RESUMEN

Mutations in the small GTPase protein KRAS are one of the leading drivers of cancers including lung, pancreatic, and colorectal, as well as a group of developmental disorders termed "Rasopathies". Recent breakthroughs in the development of mutant-specific KRAS inhibitors include the FDA approved drug Lumakras (Sotorasib, AMG510) for KRAS G12C-mutated non-small cell lung cancer (NSCLC), and MRTX1133, a promising clinical candidate for the treatment of KRAS G12D-mutated cancers. However, there are currently no FDA approved inhibitors that target KRAS mutations occurring at non-codon 12 positions. Herein, we focused on the KRAS mutant A146T, found in colorectal cancers, that exhibits a "fast-cycling" nucleotide mechanism as a driver for oncogenic activation. We developed a novel high throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay that leverages the reduced nucleotide affinity of KRAS A146T. As designed, the assay is capable of detecting small molecules that act to allosterically modulate GDP affinity or directly compete with the bound nucleotide. A pilot screen was completed to demonstrate robust statistics and reproducibility followed by a primary screen using a diversity library totaling over 83,000 compounds. Compounds yielding >50% inhibition of TR-FRET signal were selected as hits for testing in dose-response format. The most promising hit, UNC10104889, was further investigated through a structure activity relationship (SAR)-by-catalog approach in an attempt to improve potency and circumvent solubility liabilities. Overall, we present the TR-FRET platform as a robust assay to screen fast-cycling KRAS mutants enabling future discovery efforts for novel chemical probes and drug candidates.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Transferencia Resonante de Energía de Fluorescencia , Proteínas Proto-Oncogénicas p21(ras)/genética , Reproducibilidad de los Resultados , Nucleótidos

RESUMEN

Phase three clinical trial evidence suggests that colorectal cancers with the KRAS G13D mutation may benefit from EGFR inhibitors, like cetuximab, in contrast to the other most common KRAS mutations. A mechanism to explain why this mutation behaves differently from other KRAS mutations had long been lacking. Two recent studies have reproduced KRAS G13D specific sensitivity to cetuximab in cellular models, and both have implicated the tumor suppressor NF1 as a critical variable in determining sensitivity and resistance. One study proposes a mechanism that focuses on the inhibition of active, GTP-bound wild-type RAS, which is proposed to occur to a greater extent in KRAS G13D tumors due to the inability of KRAS G13D to bind NF1 well. The other study suggests NF1 can convert GTP-bound KRAS G13D to inactive, GDP-bound KRAS G13D. Here, we report an inability to reproduce cellular and biophysical studies that suggested NF1 has strong GTPase activity on KRAS G13D. We also report additional data that further suggests only WT RAS-GTP levels are reduced with EGFR inhibition and that KRAS G13D is impaired in binding to NF1. These new experiments further support a mechanism in which cetuximab inhibits wild-type (HRAS and NRAS) signals in KRAS G13D colorectal cancers. Video Abstract.

Asunto(s)

Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Mutación/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/genética , Fenómenos Biofísicos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HCT116 , Células HEK293 , Humanos , Proteínas Mutantes/metabolismo , Neurofibromina 1/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología

