RESUMO
Regulation of the PI-3 kinase (PI3K)/Akt signalling pathway is essential for maintaining the integrity of fundamental cellular processes, cell growth, survival, death and metabolism, and dysregulation of this pathway is implicated in the development and progression of cancers. Receptor tyrosine kinases (RTKs) are major upstream regulators of PI3K/Akt signalling. The phosphatase and tensin homologue (PTEN), a well characterised tumour suppressor, is a prime antagonist of PI3K and therefore a negative regulator of this pathway. Loss or inactivation of PTEN, which occurs in many tumour types, leads to overactivation of RTK/PI3K/Akt signalling driving tumourigenesis. Cellular PTEN levels are tightly regulated by a number of transcriptional, post-transcriptional and post-translational regulatory mechanisms. Of particular interest, transcription of the PTEN pseudogene, PTENP1, produces sense and antisense transcripts that exhibit post-transcriptional and transcriptional modulation of PTEN expression respectively. These additional levels of regulatory complexity governing PTEN expression add to the overall intricacies of the regulation of RTK/PI-3 K/Akt signalling. This review will discuss the regulation of oncogenic PI3K signalling by PTEN (the regulator) with a focus on the modulatory effects of the sense and antisense transcripts of PTENP1 on PTEN expression, and will further explore the potential for new therapeutic opportunities in cancer treatment.
Assuntos
Neoplasias/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Antineoplásicos/uso terapêutico , Humanos , MicroRNAs/genética , Neoplasias/tratamento farmacológico , PTEN Fosfo-Hidrolase/genética , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
The tumor suppressor PTEN plays a critical role in the regulation of multiple cellular processes that include survival, cell cycle, proliferation, and apoptosis. PTEN is frequently mutated or deleted in various human cancer cells to promote tumorigenesis. PTEN is regulated by SUMOylation, but the SUMO E3 ligase involved in the SUMOylation of PTEN remains unclear. Here, we demonstrated that PIASxα is a SUMO E3 ligase for PTEN. PIASxα physically interacted with PTEN both in vitro and in vivo. Their interaction depended on the integrity of phosphatase and C2 domains of PTEN and the region of PIASxα comprising residues 134-347. PIASxα enhanced PTEN protein stability by reducing PTEN ubiquitination, whereas the mutation of PTEN SUMO1 conjugation sites neutralized the effect of PIASxα on PTEN protein half-life. Functionally, PIASxα, as a potential tumor suppressor, negatively regulated the PI3K-Akt pathway through stabilizing PTEN protein. Overexpression of PIASxα led to G0/G1 cell cycle arrest, thus triggering cell proliferation inhibition and tumor suppression, whereas PIASxα knockdown or deficiency in catalytic activity abolished the inhibition. Together our studies suggest that PIASxα is a novel SUMO E3 ligase for PTEN, and it positively regulates PTEN protein level in tumor suppression.
