Phosphoinositide 3-kinase Target Research Articles

Activation of PI3K‐Akt signaling pathway promotes prostate cancer cell invasion

Activated phosphoinositide 3-kinase (PI3K) and its downstream target Akt/PKB are important signaling molecules and key survival factors involved in the control of cell proliferation, apoptosis and oncogenesis. We investigated the role of the PI3K-Akt signaling pathway in the invasion of prostate cancer cell lines and activation of this pathway in primary human prostate tumors. Treatment of human prostate cancer cells viz. LNCaP, PC-3 and DU145 with PI3K pharmacological inhibitor, LY294002, potentially suppressed the invasive properties in each of these cell lines. Restoration of the PTEN gene to highly invasive prostate cancer PC-3 cells or expression of a dominant negative version of the PI3K target, Akt also significantly inhibited invasion and downregulated protein expression of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9, markers for cell invasion, indicating a central role of the PI3K-Akt pathway in this process. Immunoblot analysis of PI3K and total/activated levels of Akt showed increased protein levels of catalytic (p110alpha/beta) and regulatory (p85) subunits of PI3K and constitutive Akt activation in high-grade tumors compared to low-grade tumor and benign tissue. Immunohistochemical analyses further confirmed a progressive increase in p-Akt (p-Ser473) levels but not of total-Akt (Akt1/2) in cancer tissues compared to benign specimens. A successive increase in p-Akt expression was further noted in specimens serially obtained from individuals with time-course disease progression. Taken together, these results suggest that aberrant activation of PI3K-Akt pathway may contribute to increased cell invasiveness and facilitate prostate cancer progression.

NGF-dependent formation of ruffles in PC12D cells required a different pathway from that for neurite outgrowth

Two signaling pathways, phosphoinositide 3-kinase (PI-3k)/Akt and Ras/MAPK, are major effectors triggered by nerve growth factor (NGF). Rac1, Cdc42 and GSK-3β are reported to be targets of PI-3k in the signal transduction for neurite outgrowth. Immediately after NGF was added, broad ruffles were observed temporarily around the periphery of PC12 cells prior to neurite growth. As PC12D cells are characterized by a very rapid extension of neurites in response to various agents, the signaling pathways described above were studied in relation to the NGF-induced formation of ruffles and outgrowth of neurites. Wortmannin, an Akt inhibitor (V), and GSK-3β inhibitor (SB425286) suppressed the neurite growth in NGF-treated cells, but not in dbcAMP-treated cells. The outgrowth of neurites induced by NGF but not by dbcAMP was inhibited with the expression of mutant Ras. But upon the expression of dominant-negative Rac1, cells often extended protrusions, incomplete neurites, lacking F-actin. Intact neurites were observed in cells with dominant-negative Cdc42. These results suggest that NGF-dependent neurite outgrowth occurs via a mechanism involving activation of the Ras/PI-3K/Akt/GSK-3β pathway, while dbcAMP-dependent neurite growth might be induced in a distinct manner. However, inhibitors for GSK-3β and PI-3k (wortmannin) did not suppress the NGF-dependent formation of ruffles. In addition, the formation of ruffles was not inhibited by the expression of mutant Ras. On the other hand, it was suppressed by the expression of dominant-negative Rac1 or Cdc42. These results suggest that the NGF-induced ruffling requires activation of Rac1 and Cdc42, but does not require Ras, PI-3k, Akt and GSK-3β. Taken together, the NGF-dependent formation of ruffles might not require Ras/PI-3k/Akt/GSK-3β, but these pathways might contribute to the formation of intact neurites due to combined actions including Rac1.

