RESUMEN
Cofilin is a ubiquitous actin-binding factor required for the reorganization of actin filaments in eukaryotes. The dephosphorylation of cofilin enables its actin severing and depolymerizing activity and drives directional cell motility, thus providing a simple phosphoregulatory mechanism for actin reorganization. To date, two cofilin-specific phosphatases have been identified: Slingshot and Chronophin. These cofilin phosphatases are unrelated in sequence and regulatory properties, each potentially providing a unique mechanism for cofilin activation under varying biological circumstances.
Asunto(s)
Factores Despolimerizantes de la Actina/fisiología , Actinas/metabolismo , Fosfoproteínas Fosfatasas/fisiología , Animales , Humanos , Hidrolasas/metabolismo , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Fosforilación , Distribución TisularRESUMEN
Formation of the mitotic cleavage furrow is dependent upon both microtubules and activity of the small GTPase RhoA. GEF-H1 is a microtubule-regulated exchange factor that couples microtubule dynamics to RhoA activation. GEF-H1 localized to the mitotic apparatus in HeLa cells, particularly at the tips of cortical microtubules and the midbody, and perturbation of GEF-H1 function induced mitotic aberrations, including asymmetric furrowing, membrane blebbing, and impaired cytokinesis. The mitotic kinases Aurora A/B and Cdk1/Cyclin B phosphorylate GEF-H1, thereby inhibiting GEF-H1 catalytic activity. Dephosphorylation of GEF-H1 occurs just prior to cytokinesis, accompanied by GEF-H1-dependent GTP loading on RhoA. Using a live cell biosensor, we demonstrate distinct roles for GEF-H1 and Ect2 in regulating Rho activity in the cleavage furrow, with GEF-H1 catalyzing Rho activation in response to Ect2-dependent localization and initiation of cell cleavage. Our results identify a GEF-H1-dependent mechanism to modulate localized RhoA activation during cytokinesis under the control of mitotic kinases.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Citocinesis , Factores de Intercambio de Guanina Nucleótido/metabolismo , Mitosis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Aurora Quinasas , Membrana Celular/metabolismo , Supervivencia Celular , Regulación hacia Abajo/genética , Activación Enzimática , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Fosforilación , Unión Proteica , Transporte de Proteínas , Factores de Intercambio de Guanina Nucleótido Rho , Fracciones Subcelulares/metabolismoRESUMEN
Protrusion of the leading edge of migrating epithelial cells requires precise regulation of two actin filament (F-actin) networks, the lamellipodium and the lamella. Cofilin is a downstream target of Rho GTPase signaling that promotes F-actin cycling through its F-actin-nucleating, -severing, and -depolymerizing activity. However, its function in modulating lamellipodium and lamella dynamics, and the implications of these dynamics for protrusion efficiency, has been unclear. Using quantitative fluorescent speckle microscopy, immunofluorescence, and electron microscopy, we establish that the Rac1/Pak1/LIMK1 signaling pathway controls cofilin activity within the lamellipodium. Enhancement of cofilin activity accelerates F-actin turnover and retrograde flow, resulting in widening of the lamellipodium. This is accompanied by increased spatial overlap of the lamellipodium and lamella networks and reduced cell-edge protrusion efficiency. We propose that cofilin functions as a regulator of cell protrusion by modulating the spatial interaction of the lamellipodium and lamella in response to upstream signals.
