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

Glycogen synthase kinase-3 beta (GSK-3 beta) has been described as a proline-directed kinase which phosphorylates tau protein at several sites that are elevated in Alzheimer paired helical filaments. However, it has been claimed that GSK-3 beta can also phosphorylate the non-proline-directed KXGS motifs in the presence of heparin, including Ser262 in the repeat domain of tau, which could induce the detachment of tau from microtubules. We have analyzed the activity of recombinant GSK-3 beta and of GSK-3 beta preparations purified from tissue, using two-dimensional phosphopeptide mapping, immunoblotting with phosphorylation-sensitive antibodies, and phosphopeptide sequencing. The most prominent phosphorylation sites on tau are Ser396 and Ser404 (PHF-1 epitope), Ser46 and Thr50 in the first insert, followed by a less efficient phosphorylation of other Alzheimer phosphoepitopes (antibodies AT-8, AT-270, etc). We also show that the non-proline-directed activity at KXGS 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 addition of heparin. (C) 1999 Federation of European Biochemical Societies.