PI 3K Research Articles

Exploring the pharmacology of the leukotriene B 4 receptor BLT 1, without the confounding effects of BLT 2

Most previous studies of leukotriene B 4 (LTB 4) pharmacology using primary leukocyte cultures and myeloid cell lines do not differentiate between leukotriene BLT 1 and BLT 2 receptor activation because both receptors are often expressed by these cells. Here we show that in HeLa cells expressing BLT 1 but not BLT 2 receptors, BLT 1 receptor activation resulted in IP 3 mediated calcium release from intracellular stores initially, followed by calcium influx through cell membrane channels. BLT 1 calcium signalling was sensitive to the activity of protein kinase C (PKC), protein kinase A (PKA) and protein-tyrosine kinases (PTKs), as well as changes in membrane cholesterol levels and treatments that are known to disrupt normal membrane physiology and/or lipid rafts. Inhibition of MAP kinases, Rho-associated kinases, or phosphoinositol-3-kinases (PI 3K) had no effect on BLT 1 receptor induced calcium signalling, and the receptor was insensitive to the redox state of the extracellular compartment.

Manganese-induced integrin affinity maturation promotes recruitment of alpha V beta 3 integrin to focal adhesions in endothelial cells: evidence for a role of phosphatidylinositol 3-kinase and Src.

Integrin activity is controlled by changes in affinity (i.e. ligand binding) and avidity (i.e. receptor clustering). Little is known, however, about the effect of affinity maturation on integrin avidity and on the associated signaling pathways. To study the effect of affinity maturation on integrin avidity, we stimulated human umbilical vein endothelial cells (HUVEC) with MnCl(2) to increase integrin affinity and monitored clustering of beta 1 and beta 3 integrins. In unstimulated HUVEC, beta 1 integrins were present in fibrillar adhesions, while alpha V beta 3 was detected in peripheral focal adhesions. Clustered beta 1 and beta 3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl(2)-stimulation promoted focal adhesion and actin stress fiber formation at the basal surface of the cells, and strongly enhanced mAb LM609 staining and expression of beta 3 high affinity/LIBS epitopes at focal adhesions. MnCl(2)-induced alpha V beta 3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two pharmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), and by over-expressing a dominant negative PI 3-K mutant protein. Conversely, over-expression of active PI 3-K and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced alpha V beta 3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl(2) did not alter beta 1 integrin distribution or beta1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl(2)-induced alpha V beta 3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity alpha V beta 3 to focal adhesions. Affinity-modulated alpha V beta 3 clustering requires PI3-K signaling and is negatively regulate by Src.

Characterization of TCR-induced phosphorylation of PKCθ in primary murine lymphocytes

Protein kinase C θ (PKCθ) is a member of the “novel” PKC subfamily which plays a critical role in T-cell activation. Following T-cell stimulation, PKCθ translocates to the center of the immunological synapse where it co-localizes with the T-cell receptor (TCR). PKCθ is required for the activation of the transcription factors nuclear factor-kappaB (NF-κB) and activator protein-1 (AP-1), which regulate the production of interleukin-2 (IL-2), a necessary step for the deployment of T-cell effector functions. By using primary murine T lymphocytes we have investigated regulatory phosphorylation events on PKCθ which may impinge on its activity or its interaction with other signaling mediators. Here, we describe a TCR stimulation-induced Ser/Thr phosphorylation event on PKCθ that takes place simultaneously with its recruitment to cellular membranes and which can be detected as an electrophoretic mobility shift. By analyzing Ser and Thr point mutants, we find that phosphorylation of Ser-695 in the hydrophobic motif is one, but not the only residue involved in the mobility shift. Interestingly, Ser-695 appears to be required to trigger further phosphorylation events at adjacent Thr-692 and Thr-703 residues, which also participate in the mobility shift. We show that phosphorylation at these residues is not due to auto-phosphorylation, but requires instead Src family kinase and phosphatidylinositol 3-kinase (PI 3K) activities. We further show that activation-induced phosphorylation of PKCθ correlates with its function but not with its kinase activity, suggesting that phosphorylation of PKCθ plays a role in its interaction with upstream or downstream effectors.

