The Experts below are selected from a list of 24963 Experts worldwide ranked by ideXlab platform
Ana C Carrera - One of the best experts on this subject based on the ideXlab platform.
-
Phosphoinositide 3 kinase beta protects nuclear envelope integrity by controlling rcc1 localization and ran activity
Molecular and Cellular Biology, 2015Co-Authors: Javier Redondomunoz, Maria J Rodriguez, Vicente Perezgarcia, Jose M Valpuesta, Ana C CarreraAbstract:The nuclear envelope (NE) forms a barrier between the nucleus and the cytosol that preserves genomic integrity. The nuclear lamina and nuclear pore complexes (NPCs) are NE components that regulate nuclear events through interaction with other proteins and DNA. Defects in the nuclear lamina are associated with the development of laminopathies. As cells depleted of Phosphoinositide 3-Kinase beta (PI3Kβ) showed an aberrant nuclear morphology, we studied the contribution of PI3Kβ to maintenance of NE integrity. pik3cb depletion reduced the nuclear membrane tension, triggered formation of areas of lipid bilayer/lamina discontinuity, and impaired NPC assembly. We show that one mechanism for PI3Kβ regulation of NE/NPC integrity is its association with RCC1 (regulator of chromosome condensation 1), the activator of nuclear Ran GTPase. PI3Kβ controls RCC1 binding to chromatin and, in turn, Ran activation. These findings suggest that PI3Kβ regulates the nuclear envelope through upstream regulation of RCC1 and Ran.
-
differential requirements for dock2 and Phosphoinositide 3 kinase γ during t and b lymphocyte homing
Immunity, 2004Co-Authors: Cesar Nombelaarrieta, Ana C Carrera, Rosa Ana Lacalle, Maria C Montoya, Yuya Kunisaki, Diego Megias, M Marques, Santos Manes, Yoshinori FukuiAbstract:Abstract Chemokines guide lymphocytes from blood to secondary lymphoid organs by triggering integrin-dependent firm adhesion under vascular flow and directed migration of T and B lymphocytes within lymphoid tissue. Here, we analyze the roles of DOCK2, a mammalian homolog of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City, and Phosphoinositide-3-Kinase (PI3K) during lymphocyte recirculation. DOCK2 mediated efficient lymphocyte migration in a largely PI3K-independent manner, although a minor, PI3K-dependent pathway for migration was observed in wild-type and DOCK2-deficient lymphocytes. In T cells, this residual migration depended mainly on PI3Kγ, whereas other PI3K isoforms were implicated in B cells. In vitro adhesion assays and intravital microscopy of lymphoid organ vasculature uncovered an unexpected defect in integrin activation in DOCK2 −/− B cells, whereas lack of DOCK2 did not affect chemokine-triggered integrin activation in T cells. DOCK2 and PI3Kγ thus play distinct roles during T and B cell integrin activation and migration.
-
Control of cyclin G2 mRNA expression by forkhead transcription factors: novel mechanism for cell cycle control by Phosphoinositide 3-Kinase and forkhead.
Molecular and cellular biology, 2004Co-Authors: Lorena Martínez-gac, Miriam Marqués, Zaira García, Miguel R. Campanero, Ana C CarreraAbstract:Cyclin G2 is an unconventional cyclin highly expressed in postmitotic cells. Unlike classical cyclins that promote cell cycle progression, cyclin G2 blocks cell cycle entry. Here we studied the mechanisms that regulate cyclin G2 mRNA expression during the cell cycle. Analysis of synchronized NIH 3T3 cell cultures showed elevated cyclin G2 mRNA expression levels at G0, with a considerable reduction as cells enter cell cycle. Downregulation of cyclin G2 mRNA levels requires activation of Phosphoinositide 3-Kinase, suggesting that this enzyme controls cyclin G2 mRNA expression. Because the Phosphoinositide 3-Kinase pathway inhibits the FoxO family of forkhead transcription factors, we examined the involvement of these factors in the regulation of cyclin G2 expression. We show that active forms of the forkhead transcription factor FoxO3a (FKHRL1) increase cyclin G2 mRNA levels. Cyclin G2 has forkhead consensus motifs in its promoter, which are transactivated by constitutive active FoxO3a forms. Finally, interference with forkhead-mediated transcription by overexpression of an inactive form decreases cyclin G2 mRNA expression levels. These results show that FoxO genes regulate cyclin G2 expression, illustrating a new role for Phosphoinositide 3-Kinase and FoxO transcription factors in the control of cell cycle entry.