Asunto(s)
Factores Despolimerizantes de la Actina/fisiología , Actinas/fisiología , Células Epiteliales/fisiología , Proteínas de Microfilamentos/fisiología , Seudópodos/fisiología , Quinasas p21 Activadas/fisiología , Línea Celular , Movimiento Celular , Técnica del Anticuerpo Fluorescente , Humanos , Quinasas Lim/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Cofilin is a key regulator of actin cytoskeletal dynamics whose activity is controlled by phosphorylation of a single serine residue. We report the biochemical isolation of chronophin (CIN), a unique cofilin-activating phosphatase of the haloacid dehalogenase (HAD) superfamily. CIN directly dephosphorylates cofilin with high specificity and colocalizes with cofilin in motile and dividing cells. Loss of CIN activity blocks phosphocycling of cofilin, stabilizes F-actin structures and causes massive cell division defects. Our findings identify a physiological phospho-serine protein substrate for a mammalian HAD-type phosphatase and demonstrate that CIN is an important novel regulator of cofilin-mediated actin reorganization.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Hidrolasas/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Factores Despolimerizantes de la Actina , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/aislamiento & purificación , Proteínas de Ciclo Celular/metabolismo , División Celular/fisiología , Movimiento Celular/fisiología , Citoplasma/metabolismo , ADN Complementario/análisis , ADN Complementario/genética , Regulación hacia Abajo/fisiología , Células HeLa , Humanos , Hidrolasas/química , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/aislamiento & purificación , Fosforilación , Seudópodos/metabolismo , Interferencia de ARN , Conejos , Serina/metabolismoRESUMEN
Cofilin-actin bundles (rods), which form in axons and dendrites of stressed neurons, lead to synaptic dysfunction and may mediate cognitive deficits in dementias. Rods form abundantly in the cytoplasm of non-neuronal cells in response to many treatments that induce rods in neurons. Rods in cell lysates are not stable in detergents or with added calcium. Rods induced by ATP-depletion and released from cells by mechanical lysis were first isolated from two cell lines expressing chimeric actin-depolymerizing factor (ADF)/cofilin fluorescent proteins by differential and equilibrium sedimentation on OptiPrep gradients and then from neuronal and non-neuronal cells expressing only endogenous proteins. Rods contain ADF/cofilin and actin in a 1:1 ratio. Isolated rods are stable in dithiothreitol, EGTA, Ca(2+), and ATP. Cofilin-GFP-containing rods are stable in 500 mM NaCl, whereas rods formed from endogenous proteins are significantly less stable in high salt. Proteomic analysis of rods formed from endogenous proteins identified other potential components whose presence in rods was examined by immunofluorescence staining of cells. Only actin and ADF/cofilin are in rods during all phases of their formation; furthermore, the rapid assembly of rods in vitro from these purified proteins at physiological concentration shows that they are the only proteins necessary for rod formation. Cytoplasmic rod formation is inhibited by cytochalasin D and jasplakinolide. Time lapse imaging of rod formation shows abundant small needle-shaped rods that coalesce over time. Rod filament lengths measured by ultrastructural tomography ranged from 22 to 1480 nm. These results suggest rods form by assembly of cofilin-actin subunits, followed by self-association of ADF/cofilin-saturated F-actin.
Asunto(s)
Factores Despolimerizantes de la Actina/química , Factores Despolimerizantes de la Actina/aislamiento & purificación , Actinas/química , Actinas/aislamiento & purificación , Destrina/química , Destrina/aislamiento & purificación , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Factores Despolimerizantes de la Actina/genética , Factores Despolimerizantes de la Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Destrina/genética , Destrina/metabolismo , Células HeLa , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Ratas , Porcinos , Xenopus laevisRESUMEN
Regulation of the actin cytoskeleton by microtubules is mediated by the Rho family GTPases. However, the molecular mechanisms that link microtubule dynamics to Rho GTPases have not, as yet, been identified. Here we show that the Rho guanine nucleotide exchange factor (GEF)-H1 is regulated by an interaction with microtubules. GEF-H1 mutants that are deficient in microtubule binding have higher activity levels than microtubule-bound forms. These mutants also induce Rho-dependent changes in cell morphology and actin organization. Furthermore, drug-induced microtubule depolymerization induces changes in cell morphology and gene expression that are similar to the changes induced by the expression of active forms of GEF-H1. Furthermore, these effects are inhibited by dominant-negative versions of GEF-H1. Thus, GEF-H1 links changes in microtubule integrity to Rho-dependent regulation of the actin cytoskeleton.
