RESUMEN
The contractile phenotype of smooth muscle (SM) cells is controlled by serum response factor (SRF), which drives the expression of SM-specific genes including SM alpha-actin, SM22, and others. Myocardin is a cardiac and SM-restricted coactivator of SRF that is necessary for SM gene transcription. Growth factors inducing proliferation of SM cells inhibit SM gene transcription, in a manner dependent on the activation of extracellular signal-regulated kinases ERK1/2. In this study, we found that ERK1/2 phosphorylates mouse myocardin (isoform B) at four sites (Ser(812), Ser(859), Ser(866), and Thr(893)), all of which are located within the transactivation domain of myocardin. The single mutation of each site either to alanine or to aspartate has no effect on the ability of myocardin to activate SRF. However, the phosphomimetic mutation of all four sites to aspartate (4xD) significantly impairs activation of SRF by myocardin, whereas the phosphodeficient mutation of all four sites to alanine (4xA) has no effect. This translates to a reduced ability of the 4xD (but not of 4xA) mutant of myocardin to stimulate expression of SM alpha-actin and SM22, as assessed by corresponding promoter, mRNA, or protein assays. Furthermore, we found that phosphorylation of myocardin at these sites impairs its interaction with acetyltransferase, cAMP response element-binding protein-binding protein, which is known to promote the transcriptional activity of myocardin. In conclusion, we describe a novel mode of modulation of SM gene transcription by ERK1/2 through a direct phosphorylation of myocardin.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica , Proteínas Nucleares/fisiología , Transactivadores/fisiología , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Activación Enzimática , Ratones , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Datos de Secuencia Molecular , Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , Fenotipo , Fosforilación , Homología de Secuencia de Aminoácido , Transactivadores/metabolismoRESUMEN
Regulator of G protein signaling (RGS) proteins are united into a family by the presence of the homologous RGS domain that binds the alpha subunits of heterotrimeric G proteins and accelerates their GTPase activity. A member of this family, RGS3 regulates the signaling mediated by G(q) and G(i) proteins by binding the corresponding Galpha subunits. Here we show that RGS3 interacts with the novel partners Smad2, Smad3, and Smad4-the transcription factors that are activated through a transforming growth factor-beta (TGF-beta) receptor signaling. This interaction is mediated by the region of RGS3 outside of the RGS domain and by Smad's Mad homology 2 domain. Overexpression of RGS3 results in inhibition of Smad-mediated gene transcription. RGS3 does not affect TGF-beta-induced Smad phosphorylation, but it prevents heteromerization of Smad3 with Smad4, which is required for transcriptional activity of Smads. This translates to functional inhibition of TGF-beta-induced myofibroblast differentiation by RGS3. In conclusion, this study identifies a novel, noncanonical role of RGS3 in regulation of TGF-beta signaling through its interaction with Smads and interfering with Smad heteromerization.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Smad/metabolismo , Transcripción Genética , Actinas/metabolismo , Animales , Sitios de Unión , Células CHO , Cricetinae , Cricetulus , ADN Complementario , Proteínas de Unión al GTP/química , Proteínas Activadoras de GTPasa/química , Humanos , Inmunoprecipitación , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas RGS , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Proteína Smad4/metabolismo , Transcripción Genética/efectos de los fármacos , Transfección , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Galpha12 and Galpha13 transduce signals from G protein-coupled receptors to RhoA through RhoGEFs containing an RGS homology (RH) domain, such as p115 RhoGEF or leukemia-associated RhoGEF (LARG). The RH domain of p115 RhoGEF or LARG binds with high affinity to active forms of Galpha12 and Galpha13 and confers specific GTPase-activating protein (GAP) activity, with faster GAP responses detected in Galpha13 than in Galpha12. At the same time, Galpha13, but not Galpha12, directly stimulates the RhoGEF activity of p115 RhoGEF or nonphosphorylated LARG in reconstitution assays. In order to better understand the molecular mechanism by which Galpha13 regulates RhoGEF activity through interaction with RH-RhoGEFs, we sought to identify the region(s) of Galpha13 involved in either the GAP response or RhoGEF activation. For