SEARCH · PubMed Health

Results for “PHOSPHOINOSITIDES”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 937 records · Page 52Linked to original sources

PubMed1999

New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway.

The most recently discovered PTEN tumor suppressor gene has been found to be defective in a large number of human cancers. In addition, germ-line mutations in PTEN result in the dominantly inherited disease Cowden syndrome, which is characterized by multiple hamartomas and a high proclivity for developing cancer. A series of publications over the past year now suggest a mechanism by which PTEN loss of function results in tumors. PTEN appears to negatively control the phosphoinositide 3-kinase signaling pathway for regulation of cell growth and survival by dephosphorylating the 3 position of phosphoinositides.

Cell Division↗

PubMed1995

Wortmannin, an inhibitor of phosphoinositide 3-kinase, inhibits transcytosis in polarized epithelial cells.

Wortmannin, an inhibitor of phosphoinositide 3-kinase, inhibits both basolateral to apical and apical to basolateral transcytosis of ricin in Fisher rat thyroid (FRT) cells by 50% at 100 nM in a continuous transcytosis assay. In MDCK cells, a similar effect of wortmannin on basolateral to apical transcytosis of ricin was found, whereas apical to basolateral transcytosis was inhibited to a lesser degree. Transcytosis of dimeric IgA in MDCK cells expressing the polymeric immunoglobulin receptor was also reduced to 50% of controls, suggesting that wortmannin inhibits membrane translocation rather than sorting of specific proteins in the transcytotic pathway. This effect of wortmannin is selective, however, in that endocytosis at the basolateral domain and recycling at both the basolateral and apical membrane domains are unaffected, and apical endocytosis and apical secretion are only moderately reduced. We have shown previously that cAMP stimulates a late stage in basolateral to apical transcytosis in MDCK cells through activation of protein kinase A (Hansen, S. H., and Casanova, J.E. (1994) J. Cell Biol. 126, 677-687). Elevation of cellular cAMP still induced a 100% increase in transcytosis in wortmannin-treated cells, but transcytosis was no longer increased when compared to cells which received no drugs. In contrast, in experiments using a 17 degrees C block to accumulate ricin internalized from the basolateral surface in the apical compartment of MDCK cells, wortmannin had little effect on the stimulation of transcytosis by activators of protein kinase A observed under these conditions. The data thus suggest the existence of a wortmannin-sensitive step in the transcytotic pathway, positioned after endocytosis but prior to translocation into the protein kinase A-sensitive apical compartment, implying a role for phosphoinositide 3-kinase in an intermediate step in transcytosis in polarized epithelial cells.

Androstadienes↗

PubMed1996

Phosphoinositide 3-kinase gamma and p85/phosphoinositide 3-kinase in platelets. Relative activation by thrombin receptor or beta-phorbol myristate acetate and roles in promoting the ligand-binding function of alphaIIbbeta3 integrin.

Platelets exposed to thrombin or thrombin receptor agonist peptide (SFLLRN) activate phospholipase C and protein kinase C (PKC), and accumulate 3-phosphorylated phosphoinositides (3-PPI) as a function of the activation and relocalization of two cytoskeletally-associated phosphoinositide 3-kinases (PI 3-K): p85/PI 3-K and PI 3-Kgamma. We now report that exposure of platelets to PKC-activating beta-phorbol myristate acetate (betaPMA) does not stimulate PI 3-Kgamma, but rather stimulates p85/PI 3-K, which associates with the cytoskeleton. Wortmannin is an inhibitor of both PI 3-Ks, known to act with more potency on p85/PI 3-K. betaPMA-stimulated 3-PPI accumulation is more sensitive to wortmannin (IC50 = 1.3 nM) than is SFLLRN- or thrombin-stimulated 3-PPI accumulation (IC50 = 10 nM). The activity of p85/PI 3-K in immunoprecipitates or in cytoskeletal fractions is inhibited more potently by exposure of platelets to wortmannin than is the activity of PI 3-Kgamma. betaPMA or SFLLRN promotes the conversion of platelet integrin alphaIIb/beta3 into a fibrinogen-binding form required for platelet aggregation. Activation of alphaIIb/beta3 in response to betaPMA or SFLLRN is inhibited by wortmannin with an IC50 of 1 nM in each case. Wortmannin inhibits neither activation of alphaIIb/beta3 by ligand-induced binding site antibody (anti-LIBS6 Fab) nor anti-LIBS6 Fab-induced platelet aggregation in the presence of fibrinogen, indicating that this type of "outside-in" signaling by alphaIIb/beta3 is largely PI 3-K-independent. We conclude that p85/PI 3-K, in preference to PI 3-Kgamma, contributes to activation of alphaIIb/beta3 when the thrombin receptor or PKC is stimulated.

