Guanine nucleotide binding properties of Rac2 mutant proteins and analysis of the responsiveness to guanine nucleotide dissociation stimulator

A case-control study of the associations of retinoids and specific carotenoids with breast cancer using concentrations of these nutrients in breast adipose tissue was conducted among women attending a breast clinic in the Boston area in 1989-1992. Breast adipose tissue was collected during breast biopsy. Cases (n = 46) were women whose biopsies revealed invasive or in situ breast cancer; control subjects (n = 63) were women whose biopsies revealed benign disease. We observed inverse associations between breast adipose concentrations of retinoids and carotenoids and risk of breast cancer, although not all were statistically significant. The multivariate-adjusted odds ratio comparing women above the median value of the control group for retinol with those below or equal to the median was 0.71 (95% CI: 0.26, 1.93; NS); corresponding odds ratios were 0.61 (95% CI: 0.23, 1.64; NS) for retinyl palmitate, 0.30 (95% CI: 0.11, 0.85) for beta-carotene, 0.32 (95% CI: 0.11, 0.94) for lycopene, and 0.68 (95% CI: 0.27, 1.73; NS) for lutein/zeaxanthin. There was a nonsignificant positive correlation (r = 0.23, P = 0.15) between breast adipose tissue concentrations of retinol and dietary intake of preformed vitamin A, including supplements measured by using a food-frequency questionnaire. No correlation was found between breast adipose concentrations of carotenoids and intake of dietary carotenoids. These data suggest that higher breast adipose concentrations of retinoids and some carotenoids may be associated with decreased risk of breast cancer and that further examination of these relations is warranted.     

Inhibition by anti-RCC1 monoclonal antibodies of RCC1-stimulated guanine nucleotide exchange on Ran GTPase

Nine monoclonal antibodies to RCC1, the guanine nucleotide exchange factor on Ran GTPase, were obtained using recombinant RCC1 as the antigen. Epitopes of three monoclonal antibodies, which did not inhibit RCC1 function, were localized in the N-terminus outside the RCC1 repeat, while epitopes of the other 6 monoclonal antibodies were localized within the RCC1 repeat. Three of the latter 6 monoclonal antibodies, 2B6, 6C3, and 8D9, inhibited RCC1-stimulated nucleotide release. Two of them, 2B6 and 6C3, recognized the same amino acid residues in the N-terminus of the second RCC1 repeat, Tyr89, Ser90, Phe91, and Gly92, of which one, Gly92, is conserved in Saccharomyces cerevisiae and mutated in an rcc1(-) strain, mtr1-2. The monoclonal antibody 8D9 recognized two amino acid residues, Arg320 and Ala321, downstream of Gly319 in the N-terminus of the 6th RCC1 repeat, which corresponds to Gly92 in the second RCC1 repeat. The monoclonal antibodies which inhibited RCC1 function bound to RCC1 in homogeneous solution and stained cellular RCC1. We propose that the N-terminus of the RCC1 repeat is exposed at the surface of RCC1 on the coated plate or in fixed cells, and is involved in the RCC1-stimulated nucleotide exchange on the Ran GTPase.     

