RESUMO
Cancers represent complex autonomous systems, displaying self-sufficiency in growth signaling. Autonomous growth is fueled by a cancer cell's ability to "secrete-and-sense" growth factors (GFs): a poorly understood phenomenon. Using an integrated computational and experimental approach, here we dissect the impact of a feedback-coupled GTPase circuit within the secretory pathway that imparts secretion-coupled autonomy. The circuit is assembled when the Ras-superfamily monomeric GTPase Arf1, and the heterotrimeric GTPase Giαßγ and their corresponding GAPs and GEFs are coupled by GIV/Girdin, a protein that is known to fuel aggressive traits in diverse cancers. One forward and two key negative feedback loops within the circuit create closed-loop control, allow the two GTPases to coregulate each other, and convert the expected switch-like behavior of Arf1-dependent secretion into an unexpected dose-response alignment behavior of sensing and secretion. Such behavior translates into cell survival that is self-sustained by stimulus-proportionate secretion. Proteomic studies and protein-protein interaction network analyses pinpoint GFs (e.g., the epidermal GF) as key stimuli for such self-sustenance. Findings highlight how the enhanced coupling of two biological switches in cancer cells is critical for multiscale feedback control to achieve secretion-coupled autonomy of growth factors.
Assuntos
Células Eucarióticas , Proteômica , Transdução de Sinais , GTP Fosfo-HidrolasesRESUMO
The molecular mechanisms by which receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major signaling hubs in eukaryotes, independently relay signals across the plasma membrane have been extensively characterized. How these hubs cross-talk has been a long-standing question, but answers remain elusive. Using linear ion-trap mass spectrometry in combination with biochemical, cellular, and computational approaches, we unravel a mechanism of activation of heterotrimeric G proteins by RTKs and chart the key steps that mediate such activation. Upon growth factor stimulation, the guanine-nucleotide exchange modulator dissociates Gαiâ¢ßγ trimers, scaffolds monomeric Gαi with RTKs, and facilitates the phosphorylation on two tyrosines located within the interdomain cleft of Gαi. Phosphorylation triggers the activation of Gαi and inhibits second messengers (cAMP). Tumor-associated mutants reveal how constitutive activation of this pathway impacts cell's decision to "go" vs. "grow." These insights define a tyrosine-based G protein signaling paradigm and reveal its importance in eukaryotes.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Células COS , Chlorocebus aethiops , Receptores ErbB/metabolismo , Células HEK293 , Células HeLa , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Humanos , Fosforilação , Receptores Proteína Tirosina Quinases/fisiologia , Transdução de Sinais , Tirosina/metabolismoRESUMO
We previously showed that guanine nucleotide-binding (G) protein α subunit (Gα)-interacting vesicle-associated protein (GIV), a guanine-nucleotide exchange factor (GEF), transactivates Gα activity-inhibiting polypeptide 1 (Gαi) proteins in response to growth factors, such as EGF, using a short C-terminal motif. Subsequent work demonstrated that GIV also binds Gαs and that inactive Gαs promotes maturation of endosomes and shuts down mitogenic MAPK-ERK1/2 signals from endosomes. However, the mechanism and consequences of dual coupling of GIV to two G proteins, Gαi and Gαs, remained unknown. Here we report that GIV is a bifunctional modulator of G proteins; it serves as a guanine nucleotide dissociation inhibitor (GDI) for Gαs using the same motif that allows it to serve as a GEF for Gαi. Upon EGF stimulation, GIV modulates Gαi and Gαs sequentially: first, a key phosphomodification favors the assembly of GIV-Gαi complexes and activates GIV's GEF function; then a second phosphomodification terminates GIV's GEF function, triggers the assembly of GIV-Gαs complexes, and activates GIV's GDI function. By comparing WT and GIV mutants, we demonstrate that GIV inhibits Gαs activity in cells responding to EGF. Consequently, the cAMPâPKAâcAMP response element-binding protein signaling axis is inhibited, the transit time of EGF receptor through early endosomes are accelerated, mitogenic MAPK-ERK1/2 signals are rapidly terminated, and proliferation is suppressed. These insights define a paradigm in G-protein signaling in which a pleiotropically acting modulator uses the same motif both to activate and to inhibit G proteins. Our findings also illuminate how such modulation of two opposing Gα proteins integrates downstream signals and cellular responses.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proliferação de Células/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Fator de Crescimento Epidérmico/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Proteínas dos Microfilamentos/química , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteína Quinase C-theta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Proteínas de Transporte Vesicular/químicaRESUMO
Canonical signal transduction via heterotrimeric G proteins is spatially and temporally restricted, that is, triggered exclusively at the plasma membrane (PM), only by agonist activation of G protein-coupled receptors (GPCRs) via a process that completes within a few hundred milliseconds. Recently, a rapidly emerging paradigm has revealed a non-canonical pathway for activation of heterotrimeric G proteins by the non-receptor guanidine-nucleotide exchange factor (GEF), GIV/Girdin. This pathway has distinctive temporal and spatial features and an unusual profile of receptor engagement: diverse classes of receptors, not just GPCRs can engage with GIV to trigger such activation. Such activation is spatially and temporally unrestricted, that is, can occur both at the PM and on internal membranes discontinuous with the PM, and can continue for prolonged periods of time. Here, we provide the most complete up-to-date review of the molecular mechanisms that govern the unique spatiotemporal aspects of non-canonical G protein activation by GIV and the relevance of this new paradigm in health and disease.
