CIB1 contributes to oncogenic signalling by Ras via modulating the subcellular localisation of sphingosine kinase 1.

CIB1 (calcium and integrin binding protein 1) is a small intracellular protein with numerous interacting partners, and hence has been implicated in various cellular functions. Recent studies have revealed emerging roles of CIB1 in regulating cancer cell survival and angiogenesis, although the mechanisms involved have remained largely undefined. In investigating the oncogenic function of CIB1, we initially found that CIB1 is widely up-regulated across a diverse range of cancers, with this upregulation frequently correlating with oncogenic mutations of KRas. Consistent with this, we found that ectopic expression of oncogenic KRas and HRas in cells resulted in elevated CIB1 expression. We previously described the Ca2+-myristoyl switch function of CIB1, and its ability to facilitate agonist-induced plasma membrane localisation of sphingosine kinase 1 (SK1), a location where SK1 is known to elicit oncogenic signalling. Thus, we examined the role this may play in oncogenesis. Consistent with these findings, we demonstrated here that over-expression of CIB1 by itself is sufficient to drive localisation of SK1 to the plasma membrane and enhance the membrane-associated enzymatic activity of SK1, as well as its oncogenic signalling. We subsequently demonstrated that elevated levels of CIB1 resulted in full neoplastic transformation, in a manner dependent on SK1. In agreement with our previous findings that SK1 is a downstream mediator of oncogenic signalling by Ras, we found that targeting CIB1 also inhibited neoplastic growth of cells induced by oncogenic Ras, suggesting an important pro-tumorigenic role for CIB1. Thus, we have demonstrated for the first time a role for CIB1 in neoplastic transformation, and revealed a novel mechanism facilitating oncogenic signalling by Ras and SK1.

Enhanced expression of transferrin receptor 1 contributes to oncogenic signalling by sphingosine kinase 1.

Sphingosine kinase 1 (SK1) is a lipid kinase that catalyses the formation of sphingosine-1-phosphate (S1P). Considerable evidence has implicated elevated cellular SK1 in tumour development, progression and disease severity. In particular, SK1 has been shown to enhance cell survival and proliferation and induce neoplastic transformation. Although S1P has been found to have both cell-surface G-protein-coupled receptors and intracellular targets, the specific downstream pathways mediating oncogenic signalling by SK1 remain poorly defined. Here, using a gene expression array approach, we have demonstrated a novel mechanism whereby SK1 regulates cell survival, proliferation and neoplastic transformation through enhancing expression of transferrin receptor 1 (TFR1). We showed that elevated levels of SK1 enhanced total as well as cell-surface TFR1 expression, resulting in increased transferrin uptake into cells. Notably, we also found that SK1 activation and localization to the plasma membrane, which are critical for its oncogenic effects, are necessary for regulation of TFR1 expression specifically through engagement of the S1P G-protein coupled receptor, S1P2. Furthermore, we showed that blocking TFR1 function with a neutralizing antibody inhibits SK1-induced cell proliferation, survival and neoplastic transformation of NIH3T3 fibroblasts. Similar effects were observed following antagonism of S1P2. Together these findings suggest that TFR1 has an important role in SK1-mediated oncogenesis.

Reduced sphingosine kinase 1 activity in dengue virus type-2 infected cells can be mediated by the 3' untranslated region of dengue virus type-2 RNA.

Sphingosine kinase 1 (SphK1) is a lipid kinase with important roles including regulation of cell survival. We have previously shown reduced SphK1 activity in cells with an established dengue virus type-2 (DENV-2) infection. In this study, we examined the effect of alterations in SphK1 activity on DENV-2 replication and cell death and determined the mechanisms of the reduction in SphK1 activity. Chemical inhibition or overexpression of SphK1 after established DENV-2 infection had no effect on infectious DENV-2 production, although inhibition of SphK1 resulted in enhanced DENV-2-induced cell death. Reduced SphK1 activity was observed in multiple cell types, regardless of the ability of DENV-2 infection to be cytopathic, and was mediated by a post-translational mechanism. Unlike bovine viral diarrhea virus, where SphK1 activity is decreased by the NS3 protein, SphK1 activity was not affected by DENV-2 NS3 but, instead, was reduced by expression of the terminal 396 bases of the 3' UTR of DENV-2 RNA. We have previously shown that eukaryotic elongation factor 1A (eEF1A) is a direct activator of SphK1 and here DENV-2 RNA co-localized and co-precipitated with eEF1A from infected cells. We propose that the reduction in SphK1 activity late in DENV-2-infected cells is a consequence of DENV-2 out-competing SphK1 for eEF1A binding and hijacking cellular eEF1A for its own replication strategy, rather than a specific host or virus-induced change in SphK1 to modulate viral replication. Nonetheless, reduced SphK1 activity may have important consequences for survival or death of the infected cell.

