RESUMEN
The present study describes the physiological response associated with daily subcutaneous injection of mice with recombinant follistatin288. This systemic administration of follistatin288 increases the follistatin levels in serum, indicating that the protein enters the circulation. The data suggest that a dose-dependent increase in body lean mass also occurs, together with an increase in muscle mass, possibly as a result of an increase in the size of the muscle fibers. After thirteen weeks of treatment, metabolic changes were observed; additionally, the switching of muscle fiber types was also apparent through myosin heavy chain remodeling, implying that changes are occurring at the molecular level. Furthermore, an increase in the muscle mass was associated with a significant decrease in the body fat mass. Overall, this study raises the possibility for the use of follistatin288 as an agent to treat muscle wasting diseases and/or to restrict fat accumulation by systemic administration of the protein.
Asunto(s)
Adiposidad/efectos de los fármacos , Folistatina/administración & dosificación , Folistatina/farmacología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Animales , Metabolismo Basal/efectos de los fármacos , Disección , Folistatina/sangre , Humanos , Hipertrofia , Ratones , Ratones Endogámicos C57BL , Desarrollo de Músculos/efectos de los fármacos , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/efectos de los fármacos , Tamaño de los Órganos/efectos de los fármacos , Especificidad de Órganos/efectos de los fármacos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacologíaRESUMEN
Smooth muscle contraction is a dynamic process driven by acto-myosin interactions that are controlled by multiple regulatory proteins. Our studies have shown that members of the AP-1 transcription factor family control discrete behaviors of smooth muscle cells (SMC) such as growth, migration and fibrosis. However, the role of AP-1 in regulation of smooth muscle contractility is incompletely understood. In this study we show that the AP-1 family member JunB regulates contractility in visceral SMC by altering actin polymerization and myosin light chain phosphorylation. JunB levels are robustly upregulated downstream of transforming growth factor beta-1 (TGFß1), a known inducer of SMC contractility. RNAi-mediated silencing of JunB in primary human bladder SMC (pBSMC) inhibited cell contractility under both basal and TGFß1-stimulated conditions, as determined using gel contraction and traction force microscopy assays. JunB knockdown did not alter expression of the contractile proteins α-SMA, calponin or SM22α. However, JunB silencing decreased levels of Rho kinase (ROCK) and myosin light chain (MLC20). Moreover, JunB silencing attenuated phosphorylation of the MLC20 regulatory phosphatase subunit MYPT1 and the actin severing protein cofilin. Consistent with these changes, cells in which JunB was knocked down showed a reduction in the F:G actin ratio in response to TGFß1. Together these findings demonstrate a novel function for JunB in regulating visceral smooth muscle cell contractility through effects on both myosin and the actin cytoskeleton.
Asunto(s)
Citoesqueleto de Actina/efectos de los fármacos , Actinas/genética , Miocitos del Músculo Liso/efectos de los fármacos , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta1/farmacología , Factores Despolimerizantes de la Actina/genética , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Contracción Muscular/efectos de los fármacos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Fosfatasa de Miosina de Cadena Ligera/genética , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fosforilación/efectos de los fármacos , Polimerizacion/efectos de los fármacos , Cultivo Primario de Células , ARN Interferente Pequeño/genética , Ratas , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Vejiga Urinaria/citología , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/metabolismo , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismo , CalponinasRESUMEN
CaMKII (Ca²âº/calmodulin-dependent kinase II) is a serine/threonine phosphotransferase that is capable of long-term retention of activity due to autophosphorylation at a specific threonine residue within each subunit of its oligomeric structure. The γ isoform of CaMKII is a significant regulator of vascular contractility. Here, we show that phosphorylation of CaMKII γ at Ser²6, a residue located within the ATP-binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at Ser²6, we generated a phosphospecific Ser²6 antibody and demonstrated an increase in Ser²6 phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of Ser²6 affects the kinase activity, we mutated Ser²6 to alanine or aspartic acid. The S26D mutation mimicking the phosphorylated state of CaMKII causes a dramatic decrease in Thr²87 autophosphorylation levels and greatly reduces the catalytic activity towards an exogenous substrate (autocamtide-3), whereas the S26A mutation has no effect. These data combined with molecular modelling indicate that a negative charge at Ser²6 of CaMKII γ inhibits the catalytic activity of the enzyme towards its autophosphorylation site at Thr²87 most probably by blocking ATP binding. We propose that Ser²6 phosphorylation constitutes an important mechanism for switching off CaMKII activity.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calcio/metabolismo , Isoformas de Proteínas/metabolismo , Animales , Sitios de Unión , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Calmodulina/genética , Calmodulina/metabolismo , Hurones/genética , Hurones/metabolismo , Mutación , Fosforilación/genética , Isoformas de Proteínas/genética , Serina/genética , Serina/metabolismo , Transducción de Señal , Treonina/genética , Treonina/metabolismoRESUMEN
