From the Genetics and Aging Unit, Department of
Neurology, Massachusetts General Hospital, Harvard Medical School,
Charlestown, Massachusetts 02129 and the § Department of
Psychiatry and Neurobiology, Mount Sinai School of Medicine,
New York, New York 10029
Received for publication, September 20, 2000, and in revised form, March 9, 2001
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ABSTRACT |
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Calsenilin is a
member of the recoverin family of neuronal calcium-binding proteins
that we have previously shown to interact with presenilin 1 (PS1) and
presenilin 2 (PS2) holoproteins. The expression of calsenilin can
regulate the levels of a proteolytic product of PS2 (Buxbaum,
J. D., Choi, E. K., Luo, Y., Lilliehook, C., Crowley, A. C., Merriam, D. E., and Wasco, W. (1998) Nat. Med. 4, 1177-1181) and reverse the presenilin-mediated enhancement of calcium
signaling (Leissring, M. A., Yamasaki, T. R., Wasco, W.,
Buxbaum, J. D., Parker, I., and LaFerla, F. M. (2000)
Proc. Natl. Acad. Sci. U. S. A. 97, 8590-8593). Here, we
have used cultured mammalian cells that transiently or stably express
calsenilin to extend the characterization of calsenilin and of the
calsenilin-PS2 interaction. We have found that calsenilin has the
ability to interact with endogenous 25-kDa C-terminal fragment (CTF)
that is a product of regulated endoproteolytic cleavage of PS2 and that
the presence of the N141I PS2 mutation does not significantly alter the
interaction of calsenilin with PS2. Interestingly, when the 25-kDa PS2
CTF and the 20-kDa PS2 CTF are both present, calsenilin preferentially
interacts with the 20-kDa CTF. Increases in the 20-kDa fragment are
associated with the presence of familial Alzheimer's disease-associated mutations (Kim, T., Pettingell, W. H., Jung, Y., Kovacs, D. M., and Tanzi, R. E. (1997)
Science 277, 373-376). However, the finding that the
production of the 20-kDa fragment is regulated by the phosphorylation
of PS2 (Walter, J., Schindzielorz, A., Grunberg, J., and Haass, C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1391-1396) suggests that it is a regulated physiological event that
also occurs in the absence of the familial Alzheimer's disease-associated mutations in PS2. Finally, we have demonstrated that
calsenilin is a substrate for caspase-3, and we have used site-directed
mutagenesis to map the caspase-3 cleavage site to a region that is
proximal to the calcium binding domain of calsenilin.
The pathogenesis of Alzheimer's disease
(AD)1 is defined by the
accumulation of intracellular neurofibrillary tangles and
extracellular deposits of amyloid plaques in the brain parenchyma and
cerebral blood vessels (5). Although AD occurs sporadically, inherited factors play an important role in at least half of the cases. Genetic
studies of familial Alzheimer's disease (FAD) have led to the
identification of three early onset FAD genes, which produce the
amyloid precursor protein (APP), and the presenilin 1 (PS1) and
presenilin 2 (PS2) proteins (6, 7).
To date, more than 60 mutations in PS1 and at least 2 mutations in PS2
have been genetically linked to early onset FAD (8-10). These
mutations result in the altered processing of APP and lead to increases
in the amyloid The normal function of the presenilins is not clear; however, roles in
membrane trafficking (21), APP processing (22), Notch signaling
(23-25), neuronal plasticity (26), cell adhesion (27), the regulation
of ER calcium homeostasis (26), the unfolded protein response (28), and
programmed cell death (29) have all been suggested. In an effort to
elucidate the functional role of the presenilins, a number of
presenilin-interacting proteins have been identified (8). It has been
reported that presenilins can interact with members of the armadillo
family ( Calsenilin is a novel neuronal calcium-binding protein that interacts
with the C terminus of the presenilins which was isolated using a yeast
two-hybrid screen (1). It is a member of the recoverin family of
neuronal calcium-binding proteins, and it is 25% identical to
recoverin, 29% identical to hippocalcin, and 33% identical to
neuronal calcium sensor-1. The levels of the 20-kDa PS2 CTF are
increased in the presence of calsenilin, suggesting that calsenilin
either preferentially stabilizes the levels of this fragment or
increases its formation (1). In the present study, we have extended the
characterization of calsenilin and the calsenilin-PS2 interaction and
have found that the presence of the N141I mutation in PS2 does not
appear to alter the interaction significantly. We have shown that
calsenilin has the ability to interact with the endogenous 25-kDa PS2
CTF. Interestingly, calsenilin interacts preferentially with the 20-kDa
PS2 CTF. As noted above, this smaller fragment is elevated as a result
of the FAD-associated mutations in the presenilins, and its production
is regulated by the phosphorylation state of PS2. Finally, we report
that calsenilin itself is a substrate for caspase-3, and we have used
site-directed mutagenesis to demonstrate that this cleavage takes place
at a caspase-3 consensus motif that is proximal to the calcium binding domain of calsenilin.
Cell Culture, Transfection, and Generation of Stable Cell
Lines--
COS-7 and H4 human neuroglioma cells were maintained in
Dulbecco's modified Eagle's medium containing 4.5 mg/ml
D-glucose supplemented with 10% fetal bovine serum and 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM
L-glutamine (Sigma) at 37 °C in a 5% CO2
atmosphere. H4 cells stably expressing calsenilin were generated by
transfection with a calsenilin-pcDNA3.1/Zeo+ (Invitrogen) construct
using SuperFect (Qiagen), followed by selection and maintenance in the
presence of 250 µg/ml Zeocin (Invitrogen). For expression of PS2,
wild type or N141I mutant PS2 cDNA constructs (3) were used.
Transient transfections were carried out with SuperFect according to
the manufacturer's directions.
