Proteasome Contributes to the α-Secretase Pathway of Amyloid Precursor Protein in Human Cells

Abstract: A major histopathological hallmark in Alzheimer's disease consists of the extracellular deposition of the amyloid β-peptide (Aβ) that is proteolytically derived from the β-amyloid precursor protein (βAPP). An alternative, nonamyloidogenic cleavage, elicited by a protease called α-secretase, occurs inside the Aβ sequence and gives rise to APPα, a major secreted C-terminal-truncated form of βAPP. Here, we demonstrate that human embryonic kidney 293 (HK293) cells contain a chymotryptic-like activity that can be ascribed to the proteasome and that selective inhibitors of this enzyme reduce the phorbol 12,13-dibutyrate-sensitive APPα secretion by these cells. Furthermore, we establish that a specific proteasome blocker, lactacystin, also induces increased secretion of Aβ peptide in stably transfected HK293 cells overexpressing wild-type βAPP751. Altogether, this study represents the first identification of a proteolytic activity, namely, the proteasome, contributing likely through yet unknown intracellular relays, to the α-secretase pathway in human cells.     

Iron Levels Modulate α-Secretase Cleavage of Amyloid Precursor Protein

Abstract: The amyloid precursor protein (APP) is a membrane-spanning glycoprotein that is the source of βA4 peptides, which aggregate in Alzheimer's disease to form senile plaques. APP is cleaved within the βA4 sequence to release a soluble N-terminal derivative (APP_sol), which has a wide range of trophic and protective functions. In the current study we have examined the hypothesis that iron availability may modulate expression or processing of APP, whose mRNA contains, based on sequence homology, a putative iron response element (IRE). Radiolabeled APP and its catabolites were precipitated from lysates and conditioned medium of stably transfected HEK 293 cells using antibodies selective for C-terminal, βA4, and N-terminal domains. The relative abundance of the different APP catabolites under different conditions of iron availability was determined by quantitative densitometry after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data show a specific effect on the production of APP_sol. Using standard conditions previously established for IRE studies, it was found that iron chelation reduces APP_sol production, whereas iron level elevation augments it. No changes were observed in levels of immature and mature APP holoprotein or in the C-terminal α-secretase derivative C83, βA4, and p3 peptides. The specificity for modulatory changes in APP_sol suggests that iron acts at the level of α-secretase activity. In addition to its modulatory effects, iron at very high levels was found to inhibit maturation of APP and production of its downstream catabolites without blocking formation of immature APP. The data establish a potential physiological role for iron in controlling the processing of APP. If APP_sol were to function trophically, as suggested by other studies, the current conclusion suggests that changes in iron and iron-regulating proteins in Alzheimer's disease could contribute to neuronal degeneration by decreasing the production of APP_sol.     

Protein Kinase A Phosphorylation of the Proteasome: A Contribution to the α-Secretase Pathway in Human Cells

Abstract: The β-amyloid precursor protein undergoes a physiological cleavage by α-secretase that leads to the release of a secreted C-terminally truncated fragment called APPα and likely concomitantly reduces the formation of the amyloidogenic Aβ peptide. Here we demonstrate that APPα secretion is increased by the protein kinase A (PKA) effectors 8-bromo cyclic AMP and forskolin in human embryonic kidney cells (HK293), and that this can be prevented by a proteasome inhibitor. Furthermore, we establish that PKA effectors but not protein kinase C agonists increase the chymotrypsin-like activity and phosphorylation state of the proteasome in vitro and in vivo in HK293 cells. Altogether, this report demonstrates that the α-secretase pathway is under the control of PKA in human cells and that the proteasome likely contributes, either directly or through yet unknown intermediates, to the PKA-stimulated APPα secretion in human cells.     