Assuntos
PTEN Fosfo-Hidrolase/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteína SUMO-1/metabolismo , Sumoilação/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Células HeLa , Humanos , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Inibidoras de STAT Ativados/genética , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fase de Repouso do Ciclo Celular/fisiologia , Proteína SUMO-1/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Dendritic cells (DC) play a critical role in modulating antigen-specific immune responses elicited by T cells via engagement of the prototypic T cell costimulatory receptor CD28 by the cognate ligands CD80/CD86, expressed on DC. Although CD28 signaling in T cell activation has been well characterized, it has only recently been shown that CD80/CD86, which have no demonstrated binding domains for signaling proteins in their cytoplasmic tails, nonetheless also transduce signals to the DC. Functionally, CD80/CD86 engagement results in DC production of the pro-inflammatory cytokine IL-6, which is necessary for full T cell activation. However, ligation of CD80/CD86 by CTLA4 also induces DC production of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO), which depletes local pools of the essential amino acid tryptophan, resulting in blockade of T cell activation. Despite the significant role of CD80/CD86 in immunological processes and the seemingly opposing roles they play by producing IL-6 and IDO upon their activation, how CD80/CD86 signal remains poorly understood. We have now found that cross-linking CD80/CD86 in human DC activates the PI3K/AKT pathway. This results in phosphorylation/inactivation of its downstream target, FOXO3A, and alleviates FOXO3A-mediated suppression of IL-6 expression. A second event downstream of AKT phosphorylation is activation of the canonical NF-κB pathway, which induces IL-6 expression. In addition to these downstream pathways, we unexpectedly found that CD80/CD86-induced PI3K signaling is regulated by previously unrecognized cross-talk with NOTCH1 signaling. This cross-talk is facilitated by NOTCH-mediated up-regulation of the expression of prolyl isomerase PIN1, which in turn increases enzyme activity of casein kinase II. Subsequently, phosphatase and tensin homolog (which suppresses PI3K activity) is inactivated via phosphorylation by casein kinase II. This results in full activation of PI3K signaling upon cross-linking CD80/CD86. Similar to IL-6, we have found that CD80/CD86-induced IDO production by DC at late time points is also dependent upon the PI3K â AKT â NF-κB pathway and requires cross-talk with NOTCH signaling. These data further suggest that the same signaling pathways downstream of DC CD80/CD86 cross-linking induce early IL-6 production to enhance T cell activation, followed by later IDO production to self-limit this activation. In addition to characterizing the pathways downstream of CD80/CD86 in IL-6 and IDO production, identification of a novel cross-talk between NOTCH1 and PI3K signaling may provide new insights in other biological processes where PI3K signaling plays a major role.
Assuntos
Células Dendríticas/citologia , Indolamina-Pirrol 2,3,-Dioxigenase/química , Interleucina-6/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Animais , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Caseína Quinase II/metabolismo , Proliferação de Células , Técnicas de Cocultura , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Regulação Enzimológica da Expressão Gênica , Humanos , Interferon gama/metabolismo , Interleucina-23/metabolismo , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , RNA Interferente Pequeno/metabolismo , Linfócitos T/metabolismoRESUMO
Internalization of the Na(+)/K(+)-ATPase (the Na(+) pump) has been studied in the human lung carcinoma cell line H1299 that expresses YFP-tagged α1 from its normal genomic localization. Both real-time imaging and surface biotinylation have demonstrated internalization of α1 induced by ≥100 nm ouabain which occurs in a time scale of hours. Unlike previous studies in other systems, the ouabain-induced internalization was insensitive to Src or PI3K inhibitors. Accumulation of α1 in the cells could be augmented by inhibition of lysosomal degradation but not by proteosomal inhibitors. In agreement, the internalized α1 could be colocalized with the lysosomal marker LAMP1 but not with Golgi or nuclear markers. In principle, internalization could be triggered by a conformational change of the ouabain-bound Na(+)/K(+)-ATPase molecule or more generally by the disruption of cation homeostasis (Na(+), K(+), Ca(2+)) due to the partial inhibition of active Na(+) and K(+) transport. Overexpression of ouabain-insensitive rat α1 failed to inhibit internalization of human α1 expressed in the same cells. In addition, incubating cells in a K(+)-free medium did not induce internalization of the pump or affect the response to ouabain. Thus, internalization is not the result of changes in the cellular cation balance but is likely to be triggered by a conformational change of the protein itself. In physiological conditions, internalization may serve to eliminate pumps that have been blocked by endogenous ouabain or other cardiac glycosides. This mechanism may be required due to the very slow dissociation of the ouabain·Na(+)/K(+)-ATPase complex.
