Secondary literature sources for the PI3Ka domain

For each of the hand selected primary papers associated with the PI3Ka domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

Immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate in Epon embedded specimens.

Zini N, Mazzotti G, Valmori A, Barbieri M, Maraldi NM
J Submicrosc Cytol Pathol. 1995; 27: 115-8

A comparison between the time-consuming freeze-substitution Lowicryl HM 20 embedding procedure with the Epon embedding method for the immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate (PIP2) by means of a monoclonal antibody is described. The results indicate that the intracellular localization of the phospholipid and the efficiency of its identification are similar, opening the possibility of performing the immunoreaction also on samples that are routinely processed for pathological investigations.

Structure and function of phosphoinositide 3-kinases.

Wymann MP, Pirola L
Biochim Biophys Acta. 1998; 1436: 127-50

Phosphoinositide kinases (PI3Ks) play an important role in mitogenic signaling and cell survival, cytoskeletal remodeling, metabolic control and vesicular trafficking. Here we summarize the structure-function relationships delineating the activation process of class I PI3Ks involving various domains of adapter subunits, Ras, and interacting proteins. The resulting product, PtdIns(3,4,5)P3, targets Akt/protein kinase B (PKB), Bruton's tyrosine kinase (Btk), phosphoinositide-dependent kinases (PDK), integrin-linked kinase (ILK), atypical protein kinases C (PKC), phospholipase Cgamma and more. Surface receptor-activated PI3Ks function in mammals, insects, nematodes and slime mold, but not yeast. While many members of the class II family have been identified and characterized biochemically, it is presently unknown how these C2-domain containing PI3Ks are activated, and which PI substrate they phosphorylate in vivo. PtdIns 3-P is produced by Vps34p/class III PI3Ks and operates via the PtdIns 3-P-binding proteins early endosomal antigen (EEA1), yeast Vac1p, Vps27p, Pip1p in lysosomal protein targeting. Besides the production of D3 phosphorylated lipids, PI3Ks have an intrinsic protein kinase activity. For trimeric GTP-binding protein-activated PI3Kgamma, protein kinase activity seems to be sufficient to trigger mitogen-activated protein kinase (MAPK). Recent disruption of PI3K genes in slime mold, Caenorhabditis elegans, Drosophila melanogaster and mice further underlines the importance of PI3K signaling systems and elucidates the role of PI3K signaling in multicellular organisms.

Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate.

Whitman M, Downes CP, Keeler M, Keller T, Cantley L
Nature. 1988; 332: 644-6

The generation of second messengers from the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdInsP2) by phosphoinositidase C has been implicated in the mediation of cellular responses to a variety of growth factors and oncogene products. The first step in the production of PtdInsP2 from phosphatidylinositol (PtdIns) is catalysed by PtdIns kinase. A PtdIns kinase activity has been found to associate specifically with several oncogene products, as well as with the platelet-derived growth factor (PDGF) receptor. We have previously identified two biochemically distinct PtdIns kinases in fibroblasts, and have found that only one of these, designated type I, specifically associates with activated tyrosine kinases. We have now characterized the site on the inositol ring phosphorylated by type I PtdIns kinase, and find that this kinase specifically phosphorylates the D-3 ring position to generate a novel phospholipid, phosphatidylinositol-3-phosphate (PtdIns(3)P). In contrast, the main PtdIns kinase in fibroblasts, designated type II, specifically phosphorylates the D-4 position to produce phosphatidylinositol-4-phosphate (PtdIns(4)P), previously considered to be the only form of PtdInsP. We have also tentatively identified PtdIns(3)P as a minor component of total PtdInsP in intact fibroblasts. We propose that type I PtdIns kinase is responsible for the generation of PtdIns(3)P in intact cells, and that this novel phosphoinositide could be important in the transduction of mitogenic and oncogenic signals.

Arachidonoyl-diacylglycerol kinase. Specific in vitro inhibition by polyphosphoinositides suggests a mechanism for regulation of phosphatidylinositol biosynthesis.

Walsh JP, Suen R, Glomset JA
J Biol Chem. 1995; 270: 28647-53

We previously described the purification of a membrane-bound diacylglycerol kinase highly selective for sn-1-acyl-2-arachidonoyl diacylglycerols (Walsh, J. P., Suen, R., Lemaitre, R. N., and Glomset, J. A. (1994) J. Biol. Chem. 269, 21155-21164). This enzyme appears to be responsible for the rapid clearance of the arachidonate-rich pool of diacylglycerols generated during stimulus-induced phosphoinositide turnover. We have now shown phosphatidylinositol 4,5-bisphosphate to be a potent and specific inhibitor of arachidonoyl-diacylglycerol kinase. Kinetic analyses indicated a Ki for phosphatidylinositol 4,5-bisphosphate of 0.04 mol %. Phosphatidic acid also was an inhibitor with a Ki of 0.7 mol %. Other phospholipids had only small effects at these concentrations. A series of multiply phosphorylated lipid analogs also inhibited the enzyme, indicating that the head group phosphomonoesters are the primary determinants of the polyphosphoinositide effect. However, these compounds were not as potent as phosphatidylinositol 4,5-bisphosphate, indicating some specificity for the polyphosphoinositide additional to its total charge. Five other diacylglycerol kinases were activated to varying degrees by phosphatidylinositol 4,5-bisphosphate and phosphatidic acid, suggesting that inhibition by acidic lipids may be specific for the arachidonoyl-DAG kinase isoform. Given the presumed role of arachidonoyl-diacylglycerol kinase in the phosphoinositide cycle, this inhibition may represent a mechanism for polyphosphoinositides to regulate their own synthesis.

Phosphorylation of the platelet p47 phosphoprotein is mediated by the lipid products of phosphoinositide 3-kinase.

