From the Adolf Butenandt-Institute, Department of
Biochemistry, Laboratory for Alzheimer's and Parkinson's Disease
Research, Ludwig-Maximilians-University, 44 Schillerstrasse, 80336 Munich, Germany and ¶ Center for Molecular Biology Heidelberg, Im
Neuenheimer Feld 282, 69120 Heidelberg, Germany
Received for publication, December 11, 2000, and in revised form, January 26, 2001
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ABSTRACT |
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Alzheimer's disease is the most common form of dementia and is
pathologically characterized by the invariant accumulation of senile
plaques and neurofibrillary tangles in certain areas of the brains of
Alzheimer's disease patients (1). The major constituent of senile
plaques is the amyloid Recently, an aspartyl protease with BACE is cotranslationally modified by N-glycosylation and
further maturates by complex glycosylation as well as proteolytic removal of its prodomain by a furin-like protease (11-14). The majority of BACE molecules are localized within Golgi and endosomal compartments, where they colocalize with It has recently been reported that BACE is reinternalized from the cell
surface to early endosomes and can recycle back to the cell surface, a
process that depends on a dileucine motif in the cytoplasmic tail of
BACE (14). This signal is located close to a negatively charged domain,
which contains a potential phosphorylation site. We found that BACE is
indeed phosphorylated within its C-terminal domain and that the
biological function of BACE phosphorylation resides in the regulation
of the retrieval of reinternalized BACE from endosomes.
Cell Culture and Transfection--
Human embryonic kidney (HEK)
293 and green monkey kidney COS-7 cells were cultured in Dulbecco's
modified Eagle's medium with Glutamax (Life Technologies, Inc.)
supplemented with 10% fetal calf serum (Life Technologies). The cell
lines stably overexpressing cDNAs and Fusion Proteins--
The phosphorylation site
mutants of BACE were generated by polymerase chain reaction techniques
using the appropriate oligonucleotides. The resulting polymerase chain
reaction fragments were subcloned into the
EcoRI/XhoI restriction sites of pcDNA3.1
containing a zeocine resistance gene (Invitrogen). To generate fusion
proteins of gluthatione S-transferase and the cytoplasmic
domain of BACE, the sequence of BACE encoding amino acids 476-501 was
amplified by polymerase chain reaction using appropriate primers. The
resulting fragments were subcloned into
EcoRI/XhoI restriction sites of pGEX-5X-1
(Amersham Pharmacia Biotech), and fusion proteins were expressed in
Escherichia coli DH5 Antibodies, Metabolic Labeling, Immunoprecipitation, and
Immunoblotting--
The polyclonal antibodies 7523, 7520, and GM190
recognizing the N terminus of BACE (amino acids 46-60), the C terminus
(amino acids 482-501), and the propeptide (amino acids 22-45),
respectively, have been described previously (12). Monoclonal
To radiolabel cellular proteins, cells were incubated at 37 °C in
methionine-free serum-free medium for 45 min. Cells were then incubated
with the same medium supplemented with
[35S]methionine/[35S]cysteine (Promix;
Amersham Pharmacia Biotech) and kept at 37 °C for the times
indicated in the respective experiments. For immunoprecipitations cells
were lysed in buffer containing 1% Nonidet P-40 on ice for 10 min.
Lysates were clarified by centrifugation for 10 min at 14,000 × g and immunoprecipitated for 3 h at 4 °C. After
separation by SDS-PAGE, proteins were transferred to PVDF membrane
(Imobilon; Millipore) and analyzed by autoradiography or
phosphorimaging. Alternatively, BACE was detected by immunoblotting using the enhanced chemiluminescence technique (Amersham Pharmacia Biotech).
In Vivo Phosphorylation--
HEK 293 cells were incubated 45 min
in phosphate-free media (Sigma). Media were aspirated, and the
respective fresh media were added containing 18 MBq/ml
[32P]orthophosphate (Amersham Pharmacia Biotech). After
1 h at 37 °C, cells were incubated for an additional 1 h
in the presence or absence of protein kinase activators/inhibitors or
the phosphatase inhibitor okadaic acid at the concentrations indicated
in the respective experiments. To inhibit
N-glycosylation, cells were treated with 10 µg/ml tunicamycin for 2 h prior to the addition of
[32P]orthophosphate and during the labeling period. After
labeling, media were aspirated, and the cells were washed twice with
ice-cold PBS and immediately lysed on ice with lysis buffer containing 1% Nonidet P-40 for 10 min. Cell lysates were centrifuged for 10 min
at 14,000 × g, and supernatants were
immunoprecipitated with specific antibodies as indicated.
In Vitro Phosphorylation Assays--
In vitro
phosphorylation assays were carried out as described previously (20).
Recombinant rat casein kinase (CK)-1 Phosphoamino Acid Analysis--
Phosphoamino acid analysis was
carried out by one-dimensional high voltage electrophoresis according
to Jelinek and Weber (21). Radiolabeled proteins, electrotransferred
onto PVDF 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 8 µl of pH 2.5 buffer
(5.9% glacial acetic acid, 0.8% formic acid, 0.3% pyridine, 0.3 mM EDTA) and spotted onto 20 × 20-cm cellulose thin
layer chromatography plates (Merck) together with unlabeled
phosphoamino acids (Ser(P), Thr(P), Tyr(P); 1 µg each; Sigma).
