Comparative mechanistic and substrate specificity study of inositol polyphosphate 5-phosphatase Schizosaccharomyces pombe Synaptojanin and SHIP2

Inositol-5-phosphatases are important enzymes involved in the regulation of diverse cellular processes from synaptic vesicle recycling to insulin signaling. We describe a comparative study of two representative inositol-5-phosphatases, Schizosaccharomyces pombe synaptojanin (SPsynaptojanin) and human SH2 domain-containing inositol-5-phosphatase SHIP2. We show that in addition to Mg2+, transition metals such as Mn2+, Co2+, and Ni2+ are also effective activators of SPsynaptojanin. In contrast, Ca2+ and Cu2+ are inhibitory. We provide evidence that Mg2+ binds the same site occupied by Ca2+ observed in the crystal structure of SPsynaptojanin complexed with inositol 1,4-bisphosphate (Ins(1,4)P2). Ionizations important for substrate binding and catalysis are defined for the SPsynaptojanin-catalyzed Ins(1,4,5)P3 reaction. Kinetic analysis with four phosphatidylinositol lipids bearing a 5-phosphate and 54 water-soluble inositol phosphates reveals that SP-synaptojanin and SHIP2 possess much broader substrate specificity than previously appreciated. The rank order for SPsynaptojanin is Ins(2,4,5)P3 > phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) approximately Ins(4,5)P2 approximately Ins(1,4,5)P3 approximately Ins(4,5,6)P3 > PtdIns(3,5)P2 approximately PtdIns(3,4,5)P3 approximately Ins(1,2,4,5)P4 approximately Ins(1,3,4,5)P4 approximately Ins-(2,4,5,6)P4 approximately Ins(1,2,4,5,6)P5. The rank order for SHIP2 is Ins(1,2,3,4,5)P5 > Ins(1,3,4,5)P4 > PtdIns(3,4,5)P4 approximately PtdIns(3,5)P2 approximately Ins(1,4,5,6)P4 approximately Ins(2,4,5,6)P4. Because inositol phosphate isomers elicit different biological activities, the extended substrate specificity for SPsynaptojanin and SHIP2 suggest that these enzymes likely have multiple roles in cell signaling and may regulate distinct pathways. The unique substrate specificity profiles and the importance of 2-position phosphate in binding also have important implications for the design of potent and selective SPsynaptojanin and SHIP2 inhibitors for pharmacological investigation.     

The influence of anionic lipids on SHIP2 phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase activity

The SH2 domain containing inositol 5-phosphatase 2 (SHIP2) catalyzes the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃) to phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P₂) and participates in the insulin signalling pathway in vivo. In a comparative study of SHIP2 and the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), we found that their lipid phosphatase activity was influenced by the presence of vesicles of phosphatidylserine (PtdSer). SHIP2 PtdIns(3,4,5)P₃ 5-phosphatase activity was greatly stimulated in the presence of vesicles of PtdSer. This effect appears to be specific for di-C8 and di-C16 fatty acids of PtdIns(3,4,5)P₃ as substrate. It was not observed with inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P₄) another in vitro substrate of SHIP2, nor with Type I Ins(1,4,5)P₃/Ins(1,3,4,5)P₄ 5-phosphatase activity, an enzyme which acts on soluble inositol phosphates. Vesicles of phosphatidylcholine (PtdCho) stimulated only twofold PtdIns(3,4,5)P₃ 5-phosphatase activity of SHIP2. Both a minimal catalytic construct and the full length SHIP2 were sensitive to the stimulation by PtdSer. In contrast, PtdIns(3,4,5)P₃ 5-phosphatase activity of the Skeletal muscle and Kidney enriched Inositol Phosphatase (SKIP), another member of the mammaliam Type II phosphoinositide 5-phosphatases, was not sensitive to PtdSer. Our enzymatic data establish a specificity in the control of SHIP2 lipid phosphatase activity with PtdIns(3,4,5)P₃ as substrate which is depending on the fatty acid composition of the substrate.     