Amino Acid Sequence↗

PubMed1996

Binding to the platelet-derived growth factor receptor transiently activates the p85alpha-p110alpha phosphoinositide 3-kinase complex in vivo.

Ligand stimulation of the platelet-derived growth factor (PDGF) receptor results in its association with phosphoinositide 3-kinase activity and a corresponding synthesis of 3'-phosphorylated lipids. Early studies that examined this interaction in vivo employed anti-phosphotyrosine antiserum or antiserum against the PDGF receptor. The recent identification of multiple isoforms of both the regulatory and the catalytic subunit of the enzyme have led us to utilize antisera against p85alpha and p110alpha to characterize the association of this particular phosphoinositide 3-kinase complex with the PDGF receptor following ligand stimulation of murine fibroblasts. Both the p85alpha and p110alpha subunits rapidly associated with the ligand-activated receptor resulting in a transient, 2-fold increase in the total pool of p110alpha lipid kinase activity. This association was stable for 15 min after initial stimulation. Subsequently, both subunits began to dissociate from the receptor with similar kinetics. By 60 min this process was complete, demonstrating that p85alpha and p110alpha both associate with the receptor and dissociate from the receptor as a dimeric complex. At this time, marked PDGF receptor down-regulation was observed. Immunoprecipitation from metabolically labeled cells revealed that p85alpha is constitutively phosphorylated on serine residues in quiescent cultures. Upon PDGF stimulation, this phosphorylation upon serine residues was maintained in addition to tyrosine phosphorylation of this subunit. No phosphorylation of the p110alpha subunit was detected in either quiescent or PDGF-stimulated cells. Quantitation of Western blot analysis demonstrated that only 5% of the total pool of p85alpha associated with the PDGF receptor upon ligand stimulation. The 2-fold increase in the lipid kinase activity measured in immunoprecipitates using either anti-p85alpha or anti-p110alpha antiserum therefore reflects a far greater increase in the specific activity of the enzyme upon its association with the PDGF receptor.

3T3 Cells↗

PubMed1996

The third intracellular domain of the platelet-activating factor receptor is a critical determinant in receptor coupling to phosphoinositide phospholipase C-activating G proteins. Studies using intracellular domain minigenes and receptor chimeras.

Platelet activating factor (PAF) is a potent phospholipid mediator which elicits a diverse array of biological actions by interacting with G protein-coupled PAF receptors (PAFR). Binding of PAF to PAFRs leads to activation of G protein(s) that stimulate phosphoinositide phospholipase C and subsequent intracellular signaling responses. To identify the potential role of intracellular domains of the rat PAFR (rPAFR) in signaling, we examined effects of transfecting minigenes encompassing rPAFR intracellular domains 1 (1i), 2 (2i), and 3 (3i) on inositol phosphate (IP) production mediated by the co-transfected rPAFR cDNA. Although transfection of the rPAFR1i and rPAFR2i minigenes had no effects on PAF-stimulated signaling, transfection of the rPAFR3i minigene inhibited PAF-stimulated IP production by approximately 50% compared to controls. The rPAFR3i domain did not inhibit IP production mediated by the multifunctional rat pituitary adenylate cyclase-activating polypeptide receptor (rPACAPR), demonstrating the specificity of the competition by the rPAFR3i domain. In further experiments, the rPAFR3i domain was engineered onto the homologous domain of a monofunctional transmembrane variant of the rPACAPR (rPACAPR2) that activates only adenylyl cyclase. The rPACAPR2/rPAFR3i chimera responded to PACAP with increases in IP production which were attenuated nearly completely in cells cotransfected with the rPAFR3i domain. In contrast, PACAP had no effects on IP production in a receptor chimera expressing a mutated form of the rPAFR3i domain (rPACAPR2/rPAFR3imut). These results demonstrate the ability of the rPAFR3i domain to confer a phospholipase C-signaling phenotype to a receptor deficient in this activity and show that this activity is specific for the engineered rPAFR3i domain. These results suggest that the third intracellular loop of the rPAFR is a primary determinant in its coupling to phosphoinositide phospholipase C-activating G proteins, providing the first insight into the molecular basis of interaction of PAFRs with signal-transducing G proteins.

