Phosphotidylinositol 3 kinase 92E


Table of contents

Signaling in the PI3K pathway: PI3K, the cell cycle, cell survival, and apoptosis

PI 3-kinase has emerged as a key enzyme for regulating neuronal cell survival. However, it has not as yet been demonstrated whether activation of the endogenous pool of the enzyme, that is regulated by the p85 subunit, is sufficient to promote a survival response. It is also not known whether the FGF family of growth factors promote survival via a PI 3-kinase-dependent pathway. A cell permeable p85 binding peptide has been developed; it is able to stimulate a mitogenic response in muscle cells that is dependent on a PI 3-kinase/p70 S6 kinase pathway. This peptide can rescue cerebellar granule cells from death induced by serum deprivation and this response is comparable to a growth factor response (FGF2). Experiments with wortmannin, LY294002, and rapamycin suggest that the peptide survival response is dependent on PI 3-kinase activity, but not p70 S6 kinase activity. The peptide response is correlated with a PI 3-kinase-dependent phosphorylation of Akt, an established downstream effector in the PI 3-kinase survival cascade. In contrast to the survival response stimulated by the p85 binding peptide, the response stimulated by FGF2 is not inhibited by wortmannin or LY294002, nor is it associated with phosphorylation of Akt. Thus it is concluded that activation of the endogenous pool of PI 3-kinase that is regulated by p85 is sufficient for cell survival; however, growth factors such as FGF2 can clearly support survival in a PI 3-kinase-independent manner (Williams, 1999).

Phosphatidylinositol (PI) 3-kinase is required for G1 to S phase cell cycle progression stimulated by a variety of growth factors and is implicated in the activation of several downstream effectors, including p70(S6K). However, the molecular mechanisms by which PI 3-kinase is engaged in activation of the cell cycle machinery are not well understood. The expression of a dominant negative (DN) form of either the p110alpha catalytic or the p85 regulatory subunit of heterodimeric PI 3-kinase strongly inhibits epidermal growth factor (EGF)-induced upregulation of cyclin D1 protein in NIH 3T3(M17) fibroblasts. The PI 3-kinase inhibitors LY294002 and wortmannin completely abrogate increases in both mRNA and protein levels of cyclin D1 and phosphorylation of pRb, inducing G1 arrest in EGF-stimulated cells. By contrast, rapamycin, which potently suppresses p70(S6K) activity throughout the G1 phase, has little inhibitory effect, if any, on either of these events. PI 3-kinase, but not rapamycin-sensitive pathways, is also indispensable for upregulation of cyclin D1 mRNA and protein by other mitogens in NIH 3T3 (M17) cells and in wild-type NIH 3T3 cells as well. An enforced expression of wild-type p110 is sufficient to induce cyclin D1 protein expression in growth factor-deprived NIH 3T3(M17) cells. The p110 induction of cyclin D1 in quiescent cells is strongly inhibited by coexpression of either of the PI 3-kinase DN forms, and by LY294002, but is independent of the Ras-MEK-ERK pathway. Unlike mitogen stimulation, the p110 induction of cyclin D1 is sensitive to rapamycin. These results indicate that the catalytic activity of PI 3-kinase is necessary, and could also be sufficient, for upregulation of cyclin D1, with mTOR (mammalian target of rapamycin) signaling being differentially required, depending upon cellular conditions (Takuwa, 1999).

In T lymphocytes, the hematopoietic cytokine interleukin-2 (IL-2) uses phosphatidylinositol 3-kinase (PI 3-kinase)-induced signaling pathways to regulate E2F transcriptional activity, a critical cell cycle checkpoint. PI 3-kinase also regulates the activity of p70(s6k: see Drosophila RPS6-p70-protein kinase), the 40S ribosomal protein S6 kinase, a response that is abrogated by the macrolide rapamycin. This immunosuppressive drug is known to prevent T-cell proliferation, but the precise point at which rapamycin regulates T-cell cycle progression has yet to be elucidated. Moreover, the effects of rapamycin on IL-2 and PI 3-kinase activation of E2Fs have not been characterized; neither has the role of p70(s6k) in such activation. The present results show that IL-2- and PI 3-kinase-induced pathways for the regulation of E2F transcriptional activity include both rapamycin-resistant and rapamycin-sensitive components. Expression of a rapamycin-resistant mutant of p70(s6k) in T cells can restore rapamycin-suppressed E2F responses. Thus, the rapamycin-controlled processes involved in E2F regulation appear to be mediated by p70(s6k). However, the rapamycin-resistant p70(s6k) can not rescue rapamycin inhibition of T-cell cycle entry, consistent with the involvement of additional, rapamycin-sensitive pathways in the control of T-cell cycle progression. The present results thus show that p70(s6k) is able to regulate E2F transcriptional activity and provide direct evidence for the first time for a link between IL-2 receptors, PI 3-kinase, and p70(s6k) that regulate a crucial G1 checkpoint in T lymphocytes (Brennan, 1999).

Activation of the caspase proteases by c-Jun N-terminal kinase 1 (JNK1) has been proposed as a mechanism of apoptotic cell death. Insulin activates caspase-3 by a pathway requiring phosphatidylinositol 3'-kinase (PI3-kinase). JNK1 assays demonstrate that insulin treatment of myeloma cells induces 3-fold activation of JNK1. Inhibition of PI3-kinase with wortmannin and LY294002 blocks insulin-dependent activation of JNK1. Caspase assays demonstrate that insulin increases caspase-3 activity 3-fold and that inhibition of PI3-kinase blocks this effect. Cell death is doubled by insulin and is due to a 3-fold increase in apoptosis of cells in the G1/G0 phase of the cell cycle. Inhibition of PI3-kinase completely blocks this effect. Finally, inhibition of caspase-3 with benzyloxycarbonyl-Asp-2,6-dichlorobenzoyloxymethylketone blocks cell death due to insulin. Taken together, these findings indicate that insulin activates caspase-3 by a PI3-kinase-dependent pathway resulting in increased apoptosis and cell death (Godbout, 1999).

An examination was carried out of the effects of two insulin-like growth factors, insulin and insulin-like growth factor-I (IGF-I), against apoptosis, excitotoxicity, and free radical neurotoxicity in cortical cell cultures. Like IGF-I, insulin attenuates serum deprivation-induced neuronal apoptosis in a dose-dependent manner at 10-100 ng/mL. The anti-apoptosis effect of insulin against serum deprivation disappears upon addition of a broad protein kinase inhibitor, staurosporine, but not by calphostin C, a selective protein kinase C inhibitor. Addition of PD98059, a mitogen-activated protein kinase kinase (MAPKK) inhibitor, blocks insulin-induced activation of extracellular signal-regulated protein kinases (ERK1/2) without altering the neuroprotective effect of insulin. Cortical neurons undergo activation of phosphatidylinositol (PI) 3-kinase as early as 1 min after exposure to insulin. Inclusion of wortmannin or LY294002, selective inhibitors of PI 3-K, reverse the insulin effect against apoptosis. In contrast to the anti-apoptosis effect, neither insulin nor IGF-I protect excitotoxic neuronal necrosis following continuous exposure to 15 microM NMDA or 40 microM kainate for 24 h. Surprisingly, concurrent inclusion of 50 ng/mL insulin or IGF-I aggravates free radical-induced neuronal necrosis over 24 h following continuous exposure to 10 microM Fe2+ or 100 microM buthionine sulfoximine. Wortmannin or LY294002 also reverses this potentiation effect of insulin. These results suggest that insulin-like growth factors act as anti-apoptosis factors and pro-oxidants, depending upon the activation of PI 3-kinase (Ruy, 1999).

