Phosphorylation of tau protein by recombinant GSK-3 beta: pronounced phosphorylation at select Ser/Thr-Pro motifs but no phosphorylation at Ser262 inthe repeat domain

Glycogen synthase kinase-3 beta (GSK-3 beta) has been described as a prolin e-directed kinase which phosphorylates tau protein at several sites that ar e elevated in Alzheimer paired helical filaments. However, it has been clai med that GSK-3 beta can also phosphorylate the non-proline-directed KXGS mo tifs in the presence of heparin, including Ser262 in the repeat domain of t au, which could induce the detachment of tau from microtubules. We have ana lyzed the activity of recombinant GSK-3 beta and of GSK-3 beta preparations purified from tissue, using two-dimensional phosphopeptide mapping, immuno blotting with phosphorylation-sensitive antibodies, and phosphopeptide sequ encing. The most prominent phosphorylation sites on tau are Ser396 and Ser4 04 (PHF-1 epitope), Ser46 and Thr50 in the first insert, followed by a less efficient phosphorylation of other Alzheimer phosphoepitopes (antibodies A T-8, AT-270, etc). We also show that the non-proline-directed activity at K XGS motifs is not due to GSK-3 beta itself, but to kinase contaminations in common GSK-3 beta preparations from tissues which are activated upon addit ion of heparin. (C) 1999 Federation of European Biochemical Societies.