Identification and isolation of a 75-kDa inositol polyphosphate-5-phosphatase from human platelets

We have identified, isolated, and characterized a second inositol polyphosphate-5-phosphatase enzyme from the soluble fraction of human platelets. The enzyme hydrolyzes inositol 1,4,5-trisphosphate (Ins (1,4,5)P3) to inositol 1,4-bisphosphate (Ins(1,4)P2) with an apparent Km of 24 microM and a Vmax of 25 mumol of Ins(1,4,5)P3 hydrolyzed/min/mg of protein. The enzyme hydrolyzes inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4) at a rate of 1.3 mumol of Ins(1,3,4,5)P4 hydrolyzed/min/mg of protein with an apparent Km of 7.5 microM. The enzyme also hydrolyzes inositol 1,2-cyclic 4,5-trisphosphate (cIns(1:2,4,5)P3) and Ins(4,5)P2. We purified this enzyme 2,200-fold from human platelets. The enzyme has a molecular mass of 75,000 as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration chromatography. The enzyme requires magnesium ions for activity and is not inhibited by calcium ions. The 75-kDa inositol polyphosphate-5-phosphatase enzyme differs from the previously identified platelet inositol polyphosphate-5-phosphatase as follows: molecular size (75 kDa versus 45 kDa), affinity for Ins(1,3,4,5)P4 (Km 7.5 microM versus 0.5 microM), Km for Ins(1,4,5)P3 (24 microM versus 7.5 microM), regulation by protein kinase C, wherein the 45-kDa enzyme is phosphorylated and activated while the 75-kDa enzyme is not. The 75-kDa enzyme is inhibited by lower concentrations of phosphate (IC50 2 mM versus 16 mM for the 45-kDa enzyme) and is less inhibited by Ins(1,4)P2 than is the 45-kDa enzyme. The levels of inositol phosphates that act in calcium signalling are likely to be regulated by the interplay of these two enzymes both found in the same cell.     

Identification and characterization of a novel inositol polyphosphate 5-phosphatase

We have identified a cDNA encoding a novel inositol polyphosphate 5-phosphatase. It contains two highly conserved catalytic motifs for 5-phosphatase, has a molecular mass of 51 kDa, and is ubiquitously expressed and especially abundant in skeletal muscle, heart, and kidney. We designated this 5-phosphatase as SKIP (Skeletal muscle and Kidney enriched Inositol Phosphatase). SKIP is a simple 5-phosphatase with no other motifs. Baculovirus-expressed recombinant SKIP protein exhibited 5-phosphatase activities toward inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol (PtdIns) 4,5-bisphosphate, and PtdIns 3,4, 5-trisphosphate but has 6-fold more substrate specificity for PtdIns 4,5-bisphosphate (K(m) = 180 microM) than for inositol 1,4, 5-trisphosphate (K(m) = 1.15 mM). The ectopic expression of SKIP protein in COS-7 cells and immunostaining of neuroblastoma N1E-115 cells revealed that SKIP is expressed in cytosol and that loss of actin stress fibers occurs where the SKIP protein is concentrated. These results imply that SKIP plays a negative role in regulating the actin cytoskeleton through hydrolyzing PtdIns 4,5-bisphosphate.     

Degradation of inositol 1,3,4,5-tetrakisphosphates by porcine brain cytosol yields inositol 1,3,4-trisphosphate and inositol 1,4,5-trisphosphate

Inositol 1,3,4,5-tetrakisphosphates (Ins(1,3,4,5)P4), 32P-labelled in positions 4 and 5 were prepared enzymatically, using [4-32P]-phosphatidylinositol 4-phosphate (PtdInsP) and [5-32P]phosphatidylinositol 4,5-bisphosphate (PtdInsP2) as substrates, respectively. Degradation studies of Ins(1,3,4,5)P4, using an enriched phosphatase preparation from porcine brain cytosol, led to the formation of two inositol trisphosphate isomers which were identified as inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). This novel degradation pathway of Ins(1,3,4,5)P4 to Ins(1,4,5)P3 provides an additional source for the generation of Ins(1,4,5)P3, involving a 3-phosphatase.     

Identification and characterization of a sac domain-containing phosphoinositide 5-phosphatase

We have characterized a novel Sac domain-containing inositol phosphatase, hSac2. It was ubiquitously expressed but especially abundant in the brain, heart, skeletal muscle, and kidney. Unlike other Sac domain-containing proteins, hSac2 protein exhibited 5-phosphatase activity specific for phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. This is the first time that the Sac domain has been reported to possess 5-phosphatase activity. Its 5-phosphatase activity for phosphatidylinositol 4,5-bisphosphate (K(m) = 14.3 microm) was comparable with those of Type II 5-phosphatases. These results imply that hSac2 functions as an inositol polyphosphate 5-phosphatase.     

