RESUMO
As ectopic expression of the neuronal inositol-1,4,5-trisphosphate-3-kinase A (InsP3Kinase) in tumor cells increases the metastatic potential, InsP3Kinase is an interesting target for tumor therapy. Recently, we have identified a membrane-permeable InsP3Kinase inhibitor (BAMB-4) exhibiting an IC50-value of 20 µM. Here we characterized a new InsP3Kinase inhibitor which shows a 130-fold lower IC50 value (157 ± 57 nM) as compared to BAMB-4. We demonstrate that this nitrophenolic compound, BIP-4, is non-competitive to ATP but competitive to InsP3, thus exhibits a high selectivity for inhibition of InsP3Kinase activity. Docking analysis suggested a putative binding mode of this molecule into the InsP3Kinase active site. Determination of cellular uptake in lung cancer cells (H1299) revealed that 6% of extracellular BIP-4 is internalized by non-endosomal uptake, showing that BIP-4 is not trapped inside endo/lysosomes but is available to inhibit cellular InsP3Kinase activity. Interestingly, we found that BIP-4 mediated inhibition of InsP3Kinase activity in the two lung cancer cell lines H1299 and LN4323 inhibited proliferation and adhesion at IC50 values of 3 µM or 2 µM, respectively. InsP3Kinase inhibition did not alter ATP-induced calcium signals but significantly reduced the level of Ins(1,3,4,5,6)P5. From these data we conclude that the inhibitory effect of BIP-4 on proliferation and adhesion of lung cancer cells does not result from alterations of calcium but from alterations of inositol phosphate signals. In summary, we reveal that inhibition of cellular InsP3Kinase by BIP-4 impairs proliferation and adhesion and therefore BIP-4 might be a promising compound to reduce the metastatic potential of lung carcinoma cells.
Assuntos
Antineoplásicos/farmacologia , Inositol 1,4,5-Trifosfato/farmacologia , Neoplasias Pulmonares/patologia , Naftalimidas/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Pirazóis/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Benzamidas/química , Benzamidas/farmacologia , Benzoxazóis/química , Benzoxazóis/farmacologia , Sinalização do Cálcio , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Ensaios de Triagem em Larga Escala , Humanos , Simulação de Acoplamento Molecular , Naftalimidas/química , Pirazóis/químicaRESUMO
InsP6 (inositol hexakisphosphate), the most abundant inositol phosphate in metazoa, is pyrophosphorylated to InsP7 [5PP-InsP5 (diphosphoinositol pentakisphosphate)] by cytosolic and nuclear IP6Ks (InsP6 kinases) and to 1PP-InsP5 by another InsP6/InsP7 kinase family. MINPP1 (multiple inositol-polyphosphate phosphatase 1), the only known InsP6 phosphatase, is localized in the ER (endoplasmic reticulum) and lysosome lumina. A mechanism of cytosolic InsP6 dephosphorylation has remained enigmatic so far. In the present study, we demonstrated that IP6Ks change their kinase activity towards InsP6 at a decreasing ATP/ADP ratio to an ADP phosphotransferase activity and dephosphorylate InsP6. Enantio-selective analysis revealed that Ins(2,3,4,5,6)P5 is the main InsP5 product of the IP6K reaction, whereas the exclusive product of MINPP1 activity is the enantiomer Ins(1,2,4,5,6)P5. Whereas lentiviral RNAi-based depletion of MINPP1 at falling cellular ATP/ADP ratios had no significant impact on Ins(2,3,4,5,6)P5 production, the use of the selective IP6K inhibitor TNP [N2-(m-trifluorobenzyl),N6-(p-nitrobenzyl)purine] abolished the production of this enatiomer in different types of cells. Furthermore, by analysis of rat tissue and human blood samples all (main and minor) dephosphorylation products of InsP6 were detected in vivo. In summary, we identified IP6Ks as novel nuclear and cytosolic InsP6- (and InsP5-) dephosphorylating enzymes whose activity is sensitively driven by a decrease in the cellular ATP/ADP ratio, thus suggesting a role for IP6Ks as cellular adenylate energy 'sensors'.
Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Ácido Fítico/metabolismo , Animais , Humanos , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , RatosRESUMO
In colon enterocytes and in well-differentiated colon cancer CaCo-2 cells, InsP6 (inositol hexakisphosphate) inhibits iron uptake by forming extracellular insoluble iron/InsP6 complexes. In this study, we confirmed that CaCo-2 cells are not able to take up iron/InsP6 but, interestingly, found that the cells are able to internalize metal-free and Cr3+-bound InsP6. Thus, the inability of CaCo-2 cells to take up iron/InsP6 complexes seems to be due to the iron-bound state of InsP6. Since recently we demonstrated that the highly malignant bronchial carcinoma H1299 cells internalize and process InsP6, we examined whether these cells may be able to take up iron/InsP6 complexes. Indeed, we found that InsP6 dose-dependently increased uptake of iron and demonstrated that in the iron-bound state InsP6 is more effectively internalized than in the metal-free or Cr3+-bound state, indicating that H1299 cells preferentially take up iron/InsP6 complexes. Electron microscope and cell fraction assays indicate that after uptake H1299 cells mainly stored InsP6/iron in lysosomes as large aggregates, of which about 10% have been released to the cytosol. However, this InsP6-mediated iron transport had no significant effects on cell viability. This result together with our finding that the well-differentiated CaCo-2 cells did not, but the malignant H1299 cells preferentially took up iron/InsP6, may offer the possibility to selectively transport cytotoxic substances into tumour cells.
Assuntos
Complexos de Coordenação/metabolismo , Ferro/metabolismo , Ácido Fítico/metabolismo , Transporte Biológico , Células CACO-2 , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Cromo/metabolismo , Ferritinas/metabolismo , Humanos , Lisossomos/metabolismo , Ácido Fítico/farmacologia , Espécies Reativas de OxigênioRESUMO
Fundamental to the life and destiny of every cell is the regulation of protein synthesis through ribosome biogenesis, which begins in the nucleolus with the production of ribosomal RNA (rRNA). Nucleolar organization is a highly dynamic and tightly regulated process; the structural factors that direct nucleolar assembly and disassembly are just as important in controlling rRNA synthesis as are the catalytic activities that synthesize rRNA. Here, we report that a signaling enzyme, inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5K) is also a structural component in the nucleolus. We demonstrate that IP5K has functionally significant interactions with three proteins that regulate rRNA synthesis: protein kinase CK2, TCOF1 and upstream-binding-factor (UBF). Through molecular modeling and mutagenic studies, we identified an Arg-Lys-Lys tripeptide located on the surface of IP5K that mediates its association with UBF. Nucleolar IP5K spatial dynamics were sensitive to experimental procedures (serum starvation or addition of actinomycin D) that inhibited rRNA production. We show that IP5K makes stoichiometrically sensitive contributions to the architecture of the nucleoli in intact cells, thereby influencing the degree of rRNA synthesis. Our study adds significantly to the biological significance of IP5K; previously, it was the kinase activity of this protein that had attracted attention. Our demonstration that IP5K 'moonlights' as a molecular scaffold offers an unexpected new example of how the biological sophistication of higher organisms can arise from gene products acquiring multiple functions, rather than by an increase in gene number.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA Ribossômico/biossíntese , Sequência de Aminoácidos , Linhagem Celular Tumoral , Nucléolo Celular/enzimologia , Nucléolo Celular/metabolismo , Células HeLa , Humanos , Inositol/genética , Inositol/metabolismo , Células MCF-7 , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismoRESUMO
InsP(6) [Ins(1,2,3,4,5,6)P6; phytate] is the most abundant inositol phosphate in mammalian cells with cytosolic/nuclear concentrations of up to 50 µM. We noticed that InsP6 in culture medium at a concentration of ≤50 µM significantly stimulates H1299 tumour cell growth, whereas larger concentrations of InsP6 inhibit growth. A detailed study of the fate of 30 µM InsP6 added to H199 cells revealed a major fraction of InsP6 initially precipitates as cell-surface metal complexes, but becomes slowly re-solubilized by extracellular dephosphorylation first to InsP3 isomers and subsequently to free myo-inositol. The precipitated metal-InsP6 complex is endocytosed in a receptor-independent but intact-glycocalyx-dependent manner and appears in lysosomes, where it is immediately dephosphorylated to Ins(1,2,4,5,6)P5 and very slowly to free inositol. By RNA knockdown, we identified secreted and lysosome targeted MINPP1 (multiple inositol-polyphosphate phosphatase 1), the mammalian 3-phytase, to be essentially involved both in extracellular and in lysosomal InsP6 dephosphorylation. The results of the present study indicate that tumour cells employ this enzyme to utilize the micronutrients myo-inositol and metal-phosphate when encountering extracellular InsP6 and thus to enhance their growth potential.