Assuntos
Endocitose/efeitos dos fármacos , Lisossomos/metabolismo , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Microscopia Confocal , Potássio/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Proteólise/efeitos dos fármacos , Pirimidinas/farmacologia , Ratos , ATPase Trocadora de Sódio-Potássio/genética , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
Tissue transglutaminase (tTG) functions as a GTPase and an acyl transferase that catalyzes the formation of protein cross-links. tTG expression is frequently up-regulated in human cancer, where it has been implicated in various aspects of cancer progression, including cell survival and chemo-resistance. However, the extent to which tTG cooperates with other proteins within the context of a cancer cell, versus its intrinsic ability to confer transformed characteristics to cells, is poorly understood. To address this question, we asked what effect the ectopic expression of tTG in a non-transformed cellular background would have on the behavior of the cells. Using NIH3T3 fibroblasts stably expressing a Myc-tagged form of tTG, we found that tTG strongly protected these cells from serum starvation-induced apoptosis and triggered the activation of the PI3-kinase/mTOR Complex 1 (mTORC1)/p70 S6-kinase pathway. We determined that tTG forms a complex with the non-receptor tyrosine kinase c-Src and PI3-kinase, and that treating cells with inhibitors to block tTG function (monodansylcadaverine; MDC) or c-Src kinase activity (PP2) disrupted the formation of this complex, and prevented tTG from activating the PI3-kinase pathway. Moreover, treatment of fibroblasts over-expressing tTG with PP2, or with inhibitors that inactivate components of the PI3-kinase pathway, including PI3-kinase (LY294002) and mTORC1 (rapamycin), ablated the tTG-promoted survival of the cells. These findings demonstrate that tTG has an intrinsic capability to stimulate cell survival through a novel mechanism that activates PI3-kinase signaling events, thus highlighting tTG as a potential target for the treatment of human cancer.
Assuntos
Fibroblastos/enzimologia , Proteínas de Ligação ao GTP/metabolismo , Transglutaminases/metabolismo , Animais , Proteína Tirosina Quinase CSK , Cadaverina/análogos & derivados , Cadaverina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Morfolinas/farmacologia , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Células NIH 3T3 , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteína 2 Glutamina gama-Glutamiltransferase , Pirimidinas/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Transglutaminases/antagonistas & inibidores , Transglutaminases/genética , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
PDGF-C, which is abundant in the malignant breast tumor microenvironment, plays an important role in cell growth and survival. Because tumor-associated macrophages (TAMs) contribute to cancer malignancy, macrophage survival mechanisms are an attractive area of research into controlling tumor progression. In this study, we investigated PDGF-C-mediated signaling pathways involved in anti-apoptotic effects in macrophages. We found that the human malignant breast cancer cell line MDA-MB-231 produced high quantities of PDGF-C, whereas benign MCF-7 cells did not. Recombinant PDGF-C induced PDGF receptor α chain phosphorylation, followed by Akt and Bad phosphorylation in THP-1-derived macrophages. MDA-MB-231 culture supernatants also activated macrophage PDGF-Rα. PDGF-C prevented staurosporine-induced macrophage apoptosis by inhibiting the activation of caspase-3, -7, -8, and -9 and cleavage of poly(ADP-ribose) polymerase. Finally, TAMs isolated from the PDGF-C knockdown murine breast cancer cell line 4T1 and PDGF-C knockdown MDA-MB-231-derived tumor mass showed higher rates of apoptosis than the respective WT controls. Collectively, our results suggest that tumor cell-derived PDGF-C enhances TAM survival, promoting tumor malignancy.
