NORMAL GENOMIC

• Fujiwara H et al.
• Halenaquinone, a novel phosphatidylinositol 3-kinase inhibitor from a marine sponge, induces apoptosis in PC12 cells.
• Eur J Pharmacol. 2001; 413: 37-45
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• In nerve growth factor-treated PC12 cells, 12b-methyl-(S)-1H-benzo[6,7]phenanthro[10,1-bc]furan-3,6,8,11(2H,12bH)-tet rone (halenaquinone) caused cytotoxicity in a concentration-dependent manner (EC(50) value; 10 microM). Gel electrophoretic DNA analysis of PC12 cells treated with halenaquinone (10 microM) and 11-(acetyloxy)-1,6b,7,8,9a,10,11,11b-octahydro-1-(methoxymethyl)-9a,11b-di methyl-[1S-(1 alpha,6b alpha,9a beta,11 alpha,11b beta)]-3H-furo[4,3,2-de]indeno[4,5-h]-2-benzopyran-3,6,9-trione (wortmannin) (3 microM) showed a typical apoptotic DNA ladder. In the flow cytometric analysis, halenaquinone caused apoptosis in a concentration- and time-dependent manner (EC(50) value; 10 microM), whereas 2,3-dihydro-12b-methyl-(S)-1H-benzo[6,7]phenanthro[10,1-bc]furan-6,8,11(12 bH)-trione (xestoquinone) with the methylene group at the C-3 position failed to cause apoptosis, suggesting that the carbonyl group at the C-3 position in halenaquinone is important for exerting apoptotic effects in PC12 cells. Phosphatidylinositol 3-kinase was inhibited by halenaquinone (IC(50) value; 3 microM) as well as wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. Halenaquinone inhibited phosphatidylinositol 3-kinase activity at lower concentrations than those at which it induced apoptosis in PC12 cells. These results suggest that halenaquinone causes the death of PC12 cells through an apoptotic process and that the mechanism of halenaquinone-induced apoptosis may be partially explained by the inhibition of phosphatidylinositol 3-kinase activity.

• McFall A, Ulku A, Lambert QT, Kusa A, Rogers-Graham K, Der CJ
• Oncogenic Ras blocks anoikis by activation of a novel effector pathway independent of phosphatidylinositol 3-kinase.
• Mol Cell Biol. 2001; 21: 5488-99
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• Activated Ras, but not Raf, causes transformation of RIE-1 rat intestinal epithelial cells, demonstrating the importance of Raf-independent effector signaling in mediating Ras transformation. To further assess the contribution of Raf-dependent and Raf-independent function in oncogenic Ras transformation, we evaluated the mechanism by which oncogenic Ras blocks suspension-induced apoptosis, or anoikis, of RIE-1 cells. We determined that oncogenic versions of H-, K-, and N-Ras, as well as the Ras-related proteins TC21 and R-Ras, protected RIE-1 cells from anoikis. Surprisingly, our analyses of Ras effector domain mutants or constitutively activated effectors indicated that activation of Raf-1, phosphatidylinositol 3-kinase (PI3K), or RalGDS alone is not sufficient to promote Ras inhibition of anoikis. Treatment of Ras-transformed cells with the U0126 MEK inhibitor caused partial reversion to an anoikis-sensitive state, indicating that extracellular signal-regulated kinase activation contributes to inhibition of anoikis. Unexpectedly, oncogenic Ras failed to activate Akt, and treatment of Ras-transformed RIE-1 cells with the LY294002 PI3K inhibitor did not affect anoikis resistance or growth in soft agar. Thus, while important for Ras transformation of fibroblasts, PI3K may not be involved in Ras transformation of RIE-1 cells. Finally, inhibition of epidermal growth factor receptor kinase activity did not overcome Ras inhibition of anoikis, indicating that this autocrine loop essential for transformation is not involved in anoikis protection. We conclude that a PI3K- and RalGEF-independent Ras effector(s) likely cooperates with Raf to confer anoikis resistance upon RIE-1 cells, thus underscoring the complex nature by which Ras transforms cells.