-
increased Phosphoinositide 3 kinase activity induces a lymphoproliferative disorder and contributes to tumor generation in vivo
The FASEB Journal, 2000Co-Authors: Luis R Borlado, Clara Redondo, Beatriz Alvarez, Concepcion Jimenez, Luis M Criado, Juana M Flores, Miguel A R Marcos, Carlos Martineza, Dimitrios Balomenos, Ana C CarreraAbstract:Alterations in the cell division:cell death ratio induce multiple autoimmune and transformation processes. Phosphoinositide 3-Kinase (PI3K) controls cell division and cell death in vitro, but its effect on the function of the cellular immune system and on tumor formation in mammals is poorly characterized. Here we show that transgenic mice expressing in T lymphocytes an active form of PI3K derived from a thymic lymphoma, p65PI3K, developed an infiltrating lymphoproliferative disorder and autoimmune renal disease with an increased number of T lymphocytes exhibiting a memory phenotype and reduced apoptosis. This pathology was strikingly similar to that described in mice exhibiting heterozygous loss of the tumor suppressor PTEN, a lipid and protein phosphatase. We show that overexpression of PTEN selectively blocks p65PI3K-induced 3T3 fibroblast transformation. Moreover, the early development of T cell lymphomas in p65PI3K Tg p53−/− mice indicated that PI3K contributes to tumor development. These observation...
Luis Michea - One of the best experts on this subject based on the ideXlab platform.
-
endothelial epithelial sodium channel inhibition activates endothelial nitric oxide synthase via Phosphoinositide 3 kinase akt in small diameter mesenteric arteries
Hypertension, 2009Co-Authors: Francisco R Perez, Fabiola Venegas, Magdalena Gonzalez, Sergio Andres, Catalina Vallejos, Gloria Riquelme, Jimena Sierralta, Luis MicheaAbstract:Recent studies have shown that the epithelial sodium channel (ENaC) is expressed in vascular tissue. However, the role that ENaC may play in the responses to vasoconstrictors and NO production has yet to be addressed. In this study, the contractile responses of perfused pressurized small-diameter rat mesenteric arteries to phenylephrine and serotonin were reduced by ENaC blockade with amiloride (75.1±3.2% and 16.9±2.3% of control values, respectively; P <0.01) that was dose dependent (EC50=88.9±1.6 nmol/L). Incubation with benzamil, another ENaC blocker, had similar effects. α, β, and γ ENaC were identified in small-diameter rat mesenteric arteries using RT-PCR and Western blot with specific antibodies. In situ hybridization and immunohistochemistry localized ENaC expression to the tunica media and endothelium of small-diameter rat mesenteric arteries. Patch-clamp experiments demonstrated that primary cultures of mesenteric artery endothelial cells expressed amiloride-sensitive sodium currents. Mechanical ablation of the endothelium or inhibition of eNOS with N ω-nitro-l-arginine inhibited the reduction in contractility caused by ENaC blockers. ENaC inhibitors increased eNOS phosphorylation (Ser 1177) and Akt phosphorylation (Ser 473). The presence of the Phosphoinositide 3-Kinase inhibitor LY294002 blunted Akt phosphorylation and eNOS phosphorylation and the decrease in the response to phenylephrine caused by blockers of ENaC, indicating that the Phosphoinositide 3-Kinase/Akt pathway was activated after ENaC inhibition. Finally, we observed that the effects of blockers of ENaC were flow dependent and that the vasodilatory response to shear stress was enhanced by ENaC blockade. Our results identify a previously unappreciated role for ENaC as a negative modulator of eNOS and NO production in resistance arteries.
