RESUMEN
The purpose of the present study was to understand the mechanism by which activated protein kinase A (PKA) leads to down-regulation of cyclin D3 in lymphocytes. By using Jurkat cells as a model system, we have been able to demonstrate that cyclin D3 is reduced at the level of translation by inhibition of elongation. One of the important factors involved in translational elongation is the eukaryotic elongation factor 2 (eEF2). eEF2 promotes translation in its unphosphorylated form, and we observed a rapid phosphorylation of the eEF2-protein upon forskolin treatment. When using specific inhibitors of the eEF2-kinase prior to forskolin treatment, we were able to inhibit the increased phosphorylation of eEF2. Furthermore, inhibition of eEF2-kinase prevented the forskolin-mediated down-regulation of cyclin D3. Taken together, it appears that activation of PKA in Jurkat cells reduces the expression of cyclin D3 at the level of translational elongation by increasing the phosphorylation of eEF2 and thereby inhibiting its activity.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , AMP Cíclico/fisiología , Ciclinas/genética , Factor 2 de Elongación Peptídica/metabolismo , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas , Linfocitos T/metabolismo , Northern Blotting , Western Blotting , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/efectos de los fármacos , Cromonas/farmacología , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclina D3 , Ciclinas/efectos de los fármacos , Ciclinas/metabolismo , Cicloheximida/farmacología , Replicación del ADN/efectos de los fármacos , Regulación hacia Abajo , Quinasa del Factor 2 de Elongación , Citometría de Flujo/métodos , Fase G1/efectos de los fármacos , Fase G1/genética , Regulación de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Células Jurkat , Morfolinas/farmacología , Compuestos de Organoselenio/farmacología , Extensión de la Cadena Peptídica de Translación/fisiología , Factor 2 de Elongación Peptídica/efectos de los fármacos , Fosforilación/efectos de los fármacos , Polirribosomas/genética , Polirribosomas/metabolismo , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt , Proteína de Retinoblastoma/efectos de los fármacos , Proteína de Retinoblastoma/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 70-kDa/efectos de los fármacos , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal , Linfocitos T/efectos de los fármacos , TransfecciónRESUMEN
In this study we report a new mechanism whereby cyclic AMP (cAMP) regulates the cell-cycle machinery. We demonstrate that elevation of intracellular levels of cAMP promotes degradation of cyclin D3 in proteasomes, and that this occurs via glycogen synthase kinase-3beta (GSK-3beta)-mediated phosphorylation of cyclin D3 at Thr-283. Elevation of cAMP did not change the subcellular distribution of either cyclin D3 or GSK-3beta. However, cAMP promoted the interaction between cyclin D3 and GSK-3beta both in vitro and in vivo, indicating that GSK-3beta-mediated phosphorylation of cyclin D3 might require the association between the two proteins. These results demonstrate how cAMP enhances degradation of cyclin D3. Furthermore, we provide evidence for a novel mechanism by which GSK-3beta might phosphorylate unprimed substrates in vivo.