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1.
Int J Mol Sci ; 22(5)2021 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-33801503

RESUMEN

Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor development and metastasis. This led to the development of GGTase-I inhibitors as anti-cancer drugs blocking the protein function and membrane association of e.g., Rap subfamilies that are involved in cell differentiation and cell growth. In the present study, we developed a new NanoBiT assay to monitor the interaction of human GGTase-I and its substrate Rap1B. Different Rap1B prenylation-deficient mutants (C181G, C181S, and ΔCQLL) were designed and investigated for their interaction with GGTase-I. While the Rap1B mutants C181G and C181S still exhibited interaction with human GGTase-I, mutant ΔCQLL, lacking the entire CAAX motif (defined by a cysteine residue, two aliphatic residues, and the C-terminal residue), showed reduced interaction. Moreover, a specific, peptidomimetic and competitive CAAX inhibitor was able to block the interaction of Rap1B with GGTase-I. Furthermore, activation of both Gαs-coupled human adenosine receptors, A2A (A2AAR) and A2B (A2BAR), increased the interaction between GGTase-I and Rap1B, probably representing a way to modulate prenylation and function of Rap1B. Thus, A2AAR and A2BAR antagonists might be promising candidates for therapeutic intervention for different types of cancer that overexpress Rap1B. Finally, the NanoBiT assay provides a tool to investigate the pharmacology of GGTase-I inhibitors.


Asunto(s)
Transferasas Alquil y Aril/metabolismo , Inhibidores Enzimáticos/farmacología , Fragmentos de Péptidos/farmacología , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Proteínas de Unión al GTP rap/metabolismo , Antagonistas del Receptor de Adenosina A2/farmacología , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Humanos , Prenilación de Proteína , Especificidad por Sustrato , Xantinas/farmacología , Proteínas de Unión al GTP rap/química , Proteínas de Unión al GTP rap/genética
2.
Elife ; 72018 07 31.
Artículo en Inglés | MEDLINE | ID: mdl-30063210

RESUMEN

During development, neurons form synapses with their fate-determined targets. While we begin to elucidate the mechanisms by which extracellular ligand-receptor interactions enhance synapse specificity by inhibiting synaptogenesis, our knowledge about their intracellular mechanisms remains limited. Here we show that Rap2 GTPase (rap-2) and its effector, TNIK (mig-15), act genetically downstream of Plexin (plx-1) to restrict presynaptic assembly and to form tiled synaptic innervation in C. elegans. Both constitutively GTP- and GDP-forms of rap-2 mutants exhibit synaptic tiling defects as plx-1 mutants, suggesting that cycling of the RAP-2 nucleotide state is critical for synapse inhibition. Consistently, PLX-1 suppresses local RAP-2 activity. Excessive ectopic synapse formation in mig-15 mutants causes a severe synaptic tiling defect. Conversely, overexpression of mig-15 strongly inhibited synapse formation, suggesting that mig-15 is a negative regulator of synapse formation. These results reveal that subcellular regulation of small GTPase activity by Plexin shapes proper synapse patterning in vivo.


Asunto(s)
Proteínas de Caenorhabditis elegans/química , Proteínas del Tejido Nervioso/química , Proteínas Serina-Treonina Quinasas/química , Receptores de Superficie Celular/química , Proteínas de Unión al GTP rap/química , Animales , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Guanosina Difosfato/química , Guanosina Trifosfato/química , Mutación , Proteínas del Tejido Nervioso/genética , Neurogénesis/genética , Neuronas/química , Proteínas Serina-Treonina Quinasas/genética , Receptores de Superficie Celular/genética , Transducción de Señal/genética , Sinapsis/química , Sinapsis/genética , Sinapsis/patología , Proteínas de Unión al GTP rap/genética
3.
J Biol Chem ; 293(20): 7659-7673, 2018 05 18.
Artículo en Inglés | MEDLINE | ID: mdl-29618512