RESUMEN

The basal-like breast cancer (BLBC) subtype accounts for a disproportionately high percentage of overall breast cancer mortality. The current therapeutic options for BLBC need improvement; hence, elucidating signaling pathways that drive BLBC growth may identify novel targets for the development of effective therapies. Rho GTPases have previously been implicated in promoting tumor cell proliferation and metastasis. These proteins are inactivated by Rho-selective GTPase-activating proteins (RhoGAP), which have generally been presumed to act as tumor suppressors. Surprisingly, RNA-Seq analysis of the Rho GTPase signaling transcriptome revealed high expression of several RhoGAP genes in BLBC tumors, raising the possibility that these genes may be oncogenic. To evaluate this, we examined the roles of two of these RhoGAPs, ArhGAP11A (also known as MP-GAP) and RacGAP1 (also known as MgcRacGAP), in promoting BLBC. Both proteins were highly expressed in human BLBC cell lines, and knockdown of either gene resulted in significant defects in the proliferation of these cells. Knockdown of ArhGAP11A caused CDKN1B/p27-mediated arrest in the G1 phase of the cell cycle, whereas depletion of RacGAP1 inhibited growth through the combined effects of cytokinesis failure, CDKN1A/p21-mediated RB1 inhibition, and the onset of senescence. Random migration was suppressed or enhanced by the knockdown of ArhGAP11A or RacGAP1, respectively. Cell spreading and levels of GTP-bound RhoA were increased upon depletion of either RhoGAP. We have established that, via the suppression of RhoA, ArhGAP11A and RacGAP1 are both critical drivers of BLBC growth, and propose that RhoGAPs can act as oncogenes in cancer. Cancer Res; 76(13); 3826-37. ©2016 AACR.

Asunto(s)

Neoplasias de la Mama/patología , Carcinoma Basocelular/patología , Proteínas Activadoras de GTPasa/metabolismo , Perfilación de la Expresión Génica , Proteínas de Unión al GTP rho/metabolismo , Apoptosis , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Ciclo Celular , Movimiento Celular , Proliferación Celular , Transformación Celular Neoplásica , Senescencia Celular , Citocinesis , Femenino , Proteínas Activadoras de GTPasa/genética , Humanos , Unión Proteica , Transducción de Señal , Células Tumorales Cultivadas , Proteínas de Unión al GTP rho/genética

RESUMEN

RAS proteins (KRAS4A, KRAS4B, NRAS and HRAS) function as GDP-GTP-regulated binary on-off switches, which regulate cytoplasmic signaling networks that control diverse normal cellular processes. Gain-of-function missense mutations in RAS genes are found in ∼25% of human cancers, prompting interest in identifying anti-RAS therapeutic strategies for cancer treatment. However, despite more than three decades of intense effort, no anti-RAS therapies have reached clinical application. Contributing to this failure has been an underestimation of the complexities of RAS. First, there is now appreciation that the four human RAS proteins are not functionally identical. Second, with >130 different missense mutations found in cancer, there is an emerging view that there are mutation-specific consequences on RAS structure, biochemistry and biology, and mutation-selective therapeutic strategies are needed. In this Cell Science at a Glance article and accompanying poster, we provide a snapshot of the differences between RAS isoforms and mutations, as well as the current status of anti-RAS drug-discovery efforts.

Asunto(s)

Neoplasias/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Codón sin Sentido/genética , Humanos , Mutación Missense/genética , Isoformas de Proteínas/genética

RESUMEN

Defining the full complement of substrates for each ubiquitin ligase remains an important challenge. Improvements in mass spectrometry instrumentation and computation and in protein biochemistry methods have resulted in several new methods for ubiquitin ligase substrate identification. Here we used the parallel adapter capture (PAC) proteomics approach to study ßTrCP2/FBXW11, a substrate adaptor for the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex. The processivity of the ubiquitylation reaction necessitates transient physical interactions between FBXW11 and its substrates, thus making biochemical purification of FBXW11-bound substrates difficult. Using the PAC-based approach, we inhibited the proteasome to "trap" ubiquitylated substrates on the SCF(FBXW11) E3 complex. Comparative mass spectrometry analysis of immunopurified FBXW11 protein complexes before and after proteasome inhibition revealed 21 known and 23 putatively novel substrates. In focused studies, we found that SCF(FBXW11) bound, polyubiquitylated, and destabilized RAPGEF2, a guanine nucleotide exchange factor that activates the small GTPase RAP1. High RAPGEF2 protein levels promoted cell-cell fusion and, consequently, multinucleation. Surprisingly, this occurred independently of the guanine nucleotide exchange factor (GEF) catalytic activity and of the presence of RAP1. Our data establish new functions for RAPGEF2 that may contribute to aneuploidy in cancer. More broadly, this report supports the continued use of substrate trapping proteomics to comprehensively define targets for E3 ubiquitin ligases. All proteomic data are available via ProteomeXchange with identifier PXD001062.