Protein kinase C modulates negatively the hepatocyte growth factor-induced migration, integrin expression and phosphatidylinositol 3-kinase activation

Previously, we reported that, in hepatocyte growth factor (HGF)-induced HepG2 cells, protein kinase C (PKC) decreased the duration of intensive Erk1/Erk2 MAP kinase activation. This study shows that the inhibition of PKC enhanced significantly the HGF-induced integrin expression. Beside the prolonged activation of Erk1/Erk2, the activity of phosphatidylinositol 3-kinase (PI 3K) was required for growth factor-induced integrin expression. PI 3-kinase was activated to a higher extent in response to HGF than to epidermal growth factor (EGF), though the activation was transient in both cases. In EGF-induced cells, PI 3K activation was terminated by the loss of phosphotyrosine docking sites for PI 3K. To the contrary, the decrease of PI 3K activation, which followed the HGF-induced increase was not accompanied by the loss of phosphotyrosine docking sites and was prevented by the inhibition of PKC. The negative modulator effects of PKC on integrin expression and PI 3-kinase activation correlated with its ability to limit the HGF-induced motogen response.

Tumour necrosis factor-related apoptosis-inducing ligand sequentially activates pro-survival and pro-apoptotic pathways in SK-N-MC neuronal cells.

The SK-N-MC neuroblastoma cell line, which expresses surface tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors TRAIL-R2 and TRAIL-R4, was used as a model system to examine the effect of TRAIL on key intracellular pathways involved in the control of neuronal cell survival and apoptosis. TRAIL induced distinct short-term (1-60 min) and long-term (3-24 h) effects on the protein kinase B (PKB)/Akt (Akt), extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB), nuclear factor kappa B (NF-kappaB) and caspase pathways. TRAIL rapidly (from 20 min) induced the phosphorylation of Akt and ERK, but not of c-Jun NH2-terminal kinase (JNK). Moreover, TRAIL increased CREB phosphorylation and phospho-CREB DNA binding activity in a phosphatidylinositol 3-kinase (PI 3K)/Akt-dependent manner. At later time points (from 3 to 6 h onwards) TRAIL induced a progressive degradation of inhibitor of kappaB (IkappaB)beta and IkappaBepsilon, but not IkappaBalpha, coupled to the nuclear translocation of NF-kappaB and an increase in its DNA binding activity. In the same time frame, TRAIL started to activate caspase-8 and caspase-3, and to induce apoptosis. Remarkably, caspase-dependent cleavage of NF-kappaB family members as well as of Akt and CREB proteins, but not of ERK, became prominent at 24 h, a time point coincident with the peak of caspase-dependent apoptosis.

The elastin peptides-mediated induction of pro-collagenase-1 production by human fibroblasts involves activation of MEK/ERK pathway via PKA- and PI 3K-dependent signaling

Elastin peptides, such as κ-elastin (kE), bind to the elastin receptor at the cell surface of human dermal fibroblasts and stimulate collagenase-1 expression at the gene and protein levels. Using specific inhibitors and phosphospecific antibodies, we show here that the binding of elastin peptides to their receptor activates the extracellular signal-regulated kinase (ERK) pathway; this activation is essential for the induction of pro-collagenase-1 production. Moreover, protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI 3K) signaling were found to participate in ERK activation. Concomitantly, we demonstrate that stimulation by elastin peptides leads to enhanced DNA binding of activator protein-1 (AP-1). Our data indicate that the up-regulation of collagenase-1 following treatment of fibroblasts with elastin peptides results from a cross-talk between PKA, PI 3K and the ERK signaling pathways and that this regulation is accompanied by activation of AP-1 transcription factors.

Ceramide Promotes Stress Fiber Formation

Ceramide signaling has been implicated in such processes as programmed cell death, differentiation, and cell proliferation. Hanna et al. provide evidence that ceramide may be one of the mediators of tumor necrosis factor-α (TNF-α) signaling that results in the formation of stress fibers in Rat2 fibroblasts. Treatment of the cells with TNF-α, a cell-permeable ceramide, or sphingomyelinase led to the formation of stress fibers and the tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. TNF-α and ceramide both stimulated the interaction of phosphatidylinositol 3-kinase (PI 3K) with FAK. The formation of stress fibers and paxillin phosphorylation were completely inhibited in cells exposed to ceramide or sphingomyelinase by treatment of the cells with the PI 3K inhibitor LY 294002 and partially inhibited in cells exposed to TNF-α. TNF-α and ceramide stimulate sphingosine kinase activity, which phosphorylates sphingosine, forming the active compound sphingosine 1-phosphate. If sphingosine kinase activity is inhibited, then stress fiber formation is inhibited in response to TNF-α or ceramide. Inhibition of sphingosine kinase also leads to the reorganization of the actin cytoskeleton to form a dense cortical network. Additional experiments indicate that RhoA and Ras guanosine triphosphatases are also involved downstream of the ceramide-induced cytoskeletal rearrangements and that dominant-negative Ras can inhibit the activation of sphingosine kinase. Thus, ceramide and bioactive sphingolipids appear to be involved in the pathway from TNF-α to stress fiber formation. A. N. Hanna, L. G. Berthiaume, Y. Kikuchi, D. Begg, S. Bourgoin, D. N. Brindley, Tumor necrosis factor-α induces stess fiber formation through ceramide production: Role of sphingosine kinase. Mol. Biol. Cell 12 , 3618-3630 (2001). [Abstract] [Full Text]

