, thrombin failed to activate Rap1B. Thrombin, PAR-1-, or PAR-4-activating peptides also induced the increase of intracellular Ca(2+) concentration and the release of serotonin in a dose-dependent manner. We found that activation of Rap1B by selected doses of agonists able to elicit comparable intracellular Ca(2+) increase and serotonin release was differently dependent on secreted ADP. In the presence of the ADP scavengers apyrase or phosphocreatine-phosphocreatine kinase, activation of Rap1B induced by stimulation of either PAR-1 or PAR-4 was totally inhibited. By contrast, thrombin-induced activation of Rap1B was only minimally affected by neutralization of secreted ADP. Concomitant stimulation of both PAR-1 and PAR-4 in the presence of ADP scavengers still resulted in a strongly reduced activation of Rap1B. A similar effect was also observed upon blockade of the P2Y12 receptor for ADP, as well as in P2Y12 receptor-deficient human platelets, but not after blockade of the P2Y1 receptor. Activation of Rap1B induced by thrombin was not affected by preincubation of platelets with the anti-GPIbalpha monoclonal antibody AK2 in the absence of ADP scavengers or a P2Y12 antagonist but was totally abolished when secreted ADP was neutralized or after blockade of the P2Y12 receptor. Similarly, cleavage of the extracellular portion of GPIbalpha by the cobra venom mocarhagin totally prevented Rap1B activation induced by thrombin in the presence of apyrase and in P2Y12 receptor-deficient platelets. By contrast, inhibition of MAP kinases or p160ROCK, which have been shown to be activated upon thrombin binding to GPIb-IX-V, did not affect agonist-induced activation of Rap1B in the presence of ADP scavengers. These results indicate that although both PAR-1 and PAR-4 signal Rap1B activation, the ability of thrombin to activate this GTPase independently of secreted ADP involves costimulation of both receptors as well as binding to GPIb-IX-V.
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PMID:Contribution of protease-activated receptors 1 and 4 and glycoprotein Ib-IX-V in the G(i)-independent activation of platelet Rap1B by thrombin. 1507 82

Binding of thrombopoietin (TPO) to the cMpl receptor on human platelets potentiates aggregation induced by a number of agonists, including ADP. In this work, we found that TPO was able to restore ADP-induced platelet aggregation upon blockade of the G(q)-coupled P2Y1 purinergic receptor but not upon inhibition of the G(i)-coupled P2Y12 receptor. Moreover, TPO triggered platelet aggregation upon co-stimulation of G(z) by epinephrine but not upon co-stimulation of G(q) by the thromboxane analogue U46619. Platelet aggregation induced by TPO and G(i) stimulation was biphasic, and cyclooxygenase inhibitors prevented the second but not the first phase. In contrast to ADP, TPO was unable to induce integrin alpha(IIb)beta(3) activation, as evaluated by binding of both fibrinogen and PAC-1 monoclonal antibody. However, ADP-induced activation of integrin alpha(IIb)beta(3) was blocked by antagonists of the G(q)-coupled P2Y1 receptor but was completely restored by the simultaneous co-stimulation of cMpl receptor by TPO. Inside-out activation of integrin alpha(IIb)beta(3) induced by TPO and G(i) stimulation occurred independently of thromboxane A(2) production and was not mediated by protein kinase C, MAP kinases, or Rho-dependent kinase. Importantly, TPO and G(i) activation of integrin alpha(IIb)beta(3) was suppressed by wortmannin and Ly294002, suggesting a critical regulation by phosphatidylinositol 3-kinase. We found that TPO did not activate phospholipase C in human platelets and was unable to restore ADP-induced phospholipase C activation upon blockade of the G(q)-coupled P2Y1 receptor. TPO induced a rapid and sustained activation of the small GTPase Rap1B through a pathway dependent on phosphatidylinositol 3-kinase. In ADP-stimulated platelets, Rap1B activation was reduced, although not abolished, upon blockade of the P2Y1 receptor. However, accumulation of GTP-bound Rap1B in platelets activated by co-stimulation of cMpl and P2Y12 receptor was identical to that induced by the simultaneous ligation of P2Y1 and P2Y12 receptor by ADP. These results indicate that TPO can integrate G(i), but not G(q), stimulation and can efficiently support integrin alpha(IIb)beta(3) activation platelet aggregation by an alternative signaling pathway independent of phospholipase C but involving the phosphatidylinositol 3-kinase and the small GTPase Rap1B.
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PMID:Thrombopoietin complements G(i)- but not G(q)-dependent pathways for integrin {alpha}(IIb){beta}(3) activation and platelet aggregation. 1586 6