Asunto(s)
Actinas/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Microtúbulos/metabolismo , Factores de Intercambio de Guanina Nucleótido ras/química , Factores de Intercambio de Guanina Nucleótido ras/metabolismo , Animales , Células COS , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica , Genes Reporteros , Nucleótidos de Guanina , Células HeLa , Humanos , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Genéticos , Plásmidos/metabolismo , Pruebas de Precipitina , Estructura Terciaria de Proteína , Factores de Intercambio de Guanina Nucleótido Rho , Factores de Tiempo , Transfección , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Rac1 and Rac2, members of the small Rho GTPase family, play essential roles in coordinating directional migration and superoxide production during neutrophil responses to chemoattractants. Although earlier studies in Rac1 and Rac2 knockout mice have demonstrated unique roles for each Rac isoform in chemotaxis and NADPH oxidase activation, it is still unclear how human neutrophils use Rac1 and Rac2 to achieve their immunological responses to foreign agent stimulation. In the current study, we used TAT dominant-negative Rac1-T17N and Rac2-T17N fusion proteins to acutely alter the activity of Rac1 and Rac2 individually in human neutrophils. We demonstrate distinct activation kinetics and different roles for Rac1 and Rac2 in response to low vs high concentrations of fMLP. These observations were verified using neutrophils from mice in which Rac1 or Rac2 was genetically absent. Based on these results, we propose a model to explain how human neutrophils kill invading microbes while limiting oxidative damage to the adjacent surrounding healthy tissue through the differential activation of Rac1 and Rac2 in response to different concentrations of chemoattractant.
Asunto(s)
Quimiotaxis de Leucocito/inmunología , Neuropéptidos/metabolismo , Activación Neutrófila/inmunología , Proteínas de Unión al GTP rac/metabolismo , Animales , Quimiotaxis de Leucocito/genética , Relación Dosis-Respuesta Inmunológica , Productos del Gen tat/genética , Productos del Gen tat/fisiología , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neuropéptidos/antagonistas & inhibidores , Neuropéptidos/deficiencia , Activación Neutrófila/genética , Estrés Oxidativo/inmunología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Proteínas de Unión al GTP rac/antagonistas & inhibidores , Proteínas de Unión al GTP rac/deficiencia , Proteína de Unión al GTP rac1 , Proteína RCA2 de Unión a GTPRESUMEN
Leukocytes are key cellular components of innate immunity. These phagocytic cells respond to bacteria at sites of infection through chemotactic sensing and directed motility regulated by Rho GTPases. The development of sensitive probes of Rho GTPase dynamics has provided insights into the temporal and spatial aspects of GTPase regulation during chemotaxis and subsequent microbial phagocytosis. The resulting destruction of ingested bacteria by means of reactive oxygen species (ROS) depends on a Rac-regulated "molecular switch" that is modulated by antagonistic crosstalk involving Cdc42. Recent studies of leukocytes derived from Rac1- and Rac2-knockout mice have shown that these highly homologous GTPases have unique biological roles. An understanding of the biochemical basis for such distinct activities should provide novel insights into the molecular details of Rho GTPase function and regulation in innate immunity.
Asunto(s)
Inmunidad Innata , Proteínas de Unión al GTP rho/química , Proteínas de Unión al GTP rho/fisiología , Animales , Quimiotaxis , Humanos , Inflamación , Leucocitos/citología , Ratones , Ratones Noqueados , Modelos Biológicos , NADPH Oxidasas/metabolismo , Fagocitos/enzimología , Fagocitosis , Especies Reactivas de Oxígeno , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP cdc42/fisiología , Proteínas de Unión al GTP rac/metabolismo , Proteína RCA2 de Unión a GTPRESUMEN
The GDP dissociation inhibitors (GDIs) are pivotal regulators of Rho GTPase function. GDIs control the access of Rho GTPases to regulatory guanine nucleotide exchange factors and GTPase-activating proteins, to effector targets and to membranes where such effectors reside. We discuss here our current understanding of how Rho GTPase-GDI complexes are regulated by various proteins, lipids and enzymes that exert GDI displacement activity. We propose that phosphorylation mediated by diverse kinases might provide a means of controlling and coordinating Rho GTPase activation.