• Fleming IN, Gray A, Downes CP
• Regulation of the Rac1-specific exchange factor Tiam1 involves both phosphoinositide 3-kinase-dependent and -independent components.
• Biochem J. 2000; 351: 173-82
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• The small GTPase Rac1 is involved in regulating membrane ruffling, gene transcription, cell-cycle progression and cell transformation, and some of these events are blocked by inhibitors of phosphoinositide 3-kinase (PI 3-kinase). Moreover, Rac1 can be activated by several guanine nucleotide exchange factors, which facilitate the release of GDP. We therefore investigated the ability of PI 3-kinase lipid products to regulate Tiam1, a Rac1-specific exchange factor. Tiam1 bound to polyphosphorylated inositol lipids in the rank order PtdIns(3,4,5)P(3)>PtdIns(3,4)P(2) >>PtdIns(4,5)P(2), and this binding could be attributed to the N-terminal pleckstrin-homology (N-PH) domain. Both PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) enhanced Tiam1 guanine nucleotide exchange activity in vitro, but PtdIns(4,5)P(2) had no effect. Co-expression of a constitutively active PI 3-kinase with Tiam1 increased the amount of GTP-bound Rac1 in vivo, a response which required the N-PH domain of Tiam1. Ectopic expression of Tiam1 caused membrane ruffling in Swiss 3T3 cells that was characterized by wortmannin-sensitive and -insensitive components, which required the N-PH domain and the C-terminal PH domain of Tiam1 respectively. These results reveal novel facets of Tiam1-dependent regulation of Rac1 function.

• Sun H, King AJ, Diaz HB, Marshall MS
• Regulation of the protein kinase Raf-1 by oncogenic Ras through phosphatidylinositol 3-kinase, Cdc42/Rac and Pak.
• Curr Biol. 2000; 10: 281-4
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• Activation of the protein kinase Raf-1 is a complex process involving association with the GTP-bound form of Ras (Ras-GTP), membrane translocation and both serine/threonine and tyrosine phosphorylation (reviewed in [1]). We have reported previously that p21-activated kinase 3 (Pak3) upregulates Raf-1 through direct phosphorylation on Ser338 [2]. Here, we investigated the origin of the signal for Pak-mediated Raf-1 activation by examining the role of the small GTPase Cdc42, Rac and Ras, and of phosphatidylinositol (PI) 3-kinase. Pak3 acted synergistically with either Cdc42V12 or Rac1V12 to stimulate the activities of Raf-1, Raf-CX, a membrane-localized Raf-1 mutant, and Raf-1 mutants defective in Ras binding. Raf-1 mutants defective in Ras binding were also readily activated by RasV12. This indirect activation of Raf-1 by Ras was blocked by a dominant-negative mutant of Pak, implicating an alternative Ras effector pathway in Pak-mediated Raf-1 activation. Subsequently, we show that Pak-mediated Raf-1 activation is upregulated by both RasV12C40, a selective activator of PI 3-kinase, and p110-CX, a constitutively active PI 3-kinase. In addition, p85Delta, a mutant of the PI 3-kinase regulatory subunit, inhibited the stimulated activity of Raf-1. Pharmacological inhibitors of PI 3-kinase also blocked both activation and Ser338 phosphorylation of Raf-1 induced by epidermal growth factor (EGF). Thus, Raf-1 activation by Ras is achieved through a combination of both physical interaction and indirect mechanisms involving the activation of a second Ras effector, PI 3-kinase, which directs Pak-mediated regulatory phosphorylation of Raf-1.