Assuntos
Apoptose , Linfocinas/metabolismo , Macrófagos/metabolismo , Neoplasias/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Proteína de Morte Celular Associada a bcl/metabolismo , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Inibidores Enzimáticos/farmacologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Linfocinas/genética , Macrófagos/patologia , Masculino , Neoplasias/genética , Neoplasias/fisiopatologia , Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Proto-Oncogênicas c-akt , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Estaurosporina/farmacologia , Proteína de Morte Celular Associada a bcl/genéticaRESUMO
Serum- and glucocorticoid-inducible kinase 3 (SGK3) mediates a variety of cellular processes including membrane transport, cell proliferation, and survival, and it has been implicated in Akt-independent signaling downstream of oncogenic PIK3CA mutations (activating mutations in the α catalytic subunit of PI3K) in human cancers. However, the regulation of SGK3 is poorly understood. Here we report that SGK3 stability and kinase activation are regulated by the Hsp90-Cdc37 chaperone complex. Hsp90-Cdc37 associates with the kinase domain of SGK3 and acts in concert with a C-terminal hydrophobic motif of SGK3 to prevent Hsp70 association and ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein)-mediated degradation. Phosphorylation of hydrophobic motif triggers release of Cdc37 and concomitant association of 3-phosphoinositide dependent kinase 1 (PDK1) to activate SGK3. Our study provides new insights into regulation of SGK3 stability and activation and the rationale for application of Hsp90 inhibitors in treating SGK3-dependent cancers.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Motivos de Aminoácidos , Animais , Benzoquinonas/farmacologia , Linhagem Celular Tumoral , Cromatografia Líquida , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Estrogênios/farmacologia , Humanos , Lactamas Macrocíclicas/farmacologia , Espectrometria de Massas , Camundongos , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteólise/efeitos dos fármacos , Relação Estrutura-Atividade , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
Human plasminogen kringle 5 (K5) is known to display its potent anti-angiogenesis effect through inducing endothelial cell (EC) apoptosis, and the voltage-dependent anion channel 1 (VDAC1) has been identified as a receptor of K5. However, the exact role and underlying mechanisms of VDAC1 in K5-induced EC apoptosis remain elusive. In the current study, we showed that K5 increased the protein level of VDAC1, which initiated the mitochondrial apoptosis pathway of ECs. Our findings also showed that K5 inhibited the ubiquitin-dependent degradation of VDAC1 by promoting the phosphorylation of VDAC1, possibly at Ser-12 and Thr-107. The phosphorylated VDAC1 was attenuated by the AKT agonist, glycogen synthase kinase (GSK) 3ß inhibitor, and siRNA, suggesting that K5 increased VDAC1 phosphorylation via the AKT-GSK3ß pathway. Furthermore, K5 promoted cell surface translocation of VDAC1, and binding between K5 and VDAC1 was observed on the plasma membrane. HKI protein blocked the impact of K5 on the AKT-GSK3ß pathway by competitively inhibiting the interaction of K5 and cell surface VDAC1. Moreover, K5-induced EC apoptosis was suppressed by VDAC1 antibody. These data show for the first time that K5-induced EC apoptosis is mediated by the positive feedback loop of "VDAC1-AKT-GSK3ß-VDAC1," which may provide new perspectives on the mechanisms of K5-induced apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Fragmentos de Peptídeos/farmacologia , Plasminogênio/farmacologia , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Apoptose/genética , Western Blotting , Caspases/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Retroalimentação Fisiológica/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fragmentos de Peptídeos/genética , Fosforilação/efeitos dos fármacos , Plasminogênio/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ubiquitina/metabolismo , Canal de Ânion 1 Dependente de Voltagem/genéticaRESUMO
PI3K/Akt signaling plays an important role in the regulation of cardiomyocyte death machinery, which can cause stress-induced cardiac dysfunction. Here, we report that apoptosis regulator through modulating IAP expression (ARIA), a recently identified transmembrane protein, regulates the cardiac PI3K/Akt signaling and thus modifies the progression of doxorubicin (DOX)-induced cardiomyopathy. ARIA is highly expressed in the mouse heart relative to other tissues, and it is also expressed in isolated rat cardiomyocytes. The stable expression of ARIA in H9c2 cardiac muscle cells increased the levels of membrane-associated PTEN and subsequently reduced the PI3K/Akt signaling and the downstream phosphorylation of Bad, a proapoptotic BH3-only protein. When challenged with DOX, ARIA-expressing