Metabolic switching of PI3K-dependent lipid signals.

The lipid phosphatase, PTEN (phosphatase and tensin homologue deleted on chromosome 10), is the product of a major tumour suppressor gene that antagonizes PI3K (phosphoinositide 3-kinase) signalling by dephosphorylating the 3-position of the inositol ring of PtdIns(3,4,5)P(3). PtdIns(3,4,5)P(3) is also metabolized by removal of the 5-phosphate catalysed by a distinct family of enzymes exemplified by SHIP1 [SH2 (Src homology 2)-containing inositol phosphatase 1] and SHIP2. Mouse knockout studies, however, suggest that PTEN and SHIP2 have profoundly different biological functions. One important reason for this is likely to be that SHIP2 exists in a relatively inactive state until cells are exposed to growth factors or other stimuli. Hence, regulation of SHIP2 is geared towards stimulus dependent antagonism of PI3K signalling. PTEN, on the other hand, appears to be active in unstimulated cells and functions to maintain basal PtdIns(3,4,5)P(3) levels below the critical signalling threshold. We suggest that concomitant inhibition of cysteine-dependent phosphatases, such as PTEN, with activation of SHIP2 functions as a metabolic switch to regulate independently the relative levels of PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2).

Maintenance of PtdIns45P2 pools under limiting inositol conditions, as assessed by liquid chromatography-tandem mass spectrometry and PtdIns45P2 mass evaluation in Ras-transformed cells.

Inositol-containing molecules are involved in important cellular functions, including signalling, membrane transport and secretion. Our interest is in lysophosphatidylinositol and the glycerophosphoinositols, which modulate cell proliferation and G-protein-dependent activities such as adenylyl cyclase and phospholipase A(2). To investigate the role of glycerophosphoinositol (GroPIns) in the modulation of Ras-dependent pathways and its correlation to Ras transformation, we employed a novel liquid chromatography-tandem mass spectrometry technique to directly measure GroPIns in cell extracts. The cellular levels of GroPIns in selected parental and Ras-transformed cells, and in some carcinoma cells, ranged from 44 to 925 microM, with no consistent correlation to Ras transformation across all cell lines. Moreover, the derived cellular inositol concentrations revealed a wide range ( approximately 150 microM to approximately 100 mM) under standard [(3)H]-inositol-loading, suggesting a complex relationship between the inositol pool and the phosphoinositides and their derivatives. We have investigated these pools under specific loading conditions, designing a further HPLC analysis for GroPIns, combined with mass determinations of cellular phosphatidylinositol 4,5-bisphosphate. The data demonstrate that limiting inositol conditions identify a preferred pathway of inositol incorporation and retention into the polyphosphoinositides pool. Thus, under conditions of increased metabolic activity, such as receptor stimulation or cellular transformation, the polyphosphoinositide levels will be maintained at the expense of phosphatidylinositol and the turnover of its aqueous derivatives.

Regulation of clathrin-coated vesicle formation.

The formation of clathrin-coated pits at the plasma membrane requires the concerted action of many different molecules. The real challenge lies in determining the hierarchy of these interactions. We are using assays in both intact and permeabilized cells to dissect the temporal requirements for clathrin-coated vesicle formation, and also to examine the role of phosphorylation of the coat proteins.

The role of dynamin and its binding partners in coated pit invagination and scission.

Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain-containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission. To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits. We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.

The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells.

