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INTRODUCTION |
Phylogenetically conserved Bcl-2 family proteins play
a pivotal role in the regulation of apoptosis from virus to human (1). Members of the Bcl-2 family consist of antiapoptotic proteins such as
Bcl-2, Bcl-xL, and Bcl-w, and proapoptotic proteins such as
BAD, Bax, BOD, and Bok. It has been proposed that anti- and proapoptotic Bcl-2 proteins regulate cell death by binding to each
other and forming heterodimers (2). A delicate balance between anti-
and proapoptotic Bcl-2 family members exists in each cell and the
relative concentration of these two groups of proteins determines
whether the cell survives or undergoes apoptosis.
Myeloid cell leukemia-1
(Mcl-1)1 is a Bcl-2 family
protein and was originally cloned as a differentiation-induced early
gene that was activated in a human myeloblastic leukemia cell line (3).
Mcl-1 is expressed in a wide variety of tissues and cells including
neoplastic ones (3-5). We and others recently identified a short
splicing variant of Mcl-1 short (Mcl-1S) and designated the known Mcl-1
as Mcl-1 long (Mcl-1L) (6, 7). Mcl-1L protein exhibits antiapoptotic
activity and possesses the BH (Bcl-2 homology) 1, BH2, BH3, and transmembrane domains found in the related
Bcl-2 proteins (3, 5, 8). In contrast, Mcl-1S is a BH3 domain-only proapoptotic protein that heterodimerizes with Mcl-1L (6). Although
both Mcl-1L and Mcl-1S proteins contain BH domains found in other Bcl-2
family proteins, they are distinguished by their unusually long
N-terminal sequences containing PEST (proline, glutamic acid, serine,
and threonine) motifs, four pairs of arginine residues, and alanine-
and glycine-rich regions. In addition, the tissue expression pattern of
the Mcl-1 protein is different from that of Bcl-2 suggesting a unique
role for Mcl-1 in apoptosis regulation (4, 5).
Tankyrase 1 (TRF1-interacting,
ankyrin-elated ADP-ribose
polymerase 1) was originally isolated based on its binding
to TRF1 (telomeric repeat binding
factor-1) and contains the HPS (homopolymeric runs of histidine, proline, and serine) sequence, 24 ankyrin repeats, SAM (sterile
-motif), and the catalytic domain of poly(adenosine diphosphate-ribose) polymerase (PARP) (9). Previous studies have shown
that tankyrase 1 promotes telomere elongation in human cells by
inhibiting TRF1 through its poly(ADP-ribosyl)ation by tankyrase 1 (9, 10). In addition, tankyrase 1 poly(ADP-ribosyl)ates insulin-responsive amino
peptidase (IRAP), a resident protein of GLUT4 vesicles, and
insulin stimulates the PARP activity of tankyrase 1 through its
phosphorylation by mitogen-activated protein kinase (11). Recently,
tankyrase 1 also has been shown to poly(ADP-ribosyl)ate TAB182
(tankyrase-binding protein of
182-kDa protein) (12). ADP-ribosylation is a
post-translational modification mechanism that usually results in a
loss of protein activity presumably by enhancing protein turnover
(13-16). However, little information is available regarding the
physiological function(s) of tankyrase 1 other than as a PARP enzyme.
In the present study, we found tankyrase 1 as a specific-binding
protein of Mcl-1. Overexpression of tankyrase 1 led to the inhibition
of both the survival action of Mcl-1L and the apoptotic activity of
Mcl-1S in mammalian cells. Unlike other known tankyrase 1-interacting
proteins, tankyrase 1 did not poly(ADP-ribosyl)ate either of the Mcl-1
proteins despite its ability to decrease Mcl-1 protein levels following
coexpression. Therefore, tankyrase 1 could regulate Mcl-1-modulated
apoptosis by down-regulating the expression of Mcl-1 proteins
without the involvement of its ADP-ribosylation activity.
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EXPERIMENTAL PROCEDURES |
Yeast Two-hybrid Screening--
The open reading frame of human
Mcl-1S cDNA was fused in-frame with the GAL4-binding domain into
the pGBT9 yeast shuttle vector (Clontech, Palo
Alto, CA). This vector was used to identify Mcl-1S-interacting proteins
by screening 1.5 million transformants from a GAL4 activation domain-tagged ovarian fusion cDNA library prepared from rats primed with equine chorionic gonadotropin (17). Yeast cells were cotransformed with pGBT9-Mcl-1S and cDNAs from the ovarian library, and colonies were selected in plates deficient in tryptophan, leucine, and histidine
but containing 30 mM 3-amino-1,2,4-triazole (Sigma). Plasmids were isolated from positive colonies following transformation of Escherichia coli cells and then sequenced. Nine
independent clones encoded the rat ortholog of human tankyrase 1. Full-length cDNA coding human tankyrase 1 was fused with the
activation domain of GAL4 in a yeast shuttle vector, pGADGH.
Subsequently, the specific interaction of tankyrase 1 with Mcl-1S was
confirmed based on the activation of the GAL1-HIS3 reporter gene.
Assessment of Tankyrase 1 Interactions with Bcl-2 Family Proteins
in the Yeast Two-hybrid System--
Complementary DNAs encoding
tankyrase 1 and various pro- or antiapoptotic Bcl-2 family proteins
were subcloned into pGADGH and pGBT9 vectors, respectively. Specific
binding of different protein pairs was evaluated based on the
activation of the GAL1-HIS3 reporter gene. At least 10 different
colonies were tested for each reaction.
