RESUMEN
Active metabolism regulates oocyte cell death via calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated phosphorylation of caspase-2, but the link between metabolic activity and CaMKII is poorly understood. Here we identify coenzyme A (CoA) as the key metabolic signal that inhibits Xenopus laevis oocyte apoptosis by directly activating CaMKII. We found that CoA directly binds to the CaMKII regulatory domain in the absence of Ca(2+) to activate CaMKII in a calmodulin-dependent manner. Furthermore, we show that CoA inhibits apoptosis not only in X. laevis oocytes but also in Murine oocytes. These findings uncover a direct mechanism of CaMKII regulation by metabolism and further highlight the importance of metabolism in preserving oocyte viability.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Coenzima A/metabolismo , Oocitos/metabolismo , Xenopus laevis/metabolismo , Animales , Apoptosis/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Caspasa 2/metabolismo , Supervivencia Celular/genética , Regulación del Desarrollo de la Expresión Génica , Ratones , Oocitos/crecimiento & desarrollo , Fosforilación/genética , Unión Proteica , Transducción de Señal , Activación Transcripcional , Xenopus laevis/crecimiento & desarrolloRESUMEN
The metabolism of the Xenopus laevis egg provides a cell survival signal. We found previously that increased carbon flux from glucose-6-phosphate (G6P) through the pentose phosphate pathway in egg extracts maintains NADPH levels and calcium/calmodulin regulated protein kinase II (CaMKII) activity to phosphorylate caspase 2 and suppress cell death pathways. Here we show that the addition of G6P to oocyte extracts inhibits the dephosphorylation/inactivation of CaMKII bound to caspase 2 by protein phosphatase 1. Thus, G6P sustains the phosphorylation of caspase 2 by CaMKII at Ser-135, preventing the induction of caspase 2-mediated apoptotic pathways. These findings expand our understanding of oocyte biology and clarify mechanisms underlying the metabolic regulation of CaMKII and apoptosis. Furthermore, these findings suggest novel approaches to disrupt the suppressive effects of the abnormal metabolism on cell death pathways.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Xenopus laevis/metabolismo , Animales , Apoptosis , Caspasa 2/metabolismo , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Muerte Celular , Proliferación Celular , Espectrometría de Masas/métodos , Oocitos/metabolismo , Oxígeno/metabolismo , Péptidos/química , Fosforilación , Proteína Fosfatasa 1/metabolismo , Proteínas Recombinantes/metabolismo , Sefarosa/química , Serina/química , Xenopus/metabolismoRESUMEN
Müllerian duct development appears to be similar in many vertebrate groups, and previous studies have shown that this development is estrogen sensitive. For example, embryonic exposure to diethylstilbestrol (DES) in humans and mice, and estrogen exposure in chickens, can have multiple, usually adverse, effects on müllerian duct differentiation and growth. The current study investigates 17beta-estradiol's effects on müllerian duct development in a reptile, the turtle Trachemys scripta. In T. scripta, normal müllerian duct development proceeds cranially to caudally over developmental stages 15 to 21. To ascertain 17beta-estradiol's effect on this process, four groups of eggs were incubated at a female-producing temperature. Each group was treated with 50 mug of 17beta-estradiol or a vehicle control at one of four stages (15, 17, 19, 21). The degree of müllerian duct development was assessed by examining gross morphology and histology. Results showed that estradiol-17beta blocked development of the müllerian duct if applied before differentiation began. If applied afterwards, 17beta-estradiol caused hypertrophy in the differentiated portion, but prevented further differentiation of the müllerian duct in more caudal regions. Therefore, reptilian müllerian ducts in T. scripta are estrogen sensitive and estrogen's effects may be similar to those reported for birds.