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PubMed1998

Direct targets of phosphoinositide 3-kinase products in membrane traffic and signal transduction.

Phosphoinositide 3-kinases are a family of lipid kinases that phosphorylate the 3' position of the inositol ring on phosphatidylinositol and higher-phosphorylated polyphosphoinositides. The multiplicity of cellular functions influenced by the activity of these enzymes has captured the attention of researchers working on two important fields of cell biology--signal transduction and membrane traffic. This review discusses how the recent identification of proteins that interact directly with 3'-polyphosphoinositides has revealed novel potential interconnections between these fundamental cellular processes.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2002

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.

Binding Sites↗
PubMed2001

Synthesis and biological evaluation of L-alpha-phosphatidyl-D-3-deoxy-3-heteromethyl-myo-inositols as phosphoinositide 3-kinase inhibitors.

We have synthesized a series of 3-deoxy-3-heteromethyl derivatives of L-alpha-phosphatidyl-D-myo-inositol as part of our effort to develop specific, reversible inhibitors of phosphoinositide (PI) 3-kinase. Among various derivatives examined, phosphatidyl-D-3-deoxy-3-aminomethyl-myo-inositol displays the highest potency in inhibiting PI 3-kinase both in vitro and in cells. It effectively suppressed antigen-stimulated degranulation in mast cells (IC(50), 17 microM), suggesting a potential application of this PI 3-kinase inhibitor as a mast cell-stabilizing agent.

Androstadienes↗
PubMed2000

Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine.

The specific phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY294002 have been invaluable tools for elucidating the roles of these enzymes in signal transduction pathways. The X-ray crystallographic structures of PI3Kgamma bound to these lipid kinase inhibitors and to the broad-spectrum protein kinase inhibitors quercetin, myricetin, and staurosporine reveal how these compounds fit into the ATP binding pocket. With a nanomolar IC50, wortmannin most closely fits and fills the active site and induces a conformational change in the catalytic domain. Surprisingly, LY294002 and the lead compound on which it was designed, quercetin, as well as the closely related flavonoid myricetin bind PI3K in remarkably different orientations that are related to each other by 180 degrees rotations. Staurosporine/PI3K interactions are reminiscent of low-affinity protein kinase/staurosporine complexes. These results provide a rich basis for development of isoform-specific PI3K inhibitors with therapeutic potential.

Adenosine Triphosphate↗
PubMed2003

Isoform-specific phosphoinositide 3-kinase inhibitors as therapeutic agents.

The phosphoinositide 3-kinase (PI3K) family of enzymes consists of several closely related isoforms that are thought to have distinct biological roles. Until now, researchers have been frustrated by poor selectivity of the available pharmacological inhibitors, which are unable to distinguish adequately the activities of different PI3K isoforms. Recently published patent specifications describe new PI3K inhibitors, including several that are selective for the PI3Kdelta isoform. There is now cautious optimism that isoform-selective PI3K inhibitors will provide new avenues for therapeutic applications in a range of diseases.

Animals↗
PubMed2000

Oxidative stress and vanadate induce tyrosine phosphorylation of phosphoinositide-dependent kinase 1 (PDK1).

Phosphoinositide-dependent kinase (PDK1) regulates a number of pathways involved in responses to stress and in growth factor signaling; however, little is known concerning the mechanisms governing the activity of PDK1. In this report, we find that oxidative stress (H(2)O(2)) and vanadate induce tyrosine phosphorylation of PDK1. These effects of H(2)O(2) and vanadate were found in 293T cells and CH310T1/2 cells expressing exogenous PDK1 and in A20 lymphoma cells expressing endogenous PDK1. Exogenously expressed PDK1 was also tyrosine-phosphorylated in response to NGF treatment of 293T expressing TrkA. H(2)O(2) induced a more rapid tyrosine phosphorylation of PDK1 relative to vanadate, and only vanadate-induced tyrosine phosphorylation of PDK1 was sensitive to pretreatment of cells with wortmannin. In vitro, PDK1 could be tyrosine-phosphorylated by both the c-Src and Abl tyrosine kinases. Both H(2)O(2) and vanadate treatments increased the activity of PDK1 when the serum/glucocorticoid regulated kinase (SGK) was used as substrate. Vanadate treatment appeared to bypass the requirement for phosphatidylinositol 3,4,5-trisphosphate when Akt was used as substrate for PDK1. Tyrosine phosphorylation of PDK1 by the Abl tyrosine kinase also increased the activity of PDK1 toward SGK and Akt. These data suggest a novel mechanism through which PDK1 activity may be regulated.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2001

