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1.
Mol Cell ; 83(16): 2991-3009.e13, 2023 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-37567175

RESUMO

The PIP3/PI3K network is a central regulator of metabolism and is frequently activated in cancer, commonly by loss of the PIP3/PI(3,4)P2 phosphatase, PTEN. Despite huge research investment, the drivers of the PI3K network in normal tissues and how they adapt to overactivation are unclear. We find that in healthy mouse prostate PI3K activity is driven by RTK/IRS signaling and constrained by pathway feedback. In the absence of PTEN, the network is dramatically remodeled. A poorly understood YXXM- and PIP3/PI(3,4)P2-binding PH domain-containing adaptor, PLEKHS1, became the dominant activator and was required to sustain PIP3, AKT phosphorylation, and growth in PTEN-null prostate. This was because PLEKHS1 evaded pathway-feedback and experienced enhanced PI3K- and Src-family kinase-dependent phosphorylation of Y258XXM, eliciting PI3K activation. hPLEKHS1 mRNA and activating Y419 phosphorylation of hSrc correlated with PI3K pathway activity in human prostate cancers. We propose that in PTEN-null cells receptor-independent, Src-dependent tyrosine phosphorylation of PLEKHS1 creates positive feedback that escapes homeostasis, drives PIP3 signaling, and supports tumor progression.


Assuntos
PTEN Fosfo-Hidrolase , Neoplasias da Próstata , Animais , Humanos , Masculino , Camundongos , Homeostase , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo
2.
Sci Signal ; 4(168): ra23, 2011 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-21487106

RESUMO

Neutrophils are activated by immunoglobulin G (IgG)-containing immune complexes through receptors that recognize the Fc portion of IgG (FcγRs). Here, we used genetic and pharmacological approaches to define a selective role for the ß isoform of phosphoinositide 3-kinase (PI3Kß) in FcγR-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3Kß alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3Kß and PI3Kδ, suggesting that this pathway displays stimulus strength-dependent redundancy. Activation of PI3Kß by immune complexes involved cooperation between FcγRs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B4. Coincident activation by a tyrosine kinase-coupled receptor (FcγR) and a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (BLT1) may provide a rationale for the preferential activation of the ß isoform of PI3K. PI3Kß-deficient mice were highly protected in an FcγR-dependent model of autoantibody-induced skin blistering and were partially protected in an FcγR-dependent model of inflammatory arthritis, whereas combined deficiency of PI3Kß and PI3Kδ resulted in near-complete protection in the latter case. These results define PI3Kß as a potential therapeutic target in inflammatory disease.


Assuntos
Complexo Antígeno-Anticorpo/imunologia , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Western Blotting , Antígenos CD2/genética , Antígenos CD2/metabolismo , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Inibidores Enzimáticos/farmacologia , Feminino , Citometria de Fluxo , Rearranjo Gênico do Linfócito B/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região de Junção de Imunoglobulinas/genética , Região de Junção de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neutrófilos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Espécies Reativas de Oxigênio/metabolismo , Receptores de IgG/metabolismo , Receptores do Leucotrieno B4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia
3.
Blood ; 116(23): 4978-89, 2010 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-20813901

RESUMO

The generation of reactive oxygen species (ROS) by the nicotinamide adenine dinucleotide phosphate oxidase is an important mechanism by which neutrophils kill pathogens. The oxidase is composed of a membrane-bound cytochrome and 4 soluble proteins (p67(phox), p40(phox), p47(phox), and GTP-Rac). These components form an active complex at the correct time and subcellular location through a series of incompletely understood mutual interactions, regulated, in part, by GTP/GDP exchange on Rac, protein phosphorylation, and binding to lipid messengers. We have used a variety of assays to follow the spatiotemporal assembly of the oxidase in genetically engineered primary mouse neutrophils, during phagocytosis of both serum- and immunoglobulin G-opsonized targets. The oxidase assembles directly on serum-Staphylococcus aureus-containing phagosomes within seconds of phagosome formation; this process is only partially dependent (∼ 30%) on PtdIns3P binding to p40(phox), but totally dependent on Rac1/2 binding to p67(phox). In contrast, in response to immunoglobulin G-targets, the oxidase first assembles on a tubulovesicular compartment that develops at sites of granule fusion to the base of the emerging phagosome; oxidase assembly and activation is highly dependent on both PtdIns3P-p40(phox) and Rac2-p67(phox) interactions and delivery to the phagosome is regulated by Rab27a. These results define a novel pathway for oxidase assembly downstream of FcR-activation.


