Ectodomain Phosphorylation of beta -Amyloid Precursor Protein at Two Distinct Cellular Locations*

(Received for publication, February 15, 1996, and in revised form, September 9, 1996)

Jochen Walter , Anja Capell , Albert Y. Hung Dagger , Hanno Langen §, Martina Schnölzer , Gopal Thinakaran par , Sangram S. Sisodia par , Dennis J. Selkoe Dagger and Christian Haass **

From the Central Institute of Mental Health, Department of Molecular Biology, J5, 68159 Mannheim, Germany, § Hoffmann-LaRoche Ltd., Pharmaceutical research, Gene Technologies, 4070 Basel, Switzerland, the  Department of Cell Biology, German Cancer Research Center, 69120 Heidelberg, Germany, the Dagger  Department of Neurology and Program in Neuroscience, Harvard Medical School and Center for Neurologic Diseases, Brigham and Woman's Hospital, Boston, Massachusetts 02115, and the par  Neuropathology Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The beta -amyloid precursor protein (beta APP) is a transmembrane protein that is exclusively phosphorylated on serine residues within its ectodomain. To identify the cellular site of beta APP phosphorylation, we took advantage of an antibody that specifically detects the free C terminus of beta -secretase-cleaved beta APP containing the Swedish missense mutation (APPssw-beta ). This antibody previously established the cellular location of the beta -secretase cleavage of Swedish beta APP as a post-Golgi secretory compartment (Haass, C., Lemere, C., Capell, A., Citron, M., Seubert, P., Schenk, D., Lannfelt, L., and Selkoe, D. J. (1995) Nature Med. 1, 1291-1296). We have now localized the selective ectodomain phosphorylation of beta APP to the same compartment. Moreover, the phosphorylation sites of beta APP were identified at Ser198 and Ser206 of beta APP695 by tryptic peptide mapping, mass spectrometry, and site-directed mutagenesis. Intracellular phosphorylation of beta APP was inhibited by Brefeldin A and by incubating cells at 20 °C, thus excluding phosphorylation in the endoplasmic reticulum or trans-Golgi network. Ectodomain phosphorylation within a post-Golgi compartment occurred not only with mutant Swedish beta APP, but also with wild type beta APP. In addition to phosphorylation within a post-Golgi compartment, beta APP was also found to undergo phosphorylation at the cell surface by an ectoprotein kinase. Therefore, this study revealed two distinct cellular locations for beta APP phosphorylation.


INTRODUCTION

Alzheimer's disease (AD)1 is the most common cause of age-related mental failure. It is now widely accepted that the deposition of the amyloid beta -peptide (Abeta ) within the brain parenchyma and in cerebromeningeal blood vessels is an early and necessary feature of AD (2). Abeta is derived from the membrane-spanning beta -amyloid precursor protein (beta APP; Ref. 3). beta APP can be proteolytically processed within two general pathways: an amyloidogenic and a nonamyloidogenic processing route (summarized by Haass and Selkoe (4)). Within the latter pathway, beta APP is constitutively cleaved by a protease referred to as alpha -secretase. This cleavage occurs near the middle of the Abeta region, thus inhibiting Abeta formation (5, 6) and resulting in the secretion of APPswt-alpha (for terminology, see Fig. 2A) into the media of cultured cells (7). In the amyloidogenic pathway, beta APP is first cleaved by beta -secretase at the N terminus of the Abeta domain and subsequently by gamma -secretase at its C terminus, resulting in the constitutive secretion of Abeta (8-11).


Fig. 2. A, schematic showing the proteolytic generation of the different types of APPs from wt or Swedish beta APP cleaved by alpha - or beta -secretase. The amino acid sequence of the Swedish mutation is shown in boldface letters, and the wt amino acid sequence is shown above. The epitope used to generate antibody 192sw is underlined. Arrows indicate the sites of alpha -, beta -, or gamma -secretase cleavage. B, detection of intracellular phosphorylated APPssw-beta . Cell lysates of kidney 293 cells stably transfected with the Swedish beta APP695 cDNA and metabolically labeled with [32P]orthophosphate were immunoprecipitated with antibody C7 to detect full-length beta APP or with 192sw to detect intracellular APPssw-beta . Conditioned media were precipitated with antibody 192sw to detect secreted APPssw-beta (192sw, Medium).
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One cellular mechanism for the generation of Abeta involves reinternalization of full-length beta APP from cell surface to endosomes (12), in which the beta -secretase cleavage can occur (13). During recycling of endosomes to the cell surface (13), the resulting 12-kDa C-terminal fragment is cleaved by gamma -secretase to release Abeta . Missense mutations, found in a few families with familial autosomal dominant AD, frame the Abeta domain (reviewed by Mullan and Crawford (14)). All familial autosomal dominant AD-linked mutations found in the beta APP gene have now been shown to influence directly Abeta generation. A mutation just before the N terminus of the Abeta region at the beta -secretase cleavage site (the "Swedish" mutation; Ref. 15) results in a 3-6-fold increased production of Abeta (16-18). Missense mutations close to the alpha -secretase site also cause an increased production of Abeta , but the increase is paralleled by alternative N-terminal cleavages of Abeta (19). Mutations at the C terminus of the Abeta domain (just after the gamma -secretase site) result in the generation of longer Abeta peptides ending at amino acid 42 instead of amino acid 40 (20). The former peptides have been shown to aggregate more rapidly (21), presumably leading to an accelerated amyloid plaque formation.

