Glycogen Synthase Kinase 3β Phosphorylates Tau at Both Primed and Unprimed Sites

Glycogen synthase kinase 3β (GSK3β) phosphorylates substrates, including the microtubule-associated protein tau, at both primed and unprimed epitopes. GSK3β phosphorylation of tau negatively regulates tau-microtubule interactions; however the differential effects of phosphorylation at primed and unprimed epitopes on tau is unknown. To examine the phosphorylation of tau at primed and unprimed epitopes and how this impacts tau function, the R96A mutant of GSK3β was used, a mutation that prevents phosphorylation of substrates at primed sites. Both GSK3β and GSK3β-R96A phosphorylated tau efficiently in situ. However, expression of GSK3β-R96A resulted in significantly less phosphorylation of tau at primed sites compared with GSK3β. Conversely, GSK3β-R96A phosphorylated unprimed tau sites to a significantly greater extent than GSK3β. Prephosphorylating tau with cdk5/p25 impaired the ability of GSK3β-R96A to phosphorylate tau, whereas GSK3β-R96A phosphorylated recombinant tau to a significantly greater extent than GSK3β. Moreover, the amount of tau associated with microtubules was reduced by overexpression of GSK3β but only when tau was phosphorylated at primed sites, as phosphorylation of tau by GSK3β-R96A did not negatively regulate the association of tau with microtubules. These results demonstrate that GSK3β-mediated phosphorylation of tau at primed sites plays a more significant role in regulating the interaction of tau with microtubules than phosphorylation at unprimed epitopes.