RESUMEN
Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)). The binding affinity for PtdInsP(3), together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP(3) effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules.
Asunto(s)
Fosfatidilinositoles/metabolismo , Transducción de Señal/fisiología , Proteínas Activadoras de ras GTPasa/química , Proteínas Activadoras de ras GTPasa/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Sitios de Unión/fisiología , Cristalografía , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Ratones , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Activadoras de ras GTPasa/genéticaRESUMEN
Class I phosphoinositide 3-kinases exert important cellular effects through their two primary lipid products, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)). As few molecular targets for PtdIns(3,4)P(2) have yet been identified, a screen for PI 3-kinase-responsive proteins that is selective for these is described. This features a tertiary approach incorporating a unique, primary recruitment of target proteins in intact cells to membranes selectively enriched in PtdIns(3,4)P(2). A secondary purification of these proteins, optimized using tandem pleckstrin homology domain containing protein-1 (TAPP-1), an established PtdIns(3,4)P(2) selective ligand, yields a fraction enriched in proteins of potentially similar lipid binding character that are identified by liquid chromatography-tandem MS. Thirdly, this approach is coupled to stable isotope labeling with amino acids in cell culture using differential isotope labeling of cells stimulated in the absence and presence of the PI 3-kinase inhibitor wortmannin. This provides a ratio-metric readout that distinguishes authentically responsive components from copurifying background proteins. Enriched fractions thus obtained from astrocytoma cells revealed a subset of proteins that exhibited ratios indicative of their initial, cellular responsiveness to PI 3-kinase activation. The inclusion among these of tandem pleckstrin homology domain containing protein-1, three isoforms of Akt, switch associated protein-70, early endosome antigen-1 and of additional proteins expressing recognized lipid binding domains demonstrates the utility of this strategy and lends credibility to the novel candidate proteins identified. The latter encompass a broad set of proteins that include the gene product of TBC1D2A, a putative Rab guanine nucleotide triphosphatase activating protein (GAP) and IQ motif containing GAP1, a potential tumor promoter. A sequence comparison of the former protein indicates the presence of a pleckstrin homology domain whose lipid binding character remains to be established. IQ motif containing GAP1 lacks known lipid interacting components and a preliminary analysis here indicates that this may exemplify a novel class of atypical phosphoinositide (aPI) binding domain.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Línea Celular Tumoral , Estudios de Factibilidad , Humanos , Fragmentos de Péptidos/biosíntesis , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis , Resonancia por Plasmón de Superficie , Proteínas Activadoras de ras GTPasa/biosíntesisRESUMEN
The PTEN (phosphatase and tensin homolog) tumor suppressor is a phosphatase that inhibits phosphoinositide 3-kinase-dependent signaling by metabolizing the phosphoinositide lipid phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)) at the plasma membrane. PTEN can be mono- or polyubiquitinated, and this appears to control its nuclear localization and stability, respectively. Although PTEN phosphorylation at a cluster of C-terminal serine and threonine residues has been shown to stabilize the protein and inhibit polyubiquitination and plasma membrane localization, details of the regulation of ubiquitination are unclear. Here, we show that plasma membrane targeting of PTEN greatly enhances PTEN ubiquitination and that phosphorylation of PTEN in vitro does not affect subsequent ubiquitination. These data suggest that C-terminal phosphorylation indirectly regulates ubiquitination by controlling membrane localization. We also show that either mono- or polyubiquitination in vitro greatly reduces PTEN phosphatase activity. Finally, we show that hyperosmotic stress increases both PTEN ubiquitination and cellular PtdInsP(3) levels well before a reduction in PTEN protein levels is observed. Both PTEN ubiquitination and elevated PtdInsP(3) levels were reduced within 10 min after removal of the hyperosmotic stress. Our data indicate that ubiquitination may represent a regulated mechanism of direct reversible control over the PTEN enzyme.