Metabotropic Glutamate Receptor Subtype mGluR1α Stimulates the Secretion of the Amyloid β-Protein Precursor Ectodomain

Abstract: To examine the effects of glutamatergic neurotransmission on amyloid processing, we stably expressed the metabotropic glutamate receptor subtype 1α (mGluR1α) in HEK 293 cells. Both glutamate and the selective metabotropic agonist 1-amino-1,3-cyclopentanedicarboxylic acid (ACPD) rapidly increased phosphatidylinositol (PI) turnover four- to fivefold compared with control cells that were transfected with the expression vector alone. Increased PI turnover was effectively blocked by the metabotropic antagonist α-methyl-4-carbophenylglycine (MCPG), indicating that heterologous expression of mGluR1α resulted in efficient coupling of the receptors to G protein and phospholipase C activation. Stimulation of mGluR1α with glutamate, quisqualate, or ACPD rapidly increased secretion of the APP ectodomain (APPs); these effects were blocked by MCPG. The metabotropic receptors were coupled to APP processing by protein kinases and by phospholipase A₂ (PLA₂), and melittin, a peptide that stimulates PLA₂, potently increased APPs secretion. These data indicate that mGluR1α can be involved in the regulation of APP processing. Together with previous findings that muscarinic and serotonergic receptor subtypes can increase the secretion of the APP ectodomain, these observations support the concept that proteolytic processing of APP is under the control of several major neurotransmitters.     

Evaluation of Cathepsins D and G and EC 3.4.24.15 as Candidate β-Secretase Proteases Using Peptide and Amyloid Precursor Protein Substrates

Abstract: No single protease has emerged that possesses all the expected properties for β-secretase, including brain localization, appropriate peptide cleavage specificity, and the ability to cleave amyloid precursor protein exactly at the amino-terminus of β-amyloid peptide. We have isolated and purified a brain-derived activity that cleaves the synthetic peptide substrate SEVKMDAEF between methionine and aspartate residues, as required to generate the amino-terminus of β-amyloid peptide. Its molecular size of 55–60 kDa and inhibitory profile indicate that we have purified the metalloprotease EC 3.4.24.15. We have compared the sequence specificity of EC 3.4.24.15, cathepsin D, and cathepsin G for their ability to cleave the model peptide SEVKMDAEF or related peptides that contain substitutions reported to modulate β-amyloid peptide production. We have also tested the ability of these enzymes to form carboxy-terminal fragments from full-length, membrane-embedded amyloid precursor protein substrate or amyloid precursor protein that contains the Swedish KM → NL mutation. The correct cleavage was tested with an antibody specific for the free amino-terminus of β-amyloid peptide. Our results exclude EC 3.4.24.15 as a candidate β-secretase. Although cathepsin G cleaves the model peptide correctly, it displays poor ability to cleave the Swedish KM → NL peptide and does not generate carboxy-terminal fragments that are immunoreactive with amino-terminal-specific antiserum. Cathepsin D does not cleave the model peptide or show specificity for wild-type amyloid precursor protein; however, it cleaves the Swedish "NL peptide" and "NL precursor" substrates appropriately. Our results suggest that cathepsin D could act as β-secretase in the Swedish type of familial Alzheimer's disease and demonstrate the importance of using full-length substrate to verify the sequence specificity of candidate proteases.     

Increased Secretion of the Amino-Terminal Fragment of Amyloid Precursor Protein in Brains of Rats with a Constitutive Up-Regulation of Protein Kinase C

Abstract: Protein kinase C (PKC) activation stimulates release of secreted amyloid precursor protein (APP_s) in several cell lines. To ascertain the role of PKC in regulating APP metabolism in vivo, we used an animal model (methylazoxymethanol-treated rats; MAM rats) in which PKC is permanently hyperactivated in selected brain areas, i.e., cortex and hippocampus. A significant decrease in membrane-bound APP concentration was found in synaptosomes derived from cortex and hippocampus of MAM rats, where PKC is up-regulated, with a concomitant increase in APP_s production in soluble fractions of the same brain areas. In contrast, in a brain area not affected by MAM treatment (i.e., cerebellum), APP secretion is similar in control and MAM rats, indicating that altered metabolism of APP is restricted to only those areas in which the PKC system is up-regulated. In addition, phorbol esters or H-7 modulate APP_s release in hippocampal slices from both control and MAM rats, further supporting an in vivo role for this enzyme in regulating metabolism of mature APP.     

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.     

