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Author(s): Jaewoong Lee [sup.1] , Mark E. Robinson [sup.1] , Ning Ma [sup.2] , Dewan Artadji [sup.1] , Mohamed A. Ahmed [sup.3] , Gang Xiao [sup.3] , Teresa Sadras [sup.1] [...], Interferon-induced transmembrane protein 3 (IFITM3) has previously been identified as an endosomal protein that blocks viral infection.sup.1-3. Here we studied clinical cohorts of patients with B cell leukaemia and lymphoma, and identified IFITM3 as a strong predictor of poor outcome. In normal resting B cells, IFITM3 was minimally expressed and mainly localized in endosomes. However, engagement of the B cell receptor (BCR) induced both expression of IFITM3 and phosphorylation of this protein at Tyr20, which resulted in the accumulation of IFITM3 at the cell surface. In B cell leukaemia, oncogenic kinases phosphorylate IFITM3 at Tyr20, which causes constitutive localization of this protein at the plasma membrane. In a mouse model, Ifitm3.sup.-/- naive B cells developed in normal numbers; however, the formation of germinal centres and the production of antigen-specific antibodies were compromised. Oncogenes that induce the development of leukaemia and lymphoma did not transform Ifitm3.sup.-/- B cells. Conversely, the phosphomimetic IFITM3(Y20E) mutant induced oncogenic PI3K signalling and initiated the transformation of premalignant B cells. Mechanistic experiments revealed that IFITM3 functions as a PIP3 scaffold and central amplifier of PI3K signalling. The amplification of PI3K signals depends on IFITM3 using two lysine residues (Lys83 and Lys104) in its conserved intracellular loop as a scaffold for the accumulation of PIP3. In Ifitm3.sup.-/- B cells, lipid rafts were depleted of PIP3, which resulted in the defective expression of over 60 lipid-raft-associated surface receptors, and impaired BCR signalling and cellular adhesion. We conclude that the phosphorylation of IFITM3 that occurs after B cells encounter antigen induces a dynamic switch from antiviral effector functions in endosomes to a PI3K amplification loop at the cell surface. IFITM3-dependent amplification of PI3K signalling, which in part acts downstream of the BCR, is critical for the rapid expansion of B cells with high affinity to antigen. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3-dependent signalling complexes and amplify PI3K signalling for malignant transformation. IFITM3 shifts upon phosphorylation from acting as an antiviral effector to being a scaffold for PIP3 and thereby amplifies PI3K signalling, which can be co-opted for malignant transformation in B cell leukaemia and lymphoma.

Targeting and retention of resident integral membrane proteins of the Golgi apparatus underly the function of the Golgi in glycoprotein and glycolipid processing and sorting. In yeast, steady-state Golgi localization of multiple mannosyltransferases requires recognition of their cytosolic domains by the peripheral Golgi membrane protein Vps74, an orthologue of human GOLPH3/GPP34/GMx33/MIDAS (mitochondrial DNA absence sensitive factor). We show that targeting of Vps74 and GOLPH3 to the Golgi apparatus requires ongoing synthesis of phosphatidylinositol (PtdIns) 4-phosphate (PtdIns4P) by the Pik1 PtdIns 4-kinase and that modulation of the levels and cellular location of Ptdlns4P leads to mislocalization of these proteins. Vps74 and GOLPH3 bind specifically to PtdIns4P, and a sulfate ion in a crystal structure of GOLPH3 indicates a possible phosphoinositide-binding site that is conserved in Vps74. Alterations in this site abolish phosphoinositide binding in vitro and Vps74 function in vivo. These results implicate Pikl signaling in retention of Golgi-resident proteins via Vps74 and show that GOLPH3 family proteins are effectors of Golgi PtdIns 4-kinases. doi/10.1083/jcb.200909063

The large GTPase dynamin plays a key role in clathrin-mediated endocytosis in animal cells, although its mechanism of action remains unclear. Dynamins 1, 2, and 3 contain a pleckstrin homology (PH) domain that binds phosphoinositides with a very low affinity ([K.sub.D] > 1 mM), and this interaction appears to be crucial for function. These observations prompted the suggestion that an array of PH domains drives multivalent binding of dynamin oligomers to phosphoinositide-containing membranes. Although in vitro experiments reported here are consistent with this hypothesis, we find that PH domain mutations that abolish dynamin function do not alter localization of the protein in transfected cells, indicating that the PH domain does not play a simple targeting role. An alternative possibility is suggested by the geometry of dynamin helices resolved by electron microscopy. Even with one phosphatidylinositol-4,5-bisphosphate [Ptdlns(4,5)[P.sub.2]] molecule bound per PH domain, these dynamin assemblies will elevate the concentration of Ptdlns(4,5)[P.sub.2] at coated pit necks, and effectively cluster (or sequester) this phosphoinositide. In vitro fluorescence quenching studies using labeled phosphoinositides are consistent with dynamin-induced Ptdlns(4,5)[P.sub.2] clustering. We therefore propose that the ability of dynamin to alter the local distribution of Ptdlns(4,5)[P.sub.2] could be crucial for the role of this GTPase in promoting membrane scission during clathrin-mediated endocytosis. Ptdlns(4,5)[P.sub.2] clustering could promote vesicle scission through direct effects on membrane properties, or might play a role in dynamin's ability to regulate actin polymerization. endocytosis | phosphoinositide | vesicle | GTPase | actin

