RESUMO
The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8beta chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8alphabeta, but not CD8alphaalpha or soluble CD8alphabeta, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8beta endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8beta constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2K(d), and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8beta, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56(lck). In addition, the cytoplasmic portion of CD8beta mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8alphabeta partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56(lck) in rafts, which in turn phosphorylates CD3 and initiates T cell activation.
Assuntos
Antígenos CD8/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Microdomínios da Membrana/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Complexo CD3/metabolismo , Células CHO , Cálcio/metabolismo , Cricetinae , Dados de Sequência Molecular , FosforilaçãoRESUMO
Insulin-like growth factor (IGF) signaling plays an important role in various human cancers. Therefore, the role of insulin-like growth factor I (IGF-I) signaling in growth and survival of acute myeloid leukemia (AML) cells was investigated. Expression of the IGF-I receptor (IGF-IR) and its ligand IGF-I were detected in a panel of human AML blasts and cell lines. IGF-I and insulin promoted the growth of human AML blasts in vitro and activated the phosphoinositide 3-kinase (PI3K)/Akt and the extracellular signal-regulated kinase (Erk) pathways. IGF-I-stimulated growth of AML blasts was blocked by an inhibitor of the PI3K/Akt pathway. Moreover, downregulation of the class Ia PI3K isoforms p110beta and p110delta by RNA interference impaired IGF-I-stimulated Akt activation, cell growth and survival in AML cells. Proliferation of a panel of AML cell lines and blasts isolated from patients with AML was inhibited by the IGF-IR kinase inhibitor NVP-AEW541 or by an IGF-IR neutralizing antibody. In addition to its antiproliferative effects, NVP-AEW541 sensitized primary AML blasts and cell lines to etoposide-induced apoptosis. Together, our data describe a novel role for autocrine IGF-I signaling in the growth and survival of primary AML cells. IGF-IR inhibitors in combination with chemotherapeutic agents may represent a novel approach to target human AML.
Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Leucemia Mieloide/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Doença Aguda , Anticorpos/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Comunicação Autócrina , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Citarabina/farmacologia , Regulação para Baixo , Etoposídeo/farmacologia , Humanos , Leucemia Mieloide/metabolismo , Receptor IGF Tipo 1/imunologia , Receptor IGF Tipo 1/metabolismoRESUMO
The impact of the 3-hydroxy-3methylglutaryl CoA reductase inhibitor simvastatin on human small-cell lung cancer (SCLC) cell growth and survival was investigated. Simvastatin profoundly impaired basal and growth factor-stimulated SCLC cell growth in vitro and induced apoptosis. SCLC cells treated with simvastatin were sensitized to the effects of the chemotherapeutic agent etoposide. Moreover, SCLC tumour growth in vivo was inhibited by simvastatin. These responses correlated with the inhibition of stem cell factor (SCF)-stimulated activation of extracellular signal-regulated kinase (Erk), protein kinase B (PKB) and ribosomal S6 kinase by simvastatin. Constitutive activation of the Erk pathway was sufficient to rescue SCLC cell from the effects of simvastatin. The drug did not directly affect activation of c-Kit or its localization to lipid rafts, but in addition to its ability to block Ras membrane localization, it selectively downregulated H-Ras protein levels at the post-translational level. Downregulation of either H- or K-Ras by RNA interference (RNAi) did not impair Erk activation by growth factors, whereas an RNAi specific for N-Ras inhibited activation of Erk, PKB and SCLC cell growth. Together our data demonstrate that inhibiting Ras signalling with simvastatin potently disrupts growth and survival in human SCLC cells.