Molecular targets of FTY720 (fingolimod).

FTY720 is a recently approved first line therapy for relapsing forms of multiple sclerosis. In this context, FTY720 is a pro-drug, with its anti-multiple sclerosis, immunosuppressive effects largely elicited following its phosphorylation by sphingosine kinase 2 and subsequent modulation of G protein-coupled sphingosine 1-phosphate (S1P) receptor 1 that induces lymphopenia by altering lymphocyte trafficking. A number of other biological effects of FTY720 have, however, been described, including considerable evidence that this drug also has anti-cancer properties. These other effects of FTY720 are independent of S1P receptors, and appear facilitated by modulation of a range of other recently described protein targets by nonphosphorylated FTY720. Here, we review the direct targets of FTY720 that contribute to its anti-cancer properties. We also discuss other recently described protein effectors that, in combination with S1P receptors, appear to contribute to its immunosuppressive effects.

Guanine nucleotides regulate sphingosine kinase 1 activation by eukaryotic elongation factor 1A and provide a mechanism for eEF1A-associated oncogenesis.

Sphingosine kinase 1 (SK1) catalyses the formation of bioactive phospholipid sphingosine 1-phosphate (S1P). Elevated cellular SK1 activity and S1P levels enhance cell proliferation and survival, and are strongly implicated in tumourigenesis. Regulation of SK1 activity can occur through various mechanisms, including phosphorylation and protein-protein interactions. We have previously shown that eukaryotic elongation factor 1A (eEF1A) interacts with and directly activates SK1, but the mechanisms regulating this were undefined. Notably, eEF1A has GTPase activity and can exist in GTP- or GDP-bound forms, which are associated with distinct structural conformations of the protein. Here, we show that the guanine nucleotide-bound state of eEF1A regulates its ability to activate SK1, with eEF1A.GDP, but not eEF1A.GTP, enhancing SK1 activity in vitro. Furthermore, we show that enhancing cellular eEF1A.GDP levels through expression of a guanine nucleotide dissociation inhibitor of eEF1A, translationally controlled tumour protein (TCTP), increased SK1 activity in cells. We also examined a truncated isoform of eEF1A1, termed prostate tumour inducer-1 (PTI-1), which can induce neoplastic cell transformation through undefined mechanisms. PTI-1 lacks the G protein domain of eEF1A1 and is therefore unable to undergo the GTP-binding-induced conformational change. Notably, we found that PTI-1 can directly activate SK1 and that this seems to be essential for neoplastic transformation induced by PTI-1, as chemical SK1 inhibitors or overexpression of a dominant-negative SK1 blocked this process. Thus, this study defines the mechanism regulating eEF1A-mediated SK1 activation, and also establishes SK1 as being integral for PTI-1-induced oncogenesis.

A point mutant of human sphingosine kinase 1 with increased catalytic activity.

Sphingosine kinase (SK) catalyses the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in mediating such fundamental biological processes as cell growth and survival. Very little is currently known regarding the structure or mechanisms of catalysis and activation of SK. Here we have tested the functional importance of Gly(113), a highly conserved residue of human sphingosine kinase 1 (hSK), by site-directed mutagenesis. Surprisingly, a Gly(113)-->Ala substitution generated a mutant that had 1.7-fold greater catalytic activity than wild-type hSK (hSK(WT)). Our data suggests that the Gly(113)-->Ala mutation increases catalytic efficiency of hSK, probably by inducing a conformational change that increases the efficiency of phosphoryl transfer. Interestingly, hSK(G113A) activity could be stimulated in HEK293T cells by cell agonists to a comparable extent to hSK(WT).

An oncogenic role of sphingosine kinase.

Sphingosine kinase (SphK) is a highly conserved lipid kinase that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P). S1P/SphK has been implicated as a signalling pathway to regulate diverse cellular functions [1-3], including cell growth, proliferation and survival [4-8]. We report that cells overexpressing SphK have increased enzymatic activity and acquire the transformed phenotype, as determined by focus formation, colony growth in soft agar and the ability to form tumours in NOD/SCID mice. This is the first demonstration that a wild-type lipid kinase gene acts as an oncogene. Using a chemical inhibitor of SphK, or an SphK mutant that inhibits enzyme activation, we found that SphK activity is involved in oncogenic H-Ras-mediated transformation, suggesting a novel signalling pathway for Ras activation. The findings not only point to a new signalling pathway in transformation but also to the potential of SphK inhibitors in cancer therapy.

Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes.

Sphingosine 1-phosphate (S1P) is a novel lipid messenger that has important roles in a wide variety of mammalian cellular processes including growth, differentiation and death. Basal levels of S1P in mammalian cells are generally low, but can increase rapidly and transiently when cells are exposed to mitogenic agents and other stimuli. This increase is largely due to increased activity of sphingosine kinase (SK), the enzyme that catalyses its formation. In the current study we have purified, cloned and characterized the first human SK to obtain a better understanding of its biochemical activity and possible activation mechanisms. The enzyme was purified to homogeneity from human placenta using ammonium sulphate precipitation, anion-exchange chromatography, calmodulin-affinity chromatography and gel-filtration chromatography. This resulted in a purification of over 10(6)-fold from the original placenta extract. The enzyme was cloned and expressed in active form in both HEK-293T cells and Escherichia coli, and the recombinant E. coli-derived SK purified to homogeneity. To establish whether post-translational modifications lead to activation of human SK activity we characterized both the purified placental enzyme and the purified recombinant SK produced in E. coli, where such modifications would not occur. The premise for this study was that post-translational modifications are likely to cause conformational changes in the structure of SK, which may result in detectable changes in the physico-chemical or catalytic properties of the enzyme. Thus the enzymes were characterized with respect to substrate specificity and kinetics, inhibition kinetics and various other physico-chemical properties. In all cases, both the native and recombinant SKs displayed remarkably similar properties, indicating that post-translational modifications are not required for basal activity of human SK.

Expression of a catalytically inactive sphingosine kinase mutant blocks agonist-induced sphingosine kinase activation. A dominant-negative sphingosine kinase.

Sphingosine kinase (SK) catalyzes the formation of sphingosine 1-phosphate (S1P), a lipid messenger that plays an important role in a variety of mammalian cell processes, including inhibition of apoptosis and stimulation of cell proliferation. Basal levels of S1P in cells are generally low but can increase rapidly when cells are exposed to various agonists through rapid and transient activation of SK activity. To date, elucidation of the exact signaling pathways affected by these elevated S1P levels has relied on the use of SK inhibitors that are known to have direct effects on other enzymes in the cell. Furthermore, these inhibitors block basal SK activity, which is thought to have a housekeeping function in the cell. To produce a specific inhibitor of SK activation we sought to generate a catalytically inactive, dominant-negative SK. This was accomplished by site-directed mutagenesis of Gly(82) to Asp of the human SK, a residue identified through sequence similarity to the putative catalytic domain of diacylglycerol kinase. This mutant had no detectable SK activity when expressed at high levels in HEK293T cells. Activation of endogenous SK activity by tumor necrosis factor-alpha (TNFalpha), interleukin-1beta, and phorbol esters in HEK293T cells was blocked by expression of this inactive sphingosine kinase (hSK(G82D)). Basal SK activity was unaffected by expression of hSK(G82D). Expression of hSK(G82D) had no effect on TNFalpha-induced activation of protein kinase C and sphingomyelinase activities. Thus, hSK(G82D) acts as a specific dominant-negative SK to block SK activation. This discovery provides a powerful tool for the elucidation of the exact signaling pathways affected by elevated S1P levels following SK activation. To this end we have employed the dominant-negative SK to demonstrate that TNFalpha activation of extracellular signal-regulated kinases 1 and 2 (ERK1,2) is dependent on SK activation.

The tricarboxylic acid cycle of Helicobacter pylori.

The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter pylori were investigated in situ and in cell extracts using [1H]- and [13C]-NMR spectroscopy and spectrophotometry. NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase, and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle, including alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, and succinate dehydrogenase also are shown. The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and alpha-ketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of alpha-ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational glyoxylate bypass, owing to the absence of isocitrate lyase activity; nor a gamma-aminobutyrate shunt, owing to the absence of both gamma-aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino acid sequences with those of other, more extensively studied enzymes.

Stereochemical course of hydrolysis catalysed by alpha-L-rhamnosyl and alpha-D-galacturonosyl hydrolases from Aspergillus aculeatus.

