Regulation and localization of tyrosine216 phosphorylation of glycogen synthase kinase-3β in cellular and animal models of neuronal degeneration

Inactivation of glycogen synthase kinase-3 beta (GSK3 beta) by S-9 phosphorylation is implicated in mechanisms of neuronal survival. Phosphorylation of a distinct site, Y-216, On GSK3 beta is necessary for its activity; however, whether this site can be regulated in cells is unknown. Therefore we examined the regulation of Y-216 phosphorylation on GSK3 beta in models of neurodegeneration. Nerve growth factor withdrawal from differentiated PC12 cells and staurosporine treatment of SH-SY5Y cells led to increased phosphorylation at Y-216. GSK3 beta activity, and cell death. Lithium and insulin, agents that lead to inhibition of GSK3 beta and adenoviral-mediated transduction of dominant negative GSK3 beta constructs, prevented cell death by the proapoptotic stimuli. Inhibitors induced S9 phosphorylation and inactivation of GSK3 beta but did not affect Y-216 phosphorylation, suggesting that S9 phosphorylation is sufficient to override GSK3 beta activation by Y-216 phosphorylation. Under the conditions examined, increased Y-216 phosphorylation on GSK3 beta was not an autophosphorylation response. In resting cells, Y-216 phosphorylation was restricted to GSK3 beta present at focal adhesion sites. However, after staurosporine, a dramatic alteration in the immunolocalization pattern was observed, and Y-216-phosphorylated GSK3 beta selectively increased within the nucleus. In rats. Y-216 phosphorylation was increased in degenerating cortical neurons induced by ischemia. Taken together, these results suggest that Y-216 phosphorylation of GSK3 beta represents an important mechanism by which cellular insults can lead to neuronal death.