RESUMEN
Phosphorylation analysis by using phos-tag technique has been reported to be suitable for highly sensitive quantification of smooth muscle myosin regulatory light chain (LC20 ) phosphorylation. However, there is another factor that will affect the sensitivity of phosphorylation analysis, that is, protein extraction. Here, we optimized the conditions for total protein extraction out of trichloroacetic acid (TCA)-fixed tissues. Standard SDS sample buffer extracted less LC20 , actin and myosin phosphatase targeting subunit 1 (MYPT1) from TCA/acetone treated ciliary muscle strips. On the other hand, sample buffer containing urea and thiourea in addition to lithium dodecyl sulfate (LDS) or SDS extracted those proteins more efficiently, and thus increased the detection sensitivity up to 4-5 fold. Phos-tag SDS-PAGE separated dephosphorylated and phosphorylated LC20 s extracted in LDS/urea/thiourea sample buffer to the same extent as those in standard SDS buffer. We have concluded that LDS (or SDS) /urea/thiourea sample buffer is suitable for highly sensitive phosphorylation analysis in smooth muscle, especially when it is treated with TCA/acetone.
Asunto(s)
Electroforesis en Gel de Poliacrilamida/métodos , Músculo Liso/química , Tiourea/química , Urea/química , Acetona/química , Animales , Arterias Mesentéricas/química , Ratones , Fosfoproteínas/análisis , Fosfoproteínas/química , Fosforilación , Ácido Tricloroacético/químicaRESUMEN
Ciliary muscle is a smooth muscle characterized by a rapid response to muscarinic receptor stimulation and sustained contraction. Although it is evident that these contractions are Ca(2+)-dependent, detailed molecular mechanisms are still unknown. In order to elucidate the role of Ser/Thr protein phosphatase 2A (PP2A) in ciliary muscle contraction, we examined the effects of okadaic acid and other PP2A inhibitors on contractions induced by carbachol (CCh) and ionomycin in bovine ciliary muscle strips (BCM). Okadaic acid inhibited ionomycin-induced contraction, while it did not cause significant changes in CCh-induced contraction. Fostriecin showed similar inhibitory effects on the contraction of BCM. On the other hand, rubratoxin A inhibited both ionomycin- and CCh-induced contractions. These results indicated that PP2A was involved at least in ionomycin-induced Ca(2+)-dependent contraction, and that BCM had a unique regulatory mechanism in CCh-induced contraction.
Asunto(s)
Cuerpo Ciliar/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Ácido Ocadaico/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/fisiología , Animales , Calcio/fisiología , Carbacol/antagonistas & inhibidores , Carbacol/farmacología , Bovinos , Técnicas In Vitro , Ionomicina/antagonistas & inhibidores , Ionomicina/farmacología , Micotoxinas/farmacología , Polienos/farmacología , Pironas/farmacologíaRESUMEN
Protein phosphatase 1gamma, a serine/threonine phosphatase, is a metalloprotein that coordinates two Mn(2+) in the active site when expressed in Escherichia coli in a buffer containing MnCl(2). Herein, we report on the oxidatively induced copper for manganese exchange in protein phosphatase 1gamma, thus enabling firm confirmation of the four histidine (His) amino acid residues (His66, His125, His173, and His248) involved in metal coordination. By exchanging manganese with copper the oxidation yields for the peptides increased dramatically, thus simplifying detection of the oxidized peptides and analysis of the oxidation sites within the oxidized peptides. We also found that when copper was added during the oxidation process a new metal coordination center was formed at cysteine 39, 105, 140, and 155.
Asunto(s)
Cobre/química , Histidina/química , Manganeso/química , Proteína Fosfatasa 1/química , Especies Reactivas de Oxígeno/química , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Previously we reported that a uni-axial cyclic stretch treatment of rat 3Y1 fibroblasts induced focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation (Wang et al., 2001) [Wang, J.G., Miyazu, M., Matsushita, E., Sokabe, M., Naruse, K., 2001. Uni-axial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation. Biochem. Biophys. Res. Comm. 288, 356-361]. In the present study, we investigated whether stretch-induced MAPK activation leads to proliferation of fibroblasts. 3Y1 fibroblasts were subjected to a uni-axial cyclic stretch treatment (1 Hz, 120% in length) and the bromodeoxyuridine (BrdU) incorporation was measured to access cell proliferation. BrdU incorporation increased in a time-dependent manner and became significant within 6 hours. To investigate the involvement of FAK, we transiently expressed FAK mutants that lacked tyrosine phosphorylation site (s) (F397Y, F925Y, F397/925Y). Transient expression of wild-type FAK or mock vector did not inhibit the stretch-induced BrdU incorporation, however, the FAK mutants significantly blocked BrdU incorporation. Treatment of the cells with MAPK inhibitors, PD98059 or SB203580, blocked extracellular signal-regulated kinase (ERK) phosphorylation and p38 MAPK phosphorylation, respectively, and also blocked stretch-induced BrdU incorporation. These results suggest that the stretch-induced FAK activation followed by MAPK activation plays an important role in the stretch-induced proliferation of 3Y1 fibroblasts.
Asunto(s)
Proliferación Celular , Fibroblastos/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/fisiología , Animales , Bromodesoxiuridina/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Flavonoides/farmacología , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Imidazoles/farmacología , Immunoblotting , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Piridinas/farmacología , Ratas , Estrés Mecánico , Tirosina/metabolismoRESUMEN
The effect of uni-axial cyclic mechanical stretch on the activation of the transcription factor nuclear factor kappaB (NF-kappaB) was investigated in a human fibroblast cell line (TIG-1). In response to uni-axial cyclic stretch, NF-kappaB was found to be translocated into the nucleus. The NF-kappaB was first detectable 2 min after the onset of stretch and then peaked at 4 min and returned to the basal level within 10 min. To investigate whether NF-kappaB is activated following the translocation into the nucleus, we measured the luciferase activity in the cells transfected with pNF-kappaB-luciferase. The activity of luciferase increased 4 min after the initiation of cyclic stretch, peaked at 15 min (6.4-fold increase), and decreased gradually. We examined the involvement of the stretch-activated (SA) channel in the stretch-induced NF-kappaB activation. The application of Gd3+, a blocker of the SA channel, or the removal of extracellular Ca2+ inhibited both the translocation into the nucleus and the activation of NF-kB, which suggests that NF-kappaB is activated by uni-axial cyclic stretch via SA channel activation in human lung fibroblasts.