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.     

Short-term down-regulation of the brain-specific, PtdIns(3,4,5)P3/Ins(1,3,4,5)P4-binding, adapter protein, p42IP4/centaurin-alpha 1 in rat brain after acoustic and electric stimulation

The protein p42(IP4), expressed mainly in brain, specifically recognizes two second messenger molecules, Ins(1,3,4,5)P(4) (IP(4)), a water soluble metabolite of IP(3) and the lipid PtdIns(3,4,5)P(3) (PIP(3)), the product of the growth factor-activated enzyme PI-3-kinase. Here, we studied whether there is short-term regulation of the expression level of p42(IP4) in limbic brain areas following acoustic and electric stimulation. The stimuli down-regulated the mRNA and protein levels within 2h in amygdala, hypothalamus and cingulate/retrospenial cortex. p42(IP4) mRNA decreased by about 50% for about 24h, but recovered to control values after 72 h. The present results are the first indication of a specific role of p42(IP4) in the short-term regulation of a behavioral response. They indicate that p42(IP4), an adapter protein in PIP(3)-dependent cellular signaling, may play an important role in the signal transduction pathways regulating plasticity in neuronal cells.     

Soluble and particulate Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase in bovine brain

Ins(1,4,5)P3 5-phosphatase catalyses the dephosphorylation of Ins(1,4,5)P3 in the 5 position. At 1 microM Ins(1,4,5)P3, 10-15% of total activity of a bovine brain homogenate was measured in the soluble fraction, whereas 85-90% was in the particulate fraction. Particulate activity could be solubilized by cholate or, to a lower extent, by 2 M KCl. Two soluble enzymes (type I and type II) could be fractionated by DEAE-Sephacel chromatography. Soluble activities have been further purified by blue-Sepharose, Sephacryl S-200 and phosphocellulose chromatography. Specific activities reached 10-30 mumol.min-1 mg protein-1 for type I and were 10-20 times lower for type II. Type I and type II Ins(1,4,5)P3 5-phosphatase displayed different Km values and molecular masses, as estimated by gel filtration. Type I dephosphorylated both Ins(1,4,5)P3 and Ins(1,3,4,5)P4; in contrast, type II specifically dephosphorylated Ins(1,4,5)P3 but not Ins(1,3,4,5)P4. Type I Ins(1,4,5)P3 5-phosphatase eluted as a single peak of activity with an apparent molecular mass of 51 kDa when gel filtration was performed in the presence of cholate. This molecular mass is identical to the molecular mass estimated for the particulate Ins(1,4,5)P3 5-phosphatase that was solubilized by cholate. Km values for Ins(1,4,5)P3 and Ins(1,3,4,5)P4 obtained with type I Ins(1,4,5)P3 5-phosphatase were 11 microM and 1 microM, respectively. Similar values were obtained with particulate Ins(1,4,5)P3 5-phosphatase. In conclusion, the catalytic domains of type I and particulate Ins(1,4,5)P3 5-phosphatase activity may be very similar, if not identical, but different from type II phosphatase.     

Distinct binding sites for Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in bovine parathyroid glands

We utilized high specific activity, [32P]-labelled ligands to measure the binding of Ins(1,3,4,5)P4 and Ins(1,4,5)P3 to membranes prepared from bovine parathyroid glands. [32P]Ins(1,3,4,5)P4 bound rapidly and reversibly to parathyroid membranes, and the binding data could be fitted by the interaction of the ligand with two sites, one with Kd = 6.8 x 10(-9) M and Bmax = 26 fmol/mg protein and a second, lower affinity site, with Kd = 4.1 x 10(-7) M and Bmax = 400 fmol/mg protein. InsP5 was 10-20 fold less potent than InsP4, and Ins(1,3,4)P3 and Ins(1,4,5)P3 were nearly 1000-fold less potent in displacing [32P]Ins(1,3,4,5)P4. [32P]Ins(1,4,5)P3, on the other hand, bound to a single class of sites with Kd = 7.6 x 10(-9) M and Bmax = 34 fmol/mg. While the binding of [32P]Ins(1,4,5)P3 increased markedly on raising pH from 5 to 8, the binding of [32P]Ins(1,3,4,5)P4 decreased by 75% over this range of pH. Thus, [32P]-labelled Ins(1,3,4,5)P4 and Ins(1,4,5)P3 may be used to identify distinct binding sites which may represent physiologically relevant intracellular receptors for InsP3 and InsP4 in parathyroid cells.     