D2Receptors Regulate Dopamine Transporter Function via an Extracellular Signal-Regulated Kinases 1 and 2-Dependent and Phosphoinositide 3 Kinase-Independent Mechanism

The dopamine transporter (DAT) terminates dopamine (DA) neurotransmission by reuptake of DA into presynaptic neurons. Regulation of DA uptake by D(2) dopamine receptors (D(2)R) has been reported. The high affinity of DA and other DAT substrates for the D(2)R, however, has complicated investigation of the intracellular mechanisms mediating this effect. The present studies used the fluorescent DAT substrate, 4-[4-(diethylamino)-styryl]-N-methylpyridinium iodide (ASP(+)) with live cell imaging techniques to identify the role of two D(2)R-linked signaling pathways, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and phosphoinositide 3 kinase (PI3K) in mediating D(2)R regulation of DAT. Addition of the D(2)/D(3) receptor agonist quinpirole (0.1-10 muM) to human embryonic kidney cells coexpressing human DAT and D(2) receptor (short splice variant, D(2S)R) induced a rapid, concentration-dependent and pertussis toxin-sensitive increase in ASP(+) accumulation. The D(2)/D(3) agonist (S)-(+)-(4aR, 10bR)-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin-9-ol hydrochloride (PD128907) also increased ASP(+) accumulation. D(2S)R activation increased phosphorylation of ERK1/2 and Akt, a major target of PI3K. The mitogen-activated protein kinase kinase inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) prevented the quinpirole-evoked increase in ASP(+) accumulation, whereas inhibition of PI3K was without effect. Fluorescence flow cytometry and biotinylation studies revealed a rapid increase in DAT cell-surface expression in response to D(2)R stimulation. These experiments demonstrate that D(2S)R stimulation increases DAT cell surface expression and therefore enhances substrate clearance. Furthermore, they show that the increase in DAT function is ERK1/2-dependent but PI3K-independent. Our data also suggest the possibility of a direct physical interaction between DAT and D(2)R. Together, these results suggest a novel mechanism by which D(2S)R autoreceptors may regulate DAT in the central nervous system.

A selective inhibitor of the p110δ isoform of PI 3-kinase inhibits AML cell proliferation and survival and increases the cytotoxic effects of VP16

Current therapy for acute myeloid leukaemia (AML) is suboptimal with a high incidence of relapse. There is strong evidence that constitutive phosphoinositide 3-kinase (PI3K) activity plays a significant role in the pathophysiology of AML. PI3K products are derived from the activity of a number of PI3K catalytic isoforms (class I, II and III) but the relative contribution of these enzymes in AML remains unknown. As non-isoform-selective inhibitors of PI3K such as LY294002 may produce unwanted toxicity to normal tissues, we have investigated the role of the leukocyte-restricted p110delta PI3K isoform in 14 cases of AML. p110delta was detected in all cases whereas the expression levels of the other class I PI3Ks varied more widely, and were often undetectable. The p110delta-selective compound IC87114 inhibited constitutive phosphorylation of the PI3K target Akt/PKB and reduced cell number to a mean of 66+/-5% (range 14-88%). In eight cases, the combination of IC87114 and VP16 (a topoisomerase II inhibitor) was synergistic in reducing viable cell number, and was associated with a reduction in constitutive NF-kappaB activity. IC87114 did not have direct adverse effects or enhance the activity of VP16 on the proliferation and survival of normal haemopoietic progenitors. Overall, our results identify the p110delta isoform as a potential therapeutic target in AML and support a clinical approach to use isoform-selective over broad-spectrum PI3K inhibitors.

Competitive displacement of phosphoinositide 3-kinase from β-adrenergic receptor kinase-1 improves postinfarction adverse myocardial remodeling

Adverse remodeling after myocardial infarction (MI) determines the progression of heart failure. Failing hearts are characterized by downregulation of beta-adrenergic receptor (beta-AR) signaling in part because of increased beta-AR kinase 1 activity. Our previous studies have shown that overexpression of the phosphoinositide kinase (PIK) domain of phosphoinositide 3-kinase (PI3K), prevents beta-AR downregulation and enhances adrenergic agonist responsiveness by inhibiting the targeting of PI3K to the beta-AR complex. To investigate whether preventing beta-AR downregulation in the heart ameliorates cardiac function post-MI, transgenic mice with cardiac-specific overexpression of the PIK domain peptide (TgPIK) underwent left coronary artery ligation and were subsequently followed by serial echocardiography at 4, 8, 12, 16, and 20 wk. Despite having similar infarction sizes, TgPIK mice showed better systolic function, less cardiac dilatation, and improved hemodynamic response to dobutamine compared with littermate controls after MI. To test that displacement of PI3K from the beta-AR complex, but not the total loss of PI3K-gamma, is critical for amelioration of cardiac function, mice lacking the PI3K-gamma (PI3K-gamma-KO) underwent MI, and their cardiac function was assessed 20 wk post-MI. Serial echocardiographic measurements showed severe reduction in contractile performance in PI3K-gamma-KO compared with TgPIK mice. Furthermore, significant beta-AR downregulation and desensitization were only seen in infarcted wild-type and PI3K-gamma-KO mice and not in TgPIK mice. Together, these results demonstrate that adverse remodeling of the ventricle after MI can be attenuated by a strategy that prevents recruitment of PI3K to the plasma membrane and restores normal beta-AR function.

