Phosphorylation-dependent regulation of the interaction of amyloid precursor protein with Fe65 affects the production of beta-amyloid.
Neuronal Fe65 is an adapter protein that interacts with the cytoplasmic domain of the beta-amyloid precursor protein (APP). Although the interaction has been reported to occur between the second phosphotyrosine interaction domain of Fe65 and the YENPTY motif in the cytoplasmic domain of APP, the regulatory mechanism and biological function of this interaction remain unknown. We report here that (i) a single amino acid mutation at the Thr-668 residue of APP695, located 14 amino acids toward the amino-terminal end from the (682)YENPTY(687) motif, reduced the interaction between members of the Fe65 family of proteins and APP, whereas interaction of APP with the phosphotyrosine interaction domain of other APP binders such as X11-like and mammalian disabled-1 was not influenced by this mutation; (ii) the phosphorylation of APP at Thr-668 diminished the interaction of APP with Fe65 by causing a conformational change in the cytoplasmic domain that contains the Fe65-binding motif, YENPTY; and (iii) the expression of Fe65 slightly suppressed maturation of APP and decreased production of beta-amyloid (Abeta). Mutation at Thr-668 of APP abolished the effect of Fe65 on APP maturation. This mutation blocked the Fe65-dependent suppression of Abeta production and resulted in the release of increased levels of Abeta in the presence of Fe65. We previously reported that during maturation of APP in neurons, the protein is specifically phosphorylated at Thr-668 and undergoes O-glycosylation. The present results suggest that the phosphorylation of O-glycosylated mature APP at Thr-668 causes a conformational change in its cytoplasmic domain that prevents binding of Fe65 in neurons and may lead to an alteration in the production of Abeta.