Identification of regulators for Ypt1 GTPase nucleotide cycling

Small GTPases of the Ypt/Rab family are involved in the regulation of vesicular transport. Cycling between the GDP- and GTP-bound forms and the accessory proteins that regulate this cycling are thought to be crucial for Ypt/Rab function. Guanine nucleotide exchange factors (GEFs) stimulate both GDP loss and GTP uptake, and GTPase-activating proteins (GAPs) stimulate GTP hydrolysis. Little is known about GEFs and GAPs for Ypt/Rab proteins. In this article we report the identification and initial characterization of two factors that regulate nucleotide cycling by Ypt1p, which is essential for the first two steps of the yeast secretory pathway. The Ypt1p-GEF stimulates GDP release and GTP uptake at least 10-fold and is specific for Ypt1p. Partially purified Ypt1p-GEF can rescue the inhibition caused by the dominant-negative Ypt1p-D124N mutant of in vitro endoplasmic reticulum-to-Golgi transport. This mutant probably blocks transport by inhibiting the GEF, suggesting that we have identified the physiological GEF for Ypt1p. The Ypt1p-GAP stimulates GTP hydrolysis by Ypt1p up to 54-fold, has a higher affinity for the GTP-bound form of Ypt1p than for the GDP-bound form, and is specific to a subgroup of exocytic Ypt proteins. The Ypt1p-GAP activity is not affected by deletion of two genes that encode known Ypt GAPs, GYP7 and GYP1, nor is it influenced by mutations in SEC18, SEC17, or SEC22, genes whose products are involved in vesicle fusion. The GEF and GAP activities for Ypt1p localize to particulate cellular fractions. However, contrary to the predictions of current models, the GEF activity localizes to the fraction that functions as the acceptor in an endoplasmic reticulum-to-Golgi transport assay, whereas the GAP activity cofractionates with markers for the donor. On the basis of our current and previous results, we propose a new model for the role of Ypt/Rab nucleotide cycling and the factors that regulate this process.     

Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2

A human complementary DNA was isolated that encodes a widely expressed protein, hSos1, that is closely related to Sos, the product of the Drosophila son of sevenless gene. The hSos1 protein contains a region of significant sequence similarity to CDC25, a guanine nucleotide exchange factor for Ras from yeast. A fragment of hSos1 encoding the CDC25-related domain complemented loss of CDC25 function in yeast. This hSos1 domain specifically stimulated guanine nucleotide exchange on mammalian Ras proteins in vitro. Mammalian cells overexpressing full-length hSos1 had increased guanine nucleotide exchange activity. Thus hSos1 is a guanine nucleotide exchange factor for Ras. The hSos1 interacted with growth factor receptor-bound protein 2 (GRB2) in vivo and in vitro. This interaction was mediated by the carboxyl-terminal domain of hSos1 and the Src homology 3 (SH3) domains of GRB2. These results suggest that the coupling of receptor tyrosine kinases to Ras signaling is mediated by a molecular complex consisting of GRB2 and hSos1.     

Isolation of a gene encoding a developmentally regulated T cell-specific protein with a guanine nucleotide triphosphate-binding motif

In this study, we describe a novel full length cDNA clone designated Tgtp that encodes a predicted 415-amino acid a T cell-specific guanine nucleotide triphosphate-binding protein (TGTP) bearing the characteristic motifs of a guanine nucleotide triphosphate (GTP) binding protein. Tgtp is expressed preferentially, if not exclusively, in T cells, and is up-regulated in both unfractionated and in purified CD4+8+ thymocytes upon TCR cross-linking. In contrast, expression of Tgtp is peripheral T cells is maintained at relatively high levels and is not grossly affected by TCR cross-linking. Antiserum generated against synthetic peptides from the predicted TGTP amino acid sequence recognized a single protein with a molecular mass of approximately 50 kDa, corresponding well with the computed molecular mass of 47 kDa. The only known relative of Tgtp is MUSGTP, which is reportedly expressed in B cells and bears a GTP binding motif. Thus, the discovery of Tgtp resolves a subfamily of molecules with GTP binding motifs and apparent lymphoid lineage-restricted expression. Given the restricted expression pattern in T cells, the up-regulated expression observed in response to TCR signaling in immature thymocytes, and the presence of the motifs characteristic of GTP binding proteins, we suggest that TGTP may have an important function in T cell development and/or T cell activation.     