• Wennstrom S, Downward J
• Role of phosphoinositide 3-kinase in activation of ras and mitogen-activated protein kinase by epidermal growth factor.
• Mol Cell Biol. 1999; 19: 4279-88
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• The paradigm for activation of Ras and extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase by extracellular stimuli via tyrosine kinases, Shc, Grb2, and Sos does not encompass an obvious role for phosphoinositide (PI) 3-kinase, and yet inhibitors of this lipid kinase family have been shown to block the ERK/MAP kinase signalling pathway under certain circumstances. Here we show that in COS cells activation of both endogenous ERK2 and Ras by low, but not high, concentrations of epidermal growth factor (EGF) is suppressed by PI 3-kinase inhibitors; since Ras activation is less susceptible than ERK2 activation, PI 3-kinase-sensitive events may occur both upstream of Ras and between Ras and ERK2. However, strong elevation of PI 3-kinase lipid product levels by expression of membrane-targeted p110alpha is by itself never sufficient to activate Ras or ERK2. PI 3-kinase inhibition does not affect EGF-induced receptor autophosphorylation or adapter protein phosphorylation or complex formation. The concentrations of EGF for which PI 3-kinase inhibitors block Ras activation induce formation of Shc-Grb2 complexes but not detectable EGF receptor phosphorylation and do not activate PI 3-kinase. The activation of Ras by low, but mitogenic, concentrations of EGF is therefore dependent on basal, rather than stimulated, PI 3-kinase activity; the inhibitory effects of LY294002 and wortmannin are due to their ability to reduce the activity of PI 3-kinase to below the level in a quiescent cell and reflect a permissive rather than an upstream regulatory role for PI 3-kinase in Ras activation in this system.

• Hanna AN, Chan EY, Xu J, Stone JC, Brindley DN
• A novel pathway for tumor necrosis factor-alpha and ceramide signaling involving sequential activation of tyrosine kinase, p21(ras), and phosphatidylinositol 3-kinase.
• J Biol Chem. 1999; 274: 12722-9
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• Treatment of confluent rat2 fibroblasts with C2-ceramide (N-acetylsphingosine), sphingomyelinase, or tumor necrosis factor-alpha (TNFalpha) increased phosphatidylinositol (PI) 3-kinase activity by 3-6-fold after 10 min. This effect of C2-ceramide depended on tyrosine kinase activity and an increase in Ras-GTP levels. Increased PI 3-kinase activity was 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 was inhibited by tyrosine kinase inhibitors (genistein and PP1). The stimulation of PI 3-kinase by sphingomyelinase and C2-ceramide was not observed in fibroblasts expressing dominant-negative Ras (N17) and the stimulation by TNFalpha was decreased by 70%. PI 3-kinase activation by C2-ceramide was not modified by inhibitors of acidic and neutral ceramidases, and it was 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 fibroblasts proliferation.

• Valverde AM, Navarro P, Benito M, Lorenzo M
• H-ras induces glucose uptake in brown adipocytes in an insulin- and phosphatidylinositol 3-kinase-independent manner.
• Exp Cell Res. 1998; 243: 274-81
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• Fetal brown adipocytes (parental cells) expressed mainly Glut4 mRNA glucose transporter, the expression of Glut1 mRNA being much lower. At physiological doses, insulin stimulation for 15 min increased 3-fold glucose uptake and doubled the amount of Glut4 protein located at the plasma membrane. Moreover, phosphatidylinositol (PI) 3-kinase activity was induced by the presence of insulin in those cells, glucose uptake being precluded by PI 3-kinase inhibitors such as wortmannin or LY294002. H-raslys12-transformed brown adipocytes showed a 10-fold higher expression of Glut1 mRNA and protein than parental cells, Glut4 gene expression being completely down-regulated. Glucose uptake increased by 10-fold in transformed cells compared to parental cells; this uptake was unaltered in the presence of insulin and/or wortmannin. Transient transfection of parental cells with a dominant form of active Ras increased basal glucose uptake by 5-fold, no further effects being observed in the presence of insulin. However, PI 3-kinase activity (immunoprecipitated with anti-alphap85 subunit of PI 3-kinase) remained unaltered in H-ras permanent and transient transfectants. Our results indicate that activated Ras induces brown adipocyte glucose transport in an insulin-independent manner, this induction not involving PI 3-kinase activation.