Toker A, Bachelot C, Chen CS, Falck JR, Hartwig JH, Cantley LC, Kovacsovics TJ
J Biol Chem. 1995; 270: 29525-31

Platelet stimulation by thrombin or the thrombin receptor activating peptide (TRAP) results in the activation of phosphoinositide 3-kinase and the production of the novel polyphosphoinositides phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3). We have shown previously that these lipids activate calcium-independent protein kinase C (PKC) isoforms in vitro (Toker, A., Meyer, M., Reddy, K. K., Falck, J. R., Aneja, R., Aneja, S., Parra, A., Burns, D. J., Ballas, L. M. and Cantley, L. C. (1994) J. Biol. Chem. 269, 32358-32367). Activation of platelet PKC in response to TRAP is detected by the phosphorylation of the major PKC substrate in platelets, the p47 phosphoprotein, also known as pleckstrin. Here we provide evidence for two phases of pleckstrin phosphorylation in response to TRAP. A rapid phase of pleckstrin phosphorylation (< 1 min) precedes the peak of PtdIns-3,4-P2 production and is unaffected by concentrations of wortmannin (10-100 nM) that block production of this lipid. However prolonged phosphorylation of pleckstrin (> 2 min) is inhibited by wortmannin concentrations that block PtdIns-3,4-P2 production. Phorbol ester-mediated pleckstrin phosphorylation was not affected by wortmannin and wortmannin had no effect on purified platelet PKC activity. Phosphorylation of pleckstrin could be induced using permeabilized platelets supplied with exogenous gamma-32P[ATP] and synthetic dipalmitoyl PtdIns-3,4,5-P3 and dipalmitoyl PtdIns-3,4-P2 micelles, but not with dipalmitoyl phosphatidylinositol 3-phosphate or phosphatidylinositol 4,5-bisphosphate. These results suggest two modes of stimulating pleckstrin phosphorylation: a rapid activation of PKC (via diacylglycerol and calcium) followed by a slower activation of calcium-independent PKCs via PtdIns-3,4-P2.

Novel protein kinase/phosphatidylinositol 3-kinase complex essential for receptor-mediated protein sorting to the vacuole in yeast.

Stack JH, Herman PK, DeWald DB, Marcusson EG, Lin Cereghino J, Horazdovsky BF, Emr SD
Cold Spring Harb Symp Quant Biol. 1995; 60: 157-70

Vps34p required for yeast vacuolar protein sorting is a multiple specificity kinase that exhibits both protein kinase and phosphatidylinositol-specific PI 3-kinase activities.

Stack JH, Emr SD
J Biol Chem. 1994; 269: 31552-62

The Vps15 protein kinase and the Vps34 phosphatidylinositol 3-kinase have been shown to function as a membrane-associated complex which facilitates the delivery of proteins to the vacuole in yeast. Biochemical characterization of the autophosphorylation reaction catalyzed by Vps15p demonstrates that it is a functional serine/threonine protein kinase. In addition, we show that the Vps34 phosphatidylinositol 3-kinase undergoes an autophosphorylation event both in vivo and in vitro, indicating that it represents a novel multiple specificity kinase capable of phosphorylating both protein and lipid substrates. Vps34p is phosphorylated predominately on serine in vivo and is able to phosphorylate serine, threonine, and tyrosine residues in vitro. Mutant Vps34 proteins containing alterations in conserved amino acids in the lipid kinase domain are severely defective for both PI 3-kinase activity and autophosphorylation. Characterization of the PI 3-kinase activity of Vps34p demonstrates that it, unlike the mammalian p110 PI 3-kinase, is highly resistant to the PI 3-kinase inhibitors wortmannin and LY294002. We also find that Vps34p is a phosphatidylinositol-specific 3-kinase, as it is able to utilize phosphatidylinositol (PtdIns) but not PtdIns(4)P or PtdIns(4,5)P2 as substrates in an in vitro PI kinase reaction. The substrate specificity, wortmannin resistance, and other biochemical characteristics of its PtdIns 3-kinase activity suggest that Vps34p is quite similar to a PtdIns-specific 3-kinase activity recently characterized from mammalian cells. These data indicate the existence of a family of PI 3-kinases composed of p110-like PI 3-kinases and Vps34p-like PtdIns-specific 3-kinases. On the basis of the role for Vps34p in vacuolar protein sorting, we propose that the production of a specific phosphoinositide, PtdIns(3)P, is involved in regulating intracellular protein sorting reactions in eukaryotic cells.

Vesicle-mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast.

Stack JH, DeWald DB, Takegawa K, Emr SD
J Cell Biol. 1995; 129: 321-34

A membrane-associated complex composed of the Vps15 protein kinase and the Vps34 phosphatidylinositol 3-kinase (PtdIns 3-kinase) is essential for the delivery of proteins to the yeast vacuole. An active Vps15p is required for the recruitment of Vps34p to the membrane and subsequent stimulation of Vps34p PtdIns 3-kinase activity. Consistent with this, mutations altering highly conserved residues in the lipid kinase domain of Vps34p lead to a dominant-negative phenotype resulting from titration of activating Vps15 proteins. In contrast, catalytically inactive Vps15p mutants do not produce a dominant mutant phenotype because they are unable to associate with Vps34p in a wild-type manner. These data indicate that an intact Vps15p protein kinase domain is necessary for the association with and activation of Vps34p, and they demonstrate that a functional Vps15p-Vps34p complex is absolutely required for the efficient delivery of proteins to the vacuole. Analysis of a temperature-conditional allele of VPS15, in which a shift to the nonpermissive temperature leads to a decrease in cellular PtdIns(3)P levels, indicates that the loss of Vps15p function leads to a defect in activation of Vps34p. In addition, characterization of a temperature-sensitive allele of VPS34 demonstrates that inactivation of Vps34p leads to the immediate missorting of soluble vacuolar proteins (e.g., carboxypeptidase Y) without an apparent defect in the sorting of the vacuolar membrane protein alkaline phosphatase. This rapid block in vacuolar protein sorting appears to be the result of loss of PtdIns 3-kinase activity since cellular PtdIns(3)P levels decrease dramatically in vps34 temperature-sensitive mutant cells that have been incubated at the nonpermissive temperature. Finally, analysis of the defects in cellular PtdIns(3)P levels in various vps15 and vsp34 mutant strains has led to additional insights into the importance of PtdIns(3)P intracellular localization, as well as the roles of Vps15p and Vps34p in vacuolar protein sorting.

Phox homology domains specifically bind phosphatidylinositol phosphates.

Song X, Xu W, Zhang A, Huang G, Liang X, Virbasius JV, Czech MP, Zhou GW
Biochemistry. 2001; 40: 8940-4

The recruitment of specific cytosolic proteins to intracellular membranes through binding phosphorylated derivatives of phosphatidylinositol (PtdIns) controls such processes as endocytosis, regulated exocytosis, cytoskeletal organization, and cell signaling. Protein modules such as FVYE domains and PH domains that bind specifically to PtdIns 3-phosphate (PtdIns-3-P) and polyphosphoinositides, respectively, can direct such membrane targeting. Here we show that two representative Phox homology (PX) domains selectively bind to specific phosphatidylinositol phosphates. The PX domain of Vam7p selectively binds PtdIns-3-P, while the PX domain of the CPK PI-3 kinase selectively binds PtdIns-4,5-P(2). In contrast, the PX domain of Vps5p displays no binding to any PtdInsPs that were tested. In addition, the double mutant (Y42A/L48Q) of the PX domain of Vam7p, reported to cause vacuolar trafficking defects in yeast, has a dramatically decreased level of binding to PtdIns-3-P. These data reveal that the membrane targeting function of the Vam7p PX domain is based on its ability to associate with PtdIns-3-P, analogous to the function of FYVE domains.