Recently, we (1) and others (22) showed that the increased production of Abeta from beta APP molecules bearing the Swedish mutation is due to a cellular mechanism distinct from that principally involved in Abeta generation from wild type beta APP. beta -Secretase cleavage of beta APP appears to generally occur within the endocytic pathway (13). During reinternalization, only small amounts of full-length, uncleaved beta APP molecules are available, because substantial quantities of beta APP have already been cleaved by alpha -secretase. However, in the case of Swedish mutant beta APP, we found that beta -secretase cleavage occurs at an earlier time point in beta APP trafficking, namely within the secretory pathway on the way to the cell surface, predominantly in a post-Golgi compartment, most likely secretory vesicles (1). Therefore, beta -secretase cleavage of Swedish mutant beta APP, in contrast to the principal beta -secretase cleavage of wild type beta APP, occurs in competition with alpha -secretase cleavage in the secretory pathway.

beta APP matures by undergoing N'- and O'-glycosylation, sulfation, and phosphorylation during transport from the endoplasmic reticulum to the cell surface (7, 8, 23). Protein phosphorylation is known to be involved in the regulation of cellular processes such as differentiation, metabolism, and signal transduction (for review, see Ref. 24). Besides many intracellular phosphoproteins, some phosphorylated secretory proteins have been described, e.g. fibronectin (25), fibrinogen (26), prolactin (27), chromogranin B, secretogranin II (28), and L-29, a soluble lectin (29). Although the cellular locus for phosphorylation of most of these secretory proteins is not identified, it has been shown that chromogranin B and secretogranin II are phosphorylated in the secretory pathway within the trans-cisternae of the Golgi (28).

In addition to numerous intracellular protein kinases, ectoprotein kinases acting at the surface of intact cells have been characterized (30-32). These enzymes use extracellular ATP as cosubstrate to phosphorylate endogenous cell surface proteins as well as soluble proteins and have been implicated in a number of biological phenomena, including cell growth inhibition (33), long-term potentiation in neurons and synaptogenesis (34, 35), and parasite-host interactions (36, 37). Ubiquitously occurring casein kinase-like ectoprotein kinases can be released from the cell surface upon interaction with extracellular protein substrates (38, 39), thus allowing them to act at a distance to their cellular origin.

In this study, we have determined the subcellular locations of the phosphorylation of beta APP. We used an antibody (192sw (40); see Fig. 2A) specifically detecting APPssw-beta , the derivative we found to be generated in high quantities within a well defined post-Golgi secretory compartment (1). Through biochemical and cell biological experiments we demonstrate that intracellular phosphorylation of Swedish beta APP as well as wild type beta APP occurs within this compartment, i.e. after the trans-Golgi, most likely within secretory vesicles. Ectodomain phosphorylation was mapped to Ser198 and Ser206 of beta APP695, which represent potential phosphorylation sites for casein kinase (CK)-2 and CK-1, respectively. Further, we show that beta APP can be phosphorylated by an ectoprotein kinase activity on the cell surface. Therefore, our data demonstrate that beta APP undergoes ectodomain phosphorylation at two distinct cellular locations.


MATERIALS AND METHODS

Cell Culture, Metabolic Labeling, and Drug Treatment

Kidney 293 cells were stably transfected with the wt beta APP695 cDNA (9, 41) or with the beta APP695 cDNA containing the Swedish double mutation (18). Chinese hamster ovary cells stably transfected with the amyloid precursor-like protein 2 (APLP2) cDNA have been described previously (42). Metabolic labeling and treatment of cells with 10 µg/ml of Brefeldin A (BFA; solubilized in ethanol) was carried out as described earlier (23, 43, 44). Ethanol was added in identical concentrations to the corresponding control cells.

Incubation of Cells at 20 °C

Cells were incubated at 20 °C as described (1, 45). Briefly, cells were metabolically labeled with 150 µCi of [35S]methionine or 1.5 mCi of [32P]orthophosphate for 3 h in methionine-free or sodium phosphate-free Dulbecco's minimal essential medium buffered with 10 mM HEPES. Tissue culture dishes were sealed with Parafilm and incubated in a water bath at 20 °C or 37 °C. The temperature was controlled carefully throughout the experiment.

Immunoprecipitation, Antibodies, and Electrophoresis

Immunoprecipitations were carried out as described earlier (7, 43). The following antibodies were used (see Fig. 2A). Antibody C7 was raised to the last 20 amino acids of beta APP and recognizes full-length beta APP (46). Antibody B5, which recognizes all forms of APPs, was raised to a fusion protein containing amino acids 444-592 of beta APP695 (70). Antibody 1736 (raised to beta APP-(595-611)) specifically identifies APPswt-alpha and APPssw-alpha (12) but not APPswt-beta or wt/Swedish full-length beta APP. Antibody 192sw was raised against the free C terminus of APPswt-beta (40). This antibody specifically recognizes APPswt-beta but not APPswt/sw-alpha or wt/Swedish full-length beta APP (1, 40). APLP2 was immunoprecipitated with antibody D2-1 raised to full-length mouse APLP2 (42).

