(Received for publication, February 15, 1996, and in revised form, September 9, 1996)
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
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
Neuropathology Laboratory, Johns Hopkins University School of
Medicine, Baltimore, Maryland 21205-2196
The -amyloid precursor protein (
APP) is a
transmembrane protein that is exclusively phosphorylated on serine
residues within its ectodomain. To identify the cellular site of
APP
phosphorylation, we took advantage of an antibody that specifically
detects the free C terminus of
-secretase-cleaved
APP containing
the Swedish missense mutation (APPssw-
).
This antibody previously established the cellular location of the
-secretase cleavage of Swedish
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
APP to the same compartment. Moreover,
the phosphorylation sites of
APP were identified at
Ser198 and Ser206 of
APP695 by tryptic
peptide mapping, mass spectrometry, and site-directed mutagenesis.
Intracellular phosphorylation of
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
APP, but also with wild type
APP. In addition to
phosphorylation within a post-Golgi compartment,
APP was also found
to undergo phosphorylation at the cell surface by an ectoprotein
kinase. Therefore, this study revealed two distinct cellular locations
for
APP phosphorylation.
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 -peptide (A
) within the brain
parenchyma and in cerebromeningeal blood vessels is an early and
necessary feature of AD (2). A
is derived from the membrane-spanning
-amyloid precursor protein (
APP; Ref. 3).
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,
APP is constitutively cleaved by a
protease referred to as
-secretase. This cleavage occurs near the
middle of the A
region, thus inhibiting A
formation (5, 6) and
resulting in the secretion of APPswt-
(for terminology, see Fig. 2A) into the media of cultured
cells (7). In the amyloidogenic pathway,
APP is first cleaved by
-secretase at the N terminus of the A
domain and subsequently by
-secretase at its C terminus, resulting in the constitutive secretion of A
(8-11).
One cellular mechanism for the generation of A involves
reinternalization of full-length
APP from cell surface to endosomes (12), in which the
-secretase cleavage can occur (13). During recycling of endosomes to the cell surface (13), the resulting 12-kDa
C-terminal fragment is cleaved by
-secretase to release A
.
Missense mutations, found in a few families with familial autosomal
dominant AD, frame the A
domain (reviewed by Mullan and Crawford
(14)). All familial autosomal dominant AD-linked mutations found in the
APP gene have now been shown to influence directly A
generation.
A mutation just before the N terminus of the A
region at the
-secretase cleavage site (the "Swedish" mutation; Ref. 15)
results in a 3-6-fold increased production of A
(16-18). Missense
mutations close to the
-secretase site also cause an increased
production of A
, but the increase is paralleled by alternative
N-terminal cleavages of A
(19). Mutations at the C terminus of the
A
domain (just after the
-secretase site) result in the
generation of longer A
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 A from
APP molecules bearing the Swedish mutation is due to a
cellular mechanism distinct from that principally involved in A
generation from wild type
APP.
-Secretase cleavage of
APP
appears to generally occur within the endocytic pathway (13). During
reinternalization, only small amounts of full-length, uncleaved
APP
molecules are available, because substantial quantities of
APP have
already been cleaved by
-secretase. However, in the case of Swedish
mutant
APP, we found that
-secretase cleavage occurs at an
earlier time point in
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,
-secretase cleavage of Swedish mutant
APP, in contrast to the
principal
-secretase cleavage of wild type
APP, occurs in
competition with
-secretase cleavage in the secretory pathway.
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 APP. We used an antibody (192sw (40); see Fig.
2A) specifically detecting
APPssw-
, 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
APP as well as wild type
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
APP695, which represent
potential phosphorylation sites for casein kinase (CK)-2 and CK-1,
respectively. Further, we show that
APP can be phosphorylated by an
ectoprotein kinase activity on the cell surface. Therefore, our data
demonstrate that
APP undergoes ectodomain phosphorylation at two
distinct cellular locations.
Kidney
293 cells were stably transfected with the wt APP695
cDNA (9, 41) or with the
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.
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 ElectrophoresisImmunoprecipitations 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
APP and recognizes full-length
APP (46). Antibody B5, which
recognizes all forms of APPs, was raised to a fusion
protein containing amino acids 444-592 of
APP695 (70).
