Allosteric activation of PTEN phosphatase by phosphatidylinositol 4,5-bisphosphate

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor that is lost in many human tumors and encodes a phosphatidylinositol phosphate phosphatase specific for the 3-position of the inositol ring. Here we report a novel mechanism of PTEN regulation. Binding of di-C8-phosphatidylinositol 4,5-P2 (PI(4,5)P2) to PTEN enhances phosphatase activity for monodispersed substrates, PI(3,4,5)P3 and PI(3,4)P2. PI(5)P also is an activator, but PI(4)P, PI(3,4)P2, and PI(3,5)P2 do not activate PTEN. Activation by exogenous PI(4,5)P2 is more apparent with PI(3,4)P2 as a substrate than with PI(3,4,5)P3, probably because hydrolysis of PI(3,4)P2 yields PI(4)P, which is not an activator. In contrast, hydrolysis of PI(3,4,5)P3 yields a potent activator, PI(4,5)P2, creating a positive feedback loop. In addition, neither di-C4-PI(4,5)P2 nor inositol trisphosphate-activated PTEN. Hence, the interaction between PI(4,5)P2 and PTEN requires specific, ionic interactions with the phosphate groups on the inositol ring as well as hydrophobic interactions with the fatty acid chains, likely mimicking the physiological interactions that PTEN has with the polar surface head groups and the hydrophobic core of phospholipid membranes. Mutations of the apparent PI(4,5)P2-binding motif in the PTEN N terminus severely reduced PTEN activity. In contrast, mutation of the C2 phospholipid-binding domain had little effect on PTEN activation. These results suggest a model in which a PI(4,5)P2 monomer binds to PTEN, initiates an allosteric conformational change and, thereby, activates PTEN independent of membrane binding.     

Dissection of the steps of phospholipase C beta 2 activity that are enhanced by G beta gamma subunits

Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze the hydrolysis of phosphatidylinositol 4,5 bisphosphate in a two step reaction that involves a cyclic intermediate. The PLCbetafamily are activated by both the alpha and betagamma subunits of heterotrimeric G proteins. To determine which catalytic step is affected by Gbetagamma subunits, we compared the change in PLCbeta(2) activity catalysis toward monomeric short-chain phosphatidylinositol (PI) substrates and monomeric water-soluble cyclic inositol phosphates as well as long-chain PI in bilayer and micellar interfaces in the absence and presence of Gbetagammasubunits. Unlike other PLC enzymes, no cyclic products were detected for either wild-type PLCbeta(2) or a chimeric protein composed of the PH domain of PLCbeta(2) and the catalytic domain of PLCdelta(1). Using cIP as a substrate to examine the second step of the reaction, we found that the presence of Gbetagamma subunits stimulated this step by a higher level than that for the overall reaction (k(cat) 1.5-fold (cIP) as opposed to 1.20-fold for soluble diC(4)PI). Detergents above their CMC can generate the same kinetic activation of PLCbeta(2) as Gbetagamma, suggesting that hydrophobic compounds stabilize the activated state of the enzyme. The most pronounced effect of Gbetagamma is that it relieves competitive product inhibition. Taken together, our results show that activation of PLCbeta(2) occurs through enhancement in the catalytic rate of hydrolysis of the cyclic intermediate and increased product release, and that hydrophobic interactions play a key role.     

Kinetic analysis of the formation of inositol 1:2-cyclic phosphate in carbachol-stimulated pancreatic minilobules. Half is formed by direct phosphodiesteratic cleavage of phosphatidylinositol

