RESUMEN
G-protein signaling modulator-3 (GPSM3), also known as G18 or AGS4, is a member of the Gα(i/o)-Loco (GoLoco) motif containing proteins. GPSM3 acts through its two GoLoco motifs to exert GDP dissociation inhibitor activity over Gα(i) subunits; recently revealed is the existence of an additional regulatory site within GPSM3 directed toward monomeric Gß subunits during their biosynthesis. Here, using in silico and proteomic approaches, we have found that GPSM3 also interacts directly with numerous members of the 14-3-3 protein family. This interaction is dependent on GPSM3 phosphorylation, creating a mode II consensus 14-3-3 binding site. 14-3-3 binding to the N-terminal disordered region of GPSM3 confers stabilization from protein degradation. The complex of GPSM3 and 14-3-3 is exclusively cytoplasmic, and both moieties mutually control their exclusion from the nucleus. Phosphorylation of GPSM3 by a proline-directed serine/threonine kinase and the resultant association of 14-3-3 is the first description of post-translational regulation of GPSM3 subcellular localization, a process that likely regulates important spatio-temporal aspects of G-protein-coupled receptor signaling modulation by GPSM3.
Asunto(s)
Proteínas 14-3-3/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Proteolisis , Transducción de Señal/fisiología , Proteínas 14-3-3/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/genética , Células HEK293 , Humanos , Fosforilación/fisiología , Estabilidad Proteica , Transporte de Proteínas/fisiologíaRESUMEN
Regulation of the assembly and function of G-protein heterotrimers (Gα·GDP/Gßγ) is a complex process involving the participation of many accessory proteins. One of these regulators, GPSM3, is a member of a family of proteins containing one or more copies of a small regulatory motif known as the GoLoco (or GPR) motif. Although GPSM3 is known to bind Gα(i)·GDP subunits via its GoLoco motifs, here we report that GPSM3 also interacts with the Gß subunits Gß1 to Gß4, independent of Gγ or Gα·GDP subunit interactions. Bimolecular fluorescence complementation studies suggest that the Gß-GPSM3 complex is formed at, and transits through, the Golgi apparatus and also exists as a soluble complex in the cytoplasm. GPSM3 and Gß co-localize endogenously in THP-1 cells at the plasma membrane and in a juxtanuclear compartment. We provide evidence that GPSM3 increases Gß stability until formation of the Gßγ dimer, including association of the Gß-GPSM3 complex with phosducin-like protein PhLP and T-complex protein 1 subunit eta (CCT7), two known chaperones of neosynthesized Gß subunits. The Gß interaction site within GPSM3 was mapped to a leucine-rich region proximal to the N-terminal side of its first GoLoco motif. Both Gß and Gα(i)·GDP binding events are required for GPSM3 activity in inhibiting phospholipase-Cß activation. GPSM3 is also shown in THP-1 cells to be important for Akt activation, a known Gßγ-dependent pathway. Discovery of a Gß/GPSM3 interaction, independent of Gα·GDP and Gγ involvement, adds to the combinatorial complexity of the role of GPSM3 in heterotrimeric G-protein regulation.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Complejos Multiproteicos/metabolismo , Animales , Células COS , Chaperonina con TCP-1/genética , Chaperonina con TCP-1/metabolismo , Chlorocebus aethiops , Activación Enzimática/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades beta de la Proteína de Unión al GTP/genética , Inhibidores de Disociación de Guanina Nucleótido/genética , Células HEK293 , Humanos , Complejos Multiproteicos/genética , Fosfolipasa C beta/genética , Fosfolipasa C beta/metabolismoRESUMEN
BACKGROUND: Regulator of G-protein signaling (RGS) proteins have been well-described as accelerators of Galpha-mediated GTP hydrolysis ("GTPase-accelerating proteins" or GAPs). However, RGS proteins with complex domain architectures are now known to regulate much more than Galpha GTPase activity. RGS14 contains tandem Ras-binding domains that have been reported to bind to Rap- but not Ras GTPases in vitro, leading to the suggestion that RGS14 is a Rap-specific effector. However, more recent data from mammals and Drosophila imply that, in vivo, RGS14 may instead be an effector of Ras. METHODOLOGY/PRINCIPAL FINDINGS: Full-length and truncated forms of purified RGS14 protein were found to bind indiscriminately in vitro to both Rap- and Ras-family GTPases, consistent with prior literature reports. In stark contrast, however, we found that in a cellular context RGS14 selectively binds to activated H-Ras and not to Rap isoforms. Co-transfection / co-immunoprecipitation experiments demonstrated the ability of full-length RGS14 to assemble a multiprotein complex with components of the ERK MAPK pathway in a manner dependent on activated H-Ras. Small interfering RNA-mediated knockdown of RGS14 inhibited both nerve growth factor- and basic fibrobast growth factor-mediated neuronal differentiation of PC12 cells, a process which is known to be dependent on Ras-ERK signaling. CONCLUSIONS/SIGNIFICANCE: In cells, RGS14 facilitates the formation of a selective Ras.GTP-Raf-MEK-ERK multiprotein complex to promote sustained ERK activation and regulate H-Ras-dependent neuritogenesis. This cellular function for RGS14 is similar but distinct from that recently described for its closely-related paralogue, RGS12, which shares the tandem Ras-binding domain architecture with RGS14.