Assuntos
Proliferação de Células , Monoéster Fosfórico Hidrolases/metabolismo , Ácido Fítico/metabolismo , Citosol/metabolismo , Endocitose , Endossomos/metabolismo , Cinética , Lisossomos/metabolismo , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Células Tumorais CultivadasRESUMO
The synchronization of intraerythrocytic maturation of Plasmodium parasites is an important factor in the malaria infection process. Synchronization is mediated by inositol phosphate (InsP(x))-induced Ca(2+)-release from internal stores. To further investigate the InsP(x) metabolism in these parasites a Plasmodium protein possessing inositol phosphate kinase (IPK) activity was recombinantly expressed, purified and enzymatically characterized for the first time. Its main activity is the conversion of the Ca(2+)-releasing second messenger Ins(1,4,5)P(3) to Ins(1,3,4,5)P(4), an important factor in chromatin remodeling and also in Ca(2+)-release. This protein possesses several additional IPK activities pointing to a potential role as inositol phosphate multikinase. Interestingly, we have also identified three putative subdomains of histone deacetylase in this protein possibly linking InsP(x)- and acetylation-mediated transcription regulation. Furthermore, we examined the inhibitory potential of >40 polyphenolic substances against its kinase activity. Because of the important role of InsP(x)-induced Ca(2+)-release in the development of Plasmodium parasites, IPKs are interesting targets for novel antimalarial approaches.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Plasmodium/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plasmodium/genética , Polifenóis/farmacologia , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
Human inositol phosphate multikinase (IPMK) is a multifunctional protein in cellular signal transduction, namely, a multispecific inositol phosphate kinase, phosphatidylinositol 3-kinase, and a scaffold within the mTOR-raptor complex. To fulfill these nuclear and cytoplasmic functions, intracellular targeting of IPMK needs to be regulated. We show here that IPMK, which has been considered to be a preferentially nuclear protein, is a nucleocytoplasmic shuttling protein, whose nuclear export is mediated by classical nuclear export receptor CRM1. We identified a functional nuclear export signal (NES) additionally to its previously described nuclear import signal (NLS). Furthermore, we describe a mechanism by which the activity of the IPMK-NLS is controlled. Protein kinase CK2 binds endogenous IPMK and phosphorylates it at serine 284. Interestingly, this phosphorylation can decrease nuclear localization of IPMK cell type specifically. A controlled nuclear import of IPMK may direct its actions either toward nuclear inositol phosphate (InsPx) metabolism or cytoplasmic actions on InsPx, phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2], as well as mTOR-raptor.
