Regulation of Secretion of Alzheimer Amyloid Precursor Protein by the Mitogen-Activated Protein Kinase Cascade

Abstract: 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.     

L-glutamate and phorbol ester stimulate the release of secretory amyloid precursor protein from rat cortical synaptosomes

Treatment of rat cortical synaptosomes with micromolar concentrations of L-glutamate stimulated the release of the secreted form of amyloid precursor protein in a concentration-dependent, however biphasic manner as assayed by semiquantitative Western blot analysis. The secreted amyloid precursor protein released from synaptosomes into the incubation medium was highest in the presence of 500 microM L-glutamate (about 64% over the level assayed in the incubation medium in the absence of any drug). In contrast, direct stimulation of protein kinase C by phorbol-12-myristate-13-acetate resulted in a concentration-independent increase in secretory amyloid precursor protein release by about 100% already detectable at a concentration of 0.1 microM but with no significant change at higher concentrations up to 10 microM. The presented data show that there is a constitutive release of secretory amyloid precursor protein from synaptosomes and suggest that (i) processing of amyloid precursor protein at the synaptic level is controlled by L-glutamate presumably via activation of protein kinase C, and (ii) isolated cortical synaptosomes represent a useful experimental approach to selectively study amyloid precursor protein metabolism at the synaptic level.     

Large quantities of Abeta peptide are constitutively released during amyloid precursor protein metabolism in vivo and in vitro

The metabolism of the amyloid precursor protein (APP) has been extensively investigated because its processing generates the amyloid-β-peptide (Aβ), which is a likely cause of Alzheimer disease. Much prior research has focused on APP processing using transgenic constructs and heterologous cell lines. Work to date in native neuronal cultures suggests that Aβ is produced in very large amounts. We sought to investigate APP metabolism and Aβ production simultaneously under more physiological conditions in vivo and in vitro using cultured rat cortical neurons and live pigs. We found in cultured neurons that both APP and Aβ are secreted rapidly and at extremely high rates into the extracellular space (2-4 molecules/neuron/s for Aβ). Little APP is degraded outside of the pathway that leads to extracellular release. Two metabolic pools of APP are identified, one that is metabolized extremely rapidly (t1/2;) = 2.2 h), and another, surface pool, composed of both synaptic and extrasynaptic elements, that turns over very slowly. Aβ release and accumulation in the extracellular medium can be accounted for stoichiometrically by the extracellular release of β-cleaved forms of the APP ectodomain. Two α-cleavages of APP occur for every β-cleavage. Consistent with the results seen in cultured neurons, an extremely high rate of Aβ production and secretion from the brain was seen in juvenile pigs. In summary, our experiments show an enormous and rapid production and extracellular release of Aβ and the soluble APP ectodomain. A small, slowly metabolized, surface pool of full-length APP is also identified.     

Regulation of Amyloid Precursor Protein Cleavage

Abstract : Multiple lines of evidence suggest that increased production and/or deposition of the β-amyloid peptide, derived from the amyloid precursor protein, contributes to Alzheimer's disease. A growing list of neuro-transmitters, growth factors, cytokines, and hormones have been shown to regulate amyloid precursor protein processing. Although traditionally thought to be mediated by activation of protein kinase C, recent data have implicated other signaling mechanisms in the regulation of this process. Moreover, novel mechanisms of regulation involving cholesterol-, apolipoprotein E-, and stress-activated pathways have been identified. As the phenotypic changes associated with Alzheimer's disease encompass many of these signaling systems, it is relevant to determine how altered cell signaling may be contributing to increasing brain amyloid burden. We review the myriad ways in which first messengers regulate amyloid precursor protein catabolism as well as the signal transduction cascades that give rise to these effects.     