Inhibition of β-Amyloid Production by Activation of Protein Kinase C

The cellular factors regulating the generation of β-amyloid from the amyloid precursor protein (APR) are unknown. Activation of protein kinase C (PKC) by phorbol ester treatment inhibited the generation of the 4-kDa β-amyloid peptide in transfected COS cells, a human glioma cell line, and human cortical astrocytes. An analogue of diacylglycerol, the endogenous cellular activator of PKC, also inhibited the generation of β-amyloid. Activation of PKC increased the level of secreted APP in transfected COS cells but did not significantly affect the level of secreted APP in primary human astrocytes or in the glioma cell line. Cell-associated APP and the secreted APP derivative, but not β-amyloid, were phosphorylated on serine residues. Activation of PKC did not increase the level of APP phosphorylation, suggesting that PKC modulates the proteolytic cleavage of APP indirectly by phosphorylation of other substrates. These results indicate that PKC activation inhibits β-amyloid production by altering APP processing and suggest that β-amyloid production can be regulated by the phospholipase C-diacylglycerol signal transduction pathway.     

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.     

Mitogen-Activated Protein Kinase-Dependent and Protein Kinase C-Dependent Pathways Link the m1 Muscarinic Receptor to β-Amyloid Precursor Protein Secretion

Abstract: Full and functionally selective M1 muscarinic agonists (carbachol and AF102B, respectively) activate secretion of the soluble form of amyloid precursor protein (APPs) in PC12 cells expressing the m1 muscarinic receptor (PC12M1 cells). This activation is further augmented by neurotrophins such as nerve growth factor and basic fibroblast growth factor. Muscarinic stimulation activates two transduction pathways that lead to APPs secretion: protein kinase C (PKC)-dependent and mitogen-activated protein kinase (MAPK)-dependent pathways. These pathways operate in parallel and converge with transduction pathways of neurotrophins, resulting in enhancement of APPs secretion when both muscarinic agonist and neurotrophins stimulate PC12M1 cells. These conclusions are supported by the following findings: (a) Only partial blockade of APPs secretion is observed when PKC, p21^ras, or MAPK is fully inhibited by their respective specific inhibitors, GF109203X, S-trans,trans -farnesylthiosalicylic acid, and PD98059. (b) K252a, which blocks PKC and phorbol 12-myristate 13-acetate-induced APPs secretion, enhances both muscarinic-stimulated MAPK activation and APPs secretion. (c) Activation of MAPK in PC12M1 cells by muscarinic agonists is Ras-dependent but PKC-independent and is enhanced synergistically by neurotrophins. These results suggest that muscarinic stimulation of APPs secretion is mediated by at least two independent pathways that converge and enhance the signal for APPs secretion at the convergence point.     

α-Secretase-Derived Product of β-Amyloid Precursor Protein Is Decreased by Presenilin 1 Mutations Linked to Familial Alzheimer's Disease

Abstract: Recent reports indicate that missense mutations on presenilin (PS) 1 are likely responsible for the main early-onset familial forms of Alzheimer's disease (FAD). Consensual data obtained through distinct histopathological, cell biology, and molecular biology approaches have led to the conclusion that these PS1 mutations clearly trigger an increased production of the 42-amino-acid-long species of β-amyloid peptide (Aβ). Here we show that overexpression of wild-type PS1 in HK293 cells increases Aβ40 secretion. By contrast, FAD-linked mutants of PS1 trigger increased secretion of both Aβ40 and Aβ42 but clearly favor the production of the latter species. We also demonstrate that overexpression of the wild-type PS1 augments the α-secretase-derived C-terminally truncated fragment of β-amyloid precursor protein (APPα) recovery, whereas transfectants expressing mutated PS1 secrete drastically lower amounts of APPα when compared with cells expressing wild-type PS1. This decrease was also observed when comparing double transfectants overexpressing wild-type β-amyloid precursor protein and either PS1 or its mutated congener M146V-PS1. Altogether, our data indicate that PS mutations linked to FAD not only trigger an increased ratio of Aβ42 over total Aβ secretion but concomitantly down-regulate the production of APPα.     

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.     