Assuntos
Caseína Quinase II/metabolismo , Núcleo Celular/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Humanos , Dados de Sequência Molecular , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/química , Sinais Direcionadores de Proteínas , Alinhamento de SequênciaRESUMO
The inositol 5-phosphatase SHIP1 is a negative regulator of signaling processes in hematopoietic cells. SHIP1 mediates its regulatory function after relocalization from the cytoplasm to the plasma membrane where it converts its substrate PI(3,4,5)P(3) to PI(3,4)P(2) thereby terminating PI3-kinase mediated signaling. In addition, SHIP1 converts Ins(1,3,4,5)P(4) to Ins(1,3,4)P(3) thereby regulating inositol phosphate metabolism. Here we report, that SHIP1 can be detected in nuclear puncta of Jurkat cells by confocal microscopy after expression of SHIP1 from a tetracycline inducible vector. SHIP1-containing nuclear puncta partially co-localize with FLASH, a multifunctional nuclear protein that has been linked to apoptotic signaling and transcriptional control. Nuclear localization was confirmed for endogenously expressed SHIP1 in the myeloid leukemia cell line TF1. In addition, enzymatically active SHIP1 was found in nuclear fractions of Jurkat cells with a similar specific activity as cytoplasmic SHIP1. Further analysis revealed that SHIP1 is a nucleocytoplasmic shuttling protein which is actively imported into and exported out of the nucleus. Nuclear import is mediated by two canonical nuclear localization signals (NLS) i.e. K(327)KSK and K(547)KLR. Mutational inactivation of each NLS motif inhibited nuclear import and reduced the proliferation of cells indicating a functional role of nuclear SHIP1 for cell growth. Our data indicate that SHIP1 is partly localized in the nucleus and suggest that SHIP1 plays a role for nuclear phosphoinositide and/or nuclear inositol phosphate signaling.
Assuntos
Núcleo Celular/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Motivos de Aminoácidos , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Inositol Polifosfato 5-Fosfatases , Mutagênese Sítio-Dirigida , Sinais de Localização Nuclear/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/análise , Monoéster Fosfórico Hidrolases/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
The parasitic protozoan Entamoeba histolytica is able to invade human tissues by secreting proteolytic enzymes. This secretion is regulated by inositol phosphate-mediated Ca(2+) release from internal stores. To further investigate the inositol phosphate metabolism of Entamoeba histolytica four putative inositol phosphate kinase genes (ehipk1-4) were identified and their expression analyzed by real-time quantitative PCR using RNA of trophozoites. Furthermore inositol phosphate kinase EhIPK1 was recombinantly expressed, purified and enzymatically characterized. Its main activity is the conversion of InsP(6) to 5PP-Ins(1,2,3,4,6)P(5), one of the main inositol phosphates found in Entamoeba histolytica. Remarkably, EhIPK1 possesses several additional enzymatic activities, e.g. the phosphorylation of the Ca(2+)-releasing second messenger Ins(1,4,5)P(3).We were able to identify several compounds with inhibitory potential against EhIPK1. Because of the important role of inositol phosphates in the invasion of human tissues by Entamoeba histolytica, inositol phosphate metabolizing enzymes are interesting targets for novel therapeutic approaches.
Assuntos
Entamoeba histolytica/enzimologia , Fosfatos de Inositol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sequência de Aminoácidos , Entamoeba histolytica/genética , Inibidores Enzimáticos/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Inositol-1,4,5-trisphosphate 3-kinase-A (itpka) accumulates in dendritic spines and seems to be critically involved in synaptic plasticity. The protein possesses two functional activities: it phosphorylates inositol-1,4,5-trisphosphate (Ins(1,4,5)P(3)) and regulates actin dynamics by its F-actin bundling activity. To assess the relevance of these activities for neuronal physiology, we examined the effects of altered itpka levels on cell morphology, Ins(1,4,5)P(3) metabolism and dendritic Ca(2+) signaling in hippocampal neurons. Overexpression of itpka increased the number of dendritic protrusions by 71% in immature primary neurons. In mature neurons, however, the effect of itpka overexpression on formation of dendritic spines was weaker and depletion of itpka did not alter spine density and synaptic contacts. In synaptosomes of mature neurons itpka loss resulted in decreased duration of Ins(1,4,5)P(3) signals and shorter Ins(1,4,5)P(3)-dependent Ca(2+) transients. At synapses of itpka deficient neurons the levels of Ins(1,4,5)P(3)-5-phosphatase (inpp5a) and sarcoplasmic/endoplasmic reticulum calcium ATPase pump-2b (serca2b) were increased, indicating that decreased duration of Ins(1,4,5)P(3) and Ca(2+) signals results from compensatory up-regulation of these proteins. Taken together, our data suggest a dual role for itpka. In developing neurons itpka has a morphogenic effect on dendrites, while the kinase appears to play a key role in shaping Ca(2+) transients at mature synapses.
