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INTRODUCTION |
It is now accepted that Ca2+ is one of the most
versatile second messengers relaying information within cells to
regulate their activities such as muscle contraction, secretory events,
cell cycle, differentiation, gene expression, and apoptosis.
Ca2+ plays its pivotal role through specific classes of
Ca2+-binding proteins, most of which possess
Ca2+-binding motifs such as endonexin folds, C2 regions, or
EF-hands. Many EF-hand type Ca2+-binding proteins have been
identified, and they have been classified into dozens of families based
on amino acid sequence similarities and number of EF-hand motifs in
their molecules (1).
Recently, we classified a new family of proteins possessing domains
with five EF-hand-like motifs, and we proposed the name "penta-EF-hand (PEF)"1 as
a collective name for these domains (2). The PEF domain was originally
found in the Ca2+-binding domain of the small subunit of
calpain, an intracellular Ca2+-dependent
cysteine protease, by x-ray crystallography (3, 4). Later studies
revealed that the PEF domains are present in several other
Ca2+-binding proteins such as the calpain large subunit,
sorcin (5), grancalcin (6), and apoptosis-linked
gene 2 (ALG-2) (7). Whereas sorcin and grancalcin
exist as homodimers (8, 9), calpains exist as heterodimers of the large
catalytic and small regulatory subunits (10). The bacterially expressed
recombinant PEF domain of the calpain small subunit forms a homodimer
without the large subunit (11). X-ray crystallographic studies have also revealed that the dimers are formed through a pair of fifth EF-hands (EF-5s) that have lost their Ca2+-binding
capacities due to two-residue insertions (3, 4). Therefore, it has been
proposed that PEF proteins may form dimers with each other through
EF-5, which provides a new interface for the interaction with possible targets.
The calpain small subunit, sorcin, grancalcin, and ALG-2 have
hydrophobic domains with variable lengths in the N-terminal regions. In the case of calpains, the hydrophobic N-terminal domains bind to the membranes and play an important role in the change of
subcellular localization induced by Ca2+ (12). The
N-terminal region of sorcin is required to interact with the
membrane-localized annexin VII in a
Ca2+-dependent manner (13). Thus, the
N-terminal regions of PEF proteins are thought to interact with
phospholipids and/or target proteins on membranes.
Previously, we reported a novel PEF protein, peflin (PEF
protein with a long N-terminal hydrophobic
domain), which was cloned after a homology search for other
PEF proteins (14). Peflin is most similar to ALG-2 in the PEF domain
and has the longest N-terminal hydrophobic region of proteins in the
PEF family. Peflin is expressed in several human cell lines, but its
target protein and function have not been determined yet.
In this study, using a monoclonal antibody (MoAb) specific
to human peflin, we demonstrated that peflin was co-immunoprecipitated with ALG-2. The peflin/ALG-2 heterodimer dissociated in a
Ca2+-dependent manner. The N-terminal
hydrophobic domain of peflin was not essential for the
heterodimerization. Peflin co-localized with ALG-2 in the cytoplasm and
changed the subcellular localization in a
Ca2+-dependent manner.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Jurkat cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum,
L-glutamine (0.3 mg/ml), penicillin (100 units/ml) and
streptomycin (100 µg/ml) at 37 °C under humidified air containing
5% CO2. Human embryonic kidney (HEK) 293 cells were
cultured in Dulbecco's modified Eagle's medium supplemented as above.
Preparation of Anti-peflin Monoclonal
Antibody--
BALB/c female mice were immunized three times
with His-tagged N-terminal truncated peflin (His-peflin
N) prepared
as described previously (14). Hybridomas were generated by polyethylene
glycol-mediated fusion of donor splenocytes to the P3 myeloma cell
line. Positive hybridomas were identified by enzyme-linked
immunosorbent assay and cloned by limited dilution. Cloned hybridomas
were transplanted intraperitoneally to BALB/c mice. The IgG
fraction was prepared from ascites and purified by the ammonium sulfate
precipitation method. Western blotting was performed as described
previously (14).
