From the T-cell Molecular Biology Unit,
Laboratory of Cellular and Molecular Immunology, NIAID, National
Institutes of Health, Bethesda, Maryland 20892 and the
§ Basel Institute for Immunology, Grenzacherstrasse 487, Postfach CH-4005, Basel, Switzerland
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ABSTRACT |
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ALG-2 is a 22-kDa calcium-binding protein necessary for cell death induced by different stimuli in 3DO T-cell hybridoma. 3DO cell clones depleted of ALG-2 protein exhibit normal caspases activation, suggesting that ALG-2 function is required downstream or is independent of caspase proteases activity for apoptosis to occur. Using the yeast two-hybrid screening system, we have isolated and characterized the mouse cDNA encoding for ALG-2 interacting protein 1 (AIP1), a novel protein that interacts with ALG-2. ALG-2 and AIP1 colocalize in the cytosol and the presence of calcium is an indispensable requisite for their association. Sequence alignment shows that AIP1 is highly similar to BRO1, a yeast protein related to components of the Pkc1p-MAP kinase cascade.
Overexpression of a truncated form of AIP1 protects two different cell
types from death induced by trophic factors withdrawal; thus, our data
indicate that AIP1 cooperates with ALG-2 in executing the
calcium-dependent requirements along the cell death pathway.
Programmed cell death
(PCD)1 is a physiologically
regulated cell type-specific deletion that takes place during various
developmental stages of multicellular organisms and is essential for
the establishment and the maintenance of cellular homeostasis (1). PCD
occurs by apoptosis, which refers to the morphological changes that can be observed in cells undergoing PCD. These include plasma membrane blebbing, cell shrinkage, chromatin condensation, and DNA degradation (2). The process eventually culminates with the fragmentation of the
cells into apoptotic bodies that, in vivo, are rapidly phagocitized by the surrounding cells (3).
The suicide signal can be received and transduced into the cell through
death receptors that initiate a signaling cascade, which shortly leads
to cell demise (4). A number of molecules have been identified that
either regulate the death pathway or are able to modulate the death
signal. Most attention has recently focused on the interleukin-1 In addition to proteases activation, numerous evidence demonstrates
that alterations in intracellular calcium play an important role during
apoptosis. For some time it has been proposed that DNA fragmentation, a
general hallmark of apoptosis, is a
Ca2+-dependent event that requires a
Ca2+-activated endonuclease (13, 14). Induction of
apoptosis triggers an increase of [Ca2+]i as well
(15, 16), and intracellular chelators of Ca2+ inhibit cell
death (17). As consequence of Fas stimulation, calcium is immediately
mobilized from intracellular stores (17), and lymphoid cells rendered
deficient for the calcium release channels are resistant to apoptosis
induced by different stimuli (18, 19). Of interest, and consistent with
the literature, the antiapoptotic protein Bcl-2 decreases calcium
release from the endoplasmic reticulum stores (20). Considering these
data altogether, the existence of a Ca2+-sensitive step(s)
along the cell death pathway is clearly apparent. Although a precise
picture of how Ca2+ exerts these effects at the molecular
level is still not available, it is likely that transduction of
Ca2+-regulated signals occurs through
Ca2+-binding proteins.
Members of the EF hand Ca2+-binding protein family exhibit
Ca2+ affinities and binding kinetics compatible with
concentration and time range of Ca2+ wave (21). The
functional and structural Ca2+-binding unit is the
helix-loop-helix motif commonly called EF hand domain. One of them,
ALG-2, is a 22-kDa protein that was identified during a screening for
genes involved in apoptosis (22, 23). T-cell hybridoma clones depleted
of ALG-2 via transfection of the antisense cDNA are resistant to
cell death induced by diverse stimuli, including T-cell receptor and
Fas stimulation as well as dexamethasone, staurosporine, and ceramide
treatment. Nonetheless, in these ALG-2-deficient clones caspases are
normally activated upon induction of cell death, as determined by
cleavage of poly(ADP-ribose)polymerase and of a fluorogenic
substrate (24).