• van Weering DH, de Rooij J, Marte B, Downward J, Bos JL, Burgering BM
• Protein kinase B activation and lamellipodium formation are independent phosphoinositide 3-kinase-mediated events differentially regulated by endogenous Ras.
• Mol Cell Biol. 1998; 18: 1802-11
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• Regulation of phosphoinositide 3-kinase (PI 3-kinase) can occur by binding of the regulatory p85 subunit to tyrosine-phosphorylated proteins and by binding of the p110 catalytic subunit to activated Ras. However, the way in which these regulatory mechanisms act to regulate PI 3-kinase in vivo is unclear. Here we show that several growth factors (basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], and epidermal growth factor [EGF; to activate an EGF receptor-Ret chimeric receptor]) all activate PI 3-kinase in vivo in the neuroectoderm-derived cell line SKF5. However, these growth factors differ in their ability to activate PI 3-kinase-dependent signaling. PDGF and EGF(Ret) treatment induced PI 3-kinase-dependent lamellipodium formation and protein kinase B (PKB) activation. In contrast, bFGF did not induce lamellipodium formation but activated PKB, albeit to a small extent. PDGF and EGF(Ret) stimulation resulted in binding of p85 to tyrosine-phosphorylated proteins and strong Ras activation. bFGF, however, induced only strong activation of Ras. In addition, while RasAsn17 abolished bFGF activation of PKB, PDGF- and EGF(Ret)-induced PKB activation was only partially inhibited and lamellipodium formation was unaffected. Interestingly, in contrast to activation of only endogenous Ras (bFGF), ectopic expression of activated Ras did result in lamellipodium formation. From this we conclude that, in vivo, p85 and Ras synergize to activate PI 3-kinase and that strong activation of only endogenous Ras exerts a small effect on PI 3-kinase activity, sufficient for PKB activation but not lamellipodium formation. This differential sensitivity to PI 3-kinase activation could be explained by our finding that PKB activation and lamellipodium formation are independent PI 3-kinase-induced events.

• Spear N, Estevez AG, Barbeito L, Beckman JS, Johnson GV
• Nerve growth factor protects PC12 cells against peroxynitrite-induced apoptosis via a mechanism dependent on phosphatidylinositol 3-kinase.
• J Neurochem. 1997; 69: 53-9
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• Nerve growth factor (NGF) prevents apoptosis induced by the oxidant peroxynitrite in undifferentiated PC12 rat pheochromocytoma cells. Previous studies have shown that activation of phosphatidylinositol 3-kinase (PI 3-kinase) by NGF via the TrkA receptor tyrosine kinase protects PC12 cells from serum deprivation-induced apoptosis. We found that two PI 3-kinase inhibitors, wortmannin and LY294002, eliminated the protection NGF provided against peroxynitrite-induced apoptosis at concentrations consistent with their effectiveness as PI 3-kinase inhibitors. When the activity of PI 3-kinase was assayed in phosphotyrosine immunoprecipitates after treatment of PC12 cells with peroxynitrite, PI 3-kinase activity was reduced by 50% of that detected in control cells, whereas PI 3-kinase activity in NGF-treated cells was unaffected by peroxynitrite. If an antibody against PI 3-kinase was used to immunoprecipitate the enzyme, treatment with peroxynitrite had no effect on activity. Therefore, peroxynitrite appeared to disrupt interactions between PI 3-kinase and phosphotyrosine proteins, rather than directly inhibiting the enzyme. NGF also activates p21Ras-dependent pathways, but this did not appear to be required for NGF to exert its protective effect against peroxynitrite. PC12 cells expressing a dominant inhibitory mutant of p21Ras were equally susceptible to peroxynitrite-induced apoptosis, which was prevented by NGF. Wortmannin was also able to block the protective effect of NGF in the p21Ras mutant cell line. Although many signaling pathways are activated by NGF, these results suggest that a PI 3-kinase-dependent pathway is important for inhibiting peroxynitrite-induced apoptosis.

• King WG, Mattaliano MD, Chan TO, Tsichlis PN, Brugge JS
• Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.
• Mol Cell Biol. 1997; 17: 4406-18
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• Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.