Metabolic Labeling and Immunoprecipitation--
Jurkat cells
(1 × 107) were incubated in a 60-mm dish containing
1.5 ml of a methionine/cysteine-free medium for metabolic labeling (Sigma) supplemented with PBS-dialyzed fetal bovine serum to
10% and 35S-labeled amino acid mixture (100 µCi/ml, 70%
methionine and 30% cysteine) at 37 °C for 4 h under humidified
air containing 5% CO2. Cells were washed with PBS and
lysed in buffer A (20 mM HEPES, pH 7.4, 150 mM
NaCl, 1.5 mM MgCl2, 0.2% Nonidet P-40, 0.1 mM pefabloc, 25 µM leupeptin, 10 µM E-64, and 1 µM pepstatin) containing 5 mM EGTA or 0.01 mM CaCl2. Aliquots
were incubated with indicated antibodies for 4 h at 4 °C and
further incubated with protein G-Sepharose 4FF (Amersham Pharmacia
Biotech) overnight. Immunocomplexes were washed three times with buffer
A and subjected to SDS-PAGE and analyzed by autoradiography using a BAS
2000 system (Fuji Film, Kanagawa, Japan). Anti-human RECK MoAb 32C10A
(15) was used as a negative control antibody for immunoprecipitation.
Anti-FLAG MoAb M2 was obtained from Stratagene (La Jolla, CA).
Anti-mouse ALG-2 polyclonal antibody (PoAb) raised in rabbits was
affinity-purified using recombinant human ALG-2 as described previously
(16).
Expression Vectors and Transfection--
An EcoRI
fragment of the full-length peflin cDNA was inserted into a
eukaryotic expression vector, pCXN2 (a derivative of pCAGGS, a kind
gift from Dr. J. Miyazaki; Ref. 17), and a BamHI fragment of
either a full-length or an N-terminal truncated peflin (peflin
N:
amino acids 116-284) was inserted in-frame into a pCMV-tag2 vector
(Stratagene) for expression as FLAG-tagged protein. A human ALG-2
cDNA was cloned from Jurkat cells by the reverse
transcription-polymerase chain reaction method, and a
BglII/BamHI fragment was inserted into pCXN2 and
pCMV-tag2. One day after HEK293 cells (1 × 106
cells/60-mm dish) had been seeded, the cells were transfected with the
expression plasmid DNAs by the conventional calcium phosphate precipitation method. After 48 h, cells were collected and
analyzed by the immunoprecipitation and/or Western blotting methods,
where aliquots of immunoprecipitated proteins and cell lysates were subjected to SDS-PAGE using comparable amounts of the relevant samples.
The DNA transfection efficiency monitored by the expression of a green
fluorescent protein construct, pCMV-EGFP (obtained from
CLONTECH), was about 20% under a similar condition
in separate experiments.
Immunofluorescent Staining--
Cytospin preparations of Jurkat
cell suspension (2 × 105 cells/0.2 ml) were prepared
by centrifugation using an SC-2 adapter (Tomy Seiko, Tokyo, Japan),
fixed in 4% paraformaldehyde, and permeabilized in 0.1% Triton
X-100/PBS. After blocking with 1% bovine serum albumin in 0.1% Tween
20/PBS, cover glasses were incubated with primary antibodies
(anti-peflin MoAb and anti-ALG-2 PoAb) at 4 °C overnight and with
secondary antibodies (fluorescein isothiocyanate-conjugated anti-mouse
IgG for peflin and rhodamine-conjugated anti-rabbit IgG for ALG-2) at
room temperature for 30 min. Immunofluorescences were analyzed by an
MRC-1024 Laser Scanning Confocal Imaging System (Bio-Rad).
Subcellular Fractionation--
Subcellular fractionation was
performed by lysing cells with a Dounce homogenizer in buffer B (10 mM Tris-HCl, pH 7.5, 10 mM KCl, 3 mM MgCl2, 1 mM dithiothreitol, and
the protease inhibitors as described above), followed by centrifugation
at 1,000 × g (4000 rpm by a Sakuma M-150 rotor) for 10 min at 4 °C producing a pellet (see Fig. 7A,
P1). The supernatant was further centrifuged at 10,000 × g (13,000 rpm by a Sakuma M-150 rotor) for 10 min at 4 °C producing a second pellet (see Fig. 7A,
P2) and then at 100, 000 × g (60,000 rpm by
a Beckman TLA 100 rotor) for 30 min at 4 °C producing a third pellet
and a supernatant (see Fig. 7A, P3 and
S, respectively). Nuclei were purified essentially as
described by Dignam and colleagues (18). Briefly, the crude nuclear
fraction (P1) was homogenized in buffer B containing 0.1%
Triton X-100 and 0.2 M sucrose, layered onto a cushion of
buffer A containing 2 M sucrose, and centrifuged at
20,000 × g (18,000 rpm by a Beckman TLS-55 rotor) for
30 min at 4 °C.