Cloning and characterization of GEF-H1, a microtubule-associated guanine nucleotide exchange factor for Rac and Rho GTPases

The Rho-related small GTPases are critical elements involved in regulation of signal transduction cascades from extracellular stimuli to cell nucleus and cytoskeleton. The Dbl-like guanine nucleotide exchange factors (GEF) have been implicated in direct activation of these GTPases. Here we have identified a new member of the Dbl family, GEF-H1, by screening a human HeLa cell cDNA library. GEF-H1 encodes a 100-kDa protein containing the conserved structural array of a Dbl homology domain in tandem with a pleckstrin homology domain and is most closely related to the lfc oncogene, but additionally it contains a unique coiled-coil domain at the carboxyl terminus. Biochemical analysis reveals that GEF-H1 is capable of stimulating guanine nucleotide exchange of Rac and Rho but is inactive toward Cdc42, TC10, or Ras. Moreover, GEF-H1 binds to Rac and Rho proteins in both the GDP- and guanosine 5'-3-O-(thio)triphosphate-bound states without detectable affinity for Cdc42 or Ras. Immunofluorescence reveals that GEF-H1 colocalizes with microtubules through the carboxyl-terminal coiled-coil domain. Overexpression of GEF-H1 in COS-7 cells results in induction of membrane ruffles. These results suggest that GEF-H1 may have a direct role in activation of Rac and/or Rho and in bringing the activated GTPase to specific target sites such as microtubules.     

Distinct subclasses of small GTPases interact with guanine nucleotide exchange factors in a similar manner

The Ras-related GTPases are small, 20- to 25-kDa proteins which cycle between an inactive GDP-bound form and an active GTP-bound state. The Ras superfamily includes the Ras, Rho, Ran, Arf, and Rab/YPT1 families, each of which controls distinct cellular functions. The crystal structures of Ras, Rac, Arf, and Ran reveal a nearly superimposible structure surrounding the GTP-binding pocket, and it is generally presumed that the Rab/YPT1 family shares this core structure. The Ras, Rac, Ran, Arf, and Rab/YPT1 families are activated by interaction with family-specific guanine nucleotide exchange factors (GEFs). The structural determinants of GTPases required for interaction with family-specific GEFs have begun to emerge. We sought to determine the sites on YPT1 which interact with GEFs. We found that mutations of YPT1 at position 42, 43, or 49 (effector loop; switch I), position 69, 71, 73, or 75 (switch II), and position 107, 109, or 115 (alpha-helix 3-loop 7 [alpha3-L7]) are intragenic suppressors of dominant interfering YPT1 mutant N22 (YPT1-N22), suggesting these mutations prevent YPT1-N22 from binding to and sequestering an endogenous GEF. Mutations at these positions prevent interaction with the DSS4 GEF in vitro. Mutations in the switch II and alpha3-L7 regions do not prevent downstream signaling in yeast when combined with a GTPase-defective (activating) mutation. Together, these results show that the YPT1 GTPase interacts with GEFs in a manner reminiscent of that for Ras and Arf in that these GTPases use divergent sequences corresponding to the switch I and II regions and alpha3-L7 of Ras to interact with family-specific GEFs. This finding suggests that GTPases of the Ras superfamily each may share common features of GEF-mediated guanine nucleotide exchange even though the GEFs for each of the Ras subfamilies appear evolutionarily unrelated.     

Insulin stimulates guanine nucleotide exchange on Rab4 via a wortmannin-sensitive signaling pathway in rat adipocytes

Rab4, a member of the Rab family of Ras-related small GTP-binding proteins, has been shown to be associated with GLUT4-containing vesicles and implicated in the insulin action on glucose transport in rat adipocytes. In the present study, we investigated the insulin effects on the guanine nucleotide exchange on Rab4. In electrically permeabilized rat adipocytes, the amount of [35S]guanosine 5'-O-(3-thiotrisphosphate) (GTPgammaS) bound to Rab4 increased in a time-dependent manner during 45 min of the incubation period. Addition of insulin resulted in about a 2-fold stimulation of the binding of [35S]GTPgammaS to Rab4, indicating that insulin stimulated the guanine nucleotide exchange on the GTPase. Pretreatment of the cells with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, completely abolished the stimulatory effect of insulin on [35S]GTPgammaS binding to Rab4. Wortmannin also attenuated the nucleotide binding to Rab4 in the basal cells, suggesting that phosphatidylinositol 3-kinase activity may be essential for regulation of guanine nucleotide exchange on the GTPase and insulin may up-regulate the exchange activity by stimulating the lipid kinase. Insulin-induced subcellular redistribution of Rab4 from the microsomal fraction to the soluble fraction was also inhibited by wortmannin. These results suggest that insulin stimulates the guanine nucleotide exchange on Rab4 via a phosphatidylinositol 3-kinase-dependent signaling pathway and that Rab4 is one of possible targets of insulin action on intracellular vesicle traffic in rat adipocytes.     