New Players in TLR-Mediated Innate Immunity: PI3K and Small Rho GTPases

Toll-like receptors (TLRs) play a crucial role in the innate immune system as a first line of defense against pathogens. TLR activation in phagocytes produces pro-inflammatory cytokines and chemokines that contribute directly to elimination of infectious agents and activation of adaptive immune responses. However, a sustained inflammatory response can result in tissue damage and generalized sepsis. This review summarizes the complex and sometimes conflicting links of TLR signaling with two important regulators of immune cells functions: phosphoinositide 3-kinases (PI3Ks) and small GTPases of the Rho family. A unified model of hierarchical organization of these signaling participants is still premature, given that the tools for delineating how control of TLRmediated pathways is achieved are just emerging. Critical progress in our understanding of spatial-temporal propagation of TLR signaling will certainly be provided in the near future by pharmacological targeting of PI3Ks using recently characterized, second-generation PI3K inhibitors in combination with gene-targeting strategies for PI3K subunits and Rho GTPases targeted to the murine myeloid compartment.

Phosphoinositide 3-Kinase Accelerates Postoperative Tumor Growth by Inhibiting Apoptosis and Enhancing Resistance to Chemotherapy-induced Apoptosis: NOVEL ROLE FOR AN OLD ENEMY

Tumor removal remains the principal treatment modality in the management of solid tumors. The process of tumor removal may potentiate the resurgent growth of residual neoplastic tissue. Herein, we describe a novel murine model in which flank tumor cytoreduction is followed by accelerated local tumor recurrence. This model held for primary and recurrent tumors generated using a panel of human and murine (LS174T, DU145, SW480, SW640, and 3LL) cell lines and replicated accelerated tumor growth following excisional surgery. In investigating this further, epithelial cells were purified from LS174T primary and corresponding recurrent tumors for comparison. Baseline as well as tumor necrosis factor apoptosis-inducing ligand (TRAIL)-induced apoptosis were significantly reduced in recurrent tumor epithelia. Primary and recurrent tumor gene expression profiles were then compared. This identified an increase and reduction in the expression of p110gamma and p85alpha class Ia phosphoinositide 3-kinase (PI3K) subunits in recurrent tumor epithelia. These changes were further confirmed at the protein level. The targeting of PI3K ex vivo, using LY294002, restored sensitivity to TRAIL in recurrent tumor epithelia. In vivo, adjuvant LY294002 prolonged survival and significantly attenuated recurrent tumor growth by greatly enhancing apoptosis levels. Hence, PI3K plays a role in generating the antiapoptotic and chemoresistant phenotype associated with accelerated local tumor recurrence.

Relaxin Stimulates Multiple Signaling Pathways: Activation of cAMP, PI3K, and PKCζ in THP‐1 Cells