Hepatitis C virus (HCV) reorganizes cellular membranes to establish sites of replication. The required host pathways and the mechanism of cellular membrane reorganization are poorly characterized. Therefore, we interrogated a customized small interfering RNA (siRNA) library that targets 140 host membrane-trafficking genes to identify genes required for both HCV subgenomic replication and infectious virus production. We identified 7 host cofactors of viral replication, including Cdc42 and Rock2 (actin polymerization), EEA1 and Rab5A (early endosomes), Rab7L1, and PI3-kinase C2gamma and PI4-kinase IIIalpha (phospholipid metabolism). Studies of drug inhibitors indicate actin polymerization and phospholipid kinase activity ate required for HCV replication. We found extensive co-localization of the HCV replicase markers NS5A and double-stranded RNA with Rab5A and partial co-localization with Rab7L1. PI4K-IIIalpha co-localized with NS5A and double-stranded RNA in addition to being present in detergent-resistant membranes containing NS5A. In a comparison of type II and type III P14-kinases, PI4Ks were not required for HCV entry, and only PI4K-IIIalpha was required for HCV replication. Although PI4K-IIIalpha siRNAs decreased HCV replication and virus production by almost 100%, they had no effect on initial HCV RNA translation, suggesting that PI4K-IIIalpha functions at a posttranslational stage. Electron microscopy identified the presence of membranous webs, which are thought to be the site of HCV replication, in HCV-infected cells. Pretreatment with PI4K-IIIalpha siRNAs greatly reduced the accumulation of these membranous web structures in HCV-infected cells. We propose that PI4K-IIIalpha plays an essential role in membrane alterations leading to the formation of HCV replication complexes. antivirals | membranous web | PI4K-IIIa | PIK4CA | RNAi

The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancers. Here we used gene targeting to 'knock in' PIK3CA mutations into human breast epithelial cells to identify new therapeutic targets associated with oncogenic PIK3CA. Mutant PIK3CA knockin cells were capable of epidermal growth factor and mTOR-independent cell proliferation that was associated with AKT, ERK, and GSK3[beta] phosphorylation. Paradoxically, the GSK3[beta] inhibitors lithium chloride and SB216763 selectively decreased the proliferation of human breast and colorectal cancer cell lines with oncogenic PIK3CA mutations and led to a decrease in the GSK3[beta] target gene CYCLIN D1. Oral treatment with lithium preferentially inhibited the growth of nude mouse xenografts of HCT-116 colon cancer cells with mutant PIK3CA compared with isogenic HCT-116 knockout cells containing only wild-type PIK3CA. Our findings suggest GSK3[beta] is an important effector of mutant PIK3CA, and that lithium, an FDA-approved therapy for bipolar disorders, has selective antineoplastic properties against cancers that harbor these mutations. GSK3[beta] | lithium | mTOR | phosphatidylinositol 3-kinase | cancer

D-serine is a physiologic coagonist with glutamate at NMDA-subtype glutamate receptors. As D-serine is localized in gila, synaptically released glutamate presumably stimulates the gila to form and release D-serine, enabling glutamate/D-serine cotransmission. We show that serine racemase (SR), which generates D-serine from L-serine, is physiologically inhibited by phosphatidylinositol (4,5)-bisphosphate (PIP2) presence in membranes where SR is localized. Activation of metabotropic glutamate receptors (mGluR5) on gila leads to phospholipase C-mediated degradation of PIP2, relieving SR inhibition. Thus mutants of SR that cannot bind PIP2 lose their membrane localizations and display a 4-fold enhancement of catalytic activity. Moreover, mGluR5 activation of SR activity is abolished by inhibiting phospholipase C. D-serine | metabotropic glutamate receptor | NMDA transmission | phosphatidylinositol (4,5)-bisphosphate

Deregulation of the PI3K signaling pathway is observed in many human cancers and occurs most frequently through loss of PTEN phosphatase tumor suppressor function or through somatic activating mutations in the Class IA PI3K, PIK3CA. Tumors harboring activated p 110[alpha], the protein product of PIK3CA, require p110[alpha] activity for growth and survival and hence are expected to be responsive to inhibitors of its lipid kinase activity. Whether PTEN-deficient cancers similarly depend on p110[alpha] activity to sustain activation of the PI3K pathway has been unclear. In this study, we used a single-vector lentiviral inducible shRNA system to selectively inactivate the three Class IA PI3Ks, PIK3CA, PIK3CB, and PIK3CD, to determine which PI3K isoforms are responsible for driving the abnormal proliferation of PTEN-deficient cancers. Down-regulation of PIK3CA in colorectal cancer cells harboring mutations in PIK3CA inhibited downstream PI3K signaling and cell growth. Surprisingly, PIK3CA depletion affected neither PI3K signaling nor cell growth in 3 PTEN-deficient cancer cell lines. In contrast, down-regulation of the PIK3CB isoform, which encodes p110[beta], resulted in pathway inactivation and subsequent inhibition of growth in both cell-based and in vivo settings. This essential function of PIK3CB in PTEN-deficient cancer cells required its lipid kinase activity. Our findings demonstrate that although p110[alpha] activation is required to sustain the proliferation of established PIK3CA-mutant tumors, PTEN-deficient tumors are dependent instead on p110[beta] signaling. This unexpected finding demonstrates the need to tailor therapeutic approaches to the genetic basis of PI3K pathway activation to achieve optimal treatment response.