Presenilins, the Endoplasmic Reticulum, and Neuronal Apoptosis in Alzheimer's Disease

Abstract: Many cases of autosomal dominant inherited forms of early-onset Alzheimer's disease are caused by mutations in the genes encoding presenilin-1 (PS-1; chromosome 14) and presenilin-2 (PS-2; chromosome 1). PSs are expressed in neurons throughout the brain wherein they appear to be localized primarily to the endoplasmic reticulum (ER) of cell bodies and dendrities. PS-1 and PS-2 show high homology and are predicted to have eight transmembrane domains with the C terminus, N terminus, and a loop domain all on the cytosolic side of the membrane; an enzymatic cleavage of PSs occurs at a site near the loop domain. The normal function of PSs is unknown, but data suggest roles in membrane trafficking, amyloid precursor protein processing, and regulation of ER calcium homeostasis. Homology of PSs to the C. elegans gene sel-12 , which is involved in Notch signaling, and phenotypic similarities of PS-1 and Notch knockout mice suggest a developmental role for PSs in the nervous system. When expressed in cultured cells and transgenic mice, mutant PSs promote increased production of a long form of amyloid β-peptide (Aβ1-42) that may possess enhanced amyloidogenic and neurotoxic properties. PS mutations sensitize cultured neural cells to apoptosis induced by trophic factor withdrawal, metabolic insults, and amyloid β-peptide. The mechanism responsible for the proapoptotic action of mutant PSs may involve perturbed calcium release from ER stores and increased levels of oxidative stress. Recent studies of apoptosis in many different cell types suggest that ER calcium signaling can modulate apoptosis. The evolving picture of PS roles in neuronal plasticity and Alzheimer's disease is bringing to the forefront the ER, an organelle increasingly recognized as a key regulator of neuronal plasticity and survival.     

Protein Kinase C Activation Potentiates the Rapid Secretion of the Amyloid Precursor Protein from Rat Cortical Synaptosomes

Abstract : In this study we have used the presynaptic-rich rat cerebrocortical synaptosomal preparation to investigate the proteolytic cleavage of the amyloid precursor protein (AβPP) by the α-secretase pathway within the βA4 domain to generate a soluble secreted N-terminal fragment (AβPP_s). AβPP was detected in crude cortical synaptosomal membranes, although at a lower density than that observed in whole-tissue homogenates. Protein kinase C (PKC) activation induced a translocation of the conventional PKC isoform β1 and novel PKCε from cytosol to membrane fractions, but there was no alteration in the proportion of AβPP associated with the Tritonsoluble and -insoluble fractions. AβPP_s was constitutively secreted from cortical synaptosomes, with this secretion being enhanced significantly by the direct activation of PKC with phorbol ester. The PKC-induced secretion of AβPP_s was only partially blocked by the PKC inhibitor GF109203X (2.5 μ M ), whereas the phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein was significantly inhibited by GF109203X. The differential sensitivities of the MARCKS phosphorylation and AβPP_s secretion to GF109203X may imply that different PKC isoforms are involved in these two events in the synaptosomal system. These findings strongly suggest that the α-secretase activity leading to the secretion of AβPP_s can occur at the level of the presynaptic terminal.     

Intracellular Cyclic AMP Inhibits Constitutive and Phorbol Ester-Stimulated Secretory Cleavage of Amyloid Precursor Protein

Abstract: α-Secretase cleaves the full-length Alzheimer's amyloid precursor protein (APP) within the amyloid β peptide sequence, thus precluding amyloid formation. The resultant soluble truncated APP is constitutively secreted. This nonamyloidogenic processing of APP is increased on stimulation of the phospholipase C/protein kinase C pathway by phorbol esters. Here we used C6 cells transfected with APP₇₅₁ to examine whether the α-secretase cleavage is regulated by the adenylate cyclase signal transduction pathway. Forskolin, an activator of adenylate cyclase, inhibited both the constitutive and phorbol ester-stimulated secretion of nexin II (NXII), the secreted product of the α-secretase cleavage of APP₇₅₁. At 1 µ M , forskolin inhibited secretion of NXII by ∼50% without affecting either the intracellular levels of total APP or the secretion of secretory alkaline phosphatase. In contrast, 1,9-dideoxyforskolin, an inactive analogue of forskolin, did not affect secretion of NXII. These results indicated that forskolin specifically inhibited the α-secretase cleavage of APP₇₅₁. Forskolin treatment increased the intracellular concentration of cyclic AMP (cAMP), suggesting that the forskolin effects on APP cleavage may be mediated by cAMP. In support of this suggestion, both dibutyryl cAMP, a cAMP analogue, and isoproterenol, an activator of adenylate cyclase, also inhibited secretion of NXII. These data indicate that forskolin inhibition of the nonamyloidogenic cleavage of APP is mediated by the second messenger cAMP, which together with the protein kinase C signal transduction pathway modulates the secretory cleavage of APP.     