Relaxin has been shown previously to stimulate cyclic AMP production and the activation of MAPK. We reported that phosphoinositide-3 kinase (PI3K) activity is required for biphasic stimulation of cAMP by relaxin and that relaxin treatment increased PI3K activity in THP-1 cells. A downstream target of PI3K is protein kinase C zeta (PKCzeta). Relaxin stimulated translocation of PKCzeta to the plasma membrane in THP-1, MCF-7, pregnant human myometrial (PHM1-31), and mouse mesangial (MMC) cells. PKCzeta translocation is PI3K dependent and independent of cAMP production. Pharmacological and antisense approaches, utilized to inhibit or knock down PKCzeta, resulted in a 40% inhibition of relaxin-stimulated cAMP production. The stimulation of PKCzeta by relaxin therefore is downstream of PI3K leading to increased cAMP production. To determine the role of PI3K/PKCzeta stimulation by relaxin on downstream-mediated events, we examined the increase in vascular endothelial growth factor (VEGF) gene expression by relaxin. Treatment of THP-1 or MMC cells with the PI3K inhibitor, LY294002, abolished the relaxin-mediated stimulation of VEGF transcript levels. In summary, relaxin has pleiotropic signaling effects in THP-1 cells activating ERK1/2, cAMP, PI3K, and PKCzeta. We have described a novel bifurcated pathway by which relaxin stimulates Gs alpha and PI3K/PKCzeta leading to increased cAMP production and increased VEGF gene expression. Some, but not all, of these pathways are detected in other cell lines which may cause the unique diversity of downstream responses from this interesting hormone.

Regulation of Interleukin-5–Induced β2-Integrin Adhesion of Human Eosinophils by Phosphoinositide 3-Kinase

We examined the role of phosphoinositide 3-kinase (PI3K) in integrin-mediated eosinophil adhesion. Deltap85, a dominant-negative form of the class IA PI3K adaptor subunit, was fused to an HIV-TAT protein transduction domain (TAT-Deltap85). Recombinant TAT-Deltap85 inhibited interleukin (IL)-5-stimulated phosphorylation of protein kinase B, a downstream target of PI3K. beta(2)-Integrin-dependent adhesion caused by IL-5 to the plated intracellular adhesion molecule-1 surrogate, bovine serum albumin, was inhibited by TAT-Deltap85 in a concentration-dependent manner. Similarly, two PI3K inhibitors, wortmannin and LY294002, blocked eosinophil adhesion to plated bovine serum albumin. By contrast, beta(1)-integrin-mediated eosinophil adhesion to vascular cell adhesion moelcule-1 was not blocked by TAT-Deltap85, wortmannin, or LY294002. Rottlerin, a protein kinase C (PKC)-delta inhibitor, also blocked beta(2)-integrin adhesion of eosinophils caused by IL-5, whereas beta(1) adhesion to vascular cell adhesion molecule-1 was not affected. IL-5 caused translocation of PKCdelta from the cytosol to cell membrane; inhibition of PI3K by wortmannin blocked translocation of PKCdelta. Western blot analysis demonstrated that extracellular signal-regulated kinase phosphorylation, a critical intermediary in adhesion elicited by IL-5, was blocked by inhibition of either PI3K or PKC-delta. These data suggest that extracellular signal-regulated kinase-mediated adhesion of beta(2)-integrin caused by IL-5 is mediated in human eosinophils by a class IA PI3K through activation of a PKCdelta pathway.

Platelet-activating factor induces cardioprotection in isolated rat heart akin to ischemic preconditioning: role of phosphoinositide 3-kinase and protein kinase C activation

Ischemic preconditioning (IP) is a cardioprotective mechanism against myocellular death and cardiac dysfunction resulting from reperfusion of the ischemic heart. At present, the precise list of mediators involved in IP and the pathways of their mechanisms of action are not completely known. The aim of the present study was to investigate the role of platelet-activating factor (PAF), a phospholipid mediator that is known to be released by the ischemic-reperfused heart, as a possible endogenous agent involved in IP. Experiments were performed on Langendorff-perfused rat hearts undergoing 30 min of ischemia followed by 2 h of reperfusion. Treatment with a low concentration of PAF (2 x 10(-11) M) before ischemia reduced the extension of infarct size and improved the recovery of left ventricular developed pressure during reperfusion. The cardioprotective effect of PAF was comparable to that observed in hearts in which IP was induced by three brief (3 min) periods of ischemia separated by 5-min reperfusion intervals. The PAF receptor antagonist WEB-2170 (1 x 10(-9) M) abrogated the cardioprotective effect induced by both PAF and IP. The protein kinase C (PKC) inhibitor chelerythrine (5 x 10(-6) M) or the phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 (5 x 10(-5) M) also reduced the cardioprotective effect of PAF. Western blot analysis revealed that following IP treatment or PAF infusion, the phosphorylation of PKC-epsilon and Akt (the downstream target of PI3K) was higher than that in control hearts. The present data indicate that exogenous applications of low quantities of PAF induce a cardioprotective effect through PI3K and PKC activation, similar to that afforded by IP. Moreover, the study suggests that endogenous release of PAF, induced by brief periods of ischemia and reperfusion, may participate to the triggering of the IP of the heart.