In this paper we report the cloning of AIP1, a novel gene
that interacts with ALG-2 in a calcium-regulated fashion.
Overexpression of a deletion mutant of AIP1 protects HeLa and COS cells
from apoptosis induced by serum starvation; thus AIP1 might mediate, at
least in part, the ALG-2 requirement for apoptosis.
Two-hybrid Screen and
For library screening, yeast Y 190 expressing GAL4-ALG-2 fused protein
was sequentially transformed with a mouse liver cDNA library cloned
in the pGAD10 vector (CLONTECH). 2 × 106 clones were analyzed. Transformed yeast were selected
on synthetic dropout/agar plates lacking leucine, tryptophan, and
histidine in the presence of 50 mM 3-aminotriazol (Sigma)
and grown for 5 days at 30 °C. Colonies positive for growth on
selective media were blotted on filter paper (Whatman No. 5),
permeabilized in nitrogen liquid, and placed on another filter soaked
in Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl,
1 mM MgSO4, 37.5 mM cDNA Cloning and Northern Blot--
The cDNA insert
contained in clone TH 28 was used to screen phage murine liver and a
3DO T-cell hybridoma cDNA libraries. Nucleotide sequences of
several cDNA clones, as well as the cDNA insert of clone TH 28, were determined on both strands with Sequenase kit (Amersham Pharmacia Biotech).
The cDNA insert contained in clone TH 28 was also used to probe a
mouse multiple tissue Northern blot (CLONTECH)
according to the manufacturer's protocol.
Plasmids--
The cDNA insert of clone TH 28 was
cloned in pcDNA3 (Invitrogen) with an HA epitope the provides a
Kozak consensus sequence and a methionine for translation. AIP1
full-length (3.8 kb) was cloned in pcDNA3 joining a 1.6-kb
EcoRI/BsrGI fragment from a liver cDNA clone with a
2.4-kb BsrGI/NotI fragment from clone TH 28. Most
of the other plasmid constructs used were made by polymerase chain
reaction performed with primers containing appropriate restriction
sites or epitope tags as needed. GST fusion proteins were made in pGEX
vectors (Amersham Pharmacia Biotech). Plasmid constructs were confirmed
by partial sequencing and immunoblot analysis.
In Vitro Transcription/Translation--
The cDNA encoding
for full-length AIP1 was transcribed and translated in vitro
in the presence of L-[4,5-3H]leucine with the
TNT-coupled lysate system (Promega) following the manufacturer's instructions.
Cell Culture and Antibodies--
293T, L66, HeLa, and COS cells
were cultured in Dulbecco's modified Eagle's medium, 10% fetal calf
serum. 3DO cells were cultured in RPMI, 5% fetal calf serum. The
polypeptide DEIKKERESLENLK was used to generate rabbit polyclonal
antisera. The whole rabbit serum was affinity-purified on
CNBr-activated Sepharose beads (Amersham Pharmacia Biotech) coupled
with the antigenic peptide.
HeLa and COS7 cells were transfected with lipofectAMINE (Life
Technologies, Inc.) following the manufacturer's indications. 293T
cells were transfected by the calcium phosphate method.
Immunoblot Analysis and Coprecipitation--
Cell lysates were
made in RIPA buffer, and protein concentration was determined by
Bio-Rad protein assay; 10-20 µg of protein was subjected to
polyacrylamide gel electrophoresis. Proteins were then transferred to
nitrocellulose membrane and incubated with primary antibody followed by
a secondary antibody horseradish peroxidase-conjugated (Promega). Blots
were developed using SuperSignal (Pierce) and visualized by exposure to
autoradiography film. For coimmunoprecipitation experiments, cells were
lysed in lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and a
protease inhibitor mixture). Lysates were divided and
immunoprecipitated with the indicated antibodies; immunocomplexes were
bound to protein A-agarose beads and resolved by SDS-PAGE as described above.
