Effects of FTDP-17 mutations on the in vitro phosphorylation of tau by glycogen synthase kinase 3 beta identified by mass spectrometry demonstrate certain mutations exert long-range conformational changes

In vitro phosphorylation of recombinant wild-type 2N4R tau and FTDP-17 exon ic mutant forms P301L, V337M and R406W by glycogen synthase kinase 3 beta ( GSK3 beta) was examined by two dimensional phosphopeptide mapping analysis on thin layer cellulose plates, Comparison of these peptide maps with those generated from wild-type 1N4R tau isoform from which the phosphopeptide co nstituents and sites of phosphorylation had been determined previously. ena bled us to monitor directly changes in phosphorylation of the individual ta u proteins. No differences were found in the phosphorylation of wild-type, P301L or V337M tau by GSK3 beta but the R406W mutant showed at least two cl ear differences from the other three tau proteins. The peptides, identified by mass spectrometry corresponding to phosphorylation at both threonine 23 1 and serine 235 (spot 3), serines 396, 400 and 404 (spot 6a) and serines 1 95 and 199 (spot 6b) were absent from the R406W peptide map. The findings i mply that the R406W mutation in tau exerts Long-range conformational effect s on the structure of tau, (C) 2001 Published bg Elsevier Science B.V. on b ehalf of the Federation of European Biochemical Societies.