Ectodomain phosphorylation of beta-amyloid precursor protein at two distinct cellular locations

The beta-amyloid precursor protein (betaAPP) is a transmembrane protein that is exclusively phosphorylated on serine residues within its ectodomain. To identify the cellular site of betaAPP phosphorylation, we took advantage of an antibody that specifically detects the free C terminus of beta-secretase-cleaved betaAPP containing the Swedish missense mutation (APPssw-beta). This antibody previously established the cellular location of the beta-secretase cleavage of Swedish betaAPP as a post-Golgi secretory compartment (Haass, C., Lemere, C., Capell, A., Citron, M., Seubert, P., Schenk, D., Lannfelt, L., and Selkoe, D. J. (1995) Nature Med. 1, 1291-1296). We have now localized the selective ectodomain phosphorylation of betaAPP to the same compartment. Moreover, the phosphorylation sites of betaAPP were identified at Ser198 and Ser206 of betaAPP695 by tryptic peptide mapping, mass spectrometry, and site-directed mutagenesis. Intracellular phosphorylation of betaAPP was inhibited by Brefeldin A and by incubating cells at 20 degrees C, thus excluding phosphorylation in the endoplasmic reticulum or trans-Golgi network. Ectodomain phosphorylation within a post-Golgi compartment occurred not only with mutant Swedish betaAPP, but also with wild type betaAPP. In addition to phosphorylation within a post-Golgi compartment, betaAPP was also found to undergo phosphorylation at the cell surface by an ectoprotein kinase. Therefore, this study revealed two distinct cellular locations for betaAPP phosphorylation.