The phosphoinositide phosphatase Sac1p controls trafficking of the yeast Chs3p chitin synthase.

Schorr M, Then A, Tahirovic S, Hug N, Mayinger P
Curr Biol. 2001; 11: 1421-6

Phosphoinositide phosphatases play an essential but as yet not well-understood role in lipid-based signal transduction. Members of a subfamily of these enzymes share a specific domain that was first identified in the yeast Sac1 protein [1]. Sac1 homology domains were shown to exhibit 3- and 4-phosphatase activity in vitro [2, 3] and were also found, in addition to rat and yeast Sac1p, in yeast Inp/Sjl proteins [4, 5] and mammalian synaptojanins [6]. Despite the detailed in vitro characterization of the enzymatic properties of yeast Sac1p, the exact cellular function of this protein has remained obscure. We report here that Sac1p has a specific role in secretion and acts as an antagonist of the phosphatidylinositol 4-kinase Pik1p in Golgi trafficking. Elimination of Sac1p leads to excessive forward transport of chitin synthases and thus causes specific cell wall defects. Similar defects in membrane trafficking are caused by the overexpression of PIK1. Taken together, these findings provide strong evidence that the generation of PtdIns(4)P is sufficient to trigger forward transport from the Golgi to the plasma membrane and that Sac1p is critically required for the termination of this signal.

Thrombopoietin induces activation of the phosphatidylinositol-3' kinase pathway and formation of a complex containing p85PI3K and the protooncoprotein p120CBL.

Sattler M, Salgia R, Durstin MA, Prasad KV, Griffin JD
J Cell Physiol. 1997; 171: 28-33

Thrombopoietin (TPO) promotes megakaryocyte growth and development. Its receptor, c-MPL, is restricted to cells of megakaryocytic lineage and stem cells. We have previously shown that activation of c-MPL by thrombopoietin rapidly activates at least two cytoplasmic tyrosine kinases, JAK2 and TYK2, after ligand binding. Phosphatidylinositol-3' kinase (PI3K) has been shown to play an important role in downstream signaling for many receptors. Thrombopoietin was found to also rapidly activate phosphatidylinositol-3' kinase, and the phosphatidylinositol-3' kinase inhibitor wortmannin decreased proliferation of thrombopoietin-stimulated cells, implying that phosphatidylinositol-3' kinase may have a regulatory role in thrombopoietin signaling. In immunoprecipitation studies, the regulatory subunit of phosphatidylinositol-3' kinase, p85PI3K, associated with several tyrosine phosphoproteins, and the major phosphoprotein was a 120 kDa protein identified as p120CBL. The phosphatidylinositol-3' kinase-enzyme activity in p120CBL immunoprecipitates was elevated in thrombopoietin-stimulated cells as compared to immunoprecipitates from unstimulated cells. p120CBL may be involved in signaling pathways activated by c-MPL which involve phosphatidylinositol-3' kinase.

Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras.

Rodriguez-Viciana P, Warne PH, Khwaja A, Marte BM, Pappin D, Das P, Waterfield MD, Ridley A, Downward J
Cell. 1997; 89: 457-67

The pathways by which mammalian Ras proteins induce cortical actin rearrangement and cause cellular transformation are investigated using partial loss of function mutants of Ras and activated and inhibitory forms of various postulated target enzymes for Ras. Efficient transformation by Ras requires activation of other direct effectors in addition to the MAP kinase kinase kinase Raf and is inhibited by inactivation of the PI 3-kinase pathway. Actin rearrangement correlates with the ability of Ras mutants to activate PI 3-kinase. Inhibition of PI 3-kinase activity blocks Ras induction of membrane ruffling, while activated PI 3-kinase is sufficient to induce membrane ruffling, acting through Rac. The ability of activated Ras to stimulate PI 3-kinase in addition to Raf is therefore important in Ras transformation of mammalian cells and essential in Ras-induced cytoskeletal reorganization.

Transformation by v-Src: Ras-MAPK and PI3K-mTOR mediate parallel pathways.

Penuel E, Martin GS
Mol Biol Cell. 1999; 10: 1693-703

An increase in the level of active, GTP-bound Ras is not necessary for transformation of chicken embryo fibroblasts (CEF) by v-Src. This suggests that other Ras-independent pathways contribute to transformation by v-Src. To address the possibility that activation of phosphatidylinositol-3-kinase (PI3K) and the mammalian target of rapamycin (mTOR/FRAP), represents one of these pathways, we have examined the effect of simultaneous inhibition of the Ras-MAPK and PI3K-mTOR pathways on transformation of CEF by v-Src. Transformation was assessed by the standard parameters of morphological alteration, increased hexose uptake, loss of density inhibition, and anchorage-independent growth. Inhibition of the Ras-MAPK pathway by expression of the dominant-negative Ras mutant HRasN17 or by addition of the MAPK kinase (MEK) inhibitor PD98059 reduced several of these parameters but failed to block transformation. Similarly, inhibition of the PI3K-mTOR pathway by addition of the PI3K inhibitor 2-[4-morpholinyl]-8-phenyl-4H-1-benzopyran-4-one (LY294002) or the mTOR inhibitor rapamycin, although reducing several parameters of transformation, also failed to block transformation. However, simultaneous inhibition of signaling by the Ras-MAPK pathway and the PI3K-mTOR pathway essentially blocked transformation. These data indicate that transformation of CEF by v-Src is mediated by two parallel pathways, the Ras-MAPK pathway and the PI-3K-mTOR pathway, which both contribute to transformation. The possibility that simultaneous activation of other pathways is also required is not excluded.

Synthesis of new cyclitol compounds that influence the activity of phosphatidylinositol 4-kinase isoform, PI4K230.

Pelyvas IF, Toth ZG, Vereb G, Balla A, Kovacs E, Gorzsas A, Sztaricskai F, Gergely P
J Med Chem. 2001; 44: 627-32

The synthesis, chemical derivatization, and investigation of the inhibitory properties of novel cyclitol derivatives on the phosphatidylinositol 4-kinase enzymes PI4K55 and PI4K230 involved in the phosphatidylinositol cycle are reported. Some of the prepared cyclitol derivatives (i.e. 9, 11, 12, and 14) proved to be very powerful and specific irreversible inhibitors of PI4K230 at or below a concentration of 1 mM.