Assuntos
Doenças Cardiovasculares/metabolismo , Diabetes Mellitus/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neoplasias/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Proteínas de Transporte Vesicular/metabolismo , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Doenças Cardiovasculares/terapia , Membrana Celular/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/patologia , Diabetes Mellitus/terapia , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Membranas Intracelulares , Proteínas dos Microfilamentos/genética , Modelos Moleculares , Terapia de Alvo Molecular , Neoplasias/genética , Neoplasias/patologia , Neoplasias/terapia , Mapeamento de Interação de Proteínas , Receptores Acoplados a Proteínas G/genética , Fatores de Tempo , Proteínas de Transporte Vesicular/genéticaRESUMO
Environmental cues are transmitted to the interior of the cell via a complex network of signaling hubs. Receptor tyrosine kinases (RTKs) and trimeric G proteins are two such major signaling hubs in eukaryotes. Conventionally, canonical signal transduction via trimeric G proteins is thought to be triggered exclusively by G protein-coupled receptors. Here we used molecular engineering to develop modular fluorescent biosensors that exploit the remarkable specificity of bimolecular recognition, i.e., of both G proteins and RTKs, and reveal the workings of a novel platform for activation of G proteins by RTKs in single living cells. Comprised of the unique modular makeup of guanidine exchange factor Gα-interacting vesicle-associated protein (GIV)/girdin, a guanidine exchange factor that links G proteins to a variety of RTKs, these biosensors provide direct evidence that RTK-GIV-Gαi ternary complexes are formed in living cells and that Gαi is transactivated within minutes after growth factor stimulation at the plasma membrane. Thus, GIV-derived biosensors provide a versatile strategy for visualizing, monitoring, and manipulating the dynamic association of Gαi with RTKs for noncanonical transactivation of G proteins in cells and illuminate a fundamental signaling event regulated by GIV during diverse cellular processes and pathophysiologic states.
Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Ligação ao GTP , Receptores Proteína Tirosina Quinases , Receptores de Fatores de Crescimento , Transdução de Sinais , Animais , Células COS , Chlorocebus aethiops , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
In eukaryotes, receptor tyrosine kinases (RTKs) and trimeric G proteins are two major signaling hubs. Signal transduction via trimeric G proteins has long been believed to be triggered exclusively by G protein-coupled receptors (GPCRs). This paradigm has recently been challenged by several studies on a multimodular signal transducer, Gα-Interacting Vesicle associated protein (GIV/Girdin). We recently demonstrated that GIV's C terminus (CT) serves as a platform for dynamic association of ligand-activated RTKs with Gαi, and for noncanonical transactivation of G proteins. However, exogenous manipulation of this platform has remained beyond reach. Here we developed cell-permeable GIV-CT peptides by fusing a TAT-peptide transduction domain (TAT-PTD) to the minimal modular elements of GIV that are necessary and sufficient for activation of Gi downstream of RTKs, and used them to engineer signaling networks and alter cell behavior. In the presence of an intact GEF motif, TAT-GIV-CT peptides enhanced diverse processes in which GIV's GEF function has previously been implicated, e.g., 2D cell migration after scratch-wounding, invasion of cancer cells, and finally, myofibroblast activation and collagen production. Furthermore, topical application of TAT-GIV-CT peptides enhanced the complex, multireceptor-driven process of wound repair in mice in a GEF-dependent manner. Thus, TAT-GIV peptides provide a novel and versatile tool to manipulate Gαi activation downstream of growth factors in a diverse array of pathophysiologic conditions.
