Loss of PTEN selectively desensitizes upstream IGF1 and insulin signaling.

Many tumors have chronically elevated activity of PI 3-kinase-dependent signaling pathways, caused largely by oncogenic mutation of PI 3-kinase itself or loss of the opposing tumor suppressor lipid phosphatase, PTEN. Several PI 3-kinase-dependent feedback mechanisms have been identified that may affect the sensitivity of upstream receptor signaling, but the events required to initiate an inhibited state have not been addressed. We show that in a variety of cell types, loss of PTEN via experimental knockdown or in tumor cell lines correlates with a block in insulin-like growth factor 1 (IGF1)/insulin signaling, without affecting the sensitivity of platelet-derived growth factor or epidermal growth factor signaling. These effects on IGF/insulin signaling include a reduction of up to five- to tenfold in IGF-stimulated PI 3-kinase activation, a failure to activate the ERK kinases and, in some cells, reduced expression of insulin receptor substrate 1, and both IGF1 and insulin receptors. These data indicate that chronically elevated PI 3-kinase-dependent signaling to the degree seen in many tumors causes a selective loss of sensitivity in IGF1/insulin signaling that could significantly reduce the selective advantage of deregulated activation of IGF1/IGF1-R signaling in tumor development.

Understanding PTEN regulation: PIP2, polarity and protein stability.

The PTEN tumour suppressor is a lipid and protein phosphatase that inhibits phosphoinositide 3-kinase (PI3K)-dependent signalling by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)). Here, we discuss the concept of PTEN as an 'interfacial enzyme', which exists in a high activity state when bound transiently at membrane surfaces containing its substrate and other acidic lipids, such as PtdIns(4,5)P(2) and phosphatidylserine (PtdSer). This mechanism ensures that PTEN functions in a spatially restricted manner, and may explain its involvement in forming the gradients of PtdInsP(3), which are necessary for generating and/or sustaining cell polarity during motility, in developing neurons and in epithelial tissues. Coordinating PTEN activity with alternative mechanisms of PtdInsP(3) metabolism, by the tightly regulated SHIP 5-phoshatases, synthesizing the independent second messenger PtdIns(3,4)P(2), may also be important for cellular polarization in some cell types. Superimposed on this interfacial mechanism are additional post-translational regulatory processes, which generally act to reduce PTEN activity. Oxidation of the active site cysteine residue by reactive oxygen species and phosphorylation of serine/threonine residues at sites in the C-terminus of the protein inhibit PTEN. These phosphorylation sites also appear to play a role in regulating both stability and localization of PTEN, as does ubiquitination of PTEN. Because genetic studies in mice show that the level of expression of PTEN in an organism profoundly influences tumour susceptibility, factors that regulate PTEN, localization, activity and turnover should be important in understanding its biological functions as a tumour suppressor.

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).

The regulation of membrane to cytosol partitioning of signalling proteins by phosphoinositides and their soluble headgroups.

Inositol phospholipids [PIs (phosphoinositides)] represent a group of membrane-tethered signalling molecules which differ with respect to the number and distribution of monoester phosphate groups around the inositol ring. They function by binding to proteins which possess one of several domains that bind a particular PI species, often with high affinity and specificity. PH (pleckstrin homology) domains for example possess ligand-binding pockets that are often lined with positively charged residues and which bind PIs with varying degrees of specificity. Several PH domains bind not only PIs, but also their cognate headgroups, many of which occur naturally in cells as relatively abundant cytosolic inositol phosphates. The subcellular distributions of proteins possessing such PH domains are therefore determined by the relative levels of competing membrane-bound and soluble ligands. A classic example of the latter is the PH domain of phospholipase Cdelta1, which binds both phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate. We have shown that the N-terminal PH domain of the Rho family guanine nucleotide-exchange factor, Tiam 1, binds PI ligands promiscuously allowing multiple modes of regulation. We also recently analysed the ligand-binding specificity of the PH domain of PI-dependent kinase 1 and found that it could bind abundant inositol polyphosphates such as inositol hexakisphosphate. This could explain the dual distribution of this key signalling component, which needs to access substrates at both the plasma membrane and in the cytosol.

The inhibition of phosphoinositide synthesis and muscarinic-receptor-mediated phospholipase C activity by Li+ as secondary, selective, consequences of inositol depletion in 1321N1 cells.