The regulation of M-type (KCNQ [Kv7]) [K.sup.+] channels by phosphatidylinositol 4,5-bisphosphate ([PIP.sub.2]) has perhaps the best correspondence to physiological signaling, but the site of action and structural motif of [PIP.sub.2] on these channels have not been established. Using single-channel recordings of chimeras of Kv7.3 and 7.4 channels with highly differential [PIP.sub.2] sensitivities, we localized a carboxy-terminal inter-helix linker as the primary site of [PIP.sub.2] action. Point mutants within this linker in Kv7.2 and Kv7.3 identified a conserved cluster of basic residues that interact with the lipid using electrostatic and hydrogen bonds. Homology modeling of this putative [PIP.sub.2]-binding linker in Kv7.2 and Kv7.3 using the solved structure of Kir2.1 and Kir3.1 channels as templates predicts a structure of Kv7.2 and 7.3 very similar to the Kit channels, and to the seven-[beta]-sheet barrel motif common to other [PIP.sub.2]-binding domains. Phosphoinositide-docking simulations predict affinities and interaction energies in accord with the experimental data, and furthermore indicate that the precise identity of residues in the interacting pocket alter channel-[PIP.sub.2] interactions not only by altering electrostatic energies, but also by allosterically shifting the structure of the lipid-binding surface. The results are likely to shed light on the general structural mechanisms of phosphoinositide regulation of ion channels.

Phosphatidylinositol-4,5-bisphosphate [Ptdlns(4,5)[P.sub.2]] is a signaling phospholipid implicated in a wide variety of cellular functions. At synapses, where normal Ptdlns(4,5)[P.sub.2] balance is required for proper neurotransmission, the phosphoinositide phosphatase synaptojanin 1 is a key regulator of its metabolism. The underlying gene, SYNJ1, maps to human chromosome 21 and is thus a candidate for involvement in Down's syndrome (DS), a complex disorder resulting from the overexpression of trisomic genes. Here, we show that Ptdlns(4,5)[P.sub.2] metabolism is altered in the brain of Ts65Dn mice, the most commonly used model of DS. This defect is rescued by restoring Synj1 to disomy in Ts65Dn mice and is recapitulated in transgenic mice overexpressing Synj1 from BAC constructs. These transgenic mice also exhibit deficits in performance of the Morris water maze task, suggesting that Ptdlns(4,5)[P.sub.2] dyshomeostasis caused by gene dosage imbalance for Synj1 may contribute to brain dysfunction and cognitive disabilities in DS. Alzheimer's disease | inositol 5-phosphatase | phosphatidylinositol-4,5-bisphosphate | phosphatidylinositol phosphate kinase | synapse

Large conductance, calcium- and voltage-gated potassium (BK) channels are ubiquitous and critical for neuronal function, immunity, and smooth muscle contractility. BK channels are thought to be regulated by phosphatidylinositol 4,5-bisphosphate (PI[P.sub.2]) only through phospholipase C (PLC)-generated PI[P.sub.2] metabolites that target [Ca.sup.2+] stores and protein kinase C and, eventually, the BK channel. Here, we report that PI[P.sub.2] activates BK channels independently of PI[P.sub.2] metabolites. PI[P.sub.2] enhances [Ca.sup.2+]-driven gating and alters both open and closed channel distributions without affecting voltage gating and unitary conductance. Recovery from activation was strongly dependent on PI[P.sub.2] acyl chain length, with channels exposed to water-soluble diC4 and diC8 showing much faster recovery than those exposed to PI[P.sub.2] (diC16). The PI[P.sub.2]-channel interaction requires negative charge and the inositol moiety in the phospholipid headgroup, and the sequence RKK in the S6-S7 cytosolic linker of the BK channel-forming (cbv1) subunit. PI[P.sub.2]-induced activation is drastically potentiated by accessory [[beta].sub.1] (but not [[beta].sub.4]) channel subunits. Moreover, PI[P.sub.2] robustly activates BK channels in vascular myocytes, where [[beta].sub.1] subunits are abundantly expressed, but not in skeletal myocytes, where these subunits are barely detectable. These data demonstrate that the final PI[P.sub.2] effect is determined by channel accessory subunits, and such mechanism is subunit specific. In HEK293 cells, co-transfection of cbv1+[[beta].sub.1] and PI4-kinaseII[alpha] robustly activates BK channels, suggesting a role for endogenous PI[P.sub.2] in modulating channel activity. Indeed, in membrane patches excised from vascular myocytes, BK channel activity runs down and Mg-ATP recovers it, this recovery being abolished by PI[P.sub.2] antibodies applied to the cytosolic membrane surface. Moreover, in intact arterial myocytes under physiological conditions, PLC inhibition on top of blockade of downstream signaling leads to drastic BK channel activation. Finally, pharmacological treatment that raises PI[P.sub.2] levels and activates BK channels dilates de-endothelized arteries that regulate cerebral blood flow. These data indicate that endogenous PI[P.sub.2] directly activates vascular myocyte BK channels to control vascular tone.

Nuclear PI3K and its downstream effectors play essential roles in a variety of cellular activities including cell proliferation, survival, differentiation, and pre-mRNA splicing. Aly is a nuclear speckle protein implicated in mRNA export. Here we show that Aly is a physiological target of nuclear PI3K signaling, which regulates its subnuclear residency, cell proliferation, and mRNA export activities through nuclear Akt phosphorylation and phosphoinositide association. Nuclear Akt phosphorylates Aly on threonine-219, which is required for its interaction with Akt. Aly binds phosphoinositides, and this action is regulated by Akt-mediated phosphorylation. Phosphoinositide binding but not Akt phosphorylation dictates Aly's nuclear speckle residency. Depletion of Aly results in cell growth suppression and mRNA export reduction. Inhibition of Aly phosphorylation substantially decreases cell proliferation and mRNA export. Furthermore, disruption of phosphoinositide association with Aly also significantly reduces these activities. Thus, nuclear PI3K signaling mediates both cell proliferation and mRNA export functions of Aly. nuclear PI 3-kinase | nuclear speckle | nuclear Akt | T219 phosphorylation