Asunto(s)
Membrana Celular/enzimología , Núcleo Celular/enzimología , Fosfohidrolasa PTEN/metabolismo , Ubiquitinación/fisiología , Transporte Activo de Núcleo Celular/fisiología , Línea Celular Tumoral , Membrana Celular/genética , Núcleo Celular/genética , Humanos , Presión Osmótica/fisiología , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilación/fisiologíaAsunto(s)
Transformación Celular Neoplásica , Regulación hacia Abajo , Fosfohidrolasa PTEN/metabolismo , Animales , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Ubiquitina-Proteína Ligasas Nedd4 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Dominios PDZ , Fosfohidrolasa PTEN/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , Transducción de Señal/fisiología , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
The tumour suppressor PTEN can inhibit cell proliferation and migration as well as control cell growth, in different cell types. PTEN functions predominately as a lipid phosphatase, converting PtdIns(3,4,5)P(3) to PtdIns(4,5)P(2), thereby antagonizing PI(3)K (phosphoinositide 3-kinase) and its established downstream effector pathways. However, much is unclear concerning the mechanisms that regulate PTEN movement to the cell membrane, which is necessary for its activity towards PtdIns(3,4,5)P(3) (Refs 3, 4, 5). Here we show a requirement for functional motor proteins in the control of PI3K signalling, involving a previously unknown association between PTEN and myosinV. FRET (Förster resonance energy transfer) measurements revealed that PTEN interacts directly with myosinV, which is dependent on PTEN phosphorylation mediated by CK2 and/or GSK3. Inactivation of myosinV-transport function in neurons increased cell size, which, in line with known attributes of PTEN-loss, required PI(3)K and mTor. Our data demonstrate a myosin-based transport mechanism that regulates PTEN function, providing new insights into the signalling networks regulating cell growth.
Asunto(s)
Tamaño de la Célula , Miosina Tipo V/metabolismo , Neuronas/citología , Fosfohidrolasa PTEN/metabolismo , Alanina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Ácido Aspártico/metabolismo , Sitios de Unión/genética , Células Cultivadas , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Miosina Tipo V/química , Neuronas/metabolismo , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/fisiología , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica/genética , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Transducción de Señal/genética , Transducción de Señal/fisiología , TransfecciónRESUMEN
Inositol hexakisphosphate kinases (IP6Ks) phosphorylate inositol hexakisphosphate (InsP(6)) to yield 5-diphosphoinositol pentakisphosphate (5-[PP]-InsP(5) or InsP(7)). In this study, we report the characterization of a selective inhibitor, N(2)-(m-(trifluoromethy)lbenzyl) N(6)-(p-nitrobenzyl)purine (TNP), for these enzymes. TNP dose-dependently and selectively inhibited the activity of IP6K in vitro and inhibited InsP(7) and InsP(8) synthesis in vivo without affecting levels of other inositol phosphates. TNP did not inhibit either human or yeast Vip/PPIP5K, a newly described InsP(6)/InsP(7) 1/3-kinase. Overexpression of IP6K1, -2, or -3 in cells rescued TNP inhibition of InsP(7) synthesis. TNP had no effect on the activity of a large number of protein kinases, suggesting that it is selective for IP6Ks. TNP reversibly reduced InsP(7)/InsP(8) levels. TNP in combination with genetic studies was used to implicate the involvement of two pathways for synthesis of InsP(8) in yeast. TNP induced a fragmented vacuole phenotype in yeast, consistent with inhibition of Kcs1, a Saccharomyces cerevisiae IP6K. In addition, it also inhibited insulin release from Min6 cells in a dose-dependent manner further implicating InsP(7) in this process. TNP thus provides a means of selectively and rapidly modulating cellular InsP(7) levels, providing a new and versatile tool to study the biological function and metabolic relationships of inositol pyrophosphates.