Assuntos
Cálcio/metabolismo , Neurônios/citologia , Neurônios/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Sinalização do Cálcio , Células Cultivadas , Cerebelo/metabolismo , Espinhas Dendríticas/enzimologia , Hipocampo/enzimologia , Hipocampo/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Inositol Polifosfato 5-Fosfatases , Camundongos , Camundongos Knockout , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ratos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Sinaptossomos/metabolismo , TransfecçãoRESUMO
Cell migration is one of the hallmarks of metastatic disease and thus identification of migration promoting proteins is crucial for the understanding of metastasis formation. Here we show that the neuron-specific, F-actin bundling inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) is ectopically expressed in tumor cells and critically involved in migration. Down-regulation of ITPKA expression in transformed cell-lines with ectopic expression of ITPKA significantly decreased migration and the number of linear and branched cell protrusion. Conversely, up-regulation of ITPKA in tumor cell lines with low endogenous ITPKA expression increased migration and formation of cell processes. In vitro, ITPKA alone induced the formation of linear actin filaments, whereas ITPKA mediated formation of branched protrusions seems to result from interaction between ITPKA and the F-actin cross-linking protein filamin C. Based on these actin-modulating and migration-promoting effects of ITPKA we examined its expression in clinical samples of different tumor entities, starting with the analysis of multiple tumor tissue arrays. As in lung adenocarcinoma specimens, the highest ITPKA expression rate was found, this tumor entity was examined in more detail. ITPKA was expressed early in adenocarcinoma progression (pN0) and was largely maintained in invasive and metastatic tumor cell populations (pN1/2, lymph node metastases). Together with our result that high expression of ITPKA increases motility of tumor cells we conclude that the observed expression of ITPKA early in tumor development increases the metastatic potential of lung adenocarcinoma cells. Therefore, we suggest that ITPKA may be a promising therapeutic molecular target for anti metastatic therapy of lung cancer.
Assuntos
Movimento Celular , Neoplasias/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Adenocarcinoma/enzimologia , Adenocarcinoma de Pulmão , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Proteínas Contráteis/metabolismo , Feminino , Filaminas , Humanos , Neoplasias Pulmonares/enzimologia , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
Under physiologic conditions, the inositol-1,4,5-trisphosphate (InsP(3))-metabolizing, F-actin-bundling InsP(3)-kinase-A (ITPKA) is expressed only in neurons. Tumor cells that have gained the ability to express ITPKA show an increased metastatic potential due to the migration-promoting properties of ITPKA. Here we investigated the mechanism how tumor cells have gained the ability to reexpress ITPKA by using a breast cancer cell line (T47D) with no expression and a lung carcinoma cell line (H1299) with ectopic ITPKA expression. Cloning of a 1,250-bp ITPKA promoter fragment revealed that methylation of CpG islands was reduced in H1299 as compared with T47D cells, but DNA demethylation did not alter the expression of ITPKA. Instead, we showed that the repressor-element-1-silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF), which suppresses expression of neuronal genes in nonneuronal tissues, regulates expression of ITPKA. Knockdown of REST/NRSF induced expression of ITPKA in T47D cells, whereas its overexpression in H1299 cells strongly reduced the level of ITPKA. In T47D cells, REST/NRSF was bound to the RE-1 site of the ITPKA promoter and strongly reduced its activity. In H1299 cells, in contrast, expressing comparable REST/NRSF levels as T47D cells, REST/NRSF only slightly reduced ITPKA promoter activity. This reduced suppressor activity most likely results from expression of a dominant-negative isoform of REST/NRSF, REST4, which impairs binding of REST/NRSF to the RE-1 site. Thus, ITPKA may belong to the neuronal metastasis-promoting proteins whose ectopic reexpression in tumor cells is associated with impaired REST/NRSF activity. Mol Cancer Res; 9(4); 1-10. ©2011 AACR.