Heptahelical receptors are coupled to heterotrimeric GTP-binding proteins (G-proteins) which transduce most signals through their alpha and betagamma subunits to effectors, enzymes and ion channels. Of the 367 heptahelical receptors for endogenous ligands, about 330 are potential targets for drug discovery with agonist, antagonist or inverse agonist properties. The term G-protein-coupled receptors (GPCRs) is a broader functional definition rather than a structural one referring to heptahelical receptors specifically. Non-heptahelical putative GPCRs include some transmembrane receptors with tyrosine-kinase activity on their cytosolic endings (EGF, insulin and IGF-1 receptors), other transmembrane receptors (mannose-6-phosphate/IGF-2 receptor and integrin-associated protein IAP or CD47), and some receptors belonging to the class of glycosylphosphatidylinositol (GPI)-anchored proteins and located on the outer face of the plasma membrane. Also, activators of G-protein signaling (AGS) proteins that regulate vesicular trafficking activate heterotrimeric G-proteins in the Golgi independently of receptor activation. Main effectors activated through their direct interactions with alpha subunits or betagamma dimers of heterotrimeric G-proteins include adenylylcyclases, cGMP-phosphodiesterase, phospholipases Cbeta, phosphoinositide 3-kinase gamma, Ca(V2) calcium channels, GIRK/Kir3 potassium channels, and guanine nucleotide exchange factors RasGEF and RhoGEF leading to small G-proteins and MAP-kinases activation. Current signaling cascades leading to final cell responses are depicted.
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PMID:Heptahelical and other G-protein-coupled receptors (GPCRs) signaling. 1645 39

The highly conserved small Rho G-protein, Cdc42p plays a critical role in cell polarity and cytoskeleton organization in all eukaryotes. In the yeast Saccharomyces cerevisiae, Cdc42p is important for cell polarity establishment, septin ring assembly, and pheromone-dependent MAP-kinase signaling during the yeast mating process. In this study, we further investigated the role of Cdc42p in the mating process by screening for specific mating defective cdc42 alleles. We have identified and characterized novel mating defective cdc42 alleles that are unaffected in vegetative cell polarity. Replacement of the Cdc42p Val36 residue with Met resulted in a specific cell fusion defect. This cdc42[V36M] mutant responded to mating pheromone but was defective in cell fusion and in localization of the cell fusion protein Fus1p, similar to a previously isolated cdc24 (cdc24-m6) mutant. Overexpression of a fast cycling Cdc42p mutant suppressed the cdc24-m6 fusion defect and conversely, overexpression of Cdc24p suppressed the cdc42[V36M] fusion defect. Taken together, our results indicate that Cdc42p GDP-GTP cycling is critical for efficient cell fusion.
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PMID:Cdc42p GDP/GTP cycling is necessary for efficient cell fusion during yeast mating. 1657 78

Activating mutations of the FMS-like tyrosine kinase-3 (FLT3) receptor occur in approximately 30% of acute myeloid leukemia (AML) patients and, at least for internal tandem duplication (ITD) mutations, are associated with poor prognosis. FLT3 mutations trigger downstream signaling pathways including RAS-MAP/AKT kinases and signal transducer and activator of transcription-5 (STAT5). We find that FLT3/ITD mutations start a cycle of genomic instability whereby increased reactive oxygen species (ROS) production leads to increased DNA double-strand breaks (DSBs) and repair errors that may explain aggressive AML in FLT3/ITD patients. Cell lines transfected with FLT3/ITD and FLT3/ITD-positive AML cell lines and primary cells demonstrate increased ROS. Increased ROS levels appear to be produced via STAT5 signaling and activation of RAC1, an essential component of ROS-producing NADPH oxidases. A direct association of RAC1-GTP binding to phosphorylated STAT5 (pSTAT5) provides a possible mechanism for ROS generation. A FLT3 inhibitor blocked increased ROS in FLT3/ITD cells resulting in decreased DSB and increased repair efficiency and fidelity. Our study suggests that the aggressiveness of the disease and poor prognosis of AML patients with FLT3/ITD mutations could be the result of increased genomic instability that is driven by higher endogenous ROS, increased DNA damage, and decreased end-joining fidelity.
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PMID:Internal tandem duplication of FLT3 (FLT3/ITD) induces increased ROS production, DNA damage, and misrepair: implications for poor prognosis in AML. 1819 5