Assuntos
Peptídeos Penetradores de Células/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Produtos do Gene tat/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Transdução de Sinais/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Animais , Peptídeos Penetradores de Células/uso terapêutico , Transferência Ressonante de Energia de Fluorescência , Produtos do Gene tat/química , Produtos do Gene tat/genética , Engenharia Genética/métodos , Células HeLa , Humanos , Camundongos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Reação em Cadeia da Polimerase , Transdução Genética/métodos , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genéticaRESUMO
Aim: Globally, neurodegeneration accounts for significant morbidity and mortality among the elderly. Millions of people are afflicted with neurodegenerative diseases, with the most notable cases attributed to Alzheimer's, Huntington's, amyotrophic lateral sclerosis and Parkinson's diseases. Sensitive assays that can detect proteopathic anomalies indicative of early neurodegeneration have remained elusive. Therefore, there is an urgent need for sensitive diagnostic and prognostic biomarker assays that can guide the therapeutic regimen in the clinic. Materials & methods: Single molecule array digital immunoassay platform has sensitivity about 1000-fold higher than traditional ligand binding assays. Consequently, we are now beginning to implement ultrasensitive techniques in bioanalysis. Conclusion: In the current study, we evaluated single molecule array technology and report specifications to quantitate neurofilament light chain, a bona-fide biomarker for neurodegeneration. Preliminary neurofilament light screening results from 100 human geriatric cerebrospinal fluid samples displayed huge biological variation and warrants further investigation.
Assuntos
Imunoensaio/métodos , Testes Imunológicos/métodos , Proteínas de Neurofilamentos/metabolismo , HumanosRESUMO
A long-held tenet of heterotrimeric G protein signal transduction is that it is triggered by G protein-coupled receptors (GPCRs) at the PM. Here, we demonstrate that Gi is activated in the Golgi by GIV/Girdin, a non-receptor guanine-nucleotide exchange factor (GEF). GIV-dependent activation of Gi at the Golgi maintains the finiteness of the cyclical activation of ADP-ribosylation factor 1 (Arf1), a fundamental step in vesicle traffic in all eukaryotes. Several interactions with other major components of Golgi trafficking-e.g., active Arf1, its regulator, ArfGAP2/3, and the adaptor protein ß-COP-enable GIV to coordinately regulate Arf1 signaling. When the GIV-Gαi pathway is selectively inhibited, levels of GTP-bound Arf1 are elevated and protein transport along the secretory pathway is delayed. These findings define a paradigm in non-canonical G protein signaling at the Golgi, which places GIV-GEF at the crossroads between signals gated by the trimeric G proteins and the Arf family of monomeric GTPases.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Complexo de Golgi/metabolismo , Proteínas dos Microfilamentos/genética , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/genética , Fatores de Ribosilação do ADP/metabolismo , Animais , Sítios de Ligação/genética , Células COS , Linhagem Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Proteína Coatomer/metabolismo , Ativação Enzimática , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas dos Microfilamentos/antagonistas & inibidores , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Proteínas de Transporte Vesicular/antagonistas & inibidoresRESUMO
GIV/Girdin is a multimodular signal transducer and a bona fide metastasis-related protein. As a guanidine exchange factor (GEF), GIV modulates signals initiated by growth factors (chemical signals) by activating the G protein Gαi. Here we report that mechanical signals triggered by the extracellular matrix (ECM) also converge on GIV-GEF via ß1 integrins and that focal adhesions (FAs) serve as the major hubs for mechanochemical signaling via GIV. GIV interacts with focal adhesion kinase (FAK) and ligand-activated ß1 integrins. Phosphorylation of GIV by FAK enhances PI3K-Akt signaling, the integrity of FAs, increases cell-ECM adhesion, and triggers ECM-induced cell motility. Activation of Gαi by GIV-GEF further potentiates FAK-GIV-PI3K-Akt signaling at the FAs. Spatially restricted signaling via tyrosine phosphorylated GIV at the FAs is enhanced during cancer metastasis. Thus GIV-GEF serves as a unifying platform for integration and amplification of adhesion (mechanical) and growth factor (chemical) signals during cancer progression.
