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
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
Signals propagated by receptor tyrosine kinases (RTKs) can drive cell migration and proliferation, two cellular processes that do not occur simultaneously--a phenomenon called "migration-proliferation dichotomy." We previously showed that epidermal growth factor (EGF) signaling is skewed to favor migration over proliferation via noncanonical transactivation of Gαi proteins by the guanine exchange factor (GEF) GIV. However, what turns on GIV-GEF downstream of growth factor RTKs remained unknown. Here we reveal the molecular mechanism by which phosphorylation of GIV by cyclin-dependent kinase 5 (CDK5) triggers GIV's ability to bind and activate Gαi in response to growth factors and modulate downstream signals to establish a dichotomy between migration and proliferation. We show that CDK5 binds and phosphorylates GIV at Ser1674 near its GEF motif. When Ser1674 is phosphorylated, GIV activates Gαi and enhances promigratory Akt signals. Phosphorylated GIV also binds Gαs and enhances endosomal maturation, which shortens the transit time of EGFR through early endosomes, thereby limiting mitogenic MAPK signals. Consequently, this phosphoevent triggers cells to preferentially migrate during wound healing and transmigration of cancer cells. When Ser1674 cannot be phosphorylated, GIV cannot bind either Gαi or Gαs, Akt signaling is suppressed, mitogenic signals are enhanced due to delayed transit time of EGFR through early endosomes, and cells preferentially proliferate. These results illuminate how GIV-GEF is turned on upon receptor activation, adds GIV to the repertoire of CDK5 substrates, and defines a mechanism by which this unusual CDK orchestrates migration-proliferation dichotomy during cancer invasion, wound healing, and development.
Assuntos
Movimento Celular , Proliferação de Células , Quinase 5 Dependente de Ciclina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Animais , Receptores ErbB/metabolismo , Humanos , Proteínas dos Microfilamentos/química , Dados de Sequência Molecular , Morfogênese , Fosforilação , Transporte Proteico , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteínas de Transporte Vesicular/química , CicatrizaçãoRESUMO
Gα-interacting, vesicle-associated protein (GIV/Girdin) is a multidomain signal transducer that enhances PI3K-Akt signals downstream of both G-protein-coupled receptors and growth factor receptor tyrosine kinases during diverse biological processes and cancer metastasis. Mechanistically, GIV serves as a non-receptor guanine nucleotide exchange factor (GEF) that enhances PI3K signals by activating trimeric G proteins, Gαi1/2/3. Site-directed mutations in GIV's GEF motif disrupt its ability to bind or activate Gi and abrogate PI3K-Akt signals; however, nothing is known about how GIV's GEF function is regulated. Here we report that PKCθ, a novel protein kinase C, down-regulates GIV's GEF function by phosphorylating Ser(S)1689 located within GIV's GEF motif. We demonstrate that PKCθ specifically binds and phosphorylates GIV at S1689, and this phosphoevent abolishes GIV's ability to bind and activate Gαi. HeLa cells stably expressing the phosphomimetic mutant of GIV, GIV-S1689âD, are phenotypically identical to those expressing the GEF-deficient F1685A mutant: Actin stress fibers are decreased and cell migration is inhibited whereas cell proliferation is triggered, and Akt (a.k.a. protein kinase B, PKB) activation is impaired downstream of both the lysophosphatidic acid receptor, a G-protein-coupled receptor, and the insulin receptor, a receptor tyrosine kinase. These findings indicate that phosphorylation of GIV by PKCθ inhibits GIV's GEF function and generates a unique negative feedback loop for downregulating the GIV-Gi axis of prometastatic signaling downstream of multiple ligand-activated receptors. This phosphoevent constitutes the only regulatory pathway described for terminating signaling by any of the growing family of nonreceptor GEFs that modulate G-protein activity.