Asunto(s)

Factores de Intercambio de Guanina Nucleótido/metabolismo , Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Proteínas con Repetición de beta-Transducina/fisiología , Células HEK293 , Humanos , Mutagénesis , Mutagénesis Sitio-Dirigida , Fenotipo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteoma , Proteómica , ARN Interferente Pequeño/metabolismo , Complejo Shelterina , Proteínas de Unión a Telómeros/metabolismo , Ubiquitina/química

RESUMEN

G-protein-coupled receptors (GPCRs) regulate the organisation of the actin cytoskeleton by activating the Rac subfamily of small GTPases. The guanine-nucleotide-exchange factor (GEF) P-Rex1 is engaged downstream of GPCRs and phosphoinositide 3-kinase (PI3K) in many cell types, and promotes tumorigenic signalling and metastasis in breast cancer and melanoma, respectively. Although P-Rex1-dependent functions have been attributed to its GEF activity towards Rac1, we show that P-Rex1 also acts as a GEF for the Rac-related GTPase RhoG, both in vitro and in GPCR-stimulated primary mouse neutrophils. Furthermore, loss of either P-Rex1 or RhoG caused equivalent reductions in GPCR-driven Rac activation and Rac-dependent NADPH oxidase activity, suggesting they both function upstream of Rac in this system. Loss of RhoG also impaired GPCR-driven recruitment of the Rac GEF DOCK2, and F-actin, to the leading edge of migrating neutrophils. Taken together, our results reveal a new signalling hierarchy in which P-Rex1, acting as a GEF for RhoG, regulates Rac-dependent functions indirectly through RhoG-dependent recruitment of DOCK2. These findings thus have broad implications for our understanding of GPCR signalling to Rho GTPases and the actin cytoskeleton.

Asunto(s)

Citoesqueleto de Actina/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Neutrófilos/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Neoplasias de la Mama , Carcinogénesis , Movimiento Celular/genética , Polaridad Celular/genética , Células Cultivadas , GTP Fosfohidrolasas/genética , Factores de Intercambio de Guanina Nucleótido/genética , Melanoma , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Metástasis de la Neoplasia , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/genética , Proteínas de Unión al GTP rho

RESUMEN

Persistent cellular migration requires efficient protrusion of the front of the cell, the leading edge where the actin cytoskeleton and cell-substrate adhesions undergo constant rearrangement. Rho family GTPases are essential regulators of the actin cytoskeleton and cell adhesion dynamics. Here, we examined the role of the RhoGEF TEM4, an activator of Rho family GTPases, in regulating cellular migration of endothelial cells. We found that TEM4 promotes the persistence of cellular migration by regulating the architecture of actin stress fibers and cell-substrate adhesions in protruding membranes. Furthermore, we determined that TEM4 regulates cellular migration by signaling to RhoC as suppression of RhoC expression recapitulated the loss-of-TEM4 phenotypes, and RhoC activation was impaired in TEM4-depleted cells. Finally, we showed that TEM4 and RhoC antagonize myosin II-dependent cellular contractility and the suppression of myosin II activity rescued the persistence of cellular migration of TEM4-depleted cells. Our data implicate TEM4 as an essential regulator of the actin cytoskeleton that ensures proper membrane protrusion at the leading edge of migrating cells and efficient cellular migration via suppression of actomyosin contractility.