Pulse-chase Experiments

Pulse-chase experiments were carried out as described (43). Briefly, cells stably transfected with the Swedish beta APP mutation were pulse-labeled with [35S]methionine for 5 min in methionine and serum-free media. Cells were than chased for the indicated time points in media containing excess amounts of methionine and 10% fetal calf serum. Cell lysates were immunoprecipitated with antibody C7 (to detect full-length beta APP) and antibody 192sw (to detect intracellular APPssw-beta ). Media were immunoprecipitated with antibody 192sw (to detect secreted APPssw-beta ).

Phosphoamino Acid Analysis

Phosphoamino acid analysis was carried out by two-dimensional high voltage electrophoresis (47). Radiolabeled proteins electrotransferred onto polyvinylidene difluoride-membrane were hydrolyzed in 6 M HCl for 90 min at 110 °C. Subsequently, supernatants were dried in a SpeedVac concentrator, and pellets were dissolved in 5 µl of pH 1.9 buffer (7.8% acetic acid, 2.5% formic acid) and spotted onto cellulose-TLC plates together with unlabeled phosphoamino acids (Ser(P), Thr(P), and Tyr(P); 1 µg each). High voltage electrophoresis was carried out for 20 min (pH 1.9 buffer) at 1.5 kV and for 16 min (pH 3.5 buffer; 5% acetic acid, 0.5% pyridine) at 1.3 kV, respectively. Radioactive phosphoamino acids were identified by autoradiography and comparison with ninhydrin-stained standards.

Phosphopeptide Mapping by Tryptic Digestion

In vivo, 32P-phosphorylated beta APP was isolated by immunoprecipitation and SDS-PAGE and transferred to nitrocellulose membrane (Schleicher & Schüll). Digestion of radiolabeled beta APP was carried out for 24 h at 37 °C with 0.5 mg/ml trypsin (Ref. 48; Boehringer Mannheim, sequencing grade). The tryptic digest was separated on Tris/Tricine gradient gels (10-20%; Novex), and radiolabeled peptides were visualized by autoradiography.

Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry

Approximately 10 µg of unlabeled beta APP together with a trace of 32P-labeled beta APP were digested with trypsin, and the resulting peptides were separated on 10-20% Tris/Tricine gels as described. Radiolabeled peptide bands were cut out from the gel, extracted twice for 10 min with 100 µl of 0.1% aqueous trifluoroacetic acid followed by 100 µl of 60% acetonitrile. The combined supernatants were subjected to a 5-mm micro precolumn (LC Packings) packed with Poros R2 (Perseptive Biosystems). The peptides were eluted in 10 µl with a step gradient of 80% acetonitrile, 0.1% trifluoroacetic acid. Molecular masses (isotopic average) of the eluted peptides were determined by a Vision 2000 (Finnigan) mass spectrometer equipped with a nitrogen laser and operated in reflection mode at an accelerating voltage of 5000 V. 1 µl of the peptide solution was crystallized in matrices consisting of 1% 2,4-dihydroxybenzoic acid in 0.1% aqueous trifluoroacetic acid. All peptide spectra were externally calibrated by using the monoisotopic masses of sodium (Mr 23.0) and fullerene C70 (Mr 840.0). Peptides were identified by computer-assisted analysis using the Swiss-Prot sequence data bank and the special program package HUSAR (developed at the Department of Molecular Biophysics, German Cancer Research Center, Heidelberg).

Phosphorylation of Cell Surface Proteins by Ectoprotein Kinase

Phosphorylation was carried out as described earlier (30). Briefly, subconfluent monolayer cell cultures (5-7 × 104 cells/cm2), grown in Dulbecco's minimum essential medium (10% fetal calf serum) were washed twice with prewarmed (37 °C) isotonic phosphorylation buffer (30 mM Tris, pH 7.3, 70 mM NaCl, 5 mM magnesium acetate, 0.5 mM EDTA, 5 mM KH2PO4/K2HPO4, 290 ± 10 mos M) and incubated for 5 min at 37 °C in the same buffer.

Phosphorylation was started by the addition of 0.5-1.5 µM [gamma -32P]ATP and allowed to proceed for 0-30 min at 37 °C. Reactions were terminated by removing cell supernatants followed immediately by two washes of the cells with ice-cold phosphorylation buffer containing 2 mM unlabeled ATP. Subsequently, cells were lysed in presence of 2 mM ATP for 7 min on ice. Cell lysates (prepared as described by Haass et al. (43)) were centrifuged for 10 min at 14,000 × g, and cellular beta APP was isolated by immunoprecipitation as described above and separated by SDS-PAGE. Radiolabeled proteins were detected by autoradiography of dried gels. Cell viability during phosphorylation assays was evaluated by several criteria (49).

In Vitro Mutagenesis

The beta APP cDNA construct containing a stop codon at the alpha -secretase cleavage site was described previously (23). The C-terminal deletion construct of beta APP was described by Haass et al. (44). A cDNA construct containing a stop codon at the beta -secretase site of wt beta APP was generated as described (23) using the following annealed oligonucleotides: GATCTCTGAAGTGAAGATGTAGGCAG (stop beta -wt sense) and AATTCTGCCTACATCTTCACTTCAGA (stop beta -wt antisense).

The cDNA construct containing a stop codon at the beta -secretase site of Swedish beta APP was generated as described (23) using the following annealed oligonucleotides: GATCTCTGAAGTGAATCTGTAGGCAG (stop beta -sw sense) and AATTCTGCCTACAGATTCACTTCAGA (stop beta -sw antisense).