Proteolytic Degradation of Alzheimer's Disease Amyloid β-Peptide by a Metalloproteinase from Microglia Cells

Abstract: The cerebral deposition of amyloid β-peptide (Aβ) is a histopathological characteristic of Alzheimer's disease. Because an impaired clearance of Aβ might be involved in the disease, we investigated the proteolytic degradation of synthetic Aβ (40-residue peptide) in cultures of glial cells and characterized a protease involved. Whereas rat astrocytes had a very low degradation capacity, cultivated rat microglia cells cleaved Aβ. Microglia activity was considerably enhanced by stimulation with lipopolysaccharide and to a lesser extent by phorbol esters. Most of the Aβ-degrading activity was released into the medium. By use of selective inhibitors the protease was characterized as a metalloprotease of ∼200 kDa that was different from neutral endopeptidase (a neuropeptide-degrading enzyme), matrix metalloproteases, or macrophage elastase. Its activity was efficiently reduced by four hydroxamic acid-based zinc-metalloprotease inhibitors that have been shown to inhibit membrane protein secretases (disintegrins). We conclude that activated microglia cells might impair amyloid plaque formation by release of a metalloprotease that degrades soluble Aβ before polymerization.     

Differential involvement of protein kinase C alpha and epsilon in the regulated secretion of soluble amyloid precursor protein.

We investigated the differential role of protein kinase C (PKC) isoforms in the regulated proteolytic release of soluble amyloid precursor protein (sAPPalpha) in SH-SY5Y neuroblastoma cells. We used cells stably transfected with cDNAs encoding either PKCalpha or PKCepsilon in the antisense orientation, producing a reduction of the expression of PKCalpha and PKCepsilon, respectively. Reduced expression of PKCalpha and/or PKCepsilon did not modify the response of the kinase to phorbol ester stimulation, demonstrating translocation of the respective isoforms from the cytosolic fraction to specific intracellular compartments with an interesting differential localization of PKCalpha to the plasma membrane and PKCepsilon to Golgi-like structures. Reduced expression of PKCalpha significantly impaired the secretion of sAPPalpha induced by treatment with phorbol esters. Treatment of PKCalpha-deficient cells with carbachol induced a significant release of sAPPalpha. These results suggest that the involvement of PKCalpha in carbachol-induced sAPPalpha release is negligible. The response to carbachol is instead completely blocked in PKCepsilon-deficient cells suggesting the importance of PKCepsilon in coupling cholinergic receptors with APP metabolism.     

Secretory processing of the Alzheimer amyloid beta/A4 protein precursor is increased by protein phosphorylation.

The 39-43 residue polypeptide (amyloid beta protein, beta A4) deposited as amyloid in Alzheimer's disease (AD) is derived from a set of 695-770 residue precursors referred to as the amyloid beta A4 protein precursor (beta APP). In each of the 695, 751, and 770 residue precursors, the 43 residue beta A4 is an internal peptide that begins 99 residues from the COOH-terminus of the beta APP. Each holoform is normally cleaved within the beta A4 to produce a large secreted derivative as well as a small membrane associated fragment. Neither of these derivatives can produce amyloid because neither contains the entire beta A4 peptide. In this study, we employ cells stably transfected with full length beta APP695, beta APP751, or beta APP770 expression constructs to show that phorbol ester activation of protein kinase C substantially increases the production of secreted forms from each isoform. By increasing processing of beta APP in the secretory pathway, PKC phosphorylation may help to prevent amyloid deposition.     

Ginkgo biloba extract (Egb 761) inhibits beta-amyloid production by lowering free cholesterol levels

Ginkgo biloba extract (EGb 761) can improve cognitive function in patients with Alzheimer's disease, but the molecular mechanisms underlying this effect remain undefined. Because free cholesterol may be involved in the production of β-amyloid precursor protein and amyloid β-peptide, key events in the development of Alzheimer's disease, we examined EGb 761 in relation to cholesterol and amyloidogenesis. In aging rats, EGb 761 treatment lowered circulating free cholesterol and inhibited the production of brain β-amyloid precursor protein and amyloid β-peptide. Exposure of PC12 cells to EGb 761 decreased the processing of β-amyloid precursor protein and abolished cholesterol-induced overproduction of this protein. Exposure of human NT2 cells to EGb 761 decreased free cholesterol influx and increased free cholesterol efflux. Our findings indicate that free circulating and intracellular cholesterol levels affect the processing of β-amyloid precursor protein and amyloidogenesis. Our findings also provide the first demonstration that EGb 761 can influence these mechanisms.     