Withdrawal of trophic factors necessary for Schwann cell survival regulates Schwann cell number during development and after nerve injury. Signaling pathways involved in Schwann cell survival by prosaposin [the precursor of sphingolipid activator proteins (saposin A-D)], prosaptides (peptides incorporating the neurotrophic sequence of prosaposin), and insulinlike growth factor-I (IGF-I) have been identified. When postnatal Schwann cells are placed in low serum medium, cells undergo abrupt shrinkage, condensation of nuclei occurs, and smooth rounded apoptotic bodies appear. Dose-response studies of cell death, measured by lactate dehydrogenase (LDH) release, demonstrates that both prosaptide TX14(A) and IGF-I dose-dependently reduce cell death in primary Schwann cells. There is a 10- and 14-fold increase in apoptosis after 4 and 24 hr in low serum medium, respectively, that is reduced by prosaposin, TX14(A), or IGF-I. Phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin or LY294002, block the survival effects of both TX14(A) and IGF-I. In contrast, only TX14(A) anti-apoptotic activity is blocked by the MEK inhbitor, PD98059, although TX14(A) and IGF-I are potent activators of extracellular regulated kinases in Schwann cells. Phosphorylation of the PI3K signaling target, Akt, was measured; TX14(A) and IGF-I increase Akt activity by 12-fold and 22-fold, respectively; this is activity that is inhibited by LY294002. These findings indicate that prosaposin and IGF-I use the PI3K/Akt pathway to induce survival of Schwann cells (Campana, 1999).

Phosphatidylinositol 3'-kinase (PI 3-kinase) catalyzes the formation of 3' phosphoinositides and has been implicated in an intracellular signaling pathway that inhibits apoptosis in both neuronal and hemopoietic cells. Two potential downstream mediators of PI 3-kinase, the serine/threonine p70 S6-kinase (S6-kinase) and the antiapoptotic protein B cell lymphoma-2 (Bcl-2: Drosophila homolog death executioner Bcl-2 homologue) were investigated. Stimulation of factor-dependent cell progenitor (FDCP) cells with either IL-4 or insulin-like growth factor (IGF)-I induces a 10-fold increase in the activity of both PI 3-kinase and S6-kinase. Rapamycin blocks 90% of the S6-kinase activity but does not affect PI 3-kinase, whereas wortmannin and LY294002 inhibit the activity of both S6-kinase and PI 3-kinase. However, wortmannin and LY294002, but not rapamycin, blocks the ability of IL-4 and IGF-I to promote cell survival. IL-3, IL-4, and IGF-I increase expression of Bcl-2 by >3-fold. Pretreatment with inhibitors of PI 3-kinase, but not rapamycin, abrogate expression of Bcl-2 caused by IL-4 and IGF-I, but not by IL-3. None of the cytokines affected expression of the proapoptotic protein Bax, suggesting that all three cytokines were specific for Bcl-2. These data establish that inhibition of PI 3-kinase, but not S6-kinase, blocks the ability of IL-4 and IGF-I to increase expression of Bcl-2 and protect promyeloid cells from apoptosis. The requirement for PI 3-kinase to maintain Bcl-2 expression depends upon the ligand that activates the cell survival pathway (Minshall, 1999).

In Baf-3 cells, IL-3 and IGF-1 both inhibit cell death. These growth factors act at least on two different pathways involved in the inhibition of apoptosis. They both upregulate Bcl-X at the mRNA and protein levels and also activate a pathway that inhibits apoptosis in the absence of protein synthesis. Recently, these two growth factors have been shown to activate the PI3-kinase-AKT pathway, which leads to the phosphorylation of the pro-apoptotic Bcl-XL regulator Bad. This study investigated the role of PI3-kinase in the regulation of Bcl-X expression and in the survival of Baf-3 cells. PI3-kinase activation is involved in the upregulation of Bcl-X mRNA induced by both IL-3 and IGF-1. Moreover, PI3-kinase activity is also necessary for inhibition of apoptosis and caspase regulation by IGF-1 but not IL-3 (Leverrier, 1999).

In vivo, apoptotic cells are removed by surrounding phagocytes, a process thought to be essential for tissue remodeling and the resolution of inflammation. Although apoptotic cells are known to be efficiently phagocytosed by macrophages, the mechanisms whereby their interaction with the phagocytes triggers their engulfment have not been described in mammals. Primary murine bone marrow-derived macrophages (using alphavß3 integrin for apoptotic cell uptake) extend lamellipodia to engulf apoptotic cells and form an actin cup where phosphotyrosine accumulates. Rho GTPases and PI 3-kinases have been widely implicated in the regulation of the actin cytoskeleton. Inhibition of Rho GTPases by Clostridium difficile toxin B prevents apoptotic cell phagocytosis and inhibits the accumulation of both F-actin and phosphotyrosine. Importantly, the Rho GTPases Rac1 and Cdc42 are required for apoptotic cell uptake whereas Rho inhibition enhances uptake. The PI 3-kinase inhibitor LY294002 also prevents apoptotic cell phagocytosis but has no effect on the accumulation of F actin and phosphotyrosine. These results indicate that both Rho GTPases and PI 3-kinases are involved in apoptotic cell phagocytosis but that they play distinct roles in this process (Leverrier, 2001).

Treatment of confluent rat2 fibroblasts with C2-ceramide (N-acetylsphingosine), sphingomyelinase, or tumor necrosis factor-alpha (TNFalpha) increases phosphatidylinositol (PI) 3-kinase activity by 3-6-fold after 10 min. This effect of C2-ceramide depends on tyrosine kinase activity and an increase in Ras-GTP levels. Increased PI 3-kinase activity is also accompanied by its translocation to the membrane fraction, increases in tyrosine phosphorylation of the p85 subunit, and physical association with Ras. Activation of PI 3-kinase by TNFalpha, sphingomyelinase, and C2-ceramide is inhibited by tyrosine kinase inhibitors (genistein and PP1). The stimulation of PI 3-kinase by sphingomyelinase and C2-ceramide is not observed in fibroblasts expressing dominant-negative Ras (N17) and the stimulation by TNFalpha is decreased by 70%. PI 3-kinase activation by C2-ceramide is not modified by inhibitors of acidic and neutral ceramidases, and it is not observed with the relatively inactive analog, dihydro-C2-ceramide. It is proposed that activation of Ras and PI 3-kinase by ceramide can contribute to signaling effects of TNFalpha that occur downstream of sphingomyelinase activation and result in increased fibroblast proliferation (Hanna, 1999).