Antibody 1736 (raised to
APP-(595-611)) specifically identifies
APPswt-
and
APPssw-
(12) but not
APPswt-
or wt/Swedish full-length
APP. Antibody 192sw was raised against the free C terminus of
APPswt-
(40). This antibody
specifically recognizes APPswt-
but
not APPswt/sw-
or wt/Swedish
full-length
APP (1, 40). APLP2 was immunoprecipitated with antibody
D2-1 raised to full-length mouse APLP2 (42).
Pulse-chase experiments were
carried out as described (43). Briefly, cells stably transfected with
the Swedish 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
APP) and antibody 192sw (to detect intracellular
APPssw-
). Media were
immunoprecipitated with antibody 192sw (to detect secreted
APPssw-
).
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 DigestionIn
vivo, 32P-phosphorylated APP was isolated by
immunoprecipitation and SDS-PAGE and transferred to nitrocellulose
membrane (Schleicher & Schüll). Digestion of radiolabeled
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.
Approximately 10 µg of unlabeled APP together
with a trace of 32P-labeled
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 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 [-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
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).
The APP cDNA construct
containing a stop codon at the
-secretase cleavage site was
described previously (23). The C-terminal deletion construct of
APP
was described by Haass et al. (44). A cDNA construct
containing a stop codon at the
-secretase site of wt
APP was
generated as described (23) using the following annealed
oligonucleotides: GATCTCTGAAGTGAAGATGTAGGCAG (stop
-wt sense) and
AATTCTGCCTACATCTTCACTTCAGA (stop
-wt antisense).
The cDNA construct containing a stop codon at the -secretase
site of Swedish
APP was generated as described (23) using the
following annealed oligonucleotides: GATCTCTGAAGTGAATCTGTAGGCAG (stop
-sw sense) and AATTCTGCCTACAGATTCACTTCAGA (stop
-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.
In order to obtain a
general validation of ectodomain phosphorylation of APP (23), we
examined the phosphorylation of the highly related APLP2. APLP2, APLP1,
and
APP are members of a conserved gene family of homologous
proteins (52-55). APLP2 is particularly similar to
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
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.
Stability of Ectodomain Phosphorylation of
To assess
the stability of APP phosphorylation, we examined protein
phosphatase activity in AD brain extracts as well as in the conditioned
media of cultured cells (57, 58). No
APP dephosphorylating activity
was detected in any of the AD brain extracts, both those from temporal
and occipital regions, indicating a relative resistance of
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
APP is
relatively resistant to protein phosphatase activities, suggesting a
long lasting biological function of phosphorylated APPs
molecules.
Little
is known about the cell biology of ectodomain phosphorylation. In order
to determine the subcellular locus for APP phosphorylation, we used
an antibody (192sw; Fig. 2A) that
specifically recognizes APPssw-
,
which we previously detected in high quantities within the lysates of
kidney 293 cells stably transfected with the Swedish
APP cDNA
(1). To determine if ectodomain phosphorylation also occurs on
intracellular APPssw-
, we
radiolabeled kidney 293 cells expressing Swedish mutant
APP with
[32P]orthophosphate. Upon immunoprecipitation of cell
lysates and media we detected phosphorylated intracellular and secreted
APPssw-
as well as phosphorylated
intracellular full-length
APP (Fig. 2B). This result
indicates the occurrence of intracellular phosphorylation of the
APP-ectodomain. To prove that
APPssw-
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
APP) or with
antibody 192sw (to detect intracellular APPssw-
). In addition, conditioned
media were immunoprecipitated with antibody 192sw to detect secreted
APPssw-
. As shown in Fig.
3, full-length
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
APP declines due to the secretion of APPs.
Consistent with our previous results (1), the highest level of
intracellular APPssw-
was detected
after 45 min. After this time point the levels of intracellular
APPssw-
declined, and an increase of
secreted APPssw-
in the media was
observed (Fig. 3). The precursor product relationship clearly indicates
that intracellular APPssw-
is
produced de novo and not due to a fluid phase mediated
uptake of secreted species.