Activation of muscarinic cholinergic receptors enhances the volume-sensitive efflux of myo-inositol from SH-SY5Y neuroblastoma cells

A mechanism used by cells to regulate their volume under hypo-osmotic conditions is the release of organic osmolytes, one of which is myo-inositol. The possibility that activation of phospholipase-C-linked receptors can regulate this process has been examined for SH-SY5Y neuroblastoma cells. Incubation of cells with hypo-osmolar buffers (160-250 mOsm) led to a biphasic release of inositol which persisted for up to 4 h and could be inhibited by inclusion of anion channel blockers - results which indicate the involvement of a volume-sensitive organic anion channel. Inclusion of oxotremorine-M, a muscarinic cholinergic agonist, resulted in a marked increase (80-100%) in inositol efflux under hypo-osmotic, but not isotonic, conditions. This enhanced release, which was observed under all conditions of hypo-osmolarity tested, could be prevented by inclusion of atropine. Incubation of the cells with either the calcium ionophore, ionomycin, or the phorbol ester, phorbol 12-myristate 13-acetate, partially mimicked the stimulatory effect of muscarinic receptor activation when added singly, and fully when added together. The ability of oxotremorine-M to facilitate inositol release was inhibited by removal of extracellular calcium, depletion of intracellular calcium or down-regulation of protein kinase C. These results indicate that activation of muscarinic cholinergic receptors can regulate osmolyte release in this cell line.     

Neurotoxicity of a Carboxyl-Terminal Fragment of the Alzheimer's Amyloid Precursor Protein

Abstract: We have previously shown that a recombinant carboxyl-terminal 105-amino-acid fragment (CT105) of the amyloid precursor protein (APP) induced strong non-selective inward currents in Xenopus oocytes. Here we investigated the toxic effect of CT105 peptide on the cultured mammalian cells. The CT105 peptide induced a significant lactate dehydrogenase (LDH) release from cultured rat cortical neurons and PC12 cells in a concentration (from 10 µ M )- and time (from 48 h)-dependent manner. The toxic effect of CT105 was more potent than that of any fragments of amyloid β protein (Aβ). However, CT105 peptide did not affect the viability of U251 human glioblastoma cells. In contrast to CT105, Aβ increased LDH release only slightly even at 50 µ M but significantly inhibited 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction at submicromolar concentrations. Among the various neuroprotective drugs tested, only cholesterol, which alters membrane fluidity, could attenuate the cytotoxicity of CT105 significantly. The CT105 peptide formed multiple self-aggregates on solubilization. Pretreatment with a sublethal concentration of CT105 did not significantly alter the susceptibility of cells to hydrogen peroxide and glutamate. Endogenous CT peptides were found not only in the cell lysates but also in the conditioned medium of PC12 cells. These results imply that CT peptide can directly attack the cell membrane probably by making pores or nonselective ion channels, whereas Aβ impairs the intracellular metabolic pathway first. Thus, it is thought that both CT and Aβ, which are formed during the processing of APP, may participate in the neuronal degeneration in Alzheimer's disease by different mechanisms.     

Enhanced generation of Alzheimer's amyloid-β following chronic exposure to phorbol ester correlates with differential effects on alpha and epsilon isozymes of protein kinase C