• Lopez-Ilasaca M et al.
• Requirement of phosphatidylinositol-3 kinase for activation of JNK/SAPKs by PDGF.
• Biochem Biophys Res Commun. 1997; 232: 273-7
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• The molecular mechanism by which cell surface receptors stimulate the serine/threonine kinase activity of c-Jun N-terminal kinases (JNKs) was investigated using a transient cotransfection experiments in COS-7 cells. Our data demonstrate that JNK activity is potently induced by platelet derived growth factor (PDGF) upon expression of beta PDGFR wild type (beta RWT). However, PDGF failed to mediate JNK activation in cells expressing beta PDGFR mutant lacking the binding site for phosphatidylinositol-3 (PI-3) kinase but not for phospholipase C gamma (PLC gamma) or Syp. Consistent with this result, a PI-3 kinase inhibitor, wortmannin inhibited activation of JNK by PDGF. Furthermore, overexpression of P110 the catalytic domain of PI-3 kinase was sufficient for activation of JNKs which could be efficiently inhibited by dominant negative forms of Ras, Rac but not of RhoA or Cdc42. Taken together all of these findings suggest that activation of JNK by PDGF involves receptor association with PI-3 kinase activity, which in turn acts on a ras- and rac-dependent pathway.

• Logan SK, Falasca M, Hu P, Schlessinger J
• Phosphatidylinositol 3-kinase mediates epidermal growth factor-induced activation of the c-Jun N-terminal kinase signaling pathway.
• Mol Cell Biol. 1997; 17: 5784-90
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• The signaling events which mediate activation of c-Jun N-terminal kinase (JNK) are not yet well characterized. To broaden our understanding of upstream mediators which link extracellular signals to the JNK pathway, we investigated the role of phosphatidylinositol (PI) 3-kinase in epidermal growth factor (EGF)-mediated JNK activation. In this report we demonstrate that a dominant negative form of PI 3-kinase as well as the inhibitor wortmannin blocks EGF-induced JNK activation dramatically. However, wortmannin does not have an effect on JNK activation induced by UV irradiation or osmotic shock. In addition, a membrane-targeted, constitutively active PI 3-kinase (p110beta) was shown to produce in vivo products and to activate JNK, while a kinase-mutated form of this protein showed no activation. On the basis of these experiments, we propose that PI 3-kinase activity plays a role in EGF-induced JNK activation in these cells.

• Exley M, Varticovski L
• Evidence for phosphatidylinositol 3-kinase-dependent T cell antigen receptor (TCR) signal transduction.
• Mol Immunol. 1997; 34: 221-6
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• Recent evidence implicates PI 3-kinase in TCR signal transduction. The fungal metabolite wortmannin is a specific inhibitor of PI 3-kinase both in vitro and in vivo when used at nanomolar concentrations. Therefore, we examined the effect of wortmannin on stimulation of primary T cells and T cell lines. Wortmannin had a dose-dependent inhibitory effect on TCR-dependent primary T cell proliferation with IC50 in the nanomolar range. Furthermore, activation of T cell lines independently of antigen presenting cells and, therefore of any CD28 co-stimulatory signaling, was also sensitive to wortmannin. As expected, phorbol ester stimulation bypassed PI 3-kinase signal transduction. Importantly, the effect of wortmannin correlated with inhibition of activation of PI 3-kinase in stimulated T cells. The earliest step in T cell activation, tyrosine kinase activation, was not significantly affected by wortmannin. We conclude that a wortmannin-sensitive enzyme, probably PI 3-kinase, acting downstream of tyrosine kinases, but independently of the phorbol ester activated pathway, is necessary for stimulation of T cells via the TCR, and that this requirement is independent of any role of PI 3-kinase in co-stimulation via CD28 coreceptor. PI 3-kinase is most probably involved in generation of 3-phosphorylated lipid products, and is not merely an adaptor.