Mapping of Phosphorylation Sites within
To determine
which amino acids were phosphorylated in Swedish mutant APP, we
performed phosphoamino acid analysis of intracellular as well as
secreted APPssw-
. Both species are
phosphorylated exclusively on serine residues (Fig. 4).
This result is in line with recent studies showing that wt
APP is
constitutively phosphorylated solely on serine residues (23). It also
confirms that phosphorylation of intracellular APPssw-
is an amino acid
phosphorylation, not an incorporation of phosphate into sugar moieties
of
APP.
In order to identify the site(s) of APP phosphorylation, we
performed tryptic peptide mapping of in vivo phosphorylated
APP molecules. Kidney 293 cells stably transfected with wild type
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
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
APP, since no phosphate incorporation
occurred in cells expressing the N-terminal deletion construct (Fig.
5B). Phosphorylated full-length
APP as well as the
phosphorylated C-terminal deleted
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
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
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
APP and presumably
represent peptides of autocatalytically cleaved trypsin, migrating
close to the phosphorylated
APP tryptic peptide. In contrast, the
mass of 4877.3 matches that of the sequence of amino acids 181-224 of
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
APPs was immunoprecipitated from conditioned medium with antibody B5. Phosphate incorporation was quantified by
phosphor imaging. As shown in Fig. 5E, phosphorylation of
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
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).
Phosphorylation of
Ectodomain phosphorylation of APP was found on all
types of secreted APPs molecules, regardless of whether
Swedish or wt
APP was cleaved at either the
- or the
-secretase site. To produce APPs molecules with defined
C termini corresponding to
- or
-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-
,
APPswt-
, and
APPssw-
were each secreted as
phosphorylated species. Thus, membrane insertion of
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
APP.
To determine whether phosphorylation of APP occurs in the same
compartment as the
-secretase cleavage of Swedish
APP (1), we
investigated the effect of BFA on phosphorylation of Swedish
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
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
APP) or antibody 192sw (to detect intracellular
APPssw-
), and conditioned media were
precipitated with antibody 192sw (to detect secreted
APPssw-
). As reported previously, BFA
treatment not only inhibited the maturation of full-length
APP but
also completely inhibited the generation of intracellular
APPssw-
and its secretion (Fig.
7A; Refs. 1 and 44)). Treatment with BFA also
resulted in an inhibition of
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
APP molecules that escaped the BFA block at the beginning of the
experiment.
To determine whether ectodomain phosphorylation of APP occurs within
the trans-Golgi network, kidney 293 cells expressing Swedish
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
APP within cell
lysates; no APPssw-
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
APP was precipitated from cell lysates and phosphorylated
APPssw-
from both lysates and media
(Fig. 8B). In contrast, incubation of cells at 20 °C
completely inhibited phosphorylation of full-length
APP (Fig.
8B), although large amounts of full-length
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
APP occurs
within a post-Golgi compartment, most likely secretory vesicles, and
not in the trans-Golgi network itself.
Analogous experiments were then carried out to determine the cellular
locus of the ectodomain phosphorylation of wt APP. When kidney 293 cells expressing wt
APP were labeled at 37 °C with
[35S]methionine, antibody C7 precipitated the expected
doublet of full-length
APP from cell lysates representing the
immature and mature forms of the precursor (Fig. 8C).
Precipitation with antibody 1736, which specifically identifies
APPswt-
and does not cross-react with
full-length
APP or APPswt-
,
results in the detection of intracellular
APPswt-
from cell lysates as well as
secreted APPswt-
from conditioned
media (Fig. 8C). The detection of intracellular APPswt-
is in good agreement with
data published previously (51, 59, 60), indicating
-secretase
cleavage within the secretory pathway. When cells were incubated at
20 °C, an accumulation of mature
APP was observed; however, the
generation of intracellular APPswt-
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
APP, and precipitation of cell lysates
with antibody 1736, specific for APPs-
, resulted in the
detection of intracellular phosphorylated APPswt-
(Fig. 8D).
However, incubating the cells at 20 °C completely inhibited
phosphorylation of wild type
APP; no phosphorylated full-length
APP or intracellular and secreted
APPswt-
was detected (Fig.
