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

Abstract

Glycogen synthase kinase 3␤ (GSK3␤) phosphorylates substrates, including the microtubule-associated pro-tein tau, at both primed and unprimed epitopes. GSK3␤ phosphorylation of tau negatively regulates tau-micro-tubule 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 GSK3␤ was used, a mutation that prevents phosphorylation of substrates at primed sites. Both GSK3␤ and GSK3␤-R96A phosphoryl-ated tau efficiently in situ. However, expression of GSK3␤-R96A resulted in significantly less phosphoryla-tion of tau at primed sites compared with GSK3␤. Con-versely, GSK3␤-R96A phosphorylated unprimed tau sites to a significantly greater extent than GSK3␤. Pre-phosphorylating tau with cdk5/p25 impaired the ability of GSK3␤-R96A to phosphorylate tau, whereas GSK3␤-R96A phosphorylated recombinant tau to a significantly greater extent than GSK3␤. Moreover, the amount of tau associated with microtubules was reduced by overex-pression of GSK3␤ but only when tau was phosphoryl-ated at primed sites, as phosphorylation of tau by GSK3␤-R96A did not negatively regulate the association of tau with microtubules. These results demonstrate that GSK3␤-mediated phosphorylation of tau at primed sites plays a more significant role in regulating the in-teraction of tau with microtubules than phosphoryla-tion at unprimed epitopes.