Assuntos
Isoenzimas/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Proteína Quinase C/metabolismo , Transdução de Sinais/genética , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Actinas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Proteínas dos Microfilamentos/genética , Mutação de Sentido Incorreto/genética , Fosforilação , Proteína Quinase C-theta , Proteínas de Transporte Vesicular/genéticaRESUMO
Insulin resistance (IR) is a metabolic disorder characterized by impaired glucose uptake in response to insulin. The current paradigm for insulin signaling centers upon the insulin receptor (InsR) and its substrate IRS1; the latter is believed to be the chief conduit for post-receptor signaling. We recently demonstrated that GIV, a Guanidine Exchange Factor (GEF) for the trimeric G protein, Gαi, is a major hierarchical conduit for the metabolic insulin response. By virtue of its ability to directly bind the InsR, IRS1 and PI3K, GIV enhances the InsR-IRS1-Akt-AS160 (RabGAP) signaling cascade and cellular glucose uptake via its GEF function. Phosphoinhibition of GIV-GEF by the fatty-acid/PKCθ pathway inhibits the cascade and impairs glucose uptake. Here we show that GIV directly and constitutively binds the exocyst complex subunit Exo-70 and also associates with GLUT4-storage vesicles (GSVs) exclusively upon insulin stimulation. Without GIV or its GEF function, membrane association of Exo-70 as well as exocytosis of GSVs in response to insulin are impaired. Thus, GIV is an essential component within the insulin signaling cascade that couples upstream signal transducers within the InsR and G-Protein signaling cascade to downstream vesicular trafficking events within the exocytic pathway. These findings suggest a role of GIV in coordinating key signaling and trafficking events of metabolic insulin response.
Assuntos
Transportador de Glucose Tipo 4/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Linhagem Celular , Exocitose , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Humanos , Insulina/metabolismo , Ligação Proteica , Transporte Proteico , Ratos , Transdução de SinaisRESUMO
For a sperm to successfully fertilize an egg, it must first undergo capacitation in the female reproductive tract and later undergo acrosomal reaction (AR) upon encountering an egg surrounded by its vestment. How premature AR is avoided despite rapid surges in signaling cascades during capacitation remains unknown. Using a combination of conditional knockout (cKO) mice and cell-penetrating peptides, we show that GIV (CCDC88A), a guanine nucleotide-exchange modulator (GEM) for trimeric GTPases, is highly expressed in spermatocytes and is required for male fertility. GIV is rapidly phosphoregulated on key tyrosine and serine residues in human and murine spermatozoa. These phosphomodifications enable GIV-GEM to orchestrate two distinct compartmentalized signaling programs in the sperm tail and head; in the tail, GIV enhances PI3KâAkt signals, sperm motility and survival, whereas in the head it inhibits cAMP surge and premature AR. Furthermore, GIV transcripts are downregulated in the testis and semen of infertile men. These findings exemplify the spatiotemporally segregated signaling programs that support sperm capacitation and shed light on a hitherto unforeseen cause of infertility in men.
Assuntos
Fertilidade , Regulação da Expressão Gênica , Proteínas dos Microfilamentos/genética , Transdução de Sinais/genética , Capacitação Espermática/genética , Proteínas de Transporte Vesicular/genética , Animais , Regulação para Baixo , Feminino , Fertilidade/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Fosforilação , Espermatócitos/metabolismo , Espermatozoides/metabolismo , Testículo/citologia , Testículo/patologiaRESUMO
Human vaccinia-related kinase (VRK) 1 is a novel serine-threonine kinase that regulates several transcription factors, nuclear envelope assembly, and chromatin condensation and is also required for cell cycle progression. The regulation of this kinase family is unknown. Mass spectrometry has permitted the identification of Ran as an interacting and regulatory protein of the VRK serine-threonine kinase activities. The stable interaction has been validated by pulldown of endogenous proteins as well as by reciprocal immunoprecipitations. The three members of the VRK family stably interact with Ran, and the interaction was not affected by the bound nucleotide, GDP or GTP. The interaction was stronger with the RanT24N that is locked in its inactive conformation and cannot bind nucleotides. None of