Phosphatidylinositol lipids play diverse physiological roles, and their concentrations are tightly regulated by various kinases and phosphatases. The enzymatic activity of Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP), recently identified as a member of the PTEN (phosphatase and tensin homolog deleted on chromosome 10) family of phosphatidylinositol phosphatases, is regulated by its own voltage-sensor domain in a voltagedependent manner. However, a detailed mechanism of Ci-VSP regulation and its substrate specificity remain unknown. Here we determined the in vitro substrate specificity of Ci-VSP by measuring the phosphoinositide phosphatase activity of the Ci-VSP cytoplasmic phosphatase domain. Despite the high degree of identity shared between the active sites of PTEN and Ci-VSP, Ci-VSP dephosphorylates not only the PTEN substrate, phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)[P.sub.3], but also, unlike PTEN, phosphatidylinositol 4,5-bisphosphate [PI(4,5)[P.sub.2]]. Enzymatic action on PI(4,5)[P.sub.2] removes the phosphate at position 5 of the inositol ring, resulting in the production of phosphatidylinositol 4-phosphate [PI(4)P]. The active site Cys-Xs-Arg (CXsR) sequence of Ci-VSP differs with that of PTEN only at amino acid 365 where a glycine residue in Ci-VSP is replaced by an alanine in PTEN. Ci-VSP with a G365A mutation no longer dephosphorylates PI(4,5)[P.sub.2] and is not capable of inducing depolarization-dependent rundown of a PI(4,5)[P.sub.2]-dependent potassium channel. These results indicate that Ci-VSP is a PI(3,4,5)[P.sub.3]/PI(4,5)[P.sub.2] phosphatase that uniquely functions in the voltage-dependent regulation of ion channels through regulation of PI(4,5)[P.sub.2] levels. phosphoinositide | PI(4,5)[P.sub.2] | voltage sensor | PI(3,4,5)[P.sub.3] | substrate specificity

The Saccharomyces cerevisiae phosphatidylcholine/phosphatidylinositol transfer protein Sec14p is required for Golgi apparatusderived vesicular transport through coordinate regulation of phospholipid metabolism. Sec14p is normally essential. The essential requirement for SEC14 can be bypassed by inactivation of (i) the CDP-choline pathway for phosphatidylcholine synthesis or (ii) KES1, which encodes an oxysterol binding protein. A unique screen was used to determine genome-wide genetic interactions for the essential gene SEC14 and to assess whether the two modes of 'sec14 bypass' were similar or distinct. The results indicate that inactivation of the CDP-choline pathway allows cells with inactivated SEC14 to live through a mechanism distinct from that of inactivation of KES1. We go on to demonstrate an important biological function of Kes1p. Kes1p regulates Golgi apparatus-derived vesicular transport by inhibiting the function of Pik1p-generated Golgi apparatus phosphatidylinositol-4-phosphate (PI-4P). Kes1p affects both the availability and level of Golgi apparatus PI-4P. A set of potential PI-4P-responsive proteins that include the Rab GTPase Ypt31p and its GTP exchange factor are described. KES1 | Saccharomyces cerevisiae | SEC14 | vesicular transport

Polarization of chemotaxing cells depends on positive feedback loops that amplify shallow gradients of chemoattractants into sharp intracellular responses. In particular, reciprocal activation of phosphatidylinositol 3-kinases (PI3Ks) and small GTPases like Rac leads to accumulation, at the leading edge, of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP3). Mice carrying a 'knockin' allele of the G protein-coupled receptor (GPCR)-activated PI3K [gamma] encoding a plasma membrane-targeted protein appeared normal, but their leukocytes showed GPCR-uncoupled PIP3 accumulation. In vivo, the mutation increased proliferation and decreased apoptosis, leading to leukocytosis and delayed resolution of inflammation in wound healing. Mutant leukocytes showed significantly impaired directional cell migration in response to chemoattractants. Stimulated mutant macrophages did not polarize PIP3 and showed a shortened Rac activation because of enhanced PI3K-dependent activation of RacGAPs. Together with the finding that chemoattractants stimulate a PIP3-dependent GAP activation in wild-type macrophages, these results identify a molecular mechanism involving PI3K- and RacGAP-dependent negative control of Rac that limits and fine-tunes feedback loops promoting cell polarization and directional motility. chemotaxis | signal transduction | inflammation

All eukaryotic cells contain the phospholipid phosphatidylinositol 4, 5-bisphosphate (PI[P.sub.2]) that serves multiple roles in signal transduction cascades. Type I phosphatidylinositol-4-phosphate 5-kinase (PIP5KI) catalyzes the synthesis of PIP2 by phosphorylating phos-phatidylinositol 4 phosphate. Although the classical isoforms of PIP5KI (designated as [alpha], [beta], and [gamma],) all generate the same phospholipid product, they have significantly dissimilar primary structures and expression levels in different tissues, and they appear to localize within different compartments within the cell. Therefore, it appears likely that PIP5KI isoforms have overlapping, but not identical, functions. Here we show that targeted disruption of PIP5KI[gamma], causes widespread developmental and cellular defects. PIP5KI[gamma]-null embryos have myocardial developmental defects associated with impaired intracellular junctions that lead to heart failure and extensive prenatal lethality at embryonic day 11.5 of development. Loss of PIPSKI[gamma], also results in neural tube closure defects that were associated with impaired PI[P.sub.2] production, adhesion junction formation, and neuronal cell migration. These data, along with those of other PIP5KI isoforms, indicate that individual PIP5KI isoenzymes fulfill specific roles in embryonic development. phosphoinositide | phospholipid | phosphatidylinositol 4,5-bisphosphate