High voltage electrophoresis was carried out for 45 min at 20 mA.
Radiolabeleled phosphoamino acids were localized by autoradiography and
identified by comparison with comigrating phosphoamino acids after
ninhydrine staining.
Deglycosylation Experiments--
BACE was immunoprecipitated
from cell lysates as described above, and precipitates were incubated
in the presence of endoglycosidase H (endo H; Roche Molecular
Biochemicals) or PNGase F (Roche Molecular Biochemicals) for 14 h
at 37 °C in the appropriate buffer. Reactions were stopped by the
addition of SDS sample buffer, and reaction mixtures were separated by
SDS gel electrophoresis.
Immunocytochemistry--
Cells stably expressing BACE cDNAs
were grown on polylysine-coated glass coverslips to 50-80%
confluence. Cells were fixed in 4% paraformaldehyde/PBS at room
temperature and processed for immunofluorescence as described
previously (12). Bound primary antibodies were detected by Alexa 488- or Alexa 594-conjugated secondary antibodies (Molecular Probes, Inc.,
Eugene, OR). In some experiments, cells were incubated in the presence
of 10 µg/ml brefeldin A (BFA) for 30 min at 37 °C before fixation.
Antibody Uptake Assays--
Cells grown on polylysine-coated
glass coverslips were washed twice with ice-cold PBS and incubated for
20 min on ice in serum-free medium (Opti-MEM; Life Technologies, Inc.)
containing the indicated antibodies. Cells were then washed three times
with ice-cold PBS and subsequently incubated at 37 °C or 18 °C in
Dulbecco's modified Eagle's medium with Glutamax supplemented with
10% fetal calf serum for various time periods. After two washes with
PBS, cells were fixed in 4% paraformaldehyde/PBS and processed for immunofluorescence.
Cells were analyzed using a Leica DMRB fluorescence microscope, and
photographs were taken with an RT monochrome spot camera (Diagnostic Instruments) and processed with Metaview program (Visitron Systems).
BACE Is Phosphorylated at Serine 498--
To investigate
whether BACE is posttranslationally modified by phosphorylation, we
used HEK 293 cells stably overexpressing human BACE. HEK 293 cells were
used previously to identify BACE in a functional screening assay and to
analyze its proteolytic function (7). The same cell line was also used
to successfully investigate the effects of the Swedish
After in vivo labeling with
[32P]orthophosphate, cell lysates were immunoprecipitated
with antibody 7523 directed to an N-terminal sequence after the
prodomain (Fig. 1A). Analysis
of immunoprecipitated BACE by autoradiography revealed that BACE
undergoes phosphorylation (Fig. 1B). Incorporation of
[32P]orthophosphate is dependent on the presence of the
phosphatase inhibitor okadaic acid (Fig. 1B). However,
longer exposure of the autoradiograms revealed weakly radiolabeled BACE
also in the absence of okadaic acid (data not shown). To prove if BACE
is phosphorylated within its cytoplasmic tail or within its ectodomain as previously demonstrated for its substrate
Phosphoamino acid analysis of 32P-labeled BACE revealed
that it is phosphorylated on serine residues (Fig. 1C). The
cytoplasmic domain of BACE contains a single serine residue at position
498 close to the C terminus (Fig. 1A). To confirm that
serine 498 is indeed phosphorylated in vivo, we generated a
cell line stably expressing BACE containing a serine to alanine
substitution (BACE S498A). This mutation completely blocks
phosphorylation of BACE, thus demonstrating that serine 498 is the sole
in vivo phosphorylation site (Fig. 1D).
Phosphorylation Occurs Selectively after Full Maturation of
BACE--
The results shown in Fig. 1 indicate that selectively mature
BACE (70 kDa) is phosphorylated but not the immature 66-kDa form of
BACE. If that is the case, phosphorylated BACE should be
resistant to endo H treatment (12, 14). To prove this, cells expressing BACE carrying a Myc tag at the C terminus (12) were labeled with
[32P]orthophosphate in the presence of okadaic acid, and
BACE was isolated from cell lysates by immunoprecipitation with the
Casein Kinase 1 Phosphorylates BACE--
To identify a protein
kinase involved in phosphorylation of BACE, we first carried out
in vitro phosphorylation assays using fusion proteins of GST
with the cytoplasmic domain of BACE WT (CT-WT) or the S498A mutation
(CT-S498A). The fusion proteins were incubated with four selected
purified protein kinases in the presence of [
We next analyzed the involvement of CK-1 in the phosphorylation of BACE
by cellular extracts. Lysates of HEK 293 cells were incubated with
[ Phosphorylation of the Cytoplasmic Domain Affects the Subcellular
Localization of BACE--
We next examined the subcellular
localization of BACE in stably transfected HEK 293 cells. Cells grown
on glass coverslips were fixed and costained with polyclonal antibody
7523 against BACE and with monoclonal antibody against giantin, a Golgi
marker protein (30). BACE WT and giantin partially colocalize in
juxtanuclear structures (Fig. 4,
a and b). Consistent with previous results (4, 5,
7, 12, 14), additional staining was observed in juxtanuclear and
vesicular structures that did not overlap with the Golgi marker giantin
(Fig. 4, a and b). To prove whether this
additional staining is due to localization of BACE in post-Golgi compartments, cells were treated with BFA for 30 min at 37 °C. BFA
treatment is known to result in the fusion of Golgi compartments with
the ER, while the trans-Golgi network (TGN) and other post-Golgi compartments fuse with endosomes to form vesicular/tubular structures (31). Treatment with BFA resulted in reticular staining of giantin, indicative for a redistribution of the Golgi marker protein giantin to
the ER (Fig. 4d). In contrast, BACE was detected in
juxtanuclear and peripheral vesicular structures after BFA treatment
(Fig. 4c). These results indicate that BACE is not only
localized in the ER and Golgi but also in post-Golgi compartments.