RESUMO
Recent studies have shown that inositol 1,4,5-trisphosphate 3-kinase isoform B (IP3KB) possesses important roles in the development of immune cells. IP3KB can be targeted to multiple cellular compartments, among them nuclear localization and binding in close proximity to the plasma membrane. The B isoform is the only IP3K that is almost ubiquitously expressed in mammalian cells. Detailed mechanisms of its targeting regulation will be important in understanding the role of Ins(1,4,5)P(3) phosphorylation on subcellular calcium signaling and compartment-specific initiation of pathways leading to regulatory active higher phosphorylated inositol phosphates. Here, we identified an exportin 1-dependent nuclear export signal ((134)LQRELQNVQV) and characterized the amino acids responsible for nuclear localization of IP3KB ((129)RKLR). These two targeting domains regulate the amount of nuclear IP3KB in cells. We also demonstrated that the localization of IP3KB at the plasma membrane is due to its binding to cortical actin structures. Intriguingly, all three of these targeting activities reside in one small polypeptide segment (amino acids 104-165), which acts as a multitargeting domain (MTD). Finally, a hitherto unknown subnuclear localization of IP3KB could be demonstrated in rapidly growing H1299 cells. IP3KB is specifically enriched at nuclear invaginations extending perpendicular between the apical and basal surface of the nucleus of these flat cells. Such nuclear invaginations are known to be involved in Ins(1,4,5)P(3)-mediated Ca(2+) signaling of the nucleus. Our findings indicate that IP3KB not only regulates cytoplasmic Ca(2+) signals by phosphorylation of subplasmalemmal and cytoplasmic Ins(1,4,5)P(3) but may also be involved in modulating nuclear Ca(2+) signals generated from these nuclear envelope invaginations.
Assuntos
Citoesqueleto de Actina/metabolismo , Sinalização do Cálcio/fisiologia , Membrana Celular/enzimologia , Membrana Nuclear/enzimologia , Sinais de Exportação Nuclear/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Citoesqueleto de Actina/genética , Transporte Ativo do Núcleo Celular/fisiologia , Cálcio/metabolismo , Membrana Celular/genética , Células HeLa , Humanos , Inositol 1,4,5-Trifosfato/genética , Inositol 1,4,5-Trifosfato/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Carioferinas/genética , Carioferinas/metabolismo , Membrana Nuclear/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína Exportina 1RESUMO
CD4(+)CD25(hi)Foxp3(+) regulatory T cells (T(regs)) are critical mediators of self-tolerance, which is crucial for the prevention of autoimmune disease, but T(regs) can also inhibit antitumor immunity. T(regs) inhibit the proliferation of CD4(+)CD25(-) conventional T cells (T(cons)), as well as the ability of these cells to produce effector cytokines; however, the molecular mechanism of suppression remains unclear. Here, we showed that human T(regs) rapidly suppressed the release of calcium ions (Ca(2+)) from intracellular stores in response to T cell receptor (TCR) activation in T(cons). The inhibition of Ca(2+) signaling resulted in decreased dephosphorylation, and thus decreased activation, of the transcription factor nuclear factor of activated T cells 1 (NFAT1) and reduced the activation of nuclear factor κB (NF-κB). In contrast, Ca(2+)-independent events in T(cons), such as TCR-proximal signaling and activation of the transcription factor activator protein 1 (AP-1), were not affected during coculture with T(regs). Despite suppressing intracellular Ca(2+) mobilization, coculture with T(regs) did not block the generation of inositol 1,4,5-trisphosphate in TCR-stimulated T(cons). The T(reg)-induced suppression of the activity of NFAT and NF-κB and of the expression of the gene encoding the cytokine interleukin-2 was reversed in T(cons) by increasing the concentration of intracellular Ca(2+). Our results elucidate a previously unrecognized and rapid mechanism of T(reg)-mediated suppression. This increased understanding of T(reg) function may be exploited to generate possible therapies for the treatment of autoimmune diseases and cancer.