Protein Extraction--
Cells were harvested in
phosphate-buffered saline (PBS) containing protease inhibitors and
centrifuged at 14,000 rpm at 4 °C for 10 min. The supernatant was
discarded, and the pellet was resuspended in modified RIPA buffer
containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl,
2 mM EDTA, 1% Triton X-100, 1% Nonidet P-40, 0.25%
sodium deoxycholate, and protease inhibitors. The lysed cells were
rocked at 4 °C for 1 h and centrifuged at 14,000 rpm at 4 °C
for 10 min to remove cell debris. Protein concentration was determined
with a BCA protein assay kit (Pierce). These lysates were used for
coimmunoprecipitation and Western blot analysis.
Coimmunoprecipitation and Western Blot Analysis--
Aliquots of
cell lysates were precleared with protein A conjugated with magnetic
beads (PerCeptive Diagnostics) for 2 h at 4 °C and
immunoprecipitated using polyclonal antibodies to PS2 (anti-338) or
calsenilin (anti-45). Rabbit-IgG (Pierce) was used as a negative
control. After overnight incubation at 4 °C, samples were incubated
with protein A-magnetic beads (30 µl/sample) for 2 h at 4 °C,
and the immunoprecipitates were subjected to immunoblotting with the
complementary antibody. For Western blot analysis, 50 µg of protein
from each cell lysate was separated on 4-20% gradient or 14%
Tris-glycine gel (Novex) under reducing conditions, and transferred to
polyvinylidene difluoride membrane (Bio-Rad) using a semidry
electrotransfer system (Hoefer). The membrane was blocked with 5%
non-fat dry milk in TBST (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.05% Tween 20) for 2 h at room temperature,
probed with the primary antibody (anti-45 at 1:3,000; anti-338 at
1:5,000; anti-PS2Loop (18) at 1:2000, or anti-G2L (14) at 1:1000) in TBST overnight at 4 °C, and incubated with the appropriate secondary antibody conjugated to horse-radish peroxidase. Bound antibodies were
visualized by chemiluminescent substrate as described by the
manufacturer (Kirkegaard & Perry Laboratories).
Induction of Apoptosis--
For the induction of apoptosis, H4
cells stably expressing calsenilin were seeded at 5 × 106 cells for each 150-mm dish. After 48 h, cells were
washed with PBS, and medium containing either 1 µM
staurosporine (Sigma) or increasing concentrations of MG132
(Calbiochem) was added. For the inhibition of apoptosis, cells were
pretreated with 100 µM zVAD-FMK (Enzyme System Products)
for 1 h before induction of apoptosis. Cells were harvested at the
indicated time points and lysed as described above. For the detection
of apoptosis, anti-PARP (1:1000, PharMingen or New England
BioLabs) and anti-caspase-3 (1:2,000, PharMingen) antibodies were used.
Subcellular Fractionation--
For biochemical fractionation,
COS-7 cells transiently transfected with calsenilin were harvested,
washed twice with ice-cold PBS, and resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 250 mM sucrose, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, 5 µg/ml leupeptin). When indicated, cells were also transiently transfected with wild type PS2. The extraction was carried out in the presence of 3 mM
CaCl2 or 3 mM EGTA when appropriate. After
resuspension, cells were lysed by sonicating three times for 30 s
at 4 °C. Nuclei and unbroken cells were removed by centrifugation at
1,000 × g for 5 min at 4 °C. Postnuclear
supernatant was then centrifuged at 100,000 × g for
1 h at 4 °C to separate the membrane pellet and cytosolic fraction, and complete lysis was confirmed by microscopy. The membrane
pellets were suspended in lysis buffer containing 0.5% Nonidet P-40 by
rocking for 1 h at 4 °C followed by centrifugation at 14,000 rpm for 10 min at 4 °C. The supernatant, containing solubilized
membrane proteins, was considered the membrane fraction. Equalized
proteins of soluble cytosol and membrane pellet fractions were analyzed
by Western blot analysis. For Nycodenz gradient fractionation, cells
were prepared as described (42) with some modification. Briefly, cells
were washed twice with ice-cold PBS and homogenized in H buffer (10 mM triethanolamine, 10 mM acetic acid, 250 mM sucrose, 1 mM EDTA, and 1 mM
dithiothreitol, and protease inhibitors as described above) using a
25-gauge needle and a tight pestle metal Dounce homogenizer. The
postnuclear supernatant was separated by density gradient
centrifugation using step gradients consisting of 24, 19.33, 14.66, and
10% isotonic Nycodenz solutions (0.75% NaCl, 10 mM Tris,
pH 7.4, 3 mM KCl, and 1 mM EDTA) and fractionated with a density gradient fractionator. 22 0.6-ml fractions were collected from each gradient and analyzed by Western blot analysis
using calsenilin, PS2, calnexin (1:2,000, StressGen), and 14-3-3 (1:2,000, StressGen) antibodies.
Immunofluorescence and Confocal Microscopy--
H4 cells stably
expressing calsenilin were cultured on four-chamber glass slides the
day before transfection. Cells were then transiently transfected with
vector, wild type, or N141I PS2 constructs. After 24 h, cells were
washed briefly with PBS and the fixed for 20 min with PBS containing
4% paraformaldehyde at room temperature. After fixation, cells were
washed three times, permeabilized with blocking solution (0.2% Triton
X-100 and 4% normal goat serum in PBS) for 1 h, and rinsed three
times with PBS. After overnight incubation with primary antibodies
against calsenilin (polyclonal Ab 45, 1:200), PS2 (polyclonal Ab 338, 1:200), PDI (monoclonal Ab, 1:200, StressGen), GM130 (monoclonal
Ab, 1:200, StressGen), or Site-directed Mutagenesis and in Vitro Cleavage by
Caspase-3--
Mutant calsenilin constructs were generated using the
QuickChangeTM site-directed mutagenesis kit (Stratagene)
with oligonucleotide primers designed to alter Asp61
or Asp64 to an alanine. Mutant constructs were cloned into
pcDNA3.1/Zeo+ and fully verified by sequencing. For in
vitro cleavage, 15-25 µg of protein from total lysates was
incubated for 4 h at 37 °C in 25 µl of caspase assay buffer
(20 mM HEPES, 100 mM NaCl, 10 mM
dithiothreitol, 10 mM MgCl2, 1 mM
EDTA, 0.1% CHAPS, 10% sucrose, pH 7.4) in the presence or absence of
active recombinant human caspase-3 (PharMingen). Reactions were
terminated by the addition of SDS-containing sample buffer and
analyzed by Western blot.