The issue as to whether there is direct phosphodiesteratic cleavage of phosphatidylinositol (PI), in addition to that of phosphatidylinositol 4,5-bisphosphate (PIP2), on agonist stimulation of cells has been controversial. In an attempt to resolve this issue, we have studied the kinetics of the formation and breakdown of the cyclic inositol phosphates. This approach is fairly straightforward, since the turnover of the cyclic inositol phosphates is very slow as compared to that of the noncyclic inositol phosphates and proceeds from inositol 1:2-cyclic 4,5-trisphosphate to inositol 1:2-cyclic phosphate (I(c1:2)P) directly by dephosphorylation without any branching pathways, in contrast to the multiple branchpoints of the noncyclic inositol phosphate pathway. Mouse pancreatic minilobules were prelabeled with [3H]inositol for 30 min, followed by washing to remove free inositol. They were then stimulated with carbachol for 30 min. The inositol cyclic polyphosphates reached steady state at 10-15 min, and I(c1:2)P reached steady state at 25 min. We blocked the action of carbachol by addition of an excess of atropine at 30 min, and the rate of disappearance of the three cyclic inositol phosphates was measured. From these data, the contribution of the inositol cyclic polyphosphate pathway to I(c1:2)P was calculated, which was 40-50% of total I(c1:2)P formation. Thus, 40-50% of the I(c1:2)P formed must have been derived from direct phosphodiesteratic cleavage of PI. This approach should prove useful in measuring the relative contributions of PI hydrolysis and PI phosphorylation (phosphatidylinositol 4,5-bisphosphate hydrolysis) in the overall PI cascade.     

Regulation of gating and rundown of HCN hyperpolarization-activated channels by exogenous and endogenous PIP2

The voltage dependence of activation of the HCN hyperpolarization-activated cation channels is shifted in inside-out patches by -40 to -60 mV relative to activation in intact cells, a phenomenon referred to as rundown. Less than 20 mV of this hyperpolarizing shift can be due to the influence of the canonical modulator of HCN channels, cAMP. Here we study the role of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in HCN channel rundown, as hydrolysis of PI(4,5)P(2) by lipid phosphatases is thought to underlie rundown of several other channels. We find that bath application of exogenous PI(4,5)P(2) reverses the effect of rundown, producing a large depolarizing shift in HCN2 activation. A synthetic short chain analogue of PI(4,5)P(2), dioctanoyl phosphatidylinositol 4,5-bisphosphate, shifts the HCN2 activation curve to more positive potentials in a dose-dependent manner. Other dioctanoyl phosphatidylinositides with one or more phosphates on the lipid headgroup also shift activation, although phosphatidylinositol (PI) is ineffective. Several lines of evidence suggest that HCN2 is also regulated by endogenous PI(4,5)P(2): (a) blockade of phosphatases slows the hyperpolarizing shift upon patch excision; (b) application of an antibody that binds and depletes membrane PIP(2) causes a further hyperpolarizing shift in activation; (c) the shift in activation upon patch excision can be partially reversed by MgATP; and (d) the effect of MgATP is blocked by wortmannin, an inhibitor of PI kinases. Finally, recordings from rabbit sinoatrial cells demonstrate that diC(8) PI(4,5)P(2) delays the rundown of native HCN currents. Thus, both native and recombinant HCN channels are regulated by PI(4,5)P(2).     

Allosteric interactions within subsites of a monomeric enzyme: kinetics of fluorogenic substrates of PI-specific phospholipase C

Two novel water-soluble fluorescein myo-inositol phosphate (FLIP) substrates, butyl-FLIP and methyl-FLIP, were used to examine the kinetics and subsite interactions of Bacillus cereus phosphatidylinositol-specific phospholipase C. Butyl-FLIP exhibited sigmoidal kinetics when initial rates are plotted versus substrate concentration. The data fit a Hill coefficient of 1.2-1.5, suggesting an allosteric interaction between two sites. Two substrate molecules bind to this enzyme, one at the active site and one at a subsite, causing an increase in activity. The kinetic behavior is mathematically similar to that of well-known cooperative multimeric enzymes even though this phosphatidylinositol-specific phospholipase C is a small, monomeric enzyme. The less hydrophobic substrate, methyl-FLIP, binds only to the active site and not the activator site, and thus exhibits standard hyperbolic kinetics. An analytical expression is presented that accounts for the kinetics of both substrates in the absence and presence of a nonsubstrate short-chain phospholipid, dihexanoylphosphatidylcholine. The fluorogenic substrates detect activation at much lower concentrations of dihexanoylphosphatidylcholine than previously reported.     