Assuntos
NADPH Oxidases/metabolismo , Neutrófilos/enzimologia , Fagocitose/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Ativação Enzimática/fisiologia , Humanos , Camundongos , Camundongos Knockout , Neutrófilos/imunologia , Fosfatos de Fosfatidilinositol/imunologia , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Receptores Fc/imunologia , Proteínas rac de Ligação ao GTP/imunologia
4.
Blood ; 116(26): 6027-36, 2010 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-20861461

RESUMO

The neutrophil nicotinamide adenine dinucleotide phosphate-oxidase is a multisubunit enzyme (comprising gp91(phox), p22(phox), p67(phox), p40(phox), p47(phox), and Rac) that plays a vital role in microbial killing. The recent discovery of a chronic granulomatous disease patient who expresses a mutant p40(phox) subunit, together with the development of mouse models of p40(phox) function, indicate phosphatidylinositol 3-phosphate binding to the PX domain of p40(phox) is an important signal for oxidase activation. However, the presence of other conserved residues and domains in p40(phox) suggest further regulatory roles for this protein. To test this, we introduced wild-type and mutated versions of p40(phox) into fully differentiated mouse neutrophils by retroviral transduction of p40(phox)(-/-) bone marrow progenitors and repopulation of the bone marrow compartment in radiation chimaeras. Phosphorylation of p40(phox) on threonine 154, but not serine 315, was required for full oxidase activation in response to formylated bacterial peptide fMLP, serum-opsonized S aureus, and immunoglobulin-opsonized sheep red blood cells. A functional SH3 domain was not required for oxidase activation, and deletion of the entire domain resulted in enhanced oxidase responses. Phosphorylation of threonine 154 in response to S aureus was mediated by protein kinase Cδ and was required for full translocation of p47(phox) to phagosomes. These results define an important new element in the physiological activation of the oxidase.


Assuntos
NADPH Oxidases/metabolismo , Neutrófilos/enzimologia , Fosfoproteínas/fisiologia , Proteína Quinase C-delta/fisiologia , Infecções Estafilocócicas/metabolismo , Treonina , Animais , Western Blotting , Medula Óssea/metabolismo , Eritrócitos/metabolismo , Feminino , Masculino , Camundongos , Camundongos Knockout/microbiologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Fagossomos/metabolismo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Retroviridae/genética , Ovinos , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus , Irradiação Corporal Total
5.
Blood ; 112(13): 5202-11, 2008 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-18755982

RESUMO

Phagocytosis and activation of the NADPH oxidase are important mechanisms by which neutrophils and macrophages engulf and kill microbial pathogens. We investigated the role of PI3K signaling pathways in the regulation of the oxidase during phagocytosis of Staphylococcus aureus and Escherichia coli by mouse and human neutrophils, a mouse macrophage-like cell line and a human myeloid-like cell line. Phagocytosis of these bacteria was promoted by serum, independent of serum-derived antibodies, and effectively abolished in mouse neutrophils lacking the beta(2)-integrin common chain, CD18. A combination of PI3K isoform-selective inhibitors, mouse knock-outs, and RNA-interference indicated CD18-dependent activation of the oxidase was independent of class I and II PI3Ks, but substantially dependent on the single class III isoform (Vps34). Class III PI3K was responsible for the synthesis of PtdIns(3)P on phagosomes containing either bacteria. The use of mouse neutrophils carrying an appropriate knock-in mutation indicated that PtdIns(3)P binding to the PX domain of their p40(phox) oxidase subunit is important for oxidase activation in response to both S aureus and E coli. This interaction does not, however, account for all the PI3K sensitivity of these responses, particularly the oxidase response to E coli, suggesting that additional mechanisms for PtdIns(3)P-regulation of the oxidase must exist.


Assuntos
Antígenos CD18/fisiologia , Escherichia coli/imunologia , NADPH Oxidases/metabolismo , Neutrófilos/enzimologia , Fagocitose , Fosfatidilinositol 3-Quinases/fisiologia , Staphylococcus aureus/imunologia , Animais , Linhagem Celular , Ativação Enzimática , Humanos , Camundongos , Neutrófilos/imunologia , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoproteínas/metabolismo
6.
Dev Biol ; 313(1): 384-97, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18037397

RESUMO

Growth cones are dynamic membrane structures that migrate to target tissue by rearranging their cytoskeleton in response to environmental cues. The lipid phosphatidylinositol (4,5) bisphosphate (PIP(2)) resides on the plasma membrane of all eukaryotic cells and is thought to be required for actin cytoskeleton rearrangements. Thus PIP(2) is likely to play a role during neuron development, but this has never been tested in vivo. In this study, we have characterized the PIP(2) synthesizing enzyme Type I PIP kinase (ppk-1) in Caenorhabditis elegans. PPK-1 is strongly expressed in the nervous system, and can localize to the plasma membrane. We show that PPK-1 purified from C. elegans can generate PIP(2)in vitro and that overexpression of the kinase causes an increase in PIP(2) levels in vivo. In developing neurons, PPK-1 overexpression leads to growth cones that become stalled, produce ectopic membrane projections, and branched axons. Once neurons are established, PPK-1 overexpression results in progressive membrane overgrowth and degeneration during adulthood. These data suggest that overexpression of the Type I PIP kinase inhibits growth cone collapse, and that regulation of PIP(2) levels in established neurons may be important to maintain structural integrity and prevent neuronal degeneration.


Assuntos
Axônios/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Cones de Crescimento/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Membrana Celular/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética
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