Enteropathogenic Escherichia coli subverts phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate upon epithelial cell infection

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] are phosphoinositides (PIs) present in small amounts in the inner leaflet of the plasma membrane (PM) lipid bilayer of host target cells. They are thought to modulate the activity of proteins involved in enteropathogenic Escherichia coli (EPEC) infection. However, the role of PI(4,5)P₂ and PI(3,4,5)P₃ in EPEC pathogenesis remains obscure. Here we show that EPEC induces a transient PI(4,5)P₂ accumulation at bacterial infection sites. Simultaneous actin accumulation, likely involved in the construction of the actin-rich pedestal, is also observed at these sites. Acute PI(4,5)P₂ depletion partially diminishes EPEC adherence to the cell surface and actin pedestal formation. These findings are consistent with a bimodal role, whereby PI(4,5)P₂ contributes to EPEC association with the cell surface and to the maximal induction of actin pedestals. Finally, we show that EPEC induces PI(3,4,5)P₃ clustering at bacterial infection sites, in a translocated intimin receptor (Tir)-dependent manner. Tir phosphorylated on tyrosine 454, but not on tyrosine 474, forms complexes with an active phosphatidylinositol 3-kinase (PI3K), suggesting that PI3K recruited by Tir prompts the production of PI(3,4,5)P₃ beneath EPEC attachment sites. The functional significance of this event may be related to the ability of EPEC to modulate cell death and innate immunity.     

Differential Regulation of Proton-Sensitive Ion Channels by Phospholipids: A Comparative Study between ASICs and TRPV1

Protons are released in pain-generating pathological conditions such as inflammation, ischemic stroke, infection, and cancer. During normal synaptic activities, protons are thought to play a role in neurotransmission processes. Acid-sensing ion channels (ASICs) are typical proton sensors in the central nervous system (CNS) and the peripheral nervous system (PNS). In addition to ASICs, capsaicin- and heat-activated transient receptor potential vanilloid 1 (TRPV1) channels can also mediate proton-mediated pain signaling. In spite of their importance in perception of pH fluctuations, the regulatory mechanisms of these proton-sensitive ion channels still need to be further investigated. Here, we compared regulation of ASICs and TRPV1 by membrane phosphoinositides, which are general cofactors of many receptors and ion channels. We observed that ASICs do not require membrane phosphatidylinositol 4-phosphate (PI(4)P) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) for their function. However, TRPV1 currents were inhibited by simultaneous breakdown of PI(4)P and PI(4,5)P₂. By using a novel chimeric protein, CF-PTEN, that can specifically dephosphorylate at the D3 position of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃), we also observed that neither ASICs nor TRPV1 activities were altered by depletion of PI(3,4,5)P₃ in intact cells. Finally, we compared the effects of arachidonic acid (AA) on two proton-sensitive ion channels. We observed that AA potentiates the currents of both ASICs and TRPV1, but that they have different recovery aspects. In conclusion, ASICs and TRPV1 have different sensitivities toward membrane phospholipids, such as PI(4)P, PI(4,5)P₂, and AA, although they have common roles as proton sensors. Further investigation about the complementary roles and respective contributions of ASICs and TRPV1 in proton-mediated signaling is necessary.     

The inositol polyphosphate 5-phosphatase, PIPP, Is a novel regulator of phosphoinositide 3-kinase-dependent neurite elongation