The serine to alanine mutations at amino acids 198 and 206 were carried out as described (50) using the following oligonucleotides: CTCCGCATCAGCGGCATCCACATTGTC (S198A) and CCACCAGACATCGGCGTCATCCTCCTC (S206A).

The corresponding cDNAs were stably transfected into kidney 293 cells as described (1, 51), and single cell clones were isolated using cloning cylinders (51).

All mutations were confirmed by sequencing both DNA strands.


RESULTS

Ectodomain Phosphorylation of APLP2

In order to obtain a general validation of ectodomain phosphorylation of beta APP (23), we examined the phosphorylation of the highly related APLP2. APLP2, APLP1, and beta APP are members of a conserved gene family of homologous proteins (52-55). APLP2 is particularly similar to beta APP because it shares some of its characteristic biochemical properties and also matures through the constitutive secretory pathway, where its ectodomain is secreted into culture media (42, 55, 56). To analyze the potential phosphorylation of APLP2, Chinese hamster ovary cells stably transfected with the APLP2 cDNA were metabolically labeled with [35S]methionine or [32P]orthophosphate. Conditioned media were precipitated with antibody D2-1 raised against full-length mouse APLP2 (42). As shown in Fig. 1, immunoprecipitation of conditioned media from [35S]methionine labeled cells resulted in the detection of the two major APLP2 species. In close agreement with the data reported (42), we observed a high molecular weight species that corresponds to the chondroitin sulfate glycosaminoglycan-modified form of APLP2 and a lower molecular weight species representing the unmodified form of APLP2. Both forms of APLP2 were also observed after labeling with [32P]orthophosphate (Fig. 1). These results show that APLP2, similar to beta APP, is phosphorylated within its ectodomain, indicating that ectodomain phosphorylation of secreted derivatives of proteins belonging to the APP gene family is a general phenomenon.


Fig. 1. APLP2 is phosphorylated on its ectodomain. Chinese hamster ovary cells stably transfected with the APLP2 cDNA were metabolically labeled with [35S]methionine or [32P]orthophosphate. Conditioned media were immunoprecipitated with antibody D2-1. As a control, APPs-alpha was immunoprecipitated from beta APP-transfected kidney 293 cells labeled with [32P]orthophosphate. APLP2CSGAG indicates the chondroitin sulfate glycosaminoglycan-modified form of APLP2.
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Stability of Ectodomain Phosphorylation of beta APP

To assess the stability of beta APP phosphorylation, we examined protein phosphatase activity in AD brain extracts as well as in the conditioned media of cultured cells (57, 58). No beta APP dephosphorylating activity was detected in any of the AD brain extracts, both those from temporal and occipital regions, indicating a relative resistance of beta APP to protein phosphatase activity. In addition, APPs did not undergo dephosphorylation in conditioned media (data not shown). These experiments demonstrate that ectodomain phosphorylation of beta APP is relatively resistant to protein phosphatase activities, suggesting a long lasting biological function of phosphorylated APPs molecules.

Intracellular Phosphorylation of Swedish Mutant beta APP

Little is known about the cell biology of ectodomain phosphorylation. In order to determine the subcellular locus for beta APP phosphorylation, we used an antibody (192sw; Fig. 2A) that specifically recognizes APPssw-beta , which we previously detected in high quantities within the lysates of kidney 293 cells stably transfected with the Swedish beta APP cDNA (1). To determine if ectodomain phosphorylation also occurs on intracellular APPssw-beta , we radiolabeled kidney 293 cells expressing Swedish mutant beta APP with [32P]orthophosphate. Upon immunoprecipitation of cell lysates and media we detected phosphorylated intracellular and secreted APPssw-beta as well as phosphorylated intracellular full-length beta APP (Fig. 2B). This result indicates the occurrence of intracellular phosphorylation of the beta APP-ectodomain. To prove that APPssw-beta was indeed produced de novo and not taken up by fluid phase endocytosis, we pulse-labeled kidney 293 cells stably transfected with the Swedish cDNA. The cells were then chased in the presence of excess unlabeled methionine. Aliquots of the cell lysates were immunoprecipitated either with antibody C7 (to detect maturation of full-length beta APP) or with antibody 192sw (to detect intracellular APPssw-beta ). In addition, conditioned media were immunoprecipitated with antibody 192sw to detect secreted APPssw-beta . As shown in Fig. 3, full-length beta APP is processed within 45 min from immature N'-glycosylated form to mature N'- and O'-glycosylated form. Shortly after, the amount of full-length beta APP declines due to the secretion of APPs. Consistent with our previous results (1), the highest level of intracellular APPssw-beta was detected after 45 min. After this time point the levels of intracellular APPssw-beta declined, and an increase of secreted APPssw-beta in the media was observed (Fig. 3). The precursor product relationship clearly indicates that intracellular APPssw-beta is produced de novo and not due to a fluid phase mediated uptake of secreted species.


Fig. 3. Pulse-chase experiment showing that APPssw-beta is generated as soon as fully matured (N'/O') beta APP occurs after 45 min. Shortly after that, the amount of intracellular APPssw-beta declines due to its secretion into the media. Full-length beta APP was detected with antibody C7 (top panel), and intracellular (middle panel) as well as secreted APPssw-beta (bottom panel) was precipitated with antibody 192sw.
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Mapping of Phosphorylation Sites within beta APP

To determine which amino acids were phosphorylated in Swedish mutant beta APP, we performed phosphoamino acid analysis of intracellular as well as secreted APPssw-beta . Both species are phosphorylated exclusively on serine residues (Fig. 4). This result is in line with recent studies showing that wt beta APP is constitutively phosphorylated solely on serine residues (23). It also confirms that phosphorylation of intracellular APPssw-beta is an amino acid phosphorylation, not an incorporation of phosphate into sugar moieties of beta APP.