Thrombopoietin-Mediated Activation of the mTOR/S6K/4E-BP Signaling Pathway Is Required for Proliferation in Megakaryocyte Progenitors.

Translational regulation plays a central role in cell proliferation, survival and cell differentiation through activation of the target of rapamycin (mTOR) signaling pathway. mTOR controls the phosphorylation status of proteins involved in initiating translational control, including ribosomal S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4E-BP). Recently, the mTOR and phosphoinositide 3-kinase (PI3-K) pathways have been linked through the tumor suppressor complex TSC1/2. The PI3-K target PKB inactivates the TSC1/2 complex which acts as a GAP for the Ras homologue Rheb to suppress mTOR signaling. As the regulation of cell number and cell size are important factors during megakaryopoiesis, we investigated the role of mTOR signaling in thrombopoietin (TPO) induced proliferation and differentiation in primary human megakaryocyte progenitors and in megakaryoblastic MO7e cells. Preincubation of isolated CD34+ cells, primary cultured CD61+ cells and MO7e cells with the mTOR inhibitor rapamycin (10 nM) resulted in highly specific inhibition of TPO-mediated 4E-BP1 (S65) phosphorylation, S6K (T389) phosphorylation and its substrate S6 (S235/236), without affecting PKB (S473) phosphorylation. Activation of the mTOR signaling pathway by TPO was dependent on the PI3-K pathway as LY294002 (10 μM) inhibited phosphorylation of 4E-BP1, S6K and S6. Treatment of MO7e cells with rapamycin inhibited TPO-induced proliferation and cell cycling by reducing cells in S-phase and blocking cells in G1. Rapamycin did not induce apoptosis as measured by cells in sub-G0 phase and by Annexin V expression. Suspension cultures of CD34+ cells treated with rapamycin resulted in a 2.3-fold reduction in overall cell proliferation (p=0.01) and a reduction in the percentage of CD61+ megakaryocytic cells (55.2±7.93 vs 35.0±5.42, p=0.08) generated after 7 days. The mean fluorescence intensity of CD61 and CD42 expressing cells was however not decreased. In addition, polyploidisation levels of the CD61 expressing cells cultured in the presence of rapamycin were not decreased, indicating rapamycin inhibited megakaryocyte proliferation, not differentiation. Further analysis revealed that transforming growth factor β1 (TGF-β1), which inhibits proliferation of megakaryocyte progenitors, downregulated TPO-induced S6K/S6 phosphorylation. Concluding, we have shown that the mTOR pathway is activated by TPO and plays a role in regulating proliferation in megakaryocyte progenitors. Part of the effect of the PI3-K/PKB and TGF-β pathways in regulating proliferation may be mediated by the mTOR/S6K/4E-BP1 signaling pathway.

Phosphoinositide 3-kinases as targets for therapeutic intervention.

Phosphoinositide 3-kinases (PI3K) represent a family of intracellular signaling proteins, which control a variety of important cellular functions such as proliferation, apoptosis and migration. Recent findings suggest an involvement of PI3K in the pathogenesis of numerous diseases including cancer, heart failure and autoimmune/inflammatory disorders. This review summarizes the current knowledge of the emerging therapeutic value of PI3K as targets for the intervention of several pathological disorders and in particular focusing on oncogenesis. A brief introduction on the molecular and biochemical features of these signaling proteins will be followed by a depiction of signaling interactions of PI3K in a cellular context. PI3K dependent signaling involved in the control of cell growth, proliferation, survival and cytoskeletal remodeling and the link to cellular dysfunctions will be discussed. Further we will summarize the phenotypic consequences by genetic targeting PI3K signaling in mice. In its final part this review outlines challenges and activities considering PI3K as targets for therapeutic intervention and progress in the development of first generation small molecule inhibitors.

