In Vitro Phosphorylation of the Cytoplasmic Domain of the Amyloid Precursor Protein by Glycogen Synthase Kinase‐3β

Abstract

The two pathological lesions found in the brains of Alzheimer's disease patients, neurofibrillary tangles and neuritic plaques, are likely to be formed through a common pathway. Neurofibrillary tangles are intracellular aggregates of paired helical filaments, the main component of which is hyperphosphorylated forms of the microtubule-associated protein tau. Extracellular neuritic plaques and diffuse and vascular amyloid deposits are aggregates of beta-amyloid protein, a 4-kDa protein derived from the amyloid precursor protein (APP). Using conditions in vitro under which two proline-directed protein kinases, glycogen synthase kinase-3beta (GSK-3beta) and mitogen-activated protein kinase (MAPK), were able to hyperphosphorylate tau, GSK-3beta but not MAPK phosphorylated recombinant APPcyt. The sole site of phosphorylation in APPcyt by GSK-3beta was determined by phosphoamino acid analysis and phosphorylation of APPcyt mutant peptides to be Thr743 (numbering as for APP770). This site was confirmed by endoproteinase Glu-C digestion of APPcyt and peptide sequencing. The ability of GSK-3beta to phosphorylate APPcyt and tau provides a putative link between the two lesions and indicates a critical role of GSK-3beta in the pathogenesis of Alzheimer's disease.