8D). Taken together, these data show that intracellular
ectodomain phosphorylation of wild type as well as Swedish
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
APP.
Because mature full-length APP is also present at the
cell surface, we examined whether membrane-bound
APP can be a
substrate for ectoprotein kinases. Intact kidney cells, transfected
with wild type
APP cDNA, were incubated in the presence of 1 µM [
-32P]ATP in the cell supernatant,
allowing specific detection of ectoprotein kinase activity (30).
Full-length
APP was then precipitated from cell lysates and
APPswt-
from cell supernatants. As
shown in Fig. 9A (wt) cell
surface-bound full-length
APP was phosphorylated by ectoprotein
kinase activity. Moreover, phosphorylated
APPswt-
was recovered from cell
supernatants (Fig. 9A, Media). Similar experiments with kidney 293 cells expressing Swedish
APP showed that
cell surface
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
APP, which inserts in cell membranes but does not undergo
reinternalization (13, 44). As with full-length
APP (Fig.
9A), the C-terminal truncated form of
APP was also
phosphorylated (Fig. 9B,
C), indicating that
reinternalization of
APP is not necessary for its phosphorylation. Again, phosphorylated APPswt-
was
recovered from cell supernatants (Fig. 9B,
Media). To prove whether phosphorylated
APPswt-
does exclusively derive from
its phosphorylated precursor or if soluble
APPswt-
can be phosphorylated after
proteolytic cleavage, cell-free supernatant containing
APPswt-
was incubated with
[
-32P]ATP either in the absence or in the presence of
untransfected intact kidney 293 cells. As shown in Fig. 9C,
APPswt-
was phosphorylated only in
the presence of intact cells, indicating that soluble
APPswt-
could serve as a substrate
for ectoprotein kinase. Thus, neither membrane insertion nor
reinternalization is necessary for
APP phosphorylation. However,
APP was not phosphorylated in the absence of cells (Fig.
9C, Cells), showing that ectoprotein kinase
activity is not cosecreted with
APPs-species. As
revealed by two-dimensional phosphoamino acid analysis, phosphorylation
of
APP by ectoprotein kinase occurs exclusively on serine residues
(Fig. 9D). The results clearly demonstrate that cell
surface-bound
APP and its soluble derivatives can be phosphorylated
by membrane-associated ectoprotein kinase on the surface of intact
cells.
In summary, our data show that full-length APP and its
- and
-secretase-cleaved derivatives can be phosphorylated at two different subcellular locations. In both cases,
APP is exclusively phosphorylated on its ectodomain but not in the cytoplasmic tail. Ectodomain phosphorylation of
APP has been demonstrated previously (23) and was further supported by the data presented here;
APP can
be phosphorylated on the cell surface by incubating cells with
[
-32P]ATP, and secreted APPS derived from
recombinant cDNA constructs with stop codons inserted at the
-
and
-secretase site of mutant and wild type
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
APP.
Intracellular APPs and full-length
APP molecules are phosphorylated within a post-Golgi compartment, most likely secretory vesicles. This is the cellular compartment to which we have localized the
-secretase activity cleaving Swedish
APP (1). Therefore, phosphorylation of APPs occurs during or immediately before
or after the secretory cleavages of
APP.
The in vivo phosphorylation sites of 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 APP: an ectoprotein kinase activity at the cell
surface. In contrast to the intracellular phosphorylation of
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
APP. Since
-secretase activity is present within cell lysates
(51, 59, 60), as well as on the cell surface (12, 62), full-length
surface
APP will contribute to the pool of phosphorylated
APPs molecules in conditioned media. In addition, secreted
derivatives of
APP (APPswt and
APPssw) released by
- or
-secretase into the cell supernatant also serve as substrates for
ectoprotein kinase. Our study demonstrates for the first time the
unusual phenomenon that
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
APP is phosphorylated by different protein kinases on
two or more sites within the same molecule. The functional consequences
of this complex regulation of
APP ectodomain phosphorylation are
unknown so far. However, one might speculate that extracellular
function(s) of
APP, e.g. the modulation of neuronal
excitability by APPs (69), could be regulated by selective
ectodomain phosphorylation.