Immunofluorescence and Subcellular Fractionation--
L66 cells
were grown in chamber slides, fixed in 4% paraformaldehyde for 15 min
at room temperature, and then permeabilized in phosphate-buffered
saline, 0.5% saponine. AIP1 was visualized by incubating the cells
with the affinity-purified anti-AIP1 antiserum for 30 min, washing
several times with phosphate-buffered saline, 0.5% saponine, and then
incubating for 30 min with a fluorescein isothiocyanate-conjugated
mouse anti-rabbit. L66 cells hybridized in the presence of an excess
(10 µg) of immunogenic peptide served as negative control. All steps
were done at room temperature; the slides were analyzed with a confocal
laser scanning microscope.
For subcellular fractionation, cells were homogenized with a glass
Pyrex homogenizer in lysis buffer: 20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, and a mixture of protease inhibitors. Nuclei were
removed by centrifuging the homogenates at 1,000 × g
for 10 min, and the supernatant was centrifuged at 130,000 × g for 60 min to obtain the cytosolic and the membrane fraction.
In Vitro Binding--
Immobilized GST-ALG-2 fusion protein (1 µg) was washed three times in wash buffer (20 mM HEPES,
pH 7.5, 150 mM NaCl) and added to 500 µl of binding
buffer, which consisted of wash buffer supplemented with 1 mM EGTA and either CaCl2 or MgCl2.
The amount of each of these reagents needed to obtain a given
concentration of free cation was calculated with Free Calcium program
(25).
cDNAs encoding FLAG-tagAIP1 and ALG-2 proteins were in
vitro transcribed/translated as described above. The lysate (5 µl) was added to the immobilized GST-ALG-2 protein resuspend in
binding buffer and incubated for 1 h at room temperature with
gentle rotation. In the experiment shown in Fig. 4b,
right panel, 5 µl of in vitro synthesized
FLAG-AIP1 and ALG-2 proteins were incubated in binding buffer
containing the indicated cation and were immunoprecipitated with an
anti-FLAG antibody (Kodak) and protein AG-Sepharose beads (Pierce).
The beads were then washed six times in binding buffer, resuspend in
loading buffer, and the sample analyzed by SDS-PAGE followed by autoradiography.
Cell Death Assay--
COS and HeLa cells were transfected with 2 µg of pcDNA3 expressing the indicated cDNA together with 0.2 µg of pCMV- Two-hybrid Screening--
To identify ALG-2 interacting proteins,
we performed a two-hybrid screen with the full-length ALG-2 cDNA
fused to the GAL4 DNA binding domain (GAL4BD). A plasmid library of
fusion between the GAL4 transcription activation domain (GAL4AD) and
cDNAs from mouse liver was screened for interaction with
GAL4BD-ALG-2 fusion protein in the yeast reporter strain Y190. A total
of fourteen clones were isolated that activated the AIP1 Cloning and Expression--
On Northern blots clone TH
28 detects a ~4-kb transcript ubiquitously expressed, and an
additional, less abundant, 7-kb transcript (Fig.
1a). The 3DO T-cell hydridoma
expresses only the 4-kb mRNA, and the levels of transcription are
not regulated by T-cell receptor stimulation or glucocorticoid
treatment (data not shown).
Using clone TH 28 as probe, we screened a mouse liver
cDNA phage library and cloned the full-length cDNA, that we
named AIP1 for ALG-2 Interacting
Protein 1. The nucleotide sequences of several phage clones indicated that AIP1 consists of 840 amino acids (Fig. 1b). Accordingly, the in vitro
transcription/translation of full-length AIP1 cDNA generates a
single polypeptide of ~105 kDa (Fig. 1c). A BLAST search
revealed that several homologues of A21P1 have been cloned:
YNK1 in the nematode Caenorhabditis elegans (26), palA in the filamentous fungus Aspergillus
nidulans (27), and BRO1 in the yeast
Saccharomyces cerevisiae (28). Functional and genetic
evidence suggests that Bro-1 and palA participate in signal transduction pathways.