The spatial activation of phosphoinositide 3-kinase (PI3-kinase) signaling at the axon growth cone generates phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P₃), which localizes and facilitates Akt activation and stimulates GSK-3β inactivation, promoting microtubule polymerization and axon elongation. However, the molecular mechanisms that govern the spatial down-regulation of PtdIns(3,4,5)P₃ signaling at the growth cone remain undetermined. The inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P₂) and/or PtdIns(3,4,5)P₃. We demonstrate here that PIPP, an uncharacterized 5-phosphatase, hydrolyzes PtdIns(3,4,5)P₃ forming PtdIns(3,4)P₂, decreasing Ser473-Akt phosphorylation. PIPP is expressed in PC12 cells, localizing to the plasma membrane of undifferentiated cells and the neurite shaft and growth cone of NGF-differentiated neurites. Overexpression of wild-type, but not catalytically inactive PIPP, in PC12 cells inhibited neurite elongation. Targeted depletion of PIPP using RNA interference (RNAi) resulted in enhanced neurite differentiation, associated with neurite hyperelongation. Inhibition of PI3-kinase activity prevented neurite hyperelongation in PIPP-deficient cells. PIPP targeted-depletion resulted in increased phospho-Ser473-Akt and phospho-Ser9-GSK-3β, specifically at the neurite growth cone, and accumulation of PtdIns(3,4,5)P₃ at this site, associated with enhanced microtubule polymerization in the neurite shaft. PIPP therefore inhibits PI3-kinase-dependent neurite elongation in PC12 cells, via regulation of the spatial distribution of phospho-Ser473-Akt and phospho-Ser9-GSK-3β signaling.     

Phosphatidylinositol 3,4,5-trisphosphate Localization in Recycling Endosomes Is Necessary for AP-1B–dependent Sorting in Polarized Epithelial Cells

Polarized epithelial cells coexpress two almost identical AP-1 clathrin adaptor complexes: the ubiquitously expressed AP-1A and the epithelial cell–specific AP-1B. The only difference between the two complexes is the incorporation of the respective medium subunits μ1A or μ1B, which are responsible for the different functions of AP-1A and AP-1B in TGN to endosome or endosome to basolateral membrane targeting, respectively. Here we demonstrate that the C-terminus of μ1B is important for AP-1B recruitment onto recycling endosomes. We define a patch of three amino acid residues in μ1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃]. We found this lipid enriched in recycling endosomes of epithelial cells only when AP-1B is expressed. Interfering with PI(3,4,5)P₃ formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B–dependent cargo to the apical plasma membrane. In conclusion, PI(3,4,5)P₃ formation in recycling endosomes is essential for AP-1B function.     

Profilin binding to sub-micellar concentrations of phosphatidylinositol (4,5) bisphosphate and phosphatidylinositol (3,4,5) trisphosphate

Profilin is a small (12-15 kDa) actin binding protein which promotes filament turnover. Profilin is also involved in the signaling pathway linking receptors in the cell membrane to the microfilament system within the cell. Profilin is thought to play critical roles in this signaling pathway through its interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] (P.J. Lu, W.R. Shieh, S.G. Rhee, H.L. Yin, C.S. Chen, Lipid products of phosphoinositide 3-kinase bind human profilin with high affinity, Biochemistry 35 (1996) 14027-14034). To date, profilin's interaction with polyphosphoinositides (PPI) has only been studied in micelles or small vesicles. Profilin binds with high affinity to small clusters of PI(4,5)P₂ molecules. In this work, we investigated the interactions of profilin with sub-micellar concentrations of PI(4,5)P₂ and PI(3,4,5)P₃. Fluorescence anisotropy was used to determine the relevant dissociation constants for binding of sub-micellar concentrations of fluorescently labeled PPI lipids to profilin and we show that these are significantly different from those determined for profilin interaction with micelles or small vesicles. We also show that profilin binds more tightly to sub-micellar concentrations of PI(3,4,5)P₃ (K_D = 720 μM) than to sub-micellar concentrations of PI(4,5)P₂ (K_D = 985 μM). Despite the low affinity for sub-micellar concentration of PI(4,5)P₂, profilin was shown to bind to giant unilamellar vesicles in presence of 0.5% mole fraction of PI(4,5)P₂ The implications of these findings are discussed.     

Uncovering the PI3Ksome: Phosphoinositide 3-Kinases and Counteracting PTEN Form a Signaling Complex with Intrinsic Regulatory Properties

Production of the phosphoinositide lipid phosphatidylinositol (3,4,5)trisphosphate [PI(3,4,5)P₃, or PIP₃] by class I phosphoinositide 3-kinases (PI3Ks) is a major signaling mechanism whose deregulation contributes to serious diseases, including cancer. New findings suggest that tyrosine kinase receptor engagement results in the assembly of hetero-oligomeric PI3K complexes in which PI3Kα first activates PI3Kβ, and PI3K catalytic activity then promotes recruitment and activation of the PIP₃-removing tumor suppressor PTEN. Thus, PIP₃ production is fine-tuned through formation of an intrinsically regulated “PI3Ksome.”     