Asunto(s)
Difosfatos/metabolismo , Inhibidores Enzimáticos , Fosfatos de Inositol/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Purinas , Secuencia de Aminoácidos , Animales , Línea Celular , Difosfatos/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , Fosfatos de Inositol/química , Insulina/metabolismo , Datos de Secuencia Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Purinas/química , Purinas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Tapsigargina/metabolismo , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
Influenza A virus NS1 protein stimulates host-cell phosphoinositide 3-kinase (PI3K) signaling by binding to the p85beta regulatory subunit of PI3K. Here, in an attempt to establish a mechanism for this activation, we report further on the functional interaction between NS1 and p85beta. Complex formation was found to be independent of NS1 RNA binding activity and is mediated by the C-terminal effector domain of NS1. Intriguingly, the primary direct binding site for NS1 on p85beta is the inter-SH2 domain, a coiled-coil structure that acts as a scaffold for the p110 catalytic subunit of PI3K. In vitro kinase activity assays, together with protein binding competition studies, reveal that NS1 does not displace p110 from the inter-SH2 domain, and indicate that NS1 can form an active heterotrimeric complex with PI3K. In addition, it was established that residues at the C terminus of the inter-SH2 domain are essential for mediating the interaction between p85beta and NS1. Equivalent residues in p85alpha have previously been implicated in the basal inhibition of p110. However, such p85alpha residues were unable to substitute for those in p85beta with regards NS1 binding. Overall, these data suggest a model by which NS1 activates PI3K catalytic activity by masking a normal regulatory element specific to the p85beta inter-SH2 domain.
Asunto(s)
Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Bovinos , Línea Celular , Activación Enzimática , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ARN Viral/metabolismo , Alineación de Secuencia , Relación Estructura-Actividad , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/aislamiento & purificaciónRESUMEN
Activation of class Ia PI3K (phosphoinositide 3-kinase) produces PtdInsP3, a vital intracellular mediator whose degradation generates additional lipid signals. In the present study vanadate analogues that inhibit PTPs (protein tyrosine phosphatases) were used to probe the mechanisms which regulate the concentrations of these molecules allowing their independent or integrated function. In 1321N1 cells, which lack PtdInsP3 3-phosphatase activity, sodium vanadate or a cell permeable derivative, bpV(phen) [potassium bisperoxo(1,10-phenanthroline)oxovanadate (V)], increased the recruitment into anti-phosphotyrosine immunoprecipitates of PI3K activity and of the p85 and p110a subunits of class Ia PI3K and enhanced the recruitment of PI3K activity stimulated by PDGF (platelet-derived growth factor). However, neither inhibitor much increased cellular PtdInsP3 concentrations, but both diminished dramatically the accumulation of PtdInsP3 stimulated by PDGF or insulin and markedly increased the control and stimulated concentrations of PtdIns(3,4)P2. These actions were accounted for by the ability of PTP inhibitors to stimulate the activity of endogenous PtdInsP3 5-phosphatase(s), particularly SHIP2 (Src homology 2 domain containing inositol polyphosphate 5-phosphatase 2) and to inhibit types I and II PtdIns(3,4)P2 4-phosphatases. Thus bpV(phen) promoted the translocation of SHIP2 from the cytosol to a Triton X-100-insoluble fraction and induced a marked (5-10-fold) increase in SHIP2 specific activity mediated by enhanced tyrosine phosphorylation. The net effect of these inhibitors was, therefore, to switch the signal output of class I PI3K from PtdInsP3 to PtdIns(3,4)P2. A key component controlling this shift in the balance of lipid signals is the activation of SHIP2 by increased tyrosine phosphorylation, an effect observed in HeLa cells in response to both PTP inhibitors and epidermal growth factor.