Characterization of p150, an adaptor protein for the human phosphatidylinositol (PtdIns) 3-kinase. Substrate presentation by phosphatidylinositol transfer protein to the p150.Ptdins 3-kinase complex.

Panaretou C, Domin J, Cockcroft S, Waterfield MD
J Biol Chem. 1997; 272: 2477-85

Genetic and biochemical studies have shown that the phosphatidylinositol (PtdIns) 3-kinase encoded by the yeast VPS34 gene is required for the efficient sorting and delivery of proteins to the vacuole. A human homologue of the yeast VPS34 gene product has recently been characterized as part of a complex with a cellular protein of 150 kDa (Volinia, S., Dhand, R., Vanhaesebroeck, B., MacDougall, L. K., Stein, R., Zvelebil, M. J., Domin, J., Panaretou, C., and Waterfield, M. D. (1995) EMBO J. 14, 3339-3348). Here, cDNA cloning is used to show that the amino acid sequence of this protein, termed p150, is 29.6% identical and 53% similar to the yeast Vps15p protein, an established regulator of Vps34p. Northern blot analysis showed a ubiquitous tissue distribution for p150 similar to that previously observed with PtdIns 3-kinase. Recombinant p150 associated with PtdIns 3-kinase in vitro in a stable manner, resulting in a 2-fold increase in lipid kinase activity. Addition of phosphatidylinositol transfer protein (PI-TP) further stimulated the lipid kinase activity of the p150.PtdIns 3-kinase complex 3-fold. A PtdIns 3-kinase activity could also be co-immunoprecipitated from human cell lysates using anti-PI-TP antisera. This observation demonstrates that an interaction between a PtdIns 3-kinase and PI-TP occurs in vivo, which further implicates lipid transfer proteins in the regulation of PtdIns 3-kinase activity. These results suggest that the Vps15p.Vps34p complex has been conserved from yeast to man and in both species is involved in protein trafficking.

A wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids.

Nakanishi S, Catt KJ, Balla T
Proc Natl Acad Sci U S A. 1995; 92: 5317-21

The synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], the immediate precursor of intracellular signals generated by calcium-mobilizing hormones and growth factors, is initiated by the conversion of phosphatidylinositol to phosphatidylinositol 4-phosphate [PtdIns(4)P] by phosphatidylinositol 4-kinase (PtdIns 4-kinase). Although cells contain several PtdIns 4-kinases, the enzyme responsible for regulating the synthesis of hormone-sensitive PtdIns(4,5)P2 pools has not been identified. In this report we describe the inhibitory effect of micromolar concentrations of wortmannin (WT) on the synthesis of hormone-sensitive PtdIns(4)P and PtdIns(4,5)P2 pools in intact adrenal glomerulosa cells, and the presence of a WT-sensitive PtdIns 4-kinase in adrenocortical extracts. In addition to its sensitivity to the PtdIns 3-kinase inhibitor WT, this enzyme is distinguished from the recognized membrane-bound PtdIns 4-kinases by its molecular size and weak membrane association. Inhibition of this PtdIns 4-kinase by WT results in rapid loss of the hormone-sensitive PtdIns(4,5)P2 pool in angiotensin II-stimulated glomerulosa cells. Consequently, WT treatment inhibits the sustained but not the initial increases in inositol 1,4,5-trisphosphate and cytoplasmic [Ca2+] in a variety of agonist-stimulated cells, including adrenal glomerulosa cells, NIH 3T3 fibroblasts, and Jurkat lymphoblasts. These results indicate that a specific WT-sensitive PtdIns 4-kinase is critical for the maintenance of the agonist-sensitive polyphosphoinositide pool in several cell types.

Selective recognition of phosphatidylinositol 3,4,5-trisphosphate by a synthetic peptide.

Lu PJ, Chen CS
J Biol Chem. 1997; 272: 466-72

The present study takes a novel approach to explore the mode of action of phosphoinositide 3-kinase lipid products by identifying a synthetic peptide W-NG(28-43) (WAAKIQASFRGHMARKK) that displays discriminative affinity with phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). This PtdIns(3,4,5)P3-binding peptide was discovered by a gel filtration-based binding assay and exhibits a high degree of stereochemical selectivity in phosphoinositide recognition. It forms a 1:1 complex with PtdIns(3,4,5)P3 with Kd of 2 microM, but binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) with substantially lower affinity (5- and 40-fold, respectively) despite the largely shared structural motifs with PtdIns(3,4,5)P3. Other phospholipids examined, including phosphatidylserine, phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine, show low or negligible affinity with the peptide. Several lines of evidence indicate that this phosphoinositide-peptide interaction is not due to nonspecific electrostatic interactions or phospholipid aggregation, and requires a cooperative action among the hydrophobic and basic residues to exert the selective recognition. CD data suggest that the peptide acquires an ordered structure upon binding to PtdIns(3,4,5)P3. Further, we demonstrate that PtdIns(3,4,5)P3 enhances the phosphorylation rate of this binding peptide by protein kinase C (PKC)-alpha in a dose-dependent manner. In view of the findings that this stimulatory effect is not noted with other PKC peptide substrates lacking affinity with PtdIns(3,4,5)P3 and that PKC-alpha is not susceptible to PtdIns(3,4,5)P3 activation, the activity enhancement is thought to result from the substrate-concentrating effect of the D-3 phosphoinositide, i.e. the presence of PtdIns(3,4,5)P3 allows the peptide to bind to the same vesicles/micelles to which PKC is bound. Moreover, it is noteworthy that neurogranin, the full-length protein of W-NG(28-43) and a relevant PKC substrate in the forebrain, binds PtdIns(3,4,5)P3 with high affinity. Taken together, it is plausible that, in addition to PKC activation, PtdIns(3,4,5)P3 provides an alternative mechanism to regulate PKC activity in vivo by recruiting and concentrating its target proteins at the interface to facilitate the subsequent PKC phosphorylation.

The phosphatidylinositol 4-phosphate 5-kinase family.

