Signaling by distinct classes of phosphoinositide 3-kinases

Many signaling pathways converge on and regulate phosphoinositide 3-kinase (PI3K) enzymes whose inositol lipid products are key mediators of intracellular signaling. Different PI3K isoforms generate specific lipids that bind to FYVE and pleckstrin homology (PH) domains in a variety of proteins, affecting their localization, conformation, and activities. Here we review the activation mechanisms of the different types of PI3Ks and their downstream actions, with focus on the PI3Ks that are acutely triggered by extracellular stimulation.     

Mariannaepyrone--a new inhibitor of thromboxane A2 induced platelet aggregation

Mariannaeapyrone ((E)-2-(1,3,5,7-tetramethyl-5-nonenyl)-3,5-dimethyl-6-hydroxy-4H-pyran-4-one) is a new fungal metabolite isolated from fermentations of the common mycophilic deuteromycete Mariannaea elegans. The chemical structure of the 4-pyrone was determined by spectroscopic techniques. Mariannaeapyrone is a selective inhibitor of the thromboxane A2 induced aggregation of human platelets, whereas only weak cytotoxic and antimicrobial effects could be observed.     

A phosphoinositide 3-kinase-AKT-nitric oxide-cGMP signaling pathway in stimulating platelet secretion and aggregation

Phosphoinositide 3-kinase (PI3K) and Akt play important roles in platelet activation. However, the downstream mechanisms mediating their functions are unclear. We have recently shown that nitric-oxide (NO) synthase 3 and cGMP-dependent protein kinase stimulate platelet secretion and aggregation. Here we show that PI3K-mediated Akt activation plays an important role in agonist-stimulated platelet NO synthesis and cGMP elevation. Agonist-induced elevation of NO and cGMP was inhibited by Akt inhibitors and reduced in Akt-1 knock-out platelets. Akt-1 knock-out or Akt inhibitor-treated platelets showed reduced platelet secretion and aggregation in response to low concentrations of agonists, which can be reversed by low concentrations of 8-bromo-cGMP or sodium nitroprusside (an NO donor). Similarly, PI3K inhibitors diminished elevation of cGMP and inhibited platelet secretion and the second wave platelet aggregation, which was also partially reversed by 8-bromo-cGMP. These results indicate that the NO-cGMP pathway is an important downstream mechanism mediating PI3K and Akt signals leading to platelet secretion and aggregation. Conversely, the PI3K-Akt pathway is the major upstream mechanism responsible for activating the NO-cGMP pathway in platelets. Thus, this study delineates a novel platelet activation pathway involving sequential activation of PI3K, Akt, nitric-oxide synthase 3, sGC, and cGMP-dependent protein kinase.     

A critical role for phospholipase Cgamma2 in alphaIIbbeta3-mediated platelet spreading

The interaction of fibrinogen with the integrin alphaIIbbeta3 plays a crucial role in platelet adhesion and platelet activation leading to the generation of intracellular signals that nucleate the reorganization of the cytoskeleton. Presently, we have only a limited understanding of the signaling cascades and effector proteins through which changes in the cytoskeletal architecture are mediated. The present study identifies phospholipase Cgamma2 (PLCgamma2) as an important target of the Src-dependent signaling cascade regulated by alphaIIbbeta3. Real time phasecontrast microscopy is used to show that formation of filopodia and lamellapodia in murine platelets on a fibrinogen surface is dramatically inhibited in the absence of PLCgamma2. Significantly, the formation of these structures is mediated by Ca2+ elevation and activation of protein kinase C, both directly regulated by PLC activity. With the involvement of Syk, SLP-76, and Btk, alphaIIbbeta3-induced PLCgamma2 activation partly overlaps with the pathway used by the collagen receptor glycoprotein VI. Important differences, however, exist between the two signaling cascades in that activation of PLCgamma2 by alphaIIbbeta3 is unaltered in murine platelets, which lack the FcR gamma-chain or the adaptor LAT, but is abolished in the presence of cytochalasin D. Therefore, PLCgamma2 plays not only a crucial role in activation of alphaIIbbeta3 by collagen receptors but also in alphaIIbbeta3-mediated responses.     