To examine if phosphorylation of BACE affects its accumulation in these
compartments, we generated cDNA constructs encoding BACE
derivatives in which serine residue 498 is substituted by alanine or by
aspartate residues in order to mimic unphosphorylated or phosphorylated
BACE molecules, respectively. HEK 293 cells stably expressing BACE WT,
BACE S498D, or BACE S498A were permeablized and costained with antibody
7523 and antibodies against giantin. As shown above (Fig. 4), BACE WT
and giantin partially colocalize in juxtanuclear structures (Fig.
5, a and b). A very
similar localization was observed for BACE S498D, which
mimics phosphorylated BACE (Fig. 5, c and
d). In contrast, nonphosphorylatable BACE S498A showed less
juxtanuclear staining. Rather, BACE S498A accumulated in peripheral
vesicular structures near the plasma membrane, particularly in cellular processes (Fig. 5e). Very similar data were
obtained with independent cell clones and transiently transfected HEK
293 cells (data not shown). These data indicate that some aspects of
subcellular sorting of BACE are affected by
phosphorylation/dephosphorylation of serine residue 498.
To prove that the distinct staining pattern of the BACE mutants
is not simply due to impaired maturation, we performed pulse-chase experiments. Immunoprecipitation with antibody 7520 against the C
terminus of BACE demonstrate that the phosphorylation site mutants BACE
S498A and BACE S498D mature with similar kinetics as the WT protein by
complex N-glycosylation as indicated by the molecular mass
shift from 66 to 70 kDa (Fig.
6A, left
panel). To analyze proteolytic cleavage of the prodomain of
BACE, cell lysates were immunoprecipitated with antibody GM190 directed
against the prodomain. Similar to our previous results (12), the
prodomain is predominantly observed in the immature 66-kDa BACE species
(Fig. 6A, right panel). The gradual
disappearance of prodomain-containing forms indicates efficient
maturation of BACE S498A and BACE S498D similar to BACE WT (Fig.
6A, right panel). Moreover, the mature
70-kDa forms of mutant BACE S498A and S498D are both resistant to endo
H cleavage, while PNGase F efficiently deglycosylated the 70- and
66-kDa forms (Fig. 6B). These data demonstrate that the
mutations in the cytoplasmic domain of BACE do not interfere with
maturation by complex N-glycosylation, indicating unimpaired
forward transport to late Golgi compartments and beyond. Therefore, the
altered subcellular localization of the nonphosphorylated mutant BACE
S498A is not due to impaired maturation.
Phosphorylation Regulates Retrieval of BACE from Endocytosed
Vesicles--
Recently, it was reported that cell surface-located BACE
is internalized into endosomes (14). We therefore first investigated whether phosphorylation of BACE might regulate its reinternalization from the cell surface. HEK 293 cells stably expressing BACE WT or the
phosphorylation site mutant BACE S498A or BACE S498D were incubated for
20 min on ice with antibody 7523 directed toward the ectodomain of
BACE. After removal of nonspecifically bound antibodies, cells were
returned to 37 °C for the time points indicated and processed for
immunocytochemistry. At time point 0 min, cells expressing BACE WT or
the S498D or S498A variants revealed strong staining of the cell
surface (Fig. 7, a,
e, and i). In contrast, untransfected HEK 293 cells were not labeled by antibody 7523, demonstrating specific
detection of exogenous BACE at the cell surface (Fig. 7n).
Staining of cells after 30 min at 37 °C revealed that all BACE
variants are efficiently internalized from the cell surface (Fig.
7, b, f, and k). Again,
untransfected cells were not stained, demonstrating that
antibodies were not internalized by fluid phase endocytosis (Fig.
7o). Staining cells after 1 and 2 h at 37 °C
revealed significant differences in the subcellular localization of
internalized BACE S498A as compared with BACE WT and BACE S498D. Cells
expressing BACE WT or the BACE S498D mutant that mimics phosphorylated
BACE revealed intensive juxtanuclear localization indicative for late
endosomal compartments and/or TGN (Fig. 7, b-d and
f-h). In contrast, the nonphosphorylated mutant S498A
showed predominant localization in vesicles near the plasma membrane,
which occasionally appeared to accumulate in cellular processes (Fig.
7, k-m).
To identify the subcellular compartment to which BACE is targeted after
internalization from the cell surface, we performed antibody uptake
assays as described above. To detect early endosomes, cells were
costained with monoclonal antibodies recognizing the early endosome
antigen 1 (EEA1) (32). Staining of cells after 15 min showed
internalization of BACE into some EEA1-positive compartments. No
significant differences in the staining pattern between BACE WT and the
phosphorylation site mutants (S498A and S498D) were detected at this
time point, demonstrating that internalized BACE is targeted to early
endosomal compartments independent of its phosphorylation state (Fig.