Fig. 4. Serine phosphorylation of intracellular APPssw-beta (cell lysate) and secreted APPssw-beta (media). Kidney 293 cells stably transfected with the Swedish beta APP cDNA were metabolically labeled with [32P]orthophosphate. APPssw-beta was precipitated from cell lysates and conditioned media with antibody 192sw, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membrane. The bands were excised and subjected to two-dimensional phosphoamino acid analysis by separation with high voltage electrophoresis along the horizontal axis at pH 1.9 and along the vertical axis at pH 3.5. The mobilities of ninhydrin-stained phosphoamino acid standards are indicated. The origin is denoted by an arrowhead.
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In order to identify the site(s) of beta APP phosphorylation, we performed tryptic peptide mapping of in vivo phosphorylated beta APP molecules. Kidney 293 cells stably transfected with wild type beta APP695 or cDNA constructs deleting large portions of the N-terminal half (AX construct (23) (Fig. 5A) or the C-terminal half (XB construct (23)) were labeled with [32P]orthophosphate or [35S]methionine. Secreted forms of the respective beta APP molecules were immunoprecipitated with antibody 1736. In agreement with data published earlier (23), we found that phosphorylation occurs exclusively within the N-terminal portion of beta APP, since no phosphate incorporation occurred in cells expressing the N-terminal deletion construct (Fig. 5B). Phosphorylated full-length beta APP as well as the phosphorylated C-terminal deleted beta APP (XB) were digested with trypsin, and the digestion products were separated on a 10-20% Tris/Tricine gel. A single phosphorylated peptide of approximately 4.8 kDa was detected for both full-length beta APP and XB constructs (Fig. 5C). Computer analysis of the potentially generated tryptic peptides revealed that the radiolabeled peptide could represent solely the amino acid sequence from 181-224 of beta APP695. To prove this in more detail, the radiolabeled ~4.8-kDa peptide was eluted from Tris/Tricine gel and subjected to matrix-assisted laser desorption/ionization-mass spectrometry (see "Materials and Methods"). Three monoisotopic masses of 2286.5, 3673.5, and 4877.3 (± 10) were detected in the eluate. The masses of 2286.5 and 3673.5 could not be matched to tryptic peptides of beta APP and presumably represent peptides of autocatalytically cleaved trypsin, migrating close to the phosphorylated beta APP tryptic peptide. In contrast, the mass of 4877.3 matches that of the sequence of amino acids 181-224 of beta APP695 in a double phosphorylated form (4714.7 + 160 Da). Since the amino acid sequence of this peptide contains four serine residues, we searched for putative phosphor acceptor sites by computer-assisted analysis. Serine residues 198 and 206 were identified within an acidic sequence of this peptide, representing potential phosphorylation sites for CK-2 and CK-1, respectively (Fig. 5D). These serines were therefore mutagenized to alanines, and the corresponding cDNA constructs were stably transfected into kidney 293 cells. Single cell clones were metabolically labeled with [32P]orthophosphate or [35S]methionine, and secreted beta APPs was immunoprecipitated from conditioned medium with antibody B5. Phosphate incorporation was quantified by phosphor imaging. As shown in Fig. 5E, phosphorylation of beta APP containing the S198A mutation was reduced by about 80%, while that of the S206A mutation was reduced by about 15%. Similar data were obtained after immunoprecipitation of full-length beta APP from cell lysates (data not shown). Taken together, these data might therefore indicate that both serines represent in vivo phosphorylation sites (see "Discussion" for details).


Fig. 5. Identification of the phosphorylation sites of beta APP within its ectodomain. A, schematic of wild type beta APP (WT) and the AX and XB constructs, missing large portions of the N-terminal and the C-terminal half of the beta APP ectodomain, respectively. The Abeta -domain is represented by a striped bar, and vertical lines represent cellular membranes. The numbers above denote amino acid residues with the restriction sites used to generate the constructs indicated (23). B, kidney 293 cells stably transfected with wild type (WT) beta APP695, AX, or XB were labeled with [35S]methionine (35S) and [32P]orthophosphate (32P), respectively, and conditioned media were immunoprecipitated with antibody 1736. Radiolabeled proteins were visualized by autoradiography after separation by SDS-PAGE. C, phosphopeptide map of radiolabeled, secreted forms of wild type (WT) beta APP or XB. After SDS-PAGE, proteins were transferred to nitrocellulose membrane and digested with trypsin as described under "Materials and Methods." The resulting tryptic peptides were separated on a 10-20% Tris/Tricine gel and analyzed by autoradiography. The position of the phosphorylated 4.8-kDa peptide is marked by an arrowhead. D, amino acid sequence of the phosphorylated tryptic peptide (amino acids 181-224), which was identified by mass spectrometry and computer-assisted analysis (see "Materials and Methods"). The serine residues representing potential phosphorylation sites of CK-1 (Ser206) and CK-2 (Ser198) are shown in boldface letters. E, quantification of in vivo phosphorylation of wild type beta APP (WT) and beta APP carrying serine to alanine mutations at positions 198 (S198A) and 206 (S206A). Kidney 293 cells stably expressing wild type or mutated forms of beta APP (S198A, S206A) were labeled with [35S]methionine or [32P]orthophosphate for 2 h. Quantification of protein expression and phosphate incorporation in the different forms of beta APP were carried out by phosphor imaging. Bars represent means ± S.E. of three independent experiments.
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Phosphorylation of beta APP Occurs within Golgi-derived Vesicles