Amino Acid Sequence↗

PubMed1996

D3 phosphoinositides and outside-in integrin signaling by glycoprotein IIb-IIIa mediate platelet actin assembly and filopodial extension induced by phorbol 12-myristate 13-acetate.

Phorbol 12-myristate 13-acetate (PMA) uncaps a small number of the fast-growing (barbed) ends of actin filaments, thereby eliciting slow actin assembly and extension of filopodia in human blood platelets. These reactions, which also occur in response to immunologic perturbation of the integrin glycoprotein (GP) IIb-IIIa, are sensitive to the phosphoinositide 3-kinase inhibitor wortmannin. Platelets deficient in GPIIb-IIIa integrins or with GPIIb-IIIa function inhibited by calcium chelation or the peptide RGDS have diminished PMA responsiveness. The effects of PMA contrast with thrombin receptor stimulation by >/=5 microM thrombin receptor-activating peptide (TRAP), which causes rapid and massive wortmannin-insensitive actin assembly and lamellar and filopodial extension. However, we show here that wortmannin can inhibit filopod formation if the thrombin receptor is ligated using suboptimal doses (<1 microM) of TRAP. Phosphatidylinositol 3,4-bisphosphate inhibits actin filament severing and capping by human gelsolin in vitro. The findings implicate D3 polyphosphoinositides and integrin signaling in PMA-mediated platelet stimulation and implicate D3 containing phosphoinositides generated in response to protein kinase C activation and GPIIb-IIIa signaling as late-acting intermediates leading to filopodial actin assembly.

Actin Cytoskeleton↗

PubMed1997

The lipid products of phosphoinositide 3-kinase increase cell motility through protein kinase C.

Phosphoinositide 3-kinase has been implicated as an activator of cell motility in a variety of recent studies, yet the role of its lipid product, phosphatidylinositol 1,4,5-trisphosphate (PtdIns-3,4,5-P3), has yet to be elucidated. In this study, three independent preparations of PtdIns-3,4,5-P3 were found to increase the motility of NIH 3T3 cells when examined utilizing a microchemotaxis chamber. Dipalmitoyl L-alpha-phosphatidyl-D-myo-inositol 3,4,5-triphosphate (Di-C16-PtdIns-3,4,5-P3) also produced actin reorganization and membrane ruffling. Cells pretreated with 12-O-tetradecanoylphorbol-13-acetate to cause down-regulation of protein kinase C (PKC) exhibited complete inhibition of cell motility induced by Di-C16-PtdIns-3,4,5-P3. These results are consistent with previous observations that PtdIns-3,4,5-P3 activates Ca2+-independent PKC isoforms in vitro and in vivo and provide the first demonstration of an in vivo role for the lipid products of the phosphoinositide 3-kinase. PtdIns-3,4,5-P3 appears to directly initiate cellular motility via activation of a PKC family member.

3T3 Cells↗

PubMed1998

A type II phosphoinositide 3-kinase is stimulated via activated integrin in platelets. A source of phosphatidylinositol 3-phosphate.

We have observed that aggregation of human platelets, caused by activation of integrin alphaIIb beta3 and its consequent binding of fibrinogen, stimulates a novel pathway for synthesis of phosphatidylinositol 3,4bisphosphate, thereby activating protein kinase B/Akt. Such synthesis depends upon both the generation of phosphatidylinositol 3-phosphate (PtdIns3P), which is sensitive to wortmannin (IC50 7 nM) and calpain inhibitors, and the phosphorylation of PtdIns3P by PtdIns3P 4-kinase. We now report that a recently characterized C2 domain-containing phosphoinositide 3-kinase isoform (HsC2-PI3K) is present in platelets and a leukemic cell line (CHRF-288) derived from megakaryoblasts, and is likely to be responsible for the stimulated synthesis of PtdIns3P observed in platelets. HsC2-PI3K, identifiable by Western blotting and immunoprecipitatable activity, is sensitive to wortmannin (IC50 6-10 nM), requires Mg2+, and shows strong preference for PtdIns over PtdIns4P or phosphatidylinositol 4,5-bisphosphate as substrate. HsC2-PI3K is activated severalfold when platelets aggregate in an alphaIIb beta3-dependent manner or when platelet or CHRF-288 lysates are incubated with Ca2+. Activation is prevented by calpain inhibitors. CHRF-288, which cannot undergo activation of alphaIIb beta3 and thereby aggregate in response to platelet agonists, do not generate PtdIns3P or activate HsC2-PI3K under conditions that stimulate other phosphoinositide 3-kinases. HsC2-PI3K may thus be an important effector for integrin-dependent signaling.

