Calsenilin Is a Substrate for Caspase-3 That Preferentially Interacts with the Familial Alzheimer's Disease-associated C-terminal Fragment of Presenilin 2*

Eun-Kyoung ChoiDagger , Nikhat F. ZaidiDagger , Janice S. MillerDagger , Annette C. CrowleyDagger , David E. MerriamDagger , Christina Lilliehook§, Joseph D. Buxbaum§, and Wilma WascoDagger

From the Dagger  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


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -peptide (Abeta ), 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.

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 (beta -catenin, NPRAP, and p0071) (30-32), GSK-3beta (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 beta -catenin, calmyrin, Bcl-XL, rab11, sorcin, and GSK-3beta , 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-3beta 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.

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.

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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 beta -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).

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.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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).


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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.

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.


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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.

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).


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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.

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 beta -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 (alpha PS2), anti-45 calsenilin (alpha Calsenilin), anti-calnexin (alpha Calnexin), or anti-14-3-3 (alpha 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 beta -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, beta -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 beta -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.

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.


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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.

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.


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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

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 alpha -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.

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 beta -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.

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, beta -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.

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 Abeta . Recent data demonstrate that while expression of full-length presenilin constructs that contain FAD mutations is associated with an increase in the production of Abeta , expression of constructs that contain an FAD mutation but lack the C-terminal domain of the protein do not alter Abeta 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 Abeta .

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.

    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).

    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.

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; FAD, familial Alzheimer's disease; APP, amyloid beta -protein precursor; PS, presenilin; Abeta , amyloid beta -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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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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