Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (<em>PtdIns</em>)-4,5-P2 to <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em>. This, in turn, recruits and activates cytosolic effectors with <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em>-binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110 gamma/p101 PI3K gamma is activated by G beta gamma on stimulation of G protein-coupled receptors. It is currently unknown whether in living cells G beta gamma acts as a membrane anchor or an allosteric activator of PI3K gamma, and which role its noncatalytic p101 subunit plays in its activation by G beta gamma. Using GFP-tagged PI3K gamma subunits expressed in HEK cells, we show that G beta gamma recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein-mediated activation of PI3K gamma in living cells, as assessed by use of GFP-tagged <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em>-binding PH domains. Furthermore, membrane-targeted p110 gamma displayed basal enzymatic activity, but was further stimulated by G beta gamma, even in the absence of p101. Therefore, we conclude that in vivo, G beta gamma activates PI3K gamma by a mechanism assigning specific roles for both PI3K gamma subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of G beta gamma with p110 gamma contributes to activation of PI3K gamma.

Phosphoinositide-3-OH kinase (PI(3)K), activated through growth factor stimulation, generates a lipid second messenger, phosphatidylinositol-<em>3,4,5</em>-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>). <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> is instrumental in signalling pathways that trigger cell activation, cytoskeletal rearrangement, survival and other reactions. However, some targets of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> are yet to be discovered. We demonstrate that SWAP-70, a unique signalling protein, specifically binds <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. On stimulation by growth factors, cytoplasmic SWAP-70, which is dependent on PI(3)K but independent of Ras, moved to cell membrane rearrangements known as ruffles. However, mutant SWAP-70 lacking the ability to bind <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> blocked membrane ruffling induced by epidermal growth factor or platelet-derived growth factor. SWAP-70 shows low homology with Rac-guanine nucleotide exchange factors (GEFs), and catalyses <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>-dependent guanine nucleotide exchange to Rac. SWAP-70-deficient fibroblasts showed impaired membrane ruffling after stimulation with epidermal growth factor, and failed to activate Rac fully. We conclude that SWAP-70 is a new type of Rac-GEF which, independently of Ras, transduces signals from tyrosine kinase receptors to Rac.

mTOR serves as a central regulator of cell growth and metabolism by forming two distinct complexes, mTORC1 and mTORC2. Although mechanisms of mTORC1 activation by growth factors and amino acids have been extensively studied, the upstream regulatory mechanisms leading to mTORC2 activation remain largely elusive. Here, we report that the pleckstrin homology (PH) domain of SIN1, an essential and unique component of mTORC2, interacts with the mTOR kinase domain to suppress mTOR activity. More importantly, <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, but not other <em>PtdIns</em>Pn species, interacts with SIN1-PH to release its inhibition on the mTOR kinase domain, thereby triggering mTORC2 activation. Mutating critical SIN1 residues that mediate <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> interaction inactivates mTORC2, whereas mTORC2 activity is pathologically increased by patient-derived mutations in the SIN1-PH domain, promoting cell growth and tumor formation. Together, our study unravels a PI3K-dependent mechanism for mTORC2 activation, allowing mTORC2 to activate AKT in a manner that is regulated temporally and spatially by <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>.

CONCLUSIONS

The SIN1-PH domain interacts with the mTOR kinase domain to suppress mTOR activity, and <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> binds the SIN1-PH domain to release its inhibition on the mTOR kinase domain, leading to mTORC2 activation. Cancer patient-derived SIN1-PH domain mutations gain oncogenicity by loss of suppressing mTOR activity as a means to facilitate tumorigenesis.

