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1.
Cell Commun Signal ; 18(1): 179, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33153459

RESUMO

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.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Mutação/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras)/genética , Fenômenos Biofísicos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HCT116 , Células HEK293 , Humanos , Proteínas Mutantes/metabolismo , Neurofibromina 1/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia
2.
J Cell Sci ; 129(7): 1287-92, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26985062

RESUMO

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.


Assuntos
Neoplasias/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Códon sem Sentido/genética , Humanos , Mutação de Sentido Incorreto/genética , Isoformas de Proteínas/genética
3.
J Cell Sci ; 127(Pt 11): 2589-600, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24659802

RESUMO

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.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neutrófilos/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Neoplasias da Mama , Carcinogênese , Movimento Celular/genética , Polaridade Celular/genética , Células Cultivadas , GTP Fosfo-Hidrolases/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Melanoma , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Metástase Neoplásica , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Proteínas rho de Ligação ao GTP
4.
SLAS Discov ; 28(1): 39-47, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36563789

RESUMO

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.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Transferência Ressonante de Energia de Fluorescência , Proteínas Proto-Oncogênicas p21(ras)/genética , Reprodutibilidade dos Testes , Nucleotídeos
5.
J Biol Chem ; 286(10): 8149-8157, 2011 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-21189248

RESUMO

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.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Transformação Celular Neoplásica/metabolismo , Isoenzimas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Substituição de Aminoácidos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Isoenzimas/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação de Sentido Incorreto , Invasividade Neoplásica , Fosforilação/genética , Proteína Quinase C/genética , Proteínas Proto-Oncogênicas/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
6.
Nat Struct Mol Biol ; 14(9): 814-23, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17704816

RESUMO

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.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Neoplasias/patologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Dados de Sequência Molecular , Conformação Proteica , Fatores de Troca de Nucleotídeo Guanina Rho , Domínios de Homologia de src
7.
Trends Biochem Sci ; 30(4): 163-5, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15817389

RESUMO

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.


Assuntos
Estrutura Terciária de Proteína , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/metabolismo , Ativação Enzimática , Modelos Moleculares , Nucleotídeos Cíclicos/metabolismo
8.
Nat Struct Mol Biol ; 11(8): 756-62, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15247908

RESUMO

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.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/química , Proteínas rac de Ligação ao GTP/química , Animais , Proteínas Reguladoras de Apoptose , Células CHO , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Linhagem Celular , Movimento Celular , Cricetinae , Proteínas do Citoesqueleto/metabolismo , Dimerização , Ativação Enzimática , Glutationa Transferase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Immunoblotting , Microscopia de Fluorescência , Mutagênese , Mutação , Fagocitose , Plasmídeos/metabolismo , Testes de Precipitina , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , Fatores de Tempo , Transgenes
9.
Curr Biol ; 15(14): R563-74, 2005 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-16051167

RESUMO

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.


Assuntos
Citoplasma/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Modelos Biológicos , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/fisiologia , Proteínas ras/fisiologia , Componentes do Gene , Humanos , Proteínas ras/metabolismo
10.
Curr Biol ; 15(4): 371-7, 2005 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-15723800

RESUMO

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.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Modelos Biológicos , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Domínios de Homologia de src/genética , Sequência de Aminoácidos , Animais , Western Blotting , Caenorhabditis elegans , Células Cultivadas , Humanos , Imunoprecipitação , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Proteínas rac de Ligação ao GTP/genética
11.
Mol Cell Biol ; 22(19): 6895-905, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12215546

RESUMO

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) that exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. Like many RhoGEFs, the in vitro catalytic activity of Dbs is not limited to a single substrate. It can catalyze the exchange of GDP for GTP on RhoA and Cdc42, both of which are expressed in most cell types. This lack of substrate specificity, which is relatively common among members of the RhoGEF family, complicates efforts to determine the molecular basis of their transforming activity. We have recently determined crystal structures of several RhoGEFs bound to their cognate GTPases and have used these complexes to predict structural determinants dictating the specificities of coupling between RhoGEFs and GTPases. Guided by this information, we mutated Dbs to alter significantly its relative exchange activity for RhoA versus Cdc42 and show that the transformation potential of Dbs correlates with exchange on RhoA but not Cdc42. Supporting this conclusion, oncogenic Dbs activates endogenous RhoA but not endogenous Cdc42 in NIH 3T3 cells. Similarly, a competitive inhibitor that blocks RhoA activation also blocks Dbs-mediated transformation. In conclusion, this study highlights the usefulness of specificity mutants of RhoGEFs as tools to genetically dissect the multiple signaling pathways potentially activated by overexpressed or oncogenic RhoGEFs. These ideas are exemplified for Dbs, which is strongly implicated in the transformation of NIH 3T3 cells via RhoA and not Cdc42.