In Vivo Binding of Tankyrase 1 with Mcl-1 Proteins--
Chinese
hamster ovary (CHO) cells (1.5 × 106) were cultured
in Dulbecco's modified Eagle's medium/F-12 supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. After 24 h of culture,
cells were transfected with 1.8 µg each of
pcDNA3-FLAG-epitope-tagged Mcl-1L, Mcl-1S, or BAD alone or together
with 4.2 µg of either HA-epitope-tagged tankyrase 1 or an empty
vector using LipofectAMINE (Invitrogen, Carlsbad, CA). CHO cells were
harvested at 24 h post-transfection and lysed in prechilled 1%
Nonidet P-40 lysis buffer containing 10% of a protease inhibitor
mixture (Sigma). Following 30 min incubation in the lysis buffer,
lysates were centrifuged at 10,000 × g for 10 min at
4 °C and supernatants were collected. Aliquots of the lysates were
precleared by incubation with mouse IgG and Protein-A-agarose (Santa
Cruz Biotechnology, Santa Cruz, CA) for 30 min at 4 °C. The
precleared lysates were then incubated with 1 µg/ml anti-mouse FLAG
M2 monoclonal antibody (Sigma) for 1 h followed by
Protein-A-agarose for an additional 1 h at 4 °C. The immune
complexes were centrifuged for 5 min at 2,000 × g and
washed five times with 1% Nonidet P-40 lysis buffer.
The immunoprecipitates and aliquots of total lysates were boiled in SDS
sample buffer for 5 min, subjected to SDS-PAGE, and electroblotted onto
0.22-µm nitrocellulose membranes. The membranes were blocked in 5%
nonfat dry milk in Tris-buffered saline solution with 1% Tween 20 for
1 h followed by incubation with 0.2 µg/ml of the anti-HA
monoclonal antibody (Sigma) for 1 h at room temperature. The blot
was then incubated for 10 min with 0.1 µg/ml anti-rabbit or mouse
IgG-horseradish peroxidase conjugate (Promega, Madison, WI) as a
secondary antibody before visualization by enhanced chemiluminescence (Amersham Biosciences). The same membrane was stripped and
incubated with 0.2 µg/ml mouse anti-FLAG M2 monoclonal antibody
(Sigma), rabbit anti-Mcl-1 polyclonal antibody (Santa Cruz
Biotechnology), and rabbit anti-BAD polyclonal antibody (Santa Cruz
Biotechnology) for 1 h at room temperature. The blot was exposed
to the same secondary antibody and visualized by enhanced chemiluminescence.
For immunoprecipitation of Mcl-1 by tankyrase 1, CHO cells (4.5 × 106) were transfected with equal amounts (7.5 µg) of
plasmids encoding HA-tankyrase 1 or FLAG-Mcl-1L. Cell lysates were
immunoprecipitated using the anti-HA monoclonal antibody, and the
precipitates were further blotted by the anti-Mcl-1 antibody or a
rabbit anti-tankyrase 1 polyclonal antibody (9).
Binding of Endogenous Tankyrase 1 and Mcl-1L--
Human myeloid
leukemia cell line K562 (ATCC, Manassas, VA) was cultured in RPMI 1640 medium with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. Cells were lysed
and precleared before incubation with 2 µg/ml mouse anti-Mcl-1
monoclonal antibody (BD Pharmingen, San Diego, CA) or mouse IgG for
2 h, followed by incubation with Protein-A-agarose for 2 h at
4 °C. The immune complexes were centrifuged for 5 min at 2,000 × g and washed five times with 1% Nonidet P-40 lysis buffer. Immunoblotting was performed using either the anti-Mcl-1 polyclonal antibody or the anti-tankyrase 1 antibody.
Regions Responsible for Interactions between Mcl-1
and Tankyrase 1--
To construct various truncated mutants
of Mcl-1 and tankyrase 1, PCR amplifications were performed using Pfu
DNA polymerase (Stratagene, La Jolla, CA). Truncated Mcl-1 and
tankyrase 1 cDNAs were generated using primers as described in
Table I. Wild type and truncated Mcl-1
cDNAs were subcloned into the pGBT9 vector whereas wild type and
mutant tankyrase 1 cDNAs were inserted into the pGADGH vector.
Specific interactions between these proteins were determined by the
activation of the GAL1-HIS3 reporter gene.
Assessment of Apoptosis in Transfected CHO Cells and
Down-regulation of Mcl-1 Proteins by Tankyrase 1--
Apoptosis was
monitored following transfection of different cDNAs as described
previously (18). CHO cells (2 × 105 cells/35-mm well)
were cultured for 24 h and transfected with the pcDNA3
expression vector with or without different cDNA inserts using
LipofectAMINE. For all experiments, the indicator plasmid pCMV
(Clontech) was included to allow the identification
of transfected cells. Inclusion of a 10-fold excess of expression
vectors as compared with the pCMV
reporter plasmid ensured that most
of the
-galactosidase-expressing cells also expressed the protein(s) under investigation. After a 4-h incubation of cells with the transfection mixtures in a serum-free medium, cells were incubated with
fresh medium containing 10% fetal bovine serum. Twenty-four hours
after transfection, cells were fixed in 0.3% glutaraldehyde and
stained with 0.4 mg/ml
5-bromo-4-chloro-3-indolyl-D-galactoside (Invitrogen) to
detect
-galactosidase expression. The number of blue cells was
counted by microscopic examination. Data were expressed as the
percentage (mean ± S.E.) of viable cells as compared with the
control group.