BACKGROUND: Protein kinase B (PKB), and the p70 and p90 ribosomal S6 kinases (p70 S6 kinase and p90 Rsk, respectively), are activated by phosphorylation of two residues, one in the 'T-loop' of the kinase domain and, the other, in the hydrophobic motif carboxy terminal to the kinase domain. The 3-phosphoinositide-dependent protein kinase 1 (PDK1) activates many AGC kinases in vitro by phosphorylating the T-loop residue, but whether PDK1 also phosphorylates the hydrophobic motif and whether all other AGC kinases are substrates for PDK1 is unknown. RESULTS: Mouse embryonic stem (ES) cells in which both copies of the PDK1 gene were disrupted were viable. In PDK1(-/-) ES cells, PKB, p70 S6 kinase and p90 Rsk were not activated by stimuli that induced strong activation in PDK1(+/+) cells. Other AGC kinases - namely, protein kinase A (PKA), the mitogen- and stress-activated protein kinase 1 (MSK1) and the AMP-activated protein kinase (AMPK) - had normal activity or were activated normally in PDK1(-/-) cells. The insulin-like growth factor 1 (IGF1) induced PKB phosphorylation at its hydrophobic motif, but not at its T-loop residue, in PDK1(-/-) cells. IGF1 did not induce phosphorylation of p70 S6 kinase at its hydrophobic motif in PDK1(-/-) cells. CONCLUSIONS: PDK1 mediates activation of PKB, p70 S6 kinase and p90 Rsk in vivo, but is not rate-limiting for activation of PKA, MSK1 and AMPK. Another kinase phosphorylates PKB at its hydrophobic motif in PDK1(-/-) cells. PDK1 phosphorylates the hydrophobic motif of p70 S6 kinase either directly or by activation of another kinase.

Essential role of phosphoinositide 3-kinase in leptin-induced K(ATP) channel activation in the rat CRI-G1 insulinoma cell line.

The mechanism by which leptin increases ATP-sensitive K(+) (K(ATP)) channel activity was investigated using the insulin-secreting cell line, CRI-G1. Wortmannin and LY 294002, inhibitors of phosphoinositide 3-kinase (PI3-kinase), prevented activation of K(ATP) channels by leptin. The inositol phospholipids phosphatidylinositol bisphosphate and phosphatidylinositol trisphosphate (PtdIns(3,4,5)P(3)) mimicked the effect of leptin by increasing K(ATP) channel activity in whole-cell and inside-out current recordings. LY 294002 prevented phosphatidylinositol bisphosphate, but not PtdIns(3,4,5)P(3), from increasing K(ATP) channel activity, consistent with the latter lipid acting as a membrane-associated messenger linking leptin receptor activation and K(ATP) channels. Signaling cascades, activated downstream from PI 3-kinase, utilizing PtdIns(3,4,5)P(3) as a second messenger and commonly associated with insulin and cytokine action (MAPK, p70 ribosomal protein-S6 kinase, stress-activated protein kinase 2, p38 MAPK, and protein kinase B), do not appear to be involved in leptin-mediated activation of K(ATP) channels in this cell line. Although PtdIns(3,4,5)P(3) appears a plausible and attractive candidate for the messenger that couples K(ATP) channels to leptin receptor activation, direct measurement of PtdIns(3,4,5)P(3) demonstrated that insulin, but not leptin, increased global cellular levels of PtdIns(3,4,5)P(3). Possible mechanisms to explain the involvement of PI 3-kinases in K(ATP) channel regulation are discussed.

The pleckstrin homology domains of protein kinase B and GRP1 (general receptor for phosphoinositides-1) are sensitive and selective probes for the cellular detection of phosphatidylinositol 3,4-bisphosphate and/or phosphatidylinositol 3,4,5-trisphosphate in vivo.

We have tested the binding specificities of the pleckstrin homology (PH) domains of protein kinase B (PKB) and GRP1 (general receptor for phosphoinositides-1), expressed as green fluorescent protein (GFP) fusion proteins [PH(PKB)GFP and PH(GRP1)GFP respectively] in HEK 293 cells and Swiss 3T3 cells, using confocal microscopy. Stimulation of HEK 293 cells with insulin caused a small, but sustained, increase in PtdIns(3,4,5)P(3) levels, detected using a radioligand displacement assay, which was mirrored by the translocation of PH(PKB)GFP and PH(GRP1)GFP from the cytosol to the plasma membrane of live, transfected cells. Similar results were obtained using Swiss 3T3 cells stimulated with platelet-derived growth factor (PDGF) and expressing either PH(PKB)GFP or PH(GRP1)GFP. Biochemical analyses confirmed the accumulation of both PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) in response to PDGF, but only the latter was present at increased levels in Swiss 3T3 cells 30 min after an oxidative stress (1 mM H(2)O(2)). Concomitantly, only PH(PKB)GFP, and not PH(GRP1)GFP, was localized at plasma membranes after 30 min of treatment with H(2)O(2). The fusion proteins appear accurately to report the spatial and temporal distribution of PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) in intact cells. These results establish the lipid selectivity of these PH domains in vivo, and further emphasize the overlapping, but distinct, second messenger roles of PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2).

