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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.

Phosphatidylinositol transfer proteins (PITPs) are believed to be lipid transfer proteins because of their ability to transfer either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane compartments, in vitro. However, the [...]

The adipocyte-derived hormone leptin and the pancreatic 13-cell-derived hormone insulin function as afferent signals to the hypothalamus in an endocrine feedback loop that regulates body adiposity. They act in hypothalamic centers to modulate the function of specific neuronal subtypes, such as neuropeptide Y (NPY) neurons, by modifying neuronal electrical activity. To investigate the intrinsic activity of these neurons and their responses to insulin and leptin, we used a combination of morphological features and immunocytochemical technique to identify the NPY neurons of hypothalamic arcuate nucleus (ARC) and record whole cell large-conductance [Ca.sup.2+]-activated potassium (BK) currents on them. We found that both of the hormones increase the peak amplitude of BK currents, shifting the steady-state activation curve to the left. The effect of both insulin and leptin can be prevented by pretreatment with inhibitors of tyrosine kinase and phosphatidylinositol 3-kinase (PI3K) but not MAPK. These data indicate that PI3Kmediated signals are the common regulators of BK channels by insulin and leptin and mediated the two hormones' identical activatory effects on ARC NPY neurons. The effect of insulin and leptin together was similar to that of insulin or leptin alone, and leptin or insulin pretreatment did not lead to insulin- or leptin-sensitizing effects, respectively. These intracellular signaling mechanisms may play key roles in regulating ARC NPY neuron activity and physiological processes such as the control of food intake and body weight, which are under the combined control of insulin and leptin. patch-clamp recording; phosphatidylinositol 3-kinase doi: 10.1152/ajpendo.00155.2009

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

Yui N, Okutsu R, Sohara E, Rai T, Ohta A, Noda Y, Sasaki S, Uchida S. FAPP2 is required for aquaporin-2 apical sorting at Trans-Golgi network in polarized MDCK cells. Am J Physiol Cell Physiol 297: C1389-C1396, 2009. First published September 30, 2009; doi: 10.1152/ajpcell.00098.2009.--FAPP2 is an adaptor protein of phosphatidylinositol-4-phosphate and is involved in the transport of some apical cargos from the trans-Golgi network (TGN). To investigate whether the regulated apical transport of aquaporin-2 (AQP2) is involved in the FAPP2-dependent apical protein-sorting machinery, we measured apical sorting of AQP2 in Madin-Darby canine kidney (MDCK) cells with or without FAPP2 knockdown. We established MDCK cell lines that stably express rat AQP2 without any tag sequence. Then, FAPP2-deficient stable cell lines were established from the AQP2-expressing cell lines by a retrovirus-mediated RNA interference system. In the established cell lines, AQP2 was detected in both apical and basolateral membranes. Forskolin increased only the apical localization of AQP2, which was not affected by basolateral treatment with 0.5% tannic acid, indicating that the forskolin-induced apical transport of AQP2 did not include the transcytotic pathway from basolateral to apical membranes but is a direct transport from TGN to the apical membranes. Using these cell lines, we tested the effect of FAPP2 knockdown on the polarized AQP2 transport to plasma membranes and found that the forskolin-induced apical transport of AQP2 was completely abolished by FAPP2 knockdown. By contrast, the basolateral localization of AQP2 was not affected by FAPP2 knockdown. AQP2 phosphorylation by forskolin was also impaired in FAPP2 knockdown MDCK cells. These results suggest that FAPP2 is necessary to generate AQP2-bearing vesicles at trans-Golgi that will undergo phosphorylation by PKA in subapical regions. phosphatidylinositol; forskolin; short hairpin RNA; biotinylation assay doi: 10.1152/ajpcell.00098.2009

