The occurrence of consensus phosphorylation sites in the intracellular domain of the Alzheimer’s amyloid precursor protein (APP), coupled with observations of their in vivo phosphorylation, prompted several workers to investigate the effects that phosphorylation of such sites could have on APP metabolism and subsequent Aβ production. However, hitherto all attempts to dissect the role played by such phosphorylation events failed to reveal substantial effects. Having decided to revisit this problem, our new approach was based on the following vectors: (1) site-directed mutagenesis of the target amino acids to mimic a specific phosphorylation state, (2) expression of wild-type and mutant APP-GFP (green fluorescent protein) fusion proteins for ease of visualization, (3) controlled low level expression to avoid ‘flooding’ cellular pathways, and (4) the use of cycloheximide to inhibit de novo protein synthesis. Using this method we were able to detect specific differences in APP processing that were correlated with the mimicked phosphorylation state of several phosphorylation sites. New combined methodologies, like the one described here, allow for the detailed analysis of key control points in the cellular metabolism of specific proteins that are central to neurodegenerative diseases and may be under the control of specific posttranslational modifications, such as reversible phosphorylation.

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