• Uribe JM, Keely SJ, Traynor-Kaplan AE, Barrett KE
• Phosphatidylinositol 3-kinase mediates the inhibitory effect of epidermal growth factor on calcium-dependent chloride secretion.
• J Biol Chem. 1996; 271: 26588-95
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• Epidermal growth factor (EGF) and carbachol both inhibit calcium-activated chloride secretion by the human colonic epithelial cell line, T84. Although the inhibitory mechanism for the carbachol effect involves the 3,4,5,6-isomer of inositol tetrakisphosphate, the mechanisms responsible for the EGF effect have not yet been fully elucidated. Here, we studied the role of phosphatidylinositol 3-kinase (PI 3-kinase) in the inhibitory effect of EGF. The PI 3-kinase inhibitor, wortmannin, slightly increased basal chloride secretion and potentiated the secretory response to thapsigargin. Wortmannin also partially reversed EGF-induced, but not carbachol-induced, inhibition of thapsigargin-stimulated chloride secretion. Wortmannin alone had no effect on carbachol- or histamine-induced chloride secretion and completely reversed EGF-induced inhibition of the secretory response to these agonists. EGF, carbachol, histamine, and thapsigargin all increased levels of the 85-kDa regulatory subunit of PI 3-kinase in antiphosphotyrosine immunoprecipitates. However, only EGF significantly increased levels of the 110-kDa catalytic subunit. Furthermore, only EGF increased PI 3-kinase activity in an in vitro kinase assay. High levels of phosphatidylinositol (3)-monophosphate were present in unstimulated cells and significantly reduced by wortmannin. EGF, but not carbachol, rapidly increased levels of phosphatidylinositol (3,4)-bisphosphate and phosphatidylinositol (3,4,5)-trisphosphate. Production of these lipids was also sensitive to wortmannin. Our data suggest that EGF activates PI 3-kinase and that its lipid products may mediate the inhibitory effect of EGF on calcium-dependent chloride secretion. Our data also suggest that a phosphatidylinositol-specific 3-kinase activity is present in unstimulated T84 cells and may regulate production of phosphatidylinositol (3)-monophosphate and basal secretory tone.

• Hu ZW, Shi XY, Lin RZ, Hoffman BB
• Alpha1 adrenergic receptors activate phosphatidylinositol 3-kinase in human vascular smooth muscle cells. Role in mitogenesis.
• J Biol Chem. 1996; 271: 8977-82
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• Activation of alpha1 adrenergic receptors stimulates mitogenesis in human vascular smooth muscle cells (HVSMCs). To examine signaling pathways by which activation of alpha1 receptors may induce mitogenesis in HVSMCs, we have found that alpha1 receptor stimulated-DNA synthesis and activation of mitogen-activated protein (MAP) kinase are blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). To determine directly if activation of alpha1 receptors stimulated PI 3-kinase, in vitro assays of kinase activity were performed in immunocomplexes precipitated by an antibody against the p85 alpha subunit of PI 3-kinase. Noradrenaline stimulated a time- and concentration-dependent activation of PI 3-kinase in the presence of a beta adrenergic receptor antagonist. Noradrenaline-stimulated PI 3-kinase activation was blocked by antagonists of alpha1 receptors and by pertussis toxin, suggesting that alpha1 receptors activate PI 3-kinase via a pertussis toxin-sensitive G protein. Direct activation of protein kinase C by a phorbol ester did not stimulate PI 3-kinase; also, a Ca2+ L-channel blocker did not inhibit noradrenaline-stimulated PI 3-kinase activity. Increased PI 3-kinase activity was detected in both anti-Ras and anti-phosphotyrosine immunoprecipitates from noradrenaline-stimulated HVSMCs. Moreover, noradrenaline stimulated formation of active Ras-GTP complexes. Because blockade of PI 3-kinase by wortmannin inhibited formation of this complex, this result suggests that Ras might be a target of PI 3-kinase. Noradrenaline stimulated tyrosine phosphorylation of the p85 subunit of PI 3-kinase, and a phosphorylated tyrosine protein could be co-immunoprecipitated with anti-p85 of PI 3-kinase. These results demonstrate that stimulation of alpha1 receptors activates PI 3-kinase in HVSMCs and that alpha1 receptor-activated PI 3-kinase is associated with an increase in active Ras-GTP and activation of tyrosine protein phosphorylation. These pathways may contribute to alpha1 receptor-stimulated mitogenic responses including activation of MAP kinase and DNA synthesis in HVSMCs.