this purpose, we generated chimeras between Galpha12 and Galpha13 subunits and characterized their biochemical activities. In both cell-based and reconstitution assays of RhoA activation, we found that replacing the carboxyl-terminal region of Galpha12 (residues 267-379) with that of Galpha13 (residues 264-377) conferred gain-of-function to the resulting chimeric subunit, Galpha12C13. The inverse chimera, Galpha13C12, exhibited basal RhoA activation which was similar to Galpha12. In contrast to GEF assays, GAP assays showed that Galpha12C13 or Galpha13C12 chimeras responded to the GAP activity of p115 RhoGEF or LARG in a manner similar to Galpha12 or Galpha13, respectively. We conclude from these results that the carboxyl-terminal region of Galpha13 (residues 264-377) is essential for its RhoGEF stimulating activity, whereas the amino-terminal alpha helical and switch regions of Galpha12 and Galpha13 are responsible for their differential GAP responses to the RH domain.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP G12-G13/química , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Proteínas RGS/química , Proteínas de Unión al GTP rho/química , Proteínas de Unión al GTP rho/metabolismo , Animales , Baculoviridae/genética , Células HeLa , Humanos , Modelos Biológicos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera/citología , Spodoptera/metabolismo , Spodoptera/virología , Proteínas de Unión al GTP rho/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
OBJECTIVE: Smooth muscle alpha-actin (SMA) is a cytoskeletal protein characteristic to vascular smooth muscle cells (VSMC), and it serves to facilitate cell contraction and migration. Bacterial lipopolysaccharide (LPS), a major mediator of septic shock secondary to infection, is known to directly affect VSMC. The objective of this study was to investigate the effect of LPS on the expression levels of SMA in VSMC. METHODS: This study was performed on cultured VSMC derived from human aorta, human coronary artery, or rat aorta. RESULTS: We show that SMA expression in VSMC, induced by endothelin-1 (ET1) or transforming growth factor-beta (TGF-beta), is potently inhibited by a LPS. This parallels a decreased migration of VSMC after LPS treatment. Downregulation of SMA by LPS is not a result of altered signaling of ET1 or TGF-beta receptors, and it is not mediated by canonical (for LPS) mechanisms, such as production of prostaglandins or nitric oxide, or secretion of other endocrine factors. On a molecular level, downregulation of SMA expression by LPS occurs at the level of transcription, as both SMA mRNA levels and SMA promoter activity are inhibited by LPS. The SMA promoter is controlled largely by two major regulatory elements-CArG boxes activated by serum response factor (SRF), and TGF-beta control elements (TCE). LPS does not affect the activity of SRF, but it potently inhibits both basal and inducible TCE activation. CONCLUSION: We show for the first time that LPS attenuates SMA transcription and protein expression in VSMC likely through inhibition of a TCE element on the SMA promoter.
Asunto(s)
Actinas/metabolismo , Regulación hacia Abajo , Lipopolisacáridos/farmacología , Miocitos del Músculo Liso/metabolismo , Choque Séptico/metabolismo , Animales , Aorta , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Vasos Coronarios , Expresión Génica/efectos de los fármacos , Humanos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Ratas , Ratas Endogámicas WKY , Choque Séptico/patología , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Regulators of G protein signalling (RGS) proteins are united into a family by the presence of the RGS domain which serves as a GTPase-activating protein (GAP) for various Galpha subunits of heterotrimeric G proteins. Through this mechanism, RGS proteins regulate signalling of numerous G protein-coupled receptors. In addition to the RGS domains, RGS proteins contain diverse regions of various lengths that regulate intracellular localization, GAP activity or receptor selectivity of RGS proteins, often through interaction with other partners. However, it is becoming increasingly appreciated that through these non-RGS regions, RGS proteins can serve non-canonical functions distinct from inactivation of Galpha subunits. This review summarizes the data implicating RGS proteins in the (i) regulation of G protein signalling by non-canonical mechanisms, (ii) regulation of non-G protein signalling, (iii) signal transduction from receptors not coupled to G proteins, (iv) activation of mitogen-activated protein kinases, and (v) non-canonical functions in the nucleus.