Sertoli cells provide the physical support and the necessary environment for germ cell development. Among the products secreted by Sertoli cells, lactate, the preferred energy substrate for spermatocytes and spermatids, is present. Considering the essential role of lactate on germ cell metabolism, it is supposed that Sertoli cells must ensure its production even in adverse conditions, such as those that would result from a decrease in glucose levels in the extracellular milieu. The aim of the present study was to investigate 1) a possible effect of glucose deprivation on glucose uptake and on the expression of glucose transporters in rat Sertoli cells and 2) the participation of different signal transduction pathways in the above-mentioned regulation. Results obtained show that decreasing glucose levels in Sertoli cell culture medium provokes 1) an increase in glucose uptake accompanied by only a slight decrease in lactate production, 2) an increase in GLUT1 and a decrease in GLUT3 expression, and 3) an activation of AMP-activated protein kinase (AMPK)-, phosphatidylinositol 3-kinase (PI3K)/PKB-, and p38 MAPK-dependent pathways. Additionally, by using specific inhibitors of these pathways, a possible participation of AMPK- and p38MAPK-dependent pathways in the regulation of glucose uptake and GLUT1 expression is shown. These results suggest that Sertoli cells adapt to conditions of glucose deprivation to ensure an adequate lactate concentration in the microenvironment where germ cell development occurs. testis; signal transduction; glucose transporter; lactate doi: 10.1152/ajpendo.00235.2009.

The inwardly rectifying [K.sup.+] (Kir) current in mammalian retinal pigment epithelial (RPE) cells, which is largely mediated by Kir7.1 channels, is stable in cells dialyzed with MgATP but runs down when intracellular ATP is depleted. A potential mechanism for this rundown is a decrease in phosphatidylinositol 4,5-bisphosphate ([PIP.sub.2]) regeneration by ATP-dependent lipid kinases. Here, we used the whole cell voltage-clamp technique to investigate the membrane PIP2 dependence of Kir channels in isolated bovine RPE cells. When RPE cells were dialyzed with ATP-ffee solution containing [PIP.sub.2] (25-50 [micro]M), rundown persisted hut was markedly reduced. Removal of [Mg.sup.2+] from the pipette solution also slowed rundown, indicating that elevated intracelhilar [Mg.sup.2+] concentration contributes to rundown. Cell dialysis with the [PIP.sub.2] scavenger neomycin in MgATP solution diminished Kir current in a voltage-dependent manner, suggesting that it acted at least in part by blocking the Kir channel. Kir current in MgATP-loaded cells was partially inhibited by bath application of quercetin (100 [micro]M), phenylarsine oxide (100 [micro]M), or wortmannin (50 [micro]M), inhibitors of phosphatidylinositol (PI) kinases, and was completely inhibited by cell dialysis with 2 mM adenosine, a PI4 kinase inhibitor. Both LY-294002 (100 [micro]M), an inhibitor of PI3 kinases, and its inactive analog LY-303511 (100 [micro]M) rapidly and reversibly inhibited Kir current, suggesting that these compounds act as direct channel blockers. We conclude that the activity of Kir channels in the RPE is critically dependent on the regeneration of membrane [PIP.sub.2] by PI4 kinases and that this may explain the dependence of these channels on hydrolyzable ATP. retinal pigment epithelium; Kir7.1; phosphatidylinositol 4-kinases; phosphatidylinositol 4,5-bisphosphate doi: 10.1152/ajpcell.00250.2009

Although phosphatidylinositol 3-kinase (PI3K) is essential for several cellular signal transductions, its role in the regulation of cystic fibrosis transmembrane conductance regulator (CFTR) activity in intestinal epithelial cells is poorly understood. Therefore, the possible involvement of PI3K in the regulation of cAMP- and cGMP-induced duodenal epithelial CFTR activation was investigated in the present study. Forskolin and 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) markedly stimulated duodenal mucosal HC[O.sub.3] secretion and short-circuit current ([I.sub.sc) in CFTR wild-type mice, which was significantly inhibited by [CFTR.sub.inh]-172, a highly potent and specific CFTR inhibitor. Forskolin and 8-Br-cGMP failed to stimulate duodenal HC[O.sup.-.sub.3] secretion and [I.sub.sc] in CFTR knockout mice. Moreover, forskolin- and 8-Br-cGMP-stimulated duodenal HC[O.sup.-.sub.3] secretion and [I.sub.sc] were significantly reduced by wortmannin and LY294002, two selective PI3K inhibitors that are structurally and mechanistically different. Forskolin and 8-Br-cGMP induced CFTR phosphorylation and shifted CFTR proteins to the plasma membrane of duodenal epithelial cells, which were inhibited by wortmannin and LY294002. Forskolin and 8-Br-cGMP not only increased the activity of PI3K but also induced the phosphorylation of Akt, a signaling molecule downstream of PI3K, which were again inhibited by wortmannin and LY294002. Together, our results obtained from functional, biochemical, and morphological studies demonstrate that PI3K pathway plays an important role in the regulation of cAMP- and cGMP-induced duodenal epithelial CFTR channel activity and intracellular trafficking. cystic fibrosis transmembrane conductance regulator; phosphatidylinositol 3-kinase; duodenal epithelial cell; adenosine 3',5'-cyclic monophosphate; guanosine 3',5'-cyclic monophosphate