Alzheimer's amyloid precursor protein (APP) sorting and processing are modulated through signal transduction mechanisms regulated by protein phosphorylation. Notably, protein kinase C (PKC) appears to be an important component in signaling pathways that control APP metabolism. PKCs exist in at least 11 conventional and unconventional isoforms, and PKCα and PKCε isoforms have been specifically implicated in controlling the generation of soluble APP and amyloid-β (Aβ) fragments of APP, although identification of the PKC substrate phospho-state-sensitive effector proteins remains challenging. In the current study, we present evidence that chronic application of phorbol esters to cultured cells in serum-free medium is associated with several phenomena, namely: (i) PKCα down-regulation; (ii) PKCε up-regulation; (iii) accumulation of APP and/or APP carboxyl-terminal fragments in the trans Golgi network; (iv) disappearance of fluorescence from cytoplasmic vesicles bearing a green fluorescent protein tagged form of APP; (v) insensitivity of soluble APP release following acute additional phorbol application; and (vi) elevated cellular APP mRNA levels and holoprotein, and secreted Aβ. These data indicate that, unlike acute phorbol ester application, which is accompanied by lowered Aβ generation, chronic phorbol ester treatment causes differential regulation of PKC isozymes and increased Aβ generation. These data have implications for the design of amyloid-lowering strategies based on modulating PKC activity.     

Basal Forebrain Cholinergic Dysfunction in Alzheimer’s Disease – Interrelationship with β-amyloid, Inflammation and Neurotrophin Signaling

Alzheimer’s disease, the most common neurodegenerative disorder of senile dementia, is characterized by two major morpho-pathological hallmarks. Deposition of extracellular neuritic, β-amyloid peptide-containing plaques (senile plaques) in cerebral cortical regions of Alzheimer patients is accompanied by the presence of intracellular neurofibrillary tangles in cerebral pyramidal neurons. Basal forebrain cholinergic dysfunction is also a consistent feature of Alzheimer’s disease, which has been suggested to cause, at least partly, the cognitive deficits observed in patients with Alzheimer’s disease. Impaired cortical cholinergic neurotransmission may also contribute to β-amyloid plaque pathology in Alzheimer’s disease by affecting expression and processing of the β-amyloid precursor protein (APP). Vice versa, low level of soluble β-amyloid has been observed to inhibit cholinergic synaptic function. Deposition of β-amyloid plaques in Alzheimer’s disease is also accompanied by a significant plaque-associated glial up-regulation of interleukin-1, which has been attributed to affect expression and metabolism of APP and to interfere with cholinergic transmission. Understanding the molecular mechanisms underlying the interrelationship between cortical cholinergic dysfunction, β-amyloid formation and deposition, as well as local inflammatory upregulation, would allow to derive potential treatment strategies to pharmacologically intervene in the disease-causing signaling cascade.     

Metabotropic Glutamate Receptors Increase Amyloid Precursor Protein Processing in Astrocytes: Inhibition by Cyclic AMP

Abstract: Neurotransmitter receptors that increase phosphatidylinositol hydrolysis generate second messengers that activate protein kinase C. Here, we used metabotropic glutamate receptor agonists to increase both phosphatidylinositol hydrolysis and secretion of the soluble extracellular fragment of amyloid precursor protein (APPs) from cortical astrocyte cultures. The increase in APPs secretion was mimicked by direct activation of protein kinase C with phorbol ester and was suppressed by the metabotropic glutamate receptor antagonist l -(+)-2-amino-3-phosphonopropionic acid or by the protein kinase C inhibitor GF109203X. Ionotropic glutamate agonists did not increase APPs secretion. Forskolin or dibutyryl cyclic AMP inhibited the increase in APPs secretion caused by metabotropic glutamate receptor agonists or by phorbol ester treatment but did not affect basal APPs levels. Therefore, glutamatergic agonists that increase protein kinase C activation or decrease cyclic AMP formation may enhance the conversion of full-length APP to nonamyloidogenic APPs in Alzheimer's disease.     

Activity of α-Secretase as the Common Final Effector of Protein Kinase C-Dependent and -Independent Modulation of Amyloid Precursor Protein Metabolism

The metabolic fate of the amyloid precursor protein (APP) is one of the key factors in the pathogenesis of Alzheimer's disease (AD). A complex cellular mechanism regulates the rate at which the precursor is cleaved by alpha-secretase and released as soluble protein in the extracellular space. We show here that alpha-secretase constitutes the common final effector of several independent means of stimulation of soluble APP (sAPP) release. The release of sAPP by alpha-secretase resembles that of several other membrane-bound proteins with soluble counterparts, a process that is sensitive to matrix metalloprotease inhibitors. The hydroxamic acid-based compound KD-IX-73-4 inhibits phorbol ester-mediated sAPP release from COS cells with an IC50 of 8 microM, consistent with previous data for the same compound against leukocyte L-selectin shedding. Beyond direct protein kinase C (PKC) activation we show that KD-IX-73-4 inhibits also receptor-mediated sAPP release induced by carbachol in SH-SY5Y cells and by bradykinin in human skin fibroblasts, with the latter being a PKC-independent mechanism. Altogether these data suggest that all pharmacological means of stimulating sAPP release converge to a hydroxamic acid-based inhibitor-sensitive proteolytic enzyme. Moreover, because KD-IX-73-4 was effective in the inhibition of stimulated but not constitutive sAPP release, these data suggest the existence of different enzymes regulating the two metabolic pathways leading to sAPP secretion.     