Underexpression of the 43 kDa inositol polyphosphate 5-phosphatase is associated with cellular transformation.

The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the second messenger molecules inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. We have underexpressed the 43 kDa 5-phosphatase by stably transfecting normal rat kidney cells with the cDNA encoding the enzyme, cloned in the antisense orientation into the tetracycline-inducible expression vector pUHD10-3. Antisense-transfected cells demonstrated a 45% reduction in Ins(1,4,5)P3 5-phosphatase activity in the total cell homogenate upon withdrawal of tetracycline, and an approximately 80% reduction in the detergent-soluble membrane fraction of the cell, as compared with antisense-transfected cells in the presence of tetracycline. Unstimulated antisense-transfected cells showed a concomitant 2-fold increase in Ins(1,4,5)P3 and 4-fold increase in Ins(1,3,4,5)P4 levels. The basal intracellular calcium concentration of antisense-transfected cells (170 +/- 25 nM) was increased 1.9-fold, compared with cells transfected with vector alone (90 +/- 25 nM). Cells underexpressing the 43 kDa 5-phosphatase demonstrated a transformed phenotype. Antisense-transfected cells grew at a 1.7-fold faster rate, reached confluence at higher density and demonstrated increased [3H]thymidine incorporation compared with cells transfected with vector alone. Furthermore, antisense-transfected cells formed colonies in soft agar and tumours in nude mice. These studies support the contention that a decrease in Ins(1,4,5)P3 5-phosphatase activity is associated with cellular transformation.     

Chemoattractant and guanosine 5''-[gamma-thio]triphosphate induce the accumulation of inositol 1,4,5-trisphosphate in Dictyostelium cells that are labelled with [3H]inositol by electroporation.

The analysis of the inositol cycle in Dictyostelium discoideum cells is complicated by the limited uptake of [3H]inositol (0.2% of the applied radioactivity in 6 h), and by the conversion of [3H]inositol into water-soluble inositol metabolites that are eluted near the position of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] on anion-exchange h.p.l.c. columns. The uptake was improved to 2.5% by electroporation of cells in the presence of [3H]inositol; electroporation was optimal at two 210 microseconds pulses of 7 kV. Cells remained viable and responsive to chemotactic signals after electroporation. The intracellular [3H]inositol was rapidly metabolized to phosphatidylinositol and more slowly to phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. More than 85% of the radioactivity in the water-soluble extract that was eluted on Dowex columns as Ins(1,4,5)P3 did not co-elute with authentic [32P]Ins(1,4,5)P3 on h.p.l.c. columns. Chromatography of the extract by ion-pair reversed-phase h.p.l.c. provided a good separation of the polar inositol polyphosphates. Cellular [3H]Ins(1,4,5)P3 was identified by (a) co-elution with authentic [32P]Ins(1,4,5)P3 and (b) degradation by a partially purified Ins(1,4,5)P3 5-phosphatase from rat brain. The chemoattractant cyclic AMP and the non-hydrolysable analogue guanosine 5'-[gamma-thio]triphosphate induced a transient accumulation of radioactivity in Ins(1,4,5)P3; we did not detect radioactivity in inositol 1,3,4-trisphosphate or inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In vitro, Ins(1,4,5)P3 was metabolized to inositol 1,4- and 4,5-bisphosphate, but not to Ins(1,3,4,5)P4 or another tetrakisphosphate isomer. We conclude that Dictyostelium has a receptor- and G-protein-stimulated inositol cycle which is basically identical with that in mammalian cells, but the metabolism of Ins(1,4,5)P3 is probably different.     

GAP1IP4BP contains a novel group I pleckstrin homology domain that directs constitutive plasma membrane association

The group I family of pleckstrin homology (PH) domains are characterized by their inherent ability to specifically bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and its corresponding inositol head-group inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In vivo this interaction results in the regulated plasma membrane recruitment of cytosolic group I PH domain-containing proteins following agonist-stimulated PtdIns(3,4,5)P(3) production. Among group I PH domain-containing proteins, the Ras GTPase-activating protein GAP1(IP4BP) is unique in being constitutively associated with the plasma membrane. Here we show that, although the GAP1(IP4BP) PH domain interacts with PtdIns(3,4, 5)P(3), it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (K(d) values of 0.5 +/- 0.2 and 0.8 +/- 0.5 microm, respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P(4), consistent with the constitutive plasma membrane association of GAP1(IP4BP) resulting from its PH domain-binding PtdIns(4,5)P(2), we show that in vivo depletion of PtdIns(4,5)P(2), but not PtdIns(3,4,5)P(3), results in dissociation of GAP1(IP4BP) from this membrane. Thus, the Ins(1,3,4,5)P(4)-binding PH domain from GAP1(IP4BP) defines a novel class of group I PH domains that constitutively targets the protein to the plasma membrane and may allow GAP1(IP4BP) to be regulated in vivo by Ins(1,3,4,5)P(4) rather than PtdIns(3,4,5)P(3).     

Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol.

The agonist-dependent hydrolysis of inositol phospholipids was investigated by studying the breakdown of prelabelled lipid or by measuring the accumulation of inositol phosphates. Stimulation of insect salivary glands with 5-hydroxytryptamine for 6 min provoked a rapid disappearance of [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and [3H]phosphatidylinositol 4-phosphate (PtdIns4P) but had no effect on the level of [3H]phosphatidylinositol (PtdIns). The breakdown of PtdIns(4,5)P2 was associated with a very rapid release of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which reached a peak 5 1/2 times that of the resting level after 5 s of stimulation. This high level was not maintained but declined to a lower level, perhaps reflecting the disappearance of PtdIns(4,5)P2. 5-Hydroxytryptamine also induced a rapid and massive accumulation of inositol 1,4-bisphosphate [Ins(1,4)P2]. The fact that these increases in Ins(1,4,5)P3 and Ins(1,4)P2 precede in time any increase in the level of inositol 1-phosphate or inositol provides a clear indication that the primary action of 5-hydroxytryptamine is to stimulate the hydrolysis of PtdIns(4,5)P2 to yield diacylglycerol and Ins(1,4,5)P3. The latter is then hydrolysed by a series of phosphomonoesterases to produce Ins(1,4)P2, Ins1P and finally inositol. The very rapid agonist-dependent increases in Ins(1,4,5)P3 and Ins(1,4)P2 suggests that they could function as second messengers, perhaps to control the release of calcium from internal pools. The PtdIns(4,5)P2 that is used by the receptor mechanism represents a small hormone-sensitive pool that must be constantly replenished by phosphorylation of PtdIns. Small changes in the size of this small energy-dependent pool of polyphosphoinositide will alter the effectiveness of the receptor mechanism and could account for phenomena such as desensitization and super-sensitivity.     

The metabolism of tris- and tetraphosphates of inositol by 5-phosphomonoesterase and 3-kinase enzymes

Phospholipase C cleaves phosphatidylinositol 4,5-bisphosphate to form both inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,2-cyclic 4,5-trisphosphate (cInsP3). The further metabolism of these inositol trisphosphates is determined by two enzymes: a 3-kinase and a 5-phosphomonoesterase. The first enzyme converts Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate (InsP4), while the latter forms inositol 1,4-bisphosphate and inositol 1,2-cyclic 4-bisphosphate from Ins(1,4,5)P3 and cInsP3, respectively. The current studies show that the 3-kinase is unable to phosphorylate cInsP3. Also, the 5-phosphomonoesterase hydrolyzes InsP4 with an apparent Km of 0.5-1.0 microM to form inositol 1,3,4-trisphosphate at a maximal velocity approximately 1/30 that for Ins(1,4,5)P3. The apparent affinity of the enzyme for the three substrates is InsP4 greater than Ins(1,4,5)P3 greater than cInsP3; however, the rate at which the phosphatase hydrolyzes these substrates is Ins(1,4,5)P3 greater than cInsP3 greater than InsP4. The 5-phosphomonoesterase and 3-kinase enzymes may control the levels of inositol trisphosphates in stimulated cells. The 3-kinase has a low apparent Km for Ins(1,4,5)P3 as does the 5-phosphomonoesterase for InsP4, implying that the formation and breakdown of InsP4 may proceed when both it and its precursor are present at low levels. Ins(1,4,5)P3 is utilized by both the 3-kinase and 5-phosphomonoesterase, while cInsP3 is utilized relatively poorly only by the 5-phosphomonoesterase. These findings imply that inositol cyclic trisphosphate may be metabolized slowly after its formation in stimulated cells.     

A salt-activated inositol 1,3,4,5-tetrakisphosphate 3-phosphatase at the inner surface of the human erythrocyte membrane.