Loijens JC, Boronenkov IV, Parker GJ, Anderson RA
Adv Enzyme Regul. 1996; 36: 115-40

The existence of a PIP5K family of enzymes has been suggested by Western blotting and purification of numerous PIP5Ks from various tissues and cell types. The erythrocyte has at least two PIP5Ks, named PIP5KI and PIP5KII, while the brain appears to have even more isoforms. The cloning of the first PIP5K, the PIP5KII alpha, is just the beginning of the molecular classification of this protein family. The PIP5KII alpha sequence has shown that these enzymes lack obvious homology to protein, sugar and other lipid kinases. The identification of two S. cerevisiae homologues, Mss4p and Fab1p, confirms that this family of kinases is widely distributed in eukaryotes. Not surprisingly, cloning experiments have identified additional isoforms. By cloning additional isoforms, insights into the structure and functions of this family of enzymes will be gained. One reason for a large family of PIP5Ks is that many forms of regulation and cellular functions have been ascribed to PIP5Ks, as summarized in Figure 10. Some of these functional links result from PtdIns[4,5]P2 being required for a given process, but the direct involvement of specific PIP5Ks is not well defined. Which PIP5K isoforms are regulated by a specific mechanism or are involved in a cellular process often is not clear. For example, which PIP5Ks produce PtdIns[4,5]P2 that is hydrolyzed by PLC or phosphorylated by the PI 3-kinase is not known. A few exceptions are PIP5KII not being able to phosphorylate PtdIns[4,5]P2 in native membranes, and PIP5KIs being stimulated by PtdA, required for secretion, and possibly regulated by G proteins of the Rho subfamily. The multiplicity of regulation and functions of each PIP5K isoform remains to be elucidated. Another factor governing the number of isoforms may be presence of multiple pools of polyphosphoinositides and the localizing of PIP5K function within cells. The polyphosphoinositides appear to be compartmentalized within cells and each pool appears to be sensitive to specific signals. These polyphosphoinositide pools may include those in the plasma membrane that are used by PLC, nuclear pools that appear to turn over separately from cytoplasmic pools and a small pool at sites of vesicle fusion with the plasma membrane. Each pool may be controlled by a specific PIP5K isoform. This would explain the diversity of PIP5K cellular roles. Another possibility is that the PIP5Ks are localized to certain areas of the cell by being part of a protein or proteolipid complex. Furthermore, the presence of PITP or PLC in the complex would potentially impart specificity and speed on the use of PtdIns[4]P and PtdIns[4,5]P2 because these lipids could be channeled quickly from one enzyme to the next. The concept of localized complexes containing particular PIP5K isoforms that control the composition of different polyphosphoinositide pools will likely be important as the family of PIP5K isoforms grows.

A specific product of phosphatidylinositol 3-kinase directly activates the protein kinase Akt through its pleckstrin homology domain.

Klippel A, Kavanaugh WM, Pot D, Williams LT
Mol Cell Biol. 1997; 17: 338-44

Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors after growth factor stimulation of cells. Activation of PI 3-kinase results in increased intracellular levels of 3' phosphorylated inositol phospholipids and the induction of signaling responses, including the activation of the protein kinase Akt, which is also known as RAC-PK or PKB. We tested the possibility that the phospholipid products of PI 3-kinase directly mediate the activation of Akt. We have previously described a constitutively active PI 3-kinase, p110, which can stimulate Akt activity. We used purified p110 protein to generate a series of 3' phosphorylated inositol phospholipids and tested whether any of these lipids could activate Akt in vitro. Phospholipid vesicles containing PI3,4 bisphosphate (P2) specifically activated Akt in vitro. By contrast, the presence of phospholipid vesicles containing PI3P or PI3,4,5P3 failed to increase the kinase activity of Akt. Akt could also be activated by synthetic dipalmitoylated PI3,4P2 or after enzymatic conversion of PI3,4,5P3 into PI3,4P2 with the signaling inositol polyphosphate 5' phosphatase SIP. We show that PI3,4P2-mediated activation is dependent on a functional pleckstrin homology domain in Akt, since a point mutation in the pleckstrin homology domain abrogated the response to PI3,4P2. Our findings show that a phospholipid product of PI 3-kinase can directly stimulate an enzyme known to be an important mediator of PI 3-kinase signaling.

Involvement of phosphatidylinositol 3-kinase in the mediation of erythropoietin-induced activation of p70S6k.

Jaster R, Bittorf T, Brock J
Cell Signal. 1997; 9: 175-9

We have previously shown that, in HCD-57 cells, erythropoietin (EPO) induces a biphasic activation of the ribosomal S6 kinase p70S6k, an enzyme playing a key role in the regulation of cell cycle progression. Here we present evidence that p70S6k is activated through both phosphatidylinositol (PI) 3-kinase-dependent and independent pathways: whereas the early phase of EPO-dependent stimulation of p70S6k activity was strongly suppressed by the potent PI 3-kinase inhibitor wortmannin, late phase was much less affected. The dose-dependent inhibition of cell growth by wortmannin indicates an important role of PI 3-kinase in the mediation of EPO-induced cell proliferation. Furthermore, our data suggest that the EPO-receptor-associated tyrosine kinase JAK2 is not essentially involved in the mediation of EPO-induced p70S6k activation.

Tyrosine kinase, phosphatidylinositol 3-kinase, and protein kinase C regulate insulin-stimulated NaCl absorption in the thick ascending limb.

Ito O, Kondo Y, Oba M, Takahashi N, Omata K, Abe K
Kidney Int. 1997; 51: 1037-41

We have previously shown a direct stimulatory effect of insulin on NaCl absorption in the medullary thick ascending limb of Henle's loop (mTAL). To further investigate the signal transduction involved, we determined whether tyrosine kinase, phosphatidylinositol 3-kinase (PI3-kinase), and/or protein kinase C (PKC) regulate insulin-stimulated NaCl absorption in the mTAL by in vitro microperfusion methods. In control experiments, insulin increased transepithelial voltage (V(te)) and net lumen-to-bath Cl- flux (J(Cl)). Genistein and methyl 2,5-dihydroxycinnamate, two specific tyrosine kinase inhibitors, abolished the effects of insulin. Wortmannin, a specific PI3-kinase inhibitor, inhibited the action of insulin. The effects of insulin also were inhibited by staurosporin and calphostin C, which are dissimilar inhibitors of PKC. These results indicate that insulin stimulates NaCl absorption in the mTAL through tyrosine kinase, PI3-kinase, and PKC-mediated mechanisms. Moreover, because we have reported previously that insulin causes no detectable change in cytosolic free Ca2+ in the mTAL cells, the present results also suggest that insulin-induced PKC activation is not related to inositol 1,4,5-triphosphate (IP3) production.

Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast.