The stereochemical course of hydrolysis catalysed by four Aspergillus aculeatus enzymes acting on alpha-L-rhamnosyl and alpha-D-galacturonosyl linkages in the hairy regions of pectins has been determined using 1H-NMR. Exogalacturonase acts with inversion of anomeric configuration (e-->a), shown by the initial release of beta-D-GalpA from the non-reducing end of polygalacturonic acid. Similarly, rhamnogalacturonan (RG) hydrolase also acts with inversion of anomeric configuration (e-->a) during hydrolysis of alpha-D-GalpA-(1-->2)-alpha-L-Rhap linkages in RG, initially releasing oligosaccharides with beta-D-GalpA at the reducing end. This result is consistent with the recently solved crystal structure of this enzyme, as well as its classification based on amino acid sequence similarity into glycosyl hydrolase family 28. alpha-L-Rhamnosidase and RG-rhamnohydrolase also act with inversion of configuration (a-->e), initially releasing beta-L-Rhap from p-nitrophenyl alpha-L-rhamnopyranoside and RG oligosaccharides, respectively. Thus, all four enzymes examined are inverting hydrolases which probably catalyse hydrolysis via single displacement mechanisms.

Action patterns and mapping of the substrate-binding regions of endo-(1-->5)-alpha-L-arabinanases from Aspergillus niger and Aspergillus aculeatus.

The substrate binding sites of endo-(1-->5)-alpha-L-arabinanases (EC 3.2.1.99) from Aspergillus niger and Aspergillus aculeatus were investigated using reduced and regular (1-->5)-alpha-L-arabino-oligosaccharides and high performance anion exchange chromatographic analysis. Calculation of bond cleavage frequencies and kcat/K(m) parameters for these substrates enabled the determination of the number of arabinofuranosyl binding subsites and the estimation of the binding affinities of each subsite. The A. aculeatus endo-arabinanase has six subsites arranged symmetrically around the catalytic site, while the A. niger endo-arabinanase has five subsites; two from the catalytic site towards the non-reducing end of the bound substrate and three toward the reducing end. The two subsites directly adjacent to the catalytic sites in both the A. niger and A. aculeatus endo-arabinanase have near-zero net free energy of binding. These results are unlike most glycopyranosyl endo-hydrolases studied which have net negative (unfavourable) energies of interaction at these two subsites, and may be related to the greater conformational flexibility of arabinofuranosyl residues than glycopyranosyl residues. The complete subsite maps are also rationalized with regard to the observed action patterns of these enzymes on linear (1-->5)-alpha-L-arabinan.

Purification and characterization of an extracellular beta-glucosidase from the filamentous fungus Acremonium persicinum and its probable role in beta-glucan degradation.

A beta-glucosidase from the culture filtrates of the filamentous fungus Acremonium persicinum has been purified by (NH4)2SO4 precipitation followed by anion-exchange and gel filtration chromatography. SDS-PAGE of the purified enzyme gave a single band with an apparent molecular mass of 128 kDa. The enzyme is a monomeric protein with an isoelectric point of 4.3 and a pH optimum of 5.5. Comparison of the N-terminal amino acid sequence revealed similarities between the A. persicinum enzyme and several other extracellular fungal beta-glucosidases including those from Trichoderma reesei, Aspergillus aculeatus, Saccharomycopsis fibuligera, and Pichia anomala. In addition to the hydrolysis of p-nitrophenyl-beta-glucoside, the enzyme was also active against several other aryl-beta-glucosides as well as a range of beta-linked oligoglucosides including laminaribiose, gentiobiose, cellobiose, and sophorose. D-Glucono-1,5-lactone and glucose are competitive inhibitors while the enzyme was also inhibited by N-bromosuccinimide, N-acetylimidazole, dicyclohexyl carbodiimide, Woodward's Reagent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4, and some metal ions. Possible roles for this enzyme in the noncellulolytic fungus A. persicinum are discussed in light of the increase in the rate of reducing sugar release from beta-glucans by (1-->3)- and (1-->6)-beta-glucanases when the beta-glucosidase is also present in the reaction mixtures.

Proteolytic inactivation of an extracellular (1-->3)-beta-glucanase from the fungus Acremonium persicinum is associated with growth at neutral or alkaline medium pH.

The filamentous fungus Acremonium persicinum released high levels of proteolytic enzyme activity into the culture fluid during growth at pH 7 or above. Almost total inhibition of this crude activity by phenylmethylsulfonyl fluoride suggested that it was mainly due to the presence of a serine protease. This protease inactivated one of three extracellular (1-->3)-beta-glucanases produced by this fungus, although the activities of the remaining two (1-->3)-beta-glucanases did not appear to be affected. Growth of A. persicinum in acidic conditions resulted in the presence of much lower extracellular proteolytic activity and no apparent (1-->3)-beta-glucanase inactivation.