Metabolism of the biologically active inositol phosphates Ins(1,4,5)P3 and Ins(1,3,4,5)P4 by ovarian follicles of Xenopus laevis.

The metabolism of biologically active inositol phosphates in developed ovarian follicles from Xenopus laevis was investigated. Techniques used were microinjection of tracer into the intact oocyte coupled by gap junctions to follicle cells, as well as addition of tracer to homogenates of ovarian follicles and to homogenates of oocytes stripped of outer follicle-cell layers. Metabolism was similar to that previously described for other types of cell and tissue, with several unusual features. Homogenates of ovarian follicles were shown to contain an apparent 3'-phosphomonoesterase capable of converting [3H]Ins(1,3,4,5)P4 predominantly into a substance with h.p.l.c. elution characteristics of Ins(1,4,5)P3. In intact ovarian follicles, little Ins(1,4,5)P3 was formed but the esterase was activated by the phorbol ester activator of protein kinase C, PMA (phorbol 12-myristate 13-acetate; 60 nM), as well as by acetylcholine (200 microM). In follicle homogenates, this enzyme also appeared to be active in converting [3H]Ins(1,3,4)P3 into a substance eluting as Ins(1,4)P2. The apparent 3'-phosphomonoesterase activity was not inhibited by intracellular (or higher) levels of Mg2+. Although PMA activated this enzyme in intact oocytes relative to 5'-phosphomonoesterase activation, it did not enhance overall metabolism, in contrast with reports on other tissues. Compared with the processing of inositol phosphates injected into the intact follicle, homogenization in simulated intracellular medium appeared to alter the activity and/or accessibility of several enzymes. The metabolism of inositol phosphates appears to occur predominantly in the follicle cells surrounding the oocyte, as collagenase treatment followed by defolliculation greatly diminished the rates of metabolism of several inositol phosphates. The presence in Xenopus ovarian follicles of a 3'-phosphomonoesterase activated by protein kinase C in addition to the well-known 3'-kinase suggests that, by forming a reversible interconversion between Ins(1,4,5)P3 and Ins(1,3,4,5)P4, this tissue may have the potential to prolong stimulatory signals on binding of appropriate agonists to receptors.     

Identification of gene structure and subcellular localization of human centaurin alpha 2, and p42IP4, a family of two highly homologous, Ins 1,3,4,5-P4-/PtdIns 3,4,5-P3-binding, adapter proteins

Proteins which recognize the two messengers phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3), a membrane lipid, and inositol 1,3,4,5-tetrakisphosphate (InsP4), a water-soluble ligand, play important roles by integrating external stimuli, which lead to differentiation, cell death or survival. p42IP4, a PtdInsP3/InsP4-binding protein, is predominantly expressed in brain. The recently described centaurin alpha2 of similar molecular mass which is 58% identical and 75% homologous to the human p42IP4 orthologue, is expressed rather ubiquitously in many tissues. Here, elucidating the gene structure for both proteins, we found the human gene for centaurin alpha2 located on chromosome 17, position 17q11.2, near to the NF1 locus, and human p42IP4 on chromosome 7, position 7p22.3. The two isoforms, which both have 11 exons and conserved exon/intron transitions, seem to result from gene duplication. Furthermore, we studied binding of the two second messengers, PtdInsP3 and InsP4, and subcellular localization of the two proteins. Using recombinant baculovirus we expressed centaurin alpha2 and p42IP4 in Sf9 cells and purified the proteins to homogeneity. Recombinant centaurin alpha2 bound both InsP4 and PtdInsP3 equally well in vitro. Furthermore, fusion proteins of centaurin alpha2 and p42IP4, respectively, with the green fluorescent protein (GFP) were expressed in HEK 293 cells to visualize subcellular distribution. In contrast to p42IP4, which was distributed throughout the cell, centaurin alpha2 was concentrated at the plasma membrane already in unstimulated cells. The protein centaurin alpha2 was released from the membrane upon addition of wortmannin, which inhibits PI3-kinase. p42IP4, however, translocated to plasma membrane upon growth factor stimulation. Thus, in spite of the high homology between centaurin alpha2 and p42IP4 and comparable affinities for InsP4 and PtdInsP3, both proteins showed clear differences in subcellular distribution. We suggest a model, which is based on the difference in phosphoinositide binding stoichiometry of the two proteins, to account for the difference in subcellular localization.     