Detection of Endogenous Calsenilin and Interaction of Calsenilin
with Endogenous PS2 CTF in H4 Human Neuroglioma Cells--
We have
reported previously that calsenilin interacts with full-length PS1 and
PS2 (1). To characterize the calsenilin-PS2 interaction further, we
generated a polyclonal antibody (anti-45) that recognizes calsenilin
when it is expressed in transfected cells (Fig.
1B, lower panel, lane
5) and endogenous calsenilin in mouse and human
brain.2 Although this
antibody was able to immunoprecipitate endogenous calsenilin from
naive H4 cell lysates (see arrow in Fig.
1A) it was necessary to use at least 1 mg of total extract
to detect immunoprecipitated endogenous calsenilin. In addition,
endogenous calsenilin could not be detected by Western blot analysis of
50 µg of protein from total lysates (Fig. 1A, lane
6). Taken together, these observations indicate that endogenous
levels of calsenilin levels are relatively low in these cells.
Accordingly, although we have successfully detected an endogenous
presenilin-calsenilin complex in brain extract,2 it was
difficult to detect this complex in cultured cells. To overcome
this detection problem, we generated an H4 human neuroglioma cell line
that stably expresses calsenilin, and we used these cells to assess the
ability of exogenous calsenilin to interact with endogenous PS2. As can
be seen in Fig. 1B, as increasing amounts of endogenous
25-kDa PS2 CTF were immunoprecipitated from increasing amounts of
starting material, increasing amounts of calsenilin were
coimmunoprecipitated. No calsenilin was detected when normal rabbit IgG
was used as a control antibody for immunoprecipitation. This
finding is significant because it demonstrates that calsenilin has the
ability to interact with physiological levels of the 25-kDa PS2 CTF.
Because the levels of the 20-kDa PS2 CTF are below the levels of
detection in naive cells, we did not observe the coimmunoprecipitation of this fragment. As can be seen in Fig.
2, the 20-kDa fragment can only be
detected after overexpression of PS2. Also, because the levels of
endogenous full-length PS2 are below the levels of detection, we did
not observe the coimmunoprecipitation of full-length PS2 which we have
observed previously after transfection with PS2 (1).
Calsenilin Preferentially Interacts with the 20-kDa PS2
CTF--
To determine if the N141I FAD-associated mutation in PS2
alters the interaction with calsenilin, wild type or N141I mutant PS2
constructs were transfected with or without calsenilin into H4 cells.
Cell lysates were prepared and analyzed by Western blot with antibodies
to PS2 or calsenilin. The PS2 antibody used for these experiments
(anti-338) was raised against a peptide located in the C-terminal
domain of PS2 (amino acids 339-350), and as shown in lanes
5 and 6 of Fig. 2A, after transfection of
PS2 it successfully and specifically detects PS2 holoprotein as well as
both of the PS2 CTFs. The results of coimmunoprecipitation analysis
demonstrate that the presence of the N141I mutation in PS2 did not
appear to alter significantly the ability of PS2 to coimmunoprecipitate
with calsenilin (Fig. 2B, top panel, lanes 5 versus 6). However as seen in Fig. 2A, although the
levels of the 25-kDa PS2 CTF in cells transfected with calsenilin alone (lane 2) appear to be equal to or greater than those of the
20-kDa CTF present in cells transfected with calsenilin and PS2
(lanes 5 and 6), only the 20-kDa CTF is
coimmunoprecipitated with calsenilin. This observation suggests that
although calsenilin has the ability to coimmunoprecipitate
with the 25-kDa CTF, when both the 25-kDa and the 20-kDa CTFs are
present, it preferentially coimmunoprecipitates with the 20-kDa PS2. A
preferential interaction with the 20-kDa CTF was also observed in COS-7
cells transiently transfected with calsenilin as well as in H4 and
COS-7 cells that stably express calsenilin (data not shown). This
preferential interaction was not dependent on the antibody used because
we obtained similar results using anti-PS2Loop (18) or anti-G2L PS2
(14) antibodies (data not shown). Treatment with zVAD-FMK, a broad
spectrum caspase inhibitor that inhibits the caspase-mediated cleavage
of PS2 (3), abrogates the ability to detect an interaction between
calsenilin and the 20-kDa PS2 CTF, confirming the caspase-derived
origin of the fragment (data not shown).
To confirm further the origin of the CTF that preferentially interacts
with calsenilin, apoptosis was induced by treating cells with
staurosporine, a protein kinase inhibitor that has been shown to cause
caspase activation (43, 44), and the resulting lysates were
immunoprecipitated with a PS2 antibody. As can be seen in Fig.
3A, treatment with
staurosporine resulted in the induction of apoptosis as evidenced by
the cleavage of PARP and procaspase-3. We also noted the associated
appearance of an ~28-kDa calsenilin band (arrow in Fig.
3A), which will be discussed in detail below. During
staurosporine-induced cell death, the amount of the 20-kDa PS2 CTF
progressively increased, while the amount of the 25-kDa PS2 CTF, which
can serve as a caspase substrate, decreased (Fig. 3, A and
B, top panel). Notably, increased levels of
coimmunoprecipitated calsenilin corresponded with increased presence of
the 20-kDa CTF (Fig. 3B, bottom panel). Taken
together, these data strongly suggest that when both fragments are
present, calsenilin interacts preferentially with the 20-kDa PS2 CTF,
as opposed to the 25-kDa PS2 CTF generated by regulated proteolytic cleavage.