Asunto(s)
Fosfatos de Fosfatidilinositol/análisis , Monoéster Fosfórico Hidrolasas/metabolismo , Dominios Homologos src , Línea Celular Tumoral , Factor de Crecimiento Epidérmico , Humanos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosforilación , Proteínas Tirosina Fosfatasas/antagonistas & inhibidoresRESUMEN
Although PTEN (phosphatase and tensin homologue deleted on chromosome 10) is one of the most commonly mutated tumour suppressors in human cancers, loss of PTEN expression in the absence of mutation appears to occur in an even greater number of tumours. PTEN is phosphorylated in vitro on Thr366 and Ser370 by GSK3 (glycogen synthase kinase 3) and CK2 (casein kinase 2) respectively, and specific inhibitors of these kinases block these phosphorylation events in cultured cells. Although mutation of these phosphorylation sites did not alter the phosphatase activity of PTEN in vitro or in cells, blocking phosphorylation of Thr366 by either mutation or GSK3 inhibition in glioblastoma cell lines led to a stabilization of the PTEN protein. Our data support a model in which the phosphorylation of Thr366 plays a role in destabilizing the PTEN protein.
Asunto(s)
Fosfohidrolasa PTEN/química , Fosfohidrolasa PTEN/metabolismo , Fosfotreonina/metabolismo , Animales , Quinasa de la Caseína II/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Glioblastoma , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Ratones , Mutación , Células 3T3 NIH , Fosfohidrolasa PTEN/genética , Fosforilación , SerinaRESUMEN
The Protein Tyrosine Phosphatase (PTP) family comprises a large and diverse group of enzymes, regulating a range of biological processes through de-phosphorylation of many proteins and lipids. These enzymes share a catalytic mechanism that requires a reduced and reactive cysteine nucleophile, making them potentially sensitive to inactivation and regulation by oxidation. Analysis of ten PTPs identified substantial differences in the sensitivity of these enzymes to oxidation in vitro. More detailed experiments confirmed the following rank order of sensitivity: PTEN and Sac1>PTPL1/FAP-1>>myotubularins. When the apparent sensitivity to oxidation of these PTPs in cells treated with hydrogen peroxide was analysed, this correlated well with the observed sensitivities to oxidation in vitro. These data suggested that different PTPs may fall into at least three different classes with respect to mechanisms of cellular redox regulation. 1. PTEN and Sac1 were readily and reversibly oxidised in vitro and in cells treated with hydrogen peroxide 2. PTPL1 appeared to be resistant to oxidation in cells, correlating with its sensitivity to reduction by glutathione in vitro 3. The myotubularin family of lipid phosphatases was almost completely resistant to oxidation in vitro and in cells. Our results show that sensitivity to reversible oxidation is not a necessary characteristic of the PTPs and imply that such sensitivity has evolved as a regulatory mechanism for some of this large family, but not others.
Asunto(s)
Familia de Multigenes , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Línea Celular , Glutatión/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Inmunoprecipitación , Cinética , Ratones , Oxidación-Reducción/efectos de los fármacos , Fosfatidilinositoles/metabolismo , Proteínas Tirosina Fosfatasas/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
BACKGROUND: Phosphatase and tensin homolog (PTEN) mediates many of its effects on proliferation, growth, survival, and migration through its PtdIns(3,4,5)P(3) lipid phosphatase activity, suppressing phosphoinositide 3-kinase (PI3K)-dependent signaling pathways. PTEN also possesses a protein phosphatase activity, the role of which is less well characterized. RESULTS: We have investigated the role of PTEN in the control of cell migration of mesoderm cells ingressing through the primitive streak in the chick embryo. Overexpression of PTEN strongly inhibits the epithelial-to-mesenchymal transition (EMT) of mesoderm cells ingressing through the anterior and middle primitive streak, but it does not affect EMT of cells located in the posterior streak. The inhibitory activity on EMT is completely dependent on targeting PTEN through its C-terminal PDZ binding site, but can be achieved by a PTEN mutant (PTEN G129E) with only protein phosphatase activity. Expression either of PTEN lacking the PDZ binding site or of the PTEN C2 domain, or inhibition of PI3K through specific inhibitors, does not inhibit EMT, but results in a loss of both cell polarity and directional migration of mesoderm cells. The PTEN-related protein TPTE, which normally lacks any detectable lipid and protein phosphatase activity, can be reactivated through mutation, and only this reactivated mutant leads to nondirectional migration of these cells in vivo. CONCLUSIONS: PTEN modulates cell migration of mesoderm cells in the chick embryo through at least two distinct mechanisms: controlling EMT, which involves its protein phosphatase activity; and controlling the directional motility of mesoderm cells, through its lipid phosphatase activity.