Gel Filtration--
Jurkat cells (2 × 108)
were washed twice with PBS and lysed in buffer B containing 5 mM EGTA, and the cytosolic fractions (100,000 × g, supernatant) were prepared as above. The cytosolic
proteins were fractionated by gel filtration using a Superdex-75 column (1.0 cm × 30 cm; Amersham Pharmacia Biotech). Fractions (0.2 ml each) were collected and analyzed by Western blotting. Recombinant human ALG-2 was prepared essentially as described previously (16) and
subjected to gel filtration using the same column.
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RESULTS |
Detection of Peflin with Monoclonal Antibody--
Previously, we
prepared anti-peflin antiserum and detected a 30-kDa protein as a major
band in the lysates of various cell lines (14). The antiserum, however,
also cross-reacted with a protein of about 40 kDa, and it remained
unknown whether the 30-kDa protein was processed from the 40-kDa
protein. In the present study, we prepared a MoAb, named P1G, which was
more specific to the peflin protein. As shown in Fig.
1A, the prepared MoAb P1G
recognized a 30-kDa protein as a single band in Jurkat cell lysates by
Western blotting, whereas the antiserum reacted additionally with other
proteins. The MoAb could detect FLAG-tagged peflin (FLAG-peflin) and
FLAG-tagged N-terminal truncated peflin (FLAG-peflin
N) exogenously
expressed in HEK293 cells as differently migrating bands at expected
positions. Thus, it was concluded that the 30-kDa protein detected with
the antiserum corresponds to an unprocessed peflin molecule. To
determine whether MoAb P1G could immunoprecipitate peflin, we performed
immunoprecipitation followed by Western blotting. The peflin protein
was immunoprecipitated with MoAb P1G (Fig. 1B, lane
3) but not with an irrelevant MoAb 32C10A against human RECK (Fig.
1B, lane 2), which is not expressed in Jurkat
cells.

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Fig. 1.
Characterization of peflin MoAb P1G.
A, Coomassie Brilliant Blue staining (CBB) and
Western blotting (WB) of total Jurkat cell extract
(top left) using anti-peflin PoAb (Po) or MoAb
(Mo) P1G and Western blotting of FLAG-peflin (lane
1) or FLAG-peflin N (lane 2) expressed in HEK293
cells using MoAb P1G (top right) are shown. Bands
corresponding to peflin (30 kDa) and a nonspecifically cross-reacting
protein are indicated by an arrow and by an
asterisk, respectively, in the top left panel.
Schematic structures of peflin proteins are depicted
(bottom). B, lysates of Jurkat cells were
immunoprecipitated with control MoAb 32C10A or anti-peflin MoAb P1G as
described under "Experimental Procedures." Cell lysate (lane
1) and immunoprecipitated proteins (lane 2, 32C10A;
lane 3, P1G) were analyzed by Western blotting using P1G.
Mouse immunoglobulin heavy and light chains of immunoprecipitated
antibodies (IgG-H, IgG-L) of MoAb P1G were also
detected by subsequent Western blotting using peroxidase-conjugated
anti-mouse IgG as a secondary antibody.
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Co-immunoprecipitation of Peflin with ALG-2--
To search for a
peflin-interacting protein, we metabolically labeled Jurkat cells with
35S-labeled amino acids, and immunoprecipitated peflin with
MoAb P1G. An autoradiogram revealed a protein band of about 22 kDa, which was co-immunoprecipitated with peflin in the presence of the
Ca2+ chelator EGTA but not in the presence of
CaCl2 (Fig. 2). PEF proteins
have the common feature of dimerization with each other (2). For
example, sorcin and grancalcin form homodimers (8, 9), and calpains
form heterodimers of the large and small subunits (10). Interestingly,
heterodimers of the calpain subunits have been reported to dissociate
in a Ca2+-dependent manner (19). We thought
that peflin might also dimerize with itself or with other PEF proteins.
We suspected the co-immunoprecipitated 22-kDa protein to be ALG-2
because it is the 22-kDa protein most similar to peflin in the PEF
protein family.

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Fig. 2.