Phosphatidylinositol-4,5-bisphosphate ionization and domain formation in the presence of lipids with hydrogen bond donor capabilities

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) is an important lipidic signaling molecule that is involved in a broad range of cellular processes. Its interaction with proteins and its lateral distribution are governed by the ionization state of the phosphomonoester groups and its ability to form intra- and intermolecular hydrogen bonds. In this study we have investigated the ionization state of PI(4,5)P(2) in ternary lipid vesicle systems that contain in addition to PI(4,5)P(2) and phosphatidylcholine (PC) either phosphatidylethanolamine (PE), phosphatidylserine (PS) or phosphatidylinositol (PI). In the presence of PE we find an increased ionization of PI(4,5)P(2), which can be attributed to increased deprotonation due to hydrogen bond formation between PE and the PI(4,5)P(2) phosphomonoester groups. However, the effect of PE on PI(4,5)P(2) ionization is significantly smaller than it had been found previously for phosphatidic acid in the presence of PE (Kooijman et al., 2005). The reduced impact of PE on PI(4,5)P(2) ionization can be attributed to competing intramolecular hydrogen bond formation between the phosphomonoester groups and neighboring hydroxyl groups. It is noteworthy that the presence of PE affects more strongly the ionization of the 5-phosphate group than that of the 4-phosphate, suggesting that the interaction of PE with the 5-phosphate is stronger. In PI(4,5)P(2)/PS/PC lipid vesicles, the presence of PS was expected to yield an increased protonation of the PI(4,5)P(2) phosphomonoester groups due to a decreased interfacial pH as a result of the increased negative interfacial charge. However, the effect of PS on PI(4,5)P(2) ionization is only minor, potentially suggesting that PS and PI(4,5)P(2) are demixed. The PI(4,5)P(2)/PI/PC vesicle system was characterized by a surprising mixing behavior that has potentially far reaching consequences: fluorescence microscopy measurements of giant unilammellar vesicles composed of PI(4,5)P(2)/PI/PC at physiological concentrations show that PI and PI(4,5)P(2) form macroscopic, fluid phase domains in contact with a fluid PC rich phase (fluid/fluid demixing). Despite the fact that PI and PI(4,5)P(2) co-localize, the effect of PI on PI(4,5)P(2) ionization behavior is only noticeable above pH 7. Apparently two opposing effects lead to the observed behavior: Due to the presence of the anionic PI, the interfacial pH drops, which is expected to lead to an enhanced protonation of the PI(4,5)P(2) phosphomonoester groups. In turn, hydrogen bond formation between PI and PI(4,5)P(2) would lead to the opposite, i.e. increased deprotonation of the phosphomonoester group. Apparently these two effects compensate each other for pH values smaller than about 7, while for higher pH values the increased interfacial pH in the presence of PI has a stronger impact than PI/PI(4,5)P(2) hydrogen bond formation. The cooperative formation of PI/PI(4,5)P(2) mixed domains has potentially important ramifications for the spatial organization of phosphoinositide mediated signaling events.     

New Functions for PI3K in the Control of Cell Division

Although cell lipids were initially envisioned as structural components of the cell, their essential contribution to initiation and regulation of cell responses is now clearly established. Among the different lipids that regulate cell responses, those produced by class I phosphoinositide 3-kinase (PI3K), phosphatidylinositol (3,4)P₂ (PIP₂), and phosphatidylinositol (3,4,5)P₃ (PIP₃), have concentrated much attention in recent years. PIP2 and PIP3 are involved in cell division and survival control, and mutations in the PI3K pathway are linked to autoimmunity and cancer. Here we discuss two novel observations: a PI3K function in the late-G₁ phase of the cell cycle and the contribution of the p85 PI3K regulatory subunit in the control of cytokinesis.     