Insulin stimulates increased catalytic activity of phosphoinositide-dependent kinase-1 by a phosphorylation-dependent mechanism.

Phosphoinositide-dependent kinase-1 (PDK-1) is a serine-threonine kinase downstream from PI 3-kinase that phosphorylates and activates other important kinases such as Akt that are essential for cell survival and metabolism. Previous reports have suggested that PDK-1 has constitutive catalytic activity that is not regulated by stimulation of cells with growth factors. We now show that insulin stimulation of NIH-3T3(IR) cells or rat adipose cells may significantly increase the intrinsic catalytic activity of PDK-1. Insulin treatment of NIH-3T3(IR) fibroblasts overexpressing PDK-1 increased both phosphorylation of recombinant PDK-1 in intact cells and PDK-1 kinase activity in an immune-complex kinase assay. Insulin stimulation of rat adipose cells also increased catalytic activity of endogenous PDK-1 immunoprecipitated from the cells. Both insulin-stimulated phosphorylation and activity of PDK-1 were inhibited by wortmannin and reversed by treatment with the phosphatase PP-2A. A mutant PDK-1 with a disrupted PH domain (W538L) did not undergo phosphorylation or demonstrate increased kinase activity in response to insulin stimulation. Similarly, a PDK-1 phosphorylation site point mutant (S244A) had no increase in kinase activity in response to insulin stimulation. Thus, the insulin-stimulated increase in PDK-1 catalytic activity may involve PI 3-kinase- and phosphorylation-dependent mechanisms. We conclude that the basal constitutive catalytic activity of PDK-1 in NIH-3T3(IR) cells and rat adipose cells can be significantly increased upon insulin stimulation.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2006

Pegylated wortmannin and 17-hydroxywortmannin conjugates as phosphoinositide 3-kinase inhibitors active in human tumor xenograft models.

Phosphoinositide 3-kinase (PI3K) is an important target for cancer chemotherapy due to the deregulation of its signaling pathway in a wide spectrum of human tumors. Wortmannin and its analogues are potent PI3K inhibitors whose therapeutic use has been impeded by inherent defects such as instability and toxicity. Pegylation of wortmannin and 17-hydroxywortmannin gives rise to conjugates with improved properties, including a higher therapeutic index. Pegylated 17-hydroxywortmannin (8, PWT-458) has been selected for further development.

Androstadienes↗
PubMed2006

Furan-2-ylmethylene thiazolidinediones as novel, potent, and selective inhibitors of phosphoinositide 3-kinase gamma.

Class I phosphoinositide 3-kinases (PI3Ks), in particular PI3Kgamma, have become attractive drug targets for inflammatory and autoimmune diseases. Here, we disclose a novel series of furan-2-ylmethylene thiazolidinediones as selective, ATP-competitive PI3Kgamma inhibitors. Structure-based design and X-ray crystallography of complexes formed by inhibitors bound to PI3Kgamma identified key pharmacophore features for potency and selectivity. An acidic NH group on the thiazolidinedione moiety and a hydroxy group on the furan-2-yl-phenyl part of the molecule play crucial roles in binding to PI3K and contribute to class IB PI3K selectivity. Compound 26 (AS-252424), a potent and selective small-molecule PI3Kgamma inhibitor emerging from these efforts, was further profiled in three different cellular PI3K assays and shown to be selective for class IB PI3K-mediated cellular effects. Oral administration of 26 in a mouse model of acute peritonitis led to a significant reduction of leukocyte recruitment.