Co-immunoprecipitation of peflin with a
22-kDa protein using MoAb P1G. After Jurkat cells had been labeled
with [35S]methionine and [35S]cysteine for
4 h, the cells were lysed in the presence of 5 mM EGTA
(E) or 0.01 mM CaCl2 (C)
as described under "Experimental Procedures." Whole cell extracts
were immunoprecipitated with MoAb 32C10A (control) or MoAb
P1G (peflin). Aliquots of immunoprecipitated proteins
(Ppt) and cell lysates (Lysate) were subjected to
SDS-PAGE using comparable amounts of the relevant samples,
autoradiographed by exposing on an imaging plate for 24 h
(Ppt) or 1 h (Lysate), and analyzed by a
FUJIX Bioimaging Analyzer Station BAS 2000. Asterisks and an
arrow indicate nonspecifically precipitated proteins and a
22-kDa protein, respectively. Relative radioactivities of the 30-kDa
and 22-kDa bands after subtraction of backgrounds are 556 and 132 photostimulated luminescence units, respectively.
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To investigate the interaction of peflin with ALG-2 in Jurkat cells, we
performed a combined immunoprecipitation-Western blotting analysis. As
shown in Fig. 3, ALG-2 was detected in
the immunoprecipitates using anti-peflin MoAb P1G in the presence of
EGTA but not in the presence of CaCl2. Anti-ALG-2 PoAb also
immunoprecipitated peflin in the presence of EGTA in a complementary
experiment. The immunoprecipitation experiments were repeated at least
three times under different conditions by varying the concentrations of
antibodies and protein G. The figures show representative results. Efficiency of the immunoprecipitation of ALG-2 with anti-ALG-2 PoAb was
poor, particularly in the presence of Ca2+ (see
"Discussion" below).

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Fig. 3.
Identification of a 22-kDa peflin-interacting
protein as ALG-2. Whole Jurkat cell extracts in the presence of 5 mM EGTA (E) or 0.01 mM
CaCl2 (C) were immunoprecipitated
(IP) with MoAb 32C10A (control), MoAb P1G
(peflin), or anti-ALG-2 PoAb (ALG-2).
Co-immunoprecipitated proteins (Ppt) and cell lysates
(Lysate) were detected by Western blotting (WB)
using respective antibodies. Comparable amounts of the relevant samples
were adjusted with volumes.
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Next, we examined the interaction between the two PEF proteins using
HEK293 cells co-transfected with FLAG-tagged peflin and ALG-2
expression vectors. ALG-2 was detected in the immunoprecipitates of
FLAG-peflin using anti-FLAG MoAb M2 (Fig.
4A). ALG-2 was also co-immunoprecipitated with N-terminal truncated peflin
(FLAG-peflin
N), indicating that the PEF domain of peflin is the site
of this interaction. This was confirmed by complementary
co-immunoprecipitation of FLAG-ALG-2 with untagged peflin (Fig.
4B). On the other hand, untagged peflin was not
co-immunoprecipitated with FLAG-peflin (Fig. 4C), suggesting
no possibility of peflin/peflin interaction. In contrast, untagged
ALG-2 was precipitated with FLAG-tagged ALG-2 regardless of the
presence of either EGTA or CaCl2 as reported previously
(Fig. 4D and Ref. 20).

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Fig. 4.
Examination of peflin/ALG-2 interaction.
HEK293 cells were co-transfected with expression vectors as indicated.
A, FLAG-peflin or FLAG-peflin N, and ALG-2. B,
FLAG-ALG-2 and peflin. C, FLAG-peflin and peflin.
D, FLAG-ALG-2 and ALG-2. After 48 h, cells were lysed
in the presence of 5 mM EGTA (E) or 0.01 mM CaCl2 (C) and immunoprecipitated
(IP) with anti-FLAG MoAb M2. The immunoprecipitates were
analyzed by Western blotting (WB) with anti-peflin MoAb P1G
or anti-ALG-2 PoAb. Mouse immunoglobulin light chains
(IgG-L) of MoAb M2 in the immunoprecipitates were also
detected by subsequent Western blotting using peroxidase-conjugated
anti-mouse IgG as a secondary antibody.
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Gel Filtration Analysis of Peflin/ALG-2 Heterodimer--
To
further examine whether peflin forms a complex with ALG-2, we performed
gel chromatography of the soluble fraction of Jurkat cells in the
presence of EGTA. Peflin and ALG-2 were co-eluted in the fractions
corresponding to 40-50 kDa (Fig.