Asunto(s)

Actomiosina/metabolismo , Movimiento Celular/fisiología , Endotelio Vascular/citología , Factores de Intercambio de Guanina Nucleótido Rho/fisiología , Endotelio Vascular/metabolismo , Adhesiones Focales , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neovascularización Fisiológica/fisiología

RESUMEN

Spatio-temporal activation of Rho GTPases is essential for their function in a variety of biological processes and is achieved in part by regulating the localization of their activators, the Rho guanine nucleotide exchange factors (RhoGEFs). In this study, we provide the first characterization of the full-length protein encoded by RhoGEF TEM4 and delineate its domain structure, catalytic activity, and subcellular localization. First, we determined that TEM4 can stimulate guanine nucleotide exchange on RhoA and the related RhoB and RhoC isoforms. Second, we determined that TEM4, like other Dbl RhoGEFs, contains a functional pleckstrin homology (PH) domain immediately C-terminal to the catalytic Dbl homology (DH) domain. Third, using immunofluorescence analysis, we showed that TEM4 localizes to the actin cytoskeleton through sequences in the N-terminus of TEM4 independently of the DH/PH domains. Using site-directed mutagenesis and deletion analysis, we identified a minimal region between residues 81 and 135 that binds directly to F-actin and has an ∼90-fold higher affinity for ATP-loaded F-actin. Finally, we demonstrated that a single point mutation (R130D) within full-length TEM4 abolishes actin binding and localization of TEM4 to the actin cytoskeleton, as well as dampens the in vivo activity of TEM4 towards RhoC. Taken together, our data demonstrate that TEM4 contains a novel actin binding domain and binding to actin is essential for TEM4 subcellular localization and activity. The unique subcellular localization of TEM4 suggests a spatially-restricted activity and expands the diversity of mechanisms by which RhoGEF function can be regulated.

Asunto(s)

Actinas/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Secuencia de Aminoácidos , Arginina/metabolismo , Citoesqueleto/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Factores de Intercambio de Guanina Nucleótido Rho , Relación Estructura-Actividad , Fracciones Subcelulares/metabolismo

RESUMEN

Our recent studies implicated key and distinct roles for the highly related RalA and RalB small GTPases (82% sequence identity) in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis and invasive and metastatic growth, respectively. How RalB may promote PDAC invasion and metastasis has not been determined. In light of known Ral effector functions in regulation of actin organization and secretion, we addressed a possible role for RalB in formation of invadopodia, actin-rich membrane protrusions that contribute to tissue invasion and matrix remodeling. We determined that a majority of KRAS mutant PDAC cell lines exhibited invadopodia and that expression of activated K-Ras is both necessary and sufficient for invadopodium formation. Invadopodium formation was not dependent on the canonical Raf-MEK-ERK effector pathway and was instead dependent on the Ral effector pathway. However, this process was more dependent on RalB than on RalA. Surprisingly, RalB-mediated invadopodium formation was dependent on RalBP1/RLIP76 but not Sec5 and Exo84 exocyst effector function. Unexpectedly, the requirement for RalBP1 was independent of its best known function as a GTPase-activating protein for Rho small GTPases. Instead, disruption of the ATPase function of RalBP1 impaired invadopodium formation. Our results identify a novel RalB-mediated biochemical and signaling mechanism for invadopodium formation.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/metabolismo , Extensiones de la Superficie Celular/enzimología , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de Unión al GTP ral/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Extensiones de la Superficie Celular/ultraestructura , Activación Enzimática , Humanos , Invasividad Neoplásica/ultraestructura , Neoplasias Pancreáticas/patología , Transducción de Señal