the kinases phosphorylated Ran or RCC1. VRK1 does not directly interact with RCC1, but if Ran is present they can be isolated as a complex. The main effect of the interaction of inactive RanGDP with VRK1 is the inhibition of its kinase activity, which was detected by a reduction in VRK1 autophosphorylation and a reduction in phosphorylation of histone H3 in residues Thr-3 and Ser-10. The kinase activity inhibition can be relieved by the interaction with the constitutively active RanGTP or RanL43E, which locks Ran in its GTP-bound active conformation. In this complex, the interaction with VRK proteins does not alter the effect of its guanine exchange factor, RCC1. Ran is a novel negative regulator of nuclear VRK1 and VRK2 kinase activity, which may vary in different subcellular localizations generating an asymmetric intracellular distribution of kinase activity depending on local protein interactions.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteína ran de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Núcleo Celular/enzimologia , Primers do DNA/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteína ran de Ligação ao GTP/genéticaRESUMO
Cells perceive and respond to the extracellular matrix via integrin receptors; their dysregulation has been implicated in inflammation and cancer metastasis. Here we show that a guanine nucleotide-exchange modulator of trimeric-GTPase Gαi, GIV (a.k.a Girdin), directly binds the integrin adaptor Kindlin-2. A non-canonical short linear motif within the C terminus of GIV binds Kindlin-2-FERM3 domain at a site that is distinct from the binding site for the canonical NPxY motif on the -integrin tail. Binding of GIV to Kindlin-2 allosterically enhances Kindlin-2's affinity for ß1-integrin. Consequently, integrin activation and clustering are maximized, which augments cell adhesion, spreading, and invasion. Findings elucidate how the GIVâ¢Kindlin-2 complex has a 2-fold impact: it allosterically synergizes integrin activation and enables ß1-integrins to indirectly access and modulate trimeric GTPases via the complex. Furthermore, Cox proportional-hazard models on tumor transcriptomics provide trans-scale evidence of synergistic interactions between GIVâ¢Kindlin-2â¢ß1-integrin on time to progression to metastasis.
RESUMO
Cellular proliferation, differentiation, and morphogenesis are shaped by multiple signaling cascades, and their dysregulation plays an integral role in cancer progression. Three cascades that contribute to oncogenic potential are those mediated by Wnt proteins and the receptor Frizzled (FZD), growth factor receptor tyrosine kinases (RTKs), and heterotrimeric G proteins and associated GPCRs. Daple is a guanine nucleotide exchange factor (GEF) for the G protein Gαi Daple also binds to FZD and the Wnt/FZD mediator Dishevelled (Dvl), and it enhances ß-catenin-independent Wnt signaling in response to Wnt5a-FZD7 signaling. We identified Daple as a substrate of multiple RTKs and non-RTKs and, hence, as a point of convergence for the three cascades. We found that phosphorylation near the Dvl-binding motif in Daple by both RTKs and non-RTKs caused Daple/Dvl complex dissociation and augmented the ability of Daple to bind to and activate Gαi, which potentiated ß-catenin-independent Wnt signals and stimulated epithelial-mesenchymal transition (EMT) similarly to Wnt5a/FZD7 signaling. Although Daple acts as a tumor suppressor in the healthy colon, the concurrent increased abundance of Daple and epidermal growth factor receptor (EGFR) in colorectal tumors was associated with poor patient prognosis. Thus, the Daple-dependent activation of Gαi and the Daple-dependent enhancement of ß-catenin-independent Wnt signals are not only stimulated by Wnt5a/FZD7 to suppress tumorigenesis but also hijacked by growth factor-activated RTKs to enhance tumor progression. These findings identify a cross-talk paradigm among growth factor RTKs, heterotrimeric G proteins, and the Wnt/FZD pathway in cancer.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas Heterotriméricas de Ligação ao GTP/genética , Receptores Proteína Tirosina Quinases/genética , Proteínas Wnt/genética , Via de Sinalização Wnt/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Proteínas Desgrenhadas/genética , Proteínas Desgrenhadas/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Estimativa de Kaplan-Meier , Fosforilação , Ligação Proteica , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Wnt/metabolismoRESUMO