Assuntos
Carcinoma de Células Pequenas/tratamento farmacológico , Substâncias de Crescimento/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Sinvastatina/farmacologia , Proteínas ras/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Sequência de Bases , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Etoposídeo/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , MAP Quinase Quinase Quinases/efeitos dos fármacos , MAP Quinase Quinase Quinases/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-kit/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Regulação para Cima , Proteínas ras/efeitos dos fármacos , Proteínas ras/genéticaRESUMO
Central nervous system (CNS) atypical teratoid/rhabdoid tumours (AT/RT) are among the paediatric malignant tumours with the worst prognosis and fatal outcome. Insulin-like growth factor I receptor (IGF-IR) protects cancer cells from apoptosis induced by a variety of anticancer drugs and radiation. In the present study, IGF-IR was expressed in 8/8 primary AT/RT as detected by immunohistochemistry. Moreover, we found IGF-I and IGF-II mRNA in BT-16 CNS AT/RT cells and IGF-II mRNA in BT-12 CNS AT/RT cells, and autophosphorylated IGF-IR in both cell lines, indicating the potential presence of an autocrine/paracrine IGF-I/II/IGF-IR loop in CNS AT/RT. IGF-IR antisense oligonucleotide treatment of human CNS AT/RT cells resulted in significant down-regulation of IGF-IR mRNA and protein expression, induction of apoptosis, and chemosensitisation to doxorubicin and cisplatin. These studies provide evidence for the influence of IGF-IR on cellular responses to chemotherapy and raise the possibility that curability of selected CNS AT/RT may be improved by pharmaceutical strategies directed towards the IGF-IR.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Oligorribonucleotídeos Antissenso/uso terapêutico , Receptor IGF Tipo 1/efeitos dos fármacos , Tumor Rabdoide/tratamento farmacológico , Teratoma/tratamento farmacológico , Antineoplásicos/uso terapêutico , Neoplasias do Sistema Nervoso Central/patologia , Criança , Pré-Escolar , Cisplatino/uso terapêutico , Regulação para Baixo , Doxorrubicina/uso terapêutico , Feminino , Humanos , Lactente , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Receptor IGF Tipo 1/metabolismo , Tumor Rabdoide/patologia , Teratoma/patologiaRESUMO
The class II phosphoinositide 3-kinases (PI3K) PI3K-C2alpha and PI3K-C2beta are two recently identified members of the large PI3K family. Both enzymes are characterized by the presence of a C2 domain at the carboxy terminus and, in vitro, preferentially utilize phosphatidylinositol and phosphatidylinositol 4-monophosphate as lipid substrates. Little is understood about how the catalytic activity of either enzyme is regulated in vivo. In this study, we demonstrate that PI3K-C2alpha and PI3K-C2beta represent two downstream targets of the activated epidermal growth factor (EGF) receptor in human carcinoma-derived A431 cells. Stimulation of quiescent cultures with EGF resulted in the rapid recruitment of both enzymes to a phosphotyrosine signaling complex that contained the EGF receptor and Erb-B2. Ligand addition also induced the appearance of a second, more slowly migrating band of PI3K-C2alpha and PI3K-C2beta immunoreactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both PI3K enzymes can utilize Ca(2+) as an essential divalent cation in lipid kinase assays and since the catalytic activity of PI3K-C2alpha is refractory to the inhibitor wortmannin, these properties were used to confirm the recruitment of each PI3K isozyme to the activated EGF receptor complex. To examine this interaction in greater detail, PI3K-C2beta was chosen for further investigation. EGF and platelet-derived growth factor also stimulated the association of PI3K-C2beta with their respective receptors in other cells, including epithelial cells and fibroblasts. The use of EGF receptor mutants and phosphopeptides derived from the EGF receptor and Erb-B2 demonstrated that the interaction with recombinant PI3K-C2beta occurs through E(p)YL/I phosphotyrosine motifs. The N-terminal region of PI3K-C2beta was found to selectively interact with the EGF receptor in vitro, suggesting that it mediates the association of this PI3K with the receptor. However, the mechanism of this interaction remains unclear. We conclude that class II PI3K enzymes may contribute to the generation of 3' phosphoinositides following the activation of polypeptide growth factor receptors in vivo and thus mediate certain aspects of their biological activity.