Uptake and phosphorylation of phosphatidylinositol by rat liver nuclei. Role of phosphatidylinositol transfer protein

The incorporation of phosphatidyl[2-3H]inositol ([3H]PI) from vesicles or microsomal membranes into rat liver nuclei is greatly stimulated by phosphatidylinositol transfer protein (PI-TP). The nuclei are able to phosphorylate [3H]PI, with the production of phosphatidylinositol 4-phosphate (PIP). Recovery of tritiated inositol trisphosphate, inositol phosphate, glycerophosphoinositol and inositol, suggests that in isolated nuclei a large set of enzymes of the PI cycle is present, similar to the enzymes involved in the plasma membrane PI cycle. Incubation with [gamma-32P]ATP shows that isolated nuclei are able to phosphorylate endogenous PI to PIP and phosphatidylinositol 4,5-bisphosphate (PIP2). In the presence of exogenous PI and detergent the synthesis of PIP is increased, indicating that in nuclei the PI pool is suboptimal for the PI-kinase activity. The present study suggests that PI-TP may be involved in providing substrates for PI metabolism at the nuclear level.     

Hydrolysis of inositol phosphates by plant cell extracts.

A gel-filtered soluble fraction prepared from suspension-cultured Nicotiana tabacum cells hydrolysed inositol mono-, bis- and tris-phosphates. At a concentration of 7.5 microM the rates of hydrolysis followed the sequence Ins(1,4,5)P3 greater than Ins(1,4)P2 greater than Ins(4)P congruent to Ins(1)P. The major products of Ins(1,4,5)P3 hydrolysis identified by h.p.l.c. were Ins(1,4)P2 and Ins(4,5)P2. Ins(1,4)P2 was hydrolysed exclusively to Ins(4)P. The inclusion of Ca2+ in the incubation buffer markedly stimulated the hydrolysis of all the inositol phosphate substrates. Under identical conditions, Ca2+ inhibited the hydrolysis of inositol phosphates by soluble extracts prepared from rat brain. Half-maximal stimulation of Ins(1,4)P2 hydrolysis was obtained at free [Ca2+] of 0.6 and 1.2 microM when the Mg2+ concentration in the incubations was 0.3 and 1.0 mM respectively. This effect of Ca2+ was exerted solely by increasing the Vmax. of hydrolysis without affecting the Km for Ins(1,4)P2. Again, in contrast with brain, the hydrolysis of inositol bis- or mono-phosphates was insensitive to high concentrations of Li+. We conclude that plants contain specific Li+-insensitive inositol phosphate phosphatases that are regulated by low concentrations of Ca2+ in a manner which is different from that observed in mammalian tissues.     

Phorbol esters and dioctanoylglycerol block anti-IgM-stimulated phosphoinositide hydrolysis in the murine B cell lymphoma WEHI-231

Cross-linking of membrane IgM (mIgM) on both normal resting B cells and on the murine B cell lymphoma WEHI-231 activates the phosphoinositide signal transduction pathway. The initial event in this pathway is the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2), which results in the generation of two second-messengers: inositol trisphosphate (InsP3), which can cause the release of Ca2+ from intracellular stores, and diacylglycerol (DG), which activates protein kinase C. In examining the effects of exogenous activation of protein kinase C on WEHI-231 cells, we found that phorbol esters blocked some of the biologic effects of anti-IgM on WEHI-231 cells. The mechanism of this effect was investigated. Phorbol ester treatment of WEHI-231 cells blocked the ability of anti-IgM to stimulate production of inositol phosphates and accumulation of phosphatidic acid, the phosphorylated product of DG. Phorbol esters also blocked the ability of anti-IgM to cause an increase in intracellular Ca2+. Thus, it is clear that phorbol esters block anti-IgM-stimulated PtdInsP2 hydrolysis in WEHI-231 cells. In addition, a synthetic DG, dioctanoylglycerol (diC8), also blocked anti-IgM-stimulated inositol phosphate production and the anti-IgM-stimulated rise in cytoplasmic Ca2+. The ability of phorbol esters and diC8 to block mIgM-mediated signaling may reflect a feedback inhibition mechanism by which activated protein kinase C limits the magnitude and duration of receptor signaling.     