Conditions are described for culture of 1321N1 cells under which cellular inositol is decreased from approximately 20 mM to < 0.5 mM but phosphoinositide concentrations are unaffected. The effects of the muscarinic-receptor agonist carbachol (1 mM) and/or LiCl (10 mM) on phosphoinositide turnover in these or in inositol-replete cells was examined after steady-state [3H]inositol labelling of phospholipid pools. In both inositol-replete and -depleted cells, carbachol stimulated similar initial (0-15 min) rates of phospholipase C (PLC) activity, in the presence of Li+. Subsequently (> 30-60 min) stimulated PLC activity and [3H]PtdIns concentrations declined dramatically only in depleted cells. In inositol-depleted cells, carbachol alone evoked increased concentrations of [3H]inositol, [3H]InsP1, [3H]InsP2, [3H]InsP3 and [3H]InsP4, which were largely sustained over 90 min, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased only to approximately 82, 84 and 93% of control respectively. In the presence of Li+ in these cells, the stimulated rise in [3H]inositol was prevented and, although accumulation of [3H]InsP1, [3H]InsP2 and [3H]InsP3 was initially (0-30 min) potentiated, rates of accumulation of [3H]InsP1 and concentrations of [3H]polyphosphates later (> 30-60 min) declined, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased respectively to approximately 39, 48 and 81% of control. After 60 min in the presence of both carbachol and Li+, stimulated PLC activity was decreased by approximately 70% compared with the initial rate in depleted cells. This decreased PLC activity was reflected by changes in the stimulated concentrations of [3H]Ins(1,3,4)P3 but not of [3H]Ins(1,4,5)P3, but effects of Li+ on the latter may have been obscured by the demonstrated, concomitant and equal stimulated accumulation of [3H]inositol 1:2cyclic,4,5-trisphosphate. These data suggest that receptor-mediated PLC activity is selectively impaired by Li+ as a secondary consequence of inositol monophosphatase inhibition in cells which are highly dependent on inositol re-cycling, but imply that, although Li+ attenuation of PLC activity correlates closely with parameters indicative of limiting inositol supply, it is not readily attributed to decreased PtdInsP2 availability. The potential for complex regulation of PLC and PtdIns synthase is discussed.

Analysis of [3H]inositol phosphate formation and metabolism in cerebral-cortical slices. Evidence for a dual metabolism of inositol 1,4-bisphosphate.

Muscarinic-receptor-mediated phosphoinositide hydrolysis in rat cerebral cortex was investigated by analysis of the kinetics of [3H]inositol phosphate formation and degradation in myo-[2-3H]inositol-labelled tissue slices. Carbachol stimulated rapid (5 s) increases in the concentrations of [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4 and [3H]Ins(1,4)P2. Stimulated accumulation of [3H]Ins(1,3,4)P3, [3H]Ins(1,3)P2 and [3H]Ins(3,4)P2 and [3H]Ins(1/3)P or of [3H]Ins(4)P occurred only subsequently and with a sequence indicating formation by successive dephosphorylation of [3H]Ins(1,3,4,5)P4 or of Ins(1,4)P2 respectively. A similar sequence was inferred from the order of rapidity with which the accumulations of [3H]inositol polyphosphates, resulting from sustained (5 min) carbachol stimulation in the presence of LiCl, were reversed when muscarinic receptors were subsequently blocked with atropine. During this latter period of receptor blockade, radiolabel lost from [3H]inositol polyphosphates was quantitively recovered as [3H]inositol monophosphates owing to effective inhibition of monophosphatase by Li+, and the rate of poly- into mono-phosphate conversion was similar to agonist-stimulated rates of monophosphate accumulation. This implies that, even during persistent stimulation, polyphosphoinositide, not PtdIns, is the substrate for phosphoinositidase C. Quantitative comparison of the degradation of [3H]inositol poly- to mono-phosphates after receptor blockade unexpectedly suggests the dual hydrolysis of [3H]Ins(1,4)P2 to [3H]Ins(1)P and [3H]Ins(4)P. This result advises cautious interpretation of the origin of [3H]Ins(1)P in stimulated tissue, but, with other data presented, allows calculation from the observed ratio of [3H]Ins(1/3)P:[3H]Ins(4)P that a minimum of approx. 50% of the [3H]Ins(1,4,5)P3 produced during persistent muscarinic-receptor stimulation is metabolized by Ins(1,4,5)P3 3-kinase.