3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates many kinases belonging to the AGC subfamily. PDK1 possesses a C-terminal pleckstrin homology (PH) domain that interacts with <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>/<em>PtdIns</em>(3,4)P2 and with lower affinity to <em>PtdIns</em>(4,5)P2. We describe the crystal structure of the PDK1 PH domain, in the absence and presence of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and Ins(1,<em>3,4,5</em>)P4. The structures reveal a 'budded' PH domain fold, possessing an N-terminal extension forming an integral part of the overall fold, and display an unusually spacious ligand-binding site. Mutagenesis and lipid-binding studies were used to define the contribution of residues involved in phosphoinositide binding. Using a novel quantitative binding assay, we found that Ins(1,<em>3,4,5</em>,6)P5 and InsP6, which are present at micromolar levels in the cytosol, interact with full-length PDK1 with nanomolar affinities. Utilising the isolated PDK1 PH domain, which has reduced affinity for Ins(1,<em>3,4,5</em>,6)P5/InsP6, we perform localisation studies that suggest that these inositol phosphates serve to anchor a portion of cellular PDK1 in the cytosol, where it could activate its substrates such as p70 S6-kinase and p90 ribosomal S6 kinase that do not interact with phosphoinositides.

BACKGROUND

Leptin and insulin are long-term regulators of body weight. They act in hypothalamic centres to modulate the function of specific neuronal subtypes, by altering transcriptional control of releasable peptides and by modifying neuronal electrical activity. A key cellular signalling intermediate, implicated in control of food intake by these hormones, is the enzyme phosphoinositide 3-kinase. In this study we have explored further the linkage between this enzyme and other cellular mediators of leptin and insulin action on rat arcuate nucleus neurones and the mouse hypothalamic cell line, GT1-7.

RESULTS

Leptin and insulin increased the levels of various phosphorylated signalling intermediates, associated with the JAK2-STAT3, MAPK and PI3K cascades in the arcuate nucleus. Inhibitors of PI3K were shown to reduce the hormone driven phosphorylation through the PI3K and MAPK pathways. Using isolated arcuate neurones, leptin and insulin were demonstrated to increase the activity of KATP channels in a PI3K dependent manner, and to increase levels of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. KATP activation by these hormones in arcuate neurones was also sensitive to the presence of the actin filament stabilising toxin, jasplakinolide. Using confocal imaging of fluorescently labelled actin and direct analysis of G- and F-actin concentration in GT1-7 cells, leptin was demonstrated directly to induce a re-organization of cellular actin, by increasing levels of globular actin at the expense of filamentous actin in a PI3-kinase dependent manner. Leptin stimulated PI3-kinase activity in GT1-7 cells and an increase in <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> could be detected, which was prevented by PI3K inhibitors.

CONCLUSIONS

Leptin and insulin mediated phosphorylation of cellular signalling intermediates and of KATP channel activation in arcuate neurones is sensitive to PI3K inhibition, thus strengthening further the likely importance of this enzyme in leptin and insulin mediated energy homeostasis control. The sensitivity of leptin and insulin stimulation of KATP channel opening in arcuate neurones to jasplakinolide indicates that cytoskeletal remodelling may be an important contributor to the cellular signalling mechanisms of these hormones in hypothalamic neurones. This hypothesis is reinforced by the finding that leptin induces actin filament depolymerization, in a PI3K dependent manner in a mouse hypothalamic cell line.

PTEN is a novel tumour suppressor gene that encodes a dual-specificity phosphatase with homology to adhesion molecules tensin and auxillin. It recently has been suggested that PTEN dephosphorylates phosphatidylinositol <em>3,4,5</em>-trisphosphate [<em>PtdIns</em>(3, 4,5)<em>P3</em>], which mediates growth factor-induced activation of intracellular signalling, in particular through the serine-threonine kinase Akt, a known cell survival-promoting factor. PTEN has been mapped to 10q23.3, a region disrupted in several human tumours including haematological malignancies. We have analysed PTEN in a series of primary acute leukaemias and non-Hodgkin's lymphomas (NHLs) as well as in cell lines. We have also examined whether a correlation could be found between PTEN and Akt levels in these samples. We show here that the majority of cell lines studied carries PTEN abnormalities. At the structural level, we found mutations and hemizygous deletions in 40% of these cell lines, while a smaller number of primary haematological malignancies, in particular NHLs, carries PTEN mutations. Moreover, one-third of the cell lines had low PTEN transcript levels, and 60% of these samples had low or absent PTEN protein, which could not be attributed to gene silencing by hypermethylation. In addition, we found that PTEN and phosphorylated Akt levels are inversely correlated in the large majority of the examined samples. These findings suggest that PTEN plays a role in the pathogenesis of haematological malignancies and that it might be inactivated through a wider range of mechanisms than initially considered. The finding that PTEN levels inversely correlate with phosphorylated Akt supports the hypothesis that PTEN regulates <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>and suggests a role for PTEN in apoptosis.