RESUMEN

The Rho GTPase guanine nucleotide exchange factor Ect2 is genetically and biochemically linked to the PKCι oncogene in non-small cell lung cancer (NSCLC). Ect2 is overexpressed and mislocalized to the cytoplasm of NSCLC cells where it binds the oncogenic PKCι-Par6 complex, leading to activation of the Rac1 small GTPase. Here, we identify a previously uncharacterized phosphorylation site on Ect2, threonine 328, that serves to regulate the oncogenic activity of Ect2 in NSCLC cells. PKCι directly phosphorylates Ect2 at Thr-328 in vitro, and RNAi-mediated knockdown of either PKCι or Par6 leads to a decrease in phospho-Thr-328 Ect2, indicating that PKCι regulates Thr-328 Ect2 phosphorylation in NSCLC cells. Both wild-type Ect2 and a phosphomimetic T328D Ect2 mutant bind the PKCι-Par6 complex, activate Rac1, and restore transformed growth and invasion when expressed in NSCLC cells made deficient in endogenous Ect2 by RNAi-mediated knockdown. In contrast, a phosphorylation-deficient T328A Ect2 mutant fails to bind the PKCι-Par6 complex, activate Rac1, or restore transformation. Our data support a model in which PKCι-mediated phosphorylation regulates Ect2 binding to the oncogenic PKCι-Par6 complex thereby activating Rac1 activity and driving transformed growth and invasion.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Transformación Celular Neoplásica/metabolismo , Isoenzimas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sustitución de Aminoácidos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , Isoenzimas/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mutación Missense , Invasividad Neoplásica , Fosforilación/genética , Proteína Quinasa C/genética , Proteínas Proto-Oncogénicas/genética , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo

RESUMEN

There is now considerable and increasing evidence for a causal role for aberrant activity of the Ras superfamily of small GTPases in human cancers. These GTPases function as GDP-GTP-regulated binary switches that control many fundamental cellular processes. A common mechanism of GTPase deregulation in cancer is the deregulated expression and/or activity of their regulatory proteins, guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound state and GTPase-activating proteins (GAPs) that return the GTPase to its GDP-bound inactive state. In this Review, we assess the association of GEFs and GAPs with cancer and their druggability for cancer therapeutics.

Asunto(s)

Proteínas Activadoras de GTPasa/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Animales , Brefeldino A/uso terapéutico , Descubrimiento de Drogas , Proteínas Activadoras de GTPasa/fisiología , Factores de Intercambio de Guanina Nucleótido/fisiología , Humanos , Neoplasias/etiología , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T

RESUMEN

Autoinhibition of the Rho guanine nucleotide exchange factor ASEF is relieved by interaction with the APC tumor suppressor. Here we show that binding of the armadillo repeats of APC to a 'core APC-binding' (CAB) motif within ASEF, or truncation of the SH3 domain of ASEF, relieves autoinhibition, allowing the specific activation of CDC42. Structural determination of autoinhibited ASEF reveals that the SH3 domain forms an extensive interface with the catalytic DH and PH domains to obstruct binding and activation of CDC42, and the CAB motif is positioned adjacent to the SH3 domain to facilitate activation by APC. In colorectal cancer cell lines, full-length, but not truncated, APC activates CDC42 in an ASEF-dependent manner to suppress anchorage-independent growth. We therefore propose a model in which ASEF acts as a tumor suppressor when activated by APC and inactivation of ASEF by mutation or APC truncation promotes tumorigenesis.

Asunto(s)

Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Neoplasias/patología , Proteína de Unión al GTP cdc42/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Datos de Secuencia Molecular , Conformación Proteica , Factores de Intercambio de Guanina Nucleótido Rho , Dominios Homologos src

RESUMEN

Dbl-related oncoproteins are guanine nucleotide exchange factors specific for Rho-family GTPases and typically possess tandem Dbl homology (DH) and pleckstrin homology domains that act in concert to catalyze exchange. Because the ability of many Dbl-family proteins to catalyze exchange is constitutively activated by truncations N-terminal to their DH domains, it has been proposed that the activity of Dbl-family proteins is regulated by auto-inhibition. However, the exact mechanisms of regulation of Dbl-family proteins remain poorly understood. Here we show that the Dbl-family protein, Tim, is auto-inhibited by a short, helical motif immediately N-terminal to its DH domain, which directly occludes the catalytic surface of the DH domain to prevent GTPase activation. Similar to the distantly related Vav isozymes, auto-inhibition of Tim is relieved by truncation, mutation, or phosphorylation of the auto-inhibitory helix. A peptide comprising the helical motif inhibits the exchange activity of Tim in vitro. Furthermore, substitutions within the most highly conserved surface of the DH domain designed to disrupt interactions with the auto-inhibitory helix also activate the exchange process.