Assuntos
Transformação Celular Neoplásica/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Células 3T3 , Animais , Fibroblastos/citologia , Fibroblastos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Fatores de Troca de Nucleotídeo Guanina Rho , Relação Estrutura-Atividade , Especificidade por Substrato , Transfecção , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/genética
12.
Biochem J ; 400(3): 563-72, 2006 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17007612

RESUMO

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.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Nucleotídeos de Guanina/metabolismo , Sequência de Aminoácidos , Animais , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas rho de Ligação ao GTP/metabolismo
13.
Structure ; 12(6): 1078-86, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15274927

RESUMO

Dbl proteins are guanine nucleotide exchange factors for Rho GTPases, containing adjacent Dbl homology (DH) and pleckstrin homology (PH) domains. This domain architecture is virtually invariant and typically required for full exchange potential. Several structures of DH/PH fragments bound to GTPases implicate the PH domain in nucleotide exchange. To more fully understand the functional linkage between DH and PH domains, we have determined the crystal structure of the DH/PH fragment of Dbs without bound GTPase. This structure is generally similar to previously determined structures of Dbs bound to GTPases albeit with greater apparent mobility between the DH and PH domains. These comparisons suggest that the DH and PH domains of Dbs are spatially primed for binding GTPases and small alterations in intradomain conformations that may be elicited by subtle biological responses, such as altered phosphoinositide levels, are sufficient to enhance exchange by facilitating interactions between the PH domain and GTPases.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/química , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Cristalografia por Raios X , Elétrons , GTP Fosfo-Hidrolases/química , Lipídeos/química , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Fatores de Troca de Nucleotídeo Guanina Rho
14.
Cancer Res ; 76(13): 3826-37, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27216196

RESUMO

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.


Assuntos
Neoplasias da Mama/patologia , Carcinoma Basocelular/patologia , Proteínas Ativadoras de GTPase/metabolismo , Perfilação da Expressão Gênica , Proteínas rho de Ligação ao GTP/metabolismo , Apoptose , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Ciclo Celular , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica , Senescência Celular , Citocinese , Feminino , Proteínas Ativadoras de GTPase/genética , Humanos , Ligação Proteica , Transdução de Sinais , Células Tumorais Cultivadas , Proteínas rho de Ligação ao GTP/genética
15.
Mol Cell Biol ; 35(1): 167-81, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25332235

RESUMO

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.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Proteínas Contendo Repetições de beta-Transducina/fisiologia , Células HEK293 , Humanos , Mutagênese , Mutagênese Sítio-Dirigida , Fenótipo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteoma , Proteômica , RNA Interferente Pequeno/metabolismo , Complexo Shelterina , Proteínas de Ligação a Telômeros/metabolismo , Ubiquitina/química
16.
Comb Chem High Throughput Screen ; 6(4): 409-18, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12769685

RESUMO

Ras and Rho GTPases are signaling proteins that regulate a variety of physiological events and are intimately linked to the progression of cancer. Recently, a variety of fluorescence-based assays have been refined to monitor activation of these GTPases. This review summarizes current fluorescence-based techniques for studying Ras superfamily GTPases with an emphasis on practical examples and high-throughput applications. These techniques are not only useful for biochemical characterization of Ras superfamily members, but will also facilitate the discovery of small molecule therapeutics designed to inhibit signal transduction mediated by GTPases.


Assuntos
GTP Fosfo-Hidrolases/análise , Proteínas ras/análise , Transferência de Energia , Fluorescência , GTP Fosfo-Hidrolases/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Humanos , Espectrometria de Fluorescência , Proteínas ras/metabolismo
17.
PLoS One ; 8(6): e66260, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23825001

RESUMO

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.


Assuntos
Actomiosina/metabolismo , Movimento Celular/fisiologia , Endotélio Vascular/citologia , Fatores de Troca de Nucleotídeo Guanina Rho/fisiologia , Endotélio Vascular/metabolismo , Adesões Focais , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Neovascularização Fisiológica/fisiologia
18.
PLoS One ; 7(7): e41876, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22911862

RESUMO

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.


Assuntos
Actinas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Arginina/metabolismo , Citoesqueleto/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Fatores de Troca de Nucleotídeo Guanina Rho , Relação Estrutura-Atividade , Frações Subcelulares/metabolismo
19.
Mol Cell Biol ; 32(8): 1374-86, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22331470

RESUMO

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.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Extensões da Superfície Celular/enzimologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Extensões da Superfície Celular/ultraestrutura , Ativação Enzimática , Humanos , Invasividade Neoplásica/ultraestrutura , Neoplasias Pancreáticas/patologia , Transdução de Sinais
20.
Nat Rev Cancer ; 10(12): 842-57, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21102635

RESUMO

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.


Assuntos
Proteínas Ativadoras de GTPase/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Animais , Brefeldina A/uso terapêutico , Descoberta de Drogas , Proteínas Ativadoras de GTPase/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Neoplasias/etiologia , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T
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