RESUMEN

Rap1 proteins are members of the Ras subfamily of small GTPases involved in many biological responses, including adhesion, cell proliferation, and differentiation. Like all small GTPases, they work as molecular allosteric units that are active in signaling only when associated with the proper membrane compartment. Prenylation, occurring in the cytosol, is an enzymatic posttranslational event that anchors small GTPases at the membrane, and prenyl-binding proteins are needed to mask the cytoplasm-exposed lipid during transit to the target membrane. However, several of these proteins still await discovery. In this study, we report that cyclase-associated protein 1 (CAP1) binds Rap1. We found that this binding is GTP-independent, does not involve Rap1's effector domain, and is fully contained in its C-terminal hypervariable region (HVR). Furthermore, Rap1 prenylation was required for high-affinity interactions with CAP1 in a geranylgeranyl-specific manner. The prenyl binding specifically involved CAP1's C-terminal hydrophobic ß-sheet domain. We present a combination of experimental and computational approaches, yielding a model whereby the high-affinity binding between Rap1 and CAP1 involves electrostatic and nonpolar side-chain interactions between Rap1's HVR residues, lipid, and CAP1 ß-sheet domain. The binding was stabilized by the lipid insertion into the ß-solenoid whose interior was occupied by nonpolar side chains. This model was reminiscent of the recently solved structure of the PDEδ-K-Ras complex; accordingly, disruptors of this complex, e.g. deltarasin, blocked the Rap1-CAP1 interaction. These findings indicate that CAP1 is a geranylgeranyl-binding partner of Rap1.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Diterpenos/metabolismo , Prenilación de Proteína , Células Epiteliales Tiroideas/metabolismo , Proteínas de Unión al GTP rap/metabolismo , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Diterpenos/química , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación Proteica , Ratas , Proteínas de Unión al GTP rap/química , Proteínas de Unión al GTP rap/genética
4.
Nat Commun ; 8(1): 1744, 2017 11 23.
Artículo en Inglés | MEDLINE | ID: mdl-29170462

RESUMEN

Activation of transmembrane receptor integrin by talin is essential for inducing cell adhesion. However, the pathway that recruits talin to the membrane, which critically controls talin's action, remains elusive. Membrane-anchored mammalian small GTPase Rap1 is known to bind talin-F0 domain but the binding was shown to be weak and thus hardly studied. Here we show structurally that talin-F0 binds to human Rap1b like canonical Rap1 effectors despite little sequence homology, and disruption of the binding strongly impairs integrin activation, cell adhesion, and cell spreading. Furthermore, while being weak in conventional binary binding conditions, the Rap1b/talin interaction becomes strong upon attachment of activated Rap1b to vesicular membranes that mimic the agonist-induced microenvironment. These data identify a crucial Rap1-mediated membrane-targeting mechanism for talin to activate integrin. They further broadly caution the analyses of weak protein-protein interactions that may be pivotal for function but neglected in the absence of specific cellular microenvironments.


Asunto(s)
Integrinas/metabolismo , Talina/química , Talina/metabolismo , Proteínas de Unión al GTP rap/química , Proteínas de Unión al GTP rap/metabolismo , Secuencia de Aminoácidos , Animales , Adhesión Celular/fisiología , Línea Celular , Membrana Celular/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Ratones , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Talina/genética
5.
J Cell Sci ; 130(18): 3158-3172, 2017 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-28778987

RESUMEN

Macropinocytosis involves the uptake of large volumes of fluid, which is regulated by various small GTPases. The Dictyostelium discoideum protein GflB is a guanine nucleotide exchange factor (GEF) of Rap1, and is involved in chemotaxis. Here, we studied the role of GflB in macropinocytosis, phagocytosis and cytokinesis. In plate culture of vegetative cells, compared with the parental strain AX2, gflB-knockout (KO) cells were flatter and more polarized, whereas GflB-overproducing cells were rounder. The gflB-KO cells exhibited impaired crown formation and retraction, particularly retraction, resulting in more crowns (macropinocytic cups) per cell and longer crown lifetimes. Accordingly, gflB-KO cells showed defects in macropinocytosis and also in phagocytosis and cytokinesis. F-actin levels were elevated in gflB-KO cells. GflB localized to the actin cortex most prominently at crowns and phagocytic cups. The villin headpiece domain (VHP)-like N-terminal domain of GflB directly interacted with F-actin in vitro Furthermore, a domain enriched in basic amino acids interacted with specific membrane cortex structures such as the cleavage furrow. In conclusion, GflB acts as a key local regulator of actin-driven membrane protrusion possibly by modulating Rap1 signaling pathways.