H9c2 cells exhibited enhanced apoptosis, which was reversed by the siRNA-mediated silencing of Bad. ARIA-deficient mice exhibited normal heart morphology and function. However, DOX-induced cardiac dysfunction was significantly ameliorated in conjunction with reduced cardiomyocyte death and cardiac fibrosis in ARIA-deficient mice. Phosphorylation of Akt and Bad was substantially enhanced in the heart of ARIA-deficient mice even after treatment with DOX. Moreover, repressing the PI3K by cardiomyocyte-specific expression of dominant-negative PI3K (p110α) abolished the cardioprotective effects of ARIA deletion. Notably, targeted activation of ARIA in cardiomyocytes but not in endothelial cells reduced the cardiac PI3K/Akt signaling and exacerbated the DOX-induced cardiac dysfunction. These studies, therefore, revealed a previously undescribed mode of manipulating cardiac PI3K/Akt signaling by ARIA, thus identifying ARIA as an attractive new target for the prevention of stress-induced myocardial dysfunction.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Miócitos Cardíacos/enzimologia , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Antibióticos Antineoplásicos/toxicidade , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/genética , Cardiomiopatias/induzido quimicamente , Linhagem Celular , Modelos Animais de Doenças , Doxorrubicina/toxicidade , Homeostase/fisiologia , Proteínas de Membrana , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Prolactin (PRL) regulates activity of nociceptors and causes hyperalgesia in pain conditions. PRL enhances nociceptive responses by rapidly modulating channels in nociceptors. The molecular mechanisms underlying PRL-induced transient signaling in neurons are not well understood. Here we use a variety of cell biology and pharmacological approaches to show that PRL transiently enhanced capsaicin-evoked responses involve protein kinase C ε (PKCε) or phosphatidylinositol 3-kinase (PI3K) pathways in female rat trigeminal (TG) neurons. We next reconstituted PRL-induced signaling in a heterologous expression system and TG neurons from PRL receptor (PRLR)-null mutant mice by expressing rat PRLR-long isoform (PRLR-L), PRLR-short isoform (PRLR-S), or a mix of both. Results show that PRLR-S, but not PRLR-L, is capable of mediating PRL-induced transient enhancement of capsaicin responses in both male and female TG neurons. However, co-expression of PRLR-L with PRLR-S (1:1 ratio) leads to the inhibition of the transient PRL actions. Co-expression of PRLR-L deletion mutants with PRLR-S indicated that the cytoplasmic site adjacent to the trans-membrane domain of PRLR-L was responsible for inhibitory effects of PRLR-L. Furthermore, in situ hybridization and immunohistochemistry data indicate that in normal conditions, PRLR-L is expressed mainly in glia with little expression in rat sensory neurons (3-5%) and human nerves. The predominant PRLR form in TG neurons/nerves from rats and humans is PRLR-S. Altogether, PRL-induced transient signaling in sensory neurons is governed by PI3K or PKCε, mediated via the PRLR-S isoform, and transient effects mediated by PRLR-S are inhibited by presence of PRLR-L in these cells.
Assuntos
Isoformas de Proteínas , Receptores da Prolactina/metabolismo , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais/genética , Nervo Trigêmeo/metabolismo , Animais , Células CHO , Células Cultivadas , Cricetulus , Feminino , Humanos , Masculino , Camundongos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C-épsilon/genética , Proteína Quinase C-épsilon/metabolismo , Ratos , Receptores da Prolactina/genética , Células Receptoras Sensoriais/citologia , Dente/metabolismo , Dente/fisiologia , Nervo Trigêmeo/citologiaRESUMO
Epithelial cell polarization involves several kinase signaling cascades that eventually divide the surface membrane into an apical and a basolateral part. One kinase, which is activated during the polarization process, is phosphoinositide 3-kinase (PI3K). In MDCK cells, the basolateral potassium channel Kv7.1 requires PI3K activity for surface-expression during the polarization process. Here, we demonstrate that Kv7.1 surface expression requires tonic PI3K activity as PI3K inhibition triggers endocytosis of these channels in polarized MDCK. Pharmacological inhibition of SGK1 gave similar results as PI3K inhibition, whereas overexpression of constitutively active SGK1 overruled it, suggesting that SGK1 is the primary downstream target of PI3K in this process. Furthermore, knockdown of the ubiquitin ligase Nedd4-2 overruled PI3K inhibition, whereas a Nedd4-2 interaction-deficient Kv7.1 mutant was resistant to both PI3K and SGK1 inhibition. Altogether, these data suggest that a PI3K-SGK1 pathway stabilizes Kv7.1 surface expression by inhibiting Nedd4-2-dependent endocytosis and thereby demonstrates that Nedd4-2 is a key regulator of Kv7.1 localization and turnover in epithelial cells.