Androstadienes↗

PubMed1998

Ras isoforms vary in their ability to activate Raf-1 and phosphoinositide 3-kinase.

Ha-, N-, and Ki-Ras are ubiquitously expressed in mammalian cells and can all interact with the same set of effector proteins. We show here, however, that in vivo there are marked quantitative differences in the ability of Ki- and Ha-Ras to activate Raf-1 and phosphoinositide 3-kinase. Thus, Ki-Ras both recruits Raf-1 to the plasma membrane more efficiently than Ha-Ras and is a more potent activator of membrane-recruited Raf-1 than Ha-Ras. In contrast, Ha-Ras is a more potent activator of phosphoinositide 3-kinase than Ki-Ras. Interestingly, the ability of Ha-Ras to recruit Raf-1 to the plasma membrane is significantly increased when the Ha-Ras hypervariable region is shortened so that the spacing of the Ha-Ras GTPase domains from the inner surface of the plasma membrane mimicks that of Ki-Ras. Importantly, these data show for the first time that the activation of different Ras isoforms can have distinct biochemical consequences for the cell. The mutation of specific Ras isoforms in different human tumors can, therefore, also be rationalized.

Amino Acid Sequence↗

PubMed1998

Chimeric receptors composed of phosphoinositide 3-kinase domains and FCgamma receptor ligand-binding domains mediate phagocytosis in COS fibroblasts.

Receptors for the Fc portion of IgG (FcgammaR) initiate phagocytosis of IgG-opsonized particles by a process involving the assembly of a multi-molecular signaling complex. Several members of this complex have been identified, including Src family kinases, Syk/ZAP 70 family kinases, and phosphoinositide 3-kinase (PI3-K). To test directly the role of PI3-K in mediating phagocytosis, we assessed the phagocytic ability of chimeric receptors composed of FcgammaR extracellular and transmembrane domains fused to regions of the p85 subunit of PI3-K. We found that chimeric receptors with cytoplasmic tails composed of the entire p85 subunit of PI3-K or the inter-Src homology 2 portion of p85 triggered phagocytosis in transfected COS fibroblasts. These two chimeras also showed phosphoinositide kinase activity in vitro when immunoadsorbed. In contrast, a chimera containing only the carboxyl-terminal Src homology 2 domain of p85 that does not interact with the catalytic p110 subunit of PI3-K did not trigger phagocytosis, nor did it show kinase activity in vitro. These data suggest that localization and direct activation of PI3-K at the site of particle attachment is sufficient to trigger the process of phagocytosis.

Animals↗

PubMed1998

Lipid products of phosphoinositide 3-kinase interact with Rac1 GTPase and stimulate GDP dissociation.

A number of reports suggest that under different conditions leading to cytoskeleton reorganization the GTPase Rac1 and possibly RhoA are downstream targets of phosphoinositide 3-kinase (PI 3-kinase). In order to gain more insight into this particular signaling pathway, we have addressed the question of a possible direct interaction of PI 3-kinase products with the Rho family GTPases RhoA, Rac1, and Cdc42. Using recombinant proteins, we found that Rac1 and, to a lesser extent, RhoA but not Cdc42 were capable to selectively bind to phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in a mixture of crude brain phosphoinositides. Nucleotide-depleted Rac1 was the most efficient, but the GDP- and GTP-bound forms retained significant PtdIns(3,4,5)P3 binding activity. This protein-lipid association involved electrostatic as well as hydrophobic interactions, since both phosphate groups located at specific positions of the inositol ring and fatty-acyl chains were absolutely required. Based on the sequence of Rac1, two potential binding sites were identified, one at the C terminus and one in the extra alpha-helical domain. Deletion of these two domains resulted in a complete loss of binding to PI 3-kinase products. Finally, PtdIns(3, 4,5)P3 strongly stimulated GDP dissociation from Rac1 in a dose-dependent manner. In agreement, data obtained in intact cells suggest that PtdIns(3,4,5)P3 might target Rac1 to peculiar membrane domains, allowing formation of specific clusters containing not only small GTPases but other partners bearing pleckstrin homology domains such as specific exchange factors required for Rac1 and RhoA activation.