Subcellular Localization of Calsenilin--
It is possible that
the preferential interaction between calsenilin and the 20-kDa PS2 CTF
is a result of differential subcellular localization of the 25-kDa PS2
CTF and the 20-kDa PS2 CTF. To evaluate this possibility, we
characterized the subcellular localization of calsenilin, relative to
each of the two PS2 CTFs. First, biochemical fractionation of cells
that were transiently transfected with calsenilin was carried out by
differential centrifugation. As shown in Fig.
4A, when calsenilin was
expressed by itself, it was primarily found in the cytosol. However,
when it was coexpressed with PS2, a membrane protein, there was a
significant shift in the subcellular localization of calsenilin from
the cytosol to the membrane. This observation suggests that the
association of calsenilin with the membrane occurred via an interaction
with PS2. To investigate the significance of calcium in the interaction of calsenilin with the membrane, fractions were prepared in the presence of excess calcium or EGTA. As can be seen in Fig.
4B, calcium did not appear to have a significant effect on
the interaction of calsenilin with the membrane, suggesting that the
binding to the membrane, presumably by an interaction with the
presenilins, is a calcium-independent process. To assess directly
whether calcium regulates the interaction of calsenilin with PS2, we
also carried out coimmunoprecipitation in the presence or absence of
calcium, and we found that the interaction of calsenilin and PS2 is
independent of calcium (data not shown).
To determine whether membrane-associated calsenilin and the 20-kDa PS2
CTF are localized to the same subcellular compartment(s) as the 25-kDa
PS2 CTF, total membrane fractions from H4 cells stably expressing
calsenilin in the presence or absence of transiently expressed wild
type PS2 were separated using Nycodenz discontinuous density gradient
centrifugation. As can be seen in Fig. 5,
the majority of calsenilin was found in fractions 2-7, which is where the Golgi marker 14-3-3 localized, and lesser levels were observed in
fractions 15-20, which is where the ER protein calnexin localized. The
finding that both of the PS2 CTFs were detected in the ER fractions
suggests 1) that the interaction between either of the PS2 CTFs and
calsenilin is likely to take place in this compartment and 2) that the
preferential interaction of calsenilin and the 20-kDa PS2 CTF is
unlikely to be due to differential subcellular localization of the two
fragments. The results presented in Fig. 5 also indicate that in the
Golgi, calsenilin interacts with PS2 holoprotein because this is the
only species of PS2 detected in the Golgi fractions. To confirm the
fractionation results, we carried out immunofluorescence analysis of
calsenilin in stable cells expressing calsenilin, which were
transfected with either vector or wild type PS2. PDI and
A final possible explanation for the preferential interaction is that
the interaction of calsenilin with the 20-kDa PS2 CTF stabilizes the
fragment and that this increased stability results in an apparent
preferential interaction. To address this possibility we carried out
pulse-chase analysis in the presence or absence of transfected
calsenilin and found that the expression of calsenilin did not result
in significant differences in the stability of the PS2 CTFs (data not
shown). This observation suggests that the preferential interaction of
calsenilin with the 20-kDa CTF is not caused by a change in the
stability of the fragment.
Calsenilin Is a Substrate for Caspase-3-mediated Cleavage--
The
polyclonal calsenilin antibody that we have generated detects a series
of multiple bands when they are used for Western blot analysis (Figs.
1B and 3). These multiple bands are only observed after
transfection with calsenilin, and not with vector, which indicates that
they are all forms of calsenilin. To determine whether the multiple
bands are a result of calsenilin being targeted to the
ubiquitin-proteasome pathway, cells were treated with increasing concentrations of the proteasome inhibitor MG132. Because proteasome inhibitors can also induce apoptosis (45, 46), these experiments were
carried out in the presence or absence of the caspase inhibitor zVAD-FMK. Although a series of higher molecular weight bands were detected during MG132 treatment, it is unlikely that calsenilin is
degraded in the ubiquitin-proteasome pathway because these bands were
not changed. Interestingly, a ~28-kDa calsenilin band was detected
during MG132-induced cell death, and the generation of this band was
abolished by zVAD-FMK treatment (Fig.
7A). This observation, along
with the fact that this band appears after staurosporine treatment
(Fig. 3A), indicates that it is generated during apoptosis.
Similar results were observed after treating with ALLN, which is also a
proteasome inhibitor (data not shown). At least two other bands, both
of which migrate between the largest band and the ~28-kDa band, may
be generated by endoproteolysis or post-translational modification of
calsenilin. These bands were detected routinely and were not affected
by treatment with two different apoptotic agents (Figs. 3 and 7).
Although the exact nature of these bands remains unclear, the fact that
they are specifically detected in cells transfected with calsenilin
indicates that they are calsenilin derivatives.
Caspase-3 is partially or totally responsible for the proteolysis of
many proteins during the execution phase of apoptosis. To determine
whether caspase-3 is the enzyme responsible for the generation of the
~28-kDa fragment of calsenilin, total lysates from cells stably
expressing calsenilin were incubated with increasing amounts of
purified, active recombinant caspase-3. As can be seen in Fig.
7B, this treatment resulted in the generation of an
~28-kDa calsenilin cleavage fragment, and the increase in the
fragment was dependent on the amount of enzyme added. These data
suggest that calsenilin is a substrate for caspase-3. Because
calsenilin also has several caspase-6 or caspase-8 consensus motifs, we
tested for the cleavage of calsenilin by recombinant caspase-6 or
caspase-8; however, no cleavage products were observed (data not shown).
The calsenilin amino acid sequence contains a caspase-3 consensus motif
(DXXD) at Asp61-Asp64, which is
located at the junction of the N-terminal domain that is specific for
calsenilin and the calcium binding domain that is common to all members
of the recoverin superfamily (Fig.