The localization of the human erythrocyte membrane Ins(1,3,4,5)P4 3-phosphatase was investigated by saponin permeabilization of resealed 'isoionic' erythrocyte ghosts. This enzyme is active at the inner face of the plasma membrane, at the same site as a specific 5-phosphatase that degrades both Ins (1,4,5)P3 and Ins(1,3,4,5)P4. In the presence of EDTA, Ins(1,4,5)P3 was the only product of Ins(1,3,4,5)P4 metabolism. However, when Mg2+ was present both the 5-phosphatase and the 3-phosphatase attacked Ins (1,3,4,5)P4, directly forming Ins(1,3,4)P3 and Ins(1,4,5)P3;some Ins(1,4)P2 was also formed as a product of 5-phosphatase attack on the liberated Ins(1,4,5)P3. The Ins(1,3,4,5)P4 3-phosphatase was potently activated by KCl, thus making the route of metabolism of Ins(1,3,4,5)P4 by erythrocyte ghosts strikingly sensitive to variations in ionic strength: at 'cytosolic' K+ and Mg2+ levels, 3-phosphatase activity slightly predominated over 5-phosphatase. Ins(1,3,4,5)P4 3-phosphatase was potently inhibited by Ins-(1,3,4,5,6)P5 and InsP6 at levels lower than those often observed within cells. This leaves open the question as to whether the cellular function of inositol polyphosphate 3-phosphatase is to participate in a physiological cycle that interconverts Ins(1,3,4,5)P4 and Ins(1,4,5)P3 or to metabolize other inositol polyphosphates in the cytosol compartment of cells.     

The isolation and characterization of a cDNA encoding phospholipid-specific inositol polyphosphate 5-phosphatase

We report the cDNA cloning and characterization of a novel human inositol polyphosphate 5-phosphatase (5-phosphatase) that has substrate specificity unlike previously described members of this large gene family. All previously described members hydrolyze water soluble inositol phosphates. This enzyme hydrolyzes only lipid substrates, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate. The cDNA isolated comprises 3110 base pairs and predicts a protein product of 644 amino acids and M(r) = 70,023. We designate this 5-phosphatase as type IV. It is a highly basic protein (pI = 8.8) and has the greatest affinity toward phosphatidylinositol 3,4,5-trisphosphate of known 5-phosphatases. The K(m) is 0.65 micrometer, 1/10 that of SHIP (5.95 micrometer), another 5-phosphatase that hydrolyzes phosphatidylinositol 3,4,5-trisphosphate. The activity of 5-phosphatase type IV is sensitive to the presence of detergents in the in vitro assay. Thus the enzyme hydrolyzes lipid substrates in the absence of detergents or in the presence of n-octyl beta-glucopyranoside or Triton X-100, but not in the presence of cetyltriethylammonium bromide, the detergent that has been used in other studies of the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Remarkably SHIP, a 5-phosphatase previously characterized as hydrolyzing only substrates with d-3 phosphates, also readily hydrolyzed phosphatidylinositol 4,5-bisphosphate in the presence of n-octyl beta-glucopyranoside but not cetyltriethylammonium bromide. We used antibodies prepared against a peptide predicted by the cDNA to identify the 5-phosphatase type IV enzyme in human tissues and find that it is highly expressed in the brain as determined by Western blotting. We also performed Western blotting of mouse tissues and found high levels of expression in the brain, testes, and heart with lower levels of expression in other tissues. mRNA was detected in many tissues and cell lines as determined by Northern blotting.     

Kinetic and inhibitor profiles of soluble and particulate inositol 1,4-5-trisphosphate 5-phosphatase from GH3 and IMR-32 cells.