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

Autophagy is a cellular process of 'self-eating.' During autophagy, a portion of cytoplasm is enveloped in double membrane-bound structures called autophagosomes, which undergo maturation and fusion with lysosomes for degradation. At the core of the molecular machinery of autophagy is a specific family of genes or proteins called Atg. Originally identified in yeast, Atg orthologs are now being discovered in mammalian cells and have been shown to play critical roles in autophagy. Traditionally, autophagy is recognized as a cellular response to nutrient deprivation or starvation whereby cells digest cytoplasmic organelles and macromolecules to recycle nutrients for self-support. However, studies during the last few years have indicated that autophagy is a general cellular response to stress. Interestingly, depending on experimental conditions, especially stress levels, autophagy can directly induce cell death or act as a mechanism of cell survival. In this review, we discuss the molecular machinery, regulation, and function of autophagy. In addition, we analyze the recent findings of autophagy in renal systems and its possible role in renal pathophysiology. apoptosis; Atg; cytoprotection; mammalian target of rapamycin; phosphatidylinositol 3-kinase

Endothelial activation and surface expression of cell adhesion molecules (CAMs) is critical for binding and recruitment of circulating leukocytes in tissues during the inflammatory response. Endothelial CAM expression plays a critical role in the intestinal microvasculature in inflammatory bowel disease (IBD), as blockade of leukocyte [[alpha].sub.4]-integrin binding by gut endothelial CAM ligands has therapeutic benefit in IBD. Mechanisms underlying expression of vascular cell adhesion molecule (VCAM)- 1, a ligand for [[alpha].sub.4]-integrin in primary cultures of human intestinal microvascular endothelial cells (HIMEC) has not been defined. We investigated the effect of curcumin, phosphatidylinositol 3-kinase (PI 3-kinase)/prorein kinase B (Akt), and mitogen-activated protein kinase (MAPK) inhibitors on VCAM-1 expression and function in HIMEC. CAM expression was assessed and HIMEC-leukocyte adhesion was visualized under static and flow conditions. Western blotting and in vitro kinase assays were used to assess Akt and MAPK activation. Nuclear factor-[kappa]B (NF-[kappa]B) activation and nuclear translocation of its p65 subunit were determined. Tumor necrosis factor (TNF)-[alpha]/lipopolysaccharide (LPS)-induced VCAM-1 expression in HIMEC was suppressed by Akt small-interfering RNA, curcumin, and inhibitors of NF-[kappa]B (SN-50), p38 MAPK (SB-203580) and PI 3-kinase/Akt (LY-294002). VCAM-1 induction was partially suppressed by p44/42 MAPK (PD-098059) but unaffected by c-Jun N[H.sub.2]-terminal kinase (SP-600125) inhibition. Curcumin inhibited Akt/MAPK/NF-[kappa]B activity and prevented nuclear translocation of the p65 NF-[kappa]B subunit following TNF-[alpha]./LPS. At physiological shear stress, curcumin attenuated leukocyte adhesion to TNF-[alpha]/LPS-activated HIMEC monolayers. In conclusion, curcumin inhibited the expression of VCAM-1 in HIMECs through blockade of Akt, p38 MAPK, and NF-[kappa]B. Curcumin may represent a novel therapeutic agent targeting endothelial activation in IBD. phosphatidylinositol 3-kinase; protein kinase B; mitogen-activated protein kinase; nuclear factor-[kappa]B