Isakoff SJ, Cardozo T, Andreev J, Li Z, Ferguson KM, Abagyan R, Lemmon MA, Aronheim A, Skolnik EY
EMBO J. 1998; 17: 5374-87

Phosphatidylinositol 3-kinase (PI3K) mediates a variety of cellular responses by generating PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These 3-phosphoinositides then function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) domains in these signaling molecules. We have established a novel assay in the yeast Saccharomyces cerevisiae to identify proteins that bind PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in vivo which we have called TOPIS (Targets of PI3K Identification System). The assay uses a plasma membrane-targeted Ras to complement a temperature-sensitive CDC25 Ras exchange factor in yeast. Coexpression of PI3K and a fusion protein of activated Ras joined to a PH domain known to bind PtdIns(3,4)P2 (AKT) or PtdIns(3,4,5)P3 (BTK) rescues yeast growth at the non-permissive temperature of 37 degreesC. Using this assay, we have identified several amino acids in the beta1-beta2 region of PH domains that are critical for high affinity binding to PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3, and we have proposed a structural model for how these PH domains might bind PI3K products with high affinity. From these data, we derived a consensus sequence which predicts high-affinity binding to PtdIns(3, 4)P2 and/or PtdIns(3,4,5)P3, and we have identified several new PH domain-containing proteins that bind PI3K products, including Gab1, Dos, myosinX, and Sbf1. Use of this assay to screen for novel cDNAs which rescue yeast at the non-permissive temperature should provide a powerful approach for uncovering additional targets of PI3K.

Occurrence of inosine kinase as a distinct enzyme in Spirulina platensis.

Ipata PL, Gualerzi C, Scolozzi C, Tozzi MG, Trinei M, Barsacchi D
Biochem Biophys Res Commun. 1995; 209: 547-53

Among a series of purine nucleosides, inosine was found to be phosphorylated at the highest rate by crude extracts of the cyanobacterium Spirulina platensis. The inosine phosphorylating activity could be separated from hypoxanthine-guanine phosphoribosyl transferase. This result shows that IMP formation may occur via the direct phosphorylation of inosine at its 5'-position, rather than via inosine phosphorolysis, followed by hypoxanthine phosphoribosylation, and provides unequivocal evidence for the occurrence of inosine kinase in nature.

Phosphoinositide 4- and 5-kinases and the cellular roles of phosphatidylinositol 4,5-bisphosphate.

Hsuan JJ, Minogue S, dos Santos M
Adv Cancer Res. 1998; 74: 167-216

PIPkins1, their substrates and their products: new functions for old enzymes.

Hinchliffe KA, Ciruela A, Irvine RF
Biochim Biophys Acta. 1998; 1436: 87-104

The phosphatidylinositolphosphate kinases (PIPkins) are a unique family of enzymes that catalyse the production of phosphorylated inositol lipids. Recent advances have revealed that, due to their ability to utilise a number of different lipid substrates (at least in vitro), this family is potentially able to generate several distinct, physiologically important inositol lipids. Despite their importance, however, our understanding of the regulation of the PIPkins and of their physiological role in cellular signalling and regulation is still poor. Here we describe in turn the diverse physiological functions of the known substrates and major products of the PIPkins. We then examine what is known about the members of the PIPkin family themselves, and their characteristics and regulation.

Ras and its effectors.

Herrmann C, Nassar N
Prog Biophys Mol Biol. 1996; 66: 1-41

Normal activation of p70 S6 kinase by insulin in cells overexpressing dominant negative 85kD subunit of phosphoinositide 3-kinase.

Hara K, Yonezawa K, Sakaue H, Kotani K, Kotani K, Kojima A, Waterfield MD, Kasuga M
Biochem Biophys Res Commun. 1995; 208: 735-41

The role of heteromeric phosphoinositide (PI) 3-kinase activity in insulin signal transduction was studied by investigating the effects of (i) overexpression of a dominant negative mutant p85 (delta p85) that lacks the binding site for p110 (delta p85-overexpressing cells) and (ii) inhibition of PI 3-kinase activity by wortmannin (wortmannin-treated cells). The insulin-induced association of PI 3-kinase activity with insulin receptor sustrate-1 (IRS-1) was inhibited in both wortmannin-treated cells and delta p85-overexpressing cells. However, whereas insulin-induced activation of p70 S6 kinase was completely abolished in wortmannin-treated cells, it appeared normal in delta p85-overexpressing cells. These results raise the possibility that a wortmannin-sensitive pathway independent of heteromeric PI 3-kinase is involved in the activation of p70 S6 kinase by insulin.

Association of phosphatidylinositol 3 kinase to protein kinase C zeta during interleukin-2 stimulation.

Gomez J, Martinez C, Garcia A, Rebollo A
Eur J Immunol. 1996; 26: 1781-7

Interleukin-2 induces a serine-phosphorylated phosphatidylinositol 3 kinase activity in the mouse T cell line TS1 alpha beta. Moreover, protein kinase C (PKC) zeta directly or indirectly associates with the phosphatidylinositol 3 kinase and the association appears to be necessary for the serine-phosphorylated phosphatidylinositol 3 kinase activity, since release of zeta PKC by competition of binding with peptides spanning the p110 sequence from amino acids 907 to 925 abolishes the serine-phosphorylated phosphatidylinositol 3 kinase activity. This kinase activity is also blocked when zeta PKC expression is inhibited by antisense oligonucleotide. Inhibition of phosphatidylinositol 3 kinase activity by wortmannin does not abolish zeta PKC association.

Both phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase products are increased by antigen receptor signaling in B cells.

Gold MR, Aebersold R
J Immunol. 1994; 152: 42-50

The effects of Ag binding on B cell development and activation are mediated by intracellular signals initiated by the B cell AgR. In this report, we show that the B cell AgR regulates the production of inositol phospholipids involved in two different signal transduction pathways, the phosphatidylinositol 3-kinase (PtdIns 3-kinase) pathway and the phospholipase C (PLC) pathway. Phosphatidylinositol 3-phosphate (PtdIns3P), phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2], and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] are produced by PtdIns 3-kinase, an enzyme that appears to be an essential component of tyrosine kinase-mediated signaling. Both PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are likely to function as second messengers in vivo because they can activate the zeta isoform of protein kinase C (PKC) in vitro. We show that cross-linking of the B cell AgR with anti-Ig antibodies caused a five- to sixfold increase in the levels of PtdIns(3,4)P2 in both the mature B cell line BAL 17 and the immature B cell line WEHI-231. PtdIns(3,4)P2 levels increased within 15 s of anti-Ig addition and remained elevated for at least 5 min. AgR cross-linking also caused a slower increase in PtdIns3P levels (approximately 50% over control) and a small, transient increase in PtdIns(3,4,5)P3 levels. Thus, the B cell AgR activates the PtdIns 3-kinase pathway. The other inositol phospholipid signaling pathway involves PLC, which cleaves phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], yielding second messengers that increase intracellular calcium and activate other isoforms of PKC. We analyzed the effects of AgR signaling on PtdIns(4,5)P2 and its precursor, phosphatidylinositol 4-phosphate (PtdIns4P). Consistent with its ability to activate PLC, AgR ligation decreased the levels of PtdIns(4,5)P2. In contrast, AgR cross-linking increased the levels of PtdIns4P. Increased synthesis of PtdIns4P followed by phosphorylation at the D-5 position may prevent depletion of PtdIns(4,5)P2. Thus, signaling by the B cell AgR increases the levels of PtdIns 4-kinase products and PtdIns 3-kinase products. The simplest interpretation of our results is that the B cell AgR activates both PtdIns 3-kinase and PtdIns 4-kinase.