A simple procedure for assay of Ins(1,4,5)P3 5-phosphatase is described. The reaction products [( 3H]Ins(1,4)P2, [3H]InsP and myo-[3H]inositol) are completely separated from one another, with quantitative yield, on Amprep SAX (100 mg) minicolumns. [3H]Ins(1,4,5)P3 [and [3H]Ins(1,3,4,5)P4] are adsorbed to the columns but not released to any appreciable extent by the elution conditions used. In GH3 cells, the stepwise dephosphorylation of [3H]Ins(1,4,5)P3 to myo-[3H]inositol was demonstrated, and was inhibited by 2.3-bisphosphoglycerate. The Km of the soluble form of the enzyme was lower in GH3 cells (8-13 microM) than in IMR-32 cells (26-32 microM) or in rat cerebral-cortical samples (22 microM. The Km of the particulate form of the enzyme was similar in all three preparations (10-16 microM). The pH profiles of the two soluble 5-phosphatases differed, with a wider pH optimum for the GH3-cell activity than for the IMR-32-cell activity. The soluble and particulate GH3 enzymes were more sensitive than the corresponding IMR-32 enzymes to inhibition by p-hydroxymercuribenzoate, whereas there were no differences in their sensitivities to glucose 6-phosphate, 2,3-bisphosphoglycerate, fructose 1.6- and 2.6-bisphosphate and non-radioactive Ins(1,3,4,5)P4. Dialysis of the soluble fractions and washing of the particulate fractions did not affect the inhibitor sensitivities, except for the soluble IMR-32 fraction and p-hydroxymercuribenzoate. The Km value of the soluble GH3 5-phosphatase activity was lower, and the inhibition by Ins(1,3,4,5)P4 greater, after adsorption to and elution from phosphocellulose. It is concluded that there are qualitative differences in the properties of the soluble 5-phosphatase activity from GH3 and IMR-32 cells.     

Identification of the bovine brain Ins(1,4,5)P3 5-phosphatase after SDS-polyacrylamide gel electrophoresis

Ins(1,4,5)P3 5-phosphatase catalyzes the dephosphorylation of Ins(1,4,5)P3 in the 5-position. In a high speed soluble fraction of bovine brain, there are two soluble 5-phosphatases: type I and type II. The purified Ins(1,4,5)P3 5-phosphatase type I exhibits a major silver-stained band of 43 kDa on denaturing (SDS) gels. It is possible to extract the 5-phosphatase activity form a duplicate lane after gel electrophoresis. The 43 kDa region contains the extractable Ins(1,4,5)P3 5-phosphatase activity.     

Purification and characterization of membrane-bound inositolpolyphosphate 5-phosphatase

Membrane-bound inositolpolyphosphate 5-phosphatase was solubilized and highly purified from a microsomal fraction of rat liver. Its physiochemical and enzymological properties were compared with those of highly purified preparations of two types of soluble enzyme (soluble Type I and Type II) from rat brain. The molecular masses of the membrane-bound and soluble Type I enzymes were 32 kDa, while that of soluble Type II enzyme was 69 kDa, as determined by molecular sieve chromatography. The membrane-bound and soluble Type I enzymes showed similar broad peaks on isoelectric focusing (pI 5.8-6.4), while soluble Type II enzyme showed multiple peaks in the region between pI 4.0-5.8. All three enzymes required divalent cation for activity. Mg2+ was the most effective for both the membrane-bound and soluble Type I enzymes, while Co2+ enhanced soluble Type II enzyme activity about 1.5-fold relative to Mg2+ at 1 mM. The optimal pH of both the membrane-bound and soluble Type I enzymes was 7.8, while that of soluble Type II was 6.8. The Km values for inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] of all three enzymes were similar (5-8 microM), but those for inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] were quite different, the Km values of membrane-bound and soluble Type I enzymes being 0.8 microM, while that of soluble Type II was 130 microM. These similarities between the membrane-bound and soluble Type I enzymes suggest that these two molecules may be the same protein, and that concentrations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, both of which are considered to play critical roles in the regulation of intracellular Ca2+-concentration, may be differently regulated by two functionally distinct enzymes.     

Interaction of the brain-specific protein p42IP4/centaurin-alpha1 with the peptidase nardilysin is regulated by the cognate ligands of p42IP4, PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4, with stereospecificity