Phorbol Esters but Not the Cholinergic Agonists Oxotremorine-M and Carbachol Increase Release of the Amyloid Precursor Protein in Cultured Rat Cortical Neurons

Abstract: Conventional secretory processing of the amyloid precursor protein is nonamyloidogenic, releasing carboxyl-terminus-truncated amyloid precursor protein derivatives while cleaving the amyloid β-peptide within its sequence. Alternative processing routes are potentially amyloidogenic, yielding the amyloid β-peptide segment intact. In continuous cell lines, secretory processing of the amyloid precursor protein is regulated by both protein kinase C and muscarinic receptor stimulation. However, the first and second messenger systems that regulate amyloid precursor protein release in central neurons are still under investigation. In the present investigation, we examined whether or not first and second messengers of cholinergic neurotransmission increase production of soluble derivatives of the amyloid precursor protein in primary cultures of rat cortical neurons. Activation of protein kinase C by the phorbol esters phorbol 12,13-dibutyrate and phorbol 12-myristate 13-acetate increased production of the soluble form of the amyloid precursor protein dramatically. In contrast, activation of muscarinic receptors by oxotremorine-M or carbachol did not result in a significant increase in amyloid precursor protein release. Similarly, chemically induced depolarization using 35 m M KCI did not alter production of soluble amyloid precursor protein derivatives. Our data suggest that although protein kinase C stimulation plays an important role in regulating release of the amyloid precursor protein, cholinergic neurotransmission does not regulate its release in cultured rat cortical neurons.     

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.     

α2-Macroglobulin as a β-Amyloid Peptide-Binding Plasma Protein

Abstract: The β-amyloid peptide (Aβ) is a normal proteolytic processing product of the amyloid precursor protein, which is constitutively expressed by many, if not most, cells. For reasons that are still unclear, Aβ is deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimer's disease (AD). The factor(s) responsible for the clearance of soluble Aβ from biological fluids or tissues are poorly understood. We now report that human α₂-macroglobulin (α₂M), a major circulating endoproteinase inhibitor, which has recently been shown to be present in senile plaques in AD, binds ¹²⁵I-Aβ_(1–42) with high affinity (apparent dissociation constant of 3.8 × 10^?10M). Approximately 1 mol of Aβ is bound per mole of α₂M. Both native and methylamine-activated α₂M bind ¹²⁵I-Aβ_(1–42). The binding of ¹²⁵I-Aβ_(1–42) to α₂M is enhanced by micromolar concentrations of Zn²⁺ (but not Ca²⁺) and is inhibited by noniodinated Aβ_(1–42) and Aβ_(1–40) but not by the reverse peptide Aβ_(40-1) or the cytokines interleukin 1β or interleukin 2. α₁-Antichymotrypsin, another plaque-associated protein, inhibits both the binding of ¹²⁵I-Aβ_(1–42) to α₂M as well as the degradation of ¹²⁵I-Aβ_(1–42) by proteinase-activated α₂M. Moreover, the binding of ¹²⁵I-Aβ_(1–42) to α₂M protects the peptide from proteolysis by exogenous trypsin. These data suggest that α₂M may function as a carrier protein for Aβ and could serve to either facilitate or impede clearance of Aβ from tissues such as the brain.     

Human Brain βA4 Amyloid Protein Precursor of Alzheimer''s Disease: Purification and Partial Characterization

The major component of the amyloid deposition that characterizes Alzheimer's disease is the 4-kDa beta A4 protein, which is derived from a much larger amyloid protein precursor (APP). A procedure for the complete purification of APP from human brain is described. The same amino terminal sequence of APP was found in two patients with Alzheimer's disease and one control subject. Two major forms of APP were identified in human brain with apparent molecular masses of 100-110 kDa and 120-130 kDa. Soluble and membrane fractions of brain contained nearly equal amounts of APP in both humans and rats. Immunoprecipitation with carboxyl terminus-directed antibodies indicates that the soluble forms of APP are truncated. Carboxyl terminus truncation of membrane-associated forms of human brain APP was also found to occur during postmortem autolysis. The availability of purified human brain APP will facilitate the investigation of its normal function and the events that lead to its abnormal cleavage in patients with Alzheimer's disease.     