Asunto(s)
Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/fisiología , Proteínas Quinasas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Activadores de Enzimas/farmacología , Factores de Intercambio de Guanina Nucleótido/farmacología , Humanos , Lípidos/farmacología , Fosforilación , Proteínas/fisiología , Proteínas Supresoras de Tumor , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Proteínas de Unión al GTP rho/química , Proteínas de Unión al GTP rho/fisiología , Inhibidor gamma de Disociación del Nucleótido Guanina rho , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
Anthrax lethal factor (LF), secreted by Bacillus anthracis, interacts with protective antigen to form a bipartite toxin (lethal toxin [LT]) that exerts pleiotropic biological effects resulting in subversion of the innate immune response. Although the mitogen-activated protein kinase kinases (MKKs) are the major intracellular protein targets of LF, the pathology induced by LT is not well understood. The statin family of HMG-coenzyme A reductase inhibitors have potent anti-inflammatory effects independent of their cholesterol-lowering properties, which have been attributed to modulation of Rho family GTPase activity. The Rho GTPases regulate vesicular trafficking, cytoskeletal dynamics, and cell survival and proliferation. We hypothesized that disruption of Rho GTPase function by statins might alter LT action. We show here that statins delay LT-induced death and MKK cleavage in RAW macrophages and that statin-mediated effects on LT action are attributable to disruption of Rho GTPases. The Rho GTPase-inactivating toxin, toxin B, did not significantly affect LT binding or internalization, suggesting that the Rho GTPases regulate trafficking and/or localization of LT once internalized. The use of drugs capable of inhibiting Rho GTPase activity, such as statins, may provide a means to attenuate intoxication during B. anthracis infection.
Asunto(s)
Antígenos Bacterianos/toxicidad , Toxinas Bacterianas/toxicidad , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Proteínas de Unión al GTP rho/metabolismo , Animales , Macrófagos/efectos de los fármacos , RatonesRESUMEN
The signal transduction pathways involved in neuronal death are not well understood. Neuroglobin (Ngb), a recently discovered vertebrate globin expressed predominantly in the brain, shows increased expression in neurons in response to oxygen deprivation and protects neurons from ischemic and hypoxic death. The mechanism of this neuroprotection is unclear. We examined the surface distribution of raft membrane microdomains in cortical neuron cultures during hypoxia using the raft marker cholera toxin B (CTx-B) subunit. Mechanistically, we demonstrate that hypoxia induces rapid polarization of somal membranes and aggregation of microdomains with the subjacent mitochondrial network. This signaling complex is formed well before neurons commit to die, consistent with an early role in death signal transduction. Neurons from Ngb-overexpressing transgenic (Ngb-Tg) mice do not undergo microdomain polarization or mitochondrial aggregation in response to, and are resistant to death from hypoxia. We link the protective actions of Ngb to inhibition of Pak1 kinase activity and Rac1-GDP-dissociation inhibitor disassociation, and inhibition of actin assembly and death-signaling module polarization.