Although the hormone-stimulated synthesis of 3-phosphorylated inositol lipids is known to form an intracellular signalling system, there is no consensus on the crucial receptor-regulated event in this pathway and it is still not clear which of the intermediates represent potential output signals. We show here that the key step in the synthesis of 3-phosphorylated inositol lipids in 3T3 cells stimulated by platelet-derived growth factor is the activation of a phosphatidylinositol(4,5)-bisphosphate (3)-hydroxy (<em>PtdIns</em>(4,5)P2 3-OH) kinase. A similar conclusion has been applied to explain the actions of formyl-Met-Leu-Phe on neutrophils, and it may be that receptors that couple through intrinsic tyrosine kinases or through G proteins stimulate the same step in 3-phosphorylated inositol lipid metabolism. The close parallel between these two mechanisms for the activation of <em>PtdIns</em>(4,5)P2 3-OH kinase and those described for the activation of another key signalling enzyme, phospholipase C (ref. 7), focuses attention on the product of the <em>PtdIns</em>(4,5)P2 3-OH kinase, <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, as a possible new second messenger.

The cellular effects of stromal cell-derived factor-1 (SDF-1) are mediated primarily by binding to the CXC chemokine receptor-4. We report in this study that SDF-1 and its peptide analogues induce a concentration- and time-dependent accumulation of phosphatidylinositol-(<em>3,4,5</em>)-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>) in Jurkat cells. This SDF-1-stimulated generation of D-3 phosphoinositide lipids was inhibited by pretreatment of the cells with an SDF-1 peptide antagonist or an anti-CXCR4 Ab. In addition, the phosphoinositide 3 (PI 3)-kinase inhibitors wortmannin and LY294002, as well as the Gi protein inhibitor pertussis toxin, also inhibited the SDF-1-stimulated accumulation of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. The effects of SDF-1 on D-3 phosphoinositide lipid accumulation correlated well with activation of the known PI 3-kinase effector protein kinase B, which was also inhibited by wortmannin and pertussis toxin. Concentrations of PI 3-kinase inhibitors, sufficient to inhibit <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> accumulation, also inhibited chemotaxis of Jurkat and peripheral blood-derived T lymphocytes in response to SDF-1. In contrast, SDF-1-stimulated actin polymerization was only partially inhibited by PI 3-kinase inhibitors, suggesting that while chemotaxis is fully dependent on PI 3-kinase activation, actin polymerization requires additional biochemical inputs. Finally, SDF-1-stimulated extracellular signal-related kinase (ERK)-1/2 mitogen-activated protein kinase activation was inhibited by PI 3-kinase inhibitors. In addition, the mitogen-activated protein/ERK kinase inhibitor PD098059 partially attenuated chemotaxis in response to SDF-1. Hence, it appears that ERK1/2 activation is dependent on PI 3-kinase activation, and both biochemical events are involved in the regulation of SDF-1-stimulated chemotaxis.

BACKGROUND

In a specialized epithelial cell such as the Drosophila photoreceptor, a conserved set of proteins is essential for the establishment of polarity, its maintenance, or both--in Drosophila, these proteins include the apical factors Bazooka, D-atypical protein kinase C, and D-Par6 together with D-Ecadherin. However, little is known about the mechanisms by which such apical factors might regulate the differentiation of the apical membrane into functional domains such as an apical-most stack of microvilli or more lateral sub-apical membrane.

RESULTS

We show that in photoreceptors Bazooka (D-Par3) recruits the tumor suppressor lipid phosphatase PTEN to developing cell-cell junctions (Zonula Adherens, za). za-localized PTEN controls the spatially restricted accumulation of optimum levels of the lipid <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> within the apical membrane domain. This in turn finely tunes activation of Akt1, a process essential for proper morphogenesis of the light-gathering organelle, consisting of a stack of F-actin rich microvilli within the apical membrane.