Refined crystal structures of guanine nucleotide complexes of adenylosuccinate synthetase from Escherichia coli

Structures of adenylosuccinate synthetase from Escherichia coli complexed with guanosine-5'-(beta,gamma-imido) triphosphate and guanosine-5'-(beta,gamma-methylene)triphosphate in the presence and the absence of Mg2+ have been refined to R-factors below 0.2 against data to a nominal resolution of 2.7 A. Asp333 of the synthetase hydrogen bonds to the exocyclic 2-amino and endocyclic N1 groups of the guanine nucleotide base, whereas the hydroxyl of Ser414 and the backbone amide of Lys331 hydrogen bond to the 6-oxo position. The side chains of Lys331 and Pro417 pack against opposite faces of the guanine nucleotide base. The synthetase recognizes neither the N7 position of guanine nucleotides nor the ribose group. Electron density for the guanine-5'-(beta,gamma-imido) triphosphate complex is consistent with a mixture of the triphosphate nucleoside and its hydrolyzed diphosphate nucleoside bound to the active site. The base, ribose, and alpha-phosphate positions overlap, but the beta-phosphates occupy different binding sites. The binding of guanosine-5'-(beta,gamma-methylene)triphosphate to the active site is comparable with that of guanosine-5'-(beta, gamma-imido)triphosphate. No electron density, however, for the corresponding diphosphate nucleoside is observed. In addition, electron density for bound Mg2+ is absent in these nucleotide complexes. The guanine nucleotide complexes of the synthetase are compared with complexes of other GTP-binding proteins and to a preliminary structure of the complex of GDP, IMP, Mg2+, and succinate with the synthetase. The enzyme, under conditions reported here, does not undergo a conformational change in response to the binding of guanine nucleotides, and minimally IMP and/or Mg2+ must be present in order to facilitate the complete recognition of the guanine nucleotide by the synthetase.     

Effects of guanine nucleotide depletion on cell cycle progression in human T lymphocytes

Depletion of guanine nucleotide pools after inhibition of inosine monophosphate dehydrogenase (IMPDH) potently inhibits DNA synthesis by arresting cells in G1 and has been shown to induce the differentiation of cultured myeloid and erythroid cell lines, as well as chronic granulocytic leukemic cells after blast transformation. Inhibitors of IMPDH are also highly effective as immunosuppressive agents. The mechanism underlying these pleiotropic effects of depletion of guanine nucleotides is unknown. We have examined the effects of mycophenolic acid (MPA), a potent IMPDH inhibitor, on the cell cycle progression of activated normal human T lymphocytes. MPA treatment resulted in the inhibition of pRb phosphorylation and cell entry into S phase. The expression of cyclin D3, a major component of the cyclin-dependent kinase (CDK) activity required for pRb phosphorylation, was completely abrogated by MPA treatment of T cells activated by interleukin-2 (IL-2) and leucoagglutinin (PHA-L), whereas the expression of cyclin D2, CDK6, and CDK4 was more mildly attenuated. The direct kinase activity of a complex immunoprecipitated with anti-CDK6 antibody was also inhibited. In addition, MPA prevented the IL-2-induced elimination of p27(Kip1), a CDK inhibitor, and resulted in the retention of high levels of p27(Kip1) in IL-2/PHA-L-treated T cells bound to CDK2. These results indicate that inhibition of the de novo synthesis of guanine nucleotides blocks the transition of normal peripheral blood T lymphocytes from G0 to S phase in early- to mid-G1 and that this cell cycle arrest results from inhibition of the induction of cyclin D/CDK6 kinase and the elimination of p27(Kip1) inhibitory activity.     