Cell Activation-Induced Phosphoinositide 3-Kinase Alpha/Beta Dimerization Regulates PTEN Activity

The phosphoinositide 3-kinase (PI3K)/PTEN (phosphatase and tensin homolog) pathway is one of the central routes that enhances cell survival, division, and migration, and it is frequently deregulated in cancer. PI3K catalyzes formation of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P₃] after cell activation; PTEN subsequently reduces these lipids to basal levels. Activation of the ubiquitous p110α isoform precedes that of p110β at several points during the cell cycle. We studied the potential connections between p110α and p110β activation, and we show that cell stimulation promotes p110α and p110β association, demonstrating oligomerization of PI3K catalytic subunits within cells. Cell stimulation also promoted PTEN incorporation into this complex, which was necessary for PTEN activation. Our results show that PI3Ks dimerize in vivo and that PI3K and PTEN activities modulate each other in a complex that controls cell PI(3,4,5)P₃ levels.     

The 3-Phosphorylated Phosphoinositide Response of 3T3-L1 Preadipose Cells Exposed to Insulin,Insulin-Like Growth Factor-1, or Platelet-Derived Growth Factor

We compared the pattern of 3-phosphorylated phosphoinositides produced by confluent 3T3-L1 preadipose cells upon exposure to growth factors that either induce differentiation (insulin, insulin-like growth factor-1) or do not (platelet-derived growth factor). Following addition of insulin or insulin-like growth factor-1, PI(3,4,5)P3 rapidly rose, on average, to levels tenfold over basal. PI(3,4)P₂ either did not change (after insulin) or slightly increased (1.5 fold). Time course studies with insulin demonstrated that the rise in PI(3,4,5)P₃ peaked by 1 minute, and levels then remained steady over 30 minutes. Dose-response experiments showed that insulin at a concentration of 1 nM was sufficient for the PI(3,4,5)P₃ response. Insulin failed to increase PI(3,4)P₂ at any of the time points or at any of the doses used. In contrast, after addition of platelet-derived growth factor, both PI(3,4)P₂ and PI(3,4,5)P₃ rose concurrently and to comparable extents. These data suggest that one possible mechanism contributing to insulin/insulin-like growth factor-1-induced 3T3-L1 preadipose cell differentiation is a distinct pattern of 3-phosphorylated phosphoinositide accumulation, defined by a prominent increase in PI(3,4,5)P₃ with no (in the case of insulin), or a minimal (in the case of IGF-1), rise in PI(3,4)P₂.     

The inositol 5-phosphatase SHIP2 regulates endocytic clathrin-coated pit dynamics

Phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P₂) and its phosphorylated product PI 3,4,5-triphosphate (PI(3,4,5)P₃) are two major phosphoinositides concentrated at the plasma membrane. Their levels, which are tightly controlled by kinases, phospholipases, and phosphatases, regulate a variety of cellular functions, including clathrin-mediated endocytosis and receptor signaling. In this study, we show that the inositol 5-phosphatase SHIP2, a negative regulator of PI(3,4,5)P₃-dependent signaling, also negatively regulates PI(4,5)P₂ levels and is concentrated at endocytic clathrin-coated pits (CCPs) via interactions with the scaffold protein intersectin. SHIP2 is recruited early at the pits and dissociates before fission. Both knockdown of SHIP2 expression and acute production of PI(3,4,5)P₃ shorten CCP lifetime by enhancing the rate of pit maturation, which is consistent with a positive role of both SHIP2 substrates, PI(4,5)P₂ and PI(3,4,5)P₃, on coat assembly. Because SHIP2 is a negative regulator of insulin signaling, our findings suggest the importance of the phosphoinositide metabolism at CCPs in the regulation of insulin signal output.     

Alterations in the MA and NC Domains Modulate Phosphoinositide-Dependent Plasma Membrane Localization of the Rous Sarcoma Virus Gag Protein

Retroviral Gag proteins direct virus particle assembly from the plasma membrane (PM). Phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] plays a role in PM targeting of several retroviral Gag proteins. Here we report that depletion of intracellular PI(4,5)P₂ and phosphatidylinositol-(3,4,5)-triphosphate [PI(3,4,5)P₃] levels impaired Rous sarcoma virus (RSV) Gag PM localization. Gag mutants deficient in nuclear trafficking were less sensitive to reduction of intracellular PI(4,5)P₂ and PI(3,4,5)P₃, suggesting a possible connection between Gag nuclear trafficking and phosphoinositide-dependent PM targeting.     