Stereochemical course of hydrolysis catalyzed by arabinofuranosyl hydrolases.

The stereochemical course of hydrolysis catalyzed by various enzymes acting on arabinofuranosyl linkages has been determined. 1H-NMR analysis of the action of endo-(1-->5)-alpha-L-arabinanases from Aspergillus niger and Aspergillus aculeatus showed that both hydrolyze linear arabinan with inversion of configuration, and may therefore act via a single displacement mechanism. This is consistent with the A. niger enzyme's classification in glycosyl hydrolase family 43. The catalytic mechanisms of alpha-L-arabinofuranosidases from A. niger, A. aculeatus, Aspergillus awamori, Humicola insolens, Penicillium capsulatum and Bacillus subtilis were investigated using both 1H-NMR and high performance anion exchange chromatography to follow glycosyl transfer reactions to methanol. In all cases these enzymes catalyzed the reaction with retention of configuration, and therefore probably operate via double displacement hydrolytic mechanisms. From the results with arabinofuranosidase A and B from A. niger we predict that all members of glycosyl hydrolase family 51 and 54 catalyze hydrolysis with net retention of anomeric configuration. Similar studies with (1-->4)-beta-D-arabinoxylan arabinohydrolases from A. awamori, Trichoderma reesei and Bifidobacterium adolescentis only enabled their tentative classification as inverting enzymes on the basis of their lack of glycosyl transfer to methanol.

Purification and characterization of an extracellular (1 --> 6)-beta-glucanase from the filamentous fungus Acremonium persicinum.

An endo-(1 --> 6)-beta-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 --> 6)-beta-glucans (pustulan and lutean), initially yielding a series of (1 --> 6)-beta-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as beta-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent bydrolysis of (1 --> 6)-beta- and some (1 --> 3)-beta-linkages in this substrate. K(m) values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Reagent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.

Purification and characterization of three extracellular (1-->3)-beta-D-glucan glucohydrolases from the filamentous fungus Acremonium persicinum.

Three (1-->3)-beta-D-glucanases (GNs) were isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. Homogeneity of the purified proteins was confirmed by SDS/PAGE, isoelectric focusing and N-terminal amino acid sequencing. All three GNs (GN I, II and III) are non-glycosylated, monomeric proteins with apparent molecular masses, estimated by SDS/PAGE, of 81, 85 and 89 kDa respectively. pI values for the three enzymes are 5.3, 5.1, and 4.4 respectively. The pH optimum for GN I is 6.5, and 5.0 for GN II and III. All three purified enzymes displayed stability over the pH range 4.5-10.0. Optimum activities for GN I, II and III were recorded at 65, 55 and 60 degrees C respectively, with both GN II and III having short-term stability up to 50 degrees C and GN I up to 55 degrees C. The purified GNs have high specificity for (1-->3)-beta-linkages and hydrolysed a range of (1-->3)-beta- and (1-->3)(1-->6)-beta-D-glucans, with laminarin from Laminaria digitata being the most rapidly hydrolysed substrate of those tested. K(m) values for GN I, II, and III against L. digitata laminarin were 0.1, 0.23 and 0.22 mg/ml respectively. D-Glucono-1,5-lactone does not inhibit any of the three GNs, some metals ions are mild inhibitors, and N-bromosuccinimide and KMnO4 are strong inhibitors. All three GNs acted in an exo-hydrolytic manner, determined by the release of alpha-glucose as the initial and major product of hydrolysis of (1-->3)-beta-D-glucans, and confirmed by viscometric analysis and the inability to cleave periodate-oxidized laminarin, and may be classified as (1-->3)-beta-D-glucan glucohydrolases (EC 3.2.1.58).

Noncellulolytic fungal beta-glucanases: their physiology and regulation.

The occurrence, regulation, and action of fungal enzymes capable of degrading noncellulosic beta-glucans, especially 1,3-beta- and 1,6-beta-glucans, are reviewed. Special consideration is given to their roles in both metabolic and morphogenetic events in the fungal cell, including cell wall extension, hyphal branching, sporulation, budding, and autolysis. Also examined are the protocols currently available for their purification, with some of the properties of purified beta-glucanases discussed in terms of their potential applications in industrial, agricultural, and medical fields.