Activation of protein kinase C (PKC) regulates the processing of Alzheimer amyloid precursor protein (APP) into its soluble form (sAPP) and amyloid β‐peptide (Aβ). However, little is known about the intermediate steps between PKC activation and modulation of APP metabolism. Using a specific inhibitor of mitogen‐activated protein (MAP) kinase kinase activation (PD 98059), as well as a dominant negative mutant of MAP kinase kinase, we show in various cell lines that stimulation of PKC by phorbol ester rapidly induces sAPP secretion through a mechanism involving activation of the MAP kinase cascade. In PC12‐M1 cells, activation of MAP kinase by nerve growth factor was associated with stimulation of sAPP release. Conversely, M1 muscarinic receptor stimulation, which is known to act in part through a PKC‐independent pathway, increased sAPP secretion mainly through a MAP kinase‐independent pathway. Aβ secretion and its regulation by PKC were not affected by PD 98059, supporting the concept of distinct secretory pathways for Aβ and sAPP formation.

Amyloid ␤ peptide (A␤) is a short peptide that is the major constituent of the amyloid plaques and cerebrovascular amyloid deposits found in Alzheimer's disease. The lack of availability of a cell-free system in which to study A␤ formation has limited our understanding of the molecular mechanisms involved in its production. We report here the reconstitution of such a cell-free system. The reconstituted A␤ formation was temperature-dependent and required ATP. Preincubation with purified protein kinase C (PKC) induced a pronounced inhibition of A␤ formation, similar to that observed in intact cells upon stimulation of PKC. The calmodulin antagonists W-7 and trifluoperazine inhibited A␤ formation and enhanced the action of PKC in both the cell-free system and intact cells. A role for the calcium/calmodulin-activated protein phosphatase calcineurin in the regulation of A␤ formation was demonstrated using a specific peptide inhibitor of calcineurin in vitro as well as cyclosporin A, a cell-permeant inhibitor of calcineurin, in intact cells. Our results suggest that a single substrate might mediate opposing actions of PKC and calcineurin in the regulation of A␤ formation.One of the salient features of Alzheimer's disease (AD) 1 neuropathology is deposition of the amyloid ␤ peptide (A␤) in brain parenchyma and cerebral vessels. This 40 -42-amino acid peptide is derived from the Alzheimer amyloid protein precursor (APP) (for a review, see Ref. 1). Mutations in APP have been found to cosegregate with affected status in families with earlyonset AD (1). These mutations affect the levels (2-4), length (5), or primary sequence (6) of the A␤ formed. In all of these cases, it has been argued that the mutations would lead to increased A␤ deposition in the brain in a manner sufficient to cause AD. Thus, an understanding of the mechanisms by which A␤ is formed and the means by which A␤ production is controlled may identify avenues for the development of therapies for AD (7).Numerous studies have demonstrated the formation of A␤ by intact cells (8 -10) and its regulation by a number of signal transduction pathways (reviewed in Ref.11). Activation of protein kinase C (PKC) and/or inhibition of protein phosphatase 1 or 2A dramatically inhibit A␤ formation (12)(13)(14). Raising intracellular calcium can, according to the experimental conditions, either inhibit or dramatically stimulate A␤ formation (15, 16). The mechanisms by which these various signal transduction cascades are able to regulate A␤ formation are currently unknown. The availability of a cell-free system in which to study A␤ formation would greatly facilitate the understanding of the molecular mechanisms involved.We made use of a Balch homogenizer to prepare cracked cells (17). A␤ production in these broken cell preparations was found to depend on ATP and temperature. By introducing purified PKC, a peptide inhibitor of the calcium/calmodulin-dependent protein phosphatase calcineurin, or a calmodulin antagonist (W-7 or trifluoperazine), it was demonstrated that PKC and c...