Nerve growth factor (NGF) is a required differentiation and survival factor for sympathetic sensory neurons and a majority of neural crest-derived sensory neurons in the developing vertebrate peripheral nervous system. Although much is known about the function of NGF, the intracellular signaling cascade that it uses continues to be a subject of intense study. p21 ras signaling is considered necessary for sensory neuron survival. As yet, how additional intermediates downstream or in parallel may function has not been fully understood. Two intracellular signaling cascades, extra cellular regulated kinase (erk) and phosphatidylinositol-3 (PI 3) kinase, transduce NGF signaling in the pheochromocytoma cell line PC12. To elucidate the role these cascades play in survival and differentiation, a combination of recombinant adenoviruses and chemical inhibitors were used to perturb these pathways in sensory neurons from wild-type mice and mice deficient for neurofibromin in which the survival and differentiation pathway is constitutively active. ras activity is both necessary and sufficient for the survival of embryonic sensory neurons. Downstream of ras, however, the erk cascade is neither required nor sufficient for neuron survival or overall differentiation. Instead, the activity of PI 3 kinase is necessary for the survival of the wild-type and neurofibromin-deficient neurons. Therefore, it is concluded that in sensory neurons, NGF acts via a signaling pathway, which includes both ras and PI 3 kinase (Klesse, 1998).

The IL-7R-activated signal that promotes survival and proliferation of T cell progenitors is defined in this study. The signal is distinct from the signals that induce differentiation. IL-7 activates PKB and STAT5 in human thymocytes. Chimeric receptors with a cytoplasmic domain of the IL-7R that is no longer able to activate PI-3K/PKB and STAT5 were introduced into T cell precursors and the transduced cells were tested in a fetal thymic organ culture. The T cell precursor activity of progenitors expressing dominant-negative forms of PI-3K or STAT5B was also examined. These experiments reveal that PI-3K/PKB activation is essential for the survival and proliferation of T cell precursors and suggest that STAT5 activated by IL-7 mediates T cell differentiation (Pallard, 1999).

The involvement of Ras in the activation of multiple early signaling pathways is well understood, but it is less clear how the various Ras effectors interact with the cell cycle machinery to cause G(1) progression. Ras-mediated activation of extracellular-regulated kinase/mitogen-activated protein kinase has been implicated in cyclin D(1) up-regulation, but there is little extracellular-regulated kinase activity during the later stages of G(1), when cyclin D(1) expression becomes maximal, implying that other effector pathways may also be important in cyclin D1 induction. The involvement of Ras effectors from the phosphatidylinositol (PI) 3-kinase and Ral-GDS families in G1 progression has been addressed and this involvement is compared to that of the Raf/mitogen-activated protein kinase pathway. PI 3-kinase activity is required for the expression of endogenous cyclin D1 and for S phase entry following serum stimulation of quiescent NIH 3T3 fibroblasts. Activated PI 3-kinase induces cyclin D1 transcription and E2F activity, at least in part mediated by the serine/threonine kinase Akt/PKB, and to a lesser extent the Rho family GTPase Rac. In addition, both activated Ral-GDS-like factor and Raf stimulate cyclin D1 transcription and E2F activity and act in synergy with PI 3-kinase. Therefore, multiple cooperating pathways mediate the effects of Ras on progression through the cell cycle (Gille, 1999).

Recent evidence indicates that phosphatidylinositol 3-kinase (PI3K) is a central regulator of mitosis, apoptosis and oncogenesis. Nevertheless, the mechanisms by which PI3K regulates proliferation are not well characterized. Mitogens stimulate entry into the cell cycle by inducing the expression of immediate early genes (IEGs) that in turn trigger the expression of G1 cyclins. A novel PI3K- regulated transcriptional cascade is described that is critical for mitogen regulation of the IEG, c-fos. PI3K activates gene expression by transactivating SRF-dependent transcription, independent of the previously described Rho and ETS TCF pathways. PI3K-stimulated cell cycle progression requires transactivation of SRF and expression of dominant- negative PI3K blocks mitogen-stimulated cell cycle progression. Furthermore, dominant-interfering SRF mutants attenuate mitogen-stimulated cell cycle progression, but are without effect on MEK-stimulated cell cycle entry. Moreover, expression of constitutively active SRF is sufficient for cell cycle entry. Thus, a novel SRF-dependent mitogenic cascade is described that is critical for PI3K- and growth factor-mediated cell cycle progression (Poser, 2000).

What are the mitogenic targets of PI3K-stimulated gene expression? Cyclin D1 transcription is induced by mitogens and cyclin D1 is thought to play a key role in mitogen-stimulated G1 progression. Recent evidence indicates that mitogens induce the expression and transcription of cyclin D1 in some cells via PI3K signaling. Interestingly, growth factor-induced cyclin D1 transcription is blocked by expression of dominant-negative PI3K or SRF. Because the cyclin D1 promoter does not contain an SRE, the requirement for SRF for cyclin D1 transcription is the result of the indirect expression of SRF-regulated IEG transcription factors. For example, fibroblasts deficient for the SRF-regulated IEGs, c-fos and FosB, have a defect in mitogen-induced proliferation and cyclin D1 transcription. Taken together, these results suggest that a mitotic target of PI3K-stimulated SRF-mediated transcription is cyclin D1, which in turn mediates repression of Rb and activation of E2F (Poser, 2000).

Neurogenesis in the retina requires the concerted action of three different cellular processes: proliferation, differentiation, and apoptosis. Class IA phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit. p110alpha has been shown to regulate cell division and survival. Little is known of its function in development, however, since p110alpha knockout mice exhibit CNS defects, but death at early embryonic stages impairs further study. The role of PI3K in mouse retina development was examined by expressing an activating form of PI3K regulatory subunit, p65PI3K, as a transgene in the retina. Mice expressing p65 PI3K show severely disrupted retina morphogenesis, with ectopic cell masses in the neuroepithelium that evolve into infoldings of adult retinal cell layers. These changes correlate with an altered cell proliferation/cell death balance at early developmental stages. Nonetheless, the most affected cell layer in adult retina is that of photoreceptors: this correlates with selectively increased survival of these cells at the developmental stages at which cell division has ceased. These results demonstrate the relevance of accurate PI3K regulation for normal retinal development, supporting class IA PI3K involvement in induction of cell division at early stages of neurogenesis. These data also show that, even after cell division decline, PI3K activation mediates survival of differentiated neurons in vivo (Pimentel, 2002).