Androstadienes↗

PubMed1999

Identification of multiple phosphoinositide-specific phospholipases D as new regulatory enzymes for phosphatidylinositol 3,4, 5-trisphosphate.

In the course of delineating the regulatory mechanism underlying phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) metabolism, we have discovered three distinct phosphoinositide-specific phospholipase D (PI-PLD) isozymes from rat brain, tentatively designated as PI-PLDa, PI-PLDb, and PI-PLDc. These enzymes convert [3H]PI(3,4,5)P3 to generate a novel inositol phosphate, D-myo-[3H]inositol 3,4,5-trisphosphate ([3H]Ins(3,4,5)P3) and phosphatidic acid. These isozymes are predominantly associated with the cytosol, a notable difference from phosphatidylcholine PLDs. They are partially purified by a three-step procedure consisting of DEAE, heparin, and Sephacryl S-200 chromatography. PI-PLDa and PI-PLDb display a high degree of substrate specificity for PI(3,4, 5)P3, with a relative potency of PI(3,4,5)P3 >> phosphatidylinositol 3-phosphate (PI(3)P) or phosphatidylinositol 4-phosphate (PI(4)P) > phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) > phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). In contrast, PI-PLDc preferentially utilizes PI(3)P as substrate, followed by, in sequence, PI(3,4,5)P3, PI(4)P, PI(3,4)P2, and PI(4,5)P2. Both PI(3, 4)P2 and PI(4,5)P2 are poor substrates for all three isozymes, indicating that the regulatory mechanisms underlying these phosphoinositides are different from that of PI(3,4,5)P3. None of these enzymes reacts with phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine. All three PI-PLDs are Ca2+-dependent. Among them, PI-PLDb and PI-PLDc show maximum activities within a sub-microM range (0.3 and 0.9 microM Ca2+, respectively), whereas PI-PLDa exhibits an optimal [Ca2+] at 20 microM. In contrast to PC-PLD, Mg2+ has no significant effect on the enzyme activity. All three enzymes require sodium deoxycholate for optimal activities; other detergents examined including Triton X-100 and Nonidet P-40 are, however, inhibitory. In addition, PI(4,5)P2 stimulates these isozymes in a dose-dependent manner. Enhancement in the enzyme activity is noted only when the molar ratio of PI(4,5)P2 to PI(3,4, 5)P3 is between 1:1 and 2:1.

Animals↗

PubMed1999

Involvement of PITPnm, a mammalian homologue of Drosophila rdgB, in phosphoinositide synthesis on Golgi membranes.

Phosphatidylinositol transfer protein (PITP) is involved in phospholipase C-mediated signaling and membrane trafficking. We previously reported cloning and characterization of a gene encoding for membrane-bound PITP, named PITPnm, that is a mammalian homologue of the Drosophila retinal degeneration B (rdgB) gene (Aikawa, Y., Hara, H., and Watanabe, T. (1997) Biochem. Biophys. Res. Commun. 236, 559-564). Here we report the subcellular localization of PITPnm protein and provide evidence for its involvement in phosphatidylinositol 4-phosphate (PtdIns 4-P) synthesis. PITPnm is an integral membrane protein that largely localized in close association with membranes of Golgi vacuoles and the endoplasmic reticulum (ER). The amino terminus region of PITPnm was exposed to cytoplasmic side. Interaction with various phosphoinositides was observed in the amino terminus region spanning from 196 amino acids to 257 amino acids of PITPnm. At the amino terminus regions of 1-372 amino acids, PITPnm formed a complex with type III PtdIns 4-kinase. The transmembrane and carboxyl-terminal portions (residues 418-1242) functioned to retain the PITPnm in the Golgi vacuole. These results suggest that PITPnm plays a role in phosphoinositide synthesis on the Golgi vacuoles and possibly in the PtdIns signaling pathway in mammalian cells.

1-Phosphatidylinositol 4-Kinase↗

PubMed1999

SYK is upstream of phosphoinositide 3-kinase in B cell receptor signaling.

