The cytoplasmic domain of Alzheimer's amyloid precursor protein is phosphorylated at Thr654, Ser655, and Thr668 in adult rat brain and cultured cells.
BACKGROUND
The cytoplasmic domain of the Alzheimer's disease amyloid precursor protein (APP) is phosphorylated in vitro at Thr654 and Ser655, and both in vitro and in intact cells at Thr668 (numbering for APP695 isoform).


METHODS
We have developed phosphorylation state-specific antibodies to each of the sites, and we have used these to analyze the phosphorylation of APP in adult rat brain and in cultured cell lines.


RESULTS
We demonstrate that all three sites in APP are phosphorylated in adult rat brain. Phosphorylation at Thr654, Ser655, and Thr668 was also observed in several cultured cell lines. In PC12 cells, phosphorylation at Ser655 was increased more than 10-fold by treatment with okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, but was not affected by activators of protein kinase C. In HeLa cells, phosphorylation at Thr668 was regulated in a cell cycle-dependent manner with near-stoichiometric phosphorylation being observed at the G2/M phase of the cell cycle. In general, phosphorylation at Ser655 was found to be highest in mature APP isoforms, whereas phosphorylation of Thr668 was highest in immature APP isoforms in cultured cells.


CONCLUSIONS
The results demonstrate that phosphorylation of the cytoplasmic domain of APP occurs at Thr654, Ser655, and Thr668 under physiological conditions. The further characterization of APP phosphorylation using phosphorylation-specific antibodies may help in the elucidation of the biological function of APP.
Oishi
M
M
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York, USA.
Nairn
A C
AC
Czernik
A J
AJ
Lim
G S
GS
Isohara
T
T
Gandy
S E
SE
Greengard
P
P
Suzuki
T
T
D000544:Alzheimer Disease / Q000378:metabolism*; D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats; D012694:Serine / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*
D016564:Amyloid beta-Protein Precursor; D000906:Antibodies; D013912:Threonine; D012694:Serine