Asunto(s)
Dictyostelium/citología , Dictyostelium/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Pinocitosis , Proteínas Protozoarias/metabolismo , Proteínas de Unión al GTP rap/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Movimiento Celular , Forma de la Célula , Extensiones de la Superficie Celular/metabolismo , Citocinesis , Técnicas de Inactivación de Genes , Proteínas Fluorescentes Verdes/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Morfogénesis , Fagocitosis , Unión Proteica , Dominios Proteicos , Proteínas Protozoarias/química , Fracciones Subcelulares/metabolismo , Proteínas de Unión al GTP rap/química
6.
J Mol Biol ; 428(24 Pt B): 4929-4945, 2016 12 04.
Artículo en Inglés | MEDLINE | ID: mdl-27760305

RESUMEN

Two isoforms of the small GTPase Rap1, Rap1A and Rap1B, participate in cell adhesion; Rap1A promotes steady state adhesion, while Rap1B regulates dynamic changes in cell adhesion. These events depend on the prenylation of Rap1, which promotes its membrane localization. Here, we identify previously unsuspected differences in the regulation of prenylation of Rap1A versus Rap1B, due in part to their different phosphorylation-dependent interactions with the chaperone protein SmgGDS-607. Previous studies indicate that the activation of Gαs protein-coupled receptors (GPCRs) phosphorylates S-179 and S-180 in the polybasic region (PBR) of Rap1B, which inhibits Rap1B binding to SmgGDS-607 and diminishes Rap1B prenylation and membrane localization. In this study, we investigate how phosphorylation in the PBR of multiple small GTPases, including K-Ras4B, RhoA, Rap1A, and Rap1B, affects their binding to SmgGDS, with emphasis on differences between Rap1A and Rap1B. We identify the amino acids in SmgGDS-607 necessary for binding of Rap1A and Rap1B, and present homology models examining the binding between Rap1A or Rap1B and SmgGDS-607. Unlike Rap1B, phosphorylation in the PBR of Rap1A does not detectably inhibit its prenylation or its binding to SmgGDS-607. Activation of GPCRs suppresses Rap1A prenylation, but unlike this effect on Rap1B, the GPCR-mediated suppression of Rap1A prenylation can occur independently of Rap1A phosphorylation and does not detectably diminish Rap1A membrane localization. These data demonstrate unexpected evolutionarily conserved differences in the ability of GPCRs to regulate the prenylation of Rap1B compared to Rap1A.


Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Prenilación , Procesamiento Proteico-Postraduccional , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Secuencia de Aminoácidos , Línea Celular , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Alineación de Secuencia , Proteínas de Unión al GTP rap/química , Proteínas de Unión al GTP rap1/química
7.
Tumour Biol ; 37(6): 7085-93, 2016 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-27012552

RESUMEN

Rap2B GTPase, a member of Ras-related protein superfamily, was first discovered from a platelet cDNA library in the early 1990s. Since then, it has been reported to play an important role in regulating cellular processes including cytoskeletal organization, cell growth, and proliferation. It can be stimulated and suppressed by a wide range of external and internal inducers, circulating between GTP-bound active state and GDP-bound inactive state. Increasing focus on Ras signaling pathway reveals critical effects of Rap2B on tumorigenesis. In particular, Rap2B behaves in a p53-dependent manner in regulation of apoptosis and migration. Apart from being an oncogenic activator, Rap2B has been found to participate in many other physiological events via diverse downstream effectors. In this review, we present recent studies on the structure, regulation, and multiple biological functions of Rap2B, shedding light on its potential status in treatment of cancer as well as other diseases.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias/patología , Proteínas de Unión al GTP rap/química , Proteínas de Unión al GTP rap/metabolismo , Animales , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rap/genética
8.
Biochem Biophys Res Commun ; 462(1): 46-51, 2015 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-25935485