Distinct phosphatidylinositol 3-kinase lipid products accumulate upon oxidative and osmotic stress and lead to different cellular responses.

Signaling by phosphatidylinositol (PI) 3-kinases is mediated by 3-phosphoinositides, which bind to Pleckstrin homology (PH) domains that are present in a wide spectrum of proteins. PH domains can be classified into three groups based on their different lipid binding specificities. Distinct 3-phosphoinositides can accumulate upon PI 3-kinase activation in cells in response to different stimuli and mediate specific cellular responses. In Swiss 3T3 mouse fibroblasts, oxidative stress induced by 1 mM H(2)O(2) caused almost exclusive accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3, 4)P(2)), whereas osmotic stress increased both phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and PtdIns(3,4)P(2) levels. The increase in PtdIns(3,4)P(2) levels, caused by oxidative stress, correlated with the activation of protein kinase B, which has a promiscuous PH domain that binds both PtdIns(3,4,5)P(3) and PtdIns(3, 4)P(2). p70 S6 kinase, another signaling component downstream of PI 3-kinase, however, was not activated by this oxidative stress-induced increase in PtdIns(3,4)P(2) levels. Increased PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) levels in response to osmotic stress did not correlate with protein kinase B activation, because of concomitant activation of an inhibitory pathway, but p70 S6 kinase was activated by osmotic stress. These results demonstrate that PtdIns(3,4)P(2) can accumulate independently of PtdIns(3,4, 5)P(3) and exerts a pattern of cellular responses that is distinct from that induced by accumulation of PtdIns(3,4,5)P(3).

The lipid phosphatase activity of PTEN is critical for its tumor supressor function.

Since their discovery, protein tyrosine phosphatases have been speculated to play a role in tumor suppression because of their ability to antagonize the growth-promoting protein tyrosine kinases. Recently, a tumor suppressor from human chromosome 10q23, called PTEN or MMAC1, has been identified that shares homology with the protein tyrosine phosphatase family. Germ-line mutations in PTEN give rise to several related neoplastic disorders, including Cowden disease. A key step in understanding the function of PTEN as a tumor suppressor is to identify its physiological substrates. Here we report that a missense mutation in PTEN, PTEN-G129E, which is observed in two Cowden disease kindreds, specifically ablates the ability of PTEN to recognize inositol phospholipids as a substrate, suggesting that loss of the lipid phosphatase activity is responsible for the etiology of the disease. Furthermore, expression of wild-type or substrate-trapping forms of PTEN in HEK293 cells altered the levels of the phospholipid products of phosphatidylinositol 3-kinase and ectopic expression of the phosphatase in PTEN-deficient tumor cell lines resulted in the inhibition of protein kinase (PK) B/Akt and regulation of cell survival.

Disruption by lithium of phosphoinositide signalling in cerebellar granule cells in primary culture.

Mild depolarisation (20 mM KCl) synergistically enhances the ability of a muscarinic agonist to activate phosphoinositide turnover and to elevate inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in cerebellar granule cells in primary culture. The effects of lithium on this intense stimulation of phosphoinositide turnover was studied. Lithium causes depletion of cytoplasmic inositol and phosphoinositides, which results in the inhibition of phosphoinositide turnover within 15 min and the return of Ins(1,4,5)P3 to basal levels at this time. This inhibition could not be reversed by culturing and preincubating cerebellar granule cells in concentrations of inositol similar to those detected in the CSF. Inositol concentrations substantially in excess of those in the CSF not only reversed the effects of lithium on stimulated Ins(1,4,5)P3 levels, but significantly enhanced this level in comparison with stimulation in the absence of lithium. sn-1,2-Diacylglycerol elevation during stimulated phosphoinositide turnover was also disrupted by lithium, but in contrast to Ins(1,4,5)3, the presence of lithium resulted in a transient enhancement of the elevation evoked by carbachol plus mild KCl depolarisation, which was reversed by 500 microM inositol, but not by 200 microM inositol. The implications of these phenomena in relation to the mechanism of action of lithium in the treatment of manic depression are discussed.