Asunto(s)

Proteínas de Ciclo Celular/química , Factores de Intercambio de Guanina Nucleótido/química , Péptidos y Proteínas de Señalización Intracelular/química , Secuencias de Aminoácidos/genética , Animales , Catálisis , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Activación Enzimática/genética , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Mutación , Estructura Terciaria de Proteína/genética , Proteínas Proto-Oncogénicas c-vav/química , Proteínas Proto-Oncogénicas c-vav/genética , Proteínas Proto-Oncogénicas c-vav/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

Dbl family GEFs (guanine nucleotide-exchange factors) for the Rho GTPases almost invariably contain a PH (pleckstrin homology) domain adjacent to their DH (Dbl homology) domain. The DH domain is responsible for GEF activity, and the PH domain plays a regulatory role that remains poorly understood. We demonstrated previously that Dbl family PH domains bind phosphoinositides with low affinity and cannot function as independent membrane targeting modules. In the present study, we show that dimerization of a Dbs (Dbl's big sister) DH/PH domain fragment is sufficient to drive it to the plasma membrane through a mechanism involving PH domain-phosphoinositide interactions. Thus, the Dbs PH domain could play a significant role in membrane targeting if it co-operates with other domains in the protein. We also show that mutations that prevent phosphoinositide binding by the Dbs PH domain significantly impair cellular GEF activity even in chimaeric proteins that are robustly membrane targeted by farnesylation or by the PH domain of phospholipase C-delta1. This finding argues that the Dbs PH domain plays a regulatory role that is independent of its ability to aid membrane targeting. Thus, we suggest that the PH domain plays dual roles, contributing independently to membrane localization of Dbs (as part of a multi-domain interaction) and allosteric regulation of the DH domain.

Asunto(s)

Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Nucleótidos de Guanina/metabolismo , Secuencia de Aminoácidos , Animales , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas de Unión al GTP rho/metabolismo

RESUMEN

Ras proteins function as signaling hubs that are activated by convergent signaling pathways initiated by extracellular stimuli. Activated Ras in turn regulates a diversity of downstream cytoplasmic signaling cascades. Ras proteins are founding members of a large superfamily of small GTPases that have significant sequence and biochemical similarities. Recent observations have established a complex signaling interplay between Ras and other members of the family. A key biochemical mechanism facilitating this crosstalk involves guanine nucleotide exchange factors (GEFs), which serve as regulators and effectors, as well as signaling integrators, of Ras signaling.

Asunto(s)

Citoplasma/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Modelos Biológicos , Sistemas de Mensajero Secundario/fisiología , Transducción de Señal/fisiología , Proteínas ras/fisiología , Componentes del Gen , Humanos , Proteínas ras/metabolismo

RESUMEN

Dbl homology (DH) domains are almost always followed immediately by pleckstrin homology (PH) domains in Dbl family proteins, and these DH-PH fragments directly activate GDP-bound Rho GTPases by catalyzing the exchange of GDP for GTP. New crystal structures of the DH-PH domains from leukemia-associated Rho guanine nucleotide exchange factor (RhoGEF) and PDZ-RhoGEF bound to RhoA reveal how DH-PH domains cooperate to specifically activate Rho GTPases.

Asunto(s)

Estructura Terciaria de Proteína , Proteína de Unión al GTP rhoA/química , Proteína de Unión al GTP rhoA/metabolismo , Activación Enzimática , Modelos Moleculares , Nucleótidos Cíclicos/metabolismo

RESUMEN

Guanine nucleotide-exchange factors (GEFs) are directly responsible for the activation of Rho-family GTPases in response to diverse extracellular stimuli, and ultimately regulate numerous cellular responses such as proliferation, differentiation and movement. With 69 distinct homologues, Dbl-related GEFs represent the largest family of direct activators of Rho GTPases in humans, and they activate Rho GTPases within particular spatio-temporal contexts. The failure to do so can have significant consequences and is reflected in the aberrant function of Dbl-family GEFs in some human diseases.