RESUMEN

Rap1B is a small GTPase involved in the regulation of numerous cellular processes including synaptic plasticity, one of the bases of memory. Like other members of the Ras family, the active GTP-bound form of Rap1B can bind to a large number of effector proteins and so transmit signals to downstream components of the signaling pathways. The structure of Rap1B bound only to a nucleotide has yet to be solved, but might help reveal an inactive conformation that can be stabilized by a small molecule drug. Unlike other Ras family proteins such as H-Ras and Rap2A, Rap1B crystallizes in an intermediate state when bound to a non-hydrolyzable GTP analog. Comparison with H-Ras and Rap2A reveals conservative mutations relative to Rap1B, distant from the bound nucleotide, which control how readily the protein may adopt the fully activated form in the presence of GTP. High resolution crystallographic structures of mutant proteins show how these changes may influence the hydrogen bonding patterns of the key switch residues.


Asunto(s)
Mutación , Estructura Terciaria de Proteína , Proteínas de Unión al GTP rap/química , Proteínas de Unión al GTP rap/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Cristalografía por Rayos X , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Ratas , Homología de Secuencia de Aminoácido , Proteínas de Unión al GTP rap/metabolismo
9.
Elife ; 2: e01279, 2013 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-24137545

RESUMEN

Plexins are cell surface receptors that bind semaphorins and transduce signals for regulating neuronal axon guidance and other processes. Plexin signaling depends on their cytoplasmic GTPase activating protein (GAP) domain, which specifically inactivates the Ras homolog Rap through an ill-defined non-canonical catalytic mechanism. The plexin GAP is activated by semaphorin-induced dimerization, the structural basis for which remained unknown. Here we present the crystal structures of the active dimer of zebrafish PlexinC1 cytoplasmic region in the apo state and in complex with Rap. The structures show that the dimerization induces a large-scale conformational change in plexin, which opens the GAP active site to allow Rap binding. Plexin stabilizes the switch II region of Rap in an unprecedented conformation, bringing Gln63 in Rap into the active site for catalyzing GTP hydrolysis. The structures also explain the unique Rap-specificity of plexins. Mutational analyses support that these mechanisms underlie plexin activation and signaling. DOI:http://dx.doi.org/10.7554/eLife.01279.001.


Asunto(s)
Moléculas de Adhesión Celular/química , Guanosina Trifosfato/química , Proteínas del Tejido Nervioso/química , Proteínas de Pez Cebra/química , Pez Cebra/genética , Proteínas de Unión al GTP rap/química , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Cristalografía por Rayos X , Regulación de la Expresión Génica , Guanosina Trifosfato/metabolismo , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/metabolismo
10.
J Phys Chem B ; 117(39): 11473-89, 2013 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-24041016

RESUMEN

Continuum electrostatics methods are commonly used to calculate electrostatic potentials in proteins and at protein-protein interfaces to aid many types of biophysical studies. Despite their ubiquity throughout the biophysical literature, these calculations are difficult to test against experimental data to determine their accuracy and validity. To address this, we have calculated the Boltzmann-weighted electrostatic field at the midpoint of a nitrile bond placed at a variety of locations on the surface of the protein RalGDS, both in its monomeric form as well as when docked to four different constructs of the protein Rap, and compared the computation results to vibrational absorption energy measurements of the nitrile oscillator. This was done by generating a statistical ensemble of protein structures using enhanced molecular dynamics sampling with the Amber03 force field, followed by solving the linear Poisson-Boltzmann equation for each structure using the Applied Poisson-Boltzmann Solver (APBS) software package. Using a two-stage focusing strategy, we examined numerous second stage box dimensions, grid point densities, box locations, and compared the numerical result to the result obtained from the sum of the numeric reaction field and the analytic Coulomb field. It was found that the reaction field method yielded higher correlation with experiment for the absolute calculation of fields, while the numeric solutions yielded higher correlation with experiment for the relative field calculations. Finer grid spacing typically improved the calculation, although this effect was less pronounced in the reaction field method. These sorts of calculations were also very sensitive to the box location, particularly for the numeric calculations of absolute fields using a 10(3) Å(3) box.