8, a-f). After 30 min,
internalized BACE WT and BACE S498D were efficiently targeted to
juxtanuclear structures (as described above; Fig. 8), showing less
colocalization with the EEA1-positive compartments (Fig. 8,
g-k). In contrast, the nonphosphorylated mutant BACE S498A
showed less pronounced accumulation in juxtanuclear structures (as
described above) but appeared to be retained at least partially in EEA1
positive endosomal compartments (Fig. 8, l and
m).
We next sought to identify the cellular trafficking step of BACE, which
is regulated by its phosphorylation. It was shown previously that
incubation of cells at 18 °C inhibits both forward transport from
the ER to Golgi and retrograde transport from endocytosed vesicles to
late endosomal compartments and the TGN but allows endocytosis (33,
34). Cells were incubated with antibody 7523 on ice as described above.
After washing, the cells were returned to 37 or 18 °C and incubated
for an additional 1 h. Internalized BACE was then detected with
fluorescence-labeled secondary antibody. After incubation at 37 °C,
the above-described differences in the subcellular localization of BACE
WT, BACE S498D, and BACE S489A were observed. While BACE WT and BACE
S498D accumulated in juxtanuclear compartments, BACE S498A was
predominantly localized in peripheral endocytosed vesicles (Fig.
9, a, c, and
e), identified as early endosomes (see Fig. 8). Costaining
of these cells with the monoclonal antibodies IG7/5A3 against
When cells were incubated at 18 °C, all variants of BACE were
readily internalized, but, in contrast to incubation at 37 °C, transport of reinternalized BACE WT and BACE S498D to juxtanuclear structures was inhibited (Fig. 9, g and i).
Rather, at 18 °C all variants of BACE accumulated in peripheral
vesicles, and BACE WT and S498D showed a very similar localization as
the nonphosphorylated S498A variant of BACE (Fig. 9, g,
i, and l). Costaining of these cells with In this study, we analyzed the subcellular trafficking of BACE
dependent on its phosphorylation state. A single phosphorylation site
was identified by mutagenesis of serine residue 498 in the C-terminal
domain of BACE (Fig. 1A). By testing several protein kinases
in vitro, we found that CK-1 can phosphorylate BACE at the
in vivo phosphorylation site at serine residue 498. HD,
which preferentially inhibits CK-1 beside glycogen synthase kinase 3 In order to investigate the cellular function of
phosphorylation/dephosphorylation on the trafficking of BACE, we
generated mutants in which the phosphorylation site at serine
residue 498 has been substituted by an alanine or an aspartate residue
to mimic nonphosphorylated and phosphorylated forms of BACE,
respectively. The major advantage of this strategy is to circumvent the
use of agents modulating kinase or phosphatase activities that might cause nonspecific or indirect effects (42, 43). BACE carrying the S498D
substitution was predominantly localized in juxtanuclear compartments,
where it partially colocalizes with the Golgi marker protein giantin. A
very similar distribution was observed for BACE WT, which is consistent
with previous results (7, 12, 14). In contrast, the nonphosphorylated
form BACE S498A showed less localization in juxtanuclear structures but
pronounced localization in vesicular compartments including EEA1
positive early endosomes. It was demonstrated previously that the
complete deletion of the cytoplasmic domain of BACE results in its
retention within the ER and in impaired maturation (12). However, the
phosphorylation site mutants BACE S498A and BACE S498D mature normally
by complex N-glycosylation and proteolytic removal of the
prodomain as compared with BACE WT. Therefore, the significant
differences in subcellular localization of the mutant variants of BACE
are due to distinct sorting. Consistent with previous results (12, 14),
we found that BACE is transported to the cell surface and that BACE is internalized into EEA1-positive early endosomal compartments. BACE WT
as well as the mutant derivatives S498A and S498D were efficiently
endocytosed, indicating that phosphorylation of BACE does not determine
its reinternalization from the cell surface. Rather, we found that
phosphorylation of BACE is functionally required for efficient
retrieval of the enzyme from early endosomes to later endosomal and/or
TGN compartments from which BACE might be recycled into the secretory pathway.
Interestingly, phosphorylation of reinternalized BACE affected its
colocalization with its protein substrate The regulatory mechanism of subcellular trafficking of BACE is highly
reminiscent to that of furin. Retrieval of furin from endosomal to TGN
compartments was also shown to be dependent on phosphorylation/dephosphorylation of its cytoplasmic domain (45). Therefore, the subcellular localization of BACE is regulated in a
remarkably similar fashion like furin, a protease that has recently been demonstrated to be required for propeptide cleavage of BACE (11-13).
-Secretase (BACE) is a transmembrane aspartyl
protease, which generates the N terminus of Alzheimer's disease
amyloid
-peptide. Here, we report that BACE can be phosphorylated
within its cytoplasmic domain at serine residue 498 by casein kinase
1. Phosphorylation exclusively occurs after full maturation of
BACE by propeptide cleavage and complex N-glycosylation.
Phosphorylation/dephosphorylation affects the subcellular localization
of BACE. BACE wild type and an S498D mutant that mimics phosphorylated
BACE are predominantly located within juxtanuclear Golgi compartments
and endosomes, whereas nonphosphorylatable BACE S498A accumulates in
peripheral EEA1-positive endosomes. Antibody uptake assays revealed
that reinternalization of BACE from the cell surface is independent of
its phosphorylation state. After reinternalization, BACE wild type as
well as BACE S498D are efficiently retrieved from early endosomal
compartments and further targeted to later endosomal compartments
and/or the trans-Golgi network. In contrast, nonphosphorylatable BACE
S498A is retained within early endosomes. Our results therefore demonstrate regulated trafficking of BACE within the secretory and
endocytic pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-peptide
(A
),1 which derives from
the
-amyloid precursor protein (
APP) by endoproteolytic
processing (2).