Assuntos
Receptores ErbB/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Humanos , Fosfatos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotirosina/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor ErbB-2/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Células Tumorais CultivadasRESUMO
Here, we show that fibroblast growth factor-2 (FGF-2) induces proliferation of H-510 and H-69 small cell lung cancer (SCLC) cells. However, the optimal response to FGF-2 was obtained at 10-fold lower concentrations in H-510 cells. This correlated with the selective activation of the mitogen-activated protein kinase kinase (MEK) pathway in H-510, but not H-69 cells. Moreover, inhibition of MEK with PD098059 blocked FGF-2-induced proliferation in H-510 cells only. Similarly, ribosomal protein S6 kinase 2 (S6K2), a recently identified homologue of S6K1 was activated by FGF-2 in H-510, but not H-69 cells. This activation was independent of phosphatidylinositol-3 kinase, but was sensitive to inhibition of the MEK pathway. These data suggest that S6K2 is a novel downstream target of MEK. The potency of FGF-2 in H-510 cells might reflect this additional MEK/S6K2 signalling. In contrast to S6K2, S6K1 was activated in both SCLC cell lines. Inhibition of the mammalian target of rapamycin with 10 ng/ml rapamycin blocked S6K1 activation and proliferation of both lines. However, even at 100 ng/ml, rapamycin only partially inhibited S6K2. Strikingly, this correlated with inhibition of MEK signalling. Our data indicate that S6K1, and possibly S6K2, are involved in FGF-2-induced SCLC cell growth, a notion supported by the overexpression and higher baseline activity of both isoforms in SCLC lines, as compared to normal human type-II pneumocytes.
Assuntos
Carcinoma de Células Pequenas/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neoplasias Pulmonares/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Divisão Celular/efeitos dos fármacos , Ativação Enzimática , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitógenos/metabolismo , Mitógenos/farmacologia , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases , Proteínas Quinases/metabolismo , Proteínas Quinases/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Quinases S6 Ribossômicas/fisiologia , Serina-Treonina Quinases TOR , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Medulloblastoma is the most common malignant childhood brain tumor and is associated with a poor outcome. There is an urgent need to develop novel targeted therapeutic approaches for medulloblastoma, which will arise from an enhanced understanding of the disease at the molecular level. Medulloblastoma has been recognized to be a heterogeneous disease, and no recurrent cancer gene mutations have been found, although many of the mutations described so far affect key intracellular signaling pathways, such as sonic hedgehog (SHH) and Wnt/ß-catenin. The PI3K/AKT/mTOR (PAM) signaling pathway controls key cellular responses, such as cell growth and proliferation, survival, migration and metabolism. Over the last decades, it has been recognized that this intracellular signaling pathway is frequently activated by genetic and epigenetic alterations in malignant brain tumors, including medulloblastoma. Clinical trials have started to evaluate the safety and efficacy of agents targeting this pathway in malignant brain tumors. Due to the complexity of the PAM signaling pathway, there remain significant difficulties in the development of novel therapeutic approaches. The future challenges in developing effective treatments for cancer patients include the development of predictive biomarkers and combinatorial approaches to effectively target multiple signal transduction pathways. In this review article, we will summarize the current knowledge about the role of PAM signaling in medulloblastoma and discuss the strategies that are currently being evaluated with targeted agents against this pathway.
Assuntos
Meduloblastoma/tratamento farmacológico , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Serina-Treonina Quinases TOR/genética , Humanos , Meduloblastoma/genética , Meduloblastoma/patologia , Terapia de Alvo Molecular , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , beta Catenina/genéticaRESUMO
Chemotherapeutic drug resistance is one of the major causes for treatment failure in high-risk neuroblastoma (NB), the most common extra cranial solid tumor in children. Poor prognosis is typically associated with MYCN amplification. Here, we utilized a loss-of-function kinome-wide RNA interference screen to identify genes that cause cisplatin sensitization. We identified fibroblast growth factor receptor 2 (FGFR2) as an important determinant of cisplatin resistance. Pharmacological inhibition of FGFR2 confirmed the importance of this kinase in NB chemoresistance. Silencing of FGFR2 sensitized NB cells to cisplatin-induced apoptosis, which was regulated by the downregulation of the anti-apoptotic proteins BCL2 and BCLXL. Mechanistically, FGFR2 was shown to activate protein kinase C-δ to induce BCL2 expression. FGFR2, as well as the ligand fibroblast growth factor-2, were consistently expressed in primary NB and NB cell lines, indicating the presence of an autocrine loop. Expression analysis revealed that FGFR2 correlates with MYCN amplification and with advanced stage disease, demonstrating the clinical relevance of FGFR2 in NB. These findings suggest a novel role for FGFR2 in chemoresistance and provide a rational to combine pharmacological inhibitors against FGFR2 with chemotherapeutic agents for the treatment of NB.