• Kwon HJ et al.
• Morphology of ras-transformed cells becomes apparently normal again with tyrosine kinase inhibitors without a decrease in the ras-GTP complex.
• J Biochem (Tokyo). 1995; 118: 221-8
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• Radicicol, an inhibitor of protein-tyrosine kinase, was found to cause morphological reversion of v-Ha-ras-transformed NIH3T3 fibroblasts and T24 human urinary bladder carcinoma cells that contain an activated ras mutation. The network of actin stress fibers was restored during the treatment with radicicol. A similar morphological change was observed with another protein-tyrosine kinase inhibitor, herbimycin A. Radicicol did not cause any changes in the proportion of the active GTP binding form of p21ras or its subcellular localization. These results rule out the possibility that the morphological reversion by radicicol is due to direct or indirect inhibition of the p21ras function. Cycloheximide and actinomycin D inhibited the morphological change by radicicol, suggesting that the induced transcription of a gene(s) followed by de novo protein synthesis is required for suppression of the transformed phenotype in ras-transformed cells by tyrosine kinase inhibitors.

• Kiss Z, Crilly KS
• Ha-Ras stimulates uptake and phosphorylation of ethanolamine: inhibition by wortmannin.
• FEBS Lett. 1995; 357: 279-82
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• Transformation of NIH 3T3 fibroblasts by Ha-Ras resulted in large increases in the phosphorylation of both [14C]ethanolamine (Etn) and [14C]choline (Cho) when these precursors were added to the medium. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), preferentially decreased phosphorylation of externally added Etn in the Ha-Ras transformed, but not in the untransformed, fibroblasts. However, wortmannin had no effect on the phosphorylation of Etn formed endogenously by phorbol ester-stimulated hydrolysis of phosphatidylethanolamine. Data suggest that interaction of mutated Ras with PI3K leads to specific stimulation of Etn uptake, followed by nearly quantitative phosphorylation of Etn by a Ras-activated Cho/Etn kinase.

• Cantrell D, Izquierdo-Pastor M, Reif K, Woodrow M
• Signal transduction by the T-cell antigen receptor: regulation and function of p21ras and PtdIns-3 kinase.
• Chem Immunol. 1994; 59: 115-27

• Hara K et al.
• 1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells.
• Proc Natl Acad Sci U S A. 1994; 91: 7415-9
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• Insulin stimulation drives the formation of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and 1-phosphatidylinositol 3-kinase (PI 3-kinase; ATP:1-phosphatidyl-1D-myo-inositol 3-phosphotransferase, EC 2.7.1.137), a heterodimer consisting of regulatory 85-kDa (p85) and catalytic 110-kDa (p110) subunits. This interaction takes place via the phosphorylated YMXM motifs of IRS-1 and the Src homology region 2 (SH2) domains of p85. In this study, the stable overexpression in a Chinese hamster ovary (CHO) cell line of a mutant p85 alpha (delta p85) protein, which lacks a binding site for p110, disrupted the complex formation between IRS-1 and the catalytic subunit of PI 3-kinase in intact cells during insulin stimulation. Activation of insulin receptor kinase and the tyrosine phosphorylation of IRS-1 remained unaffected. In this cell line, both insulin-stimulated accumulation of phosphatidylinositol 3,4,5-trisphosphate and the insulin-stimulated glucose uptake due to the translocation of GLUT1 glucose transporters were markedly impaired, whereas neither phorbol 12-myristate 13-acetate-stimulated glucose uptake nor the insulin-stimulated activation of RAS was impaired. These results suggest that PI 3-kinase is required for glucose transport in insulin signaling in CHO cells.

• Jhun BH et al.
• Microinjection of the SH2 domain of the 85-kilodalton subunit of phosphatidylinositol 3-kinase inhibits insulin-induced DNA synthesis and c-fos expression.
• Mol Cell Biol. 1994; 14: 7466-75
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• We have investigated the functional role of the SH2 domain of the 85-kDa subunit (p85) of the phosphatidylinositol 3-kinase in the insulin signal transduction pathway. Microinjection of a bacterial fusion protein containing the N-terminal SH2 domain of p85 inhibited insulin- and other growth factor-induced DNA synthesis by 90% and c-fos protein expression by 80% in insulin-responsive rat fibroblasts. The specificity of the fusion protein was examined by in vitro precipitation experiments, which showed that the SH2 domain of p85 can independently associate with both insulin receptor substrate 1 and the insulin receptor itself in the absence of detectable binding to other phosphoproteins. The microinjection results were confirmed through the use of an affinity-purified antibody directed against p85, which gave the same phenotype. Additional studies were carried out in another cell line expressing mutant insulin receptors which lack the cytoplasmic tyrosine residues with which p85 interacts. Microinjection of the SH2 domain fusion protein also inhibited insulin signaling in these cells, suggesting that association of p85 with insulin receptor substrate 1 is a key element in insulin-mediated cell cycle progression. In addition, coinjection of purified p21ras protein with the p85 fusion protein or the antibody restored DNA synthesis, suggesting that ras function is either downstream or independent of p85 SH2 domain interaction.

