Abstract
Glycogen synthase kinase-3 (GSK-3) plays a critical role in neuronal apoptosis. The two mammalian isoforms of the kinase, GSK-3α and GSK-3β, are inhibited by phosphorylation at Ser-21 and Ser-9, respectively. Depolarization, which is vital for neuronal survival, causes both an increase in Ser-21/9 phosphorylation and an inhibition of GSK-3α/β. However, the role of GSK-3 phosphorylation in depolarization-dependent neuron survival and the signaling pathway contributing to GSK-3 phosphorylation during depolarization remain largely unknown. Using several approaches, we showed that both isoforms of GSK-3 are important for mediating neuronal apoptosis. Nonphosphorylatable GSK-3α/β mutants (S21A/S9A) promoted apoptosis, whereas a peptide encompassing Ser-9 of GSK-3β protected neurons in a phosphorylation-dependent manner; these results indicate a critical role for Ser-21/9 phosphorylation on depolarization-dependent neuron survival. We found that Ser-21/9 phosphorylation of GSK-3 was mediated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) but not by Akt/PKB, PKA, or p90(RSK). CaMKII associated with and phosphorylated GSK-3α/β. Furthermore, the pro-survival effect of CaMKII was mediated by GSK-3 phosphorylation and inactivation. These findings identify a novel Ca(2+)/calmodulin/CaMKII/GSK-3 pathway that couples depolarization to neuronal survival.
Highlights
(CGNs),3 suggesting the importance of afferent input-related factors for survival of CGNs in vivo (7)
Various studies have demonstrated that Glycogen synthase kinase-3 (GSK-3) promotes neuronal apoptosis that is induced by a diverse array of insults (21, 26 –30), and dysregulated GSK-3 activity has been implicated in cell death during the pathogenesis of both Parkinson (31, 32) and Alzheimer disease (33)
We have provided evidence that both GSK-3␣ and GSK-3 play a crucial role in neuronal apoptosis and that Ser-21/9 phosphorylation of GSK-3␣/ is critical for depolarization maintenance of CGN survival
Summary
Reagents—AR-A014418, U0126, PD98059, KN62, wortmannin, LY294002, CaMKIINtide (calmodulin kinase IINtide), myr-CaMKIINtide (myristoylated calmodulin kinase IINtide), and protein A plus protein G-agarose were obtained from EMD Biosciences. GFP- GSK-3 substrate (collapsin response mediator protein 2, positive neurons with intact nuclei showing diffuse Hoechst CRMP2) at Thr-514 (42, 43) was decreased by depolarization, staining and no propidium iodide staining were scored as via- whereas the total CRMP2 protein level remained unchanged ble, and dying or dead neurons displayed condensed nuclei and (Fig. 1A, top panel), indicating that endogenous GSK-3 activity fragmented nuclei with stronger fluorescence of Hoechst and was reduced by depolarization.
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