Wnt proteins preferentially activate either ß-catenin-dependent or ß-catenin-independent signals, but the activity of a particular Wnt also depends on cellular context and receptor availability. We previously reported that Wnt-3a induces neural differentiation of human embryonic stem cell-derived neural stem cells (NSCs) in a ß-catenin-independent manner by activating a signal involving JNK and the AP-1 family member ATF-2. Here, we report the results of a gene silencing approach to identify the Wnt receptors that mediate this response to Wnt-3a. Silencing of ROR2 increased neuronal differentiation, as measured by expression of the genes DCX, NEUROD1, and NGN1, suggesting ROR2 signals normally prevent differentiation. Silencing of the other Wnt receptors singly did not affect Wnt-3a-induced neuronal differentiation. However, pairwise silencing of ROR1 and FZD4 or FZD5 and of LRP6 and FZD4 or FZD5 inhibited neuronal differentiation, as detected by reductions in the expression of neuronal genes and immunocytochemical detection of DCX, NEUROD1 and DCX. Ectopic expression of these receptors in HEK 293 cells increased ATF2-dependent transcription. In addition, ROR1 coimmunoprecipitated with FZD4 and LRP6 in transfected HEK 293 cells and colocalized with FZD4 and with LRP6 at the cell surface of transfected L cells. Wnt-3a did not appear to affect these interactions but did alter the interactions between LRP6 and FZD4/5. Together, these observations highlight roles for ROR1, LRP6, FZD4, and FZD5 in neural stem cell differentiation and provide support for a model in which dynamic interactions among these receptors mediate Wnt-3a activation of ATF2 signaling.
Assuntos
Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Receptores Wnt/metabolismo , Proteína Wnt3A/farmacologia , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Sox2 is a pluripotency transcription factor that as an oncogene can also regulate cell proliferation. Therefore, genes implicated in several different aspects of cell proliferation, such as the VRK1 chromatin-kinase, are candidates to be targets of Sox2. Sox 2 and VRK1 colocalize in nuclei of proliferating cells forming a stable complex. Sox2 knockdown abrogates VRK1 gene expression. Depletion of either Sox2 or VRK1 caused a reduction of cell proliferation. Sox2 up-regulates VRK1 expression and both proteins cooperate in the activation of CCND1. The accumulation of VRK1 protein downregulates SOX2 expression and both proteins are lost in terminally differentiated cells. Induction of neural differentiation with retinoic acid resulted in downregulation of Sox2 and VRK1 that inversely correlated with the expression of differentiation markers such as N-cadherin, Pax6, mH2A1.2 and mH2A2. Differentiation-associated macro histones mH2A1.2and mH2A2 inhibit CCND1 and VRK1 expression and also block the activation of the VRK1 promoter by Sox2. VRK1 is a downstream target of Sox2 and both form an autoregulatory loop in epithelial cell differentiation.
Assuntos
Ciclo Celular/genética , Diferenciação Celular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Oncogenes/genética , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição SOXB1/genética , Biomarcadores/metabolismo , Caderinas/genética , Carcinogênese/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Ciclina D1/genética , Regulação para Baixo/genética , Epitélio/metabolismo , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Fator de Transcrição PAX6/genética , Regiões Promotoras Genéticas/genética , Regulação para Cima/genéticaRESUMO
Canonical signal transduction via heterotrimeric G proteins is spatiotemporally restricted, i.e., triggered exclusively at the plasma membrane, only by agonist activation of G protein-coupled receptors via a finite process that is terminated within a few hundred milliseconds. Recently, a rapidly emerging paradigm has revealed a noncanonical pathway for activation of heterotrimeric G proteins via the nonreceptor guanidine-nucleotide exchange factor, GIV/Girdin. Biochemical, biophysical, and functional studies evaluating this pathway have unraveled its unique properties and distinctive spatiotemporal features. As in the case of any new pathway/paradigm, these studies first required an in-depth optimization of tools/techniques and protocols, governed by rationale and fundamentals unique to the pathway, and more specifically to the large multimodular GIV protein. Here we provide the most up-to-date overview of protocols that have generated most of what we know today about noncanonical G protein activation by GIV and its relevance in health and disease. © 2016 by John Wiley & Sons, Inc.