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 activation of Kv 1.3 potassium channel has obligatory roles in immune responses of T lymphocytes. Stromal cell-derived factor-1[alpha] (SDF-1[alpha] binds to C-X-C chemokine receptor type 4, activates phosphoinositide 3-kinase, and plays essential roles in cell migration of T lymphocytes. In this study, the effects of phosphoinositides and SDF-1[alpha] on Kvl.3 current activity were examined in the Jurkat T cell line using whole cell patch-clamp techniques. The internal application of 10 p,M phosphatidylinositol 4,5-bisphosphate ([PIP.sub.2]) or 10 [micro]M phosphatidylinositol-3,4,5-trisphosphate ([PIP.sub.3]) significantly reduced Kv1.3 current, but that of 10 [micro]M phosphatidylinositol-4-monophosphate (PIP) did not. The coapplication of 10 [micro]g/ml anti-[PIP.sub.3] antibody with [PIP.sub.2] from the pipette did not change the reduction of Kvl.3 current by PI[P.sub.2], but the coapplication of the antibody with [PIP.sub.3] eliminated the reduction. The heat-inactivated anti-[PIP.sub.3] antibody had no effect on [PIP.sub.3]-induced inhibition. These results suggest that [PIP.sub.2] per se can reduce Kvl.3 current as well as [PIP.sub.3] External application of 1 [micro]M Akt-kinase inhibitor VIII did not reverse the effect of intracellular [PIP.sub.3] External application of 10 and 30 ng/ml SDF-l[alpha] significantly reduced Kvl.3 current. Internal application of anti-[PIP.sub.3] antibody reversed the SDF-1[alpha]-induced reduction. These results suggest that, in Jurkat T cells, [PIP.sub.2], [PIP.sub.3], and SDF-1[alpha] reduce Kv1.3 channel activity and that the reduction by SDF-1[alpha] may be mediated by the enhancement of [PIP.sub.3] production. These novel inhibitory effects of phosphoinositides and SDF-1[alpha] on Kv1.3 current may have a significant function as a downregulation mechanism of Kv1.3 activity for the maintenance of T lymphocyte activation in immune responses. potassium channel; T lymphocytes

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

In the intestinal epithelium, activation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT pathways, via growth factor-mediated signaling, has been shown to regulate cell proliferation and inhibit apoptosis. An immune-activated receptor critical for Th2 immune responses, IL-4R[alpha] can also activate PI3-kinase via insulin receptor substrate (IRS)-dependent signaling. Here, using the intestinal goblet cell-specific gene RELM[beta], we investigated the effect of PI3-kinase activation via Th2 immune responses on the goblet cell phenotype. IL-13 stimulation activated PI3-kinase and AKT signal transduction in LS 174T cells. Not only did pharmacological inhibition of PI3-kinase and AKT 1/2 inhibit RELM[beta] induction by IL-13, but AKT inhibition also significantly reduced constitutive basal expression of RELM[beta], a response reproduced by the simultaneous pharmacological inhibition of both epidermal growth factor receptor and IGF-I receptor signaling. In vivo, the disruption of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), an inhibitor of PI3-kinase activation, led to the activation of RELM[beta] expression in the small intestine. Furthermore, induction of an intestinal Th2 immune response by infection with a small intestinal nematode parasite, Heligmosomoides polygyrus, led to enhanced epithelial cell proliferation, activation of AKT as demonstrated by the loss of Foxol nuclear localization, and robust induction of RELM[3 expression in wild-type, but not IL-4R[alpha] knockout, mice. These results demonstrate that Th2 immune responses can regulate goblet cell responses by activation of PI3-kinase and AKT pathways via IL-4R[alpha]. phosphatidylinositol 3-kinase; epithelium; intestine

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2008.07.002 Byline: Odile Lecompte (1)(3)(4)(5)(6), Olivier Poch (1)(3)(4)(5)(6), Jocelyn Laporte (2)(3)(4)(5)(6)(7) Abstract: Phosphoinositides are lipid second messengers that are essential for many cellular processes, including signal transduction and cell compartmentalization. Among them, phosphatidylinositol 5-phosphate (PtdIns5P) is the least characterized, although several proteins involved in its regulation are implicated in human diseases. We studied the distribution of 32 PtdIns5P-metabolizing proteins in 39 eukaryotic genomes. Phylogenetic profiles identify four groups of co-evolving proteins, confirming known protein complexes and revealing new ones. The complexes comprise a phosphatase, a kinase and a regulator; this indicates that physical interactions between the three partners are necessary for the acute spatial regulation of PtdIns5P turnover. By examining PtdIns5P metabolism in this new perspective, we propose a role for PtdIns5P in membrane trafficking from late endosomal compartments to the plasma membrane. Author Affiliation: (1) Department of Structural Biology and Genomics, rue Laurent Fries, Illkirch, F-67400 France (2) Department of Neurobiology and Genetics, rue Laurent Fries, Illkirch, F-67400 France (3) Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGMBC), rue Laurent Fries, Illkirch, F-67400 France (4) Institut National de la Sante et de la Recherche Medicale (Inserm), U596, Illkirch, rue Laurent Fries, F-67400 France (5) Centre National de la Recherche Scientifique (CNRS), UMR7104, rue Laurent Fries, Illkirch, F-67400 France (6) Universite Louis Pasteur, Strasbourg, F-67000 France (7) College de France, chaire de genetique humaine, rue Laurent Fries, Illkirch, F-67400 France