8A). Cleavage at this site
would result in the production of a small (~60 amino acids)
N-terminal fragment, and a larger (~190 amino acids) C-terminal
fragment that would be predicted to migrate at ~28 kDa. To determine
if calsenilin cleavage by caspase-3 takes place at the identified
consensus motif, site-directed mutagenesis was used to change the
candidate aspartate residues to alanine residues. The resulting D61A
and D64A mutant constructs were transfected into H4 cells, and an
in vitro caspase cleavage assay was carried out on the cell
lysates. As shown in Fig. 8B, both substitutions blocked
cleavage by caspase-3. This finding indicates that the caspase-3
cleavage of calsenilin takes place at the predicted site.
Interestingly, cleavage at this site serves to separate the calcium
binding domain from the N-terminal domain of the molecule. We also
confirmed this cleavage during staurosporine-induced apoptosis in
cultured cells, and as can be seen in Fig. 8C, cleavage is abolished by the alanine substitution in the caspase-consensus motif in
this assay as well as in the in vitro caspase assay.
In a previous report, we described the initial isolation and
characterization of calsenilin, a novel calcium-binding protein that
interacts with the C terminus of the presenilins (1). More recently we
demonstrated that calsenilin can reverse the potentially pathogenic
effects that mutant presenilins have on calcium signals evoked by
inositol 1,4,5-triphosphate enhancement (2). Calsenilin is a member of
the recoverin family of calcium-binding proteins (47). This family also
includes hippocalcin (48), neuronal calcium sensor-1 (49), neurocalcin
(50), frequenin (51), visinin (52), visinin-like protein (53),
S-modulin (54), and guanylyl cyclase-activating protein (55). Although the cellular function of the majority of proteins in this family remains unknown, it is clear that recoverin acts to inhibit rhodopsin kinase and that the guanylyl cyclase-activating proteins regulate photoreceptor guanylyl cyclases (56). In addition, frequenin has been
shown to be a positive regulator of neurotransmitter release in
Xenopus and Drosophila, whereas neuronal calcium
sensor-1, the mammalian homolog of frequenin, regulates neurosecretion
from dense core granules (57). Calsenilin appears to be a unique member
of the family because it has a novel N-terminal domain that is absent
in the other proteins. Since our initial report, calsenilin has also
been identified as a transcriptional repressor that was termed
downstream-regulatory-element antagonist modulator (DREAM) (58).
Although these investigators found DREAM to be localized to the nuclear
fraction of mammalian cells, we have not observed significant
association of calsenilin with this fraction. Interestingly, a recent
study identifying calsenilin as a potassium channel Perhaps the most essential physiological roles performed by
calcium-binding proteins are to act as calcium sensors that transduce calcium signals via specific interactions with intracellular target protein (60). Interestingly, the biochemical studies presented here
indicate that the association of calsenilin with the presenilins is
independent of calcium. Likewise, the interaction of calsenilin with
the potassium channel and the association of neuronal calcium sensor-1
with membranes are also calcium-independent (49, 59). Calcium does
appear to be required for the modulatory effects of calsenilin on the
activity of the potassium channel (59). Whether calcium is similarly
required for modulation of presenilin function by calsenilin awaits
conclusive elucidation of presenilin function. Although the functional
significance of calcium binding to calsenilin remains unclear, we have
reported recently that the enhancement of calcium signaling by PS1
mutation is reversed by calsenilin coexpression (2).
In our original study, we demonstrated that calsenilin interacted with
the PS1 and PS2 holoproteins, but we did not address the ability of
calsenilin to interact with the presenilin CTFs. Importantly, in this
study, we have demonstrated that calsenilin has the ability to interact
with endogenous levels of the 25-kDa PS2 CTF. Given reports that in the
cell the majority of presenilins appear to exist as N- and C-terminal
fragments, and the resulting speculation that these fragments are
likely to be the functional forms of the protein, this is an important
finding. In naive cells, the 20-kDa PS2 CTF is not present at
detectable levels, therefore we used transfected cells and/or cells
that were treated with pro-apoptotic reagents to assess the ability of
calsenilin to interact with the 20-kDa PS2 CTF. Interestingly, we have
found that when both the 25-kDa PS2 CTF and the 20-kDa PS2 CTF are
present, calsenilin interacts preferentially with the 20-kDa PS2 CTF,
even when the larger fragment appears to be present at equal or higher levels. This behavior is quite different from that of other interacting proteins, including Differential subcellular localization of the two PS2 CTFs in relation
to calsenilin does not appear to account for the preferential interaction with the smaller fragment, nor does calsenilin appear to
alter the stability of either of the PS2 CTFs significantly. There are
several other possible explanations for the preferential interaction.
Calsenilin may genuinely prefer to bind the 20-kDa PS2 CTF after it is
generated, or it may bind to full-length PS2 and induce caspase
activation to produce increased amounts of the caspase fragment, which
may then compete with PS2 holoprotein for binding with calsenilin. It
is also possible that in vivo the interaction between
calsenilin and the 20-kDa PS2 CTF may be downstream of caspase cleavage
and be independent of an interaction with full-length PS2. Another
possible explanation for the preferential interaction of calsenilin
with the 20-kDa CTF is that the interaction may be regulated by
post-translational modification of PS2. The phosphorylation of PS1
regulates the physiological and/or pathological properties of its
fragments (61, 62), and the phosphorylation of PS2 can regulate its
cleavage by caspase (4). Likewise, the interaction with calsenilin
might be regulated by the phosphorylation status of PS2. We tested this
possibility using stable cells expressing wild type PS2 CTF or PS2 CTF
that contain a mutated phosphorylation site (kindly provided by J. Walter and C. Haass) and found that the phosphorylation status of the
PS2 CTF did not appear to affect the interaction with calsenilin (data
not shown).