-Secretase cleaves
APP at the N terminus of the
A
-domain, resulting in the generation of soluble
APPS-
and a membrane-associated C-terminal
fragment bearing the complete A
-domain. Subsequent cleavage of this
fragment by
-secretase, which appears to be identical with the
presenilins, results in the release and secretion of A
(3).
-secretase activity was
identified in human embryonic kidney (HEK) 293 cells and was initially
called BACE (
-site APP-cleaving enzyme, Asp2, or memapsin 2)
(4-8) (Fig. 1A). A close homologue was also identified and termed as BACE-2, Asp1, DRAP, or memapsin 1 (4, 5,
9).2 Both enzymes are type I
membrane proteins sharing significant homology with other members of
the aspartyl protease family (5-8). While BACE-2 is predominantly
expressed in peripheral tissues, BACE is highly expressed in neurons,
the major site of A
generation. However, it appears that
APP is
not the only substrate for BACE. In fact, mouse
APP is a very poor
substrate for
-secretase activity (10). "Humanizing" the A
domain of mouse
APP by three amino acid substitutions resulted in
efficient cleavage by
-secretase (10), suggesting that
APP may
not be the exclusive substrate for BACE. However, other physiological
substrates of BACE remain to be identified.
APP (4, 5, 7, 12, 14). The
acidic pH optimum of BACE (5-8) indicates that it is predominantly
active within late Golgi compartments and/or endosomes/lysosomes. This
is consistent with previous findings demonstrating that
-secretase
cleavage of
APP can occur in all of these acidic compartments
(15-18).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
APP695 (19) and BACE
carrying Myc epitopes at the C terminus (12) have been described
previously. Transfection of cells with BACE cDNAs was carried out
using Fugene reagent (Roche Molecular Biochemicals) according to the
supplier's instructions. Single cell clones were generated by
selection in 200 µg/ml zeocine (Invitrogen).
and purified on GSH-Sepharose according to the supplier's instructions.
-giantin antibodies and monoclonal
-
APP antibodies were
generously provided by Drs. H.-P. Hauri and E. H. Koo,
respectively. Monoclonal
-EEA1 antibody was from Transduction
Laboratories, and monoclonal antibody 9E10 developed by J. M. Bishop was obtained from the Developmental Studies Hybridoma Bank
(University of Iowa).
(New England Biolabs),
recombinant
-subunit of human CK-2 (New England Biolabs), and the
catalytic subunit of protein kinase A purified from bovine heart (gift
from Dr. V. Kinzel) were used for in vitro phosphorylation
assays in a buffer containing 20 mM Tris, pH 7.5, 5 mM magnesium acetate, 5 mM dithiothreitol.
Protein kinase C purified from rat brain (Biomol) was assayed in a
similar buffer supplemented with 1 µM phorbol
12,13-dibutyrate, 0.5 mM calcium chloride, and 100 µg/ml
phosphatidylserine under mixed micellar conditions. As substrates,
fusion proteins of GST and the C terminus of BACE (see above) were
used. Phosphorylation reactions were started by the addition of 10 µM [
-32P]ATP and allowed to proceed for
10 min at 32 °C. To control the kinase activities, parallel
phosphorylation reactions were carried out using phosvitin (1 mg/ml;
Sigma) or histone (0.5 mg/ml; Sigma) as protein substrates. Reactions
were stopped by the addition of SDS sample buffer. Alternatively, cell
extracts were used to phosphorylate fusion proteins of GST and BACE.
HEK 293 cells were lysed in a buffer containing 20 mM Tris,
pH 7.5, 5 mM magnesium acetate, 5 mM
dithiothreitol, and 0.5% Triton X-100. Lysates were centrifuged for 10 min at 14,000 × g, and fusion proteins of GST and
BACE-CT were added to the supernatant. Phosphorylation reactions were started by the addition of [
-32P]ATP and allowed
to proceed for 15 min in the presence or absence of 10 µM
hymenialdisine (HD) and 1 µM okadaic acid. GST fusion proteins were isolated by precipitation with GSH-Sepharose (Amersham Pharmacia Biotech) for 2 h at 4 °C. Precipitates were washed
five times with PBS and eluted by the addition of SDS sample buffer and
separated by SDS-gel electrophoresis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
APP mutation
on
-secretase cleavage (16), thus demonstrating that HEK 293 cells
represent a valid model system to study the cell biology of BACE.
APP (22-24), we
generated a truncated derivative lacking the cytoplasmic domain
(BACE
C; Fig. 1A (12)). Although high levels of BACE
C
were expressed, the deletion of the cytoplasmic tail of BACE completely
abolished phosphorylation (Fig. 1B). A soluble derivative of
BACE lacking the transmembrane domain (12) is also not phosphorylated
by cultured cells (data not shown), further supporting the finding that
the ectodomain of BACE is not phosphorylated. These results therefore
demonstrate exclusive phosphorylation of BACE within its cytoplasmic
tail.