To assess Mcl-1 protein levels following tankyrase 1 coexpression, CHO
cells were transfected with different cDNAs and cell lysates were
prepared at 24 h after transfection. Equal amounts of the lysates
were subjected to SDS-PAGE. The same gel was immunoblotted with the
anti-FLAG M2, the anti-tankyrase 1, and the anti-
-actin monoclonal
antibodies (Sigma).
Purification of Recombinant Mcl-1L, Mcl-1S, and Tankyrase 1 Proteins--
N-terminal FLAG-tagged Mcl-1 proteins were expressed
in E. coli (BL21 codon plus) using the pET-21a Vector
(Novagen, Madison, WI) and cell lysates were prepared by a French
presser. Recombinant Mcl-1 proteins were purified using an anti-FLAG M2
affinity gel and eluted using the 3× FLAG peptide (Sigma). Purified
Mcl-1 proteins were monitored using SDS-PAGE followed by Coomassie Blue
staining. Baculovirus-derived tankyrase 1 and TRF1 were prepared as
described (9).
In Vitro Interactions between Recombinant Tankyrase 1 and Mcl-1
Proteins--
Tankyrase 1 (100 ng) was incubated with either 100 ng of
Mcl-1L or Mcl-1S in 100 µl of 1% Nonidet P-40 lysis buffer. After 1 h at 4 °C, the mixtures were subjected to immunoprecipitation using the anti-FLAG M2 affinity gel. The immune complexes were centrifuged for 5 min at 2,000 × g and washed five
times with the lysis buffer and boiled in the SDS sample buffer.
Immunoblotting was done using rabbit antibodies against tankyrase 1 or
Mcl-1.
PARP Assay--
PARP activity was determined using
baculovirus-derived tankyrase 1 as previously described (9). For each
reaction, purified tankyrase 1 (2 or 4 µg) was mixed with equal or
increasing amounts of the target protein (Mcl-1L, Mcl-1S, and/or TRF1)
and incubated for 30 min at 25 °C in an assay buffer containing 50 mM Tris (pH 8.0), 4 mM MgCl2, 0.2 mM dithiothreitol, and 1.3 µM
[32P]NAD+ (4 µCi, 30 Ci/mmol). Reactions
were stopped by the addition of trichloroacetic acid to 25%.
Acid-insoluble proteins were collected by centrifugation, rinsed in 5%
trichloroacetic acid, suspended in the sample loading buffer (1 M Tris base, 12% SDS, 0.2% bromphenol blue, and 0.2 M dithiothreitol), and fractionated on an 10%
SDS-polyacrylamide gel. The gel was dried and exposed on a PhosphorImager.
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RESULTS |
Specific Interactions of Tankyrase 1 with Mcl-1L and Mcl-1S but Not
Other Bcl-2 Family Proteins in Yeast and Mammalian Cells--
We
performed a yeast two-hybrid screening using full-length Mcl-1S as bait
to identify interacting proteins from the ovary cDNA library.
Sequence analysis revealed that nine strongly interacting clones encode
a rat ortholog of human tankyrase 1 with 330 amino acids truncated at
the C terminus. The yeast two-hybrid system was used to determine the
interactions of tankyrase 1 with Mcl-1 and other Bcl-2 family members.
Human tankyrase 1 interacted strongly with both Mcl-1L and Mcl-1S (Fig.
1). In contrast, no interaction was
detectable between tankyrase 1 and different Bcl-2 family proteins
tested including antiapoptotic Bcl-2, Bcl-w, Bcl-xL, Bfl-1,
Ced-9, and BHRF-1, as well as proapoptotic Bax, Bak, Bik, Bod-L, BAD,
Diva, Nix, and Bok-L (Fig. 1).

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Fig. 1.
Specific interaction of tankyrase 1 with both
Mcl-1L and Mcl-1S, but not other Bcl-2 family proteins, in the yeast
two-hybrid system. Yeast cells were grown in selective media
containing 30 mM 3-aminotriazole and lacking Trp, Leu, and
His. Prominent growth was found in colonies expressing tankyrase 1 fused to the GAL4 activation domain and Mcl-1L or Mcl-1S fused to the
GAL4-binding domain. Other Bcl-2 proteins tested include the
antiapoptotic Bcl-2, Bcl-w, Bcl-xL, Bfl-1, Ced-9, and
BHRF-1 as well as the proapoptotic Bax, Bak, Bik, Bod-L, BAD,
Diva, Nix, and Bok-L. No growth of yeast colonies was found in cells
expressing only the Bcl-2 family proteins, thus ruling out
self-activation of these constructs. The result is a representative of
10 different colonies tested.
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In vivo interactions of tankyrase 1 with Mcl-1 proteins were
confirmed in CHO cells transiently transfected with tankyrase 1 cDNA and Mcl-1 cDNAs. Cell lysate was precipitated using the M2
antibody against the FLAG epitope, followed by blotting using the HA
antibodies. As shown in Fig.
2A (panel a,
lanes 4 and 6), HA-tagged tankyrase 1 was
coimmunoprecipitated with FLAG-tagged Mcl-1L or Mcl-1S. In contrast,
tankyrase 1 failed to coprecipitate with FLAG-tagged BAD protein (Fig.
2A, panel a, lane 2), consistent with
a lack of interaction observed in the yeast two-hybrid system. Of
interest, the amount of Mcl-1L or Mcl-1S protein was lower when the
Mcl-1 proteins were coexpressed with tankyrase 1 as shown in Fig.