Assuntos
Imunofluorescência/métodos , Fatores de Troca do Nucleotídeo Guanina/análise , Immunoblotting/métodos , Imunoprecipitação/métodos , Animais , Biofísica/métodos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Transdução de SinaisRESUMO
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
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
Insulin resistance (IR) is a metabolic disorder characterized by impaired insulin signaling and cellular glucose uptake. The current paradigm for insulin signaling centers upon the insulin receptor (InsR) and its substrate IRS1; the latter is believed to be the sole conduit for postreceptor signaling. Here we challenge that paradigm and show that GIV/Girdin, a guanidine exchange factor (GEF) for the trimeric G protein Gαi, is another major hierarchical conduit for the metabolic insulin response. By virtue of its ability to directly bind InsR, IRS1, and phosphoinositide 3-kinase, GIV serves as a key hub in the immediate postreceptor level, which coordinately enhances the metabolic insulin response and glucose uptake in myotubes via its GEF function. Site-directed mutagenesis or phosphoinhibition of GIV-GEF by the fatty acid/protein kinase C-theta pathway triggers IR. Insulin sensitizers reverse phosphoinhibition of GIV and reinstate insulin sensitivity. We also provide evidence for such reversible regulation of GIV-GEF in skeletal muscles from patients with IR. Thus GIV is an essential upstream component that couples InsR to G-protein signaling to enhance the metabolic insulin response, and impairment of such coupling triggers IR. We also provide evidence that GIV-GEF serves as therapeutic target for exogenous manipulation of physiological insulin response and reversal of IR in skeletal muscles.
Assuntos
Reguladores de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Resistência à Insulina/fisiologia , Proteínas dos Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Células Cultivadas , Ácidos Graxos/metabolismo , Feminino , Humanos , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Músculo Esquelético/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/metabolismo , Transdução de SinaisRESUMO
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
A long-standing issue in the field of signal transduction is to understand the cross-talk between receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major and distinct signaling hubs that control eukaryotic cell behavior. Although stimulation of many RTKs leads to activation of trimeric G proteins, the molecular mechanisms behind this phenomenon remain elusive. We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins. Using a combination of homology modeling, protein-protein interaction, and kinase assays, we demonstrate that a stretch of â¼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands. Using protein-protein interaction assays, we demonstrated that both SH2 and GEF domains of GIV are required for the formation of a ligand-activated ternary complex between GIV, Gαi, and growth factor receptors and for activation of Gαi after growth factor stimulation. Expression of a SH2-deficient GIV mutant (Arg 1745âLeu) that cannot bind RTKs impaired all previously demonstrated functions of GIV-Akt enhancement, actin remodeling, and cell migration. The mechanistic and structural insights gained here shed light on the long-standing questions surrounding RTK/G protein cross-talk, set a novel paradigm, and characterize a unique pharmacological target for uncoupling GIV-dependent signaling downstream of multiple oncogenic RTKs.
Assuntos
Receptores ErbB/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Proteínas dos Microfilamentos/química , Proteínas de Transporte Vesicular/química , Sequência de Aminoácidos , Animais , Movimento Celular , Receptores ErbB/genética , Receptores ErbB/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Homologia Estrutural de Proteína , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
DNA damage immediate cellular response requires the activation of p53 by kinases. We found that p53 forms a basal stable complex with VRK1, a Ser-Thr kinase that responds to UV-induced DNA damage by specifically phosphorylating p53. This interaction takes place through the p53 DNA binding domain, and frequent DNA-contact mutants of p53, such as R273H, R248H or R280K, do not disrupt the complex. UV-induced DNA damage activates VRK1, and is accompanied by phosphorylation of p53 at Thr-18 before it accumulates. We propose that the VRK1-p53 basal complex is an early-warning system for immediate cellular responses to DNA damage.