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Fig. 1.
Phosphorylation of BACE at serine residue 498 within the cytoplasmic domain. A, schematic of BACE.
The aspartyl protease active site motifs (DTGS and DSGT) are indicated
by asterisks. The signal peptide (SP) is
indicated by a black box, and the propeptide
(pro) is shown by a hatched box. The
amino acid sequence of the cytoplasmic domain is given in single letter
code, and the phosphorylation site (Ser498) is shown in
boldface type. The epitopes of antibodies 7520, 7523, and GM190 are indicated. B, HEK cells stably
expressing wild-type human BACE (WT) or a truncated
derivative lacking the cytoplasmic domain ( C) were
labeled with [32P]orthophosphate in the presence or
absence of 0.2 µM okadaic acid (oa). BACE was
isolated by immunoprecipitation, separated by SDS-PAGE, and transferred
to a PVDF membrane. Radiolabeled proteins were visualized by
phosphorimaging (32P), and BACE was detected by
Western immunoblot with antibody 7523 (WB). Mature
(m) and immature (im) forms of BACE are indicated
by arrowheads. C, phosphoamino acid analysis of
radiolabeled BACE. The arrowheads indicate migrations of
standard phosphoamino acids (P-Ser,
P-Thr, P-Tyr) and origin of sample application
(ori). D, phosphorylation of BACE WT and BACE
S498A was analyzed as described for B. Substitution of
Ser498 by Ala completely abolished phosphate
incorporation.
-Myc antibody 9E10. Immunoprecipitates were incubated in
vitro in the presence or absence of endo H or PNGase F and
separated by SDS-PAGE. Immunoblot analysis with antibody 7523 revealed
that endo H deglycosylated exclusively the immature 66-kDa form of BACE
(Fig. 2A, left
panel), consistent with the specificity of endo H for
immature, biantennary or high mannose, N-linked glycans. We
also identified the immature forms of BACE using antibody GM190
directed against the prodomain (12) and found that endo H selectively
deglycosylates prodomain-containing immature forms of BACE (Fig.
2A, left panel). Autoradiography of
the same membrane revealed that phosphorylated BACE is completely resistant to endo H treatment (Fig. 2A, left
panel). In contrast to endo H, treatment with PNGase F,
which removes all types of N-linked glycans, resulted in
complete deglycosylation of both phosphorylated mature and
unphosphorylated immature BACE as demonstrated by a significant
molecular mass shift of 32P-labeled BACE from 70 to
50 kDa (Fig. 2A, right panel). These results indicate that selectively fully mature BACE is phosphorylated. To confirm this in living cells, HEK 293 cells were labeled with [32P]orthophosphate in the presence or absence of
tunicamycin to inhibit cotranslational N-glycosylation. BACE
was immunoprecipitated with the
-Myc antibody 9E10 and separated by
SDS-PAGE. Consistent with previous results (12), tunicamycin treatment
resulted in the accumulation of unglycosylated 50-kDa forms
of BACE (Fig. 2B). Detection with antibody GM190
demonstrates accumulation of substantial levels of the unglycosylated
prodomain-containing 50-kDa form of BACE after tunicamycin treatment
(Fig. 2B). Autoradiography of the same membrane revealed
that the unglycosylated form is not phosphorylated, in contrast to the
fully mature BACE at 70 kDa (Fig. 2B). Taken together, these
experiments demonstrate that phosphorylation of BACE occurs selectively
after full maturation by removal of the prodomain and complex
N-glycosylation.
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Fig. 2.
Selective phosphorylation of mature
BACE. A, cells stably expressing BACE WT were labeled
with [32P]orthophosphate in the presence of 0.2 µM okadaic acid. BACE was isolated by immunoprecipitation
and incubated in the presence (+) or absence ( ) of endo H or PNGase
F. Proteins were then separated by SDS-PAGE and transferred to a PVDF
membrane. After detection of 32P-phosphorylated BACE by
phosphorimaging (32P), membranes were subjected to
immunoblotting with antibody 7523. To specifically detect immature
forms of BACE, the same membrane was reprobed with antibody GM190
directed against the prodomain. The phosphorylated form of BACE is
resistant to treatment with endo H, while it is sensitive to PNGase F. B, HEK cells were incubated with
[32P]orthophosphate in the presence of 0.2 µM okadaic acid and in the presence or absence of
tunicamycin. BACE was isolated by immunoprecipitation, separated by
SDS-PAGE, and transferred to a PVDF membrane. The membrane was first
subjected to autoradiography to visualize 32P-labeled
proteins (32P) and then probed with antibody 7523 and with antibody GM190. Note that BACE is exclusively phosphorylated
after full maturation by complex N-glycosylation and
proteolytic removal of its prodomain.
-32P]
ATP. Since the phosphorylation site of BACE is located within an acidic
motif typical for substrate recognition by CKs (25) (see Fig.
1A), we used CK-1 and CK-2 in initial experiments. We also
analyzed protein kinases A and C, since these enzymes have been shown
to affect endoproteolytic processing of
APP (26-28). The fusion
protein CT-WT was readily phosphorylated by CK-1, while CK-2, protein
kinase A, and protein kinase C were not efficient (Fig.
3A, left
panel), although all kinases were catalytically active under
the assay conditions (Fig. 3B). CK-1-mediated
phosphorylation of the cytoplasmic domain of BACE was completely
abolished when CT-S498A was used as a substrate (Fig. 3A,
right panels).