Constitutive and Protein Kinase C-Regulated Secretory Cleavage of Alzheimer's β-Amyloid Precursor Protein: Different Control of Early and Late Events by the Proteasome

Abstract: The physiological processing of the β-amyloid precursor protein (βAPP) by a protease called α-secretase gives rise to APPα, a C-terminally truncated fragment of βAPP with known neurotrophic and cytoprotective properties. Several lines of evidence indicate that protein kinase C (PKC)-mediated events regulate this physiological pathway. We show here that the proteasome multicatalytic complex modulates the phorbol 12,13-dibutyrate-stimulated APPα secretion at several levels in human kidney 293 (HK293) cells. Two blocking agents of the proteasome, namely, Z -IE(Ot-Bu)A-leucinal and lactacystin, elicit a dual effect on PKC-regulated APPα secretion by metabolically labeled HK293 cells. Thus, short periods of preincubation (2–5 h) of the cells with the inhibitors trigger a drastic potentiation of APPα recovery, whereas long-term treatment of the cells (15–20 h) with the blocking agents leads to an overall decrease in the secretion of APPα. Such a dual effect was not observed on constitutive APPα secretion and intracellular formation generated by HK293 cells, which both only increase upon inhibitor treatments. Similar effects on the constitutive and PKC-regulated APPα secretion were observed with PC12 cells. Altogether, these data suggest distinct mechanisms underlying basal and PKC-regulated APPα production, indicating that this multicatalytic complex appears as a key contributor of the α-secretase pathway.     

An Etiological Role of Amyloidogenic Carboxyl-Terminal Fragments of the β-Amyloid Precursor Protein in Alzheimer's Disease

Abstract: Amyloid β protein (Aβ), 39–43 amino acids long, is the principal constituent of the extracellular amyloid deposits in brain that are characteristic of Alzheimer's disease (AD). Several lines of evidence indicate that Aβ may play an important role in the pathogenesis of AD. However, there are several discrepancies between the production of Aβ and the development of the disease. Thus, Aβ may not be the sole active fragment of β-amyloid precursor protein (βAPP) in the neurotoxicity associated with AD. Consequently, the possible effects of other cleaved products of βAPP need to be explored. The recent concentration on other potentially amyloidogenic products of βAPP has produced interesting candidates, the most promising of which are the amyloidogenic carboxyl-terminal (CT) fragments of βAPP. This review discusses a possible etiological role of CT fragments of βAPP in AD.     

Amyloid Precursor Protein Metabolism in Primary Cell Cultures of Neurons, Astrocytes, and Microglia

Abstract: Amyloid precursor protein (APP) gives rise by proteolytic processing to the amyloid β peptide (Aβ) found abundantly in cerebral senile plaques of individuals with Alzheimer's disease. APP is highly expressed in the brain. To assess the source of cerebral Aβ, the metabolism of APP was investigated in the major cell types of the newborn rat cerebral cortex by pulse/chase labeling and immunoprecipitation of the APP and APP metabolic fragments. We describe a novel C-terminally truncated APP isoform that appears to be made only in neurons. The synthesis, degradation, and metabolism of APP were quantified by phosphorimaging in neurons, astrocytes, and microglia. The results show that although little APP is metabolized through the amyloidogenic pathways in each of the three cultures, neurons appear to generate more Aβ than astrocytes or microglia.     

In Vitro Phosphorylation of the Cytoplasmic Domain of the Amyloid Precursor Protein by Glycogen Synthase Kinase-3β

Abstract: The two pathological lesions found in the brains of Alzheimer's disease patients, neurofibrillary tangles and neuritic plaques, are likely to be formed through a common pathway. Neurofibrillary tangles are intracellular aggregates of paired helical filaments, the main component of which is hyperphosphorylated forms of the microtubule-associated protein τ. Extracellular neuritic plaques and diffuse and vascular amyloid deposits are aggregates of β-amyloid protein, a 4-kDa protein derived from the amyloid precursor protein (APP). Using conditions in vitro under which two proline-directed protein kinases, glycogen synthase kinase-3β (GSK-3β) and mitogen-activated protein kinase (MAPK), were able to hyperphosphorylate τ, GSK-3β but not MAPK phosphorylated recombinant APP_cyt. The sole site of phosphorylation in APP_cyt by GSK-3β was determined by phosphoamino acid analysis and phosphorylation of APP_cyt mutant peptides to be Thr⁷⁴³ (numbering as for APP₇₇₀). This site was confirmed by endoproteinase Glu-C digestion of APP_cyt and peptide sequencing. The ability of GSK-3β to phosphorylate APP_cyt and τ provides a putative link between the two lesions and indicates a critical role of GSK-3β in the pathogenesis of Alzheimer's disease.