Fab1p is essential for PtdIns(3)P 5-kinase activity and the maintenance of vacuolar size and membrane homeostasis.

Gary JD, Wurmser AE, Bonangelino CJ, Weisman LS, Emr SD
J Cell Biol. 1998; 143: 65-79

The Saccharomyces cerevisiae FAB1 gene encodes a 257-kD protein that contains a cysteine-rich RING-FYVE domain at its NH2-terminus and a kinase domain at its COOH terminus. Based on its sequence, Fab1p was initially proposed to function as a phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinase (). Additional sequence analysis of the Fab1p kinase domain, reveals that Fab1p defines a subfamily of putative PtdInsP kinases that is distinct from the kinases that synthesize PtdIns(4,5)P2. Consistent with this, we find that unlike wild-type cells, fab1Delta, fab1(tsf), and fab1 kinase domain point mutants lack detectable levels of PtdIns(3,5)P2, a phosphoinositide recently identified both in yeast and mammalian cells. PtdIns(4,5)P2 synthesis, on the other hand, is only moderately affected even in fab1Delta mutants. The presence of PtdIns(3)P in fab1 mutants, combined with previous data, indicate that PtdIns(3,5)P2 synthesis is a two step process, requiring the production of PtdIns(3)P by the Vps34p PtdIns 3-kinase and the subsequent Fab1p- dependent phosphorylation of PtdIns(3)P yielding PtdIns(3,5)P2. Although Vps34p-mediated synthesis of PtdIns(3)P is required for the proper sorting of hydrolases from the Golgi to the vacuole, the production of PtdIns(3,5)P2 by Fab1p does not directly affect Golgi to vacuole trafficking, suggesting that PtdIns(3,5)P2 has a distinct function. The major phenotypes resulting from Fab1p kinase inactivation include temperature-sensitive growth, vacuolar acidification defects, and dramatic increases in vacuolar size. Based on our studies, we hypothesize that whereas Vps34p is essential for anterograde trafficking of membrane and protein cargoes to the vacuole, Fab1p may play an important compensatory role in the recycling/turnover of membranes deposited at the vacuole. Interestingly, deletion of VAC7 also results in an enlarged vacuole morphology and has no detectable PtdIns(3,5)P2, suggesting that Vac7p functions as an upstream regulator, perhaps in a complex with Fab1p. We propose that Fab1p and Vac7p are components of a signal transduction pathway which functions to regulate the efflux or turnover of vacuolar membranes through the regulated production of PtdIns(3,5)P2.

Novel quinone antiproliferative inhibitors of phosphatidylinositol-3-kinase.

Frew T, Powis G, Berggren M, Gallegos A, Abraham RT, Ashendel CL, Zalkow LH, Hudson C, Gruszecka-Kowalik E, Burgess EM, et al.
Anticancer Drug Des. 1995; 10: 347-59

The inhibition of phosphatidylinositol-3-kinase (PtdIns-3-kinase), protein kinase C and c-Src protein tyrosine kinase by a series of halogenated naphthoquinones and quinoline quinones related to the plant-derived naphthoquinones juglone and methyljuglone, which inhibit protein kinase C, has been investigated. Some of the compounds inhibited PtdIns-3-kinase at micromolar concentrations and below. PtdIns-3-kinase inhibition was time dependent and could be prevented by endogenous thiol. The compounds were only weak inhibitors of PtdIns-4-kinase. Some of the compounds inhibited protein kinase C, but c-Src protein tyrosine kinase was only weakly inhibited. In intact cells, PtdIns-3-kinase was only partly inhibited by concentrations of the halogenated quinones that inhibited cell growth. Some halogenated quinones showed in vivo antitumor activity without accompanying toxicity, while methyljuglone was without in vivo antitumor activity. Halogenated quinones may have multiple biochemical effects in the cell that could contribute to their cytotoxic and antitumor effects. Inhibition of PtdIns-3-kinase by the halogenated quinones may provide a lead for the development of more potent and specific inhibitors.

A multiwell assay for inhibitors of phosphatidylinositol-3-kinase and the identification of natural product inhibitors.

Frew T, Powis G, Berggren M, Abraham RT, Ashendel CL, Zalkow LH, Hudson C, Qazia S, Gruszecka-Kowalik E, Merriman R, et al.
Anticancer Res. 1994; 14: 2425-8

A convenient and reliable multisample assay for the screening of inhibitors of the growth factor signalling enzyme phosphatidylinositol-3-kinase (PtdIns-3-K) has been developed. Four natural product inhibitors of Ptdlns-3-K have been identified with IC50 values for hypericin 0.18 microM, emodin 3.3 microM, asperuloside 2.0 microM and uttronin A 1.1 microM.

Signal transduction. A target for PI(3) kinase.

Downward J
Nature. 1995; 376: 553-4

Osmotic stress activates phosphatidylinositol-3,5-bisphosphate synthesis.

Dove SK, Cooke FT, Douglas MR, Sayers LG, Parker PJ, Michell RH
Nature. 1997; 390: 187-92

Inositol phospholipids play multiple roles in cell signalling systems. Two widespread eukaryotic phosphoinositide-based signal transduction mechanisms, phosphoinositidase C-catalysed phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis and 3-OH kinase-catalysed PtdIns(4,5)P2 phosphorylation, make the second messengers inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) sn-1,2-diacylglycerol and PtdIns(3,4,5)P3. In addition, PtdIns(4,5)P2 and PtdIns3P have been implicated in exocytosis and membrane trafficking. We now show that when the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe are hyperosmotically stressed, they rapidly synthesize phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2) by a process that involves activation of a PtdIns3P 5-OH kinase. This PtdIns(3,5)P2 accumulation only occurs in yeasts that have an active vps34-encoded PtdIns 3-OH kinase, showing that this latter kinase makes the PtdIns3P needed for PtdIns(3,5)P2 synthesis and indicating that PtdIns(3,5)P2 may have a role in sorting vesicular proteins. PtdIns(3,5)P2 is also present in mammalian and plant cells: in monkey Cos-7 cells, its labelling is inversely related to the external osmotic pressure. The stimulation of a PtdIns3P 5-OH kinase-catalysed synthesis of PtdIns(3,5)P2, a molecule that might be a new type of phosphoinositide 'second messenger, thus appears to be central to a widespread and previously uncharacterized regulatory pathway.