CONCLUSIONS

Spatially localized <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> mediates directional sensing during neutrophil and Dictyostelium chemotaxis. We conclude that a conserved mechanism also operates during photoreceptor epithelial cell morphogenesis in order to achieve normal differentiation of the apical membrane.

We show that matrices carrying the tethered homologs of natural phosphoinositides can be used to capture and display multiple phosphoinositide binding proteins in cell and tissue extracts. We present the mass spectrometric identification of over 20 proteins isolated by this method, mostly from leukocyte extracts: they include known and novel proteins with established phosphoinositide binding domains and also known proteins with surprising and unusual phosphoinositide binding properties. One of the novel <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> binding proteins, ARA<em>P3</em>, has an unusual domain structure, including five predicted PH domains. We show that it is a specific <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>/<em>PtdIns</em>(3,4)P2-stimulated Arf6 GAP both in vitro and in vivo, and both its Arf GAP and Rho GAP domains cooperate in mediating PI3K-dependent rearrangements in the cell cytoskeleton and cell shape.

Several pleckstrin-homology (PH) domains with the ability to bind phosphatidylinositol (<em>3,4,5</em>)-trisphosphate [<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>, PI<em>P3</em>] were expressed as green fluorescent protein (GFP) fusion proteins to determine their effects on various cellular responses known to be activated by PI<em>P3</em>. These proteins comprised the PH domains of Akt, ARNO, Btk or GRP1, and were found to show growth-factor-stimulated and wortmannin-sensitive translocation from the cytosol to the plasma membrane in several cell types, indicating their ability to recognize PI<em>P3</em>. Remarkably, although overexpressed Akt-PH-GFP and Btk-PH-GFP were quite potent in antagonizing the PI<em>P3</em>-mediated activation of the Akt protein kinase, such inhibition was not observed with the other PH domains. By contrast, expression of the PH domains of GRP1 and ARNO, but not of Akt or Btk, inhibited the attachment and spreading of freshly seeded cells to culture dishes. Activation of PLCgamma by epidermal growth factor (EGF) was attenuated by the PH domains of GRP1, ARNO and Akt, but was significantly enhanced by the Btk PH domain. By following the kinetics of expression of the various GFP-fused PH domains for several days, only the PH domain of Akt showed a lipid-binding-dependent self-elimination, consistent with its interference with the anti-apoptotic Akt signaling pathway. Mutations of selective residues that do not directly participate in PI<em>P3</em> binding in the GRP1-PH and Akt-PH domain were able to reduce the dominant-negative effects of these constructs yet retain their lipid binding. These data suggest that interaction with and sequestration of PI<em>P3</em> may not be the sole mechanism by which PH domains interfere with cellular responses and that their interaction with other membrane components, most probably with proteins, allows a more specific participation in the regulation of specific signaling pathways.

OBJECTIVE

Increased cellular production of ceramide has been implicated in the pathogenesis of insulin resistance and in the impaired utilisation of glucose. In this study we have used L6 muscle cells to investigate the mechanism by which the short-chain ceramide analogue, C2-ceramide, promotes a loss in insulin sensitivity leading to a reduction in insulin stimulated glucose transport and glycogen synthesis.

METHODS

L6 muscle cells were pre-incubated with C2-ceramide and the effects of insulin on glucose transport, glycogen synthesis and the activities of key molecules involved in proximal insulin signalling determined.

RESULTS

Incubation of L6 muscle cells with ceramide (100 micromol/l) for 2 h led to a complete loss of insulin-stimulated glucose transport and glycogen synthesis. This inhibition was not due to impaired insulin receptor substrate 1 phosphorylation or a loss in phosphoinositide 3-kinase activation but was caused by a failure to activate protein kinase B. This defect could not be attributed to inhibition of 3-phosphoinositide-dependent kinase-1, or to impaired binding of phosphatidylinositol <em>3,4,5</em> triphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>) to the PH domain of protein kinase B, but results from the inability to recruit protein kinase B to the plasma membrane. Expression of a membrane-targetted protein kinase B led to its constitutive activation and an increase in glucose transport that was not inhibited by ceramide.