The mechanism of muscarinic receptor-stimulated phosphatidylinositol resynthesis in 1321N1 astrocytoma cells and its inhibition by Li+.

The coupling of muscarinic receptor-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C to resynthesis of phosphatidylinositol (PtdIns) and the ability of Li+ to inhibit this after cellular inositol depletion were studied in 1321N1 astrocytoma cells cultured in medium +/- inositol (40 microM). In inositol-replete cells, 1 mM carbachol/10 mM LiCl evoked an initial (0-30 min) approximately > or = 20-fold activation of phospholipase C, whereas prolonged (> 60 min) stimulation turned over PtdIns equal to the cellular total mass, involving approximately 80% of the cellular PtdIns pool without reducing PtdIns concentrations significantly. PtdIns resynthesis was achieved by a similar, initial agonist activation of PtdIns synthase. The dose dependency for carbachol stimulation of PtdIns synthase and phospholipase C was similar (EC50 approximately 20 microM) as was the relative intrinsic activity of muscarinic receptor partial agonists. This demonstrates the tight coupling of phosphoinositide hydrolysis to resynthesis and suggests this is achieved by a direct mechanism. In inositol-replete or depleted cells basal concentrations of inositol and CMP-phosphatidate were respectively approximately 20 mM or < or = 100-500 microM and approximately 0.1 or approximately > or = 1-10 pmol/mg of protein. Comparison of the effects of agonist +/- Li+ on the concentrations of these cosubstrates for PtdIns synthase suggest that accelerated activity of this enzyme is differentially driven by stimulated increases in the amounts of CMP-phosphatidate or inositol in inositol-replete or depleted cells, respectively. Thus, the preferential capacity of Li+ to impair stimulated phosphoinositide turnover in systems expressing low cellular inositol can be attributed to its ability to attenuate the stimulated rise in inositol concentrations on which such systems selectively depend to trigger accelerated PtdIns resynthesis.

Thrombin receptors modulate insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate accumulation in 1321N1 astrocytoma cells.

Thrombin and insulin receptor signaling via phosphoinositide (PI)-specific phospholipase C (PLC) and PI 3-kinase was studied in [3H]inositol-labelled 1321N1 cells. Thrombin stimulated a dramatic, transient activation of PLC which is probably mediated via receptors of the 'tethered-ligand' type, since it was both reproduced by, and abolished following, pretreatment of cells with a synthetic peptide (SFLLRN) corresponding to the ligand domain of the human thrombin receptor. However, neither thrombin nor SFLLRN stimulated PI 3-kinase. By contrast, insulin did not influence [3H]InsP3 concentration but stimulated accumulation of [3H]PtdIns(3,4,5)P3 and [3H]PtdIns(3,4)P2, the relative steady-state concentrations of which may indicate degradation of [3H]PtdIns(3,4,5)P3 by 5- and 3-phosphatases. The independent coupling of thrombin and insulin receptors to PLC and PI 3-kinase respectively in 1321N1 cells allowed interactions between these systems to be examined. Thus insulin-stimulated [3H]PtdIns(3,4,5)P3 accumulation was attenuated on co-stimulation of the thrombin receptor, whereas concentrations of [3H]PtdIns(3,4)P2 were transiently enhanced but then reduced. These results indicate that thrombin receptors in 1321N1 cells do not activate PI 3-kinase, but can modulate signalling by this enzyme.

Rapid accumulation and sustained turnover of inositol phosphates in cerebral-cortex slices after muscarinic-receptor stimulation.

The rapid kinetics of [3H]inositol phosphate accumulation and turnover were examined in rat cerebral-cortex slices after muscarinic-receptor stimulation. Markedly increased [3H]inositol polyphosphate concentrations were observed to precede significant stimulated accumulation of [3H]inositol monophosphate. New steady-state accumulations of several 3H-labelled products were achieved after 5-10 min of continued agonist stimulation, but were rapidly and effectively reversed by subsequent receptor blockade. The results show that muscarinic-receptor activation involves phosphoinositidase C-catalysed hydrolysis initially of polyphosphoinositides rather than of phosphatidylinositol. Furthermore, prolonged carbachol stimulation is shown not to cause receptor desensitization, but to allow persistent hydrolysis of [3H]phosphatidylinositol bisphosphate and permit sustained metabolic flux through the inositol tris-/tetrakis-phosphate pathway.