Assuntos
Endocitose/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Canal de Potássio KCNQ1/biossíntese , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Cães , Endocitose/efeitos dos fármacos , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/genética , Canal de Potássio KCNQ1/genética , Células Madin Darby de Rim Canino , Mutação , Ubiquitina-Proteína Ligases Nedd4 , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Deregulation of ErbB receptor-tyrosine kinases is a hallmark of many human cancers. Conserved in the ErbB family is a cluster of basic amino acid residues in the cytoplasmic juxtamembrane region. We found that charge-silencing mutagenesis within this juxtamembrane region of the epidermal growth factor receptor (EGFR) results in the generation of a mutant receptor (EGFR Mut R1-6) that spontaneously transforms NIH 3T3 cells in a ligand-independent manner. A similar mutant with one additional basic residue, EGFR Mut R1-5, fails to exhibit ligand-independent transformation. The capacity of EGFR Mut R1-6 to mediate this transformation is maintained when this mutant is retained in the endoplasmic reticulum via a single point mutation, L393H, which we describe. We show that EGFR Mut R1-6 with or without L393H exhibits enhanced basal tyrosine phosphorylation when ectopically expressed, and the ligand-independent transforming activity of EGFR Mut R1-6 is sensitive to inhibition of EGFR kinase activity and is particularly dependent on PI3K and mTOR activity. Similar to EGFR Mut R1-6/L393H in NIH 3T3 cells, EGFR variant type III, a highly oncogenic mutant form of EGFR linked to human brain cancers, confers transforming activity while it is wholly endoplasmic reticulum-retained in U87 cells. Our findings highlight the importance of the polybasic juxtamembrane sequence in regulating the oncogenic potential of EGFR signaling.
Assuntos
Neoplasias Encefálicas/genética , Transformação Celular Neoplásica/genética , Retículo Endoplasmático/metabolismo , Receptores ErbB/genética , Animais , Neoplasias Encefálicas/patologia , Membrana Celular/genética , Membrana Celular/metabolismo , Elafina/metabolismo , Retículo Endoplasmático/genética , Receptores ErbB/metabolismo , Humanos , Ligantes , Camundongos , Mutação , Células NIH 3T3 , Transdução de SinaisRESUMO
In fibrotic liver, connective tissue growth factor (CTGF) is constantly expressed in activated hepatic stellate cells (HSCs) and acts downstream of TGF-ß to modulate extracellular matrix production. Distinct from other cell types in which Smad signaling plays major role in regulating CTGF production, TGF-ß stimulated CTGF expression in activated HSCs is only in part dependent on Smad3. Other signaling molecules like MAPKs and PI3Ks may also participate in this process, and the underlying mechanisms have yet to be clarified. In this study, we report involvement of Stat3 activation in modulating CTGF production upon TGF-ß challenge in activated HSCs. Stat3 is phosphorylated via JAK1 and acts as a critical ALK5 (activin receptor-like kinase 5) downstream signaling molecule to mediate CTGF expression. This process requires de novo gene transcription and is additionally modulated by MEK1/2, JNK, and PI3K pathways. Cell-specific knockdown of Smad3 partially decreases CTGF production, whereas it has no significant influence on Stat3 activation. The total CTGF production induced by TGF-ß in activated HSCs is therefore, to a large extent, dependent on the balance and integration of the canonical Smad3 and Stat3 signaling pathways.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/biossíntese , Regulação da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/metabolismo , Sistema de Sinalização das MAP Quinases , Fator de Transcrição STAT3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Linhagem Celular Transformada , Fator de Crescimento do Tecido Conjuntivo/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Células Estreladas do Fígado/patologia , Humanos , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Transcrição STAT3/genética , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