Induction of Hypoxia-Inducible-Factor 1 by Nitric Oxide Is Mediated via the PI 3K Pathway

Adaptation to hypoxic stress provokes activation of the hypoxia-inducible-factor-1 (HIF-1) which mediates gene expression of, e.g., erythropoietin or vascular endothelial growth factor. Detailed information on signaling pathways that stabilize HIF-1 is missing, but reactive oxygen species degrade the HIF-1α subunit, whereas phosphorylation causes its stabilization. It was believed that hypoxia resembles the only HIF-1 inducer but recent evidence characterized other activators of HIF-1 such as nitric oxide (NO). Herein, we concentrated on NO-evoked HIF-1 induction as a heretofore unappreciated inflammatory response in association with massive NO formation. We demonstrated that S-nitrosoglutathione induces HIF-1α accumulation and concomitant DNA binding. The response was attenuated by the kinase inhibitor genistein and blockers of phosphatidylinositol 3-kinase such as Ly 294002 or wortmannin. Whereas mitogen-activated protein kinases were not involved, we noticed phosphorylation/activation of Akt in correlation with HIF-1α stabilization. NO appears to regulate HIF-1α via the PI 3K/Akt pathway under normoxic conditions.

Natural variants of human p85 alpha phosphoinositide 3-kinase in severe insulin resistance: a novel variant with impaired insulin-stimulated lipid kinase activity.

Phosphoinositide 3-kinase (PI 3K) plays a central part in the mediation of insulin-stimulated glucose disposal. No genetic studies of this enzyme in human syndromes of severe insulin resistance have been previously reported. Phosphoinositide 3-kinase p85 alpha regulatory subunit cDNA was examined in 20 subjects with syndromes of severe insulin resistance by single strand conformational polymorphism and restriction fragment length polymorphism analyses. Insulin-stimulated phosphoinositide 3-kinase activity and recruitment into phosphotyrosine complexes of variants of p85 alpha were studied in transiently transfected HEK293 cells. Phosphopeptide binding characteristics of wild-type and mutant p85 alpha-GST fusion proteins were examined by surface plasmon resonance. The common p85 alpha variant, Met326I1e, was identified in 9 of the 20 subjects. Functional studies of the Met326Ile variant showed it to have equivalent insulin-stimulated lipid kinase activity and phosphotyrosine recruitment as wild-type p85 alpha. A novel heterozygous mutation, Arg409Gln, was detected in one subject. Within the proband's family, carriers of the mutation had a higher median fasting plasma insulin (218 pmol/l) compared with wild-type relatives (72 mol/l) (n = 8 subjects, p = 0.06). The Arg409Gln p85 alpha subunit was associated with lower insulin-stimulated phosphoinositide 3-kinase activity compared with wild-type (mean reduction 15%, p < 0.05, n = 5). The recruitment of Arg409Gln p85 alpha into phosphotyrosine complexes was not significantly impaired. GST fusion proteins of wild-type and mutant p85 alpha showed identical binding to phosphopeptides in surface plasmon resonance studies. Mutations in p85 alpha are uncommon in subjects with syndromes of severe insulin resistance. The Met326Ile p85 alpha variant appears to have no functional effect on the insulin-stimulated phosphoinositide 3-kinase activity. The impaired phosphoinositide 3-kinase activity of the Arg409Gln mutant suggests that it could contribute to the insulin resistance seen in this family.

PI 3-kinase signalling in platelets: the significance of synergistic, autocrine stimulation

Phosphoinositide 3-kinases (PI 3Ks) play a key role in regulation of intracellular signalling and cellular function, including cell proliferation, apoptosis, chemotaxis, membrane trafficking and platelet activation. The PI 3Ks are grouped into three classes on the basis on their structure and in vitro substrate specificity. Class I are activated by a variety of agonists which mediate their effect through tyrosine kinase-linked or G-proteinlinked receptors. In vivo class I PI 3Ks seem to preferentially phosphorylate the D3 hydroxyls of the inositol moiety of PtdIns(4,5)P 2 to produce PtdIns(3,4,5)P 3 . However, class II PI 3Ks preferentially phosphorylate the D3 hydroxyl of PtdIns and PtdIns(4)P to produce PtdIns(3)P and PtdIns(3,4)P 2 , respectively. The late accumulation of PtdIns(3,4)P 2 has been suggested to play an important role in irreversible platelet aggregation. In human platelets the class II PI 3K isoform HsC2-PI 3K is activated in an integrin alpha Ib beta 3 +fibrinogen dependent manner. Class III PI 3Ks phosphorylate PtdIns to produce PtdIns(3)P, which play a crucial role in vesicular trafficking. Recent work has suggested that crosstalk between individual receptors and their downstream signal pathways play a central role in PI 3K signalling responses. In this review, we will concentrate on recent advances regarding the regulation of platelet PI 3Ks. I