Asunto(s)
Movimiento Celular/fisiología , Gástrula/enzimología , Mesodermo/enzimología , Fosfohidrolasa PTEN/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animales , Embrión de Pollo , Estructura Terciaria de ProteínaRESUMEN
PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumour suppressor that functions as a PtdIns(3,4,5)P3 3-phosphatase to inhibit cell proliferation, survival and growth by antagonizing PI3K (phosphoinositide 3-kinase)-dependent signalling. Recent work has begun to focus attention on potential biological functions of the protein phosphatase activity of PTEN and on the possibility that some of its functions are phosphatase-independent. We discuss here the structural and regulatory mechanisms that account for the remarkable specificity of PTEN with respect to its PtdIns substrates and how it avoids the soluble headgroups of PtdIns that occur commonly in cells. Secondly we discuss the concept of PTEN as a constitutively active enzyme that is subject to negative regulation both physiologically and pathologically. Thirdly, we review the evidence that PTEN functions as a dual specificity phosphatase with discrete lipid and protein substrates. Lastly we present a current model of how PTEN may participate in the control of cell migration.
Asunto(s)
Fosfohidrolasa PTEN/fisiología , Regulación Alostérica , Animales , Humanos , Modelos Biológicos , Fosfohidrolasa PTEN/química , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Especificidad por SustratoRESUMEN
Phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5)P3] is a lipid second messenger, produced by Type I phosphoinositide 3-kinases (PI 3-kinases), which mediates intracellular responses to many growth factors. Although PI 3-kinases are implicated in events at both the plasma membrane and intracellular sites, including the nucleus, direct evidence for the occurrence of PtdIns(3,4,5)P3 at non-plasma membrane locations is limited. We made use of the pleckstrin homology (PH) domain of general receptor for phosphoinositides (Grp1) to detect PtdIns(3,4,5)P3 in an on-section labeling approach by quantitative immunogold electron microscopy. Swiss 3T3 cells contained low levels of PtdIns(3,4,5)P3 that increased up to 15-fold upon stimulation with platelet-derived growth factor (PDGF). The signal was sensitive to PI 3-kinase inhibitors and present mainly at plasma membranes, including lamellipodia, and in a surprisingly large pool within the nuclear matrix. Comparatively little labeling was observed in endomembranes. A similar distribution of PtdIns(3,4,5)P3 was observed in U87MG cells, which lack the PtdIns(3,4,5)P3 phosphatase, PTEN. Re-expression of PTEN into U87MG cells ablated plasma membrane PtdIns(3,4,5)P3, but not the nuclear pool of this lipid even when PTEN was targeted to nuclei. These data have important implications for the versatility of PI 3-kinase signaling and for the proposed functions of PTEN in the nucleus.
Asunto(s)
Núcleo Celular/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Células 3T3 , Animales , Western Blotting , Línea Celular Tumoral , Membrana Celular/metabolismo , Núcleo Celular/ultraestructura , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
In obesity and diabetes, the ability of hypothalamic neurons to sense and transduce changes in leptin and insulin levels is compromised. The effects of both hormones require intracellular signalling via the PI3-kinase pathway, which is inhibited by the phosphatase PTEN. We show that leptin-stimulated F-actin depolymerization in mouse hypothalamic cells is inhibited by PTEN, a process involving independent effects of both its lipid and protein phosphatase activities. Potentially mediating this F-actin depolymerization, leptin, but not insulin, stimulated the phosphorylation of PTEN in a CK2 dependent manner, and inhibited its phosphatase activity. Similarly, hyperpolarization of mouse pancreatic beta-cells by leptin also requires coincident PtdIns(3,4,5)P3 generation and actin depolymerization, and could be inhibited by mechanisms requiring both the lipid and protein phosphatase activities of PTEN. These results demonstrate a critical role for PTEN in leptin signalling and indicate a mechanism by which leptin and insulin can produce PI3K dependent differential cellular outputs.
