Regulation and localization of tyrosine(216) phosphorylation of glycogen synthase kinase-3β in cellular and animal models of neuronal degeneration

Inactivation of glycogen synthase kinase-3β (GSK3β) by S(9) phosphorylation is implicated in mechanisms of neuronal survival. Phosphorylation of a distinct site, Y(216), on GSK3β 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 GSK3β 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), GSK3β activity, and cell death. Lithium and insulin, agents that lead to inhibition of GSK3β and adenoviral-mediated transduction of dominant negative GSK3β constructs, prevented cell death by the proapoptotic stimuli. Inhibitors induced S(9) phosphorylation and inactivation of GSK3β but did not affect Y(216) phosphorylation, suggesting that S(9) phosphorylation is sufficient to override GSK3β activation by Y(216) phosphorylation. Under the conditions examined, increased Y(216) phosphorylation on GSK3β was not an autophosphorylation response. In resting cells, Y(216) phosphorylation was restricted to GSK3β present at focal adhesion sites. However, after staurosporine, a dramatic alteration in the immunolocalization pattern was observed, and Y(216)-phosphorylated GSK3β 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 GSK3β represents an important mechanism by which cellular insults can lead to neuronal death.