Mass measurements of inositol(1,4,5)trisphosphate in rat cerebral cortex slices using a radioreceptor assay: effects of neurotransmitters and depolarization.

The specific binding of [3H] and [32P]Ins(1,4,5)P3 to a particulate preparation of bovine adrenal cortex has been used as a radioreceptor assay to determine the concentration of Ins(1,4,5)P3 in agonist- and depolarization-stimulated rat cerebral cortex slices. The resting concentration of Ins(1,4,5)P3 in slices that had been preincubated in a physiological medium was 18.8 +/- 2.6 pmol/mg prot. Carbachol evoked a rapid and dose-related increase in the concentration of Ins(1,4,5)P3. Maximal stimulation (80%) was already seen at the earliest point (10 sec) examined and was maintained for at least 5 min. The EC50 for carbachol was 75 +/- 17 microM and the response was totally suppressed by the muscarinic antagonist atropine. A direct comparison in the same slices was made between mass determinations and [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 accumulation determined by h.p.l.c. Although an identical time course was observed for cold and radiolabelled Ins(1,4,5)P3, the greater stimulation of [3H]Ins(1,4,5)P3 may indicate changes in specific radioactivity. Of a variety of other receptor agonists studied, only the glutamate receptor agonist quisqualate, and noradrenaline significantly increased the mass of Ins(1,4,5)P3 in cerebral cortical slices. However, depolarizing concentrations of K+ were as effective as carbachol at elevating this second messenger.

Evidence for a model of integrated inositol phospholipid pools implies an essential role for lipid transport in the maintenance of receptor-mediated phospholipase C activity in 1321N1 cells.

The compartmentation of inositol phospholipids was examined by using a combination of radiolabelling approaches in intact and permeabilized 1321N1 astrocytoma cells. A 'chase' protocol was developed with whole cells in which phosphoinositide (PI) pools were labelled to steady state with [3H]inositol and the cellular [3H]inositol pool was then diluted selectively with non-radioactive inositol. In these cells muscarinic-receptor-stimulated phospholipase C (PLC) hydrolysed [3H]PI at approx. 1-2%/min. However, after the chase procedure the relative specific radioactivity of [3H]Ins(1,3,4)P3, a rapidly metabolized and sensitive marker of PLC activity, decreased only after more than 5 min and over a time course similar to that during which the labelling of each [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 declined by at least 50%. These results demonstrate a large receptor-responsive [3H]PI pool that is accessed by stimulated PLC without apparent metabolic compartmentation, despite its probable distribution between different membrane fractions. Support for this was obtained in intact cells by using an acute [3H]inositol labelling method in which increases in the specific radioactivity of [3H]inositol phosphates stimulated by carbachol occurred only in parallel with similar increases in the labelling of the bulk of cellular [3H]PI. In [3H]inositol-prelabelled cells permeabilized to deplete cytosolic proteins, carbachol and guanosine 5'-[gamma-thio]triphosphate stimulated the endogenous PLC to degrade only approx. 5% of [3H]PI. This was increased to approx. 30% in the presence of exogenous PtdIns transfer protein, which, at a concentration approx. 5-10% of that in 1321N1 cell cytosol, was sufficient to support PLC activity comparable with that observed in response to carbachol in whole cells. These and earlier results in 1321N1 cells suggest a model of integrated PI pools involving an obligatory role for lipid transport. Given the multifunctional capacity of PI in cellular signalling mechanisms, this model has important implications, particularly for the hypothesis that the ability of Li+ ions to influence these selectively might account for its therapeutic actions.

Accumulation of inositol polyphosphate isomers in agonist-stimulated cerebral-cortex slices. Comparison with metabolic profiles in cell-free preparations.