Regulation and cellular functions of class II phosphoinositide 3-kinases

Class II isoforms of PI3K (phosphoinositide 3-kinase) are still the least investigated and characterized of all PI3Ks. In the last few years, an increased interest in these enzymes has improved our understanding of their cellular functions. However, several questions still remain unanswered on their mechanisms of activation, their specific downstream effectors and their contribution to physiological processes and pathological conditions. Emerging evidence suggests that distinct PI3Ks activate different signalling pathways, indicating that their functional roles are probably not redundant. In the present review, we discuss the recent advances in our understanding of mammalian class II PI3Ks and the evidence suggesting their involvement in human diseases.     

Activation of P2Y2 receptor induces c-FOS protein through a pathway involving mitogen-activated protein kinases and phosphoinositide 3-kinases in HeLa cells

The effects of P2Y2 purinoceptor activation on c-Fos expression and the signaling pathways evoked by extracellular ATP/UTP in HeLa cells were investigated. We found that P2Y2 activation induced c-Fos protein and phosphorylated the extracellular signal-regulated kinases 1 and 2 (ERK1/2). The P2Y2-stimulated c-Fos induction was partly blocked (a) by U73122, a phospholipase C inhibitor, (b) by G?6976, a conventional PKC inhibitor, (c) by PD098059, a mitogen-activated protein kinase kinase inhibitor, and, moreover, (d) by the inhibitors of phosphoinositide 3-kinases (PI3K), LY294002 and wortmannin. When G?6976 and PD098059, or G?6976 and wortmannin, were combined there was a totally inhibition of P2Y2-induced c-Fos increase. Either U73122 or G?6976 did not inhibit ERK1/2 phosphorylation induced by ATP/UTP, while it was inhibited by LY294002 (or wortmannin) and by staurosporine. Additionally, wortmannin inhibited the cytosol-to-membrane translocation of PKC- epsilon induced by ATP/UTP. These data indicated that agonist-induced PI3K and downstream PKC- epsilon activation mediated the effect of ATP/UTP on ERK1/2 activation. To test the biological consequences of ERK1/2 activation, the effect of P2Y2 on cell functions were examined. P2Y2 stimulation increased cell proliferation and this effect was attenuated by PD098059 in a dose-dependent manner, thereby indicating that the ERK pathway mediates mitogenic signaling by P2Y2. In conclusion, the activation of conventional PKCs through P2Y2 receptor acts in concert with ERK and PI3K/PKC- epsilon pathways to induce c-Fos protein and HeLa cell proliferation.     

3D pharmacophore models for thromboxane A2 receptor antagonists

Thromboxane A₂ (TXA₂) is an endogenous arachidonic acid derivative closely correlated to thrombosis and other cardiovascular diseases. The action of TXA₂ can be effectively inhibited with TXA₂ receptor antagonists (TXRAs). Previous studies have attempted to describe the interactions between the TXA₂ receptor and its ligands, but their conclusions are still controversial. In this study, ligand-based computational drug design is used as a new and effective way to investigate the structure-activity relationship of TXRAs. Three-dimensional pharmacophore models of TXRAs were built with HypoGenRefine and HipHop modules in CATALYST software. The optimal HypoGenRefine model was developed on the basis of 25 TXRAs. It consists of two hydrophobic groups, one aromatic ring, one hydrogen-bond acceptor and four excluded volumes. The optimal HipHop model contains two hydrophobic groups and two hydrogen-bond acceptors. These models describe the key structure-activity relationship of TXRAs, can predict their activities, and can thus be used to design novel antagonists. Figure Optimal three-dimensional pharmacophore models of TXA₂ receptor antagonists (TXRAs) built with HypoGenRefine (a) and HipHop (b) modules. a Hypo-1 model mapped with compounds 11 (purple), and 20 (green). b Hypo-2 model mapped with compounds 31 (green) and 64 (yellow). Spheres: Green Hydrogen bond acceptors (HBA), cyanhydrophobic (H), orange aromatic rings (RA), black excluded volumes (EV) Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Production of platelet thromboxane A2 inactivates purified human platelet thromboxane synthase.

