Fyn/NMDA Signaling Involves The Regulation Of CREB Activity By GSK-3β

GSK-3β is a ubiquitously expressed, constitutively active, proline-directed serine/threonine kinase involved in a variety of cellular processes. Dysregulation of GSK-3β plays a key role in the mechanisms of many neurodegenerative diseases. Overactivity GSK-3P attributes to down-regulate the activity of the transcriptional factor CREB, and impairs the learning and memory abilities of animals. But the actual mechanism how GSK-3β down-regulate the CREB activity is not clear. Here, we found that the Fyn/NMDA receptor signaling involves in the GSK-3p-mediated down-regulation of CREB activity. In this study, we treated N2a neuroma cell with wortmannin or transfecting with wtGSK-3β plasmid, then treated the cells with PP2for1h. Finally we detected the activities of GSK-3β, Fyn, PKA, CREB and the level of pNR2B and the pNR2B-PSD95complex by Western blotting. Results showed that:(1) In N2a cells, the activity of Fyn decreased to75%of the control level by treated different concentration of PP2(2.5μM,5μM,10μM).(2) Compared with the control, the activity of GSK-3β increased significantly by2.5μM,5μM,10μM wortmannin or transfecting with wtGSK-3β plasmid in N2a cells, and it wasn’t affected by the treatment of PP2.(3) The overactivity of GSK-3β up-regulated the Fyn activity in N2a cells than control, and the fyn activity turned to normal after the N2a cells treated with2.5μM PP2for1h.(4) Compared with the control, the overactivity of GSK-3β also up-regulated the level of pNR2B and the pNR2B-PSD95complex, after treated with2.5μM PP2for1h, the level of pNR2B and the pNR2B-PSD95complex decreased dramatically.(5) The overactivity of GSK-3β down-regulated the activity of PKA, and2.5μM PP2could reversed this action.(6) The phosphorylation of CREB at Ser133, which reflected the activity of CREB, decreased to50%of the normal level by GSK-3β overactivity, while2.5μM PP2treatment could recover it1h later. In a conclusion, with the GSK-3β-mediated stimulation of Fyn activity, the overproduced pNR2B, a substrate of Fyn and the pNR2B-PSD95complex, could decrease PKA activity and then down-regulate the CREB activity. This study implied a new mechanism of the CREB activity regulated by GSK-3β, and offered some evidences of treating some CNS disorders including AD, PD.