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factor de Crecimiento Nervioso/antagonistas & inhibidores , Proteínas RGS/fisiología , Proteínas ras/metabolismo , Animales , Sitios de Unión , Diferenciación Celular , Quinasas MAP Reguladas por Señal Extracelular , Factor 2 de Crecimiento de Fibroblastos/fisiología , Humanos , Ratones , Proteínas Quinasas Activadas por Mitógenos , Complejos Multiproteicos , Factor de Crecimiento Nervioso/fisiología , Neuritas , Células PC12 , Unión Proteica , Ratas , Quinasas rafRESUMEN
Recently, we identified a novel signaling pathway involving Epac, Rap, and phospholipase C (PLC)epsilon that plays a critical role in maximal beta-adrenergic receptor (betaAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we demonstrate that PLCepsilon phosphatidylinositol 4,5-bisphosphate hydrolytic activity and PLCepsilon-stimulated Rap1 GEF activity are both required for PLCepsilon-mediated enhancement of sarcoplasmic reticulum Ca2+ release and that PLCepsilon significantly enhances Rap activation in response to betaAR stimulation in the heart. Downstream of PLCepsilon hydrolytic activity, pharmacological inhibition of PKC significantly inhibited both betaAR- and Epac-stimulated increases in CICR in PLCepsilon+/+ myocytes but had no effect in PLCepsilon-/- myocytes. betaAR and Epac activation caused membrane translocation of PKCepsilon in PLCepsilon+/+ but not PLCepsilon-/- myocytes and small interfering RNA-mediated PKCepsilon knockdown significantly inhibited both betaAR and Epac-mediated CICR enhancement. Further downstream, the Ca2+/calmodulin-dependent protein kinase II (CamKII) inhibitor, KN93, inhibited betaAR- and Epac-mediated CICR in PLCepsilon+/+ but not PLCepsilon-/- myocytes. Epac activation increased CamKII Thr286 phosphorylation and enhanced phosphorylation at CamKII phosphorylation sites on the ryanodine receptor (RyR2) (Ser2815) and phospholamban (Thr17) in a PKC-dependent manner. Perforated patch clamp experiments revealed that basal and betaAR-stimulated peak L-type current density are similar in PLCepsilon+/+ and PLCepsilon-/- myocytes suggesting that control of sarcoplasmic reticulum Ca2+ release, rather than Ca2+ influx through L-type Ca2+ channels, is the target of regulation of a novel signal transduction pathway involving sequential activation of Epac, PLCepsilon, PKCepsilon, and CamKII downstream of betaAR activation.
Asunto(s)
Señalización del Calcio/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calcio/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Miocardio/enzimología , Fosfoinositido Fosfolipasa C/metabolismo , Animales , Bencilaminas/farmacología , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Membrana Celular/enzimología , Membrana Celular/genética , Activación Enzimática/fisiología , Factores de Intercambio de Guanina Nucleótido/genética , Ratones , Ratones Noqueados , Miocardio/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/enzimología , Fosfatidilinositol 4,5-Difosfato , Fosfoinositido Fosfolipasa C/genética , Fosforilación/fisiología , Proteína Quinasa C-epsilon/genética , Proteína Quinasa C-epsilon/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas/fisiología , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/enzimología , Retículo Sarcoplasmático/genética , Sulfonamidas/farmacologíaAsunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Miocardio/metabolismo , Animales , Señalización del Calcio/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Estimulación Eléctrica , Ratones , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , RatasRESUMEN
Phospholipase Cepsilon (PLCepsilon) has been suggested to transduce signals from small GTPases, but its biological function has not yet been clarified. Using astrocytes from PLCepsilon-deficient mice, we demonstrate that endogenous G protein-coupled receptors (GPCRs) for lysophosphatidic acid, sphingosine 1-phosphate, and thrombin regulate phosphoinositide hydrolysis primarily through PLCepsilon. Stimulation by lysophospholipids occurs through G(i), whereas thrombin activates PLC through Rho. Further studies reveal that PLCepsilon is required for thrombin- but not LPA-induced sustained ERK activation and DNA synthesis, providing a novel mechanism for GPCR and Rho signaling to cell proliferation. The requirement for PLCepsilon in this pathway can be explained by its role as a guanine nucleotide exchange factor for Rap1. Thus, PLCepsilon serves to transduce mitogenic signals through a mechanism distinct from its role in generation of PLC-derived second messengers.