Assuntos
Sinalização do Cálcio/imunologia , Tolerância Imunológica , Ativação Linfocitária , NF-kappa B/imunologia , Fatores de Transcrição NFATC/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/terapia , Cálcio/imunologia , Cálcio/metabolismo , Células Cultivadas , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-2/imunologia , Interleucina-2/metabolismo , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/terapia , Fosfatos de Fosfatidilinositol/imunologia , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Fator de Transcrição AP-1/imunologia , Fator de Transcrição AP-1/metabolismoRESUMO
Cellular migration is an essential prerequisite for metastatic dissemination of cancer cells. This study demonstrates that the neuron/testis-specific F-actin-targeted inositol 1,4,5-trisphosphate 3-kinase-A (ITPKA) is ectopically expressed in different human tumor cell lines and during tumor progression in the metastatic tumor model Balb-neuT. High expression of ITPKA increases invasive migration in vitro and metastasis in a xenograft SCID mouse model. Mechanistic studies show that ITPKA promotes migration of tumor cells by two different mechanisms as follows: growth factor independently high levels of ITPKA induce the formation of large cellular protrusions by directly modulating the actin cytoskeleton. The F-actin binding activity of ITPKA stabilizes and bundles actin filaments and thus increases the levels of cellular F-actin. In growth factor-stimulated cells, the catalytically active domain enhances basal ITPKA-induced migration by activating store-operated calcium entry through production of inositol 1,3,4,5-tetrakisphosphate and subsequent inhibition of inositol phosphate 5-phosphatase. These two functional activities of ITPKA stimulating tumor cell migration place the enzyme among the potential targets of anti-metastatic therapy.
Assuntos
Movimento Celular , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Cálcio/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Células Hep G2 , Humanos , Fosfatos de Inositol/metabolismo , Camundongos , Camundongos SCID , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/antagonistas & inibidores , Transplante de Neoplasias , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Transplante HeterólogoRESUMO
In the present study, effects of increased IP3K-A [Ins(1,4,5)P(3) 3-kinase-A] expression were analysed. H1299 cells overexpressing IP3K-A formed branching protrusions, and under three-dimensional culture conditions, they exhibited a motile fibroblast-like morphology. They lost the ability to form actin stress fibres and showed increased invasive migration in vitro. Furthermore, expression levels of the mesenchymal marker proteins vimentin and N-cadherin were increased. The enzymatic function of IP3K-A is to phosphorylate the calcium-mobilizing second messenger Ins(1,4,5)P(3) to (Ins(1,3,4,5)P(4). Accordingly, cells overexpressing IP3K-A showed reduced calcium release and altered concentrations of InsPs, with decreasing concentrations of Ins(1,4,5)P(3), InsP(6) and Ins(1,2,3,4,5)P(5), and increasing concentrations of Ins(1,3,4,5)P(4). However, IP3K-A-induced effects on cell morphology do not seem to be dependent on enzyme activity, since a protein devoid of enzyme activity also induced the formation of branching protrusions. Therefore we propose that the morphological changes induced by IP3K-A are mediated by non-enzymatic activities of the protein.