5B, top and middle, fractions 9-14) greater than the
calculated molecular masses (peflin, 30 kDa; ALG-2, 22 kDa). On the
other hand, recombinant human ALG-2 was detected in the fractions
eluting later than in those by Jurkat ALG-2 (Fig. 5B,
bottom, fractions 13-16). Recombinant human peflin was insoluble and could not be applied to the column.

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Fig. 5.
Co-elution of peflin and ALG-2 in gel
filtration column chromatography. Preparation of the soluble
fraction of Jurkat cells and recombinant ALG-2 (rALG-2), and
gel filtration were performed as described under "Experimental
Procedures." A, 100,000 × g supernatant
was applied to a Superdex-75 column (1.0 × 30 cm), and 0.2-ml
fractions were collected. The peak positions of three molecular mass
markers are indicated with arrows. BSA,
bovine serum albumin (67 kDa); OVA, ovalbumin (43 kDa);
CHY, chymotrypsinogen (25 kDa). B, Western
blotting of each fraction with anti-peflin MoAb P1G or anti-ALG-2 PoAb.
Fraction numbers in panel A are indicated above
each lane.
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Subcellular Localization of Peflin and ALG-2--
We investigated
the localization of peflin and ALG-2 using Jurkat cells.
Double-immunofluorescent staining was performed using both anti-peflin
MoAb P1G and anti-ALG-2 PoAb and analyzed by confocal laser scanning
microscopy. Immunofluorescence was detected in the cytoplasm for peflin
and in both the cytoplasm and the nucleus for ALG-2 (Fig.
6).

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Fig. 6.
Subcellular localization
of peflin and ALG-2 in indirect immunofluorescent staining. Jurkat
cells were double-immunostained for peflin (green) with
anti-peflin MoAb P1G or for ALG-2 (red) with anti-ALG-2 PoAb
by detecting with secondary fluorescein isothiocyanate-conjugated
anti-mouse IgG antibody and secondary rhodamine-conjugated anti-rabbit
IgG antibody, respectively, as described under "Experimental
Procedures." Immunofluorescences were visualized under a confocal
laser-scanning microscope, and a merged image was obtained. Scale
bar indicates 10 µm.
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As shown in Fig. 7A, peflin
was recovered in the cytosolic fraction (S) using a lysis
buffer containing 3 mM MgCl2 but containing neither EGTA nor CaCl2 by subcellular fractionation based
on the differential centrifugation method. In contrast, ALG-2 was
recovered in the crude nuclear fraction (P1), as well as in
the cytosolic fraction. The crude nuclear fraction was subjected to
centrifugation on a 2 M sucrose cushion. ALG-2 was detected
in the purified nuclei (N), agreeing with the result of
immunofluorescent staining (Fig. 6). In the presence of 0.01 mM CaCl2, however, almost all peflin and ALG-2
were recovered in the crude nuclear fraction (Fig. 7, B and
C, P1), but the purified nuclei contained a smaller amount of peflin and most of the peflin protein was recovered in the membrane/cytoskeletal fraction above a 2 M sucrose cushion
(data not shown). In contrast, under the same conditions, roughly equal amounts of ALG-2 were recovered in the membrane/cytoskeletal fraction and in the purified nuclei (data not shown). Inclusion of 0.1% Triton
X-100 in a buffer containing 5 mM EGTA or 0.01 mM CaCl2 partially solubilized peflin and ALG-2
(Fig. 7, B and C), but some of the PEF proteins
were resistant to the detergent. A higher concentration of Triton X-100
(1%) gave similar results (data not shown). These results suggested
that peflin and ALG-2 also changed their subcellular distribution with
Ca2+ as reported for other PEF proteins, probably from the
cytosol to the membrane and detergent-insoluble (cytoskeletal)
fractions.

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Fig. 7.
Subcellular fractionation of peflin and
ALG-2. A, Jurkat cells were homogenized in a low salt
buffer and fractionated into cytosolic (S), light membrane
(P3), heavy membrane (P2), and crude nuclear
(P1) fractions by the differential centrifugation method as
described under "Experimental Procedures." The crude nuclear
fraction was re-homogenized and centrifuged on a 2 M
sucrose cushion to obtain the purified nuclear fraction (N).
Volumes for SDS-PAGE were adjusted to compare the relative amounts of
the PEF proteins in the subcellular fractions. Peflin and ALG-2 were
detected by Western blotting using anti-peflin P1G or anti-ALG-2 PoAb.