RanBPM, a novel interaction partner of the brain-specific protein p42IP4/centaurin alpha-1

The protein p42^IP4 (aka Centaurin α-1) is highly enriched in the brain and has specific binding sites for the membrane lipid phosphatidylinositol 3,4,5-trisphosphate and the soluble messenger inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P₄; IP4). p42^IP4 shuttles between plasma membrane, cytosol and cell nucleus. However, the molecular function of p42^IP4 is still largely unclear. Here, we report a novel interaction partner for p42^IP4, Ran binding protein in microtubule-organizing center (RanBPM). RanBPM is ubiquitously expressed and seems to act as scaffolding and modulator protein. In our studies, we established this interaction in vitro and in vivo . The in vivo interaction was demonstrated with endogenous RanBPM from rat brain. Both proteins co-localize in transfected HEK 293 cells. We could show that the interaction does not require additional proteins. D-Ins(1,3,4,5)P₄, a specific ligand for p42^IP4, is a concentration-dependent and stereoselective inhibitor of this interaction; the l -isoform is much less effective. We found that mainly the SPRY domain of RanBPM mediates the p42^IP4-RanBPM association. The ARFGAP domain of p42^IP4 is important for the interaction, without being the only interaction site. Recently, p42^IP4 and RanBPM were shown to be involved in dendritic differentiation. Thus, we hypothesize that RanBPM could act as a modulator together with p42^IP4 in synaptic plasticity.     

Translocation between membranes and cytosol of p42IP4, a specific inositol 1,3,4,5-tetrakisphosphate/phosphatidylinositol 3,4, 5-trisphosphate-receptor protein from brain, is induced by inositol 1,3,4,5-tetrakisphosphate and regulated by a membrane-associated 5-phosphatase.

The highly conserved 42-kDa protein, p42IP4 was identified recently from porcine brain. It has also been identified similarly in bovine, rat and human brain as a protein with two pleckstrin homology domains that binds Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3 with high affinity and selectivity. The brain-specific p42IP4 occurs both as membrane-associated and cytosolic protein. Here, we investigate whether p42IP4 can be translocated from membranes by ligand interaction. p42IP4 is released from cerebellar membranes by incubation with Ins(1,3,4,5)P4. This dissociation is concentration-dependent (> 100 nM), occurs within a few minutes and and is ligand-specific. p42IP4 specifically associates with PtdIns(3, 4,5)P3-containing lipid vesicles and can dissociate from these vesicles by addition of Ins(1,3,4,5)P4. p42IP4 is only transiently translocated from the membranes as Ins(1,3,4,5)P4 can be degraded by a membrane-associated 5-phosphatase to Ins(1,3,4)P3. Then, p42IP4 re-binds to the membranes from which it can be re-released by re-addition of Ins(1,3,4,5)P4. Thus, Ins(1,3,4,5)P4 specifically induces the dissociation from membranes of a PtdIns(3,4,5)P3 binding protein that can reversibly re-associate with the membranes. Quantitative analysis of the inositol phosphates in rat brain tissue revealed a concentration of Ins(1,3,4,5)P4 comparable to that required for p42IP4 translocation. Thus, in vivo p42IP4 might interact with membranes in a ligand-controlled manner and be involved in physiological processes induced by the two second messengers Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3.     