Asunto(s)
Globinas/fisiología , Hipoxia/metabolismo , Proteínas del Tejido Nervioso/fisiología , Neuronas/patología , Transducción de Señal , Actinas/antagonistas & inhibidores , Animales , Corteza Cerebral , Microdominios de Membrana/metabolismo , Microdominios de Membrana/ultraestructura , Ratones , Ratones Transgénicos , Neuroglobina , Quinasas p21 Activadas/antagonistas & inhibidoresRESUMEN
Actin in migrating cells is regulated by Rho GTPases. However, Rho proteins might also affect microtubules (MTs). Here, we used time-lapse microscopy of PtK1 cells to examine MT regulation downstream of Rac1. In these cells, "pioneer" MTs growing into leading-edge protrusions exhibited a decreased catastrophe frequency and an increased time in growth as compared with MTs further from the leading edge. Constitutively active Rac1(Q61L) promoted pioneer behavior in most MTs, whereas dominant-negative Rac1(T17N) eliminated pioneer MTs, indicating that Rac1 is a regulator of MT dynamics in vivo. Rac1(Q61L) also enhanced MT turnover through stimulation of MT retrograde flow and breakage. Inhibition of p21-activated kinases (Paks), downstream effectors of Rac1, inhibited Rac1(Q61L)-induced MT growth and retrograde flow. In addition, Rac1(Q61L) promoted lamellipodial actin polymerization and Pak-dependent retrograde flow. Together, these results indicate coordinated regulation of the two cytoskeletal systems in the leading edge of migrating cells.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Movimiento Celular/fisiología , Polaridad Celular/fisiología , Células Eucariotas/metabolismo , Microtúbulos/metabolismo , Seudópodos/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Microscopía por Video , Mutación/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Quinasas p21 Activadas , Proteína de Unión al GTP rac1/genéticaRESUMEN
p21-activated kinases are important signaling kinases for which no specific chemical inhibitors are known. In this issue of Chemistry & Biology, Deacon et al. target allosteric transitions undergone during PAK activation to identify a selective inhibitor (Deacon et al., 2008).
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Quinasas p21 Activadas/antagonistas & inhibidores , Quinasas p21 Activadas/metabolismo , Animales , Disulfuros/química , Disulfuros/metabolismo , Disulfuros/farmacología , Activación Enzimática/efectos de los fármacos , Homeostasis/efectos de los fármacos , Ratones , Conformación Proteica/efectos de los fármacos , Especificidad por SustratoRESUMEN
Rho GTPases (Rac, Rho, and Cdc42) play important roles in regulating cell function through their ability to coordinate the actin cytoskeleton, modulate the formation of signaling reactive oxidant species, and control gene transcription. Activation of Rho GTPase signaling pathways requires the regulated release of Rho GTPases from RhoGDI complexes, followed by their reuptake after membrane cycling. We show here that Src kinase binds and phosphorylates RhoGDI both in vitro and in vivo at Tyr156. Analysis of Rho GTPase-RhoGDI complexes using in vitro assays of complexation and in vivo by coimmunoprecipitation analysis indicates that Src-mediated phosphorylation of Tyr156 causes a dramatic decrease in the ability of RhoGDI to form a complex with RhoA, Rac1, or Cdc42. Phosphomimetic mutation of Tyr156-->Glu results in the constitutive association of RhoGDI(Y156E) with the plasma membrane and/or associated cortical actin. Substantial cortical localization of tyrosine-phosphorylated RhoGDI is also observed in fibroblasts expressing active Src, where it is most evident in podosomes and regions of membrane ruffling. Expression of membrane-localized RhoGDI(Y156E) mutant is associated with enhanced cell spreading and membrane ruffling. These results suggest that Src-mediated RhoGDI phosphorylation is a novel physiological mechanism for regulating Rho GTPase cytosol membrane-cycling and activity.
Asunto(s)
Estructuras de la Membrana Celular/metabolismo , Citosol/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Familia-src Quinasas/metabolismo , Animales , Línea Celular Transformada , Fibroblastos/citología , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Fosforilación , Fosfotirosina/metabolismo , Unión Proteica , Transporte de Proteínas , Inhibidor alfa de Disociación del Nucleótido Guanina rho , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
In addition to their role in bacterial killing by leukocytes, reactive oxygen species (ROS) have been increasingly recognized as important components of signaling and host defense in other cell types. The formation of ROS in both phagocytic- and non-phagocytic cells involves membrane-localized NADPH oxidases (Noxs). Nox proteins show structural homology to the cytochrome b(558) of leukocytes but, until recently, their regulation has been poorly understood. Here, we describe our current understanding of Nox function, and discuss emerging paradigms for regulation of Nox activity by Rac GTPase and/or other cytosolic components.