We have shown previously that cardiac G protein-gated inwardly rectifying [K.sup.+] (GIRK) channels are inhibited by Gq protein-coupled receptors (GqPCRs) via phosphatidylinositol 4,5-bisphosphate (PI[P.sub.2]) depletion in a receptor-specific manner. To investigate the mechanism of receptor specificity, we examined whether the activation of GqPCRs induces localized PI[P.sub.2] depletion. When we applied endothelin-1 to the bath, GIRK channel activities recorded in cell-attached patches were not changed, implying that PI[P.sub.2] signal is not diffusible but is a localized signal. To test this possibility, we directly measured lateral diffusion by introducing fluorescence-labeled phosphoinositides to a small area of the membrane with patch pipettes. After pipettes were attached, phosphatidylinositol 4-monophosphate or phosphatidylinositol diffused rapidly to the entire membrane, whereas PI[P.sub.2] was confined to the membrane patch inside the pipette. The confinement of PI[P.sub.2] was disrupted after cytochalasin D treatment, suggesting that the cytoskeleton is responsible for the low mobility of PI[P.sub.2]. The diffusion coefficient (D) of PI[P.sub.2] in the plasma membrane measured with the fluorescence recovery after photobleaching technique was 0.00039/[micro]m2/s (n = 6), which is markedly lower than D of phosphatidylinositol (5.8 /[micro][m.sup.2]/s, n = 5). Simulation of PI[P.sub.2] concentration profiles by the diffusion model confirms that when D is small, the kinetics of PI[P.sub.2] depletion at different distances from phospholipase C becomes similar to the characteristic kinetics of GIRK inhibition by different agonists. These results imply that PI[P.sub.2] depletion is localized adjacent to GqPCRs because of its low mobility, and that spatial proximity of GqPCR and the target protein underlies the receptor specificity of PI[P.sub.2]-mediated signaling.

Among various second messengers, phosphatidylinositol 3,4,5-triphosphate (PI [P.sub.3]) and phosphatidylinositol 3,4-bisphosphate [PI (3,4)[P.sub.2]] regulate a variety of cellular processes, such as cell survival, polarization, and proliferation. Many of these functions are achieved via activation of serine/threonine kinase Akt. To investigate the spatiotemporal regulation of these lipids, we constructed a genetically targetable phosphoinositide (PI) indicator by sandwiching pleckstrin homology (PH) domain of Akt and a 'pseudoligand' containing acidic amino acid residues, between cyan and yellow mutants of GFP. In living cells, elevations in PI [P.sub.3] and PI(3,4)[P.sub.2] by growth factor-induced activation of phosphatidylinositol 3-kinase (PI3K) resulted in a change in fluorescence resonance energy transfer (FRET) between the fluorescent proteins, increasing yellow to cyan emission ratios by 10-30%. This response can be reversed by inhibiting PI3K and abolished by mutating the critical residues responsible for PI binding. Differential dynamics of PIs were observed at plasma membrane of NIH 3T3 cells, stimulated by various growth factors. On the other hand, the nuclear targeted indicator showed no response within an hour after platelet-derived growth factor stimulation, suggesting that no appreciable amounts of accessible PI [P.sub.3] and PI(3,4) [P.sub.2] were produced in the nucleus. Furthermore, simultaneous imaging of a plasma membrane-targeted PI indicator and a nuclear-targeted Akt activity reporter revealed a gradual and sustained accumulation of Akt activity in the nucleus after rapid and transient production of PI[P.sub.3] and PI(3,4)[P.sub.2] at plasma membrane in the same cell. Thus, signal propagation from the lipid messengers at plasma membrane to the effectors in the nucleus is precisely controlled by kinases as well as lipid and protein phosphatases. FRET

Phosphatidylionsitol 4,5-bisphosphate (PI[P.sub.2]), a substrate of phospholipase C, has recently been recognized to regulate membrane-associated proteins and act as a signal molecule in phospholipase C-linked Gq-coupled receptor (GqPCR) pathways. However, it is not known whether PI[P.sub.2] depletion induced by GqPCRs can act as receptor-specific signals in native cells. We investigated this issue in cardiomyocytes where PI[P.sub.2]-dependent ion channels, G protein-gated inwardly rectifying [K.sup.+] (GIRK) and inwardly rectifying background [K.sup.+] (IRK) channels, and various GqPCRs are present. The GIRK current was recorded by using the patch-clamp technique during the application of 10 [micro]M acetylcholine. The extent of receptor-mediated inhibition was estimated as the current decrease over 4 min while taking the GIRK current ([I.sub.GIRK]) value during a previous stimulation as the control. Each GqPCR agonist inhibited [I.sub.GIRK] with different potencies and kinetics. The extents of inhibition induced by phenylephrine, angiotensin II, endothelin-1, prostaglandin F2[alpha], and bradykinin at supramaximal concentrations were (mean [+ or -] SE) 32.1 [+ or -] 0.6%, 21.9 [+ or -] 1.4%, 86.4 [+ or -] 1.6%, 63.7 [+ or -] 4.9%, and 5.7 [+ or -] 1.9%, respectively. GqPCR-induced inhibitions of [I.sub.GIRK] were not affected by protein kinase C inhibitor (calphostin C) but potentiated and became irreversible when the replenishment of PI[P.sub.2] was blocked by wortmannin (phosphatidylinositol kinase inhibitor). Loading the cells with PI[P.sub.2] significantly reduced endothelin-1 and prostaglandin F2[alpha]-induced inhibition of [I.sub.GIRK]. On the contrary, GqPCR-mediated inhibitions of inwardly rectifying background [K.sup.+] currents were observed only when GqPCR agonists were applied with wortmannin, and the effects were not parallel with those on [I.sub.GIRK]. These results indicate that GqPCR-induced inhibition of ion channels by means of PI[P.sub.2] depletion occurs in a receptor-specific manner. phospholipase C | cardiac myocyte | phosphatidylinositol 4-kinase | Gq-coupled receptor | G protein-gated inwardly rectifying [K.sup.+] channels