Regulation of protein synthesis by cholecystokinin in rat pancreatic acini involves PHAS-I and the p70 S6 kinase pathway

Background & Aims: Cholecystokinin (CCK) stimulates protein synthesis in pancreatic acini at the translational level, although the signaling mechanisms involved remain uncharacterized. Two intermediates controlling translation are p70 S6 kinase and PHAS-I. We previously showed that CCK activates p70 S6K in pancreatic acini through phosphatidylinositol 3-kinase (PI 3K). In the present study we investigated the role of PI 3K, p70 S6K, and PHAS-I in mediating CCK-stimulated protein synthesis. Methods: Protein synthesis was measured by [ 35S]methionine incorporation into pancreatic protein using acini from rats with streptozotocin-induced diabetes. p70 S6 K activity was measured. PHAS-I was identified by Western analysis. PHAS-I/eIF-4E association was measured as the amount of PHAS-I recovered after purification of translation factor eIF-4E by 7-methyl guanosine triphosphate–Sepharose. Results: Rapamycin and PI 3K inhibitors, wortmannin and LY294002, blocked CCK-stimulated p70 S6K activity. Rapamycin inhibited basal protein synthesis and blocked the increase to all CCK concentrations. Wortmannin and LY294002 dose-dependently inhibited basal and CCK-stimulated protein synthesis and also blocked insulin-stimulated protein synthesis. CCK dose-dependently increased PHAS-I phosphorylation via a rapamycin- and LY294002-sensitive pathway and decreased the amount of PHAS-I associated with eIF-4E. Rapamycin and LY294002 eliminated this effect of CCK. Conclusions: CCK stimulation of protein synthesis in pancreatic acini is sensitive to rapamycin and PI 3K inhibitors and involves PHAS-I phosphorylation and its association with eIF-4E. GASTROENTEROLOGY 1998;115:733-742

Phosphoinositide 3-Kinase Inhibition Spares Actin Assembly in Activating Platelets but Reverses Platelet Aggregation

Platelet stimulation by thrombin leads to the activation of phosphoinositide 3-kinase (PI 3K) and to the production of the D3 phosphoinositides, phosphatidylinositol 3,4-bisphosphate (PdtIns-3,4P2) and 3,4,5-trisphosphate (PdtIns-3,4,5-P3). Because changes in the levels of these phosphoinositides correlate with the kinetics of actin assembly, they have been proposed to mediate actin assembly, causing cell shape changes. Wortmannin and LY294002, two unrelated inhibitors of PI 3-K, were used to investigate the role of PI 3-K in platelet actin assembly and aggregation. Both PI 3-K inhibitors abrogated the production of PdtIns-3,4-P2 and PdtIns-3,4,5-P3 in thrombin receptor-activating peptide (TRAP)-stimulated cells. However, neither wortmannin nor LY294002 altered the kinetics of actin assembly or the exposure of nucleation sites in TRAP-stimulated cells. In contrast, PI 3-K inhibitors showed a specific inhibitory pattern of cell aggregation, characterized by a primary phase of aggregation followed by progressive disaggregation. Flow cytometry analysis with the PAC1 monoclonal antibody or with FITC-labeled fibrinogen indicated that wortmannin inhibited the maintenance of the platelet integrin GPIIb-IIIa in its active state. Wortmannin also inhibited, in a dose-dependent manner, platelet aggregation induced by the binding of the monoclonal antibodies P256 and LIBS-6 to GPIIb-IIIa. LIBS Fab-induced aggregation also led to the production of PdtIns-3,4-P2. Platelet secretion, as evidenced by the release of preloaded 14C-5-hydroxy-tryptamine secretion or P-selectin up-regulation, was not affected by PI 3-K inhibition. These results demonstrate that the generation of D3 phosphoinositides is not required for actin assembly in TRAP-activated platelets. However, PI 3-K stimulation is necessary for prolonged GPIIb-IIIa activation and irreversible platelet aggregation. PI 3-K stimulation downstream of GPIIb-IIIa engagement may provide positive feedback required to sustain active GPIIb-IIIa.