Regulation of the Saccharomyces cerevisiae Vps34p phosphatidylinositol 3-kinase.

DeWald DB, Wurmser AE, Emr SD
Biochem Soc Trans. 1997; 25: 1141-6

Changes in phosphatidylinositol metabolism in response to hyperosmotic stress in Daucus carota L. cells grown in suspension culture.

Cho MH, Shears SB, Boss WF
Plant Physiol. 1993; 103: 637-47

Carrot (Daucus carota L.) cells plasmolyzed within 30 s after adding sorbitol to increase the osmotic strength of the medium from 0.2 to 0.4 or 0.6 osmolal. However, there was no significant change in the polyphosphorylated inositol phospholipids or inositol phosphates or in inositol phospholipid metabolism within 30 s of imposing the hyperosmotic stress. Maximum changes in phosphatidylinositol 4-monophosphate (PIP) metabolism were detected at 5 min, at which time the cells appeared to adjust to the change in osmoticum. There was a 30% decrease in [3H]inositol-labeled PIP. The specific activity of enzymes involved in the metabolism of the inositol phospholipids also changed. The plasma membrane phosphatidylinositol (PI) kinase decreased 50% and PIP-phospholipase C (PIP-PLC) increased 60% compared with the control values after 5 min of hyperosmotic stress. The PIP-PLC activity recovered to control levels by 10 min; however, the PI kinase activity remained below the control value, suggesting that the cells had reached a new steady state with regard to PIP biosynthesis. If cells were pretreated with okadaic acid, the protein phosphatase 1 and 2A inhibitor, the differences in enzyme activity resulting from the hyperosmotic stress were no longer evident, suggesting that an okadaic acid-sensitive phosphatase was activated in response to hyperosmotic stress. Our work suggests that, in this system, PIP is not involved in the initial response to hyperosmotic stress but may be involved in the recovery phase.

Inactivation of two Dictyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis, lysosome to postlysosome transport, and actin cytoskeleton organization.

Buczynski G, Grove B, Nomura A, Kleve M, Bush J, Firtel RA, Cardelli J
J Cell Biol. 1997; 136: 1271-86

Phosphatidylinositide 3-kinases (PI3-kinases) have been implicated in controlling cell proliferation, actin cytoskeleton organization, and the regulation of vesicle trafficking between intracellular organelles. There are at least three genes in Dictyostelium discoideum. DdPIK1, DdPIK2, and DdPIK3, encoding proteins most closely related to the mammalian 110-kD PI-3 kinase in amino acid sequence within the kinase domain. A mutant disrupted in DdPIK1 and DdPIK2 (delta ddpik1/ddpik2) grows slowly in liquid medium. Using FITC-dextran (FD) as a fluid phase marker, we determined that the mutant strain was impaired in pinocytosis but normal in phagocytosis of beads or bacteria. Microscopic and biochemical approaches indicated that the transport rate of fluid-phase from acidic lysosomes to non-acidic postlysosomal vacuoles was reduced in mutant cells resulting in a reduction in efflux of fluid phase. Mutant cells were also almost completely devoid of large postlysosomal vacuoles as determined by transmission EM. However, delta ddpik1/ddpik2 cells functioned normally in the regulation of other membrane traffic. For instance, radiolabel pulse-chase experiments indicated that the transport rates along the secretory pathway and the sorting efficiency of the lysosomal enzyme alpha-mannosidase were normal in the mutant strain. Furthermore, the contractile vacuole network of membranes (probably connected to the endosomal pathway by membrane traffic) was functionally and morphologically normal in mutant cells. Light microscopy revealed that delta ddpik1/ddpik2 cells appeared smaller and more irregularly shaped than wild-type cells; 1-3% of the mutant cells were also connected by a thin cytoplasmic bridge. Scanning EM indicated that the mutant cells contained numerous filopodia projecting laterally and vertically from the cell surface, and fluorescent microscopy indicated that these filopodia were enriched in F-actin which accumulated in a cortical pattern in control cells. Finally, delta ddpik1/ddpik2 cells responded and moved more rapidly towards cAMP. Together, these results suggest that Dictyostelium DdPIK1 and DdPIK2 gene products regulate multiple steps in the endosomal pathway, and function in the regulation of cell shape and movement perhaps through changes in actin organization.

Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: a comparison with tyrosine kinase and protein kinase C inhibition.

Agullo G, Gamet-Payrastre L, Manenti S, Viala C, Remesy C, Chap H, Payrastre B
Biochem Pharmacol. 1997; 53: 1649-57

Depending on their structure, flavonoids display more or less potent inhibitory effects on the growth and proliferation of certain malignant cells in vitro, and these effects are thought to be due to inhibition of various enzymes. We investigated the inhibitory action of fourteen flavonoids of different chemical classes on phosphatidylinositol 3-kinase alpha (PI 3-kinase alpha) activity, an enzyme recently shown to play an important role in signal transduction and cell transformation. Of the fourteen flavonoids tested, myricetin was the most potent PI 3-kinase inhibitor (IC50 = 1.8 microM), while luteolin and apigenin were also effective inhibitors, with IC50 values of 8 and 12 microM, respectively. Fisetin and quercetin, as previously reported, were also found to significantly inhibit PI 3-kinase activity. The same flavonoids were also analyzed for inhibition of epidermal growth factor receptor (EGF-R), intrinsic tyrosine kinase and bovine brain protein kinase C (PKC). At elevated doses, some of these flavonoids were found to also cause significant inhibition of PKC and tyrosine kinase activity of EGF-R. A structure-activity study indicated that the position, number and substitution of the hydroxyl group of the B ring, and saturation of the C2-C3 bond are important factors affecting flavonoid inhibition of PI 3-kinase. They may also play a significant role in specificity of inhibition and could help to provide a basis for the further design of specific inhibitors of this lipid kinase. Finally, possible relationships between the antitumoral properties of these flavonoids and their biological activities are discussed.