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

摘要

Inactivation of glycogen synthase kinase-3β (GSK3β) by S⁹ phosphorylation is implicated in mechanisms of neuronal survival. Phosphorylation of a distinct site, Y²¹⁶, on GSK3β is necessary for its activity; however, whether this site can be regulated in cells is unknown. Therefore we examined the regulation of Y²¹⁶ 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 Y²¹⁶, 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 S⁹ phosphorylation and inactivation of GSK3β but did not affect Y²¹⁶ phosphorylation, suggesting that S⁹ phosphorylation is sufficient to override GSK3β activation by Y²¹⁶ phosphorylation. Under the conditions examined, increased Y²¹⁶ phosphorylation on GSK3β was not an autophosphorylation response. In resting cells, Y²¹⁶ phosphorylation was restricted to GSK3β present at focal adhesion sites. However, after staurosporine, a dramatic alteration in the immunolocalization pattern was observed, and Y²¹⁶-phosphorylated GSK3β selectively increased within the nucleus. In rats, Y²¹⁶ phosphorylation was increased in degenerating cortical neurons induced by ischemia. Taken together, these results suggest that Y²¹⁶ phosphorylation of GSK3β represents an important mechanism by which cellular insults can lead to neuronal death.