Colocalization of Ras and Ral on the membrane is required for Ras-dependent Ral activation through Ral GDP dissociation stimulator

Ral GDP dissociation stimulator (RalGDS), a putative effector protein of Ras, stimulated the GDP/GTP exchange reaction of the post-tanslationally lipid-modified but not the unmodified form of Ral in response to epidermal growth factor in COS cells. The RalGDS action on Ral was enhanced by an active form of Ras but not a Ras mutant which was not post-translationally modified in the cells. The RalGDS activity was inhibited by acidic membrane phospholipids such as phosphatidylinositol and phosphatidylserine but not by phosphatidylcholine or phosphatidylethanolamine in vitro. The post-translationally modified form but not unmodified form of Ras, Ral, and Rap were incorporated in liposomes consisting of these phospholipids. When Ral was incorporated alone in the liposomes, RalGDS did not stimulate the dissociation of GDP from Ral. When Ral was incorporated with the GTP-bound form of Ras in the liposomes, RalGDS stimulated the dissociation of GDP from Ral, while the GDP-bound form of Ras did not affect the RalGDS action. The Ras-dependent Ral activation through RalGDS required the Ras-binding domain of RalGDS. Rap, which shared the same effector loop as Ras, also stimulated the dissociation of GDP from Ral through RalGDS in the liposomes, although Rap did not enhance the RalGDS action in COS cells. Taken together with our previous observations that Ras recruits RalGDS to the membrane, these results indicate that the post-translational modifications of Ras and Ral are important for Ras-dependent Ral activation through RalGDS and that colocalization of Ras and Ral on the membrane is necessary for Ral activation in intact cells.     

Lipid products of phosphoinositide 3-kinase interact with Rac1 GTPase and stimulate GDP dissociation

A number of reports suggest that under different conditions leading to cytoskeleton reorganization the GTPase Rac1 and possibly RhoA are downstream targets of phosphoinositide 3-kinase (PI 3-kinase). In order to gain more insight into this particular signaling pathway, we have addressed the question of a possible direct interaction of PI 3-kinase products with the Rho family GTPases RhoA, Rac1, and Cdc42. Using recombinant proteins, we found that Rac1 and, to a lesser extent, RhoA but not Cdc42 were capable to selectively bind to phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in a mixture of crude brain phosphoinositides. Nucleotide-depleted Rac1 was the most efficient, but the GDP- and GTP-bound forms retained significant PtdIns(3,4,5)P3 binding activity. This protein-lipid association involved electrostatic as well as hydrophobic interactions, since both phosphate groups located at specific positions of the inositol ring and fatty-acyl chains were absolutely required. Based on the sequence of Rac1, two potential binding sites were identified, one at the C terminus and one in the extra alpha-helical domain. Deletion of these two domains resulted in a complete loss of binding to PI 3-kinase products. Finally, PtdIns(3, 4,5)P3 strongly stimulated GDP dissociation from Rac1 in a dose-dependent manner. In agreement, data obtained in intact cells suggest that PtdIns(3,4,5)P3 might target Rac1 to peculiar membrane domains, allowing formation of specific clusters containing not only small GTPases but other partners bearing pleckstrin homology domains such as specific exchange factors required for Rac1 and RhoA activation.     

Structure determination of the Ras-binding domain of the Ral-specific guanine nucleotide exchange factor Rlf

Ral-specific guanine nucleotide exchange factors RalGDS, Rgl, and Rlf have been suggested to function as intermediates between Ras and Ral pathways by being able to bind Ras proteins through their C-terminal Ras-binding domains (RBD). The RBDs of RalGDS and of the Ser/Thr kinase c-Raf-1 have been shown to have the same tertiary structure. In contrast to the RBDs of Raf and RalGDS, which bind either Ras or Rap with high affinity, Rlf-RBD has a similar affinity for both GTP-binding proteins. To be able to compare these RBDs on a structural level, we have solved the three-dimensional structure of Rlf-RBD by NMR spectroscopy. The overall tertiary structure of Rlf-RBD shows the betabetaalphabetabetaalphabeta-fold of the ubiquitin superfamily and is very similar to that of RalGDS-RBD. The binding interface of Rlf-RBD to Ras was mapped using chemical shift analysis and indicated a binding mode similar to that in the case of Rap.Raf-RBD. However, comparison of the putatively interacting regions revealed structural differences which are proposed to be responsible for the different substrate affinities of Rlf-, RalGDS-, and Raf-RBD.     