An incomplete understanding of the molecular mechanisms responsible for myometrial activation from the quiescent pregnant state to the active contractile state during labor has hindered the development of effective therapies for preterm labor. Myometrial stretch has been implicated clinically in the initiation of labor and the etiology of preterm labor, but the molecular mechanisms involved in the human have not been determined. We investigated the mechanisms by which gestation-dependent stretch contributes to myometrial activation, by using human uterine samples from gynecologic hysterectomies and Cesarean sections. Here we demonstrate that the Ca requirement for activation of the contractile filaments in human myometrium increases with caldesmon protein content during gestation and that an increase in caldesmon phosphorylation can reverse this inhibitory effect during labor. By using phosphotyrosine screening and mass spectrometry of stretched human myometrial samples, we identify 3 stretch-activated focal adhesion proteins, FAK, p130Cas, and alpha actinin. FAK-Y397, which signals integrin engagement, is constitutively phosphorylated in term human myometrium whereas FAK-Y925, which signals downstream ERK activation, is phosphorylated during stretch. We have recently identified smooth muscle Archvillin (SmAV) as an ERK regulator. A newly produced SmAV-specific antibody demonstrates gestation-specific increases in SmAV protein levels and stretch-specific increases in SmAV association with focal adhesion proteins. Thus, whereas increases in caldesmon levels suppress human myometrium contractility during pregnancy, stretch-dependent focal adhesion signaling, facilitated by the ERK activator SmAV, can contribute to myometrial activation. These results suggest that focal adhesion proteins may present new targets for drug discovery programs aimed at regulation of uterine contractility.
Asunto(s)
Proteínas de Unión a Calmodulina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Adhesiones Focales/metabolismo , Miometrio/enzimología , Contracción Uterina , Animales , Calcio/metabolismo , Femenino , Humanos , Miometrio/metabolismo , Fosforilación , Embarazo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Útero/patologíaRESUMEN
ERK influences a number of pathways in all cells, but how ERK activities are segregated between different pathways has not been entirely clear. Using immunoprecipitation and pulldown experiments with domain-specific recombinant fragments, we show that smooth muscle archvillin (SmAV) binds ERK and members of the ERK signaling cascade in a domain-specific, stimulus-dependent, and pathway-specific manner. MEK binds specifically to the first 445 residues of SmAV. B-Raf, an upstream regulator of MEK, constitutively interacts with residues 1-445 and 446-1250. Both ERK and 14-3-3 bind to both fragments, but in a stimulus-specific manner. Phosphorylated ERK is associated only with residues 1-445. An ERK phosphorylation site was determined by mass spectrometry to reside at Ser132. A phospho-antibody raised to this site shows that the site is phosphorylated during alpha-agonist-mediated ERK activation in smooth muscle tissue. Phosphorylation of SmAV by ERK decreases the association of phospho-ERK with SmAV. These results, combined with previous observations, indicate that SmAV serves as a new ERK scaffolding protein and provide a mechanism for regulation of ERK binding, activation, and release from the signaling complex.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Músculo Liso/metabolismo , Proteínas 14-3-3/metabolismo , Secuencia de Aminoácidos , Animales , Aorta/citología , Aorta/metabolismo , Hurones , Inmunoprecipitación , Espectrometría de Masas , Datos de Secuencia Molecular , Músculo Liso/citología , Fosforilación , Proteínas Proto-Oncogénicas B-raf/metabolismo , Transducción de SeñalRESUMEN
We present here the identification and characterization of an SCP3 (small C-terminal domain phosphatase-3) homologue in smooth muscle and show, for the first time, that it dephosphorylates CaMKII [Ca(2+)/CaM (calmodulin)-dependent protein kinase II]. SCP3 is a PP2C (protein phosphatase 2C)-type phosphatase that is primarily expressed in vascular smooth muscle tissues and specifically binds to the association domain of the CaMKIIgamma G-2 variant. The dephosphorylation is site-specific, excluding the Thr(287) associated with Ca(2+)/CaM-independent activation of the kinase. As a result, the autonomous activity of CaMKIIgamma G-2 is not affected by the phosphatase activity of SCP3. SCP3 co-localizes with CaMKIIgamma G-2 on cytoskeletal filaments, but is excluded from the nucleus in differentiated vascular smooth muscle cells. Upon depolarization-induced Ca(2+) influx, CaMKIIgamma G-2 is activated and dissociates from SCP3. Subsequently, CaMKIIgamma G-2 is targeted to cortical adhesion plaques. We show here that SCP3 regulates phosphorylation sites in the catalytic domain, but not those involved in regulation of kinase activation. This selective dephosphorylation by SCP3 creates a constitutively active kinase that can then be differentially regulated by other phosphorylation-dependent regulatory mechanisms.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Modelos Biológicos , Datos de Secuencia Molecular , Fosforilación , Proteína Fosfatasa 2C , Estructura Terciaria de Proteína , Resonancia por Plasmón de SuperficieRESUMEN