CONCLUSIONS

These findings suggest that a defect in protein kinase B recruitment underpins the ceramide-induced loss in insulin sensitivity of key cell responses such as glucose transport and glycogen synthesis in L6 cells. They also suggest that a stimulated rise in <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> is necessary but not sufficient for protein kinase B activation in this system.

Receptor-mediated endocytosis via clathrin-coated vesicles has been extensively studied and, while many of the protein players have been identified, much remains unknown about the regulation of coat assembly and the mechanisms that drive vesicle formation [1]. Some components of the endocytic machinery interact with inositol polyphosphates and inositol lipids in vitro, implying a role for phosphatidylinositols in vivo [2] [3]. Specifically, the adaptor protein complex AP2 binds phosphatidylinositol-4,5-bisphosphate (<em>PtdIns</em>(4,5)P2), <em>PtdIns</em>(3)P, <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and inositol phosphates. Phosphatidylinositol binding regulates AP2 self-assembly and the interactions of AP2 complexes with clathrin and with peptides containing endocytic motifs [4] [5]. The GTPase dynamin contains a pleckstrin homology (PH) domain that binds <em>PtdIns</em>(4,5)P2 and <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> to regulate GTPase activity in vitro [6] [7]. However, no direct evidence for the involvement of phosphatidylinositols in clathrin-mediated endocytosis exists to date. Using well-characterized PH domains as high affinity and high specificity probes in combination with a perforated cell assay that reconstitutes coated vesicle formation, we provide the first direct evidence that <em>PtdIns</em>(4,5)P2 is required for both early and late events in endocytic coated vesicle formation.

Cultured adherent cells divide on the substratum, leading to formation of the cell monolayer. However, how the orientation of this anchorage-dependent cell division is regulated remains unknown. We have previously shown that integrin-dependent adhesion orients the spindle parallel to the substratum, which ensures this anchorage-dependent cell division. Here, we show that phosphatidylinositol-<em>3,4,5</em>-triphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>) is essential for this spindle orientation control. In metaphase, <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> is accumulated in the midcortex in an integrin-dependent manner. Inhibition of phosphatidylinositol-3-OH kinase (PI(3)K) reduces the accumulation of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and induces spindle misorientation. Introduction of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> to these cells restores the midcortical accumulation of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and proper spindle orientation. PI(3)K inhibition causes dynein-dependent spindle rotations along the z-axis, resulting in spindle misorientation. Moreover, dynactin, a dynein-binding partner, is accumulated in the midcortex in a <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>-dependent manner. We propose that <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> directs dynein/dynactin-dependent pulling forces on spindles to the midcortex, and thereby orients the spindle parallel to the substratum.

The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour-suppressor protein is a phosphoinositide 3-phosphatase which antagonizes phosphoinositide 3-kinase-dependent signalling by dephosphorylating <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>. Most tumour-derived point mutations of PTEN induce a loss of function, which correlates with profoundly reduced catalytic activity. However, here we characterize a point mutation at the N-terminus of PTEN, K13E from a human glioblastoma, which displayed wild-type activity when assayed in vitro. This mutation occurs within a conserved polybasic motif, a putative <em>PtdIns</em>(4,5)P2-binding site that may participate in membrane targeting of PTEN. We found that catalytic activity against lipid substrates and vesicle binding of wild-type PTEN, but not of PTEN K13E, were greatly stimulated by anionic lipids, especially <em>PtdIns</em>(4,5)P2. The K13E mutation also greatly reduces the efficiency with which anionic lipids inhibit PTEN activity against soluble substrates, supporting the hypothesis that non-catalytic membrane binding orientates the active site to favour lipid substrates. Significantly, in contrast to the wild-type enzyme, PTEN K13E failed either to prevent protein kinase B/Akt phosphorylation, or inhibit cell proliferation when expressed in PTEN-null U87MG cells. The cellular functioning of K13E PTEN was recovered by targeting to the plasma membrane through inclusion of a myristoylation site. Our results establish a requirement for the conserved N-terminal motif of PTEN for correct membrane orientation, cellular activity and tumour-suppressor function.