Asunto(s)
Proteínas RGS/fisiología , Transducción de Señal , Núcleo Celular/química , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas RGS/análisisRESUMEN
Extracellular ATP stimulates proliferation of vascular smooth muscle cells (VSMC) through activation of G protein-coupled P2Y purinergic receptors. We have previously shown that ATP stimulates a transient activation of protein kinase A (PKA), which, together with the established mitogenic signaling of purinergic receptors, promotes proliferation of VSMC (Hogarth DK, Sandbo N, Taurin S, Kolenko V, Miano JM, Dulin NO. Am J Physiol Cell Physiol 287: C449-C456, 2004). We also have shown that PKA can phosphorylate beta-catenin at two novel sites (Ser552 and Ser675) in vitro and in overexpression cell models (Taurin S, Sandbo N, Qin Y, Browning D, Dulin NO. J Biol Chem 281: 9971-9976, 2006). beta-Catenin promotes cell proliferation by activation of a family of T-cell factor (TCF) transcription factors, which drive the transcription of genes implicated in cell cycle progression including cyclin D1. In the present study, using the phosphospecific antibodies against phospho-Ser552 or phospho-Ser675 sites of beta-catenin, we show that ATP can stimulate PKA-dependent phosphorylation of endogenous beta-catenin at both of these sites without affecting its expression levels in VSMC. This translates to a PKA-dependent stimulation of TCF transcriptional activity through an increased association of phosphorylated (by PKA) beta-catenin with TCF-4. Using the PKA inhibitor PKI or dominant negative TCF-4 mutant, we show that ATP-induced cyclin D1 promoter activation, cyclin D1 protein expression, and proliferation of VSMC are all dependent on PKA and TCF activities. In conclusion, we show a novel mode of regulation of endogenous beta-catenin through its phosphorylation by PKA, and we demonstrate the importance of this mechanism for ATP-induced proliferation of VSMC.
Asunto(s)
Adenosina Trifosfato/farmacología , División Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , beta Catenina/metabolismo , Animales , Aorta , ADN Complementario/genética , Fosforilación , Ratas , Ratas Endogámicas WKY , beta Catenina/genéticaRESUMEN
Endothelin-1 (ET1) is a vasoactive peptide that stimulates hypertrophy of vascular smooth muscle cells (VSMC) through diverse signaling pathways mediated by G(q)/G(i)/G(13) heterotrimeric G proteins. We have found that ET1 stimulates the activity of cAMP-dependent protein kinase (PKA) in VSMC as profoundly as the G(s)-linked beta-adrenergic agonist, isoproterenol (ISO), but in a transient manner. PKA activation by ET1 was mediated by type-A ET1 receptors (ETA) and recruited an autocrine signaling mechanism distinct from that of ISO, involving G(i)-coupled betagamma subunits of heterotrimeric G proteins, extracellular signal-regulated kinases ERK1/2, cyclooxygenase COX-1 (but not COX-2) and prostacyclin receptors. In the functional studies, inhibition of PKA or COX-1 attenuated ET1-induced VSMC hypertrophy, suggesting the positive role of PKA in this response to ET1. Furthermore, we found that ET1 stimulates a Gbetagamma-mediated, PKA-dependent phosphorylation and inactivation of glycogen synthase kinase-3 (GSK3), an enzyme that regulates cell growth. Together, this study describes that (i) PKA can be transiently activated by G(i)-coupled agonists such as ET1 by an autocrine mechanism involving Gbetagamma/calcium/ERK/COX-1/prostacyclin signaling, and (ii) this PKA activation promotes VSMC hypertrophy, at least in part, through PKA-dependent phosphorylation and inhibition of GSK3.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Endotelina-1/farmacología , Epoprostenol/biosíntesis , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Músculo Liso Vascular/enzimología , Vasoconstrictores/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Comunicación Autocrina/efectos de los fármacos , Comunicación Autocrina/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Ciclooxigenasa 1/genética , Ciclooxigenasa 1/metabolismo , Endotelina-1/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/genética , Glucógeno Sintasa Quinasa 3/genética , Hipertrofia/enzimología , Hipertrofia/genética , Hipertrofia/patología , Isoproterenol/farmacología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/patología , Fosforilación/efectos de los fármacos , Ratas , Ratas Endogámicas WKY , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Vasoconstrictores/metabolismoRESUMEN