A final explanation for the preferential interaction of calsenilin and
the 20-kDa PS2 CTF is that other interacting proteins, which bind to
the 25-kDa PS2 CTF, sterically inhibit the ability of calsenilin to
interact with the fragment. As mentioned above, Although the molecular mechanism for the preferential interaction
between calsenilin and the 20-kDa PS2 CTF remains unclear, this
observation may still provide insight into potential effects of
calsenilin on the generation of A Finally, we have found that calsenilin itself is a substrate for
caspase cleavage, and we have used site-directed mutagenesis to
demonstrate that this cleavage occurs at a caspase-3 DXXD
consensus cleavage sequence, 61DSSD64. Notably,
the cleavage of calsenilin by caspase-3 occurs at a site that separates
the calcium binding domain that is conserved in all members of the
recoverin superfamily from the novel N-terminal domain that is specific
for calsenilin and one of its recently discovered homologs. Although
the function of this N-terminal domain remains unknown, it is tempting
to speculate that is involved in the interaction of calsenilin with
target proteins such as the presenilins. Caspase cleavage may serve to
separate the interacting domain from the calcium responsive domain of
the molecule, severing either calcium regulation of the target protein
or an interaction with a second molecule that is regulated by the
calcium binding domain.
Taken together, the data presented here demonstrate 1) that when both
of the PS2 CTFs are present, calsenilin interacts preferentially with
FAD-associated 20-kDa PS2 CTF; 2) that the PS2 N141I mutation does not
alter the interaction significantly; and 3) that cleavage of calsenilin
by caspase-3 separates the novel N-terminal domain from the calcium
binding domain. Although we have explored several possibilities to
explain the preferential interaction between calsenilin and the 20-kDa
PS2 CTF, the molecular mechanism underlying the preferred interaction
remains unclear. The cleavage of calsenilin by caspase-3 is consistent
with a role in programmed cell death, which in turn is consistent with
the manner in which the protein was isolated (1). Further investigation
of the physiological and/or pathological roles of calsenilin may help
explain the functional roles of presenilins in both the normal and
diseased brain.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-peptide (A
), which is derived from APP and is the
main component of amyloid plaques (11-14). The presenilins are
multitransmembrane domain proteins whose primary subcellular location
appears to be the membranes of the ER and Golgi. Proteolytic processing
of both proteins, which results in a 35-kDa N-terminal fragment for
both proteins, a 20-kDa CTF for PS1, and a 25-kDa CTF for PS2, has been
reported for the presenilins in mouse and human brain as well as
cultured cells (15-17). In vivo, the majority of detectable
presenilin appears in the form of the N- and C-terminal fragments that
are tightly regulated at steady-state levels and form a stable complex
after endoproteolytic processing (18). The presenilins have also been
shown to be substrates for cleavage by caspase-3-like proteases (3, 19,
20) at a site distal to the regulated cleavage site. This cleavage
results in the production of a smaller CTF. Notably, the presence of
FAD mutations in the presenilins is associated with increased levels of
these caspase-derived fragments, which are normally present at low
levels and can only be detected after presenilin transfection.
Recently, Walter et al. (4) reported that the
phosphorylation of PS2 at a site within the C-terminal domain inhibits
cleavage by caspase-3. This finding demonstrates that the
phosphorylation state of PS2 controls its cleavage by caspase and
suggests that cleavage of PS2 is a regulated biological event that
occurs physiologically in the absence of FAD mutations.
-catenin, NPRAP, and p0071) (30-32), GSK-3
(32),
filamins (actin-binding protein 280, ABP280/filamin homolog 1, Fh 1)
(33), µ-calpain (34), calmyrin (35), APP (36), Go (37),
Bcl-2 family proteins (29, 38), QM/Jif1 (39), rab11 (40), sorcin (41),
and calsenilin (1). Most interactors, including
-catenin, calmyrin, Bcl-XL, rab11, sorcin, and GSK-3
, bind to the large
hydrophilic loop domain of the presenilins, whereas calsenilin and
Go interact with the C-terminal domain. The interaction of
the armadillo proteins, GSK-3
and Go appears to be
specific for PS1, whereas µ-calpain, calmyrin, and sorcin interact
only with PS2. In contrast, only calsenilin, filamins,
Bcl-XL, and APP have the ability to interact with both PS1
and PS2. The characterization of interactors that are specific for
either PS1 or PS2 may aid in the elucidation of distinct biological
roles for the two proteins, whereas the characterization of proteins
that interact with both presenilins (such as calsenilin) may provide
information about common functions in both the normal and diseased brain.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-COP (monoclonal Ab, 1:200,
StressGen) and three washes in PBS, secondary antibodies were applied
(1:200 in PBS). Bodipy FL anti-mouse IgG (Molecular Probes) and Cy3
anti-rabbit IgG (Jackson ImmunoResearch) were used as secondary
antibodies. Control reactions omitting the primary antibodies resulted
in no labeling with the secondary antibodies (data not shown). Sections
were examined using conventional immunofluorescence microscopy and a
Bio-Rad 1024 laser confocal microscope (Hercules).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Detection of endogenous calsenilin and
interaction of calsenilin with endogenous PS2 in stable H4 human
neuroglioma cells expressing calsenilin. A,
detection of endogenous calsenilin. Naive H4 human neuroglioma cells
were lysed in RIPA buffer and used for immunoprecipitation
(IP) as described under "Experimental Procedures."
Increasing amounts of protein were immunoprecipitated with anti-45
calsenilin antibody as indicated, and samples were analyzed by Western
blot using anti-45 calsenilin antibody. Note that although increasing
amounts of endogenous calsenilin (arrow) were
immunoprecipitated with increasing amounts of total extract protein,
Western blot analysis of 50 µg of protein from total lysates was not
able to detect endogenous calsenilin (lane 6). B,
interaction of calsenilin with endogenous PS2. Increasing
amounts of total extract protein prepared from stable H4 cell lines
expressing calsenilin were immunoprecipitated with anti-338 PS2
antibody. Immunoprecipitates were then analyzed by Western blot using
anti-45 calsenilin or anti-338 PS2 antibodies. Each experiment was
repeated at least three times, and similar results were obtained.