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

We investigated the effect the loss of the CAT-2 gene (CAT-2-/-) has on lung resistance ([R.sub.L]) and tracheal isometric tension. The [R.sub.L] of CAT-2-/- mice at a maximal dose of acetylcholine (ACh) was decreased by 33.66% (P = 0.05, n = 8) compared with that of C57BL/6 (B6) mice. The isometric tension of tracheal rings from CAT-2-/- mice showed a significant decrease in carbachol (CCh)-induced force generation (33.01%, P < 0.05, n = 8) compared with controls. The isoproterenol- or the sodium nitroprusside-induced relaxation was not affected in tracheal rings from CAT-2-/- mice. The activity of iNOS and arginase in lung tissue lysates of CAT-2-/- mice was indistinguishable from that of B6 mice. Furthermore, the expression of phospholipase-C[beta] (PLC-[beta]) and phosphatidylinositol-(4)-phosphate-5-kinase-[gamma] (PIP-5K-[gamma]) was examined in the lung tissue of CAT-2-/- and B6 mice. The expression of PIP-5K-[gamma] but not PLC-[beta] was significantly reduced in CAT-2-/- compared with B6 mice. The reduced airway smooth muscle (ASM) contractility to CCh seen in the CAT-2-/- tracheal rings was completely reversed by pretreating the rings with 100 [micro]M spermine. This increase in the CAT-2-/- tracheal ring contraction upon spermine pretreatment correlated with a recovery of the expression of PIP-5K-[gamma]. Our data indicates that CAT-2 exerts control over ASM force development through a spermine-dependent pathway that directly correlates with the expression level of PIP-5K-[gamma] in the lung. lung resistance; tracheal isometric tension