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Interferência de RNA , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais/genética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Compostos de Bifenilo/farmacologia , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cisplatino/farmacologia , Ativação Enzimática/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Nitrofenóis/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Piperazinas/farmacologia , Proteína Quinase C-delta/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirróis/farmacologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Brain tumors comprise a wide variety of neoplasia classified according to their cellular origin and their morphological and histological characteristics. The transformed phenotype of brain tumor cells has been extensively studied in the past years, achieving a significant progress in our understanding of the molecular pathways leading to tumorigenesis. It has been reported that the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway is frequently altered in grade IV brain tumors resulting in uncontrolled cell growth, survival, proliferation, angiogenesis, and migration. This aberrant activation can be explained by oncogenic mutations in key components of the pathway or through abnormalities in its regulation. These alterations include overexpression and mutations of receptor tyrosine kinases (RTKs), mutations and deletions of the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) tumor suppressor gene, encoding a lipid kinase that directly antagonized PI3K activity, and alterations in Ras signaling. Due to promising results of preclinical studies investigating the PI3K/AKT pathway in grade IV brain tumors like glioblastoma and medulloblastoma, the components of this pathway have emerged as promising therapeutic targets to treat these malignant brain tumors. Although an arsenal of small molecule inhibitors that target specific components of this signaling pathway is being developed, its successful application in the clinics remains a challenge. In this article we will review the molecular basis of the PI3K/AKT signaling pathway in malignant brain tumors, mainly focusing on glioblastoma and medulloblastoma, and we will further discuss the current status and potential of molecular targeted therapies.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Terapia de Alvo Molecular , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Inibidores Enzimáticos/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Meduloblastoma/tratamento farmacológico , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Gradação de Tumores , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 QuinaseRESUMO
Quassinoids are a group of compounds extracted from plants of the Simaroubaceae family, which have been used for many years in folk medicine. These molecules gained notoriety after the initial discovery of the anti-leukemic activity of one member, bruceantin, in 1975. Currently over 150 quassinoids have been isolated and classified based on their chemical structures and biological properties investigated in vitro and in vivo. Many molecules display a wide range of inhibitory effects, including anti-inflammatory, anti-viral, anti-malarial and anti-proliferative effects on various tumor cell types. Although often the exact mechanism of action of the single agents remains unclear, some agents have been shown to affect protein synthesis in general, or specifically HIF-1α and MYC, membrane polarization and the apoptotic machinery. Considering that future research into chemical modifications is likely to generate more active and less toxic derivatives of natural quassinoids, this family represents a powerful source of promising small molecules targeting key prosurvival signaling pathways relevant for diverse pathologies. Here, we review available knowledge of functionality and possible applications of quassinoids and quassinoid derivatives, spanning traditional use to the potential impact on modern medicine as cancer therapeutics.
Assuntos
Antineoplásicos Fitogênicos/química , Neoplasias/tratamento farmacológico , Quassinas/química , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/uso terapêutico , Humanos , Medicina Tradicional , Quassinas/isolamento & purificação , Quassinas/uso terapêutico , Simaroubaceae/químicaRESUMO
Medulloblastoma (MB) is the most common malignant brain tumor in children. It is known that overexpression and/or amplification of the MYC oncogene is associated with poor clinical outcome, but the molecular mechanisms and the MYC downstream effectors in MB remain still elusive. Besides contributing to elucidate how progression of MB takes place, most importantly, the identification of novel MYC-target genes will suggest novel candidates for targeted therapy in MB. A group of 209 MYC-responsive genes was obtained from a complementary DNA microarray analysis of a MB-derived cell line, following MYC overexpression and silencing. Among the MYC-responsive genes, we identified the members of the bone morphogenetic protein (BMP) signaling pathway, which have a crucial role during the development of the cerebellum. In particular, the gene BMP7 was identified as a direct target of MYC. A positive correlation between MYC and BMP7 expression was documented by analyzing two distinct sets of primary MB samples. Functional studies in vitro using a small-molecule inhibitor of the BMP/SMAD signaling pathway reproduced the effect of the small interfering RNA-mediated silencing of BMP7. Both approaches led to a block of proliferation in a panel of MB cells and to inhibition of SMAD phosphorylation. Altogether, our findings indicate that high MYC levels drive BMP7 overexpression, promoting cell survival in MB cells. This observation suggests the potential relevance of targeting the BMP/SMAD pathway as a novel therapeutic approach for the treatment of childhood MB.