Acute Disease↗
PubMed2006

Microscopic modes and free energies of 3-phosphoinositide-dependent kinase-1 (PDK1) binding with celecoxib and other inhibitors.

Celecoxib, also known as Celebrex (approved by FDA in 1998) and remembered as the fastest-selling drug in history, was used as a cyclooxygenase-2 (COX-2) selective inhibitor having both anti-inflammatory and anticancer activities. Most recent studies have revealed that the apoptotic activity of celecoxib (and its derivatives) is actually independent of the COX-2 inhibitory activity and that celecoxib also inhibits the kinase activity of 3-phosphoinositide-dependent protein kinase-1 (PDK1), suggesting that the well-known anticancer activity of celecoxib is not due to the inhibition of COX-2, but possibly is due to the inhibition of PDK1. It is highly desirable to develop new celecoxib derivatives as PDK1-specifc inhibitors to avoid the side effects of COX-2 inhibitors. To understand how PDK1 binds with celecoxib and its derivatives, we have performed extensive molecular docking and combined molecular dynamics (MD) simulations and molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations on eight representative PDK1 inhibitors, leading to the finding of a new, more favorable binding mode which is remarkably different from the previously proposed binding mode. Based on the determined most stable binding structures, the calculated binding free energies are all in good agreement with the corresponding experimental data, and the biological activity data available for celecoxib and its derivatives can be better interpreted. The obtained new insights, concerning both the binding mode and computational protocol, will be valuable not only for future rational design of novel, more potent PDK1-specific inhibitors as promising anticancer therapeutics, but also for rational design of drugs targeting other proteins.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed1997

AtPLC2, a gene encoding phosphoinositide-specific phospholipase C, is constitutively expressed in vegetative and floral tissues in Arabidopsis thaliana.

A cDNA encoding a phosphoinositide-specific phospholipase C (PI-PLC) from the higher plant Arabidopsis thaliana was cloned and characterized. The gene corresponding to this cDNA is designated AtPLC2. The overall structure of the predicted AtPLC2 protein is similar to those of plant PI-PLCs and mammalian delta-type PI-PLCs. Northern blot analysis revealed that AtPLC2 is expressed constitutively whereas AtPLC1S, another gene for PI-PLC of Arabidopsis, is induced by environmental stresses such as dehydration and salinity, indicating that the function of AtPLC2 is distinct from that of AtPLC1S. The AtPLC2 mRNA was detected in vegetative and floral tissues. We determined the positions of these two PI-PLCs genes on Arabidopsis chromosomes by RFLP mapping using P1 genomic clones.

Amino Acid Sequence↗
PubMed1999

Alteration of phosphoinositide degradation by cytosolic and membrane-bound phospholipases after forebrain ischemia-reperfusion in gerbil: effects of amyloid beta peptide.