Asunto(s)

Factores de Intercambio de Guanina Nucleótido/química , Proteínas de Unión al GTP rho/química , Sitio Alostérico , Animales , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Membrana Dobles de Lípidos/química , Ratones , Modelos Biológicos , Modelos Moleculares , Neuronas/metabolismo , Filogenia , Unión Proteica , Estructura Terciaria de Proteína , Transducción de Señal , ras-GRF1/química

RESUMEN

CDM (CED-5, Dock180, Myoblast city) family members have been recently identified as novel, evolutionarily conserved guanine nucleotide exchange factors (GEFs) for Rho-family GTPases . They regulate multiple processes, including embryonic development, cell migration, apoptotic-cell engulfment, tumor invasion, and HIV-1 infection, in diverse model systems . However, the mechanism(s) of regulation of CDM proteins has not been well understood. Here, our studies on the prototype member Dock180 reveal a steric-inhibition model for regulating the Dock180 family of GEFs. At basal state, the N-terminal SH3 domain of Dock180 binds to the distant catalytic Docker domain and negatively regulates the function of Dock180. Further studies revealed that the SH3:Docker interaction sterically blocks Rac access to the Docker domain. Interestingly, ELMO binding to the SH3 domain of Dock180 disrupted the SH3:Docker interaction, facilitated Rac access to the Docker domain, and contributed to the GEF activity of the Dock180/ELMO complex. Additional genetic rescue studies in C. elegans suggested that the regulation of the Docker-domain-mediated GEF activity by the SH3 domain and its adjoining region is evolutionarily conserved. This steric-inhibition model may be a general mechanism for regulating multiple SH3-domain-containing Dock180 family members and may have implications for a variety of biological processes.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Modelos Biológicos , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Dominios Homologos src/genética , Secuencia de Aminoácidos , Animales , Western Blotting , Caenorhabditis elegans , Células Cultivadas , Humanos , Inmunoprecipitación , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Proteínas de Unión al GTP rac/genética

Asunto(s)

Proteínas ras/fisiología , Factores de Ribosilacion-ADP/fisiología , Animales , Membrana Celular/metabolismo , GTP Fosfohidrolasas/química , Humanos , Metabolismo de los Lípidos , Modelos Biológicos , Familia de Multigenes , Proteínas de Unión al GTP rab/fisiología , Proteína de Unión al GTP ran/fisiología , Proteínas ras/metabolismo , Proteínas de Unión al GTP rho/fisiología

RESUMEN

The members of the Dock180 superfamily of proteins are novel guanine nucleotide exchange factors (GEF) for Rho family GTPases and are linked to multiple biological processes from worms to mammals. ELMO is a critical regulator of Dock180, and the Dock180-ELMO complex functions as a bipartite GEF for Rac. We identified a mechanism wherein the PH domain of ELMO, by binding the Dock180-Rac complex in trans, stabilizes Rac in the nucleotide-free transition state. Mutagenesis studies reveal that this ELMO PH domain-dependent regulation is essential for the Dock180-ELMO complex to function in phagocytosis and cell migration. Genetic rescue studies in Caenorhabditis elegans using ELMO and its homolog CED-12 support the above observations in vivo. These data reveal a new mode of action of PH domains and a novel, evolutionarily conserved mechanism by which a bipartite GEF can activate Rac.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/química , Proteínas de Unión al GTP rac/química , Animales , Proteínas Reguladoras de la Apoptosis , Células CHO , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Línea Celular , Movimiento Celular , Cricetinae , Proteínas del Citoesqueleto/metabolismo , Dimerización , Activación Enzimática , Glutatión Transferasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Immunoblotting , Microscopía Fluorescente , Mutagénesis , Mutación , Fagocitosis , Plásmidos/metabolismo , Pruebas de Precipitina , Estructura Terciaria de Proteína , Espectrometría de Fluorescencia , Factores de Tiempo , Transgenes