We generated homozygous knockin ES cells expressing a form of 3-phosphoinositide-dependent protein kinase-1 (PDK1) with a mutation in its pleckstrin homology (PH) domain that abolishes phosphatidylinositol <em>3,4,5</em>-tris-phosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>) binding, without affecting catalytic activity. In the knockin cells, protein kinase B (PKB) was not activated by IGF1, whereas ribosomal S6 kinase (RSK) was activated normally, indicating that <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> binding to PDK1 is required for PKB but not RSK activation. Interestingly, amino acids and Rheb, but not IGF1, activated S6K in the knockin cells, supporting the idea that <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> stimulates S6K through PKB-mediated activation of Rheb. Employing PDK1 knockin cells in which either the <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> binding or substrate-docking 'PIF pocket' was disrupted, we established the roles that these domains play in regulating phosphorylation and stabilisation of protein kinase C isoforms. Moreover, mouse PDK1 knockin embryos in which either the PH domain or PIF pocket was disrupted died displaying differing phenotypes between E10.5 and E11.5. Although PDK1 plays roles in regulating cell size, cells derived from PH domain or PIF pocket knockin embryos were of normal size. These experiments establish the roles of the PDK1 regulatory domains and illustrate the power of knockin technology to probe the physiological function of protein-lipid and protein-protein interactions.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a phosphoinositide 3-phosphatase, is an important regulator of insulin-dependent signaling. The loss or impairment of PTEN results in an antidiabetic impact, which led to the suggestion that PTEN could be an important target for drugs against type II diabetes. Here we report the design and validation of a small- molecule inhibitor of PTEN. Compared with other cysteine-based phosphatases, PTEN has a much wider active site cleft enabling it to bind the <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> substrate. We have exploited this feature in the design of vanadate scaffolds complexed to a range of different organic ligands, some of which show potent inhibitory activity. A vanadyl complexed to hydroxypicolinic acid was found to be a highly potent and specific inhibitor of PTEN that increases cellular <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> levels, phosphorylation of Akt, and glucose uptake in adipocytes at nanomolar concentrations. The findings presented here demonstrate the applicability of a novel and specific chemical inhibitor against PTEN in research and drug development.

Intracellular signaling by most cell surface receptors requires the generation of two major second messengers, phosphatidylinositol-<em>3,4,5</em>-trisphosphate (<em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em>) and inositol-1,4,5-trisphosphate (I<em>P3</em>). The enzymes that produce these second messengers, phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC), utilize a common substrate, phosphatidylinositol-4,5-bisphosphate (<em>PtdIns</em>-4,5-P2). Until now, it has not been clear whether de novo <em>PtdIns</em>-4,5-P2 synthesis is necessary for <em>PtdIns</em>-<em>3,4,5</em>-<em>P3</em> and I<em>P3</em> production. Here we show that BTK, a member of the Tec family of cytoplasmic protein tyrosine kinases, associates with phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks), the enzymes that synthesize <em>PtdIns</em>-4,5-P2. Upon B cell receptor activation, BTK brings PIP5K to the plasma membrane as a means of generating local <em>PtdIns</em>-4,5-P2 synthesis. This enzyme-enzyme interaction provides a shuttling mechanism that allows BTK to stimulate the production of the substrate required by both its upstream activator, PI3K, and its downstream target, PLC-gamma2.

When PI3Ks are deregulated by aberrant surface receptors or modulators, accumulation of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> leads to increased cell growth, proliferation and contact-independent survival. The PI3K/PKB/TOR axis controls protein synthesis and growth, while <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em>-mediated activation of Rho GTPases directs cell motility. PI3K activity has been linked to the formation of tumors, metastasis, chronic inflammation, allergy and cardiovascular disease. Although increased <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> is a well-established cause of disease, it is seldom known which PI3K isoform is implied. Recent work has demonstrated that PI3Kgamma contributes to the control of cAMP levels in the cardiac system, where the protein acts as a scaffold, but not as a lipid kinase.