Asunto(s)
Hipotálamo/citología , Células Secretoras de Insulina/metabolismo , Leptina/metabolismo , Fosfohidrolasa PTEN/metabolismo , Transducción de Señal/fisiología , Actinas/metabolismo , Animales , Células Cultivadas , Hipotálamo/metabolismo , Células Secretoras de Insulina/citología , Ratones , Fosfohidrolasa PTEN/antagonistas & inhibidores , Fosfohidrolasa PTEN/genética , Técnicas de Placa-Clamp , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilación , Canales de Potasio/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de LeptinaRESUMEN
Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues is an important posttranslational modification that is linked to insulin resistance. Several phosphoserine sites on IRS1 have been identified; the majority are located proximal to the phosphotryosine-binding domain or near key receptor tyrosine kinase substrate- and/or Src-homology 2 domain-binding sites. Here we report on the characterization of a serine phosphorylation site in the N-terminal pleckstrin homology (PH) domain of IRS1. Bioinformatic tools identify serine 24 (Ser24) as a putative substrate site for the protein kinase C (PKC) family of serine kinases. We demonstrate that this site is indeed a bona fide substrate for conventional PKC. In vivo, IRS-1 is also phosphorylated on Ser24 after phorbol 12-myristate 13-acetate treatment of cells, and isoform-selective inhibitor studies suggest the involvement of PKCalpha. By comparing the pharmacological characteristics of phorbol 12-myristate 13-acetate-stimulated Ser24 phosphorylation with phosphorylation at two other sites previously linked to PKC activity (Ser307 and Ser612), we show that PKCalpha is likely to be directly involved in Ser24 phosphorylation, but indirectly involved in Ser307 and Ser612 phosphorylation. Using Ser24Asp IRS-1 mutants to mimic the phosphorylated residue, we demonstrate that the phosphorylation status of Ser24 does play an important role in regulating phosphoinositide binding to, and the intracellular localization of, the IRS1-PH domain, which can ultimately impinge on insulin-stimulated glucose uptake. Hence we provide evidence that IRS1-PH domain function is important for normal insulin signaling and is regulated by serine phosphorylation in a manner that could contribute to insulin resistance.
Asunto(s)
Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteína Quinasa C/metabolismo , Serina/fisiología , Células 3T3-L1 , Animales , Proteínas Sanguíneas/química , Dominio Catalítico , Ceramidas/farmacología , Humanos , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Resistencia a la Insulina , Ratones , Modelos Moleculares , Células 3T3 NIH , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Proteína Quinasa C-alfa/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Serina/metabolismo , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
The inositol phospholipids (PIs) comprise a family of eight species with different combinations of phosphate groups arranged around the inositol ring. PIs are among the most versatile signaling molecules known, with key roles in receptor-mediated signal transduction, actin remodeling and membrane trafficking. Recent studies have identified effector proteins and specific lipid-binding domains through which PIs signal. These lipid-binding domains can be used as probes to further our understanding of the spatial and temporal control of individual PI species. New layers of complexity revealed by the use of such probes include the occurrence of PIs at intracellular locations, the identification of phosphatidylinositol signaling hotspots and the presence of non-membrane pools of PIs in cell nuclei.
Asunto(s)
Fosfatidilinositoles/metabolismo , Membrana Celular/metabolismo , Membranas Intracelulares/metabolismo , Lípidos de la Membrana/metabolismo , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Unión Proteica , Transducción de SeñalRESUMEN
Insulin-like growth factors elicit many responses through activation of phosphoinositide 3-OH kinase (PI3K). The tuberous sclerosis complex (TSC1-2) suppresses cell growth by negatively regulating a protein kinase, p70S6K (S6K1), which generally requires PI3K signals for its activation. Here, we show that TSC1-2 is required for insulin signaling to PI3K. TSC1-2 maintains insulin signaling to PI3K by restraining the activity of S6K, which when activated inactivates insulin receptor substrate (IRS) function, via repression of IRS-1 gene expression and via direct phosphorylation of IRS-1. Our results argue that the low malignant potential of tumors arising from TSC1-2 dysfunction may be explained by the failure of TSC mutant cells to activate PI3K and its downstream effectors.