The reperfusion of previously ischemic brain is associated with exacerbation of cellular injury. Reperfusion occasionally potentates release of intracellular enzymes, influx of Ca2+, breakdown of membrane phospholipids, accumulation of amyloid precursor protein or amyloid beta-(like) proteins, and apolipoprotein E. In this study, the effect of reperfusion injury on the activity of cerebral cortex enzymes acting on phosphatidyl [3H] inositol (PI) and [14C-arachidonoyl] PI was investigated. Moreover the effect of amyloid beta25-35 on PI degradation by phospholipase(s) of normoxic brain and subjected to ischemia-reperfusion injury was determined. Brain ischemia in gerbils (Meriones unguiculatus) was induced by ligation of both common carotid arteries for 5 min and then brains were perfused for 15 min, 2 h and 7 days. Statistically significant activation of enzyme(s) involved in phosphatidylinositol degradation in gerbils subjected to ischemia-reperfusion injury was observed. Nearly all gerbils showed a higher activity of cytosolic PI phospholipase C (PLC) at 15 min after ischemia. Concomitantly, the significant enhancement of the level of DAG and AA radioactivity at this short reperfusion time confirmed the active PI degradation by phospholipase(s) in cerebral cortex and hippocampus. After a prolonged reperfusion time of 7 days after ischemia, both cytosolic and membrane-bound forms of PI-PLC were activated. The question arises if alteration of membranes by the degradation of phospholipids occurring after an ischemic episode potentiates the effect of Abeta on membrane-bound enzymes. A neurotoxic fragment of amyloid, Abeta 25-35, incubated in the presence of endogenous Ca2+, increased significantly the PI-PLC activity of normoxic brain. In its non-aggregated form, Abeta 25-35 activates PI-PLC but in the aggregated form the enzymatic activity decreased. Thus, Abeta 25-35 exerts a similar effect on the membrane-bound PI-PLC from normoxic brain or subjected to ischemia reperfusion injury. We conclude that the degradation of phosphatidylinositol by cytosolic phosphoinositide-phospholipase C may contribute to the pathophysiology of delayed neuronal death following cerebral ischemia. Thus, a specific inhibitor of this enzyme(s) may offer therapeutic strategies to protect the brain from damage triggered by ischemia. Ischemia-reperfusion injury had no effect on Abeta-evoked alterations of synaptic plasma membrane-bound PI-PLC.

Amyloid beta-Peptides↗
PubMed1996

Crystal structure of a mammalian phosphoinositide-specific phospholipase C delta.

Mammalian phosphoinositide-specific phospholipase C enzymes (PI-PLC) act as signal transducers that generate two second messengers, inositol-1,4,5-trisphosphate and diacylglycerol. The 2.4-A structure of phospholipase C delta 1 reveals a multidomain protein incorporating modules shared by many signalling proteins. The structure suggests a mechanism for membrane attachment and Ca2+-dependent hydrolysis of second-messenger precursors. The regulation and reversible membrane association of PI-PLC may serve as a model for understanding other multidomain enzymes involved in phospholipid signalling.

Amino Acid Sequence↗
PubMed2000

Pivotal role of phosphoinositide-3 kinase in regulation of cytotoxicity in natural killer cells.

The mitogen-activated protein kinase-extracellular signal-regulated kinase signaling element (MAPK-ERK) plays a critical role in natural killer (NK) cell lysis of tumor cells, but its upstream effectors were previously unknown. We show that inhibition of phosphoinositide-3 kinase (PI3K) in NK cells blocks p21-activated kinase 1 (PAK1), MAPK kinase (MEK) and ERK activation by target cell ligation, interferes with perforin and granzyme B movement toward target cells and suppresses NK cytotoxicity. Dominant-negative N17Rac1 and PAK1 mimic the suppressive effects of PI3K inhibitors, whereas constitutively active V12Rac1 has the opposite effect. V12Rac1 restores the activity of downstream effectors and lytic function in LY294002- or wortmannin-treated, but not PD98059-treated, NK cells. These results document a specific PI3K-->Rac1-->PAK1-->MEK-->ERK pathway in NK cells that effects lysis.

Cell Line↗
PubMed2004

Essential role for the p110delta phosphoinositide 3-kinase in the allergic response.

Inflammatory substances released by mast cells induce and maintain the allergic response. Mast cell differentiation and activation are regulated, respectively, by stem cell factor (SCF; also known as Kit ligand) and by allergen in complex with allergen-specific immunoglobulin E (IgE). Activated SCF receptors and high-affinity receptors for IgE (FcvarepsilonRI) engage phosphoinositide 3-kinases (PI(3)Ks) to generate intracellular lipid second messenger signals. Here, we report that genetic or pharmacological inactivation of the p110delta isoform of PI(3)K in mast cells leads to defective SCF-mediated in vitro proliferation, adhesion and migration, and to impaired allergen-IgE-induced degranulation and cytokine release. Inactivation of p110delta protects mice against anaphylactic allergic responses. These results identify p110delta as a new target for therapeutic intervention in allergy and mast-cell-related pathologies.