Phosphatases as small-molecule targets: inhibiting the endogenous inhibitors of kinases.

Inositols and their phosphorylated derivatives, phosphoinositides, play an important role in diverse cellular functions. They have been recognized as second messengers and are accurately controlled by phosphatases and kinases, such as phosphoinositide 3-kinase and the phosphatidylinositol 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10). Specific inhibitors targeting phosphoinositide 3-kinase and protein phosphatases have been described and characterized, but no small-molecule tools for phosphoinositide phosphatases are currently available. The present mini-review gives an overview of representative phosphatase inhibitors and summarizes the work that has been done recently on molecules that inhibit PTEN.

Role of phospholipase D1 in the regulation of mTOR activity by lysophosphatidic acid.

Mitogens activate protein translation through phosphorylation of p7S6 kinase (p70(S6K)) and eIF4E binding protein 1 (4E-BP1) mediated by the mammalian target of rapamycin (mTOR) or phosphoinositide 3-kinase (PI3K). A recent report (Science 294, 1942, 2001) has implicated phospholipase D (PLD) in mTOR signaling. We studied the role of PLD in the phosphorylation of p70(S6K) and 4E-BP1 induced by lysophosphatidic acid (LPA) and platelet-derived growth factor (PDGF) using fibroblasts deficient in PLD activity and also 1-butanol, which inhibits phosphatidic acid production by PLD. The reduction in PLD activity in both situations impaired the effect of LPA on mTOR signaling but did not inhibit the effect of PDGF. PDGF induced marked phosphorylation of Akt (a PI3K target) but this was not affected by PLD deficiency. LPA caused much less phosphorylation of Akt and this was dependent on PLD activity. Toxin B, which inactivates Rho GTPases, markedly impaired PLD1 activation and phosphorylation of Akt, p70(S6K), and 4E-BP1 induced by LPA but had a minimal or no effect on the actions of PDGF. These results support the hypothesis that LPA activates protein translation through the action of PLD1-generated PA on mTOR and the PI3K/Akt pathway whereas PDGF acts through P13K/Akt independent of PLD1.

Blockade of Inflammation and Airway Hyperresponsiveness in Immune-sensitized Mice by Dominant-Negative Phosphoinositide 3-Kinase–TAT

Phosphoinositide 3-kinase (PI3K) is thought to contribute to the pathogenesis of asthma by effecting the recruitment, activation, and apoptosis of inflammatory cells. We examined the role of class IA PI3K in antigen-induced airway inflammation and hyperresponsiveness by i.p. administration into mice of Δp85 protein, a dominant negative form of the class IA PI3K regulatory subunit, p85α, which was fused to HIV-TAT (TAT-Δp85). Intraperitoneal administration of TAT-Δp85 caused time-dependent transduction into blood leukocytes, and inhibited activated phosphorylation of protein kinase B (PKB), a downstream target of PI3K, in lung tissues in mice receiving intranasal FMLP. Antigen challenge elicited pulmonary infiltration of lymphocytes, eosinophils and neutrophils, increase in mucus-containing epithelial cells, and airway hyperresponsiveness to methacholine. Except for modest airway neutrophilia, these effects all were blocked by treatment with 3–10 mg/kg of TAT-Δp85. There was also significant reduction in IL-5 and IL-4 secretion into the BAL. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by systemic pretreatment with TAT-Δp85. We conclude that PI3K has a regulatory role in Th2-cell cytokine secretion, airway inflammation, and airway hyperresponsiveness in mice.

Activation of group IV cytosolic phospholipase A2 in human eosinophils by phosphoinositide 3-kinase through a mitogen-activated protein kinase-independent pathway.