The role of the PI 3-kinase cascade in regulation of cell growth is well established. PKB (protein kinase B) is a key downstream effector of the PI 3-kinase pathway and is best known for its antiapoptotic effects and the role it plays in initiation of S phase. PKB activity is high in the G2/M phase of the cell cycle in epithelial cells. Inhibition of the PI 3-kinase pathway in MDCK cells induces apoptosis at the G2/M transition, prevents activation of cyclin B-associated kinase, and prohibits entry of the surviving cells into mitosis. All of these consequences of the inhibition of PI 3-kinase are relieved by expression of a constitutively active form of PKB (caPKB), indicating that PKB plays a role in regulation of the G2/M phase. Inhibition of PI 3-kinase results in activation of Chk1, whereas constitutively active PKB inhibits the ability of Chk1 to become activated in response to treatment with hydroxyurea. Preliminary data show that PKB phosphorylates the Chk1 polypeptide in vitro on serine 280. These results not only implicate PKB activity in transition through the G2/M stage of the cell cycle, but they also suggest the existence of crosstalk between the PI 3-kinase pathway and the key regulators of the DNA damage checkpoint machinery (Shtivelman, 2002).

Neuronal precursor cells in the developing cerebellum require activity of the sonic hedgehog (Shh) and phosphoinositide-3-kinase (PI3K) pathways for growth and survival. Synergy between the Shh and PI3K signaling pathways are implicated in the cerebellar tumor medulloblastoma. A mechanism through which these disparate signaling pathways cooperate to promote proliferation of cerebellar granule neuron precursors is described. Shh signaling drives expression of mRNA encoding the Nmyc1 oncoprotein (previously N-myc), which is essential for expansion of cerebellar granule neuron precursors. The PI3K pathway stabilizes Nmyc1 protein via inhibition of GSK3-dependent Nmyc1 phosphorylation and degradation. The effects of PI3K activity on Nmyc1 stabilization are mimicked by insulin-like growth factor, a PI3K agonist with roles in central nervous system precursor growth and tumorigenesis. These findings indicate that Shh and PI3K signaling pathways converge on N-Myc to regulate neuronal precursor cell cycle progression. Furthermore, they provide a rationale for therapeutic targeting of PI3K signaling in medulloblastoma (Kenney, 2004).

Signaling in the PI3K pathway: PI3K, cell migration, branching morphogenesis, and differentiation

Overexpression of focal adhesion kinase (FAK) in Chinese hamster ovary (CHO) cells promotes their migration on fibronectin. This effect is dependent on the phosphorylation of FAK at Tyr-397. This residue was known to serve as a binding site for both Src and phosphatidylinositol 3-kinase (PI3K), implying that either one or both are required for FAK to promote cell migration. This study examined the role of PI3K in FAK-promoted cell migration. The PI3K inhibitors, wortmannin and LY294002, are able to inhibit FAK-promoted migration in a dose-dependent manner. Furthermore, a FAK mutant capable of binding Src but not PI3K was generated by a substitution of Asp residue 395 with Ala. When overexpressed in CHO cells, this differential binding mutant fails to promote cell migration although its association with Src is retained. Together, these results strongly suggest that PI3K binding is required for FAK to promote cell migration and that the binding of Src and p130(Cas) to FAK may not be sufficient for this event (Reiske, 1999).

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of colonies followed by disruption of cell-cell junctions and subsequent cell scattering. In Madin-Darby canine kidney cells, HGF/SF-induced motility involves actin reorganization mediated by Ras, but whether Ras and downstream signals regulate the breakdown of intercellular adhesions has not been established. Both HGF/SF and V12Ras induces the loss of the adherens junction proteins E-cadherin and beta-catenin from intercellular junctions during cell spreading, and the HGF/SF response is blocked by dominant-negative N17Ras. Desmosomes and tight junctions are regulated separately from adherens junctions, because they are not disrupted by V12Ras. MAP kinase, phosphatidylinositide 3-kinase (PI 3-kinase), and Rac are required downstream of Ras, because loss of adherens junctions is blocked by the inhibitors PD098059 and LY294002 or by dominant-inhibitory mutants of MAP kinase kinase 1 or Rac1. All of these inhibitors also prevent HGF/SF-induced cell scattering. Interestingly, activated Raf or the activated p110alpha subunit of PI 3-kinase alone does not induce disruption of adherens junctions. These results indicate that activation of both MAP kinase and PI 3-kinase by Ras is required for adherens junction disassembly and that this is essential for the motile response to HGF/SF (Potempa, 1998).

Phosphatidylinositol 3-kinase (PI3-kinase) enzymes are key signaling molecules in the PC12 and neuronal cell survival pathway and are also involved in the regulation of retrograde axonal transport of nerve growth factor (NGF), with sympathetic neurons more sensitive to the effects of wortmannin/LY294002 than sensory neurons. In this article, the mRNA expression of PI3-kinase isoforms was characterized in mouse sympathetic superior cervical ganglia (SCG) and sensory trigeminal ganglia (TGG) and examined in the subcellular locations of immunoreactivity of the PI3-kinase isoforms in mouse cultured SCG and dorsal root ganglion (DRG) neurons. Both the SCG and the TGG express mRNA for the p110alpha, beta, gamma, delta, and vps34p PI3-kinase isoforms, but the TGG and not the SCG express mRNA for the p170 PI3-kinase isoform. In cultured SCG and DRG neurons, p110alpha, beta, and gamma immunoreactivity is in the SCG and DRG growth cones, and predominantly in puncta throughout the growth cone varicosity. However, immunoreactivity varies: in the cell bodies, p110alpha is localized predominantly at the plasma membrane, while p110beta and gamma are localized in the perinuclear region of the cells. In addition, unlike other cell types, wortmannin has little effect on actin filament polymerization in either mouse cultured SCG or DRG neurons (Bartlett, 1999).

Expression of rat TrkA in Xenopus spinal neurons confers responsiveness of these neurons to nerve growth factor (NGF) in assays of neuronal survival and growth cone chemotropism. Mutational analysis indicates that coactivation of phospholipase C-gamma (PLC-gamma) and phosphoinositide 3-kinase (PI3-kinase) by specific cytoplasmic domains of TrkA is essential for triggering chemoattraction of the growth cone in an NGF gradient. Uniform exposure of TrkA-expressing neurons to NGF results in a cross-desensitization of turning responses induced by a gradient of netrin-1, brain-derived neurotrophic factor (BDNF), or myelin-associated glycoprotein (MAG) but not by a gradient of collapsin-1/semaphorin III/D or neurotrophin-3 (NT-3). These results, together with the effects of pharmacological inhibitors, support the notion that there are common cytosolic signaling pathways for two separate groups of guidance cues, one of which requires coactivation of PLC-gamma and PI3-kinase pathways (Ming, 1999).

Differentiation of adipocytes is an important aspect of energy homeostasis. Although the transcriptional regulation of adipocyte differentiation is relatively well characterized, the subsequent molecular events remain unclear. The activity of phosphoinositide (PI) 3-kinase precipitated with antibodies to phosphotyrosine has now been shown to increase transiently during adipocyte differentiation of 3T3-F442A and of 3T3-L1 cells. PI 3-kinase activity precipitates with antibodies to insulin receptor substrate 1 (IRS1) and association of subunits of PI 3-kinase with IRS1 are also increased at this stage of differentiation, suggesting that IRS1 contributes to PI 3-kinase activation. Inhibition of the activation of PI 3-kinase by expression of dominant negative mutant subunits of the enzyme prevents adipogenesis, as assessed by lipid accumulation and expression of key adipocyte proteins such as GLUT4, adipsin, and aP2, suggesting that PI 3-kinase activation is essential for adipocyte differentiation. However, these mutant proteins do not affect either the expression of the transcription factor PPARgamma at the mRNA or protein level or the increase in the abundance of mRNAs encoding the adipocyte marker proteins. These results demonstrate that adipocyte differentiation is regulated at the posttranscriptional level and that activation of PI 3-kinase is required for this regulation (Sakaue, 1998).