Asunto(s)
Astrocitos/citología , Receptores Acoplados a Proteínas G/metabolismo , Fosfolipasas de Tipo C/fisiología , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Astrocitos/metabolismo , Carbacol/farmacología , Proliferación Celular , GTP Fosfohidrolasas/metabolismo , Genotipo , Lisofosfolípidos/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Fosfoinositido Fosfolipasa C , Transducción de SeñalRESUMEN
Recently we demonstrated that PLC(epsilon) plays an important role in beta-adrenergic receptor (betaAR) stimulation of Ca(2+)-induced Ca(2+) release (CICR) in cardiac myocytes. Here we have reported for the first time that a pathway downstream of betaAR involving the cAMP-dependent Rap GTP exchange factor, Epac, and PLC(epsilon) regulates CICR in cardiac myocytes. To demonstrate a role for Epac in the stimulation of CICR, cardiac myocytes were treated with an Epac-selective cAMP analog, 8-4-(chlorophenylthio)-2'-O-methyladenosine-3',5'-monophosphate (cpTOME). cpTOME treatment increased the amplitude of electrically evoked Ca(2+) transients, implicating Epac for the first time in cardiac CICR. This response is abolished in PLC(epsilon)(-/-) cardiac myocytes but rescued by transduction with PLC(epsilon), indicating that Epac is upstream of PLC(epsilon). Furthermore, transduction of PLC(epsilon)(+/+) cardiac myocytes with a Rap inhibitor, RapGAP1, significantly inhibited isoproterenol-dependent CICR. Using a combination of cpTOME and PKA-selective activators and inhibitors, we have shown that betaAR-dependent increases in CICR consist of two independent components mediated by PKA and the novel Epac/(epsilon) pathway. We also show that Epac/PLC(epsilon)-dependent effects on CICR are independent of sarcoplasmic reticulum loading and Ca(2+) clearance mechanisms. These data define a novel endogenous PKA-independent betaAR-signaling pathway through cAMP-dependent Epac activation, Rap, and PLC(epsilon) that enhances intracellular Ca(2+) release in cardiac myocytes.
Asunto(s)
Señalización del Calcio/fisiología , Miocitos Cardíacos/enzimología , Receptores Adrenérgicos beta/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Ratones , Ratones Noqueados , Miocitos Cardíacos/citología , Fosfoinositido Fosfolipasa C , Retículo Sarcoplasmático/enzimología , Retículo Sarcoplasmático/genética , Transducción Genética , Fosfolipasas de Tipo C/deficiencia , Proteínas de Unión al GTP rap/metabolismoRESUMEN
Phospholipase C (PLC) epsilon is a recently identified enzyme regulated by a wide range of molecules including Ras family small GTPases, Rho A, Galpha(12/13), and Gbetagamma with primary sites of expression in the heart and lung. In a screen for human signal transduction genes altered during heart failure, we found that PLCepsilon mRNA is upregulated. Two murine models of cardiac hypertrophy confirmed upregulation of PLCepsilon protein expression or PLCepsilon RNA. To identify a role for PLCepsilon in cardiac function and pathology, a PLCepsilon-deficient mouse strain was created. Echocardiography indicated PLCepsilon(-/-) mice had decreased cardiac function, and direct measurements of left ventricular contraction demonstrated that PLCepsilon(-/-) mice had a decreased contractile response to acute isoproterenol administration. Cardiac myocytes isolated from PLCepsilon(-/-) mice had decreased beta-adrenergic receptor (betaAR)-dependent increases in Ca2+ transient amplitudes, likely accounting for the contractile deficiency in vivo. This defect appears to be independent from the ability of the betaAR system to produce cAMP and regulation of sarcoplasmic reticulum Ca2+ pool size. To address the significance of these functional deficits to cardiac pathology, PLCepsilon(-/-) mice were subjected to a chronic isoproterenol model of hypertrophic stress. PLCepsilon(-/-) mice were more susceptible than wild-type littermates to development of hypertrophy than wild-type littermates. Together, these data suggest a novel PLC-dependent component of betaAR signaling in cardiac myocytes responsible for maintenance of maximal contractile reserve and loss of PLCepsilon signaling sensitizes the heart to development of hypertrophy in response to chronic cardiac stress.