We report a novel connection between the phosphatidylinositol (PI) metabolic pathway and the DNA replication and damage checkpoint pathway discovered from an unbiased chemical genomics screen. Substrates and products of PI kinases are important signaling molecules that affect a wide range of biological processes. The full collection of yeast deletion strains was screened to identify genes that confer altered sensitivity to the natural product wortmannin, a PI kinase inhibitor. These experiments have allowed us to explore metabolomic and proteomic implications of PI synthesis and turnover. This study also uncovers other biological processes affected by wortmannin treatment, including proteasome-mediated degradation and chromatin remodeling. Bioinformatic analyses were used to reveal the relative distances among cellular processes affected by wortmannin and protein-protein interactions in the wortmannin-sensitive proteomic subnetwork. These results illustrate the great utility of using a whole-genome approach in annotating the biological effects of small molecules and have clear implications for pharmacogenomics. Furthermore, our discovery points to a route to overcoming genome instability, a result of defective DNA damage signaling/ repair and a hallmark of cancer.

Neutrophils exposed to chemoattractants polarize and accumulate polymerized actin at the leading edge. In neutrophil-like HL-60 cells, this asymmetry depends on a positive feedback loop in which accumulation of a membrane lipid, phosphatidylinositol (PI) 3,4,5-trisphosphate (PI[3,4,5] [P.sub.3]), leads to activation of Rac and/or Cdc42, and vice versa. We now report that Rac and Cdc42 play distinct roles in regulating this asymmetry. In the absence of chemoattractant, expression of constitutively active Rac stimulates accumulation at the plasma membrane of actin polymers and of GFP-tagged fluorescent probes for PI(3,4,5)[P.sub.3] (the PH domain of Akt) and activated Rac (the p21-binding domain of p21-activated kinase). Dominant negative Rac inhibits chemoattractant-stimulated accumulation of actin polymers and membrane translocation of both fluorescent probes and attainment of morphologic polarity. Expression of constitutively active Cdc42 or of two different protein inhibitors of Cdc42 fails to mimic effects of the Rac mutants on actin or PI(3,4,5)[P.sub.3]. Instead, Cdc42 inhibitors prevent cells from maintaining a persistent leading edge and frequently induce formation of multiple, short lived leading edges containing actin polymers, PI(3,4,5)[P.sub.3], and activated Rac. We conclude that Rac plays a dominant role in the PI(3,4,5)[P.sub.3]-dependent positive feedback loop required for forming a leading edge, whereas location and stability of the leading edge are regulated by Cdc42.

Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-[P.sub.2] to PtdIns-3,4,5-[P.sub.3]. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-[P.sub.3]--binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class [I.sub.B] 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 PtdIns-3,4,5-[P.sub.3]--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].

Research has been conducted on the hybrid lipid with a 2,3-diacylthreitol backbone. The results of asymmetric synthesis of this lipid are reported.

The clathrin adaptor complex AP-2 serves to coordinate clathrin-coated pit assembly with the sorting of transmembrane cargo proteins at the plasmalemma. How precisely AP-2 assembly and cargo protein recognition at sites of endocytosis are regulated has remained unclear, but recent evidence implicates phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI[4,5][P.sub.2]), in these processes. Here we have identified and functionally characterized a conserved binding site for PI(4,5)[P.sub.2] within [mu]2-adaptin, the medium chain of the clathrin adaptor complex AP-2. Mutant [mu]2 lacking a cluster of conserved lysine residues fails to bind PI(4,5)[P.sub.2] and to compete the recruitment of native clathrin/AP-2 to PI(4,5)[P.sub.2]-containing liposomes or to presynaptic membranes. Moreover, we show that expression of mutant [mu]2 inhibits receptor-mediated endocytosis in living cells. We suggest that PI(4,5)[P.sub.2] binding to [mu]2-adaptin regulates clathrin-mediated endocytosis and thereby may contribute to structurally linking cargo recognition to coat formation.

Research has been conducted on the phosphatidylinositol-4-phosphate 5-kinase activity in Candida albicans. Results indicate that this enzyme is activated by phosphatidic acid during the induced by temperature morphogenesis.

In vitro motility assay shows that Unc104 kinesin transports membrane cargo mediated by pH domain of the protein, which binds to the liposomes containing phosphatidylinositol(4,5)bisphosphate. Data indicate that Unc104 clustering in phosphatidylinositol(4,5)bisphosphate lipid rafts may activate membrane transport.