AIP1 and ALG-2 Interact in Vivo--
A rabbit antiserum was raised
against the polypeptide Asp561-Leu574 of the
AIP1 amino acidic sequence, which is also included in the TH 28 sequence. Immunoblot analysis performed on cell lysates from L66
fibroblasts and 3DO T-cell hybridoma indicates that this antiserum
recognizes the endogenous AIP1 protein as a single band migrating with
an apparent molecular mass of 105 kDa. 293T cells transfected with AIP1
cDNA exhibit a specific band that comigrates with the endogenous
AIP1 (Fig. 2a). Both signals
from murine cell lysates and transfected 293T cells were specific,
because the immunoreactivity was abolished by competition with the
immunogenic peptide (data not shown).
We then tested whether the interaction between ALG-2 and AIP1 also
occurs in mammalian cells. As shown in Fig. 2b, HA-flagged AIP1 specifically coprecipitates with ALG-2 in lysates from
293T-transfected cells. As expected, HA-flagged TH 28 coprecipitates
with ALG-2 as well. This latter observation suggests that in
overexpression experiments TH 28 can potentially inhibit the
association between ALG-2 and AIP1.
In addition, in lysates from untransfected L66 cells and the T-cell
hybridoma, the endogenous AIP1 coprecipitates with the endogenous ALG-2
(Fig. 2c). Thus, ALG-2 and AIP1 physically interact in
mammalian cells.
ALG-2 and AIP1 Colocalize in the Cytosol--
To determine the
cellular localization of AIP1, L66 fibroblasts were permeabilized and
stained with the anti-AIP1 antiserum. Fluorescence microscopy revealed
that AIP1 has a granular cytoplasmic distribution, consistent with a
cytosolic localization of the protein (Fig.
3). Subcellular fractionation experiments
confirmed this observation. Immunoblot analysis of fractionated lysates from L66 cells showed that AIP1 was present mostly in the cytosolic fraction, although a portion of the protein also distributes in the
membrane-containing fraction (Fig. 3C). In the same assay ALG-2 localizes essentially in the cytosolic fraction. Together with
the microscopic results, these data implicate that ALG-2 and AIP1
colocalize in the cytosolic compartment.
The Binding of AIP1 to ALG-2 Is
Calcium-dependent--
Intracellular free Ca2+
modulates the activity of Ca2+-binding proteins by binding
to their EF hand domain(s). Some of these proteins undergo major
conformational reorganization upon Ca2+ binding, which
regulates the interactions with target protein(s) (21). Since ALG-2 is
known to undergo such a Ca2+-dependent
conformational change (22), we asked whether Ca2+ could
modulate the interaction with AIP1. In these experiments we used
several [Ca2+], ranging from the physiological amount of
free Ca2+ present in resting cells (~50 nM)
to a concentration that is reached during Ca2+ flux from
storage compartments (1-2 µM). As shown in Fig.
4a, in vitro
transcribed/translated AIP1 and GST-ALG-2 fusion protein interact only
when at least 750 nM free Ca2+ is present in
the binding buffer. The specificity of Ca2+ was
investigated by replacing Ca2+ with another divalent
cation, Mg2+. At physiological intracellular concentrations
of free Mg2+ (900 µM), ALG-2·AIP1 complex
did not occur (Fig. 4b). Overall, these data indicate that
ALG-2 and AIP1 interact in a Ca2+-specific manner and only
when intracellular concentrations of free Ca2+ increase
above the normal resting level.
AIP1 Involvement in Cell Death--
The interaction of AIP1 with
ALG-2 prompted us to investigate the effects of AIP1 expression on
programmed cell death. To test for this, AIP1 and TH28 were
individually cotransfected with a plasmid encoding the
Work from many laboratories has implicated Ca2+
signaling in the regulation of apoptosis in mammalian cells. In
lymphoid cells, antigen-receptor engagement results in a sustained
increase in intracellular [Ca2+], followed by alternative
responses such as apoptosis or cellular activation and proliferation.