2A (panel e, lane 4 versus
5 and lane 6 versus 7). The same amount of lysate was loaded in each lane and the nonspecific 30-kDa band served as an internal control. The observed lower expression of Mcl-1L or Mcl-1S protein in the presence of tankyrase 1 suggested an increased turnover of these proteins because of their
interactions with tankyrase 1.

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Fig. 2.
In vivo dimerization of tankyrase
1 with Mcl-1 proteins in mammalian cells. A,
co-immunoprecipitation of tankyrase 1 together with Mcl-1 proteins. CHO
cells were transfected with FLAG-tagged Mcl-1L or Mcl-1S with or
without HA-tagged tankyrase 1. As a negative control, HA-tagged
tankyrase 1 was also coexpressed with FLAG-tagged BAD. Equal amounts of
total DNA (6 µg) was used for transfection, including 4.2 µg of
HA-tagged tankyrase 1 or the empty vector along with 1.8 µg of
plasmid encoding FLAG-tagged Mcl-1L, Mcl-1S, or BAD. At 24 h after
transfection, the cell lysate was used for immunoprecipitation tests
with the M2 antibodies against the FLAG epitope ( FLAG
IP). Following precipitation of FLAG-tagged Mcl-1 proteins,
immunoblotting with the HA antibody was performed to demonstrate the
co-precipitation of tankyrase 1 (panel a). The same membrane
was blotted using antibodies against Mcl-1 (panel b) and BAD
(panel c) to confirm their identity. Aliquots of the same
lysate also were used for the detection of tankyrase 1 (panel
d), Mcl-1, and BAD (panel e). B,
co-immunoprecipitation of Mcl-1 proteins together with tankyrase 1. CHO
cells were transfected with FLAG-tagged Mcl-1L with or without
HA-tagged tankyrase 1. Equal amounts of total DNA (15 µg) were used
for transfection, including 7.5 µg of HA-tagged tankyrase 1 or
FLAG-tagged Mcl-1L. Cell lysates were immunoprecipitated using the HA
antibody ( HA IP), followed by blotting with the Mcl-1
antibody (panel a) or the tankyrase 1 antibody (panel
b). Aliquots of the lysates were also blotted using the same
antibodies (panels c and d). C,
interactions between endogenous Mcl-1 and tankyrase 1 in K562 cells.
The cell lysate was immunoprecipitated with the monoclonal Mcl-1
antibody or IgG, and the precipitates ( Mcl-1 IP or IgG IP) were blotted using the polyclonal Mcl-1 antibody
(left panel) or the tankyrase 1 antibody (right
panel).
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In vivo interactions between Mcl-1 and tankyrase 1 were
further determined in CHO cells following immunoprecipitation of
tankyrase 1. Mcl-1L was coprecipitated with HA-tagged tankyrase 1 using the HA antibody, followed by immunoblotting using the Mcl-1 antibody (Fig. 2B, panel a, lane 3). Because a
nonspecific protein reacted to the Mcl-1 antibody and migrated to the
same position as the Mcl-1S in the SDS-PAGE gel (data not shown),
coprecipitation of Mcl-1S with tankyrase 1 could not be tested.
To test interactions between endogenous Mcl-1 and tankyrase 1 proteins,
we used the human leukemia cell line K562. As shown in Fig.
2C (left panel), endogenous Mcl-1L in these cells
was precipitated by an Mcl-1 antibody but not by nonimmune IgG. In addition, tankyrase 1 was found to be coprecipitated with Mcl-1L as
evidenced by an immunoreactive band above 150 kDa (Fig.
2C, right panel). The detection of endogenous
Mcl-1L and tankyrase 1 as complexes supports the physiological
interactions between these two proteins.
Tankyrase 1 Interacts with the Mcl-1 Proteins through Its Ankyrin
Domain--
To determine the region of tankyrase 1 responsible for
interaction with Mcl-1 proteins, truncated mutants of tankyrase 1 (T1 to T10) were generated and their binding to Mcl-1L and Mcl-1S was
tested in the yeast two-hybrid system. Tankyrase 1 protein has four
major domains (Fig. 3) including the HPS
sequence, 24 ankyrin repeats, SAM motif, and the catalytic domain of
PARP (9). The two Mcl-1 proteins did not interact with the HPS (T1),
SAM (T2), or PARP (T2) motif of tankyrase 1, whereas the ankyrin domain of tankyrase 1 alone (T3) exhibited a strong interaction to both Mcl-1
proteins (Fig. 3). We further truncated the ankyrin repeats of
tankyrase 1 to determine the minimal region responsible for binding to
Mcl-1. As shown in Fig. 3, the region spanning from amino acid 181 to
586 of tankyrase 1 (T5), containing 12 ankyrin repeats exhibited the
same degree of interaction as wild type tankyrase 1. Additional
truncation of ankyrin repeats (T6 to T10) resulted in a lost or weaker
interaction.

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Fig. 3.
Interactions of Mcl-1 proteins with truncated
tankyrase 1 mutants in the yeast two-hybrid system: involvement of
ankyrin repeats for Mcl-1 interaction. Yeast cells were grown
under the same conditions described in the legend of Fig. 1 and the
interactions between tankyrase 1 mutants and the Mcl-1 proteins were
examined. Either Mcl-1L or Mcl-1S coding plasmids was fused to the
GAL4-binding domain, and wild type (T) or mutant
(T1 to T10) tankyrase 1 were expressed in the
yeast vector containing the GAL4 activation domain. The truncation of
the HPS sequence, SAM, and PARP motifs of tankyrase 1 did not affect
the interactions between tankyrase 1 and the Mcl-1 proteins. In
addition, ankyrin repeats toward the C-terminal end (T3
versus T5) are dispensable for tankyrase 1 binding to the
Mcl-1 proteins. However, truncation of the N-terminal ankyrin repeats
(T7 to T10) of tankyrase 1 abolished its interaction with both Mcl-1
proteins. The absence of detectable yeast growth is indicated as
" ", whereas +++, ++, and + depict strong, medium, and weak
interactions, respectively. The data is a summary of at least 10 different colonies.