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

Phosphatidylinositol 3-kinase (PI3K) is a pivotal enzyme involved in the control of a variety of diverse metabolic functions. Glucocorticoids have been shown to attenuate PI3K signaling in some nonrenal cell types, raising the possibility that some physiological effects of glucocorticoids in renal cells may be achieved by a similar mechanism. Therefore, we tested whether glucocorticoids affect signaling through the insulin receptor substrate (IRS)-1/PI3K/Akt signaling cascade in LLC-PK1-GR101 renal epithelial cells. Treatment of cells with dexamethasone for 24 h: 1) suppressed IRS-1-associated PI3K activity and Akt phosphorylation, 2) increased the level of the PI3K p85 regulatory subunit but not the p110 catalytic subunit, and 3) induced the phosphorylation of IRS-1 on inhibitory [Ser.sup.307]. We have previously reported that glucocorticoids increase branched-chain ketoacid dehydrogenase (BCKD) activity in LLC-PK1-GR101 cells. This response was achieved, in part, by alterations in the transcription of BCKD subunits and BCKD kinase, which inactivates the enzyme complex by phosphorylation. Therefore, we tested whether inhibition of PI3K signaling would mimick glucocorticoids by increasing branched-chain amino acid degradation. Expression of a dominant negative PI3K p85 regulatory subunit (Adp85[DELTA]iSH2) increased BCKD activity, and dexamethasone did not further stimulate enzyme activity. Inhibition of PI3K using LY-294002 increased the transcription of the BCKD E2 subunit but not the E1[alpha] subunit or BCKD kinase. Thus, glucocorticoids inhibit signaling through the IRS-1/PI3K/Akt pathway with a consequence of increased branched-chain amino acid catabolism. phosphatidylinositol 3-kinase; kidney doi:10.1152/ajpcell.00617.2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2005.11.013 Byline: Robert H. Michell (a), Victoria L. Heath (a), Mark A. Lemmon (b), Stephen K. Dove (a) Abstract: Polyphosphoinositides (PPIn) are low-abundance membrane phospholipids that each bind to a distinctive set of effector proteins and, thereby, regulate a characteristic suite of cellular processes. Major functions of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P.sub.2] are in membrane and protein trafficking, and in pH control in the endosome-lysosome axis. Recently identified PtdIns(3,5)P.sub.2 effectors include a family of novel [beta]-propeller proteins, for which we propose the name PROPPINs [for [beta]-propeller(s) that binds PPIn], and possibly proteins of the epsin and CHMP (charged multi-vesicular body proteins) families. All eukaryotes, with the exception of some pathogenic protists and microsporidians, possess proteins needed for the formation, metabolism and functions of PtdIns(3,5)P.sub.2. The importance of PtdIns(3,5)P.sub.2 for normal cell function is underscored by recent evidence for its involvement in mammalian cell responses to insulin and for PtdIns(3,5)P.sub.2 dysfunction in the human genetic conditions X-linked myotubular myopathy, Type-4B Charcot-Marie-Tooth disease and fleck corneal dystrophy. Author Affiliation: (a) School of Biosciences, University of Birmingham, Birmingham, UK, B15 2TT (b) Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2005.11.003 Byline: Shamshad Cockcroft Abstract: Members of the phosphoinositide-specific phospholipase C (PLC) family have key roles in cell signalling. In response to many extracellular stimuli, such as hormones, neurotransmitters, antigens and growth factors, PLCs catalyse the hydrolysis of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P.sub.2], thereby generating two well-established second messengers, inositol (1,4,5)-trisphosphate and diacylglycerol. Eleven PLC isozymes encoded by different genes have been identified in mammals and, on the basis of their structure and sequence relationships, have been classified into five families designated PLC[beta] (1-4), PLC[gamma] (1 and 2), PLC[delta] (1, 3 and 4), PLCI[micro] (1) and PLCI[paragraph] (1). All PLCs contain the catalytic X and Y domain, in addition to other regulatory domains including the C2 domain and the EF-hand domain. In 2005, four groups independently identified an entirely new family of PLCs - I*1 and I*2 - using data mining of mammalian genomes. The properties of the PLCI* enzyme suggest that it might act as a Ca.sup.2+ sensor, in particular, functioning during formation and maintenance of the neuronal network in the postnatal brain. Author Affiliation: Department of Physiology, University College London, London WC1E 6JJ, UK

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tips.2005.11.008 Byline: Indu S. Ambudkar Abstract: The transient receptor potential canonical family (TRPC1-TRPC7) of ion channel proteins, which are activated in response to agonist-stimulated phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P.sub.2] hydrolysis, are proposed components of the elusive store-operated Ca.sup.2+ (SOC) channel. TRPC channels display distinct properties and interact to form homomeric or heteromeric channels that differ in their function and regulation. Although the exact function of TRPC channels and how they are regulated has not been established, increasing data suggest that they are localized and regulated within Ca.sup.2+ signaling microdomains. TRPC channels contribute to store-operated and store-independent Ca.sup.2+ entry mechanisms, both of which are activated by agonist-stimulated PtdIns(4,5)P.sub.2 hydrolysis. Elucidation of how cells achieve specificity and precise temporal and spatial coordination of channel activation is crucial for understanding the molecular basis of agonist-mediated stimulation of Ca.sup.2+ entry and identifying downstream physiological functions. This review will address the assembly and localization of TRPC channels and how these processes impact their function. Author Affiliation: Secretory Physiology Section, GTTB, NIDCR, NIH, Bethesda, MD 20892, USA

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

The aim of this study was to determine whether the phosphatidylinositol 3-kinase (PI3K)-dependent mammalian target of rapamycin (mTOR)-eukaryotic initiation factor 4E binding protein 1 (4E-BPI) signal pathway and $6 kinase (S6K), the major element of the mTOR pathway, play a role in the enhanced vascular endothelial cell (EC) proliferation induced by cyclic strain. Bovine aortic ECs were subjected to an average of 10% strain at a rate of 60 cycles/min for [is less than or equal to] 24 h. Cyclic strain-induced EC proliferation was reduced by pretreatment with rapamycin but not the MEK 1 inhibitor PD-98059. The P13K inhibitors wortmannin and LY-294002 also attenuated strain-induced EC proliferation and strain-induced activation of S6K. Rapamycin but not PD-98059 prevented strain-induced S6K activation, and PD-98059 but not rapamycin prevented strain-induced activation of extracellular signal-regulated kinases 1 and 2. Cyclic strain also activated 4E-BP1, which could be inhibited by PI3K inhibitors. These data suggest that the P13K-dependent S6K-mTOR-4E-BP1 signal pathway may be critically involved in strain-induced bovine aortic EC proliferation. phosphatidylinositol 3-kinase; mammalian target of rapamycin; mitogen-activated protein: $6; signal transduction; eukaryotic initiation factor binding protein: Akt