-
endothelial epithelial sodium channel inhibition activates endothelial nitric oxide synthase via Phosphoinositide 3 kinase akt in small diameter mesenteric arteries
Hypertension, 2009Co-Authors: Francisco R Perez, Fabiola Venegas, Magdalena Gonzalez, Sergio Andres, Catalina Vallejos, Gloria Riquelme, Jimena Sierralta, Luis MicheaAbstract:Recent studies have shown that the epithelial sodium channel (ENaC) is expressed in vascular tissue. However, the role that ENaC may play in the responses to vasoconstrictors and NO production has yet to be addressed. In this study, the contractile responses of perfused pressurized small-diameter rat mesenteric arteries to phenylephrine and serotonin were reduced by ENaC blockade with amiloride (75.1+/-3.2% and 16.9+/-2.3% of control values, respectively; P<0.01) that was dose dependent (EC(50)=88.9+/-1.6 nmol/L). Incubation with benzamil, another ENaC blocker, had similar effects. alpha, beta, and gamma ENaC were identified in small-diameter rat mesenteric arteries using RT-PCR and Western blot with specific antibodies. In situ hybridization and immunohistochemistry localized ENaC expression to the tunica media and endothelium of small-diameter rat mesenteric arteries. Patch-clamp experiments demonstrated that primary cultures of mesenteric artery endothelial cells expressed amiloride-sensitive sodium currents. Mechanical ablation of the endothelium or inhibition of eNOS with N(omega)-nitro-L-arginine inhibited the reduction in contractility caused by ENaC blockers. ENaC inhibitors increased eNOS phosphorylation (Ser 1177) and Akt phosphorylation (Ser 473). The presence of the Phosphoinositide 3-Kinase inhibitor LY294002 blunted Akt phosphorylation and eNOS phosphorylation and the decrease in the response to phenylephrine caused by blockers of ENaC, indicating that the Phosphoinositide 3-Kinase/Akt pathway was activated after ENaC inhibition. Finally, we observed that the effects of blockers of ENaC were flow dependent and that the vasodilatory response to shear stress was enhanced by ENaC blockade. Our results identify a previously unappreciated role for ENaC as a negative modulator of eNOS and NO production in resistance arteries.
Len R Stephens - One of the best experts on this subject based on the ideXlab platform.
-
crystal structure and functional analysis of ras binding to its effector Phosphoinositide 3 kinase γ
Cell, 2000Co-Authors: Michael E Pacold, Phillip T Hawkins, Len R Stephens, Sabine Suire, Olga Perisic, Samuel Laragonzalez, Colin T Davis, Edward H Walker, John F Eccleston, Roger WilliamsAbstract:Ras activation of Phosphoinositide 3-Kinase (PI3K) is important for survival of transformed cells. We find that PI3Kgamma is strongly and directly activated by H-Ras G12V in vivo or by GTPgammaS-loaded H-Ras in vitro. We have determined a crystal structure of a PI3Kgamma/Ras.GMPPNP complex. A critical loop in the Ras binding domain positions Ras so that it uses its switch I and switch II regions to bind PI3Kgamma. Mutagenesis shows that interactions with both regions are essential for binding PI3Kgamma. Ras also forms a direct contact with the PI3Kgamma catalytic domain. These unique Ras/PI3Kgamma interactions are likely to be shared by PI3Kalpha. The complex with Ras shows a change in the PI3K conformation that may represent an allosteric component of Ras activation.