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A Short Stretch of 25 Amino Acids toward the N Terminus of Mcl-1 Is
Sufficient for Binding to Tankyrase 1--
Mcl-1L has two PEST motifs
together with BH1, BH2, BH3, and a transmembrane domain, whereas Mcl-1S
possesses only PEST motifs and the BH3 domain (Fig.
4A) (6). Various truncated
Mcl-1 mutants (M1 to M13) were generated to determine the region
essential for Mcl-1 interaction with tankyrase 1. Because both Mcl-1L
and Mcl-1S interacted with tankyrase 1 to the same extent, the
interacting region likely resides in the common sequences of Mcl-1L and
Mcl-1S. Consistent with this notion, deletion of BH1, BH2, and the
transmembrane domains did not affect the interaction of Mcl-1 with
tankyrase 1 (Fig. 4A, M1). In addition, the BH3
domain, the only conserved region found in all Bcl-2 family members,
was not involved in tankyrase 1 binding (M2). Deletion of either one or
both of the known PEST sequences of Mcl-1 (M4 and M5) also did not
alter tankyrase 1 binding; the N-terminal region of Mcl-1 interacted
with tankyrase 1 as strongly as the full-length Mcl-1 (M5). However,
truncation of the N-terminal region of Mcl-1 from amino acid 76 to 100 (M7) abolished its interaction with tankyrase 1 (Fig. 4B).
The importance of this region was further supported by a lack of
interaction with tankyrase 1 by other Mcl-1 mutants (M8, M9,
M11, and M12) lacking amino acids 76 to 100. In
contrast, the Mcl-1 mutant (M13) containing only amino acids 76 to 100 retained a strong binding to tankyrase 1 comparable with that of wild
type Mcl-1L or Mcl-1S. Therefore, the short stretch of 25 amino acids
(VARPPPIGAEVPDVTATPARLLFFA) of Mcl-1 is sufficient to
mediate tankyrase 1 binding. According to the PESTFIND program (Pasteur
Institute, France), this interacting region also contains weak PEST
sequences (underlined).

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Fig. 4.
Determination of the regions in Mcl-1 that
mediate its association with tankyrase 1 in the yeast two-hybrid
system. The interactions between the Mcl-1 mutants (M1 to M13) and
tankyrase 1 were determined by expressing truncated Mcl-1 mutants fused
to the GAL4-binding domain and the full-length tankyrase 1 fused to the
GAL4 activation domain. A, truncation of the majority of the
C-terminal regions including the PEST, BH1, BH2, BH3, and TM domains of
Mcl-1 (M1 to M5) did not affect interaction with
tankyrase 1. However, truncation of the N-terminal region of Mcl-1
(M6) abolished interactions with tankyrase 1. B,
the extreme N terminus of Mcl-1 was further truncated (M7 to
M13) to identify sequences responsible for tankyrase 1 binding. Mutants missing amino acids 76 to 100 (M7 to
M9, M11, and M12) lost the ability to
interact with tankyrase 1, whereas an Mcl-1 mutant possessing only
amino acids 76 to 100 (M13) exhibited a strong interaction
with tankyrase 1 comparable with that of wild type Mcl-1.
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Tankyrase 1 Antagonizes Both the Survival Effect of Mcl-1L and the
Proapoptotic Action of Mcl-1S--
We investigated whether tankyrase 1 modulates the function of Mcl-1 proteins using a cell viability assay.
Overexpression of increasing amounts of tankyrase 1 itself did not
alter CHO cell viability as compared with controls (Fig.
5). In accordance with previous studies
(6-8), overexpression of Mcl-1S induced cell death and overexpression
of Mcl-1L prevented the small increases in cell death resulting from
transfection per se. When tankyrase 1 was coexpressed with
either Mcl-1L or Mcl-1S, tankyrase 1 antagonized the actions of both
Mcl-1L and Mcl-1S in a concentration-dependent manner. At a
higher dose, tankyrase 1 effectively blocked both Mcl-1L-mediated cell
survival and Mcl-1S-induced cell death. In contrast, the proapoptotic
action of BAD was not antagonized by tankyrase 1. These data suggest
that inhibition of Mcl-1 activities by tankyrase 1 is specific, likely
resulting from direct interactions of Mcl-1 proteins with tankyrase
1.

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Fig. 5.
