τ is phosphorylated by GSK-3 at several sites found in Alzheimer disease and its biological activity markedly inhibited only after it is prephosphorylated by A-kinase

Alzheimer disease is characterized by a specific type of neuronal degeneration in which the microtubule associated protein τ is abnormally hyperphosphorylated causing the disruption of the microtubule network. We have found that the phosphorylation of human τ (τ3L) by A-kinase, GSK-3 or CK-1 inhibits its microtubule assembly-promoting and microtubule-binding activities. However, the inhibition of these activities of τ by GSK-3 is significantly increased if τ is prephosphorylated by A-kinase or CK-1. The most potent inhibition is observed by combination phosphorylation of τ with A-kinase and GSK-3. Under these conditions, only very few microtubules are seen by electron microscopy. Sequencing of 32P-labeled trypsin phosphopeptides from τ prephosphorylated by A-kinase (using unlabeled ATP) and further phosphorylated by GSK-3 in the presence of [γ- 32P]ATP revealed that Ser-195, Ser-198, Ser-199, Ser-202, Thr-205, Thr-231, Ser-235, Ser-262, Ser-356 and Ser-404 are phosphorylated, whereas if τ is not prephosphorylated by A-kinase, GSK-3 phosphorylates it at Thr-181, Ser-184, Ser-262, Ser-356 and Ser-400. These data suggest that (i) prephosphorylation of τ by A-kinase makes additional and different sites accessible for phosphorylation by GSK-3; (ii) phosphorylation of τ at these additional sites further inhibits the biological activity of τ in its ability to bind to microtubules and promote microtubule assembly. Thus a combined role of A-kinase and GSK-3 should be considered in Alzheimer neurofibrillary degeneration.