Role of PRIP-1, a novel Ins(1,4,5)P3 binding protein, in Ins(1,4,5)P3-mediated Ca2+ signaling

PRIP-1 was isolated as a novel inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] binding protein with a domain organization similar to phospholipase C-delta1 (PLC-delta1) but lacking the enzymatic activity. Further studies revealed that the pleckstrin homology (PH) domain of PRIP-1 is the region responsible for binding Ins(1,4,5)P3. In this study we aimed to clarify the role of PRIP-1 at the physiological concentration in Ins(1,4,5)P3-mediated Ca2+ signaling, as we had previously used COS-1 cells overexpressing PRIP-1 (Takeuchi et al., 2000, Biochem J 349:357-368). For this purpose we employed PRIP-1 knock out (PRIP-1-/-) mice generated previously (Kanematsu et al., 2002, EMBO J 21:1004-1011). The increase in free Ca2+ concentration in response to purinergic receptor stimulation was lower in primary cultured cortical neurons prepared from PRIP-1-/- mice than in those from wild type mice. The relative amounts of [3H]Ins(1,4,5)P3 measured in neurons labeled with [3H]inositol was also lower in cells from PRIP-1-/- mice. In contrast, PLC activities in brain cortex samples from PRIP-1-/- mice were not different from those in the wild type mice, indicating that the hydrolysis of Ins(1,4,5)P3 is enhanced in cells from PRIP-1-/- mice. In vitro analyses revealed that type1 inositol polyphosphate 5-phosphatase physically interacted with a PH domain of PRIP-1 (PRIP-1PH) and its enzyme activity was inhibited by PRIP-1PH. However, physical interaction with these two proteins did not appear to be the reason for the inhibition of enzyme activity, indicating that binding of Ins(1,4,5)P3 to the PH domain prevented its hydrolyzation. Together, these results indicate that PRIP-1 plays an important role in regulating the Ins(1,4,5)P3-mediated Ca2+ signaling by modulating type1 inositol polyphosphate 5-phosphatase activity through binding to Ins(1,4,5)P3.     

A novel, specific binding protein assay for quantitation of intracellular inositol 1,3,4,5-tetrakisphosphate (InsP4) using a high-affinity InsP4 receptor from cerebellum

A membrane preparation from porcine cerebellum displays high-affinity binding sites for [3H]inositol 1,3,4,5-tetrakisphosphate ([3H]InsP4) with a dissociation constant (Kd) of 1.0 nM and a density of 220 fmol/mg protein. Specific binding was maximal in the presence of 25 mM phosphate and at pH 5.0. The receptor site was specific for InsP4, since Ins(1,3,4,5,6)P5 and Ins(1,4,5,6)P4 displaced binding of InsP4 with EC50 values of 0.2 and 0.3 microM, respectively. Ins(1,4,5)P3 and other inositol phosphates were less effective. Using this InsP4 receptor, an assay for measuring tissue content of InsP4 was developed. The detection limit of the assay was 0.1 pmol. In the same tissue samples the amount of Ins(1,4,5)P3 was determined in parallel with a similar assay using a binding protein preparation from beef liver.     

CLN3P, the Batten disease protein, localizes to membrane lipid rafts (detergent-resistant membranes)

Juvenile neuronal ceroid lipofuscinosis is an inherited pediatric neurodegenerative disorder, which occurs as a result of mutations in the CLN3 gene that is located on chromosome 16p12.1. The encoded protein, CLN3P, is a putative transmembrane protein with no known function. In this study, we demonstrate that CLN3P resides on membrane lipid raft domains (detergent-resistant membranes) and provide important new data towards possible functions of the protein.     