We have recently demonstrated that the D3-phosphoinositide phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P(3)) is critical for producing sustained calcium signals through its role in promoting the function of TEC family tyrosine kinases such as Bruton's tyrosine kinase. Although PtdIns-3,4,5-P(3) can potentially be synthesized by any of several types of phosphoinositide 3-kinases (PI3Ks), B cell receptor (BCR)-induced PtdIns-3,4,5-P(3) production is thought to occur primarily through the activation of the class Ia (p85/p110) PI3Ks. This process has been proposed to be mediated by an interaction between the Src family kinase LYN and the p85 subunit of PI3K and/or through p85 membrane recruitment mediated by CBL and/or CD19. However, calcium signaling and other PI3K-dependent signals are relatively preserved in a LYN kinase-deficient B lymphocyte cell line, suggesting that an alternative pathway for PI3K activation exists. As SYK/ZAP70 kinases are upstream from many BCR-initiated signaling events, we directly analyzed SYK-dependent accumulation of both PtdIns-3,4,5-P(3) and PtdIns-3,4-P(2) in B cell receptor signaling using both dominant negative and genetic knockout approaches. Both methods indicate that SYK is upstream of, and necessary for, a significant portion of BCR-induced PtdIns-3,4, 5-P(3) production. Whereas CD19 does not appear to be involved in this SYK-dependent pathway, the SYK substrate CBL is likely involved as the dominant negative SYK markedly attenuates CBL tyrosine phosphorylation and completely blocks the BCR-dependent association of CBL with p85 PI3K.

Animals↗

PubMed1999

Actin and phosphoinositide binding by the ActA protein of the bacterial pathogen Listeria monocytogenes.

The surface protein ActA of the pathogenic bacterium Listeria monocytogenes induces actin-driven movement of bacteria in the cytoplasm of infected host cells and serves as a model for actin-based motility in general. We generated and purified soluble recombinant fragments of ActA and assessed their ability to interact with the acidic phospholipids phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, both implicated in the regulation of actin polymerization. Purified ActA consisted of biologically active, elongated molecules with an alpha-helix and beta-sheet content of 11 and 32%, respectively. In the presence of either phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate, but not phosphatidylcholine, ActA molecules underwent a structural change that raised the alpha-helix content to 19% and lowered the beta-sheet content to 27%. Co-sedimentation experiments with phosphatidylcholine vesicles containing different acidic phospholipids demonstrated that ActA binds preferentially to D-3 phosphoinositides. The D-3 phosphoinositide binding activity was mapped to a small subregion in the N-terminal domain of ActA. This subregion comprised 19 amino acids and showed homology to cecropins. In addition, we found that amino acids 33 to 74 of ActA mediated actin binding by the whole, folded ActA molecule. These findings shed new light on ActA function.

Actins↗

PubMed1999

Expression cloning of protein targets for 3-phosphorylated phosphoinositides.

The phosphatidylinositol 3-kinase (PI 3'-K) family of lipid kinases play a critical role in cell proliferation, survival, vesicle trafficking, motility, cytoskeletal rearrangements, and oncogenesis. To identify downstream effectors of PI 3'-K, we developed a novel screen to isolate proteins that bind to the major products of PI 3'-K: phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P(2)) and PtdIns-3,4,5-trisphosphate (PtdIns-3,4,5-P(3)). This screen uses synthetic biotinylated analogs of these lipids in conjunction with libraries of radiolabeled proteins that are produced by coupled in vitro transcription/translation reactions. The feasibility of the screen was initially demonstrated using avidin-coated beads prebound to biotinylated PtdIns-3,4-P(2) and PtdIns-3,4,5-P(3) to specifically isolate the pleckstrin homology domain of the serine/threonine kinase Akt. We then demonstrated the utility of this technique in isolating novel 3'-phosphorylated phosphatidylinositol (3'-PPI)-binding proteins through the preliminary screening of in vitro transcribed/translated cDNAs from a small pool expression library derived from mouse spleen. Three proteins were isolated that bound specifically to 3'PPIs. Two of these proteins have been previously characterized as PIP3BP/p42(IP4) and the PtdIns-3,4,5-P(3)-dependent serine/threonine kinase phosphoinositide-dependent kinase 1. The third protein is a novel protein that contains only a Src homology 2 domain and a pleckstrin homology domain; this protein has a higher specificity for both PtdIns-3,4,5-P(3) and PtdIns-3,4-P(2) than for PtdIns-4, 5-bisphosphate. Transcripts of this novel gene are present in every tissue analyzed but are most prominently expressed in spleen. We have renamed this new protein PHISH for 3'-phosphoinositide-interacting Src homology-containing protein. This report demonstrates the utility of this technique for isolating and characterizing 3'-PPI-binding proteins and has broad applicability for the isolation of binding domains for other lipid products.