Assuntos
Proteína Morfogenética Óssea 7/genética , Sobrevivência Celular/genética , Neoplasias Cerebelares/genética , Genes myc , Meduloblastoma/genética , Western Blotting , Neoplasias Cerebelares/patologia , Criança , Ensaio de Imunoadsorção Enzimática , Inativação Gênica , Humanos , Meduloblastoma/patologia , Fosforilação , Proteínas Smad/metabolismoRESUMO
Gelsolin is an actin filament-capping protein that has been shown to play a key role in cell migration. Here we have studied the involvement of phosphoinositide 3-kinase (PI 3-kinase) and GTP-binding proteins (G-proteins) in the regulation of gelsolin-actin interactions in neutrophils. Inhibition of PI 3-kinase activity in vivo by wortmannin did not affect the dissociation of actin-gelsolin (1:1) complexes induced by neutrophil stimulation with N-formyl-Met-Leu-Phe. Guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) indirectly promoted the dissociation of actin-gelsolin complexes in a cell-free system using neutrophil cytosol, and this effect was blocked by the GDP dissociation inhibitor for Rho (Rho-GDI). The GTPgammaS-loaded ialpha2 and the beta1gamma2 subunits of heterotrimeric G-proteins (Gialpha2 and Gbeta1gamma2) also triggered actin-gelsolin dissociation in a Rho-GDI-sensitive manner. GTP-loaded activated Rac, but not activated Rho, induced the dissociation of cytosolic actin-gelsolin complexes. The guanine nucleotide exchange on Rac was increased by addition of GTPgammaS-loaded Gialpha2 or Gbeta1gamma2 to neutrophil cytosol. These findings suggest that activation of Rac by G-protein-coupled receptors in neutrophils triggers uncapping of actin filaments, independently of PI 3-kinase.
Assuntos
Actinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Gelsolina/metabolismo , Neutrófilos/metabolismo , Androstadienos/farmacologia , Animais , Cálcio/metabolismo , Inibidores Enzimáticos/farmacologia , Fatores de Troca do Nucleotídeo Guanina , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas/metabolismo , Coelhos , Proteínas Recombinantes/metabolismo , Wortmanina , Proteínas rac de Ligação ao GTPRESUMO
Various agonist-induced cell responses in neutrophils and fibroblasts, such as chemotaxis and cytoskeletal rearrangements, have been shown to correlate with the synthesis of PtdIns(3,4,5)P3; however, the significance of this rise in second messenger levels is not clear. We show here that wortmannin inhibits platelet-derived growth factor (PDGF)-mediated production of PtdIns(3,4,5)P3 in human foreskin fibroblasts with an IC50 of about 5 nM. A similar inhibition was observed in in vitro assays (IC50 approximately 1 nM) with phosphatidylinositol 3-kinase immunoprecipitated by antibodies directed against the 85 kDa subunit (p85). On the other hand, wortmannin did not affect PDGF-mediated phosphorylation of p85 as detected by immunoprecipitation with anti-phosphotyrosine antibodies, and did not dissociate the complex of p85 and the catalytic subunit (p110) of phosphatidylinositol 3-kinase. These results are consistent with a direct, specific inhibition of the enzyme by wortmannin at concentrations relevant for its previously reported effects on cellular responses. When stimulated with PDGF, human foreskin fibroblasts form circular structures of filamentous actin. Preincubation of these cells with wortmannin inhibits PDGF-mediated actin rearrangements, suggesting a need for PtdIns(3,4,5)P3 formation as a signal for this cell response.