-
structural determinants of Phosphoinositide 3 kinase inhibition by wortmannin ly294002 quercetin myricetin and staurosporine
Molecular Cell, 2000Co-Authors: Edward H Walker, Len R Stephens, Matthias P Wymann, Michael E Pacold, Olga Perisic, Philip T Hawkins, Roger WilliamsAbstract:The specific Phosphoinositide 3-Kinase (PI3K) inhibitors wortmannin and LY294002 have been invaluable tools for elucidating the roles of these enzymes in signal transduction pathways. The X-ray crystallographic structures of PI3Kgamma bound to these lipid kinase inhibitors and to the broad-spectrum protein kinase inhibitors quercetin, myricetin, and staurosporine reveal how these compounds fit into the ATP binding pocket. With a nanomolar IC50, wortmannin most closely fits and fills the active site and induces a conformational change in the catalytic domain. Surprisingly, LY294002 and the lead compound on which it was designed, quercetin, as well as the closely related flavonoid myricetin bind PI3K in remarkably different orientations that are related to each other by 180 degrees rotations. Staurosporine/PI3K interactions are reminiscent of low-affinity protein kinase/staurosporine complexes. These results provide a rich basis for development of isoform-specific PI3K inhibitors with therapeutic potential.
-
structural determinants of Phosphoinositide 3 kinase inhibition by wortmannin ly294002 quercetin myricetin and staurosporine
Molecular Cell, 2000Co-Authors: Edward H Walker, Len R Stephens, Matthias P Wymann, Michael E Pacold, Olga Perisic, Philip T Hawkins, Roger WilliamsAbstract:Abstract The specific Phosphoinositide 3-Kinase (PI3K) inhibitors wortmannin and LY294002 have been invaluable tools for elucidating the roles of these enzymes in signal transduction pathways. The X-ray crystallographic structures of PI3Kγ bound to these lipid kinase inhibitors and to the broad-spectrum protein kinase inhibitors quercetin, myricetin, and staurosporine reveal how these compounds fit into the ATP binding pocket. With a nanomolar IC 50 , wortmannin most closely fits and fills the active site and induces a conformational change in the catalytic domain. Surprisingly, LY294002 and the lead compound on which it was designed, quercetin, as well as the closely related flavonoid myricetin bind PI3K in remarkably different orientations that are related to each other by 180° rotations. Staurosporine/PI3K interactions are reminiscent of low-affinity protein kinase/staurosporine complexes. These results provide a rich basis for development of isoform-specific PI3K inhibitors with therapeutic potential.
-
activation of Phosphoinositide 3 kinase is required for pdgf stimulated membrane ruffling
Current Biology, 1994Co-Authors: Stefan Wennstrom, Lena Claessonwelsh, Kazuyoshi Yonezawa, Phillip T Hawkins, Frank T Cooke, Kenta Hara, Masato Kasuga, T R Jackson, Len R StephensAbstract:Abstract Background: There is substantial evidence that Phosphoinositide 3-Kinase (PI 3-Kinase) is a critical component of signalling pathways used by the cell-surface receptors for a variety of mammalian growth factors and other hormones. The physiological product of this enzyme is a highly polar membrane lipid called phosphatidylinositol (3,4,5)-trisphosphate This lipid has been postulated to act as a second-messenger in cells but its putative targets are still unknown. Results A particular rearrangement of actin filaments, which results in membrane ruffling, is elicited by the activation of PDGF β -receptors expressed in cultured porcine aortic endothelial cells. We have found that this consequence of PDGF β -receptor activation is inhibited by three independent manipulations of PI 3-Kinase activity: firstly, by the deletion of tyrosine residues in the PDGF β -receptor to which PI 3-Kinase binds; secondly, by the overexpression of a mutant 85 kD PI 3-Kinase regulatory subunit to which the catalytic kinase subunit cannot bind; and thirdly, by the addition of the fungal metabolite wortmannin, which is a potent inhibitor of the catalytic activity of PI 3-Kinase. Conclusion These results argue strongly that phosphatidylinositol (3,4,5)-trisphosphate synthesis is required for growth-factor-stimulated membrane ruffling in porcine aortic endothelial cells, and suggest that synthesis of this lipid may be part of a signalling pathway leading to direct or indirect activation of the small GTP-binding protein Rac.