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

Phosphatidylinositol 3-kinase (PI3K) is required for smooth muscle cell (SMC) proliferation. This study reports that inhibitors of PI3K also prevent SMC migration and block neointimal hyperplasia in an organ culture model of restenosis. Inhibition of neointimal formation by LY-294002 was concentration and time dependent, with 10 [micro]M yielding the maximal effect. Continuous exposure for at least the first 4-7 days of culture was essential for significant inhibition. To assess the role of matrix metalloproteinases (MMPs) in this process, we monitored MMP secretion by injured vessels in culture. Treatment with LY-294002 selectively reduced active MMP-2 in media samples according to zymography and Western blot analysis without concomitant changes in latent MMP-2. Parallel results with wortmannin indicate that MMP-2 activation is PI3K dependent. Previous research has shown a role for both furin and membrane-type 1 (MT1)-MMP (MMP-14) in the activation of MMP-2. The furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone did not prevent MMP-2 activation after balloon angioplasty. In contrast, balloon angioplasty induced a significant increase in the levels of MT1-MMP, which was suppressed by LY-294002. No change in MT1-MMP mRNA was observed with LY-294002, because equivalent amounts of this mRNA were present in both injured and noninjured vessels. These results implicate PI3K-dependent regulation of MT1-MMP protein synthesis and subsequent activation of latent MMP-2 as critical events in neointimal hyperplasia after vascular injury. matrix metalloproteinase; LY-294002: wortmannin; furin; restenosis

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.

Bile acids are cytoprotective in hepatocytes by activating phosphatidylinositol-3-kinase (PI3-K) and its downstream signal AKT. Our aim was to determine whether feeding taurocholate to C[Cl.sub.4]-treated rats reduces cholangiocyte apoptosis and whether this cytoprotective effect is dependent on PI3-K. Cholangiocyte proliferation, secretion, and apoptosis were determined in cholangiocytes from bile duct ligation (BDL), C[Cl.sub.4]-treated BDL rats, and C[Cl.sub.4]-treated taurocholate-fed rats. In vitro, we tested whether C[Cl.sub.4] induces apoptosis and whether loss of cholangiocyte proliferation and secretion is dependent on PI3-K. The C[Cl.sub.4]-induced cholangiocyte apoptosis and loss of cholangiocyte proliferation and secretion were reduced in C[Cl.sub.4]-treated rats fed taurocholate. C[Cl.sub.4]-induced cholangiocyte apoptosis, loss of cholangiocytes secretion, and proliferation were prevented by preincubation with taurocholate. Taurocholate cytoprotective effects were ablated by wortmannin. Taurocholate prevented, in vitro, C[Cl.sub.4]-induced decrease of phosphorylated AKT protein expression in cholangiocytes. The cytoprotective effects of taurocholate on C[Cl.sub.4] effects on cholangiocyte proliferation and secretion were abolished by wortmannin. Taurocholate protects cholangiocytes from C[Cl.sub.4]-induced apoptosis by a PI3-K-dependent mechanism. Bile acids are important in the prevention of drug-induced ductopenia in cholangiopathies. cytoprotection; biliary epithelium; bile acids