Asunto(s)
Leucocitos/enzimología , NADPH Oxidasas/metabolismo , Animales , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Leucocitos/inmunología , Modelos Moleculares , NADPH Oxidasas/genética , Fagocitos/metabolismo , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Acute lung injury, sepsis, lung inflammation, and ventilator-induced lung injury are life-threatening conditions associated with lung vascular barrier dysfunction, which may lead to pulmonary edema. Increased levels of atrial natriuretic peptide (ANP) in lung circulation reported in these pathologies suggest its potential role in the modulation of lung injury. Besides well recognized physiological effects on vascular tone, plasma volume, and renal function, ANP may exhibit protective effects in models of lung vascular endothelial cell (EC) barrier dysfunction. However, the molecular mechanisms of ANP protective effects are not well understood. The recently described cAMP-dependent guanine nucleotide exchange factor (GEF) Epac activates small GTPase Rap1, which results in activation of small GTPase Rac-specific GEFs Tiam1 and Vav2 and Rac-mediated EC barrier protective responses. Our results show that ANP stimulated protein kinase A and the Epac/Rap1/Tiam/Vav/Rac cascade dramatically attenuated thrombin-induced pulmonary EC permeability and the disruption of EC monolayer integrity. Using pharmacological and molecular activation and inhibition of cAMP-and cGMP-dependent protein kinases (PKA and PKG), Epac, Rap1, Tiam1, Vav2, and Rac we linked ANP-mediated protective effects to the activation of Epac/Rap and PKA signaling cascades, which dramatically inhibited the Rho pathway of thrombin-induced EC hyper-permeability. These results suggest a novel mechanism of ANP protective effects against agonist-induced pulmonary EC barrier dysfunction via inhibition of Rho signaling by Epac/Rap1-Rac and PKA signaling cascades.
Asunto(s)
Factor Natriurético Atrial/farmacología , Barrera Alveolocapilar/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Endoteliales/enzimología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Pulmón/enzimología , Proteínas de Unión al GTP rac/metabolismo , Actinas/metabolismo , Barrera Alveolocapilar/efectos de los fármacos , Barrera Alveolocapilar/patología , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , GMP Cíclico/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Pulmón/citología , Pulmón/efectos de los fármacos , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Fibras de Estrés/efectos de los fármacos , Fibras de Estrés/metabolismo , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Trombina/farmacología , Proteínas de Unión al GTP rac/antagonistas & inhibidores , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Leukocyte chemoattractants regulate many leukocyte functions, including leukocyte chemotaxis, via the Rho family of small GTPases that include RhoA, Cdc42, and Rac. Previous work has revealed mechanisms by which chemoattractants regulate RhoA and Cdc42 in mouse neutrophils, but the mechanisms for regulation of Rac remain unclear even though Rac is important for neutrophil functions. Here, we characterized P-Rex1, a Gbetagamma and PIP(3)-regulated guanine nucleotide exchange factor that was initially identified as a Rac activator in response to chemoattractants, for its roles in the regulation of Rac activity and neutrophil functions. We generated a mouse line in which the P-Rex1 gene is disrupted and found that P-Rex1 deficiency did not significantly affect Rac1 activation but diminished Rac2 activation in response to a chemoattractant fMLP in mouse neutrophils. This preference for Rac2 may partially result from the apparent higher affinity of P-Rex1 for Rac2 than for Rac1 because P-Rex1 was more readily immunoprecipitated with Rac2(S17N) than Rac1(S17N). In addition, P-Rex1 deficiency significantly attenuated fMLP-induced F actin formation and superoxide production without affecting LPS- or PMA-induced production. Furthermore, P-Rex1 deficiency caused a chemotactic defect that is primarily attributed to a reduction in the migration rate rather than directionality.