The initial steps in insulin signal transduction occur at the plasma membrane and lead to the activation of phosphatidylinositide (<em>PtdIns</em>) 3-kinase and the formation of <em>PtdIns</em>(<em>3,4,5</em>,)<em>P3</em> in the inner leaflet of the plasma membrane which is then converted to <em>PtdIns</em>(3,4)P2 by a specific phosphatase. Inhibitors of <em>PtdIns</em> 3-kinase suppress nearly all the metabolic actions of insulin indicating that <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and/or <em>PtdIns</em>(3,4)P2 are key 'second messengers' for this hormone. A major effect of insulin is its ability to stimulate the synthesis of glycogen in skeletal muscle. By 'working backwards' from glycogen synthesis, we have dissected an insulin-stimulated protein kinase cascade which is triggered by the activation of <em>PtdIns</em> 3-kinase. The first enzyme in this cascade is termed 3-phosphoinositide-dependent protein kinase (PDK1), because it is only active in the presence of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> or <em>PtdIns</em>(3,4)P2. PDK1 then activates protein kinase B (PKB) which, in turn, inactivates glycogen synthase kinase-3 (GSK3), leading to the dephosphorylation and activation of glycogen synthase and hence to an acceleration of glycogen synthesis. We review the evidence which indicates that the phosphorylation of other proteins by PKB and GSK3 is likely to mediate many of the intracellular actions of insulin.

The Vps34 (vacuolar protein sorting 34) class III PI3K (phosphoinositide 3-kinase) phosphorylates <em>PtdIns</em> (phosphatidylinositol) at endosomal membranes to generate <em>PtdIns</em>(3)P that regulates membrane trafficking processes via its ability to recruit a subset of proteins possessing <em>PtdIns</em>(3)P-binding PX (phox homology) and FYVE domains. In the present study, we describe a highly selective and potent inhibitor of Vps34, termed VPS34-IN1, that inhibits Vps34 with 25 nM IC50 in vitro, but does not significantly inhibit the activity of 340 protein kinases or 25 lipid kinases tested that include all isoforms of class I as well as class II PI3Ks. Administration of VPS34-IN1 to cells induces a rapid dose-dependent dispersal of a specific <em>PtdIns</em>(3)P-binding probe from endosome membranes, within 1 min, without affecting the ability of class I PI3K to regulate Akt. Moreover, we explored whether SGK3 (serum- and glucocorticoid-regulated kinase-3), the only protein kinase known to interact specifically with <em>PtdIns</em>(3)P via its N-terminal PX domain, might be controlled by Vps34. Mutations disrupting <em>PtdIns</em>(3)P binding ablated SGK3 kinase activity by suppressing phosphorylation of the T-loop [PDK1 (phosphoinositide-dependent kinase 1) site] and hydrophobic motif (mammalian target of rapamycin site) residues. VPS34-IN1 induced a rapid ~50-60% loss of SGK3 phosphorylation within 1 min. VPS34-IN1 did not inhibit activity of the SGK2 isoform that does not possess a <em>PtdIns</em>(3)P-binding PX domain. Furthermore, class I PI3K inhibitors (GDC-0941 and BKM120) that do not inhibit Vps34 suppressed SGK3 activity by ~40%. Combining VPS34-IN1 and GDC-0941 reduced SGK3 activity ~80-90%. These data suggest SGK3 phosphorylation and hence activity is controlled by two pools of <em>PtdIns</em>(3)P. The first is produced through phosphorylation of <em>PtdIns</em> by Vps34 at the endosome. The second is due to the conversion of class I PI3K product, <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> into <em>PtdIns</em>(3)P, via the sequential actions of the <em>PtdIns</em> 5-phosphatases [SHIP1/2 (Src homology 2-domain-containing inositol phosphatase 1/2)] and <em>PtdIns</em> 4-phosphatase [INPP4B (inositol polyphosphate 4-phosphatase type II)]. VPS34-IN1 will be a useful probe to delineate physiological roles of the Vps34. Monitoring SGK3 phosphorylation and activity could be employed as a biomarker of Vps34 activity, in an analogous manner by which Akt is used to probe cellular class I PI3K activity. Combining class I (GDC-0941) and class III (VPS34-IN1) PI3K inhibitors could be used as a strategy to better analyse the roles and regulation of the elusive class II PI3K.