Human platelet thromboxane synthase was partially purified by DEAE-cellulose, Affi-Gel Blue, and Sephacryl S-300 chromatography to a specific activity of 259 nmol of thromboxane B2/min per mg. Thromboxane synthase retained 75-90% of its enzymic activity when bound to phenyl-Sepharose. The immobilized enzyme was inactivated at pH 3.0 and inhibited by 1-benzylimidazole and U-63,557A. The ability of the enzyme to produce thromboxane A2 from prostaglandin H2 was dramatically reduced by multiple additions of prostaglandin H2. Our data suggest that the production of thromboxane A2 by the enzyme is self-limiting and that the enzyme is inactivated during the reaction.     

Identification of a putative thromboxane A2/prostaglandin H2 receptor in human platelet membranes

The binding of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist (9,11-dimethylmethano-11, 12-methano-16-(3-aza-15 alpha beta-omega-tetranor-TXA2) ([125I]PTA-OH) to membranes prepared from human platelets was characterized. [125I]PTA-OH binding to membranes from human platelets was saturable, displaceable, and dependent on protein concentration. Scatchard analysis of equilibrium binding carried out at 30 degrees C revealed one class of binding sites with a Kd of 30 +/- 4 nM and a Bmax of 1.8 +/- 0.3 pmol/mg of protein (n = 5). Kinetic analysis of the binding of [125I]PTA-OH at 0 degrees C yielded a k1 of 1.35 X 10(6) M-1 min-1 and a k-1 of 0.032 min-1, Kd = k-1/k1 = 24 nM. The potencies of a series of TXA2/PGH2 antagonists as inhibitors of [125I]PTA-OH binding was correlated with their potencies as inhibitors of platelet aggregation induced by the TXA2/PGH2 mimetic, U46619 (1 microM) (r = 0.93, p less than 0.01). A series of TXA2/PGH2 mimetics also displaced [125I]PTA-OH from its binding site, and their potencies as inhibitors of [125I]PTA-OH binding were correlated with their potencies as stimulators of platelet aggregation (r = 0.91, p less than 0.05). The IC50 values for displacement of [125I]PTA-OH by PGF2 alpha, PGD2, and the stable PGI2 analog Iloprost were greater than 25 microM, suggesting that [125I]PTA-OH does not bind to other known platelet prostaglandin receptors. These data are consistent with the notion that this binding site may represent the platelet TXA2/PGH2 receptor.     

Two PI 3-kinases and one PI 3-phosphatase together establish the cyclic waves of phagosomal PtdIns(3)P critical for the degradation of apoptotic cells

Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a signaling molecule important for many membrane trafficking events, including phagosome maturation. The level of PtdIns(3)P on phagosomes oscillates in two waves during phagosome maturation. However, the physiological significance of such oscillation remains unknown. Currently, the Class III PI 3-kinase (PI3K) Vps34 is regarded as the only kinase that produces PtdIns(3)P in phagosomal membranes. We report here that, in the nematode C. elegans, the Class II PI3K PIKI-1 plays a novel and crucial role in producing phagosomal PtdIns(3)P. PIKI-1 is recruited to extending pseudopods and nascent phagosomes prior to the appearance of PtdIns(3)P in a manner dependent on the large GTPase dynamin (DYN-1). PIKI-1 and VPS-34 act in sequence to provide overlapping pools of PtdIns(3)P on phagosomes. Inactivating both piki-1 and vps-34 completely abolishes the production of phagosomal PtdIns(3)P and disables phagosomes from recruiting multiple essential maturation factors, resulting in a complete arrest of apoptotic-cell degradation. We have further identified MTM-1, a PI 3-phosphatase that antagonizes the activities of PIKI-1 and VPS-34 by down-regulating PtdIns(3)P on phagosomes. Remarkably, persistent appearance of phagosomal PtdIns(3)P, as a result of inactivating mtm-1, blocks phagosome maturation. Our findings demonstrate that the proper oscillation pattern of PtdIns(3)P on phagosomes, programmed by the coordinated activities of two PI3Ks and one PI 3-phosphatase, is critical for phagosome maturation. They further shed light on how the temporally controlled reversible phosphorylation of phosphoinositides regulates the progression of multi-step cellular events.     