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Fig. 3.
Casein kinase-1 phosphorylates BACE.
A, fusion proteins of GST carrying the WT (CT-WT;
left panels) or the S498A mutant (CT-S498A;
right panels) cytoplasmic domain of BACE were
incubated with protein kinases CK-1, CK-2, A (PKA), and C
(PKC) in the presence of [ -32P]ATP.
Reaction mixtures were separated by SDS-PAGE and transferred to PVDF
membrane. Phosphorylated fusion proteins were detected by
autoradiography (32P). The same membrane was stained
with Coomassie (Co) to control protein loading.
B, the catalytic activities of CK-1 and CK-2 were assayed
using 1 mg/ml phosvitin, and that of protein kinase A was assayed with
0.5 mg/ml histone. The activity of rat brain protein kinase C was
controlled by detection of autophosphorylation. C, lysates
of HEK 293 cells were incubated with fusion protein CT-WT and
[
-32P]ATP in the presence or absence of the
CK-1-selective inhibitor hymenialdisine (HD). As
controls, the fusion protein CT-S498A or GST alone were used as
substrates. After the phosphorylation reaction, fusion proteins were
precipitated with GSH-Sepharose and separated by SDS-PAGE.
Phosphorylated fusion protein was detected by autoradiography
(32P). To prove equal loading, fusion proteins were
stained with Coomassie (Co). The arrowheads
indicate migration of GST fusion proteins. Hymenialdisine efficiently
inhibits phosphorylation of CT-WT.
-32P]ATP in the presence or absence of HD,
which preferentially inhibits CK-1, glycogen synthase kinase 3
, and
cyclin-dependent kinases (29), using the fusion protein
CT-WT as substrate. CT-WT was readily phosphorylated by the cellular
extracts in the absence of HD (Fig. 3C). In contrast,
phosphorylation was completely blocked by HD. As expected, the fusion
protein CT-S498A and GST alone were not phosphorylated by cellular
extracts (Fig. 3C).
View larger version (115K):
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Fig. 4.
Subcellular localization of BACE. HEK
293 cells stably transfected with cDNAs encoding BACE WT were grown
on glass coverslips and incubated for 30 min at 37 °C in the
presence (c and d) or absence (a and
b) of brefeldin A (10 µg/ml). Cells were fixed and
costained with the polyclonal antibody 7523 (a and
c) and the monoclonal anti-giantin antibody (b
and d). Note the difference in the redistribution of BACE
and giantin after BFA treatment. Bar, 10 µm.
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Fig. 5.
Phosphorylation state-dependent
subcellular localization of BACE. HEK 293 cells expressing BACE WT
(a and b), the mutant derivatives BACE S498D
(c and d), or BACE S498A (e and
f) were grown on glass coverslips and then processed for
immunocytochemistry. After fixation, cells were costained with
polyclonal antibodies 7523 against BACE (a, c,
and e) and monoclonal antibodies against giantin
(b, d, and f). Note that BACE WT and
BACE S498D showed similar subcellular distribution predominantly in
juxtanuclear compartments (arrowheads) and partial
colocalization with giantin. In contrast, BACE S498A revealed more
vesicular staining in the periphery of the cell (arrows).
Bar, 10 µm.
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Fig. 6.
Substitution of serine residue 498 does not
affect maturation of BACE. A, pulse-chase experiments
with cells expressing BACE WT, S498A, and S498D. Cells stably
expressing the respective cDNAs were pulse-labeled for 10 min with
[35S]methionine and chased in the presence of excess
amounts of unlabeled methionine for the time points indicated. BACE was
immunoprecipitated with antibody 7520 (left
panel) or with antibody GM190 (right
panel) and detected by autoradiography. B, BACE
S498A and S498D were isolated by immunoprecipitation from cells labeled
with [35S]methionine and incubated in the presence or
absence of endo H or PNGase F. After separation by SDS-PAGE,
radiolabeled proteins were detected by autoradiography.
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Fig. 7.
Phosphorylation regulates intracellular
trafficking of BACE. HEK 293 cells stably expressing BACE WT
(a-d), BACE S498D (e-h), or BACE S498A
(i-m) or untransfected cells (n and
o) were incubated for 20 min with antibody 7523 on ice.
Cells were washed and incubated at 37 °C for the time points
indicated. Internalized antibodies 7523 were detected by Alexa
488-labeled anti-rabbit secondary antibody. Bar, 25 µm.
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Fig. 8.
BACE is internalized from the cell surface
into EEA1 positive early endosomes. HEK 293 cells stably
expressing BACE WT (a, b, g, and
h), BACE S498D (c, d, i,
and k), or BACE S498A (e, f,
l, and m) were incubated for 20 min with antibody
7523 on ice. Cells were washed and incubated at 37 °C for 15 min
(a-f) or 30 min (g-m) before fixation.
Internalized antibodies 7523 were detected by Alexa 488-labeled
anti-rabbit secondary antibody (a, c,
e, g, i, and l), and early
endosomal compartments were detected with monoclonal antibodies against
EEA1 (b, d, f, h,
k, and m). Colocalization of BACE and EEA1 in
early endosomal compartments is indicated by arrowheads
(a-f, l, and m), while juxtanuclear
structures containing BACE WT (g) or BACE S498D
(i) are indicated by arrows. Bar, 10 µm.