-
a novel Phosphoinositide 3 kinase activity in myeloid derived cells is activated by g protein βγ subunits
Cell, 1994Co-Authors: Len R Stephens, T R Jackson, Alan V Smrcka, F T Cooke, Paul C Sternweis, Phillip T HawkinsAbstract:Abstract Phosphoinositide 3 kinase (PI3K) is a key signaling enzyme implicated in receptor-stimulated mitogenesis, oxidative bursting in neutrophils, membrane ruffling, and glucose uptake. A PI3K has already been purified, cloned, and shown to be regulated by receptors that act via tyrosine kinase-dependent regulatory mechanisms. We report that an immunologically, pharmacologically, and chromatographically distinct form of PI3K activity present in neutrophils and U937 cells is specifically activated by G protein βγ subunits. This data suggests PI3Ks conform to the paradigm set by receptor regulation of phosphoinositidase Cs: different receptor transduction systems specifically regulate dedicated isoforms of effector protein.
Peter J Tummino - One of the best experts on this subject based on the ideXlab platform.
-
epigallocatechin gallate egcg a major component of green tea is a dual Phosphoinositide 3 kinase mtor inhibitor
Biochemical and Biophysical Research Communications, 2011Co-Authors: Glenn S Van Aller, Jeffrey D Carson, Wei Tang, Hao Peng, Lin Zhao, Peter J TumminoAbstract:Abstract The PI3K signaling pathway is activated in a broad spectrum of human cancers, either directly by genetic mutation or indirectly via activation of receptor tyrosine kinases or inactivation of the PTEN tumor suppressor. The key nodes of this pathway have emerged as important therapeutic targets for the treatment of cancer. In this study, we show that (−)-epigallocatechin-3-gallate (EGCG), a major component of green tea, is an ATP-competitive inhibitor of both Phosphoinositide-3-Kinase (PI3K) and mammalian target of rapamycin (mTOR) with Ki values of 380 and 320 nM respectively. The potency of EGCG against PI3K and mTOR is within physiologically relevant concentrations. In addition, EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231 and A549 cells. Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site, agreeing with the finding that EGCG competes for ATP binding. Our results suggest another important molecular mechanism for the anticancer activities of EGCG.
-
epigallocatechin gallate egcg a major component of green tea is a dual Phosphoinositide 3 kinase mtor inhibitor
Biochemical and Biophysical Research Communications, 2011Co-Authors: Glenn S Van Aller, Jeffrey D Carson, Wei Tang, Hao Peng, Lin Zhao, Robert A Copeland, Peter J TumminoAbstract:Abstract The PI3K signaling pathway is activated in a broad spectrum of human cancers, either directly by genetic mutation or indirectly via activation of receptor tyrosine kinases or inactivation of the PTEN tumor suppressor. The key nodes of this pathway have emerged as important therapeutic targets for the treatment of cancer. In this study, we show that (−)-epigallocatechin-3-gallate (EGCG), a major component of green tea, is an ATP-competitive inhibitor of both Phosphoinositide-3-Kinase (PI3K) and mammalian target of rapamycin (mTOR) with Ki values of 380 and 320 nM respectively. The potency of EGCG against PI3K and mTOR is within physiologically relevant concentrations. In addition, EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231 and A549 cells. Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site, agreeing with the finding that EGCG competes for ATP binding. Our results suggest another important molecular mechanism for the anticancer activities of EGCG.
Roger Williams - One of the best experts on this subject based on the ideXlab platform.