Calcium ionophores induce apoptotic cell death in a variety of
experimental systems, suggesting that an increase in intracellular
[Ca2+] is sufficient to signal the cell to enter the
apoptotic program. It appears, therefore, that while Ca2+
is essential for a response to occur, the type of response is more
dependent on the activation of specific pathways. Yet, the critical
target(s) relating this calcium flux to cellular apoptosis are only
partially identified. ALG-2 is a Ca2+-binding protein shown
to be directly involved in the control of programmed cell death;
however, knowledge concerning the biochemical mechanisms involving
ALG-2 is still incomplete. In this study we have identified and
characterized AIP1, a protein that interacts with ALG-2 in a
Ca2+-dependent manner. The presence of
Ca2+ is essential for this binding, presumably due to
conformational requirements necessary for association which are
satisfied only when ALG-2 is in a Ca2+-loaded state. The
range of [Ca2+] needed for the association of the two
proteins in vitro is compatible with physiological levels of
cytosolic Ca2+. In fact, while in resting cells
Ca2+ is maintained at relatively low concentration (10-100
nM), extracellular flux or release from intracellular
stores can result in an increase to the 500-1000 nM range
required for the interaction. This observation suggests that AIP1 and
ALG-2 are dissociated in resting cells and associate following
stimulation that results in intracellular [Ca2+] rise.
Expression of a truncated form of AIP1 partially inhibits apoptosis
evoked by some stimuli. The simplest interpretation of these
experiments is that interaction between ALG-2 and AIP1 is required for
cell death, and the polypeptide generated by TH 28 acts as a negative
inhibitor of AIP1 by competing for binding to ALG-2. However, it is
possible that the truncated protein encoded by TH 28 might still
perform some of the functions executed by AIP1. This would explain why
the inhibition of apoptosis that we observed was only partial and
evident with only a few stimuli. More specific reagents will allow us
to precisely determine the role of this association in programmed cell
death and in cell physiology in general.
AIP1 shows a high degree of homology to BRO1, a S. cerevisiae protein of 844 amino acids that is 22% identical to
AIP1 over the entire sequence (28). Genetic and functional evidence
relates BRO1 to components of the Pkc1p-MAP kinase cascade.
BRO1 mutants, in fact, display phenotypes similar to those
caused by deletion of BCK1, a gene encoding a MAP/ERK kinase
that mediates maintenance of cell integrity. BRO1 mutations result in a
temperature-sensitive osmoremedial growth defect, which is suppressed
by Ca2+. Since BRO1 itself lacks calcium binding domains,
it is likely that, in yeast, the function of BRO1 is regulated by an
ALG-2-like protein.
The homology between AIP1 and BRO1 suggests that one possible mechanism
by which the interaction ALG-2/AIP1 might control programmed cell death
is by activation of signal transduction pathways linked to MAP kinases.
Consistent with this idea is the evidence that p38 MAP kinase is
required for apoptosis induced by trophic factors withdrawal (29).
However, further studies are needed to define whether
Ca2+-regulated signals influence, through AIP1, activation
of MAP kinases.
Involvement of other Ca2+-dependent protein
kinases and phosphatases in apoptosis has been demonstrated previously.
Expression of calcineurin, a Ca2+-dependent
protein phosphatase that functions in T-cell activation, rapidly
induces apoptosis in the absence of growth factors (30). Because
calcineurin activates the transcription factor NF-AT, it is possible
that an alteration in gene transcription influences the decision to
enter apoptosis. This is also supported by the recent evidence that
NF-AT is activated during Ca2+-dependent
apoptosis, and Bcl-2 suppresses the signaling through NF-AT by binding
and sequestering of active calcineurin (31). Whether AIP1 plays a role
in this scenario remains to be determined.