Coexpression with tankyrase 1 blocks the
antiapoptotic activity of Mcl-1L and the proapoptotic activity of
Mcl-1S: lack of involvement of the PARP domain of tankyrase
1. Quantitative cell viability analysis was performed to
test the modulatory effect of tankyrase 1 on the Mcl-1 regulation of
apoptosis. CHO cells were transfected with a total of 1.1 µg of
plasmid DNA including 1.0 µg of the pcDNA3 expression construct
and 0.1 µg of the pCMV- -gal reporter. The number of
-galactosidase-expressing cells was determined at 24 h after
transfection. Data (mean ± S.E.) are from at least three
different experiments in triplicate. For the control group, 1.0 µg of
the empty pcDNA3 vector was used. When increasing amounts (0.1, 0.3, or 0.7 µg) of tankyrase 1-expressing plasmid were used for
transfection, the empty vector was added to ensure the use of the same
amount (1.1 µg) of total plasmid. In all experiments, 0.3 µg of
plasmid encoding Mcl-1S, Mcl-1L, or BAD with or without tankyrase 1 plasmid were used. Both cell survival mediated by Mcl-1L and apoptosis
induced by Mcl-1S were dose-dependently blocked by
tankyrase 1. In contrast, tankyrase 1 was not effective in blocking the
cell killing induced by BAD. A mutant tankyrase 1 with the PARP domain
deleted (mutant Tanky; 0.7 µg) maintained the ability to inhibit the
actions of both Mcl-1L and Mcl-1S, indicating this region is
dispensable for the antagonistic actions of tankyrase 1.
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To determine the involvement of the PARP catalytic domain of tankyrase
1 in its inhibition of Mcl-1 actions, a mutant tankyrase 1 devoid of
the PARP domain (mutant Tanky; amino acids 1 to 1150) was coexpressed
with Mcl-1. Of interest, this mutant was still able to antagonize the
effects of either Mcl-1L or Mcl-1S on cell viability (Fig. 5). These
results suggest that the blockage of Mcl-1 effects by tankyrase 1 does
not require ADP-ribosylation of Mcl-1 proteins.
Tankyrase 1 Down-regulates Mcl-1L and Mcl-1S Protein
Levels--
Because coexpression with tankyrase 1 is associated
with decreased levels of the Mcl-1 proteins (Fig. 2A),
down-regulation of the Mcl-1 proteins by takyrase 1 was further
investigated. Coexpression of increasing amounts of tankyrase 1 decreased the expression of both Mcl-1L and Mcl-1S in a
concentration-dependent manner (Fig.
6, panels A and B).
In contrast, tankyrase 1 did not alter the expression of BAD.
Furthermore, mutant tankyrase 1 devoid of the PARP domain (mutant
Tanky) also effectively decreased the expression of both Mcl-1L and
Mcl-1S. These data indicate that tankyrase 1-mediated Mcl-1
down-regulation does not require the PARP enzymatic activity of
tankyrase 1. Equal loading of samples was demonstrated by Western
blotting of the same membrane using the
-actin antibody (Fig. 6,
panel C), and the nonspecific 30-kDa band that reacted with
the FLAG antibody served as an internal control (Fig. 6, panel
A). Several lower bands found in lanes containing either wild type
(lanes 3-5, 7-9, and 11-13) or
mutant tankyrase 1 (lanes 15 and 16) could result
from cleavages of tankyrase 1 by caspases or other enzymes as found for
other PARP proteins (19). Because the concentrations of plasmids used
were comparable between the experiments shown in Figs. 5 and 6, the
effects of both wild type and mutant tankyrase 1 on Mcl-1-regulated
apoptosis correlate with their down-regulation of Mcl-1 proteins.

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Fig. 6.
Down-regulation of Mcl-1 protein levels by
wild type tankyrase 1 and its PARP deletion mutant in transfected
cells. CHO cells were transfected using a total of 1.0 µg of the
pcDNA3 expression plasmid and cell lysates were prepared 24 h
later. Plasmids (0.3 µg) encoding Mcl-1S, Mcl-1L, or BAD, were used,
together with increasing concentrations (0.1, 0.3, or 0.7 µg) of the
tankyrase 1 plasmid or a plasmid (0.7 µg) encoding the mutant
tankyrase 1 (mutant Tanky) with the PARP domain deleted. Tankyrase 1 decreased the levels of Mcl-1 proteins in a
concentration-dependent manner, whereas BAD expression was
not altered by tankyrase 1 as demonstrated by immunoblotting using the
M2 antibody against the FLAG epitope (panel A). Panel
B is an immunoblot using the tankyrase 1 antibody to demonstrate
takyrase 1 expression. Equal loading of lysates is evident in
immunoblots using the -actin antibody (panel C).
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Direct Interactions between Recombinant Mcl-1 Proteins and
Tankyrase 1: Tankyrase 1 Does Not ADP-ribosylate Mcl-1
Proteins--
We evaluated the interactions between Mcl-1 and
tankyrase 1 and the possible ADP-ribosylation of Mcl-1 by tankyrase 1. To assess the functional integrity of the purified recombinant Mcl-1 proteins, their binding to recombinant tankyrase 1 protein was tested
in vitro. As shown in Fig.
7A (lower panel,
lanes 2 and 3), the recombinant Mcl-1 proteins
efficiently pulled down tankyrase 1, indicating that the purified Mcl-1
proteins are functionally active. The purity of the Mcl-1 proteins used
in this test were analyzed by SDS-PAGE followed by Coomassie Blue
staining (Fig. 7B).

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Fig. 7.
Interactions between recombinant Mcl-1
proteins and tankyrase 1 in vitro. A,
purified Mcl-1 proteins (100 ng) were incubated with tankyrase 1 (100 ng), and the mixtures were immunoprecipitated using the anti-FLAG M2
affinity gel. The immune complexes were fractionated by SDS-PAGE and
then immunoblotted using the Mcl-1 polyclonal antibody (top
panel). The same membrane was further blotted using the tankyrase
1 antibody (lower panel). B, purified recombinant
FLAG-tagged Mcl-1 proteins (1 µg) were subjected to 10% SDS-PAGE and
stained with Coomassie Blue.