Identification of a novel integrin signaling pathway involving the kinase Syk and the guanine nucleotide exchange factor Vav1

BACKGROUND:. Integrins induce the formation of large complexes of cytoskeletal and signaling proteins, which regulate many intracellular processes. The activation and assembly of signaling complexes involving focal adhesion kinase (FAK) occurs late in integrin signaling, downstream from actin polymerization. Our previous studies indicated that integrin-mediated activation of the non-receptor tyrosine kinase Syk in hematopoietic cells is independent of FAK and actin polymerization, and suggested the existence of a distinct signaling pathway regulated by Syk. RESULTS:. Multiple proteins were found to be activated by Syk, downstream of engagement of the platelet/megakaryocyte-specific integrin alphaIIbbeta3. The guanine nucleotide exchange factor Vav1 was inducibly phosphorylated in a Syk-dependent manner in cells following their attachment to fibrinogen. Together, Syk and Vav1 triggered lamellipodia formation in fibrinogen-adherent cells and both Syk and Vav1 colocalized with alphaIIbbeta3 in lamellipodia but not in focal adhesions. Additionally, Syk and Vav1 cooperatively induced activation of Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase 2 (ERK2) and the kinase Akt, and phosphorylation of the oncoprotein Cbl in fibrinogen-adherent cells. Activation of all of these proteins by Syk and Vav1 was not dependent on actin polymerization. CONCLUSIONS:. Syk and Vav1 regulate a unique integrin signaling pathway that differs from the FAK pathway in its proximity to the integrin itself, its localization to lamellipodia, and its activation, which is independent of actin polymerization. This pathway may regulate multiple downstream events in hematopoietic cells, including Rac-induced lamellipodia formation, tyrosine phosphorylation of Cbl, and activation of JNK, ERK2 and the phosphatidylinositol 3'-kinase-regulated kinase Akt.     

Vagus nerve stimulator as a treatment for intractable epilepsy

Vagus nerve stimulation was recently approved for control of medically intractable seizures. This therapy provides some relief of seizures for selective patients, however seizure freedom using this device is uncommon. Vagus nerve stimulation appears to work by calming "hyperexcited" nerve cells and reverting brain activity to its normal patterns. Many people do have significant relief in the intensity and duration of their seizures and report improved quality of life using this device.     

Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13

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.     

Non-anticoagulant heparin increases endothelial nitric oxide synthase activity: role of inhibitory guanine nucleotide proteins

Heparin, which is widely used clinically, has recently been shown to have specific properties affecting the vascular endothelium. We hypothesized that heparin stimulates endothelial nitric oxide synthase (eNOS) activity by a mechanism independent of its anticoagulant properties and dependent on an inhibitory guanine nucleotide regulatory protein (Gi). We determined the effect of both heparin and N-acetyl heparin (Non-Hep), a heparin derivative without anticoagulant properties, on eNOS activity in cultured bovine aortic endothelial cells and on endothelium-dependent relaxation in isolated vascular rings. The eNOS activity was determined by measuring both citrulline and nitric oxide (NO) metabolite formation. Heparin and Non-Hep dose-dependently increased basal eNOS activity (ED50 1.0 microgram/ml or 0.15 U/ml), an effect that was significantly inhibited by pertussis toxin (100 ng/ml), a Gi-protein inhibitor. Agonist-stimulated (acetylcholine, 10 microM) eNOS activity was potentiated following pre-treatment with both heparin and Non-Hep and reversed by pertussis toxin. Heparin and Non-Hep induced a dose-dependent relaxation in preconstricted thoracic aortic rings, an effect that was significantly inhibited by pertussis toxin, endothelial inactivation (following treatment with sodium deoxycholate) and NG-nitro-L-arginine-methyl ester (L-NAME). We conclude that heparin and non-anticoagulant heparin induce endothelium-dependent relaxation following activation of eNOS by a mechanism involving a Gi-protein. Administration of heparin derivatives without anticoagulant properties may have therapeutic implications for the preservation of eNOS in conditions characterized by endothelial dysfunction.     