Asunto(s)
Modelos Moleculares , Electricidad Estática , Factor de Intercambio de Guanina Nucleótido ral/química , Proteínas de Unión al GTP rap/química , Algoritmos , Modelos Lineales , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Nitrilos/química , Probabilidad , Conformación Proteica , Programas Informáticos , Tiocianatos/química , Vibración
11.
J Biol Chem ; 287(11): 8013-20, 2012 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-22267739

RESUMEN

The principle virulence factors in Clostridium difficile pathogenesis are TcdA and TcdB, homologous glucosyltransferases capable of inactivating small GTPases within the host cell. We present crystal structures of the TcdA glucosyltransferase domain in the presence and absence of the co-substrate UDP-glucose. Although the enzymatic core is similar to that of TcdB, the proposed GTPase-binding surface differs significantly. We show that TcdA is comparable with TcdB in its modification of Rho family substrates and that, unlike TcdB, TcdA is also capable of modifying Rap family GTPases both in vitro and in cells. The glucosyltransferase activities of both toxins are reduced in the context of the holotoxin but can be restored with autoproteolytic activation and glucosyltransferase domain release. These studies highlight the importance of cellular activation in determining the array of substrates available to the toxins once delivered into the cell.


Asunto(s)
Toxinas Bacterianas/química , Clostridioides difficile/enzimología , Enterotoxinas/química , Glucosiltransferasas/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Enterotoxinas/metabolismo , Activación Enzimática/fisiología , Glucosiltransferasas/metabolismo , Estructura Terciaria de Proteína , Uridina Difosfato Glucosa/química , Uridina Difosfato Glucosa/metabolismo , Proteínas de Unión al GTP rap/química , Proteínas de Unión al GTP rap/metabolismo
12.
Trends Cell Biol ; 21(10): 615-23, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21820312

RESUMEN

Signaling by the small G-protein Rap is under tight regulation by its GEFs and GAPs. These are multi-domain proteins that are themselves controlled by distinct upstream pathways, and thus couple different extra- and intracellular cues to Rap. The individual RapGEFs and RapGAPs are, in addition, targeted to specific cellular locations by numerous anchoring mechanisms and, consequently, may control different pools of Rap. Here, we review the various activating signals and targeting mechanisms of these proteins and discuss their contribution to the spatiotemporal regulation and biological functions of the Rap proteins.


Asunto(s)
AMP Cíclico/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Modelos Moleculares , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP rap/metabolismo , AMP Cíclico/química , Proteínas Activadoras de GTPasa/química , Factores de Intercambio de Guanina Nucleótido/química , Proteínas de Unión al GTP Monoméricas/química , Transducción de Señal , Proteínas de Unión al GTP rap/química , Proteínas de Unión al GTP rap1/química , Proteínas de Unión al GTP rap1/metabolismo
13.
FEBS Lett ; 585(12): 1707-10, 2011 Jun 23.
Artículo en Inglés | MEDLINE | ID: mdl-21570977

RESUMEN

RUN domain is present in several proteins related to the functions of Rap and Rab family GTPases. Accumulating evidence supports the hypothesis that RUN domain-containing proteins act as a component of vesicle traffic and might be responsible for an interaction with a filamentous network linked to actin cytoskeleton or microtubules. That is to say, on one hand, RUN domains associate with Rab or Rap family proteins, on the other hand, they also might interact with motor proteins such as kinesin or myosin via intervention molecules. In this review, we summarize the background and current status of RUN domain research with an emphasis on the interaction between RUN domain and motor proteins with respect to the vesicle traffic on filamentous network.