APP
(35) revealed that internalized BACE WT and S498D partially colocalized
with its protein substrate
APP in juxtanuclear structures, while
internalized BACE S498A showed less colocalization with
APP (Fig. 9,
a-f).
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Fig. 9.
Phosphorylation-dependent
retrieval of BACE from endocytosed vesicles. HEK 293 cells stably
expressing BACE WT (a, b, g, and
h), BACE S498D (c, d, i,
and k), or BACE S489A (e, f,
l, and m) were incubated for 20 min with antibody
7523 on ice. Cells were washed and incubated at 37 °C
(a-f) or 18 °C (g-m) for an additional
1 h. Cells were fixed, and internalized antibodies were visualized
with Alexa 488-labeled anti-rabbit secondary antibody. APP was
detected with monoclonal antibodies IG7/5A3 and Alexa 594-labeled
anti-mouse secondary antibody. The arrowheads indicate
juxtanuclear localization of BACE WT and BACE S498D (a and
c), and the arrows indicate localization of BACE
S498A in peripheral vesicles (e). Bar, 25 µm.
APP
antibodies demonstrates that localization of
APP in juxtanuclear
structures was not impaired under these experimental conditions (Fig.
9, g-m).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and cyclin-dependent kinases (29), significantly reduced
phosphorylation of the cytoplasmic domain of BACE in cellular extracts.
The phosphorylation site identified at serine residue 498 is preceded
by a stretch of acidic amino acid residues (Fig. 1A)
representing a canonical recognition motif for CK-1, but not for
glycogen synthase kinase 3
or cyclin-dependent kinases.
Taken together, these data indicate that CK-1 or a CK-1-like kinase is
involved in the phosphorylation of BACE in vivo.
Phosphorylation occurs exclusively on the fully maturated
BACE after propeptide removal and complex
N-glycosylation. This indicates that phosphorylation of BACE
takes place selectively after its exit from the ER, presumably in Golgi
or post-Golgi compartments. CK-1 occurs in several isoforms, and some
have been shown to be associated with the plasma membrane and synaptic
vesicles and to selectively phosphorylate a subset of membrane proteins (36). CK-1 is implicated in the regulation of vesicular trafficking in
yeast presumably by phosphorylating components of clathrin adaptor
proteins (37, 38). Our data suggest that a CK-1 isoform with a
particular subcellular distribution (i.e. in late Golgi and/or endosomal compartments) might be responsible for the highly selective phosphorylation of mature BACE. It has been reported that
CK-1 is significantly elevated in Alzheimer's disease brains (39, 40).
Moreover, A
has been shown to activate CK-1 in vitro
(41). However, it remains to be determined if phosphorylation of BACE
is altered during pathogenesis of Alzheimer's disease.
APP in juxtanuclear structures. However, overexpressing BACE WT or the phosphorylation site
mutants led to increased secretion of A
as compared with untransfected control cell lines (data not shown). This may reflect that expression of exogenous BACE leads to saturated levels of A
generation, regardless of the differences in subcellular localization of the BACE variants. Indeed, previous studies demonstrated that
APP
could be cleaved by
-secretase activity in distinct compartments, including endosomes and late Golgi compartments (16, 17, 44). We also
demonstrate that reinternalization of BACE was not affected by the
S498A or S498D mutation. In addition, all variants of BACE undergo
normal maturation by complex N-glycosylation.
These data indicate that at least two major sites of
-secretase
activity, endosomes and secretory vesicles, can be reached by BACE
independent of its phosphorylation state. However, it might be possible
that regulation of subcellular trafficking of endogenously expressed BACE by phosphorylation affects proteolytic processing of other, yet
unknown, protein substrates. In addition, we cannot yet exclude the
possibility that phosphorylation may have subtle effects on
APP processing.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. H.-P. Hauri for
anti-giantin and E. H. Koo for anti-APP antibodies and to
Dr. V. Kinzel for purified protein kinase A. We thank Drs. L. Meijer
and G. Pettit for hymenialdisine. We also thank Drs. M. Sastre and H. Steiner for helpful discussions and critically reading the
manuscript and L. Meyn for expert technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by Boehringer Ingelheim Inc. and the Deutsche Forschungsgemeinschaft.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 may be addressed. Tel.: 49 89 5996 471 or 49 89 5996 472; Fax: 49 89 5996 415; E-mail: jwalter@pbm.med.uni-muenchen.de.
To whom correspondence may be addressed. Tel.: 49 89 5996 471 or 49 89 5996 472; Fax: 49 89 5996 415; E-mail:
chaass@pbm.med.uni-muenchen.de.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M011116200
2 For reasons of simplicity, we use the terms BACE and BACE-2 in this study.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
A, amyloid
-peptide;
BFA, brefeldin A;
APP,
-amyloid precursor protein;
CK, casein kinase;
CT, cytoplasmic tail;
EEA1, early endosome antigen
1;
GST, glutathione S-transferase;
HD, hymenialdisine;
HEK, human embryonic kidney;
PVDF, polyvinylidene difluoride;
PAGE, polyacrylamide gel electrophoresis;
BACE,
-secretase;
endo H, endoglycosidase H;
PBS, phosphate-buffered saline;
ER, endoplasmic
reticulum;
TGN, trans-Golgi network;
WT, wild type;
PNGase F, peptide:N-glycanase F..
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