Insulin-like growth factors (IGFs) are potent inducers of skeletal muscle differentiation and phosphatidylinositol (PI) 3-kinase activity is essential for this process. IGF-II induces nuclear factor-kappaB (NF-kappaB) and nitric-oxide synthase (NOS) activities downstream from PI 3-kinase and these events are shown to be are critical for myogenesis. Differentiation of rat L6E9 myoblasts with IGF-II transiently induces NF-kappaB DNA binding activity, inducible nitric-oxide synthase (iNOS) expression, and nitric oxide (NO) production. IGF-II-induced iNOS expression and NO production are blocked by NF-kappaB inhibition. Both NF-kappaB and NOS activities are essential for IGF-II-induced terminal differentiation (myotube formation and expression of skeletal muscle proteins: myosin heavy chain, GLUT 4, and caveolin 3), which is totally blocked by NF-kappaB or NOS inhibitors in rat and human myoblasts. Moreover, the NOS substrate L-Arg induces myogenesis in the absence of IGFs in both rat and human myoblasts, and this effect is blocked by NOS inhibition. Regarding the mechanisms involved in IGF-II activation of NF-kappaB, PI 3-kinase inhibition prevents NF-kappaB activation, iNOS expression, and NO production. Moreover, IGF-II induces, through a PI 3-kinase-dependent pathway, a decrease in IkappaB-alpha protein content that correlates with a decrease in the amount of IkappaB-alpha associated with p65 NF-kappaB (Kaliman, 1999).

Phosphoinositide 3-kinase (PI3K) has been shown to regulate cell and organ size in Drosophila, but the role of PI3K in vertebrates in vivo is not well understood. To examine the role of PI3K in intact mammalian tissue, transgenic mice expressing constitutively active or dominant-negative mutants of PI3K in the heart have been created and characterized. Cardiac-specific expression of constitutively active PI3K results in mice with larger hearts, while dominant-negative PI3K results in mice with smaller hearts. The increase or decrease in heart size is associated with comparable increase or decrease in myocyte size. Cardiomyopathic changes, such as myocyte necrosis, apoptosis, interstitial fibrosis or contractile dysfunction, are not observed in either of the transgenic mice. Thus, the PI3K pathway is necessary and sufficient to promote organ growth in mammals (Shioi, 2000).

What are the downstream targets of PI3K that are involved in the regulation of organ size? The available information does not provide a definitive answer to this question. However, Akt, a well characterized downstream target of PI3K, is likely to be one of the major mediators of this process. Akt is necessary and sufficient for phosphorylation and subsequent inactivation of 4E-BP1, a repressor of mRNA translation. Akt can also activate p70S6K in some contexts, although activation of p70S6K might not be solely dependent on Akt. Another potential candidate is p70S6K, which is upregulated in caPI3K hearts and downregulated in dnPI3K hearts. The amount of phosphorylated S6 protein correlates with the activation of p70S6K. The inhibition of the p70S6K pathway by rapamycin at nanomolar concentrations selectively suppresses an increase in protein synthesis of cultured neonatal myocytes in response to growth factors. Interestingly, rapamycin does not inhibit other phenotypic changes associated with myocyte hypertrophy, such as re-activation of fetal genes and sarcomere organization. This raises the possibility that p70S6K may selectively regulate cell size via controlling the rate of protein synthesis. Gene disruption of p70S6K is known to result in smaller body size in mice. In Drosophila, deficiency of the S6K gene is associated with a reduction in body size associated with smaller cells (Shioi, 2000 and references therein).

The mouse submandibular gland (SMG) epithelium undergoes extensive morphogenetic branching during embryonic development as the first step in the establishment of its glandular structure. However, the specific signaling pathways required for SMG branching morphogenesis are not well understood. Using E13 mouse SMG organ cultures, it has been shown that inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase), wortmannin and LY294002, substantially inhibit branching morphogenesis in SMG. Branching morphogenesis of epithelial rudiments denuded of mesenchyme is inhibited similarly, indicating that PI 3-kinase inhibitors act directly on the epithelium. Immunostaining and Western analysis demonstrate that the p85 isoform of PI 3-kinase is expressed in epithelium at levels higher than in the mesenchyme. A target of PI 3-kinase, Akt/protein kinase B (PKB), shows decreased phosphorylation at Ser473 by Western analysis in the presence of PI 3-kinase inhibitors. The major lipid product of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (PIP3), was added exogenously to SMG via a membrane-transporting carrier in the presence of PI 3-kinase inhibitors and was found to stimulate cleft formation, the first step of branching morphogenesis. Together, these data indicate that PI 3-kinase plays a role in the regulation of epithelial branching morphogenesis in mouse SMG acting through a PIP3 pathway (Larsen, 2003).

During vertebrate gastrulation, cell polarization and migration are core components in the cellular rearrangements that lead to the formation of the three germ layers, ectoderm, mesoderm, and endoderm. The Wnt/planar cell polarity (PCP) signaling pathway has been implicated in controlling cell morphology and movement during gastrulation. However, cell polarization and directed cell migration are reduced but not completely abolished in the absence of Wnt/PCP signals; this observation indicates that other signaling pathways must be involved. Phosphoinositide 3-Kinases (PI3Ks) are shown to be required at the onset of zebrafish gastrulation in mesendodermal cells for process formation and cell polarization. Platelet Derived Growth Factor (PDGF) functions upstream of PI3K, while Protein Kinase B (PKB), a downstream effector of PI3K activity, localizes to the leading edge of migrating mesendodermal cells. In the absence of PI3K activity, PKB localization and cell polarization are strongly reduced in mesendodermal cells and are followed by slower but still highly coordinated and directed movements of these cells. Thus this study has identified a novel role of a signaling pathway comprised of PDGF, PI3K, and PKB in the control of morphogenetic cell movements during gastrulation. Furthermore, these findings provide insight into the relationship between cell polarization and directed cell migration at the onset of zebrafish gastrulation (Montero, 2003).

PI3Ks play important roles in the control of cell polarization and directed cell migration in different cell types in vitro. The observation from this study that PI3K activity is required for prechordal plate progenitor cells to polarize and that the failure to polarize in the absence of PI3K activity is accompanied by slower movements of these cells provides the first clear evidence for an involvement of PI3Ks in cell polarization and directed cell migration in the gastrula embryo. Moreover, the observation that PKB is localized to the leading edge of prechordal plate progenitors and that this localization is dependent on PI3K activity indicates that PI3Ks control cell polarization in vivo by using similar downstream effectors as previously shown in cultured cells such as mammalian lymphocytes and Dictyostelium cells. However, in contrast to those in vitro studies, PI3K activity is not required for the direction of cell migration in prechordal plate progenitors, indicating that the specific functions of PI3Ks in cell polarization and migration can substantially vary depending on the particular cellular environment (Montero, 2003).