Asunto(s)
Proteínas Motoras Moleculares/metabolismo , Proteínas de Unión al GTP rab/fisiología , Proteínas de Unión al GTP rap/fisiología , Transporte Biológico , Estructura Terciaria de Proteína , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rap/química
14.
Thromb Haemost ; 105(3): 479-86, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21136013

RESUMEN

We have recently shown that ADP-induced activation of protein kinase C (PKC) requires the co-stimulation of both P2Y1 and P2Y12 receptors. In this work, we show that inhibition of ADP-mediated phosphorylation of pleckstrin, the main PKC substrate, caused by antagonists of the P2Y12 receptor can be reversed by stimulation of the α2-adrenergic receptor by epinephrine. However, we also observed that addition of epinephrine alone caused a marked phosphorylation of pleckstrin. This effect occurred in the absence of Gq stimulation, as it was not associated to intracellular Ca2+ release. Epinephrine-induced pleckstrin phosphorylation was time- and dose-dependent, and was inhibited by the α2-adrenergic antagonist yohimbin. Phosphorylation of pleckstrin did not occur when platelet stimulation with epinephrine was performed in the presence of the ADP scavenger apyrase, and was suppressed by antagonists of both P2Y1 and P2Y12 ADP receptors. Importantly, no release of dense granules was measured in epinephrine-treated platelets. Addition of epinephrine to platelets was also able to stimulate Rap1b activation. Similarly to pleckstrin phosphorylation, however, this effect was prevented in the presence of apyrase or upon pharmacologic blockade of either P2Y1 or P2Y12 receptors. These results indicate that sub-threshold amounts of ADP in the medium are essential to allow epinephrine stimulation of α2-adrenergic receptor to elicit platelet responses, and reveal a novel synergism among strong stimulation of Gz and sub-threshold stimulation of both Gq and Gi, able to dissociate PKC activation from intracellular Ca2+ mobilisation.


Asunto(s)
Epinefrina/química , Receptores Purinérgicos P2Y12/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Proteínas Sanguíneas/química , Calcio/química , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Humanos , Fosfoproteínas/química , Fosforilación , Transducción de Señal , Yohimbina/farmacología , Proteínas de Unión al GTP rap/química
15.
EMBO J ; 29(7): 1205-14, 2010 Apr 07.
Artículo en Inglés | MEDLINE | ID: mdl-20186121

RESUMEN

The molecular mechanism by which dual-specificity RasGAPs of the Gap1 subfamily activate the GTP hydrolysis of both Rap and Ras is an unresolved phenomenon. RasGAPs and RapGAPs use different strategies to stimulate the GTPase reaction of their cognate G-proteins. RasGAPs contribute an arginine finger to orient through the Gln61 of Ras the nucleophilic water molecule. RapGAP contributes an asparagine (Asn thumb) into the active site to substitute for the missing Gln61. Here, by using steady-state kinetic assays and time-resolved Fourier-transform infrared spectroscopy (FTIR) experiments with wild type and mutant proteins, we unravel the remarkable mechanism for the specificity switch. The plasticity of GAP1(IP4BP) and RASAL is mediated by the extra GTPase-activating protein (GAP) domains, which promote a different orientation of Ras and Rap's switch-II and catalytic residues in the active site. Thereby, Gln63 in Rap adopts the catalytic role normally taken by Gln61 of Ras. This re-orientation requires specific interactions between switch-II of Rap and helix-alpha6 of GAPs. This supports the notion that the specificities of fl proteins versus GAP domains are potentially different.


Asunto(s)
Proteínas de Unión al GTP rap/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Secuencia de Aminoácidos , Proteínas Activadoras de GTPasa/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Proteínas de Unión al GTP rap/química , Proteínas de Unión al GTP rap/genética , Proteínas Activadoras de ras GTPasa/química , Proteínas Activadoras de ras GTPasa/genética , Proteínas ras/metabolismo
16.
Biol Chem ; 390(10): 1063-77, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19642867