Protein phosphatase 2A (PP2A) regulates almost all cell signaling pathways. It consists of a scaffolding A subunit to which a catalytic C subunit and one of many regulatory B subunits bind. Of the more than 80 PP2A isoforms, 10% use Aß as a scaffold. This study demonstrates the isoform-specific function of the A scaffold subunits. Polyomaviruses have shown the importance of phosphotyrosine, PI3K, and p53 in transformation. Comparisons of polyoma and SV40 small T antigens implicate Aß in the control of differentiation. Knockdown of Aß enhanced differentiation. Akt signaling regulated differentiation; its activation or inhibition promoted or blocked it, respectively. Aß bound Akt. Enhancement of PP2A Aß/Akt interaction by polyoma small T antigen increased turnover of Akt Ser-473 phosphorylation. Conversely, knockdown of Aß promoted Akt activity and reduced turnover of phosphate at Ser-473 of Akt. These data provide new insight into the regulation of Akt, a protein of extreme importance in cancer. Furthermore, our results suggest that the role for Aß in differentiation and perhaps tumor suppression may lie partly in its ability to negatively regulate Akt.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Diferenciação Celular , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células 3T3-L1 , Peptídeos beta-Amiloides/genética , Animais , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Isoenzimas , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/virologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Polyomavirus/genética , Polyomavirus/metabolismo , Proteína Fosfatase 2/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mitochondrial abnormalities are associated with cancer development, yet how oncogenic signals affect mitochondrial functions has not been fully understood. In this study, we investigate the relationship between mitochondrial alterations and PI3K/protein kinase B (AKT) signaling activation using hepatocytes and liver tissues as our experimental models. We show here that liver-specific deletion of Pten, which leads to activation of PI3K/AKT, is associated with elevated oxidative stress, increased mitochondrial mass, and augmented respiration accompanied by enhanced glycolysis. Consistent with these observations, estrogen-related receptor α (ERRα), an orphan nuclear receptor known for its role in mitochondrial biogenesis, is up-regulated in the absence of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Our pharmacological and genetic studies show that PI3K/AKT activity regulates the expression of ERRα and mitochondrial biogenesis/respiration. Furthermore, cAMP-response element-binding protein, as a downstream target of AKT, plays a role in the regulation of ERRα, independent of PKA signaling. ERRα regulates reactive oxygen species production, and ERRα knockdown attenuates proliferation and colony-forming potential in Pten-null hepatocytes. Finally, analysis of clinical datasets from liver tissues showed a negative correlation between expressions of ERRα and PTEN in patients with liver cancer. Therefore, this study has established a previously unrecognized link between a growth signal and mitochondrial metabolism.