Interestingly, a strong reduction of PI3K activity in prechordal plate progenitors not only leads to a considerable decrease in the total number of cellular processes formed, but also to a shift from pseudopodial and filopodial character to predominantly lamellipodial character in the few remaining cellular processes. Similarly, an overactivation of PI3K signaling leads to an increase in the number of cellular processes (filopods and pseudopods) and a concomitant more extensive branching of those processes. This suggests that PI3Ks promote the formation and arborization of cellular processes with filopodial and pseudopodial character in prechordal plate progenitor cells at the onset of gastrulation. A similar function of PI3K has also recently been observed in sensory neurons in vitro where PI3K signaling promotes axonal elongation and branching (Montero, 2003 and references therein).

Cell attachment and the assembly of cytoskeletal and signaling complexes downstream of integrins are intimately linked and coordinated. Although many intracellular proteins have been implicated in these processes, a new paradigm is emerging from biochemical and genetic studies that implicates integrin-linked kinase (ILK) and its interacting proteins, such as CH-ILKBP (alpha-parvin; see Drosophila Parvin), paxillin, and PINCH in coupling integrins to the actin cytoskeleton and signaling complexes. Genetic studies in Drosophila, Caenorhabditis elegans, and mice point to an essential role of ILK as an adaptor protein in mediating integrin-dependent cell attachment and cytoskeletal organization. This study demonstrates that inhibiting ILK kinase activity, or expression, results in the inhibition of cell attachment, cell migration, F-actin organization, and the specific cytoskeletal localization of CH-ILKBP and paxillin in human cells. The kinase activity of ILK is elevated in the cytoskeletal fraction and the interaction of CH-ILKBP with ILK within the cytoskeleton stimulates ILK activity and downstream signaling to PKB/Akt and GSK-3. Interestingly, the interaction of CH-ILKBP with ILK is regulated by the Pi3 kinase pathway, because inhibition of Pi3 kinase activity by pharmacological inhibitors, or by the tumor suppressor PTEN, inhibits this interaction as well as cell attachment and signaling. These data demonstrate that the kinase and adaptor properties of ILK function together, in a Pi3 kinase-dependent manner, to regulate integrin-mediated cell attachment and signal transduction (Attwell, 2003).

Directed cell migration involves signaling events that lead to local accumulation of PI(3,4,5)P3, but additional pathways act in parallel. A genetic screen in Dictyostelium discoideum to identify redundant pathways revealed a gene with homology to patatin-like phospholipase A2. Loss of this gene did not alter PI(3,4,5)P3 regulation, but chemotaxis became sensitive to reductions in PI3K activity. Likewise, cells deficient in PI3K activity were more sensitive to inhibition of PLA2 activity. Deletion of the PLA2 homolog and two PI3Ks caused a strong defect in chemotaxis and a reduction in receptor-mediated actin polymerization. In wild-type cells, chemoattractants stimulated a rapid burst in an arachidonic acid derivative. This response was absent in cells lacking the PLA2 homolog, and exogenous arachidonic acid reduced their dependence on PI3K signaling. It is proposed that PLA2 and PI3K signaling act in concert to mediate chemotaxis, and metabolites of PLA2 may be important mediators of the response (Chen, 2007).

The PTEN/PI3K pathway governs normal vascular development and tumor angiogenesis

PTEN is an important tumor suppressor gene. Hereditary mutation of PTEN causes tumor-susceptibility diseases such as Cowden disease. The Cre-loxP system was used to generate an endothelial cell-specific mutation of Pten (Tie2CrePten) in mice. Tie2CrePtenflox/+ mice display enhanced tumorigenesis due to an increase in angiogenesis driven by vascular growth factors. This effect is partially dependent on the PI3K subunits p85alpha and p110gamma. In vitro, Tie2CrePtenflox/+ endothelial cells show enhanced proliferation/migration. Tie2CrePtenflox/flox mice die before embryonic day 11.5 (E11.5) due to bleeding and cardiac failure caused by impaired recruitment of pericytes and vascular smooth muscle cells to blood vessels, and of cardiomyocytes to the endocardium. These phenotypes depend strongly on p110gamma rather than on p85alpha and are associated with decreased expression of Ang-1, VCAM-1, connexin 40, and ephrinB2 but increased expression of Ang-2, VEGF-A, VEGFR1, and VEGFR2. Pten is thus indispensable for normal cardiovascular morphogenesis and post-natal angiogenesis, including tumor angiogenesis (Hamada, 2005).

RT-PCR analyses of gene expression in whole yolk sacs from E8.5 Tie2CrePten+/+ and Tie2CrePtenflox/flox embryos show that a lack of Pten significantly reduces expression of connexin-40, Ang-1, ephrinB2, and VCAM-1, but increased expression of Ang-2, VEGF-A, VEGFR1, VEGFR2, TGF-ß, and PAI-1 . These differences were confirmed by RT-PCR analyses of VEGFR2+ cells from E9.5 Tie2CrePten+/+ and Tie2CrePtenflox/flox embryos and protein analyses of human umbilical vein endothelial cells (HUVECs) in which PTEN expression was reduced by siRNA. These results suggest that Pten deficiency leads directly to an altered VGF profile that may be responsible for the cardiovascular defects of Tie2CrePtenflox/flox mice (Hamada, 2005).

In Tie2CrePten+/+ endothelial cells, expression levels of VEGF-A and its receptors VEGFR1 and VEGFR2 is significantly increased after stimulation with VEGF-A, as expected. However, this expression is further increased in Tie2CrePtenflox/+ endothelial cells. This enhanced expression of VEGF-A and its receptors may thus partly contribute to the enhanced angiogenesis observed in Tie2CrePtenflox/+ mice (Hamada, 2005).

The precise functions of PI3K isoforms in endothelial cells have been difficult to ascertain. Because most VGF receptors have tyrosine kinase activity, class IA PI3Ks likely play major roles in cardiovasculogenesis and tumor angiogenesis. Indeed, endothelial cell growth/survival and angiogenesis are enhanced following ectopic expression of constitutively active p110alpha, the catalytic subunit of class IA PI3Ks. Consistent with this finding, the enhanced angiogenesis and accelerated tumor growth observed in Tie2CrePtenflox/+ mice, and the impaired cardiovascular morphogenesis observed in Tie2CrePtenflox/flox mice are partially resolved by loss of p85alpha, the major regulatory subunit of class IA PI3Ks (Hamada, 2005).