Ectodomain phosphorylation of beta APP was found on all types of secreted APPs molecules, regardless of whether Swedish or wt beta APP was cleaved at either the alpha - or the beta -secretase site. To produce APPs molecules with defined C termini corresponding to alpha - or beta -secretase-cleaved APPswt/sw, we stably transfected kidney 293 cells with cDNA constructs containing stop codons at sites corresponding to these scissions. These transfectants were then metabolically labeled with [35S]methionine or [32P]orthophosphate, and their conditioned media were precipitated with antibody B5, which detects all secreted APPs species. As shown in Fig. 6, APPswt-alpha , APPswt-beta , and APPssw-beta were each secreted as phosphorylated species. Thus, membrane insertion of beta APP is not necessary for its phosphorylation, and APPs can be phosphorylated regardless of which secretase activity cleaved the precursor, indicating a general cellular mechanism for the ectodomain phosphorylation of mutant and wt beta APP.


Fig. 6. Phosphorylation of truncated, soluble forms of beta APP. Stop codons were introduced into wild type or Swedish beta APP cDNA corresponding to the cleavage sites of alpha -secretase (alpha -stop), beta -secretase (beta  wt-stop), or beta -secretase of Swedish beta APP (beta  sw-stop). Kidney 293 cells stably expressing these constructs were metabolically labeled with [35S]methionine (left panel) or [32P]orthophosphate (right panel) and precipitated from conditioned media with antibody B5. The arrow marked APPs indicates APPs derived from the transfected cDNA constructs. The unmarked arrow indicates APPs from endogenous beta APP751. Differences in the amounts of APPs in these cell lines are due to different expression levels of beta APP.
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To determine whether phosphorylation of beta APP occurs in the same compartment as the beta -secretase cleavage of Swedish beta APP (1), we investigated the effect of BFA on phosphorylation of Swedish beta APP. BFA is known to cause a collapse of the Golgi network, resulting in a block of forward transport at the cis-Golgi compartment (61). Kidney 293 cells stably transfected with Swedish beta APP were metabolically labeled with either [35S]methionine or [32P]orthophosphate in the absence or presence of BFA. Cell lysates were precipitated with antibody C7 (to detect full-length beta APP) or antibody 192sw (to detect intracellular APPssw-beta ), and conditioned media were precipitated with antibody 192sw (to detect secreted APPssw-beta ). As reported previously, BFA treatment not only inhibited the maturation of full-length beta APP but also completely inhibited the generation of intracellular APPssw-beta and its secretion (Fig. 7A; Refs. 1 and 44)). Treatment with BFA also resulted in an inhibition of beta APP ectodomain phosphorylation (Fig. 7B), clearly showing that phosphorylation does not occur within the endoplasmic reticulum or the early Golgi. The trace amounts of phosphorylated species detected after BFA treatment are due to beta APP molecules that escaped the BFA block at the beginning of the experiment.


Fig. 7. Brefeldin A inhibits phosphorylation of beta APP. Cells transfected with the Swedish beta APP cDNA were metabolically labeled with [35S]methionine (left panel) or [32P]orthophosphate (right panel) in the presence (Brefeldin A) or absence (control) of BFA (10 µg/ml). Cell lysates were precipitated with antibody C7 to visualize full-length beta APP (N' and N'/O') or antibody 192sw to identify intracellular APPssw-beta . Secreted beta APPssw-beta was immunoprecipitated from conditioned media with antibody 192sw.
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To determine whether ectodomain phosphorylation of beta APP occurs within the trans-Golgi network, kidney 293 cells expressing Swedish beta APP were incubated at 20 °C. Under such conditions, membrane proteins accumulate within the trans-Golgi network (45). As reported previously (1) incubation at 20 °C resulted in the accumulation of full-length N'- and O'-glycosylated beta APP within cell lysates; no APPssw-beta was detected in cell lysates or conditioned media (Fig. 8A; Ref. 1). As shown above, after labeling with [32P]orthophosphate at 37 °C, mature phosphorylated beta APP was precipitated from cell lysates and phosphorylated APPssw-beta from both lysates and media (Fig. 8B). In contrast, incubation of cells at 20 °C completely inhibited phosphorylation of full-length beta APP (Fig. 8B), although large amounts of full-length beta APP were present as shown by labeling with [35S]methionine (Fig. 8A). Taken together, these data strongly suggest that the intracellular ectodomain phosphorylation of Swedish beta APP occurs within a post-Golgi compartment, most likely secretory vesicles, and not in the trans-Golgi network itself.