Hypoxic/ischemic conditions provoke activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 is composed of HIF-1[alpha] (subjected to protein stability regulation) and constitutively expressed HIF-1[beta]. Besides hypoxia, diverse agonists are identified that stabilize HIF-1[alpha] during normoxia. Here we used a coculture system of RAW 264.7 macrophage cells and tubular LLC-P[K.sub.1] cells to establish that lipopolysaccharide- and interferon-[gamma]-stimulated but not resting macrophages elicited HIF-1[alpha] accumulation in LLC-P[K.sub.1] cells. Via pharmacological interventions such as blockade of nitric oxide (NO) production in macrophages, scavenging of NO with the use of 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, or application of tumor necrosis factor-[alpha] (TNF-[alpha])-neutralizing antibodies, we identified NO and TNF-[alpha] as signaling molecules. Working in concert, NO and TNF-[alpha] have a stronger response when allowed direct cell-to-cell contact instead of contact with only the cell supernatant of activated macrophages. We show that signal transmission by NO with TNF-[alpha] in LLC-P[K.sub.1] cells is mediated via the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, because it is blocked by wortmannin or dominant-negative forms of PI3-K as well as protein kinase B. We conclude that NO and TNF-[alpha], derived from activated macrophages, provoke HIF-1[alpha] stabilization in LLC-P[K.sub.1] cells under normoxic conditions, which underscores HIF-1[alpha] stabilization due to intercellular regulation. nitric oxide; tumor necrosis factor-[alpha]; hypoxia-inducible factor-1; intercellular signaling; phosphatidylinositol 3-kinase; cytokine; Akt

Research has been conducted on the role of aromatic residues in ligand recognition. Results demonstrate that Src homology 3 domains recognize Pro-rich motifs via the use of hydrophobic cleft containing some aromatic residues, and the use of excited ultraviolet resonance Raman spectroscopies applied to Src and phosphatidylinositol 3-kinase in investigating the aromatic residue contribution to recognition of ligands is described.

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].

We have previously shown that PGE2 and PGI2 induce recovery of transepithelial resistance (TER) in ischemia-injured porcine ileal mucosa, associated with initial increases in [Cl.sup.-] secretion. We believe that the latter generates an osmotic gradient that stimulates resealing of tight junctions. Because of evidence implicating phosphatidylinositol 3-kinase (PI3K) in regulating tight junction assembly, we postulated that this signaling pathway is involved in PG-induced mucosal recovery. Porcine ileum was subjected to 45 min of ischemia, after which TER was monitored for a 180-min recovery period. Endogenous PG production was inhibited with indomethacin (5 [micro]M). PGE2 (1 [micro]M) and PG[I.sup.2] (1 [micro]M) stimulated recovery of TER, which was inhibited by serosal application of the osmotic agent urea (300 mosmol/kg[H.sub.2]O). The PI3K inhibitor wortmannin (10 nM) blocked recovery of TER in response to PGs or mucosal urea. Immunofluorescence imaging of recovering epithelium revealed that PGs restored occludin and zonula occludens-1 distribution to interepithelial junctions, and this pattern was disrupted by pretreatment with wortmannin. These experiments suggest that PGs stimulate recovery of paracellular resistance via a mechanism involving transepithelial osmotic gradients and PI3K-dependent restoration of tight junction protein distribution. phosphatidylinositol 3-kinase; tight junction; occludin; zonula occludens-1

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

Human vascular smooth muscle cell proliferation and migration contribute to vascular remodeling in pulmonary hypertension and atherosclerosis. The precise mechanisms that regulate structural remodeling of the vessel wall remain unknown. This study tests the hypothesis that phosphatidylinositol 3-kinase (PI3K) activation is both necessary and sufficient to mediate human pulmonary vascular smooth muscle (PVSM) cell proliferation and migration. Microinjection of human PVSM cells with a dominant-negative class IA PI3K inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis by 65% (P < 0.001; [chi square] analysis) compared with cells microinjected with control plasmid, whereas microinjection of cells with a constitutively active class IA PI3K (p110*-CA) was sufficient to induce DNA synthesis (mitotic index of p110*-CA-microinjected cells was 15% vs. 3% in control cells; P < 0.01). Transfection of PVSM cells with p110*-CA was also sufficient to promote human PVSM cell migration. In parallel experiments, stimulation of human PVSM cells with PDGF induced PI3K-dependent activation of Akt, p70 S6 kinase, and ribosomal protein S6 but not mitogen-activated protein kinase. PDGF-induced proliferation and migration was inhibited by LY-294002. These results demonstrate that PI3K signaling is both necessary and sufficient to mediate human PVSM cell proliferation and migration and suggest that the activation of PI3K may play an important role in vascular remodeling. phosphatidylinositol 3-kinase; hypertension; Akt; S6K1; ribosomal protein S6