This study sheds light on the potential roles of the class IB PI3K in cardiovascular morphogenesis and post-natal angiogenesis. Double mutant mice lacking both Pten and class IB PI3K functions were generated, and it was demonstrated that the post-natal angiogenic responses of Tie2CrePtenflox/+ mice are rescued to the same degree by loss of p110gamma, the catalytic subunit of PI3Kgamma, as by loss of p85alpha. Furthermore, compared with p85alpha deficiency, p110gamma deficiency dramatically resolves the defective cardiovasculogenesis observed in Tie2CrePtenflox/flox mice. In the p85alpha-deficient mice used in this study, only the p85alpha isoform was deleted, not its alternative splicing isoforms p55alpha and p50alpha. Moreover, p85ß and p55gamma, the alternative regulatory subunits of class IA PI3Ks, still exist in these mice. However, since p85alpha is the major regulatory subunit of class IA PI3Ks, it is believed that the class IB PI3K may have a more important function in cardiovasculogenesis than do the class IA PI3Ks (Hamada, 2005).

It was not expected that the enhanced angiogenesis induced in Tie2CrePtenflox/+ mice by RTK agonists (e.g., VEGF and Ang-1) would be partially rescued by p110gamma deficiency. Up until now, p110gamma has been postulated to be activated downstream of GPCR but not downstream of RTK. Indeed, the activation of PKB/Akt and MAPK induced by VEGF and Ang-1 is not suppressed by p110gamma deficiency in vitro. It is thus unlikely that an RTK type VGF receptor directly couples with p110gamma, an interaction noted for PDGF receptors and erythropoietin receptors. It may be that, in vivo, an unknown VGF (possibly a GPCR ligand) activates p110gamma and influences RTK signaling that is initiated by VEGF or Ang-1 and leads to angiogenesis. In endothelial cells, identified GPCR ligands include sphingosine-1-phosphate (S1P), angiotensin II, CXCL-16, and shear stress. One of the candidate GPCR ligands that may activate p110gamma in endothelial cells is sphingosine-1-phosphate (S1P). S1P induces endothelial cell proliferation, migration, and morphogenesis in vitro and in vivo. Moreover, EDG1, a GPCR-type S1P receptor, is essential for vascular maturation. This study shows that the p110gamma deficiency partially blocks enhanced angiogenesis driven not only by Ang-1 or VEGF-A but also by S1P. However, S1P-induced activation of PKB/Akt and MAPK is not suppressed by p110gamma deficiency, indicating that the major downstream target of S1P may not be p110gamma (Hamada, 2005).

Although PTEN has dual lipid and protein phosphatase activities, the results clearly demonstrate that a primary function of PTEN is to fine-tune the intracellular level of PIP3 produced by PI3Ks and thereby regulate vascular remodeling and tumor angiogenesis. The data also suggest the functional overlapping of class IA and IB PI3Ks in angiogenesis. This hypothesis is supported by the lack of an endothelial cell phenotype in mice lacking p85alpha, p85ß, p110ß, or p110gamma or in p110delta 'kinase dead' knock-in mice. Furthermore, the results demonstrate that, among the multiple PIP3 phosphatases, PTEN has an exclusive role in down-regulating PIP3 in endothelial cells. Various PI3Kgamma-specific inhibitors that are under investigation as anti-inflammatory drugs may therefore also be useful as cancer therapies targeting tumor angiogenesis (Hamada, 2005).

This study is the first report of the functional analysis of Pten and PI3Kgamma in murine endothelial cells in vivo. The normal function of the PI3K-PKB/Akt-Pten pathway in endothelial cells is required for cardiovascular development, and loss of Pten-mediated control of this pathway enhances tumor angiogenesis. Deficiency in Pten function thus contributes both to susceptibility to new tumorigenic mutations and to accelerated tumor growth. Inhibition of the PI3K pathway, including PI3Kgamma, is thus an attractive therapeutic target for the treatment of various malignancies (Hamada, 2005).

Signaling in the PI3K pathway: PI3K and transformation

An increase in the level of active, GTP-bound Ras is not necessary for transformation of chicken embryo fibroblasts (CEF) by v-Src. This suggests that other Ras-independent pathways contribute to transformation by v-Src. To address the possibility that activation of phosphatidylinositol-3-kinase (PI3K) and the mammalian target of rapamycin (mTOR/FRAP), represents one of these pathways, the effect of simultaneous inhibition of the Ras-MAPK and PI3K-mTOR pathways was examined on transformation of CEF by v-Src. Transformation was assessed by the standard parameters of morphological alteration, increased hexose uptake, loss of density inhibition, and anchorage-independent growth. Inhibition of the Ras-MAPK pathway by expression of the dominant-negative Ras mutant HRasN17 or by addition of the MAPK kinase (MEK) inhibitor PD98059 reduces several of these parameters but fails to block transformation. Similarly, inhibition of the PI3K-mTOR pathway by addition of a PI3K inhibitor or the mTOR inhibitor rapamycin, although reducing several parameters of transformation, also fails to block transformation. However, simultaneous inhibition of signaling by the Ras-MAPK pathway and the PI3K-mTOR pathway essentially blocks transformation. These data indicate that transformation of CEF by v-Src is mediated by two parallel pathways, the Ras-MAPK pathway and the PI-3K-mTOR pathway, which both contribute to transformation. The possibility is not excluded that simultaneous activation of other pathways is also required (Penuel, 1999).

Several different human virus oncoproteins, including adenovirus type 9 E4-ORF1, evolved to target the Dlg1 mammalian homolog of the membrane-associated Drosophila discs-large tumor suppressor. This fact has implicated this cellular factor in human cancer. Despite a general belief that such interactions function solely to inactivate this suspected human tumor suppressor protein, this study demonstrates that E4-ORF1 specifically requires endogenous Dlg1 to provoke oncogenic activation of phosphatidylinositol 3-kinase (PI3K) in cells. Based on these results, a model is proposed wherein E4-ORF1 binding to Dlg1 triggers the resulting complex to translocate to the plasma membrane and, at this site, to promote Ras-mediated PI3K activation. These findings establish the first known function for Dlg1 in virus-mediated cellular transformation and also surprisingly expose a previously unrecognized oncogenic activity encoded by this suspected cellular tumor suppressor gene (Frese, 2006).

It is important to point out that, though unexpected, the finding that Dlg1 possesses an oncogenic activity does not refute a substantial body of evidence supporting its designation as a candidate tumor suppressor gene. Viral oncoproteins typically disregulate normal functions of their cellular targets, so it may be pertinent that Dlg1 was recently reported to recruit PI3K into a membrane-associated complex with E-cadherin to promote both adherens junction formation and terminal differentiation in enterocytes. Since this activity is predicted to induce an antiproliferative state in cells, it may represent a tumor suppressor function for Dlg1. Therefore, an intriguing possibility presents itself: whereas Dlg1 normally functions to promote temporally and spatially restricted E-cadherin-dependent PI3K activation during adherens junction formation in cells, Dlg1 modified by E4-ORF1 instead supports constitutive and spatially unrestricted, Ras-dependent PI3K activation at the plasma membrane. This interesting model hints that E4-ORF1 may simultaneously inactivate a tumor suppressor function and stimulate an oncogenic function of Dlg1 (Frese, 2006).