The oncogenic G(12/13) subfamily of heterotrimeric G proteins transduces extracellular signals that regulate the actin cytoskeleton, cell cycle progression, and gene transcription. Previously, structural analyses of fully functional G alpha(12/13) subunits have been hindered by insufficient amounts of homogeneous, functional protein. Herein, we report that substitution of the N-terminal helix of G alpha(i1) for the corresponding region of G alpha12 or G alpha13 generated soluble chimeric subunits (G alpha(i/12) and G alpha(i/13)) that could be purified in sufficient amounts for crystallographic studies. Each chimera bound guanine nucleotides, G betagamma subunits, and effector proteins and exhibited GAP responses to p115RhoGEF and leukemia-associated RhoGEF. Like their wild-type counterparts, G alpha(i/13), but not G alpha(i/12), stimulated the activity of p115RhoGEF. Crystal structures of the G alpha(i/12) x GDP x AlF4(-) and G alpha(i/13) x GDP complexes were determined using diffraction data extending to 2.9 and 2.0 A, respectively. These structures reveal not only the native structural features of G alpha12 and G alpha13 subunits, which are expected to be important for their interactions with GPCRs and effectors such as G alpha-regulated RhoGEFs, but also novel conformational changes that are likely coupled to GTP hydrolysis in the G alpha(12/13) class of heterotrimeric G proteins.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP G12-G13/química , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Quimera/genética , Quimera/metabolismo , Cristalografía por Rayos X , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Subunidades alfa de la Proteína de Unión al GTP G12-G13/aislamiento & purificación , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Nucleótidos de Guanina/química , Nucleótidos de Guanina/genética , Nucleótidos de Guanina/metabolismo , Leucemia/metabolismo , Conformación Proteica , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Factores de Intercambio de Guanina Nucleótido RhoRESUMEN
G alpha 12/13-mediated pathways have been shown to be involved in various fundamental cellular functions in mammalian cells such as axonal guidance, apoptosis, and chemotaxis. Here, we identified a homologue of Rho-guanine nucleotide exchange factor (GEF) in Caenorhabditis elegans (CeRhoGEF), which functions downstream of gpa-12, the C. elegans homologue of G alpha 12/13. CeRhoGEF contains a PSD-95/Dlg/ZO-1 domain and a regulator of G protein signaling (RGS) domain upstream of the Dbl homology-pleckstrin homology region similar to mammalian RhoGEFs with RGS domains, PSD-95/Dlg/ZO-1-RhoGEF and leukemia-associated RhoGEF. It has been shown in mammalian cells that these RhoGEFs interact with activated forms of G alpha 12 or G alpha 13 through their RGS domains. We demonstrated by coimmunoprecipitation that the RGS domain of CeRhoGEF interacts with GPA-12 in an AIF4- activation-dependent manner and confirmed that the Dbl homology-pleckstrin homology domain of CeRhoGEF was capable of Rho-dependent signaling. These results proved conservation of the G alpha 12-RhoGEF pathway in C. elegans. Expression of DsRed or GFP under the control of the promoter of CeRhoGEF or gpa-12 revealed an overlap of their expression patterns in ventral cord motor neurons and several neurons in the head. RNA-mediated gene interference for CeRhoGEF and gpa-12 resulted in similar phenotypes such as embryonic lethality and sensory and locomotive defects in adults. Thus, the G alpha 12/13-RhoGEF pathway is likely to be involved in embryonic development and neuronal function in C. elegans.