palA is the A. nidulans gene encoding for a
798-residues protein which displays 25.6% similarity to AIP1 and
participates in the regulation of pH-dependent gene
expression (27). A homologue of AIP1 also exists in C. elegans, namely YNK1 (26), which encodes for a
798-amino acid protein. Interestingly, YNK1, palA, and AIP1 all
conserve, near their carboxyl terminus, a proline-rich region containing the PXXP consensus sequence of SH3 domain-binding
motifs. It is, therefore, likely that AIP1 is involved in a variety of complex cellular responses, of which apoptosis is only one of the
possible outcomes.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
converting enzyme-like proteases, a family of at least 10 related
mammalian cysteine proteases collectively named caspases (5). These
proteins are considered the executioners of mammalian apoptosis by
virtue of two seminal observations: (a) ectopic
overexpression of interleukin-1
converting enzyme, as well as other
members of the family, results in protease activation and induces
apoptosis (6, 7), and (b) specific inhibitors of these proteases
inhibit cell death (8, 9). Accordingly, the caspase paradigm asserts
that cells undergoing apoptosis accomplish their suicide program by
activating a hierarchy of caspases. However activation of caspases does
not always correlate with induction of apoptosis (10, 11), and in some
circumstances, caspase activity is required for protection from
apoptosis (12).
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
-Galactosidase Assays--
The
two-hybrid screening was conducted using the Matchmaker system from
CLONTECH according to the manufacturer's
instructions. Yeast strain Y190 was transformed with the corresponding
bait plasmids by lithium acetate/polyethylene glycol 4000 procedure and
selected on synthetic dropout plates lacking tryptophan. Selected colonies were then analyzed for expression of the GAL4-bait fusion protein by immunoblot analysis.
-mercaptoethanol) containing 1 mM
5-bromo-4-chloro-3-indolyl-
-D-galactoside. Colonies that
developed color were re-streaked on selective plates to allow plasmid
segregation and tested again for
-galactosidase activity. Yeast
colonies were then scored as positive when a bright color developed in
2-5 h, a negative was scored when color failed to develop within
12 h. Assays were done for 5-10 independent transformant.
-gal (CLONTECH). 24 h after
transfection the cells were treated with 1 µM etoposide or 1 µM staurosporine (Sigma) for 7 h or starved
lowering the fetal calf serum concentration to 0.1%.
-Galactosidase
activity was visualized by fixing the cells in 0.2% glutaraldehyde for 10 min followed by staining in phosphate-buffered saline containing 20 mM each K3Fe(CN)6 and
K4Fe(CN)6-H2O and 1 mg/ml
5-bromo-4-chloro-3-indolyl-
-D-galactoside for 1-3 h at
37 °C. The number of live blue cells and blue cells with apoptotic
morphology were counted in at least four fields.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
-galactosidase
reporter when approximately 2 × 106 plasmids were
transformed. Restriction mapping and sequencing of these 14 cDNAs
revealed that three clones (TH 2, TH 28, and TH 37) had the same insert containing an open reading frame
coding for 402 amino acids fused to GAL4AD. Further assays were then performed with clone TH 28 in the reporter yeast strain. As
summarized in Table I, this library clone
did not activate
-galactosidase by itself or when coexpressed with
the empty GAL4BD vector or with a control plasmid. Conversely, it
strongly interacted with GAL4BD-ALG-2, and no association was reported
with truncated forms of ALG-2 deleted at the amino or carboxyl
terminus. Hence, only the full-length ALG-2 specifically interacts with
TH 28 in yeast.
Two-hybrid interaction assay between clone TH 28 and ALG-2 constructs
-galactosidase activity was determined by a filter assay for
yeast transformed with the indicated plasmid as described under
"Experimental Procedures"; b, schematic representation of the
full-length ALG-2 and the deletion constructs used in the assay. The
black boxes represent the calcium binding domains; the numbers indicate
the amino acidic
position.
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Fig. 1.
Cloning of AIP1. a, Northern
blot analysis of AIP1 expression in different adult mouse
tissues. b, predicted amino acidic sequence of AIP1. The
polypeptide encoded by TH 28 is underlined; the
peptide DEIKKERESLENDL, double-underlined, was used as immunogen to
produce a rabbit antiserum. c, in vitro
transcription and translation of the cDNA encoding for AIP1.
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Fig. 2.