The brain-specific protein p42IP4, also called centaurin-alpha1, specifically binds phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. Here, we investigate the interaction of p42IP4/centaurin-alpha1 with nardilysin (NRDc), a member of the M16 family of zinc metalloendopeptidases. Members of this peptidase family exhibit enzymatic activity and also act as receptors for other proteins. We found that p42IP4/centaurin-alpha1 binds specifically to NRDc from rat brain. We further detected that centaurin-alpha2, a protein that is highly homologous to p42IP4/centaurin-alpha1 and expressed ubiquitously, also binds to NRDc. In vivo interaction was demonstrated by co-immunoprecipitation of p42IP4/centaurin-alpha1 with NRDc from rat brain. The acidic domain of NRDc (NRDc-AD), which does not participate in catalysis, is sufficient for the protein interaction with p42IP4. Interestingly, preincubation of p42IP4 with its cognate ligands D-Ins(1,3,4,5)P4 and the lipid diC8PtdIns(3,4,5)P3 negatively modulates the interaction between the two proteins. D-Ins(1,3,4,5)P4 and diC8PtdIns(3,4,5)P3 suppress the interaction with virtually identical concentration dependencies. This inhibition is highly ligand specific. The enantiomer L-Ins(1,3,4,5)P4 is not effective. Similarly, the phosphoinositides diC8PtdIns(3,4)P2, diC8PtdIns(3,5)P2 and diC8PtdIns(4,5)P2 all have no influence on the interaction. Further experiments revealed that endogenous p42IP4 from rat brain binds to glutathione-S-transferase (GST)-NRDc-AD. The proteins dissociate from each other when incubated with D-Ins(1,3,4,5)P4, but not with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. In summary, we demonstrate that p42IP4 binds to NRDc via the NRDc-AD, and that this interaction is controlled by the cognate cellular ligands of p42IP4/centaurin-alpha1. Thus, specific ligands of p42IP4 can modulate the recruitment of proteins, which are docked to p42IP4, to specific cellular compartments.     

Soluble and particulate inositol 1,4,5-trisphosphate 5-phosphatases show common antigenic determinants

Inositol 1,4,5-trisphosphate 5-phosphatase catalyses the dephosphorylation of the phosphate in the 5-position from inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. One particulate and two soluble enzymes were previously described in bovine brain. In this study, we have obtained a precipitating antiserum against soluble type I inositol 1,4,5-trisphosphate 5-phosphatase. The particulate, but not the soluble type II enzyme, was immunoprecipitated by the serum. Inositol 1,4,5-triphosphate 5-phosphatase activity from crude extracts of rat brain, human platelets and rat liver were immmunoprecipitated by the same antibodies, suggesting the existence of common antigenic determinant among inositol 1,4,5-trisphosphate 5-phosphatases of diverse sources.     

Recruitment of OCRL and Inpp5B to phagosomes by Rab5 and APPL1 depletes phosphoinositides and attenuates Akt signaling

Sealing of phagosomes is accompanied by the disappearance of phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2)) from their cytoplasmic leaflet. Elimination of PtdIns(4,5)P(2), which is required for actin remodeling during phagosome formation, has been attributed to hydrolysis by phospholipase C and phosphorylation by phosphatidylinositol 3-kinase. We found that two inositol 5-phosphatases, OCRL and Inpp5B, become associated with nascent phagosomes. Both phosphatases, which are Rab5 effectors, associate with the adaptor protein APPL1, which is recruited to the phagosomes by active Rab5. Knockdown of APPL1 or inhibition of Rab5 impairs association of OCRL and Inpp5B with phagosomes and prolongs the presence of PtdIns(4,5)P(2) and actin on their membranes. Even though APPL1 can serve as an anchor for Akt, its depletion accentuated the activation of the kinase, likely by increasing the amount of PtdIns(4,5)P(2) available to generate phosphatidylinositol (3,4,5)-trisphosphate. Thus, inositol 5-phosphatases are important contributors to the phosphoinositide remodeling and signaling that are pivotal for phagocytosis.     

Recovery of hepatocytes from attack by the pore former amphotericin B.

Factors underlying the transience of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] accumulation following muscarinic stimulation of RINm5F cells were examined. Transience was not due to a protein kinase C-mediated stimulation of Ins(1,4,5)P3 dephosphorylation, since pretreatment of cells with tetradecanoyl-phorbol acetate (TPA) did not alter the rate of this conversion. However, preincubation with TPA did inhibit carbamoylcholine-stimulated Ins(1,4,5)P3 formation. In permeabilized cells, the conversion of Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] was slightly enhanced in the presence of TPA or cyclic AMP, but much more markedly by raising the Ca2+ concentration from 10(-7) M to 10(-6) or 10(-5) M. In intact cells the most rapid rate of accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 occurred in the first 2 s following stimulation, whereas the levels of inositol 1,4-bisphosphate were not increased until after 5 s. This suggests that Ins(1,4,5)P3 kinase is chiefly responsible for the early disposal of Ins(1,4,5)P3 following cellular stimulation. The results are consistent with the proposal that the transient accumulation of Ins(1,4,5)P3 is due both to its enhanced metabolism via the Ca2+-calmodulin-sensitive Ins(1,4,5)P3 kinase, as well as a down-regulation of phosphatidylinositol 4,5-bisphosphate hydrolysis.