Conformation of paired helical filaments blocks dephosphorylation of epitopes shared with fetal tau except Ser199/202 and Ser202/Thr205.
To determine if the high phosphate content of paired helical filaments (PHFs) in Alzheimer's disease (AD) is a result of limited access to filament phosphorylation sites, we studied in vitro dephosphorylation of intact PHFs, PHFs with filamentous structure abolished by formic acid treatment (PHF(FA)) and fetal human tau protein. Samples were treated with alkaline phosphatase for up to 24 h at 37 degrees C and then immunoblotted with eight well characterized tau antibodies, that recognize two phosphorylation-insensitive sites and six phosphorylation-sensitive epitopes at Thr181, Ser199/202, Ser202/Thr205, Thr231, Ser262/356 and Ser396/404. Intact PHFs were effectively dephosphorylated only at the two N-terminal epitopes Ser199/202 and Ser202/Thr205, with little change in electrophoretic mobility. In contrast, PHF(FA) were dephosphorylated at all epitopes, with particular effectiveness at those in the C-terminus and with significant increase in electrophoretic mobility. The fetal tau epitopes were effectively dephosphorylated except at Thr181 and Thr231 with marked increase in mobility. The extent of dephosphorylation of PHF(FA) was equal or more effective than in fetal tau, except for Thr181 that was minimally dephosphorylated in both proteins. The results indicate that intact PHFs, but not PHF(FA) or fetal tau display differential dephosphorylation of the N- and C-terminal epitopes. The results confirm that the filamentous conformation may significantly contribute to hyperphosphorylation of PHFs in the C-terminus. The filamentous conformation, however, does not limit access to two N-terminal epitopes Ser199/202 and Ser202/Thr205. The access to these sites in AD may be limited by other factors, e.g., inhibition of phosphatase binding.
Gordon-Krajcer
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Department of Pathology, Rm. F-538, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, USA.
Yang
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Ksiezak-Reding
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H
D000368:Aged; D000369:Aged, 80 and over; D000469:Alkaline Phosphatase; D000544:Alzheimer Disease / Q000473:pathology*; D000595:Amino Acid Sequence; D001921:Brain / Q000473:pathology*; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism; D005260:Female; D005333:Fetus; D005561:Formates; D006801:Humans; D007700:Kinetics; D008875:Middle Aged; D019600:Neuropil Threads / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure*; D010766:Phosphorylation; D012694:Serine; D013912:Threonine; D016875:tau Proteins / Q000737:chemistry* / Q000378:metabolism*
D000939:Epitopes; D005561:Formates; D016875:tau Proteins; C030544:formic acid; D013912:Threonine; D012694:Serine; D000469:Alkaline Phosphatase