Characterization of a Polyphosphoinositide Phospholipase C from the Plasma Membrane of Avena sativa

A phosphoinositide-specific phospholipase C activity was identified in oat root (Avena sativa, cv Victory) plasma membranes purified by separation in an aqueous two-phase polymer system. The enzyme is highly active toward inositol phospholipids but only minimally active toward phosphatidylethanolamine and phosphatidylcholine. Activity approaches maximal levels at 200 micromolar phosphatidylinositol 4-phosphate (PIP) and is highly dependent on calcium; it is inhibited by 1 millimolar EGTA and is activated by calcium with an apparent activation constant of 2 micromolar. At 10 micromolar calcium and 200 micromolar inositol phospholipid, the enzyme is specific for phosphatidylinositol 4,5-bisphosphate (PIP₂) and PIP, which are hydrolyzed at 10 and 4 times, respectively, the rate of phosphatidylinositol (PI) hydrolysis. The principle water soluble products of hydrolysis, as determined by high performance liquid chromatography, are inositol 1,4,5-trisphosphate from PIP₂, inositol 1,4-bisphosphate from PIP, and inositol phosphate from PI.     

Identification of multiple phosphoinositide-specific phospholipases D as new regulatory enzymes for phosphatidylinositol 3,4, 5-trisphosphate

In the course of delineating the regulatory mechanism underlying phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) metabolism, we have discovered three distinct phosphoinositide-specific phospholipase D (PI-PLD) isozymes from rat brain, tentatively designated as PI-PLDa, PI-PLDb, and PI-PLDc. These enzymes convert [3H]PI(3,4,5)P3 to generate a novel inositol phosphate, D-myo-[3H]inositol 3,4,5-trisphosphate ([3H]Ins(3,4,5)P3) and phosphatidic acid. These isozymes are predominantly associated with the cytosol, a notable difference from phosphatidylcholine PLDs. They are partially purified by a three-step procedure consisting of DEAE, heparin, and Sephacryl S-200 chromatography. PI-PLDa and PI-PLDb display a high degree of substrate specificity for PI(3,4, 5)P3, with a relative potency of PI(3,4,5)P3 > phosphatidylinositol 3-phosphate (PI(3)P) or phosphatidylinositol 4-phosphate (PI(4)P) > phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) > phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). In contrast, PI-PLDc preferentially utilizes PI(3)P as substrate, followed by, in sequence, PI(3,4,5)P3, PI(4)P, PI(3,4)P2, and PI(4,5)P2. Both PI(3, 4)P2 and PI(4,5)P2 are poor substrates for all three isozymes, indicating that the regulatory mechanisms underlying these phosphoinositides are different from that of PI(3,4,5)P3. None of these enzymes reacts with phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine. All three PI-PLDs are Ca2+-dependent. Among them, PI-PLDb and PI-PLDc show maximum activities within a sub-microM range (0.3 and 0.9 microM Ca2+, respectively), whereas PI-PLDa exhibits an optimal [Ca2+] at 20 microM. In contrast to PC-PLD, Mg2+ has no significant effect on the enzyme activity. All three enzymes require sodium deoxycholate for optimal activities; other detergents examined including Triton X-100 and Nonidet P-40 are, however, inhibitory. In addition, PI(4,5)P2 stimulates these isozymes in a dose-dependent manner. Enhancement in the enzyme activity is noted only when the molar ratio of PI(4,5)P2 to PI(3,4, 5)P3 is between 1:1 and 2:1.     