Asunto(s)
Insulina/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/fisiología , Animales , Supervivencia Celular , Quimiotaxis , Fibroblastos/citología , Proteínas Sustrato del Receptor de Insulina , Factor I del Crecimiento Similar a la Insulina/fisiología , Péptidos y Proteínas de Señalización Intracelular , Ratones , Fosfoproteínas/antagonistas & inhibidores , Fosforilación , Proteínas/fisiología , Proteínas Represoras/fisiología , Proteínas Quinasas S6 Ribosómicas/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis TuberosaRESUMEN
The acquisition of resistance to apoptosis, the cell's intrinsic suicide program, is essential for cancers to arise and progress and is a major reason behind treatment failures. We show in this article that small molecule antagonists of the sigma-1 receptor inhibit tumor cell survival to reveal caspase-dependent apoptosis. sigma antagonist-mediated caspase activation and cell death are substantially attenuated by the prototypic sigma-1 agonists (+)-SKF10,047 and (+)-pentazocine. Although several normal cell types such as fibroblasts, epithelial cells, and even sigma receptor-rich neurons are resistant to the apoptotic effects of sigma antagonists, cells that can promote autocrine survival such as lens epithelial and microvascular endothelial cells are as susceptible as tumor cells. Cellular susceptibility appears to correlate with differences in sigma receptor coupling rather than levels of expression. In susceptible cells only, sigma antagonists evoke a rapid rise in cytosolic calcium that is inhibited by sigma-1 agonists. In at least some tumor cells, sigma antagonists cause calcium-dependent activation of phospholipase C and concomitant calcium-independent inhibition of phosphatidylinositol 3'-kinase pathway signaling. Systemic administration of sigma antagonists significantly inhibits the growth of evolving and established hormone-sensitive and hormone-insensitive mammary carcinoma xenografts, orthotopic prostate tumors, and p53-null lung carcinoma xenografts in immunocompromised mice in the absence of side effects. Release of a sigma receptor-mediated brake on apoptosis may offer a new approach to cancer treatment.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Receptores sigma/antagonistas & inhibidores , Animales , Apoptosis/fisiología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Señalización del Calcio/efectos de los fármacos , Carbazoles/farmacología , Caspasas/metabolismo , Bovinos , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular Tumoral , Activación Enzimática , Etilenodiaminas/farmacología , Haloperidol/farmacología , Humanos , Isoenzimas/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Desnudos , Fosfolipasa C delta , Piperazinas/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt , Fosfolipasas de Tipo C/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Receptor Sigma-1RESUMEN
Binding of the Rac1-specific guanine-nucleotide-exchange factor, Tiam1, to the plasma membrane requires the N-terminal pleckstrin homology domain. In the present study, we show that membrane-association is mediated by binding of PtdIns(4,5)P(2) to the pleckstrin homology domain. Moreover, in 1321N1 astrocytoma cells, translocation of Tiam1 to the cytosol, following receptor-mediated stimulation of PtdIns(4,5)P(2) breakdown, correlates with decreased Rac1-GTP levels, indicating that membrane-association is required for GDP/GTP exchange on Rac1. In addition, we show that platelet-derived growth factor activates Rac1 in vivo by increasing PtdIns(3,4,5)P(3) concentrations, rather than the closely related lipid, PtdIns(3,4)P(2). Finally, the data demonstrate that PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) bind to the same pleckstrin homology domain in Tiam1 and that soluble inositol phosphates appear to compete with lipids for this binding. Together, these novel observations provide strong evidence that distinct phosphoinositides regulate different functions of this enzyme, indicating that local concentrations of signalling lipids and the levels of cytosolic inositol phosphates will play crucial roles in determining its activity in vivo.