1. Basal and carbachol-stimulated accumulations of isomeric [3H]inositol mono-, bis-, tris- and tetrakis-phosphates were examined in rat cerebral-cortex slices labelled with myo-[2-3H]inositol. 2. In control samples the major [3H]inositol phosphates detected were co-eluted on h.p.l.c. with Ins(1)P, Ins(4)P (inositol 1- and 4-monophosphate respectively), Ins(1,4)P2 (inositol 1,4-bisphosphate), Ins(1,4,5)P3 (inositol 1,4,5-tris-phosphate) and Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate). 3. After stimulation to steady state with carbachol, accumulation of each of these products was markedly increased. 4. Agonist stimulation, however, also evoked much more dramatic increased accumulations of a second [3H]inositol trisphosphate, which was co-eluted on h.p.l.c. with authentic Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) and of three further [3H]inositol bisphosphates ([3H]InsP2(s]. 5. Examination of the latter by chemical degradation by periodate oxidation and/or h.p.l.c. allowed identification of these as [3H]Ins(1,3)P2, [3H]Ins(3,4)P2 and [3H]Ins(4,5)P2 (inositol 1,3-, 3,4- and 4,5-bisphosphates respectively), which respectively accounted for about 22%, 8% and 3% of total [3H]InsP2 in extracts from stimulated tissue slices. 6. By using a h.p.l.c. method which clearly resolves Ins(1,3,4,5)P4 and Ins(1,3,4,6)P4 (inositol 1,3,4,6-tetrakisphosphate), only the former isomer could be detected in extracts from either control or stimulated tissue slices. Similarly, [3H]inositol pentakis- and hexakis-phosphates were not detectable either in the presence or absence of carbachol under the radiolabelling conditions described. 7. The catabolism of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 by cell-free preparations from cerebral cortex was also studied. 8. In the presence of Mg2+, [3H]Ins(1,4,5)P3 was specifically dephosphorylated via [3H]Ins(1,4)P2 and [3H]Ins(4)P to free [3H]inositol, whereas [3H]Ins(1,3,4)P3 was degraded via [3H]Ins(3,4)P2 and, to a lesser extent, via [3H]Ins(1,3)P2 to D- and/or L-[3H]Ins(1)P and [3H]inositol. 9. In the presence of EDTA, hydrolysis of [3H]Ins(1,4,5)P3 was greater than or equal to 95% inhibited, whereas [3H]Ins(1,3,4)P3 was still degraded, but yielded only a single [3H]InsP2 identified as [3H]Ins(1,3)P2. 10. The significance of these observations with cell-free preparations is discussed in relation to the proportions of the separate isomeric [3H]inositol phosphates measured in stimulated tissue slices.

Cross-talk between phospholipase C and phosphoinositide 3-kinase signalling pathways.

1321N1 astrocytoma cells have proved a valuable model system in which to study interactions between two major PtdIns (4,5) P2-utilizing signaling pathways, since they possess receptor populations which elicit independent activation of PI 3-kinase and a G-protein-dependent PLC respectively. Activation of PLC down-regulates PI 3-kinase by at least two mechanisms involving inhibition of IRS-1-associated PI 3-kinase and acute activation of a PtdIns (3,4,5) P3 5-phosphatase. PKB, which is an important early PI 3-kinase-dependent component of insulin signalling pathways, is also down-regulated by PLC-coupled agonists. The activation of PKB by insulin appears to involve a novel PtdIns (3,4,5) P3-dependent protein kinase, which we have named PDK1. The molecular mechanisms underlying PtdIns (3,4,5) P3-stimulated phosphorylation and activation of PKB by PDK1 are currently under investigation.

The characteristics, capacity and receptor regulation of inositol uptake in 1321N1 astrocytoma cells.

The uptake of inositol into 1321N1 astrocytoma cells was studied by measurement of the accumulation of free [3H]inositol within the intracellular pool. Uptake occurs via a saturable transporter with apparent Km for inositol approximately 40 microM and Vmax approximately 180 pmol/min per mg of protein, which permits intracellular inositol concentrations to exceed those of the medium by a factor of approximately 500. At extracellular concentrations up to 500 microM, inositol uptake is highly dependent (> or = 85%) on the presence of Na+ in the medium, and at physiological extracellular inositol concentrations, allows inositol to achieve an intracellular concentration of approximately 20 mM, indicating an active process driven by the Na+ gradient. Despite this, uptake was only minimally impaired or was unaffected by ouabain (1 mM) or dinitrophenol (1 mM). Consistent with a carrier-mediated mechanism, uptake was competitively blocked by phlorhizin (K1 approximately 125 microM). Uptake was also inhibited by carbachol and histamine, which act respectively via muscarinic and H1 receptors in these cells to stimulate phospholipase C. Inhibition by carbachol was dose-dependent (EC50 approximately 3-30 microM) and blocked by atropine. Inhibition by carbachol (1 mM) was non-competitive, resulting from approximately 50% decrease in the Vmax for uptake without affecting the Km and was persistent over 30-90 min. Inhibition by carbachol and histamine was independent of extracellular Ca2+ and was reproduced by phorbol ester, but not by Ca2+ ionophore or stimulation of adenylate cyclase. These results imply that receptors which couple to phospholipase C may mediate inhibition of inositol uptake via protein kinase C. The data are discussed in relation to inositol homoeostasis in resting and stimulated cells.