Fig. 8. Incubating cells at 20 °C inhibits beta APP phosphorylation. Cells transfected with the Swedish (A, B) or wild type (C, D) cDNA were metabolically labeled with [35S]methionine (A, C) or [32P]orthophosphate (B, D) at 20 or 37 °C. Cell lysates were precipitated with antibody C7 to detect full-length wild type and Swedish beta APP, with antibody 192sw to detect intracellular APPssw-beta , or with antibody 1736 to detect intracellular APPswt-alpha . Conditioned media were precipitated with antibody 192sw to detect secreted APPssw-beta or with antibody 1736 to detect APPswt-alpha . Immunoprecipitates were separated by SDS-PAGE and radiolabeled proteins visualized by autoradiography. Differences in the amount of immunoprecipitated APPs from [35S]methionine- and [32P]orthophosphate-labeled cells are due to variabilities during immunoprecipitations, thus not allowing a quantitative comparison.
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Analogous experiments were then carried out to determine the cellular locus of the ectodomain phosphorylation of wt beta APP. When kidney 293 cells expressing wt beta APP were labeled at 37 °C with [35S]methionine, antibody C7 precipitated the expected doublet of full-length beta APP from cell lysates representing the immature and mature forms of the precursor (Fig. 8C). Precipitation with antibody 1736, which specifically identifies APPswt-alpha and does not cross-react with full-length beta APP or APPswt-beta , results in the detection of intracellular APPswt-alpha from cell lysates as well as secreted APPswt-alpha from conditioned media (Fig. 8C). The detection of intracellular APPswt-alpha is in good agreement with data published previously (51, 59, 60), indicating alpha -secretase cleavage within the secretory pathway. When cells were incubated at 20 °C, an accumulation of mature beta APP was observed; however, the generation of intracellular APPswt-alpha and consequently its secretion was completely inhibited (Fig. 8C). When cells were metabolically labeled with [32P]orthophosphate at 37 °C, we detected mature phosphorylated full-length beta APP, and precipitation of cell lysates with antibody 1736, specific for APPs-alpha , resulted in the detection of intracellular phosphorylated APPswt-alpha (Fig. 8D). However, incubating the cells at 20 °C completely inhibited phosphorylation of wild type beta APP; no phosphorylated full-length beta APP or intracellular and secreted APPswt-alpha was detected (Fig. 8D). Taken together, these data show that intracellular ectodomain phosphorylation of wild type as well as Swedish beta APP occurs within a post-Golgi compartment, most likely within secretory vesicles, suggesting that this compartment represents a general subcellular site of ectodomain phosphorylation of beta APP.

Ectodomain Phosphorylation Can Occur on the Cell Surface

Because mature full-length beta APP is also present at the cell surface, we examined whether membrane-bound beta APP can be a substrate for ectoprotein kinases. Intact kidney cells, transfected with wild type beta APP cDNA, were incubated in the presence of 1 µM [gamma -32P]ATP in the cell supernatant, allowing specific detection of ectoprotein kinase activity (30). Full-length beta APP was then precipitated from cell lysates and APPswt-alpha from cell supernatants. As shown in Fig. 9A (wt) cell surface-bound full-length beta APP was phosphorylated by ectoprotein kinase activity. Moreover, phosphorylated APPswt-alpha was recovered from cell supernatants (Fig. 9A, Media). Similar experiments with kidney 293 cells expressing Swedish beta APP showed that cell surface beta APPsw is also phosphorylated by ectoprotein kinase activity (data not shown). Cell surface phosphorylation was also investigated with cells expressing a C-terminal truncated form of beta APP, which inserts in cell membranes but does not undergo reinternalization (13, 44). As with full-length beta APP (Fig. 9A), the C-terminal truncated form of beta APP was also phosphorylated (Fig. 9B, Delta C), indicating that reinternalization of beta APP is not necessary for its phosphorylation. Again, phosphorylated APPswt-alpha was recovered from cell supernatants (Fig. 9B, Media). To prove whether phosphorylated APPswt-alpha does exclusively derive from its phosphorylated precursor or if soluble APPswt-alpha can be phosphorylated after proteolytic cleavage, cell-free supernatant containing APPswt-alpha was incubated with [gamma -32P]ATP either in the absence or in the presence of untransfected intact kidney 293 cells. As shown in Fig. 9C, APPswt-alpha was phosphorylated only in the presence of intact cells, indicating that soluble APPswt-alpha could serve as a substrate for ectoprotein kinase. Thus, neither membrane insertion nor reinternalization is necessary for beta APP phosphorylation. However, beta APP was not phosphorylated in the absence of cells (Fig. 9C, Cells), showing that ectoprotein kinase activity is not cosecreted with beta APPs-species. As revealed by two-dimensional phosphoamino acid analysis, phosphorylation of beta APP by ectoprotein kinase occurs exclusively on serine residues (Fig. 9D). The results clearly demonstrate that cell surface-bound beta APP and its soluble derivatives can be phosphorylated by membrane-associated ectoprotein kinase on the surface of intact cells.