The inositol pyrophosphate pathway in health and diseases

Inositol pyrophosphates (IPPs) are present in organisms ranging from plants, slime moulds and fungi to mammals. Distinct classes of kinases generate different forms of energetic diphosphate‐containing IPPs from inositol phosphates (IPs). Conversely, polyphosphate phosphohydrolase enzymes dephosphorylate IPPs to regenerate the respective IPs. IPPs and/or their metabolizing enzymes regulate various cell biological processes by modulating many proteins via diverse mechanisms. In the last decade, extensive research has been conducted in mammalian systems, particularly in knockout mouse models of relevant enzymes. Results obtained from these studies suggest impacts of the IPP pathway on organ development, especially of brain and testis. Conversely, deletion of specific enzymes in the pathway protects mice from various diseases such as diet‐induced obesity (DIO), type‐2 diabetes (T2D), fatty liver, bacterial infection, thromboembolism, cancer metastasis and aging. Furthermore, pharmacological inhibition of the same class of enzymes in mice validates the therapeutic importance of this pathway in cardio‐metabolic diseases. This review critically analyses these findings and summarizes the significance of the IPP pathway in mammalian health and diseases. It also evaluates benefits and risks of targeting this pathway in disease therapies. Finally, future directions of mammalian IPP research are discussed.     

Molecular characterization of At5PTase1, an inositol phosphatase capable of terminating inositol trisphosphate signaling

The inositol triphosphate (IP(3))-signaling pathway has been associated with several developmental and physiological processes in plants, but we currently know little about the regulation of this pathway. Inositol 5' phosphatases (5PTases) are enzymes that remove a 5' phosphate from several potential second messengers, including IP(3). In catalyzing the removal of a 5' phosphate from second messenger substrates, 5PTases can act to terminate signal transduction events. We describe the molecular analysis of At5PTase1, a 5PTase gene from Arabidopsis. When expressed transiently in Arabidopsis leaf tissue or ectopically in transgenic plants, At5PTase1 allowed for the increased hydrolysis of I(1,4,5)P(3) and I(1,3,4,5)P(4) substrates. At5PTase1 did not hydrolyze I(1)P, I(1,4)P(2), or PI(4,5)P(2) substrates. This substrate specificity was similar to that of the human Type I 5PTase. We identified 14 other potential At5PTase genes and constructed an unrooted phylogenetic tree containing putative Arabidopsis, human, and yeast 5PTase proteins. This analysis indicated that the Arabidopsis 5PTases were grouped in two separate branches of the tree. The multiplicity of At5PTases indicates that these enzymes may have different substrate specificities and play different roles in signal termination in Arabidopsis.     

Polyamines, PI(4,5)P2, and actin polymerization

Spermine (SPM) and spermidine (SPD) activate isolated phosphatidylinositol-4-phosphate 5-kinases (PI(4)P5K), enzymes that convert phosphatidylinositol-4-phosphate to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). PI(4,5)P2 formation is known to be involved in cellular actin reorganization and motility, functions that are also influenced by polyamines. It has not been proven that endogenous polyamines can control inositol phospholipid metabolism. We evoked large decreases in SPD and putrescine (PUT) contents in HL60 cells, using the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine (DFMO), which resulted in decreases in PI(4,5)P2 content per cell and inositol phosphate formation to 76.9 +/- 3.5% and 81.5 +/- 4.0% of control, respectively. Accurately reversing DFMO-evoked decreases in SPD content by incubating cells with exogenous SPD for 20 min rescued these decreases. DFMO treatment and SPD rescues also changed the ratio of total cellular PI(4,5)P2 to PIP suggesting involvement of a SPD-sensitive PI(4)P5K. PUT and SPM were not involved in DFMO-evoked changes in cellular PI(4,5)P2 contents. In DFMO-treated HL60 cells, the percent of total actin content that was filamentous was decreased to 59.1 +/- 5.8% of that measured in paired control HL60 cells, a finding that was rescued following reversal of DFMO-evoked decreases in SPD and PI(4,5)P2 contents. In slowly proliferating DMSO-differentiated HL60 cells, inositol phospholipid metabolism was uncoupled from SPD control. We conclude: in rapidly proliferating HL60 cells, but not in slowly proliferating differentiated HL60 cells, there are endogenous SPD-sensitive PI(4,5)P2 pools, probably formed via SPD-sensitive PI(4)P5K, that likely control actin polymerization.     