Phosphatidylinositol 3,5-bisphosphate (Ptdlns[3,5][P.sub.2]) was first identified as a nonabundant phospholipid whose levels increase in response to osmotic stress. In yeast, Fab1p catalyzes formation of Ptdlns(3,5)[P.sub.2] via phosphorylation of Ptdlns(3)P. We have identified Vac14p, a novel vacuolar protein that regulates Ptdlns(3,5) [P.sub.2] synthesis by modulating Fab1p activity in both the absence and presence of osmotic stress. We find that Ptdlns(3)P levels are also elevated in response to osmotic stress, yet, only the elevation of Ptdlns(3,5)[P.sub.2] levels are regulated by Vac14p. Under basal conditions the levels of Ptdlns(3,5) [P.sub.2] are 18-28-fold lower than the levels of Ptdlns(3)P, Ptdlns(4)P, and Ptdlns(4,5) [P.sub.2]. After a 10 min exposure to hyperosmotic stress the levels of Ptdlns(3,5) [P.sub.2] rise 20-fold, bringing it to a cellular concentration that is similar to the other phosphoinositides. This suggests that Ptdlns(3,5) [P.sub.2] plays a major role in osmotic stress, perhaps via regulation of vacuolar volume. In fact, during hyperosmotic stress the vacuole morphology of wild-type cells changes dramatically, to smaller, more highly fragmented vacuoles, whereas mutants unable to synthesize Ptdlns(3,5) [P.sub.2] continue to maintain a single large vacuole. These findings demonstrate that Vac14p regulates the levels of Ptdlns(3,5) [P.sub.2] and provide insight into why Ptdlns(3,5) [P.sub.2] levels rise in response to osmotic stress.

An SH2-domain-containing kinase has been found to regulate the phosphatidylinositol-3 kinase (PI3K) pathway negatively. It has been shown that SHK1 is necessary for proper phagocytosis and chemotaxis and it appears that SHK1 is necessary for the proper spatiotemporal control of F-actin levels in chemotaxing cells. SHK1 is localized at the plasma membrane/cortex.

Phosphatidylinositol 4-phosphate 5-kinase alpha, a downstream effector of the small G protein ARF6 in membrane ruffle formation, is discussed. When ARF6 is activated by stimulation from an agonist, it brings on recruitment of a diverse set of signaling molecules into active cytoskeletal and membrane rearrangement sites. These signaling molecules are interactive. Colocalization of ARF6 is seen in ruffling membranes formed where AlF(sub.4) and EGF stimulation exist. It is blocked by dominant-negative ARF6.

Native and recombinant G protein-gated inwardly rectifying potassium (GIRK) channels are directly activated by the (beta gamma) subunits of GTP-binding (G) proteins. The presence of phosphatidylinositol-bis-phosphate (PIP(sub 2)) is required for G protein activation. Formation (via hydrolysis of ATP) of endogenous PIP(sub 2) or application of exogenous PIP(sub 2) increases the mean open time of GIRK channels and sensitizes them to gating by internal Na(super +) ions. In the present study, we show that the activity of ATP- or PIP(sub 2)-modified channels could also be stimulated by intracellular Mg(super 2+) ions. In addition, Mg(super 2+) ions reduced the single-channel conductance of GIRK channels, independently of their gating ability. Both Na(super +) and Mg(super 2+) ions exert their gating effects independently of each other or of the activation by the G(sub beta gamma) subunits. At high levels of PIP(sub 2), synergistic interactions among Na(super +), Mg(super 2+), and G(sub beta gamma) subunits resulted in severalfold stimulated levels of channel activity. Changes in ionic concentrations and/or G protein subunits in the local environment of these K(super +) channels could provide a rapid amplification mechanism for generation of graded activity, thereby adjusting the level of excitability of the cells. KEY WORDS: G protein-gated inwardly rectifying potassium channels; phosphatidylinositol-bis-phosphate; G(sub beta gamma) gating; Mg(super 2+) gating; Na(super +) gating

The phosphatidylinositol 3-kinase (PI3K)-signaling pathway has emerged as an important component of cytokine-mediated survival of hemopoietic cells. Recently, the protein kinase PKB/akt (referred to here as PKB) has been identified as a downstream target of PI3K necessary for survival. PKB has also been implicated in the phosphorylation of Bad, potentially linking the survival effects of cytokines with the Bcl-2 family. We have shown that granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity, and we now show that when PKB activation is also completely blocked, GM-CSF is still able to stimulate phosphorylation of Bad. Interleukin 3 (IL-3), on the other hand, requires PI3K for survival, and blocking PI3K partially inhibited Bad phosphorylation. IL-4, unique among the cytokines in that it lacks the ability to activate the p21ras-mitogen-activated protein kinase (MAPK) cascade, was found to activate PKB and promote cell survival, but it did not stimulate Bad phosphorylation. Finally, although our data suggest that the MAPK pathway is not required for inhibition of apoptosis, we provide evidence that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway. Together, these results demonstrate that although PI3K may contribute to phosphorylation of Bad in some instances, there is at least one other PI3K. independent pathway involved, possibly via activation of MEK. Our data also suggest that although phosphorylation of Bad may be one means by which cytokines can inhibit apoptosis, it may be neither sufficient nor necessary for the survival effect.

PKB (also known as c-Akt or Rac-PK) was originally identified as the transforming oncogene in a retrovirus from a spontaneous thymoma in an AKR mouse(1). The oncogene product contains all [...], Protein kinase B (PKB) is a proto-oncogene that is activated in signaling pathways initiated by phosphoinositide 3-kinase. Chromatographic separation of brain cytosol revealed a kinase activity that phosphorylated and activated PKB only in the presence of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5) [P.sub.3]]. Phosphorylation occurred exclusively on threonine-308, a residue implicated in activation of PKB in vivo. PtdIns(3,4,5)[P.sub.3] was determined to have a dual role: Its binding to the pleckstrin homology domain of PKB was required to allow phosphorylation by the upstream kinase and it directly activated the upstream kinase.

The role of phosphoinositide (PI) 3-kinase in signal transduction processes was determined by investigating its action in the synthesis of 3-mono-, 3,4-bis-, and 3,4,5-triphosphates of L-alpha-phosphatidyl-D-myo-inositol from the enantiomers of 1,2:5,6-D-O-cyclohexylidene-myo-inositol. The results showed that PI 3-kinase is a signaling molecule that has modulatory effects on various biomolecules.

