Effects of FTDP-17 mutations on the in vitro phosphorylation of tau by glycogen synthase kinase 3beta identified by mass spectrometry demonstrate certain mutations exert long-range conformational changes.
In vitro phosphorylation of recombinant wild-type 2N4R tau and FTDP-17 exonic mutant forms P301L, V337M and R406W by glycogen synthase kinase 3beta (GSK3beta) 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 constituents and sites of phosphorylation had been determined previously, enabled us to monitor directly changes in phosphorylation of the individual tau proteins. No differences were found in the phosphorylation of wild-type, P301L or V337M tau by GSK3beta but the R406W mutant showed at least two clear differences from the other three tau proteins. The peptides, identified by mass spectrometry corresponding to phosphorylation at both threonine 231 and serine 235 (spot 3), serines 396, 400 and 404 (spot 6a) and serines 195 and 199 (spot 6b) were absent from the R406W peptide map. The findings imply that the R406W mutation in tau exerts long-range conformational effects on the structure of tau.
Connell
J W
JW
Department of Neuroscience, Institute of Psychiatry, King's College London, De Crespigny Park, London, UK.
Gibb
G M
GM
Betts
J C
JC
Blackstock
W P
WP
Gallo
J
J
Lovestone
S
S
Hutton
M
M
Anderton
B H
BH
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005091:Exons; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D006801:Humans; D013058:Mass Spectrometry; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D020125:Mutation, Missense; D010449:Peptide Mapping; D010766:Phosphorylation / Q000187:drug effects; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D016875:tau Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
C054369:MAPT protein, human; D008869:Microtubule-Associated Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D016875:tau Proteins; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3