Substitution of phenylalanine by proline at position 19 of amyloid β peptide results in an increased production of amyloid β peptides with alternative N-termini after protein kinase C stimulation

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Abstract
Amyloid β peptide (Aβ) is derived from the β amyloid precursor protein (AβPP) by proteolytic processing. Three proteolytic activities are involved in the processing of AβPP. β–secretase generates the N-terminus of Aβ, γ–secretase generates the C-terminus and α–secretase cleaves in the central region of the Aβ domain thus preventing Aβ generation. In addition to the major β–secretase cleavage at aspartate 1 of the Aβ domain, alternative cleavages occur both in vivo and in vitro, leading to the generation of Aβ–like peptides with truncated or elongated N-termini. To determine if the N-terminal cleavage of alternative Aβ–like molecules is mediated by the β– or α–secretory pathway, we stimulated α–secretory processing by activation of protein kinase C with phorbol esters. For this study we used a mutagenized AβPP cDNA replacing a critical phenylalanine with proline at position 690 of AβPp770. This mutation is close to the α–secretase site and inhibits the normal α–secretase cleavage at Lys 16 resulting in the production of high amounts of Aβ–like molecules and truncated forms of AβPPi. Activation of protein kinase C (PKC) in cells expressing the proline mutation did not inhibit Aβ production, but led to an increase of the production of Aβ–like molecules. This indicates that the N-terminus of alternative Aβ molecules can be produced by a proteolytic pathway which is activated after PKC stimulation.