The signalling activity of the phosphatidylinositol-3-OH kinase (PI(3)K) protein encoded by the age-1 gene controls longevity and dauer diapause in Caenorhabditis elegans. AGE-1 PI(3)K is involved in neuroendocrine signalling of the dauer pathway. Its absence leads to dauer characteristics and reduced activity decreases the rate of senescence. Phospahatidylinositol-3,4,5-triphosphate signalling acts negatively on the mutations affecting dauer formation that suppress dauer arrest and longevity age-1 phenotypes.

Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine [...]

Phosphatidylinositol 4,5-phosphate PtdIns (4,5) is an enzyme produced in the plasma membrane that mediates the actions of hormones, messengers and transmitters. Inhibition of the enzyme by wortmannin in the adrenal glomerulosa cells and adrenocortical cells resulted to rapid loss of PtdIns (4,5) P2 pool in angiotensin cells. It also inhibited increases in inositol 1,4,5-triphosphate and cytoplasmic calcium in a variety of cells. This indicates that PtdIns (4,5) is involved in the maintenance of inositolphospholipids in a wide array of cell types.

PCR technique using oligonucleotides based on conserved sequence in phosphatidylinositol (PI) 3-kinases from yeast and bovine tissues helps clone an Arabidopsis thaliana PI-3 kinase cDNA (AtVPS34), which exhibits sequence similarity with that of yeast VPS34 in the C-terminal half. The plant protein has a lipid-binding region that depends on calcium levels near N terminus and a catalytic, ATP-binding site in the C-terminus. Growth- and development-inhibited second-generation plants result from the expression of antisense constructs of the plant PI 3-kinase.

Western blotting, immunoprecipitation and phosphatidylinositol 3-kinase (PI 3-kinase) assays of focal adhesion kinase (FAK)-Src homology 2 domain with signalling molecule interactions indicate that FAK associates with the PI 3-kinase of NIM3 T3 mouse fibroblasts in a stable manner. Recombinant FAK also links to the PI 3-kinase p85 subunit and its autophosphorylation enhances PI 3-kinase binding. FAK induces phosphorylation of p85 subunit of PI 3-kinase, indicating that PI 3-kinase functions as a FAK substrate and effector.

Studies of insulin action in two insulin receptor-transfected strains of human K562 erythroleukemia cells, one in which the cells cannot produce PI-GlcNAc (IA), and another in which deacetylation of PI-GlcNAc is impaired (IVD), reveal that glycosyl-phosphatidylinositol is essential for the modulation of glycogen synthesis. While both cell lines display a similar increase in the insulin-induced autophosphorylation of the receptors while insulin-induced glycogen synthesis exhibits differences in the two cell lines. IA cells do not exhibit any insulin induction while in IVD cells the stimulation is observed at low levels.

Use of antibodies against tyrosine phosphorylated PYbeta1 peptide reveals the localization of the PYbeta1 subunit to podosomes of RSV-transformed NIH 3T3 cells, in a manner different from that of phosphotyrosine and beta1 integrins. One SH2 domain of the p85 phosphatidylinositol-3-kinase subunit binds only to the PYbeta1 integrin subunit and not to the non-phosphorylated peptide. Tyrosine phosphorylation of cytoplasmic domain of beta1 subunit influences cellular signaling pathways.

Phosphatidylinositol-3-OH kinase (PI(3)K) controls the platelet-derived growth factor receptor phosphorylation or insulin receptor signaling to pp70 (s6k). PI(3)K- controlled stimulation of pp70 (s6k) does not depend on conventional protein kinase C isoforms. Rapamycin inhibits pp70 (s6k) stimulation through all mitogens without inhibition of PI(3)K.

The entire sequence of mouse stem cell antigen Sca-2 reveals that Sca-2 belongs to the Ly-6 family which is a group of small cysteine-rich cell surface proteins. The proteins are attached to the membrane through glycosyl-phosphatidylinositol moiety. Lymphoid precursor cell expresses Sca-2 upon entry into the thymus and indicates its importance in early thymopoiesis. Sca-2 antigen is seen in immature thymocytes and is not restricted to lac T lineage.

The functional complementation of genes in Leishmania enables the isolation of virulence genes, which in turn enables the synthesis of surface glycosyl-phosphatidylinositol. Lipophosphoglycan (LPG) is a virulence determinant glycosyltransferase that appends the residues of galactofuranosyl to the LPG chain. Tropical diseases are caused by the trypanosomatid parasites found in Leishmania.

A variety of peptide growth factors and hormones, such as platelet-derived growth factor (PDGF), nerve growth factor (NGF), and insulin mediate their cellular effects by interaction with cell surface receptor [...], The VPS34 gene product (Vps34p) is required for protein sorting to the lysosome-like vacuole of the yeast Saccharomyces cerevisiae. Vps34p shares (significant sequence similarity with the catalytic subunit of bovine phosphatidylinositol (PI) 3-kinase [the 110-kilodalton (p110) subunit of PI 3-kinase], which is known to interact with activated cell surface receptor tyrosine kinases. Yeast strains deleted for the VPS34 gene or carrying vps34 point mutations lacked detectable PI 3-kinase activity and exhibited severe defects in vacuolar protein sorting. Overexpression of Vps34p resulted in an increase in PI 3-kinase activity, and this activity was specifically precipitated with antisera to Vps34p. VPS34 encodes a yeast PI 3-kinase, and this enzyme appears to regulate intracellular protein trafficking decisions.