Gene-targeting reveals physiological roles and complex regulation of the phosphoinositide 3-kinases

Phosphoinositide 3-kinases (PI3Ks) are represented by a family of eight distinct enzymes that can be divided into three classes based on their structure and function. The class I PI3Ks are heterodimeric enzymes that are regulated by recruitment to plasma membrane following receptor activation and which control numerous cellular functions, including growth, differentiation, migration, survival, and metabolism. New light has been shed on the biological role of individual members of the class I PI3Ks and their regulatory subunits through gene-targeting experiments. In addition, these experiments have brought the complexity of how PI3K activation is regulated into focus.     

Modulatory role of phosphoinositide 3-kinase in gastric acid secretion

The gastric parietal cell is responsible for the secretion of HCl into the lumen of the stomach mainly due to stimulation by histamine via the cAMP pathway. However, the participation of several other receptors and pathways have been discovered to influence both stimulation and inhibition of acid secretion (e.g., cholinergic). Here we examine the role of phosphoinositide 3-kinase (PI3K) in the modulation of acid secretion. Treatment of isolated gastric glands and parietal cells with the PI3K inhibitor, LY294002 (LY), potentiated acid secretion in response to histamine to nearly the maximal secretion obtained with histamine plus phosphodiesterase inhibitors. As cAMP levels were elevated in response to histamine plus LY, but other means of elevating cAMP (e.g., forskolin, dbcAMP) were not influenced by LY, we posited that the effect might require activation of G-protein-coupled histamine H(2) receptors, possibly through the protein kinase B pathway (also known as Akt). Study of downstream effectors of PI3K showed that histaminergic stimulation increased Akt phosphorylation, which in turn was blocked by inhibition of PI3K. Expression studies showed that high expression of active Akt decreased acid secretion, whereas dominant-negative Akt increased acid secretion. Taken together, these data suggest stimulation with histamine increases the activity of PI3K leading to increased activity of Akt and decreased levels of cAMP in the parietal cell.     

Structural insight into substrate specificity and regulatory mechanisms of phosphoinositide 3-kinases

Phosphoinositide 3-kinases (PI3Ks) are implicated in a variety of fundamental cellular processes. These enzymes catalyse phosphorylation of the 3'-OH position of myo-inositol lipids that serve as secondary messengers. The catalytic subunit for one of the family members, PI3K gamma, has been structurally characterized, independently, in complexes with kinase inhibitors and with the p21(Ras) GTPase. These atomic structures provide a basis for the rationalization of some PI3K substrate specificities and regulatory mechanisms, establishing links to functional and cellular data. Ongoing comprehensive structural and functional studies are essential to realize the promise of PI3K isozyme-specific therapeutic agents.     

Activation of phosphoinositide 3-kinases by the CCR4 ligand macrophage-derived chemokine is a dispensable signal for T lymphocyte chemotaxis

Macrophage-derived chemokine (MDC/CC chemokine ligand 22 (CCL22)) mediates its cellular effects principally by binding to its receptor CCR4, and together they constitute a multifunctional chemokine/receptor system with homeostatic and inflammatory roles in the body. We report the CCL22-induced accumulation of phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P(3)) in the leukemic T cell line CEM. CCL22 also had the ability to chemoattract human Th2 cells and CEM cells in a pertussis toxin-sensitive manner. Although the PI(3,4,5)P(3) accumulation along with the pertussis toxin-susceptible phosphorylation of protein kinase B were sensitive to the two phosphoinositide 3-kinase inhibitors, LY294002 and wortmannin, cell migration was unaffected. However, cell migration was abrogated with the Rho-dependent kinase inhibitor, Y-27632. These data demonstrate that although there is PI(3,4,5)P(3) accumulation downstream of CCR4, phosphoinositide 3-kinase activity is a dispensable signal for CCR4-stimulated chemotaxis of Th2 cells and the CEM T cell line.     