Adaptor Proteins, Signal Transducing↗

PubMed2000

The class II phosphoinositide 3-kinase PI3K-C2alpha is concentrated in the trans-Golgi network and present in clathrin-coated vesicles.

In recent years, a large family of phosphoinositide 3-kinase (PI3K) isozymes has been characterized and cloned. Several of these PI3K enzymes have overlapping tissue distributions and it remains unclear if and how their 3-phosphoinositide products elicit differential, intracellular effects. One possibility is that the PI3K enzymes display a restricted distribution within the cell to produce their 3-phospholipid products in specific, subcellular compartments. In the present study we characterize the subcellular distribution of the novel class II PI3K isozyme PI3K-C2alpha in several mammalian cell types. Differential centrifugation of COS-1 and U937 cells together with Western blot analysis demonstrated that PI3K-C2alpha is constitutively associated with phospholipid membranes. Centrifugation of rat brain homogenates and Western blotting revealed that in contrast to the class IA PI3K enzymes, PI3K-C2alpha could be co-purified with a population of clathrin-coated vesicles (CCVs). Furthermore, a PI3K activity refractory to wortmannin treatment was detected in CCV preparations consistent with the presence of the PI3K-C2alpha isozyme. These biochemical observations were supported by immunofluorescence analysis that revealed PI3K-C2alpha to have a punctate distribution and an enrichment of immunoreactivity within a perinuclear site consistent with its presence in the endoplasmic reticulum or Golgi apparatus. Dual label immunofluorescence demonstrated that in this region, the distribution of PI3K-C2alpha closely paralleled that of gamma-adaptin, a component of the AP-1 adaptor that is present in the trans-Golgi and the trans-Golgi network (TGN) resident protein TGN-46. Neither the phospholipid association nor the subcellular localization of PI3K-C2alpha was dependent upon either its COOH-terminal PX or C2 domains. Mutants lacking these domains demonstrated a similar distribution to the wild type enzyme when expressed as recombinant proteins. Treatment of cells with brefeldin A disrupted the perinuclear staining pattern of both PI3K-C2alpha and the AP-1 complex demonstrating that the localization of both molecules at the TGN is dependent upon ADP-ribosylation factor GTPase activity.

Animals↗

PubMed2000

Localization and insulin-regulated relocation of phosphoinositide 5-kinase PIKfyve in 3T3-L1 adipocytes.

The mammalian phosphoinositide kinase PIKfyve catalyzes the synthesis of phosphatidylinositol 5-P and phosphatidylinositol 3,5-P(2), thought essential in cellular functions, including membrane trafficking. To discern the intracellular loci of PIKfyve products' formation, we have examined the localization of PIKfyve protein versus enzymatic activity and a possible acutely regulated redistribution in 3T3-L1 adipocytes. Subcellular fractions of resting cells that were positive for immunoreactive PIKfyve, such as cytosol ( approximately 76%), internal structures (low density microsomal fraction (LDM), composed of recycling endosomes, GLUT4 storage compartment, Golgi, and cytoskeletal elements) ( approximately 20%), and plasma membrane (( approximately )4%), expressed enzymatically active PIKfyve. While the presence of a FYVE finger in PIKfyve predicts early endosome targeting, density gradient sedimentation, immunoadsorption, and fluorescence microscopy analyses segregated the LDM-associated PIKfyve from the membranes of the recycling endosomes and GLUT4. PIKfyve fluorescence staining largely coincided with trans-Golgi network/multivesicular body markers, indicating PIKfyve's role in the late endocytic/biosynthetic pathways. A subfraction of particulate PIKfyve resisted nonionic detergent treatment, implying association with cytoskeletal structures, previously found positive for key members of the insulin signaling cascade. Upon acute stimulation of 3T3-L1 adipocytes with insulin or pervanadate, a portion of the cytosolic PIKfyve was recruited onto LDM, which was coupled with a commensurate increase of PIKfyve lipid kinase activity and an electrophoretic mobility shift. We suggest the recruited PIKfyve specifies the site and timing of phosphoinositide signals that are relevant to the acute insulin action.

3T3 Cells↗