B, effects of Ca2+ on subcellular fractionation
of peflin and ALG-2 were examined. Cellular fractionation into cytosol
(S), membrane (P2/P3), and crude nuclear
(P1) fractions was performed in the presence of 5 mM EGTA or 0.01 mM CaCl2. Effects
of a non-ionic detergent, 0.1% Triton X-100, on solubilization of
peflin and ALG-2 were examined. C, the immunoblots shown in
Fig. 7B were scanned with a flat-bed scanner (EPSON
GT-7000 ART), and the densities were quantified using a computerized
image analysis system for Macintosh (NIH Image software, version 1.55).
Relative amounts of peflin and ALG-2 in each subcellular fraction were
expressed in histograms, respectively, where the sum of each
fraction (S, P2/P3 and P1) is
1.0.
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DISCUSSION |
We showed that peflin exists as a complex with ALG-2 in the
absence of Ca2+ but that the complex dissociates in the
presence of the divalent cation. Peflin and ALG-2 seem to interact
directly because the two proteins were eluted from the gel filtration
column at the position of a heterodimer (Fig. 5). Previously,
Missotten and colleagues (20) showed that ALG-2 forms a
homodimer by co-immunoprecipitation of transiently co-overexpressed
FLAG- and Myc-tagged proteins in HEK293 cells. In agreement with their
result, we also detected a complex of exogenously expressed FLAG-tagged
ALG-2 and untagged ALG-2, but the efficiency of the dimer formation was
lower than that of the heterodimer formation with peflin (Fig. 4,
B and D). Without co-transfection with untagged
ALG-2, no ALG-2 immunoreactive band was detected in the
immunoprecipitates of anti-FLAG MoAb M2 (data not shown). In similar
experiments, no FLAG-peflin/peflin complex was observed (Fig.
4C). On the other hand, a peflin/ALG-2 complex was
clearly observed in the co-transfection assays (Fig. 4, A
and B) and even in the endogenously expressing proteins in Jurkat cells (Fig. 3). Thus, the formation of a peflin/ALG-2
heterodimer seems dominant over an ALG-2/ALG-2 homodimer.
Since the 22-kDa protein that was co-immunoprecipitated with peflin in
35S-labeled Jurkat cells was identified as ALG-2 (Fig. 2),
it became possible to estimate an approximate molar ratio between
peflin and ALG-2 in the complex. Assuming that peflin and ALG-2
incorporate 35S-labeled amino acids with similar
efficiencies during de novo synthesis, relative specific
radioactivities can be calculated from the numbers of methionine
(excluding translation initiation Met) and cysteine residues in the
proteins (peflin: 9 Met, 5 Cys; ALG-2: 3 Met, 1 Cys). Thus, the ratio
of relative specific radioactivities of [35S]peflin and
[35S]ALG-2 is 14:4. This ratio agrees well with that of
the observed relative radioactivities of the 30-kDa band (peflin) and
the 22-kDa band (ALG-2) in the autoradiogram analyzed by a bioimaging
analyzer BAS 2000 system (3.5:1 versus 4.2:1 calculated from
photostimulated luminescence units: peflin, 556; ALG-2, 132). This fact
indicates the presence of an approximately equal molar ratio (1:0.83)
of peflin and ALG-2 in the immunoprecipitates and suggests that the majority of peflin exists as a heterodimer with ALG-2 in the cytosol in
the absence of Ca2+.
On the other hand, not all of ALG-2 forms a heterodimer with peflin.
Approximately 50% of ALG-2 is present in the 100,000 × g supernatant fraction, and the rest is found in nuclei and membrane/cytoskeletal fractions (Figs. 6 and 7). Because the amounts of
peflin in the latter fractions are quite low, non-cytosolic ALG-2 may
exist either as a homodimer or complexed with unknown macromolecules.