Insulin activates protein kinase B, inhibits glycogen synthase kinase-3 and activates glycogen synthase by rapamycin-insensitive pathways in skeletal muscle and adipose tissue.

Insulin stimulated protein kinase B alpha (PKB alpha) more than 10-fold and decreased glycogen synthase kinase-3 (GSK3) activity by 50 +/- 10% in skeletal muscle and adipocytes. Rapamycin did not prevent the activation of PKB, inhibition of GSK3 or stimulation of glycogen synthase up to 5 min. Thus rapamycin-insensitive pathways mediate the acute effect of insulin on glycogen synthase in the major insulin-responsive tissues. The small and very transient effects of EGF on phosphatidylinositol (3,4,5)P3 PKB alpha and GSK3 in adipocytes, compared to the strong and sustained effects of insulin, explains why EGF does not stimulate glucose uptake or glycogen synthesis in adipocytes.

A novel, rapid, and highly sensitive mass assay for phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and its application to measure insulin-stimulated PtdIns(3,4,5)P3 production in rat skeletal muscle in vivo.

The pivotal role of phosphatidylinositol 3-kinase (PI 3-kinase) in signal transduction has been well established in recent years. Receptor-regulated forms of PI 3-kinase are thought to phosphorylate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) at the 3-position of the inositol ring to give the putative lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4, 5)P3). Cellular levels of PtdIns(3,4,5)P3 are currently measured by time-consuming procedures involving radiolabeling with high levels of 32PO4, extraction, and multiple chromatography steps. To avoid these lengthy and hazardous procedures, many laboratories prefer to assay PI 3-kinase activity in cell extracts and/or appropriate immunoprecipitates. Such approaches are not readily applied to measurements of PtdIns(3,4,5)P3 in extracts of animal tissues. Moreover, they can be misleading since the association of PI 3-kinases in molecular complexes is not necessarily correlated with the enzyme's activity state. Direct measurements of PtdIns(3,4,5)P3 would also be desirable since its concentration may be subject to additional control mechanisms such as activation or inhibition of the phosphatases responsible for PtdIns(3,4,5)P3 metabolism. We now report a simple, reproducible isotope dilution assay which detects PtdIns(3,4,5)P3 at subpicomole sensitivity, suitable for measurements of both basal and stimulated levels of PtdIns(3,4,5)P3 obtained from samples containing approximately 1 mg of cellular protein. Total lipid extracts, containing PtdIns(3,4,5)P3, are first subjected to alkaline hydrolysis which results in the release of the polar head group Ins(1,3,4,5)P4. The latter is measured by its ability to displace [32P]Ins(1,3,4,5)P4 from a highly specific binding protein present in cerebellar membrane preparations. We show that this assay solely detects PtdIns(3,4,5)P3 and does not suffer from interference by other compounds generated after alkaline hydrolysis of total cellular lipids. Measurements on a wide range of cells, including rat-1 fibroblasts, 1321N1 astrocytoma cells, HEK 293 cells, and rat adipocytes, show wortmannin-sensitive increased levels of PtdIns(3,4,5)P3 upon stimulation with appropriate agonists. The enhanced utility of this procedure is further demonstrated by measurements of PtdIns(3,4,5)P3 levels in tissue derived from whole animals. Specifically, we show that stimulation with insulin increases PtdIns(3,4,5)P3 levels in rat skeletal muscle in vivo with a time course which parallels the activation of protein kinase B in the same samples.

Dictyostelium discoideum contains three inositol monophosphatase activities with different substrate specificities and sensitivities to lithium.