Regulation of GRP1-catalyzed ADP ribosylation factor guanine nucleotide exchange by phosphatidylinositol 3,4,5-trisphosphate

Cellular levels of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) are rapidly elevated in response to activation of growth factor receptor tyrosine kinases. This polyphosphoinositide binds the pleckstrin homology (PH) domain of GRP1, a protein that also contains 200 residues with high sequence similarity to a segment of the yeast Sec7 protein that functions as an ADP ribosylation exchange factor (ARF) (Klarlund, J., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that dioctanoyl PtdIns(3,4,5)P3 binds the PH domain of GRP1 with a Kd = 0.5 microM, an affinity 2 orders of magnitude greater than dioctanoyl-PtdIns(4,5)P2. Further, the Sec7 domain of GRP1 is found to catalyze guanine nucleotide exchange of ARF1 and -5 but not ARF6. Importantly, PtdIns(3,4,5)P3, but not PtdIns(4,5)P2, markedly enhances the ARF exchange activity of GRP1 in a reaction mixture containing dimyristoylphosphatidylcholine micelles, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and a low concentration of sodium cholate. PtdIns(3,4,5)P3-mediated ARF nucleotide exchange through GRP1 is selectively blocked by 100 microM inositol 1,3,4,5-tetrakisphosphate, which also binds the PH domain of GRP1. Taken together, these data are consistent with the hypothesis that selective recruitment of GRP1 to PtdIns(3,4,5)P3 in membranes activates ARF1 and -5, known regulators of intracellular membrane trafficking.     

Characterization of H. parasuis periplasmic nucleotide pyrophosphatase as a potential target enzyme for inhibition of growth

The periplasmic nucleotide pyrophosphatase from Haemophilus parasuis was purified 750-fold to electrophoretic homogeneity through salt fractionation and ion-exchange and affinity chromatography. The purified enzyme was monomeric with an apparent M(r) of 70,000 and catalyzed the hydrolysis of the pyrophosphate bond of NAD to yield NMN and AMP as products. The enzyme exhibited negative cooperativity in the hydrolysis of a number of pyridine dinucleotides and structurally-related pyrophosphate compounds as indicated by biphasic double-reciprocal plots and Hill coefficients of 0.5. The kinetic parameters, K(m) and Vm, determined titrimetrically and analyzed through computer programs, were used to compare the relative effectiveness of dinucleotides containing nitrogen bases other than nicotinamide or adenine to that of NAD. Effective substrate-competitive inhibition of the pyrophosphatase was observed with purine and pyrimidine nucleoside diphosphates in the low micromolar concentration range. Although less effective, N1-alkylnicotinamide chlorides also inhibited competitively with respect to the substrate, NAD. In addition to being an effective inhibitor of the purified enzyme, adenosine diphosphate also inhibited growth of H. parasuis at a low micromolar concentration. This inhibition of growth correlates well with inhibition of the periplasmic pyrophosphatase which is supported by the fact that adenosine diphosphate does not effectively inhibit growth when the pyrophosphatase is by-passed by growth on nicotinamide mononucleotide. These observations are all consistent with the periplasmic nucleotide pyrophosphatase being essential for the growth of the organism on NAD and therefore, a very important enzyme with respect to the pathogenesis of the organism. 3-Aminopyridine mononucleotide, which also inhibited growth of H. parasuis at a low micromolar concentration, did not effectively inhibit the purified pyrophosphatase and a different target enzyme needs to be considered to explain growth inhibition by this derivative.