Subcellular targeting of kinases controls their activation and access to substrates. Although Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to regulate differentiated smooth muscle cell (dSMC) contractility, the importance of targeting in this regulation is not clear. The present study investigated the function in dSMCs of a novel variant of the gamma isoform of CaMKII that contains a potential targeting sequence in its association domain (CaMKIIgamma G-2). Antisense knockdown of CaMKIIgamma G-2 inhibited extracellular signal-related kinase (ERK) activation, myosin phosphorylation, and contractile force in dSMCs. Confocal colocalization analysis revealed that in unstimulated dSMCs CaMKIIgamma G-2 is bound to a cytoskeletal scaffold consisting of interconnected vimentin intermediate filaments and cytosolic dense bodies. On activation with a depolarizing stimulus, CaMKIIgamma G-2 is released into the cytosol and subsequently targeted to cortical dense plaques. Comparison of phosphorylation and translocation time courses indicates that, after CaMKIIgamma G-2 activation, and before CaMKIIgamma G-2 translocation, vimentin is phosphorylated at a CaMKII-specific site. Differential centrifugation demonstrated that phosphorylation of vimentin in dSMCs is not sufficient to cause its disassembly, in contrast to results in cultured cells. Loading dSMCs with a decoy peptide containing the polyproline sequence within the association domain of CaMKIIgamma G-2 inhibited targeting. Furthermore, prevention of CaMKIIgamma G-2 targeting led to significant inhibition of ERK activation as well as contractility. Thus, for the first time, this study demonstrates the importance of CaMKII targeting in dSMC signaling and identifies a novel targeting function for the association domain in addition to its known role in oligomerization.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Músculo Liso Vascular/fisiología , Actinina/análisis , Actinina/metabolismo , Animales , Células COS , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/análisis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Diferenciación Celular , Hurones , MAP Quinasa Quinasa 1/análisis , MAP Quinasa Quinasa 2/análisis , Músculo Liso Vascular/citología , Oligonucleótidos Antisentido/farmacología , Fosforilación , Estructura Terciaria de Proteína , Transporte de Proteínas , Vasoconstricción , Vimentina/análisis , Vimentina/metabolismoRESUMEN
The mechanisms by which protein kinase C (PKC) and extracellular-signal-regulated kinases (ERK1/2) govern smooth-muscle contractility remain unclear. Calponin (CaP), an actin-binding protein and PKC substrate, mediates signaling through ERK1/2. We report here that CaP sequences containing the CaP homology (CH) domain bind to the C-terminal 251 amino acids of smooth-muscle archvillin (SmAV), a new splice variant of supervillin, which is a known actin- and myosin-II-binding protein. The CaP-SmAV interaction is demonstrated by reciprocal yeast two-hybrid and blot-overlay assays and by colocalization in COS-7 cells. In differentiated smooth muscle, endogenous SmAV and CaP co-fractionate and co-translocate to the cell cortex after stimulation by agonist. Antisense knockdown of SmAV in tissue inhibits both the activation of ERK1/2 and contractions stimulated by either agonist or PKC activation. This ERK1/2 signaling and contractile defect is similar to that observed in CaP knockdown experiments. In A7r5 smooth-muscle cells, PKC activation by phorbol esters induces the reorganization of endogenous, membrane-localized SmAV and microfilament-associated CaP into podosome-like structures that also contain F-actin, nonmuscle myosin IIB and ERK1/2. These results indicate that SmAV contributes to the regulation of contractility through a CaP-mediated signaling pathway, involving PKC activation and phosphorylation of ERK1/2.
Asunto(s)
Proteínas de la Membrana/fisiología , Proteínas de Microfilamentos/fisiología , Músculo Liso/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Aorta/metabolismo , Western Blotting , Células COS , Proteínas de Unión al Calcio/metabolismo , ADN Complementario/metabolismo , Activación Enzimática , Hurones , Glutatión Transferasa/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Microscopía Fluorescente , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/farmacología , Fosforilación , Unión Proteica , Proteína Quinasa C/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Transducción de Señal , Fracciones Subcelulares/metabolismo , Factores de Tiempo , Transfección , Técnicas del Sistema de Dos Híbridos , CalponinasRESUMEN
Six variants of calmodulin-dependent protein kinase IIgamma were isolated from a ferret-aorta smooth-muscle cDNA library. Variant G-2 is generated by a novel alternative polyadenylation, utilizing a site contained in an intron. The last 77 residues of the association domain are replaced with 99 residues of a unique sequence containing Src homology 3-domain-binding motifs, which alter catalytic activity. Variant C-2 has an eight-residue deletion in an ATP-binding motif and does not autophosphorylate Thr(286), but does phosphorylate exogenous substrate. Two variants, B and J, autodephosphorylate. Four variants differing only in the variable domain have differing catalytic activities, despite identical sequences in the catalytic domains. Thus structural features determined by variable and association domains are important for the catalytic activity of calmodulin-dependent protein kinase II.