Distinct signalling pathways mediate insulin and phorbol ester-stimulated eukaryotic initiation factor 4F assembly and protein synthesis in HEK 293 cells.

Stimulation of serum-starved human embryonic kidney (HEK) 293 cells with either the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), or insulin resulted in increases in the phosphorylation of 4E-BP1 and p70 S6 kinase, eIF4F assembly, and protein synthesis. All these effects were blocked by rapamycin, a specific inhibitor of mTOR. Phosphatidylinositol 3-kinase and protein kinase B were activated by insulin but not by TPA. Therefore TPA can induce eIF4F assembly, protein synthesis, and the phosphorylation of p70 S6 kinase and 4E-BP1 independently of both phosphatidylinositol 3-kinase and protein kinase B. Using two structurally unrelated inhibitors of MEK (PD098059 and U0126), we provide evidence that Erk activation is important in TPA stimulation of eIF4F assembly and the phosphorylation of p70 S6 kinase and 4E-BP1 and that basal MEK activity is important for basal, insulin, and TPA-stimulated protein synthesis. Transient transfection of constitutively active mitogen-activated protein kinase interacting kinase 1 (the eIF4E kinase) indicated that inhibition of protein synthesis and eIF4F assembly by PD098059 is not through inhibition of eIF4E phosphorylation but of other signals emanating from MEK. This report also provides evidence that increased eIF4E phosphorylation alone does not affect the assembly of the eIF4F complex or general protein synthesis.

Formation of inositol polyphosphates in airway smooth muscle after muscarinic receptor stimulation.

The effect of muscarinic-receptor stimulation on [3H]inositol mono-, bis-, tris- and tetrakisphosphate (InsP1, InsP2, InsP3 and InsP4) accumulation was examined in bovine tracheal smooth muscle slices prelabeled with myo-[3H]inositol. Carbachol (0.1 mM) caused a rapid increase in [3H]InsP3 and [3H]InsP2 followed by delayed increases in [3H]InsP1 and [3H]InsP4 accumulation. Analysis of the [3H]InsP3 isomers by HPLC showed an immediate although transient increase in [3H]Ins(1,4,5)P3 with a progressive and sustained accumulation of [3H]Ins(1,3,4)P3. [3H]Ins(1,3,4)P3 was confirmed as the predominant (greater than 80%) [3H]InsP3 isomer present at 1 and 30 min using an enzymatic method which causes selective hydrolysis of Ins(1,3,4)P3. Lithium enhanced markedly the carbachol-stimulated accumulation of [3H]InsP1 and [3H]InsP2 with a lower potency than that observed in other tissues but had no significant influence on [3H]InsP3 or [3H]InsP4 values. These data support a role for Ins(1,4,5)P3 in initiating airway smooth muscle contraction and indicate the importance of the Ins(1,3,4,5)P4/Ins(1,3,4)P4 pathway in this tissue.

A simple enzymic method to separate [3H]inositol 1,4,5- and 1,3,4-trisphosphate isomers in tissue extracts.

A novel method to separate [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 in tissue extracts is described. It is based on the selective metabolism of Ins(1,3,4)P3 by a crude cerebral supernatant in a Mg2+-free buffer followed by separation of [3H]inositol trisphosphates using conventional anion-exchange chromatography. Evaluation of the assay was performed using [3H]Ins(1,3,4)P3 standards and tissue extracts containing different proportions of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3. Parallel h.p.l.c. separations of extracts established the selective and complete metabolism of [3H]Ins(1,3,4)P3 under the above conditions and demonstrated that the enzymic method provides an accurate estimate of the trisphosphate isomers in rat cerebral cortex, parotid gland and bovine tracheal smooth muscle.