Activation of group IV cytosolic phospholipase A(2) (gIV-PLA(2)) is the essential first step in the synthesis of inflammatory eicosanoids and in integrin-mediated adhesion of leukocytes. Prior investigations have demonstrated that phosphorylation of gIV-PLA(2) results from activation of at least two isoforms of mitogen-activated protein kinase (MAPK). We investigated the potential role of phosphoinositide 3-kinase (PI3K) in the activation of gIV-PLA(2) and the hydrolysis of membrane phosphatidylcholine in fMLP-stimulated human blood eosinophils. Transduction into eosinophils of Deltap85, a dominant negative form of class IA PI3K adaptor subunit, fused to an HIV-TAT protein transduction domain (TAT-Deltap85) concentration dependently inhibited fMLP-stimulated phosphorylation of protein kinase B, a downstream target of PI3K. FMLP caused increased arachidonic acid (AA) release and secretion of leukotriene C(4) (LTC(4)). TAT-Deltap85 and LY294002, a PI3K inhibitor, blocked the phosphorylation of gIV-PLA(2) at Ser(505) caused by fMLP, thus inhibiting gIV-PLA(2) hydrolysis and production of AA and LTC(4) in eosinophils. FMLP also caused extracellular signal-related kinases 1 and 2 and p38 MAPK phosphorylation in eosinophils; however, neither phosphorylation of extracellular signal-related kinases 1 and 2 nor p38 was inhibited by TAT-Deltap85 or LY294002. Inhibition of 1) p70 S6 kinase by rapamycin, 2) protein kinase B by Akt inhibitor, or 3) protein kinase C by Ro-31-8220, the potential downstream targets of PI3K for activation of gIV-PLA(2), had no effect on AA release or LTC(4) secretion caused by fMLP. We find that PI3K is required for gIV-PLA(2) activation and hydrolytic production of AA in activated eosinophils. Our data suggest that this essential PI3K independently activates gIV-PLA(2) through a pathway that does not involve MAPK.

Sphingosine-1-phosphate stimulates smooth muscle cell migration through gα i- and pi3-kinase-dependent p38 mapk activation

Background Sphingosine-1-phosphate (S-1-P) is an extracellular mediator released in response to vessel injury. S-1-P binds to G-protein-coupled receptors, which can be Gα i-, Gα q-, or G 12/13-linked. This study examines the role of p38 mitogen-activated protein kinase (p38 MAPK) in vascular smooth muscle cell migration after stimulation with S-1-P, and pathways leading to p38 MAPK activation. S-1-P has previously been shown to stimulate migration of vascular smooth muscle cells (VSMCs) in vitro through ERK1/2 and G i. We hypothesized that S-1-P-induced VSMC migration is also dependent on p38 MAPK activation through a G i-coupled extracellular receptor and phosphoinositide 3-kinase (PI3-K). Methods VSMCs were cultured in vitro. A linear wound assay was performed in the presence of S-1-P and inhibitors of p38 MAPK (SB203580) or epidermal growth factor (EGF) receptor kinase (AG1478). Chemotaxis stimulated by S-1-P was also assayed in a modified Boyden chamber with and without SB203580 pretreatment. Western blotting was performed to examine p38 MAPK activation in response to S-1-P with and without SB203580, AG1478, or inhibitors of G i (pertussis toxin), PI3-K (Wortmannin and LY294002), or MEK1 (PD98059). Western blotting and immunoprecipitation for targets of p38 MAPK (MAPKAP kinase-2) and PI3-K (Akt) were also performed. Results S-1-P stimulated migration of VSMCs in both wound and Boyden transwell assays. This migration was inhibited by SB203580 to the level of control, whereas AG478 had no effect. S-1-P stimulated activation of p38 MAPK that peaked at 10 min, as well as activation of MAPKAP kinase-2. Activation of p38 MAPK was significantly inhibited by SB203580, pertussis toxin, Wortmannin, and LY294002, but not by PD98059 or AG1478; MAPKAP kinase-2 activation was inhibited by SB203580. Akt was activated by S-1-P at 3 to 5 min; this response was inhibited by Wortmannin and LY294002, but not by SB203580 or pertussis toxin. Conclusions S-1-P induced VSMC migration through a G i-linked and a PI3-K coupled, p38 MAPK- dependent process. PI3-K appears to function upstream of p38 MAPK, but was not G i-dependent. S-1-P-stimulated activation of p38 MAPK does not signal via transactivation of the EGF receptor. Understanding signal transduction will allow targeted molecular interventions to treat the response of a vessel to injury.