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Fig. 2.
Interaction of calsenilin with the 20-kDa PS2
CTF. A, detection of calsenilin and PS2 in
transiently transfected H4 cells. 50 µg of total extract protein
prepared from H4 cells that were transiently transfected with the
indicated constructs (vector, calsenilin, wild type PS2, or mutant
N141I PS2) was prepared as described under "Experimental
Procedures" and analyzed by Western blot using polyclonal anti-338
PS2 (top) or anti-45 calsenilin (bottom)
antibodies. In this cell line, under the conditions used for this
experiment, the levels of the 20-kDa fragment were not influenced by
the presence of the N141I mutation or of calsenilin, as they were in
the conditions used for Fig. 4 and in our previous report (1).
B, coimmunoprecipitation of calsenilin with the 20-kDa PS2
CTF. Total lysates prepared from H4 cells transfected with the
indicated constructs were immunoprecipitated (IP) with
anti-45 calsenilin antibody as described under "Experimental
Procedures" and then analyzed by Western blot with either anti-338
PS2 (top) or anti-45 calsenilin antibodies
(bottom). Note that calsenilin interacts preferentially with
the 20-kDa PS2 CTF (lanes 5 and 6) compared with
the 25-kDa PS2 CTF (lane 2). FL, full-length PS2;
Wt, wild type. These results are representative of at lease
five separate experiments.
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Fig. 3.
Calsenilin interacts preferentially with the
20-kDa PS2 CTF. A, detection of calsenilin, PS2,
and apoptosis markers after staurosporine (STS) treatment.
Cells stably expressing calsenilin were treated with 1 µM
staurosporine for the indicated intervals, and cells from each time
point were harvested, lysed, and subjected to Western blot analysis
with anti-338 PS2, anti-45 calsenilin, anti-PARP, or anti-caspase-3
antibodies as described under "Experimental Procedures." Note that
calsenilin is cleaved, and a ~28-kDa fragment is generated during
staurosporine-induced apoptosis (arrow). Anti-PARP or
anti-caspase-3 antibodies were used as markers of apoptosis.
B, preferential interaction of calsenilin with the 20-kDa
PS2 CTF. Cells from each time point described above were harvested and
lysed, and immunoprecipitation (IP) with anti-338 PS2
antibody followed by Western blot analysis with either anti-338 PS2
(top panel) or anti-45 calsenilin antibodies (bottom
panel) was carried out. Note that during the progression of
apoptosis calsenilin coimmunoprecipitated with the 20-kDa PS2 CTF but
not with the 25-kDa CTF. These results are representative of at least
three separate experiments.
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Fig. 4.
Calcium does not regulate the association of
calsenilin with the membrane. A, redistribution
of membrane-associated calsenilin in the presence of PS2. After
transfection with vector, calsenilin, wild type PS2, or calsenilin and
wild type PS2, COS-7 cells were harvested and lysed as described under
"Experimental Procedures." Soluble cytosol (C) or
membrane (M) fractions were prepared and subjected to
SDS-polyacrylamide gel electrophoresis followed by Western blot
analysis with anti-calsenilin, anti-PS2, or calnexin antibodies. Note
that there is a significant shift in the subcellular localization of
calsenilin from the cytosol to the membrane fraction in the presence of
PS2. FL, full-length PS2; Wt, wild type.
B, effect of calcium on the membrane association of
calsenilin. COS-7 cells transiently expressing calsenilin were
harvested and lysed in the presence of either 3 mM
CaCl2 or 3 mM EGTA. Soluble cytosol and
membrane fractions were prepared as described under "Experimental
Procedures" and analyzed by immunoblot with anti-45 calsenilin
antibody. These results are representative of at least three separate
experiments.
-COP/GM130
were used as ER or Golgi markers, respectively. In agreement with our
previous observations in transiently transfected cells (1) and the
above data, the immunofluorescence analysis clearly showed that in the
presence of PS2, calsenilin was associated with both the ER and the
Golgi compartments (Fig. 6).
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Fig. 5.
Subcellular localization of calsenilin.
H4 cells stably expressing calsenilin were transfected with vector
(( ) PS2) or wild type PS2 ((+) PS2)). 24 h
after transfection, membrane fractions were prepared and separated by
Nycodenz density gradient centrifugation as described under
"Experimental Procedures." Each fraction was analyzed by Western
blot using anti-338 PS2 (
PS2), anti-45
calsenilin (
Calsenilin), anti-calnexin
(
Calnexin), or anti-14-3-3
(
14-3-3) antibodies. FL,
full-length PS2. Each experiment was repeated at least three times, and
similar results were obtained.
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Fig. 6.
Immunofluorescence localization of
calsenilin. H4 cells stably expressing calsenilin were cultured on
four-chamber glass slides and were then transiently transfected with
wild type PS2. Cells were incubated with antibodies to calsenilin and
PDI (A-C), calsenilin and -COP (D-F), or
calsenilin and GM130 (G-I) and were examined by confocal
laser scanning microscopy. Calsenilin was visualized by incubation with
primary antibody (anti-45) followed by cyanine Cy3-conjugated secondary
antibody (red fluorescence, A, D, and
G). PDI,
-COP, and GM130 were visualized by incubation
with primary antibody followed by Bodipy-conjugated secondary antibody
(green fluorescence, B, E, and
H, respectively). PDI and
-COP/GM130 were used as markers
for ER and Golgi fractions, respectively. Overlays (C,
F, and I) represent digitally merged images, and
yellow fluorescence indicates colocalization of calsenilin
and marker proteins. Note that calsenilin and PS2 are localized to the
ER and Golgi. These results are representative of at least three
independent experiments.
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Fig. 7.