Determination of mass changes in phosphatidylinositol 4,5-bisphosphate and evidence for agonist-stimulated metabolism of inositol 1,4,5-trisphosphate in airway smooth muscle.

Stimulation of muscarinic receptors in bovine tracheal smooth muscle (BTSM) causes a sustained increase in muscle tone, but a transient increase in the second messenger Ins(1,4,5)P3. To examine whether this brief increase in Ins(1,4,5)P3 mass results from transient formation or is due to agonist-stimulation of Ins(1,4,5)P3 metabolism, we have studied the relationship between mass changes in PtdIns(4,5)P2 and Ins(1,4,5)P3 accumulation, and changes in [3H]InsP3, [3H]PtdIns, [3H]PtdInsP1 and [3H]PtdInsP2 in carbachol-stimulated myo-[3H]inositol-prelabelled BTSM slices. Carbachol (0.1 mM) caused a rapid transient increase in Ins(1,4,5)P3 concentration (basal, 12.9 +/- 0.8 pmol/mg of protein; 5 s carbachol treatment, 27.1 +/- 1.5 pmol/mg of protein), with values returning to basal levels by 30 s, but a sustained accumulation of total [3H]InsP3s, with [3H]Ins(1,3,4)P3 being the predominant isomer present at later time points. In contrast, PtdIns(4,5)P2 mass, determined by radioreceptor assay of Ins(1,4,5)P3 in desalted alkaline hydrolysates of acidified chloroform/methanol tissue extracts, declined rapidly (basal, 941 +/- 22 pmol/mg of protein; 120 s carbachol, 365 +/- 22 pmol/mg of protein; t1/2 14 s) and remained at this new steady-state level for at least 20 min in the continued presence of carbachol. Addition of 10 microM-atropine 2 min after carbachol caused a prompt return of PtdIns(4,5)P2 concentration to prestimulated values (t1/2 210 s). Ongoing resynthesis of PtdIns(4,5)P2 after carbachol stimulation was demonstrated in [3H]inositol-labelled tissue by observing a persistent increase in the specific radioactivity of [3H]PtdInsP2, shown to be exclusively [3H]PtdIns(4,5)P2, over a 10 min period. These findings strongly suggest the occurrence of persistent receptor-mediated increases in PtdIns(4,5)P2 hydrolysis and Ins(1,4,5)P3 formation which, in conjunction with the transient accumulation of Ins(1,4,5)P3 observed, provide evidence that regulation of the metabolism of Ins(1,4,5)P3 is a major determinant of Ins(1,4,5)P3 concentration in this tissue under agonist-stimulated conditions.

Acute regulation of the tumour suppressor phosphatase, PTEN, by anionic lipids and reactive oxygen species.

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a member of the protein tyrosine phosphatase family that is structurally adapted to facilitate the metabolism of 3-phosphoinositide lipid second messengers, especially PtdIns(3,4,5) P (3). Cellular PTEN activity is restrained by the retention of C-terminally phosphorylated enzyme in the cytosol. Dephosphorylation by as yet undefined phosphatases initiates an electrostatic switch which targets PTEN specifically to the plasma membrane, where it binds through multiple positively charged residues in both the C2 and N-terminal domains and is susceptible to feedback regulation through proteolytic degradation. PTEN also forms signalling complexes with PDZ domain-containing adaptors, such as the MAGUK (membrane-associated guanylate kinase) proteins, interactions which appear to be necessary for metabolism of localized pools of PtdIns(3,4,5) P (3) involved in regulating actin cytoskeleton dynamics. TPIP [TPTE (transmembrane phosphatase with tensin homology) and PTEN homologous inositol lipid phosphatase] is a novel gene product which exists in multiply spliced forms. TPIPalpha has PtdIns(3,4,5) P (3) 3-phosphatase activity and is localized to the endoplasmic reticulum, via two transmembrane spanning regions, where it may metabolize PtdIns(3,4,5) P (3) that appears to be unaffected by expressed PTEN. PTEN can be acutely regulated by oxidative stress and by endogenously produced reactive oxygen species. This mechanism provides a novel means to stimulate phosphoinositide 3-kinase-dependent signalling pathways, which may be important in circumstances where PtdIns(3,4,5) P (3) and oxidants are produced concurrently.