RESUMEN

RhoA and RhoC are highly related Rho GTPases, but differentially control cellular behaviour. We combined molecular, cellular, and biochemical experiments to characterise differences between these highly similar GTPases. Our findings demonstrate that enhanced expression of RhoC results in a striking increase in the migration and invasion of pancreatic carcinoma cells, whereas forced expression of RhoA decreases these actions. These isoform-specific functions correlate with differences in the cellular activity of RhoA and RhoC in human cells, with RhoC being more active than RhoA in activity assays and serum-response factor-dependent gene transcription. Subcellular localisation studies revealed that RhoC is predominantly localised in the membrane-containing fraction, whereas RhoA is mainly localised in the cytoplasmic fraction. These differences are not mediated by a different interaction with RhoGDIs. In vitro GTP/GDP binding analyses demonstrate different affinity of RhoC for GTP[S] and faster intrinsic and guanine nucleotide exchange factor (GEF)-stimulated GDP/GTP exchange rates compared to RhoA. Moreover, the catalytic domains of SopE and Dbs are efficacious GEFs for RhoC. mRNA expression of RhoC is markedly enhanced in advanced pancreatic cancer stages, and thus the differences discovered between RhoA and RhoC might provide explanations for their different influences on cell migration and tumour invasion.


Asunto(s)
Movimiento Celular , Invasividad Neoplásica , Neoplasias Pancreáticas/patología , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Línea Celular Tumoral , Estimulación Encefálica Profunda , Humanos , Neoplasias Pancreáticas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Unión al GTP rap/química , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rho/química , Proteínas de Unión al GTP rho/genética , Proteína de Unión al GTP rhoA/química , Proteína de Unión al GTP rhoA/genética , Proteína rhoC de Unión a GTP
17.
J Biol Chem ; 284(40): 27480-6, 2009 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-19651783

RESUMEN

Rap1b has been implicated in the transduction of the cAMP mitogenic response. Agonists that increase intracellular cAMP rapidly activate (i.e. GTP binding) and phosphorylate Rap1b on Ser(179) at its C terminus. cAMP-dependent protein kinase (PKA)-mediated phosphorylation of Rap1b is required for cAMP-dependent mitogenesis, tumorigenesis, and inhibition of AKT activity. However, the role of phosphorylation still remains unknown. In this study, we utilized amide hydrogen/deuterium exchange mass spectroscopy (DXMS) to assess potential conformational changes and/or mobility induced by phosphorylation. We report here DXMS data comparing exchange rates for PKA-phosphorylated (Rap1-P) and S179D phosphomimetic (Rap1-D) Rap1b proteins. Rap1-P and Rap1-D behaved exactly the same, revealing an increased exchange rate in discrete regions along the protein; these regions include a domain around the phosphorylation site and unexpectedly the two switch loops. Thus, local effects induced by Ser(179) phosphorylation communicate allosterically with distal domains involved in effector interaction. These results provide a mechanistic explanation for the differential effects of Rap1 phosphorylation by PKA on effector protein interaction.


Asunto(s)
Proteínas de Unión al GTP rap/química , Proteínas de Unión al GTP rap/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Línea Celular , Medición de Intercambio de Deuterio , Humanos , Espectrometría de Masas , Modelos Moleculares , Fosforilación , Conformación Proteica , Estructura Terciaria de Proteína
18.
Nature ; 455(7209): 124-7, 2008 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-18660803

RESUMEN

Epac proteins are activated by binding of the second messenger cAMP and then act as guanine nucleotide exchange factors for Rap proteins. The Epac proteins are involved in the regulation of cell adhesion and insulin secretion. Here we have determined the structure of Epac2 in complex with a cAMP analogue (Sp-cAMPS) and RAP1B by X-ray crystallography and single particle electron microscopy. The structure represents the cAMP activated state of the Epac2 protein with the RAP1B protein trapped in the course of the exchange reaction. Comparison with the inactive conformation reveals that cAMP binding causes conformational changes that allow the cyclic nucleotide binding domain to swing from a position blocking the Rap binding site towards a docking site at the Ras exchange motif domain.


Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , AMP Cíclico/análogos & derivados , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Tionucleótidos/química , Tionucleótidos/metabolismo , Proteínas de Unión al GTP rap/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/ultraestructura , Cristalografía por Rayos X , AMP Cíclico/química , AMP Cíclico/metabolismo , Activación Enzimática , Factores de Intercambio de Guanina Nucleótido/ultraestructura , Humanos , Ratones , Microscopía Electrónica , Modelos Moleculares , Unión Proteica , Conformación Proteica , Proteínas de Unión al GTP rap/química , Proteínas de Unión al GTP rap/ultraestructura
19.
Cell Signal ; 20(9): 1662-70, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18582561

RESUMEN

Rap1b and Rap2b are the only members of the Rap family of GTPases expressed in circulating human platelets. Rap1b is involved in the inside-out activation of integrins, while the role of Rap2b is still poorly understood. In this work, we investigated the localization of Rap proteins to specific microdomains of plasma membrane called lipid rafts, implicated in signal transduction. We found that Rap1b was not associated to lipid rafts in resting platelets, and did not translocate to these microdomains in stimulated cells. By contrast, about 20% of Rap2b constitutively associated to lipid rafts, and this percentage did not increase upon platelet stimulation. Rap2b interaction with lipid rafts also occurred in transfected HEK293T cell. Upon metabolic labelling with [(3)H]palmitate, incorporation of the label into Rap2b was observed. Palmitoylation of Rap2b did not occur when Cys176 or Cys177 were mutated to serine, or when the C-terminal CAAX motif was deleted. Contrary to CAAX deletion, Cys176 and Cys177 substitution did not alter the membrane localization of Rap2b, however, relocation of the mutants within lipid rafts was completely prevented. In intact platelets, disruption of Rap2b interaction with lipid rafts obtained by cholesterol depletion caused a significant inhibition of aggregation. Importantly, agonist-induced activation of Rap2b was concomitantly severely impaired. These results demonstrate that Rap2b, but not the more abundant Rap1b, is associated to lipid rafts in human platelets. This interaction is supported by palmitoylation of Rap2b, and is important for a complete agonist-induced activation of this GTPase.


Asunto(s)
Plaquetas/enzimología , Cisteína/metabolismo , Lipoilación , Microdominios de Membrana/enzimología , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP rap/metabolismo , Secuencia de Aminoácidos , Plaquetas/efectos de los fármacos , Línea Celular , Colesterol/deficiencia , Detergentes/farmacología , Activación Enzimática/efectos de los fármacos , Humanos , Lipoilación/efectos de los fármacos , Microdominios de Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Activación Plaquetaria/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Proteínas de Unión al GTP rap/química
20.
J Biol Chem ; 283(28): 19691-703, 2008 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-18411261

RESUMEN

Exchange proteins directly activated by cAMP (EPACs) are guanine nucleotide-exchange factors for the small GTPases Rap1 and Rap2 and represent a key receptor for the ubiquitous cAMP second messenger in eukaryotes. The cAMP-dependent activation of apoEPAC is typically rationalized in terms of a preexisting equilibrium between inactive and active states. Structural and mutagenesis analyses have shown that one of the critical determinants of the EPAC activation equilibrium is a cluster of salt bridges formed between the catalytic core and helices alpha1 and alpha2 at the N terminus of the cAMP binding domain and commonly referred to as ionic latch (IL). The IL stabilizes the inactive states in a closed topology in which access to the catalytic domain is sterically occluded by the regulatory moiety. However, it is currently not fully understood how the IL is allosterically controlled by cAMP. Chemical shift mapping studies consistently indicate that cAMP does not significantly perturb the structure of the IL spanning sites within the regulatory region, pointing to cAMP-dependent dynamic modulations as a key allosteric carrier of the cAMP-signal to the IL sites. Here, we have therefore investigated the dynamic profiles of the EPAC1 cAMP binding domain in its apo, cAMP-bound, and Rp-cAMPS phosphorothioate antagonist-bound forms using several 15N relaxation experiments. Based on the comparative analysis of dynamics in these three states, we have proposed a model of EPAC activation that incorporates the dynamic features allosterically modulated by cAMP and shows that cAMP binding weakens the IL by increasing its entropic penalty due to dynamic enhancements.


Asunto(s)
AMP Cíclico/química , Factores de Intercambio de Guanina Nucleótido/química , Modelos Moleculares , Regulación Alostérica/fisiología , AMP Cíclico/genética , AMP Cíclico/metabolismo , Entropía , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Estructura Secundaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Sistemas de Mensajero Secundario/fisiología , Proteínas de Unión al GTP rap/química , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rap1/química , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismo
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