Assuntos
Adesões Focais/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Quinase 1 de Adesão Focal/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Transdução de Sinais , Tirosina/metabolismoRESUMO
Parenchymal lung inflammation and airway and alveolar epithelial cell apoptosis are associated with cigarette smoke exposure (CSE), which contributes to chronic obstructive pulmonary disease (COPD). Epidemiological studies indicate that people exposed to chronic cigarette smoke with or without COPD are more susceptible to influenza A virus (IAV) infection. We found increased p53, PAI-1 and apoptosis in AECs, with accumulation of macrophages and neutrophils in the lungs of patients with COPD. In Wild-type (WT) mice with passive CSE (PCSE), p53 and PAI-1 expression and apoptosis were increased in AECs as was lung inflammation, while those lacking p53 or PAI-1 resisted AEC apoptosis and lung inflammation. Further, inhibition of p53-mediated induction of PAI-1 by treatment of WT mice with caveolin-1 scaffolding domain peptide (CSP) reduced PCSE-induced lung inflammation and reversed PCSE-induced suppression of eosinophil-associated RNase1 (EAR1). Competitive inhibition of the p53-PAI-1 mRNA interaction by expressing p53-binding 3'UTR sequences of PAI-1 mRNA likewise suppressed CS-induced PAI-1 and AEC apoptosis and restored EAR1 expression. Consistent with PCSE-induced lung injury, IAV infection increased p53, PAI-1 and apoptosis in AECs in association with pulmonary inflammation. Lung inflammation induced by PCSE was worsened by subsequent exposure to IAV. Mice lacking PAI-1 that were exposed to IAV showed minimal viral burden based on M2 antigen and hemagglutination analyses, whereas transgenic mice that overexpress PAI-1 without PCSE showed increased M2 antigen and inflammation after IAV infection. These observations indicate that increased PAI-1 expression promotes AEC apoptosis and exacerbates lung inflammation induced by IAV following PCSE.
Assuntos
Vírus da Influenza A/fisiologia , Influenza Humana/complicações , Lesão Pulmonar/virologia , Infecções por Orthomyxoviridae/complicações , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Fumar , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/patologia , Animais , Apoptose/efeitos dos fármacos , Caveolina 1/farmacologia , Humanos , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/patologia , Influenza Humana/virologia , Elastase de Leucócito/metabolismo , Luciferases/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/enzimologia , Pulmão/patologia , Lesão Pulmonar/etiologia , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Fragmentos de Peptídeos/farmacologia , Peroxidase/metabolismo , Regiões Promotoras Genéticas/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Wnt signaling is essential for tissue homeostasis and its dysregulation causes cancer. Wnt ligands trigger signaling by activating Frizzled receptors (FZDRs), which belong to the G-protein coupled receptor superfamily. However, the mechanisms of G protein activation in Wnt signaling remain controversial. In this study, we demonstrate that FZDRs activate G proteins and trigger non-canonical Wnt signaling via the Dishevelled-binding protein, Daple. Daple contains a Gα-binding and activating (GBA) motif, which activates Gαi proteins and an adjacent domain that directly binds FZDRs, thereby linking Wnt stimulation to G protein activation. This triggers non-canonical Wnt responses, that is, suppresses the ß-catenin/TCF/LEF pathway and tumorigenesis, but enhances PI3K-Akt and Rac1 signals and tumor cell invasiveness. In colorectal cancers, Daple is suppressed during adenoma-to-carcinoma transformation and expressed later in metastasized tumor cells. Thus, Daple activates Gαi and enhances non-canonical Wnt signaling by FZDRs, and its dysregulation can impact both tumor initiation and progression to metastasis.
Assuntos
Receptores Frizzled/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Via de Sinalização Wnt , HumanosRESUMO
Although aberrant fibrinolysis and plasminogen activator inhibitor 1 (PAI-1) are implicated in acute lung injury, the role of this serpin in the pathogenesis of wood bark smoke (WBS)-induced acute lung injury (SIALI) and its regulation in resident lung cells after exposure to smoke are unclear. A total of 22 mechanically ventilated pigs were included in this study. Immunohistochemical analyses were used to assess fibrin and PAI-1 in the lungs of pigs with SIALI in situ. Plasminogen activator inhibitor 1 was measured in bronchoalveolar lavage fluids by Western blotting. Induction of PAI-1 was determined at the protein and mRNA levels by Western and polymerase chain reaction analyses in primary porcine alveolar type II cells, fibroblasts, and pleural mesothelial cells. Plasminogen activator inhibitor 1 mRNA stability was determined by transcription chase studies. Gel shift analyses were used to characterize the mechanism regulating PAI-1 mRNA stability. Smoke-induced ALI induced PAI-1, with prominent extravascular fibrin deposition in large and small airways as well as alveolar and subpleural compartments. In pleural mesothelial cells, lung fibroblasts, and alveolar type II cells, PAI-1 mRNA was stabilized by WBS extract and contributed to induction of PAI-1. The mechanism involves dissociation of a novel 6-phospho-d-gluconate-NADP oxidoreductase-like PAI-1 mRNA binding protein from PAI-1 mRNA. Exposure to WBS induces prominent airway and mesothelial expression of PAI-1, associated with florid distribution of fibrin in SIALI in vivo Wood bark smoke components induce PAI-1 in vitro in part by stabilization of PAI-1 mRNA, a newly recognized pathway that may promote extravascular fibrin deposition and lung dysfunction in SIALI.