Critical role for scaffolding adapter Gab2 in Fc gamma R-mediated phagocytosis.

Grb2-associated binder 2 (Gab2), a member of the Dos/Gab subfamily scaffolding molecules, plays important roles in regulating the growth, differentiation, and function of many hematopoietic cell types. In this paper, we reveal a novel function of Gab2 in Fcγ receptor (FcγR)–initiated phagocytosis in macrophages. Upon FcγR activation, Gab2 becomes tyrosyl phosphorylated and associated with p85, the regulatory subunit of phosphoinositide 3-kinase (PI3K), and the protein–tyrosine phosphatidylinositol Shp-2. FcγR-mediated phagocytosis is severely impaired in bone marrow–derived macrophages from Gab2−/− mice. The defect in phagocytosis correlates with decreased FcγR-evoked activation of Akt, a downstream target of PI3K. Using confocal fluorescence microscopy, we find that Gab2 is recruited to the nascent phagosome, where de novo PI3K lipid production occurs. Gab2 recruitment requires the pleckstrin homology domain of Gab2 and is sensitive to treatment with the PI3K inhibitor wortmannin. The Grb2 binding site on Gab2 also plays an auxiliary role in recruitment to the phagosome. Because PI3K activity is required for FcγR-mediated phagocytosis, our results indicate that Gab2 acts as a key component of FcγR-mediated phagocytosis, most likely by amplifying PI3K signaling in the nascent phagosome.

Functions of PI 3-kinase in development of the nervous system

In the nervous system, receptor regulated phosphoinositide (PI) 3-kinases (PI 3-kinases) participate in fundamental cellular activities that underlie development. Activated by trophic factors, growth factors, neuregulins, cytokines, or neurotransmitters, PI 3-kinases have been implicated in neuronal and glial survival and differentiation. PI 3-kinases produce inositol lipid second messengers that bind to pleckstrin homology (PH) domains in diverse groups of signal transduction proteins, and control their enzymatic activities, subcellular membrane localization, or both. Downstream targets of the inositol lipid messengers include protein kinases and regulators of small GTPases. The kinase Akt/PKB functions as a key component of the PI 3-kinase dependent survival pathway through its phosphorylation and regulation of apoptotic proteins and transcription factors. Furthermore, since members of the Rho GTPase and Arf GTPase families have been implicated in regulation of the actin cytoskeleton, vesicular trafficking, and transcription, the downstream targets of PI 3-kinase that control these GTPases are excellent candidates to mediate aspects of PI 3-kinase dependent neuronal and glial differentiation.

Akt/protein kinase B promotes organ growth in transgenic mice.

One of the least-understood areas in biology is the determination of the size of animals and their organs. In Drosophila, components of the insulin receptor phosphoinositide 3-kinase (PI3K) pathway determine body, organ, and cell size. Several biochemical studies have suggested that Akt/protein kinase B is one of the important downstream targets of PI3K. To examine the role of Akt in the regulation of organ size in mammals, we have generated and characterized transgenic mice expressing constitutively active Akt (caAkt) or kinase-deficient Akt (kdAkt) specifically in the heart. The heart weight of caAkt transgenic mice was increased 2.0-fold compared with that of nontransgenic mice. The increase in heart size was associated with a comparable increase in myocyte cell size in caAkt mice. The kdAkt mutant protein attenuated the constitutively active PI3K-induced overgrowth of the heart, and the caAkt mutant protein circumvented cardiac growth retardation induced by a kinase-deficient PI3K mutant protein. Rapamycin attenuated caAkt-induced overgrowth of the heart, suggesting that the mammalian target of rapamycin (mTOR) or effectors of mTOR mediated caAkt-induced heart growth. In conclusion, Akt is sufficient to induce a marked increase in heart size and is likely to be one of the effectors of the PI3K pathway in mediating heart growth.