Phosphatidylinositol 3,4,5-trisphosphate is a substrate for the 75 kDa inositol polyphosphate 5-phosphatase and a novel 5-phosphatase which forms a complex with the p85/p110 form of phosphoinositide 3-kinase.

Agonist-stimulated production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], is considered the primary output signal of activated phosphoinositide (PI) 3-kinase. The physiological targets of this novel phospholipid and the identity of enzymes involved in its metabolism have not yet been established. We report here the identification of two enzymes which hydrolyze the 5-position phosphate of PtdIns(3,4,5)P3, forming phosphatidylinositol (3,4)-bisphosphate. One of these enzymes is the 75 kDa inositol polyphosphate 5-phosphatase (75 kDa 5-phosphatase), which has previously been demonstrated to metabolize inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. We have identified a second PtdIns(3,4,5)P3 5-phosphatase in the cytosolic fraction of platelets, which forms a complex with the p85/p110 form of PI 3-kinase. This enzyme is immunologically and chromatographically distinct from the platelet 43 kDa and 75 kDa 5-phosphatases and is unique in that it removes the 5-position phosphate from PtdIns(3,4,5)P3, but does not metabolize PtdIns(4,5)P2, Ins(1,4,5)P3 or Ins(1,3,4,5)P4. These studies demonstrate the existence of multiple PtdIns(3,4,5)P3 5-phosphatases within the cell.     

Activation of protein kinase C in rat basophilic leukemia cells stimulates increased production of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate: correlation with actin polymerization.

Cross-linking of the immunoglobulin E receptor on rat basophilic leukemia (RBL)1 cells by multivalent antigen activates phosphatidylinositol (PI) kinase and phosphatidylinositol 4-phosphate (PIP) kinase leading to the increased production of PIP and phosphatidylinositol 4,5-bisphosphate (PIP2). Activators of protein kinase C (PKC), such as phorbol myristate acetate (PMA) and the synthetic diacylglycerol, 1,2-dioctanoyl-sn-glycerol (diC8), were found to have the same effect even though PMA and diC8 do not cause the activation of phospholipase C. Although the kinetics are different depending on the stimulant, activation of PKC using multivalent antigen, PMA or diC8 also causes the polymerization of actin and an increase in the F-actin content of the cells. In all cases, a good correlation was observed between F-actin levels, activation of PI and PIP kinases, and the increased production of PIP and PIP2. However, in the case of antigen, there is no correlation between actin polymerization and the total amount of PIP and PIP2. Staurosporine, an inhibitor of protein kinases, blocks the F-actin response and the increased synthesis of PIP and PIP2 with similar dose dependencies. Furthermore, depletion of PKC activity through long-term exposure to PMA, inhibited both the F-actin response and the increased synthesis of PIP and PIP2 induced by either DNP-BSA or diC8. These results suggest that activation of PKC precedes the activation of PI and PIP kinases and that under certain circumstances activation of the kinases and the increased synthesis of PIP and PIP2 may be involved in the polymerization of actin in RBL cells, possibly through the interaction of the polyphosphoinositides with actin-binding proteins such as gelsolin and profilin.     

Emerging cell biological functions of phosphatidylinositol 5 phosphate 4 kinase

The generation of phosphoinositides (PIs) with spatial and temporal control is a key mechanism in cellular organization and signaling. The synthesis of PIs is mediated by PI kinases, proteins that are able to phosphorylate unique substrates at specific positions on the inositol headgroup to generate signaling molecules. Phosphatidylinositol 5 phosphate 4 kinase (PIP4K) is one such lipid kinase that is able to specifically phosphorylate phosphatidylinositol 5 phosphate, the most recently discovered PI to generate the well-known and abundant PI, phosphatidylinositol 4,5 bisphosphate [PI(4,5)P₂]. PIP4K appears to be encoded only in metazoan genomes, and several genetic studies indicate important physiological functions for these enzymes in metabolism, immune function, and growth control. PIP4K has recently been reported to localize to multiple cellular compartments, including the nucleus, plasma membrane, endosomal systems, and autophagosome. However, the biochemical activity of these enzymes that is relevant to these physiological functions remains elusive. We review recent developments in this area and highlight emerging roles for these enzymes in cellular organization.     