• Payrastre B, Gironcel D, Plantavid M, Mauco G, Breton M, Chap H
• Phosphoinositide 3-phosphatase segregates from phosphatidylinositol 3-kinase in EGF-stimulated A431 cells and fails to in vitro hydrolyse phosphatidylinositol(3,4,5)trisphosphate.
• FEBS Lett. 1994; 341: 113-8
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• Beside 4- and 5-phosphatases playing a role in the interconversion between the D-3 phosphorylated polyphosphoinositides, the only enzyme described so far to be responsible for a phosphomonesterasic activity on the D-3 position of inositol lipids is a specific 3-phosphatase that hydrolyzes PtdIns(3)P in NIH 3T3 cells. We report here the presence of a potent 3-phosphatase activity in different cell types. This activity is detected both in cytosol and membranes of A431 cells and is inhibited by VO4(-3) and Zn2+. Interestingly, the cytosolic activity from A431 cells selectively hydrolyzes in vitro PtdIns(3)P and PtdIns(3,4)P2, whereas PtdIns(3,4,5)P3 remains a very poor substrate under the same conditions. Finally, assays of phosphatidylinositol 3-kinase and 3-phosphatase activities in the pool of phosphotyrosine-containing proteins isolated from EGF-stimulated A431 cells suggest a compartmentation of these two antagonistic activities during cell activation.

• Kimura K et al.
• Neurite outgrowth of PC12 cells is suppressed by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase.
• J Biol Chem. 1994; 269: 18961-7
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• The effects of wortmannin (WT), an inhibitor of phosphatidylinositol (PI) 3-kinase, on differentiation of PC12 cells were analyzed. WT inhibited PI 3-kinase activity of PC12 cells at a concentration of 10(-7) M in vivo and in vitro. Transient inhibition of PI 3-kinase activity at the time of nerve growth factor stimulation had no effect on activation of the ras protein or neurite formation by the cells. However, continuous inhibition of PI 3-kinase blocked differentiation at the step just before neurite formation. When WT was applied to cells growing neurites, elongation of the neurites was stopped at that step. These results suggest that PI 3-kinase may be involved in neurite elongation.

• Sjolander A, Yamamoto K, Huber BE, Lapetina EG
• Association of p21ras with phosphatidylinositol 3-kinase.
• Proc Natl Acad Sci U S A. 1991; 88: 7908-12
• Display abstract

• In mammalian cells, ras genes code for 21-kDa GTP-binding proteins. Increased expression and mutations in specific amino acids have been closely linked to alterations of normal cell morphology, growth, and differentiation and, in particular, to neoplastic transformation. The signal transduction induced by these p21ras proteins is largely unknown; however, the signaling pathways of several growth factors have been reported to involve phosphatidylinositol (PtdIns) 3-kinase. In the present study of a Ha-ras-transformed epithelial cell line, we demonstrated increased PtdIns 3-kinase activity in anti-phosphotyrosine and anti-receptor (insulin and hybrid insulin-like growth factor I) immunoprecipitates of cells that had been stimulated with insulin or insulin-like growth factor I. The PtdIns 3-kinase activity was also immunoprecipitated in these experiments by the anti-Ras monoclonal antibody Y13-259. The specificity of this association with p21ras was ascertained by the neutralizing effect of the antigen peptide and the absence of PtdIns 3-kinase activity in Y13-259 immunoprecipitates from cells in which the ras gene was turned off. These data indicate that PtdIns 3-kinase activity is an important step in the cascade of reactions in p21ras signal transduction, suggesting that the alterations of the cytoskeleton and growth in ras-transformed cells could be mediated by PtdIns 3-kinase activity.