Asunto(s)
Quimiotaxis/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Neutrófilos/metabolismo , Neutrófilos/fisiología , Actinas/metabolismo , Animales , Caseínas , Línea Celular , Clonación Molecular , Activación Enzimática/fisiología , Marcación de Gen , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Inmunoprecipitación , Ratones , Ratones Noqueados , N-Formilmetionina Leucil-Fenilalanina , Neuropéptidos/metabolismo , Transporte de Proteínas/fisiología , Superóxidos/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1 , Proteína RCA2 de Unión a GTPRESUMEN
PI 3,4,5-trisphosphate [PI(3,4,5)P3; PIP3]-dependent Rac exchanger 1 (P-Rex1) is a Rac-specific guanine nucleotide exchange factor abundant in neutrophils and myeloid cells. As a selective catalyst for Rac2 activation, P-Rex1 serves as an important regulator of human neutrophil NADPH oxidase activity and chemotaxis in response to a variety of extracellular stimuli. The exchange activity of P-Rex1 is synergistically activated by the binding of PIP3 and betagamma subunits of heterotrimeric G proteins in vitro, suggesting that the association of P-Rex1 with membranes is a prerequisite for cellular activation. However, the spatial regulation of endogenous P-Rex1 has not been well defined, particularly in human neutrophils activated through G protein-coupled receptors. Upon stimulation of neutrophil chemoattractant receptors, we observed that P-Rex1 translocated from cytoplasm to the leading edge of polarized cells in a G protein betagamma subunit- and PIP3-dependent manner, where it colocalized with F-actin and its substrate, Rac2. Redistribution of P-Rex1 to the leading edge was also dependent on tyrosine kinase activity and was modulated by cell adhesion. Furthermore, we observed that activation of cAMP-dependent protein kinase A (PKA), which phosphorylates and inactivates P-Rex1, inhibited its translocation. Our data indicate that endogenous P-Rex1 translocates to areas of Rac2 and cytoskeletal activation at the leading edge in response to chemoattractant stimuli in human neutrophils and that this translocation can be negatively modulated by activation of PKA and by cell adhesion.
Asunto(s)
Factores Quimiotácticos/farmacología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Neutrófilos/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rac/metabolismo , Adhesión Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de Unión al GTP/metabolismo , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas/metabolismo , Transporte de Proteínas , Proteína RCA2 de Unión a GTPRESUMEN
The functionality of the actin cytoskeleton depends on a dynamic equilibrium between filamentous and monomeric actin. Proteins of the ADF/cofilin family are essential for the high rates of actin filament turnover observed in motile cells through regulation of actin polymerization/depolymerization cycles. Rho GTPases act through p21-activated kinase-1 (Pak-1) and Rho kinase to inhibit cofilin activity via the LIM kinase (LIMK)-mediated phosphorylation of cofilin on Ser3. We report the identification of 14-3-3zeta as a novel phosphocofilin binding protein involved in the maintenance of the cellular phosphocofilin pool. A Ser3 phosphocofilin binding protein was purified from bovine brain and was identified as 14-3-3zeta by mass spectrometry. The phosphorylation-dependent interaction between cofilin and 14-3-3zeta was confirmed in pulldown and coimmunoprecipitation experiments. Both Ser3 phosphorylation and a 14-3-3 recognition motif in cofilin are necessary for 14-3-3 binding. The expression of 14-3-3zeta increases phosphocofilin levels, and the coexpression of 14-3-3zeta with LIMK further elevates phosphocofilin levels and potentiates LIMK-dependent effects on the actin cytoskeleton. This potentiation of cofilin action appears to be a result of the protection of phosphocofilin from phosphatase-mediated dephosphorylation at Ser3 by bound 14-3-3zeta. Taken together, these results suggest that 14-3-3zeta proteins may play a dynamic role in the regulation of cellular actin structures through the maintenance of phosphocofilin levels.