Nonradioactive methods for the assay of phosphoinositide 3-kinases and phosphoinositide phosphatases and selective detection of signaling lipids in cell and tissue extracts

We describe a novel approach to quantitation of phosphoinositides in cell extracts and in vitro enzyme-catalyzed reactions using suitably tagged and/or labeled pleckstrin homology (PH) domains as probes. Stable complexes were formed between the biotinylated target lipid and an appropriate PH domain, and phosphoinositides present in samples were detected by their ability to compete for binding to the PH domain. Complexes were detected using AlphaScreen technology or time-resolved FRET. The assay procedure was validated using recombinant PI 3-kinase gamma with diC8PtdIns(4,5)P(2) as substrate and general receptor for phosphoinositides-1 (GRP1) PH domain as a PtdIns(3,4,5)P(3)-specific probe. This PI 3-kinase assay was robust, was suitable for high-throughput screening platforms, and delivered expected IC(50) values for reference compounds. The approach is adaptable to a wide range of enzymes as demonstrated by assays of the tumor suppressor protein, PTEN, a phosphoinositide 3-phosphatase, which was measured using the same reagents but with diC8PtdIns(3,4,5)P(3) as substrate. PtdIns(3,4,5)P(3) present in lipid extracts of Swiss 3T3 and HL60 cells stimulated with platelet-derived growth factor and fMLP, respectively, was also detectable at picomole sensitivity. The versatility and general utility of this approach were demonstrated by exchanging the GRP1 PH domain for that of TAPP1 (which binds PtdIns(3,4)P(2) and not PtdIns(3,4,5)P(3)). This system was used to monitor the accumulation of PtdIns(3,4)P(2) in Swiss 3T3 cells exposed to an oxidative stress. It is therefore proposed that similar procedures should be capable of measuring any known phosphoinositide present in cell and tissue extracts or produced in kinase and phosphatase assays by using one of several well-characterized protein domains with appropriate phosphoinositide-binding specificity.     

Agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane by beta-adrenergic receptor kinase 1. A role in receptor sequestration

Agonist-dependent desensitization of the beta-adrenergic receptor requires translocation and activation of the beta-adrenergic receptor kinase1 by liberated Gbetagamma subunits. Subsequent internalization of agonist-occupied receptors occurs as a result of the binding of beta-arrestin to the phosphorylated receptor followed by interaction with the AP2 adaptor and clathrin proteins. Receptor internalization is known to require D-3 phosphoinositides that are generated by the action of phosphoinositide 3-kinase. Phosphoinositide 3-kinases form a family of lipid kinases that couple signals via receptor tyrosine kinases and G-protein-coupled receptors. The molecular mechanism by which phosphoinositide 3-kinase acts to promote beta-adrenergic receptor internalization is not well understood. In the present investigation we demonstrate a novel finding that beta-adrenergic receptor kinase 1 and phosphoinositide 3-kinase form a cytosolic complex, which leads to beta-adrenergic receptor kinase 1-mediated translocation of phosphoinositide 3-kinase to the membrane in an agonist-dependent manner. Furthermore, agonist-induced translocation of phosphoinositide 3-kinase results in rapid interaction with the receptor, which is of functional importance, since inhibition of phosphoinositide 3-kinase activity attenuates beta-adrenergic receptor sequestration. Therefore, agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane is an important step in the process of receptor sequestration and links phosphoinositide 3-kinase to G-protein-coupled receptor activation and sequestration.