Assuntos
Citoesqueleto/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Cálcio/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Citoesqueleto/ultraestrutura , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Mutação , Fenótipo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Transdução de SinaisAssuntos
Antígenos CD34/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Antígenos CD34/imunologia , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células NIH 3T3 , Fosforilação , Subunidades ProteicasRESUMO
InsP6 is an intracellular signal with several proposed functions that is synthesized by IP5K [Ins(1,3,4,5,6)P5 2-kinase]. In the present study, we overexpressed EGFP (enhanced green fluorescent protein)-IP5K fusion proteins in NRK (normal rat kidney), COS7 and H1299 cells. The results indicate that there is spatial microheterogeneity in the intracellular localization of IP5K that could also be confirmed for the endogenous enzyme. This may facilitate changes in InsP6 levels at its sites of action. For example, overexpressed IP5K showed a structured organization within the nucleus. The kinase was preferentially localized in euchromatin and nucleoli, and co-localized with mRNA. In the cytoplasm, the overexpressed IP5K showed locally high concentrations in discrete foci. The latter were attributed to stress granules by using mRNA, PABP [poly(A)-binding protein] and TIAR (TIA-1-related protein) as markers. The incidence of stress granules, in which IP5K remained highly concentrated, was further increased by puromycin treatment. Using FRAP (fluorescence recovery after photobleaching) we established that IP5K was actively transported into the nucleus. By site-directed mutagenesis we identified a nuclear import signal and a peptide segment mediating the nuclear export of IP5K.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Cromatografia Líquida de Alta Pressão , Primers do DNA , Humanos , Hibridização in Situ Fluorescente , Cinética , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Mensageiro/genética , Frações Subcelulares/enzimologiaRESUMO
RAS genes, predominantly N-RAS and K-RAS, have been implicated in the pathogenesis of acute myeloid leukemia (AML), due to activating RAS mutations detectable in approximately 20% of AML patients. In the present study, RAS proteins were detected in their activated, GTP-bound form, in AML patients (n = 10) not expressing mutated forms of H-RAS, K-RAS and N-RAS. Further analysis revealed the simultaneous presence of N-RAS and K-RAS proteins in the GTP-bound state in seven out of 10 AML samples. In four out of 10 samples the levels of RAS-GTP were comparable to an AML cell line (TF-1) with an activating N-RAS mutation (Q61P). The detection of RAS-GTP in AML patients without RAS mutations further supports a functional role of RAS proteins in the pathogenesis of AML and may explain the observed effects of RAS inhibitors in some AML patients in the absence of activating RAS mutations.
Assuntos
Análise Mutacional de DNA , Regulação Neoplásica da Expressão Gênica , Genes ras/genética , Guanosina Trifosfato/química , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutação , Proteínas ras/biossíntese , Proteínas ras/genética , Adulto , Idoso , Feminino , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação , Ligação ProteicaRESUMO
The three isoforms of human Ins(1,4,5)P3 3-kinase (IP3K) show remarkable differences in their intracellular targeting. Whereas predominant targeting to the cytoskeleton and endoplasmic reticulum has been shown for IP3K-A and IP3K-B, rat IP3K-C shuttles actively between the nucleus and cytoplasm. In the present study we examined the expression and intracellular localisation of endogenous IP3K-C in different mammalian cell lines using an isoform-specific antibody. In addition, human IP3K-C, showing remarkable differences to its rat homologue in the N-terminal targeting domain, was tagged with EGFP and used to examine active transport mechanisms into and out of the nucleus. We found both a nuclear import activity residing in its N-terminal domain and a nuclear export activity sensitive to treatment with leptomycin B. Different from the rat isoform, an exportin 1-dependent nuclear export site of the human enzyme resides outside the N-terminal targeting domain in the catalytic enzyme domain. A phylogenetic survey of vertebrate IP3K sequences indicates that in each of the three isoforms a nuclear export signal has evolved in the catalytic domain either de novo (IP3K-A) or as a substitute for an earlier evolved corresponding N-terminal signal (IP3K-B and IP3K-C). In higher vertebrates, and in particular in primates, re-export of nuclear IP3K activity may be guaranteed by the mechanism discovered.