Anaphylaxis↗
PubMed2004

The serine kinase phosphoinositide-dependent kinase 1 (PDK1) regulates T cell development.

T lymphocyte activation is associated with activation of diverse AGC serine kinases (named after family members protein kinase A, protein kinase G and protein kinase C). It has been difficult to assess the function of these molecules in T cell development with simple gene-deletion strategies because different isoforms of AGC kinases are coexpressed in the thymus and have overlapping, redundant functions. To circumvent these problems, we explored the consequences of genetic manipulation of phosphoinositide-dependent kinase 1 (PDK1), a rate-limiting 'upstream' activator of AGC kinases. Here we analyzed the effect of PDK1 deletion on T lineage development. We also assessed the consequences of reducing PDK1 levels to 10% of normal. Complete PDK1 loss blocked T cell differentiation in the thymus, whereas reduced PDK1 expression allowed T cell differentiation but blocked proliferative expansion. These studies show that AGC family kinases are essential for T cell development.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2000

Inhibition of phosphoinositide 3-kinase impairs pre-commitment cell cycle traverse and prevents differentiation in erythroleukaemia cells.

During the early hours after exposure to differentiation inducing agents, Friend erythroleukaemia cells undergo alterations which commit them to cessation of growth and development of the characteristics of differentiation. Our current experiments have compared the expression and activity of phosphoinositide 3-kinase (PI 3-kinase) in control cells with cells undergoing differentiation which has been induced by dimethyl sulfoxide (DMSO). When the cultures were initiated with stationary phase cells and DMSO was added at the time of seeding, PI 3-kinase activity was stimulated in both treated and control cells during the first 3 h from seeding. This event appears to be a rate limiting step in commitment since pretreatment of cells with 10 microM LY294002 or down-regulation of p85 expression prior to adding DMSO completely prevents commitment to erythropoiesis. Accordingly, PI 3-kinase inhibition during the commitment period prevents DNA-binding of the transcription factor GATA-1, essential for erythroid differentiation. However, once cells are committed to differentiate, PI 3-kinase activity and expression dramatically decreases along with the differentiation programme, to become barely detectable after 96 h. Remarkably, LY294002 treatment leads to accumulation of cell in G1 phase and prevents DMSO-dependent cyclin D3 induction. Based on these data, we suggest that PI 3-kinase is rate limiting for the completion of the first round cycle of cell division required for initiation of erythrocytic differentiation. On the other hand, the late decrease of PI 3-kinase associated with the differentiation process seems to be part of the programmed shut off of genes not needed in mature erythrocytes.

Androstadienes↗
PubMed2000

M1 muscarinic receptors block caspase activation by phosphoinositide 3-kinase- and MAPK/ERK-independent pathways.

When PC12 cells are deprived of trophic support they undergo apoptosis. We have previously shown that survival of trophic factor-deprived PC12M1 cells can be promoted by activation of the G protein-coupled muscarinic receptors. The mechanism whereby muscarinic receptors inhibit apoptosis is poorly understood. In the present study we investigated this mechanism by examining the effect of muscarinic receptor activation on the serum deprivation-induced activity of key players in apoptosis, the caspases, in PC12M1 cells. The results showed that m1 muscarinic activation inhibits caspase activity induced by serum deprivation. This effect appeared to be caused by the prevention of activation of caspases such as caspase-2 and caspase-3, and not by the inhibition of existing activity. Muscarinic receptor activation also stimulated the mitogen-activated protein kinase/extracellular signaling-regulated kinase (MAPK/ERK) and phosphoinositide (PI) 3-kinase signaling pathways. The PI 3-kinase pathway inhibitors wortmannin and LY294002, as well as the MAPK/ERK pathway PD98059 inhibitor, did not however suppress the inhibitory effect of the muscarinic receptors on caspase activity. The results therefore suggested that the muscarinic survival effect is mediated by a pathway that leads to caspase inhibition by MAPK/ERK- and PI 3-kinase-independent signaling cascades.

Animals↗