IRBIT,a novel inositol 1,4,5-trisphosphate (IP3) receptor-binding protein,is released from the IP3 receptor upon IP3 binding to the receptor

The inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are IP(3)-gated Ca(2+) channels on intracellular Ca(2+) stores. Herein, we report a novel protein, termed IRBIT (IP(3)R binding protein released with inositol 1,4,5-trisphosphate), which interacts with type 1 IP(3)R (IP(3)R1) and was released upon IP(3) binding to IP(3)R1. IRBIT was purified from a high salt extract of crude rat brain microsomes with IP(3) elution using an affinity column with the huge immobilized N-terminal cytoplasmic region of IP(3)R1 (residues 1-2217). IRBIT, consisting of 530 amino acids, has a domain homologous to S-adenosylhomocysteine hydrolase in the C-terminal and in the N-terminal, a 104 amino acid appendage containing multiple potential phosphorylation sites. In vitro binding experiments showed the N-terminal region of IRBIT to be essential for interaction, and the IRBIT binding region of IP(3)R1 was mapped to the IP(3) binding core. IP(3) dissociated IRBIT from IP(3)R1 with an EC(50) of approximately 0.5 microm, i.e. it was 50 times more potent than other inositol polyphosphates. Moreover, alkaline phosphatase treatment abolished the interaction, suggesting that the interaction was dualistically regulated by IP(3) and phosphorylation. Immunohistochemical studies and co-immunoprecipitation assays showed the relevance of the interaction in a physiological context. These results suggest that IRBIT is released from activated IP(3)R, raising the possibility that IRBIT acts as a signaling molecule downstream from IP(3)R.     

Oligomerization controls in tissue-specific manner ligand binding of native, affinity-purified p42(IP4)/centaurin alpha1 and cytohesins-proteins with high affinity for the messengers D-inositol 1,3,4,5-tetrakisphosphate/phosphatidylinositol 3,4,5-trisphosphate

Several distinct receptor proteins for the second messengers Ins(1,3,4,5)P(4) and PtdIns(3,4,5)P(3) are already known, such as the brain-specific p42(IP4), which we have previously cloned from different species, and cytohesins. However, it is still unclear whether proteins interacting with phosphoinositide and inositolpolyphosphate second messengers are regulated differently in different tissues. Here, we investigated these native proteins for comparison also from rat lung cytosol and purified them by PtdIns(3,4,5)P(3) affinity chromatography. Proteins selectively binding Ins(1,3,4,5)P(4) with high affinity also showed high affinity and specificity towards PtdIns(3,4,5)P(3). In lung cytosol, two prominent protein bands were found in the eluate from a PtdIns(3,4,5)P(3) affinity column. We identified these proteins by mass spectrometry as the cytohesin family of Arf guanosine nucleotide exchange factors (cytohesin 1, ARNO, GRP-1) and as Bruton's tyrosine kinase. Western blot analysis indicated that p42(IP4) was present in lung only at very low concentrations. Applying the affinity purification scheme established for rat lung cytosol to cytosol from rat brain, however, yielded only p42(IP4). We identified cytohesins in rat brain by Western blotting and PCR, but cytohesins surprisingly did not bind to the PtdIns(3,4,5)P(3)-affinity column. Gel filtration experiments of brain cytosol revealed that brain cytohesins are bound to large molecular weight complexes (150 to more than 500 kDa). Thus, we hypothesize that this finding explains why brain cytohesins apparently do not bind the inositolphosphate ligand. In lung cytosol, on the other hand, cytohesins occur as dimers. Gel filtration also showed that p42(IP4) in brain cytosol occurs as a monomer. Thus, oligomerization (homomeric or heteromeric) of InsP(4)/PtdInsP(3) binding proteins can modulate their function in a tissue-dependent manner because it can modify their ability to interact with the ligands.     

Characterization of a high-affinity Ins-P4 (inositol 1,3,4,5-tetrakisphosphate) receptor from brain by an anti-peptide antiserum

From a high-affinity Ins-P₄ (inositol 1,3,4,5-P₄) receptor purified from pig cerebellum, digested with the protease Lys C peptide sequences were obtained. Synthetic peptide-3 (19 amino acid residues) was used to generate an antiserum. Reaction of the affinity-purified antibodies with the purified pig receptor protein in ELISA or Western blot was completely inhibited by peptide-3. In cerebellar membranes, the antibodies clearly recognized the 42 kDa Ins-P₄ receptor protein and two additional proteins (25 kDa, 37 kDa) which still have to be identified. The anti-peptide antibodies could selectively immunoprecipitate the Ins-P₄ receptor protein. The antiserum was used (i) to demonstrate that in brain from different species (human, pig, beef, rat, mouse and sheep) a similar 42 kDa Ins-P₄ receptor protein is contained, and (ii) to obtain indications for the existence of a related soluble form of the 42 kDa Ins-P₄ receptor besides the membrane-associated receptor.     