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Tankyrase 1 contains the catalytic domain of PARP, and ADP-ribosylates
itself (9) and its interacting proteins such as TRF1 (9), IRAP (11),
and TAB182 (12). However, our studies using PARP-deleted tankyrase 1 suggest that ADP-ribosylation is not essential for the modulation of
Mcl-1 activities and levels. We further tested whether tankyrase 1 could ADP-ribosylate Mcl-1 proteins. An in vitro
poly(ADP-ribosyl)ation assay was performed using purified recombinant
Mcl-1 proteins and tankyrase 1. Incubation of tankyrase 1 in the
presence of [32P]NAD+ resulted in
auto-ADP-ribosylation (Fig.
8A, lane 1)
consistent with previous findings (9). Coincubation of increasing
amounts of either Mcl-1L or Mcl-1S with tankyrase 1 did not lead to
ADP-ribosylation of Mcl-1L or Mcl-1S (Fig. 8A, lanes
5-10). In contrast, tankyrase 1 ADP-ribosylated its known
substrate, TRF1, in a concentration-dependent manner (Fig.
8A, lanes 2-4) (9). To test the possibility that binding between Mcl-1 proteins and tankyrase 1 could alter the PARP
activity of tankyrase 1, the ability of Mcl-1 proteins to modify
poly(ADP-ribosyl)ation by tankyase 1 was further examined. As shown in
Fig. 8B, increasing amounts of either Mcl-1L or Mcl-1S reduced the ADP-ribosylation of TRF1 by tankyrase 1 in a
concentration-dependent manner. In addition,
auto-ADP-ribosylation of tankyrase 1 was also decreased in the presence
of increasing amounts of Mcl-1 protein (Fig. 8B; shorter
exposure). These data suggest that tankyrase 1-mediated
ADP-ribosylation of Mcl-1 is unlikely to be the mechanism underlying
the down-regulation of the Mcl-1 proteins, and the Mcl-1 proteins could
affect tankyrase 1 function by modulating its ADP-ribosylation of
itself and its binding protein, TRF1.

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Fig. 8.
Tankyrase 1 does not ADP-ribosylate Mcl-1
proteins whereas Mcl-1 proteins suppress ADP-ribosylation of TRF1
and tankyrase 1. PARP reactions were performed using
[32P]NAD+ as the substrate. Samples were
fractionated on a 10% SDS-PAGE gel and
32P-poly(ADP-ribosyl)ated proteins were visualized by a
phosphoimaging. A, equal amounts of tankyrase 1 (4 µg)
were incubated without (lane 1) or with increasing amounts
(1, 2, or 4 µg) of TRF1 (lanes 2-4), Mcl-1L (lanes
5-7), or Mcl-1S (lanes 8-10). Although tankyrase 1 poly(ADP-ribosyl)ated itself and TRF1, it did not ADP-ribosylate either
Mcl-1L or Mcl-1S. B, the same amount (2 µg) of tankyrase 1 and TRF1 was incubated in the absence (lane 2) or presence
of increasing concentrations (1, 2, or 4 µg) of Mcl-1L (lanes
3-5) or Mcl-1S (lanes 6-8). Although Mcl-1 proteins
were not ADP-ribosylated by tankyrase 1, inclusion of increasing
amounts of Mcl-1 proteins decreased tankyrase 1-mediated
poly(ADP-ribosyl)ation of TRF1 and tankyrase 1 itself in a concentration-dependent manner.
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DISCUSSION |
Mcl-1, a unique Bcl-2 family protein, is transiently induced by
various growth factors and shows a rapid turnover rate (20, 21).
Earlier studies demonstrated that Mcl-1 expression is up-regulated by
epidermal growth factor, elk-1, granulocyte-macrophage
colony-stimulating factor, gonadotropins, stem cell factor,
interferon-
, and different interleukins in diverse cell lineages
(22-26). Despite numerous studies on the induction of Mcl-1, no
information is available regarding the suppression of the rapidly
induced Mcl-1 proteins. Based on yeast two-hybrid screening, we
identified an Mcl-1-interacting protein, tankyrase 1, that could
provide a novel mechanism to decrease the levels and to suppress the
actions of both Mcl-1L and its splicing variant Mcl-1S.
We demonstrated that overexpression of tankyrase 1 suppressed cell survival induced by Mcl-1L. In addition, tankyrase 1 also effectively blocked apoptosis induced by Mcl-1S. In contrast, overexpression of tankyrase 1 itself exhibited no significant effect on
CHO cell viability, consistent with a recent observation (34).
Furthermore, immunoblot studies showed that coexpression of tankyrase 1 resulted in lower levels of both Mcl-1L and Mcl-1S proteins. Thus,
tankyrase 1-mediated changes in cell viability may be related to its
ability to down-regulate Mcl-1 proteins. The finding of endogenous
interactions between Mcl-1L and tankyrase 1 in a human leukemia cell
line further supports the physiological importance of the present findings.
More than 20 known mammalian Bcl-2 family proteins share common
regulatory mechanisms to modulate apoptosis. They dimerize with other
Bcl-2 family members (1) or interact with specific interacting
proteins. For instance, BAD binding to 14-3-3 proteins suppresses the
apoptotic activity of BAD (18, 27), whereas Bim/BOD binding to the LC8
dynein light chain leads to its sequestration to the
microtubule-associated dynein motor complex (28). Likewise, Bmf
interacts with the dynein light chain 2 before being sequestered to the
myosin V actin motor complex (29), leading to apoptosis suppression.