Phosphate inhibition of spinach leaf sucrose phosphate synthase as affected by glucose-6-phosphate and phosphoglucoisomerase

The inhibition patterns of inorganic phosphate (Pi) on sucrose phosphate synthase activity in the presence and absence of the allosteric activator glucose-6-P was studied, as well as the effects of phosphoglucoisomerase on fructose-6-P saturation kinetics with and without Pi. In the presence of 5 millimolar glucose-6-P, Pi was a partial competitive inhibitor with respect to both substrates, fructose-6-P and uridine diphosphate glucose. In the absence of glucose-6-P, the inhibition patterns were more complex, apparently because of the interaction of Pi at the activation site as well as the catalytic site. In addition, substrate activation by uridine diphosphate glucose was observed in the absence of effectors. The results suggested that Pi antagonizes glucose-6-P activation of sucrose phosphate synthase by competing with the activator for binding to the modifier site.

The fructose-6-P saturation kinetics were hyperbolic in the absence of phosphoglucoisomerase activity, but became sigmoidal by the addition of excess phosphoglucoisomerase. The sigmoidicity persisted in the presence of Pi, but sucrose phosphate synthase activity was decreased. The apparent sigmoidal response may represent the physiological response of sucrose phosphate synthase to a change in hexose-P concentration because sucrose phosphate synthase operates in the cytosol in the presence of high activities of phosphoglucoisomerase. Thus, the enzymic production of an activator from a substrate represents a unique mechanism for generating sigmoidal enzyme kinetics.

     

Stimulation of phosphoinositides breakdown by the heat stable E. coli enterotoxin in rat intestinal epithelial cells

Rat intestinal epithelial cells were labelled with [32P]Pi and extracted, and the phospholipids were analysed by thin-layer chromatography. 32P-incorporation in phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-phosphate (PIP2) were measured in control and heat stable enterotoxin (ST)-treated cells. ST was found to induce rapid degradation of PIP and PIP2. The degradation of inositol lipids was accompanied by an increase of water soluble inositol phosphate (IP1, IP2, IP3) compounds. There was a two-fold increase of radioactivity in IP2 and IP3 but no significant change was observed in IP1. Phospholipase C activity was increased tenfold with substrate PIP2 in ST-pretreated cells. The present study indicates that ST triggers another second messenger system by increasing the PIP2 hydrolysis with the enzyme phospholipase C.     

Kinetics, substrate specificity, and stereospecificity of two new protein tyrosine phosphatase-like inositol polyphosphatases from Selenomonas lacticifex

Inositol polyphosphatases (IPPases) play an important role in the metabolism of inositol polyphosphates, a class of molecules involved in signal transduction. Here we characterize 2 new protein tyrosine phosphatase-like IPPases (PhyAsl and PhyBsl) cloned from Selenomonas lacticifex that can hydrolyze myo-inositol hexakisphosphate (InsP6) in vitro. To determine their preferred substrates and stereospecificity of InsP6 dephosphorylation, a combination of kinetic and high-performance ion pair chromatography studies were conducted. Despite only 33% amino acid sequence identity between them, both enzymes display strict specificity for IPP substrates and cleave InsP6 primarily at the d-3-phosphate position (>90%). Furthermore, both enzymes predominantly degrade InsP6 to Ins(2)P via identical and very specific routes of dephosphorylation (3,4,5,6,1). Despite these similarities, PhylAsl is shown to have a slight kinetic preference for the major inositol pentakisphosphate intermediate in its InsP6 hydrolysis pathway, whereas PhyBsl displays a unique and substantial preference for an inositol tetrakisphosphate intermediate.