Assuntos
Actinas/metabolismo , Fibroblastos/ultraestrutura , Fosfatos de Fosfatidilinositol/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Fator de Crescimento Derivado de Plaquetas/farmacologia , Células 3T3 , Androstadienos/farmacologia , Animais , Linhagem Celular , Citoesqueleto/ultraestrutura , Ativação Enzimática , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Técnicas de Imunoadsorção , Masculino , Camundongos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Fosfatidilinositol 3-Quinases , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , WortmaninaRESUMO
Phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) is rapidly produced upon exposure of neutrophils to the chemoattractant N-formylmethionyl-leucylphenylalanine (fMLP), and has been proposed to act as a second messenger mediating actin polymerization and respiratory-burst activity. Here we present evidence that wortmannin, a known inhibitor of respiratory-burst activity, acts on PtdIns 3-kinase, the enzyme producing PtdInsP3 from PtdIns(4,5)P2. Pretreatment of 32P-labelled human neutrophils with 100 nM wortmannin totally abolished fMLP-mediated PtdInsP3 production, raised PtdInsP2 levels, and did not affect cellular PtdInsP and PtdIns contents. The inhibitory effect on PtdInsP3 formation in intact cells was dose-dependent, with an IC50 of approximately 5 nM. Similar results were obtained with PtdIns 3-kinase immunoprecipitated by antibodies against the p85 regulatory subunit: wortmannin totally inhibited PtdIns3P production in immunoprecipitates at concentrations of 10-100 nM (IC50 approximately 1 nM). These results illustrate the direct and specific inhibition of PtdIns 3-kinase by wortmannin. Since agonist-mediated respiratory-burst activation is most sensitive to wortmannin (IC50 = 12 nM), this suggests that agonist-mediated PtdInsP3 formation is indispensable for this cell response. Neutrophils pretreated with wortmannin develop oscillatory changes in F-actin content, but actin polymerization in response to fMLP is not inhibited. This, and the absence of PtdInsP3 under these conditions, are in agreement with a modulatory role for PtdInsP3 in cytoskeletal rearrangements, but imply that PtdInsP3 production is not a primary event triggering elongation of actin filaments in neutrophils.
Assuntos
Androstadienos/farmacologia , Neutrófilos/metabolismo , Fosfatos de Fosfatidilinositol/sangue , Fosfatidilinositóis/sangue , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Autorradiografia , Humanos , Cinética , Medições Luminescentes , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Consumo de Oxigênio/efeitos dos fármacos , Fosfatos/sangue , Fosfatidilinositol 3-Quinases , Fosfolipase D/antagonistas & inibidores , Fosfolipídeos/sangue , Fosfolipídeos/isolamento & purificação , Radioisótopos de Fósforo , Acetato de Tetradecanoilforbol/farmacologia , WortmaninaRESUMO
The novel class II phosphoinositide (PI) 3-kinases are characterized by the presence of a C-terminal C2 domain, but little is known about their regulation. We find insulin causes a rapid 2-3-fold increase in the activity of PI 3-kinase C2alpha (PI3K-C2alpha) in CHO-IR cells, 3T3-L1 adipocytes, and fully differentiated L5L6 myotubes. No insulin-induced activation of PI3K-C2alpha was observed in cell types known to have low responsiveness to insulin including HEK 293 cells, 3T3-L1 preadipocytes, and undifferentiated L5L6 myoblasts. The mechanism of activation of PI3K-C2alpha by insulin differs from that of class Ia PI 3-kinases in that insulin stimulation did not cause PI3K-C2alpha to associate with IRS-1 or insulin receptor. PI3K-C2alpha existed as a doublet, and insulin stimulation caused a redistribution from the lower molecular weight band to the higher molecular weight band, suggesting phosphorylation-induced bandshift. Consistent with this, in vitro phosphatase treatment reduced the intensity of the upper band back to that seen in unstimulated cells. This suggests that insulin-induced phosphorylation could play a role in regulation of the activity of PI3K-C2alpha. The finding that insulin activates PI3K-C2alpha in cell types known to possess a wide range of responses to insulin suggests that PI3K-C2alpha is a novel component of insulin-stimulated signaling cascades.
Assuntos
Insulina/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Camundongos , Isoformas de ProteínasRESUMO
To investigate the molecular basis that makes heterodimeric CD8alphabeta a more efficient coreceptor than homodimeric CD8alphaalpha, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8beta and that this allows partitioning of CD8alphabeta, but not of CD8alphaalpha, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8beta abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3zeta in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8beta is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.