Here, we have demonstrated that both Mcl-1L and Mcl-1S are strongly
associated with tankyrase 1 in yeast and mammalian cells. Interactions
of tankyrase 1 with Mcl-1 proteins were specific because none of the
other members of the Bcl-2 family that were tested bound to tankyrase 1.
The antiapoptotic Mcl-1L protein consists of two consensus PEST
sequences as well as the BH3, BH1, BH2, and a transmembrane domain.
However, the proapoptotic splicing variant, Mcl-1S, possesses only the
PEST sequences and the BH3 domain. The common N-terminal end of both
Mcl-1 proteins contain the two PEST motifs and four pairs of arginine
residues found in rapidly degraded proteins such as c-Myc and
p53 (30). Although the deletion of these two PEST sequences in Mcl-1
does not affect the half-life of the mutant protein (20), the extreme
N-terminal end of Mcl-1 has a stretch of residues with a weak PEST
homology. We demonstrated that the binding of Mcl-1 proteins to
tankyrase 1 involves this short stretch of 25 amino acids (76 to 100).
Of interest, the same region also contains a
RPPPIG sequence that resembles the
consensus tankyrase-binding motif (RXXPDG) found in other
tankyrase 1-interacting proteins (31).
Tankyrase 1 possesses four distinct motifs: the HPS module, 24 ankyrin
motifs, SAM, and the catalytic domain of PARP (9). Tankyrase 1 is a
unique protein with structural features found in both ankyrin and PARP
family genes. Ankyrin family proteins are linkers that couple diverse
membrane proteins via ankyrin motifs to the underlying cytoskeleton
(32, 33). All known tankyrase 1-interacting proteins (TRF1, IRAP, and
TAB182) bind to tankyrase 1 through ankyrin repeat regions (9, 11, 12). Likewise, our data indicated that interactions between Mcl-1 proteins and tankyrase 1 also involved the ankyrin motifs, and the first half of
the ankyrin repeats (1 to 12) was sufficient for dimerization. Of
particular interest, TRF1 and Mcl-1 are capable of binding to
overlapping ankyrin repeats in tankyrase 1, suggesting potential competition between these tankyrase 1-interacting proteins.
Tankyrase 1 contains the catalytic domain of PARP and is known to
poly(ADP-ribosyl)ate its interacting proteins, TRF1, IRAP, and TAB182
(9, 11, 12). PARP family proteins catalyze the attachment of the
poly(ADP-ribose) moiety onto a protein acceptor using the substrate
NAD+ (nicotinamide adenine dinucleotide), and
ADP-ribosylation of proteins usually leads to protein inactivation
(13-16). However, our study demonstrated that tankyrase 1 does not
poly(ADP-ribosyl)ate either of the Mcl-1 proteins. Furthermore, a
tankyrase 1 mutant with the PARP domain deleted is still capable of
decreasing the Mcl-1 protein levels and blocking the actions of the
Mcl-1 proteins. These data suggest that the ability of tankyrase 1 to
suppress both anti- and pro-apoptotic actions of the Mcl-1 proteins is not mediated by ADP-ribosylation of these proteins. The findings also
suggest that tankyrase 1-interacting proteins are not always the
substrates for tankyrase 1. Consistent with the unique structural features and subcellular localization of tankyrase 1, the current study
suggests that tankyrase 1 is different from other PARP enzymes (9, 11,
35, 36) and could have nonenzymatic functions. Of interest, tankyrase
2, a gene with 83% sequence identity to tankyrase 1, has recently been
isolated (37, 38) and found to induce cell death in different cell
lines (34).
In addition to the tankyrase 1 regulation of Mcl-1 functions, Mcl-1
proteins could reciprocally regulate the function of tankyrase 1. When
Mcl-1L or Mcl-1S are coincubated with tankyrase 1, auto-ADP-ribosylation of tankyrase 1 decreases in a Mcl-1
concentration-dependent manner. Although the physiological
significance of the apparent decrease in poly(ADP-ribosyl)ation of
tankyrase 1 is still unclear and one cannot completely rule out the
nonspecific effects of unknown contaminants in the purified Mcl-1
preparation, Mcl-1 proteins caused a more profound suppression of the
poly(ADP-ribosyl)ation of TRF1 in the same in vitro test.
Because both Mcl-1 proteins and TRF1 interact with the ankyrin repeats
in tankyrase 1, Mcl-1 proteins could compete with TRF1 for binding to
tankyrase 1, leading to lower ADP-ribosylation of TRF1. Because
tankyrase 1-mediated telomere extension is dependent on
ADP-ribosylation of TRF1 (10), inhibition of the ADP-ribosylation of
TRF1 by Mcl-1 could prevent telomere elongation and facilitate cell
senescence. This is consistent with the ability of tankyrase 1 to
suppress the survival action of Mcl-1L, thus leading to the prevention
of cell immortalization.
Recent studies indicated that Mcl-1L is localized to the nucleus (39,
40). and is involved in cell cycle regulation by interacting with the
proliferating cell nuclear antigen (39). Mcl-1 also serves as a nuclear
chaperone for another antiapoptotic protein, fortilin (40). Together
with our observation of interactions between Mcl-1 and tankyrase 1, these findings suggest an important role of Mcl-1 in the regulation of
diverse nuclear events including cell cycle regulation and telomere
elongation. In conclusion, the observed interactions between Mcl-1
proteins and tankyrase 1 represent a novel mechanism for the regulation
of the apoptosis function of Mcl-1 proteins. Because Mcl-1 could also
regulate the function of tankyrase 1 and other nuclear proteins, Mcl-1 could play an important role in the coordinated regulation of cell
survival, proliferation, and immortalization.