PIK3CA, one of the two most frequently mutated oncogenes in human tumors, codes for p110α, the catalytic subunit of a phosphatidylinositol 3-kinase, isoform α (PI3Kα, p110α/p85). This study reports a 3.0 angstrom resolution structure of a complex between p110α and a polypeptide containing the p110α-binding domains of p85α, a protein required for its enzymatic activity. The structure shows that many of the mutations occur at residues lying at the interfaces between p110α and p85α or between the kinase domain of p110α and other domains within the catalytic subunit. Disruptions of these interactions are likely to affect the regulation of kinase activity by p85 or the catalytic activity of the enzyme, respectively. In addition to providing new insights about the structure of PI3Kα, these results suggest specific mechanisms for the effect of oncogenic mutations in p110α and p85α (Huang, 2007).

Many human cancers involve up-regulation of the phosphoinositide 3-kinase PI3Kα, with oncogenic mutations identified in both the p110α catalytic and the p85α regulatory subunits. Crystallographic and biochemical approaches were used to gain insight into activating mutations in two noncatalytic p110α domains -- the adaptor-binding and the helical domains. A structure of the adaptor-binding domain of p110α in a complex with the p85α inter-Src homology 2 (inter-SH2) domain shows that oncogenic mutations in the adaptor-binding domain are not at the inter-SH2 interface but in a polar surface patch that is a plausible docking site for other domains in the holo p110/p85 complex. Helical domain mutations were examinedm and it was found that the Glu545 to Lys545 (E545K) oncogenic mutant disrupts an inhibitory charge-charge interaction with the p85 N-terminal SH2 domain. These studies extend understanding of the architecture of PI3Ks and provide insight into how two classes of mutations that cause a gain in function can lead to cancer (Miled, 2007).

PI3K and synaptic plasticity

An understanding of emotional fear in terms of its underlying cellular and molecular mechanisms is not only an essential piece of information for pharmacological intervention of anxiety and posttraumatic stress disorders but may also provide some insight into the long-term memory formation in the brain. Accumulated evidence indicates that the amygdala is a crucial neural locus for the induction and expression of fear memory. When rats encounter a tone (conditioned stimulus [CS]) that was previously paired with noxious stimulus (unconditioned stimulus [US]), such as foot-shock, information flows from the auditory thalamus and cortex to the lateral (LA) and basolateral amygdala (BLA), where alterations of synaptic transmission are thought to subserve the encoding of fear memory. Western blot analysis of neuronal tissues taken from fear-conditioned rats shows a selective activation of phosphatidylinositol 3-kinase (PI-3 kinase) in the amygdala. PI-3 kinase is also activated in response to long-term potentiation (LTP)-inducing tetanic stimulation. PI-3 kinase inhibitors block tetanus-induced LTP as well as PI-3 kinase activation. In parallel, these inhibitors interfer with long-term fear memory while leaving short-term memory intact. Tetanus and forskolin-induced activation of mitogen-activated protein kinase (MAPK) is blocked by PI-3 kinase inhibitors, which also inhibit cAMP response element binding protein (CREB) phosphorylation. These results provide novel evidence of a requirement of PI-3 kinase activation in the amygdala for synaptic plasticity and memory consolidation, and this activation may occur at a point upstream of MAPK activation (Lin, 2001).

Recently, it has been suggested that the changes of synaptic transmission in the LA are initiated by an influx of Ca2+ into cells through NMDA receptors or L-type Ca2+ channels, leading to the activation of protein kinases. Once stimulated, protein kinases such as cAMP-dependent protein kinase (PKA) or mitogen-activated protein kinase (MAPK) can translocate to the nucleus and subsequently activate transcription factors to promote gene transcription and new protein synthesis. The demonstration of an inhibition of memory consolidation by pharmacological blockade of protein synthesis, PKA, or MAPK is consistent with this conceptual framework. Whereas there is now a beginning understanding of the cellular mechanisms that underlie learned fear, the signaling pathway is still not completely known (Lin, 2001).

Phosphatidylinositol 3-kinase (PI-3 kinase) catalyzes the transfer of gamma-phosphate of ATP to the D-3 hydroxy group of the inositol headgroup of phosphoinositides. It is first found that the levels of 3-phosphorylated phosphoinositides are increased in transformed and/or mitogen-stimulated cells, implicating a role played by the PI-3 kinase in oncogenic and mitogenic signal transduction. PI-3 kinase has also been suggested to participate in nerve growth factor (NGF)-mediated and glial cell line-derived neurotropic factors (GDNFs)-mediated survival of sympathetic and spinal cord motoneurons. Although a recent study implied that PI-3 kinase may play a role in the expression of long-term potentiation (LTP) in hippocampal dentate gyrus, the data are in conflict, since it was found that PI-3 kinase-deficient mice display an enhanced hippocampal CA1 LTP, but with normal spatial memory (Lin, 2001).

To identify a signaling pathway as being important for synaptic plasticity, at least two criteria have to be fulfilled: (1) behavioral training that induces physiological responses should be able to activate biochemical cascade; (2) this biochemical cascade has to be necessary for the response to occur, and blockade within this cascade should interrupt the response. Here, it is shown that the PI-3 kinase pathway meets these two criteria of a genuine signaling pathway needed for the induction of fear-potentiated startle and LTP in the amygdala. PI-3 kinase is selectively activated in the amygdala following fear conditioning, and pharmacological blockade of PI-3 kinase impairs fear memory in a dose-dependent manner. In in vitro slice preparation, it has been shown that bath application of PI-3 kinase inhibitors attenuates tetanus-induced L-LTP in the amygdala. Furthermore, the role of PI-3 kinase in tetanus- and forskolin-induced activation of MAPK signaling pathway is elucidated. PI-3 kinase likely contributes to L-LTP and fear memory via the activation of MAPK and cAMP response element binding protein (CREB) (Lin, 2001).

The NMDA receptor, brain-derived neurotrophic factor (BDNF), postsynaptic density protein 95 (PSD-95) and phosphatidylinositol 3-kinase (PI3K) have all been implicated in long-term potentiation. This study shows that these molecules are involved in a single pathway for synaptic potentiation. In visual cortical neurons in young rodents, the neurotrophin receptor TrkB is associated with PSD-95. When BDNF is applied to cultured visual cortical neurons, PSD-95-labeled synaptic puncta enlarge, and fluorescent recovery after photobleaching (FRAP) reveals increased delivery of green fluorescent protein-tagged PSD-95 to the dendrites. The recovery of fluorescence requires TrkB, signaling through PI3K and the serine-threonine kinase Akt, and an intact Golgi apparatus. Stimulation of NMDARs mimics the PSD-95 trafficking that is induced by BDNF but requires active BDNF and PI3K. Furthermore, local dendritic contact with a BDNF-coated microsphere induces PSD-95 FRAP throughout the dendrites of the stimulated neuron, suggesting that this mechanism induces rapid neuron-wide synaptic increases in PSD-95 and refinement whenever a few robust inputs activate the NMDAR-BDNF-PI3K pathway (Yoshii, 2007).

Table of contents

Phosphotidylinositol 3 kinase 92E: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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