Structural and Biophysical Characterization of the Interactions between Calmodulin and the Pleckstrin Homology Domain of Akt

The translocation of Akt, a serine/threonine kinase, to the plasma membrane is a critical step in the Akt activation pathway. It is established that membrane binding of Akt is mediated by direct interactions between its pleckstrin homology domain (PHD) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃). There is now evidence that Akt activation in many breast cancer cells is also modulated by the calcium-binding protein, calmodulin (CaM). Upon EGF stimulation of breast cancer cells, CaM co-localizes with Akt at the plasma membrane to enhance activation. However, the molecular details of Akt(PHD) interaction with CaM are not known. In this study, we employed NMR, biochemical, and biophysical techniques to characterize CaM binding to Akt(PHD). Our data show that CaM forms a tight complex with the PHD of Akt (dissociation constant = 100 nm). The interaction between CaM and Akt(PHD) is enthalpically driven, and the affinity is greatly dependent on salt concentration, indicating that electrostatic interactions are important for binding. The CaM-binding interface in Akt(PHD) was mapped to two loops adjacent to the PI(3,4,5)P₃ binding site, which represents a rare CaM-binding motif and suggests a synergistic relationship between CaM and PI(3,4,5)P₃ upon Akt activation. Elucidation of the mechanism by which Akt interacts with CaM will help in understanding the activation mechanism, which may provide insights for new potential targets to control the pathophysiological processes of cell survival.     

Inositol‐requiring enzyme‐1 regulates phosphoinositide signaling lipids and macrophage growth

The ER‐bound kinase/endoribonuclease (RNase), inositol‐requiring enzyme‐1 (IRE1), regulates the phylogenetically most conserved arm of the unfolded protein response (UPR). However, the complex biology and pathology regulated by mammalian IRE1 cannot be fully explained by IRE1’s one known, specific RNA target, X box‐binding protein‐1 (XBP1) or the RNA substrates of IRE1‐dependent RNA degradation (RIDD) activity. Investigating other specific substrates of IRE1 kinase and RNase activities may illuminate how it performs these diverse functions in mammalian cells. We report that macrophage IRE1 plays an unprecedented role in regulating phosphatidylinositide‐derived signaling lipid metabolites and has profound impact on the downstream signaling mediated by the mammalian target of rapamycin (mTOR). This cross‐talk between UPR and mTOR pathways occurs through the unconventional maturation of microRNA (miR) 2137 by IRE1’s RNase activity. Furthermore, phosphatidylinositol (3,4,5) phosphate (PI(3,4,5)P₃) 5‐phosphatase‐2 (INPPL1) is a direct target of miR‐2137, which controls PI(3,4,5)P₃ levels in macrophages. The modulation of cellular PI(3,4,5)P₃/PIP₂ ratio and anabolic mTOR signaling by the IRE1‐induced miR‐2137 demonstrates how the ER can provide a critical input into cell growth decisions.     

Regulation of the transient receptor potential channel TRPM3 by phosphoinositides