T3JAM, a novel protein that specifically interacts with TRAF3 and promotes the activation of JNK(1)

Previous studies suggest that localization of tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family members is important for regulating their signal transduction. During a screen for TRAF3-associated proteins that potentially alter TRAF3 subcellular localization and enable signal transduction, we identified a novel protein, T3JAM (TRAF3-interacting Jun N-terminal kinase (JNK)-activating modulator). This protein associates specifically with TRAF3 but not other TRAF family members. Coexpression of T3JAM with TRAF3 recruits TRAF3 to the detergent-insoluble fraction. More importantly, T3JAM and TRAF3 synergistically activate JNK but not nuclear factor (NF)-kappaB. Our studies indicate that T3JAM may function as an adapter molecule that specifically regulates TRAF3-mediated JNK activation.     

Development of a novel, Ins(1,4,5)P3-specific binding assay. Its use to determine the intracellular concentration of Ins(1,4,5)P3 in unstimulated and vasopressin-stimulated rat hepatocytes

The binding of [3H]Ins(1,4,5)P3 to bovine adrenocortical microsomes has been shown to be rapid, reversible and saturable. The microsomal preparation contained a single population of high affinity sites (KD = 6.82+/-2.3 nM, Bmax = 370+/-38 fmol/mg protein). The binding site was shown to exhibit positional specificity with respect to inositol trisphosphate binding, i.e. Ins(2,4,5)P3 was able to compete with [3H]Ins(1,4,5)P3 whereas Ins(1,3,4)P3 was not. Ins(1,3,4,5)P4 showed a similar affinity for the receptor as Ins(2,4,5)P3 whereas the other inositol phosphates tested, ATP, GTP and 2,3-DPG, were poor competitors. [3H]Ins(1,4,5)P3-binding was independent of free Ca2+ concentrations. The adrenocortical microsomal preparation has been incorporated into an assay which has been used to determine the basal and vasopressin-stimulated content of neutralised acid extracts of rat hepatocytes. Intracellular concentrations of Ins(1,4,5)P3 were calculated to be 0.22+/-0.15 microM basal and 2.53+/-1.8 microM at peak stimulation. This assay provides a simple, specific and quantitative method for the measurement of Ins(1,4,5)P3 concentrations in the picomolar range.     

PTEN M-CBR3, a versatile and selective regulator of inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5). Evidence for Ins(1,3,4,5,6)P5 as a proliferative signal

The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumor suppressor is a phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) 3-phosphatase that plays a crucial role in regulating many cellular processes by antagonizing the phosphoinositide 3-kinase signaling pathway. Although able to metabolize soluble inositol phosphates in vitro, the question of their significance as physiological substrates is unresolved. We show that inositol phosphates are not regulated by wild type PTEN, but that a synthetic mutant, PTEN M-CBR3, previously thought to be inactive toward inositides, can selectively regulate inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5). Transfection of U87-MG cells with PTEN M-CBR3 lowered Ins(1,3,4,5,6)P5 levels by 60% without detectable effect on PtdInsP3. Although PTEN M-CBR3 is a 3-phosphatase, levels of myo-inositol 1,4,5,6-tetrakisphosphate were not increased, whereas myo-inositol 1,3,4,6-tetrakisphospate levels increased by 80%. We have used PTEN M-CBR3 to study the physiological function of Ins(1,3,4,5,6)P5 and have found that Ins(1,3,4,5,6)P5 does not modulate PKB phosphorylation, nor does it regulate clathrin-mediated epidermal growth factor receptor internalization. By contrast, PTEN M-CBR3 expression, and the subsequent lowering of Ins(1,3,4,5,6)P5, are associated with reduced anchorage-independent colony formation and anchorage-dependent proliferation in U87-MG cells. Our results, together with previously published data, suggest that Ins(1,3,4,5,6)P5 has a role in proliferation.