Assuntos
Antígenos CD8/metabolismo , Ácido Palmítico/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Ácido 4-Aminobenzoico/imunologia , Sequência de Aminoácidos , Animais , Complexo CD3/metabolismo , Antígenos CD8/imunologia , Antígenos CD8/fisiologia , Detergentes , Ativação Enzimática/imunologia , Antígenos H-2/fisiologia , Hibridomas , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Fosforilação , Plasmodium berghei/imunologia , Estrutura Terciária de Proteína , Proteínas de Protozoários/imunologia , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The cDNA for a human Class II phosphoinositide 3-kinase (PI 3-kinase C2beta) with a C2 domain was cloned from a U937 monocyte cDNA library and the enzyme expressed in mammalian and insect cells. Like other Class II PI 3-kinases in vitro, PI 3-kinase C2beta utilizes phosphatidylinositol (PI) and PI 4-monophosphate but not PI 4, 5-biphosphate as substrates in the presence of Mg2+. Remarkably, and unlike other PI 3-kinases, the enzyme can use either Mg-ATP or Ca-ATP to generate PI 3-monophosphate. PI 3-kinase C2beta, like the Class I PI 3-kinases, but unlike PI 3-kinase C2alpha, is sensitive to low nanomolar levels of the inhibitor wortmannin. The enzyme is not regulated by the small GTP-binding protein Ras. The C2 domain of the enzyme bound anionic phospholipids such as PI and phosphatidylserine in vitro, but did not co-operatively bind Ca2+ and phospholipids. Deletion of the C2 domain increased the lipid kinase activity suggesting that it functions as a negative regulator of the catalytic domain. Although presently it is not known whether PI 3-kinase C2beta is regulated by Ca2+ in vivo, our results suggest a novel role for Ca2+ ions in phosphate transfer reactions.
Assuntos
Cálcio/metabolismo , Isoenzimas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Sequência de Bases , Catálise , Linhagem Celular , Clonagem Molecular , Primers do DNA , Humanos , Isoenzimas/química , Isoenzimas/genética , Modelos Químicos , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera , Frações Subcelulares/enzimologia , Especificidade por SubstratoRESUMO
Src homology 2 (SH2) domains exist in many intracellular proteins and have well characterized roles in signal transduction. SH2 domains bind to phosphotyrosine (Tyr(P))-containing proteins. Although tyrosine phosphorylation is essential for protein-SH2 domain interactions, the binding specificity also derives from sequences C-terminal to the Tyr(P) residue. The high affinity and specificity of this interaction is critical for precluding aberrant cross-talk between signaling pathways. The p85alpha subunit of phosphoinositide 3-kinase (PI 3-kinase) contains two SH2 domains, and it has been proposed that in competition with Tyr(P) binding they may also mediate membrane attachment via interactions with phosphoinositide products of PI 3-kinase. We used nuclear magnetic resonance spectroscopy and biosensor experiments to investigate interactions between the p85alpha SH2 domains and phosphoinositides or inositol polyphosphates. We reported previously a similar approach when demonstrating that some pleckstrin homology domains show binding specificity for distinct phosphoinositides (Salim, K., Bottomley, M. J., Querfurth, E., Zvelebil, M. J., Gout, I., Scaife, R., Margolis, R. L., Gigg, R., Smith, C. I., Driscoll, P. C., Waterfield, M. D., and Panayotou, G. (1996) EMBO J. 15, 6241-6250). However, neither SH2 domain exhibited binding specificity for phosphoinositides in phospholipid bilayers. We show that the p85alpha SH2 domain Tyr(P) binding pockets indiscriminately accommodate phosphoinositides and inositol polyphosphates. Binding of the SH2 domains to Tyr(P) peptides was only poorly competed for by phosphoinositides or inositol polyphosphates. We conclude that these ligands do not bind p85alpha SH2 domains with high affinity or specificity. Moreover, we observed that although wortmannin blocks PI 3-kinase activity in vivo, it does not affect the ability of tyrosine-phosphorylated proteins to bind to p85alpha. Consequently phosphoinositide products of PI 3-kinase are unlikely to regulate signaling through p85alpha SH2 domains.