Co-elution of cytosolic ALG-2 with peflin in the gel filtration
chromatography suggests that the majority of cytosolic ALG-2 forms a
heterodimer with peflin (Fig. 5). The results of the
immunoprecipitation experiments using anti-ALG-2 PoAb, however, do not
support this notion (Fig. 3). Whereas only a fraction (<10%) of ALG-2
was immunoprecipitable from the lysate, more than half of peflin was
co-immunoprecipitable with the antibody. The major cause of this
inconsistency may be due to the nature of the anti-ALG-2 PoAb used in
this study. The antibody was first raised in rabbits using denatured
recombinant mouse ALG-2 and was later affinity-purified using
recombinant human ALG-2 as a ligand. The obtained antibody may
recognize only a fraction of ALG-2 that retains a specific conformation
favoring interaction with peflin and may poorly recognize ALG-2
monomers and homodimers under undenatured conditions. Alternatively, the antibody may disrupt the protein-protein interaction under investigation. It is unlikely that the co-immunoprecipitation of peflin
with anti-ALG-2 PoAb was due to a cross-reactivity of the antibody with
peflin, because the antibody did not react with peflin overexpressed in
HEK293 cells by Western blotting (data not shown). Indeed, anti-FLAG
MoAb co-immunoprecipitated untagged peflin together with FLAG-tagged
ALG-2 from the lysates of HEK293 cells transfected with the tagged
ALG-2-expressing construct (Fig. 4B).
X-ray crystallographic analysis of the PEF domains of the recombinant
rat and pig calpain small subunits revealed homodimerization through
EF-5 of each molecule (3, 4). Recently, the heterodimers of recombinant
m-calpains of the large and small subunits and the homodimer of
grancalcin have been crystallized, and PEF domains have been shown to
form similar dimer structures through EF-5s (21-23). We assume that
peflin forms a heterodimer with ALG-2 by a similar protein-protein
interaction mechanism. The N-terminal hydrophobic region of peflin is
not essential for heterodimer formation as revealed by FLAG-peflin
N
(Fig. 4A). In the present study, however, we could not
investigate the potential role of the EF-5 domains of peflin and ALG-2
in their interaction. A deletion mutant lacking EF-5 (peflin
EF5)
could not be expressed in transient transfection experiments using
HEK293 cells suggesting the importance of heterodimerization with ALG-2
for stability and/or correct folding of peflin.
The results of cellular fractionation experiments suggested that peflin
and ALG-2 translocate from the cytosolic fraction to the membrane and
Triton X-100-insoluble (cytoskeletal) fractions in a
Ca2+-dependent manner (Fig. 7). In this study,
however, we could not detect a change in the immunofluorescence after
stimulation of Jurkat cells with Ca2+-ionophore, and we
could not obtain direct evidence of the Ca2+-induced
translocation of these proteins by immunofluorescent staining.
Surprisingly, ALG-2 was also found to localize in nuclei, raising the
possibility of a specific function in nuclear Ca2+
signaling. Recently, Krebs and Klemenz (24) showed the nuclear localization of ALG-2 by immunofluorescent staining of breast cancer
cells and observed disappearance of the nuclear localization of ALG-2
at the onset of mitosis.
Apoptotic pathways of ALG-2 have been partially clarified. ALG-2 was
originally identified by the method called "death trap" in T-cell
hybridoma using anti-CD3 antibody (7). An antisense ALG-2 cDNA
expression prompted survival after a variety of apoptotic stimuli, but
caspase activities were not affected (25). An ALG-2-interacting protein
named either AIP1 (ALG-2-interacting
protein 1) or Alix (ALG-2-interacting protein X) was
cloned concurrently by two independent groups (20, 26). AIP1 is a
105-kDa protein with a proline-rich C-terminal region containing 10 PXXP sequence motifs that potentially bind to SH3
domains. The N-terminal-truncated AIP1 construct exerted dominant-negative effects on the apoptosis of transfected cells induced
by starvation of trophic factors or staurosporine (26). The interaction
between AIP1 and ALG-2 requires Ca2+. In addition, AIP1 has
been reported to interact with SETA (SH3 domain-containing protein expressed in
tumorigenic astrocytes) through its C-terminal
proline-rich region, which binds to SH3-N (one of the two SH3 domains)
of SETA in a Ca2+-independent manner (27). Overexpressed
SETA proteins capable of binding to AIP1 sensitized astrocytes to UV
light-induced cell death. Thus, in resting cells, SETA/AIP1 and
peflin/ALG-2 complexes may exist separately in the cytoplasm.
After Ca2+-mobilization, ALG-2 may dissociate from peflin
and interact with SETA/AIP1 complex. In our preliminary
experiments, however, peflin-overexpressed HEK293 cells did not show
morphological changes and differences in apoptotic sensitivity upon
stimulation with Ca2+-ionophore or staurosporine compared
with control transfectants. Studies are in progress to investigate the
potential role of peflin in Ca2+-dependent
apoptosis under various conditions.