The plasma membrane delimits the cell and controls material and information exchange between itself and the environment. How different plasma-membrane processes are coordinated and how the relative abundance of plasma-membrane lipids and proteins is homeostatically maintained are not yet understood. Here, we used a quantitative genetic interaction map, or E-MAP, to functionally interrogate a set of approximately 400 genes involved in various aspects of plasma-membrane biology, including endocytosis, signaling, lipid metabolism and eisosome function. From this E-MAP, we derived a set of 57,799 individual interactions between genes functioning in these various processes. Using triplet genetic motif analysis, we identified a new component of the eisosome, Eis1, and linked the poorly characterized gene EMP70 to endocytic and eisosome function. Finally, we implicated Rom2, a GDP/GTP exchange factor for Rho1 and Rho2, in the regulation of sphingolipid metabolism.
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PMID:A plasma-membrane E-MAP reveals links of the eisosome with sphingolipid metabolism and endosomal trafficking. 2052 36

The mechanism of action of TNP-470 [O-(chloroacetyl-carbamoyl) fumagillol], which potently and selectively inhibits the proliferation of endothelial cells, is incompletely understood. Previous studies have established its binding protein and the most distal effector of its growth arrest activity as methionine aminopeptidase 2 (MetAP-2) and p21(WAF1/CIP1), respectively. However, the mechanistic steps between these two effectors have not been identified. We have found that addition of exogenous guanine and guanine-containing nucleosides to culture medium will completely reverse the cytostatic effect of TNP-470 on both cultured bovine aortic and mouse pulmonary endothelial cells. Western blotting showed that supplementation with exogenous guanosine reverses the induction of p21(WAF1/CIP1) by TNP-470. This "rescue" by guanine/guanosine was abolished when the guanine salvage pathway of nucleotide biosynthesis was inhibited with Immucillin H, suggesting that TNP-470 might reduce de novo guanine synthesis in endothelial cells. However, an analysis of inosine 5'-monophosphate dehydrogenase, the rate-limiting enzyme in de novo guanine synthesis and target of the antiangiogenic drug mycophenolic acid, showed no TNP-470-induced changes. Curiously, quantitation of cellular nucleotides confirmed that GTP levels were not reduced after TNP-470 treatment. Addition of guanosine at the start of G(1) phase causes a doubling in GTP levels that persists to the G(1)/S phase transition, where commitment to TNP-470 growth arrest occurs. Thus, guanine rescue involves an augmentation of cellular GTP beyond physiological levels rather than a restoration of a drug-induced GTP deficit. Determining the mechanism whereby this causes restoration of endothelial cell proliferation is an ongoing investigation.
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PMID:Reversal of TNP-470-induced endothelial cell growth arrest by guanine and guanine nucleosides. 2057 Oct 59

Ras proteins are laterally segregated into transient nanoclusters on the plasma membrane, a property essential for high fidelity signal transduction through the MAPK pathway. From a proteomic screen we identified nucleophosmin (NPM) and nucleolin as two novel regulators of K-Ras plasma membrane interactions that in turn influence MAP Kinase signaling. NPM and nucleolin are predominately nucleolar proteins but also possess extra-nuclear functions. We showed that a subset of NPM and nucleolin localize to the inner leaflet of the plasma membrane and specifically interact with K-Ras but not H-Ras. This interaction is independent of the activation state of K-Ras, and stabilizes K-Ras membrane levels. NPM expression also increases the fraction of K-Ras in nanoclusters. The increase in clustered K-Ras-GTP enhances signaling through the MAPK pathway. Together these results identify NPM and nucleolin as a new class of K-Ras regulators that modulate signal transduction via the MAPK pathway.
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PMID:Nucleophosmin and nucleolin regulate K-Ras signaling. 2058 19


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