Ca2+ entry through store-operated Ca2+ channels involves the interaction at ER-PM (endoplasmic reticulum-plasma membrane) junctions of STIM (stromal interaction molecule) and Orai. STIM proteins are sensors of the luminal ER Ca2+ concentration and, following depletion of ER Ca2+, they oligomerize and translocate to ER-PM junctions where they form STIM puncta. Direct binding to Orai proteins activates their Ca2+ channel function. It has been suggested that an additional interaction of the C-terminal polybasic domain of STIM1 with PM phosphoinositides could contribute to STIM1 puncta formation prior to binding to Orai. In the present study, we investigated the role of phosphoinositides in the formation of STIM1 puncta and SOCE (store-operated Ca2+ entry) in response to store depletion. Treatment of HeLa cells with inhibitors of PI3K (phosphatidylinositol 3-kinase) and PI4K (phosphatidylinositol 4-kinase) (wortmannin and LY294002) partially inhibited formation of STIM1 puncta. Additional rapid depletion of <em>PtdIns</em>(4,5)P2 resulted in more substantial inhibition of the translocation of STIM1-EYFP (enhanced yellow fluorescent protein) into puncta. The inhibition was extensive at a concentration of LY294002 (50 microM) that should primarily inhibit PI3K, consistent with a major role for <em>PtdIns</em>(4,5)P2 and <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> in puncta formation. Depletion of phosphoinositides also inhibited SOCE based on measurement of the rise in intracellular Ca2+ concentration after store depletion. Overexpression of Orai1 resulted in a recovery of translocation of STMI1 into puncta following phosphoinositide depletion and, under these conditions, SOCE was increased to above control levels. These observations support the idea that phosphoinositides are not essential but contribute to STIM1 accumulation at ER-PM junctions with a second translocation mechanism involving direct STIM1-Orai interactions.

The phosphoinositides <em>PtdIns</em>(4,5)P2 and <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> are concentrated in plasma membranes of eukaryotic cells, and excluded from endosomes, whereas <em>PtdIns</em>(3)P is formed in these latter intracellular membranes and is apparently excluded from the plasma membrane. The logic of this asymmetric disposition is now revealed by the nature of the effector proteins that selectively bind these lipids through specific modules and by the processes that they catalyze. <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> has a role in directing exocytosis, in addition to many other signaling events, whereas <em>PtdIns</em>(4,5)P2 directs endocytosis through its ability to anchor several coat proteins to the plasma membrane. Remarkably, the elimination of <em>PtdIns</em>(4,5)P2 from forming endosomes may be required for membrane fission to occur. Thus membrane insertion and retrieval can be regulated by plasma membrane concentrations of <em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> and <em>PtdIns</em>(4,5)P2, whereas <em>PtdIns</em>(3)P directs the downstream trafficking and recycling of intracellular membranes through its attraction of proteins that catalyze these processes. The phosphoinositides thereby control many cell features that depend upon protein sorting, including the composition of the plasma membrane itself, which in turn determines the cell's responses to its environment.

Phosphatidylinositol-<em>3,4,5</em>-trisphosphate (<em>PtdIns</em>(<em>3,4,5</em>)<em>P3</em> or PI<em>P3</em>) mediates signalling pathways as a second messenger in response to extracellular signals. Although primordial functions of phospholipids and RNAs have been hypothesized in the 'RNA world', physiological RNA-phospholipid interactions and their involvement in essential cellular processes have remained a mystery. We explicate the contribution of lipid-binding long non-coding RNAs (lncRNAs) in cancer cells. Among them, long intergenic non-coding RNA for kinase activation (LINK-A) directly interacts with the AKT pleckstrin homology domain and PI<em>P3</em> at the single-nucleotide level, facilitating AKT-PI<em>P3</em> interaction and consequent enzymatic activation. LINK-A-dependent AKT hyperactivation leads to tumorigenesis and resistance to AKT inhibitors. Genomic deletions of the LINK-A PI<em>P3</em>-binding motif dramatically sensitized breast cancer cells to AKT inhibitors. Furthermore, meta-analysis showed the correlation between LINK-A expression and incidence of a single nucleotide polymorphism (rs12095274: A>> G), AKT phosphorylation status, and poor outcomes for breast and lung cancer patients. PI<em>P3</em>-binding lncRNA modulates AKT activation with broad clinical implications.