Processing of Amyloid Precursor Protein in Human Primary Neuron and Astrocyte Cultures

Abstract: Increased production of amyloid β peptide (Aβ) is highly suspected to play a major role in Alzheimer's disease (AD) pathogenesis. Because Aβ deposits in AD senile plaques appear uniquely in the brain and are fairly restricted to humans, we assessed amyloid precursor protein (APP) metabolism in primary cultures of the cell types associated with AD senile plaques: neurons, astrocytes, and microglia. We find that neurons secrete 40% of newly synthesized APP, whereas glia secrete only 10%. Neuronal and astrocytic APP processing generates five C-terminal fragments similar to those observed in human adult brain, of which the most amyloidogenic higher-molecular-weight fragments are more abundant. The level of amyloidogenic 4-kDa Aβ exceeds that of nonamyloidogenic 3-kDa Aβ in both neurons and astrocytes. In contrast, microglia make more of the smallest C-terminal fragment and no detectable Aβ. We conclude that human neurons and astrocytes generate higher levels of amyloidogenic fragments than microglia and favor amyloidogenic processing compared with previously studied culture systems. Therefore, we propose that the higher amyloidogenic processing of APP in neurons and astrocytes, combined with the extended lifespan of individuals, likely promotes AD pathology in aging humans.     

Secreted Forms of β-Amyloid Precursor Protein Protect Against Ischemic Brain Injury

Abstract: The β-amyloid precursor protein (βAPP) is the source of the amyloid β-peptide that accumulates in the brain in Alzheimer's disease. A major processing pathway for βAPP involves an enzymatic cleavage within the amyloid β-peptide sequence that liberates secreted forms of βAPP (APP^Ss) into the extracellular milieu. We now report that postischemic administration of these APP^Ss intracerebroventricularly protects neurons in the CA1 region of rat hippocampus against ischemic injury. Treatment with APP^S695 or APP^S751 resulted in increased neuronal survival, and the surviving cells were functional as demonstrated by their ability to synthesize protein. These data provide direct evidence for a neuroprotective action of APP^Ss in vivo.     

Selective ectodomain phosphorylation and regulated cleavage of beta-amyloid precursor protein.

The beta-amyloid precursor protein (beta APP) is a highly conserved integral membrane protein expressed in most mammalian tissues and found at highest levels in the nervous system. Cerebral deposition of the amyloid beta-peptide (A beta), derived by proteolysis of beta APP, is an early and invariant feature of Alzheimer's disease. Protein phosphorylation by protein kinase C (PKC) has been found to regulate the metabolism of beta APP into nonamyloidogenic and amyloidogenic derivatives, but both the mechanism of these effects and the nature of beta APP phosphorylation are unknown. When labeled in vivo with [32P]orthophosphate, beta APP was phosphorylated only on serine residues in the N-terminal half of the extracellular domain, resulting in the secretion of phosphorylated soluble beta APP. PKC-mediated stimulation of beta APP secretion and concurrent inhibition of A beta release did not involve enhanced phosphorylation of beta APP and proceeded in the absence of cytoplasmic or extracellular phosphorylation of the precursor. The region of beta APP required for this indirect regulation by PKC was largely restricted to a 64 amino acid stretch around the secretory cleavage site. Moreover, in a truncated molecule designed to release soluble beta APP without the need for proteolytic cleavage, secretion was no longer regulated by PKC. Our data indicate that PKC-mediated pathways play a pivotal role in the control of beta APP metabolism and amyloid formation. However, in contrast to current postulates, this regulation is independent of beta APP phosphorylation and instead involves phosphorylation of other substrates that alter beta APP processing, such as beta APP-cleaving proteases.