Fig. 9. Cell surface phosphorylation of beta APP by ectoprotein kinase activity. Cell surface proteins of kidney 293 cells stably transfected with wt beta APP cDNA (wt) (A) or with a C-terminal truncated form of beta APP (Delta C) (B) were phosphorylated in the presence of 1 µM [gamma -32P]ATP for 20 min at 37 °C. Full-length beta APP was immunoprecipitated from cell lysates with antibody C7 (Lysate) in the case of wt beta APP (A) or with antibody B5 in the case of Delta C-beta APP (B). Secreted APPswt-alpha was precipitated from cell supernatants (Media) with antibody 1736. The relatively higher amounts of APPswt-alpha in supernatants from transfectants expressing Delta C-beta APP as compared with that from transfectants expressing wt-beta APP is due to a higher rate of alpha -secretase cleavage, which is in close agreement with previous results (13, 44). C, phosphorylation of soluble APPs-alpha by ectoprotein kinase on the surface of kidney 293 cells. APPs-alpha was collected from supernatants of kidney 293 cells stably transfected with a cDNA construct containing a stop codon corresponding to the alpha -secretase site (compare Fig. 4) for 1 h. The supernatant was taken off and split into two halves. One half was incubated with untransfected kidney 293 cells (+ Cells), and the other half was incubated in a Petri dish without cells (- Cells). Both dishes were incubated for 15 min at 37 °C in the presence of 1 µM [gamma -32P]ATP. APPs-alpha was immunoprecipitated with antibody 1736 and separated by SDS-PAGE. D, two-dimensional phosphoamino acid analysis of cell surface beta APP, showing that beta APP is exclusively phosphorylated on serine residues by ectoprotein kinase.
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DISCUSSION

In summary, our data show that full-length beta APP and its alpha - and beta -secretase-cleaved derivatives can be phosphorylated at two different subcellular locations. In both cases, beta APP is exclusively phosphorylated on its ectodomain but not in the cytoplasmic tail. Ectodomain phosphorylation of beta APP has been demonstrated previously (23) and was further supported by the data presented here; beta APP can be phosphorylated on the cell surface by incubating cells with [gamma -32P]ATP, and secreted APPS derived from recombinant cDNA constructs with stop codons inserted at the alpha - and beta -secretase site of mutant and wild type beta APP still result in the secretion of phosphorylated APPS. Moreover, APPS incubated with living cells is phosphorylated by a cell surface ectoprotein kinase. Therefore, evidence from multiple experiments proves exclusive ectodomain phosphorylation of beta APP. Intracellular APPs and full-length beta APP molecules are phosphorylated within a post-Golgi compartment, most likely secretory vesicles. This is the cellular compartment to which we have localized the beta -secretase activity cleaving Swedish beta APP (1). Therefore, phosphorylation of APPs occurs during or immediately before or after the secretory cleavages of beta APP.

The in vivo phosphorylation sites of beta APP were identified as serine residues 198 and 206 by phosphopeptide mapping, site-directed mutagenesis, and mass spectrometry. Moreover, in vivo secreted APPs was detected exclusively in double phosphorylated form. Ser198 is followed by acidic amino acid residues and therefore represents a putative phosphorylation site for CK-2 (63), while Ser206 is preceded by an acidic domain and represents a CK-1 phosphorylation site (64). However, individual mutations of Ser198 and Ser206 differently affected the phosphate incorporation. The S198A mutation resulted in a reduction of phosphorylation of about 80%, while the S206A mutation reduced phosphorylation by about 15%. This might be explained by sequential phosphorylation events, in which the first phosphorylation at Ser198 facilitates the subsequent phosphorylation at Ser206 by acidifying this domain. A similar process has been described involving protein kinases A and CK-1 (65, 66).

Interestingly, in addition to the intracellular phosphorylation, our data also demonstrate a second cellular site for phosphorylation of membrane-bound beta APP: an ectoprotein kinase activity at the cell surface. In contrast to the intracellular phosphorylation of beta APP, which appears to be a constitutive event (23), phosphorylation by ectoprotein kinases could represent a regulated mechanism. Because ATP is known to be released into the extracellular environment by a variety of cellular stimuli (for review see Refs. 67 and 68), the availability of this cosubstrate for ectoprotein kinases could represent a biological regulation mechanism for phosphorylation of cell surface beta APP. Since alpha -secretase activity is present within cell lysates (51, 59, 60), as well as on the cell surface (12, 62), full-length surface beta APP will contribute to the pool of phosphorylated APPs molecules in conditioned media. In addition, secreted derivatives of beta APP (APPswt and APPssw) released by alpha - or beta -secretase into the cell supernatant also serve as substrates for ectoprotein kinase. Our study demonstrates for the first time the unusual phenomenon that beta APP and its principal secreted derivatives can undergo selective ectodomain phosphorylation at two distinct subcellular locations. It will now be important to determine whether both mechanisms result in the phosphorylation of identical amino acid residues or if beta APP is phosphorylated by different protein kinases on two or more sites within the same molecule. The functional consequences of this complex regulation of beta APP ectodomain phosphorylation are unknown so far. However, one might speculate that extracellular function(s) of beta APP, e.g. the modulation of neuronal excitability by APPs (69), could be regulated by selective ectodomain phosphorylation.


FOOTNOTES

*   This work was supported by Deutsche Forschungsgemeinschaft Grant HA 1737-2-1 (to C. H.) and National Institutes of Health Grants HL 49552-04 and AG 12749 (to D. J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
**   To whom correspondence should be addressed: Zentralinstitut für Seelische Gesundheit, Abteilung Molekularbiologie J5, 68159 Mannheim, Germany. Tel.: 49-621-1703-884; Fax: 49-621-23429; E-mail: Haass{at}as200.zi-mannheim.de.
1    The abbreviations used are: AD, Alzheimer's disease; Abeta , amyloid beta -peptide; APP, amyloid precursor protein; beta -APP, beta -amyloid precursor protein; CK, casein kinase; wt, wild type; BFA, Brefeldin A; APLP1 and APLP2, amyloid precursor-like proteins 1 and 2, respectively; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]glycine.

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