Calsenilin is cleaved by
caspase-3. A, concentration-dependent
cleavage of calsenilin after MG132-induced apoptosis. Cells were
pretreated with vehicle (dimethyl sulfoxide) or 100 µM
zVAD-FMK for 1 h, followed by treatment with increasing
concentrations of MG132 for 24 h. 50 µg of total extract protein
was analyzed by Western blot using anti-45 calsenilin antibody as
described under "Experimental Procedures." Note that calsenilin is
cleaved during MG132-induced apoptosis, that cleavage is abolished by
zVAD-FMK treatment, and that it did not appear to undergo proteasomal
degradation. B, in vitro cleavage of
calsenilin by active recombinant caspase-3. Total lysates prepared from
cells stably expressing calsenilin were incubated in the presence or
absence of the indicated amount of active recombinant caspase-3 as
described under "Experimental Procedures." Proteins were analyzed
by Western blot with anti-45 calsenilin antibody. The major cleavage
product is indicated by an arrow. Each experiment was
repeated at least three times, and similar results were obtained.
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Fig. 8.
Identification of the caspase-3 cleavage site
of calsenilin using site-directed mutagenesis. A,
calsenilin contains a caspase-3 consensus motif. The calsenilin amino
acid sequence contains a caspase-3 consensus motif (DXXD)
beginning at aspartic acid 61. Note that caspase cleavage at this site
would result in the separation of the calcium binding domain from the
novel N-terminal domain of the molecule. B, site-directed
mutagenesis of the caspase-3 consensus site abolishes cleavage of
calsenilin in vitro. Wild type (WT) calsenilin
and two mutant constructs created by site-directed mutagenesis (D61A
and D64A) as described under "Experimental Procedures" were
transfected into H4 cells, and total lysates were used for an in
vitro caspase cleavage assay as described for Fig. 7. Proteins
were analyzed by Western blot with anti-45 calsenilin antibody
(upper panel). Cleaved PARP was used as a control for
caspase activation (lower panel). The caspase-3-mediated
cleavage product of calsenilin is indicated by an arrow.
C, site-directed mutagenesis of the caspase-3 consensus site
abolishes cleavage of calsenilin during staurosporine
(STS)-induced apoptosis. H4 cells were transfected with the
indicated constructs, and the cells were treated with 1 µM staurosporine for 8 h. Cells from each time
points were harvested, lysed, and subjected to Western blot analysis
with anti-45 calsenilin antibody. The caspase-derived calsenilin
fragment is indicated by an arrow. Each experiment was
repeated at least three times, and the results presented here are
representative of these experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit
interacting protein (59) also reports the identification of two novel
proteins that are highly homologous to calsenilin, one that contains
the novel N-terminal domain and one that does not. Notably, because the
majority of the data that we have presented here has been generated
from cells transfected with calsenilin constructs, it is clear that we
have assessed calsenilin itself and not either of the other two members
of the family.
-catenin (30) and sorcin (41), which interact
with only the larger PS1 or PS2 CTF, respectively. Although the
biological relevance of this preferential interaction remains unclear,
it is interesting in light of findings that indicate that the presence
of the FAD mutations in the presenilins results in increases in the
levels of this fragment and that the levels of the fragment are
regulated by the phosphorylation status of PS2. One predicted
consequence of increased levels of the 20-kDa CTF would that be that
along with an increase in the amount of calsenilin associated with this
fragment, there would be an accompanying decrease in the amount of
calsenilin available for interactions with the 25-kDa PS2 CTF or other
interacting proteins, such as the potassium channel.
-catenin interacts
only with the larger PS1 CTF (30), and sorcin binds only to the 25-kDa
PS2 CTF (41). These proteins would be potential candidates for
sterically inhibiting calsenilin binding to the larger presenilin
CTF.
. Recent data demonstrate that
while expression of full-length presenilin constructs that contain FAD
mutations is associated with an increase in the production of A
,
expression of constructs that contain an FAD mutation but lack the
C-terminal domain of the protein do not alter A
production (14).
Moreover, the C-terminal domain but not the large hydrophilic loop
(which is where the majority of presenilin interactors bind) is
critical for the processing pathway of holoprotein (63, 64). Therefore,
because calsenilin interacts with the C-terminal domain of the
presenilins and because calsenilin interacts preferentially with the
20-kDa PS2 CTF, whose generation is linked to the presenilin mutations,
calsenilin may also be involved in presenilin mutation-associated increases in A
.
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ACKNOWLEDGEMENTS |
---|
We thank T. Tekirian, T.-W. Kim, G. Tesco, R. Tanzi, and S. Guénette for helpful discussions. Antibodies were kindly provided by T. Iwatsubo (anti-G2L) and G. Thinakaran (anti-PS2Loop).
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FOOTNOTES |
---|
* This work was supported by a grant from the Korea Research Foundation (to E.-K. C.), National Institutes of Health Grants NS35975 and AG16361 (to W. W.) and AG15801 and AG05138 (to J. D. B.), and by Alzheimer Association grants (to W. W. and J. D. B.).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.
¶ Pew Biomedical Scholar. To whom correspondence should be addressed: Genetics and Aging Unit, Dept. of Neurology, Massachusetts General Hospital, 114 16th St., Charlestown, MA 02129. Tel.: 617-726-8307; Fax: 617-724-1823; E-mail: wasco@helix.mgh.harvard.edu.
Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.M008597200
2 N. F. Zaidi, O. Berezovska E.-K. Choi, H. Chan, C. Lillihook, B. Hyman, J. Buxbaum, and W. Wasco, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
AD, Alzheimer's
disease;
FAD, familial Alzheimer's disease;
APP, amyloid -protein
precursor;
PS, presenilin;
A
, amyloid
-peptide;
ER, endoplasmic
reticulum;
CTF, C-terminal fragment;
PBS, phosphate-buffered saline;
Ab, antibody;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PDI, protein disulfide isomerase;
COP, coatomer protein;
PARP, poly(ADP-ribose) polymerase.
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