The transient receptor potential (TRP) channel TRPM3 is a calcium-permeable cation channel activated by heat and by the neurosteroid pregnenolone sulfate (PregS). TRPM3 is highly expressed in sensory neurons, where it plays a key role in heat sensing and inflammatory hyperalgesia, and in pancreatic β cells, where its activation enhances glucose-induced insulin release. However, despite its functional importance, little is known about the cellular mechanisms that regulate TRPM3 activity. Here, we provide evidence for a dynamic regulation of TRPM3 by membrane phosphatidylinositol phosphates (PIPs). Phosphatidylinositol 4,5-bisphosphate (PI[4,5]P₂) and ATP applied to the intracellular side of excised membrane patches promote recovery of TRPM3 from desensitization. The stimulatory effect of cytosolic ATP on TRPM3 reflects activation of phosphatidylinositol kinases (PI-Ks), leading to resynthesis of PIPs in the plasma membrane. Various PIPs directly enhance TRPM3 activity in cell-free inside-out patches, with a potency order PI(3,4,5)P₃ > PI(3,5)P₂ > PI(4,5)P₂ ≈ PI(3,4)P₂ >> PI(4)P_. Conversely, TRPM3 activity is rapidly and reversibly inhibited by activation of phosphatases that remove the 5-phosphate from PIPs. Finally, we show that recombinant TRPM3, as well as the endogenous TRPM3 in insuloma cells, is rapidly and reversibly inhibited by activation of phospholipase C–coupled muscarinic acetylcholine receptors. Our results reveal basic cellular mechanisms whereby membrane receptors can regulate TRPM3 activity.     

Insights into the inhibitory mechanism of triazole-based small molecules on phosphatidylinositol-4,5-bisphosphate binding pleckstrin homology domain

BackgroundPhosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is an important regulator of several cellular processes and a precursor for other second messengers which are involved in cell signaling pathways. Signaling proteins preferably interact with PI(4,5)P₂ through its pleckstrin homology (PH) domain. Efforts are underway to design small molecule-based antagonist, which can specifically inhibit the PI(4,5)P₂/PH-domain interaction to establish an alternate strategy for the development of drug(s) for phosphoinositide signaling pathways.MethodsSurface plasmon resonance, molecular docking, circular dichroism, competitive Förster resonance energy transfer, isothermal titration calorimetric analyses and liposome pull down assay were used.ResultsIn this study, we employed 1,2,3-triazol-4-yl methanol containing small molecule (CIPs) as antagonists for PI(4,5)P₂/PH-domain interaction and determined their inhibitory effect by using competitive-surface plasmon resonance analysis (IC₅₀ ranges from 53 to 159 nM for PI(4,5)P₂/PLCδ1-PH domain binding assay). We also used phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃], phosphatidylinositol 3,4-bisphosphate [PI(3,4)P₂], PI(4,5)P₂ specific PH-domains to determine binding selectivity of the compounds. Various physicochemical analyses showed that the compounds have weak affect on fluidity of the model membrane but, strongly interact with the phospholipase C δ1 (PLCδ1)-PH domains. The 1,2,3-triazol-4-yl methanol moiety and nitro group of the compounds are essential for their exothermic interaction with the PH-domains. Potent compound can efficiently displace PLCδ1-PH domain from plasma membrane to cytosol in A549 cells.ConclusionsOverall, our studies demonstrate that these compounds interact with the PIP-binding PH-domains and inhibit their membrane recruitment.General significanceThese results suggest specific but differential binding of these compounds to the PLCδ1-PH domain and emphasize the role of their structural differences in binding parameters. These triazole-based compounds could be directly used/further developed as potential inhibitor for PH domain-dependent enzyme activity.     

Tensin2 reduces intracellular phosphatidylinositol 3,4,5-trisphosphate levels at the plasma membrane

Tensins are proposed cytoskeleton-regulating proteins. However, Tensin2 additionally inhibits Akt signalling and cell survival. Structural modelling of the Tensin2 phosphatase (PTPase) domain revealed an active site-like pocket receptive towards phosphoinositides. Tensin2-expressing HEK293 cells displayed negligible levels of plasma membrane phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃) under confocal microscopy. However, mock-transfected cells, and Tensin2 cells harbouring a putative phosphatase-inactivating mutation, exhibited significant PtdIns(3,4,5)P₃ levels, which decreased upon phosphatidylinositol 3-kinase inhibition with LY294002. In contrast, wtTensin3, mock and mutant cells were identical in membrane PtdIns(3,4,5)P₃ and Akt phosphorylation. In vitro lipid PTPase activity was however undetectable in isolated recombinant PTPase domains of both Tensins, indicating a possible loss of structural stability when expressed in isolation. In summary, we provide evidence that Tensin2, in addition to regulating cytoskeletal dynamics, influences phosphoinositide-Akt signalling through its PTPase domain.