The small ion lithium, a very effective agent in the treatment of manic depressive patients, inhibits the mammalian enzyme inositol monophosphatase, which is proposed as the biological target for the effects of lithium. In this study we investigated Dictyostelium discoideum inositol monophosphatase activity. Partial purification of the proteins in the soluble cell fraction using anion-exchange chromatography revealed the presence of at least three enzyme activities capable of degrading inositol monophosphate isomers. The first activity was similar to the monophosphatase found in mammalian cells, as it degraded Ins(4)P, Ins(1)P and to a lesser extent Ins(3)P, was dependent on MgCl2 and inhibited by LiCl in a uncompetitive [corrected] manner. The second enzyme activity was specific for Ins(4)P; the enzyme activity was not dependent on MgCl2 and not inhibited by LiCl. The third monophosphatase activity degraded especially Ins(3)P, but also Ins(4)P and Ins(1)P; increasing concentrations of MgCl2 inhibited this enzyme activity, whereas LiCl had no effect. In vivo, LiCl induces a reduction of inositol levels by about 20%. In [3H]inositol-labelled cells LiCl causes a 6-fold increase in the radioactivity of [3H]Ins(1)P, a doubling of [3H]Ins(4)P and a slight decrease in the radioactivity in [3H]Ins(3)P. These data indicate that the biological effects of lithium in Dictyostelium are not due to depletion of the inositol pool by inhibition of inositol monophosphatase activity.

Stereospecificity of inositol hexakisphosphate dephosphorylation by Paramecium phytase.

InsP6 is an abundant compound in many micro-organisms, plants and animal cells. Its function and route of synthesis are still largely unknown. Degradation of InsP6 is mediated by phytase, which in most organisms dephosphorylates InsP6 in a relatively non-specific way. In the micro-organism Paramecium, however, the enzyme has been shown to dephosphorylate InsP6 to InsP2 in a specific order, but its stereospecificity has not been established, i.e. the phosphates are removed in the sequence 6/5/4/3 or 6/5/4/1 or 4/5/6/1 or 4/5/6/3 [Freund, Mayr, Tietz and Schultz (1992) Eur. J. Biochem. 207, 359-367]. We have isolated the InsP4 intermediate and identified its absolute configuration as D-Ins(1,2,3,4)P4. Furthermore, degradation of [3,5-32P]InsP6 yielded a 32P-labelled InsP2 isomer, D-Ins(2,3)P2. These data demonstrate that Paramecium phytase removes the phosphates of InsP6 in the sequence 6/5/4/1. Knowing the stereochemical course of the enzyme, it can be used to elucidate the route of InsP6 synthesis, as it allows us to determine the specific radioactivity at individual positions of the molecular after pulse-labelling cells with [32P]P1 in vivo or [gamma-32P]ATP in vitro.

Nucleus-associated phosphorylation of Ins(1,4,5)P3 to InsP6 in Dictyostelium.

Although many cells contain large amounts of InsP6, its metabolism and function is still largely unknown. In Dictyostelium lysates, the formation of InsP6 by sequential phosphorylation of inositol via Ins(3,4,6)P3 has been described [Stevens and Irvine (1990) Nature (London) 346, 580-583]; the second messenger Ins(1,4,5)P3 was excluded as a potential substrate or intermediate for InsP6 formation. However, we observed that mutant cells labelled in vivo with [3H]inositol showed altered labelling of both [3H]Ins(1,4,5)P3 and [3H]InsP6. In this report we demonstrate that Ins(1,4,5)P3 is converted into InsP6 in vitro by nucleus-associated enzymes, in addition to the previously described stepwise phosphorylation of inositol to InsP6 that occurs in the cytosol. HPLC analysis indicates that Ins(1,4,5)P3 is converted into InsP6 via sequential phosphorylation at the 3-, 6- and 2-positions. Ins[32P]P6, isolated from cells briefly labelled with [32P]Pi, was analysed using Paramecium phytase, which removes the phosphates of InsP6 in a specific sequence. The 6-position contained significantly more 32P radioactivity than the 4- or 5-positions, indicating that the 6-position is phosphorylated after the other two positions. The results from these in vivo and in vitro experiments demonstrate a metabolic route involving the phosphorylation of Ins(1,4,5)P3 via Ins(1,3,4,5)P4 and Ins(1,3,4,5,6)P5 to InsP6 in a nucleus-associated fraction of Dictyostelium cells.