AIP1 coimmunoprecipitates with ALG-2.
a, immunoblot analysis of AIP1 protein. 5 µg of total
lysate from 293T cells transfected with the vector empty (pcDNA3)
or expressing the TH 28 and the AIP1 cDNAs were resolved by
SDS-PAGE along with 10 µg of total cell lysate from L66 fibroblast
and 3DO T-cell hybridoma (T hybr.). The immunoblot was
hybridized with the affinity-purified anti-AIP1 antiserum.
b, total cell lysates prepared from 293T cells transfected
with the indicated vectors were immunoprecipitated (IP) with
an anti-HA monoclonal antibody and analyzed by immunoblot probed with
ALG-2 antiserum. c, lysates from L66 and 3DO cells were
immunoprecipitated (IP) with
ALG-2 antiserum or preimmune
rabbit serum (PI). After gel separation, proteins were
transferred on nitrocellulose membrane and probed with anti-AIP1
antiserum followed by a protein A-horseradish peroxidase-conjugated
antibody.
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Fig. 3.
Cellular localization of AIP1. Confocal
images of L66 fibroblast stained with anti-AIP1 antibody followed by
fluorescein isothiocyanate-conjugated mouse anti-rabbit (A)
and in the presence of 10 µg of immunogenic peptide (B).
C, subcellular fractionation of lysates from L66 cells. 10 µg of proteins from unfractionated lysate or from cytosolic and
membrane fraction were resolved by SDS-PAGE and immunoblotted using the
anti-AIP1 or the ALG-2 antiserum.
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Fig. 4.
The interaction between ALG-2 and AIP1 is
Ca2+-dependent. a, in
vitro transcribed and translated AIP1 (I.V.T.) was
incubated with either GST or GST-ALG-2 fusion protein in the presence
of the indicated concentrations of free Ca2+. b,
Mg2+ cannot replace Ca2+. The experiment in the
left panel was performed similarly to a. In the
right panel, FLAG-AIP1 protein, synthesized in
vitro with no labeled ammino acids, was incubated with
[3H]leucine-labeled ALG-2 protein. Recombinant proteins
were incubated in the presence of either free Ca2+ or free
Mg2+ and were immunoprecipitated with an anti-FLAG
antibody.
-galactosidase gene in HeLa cells, which were subsequently treated
with anti-Fas and TNF. However, no differences in cell death could be
detected between experimental and control transfections in these
experiments (data not shown). Nevertheless, when apoptotis was evoked
by trophic factors withdrawal, a significant protective effect was
observed on HeLa and COS cells transfected with TH 28 (Fig.
5a). A protective effect was
also reported when cells were treated with staurosporine or etoposide.
In this latter set of experiments, the cotranfection of ALG-2 along
with TH 28 restores the susceptibility to cell death (Fig. 5,
b and c).
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Fig. 5.
Overexpression of TH 28 partially protects
from apoptosis. a, HeLa and COS cells were transfected
with the indicated plasmids together with pCMV- -gal and induced in
apoptosis by serum starvation for 16 h or treated with 1 µM etoposide (b) or 1 µM
staurosporine for 7 h (c). After treatment cells were
stained and examined by phase contrast microscopy. Numbers
are the percentage of apoptotic blue cells over the total blue cells.
Data in b and c represent mean ± S.D. of
three experiments. Data in a are representative of two
experiments done in duplicate.
DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References
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ACKNOWLEDGEMENTS |
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We thank Emanuela Lacana', Laura Tonnetti, and Kelly Ganjei for sharing reagents and helpful discussion; Marco Colonna, Susan Gilfillan, and Klaus Karjalainen for critical review of the manuscript; and members of the Laboratory of Cellular and Molecular Immunology and the Basel Institute for Immunology for comments and suggestions.
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FOOTNOTES |
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* The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche and Co. Ltd., CH-4005 Basel, Switzerland.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 41-61-605-1349; Fax: 41-61-605-1364; E-mail: vito{at}mail.bii.ch.
The abbreviations used are: PCD, programmed cell death; HA, hemagglutinin; kb, kilobase(s); GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; MAP, mitogen-activated protein.
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REFERENCES |
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