Assuntos
Hepatócitos/metabolismo , Neoplasias Hepáticas/metabolismo , Mitocôndrias Hepáticas/metabolismo , Consumo de Oxigênio , PTEN Fosfo-Hidrolase/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Ativação Enzimática/genética , Regulação Enzimológica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Hepatócitos/patologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/genética , Mitocôndrias Hepáticas/patologia , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Estrogênio/genética , Transdução de Sinais/genética , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
RLIP76 is a multifunctional protein involved in tumor growth and angiogenesis, and a promising therapeutic target in many cancers. RLIP76 harbors docking sites for many proteins, and we have found that it interacts with ARNO, a guanine nucleotide exchange factor for Arf6, and that RLIP76 regulates activation of Rac1 via Arf6, and regulates cell spreading and migration in an ARNO and Arf6-dependent manner. Here we show that ARNO interacts with the RLIP76 N-terminal domain, and this domain was required for RLIP76-dependent cell spreading and migration. We identified two sites in the RLIP76 N-terminus with differential effects on ARNO binding and downstream signaling: Ser29/Ser30 and Ser62. Ser29/30 mutation to Alanine inhibited ARNO interaction and was sufficient to block RLIP76-dependent cell spreading and migration, as well as RLIP76-dependent Arf6 activation. In contrast, RLIP76(S62A) interacted with ARNO and supported Arf6 activation. However, both sets of mutations blocked Rac1 activation. RLIP76-mediated Rac and Arf6 activation required PI3K activity. S29/30A mutations inhibited RLIP76-dependent PI3K activation, but S62A mutation did not. Together these results show that ARNO interaction with the RLIP76 N-terminus regulates cell spreading and motility via PI3K and Arf6, independent of RLIP76 control of Rac.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Movimento Celular , Proteínas Ativadoras de GTPase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/metabolismo , Fator 6 de Ribosilação do ADP , Animais , Sítios de Ligação , Células Cultivadas , Camundongos , Células NIH 3T3RESUMO
BACKGROUND: Microtubule-binding protein tau is a misfolding-prone protein associated with tauopathies. As tau undergoes cell-to-cell transmission, extracellular tau aggregates convert astrocytes into a pro-inflammatory state via integrin activation, causing them to release unknown neurotoxic factors. RESULTS: Here, we combine transcriptomics with isotope labeling-based quantitative mass spectrometry analysis of mouse primary astrocyte secretome to establish PI3K-AKT as a critical differentiator between pathogenic and physiological integrin activation; simultaneous activation of PI3K-AKT and focal adhesion kinase (FAK) in tau fibril-treated astrocytes changes the output of integrin signaling, causing pro-inflammatory gene upregulation, trans-Golgi network restructuring, and altered secretory flow. Furthermore, NCAM1, as a proximal signaling component in tau-stimulated integrin and PI3K-AKT activation, facilitates the secretion of complement C3 as a main neurotoxic factor. Significantly, tau fibrils-associated astrogliosis and C3 secretion can be mitigated by FAK or PI3K inhibitors. CONCLUSIONS: These findings reveal an unexpected function for PI3K-AKT in tauopathy-associated reactive astrogliosis, which may be a promising target for anti-inflammation-based Alzheimer's therapy.
RESUMO
The Microtubule-Associated Protein Tau is expressed in several cancers, including low-grade gliomas and glioblastomas. We have previously shown that Tau is crucial for the 2D motility of several glioblastoma cell lines, including U87-MG cells. Using an RNA interference (shRNA), we tested if Tau contributed to glioblastoma in vivo tumorigenicity and analyzed its function in a 3D model of multicellular spheroids (MCS). Tau depletion significantly increased median mouse survival in an orthotopic glioblastoma xenograft model. This was accompanied by the inhibition of MCS growth and cell evasion, as well as decreased MCS compactness, implying N-cadherin mislocalization. Intracellular Signaling Array analysis revealed a defective activation of the PI3K/AKT pathway in Tau-depleted cells. Such a defect in PI3K/AKT signaling was responsible for reduced MCS growth and cell evasion, as demonstrated by the inhibition of the pathway in control MCS using LY294002 or Perifosine, which did not significantly affect Tau-depleted MCS. Finally, analysis of the glioblastoma TCGA dataset showed a positive correlation between the amount of phosphorylated Akt-Ser473 and the expression of MAPT RNA encoding Tau, underlining the relevance of our findings in glioblastoma disease. We suggest a role for Tau in glioblastoma by controlling 3D cell organization and functions via the PI3K/AKT signaling axis.