Role of phospholipase C in Dictyostelium: formation of inositol 1,4,5-trisphosphate and normal development in cells lacking phospholipase C activity.

The micro-organism Dictyostelium uses extracellular cAMP to induce chemotaxis and cell differentiation. Signals are transduced via surface receptors, which activate G proteins, to effector enzymes. The deduced protein sequence of Dictyostelium discoideum phosphatidylinositol-specific phospholipase C (PLC) shows strong homology with the mammalian PLC-delta isoforms. To study the role of PLC in Dictyostelium, a plc- mutant was constructed by disruption of the PLC gene. No basal or stimulated PLC activity could be measured during the whole developmental programme of the plc- cells. Loss of PLC activity did not result in a visible alteration of growth or development. Further analysis showed that developmental gene regulation, cAMP-mediated chemotaxis and activation of guanylyl and adenylyl cyclase were normal. Although the cells lack PLC activity, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was present at only slightly lower concentrations compared with control cells. Mass analysis of inositol phosphates demonstrated the presence of a broad spectrum of inositol phosphates in Dictyostelium, which was unaltered in the plc- mutant. Cell labelling experiments with [3H]inositol indicated that [3H]Ins(1,4,5)P3 was formed in a different manner in the mutant than in control cells.

Dynamics and function of the inositolcycle in Dictyostelium discoideum.

The inositolcycle in Dictyostelium discoideum was studied under several conditions both in vitro and in vivo. The results are compared with the inositolcycle as it is known from higher eukaryotes: although there is a strong resemblance both cycles are different at some essential points.

Increased conversion of phosphatidylinositol to phosphatidylinositol phosphate in Dictyostelium cells expressing a mutated ras gene.

Dictyostelium discoideum cells that overexpress a ras gene with a Gly12----Thr12 mutation (Dd-ras-Thr12) have an altered phenotype. These cells were labeled with [3H]inositol and the incorporation of radioactivity into inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was analyzed and found to be higher than in control cells. In contrast, the total mass of Ins(1,4,5)P3, as assessed with an assay using a specific Ins(1,4,5)P3-binding protein, was not significantly different between control and Dd-ras-Thr12 cells. Cells were labeled with [3H]inositol and the incorporation of radioactivity in all inositol metabolites was analyzed. Increased levels of radioactivity were observed for phosphatidylinositol phosphate (PtdInsP), phosphatidylinositol bisphosphate (PtdInsP2), Ins(1,4,5)P3, inositol 1,4-bisphosphate, inositol 4,5-bisphosphate, and inositol 4-monophosphate in Dd-ras-Thr12 cells relative to control cells. Decreased levels were found for phosphatidylinositol (PtdIns) and inositol 1-monophosphate. Calculations on the substrate/product relationships [i.e., Ins(1,4,5)P3/PtdInsP2] demonstrate that the observed differences are due only to the increased conversion of PtdIns to PtdInsP; other enzyme reactions, including phospholipase C, are not significantly different between the cell lines. The activity of PtdIns kinase in vitro is not different between Dd-ras-Thr12 and control cells, suggesting that either the regulation of this enzyme is altered or that the translocation of substrate from the endoplasmic reticulum to the kinase in the plasma membrane is modified. The results suggest multiple metabolic compartments of Ins(1,4,5)P3 in Dictyostelium cells. In Dd-ras-Thr12 transformants the increased conversion of PtdIns to PtdInsP leads to increased levels of Ins(1,4,5)P3 in the compartment with a high metabolic turnover. This Ins(1,4,5)P3 compartment is suggested to be involved in the regulation of cytosolic Ca2+ levels.