-
mechanism of two classes of cancer mutations in the Phosphoinositide 3 kinase catalytic subunit
Science, 2007Co-Authors: Nabil Miled, Jonathan M. Backer, Olga Perisic, Marketa Zvelebil, Yuval Inbar, Dina Schneidmanduhovny, Haim J Wolfson, Roger WilliamsAbstract: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. We used crystallographic and biochemical approaches 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. We also examined helical domain mutations and 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 our 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.
-
crystal structure and functional analysis of ras binding to its effector Phosphoinositide 3 kinase γ
Cell, 2000Co-Authors: Michael E Pacold, Phillip T Hawkins, Len R Stephens, Sabine Suire, Olga Perisic, Samuel Laragonzalez, Colin T Davis, Edward H Walker, John F Eccleston, Roger WilliamsAbstract:Ras activation of Phosphoinositide 3-Kinase (PI3K) is important for survival of transformed cells. We find that PI3Kgamma is strongly and directly activated by H-Ras G12V in vivo or by GTPgammaS-loaded H-Ras in vitro. We have determined a crystal structure of a PI3Kgamma/Ras.GMPPNP complex. A critical loop in the Ras binding domain positions Ras so that it uses its switch I and switch II regions to bind PI3Kgamma. Mutagenesis shows that interactions with both regions are essential for binding PI3Kgamma. Ras also forms a direct contact with the PI3Kgamma catalytic domain. These unique Ras/PI3Kgamma interactions are likely to be shared by PI3Kalpha. The complex with Ras shows a change in the PI3K conformation that may represent an allosteric component of Ras activation.
-
structural determinants of Phosphoinositide 3 kinase inhibition by wortmannin ly294002 quercetin myricetin and staurosporine
Molecular Cell, 2000Co-Authors: Edward H Walker, Len R Stephens, Matthias P Wymann, Michael E Pacold, Olga Perisic, Philip T Hawkins, Roger WilliamsAbstract:The specific Phosphoinositide 3-Kinase (PI3K) inhibitors wortmannin and LY294002 have been invaluable tools for elucidating the roles of these enzymes in signal transduction pathways. The X-ray crystallographic structures of PI3Kgamma bound to these lipid kinase inhibitors and to the broad-spectrum protein kinase inhibitors quercetin, myricetin, and staurosporine reveal how these compounds fit into the ATP binding pocket. With a nanomolar IC50, wortmannin most closely fits and fills the active site and induces a conformational change in the catalytic domain. Surprisingly, LY294002 and the lead compound on which it was designed, quercetin, as well as the closely related flavonoid myricetin bind PI3K in remarkably different orientations that are related to each other by 180 degrees rotations. Staurosporine/PI3K interactions are reminiscent of low-affinity protein kinase/staurosporine complexes. These results provide a rich basis for development of isoform-specific PI3K inhibitors with therapeutic potential.
-
structural determinants of Phosphoinositide 3 kinase inhibition by wortmannin ly294002 quercetin myricetin and staurosporine
Molecular Cell, 2000Co-Authors: Edward H Walker, Len R Stephens, Matthias P Wymann, Michael E Pacold, Olga Perisic, Philip T Hawkins, Roger WilliamsAbstract:Abstract The specific Phosphoinositide 3-Kinase (PI3K) inhibitors wortmannin and LY294002 have been invaluable tools for elucidating the roles of these enzymes in signal transduction pathways. The X-ray crystallographic structures of PI3Kγ bound to these lipid kinase inhibitors and to the broad-spectrum protein kinase inhibitors quercetin, myricetin, and staurosporine reveal how these compounds fit into the ATP binding pocket. With a nanomolar IC 50 , wortmannin most closely fits and fills the active site and induces a conformational change in the catalytic domain. Surprisingly, LY294002 and the lead compound on which it was designed, quercetin, as well as the closely related flavonoid myricetin bind PI3K in remarkably different orientations that are related to each other by 180° rotations. Staurosporine/PI3K interactions are reminiscent of low-affinity protein kinase/staurosporine complexes. These results provide a rich basis for development of isoform-specific PI3K inhibitors with therapeutic potential.